摘要:

13 Alicyclic Chemistry ~ ~~ By J. M. MELLOR Department of Chemistry University of Southampton Southampton SO9 5NH 1 Introduction The stimulus of attempted synthesis of natural products -particularly those having a skeleton incorporating five- and seven-membered rings has led to further development of annelation methods.* These are discussed in this Report together with a brief review of advances in the chemistry of adamantanes and of spiro-compounds and then various stereochemical topics are reported. Discus- sion of other important advances may be found in the Chemical Society Specialist Periodical Reports.’ a valuable supplement to Rodd.’ and in reviews concerning the theory3 and application4 of pericyclic reactions a detailed analysis of the cyclobutene-butadiene intercon~ersion,~ the present status of Bredt’s Rule,6 to which further exceptions have been found,’ the use of allylic sulphoxides,* the 1,3-anionic cycloaddition of organolithium compounds9 and the acid- catalysed rearrangement of By-unsaturated ketones ’’ in synthesis and the 0.r.d.and c.d. of decalones.’ 2 Synthesis Three-and Four-membered Rings.-The important use of cyclopropylylides in the development of spiropentanes cyclobutanones and cyclopentanes [Ann. * See also Chapter 15. ’ ‘Aliphatic Alicyclic and Saturated Heterocyclic Chemistry’ ed. W. Parker (Specialist Periodical Reports) The Chemical Society London Vol. 2 1974 Vol. 3 1975. ’ ‘Rodds Chemistry of Carbon Compounds’ ed. M. F. Ansell Elsevier Amsterdam 1974. N.D. Epiotis Angew. Chem. Internat. Edn. 1974 13 751. J. B. Hendrickson Angew. Chem. Internat. Edn. 1974 13,47. L. M. Stephenson and J. I. Brauman Accounts Chem. Res. 1974 7 65. ‘ G. L. Buchanan Chem. SOC. Rev. 1974,3,41. ’ H. 0. Krabbenhoft J. R. Wiseman and C. B. Quinn J. Amer. Chem. SOC.,1974 96 258; D. Lenoir and J. Firl Annalen 1974 1467 J.C.S. Chem. Comm. 1974 858; B. L. Adams and P. Kovacic J. Org. Chem. 1974 39 3090; B. L. Adams and P. * Kovacic J. Amer. Chem. SOC. 1974 96,7014. D. A. Evans and G. C. Andrews Accounts Chem. Res. 1974 7 147. T. Kauffmann Angew. Chem. Internat. Edn. 1974,. 13 627. lo R. L. Cargill T. E. Jackson N. P. Peet and D. M. Pond Accounts Chem. Res. 1974 7 106. ‘I D. N. Kirk and W. Klyne J.C.S. Perkin I 1974 1076. 359 360 J.M. Mellor Reports (B), 1974,70,572]has now been reviewed" by Trost. Cyclopropanation of olefins may be limited by instability of precursors. Two new methods offer some synthetic advantage and have interesting mechanistic aspects. When benzal halides are decomposed by potassium t-but oxide in hydrocarbon sol- vent~~~ in the presence of a crown ether a free carbene not a carbenoid is produced and cyclopropanation may become more efficient. Olefins are cyclo- propanated in moderate yield by the reaction of diphenylsulphonium methylide in ether solventsi4 in the presence of cupric acetylacetonate. An intermediate metal-carbene complex is suggested and the aflalogy made to a possibly related mechanism of cyclopropanation by S-adenosylmethionine.x-. ... MeOzC C0,Me Et0,C C0,Et Reagents i Ni(COD),; ii CH =CHCO,Me; iii Me0,CCH =CHCO,Me (cis); iv Et0,CCH =CHCO,Et (trans) Scheme 1 Details are reported of the synthesis15 of (1) of its quantitative trimerization" by palladium(0) to give (2) and of its nickel(0)-catalysed additions" shown in Scheme 1. Dehydrobromination of (3) at -20 "C affords" the highly strained and reactive cyclopropenone (4). l2 B. M. Trost Accounts Chem. Res. 1974 7 85; see also B. M. Trost and D. E. Keeley J. Amer. Chem. SOC.,1974 96,1252. R. A. Moss and F. G. Pilkiewicz J. Amer. Chem. SOC.,1974 % 5632. l4 T. Cohen G. Herman T. M. Chapman and D. Kuhn J. Amer. Chem. Soc. 1974,96 5627. " P. Binger Synthesis 1974 190. l6 P. Binger G. Schroth and J.McMeeking Angew. Chem. Internat. Edn. 1974 13 465. P. Binger and J. McMeeking Angew. Chem. Internat. Edn. 1974 13 466. 18 M. Suda and S. Masamune J.C.S. Chem. Comm. 1974 504. Alicyclic Chemistry A novel synthesis of bicyclob~tanes'~ (Scheme 2) is limited to cases with elec- tron-withdrawing substituents and in the final step yields are moderate (-20 "/,). Me0,C C02Me C0,Me NMe C0,Me -PNMe Reagents i MeOSO,CF ; ii NaH N-methylpyrrolidone Scheme 2 Synthesis of cyclobutadiene and isolation by matrix techniques2' has been reviewed. Although the photoelectron spectrum of (5) has been recorded2' S and suggests a singlet ground state the existing spectroscopic data concerning cyclobutadiene are too conflicting to permit an unequivocal assignment to the ground state.However X-ray analysis22 establishes that (5) is rectangular (bond lengths 1.344 and 1.600 A).The larger bond length may be partly attributed to repulsive methyl-methyl interactions. '' H. K. Hall and P. Ykman J.C.S. Chem. Comm. 1974 587. 2o G. Maier Angew. Chem. Internat. Edn. 1974 13 425. 21 G. Lauer C. Muller K.-W. Schulte A. Schweig and A. Krebs Angew. Chem. Internat. Edn. 1974 13 544. 22 H. Irngartinger and H. Rodewald Angew. Chem. Internat. Edn. 1974 13 740. 362 J. M. Mellor Ref. 23 +OEt iii iv 30 Ref. 24 -cy" OEt C0,Me SMe --P p-SOMeM e 1v.659~, lv.650,0, (SMe SOMe Ref. 25 CH,Ph CH,Ph Ref. 26 Reagents i R'CH=CHR2; ii OH-; iii CH,=CHCO,Me A; iv H+; v KH THF PhCH,CH(CH,Br),; vi Me,SiCl NEt, DMF; vii CH,12 Zn-Ag; viii CH =CCNCl Scheme 3 Important new syntheses of cyclobutanones are shown in Scheme 3.23-27 Conia et dZ6 have developed methods for cyclopropanation of silyl enol ethers and hence have extended the useful route from silyl ethers of vinylcyclopropanols to cyclobutanones and cyclopentanones.Biscyclopropanation affords2*l-cyclo-propylcyclopropanols in high yield. 23 A. Sidani J. Marchand-Brynaert and L. Ghosez Angew. Chem. Internat. Edn. 1974 13 267. 24 P. Amice and J. M. Conia Tetrahedron Letters 1974 479; Bull. SOC. chim France 1974 1015. 25 K. Ogura M. Yamashita M. Suzuki and G. Tsuchihashi Tetrahedron Letters 1974 3653. 26 C. Girard P. Amice J. P. Barnier and J. M. Conia Tetrahedron Letters 1974 3329.21 J. 0.Madsen and S. 0. Lawesson Tetrahedron 1974 30,3481. 28 C. Girard and J. M. Conia Tetrahedron Letters 1974 3333. A licyclic Chemistry 363 (6) (7) (8) Opening29 of the epoxide (7) by anionic attack leads preferentially to the cyclo- butane (8) and not the cyclopentane (6). This kinetically controlled opening favoured by greater facility for a backside displacement should become an important method for cyclobutane synthesis. Reaction is stereospecific and the ring size of the product is not determined solely by the relative steric hindrance to attack at the two epoxide centres. Thus cyclization of (9)affords (10)and hence grandisol(1l),a sex attractant that is released by the male boll weevil. * *ow OH +OH 0 OTHP -+ CN (9) (10) (1 1) Five-membered Rings.-A promise of things to come rather than an immediate synthetic use is to be found in the probable synthesis of cy~lopentanone~’ from ethylene and carbon monoxide (Scheme 4) and the formation in low yield (5:<) of cyclopentenones by acid-promoted decomposition3 of the iron carbonyl complex of butadiene.I Cp,TiN=NTiCp + c TiCp -+ Reagents; i CH,=CH,; ii CO Scheme 4 Of greater immediate synthetic value are methods shown in Scheme 5.3220 In particular some efficient and elegant syntheses of cyclopentenones have been ” G. Stork and J. F. Cohen J. Amer. Chem. SOC.,1974 96 5270. 30 J. X. McDermott and G. M. Whitesides J. Amer. Chem. SOC.,1974 % 947. 3’ B. F. G. Johnson J.Lewis and D. J. Thompson Tetrahedron Letters 1974 3789. 32 T. Cuvigny M. Larcheveque and H. Normant Tetrahedron Letters 1974 1237. ” I. Kawamoto S. Muramatsu and Y. Yura Tetrahedron Letters 1974 4223. 34 A. A. Schegolev W. A. Smit G. V. Roitburd and V. F. Kucherov; Tetrahedron Letters 1974 3373. 35 P. M. McMurry and R. K. Singh J. Org. Chem. 1974 39 2317. 3h R. F. Romanet and R. H. Schlessinger J. Amer. Chem. SOC.,1974 96 3701. 37 T. Hiyama M. Tsukanaka and H. Nozaki J. Amer. Chem. Soc. 1974 % 3713. 38 P. L. Fuchs J. Amer. Chem. Soc. 1974 % 1607. 39 J. P. Marino and W. B. Mesbergen J. Amer. Chem. SOC.,1974 % 4050. O0 S Danishefsky J. Dynak E. Hatch and M. Yamamoto J. Amer. Chem. SOC.,1974 96 1256. 364 J.M. MeIlor C,H,3 . )=NC,H I I i 73% C6H ,,COCH,CH,CCI=CH 1 ii 18* Ref.32 iii 80% C6H ,COCH2CH,COCH3 0 Ref. 34 Ref. 35 SMe Ref. 37 A licy clic Chemistry EtO,C COzMe Ref. 38 :4. Ref. 39 HO S0,Ar t C0,Et Ref. 40 72 % Reagents i Li Et,NH C,H, HMPT ClCH -CCl=CH,; ii H'; iii OH- iv + s\/s v NaH CH =CMePPh,; vi Ni; vii CH,Cl, C,H,CI, -60 "C; viii RLi R = MeCH,CH=CHCH,CH,Li; ix CrO,; x 3% NaOH; xi NaH THF; ... xii MeCH=C /SMe . XIII CH,=CHCH,I; xiv R,N+Br- CHCI,, 'SOMe ' OH-; xv NaH HMPA; xvi xvii Pr,NLi; xviii CH,= CHC0,Et; xix CF,CO,H CH,Cl,; xx Dimsylsodium DMSO 95 "C Scheme 5 deveioped. Some of the syntheses in Scheme 5 have wide application and show a welcome diversification of the objectives of synthesis of five-membered rings from those of prostaglandins.In this latter area. elegant alternative routes have been developed to the Corey intermediate (12) (Scheme 6). Use4' of 6-acetoxy-OCOC,H,Ph (12) E. D. Brown R. Clarkson T. J. Leeney and G. E. Robinson J.C.S. Chem. Comm. 1974. 642. 366 J. M. Mellor Ref. 41 (13) CN CN CO,H ___* 1vi Ref. 42 f-Br Br 0 Reagents i CH,=CCICN; ii H + ; iii (CH,O) HC0,H; iv OH -; v CrO,; vi 48 % HBr AcOH; vii CH,C03H Scheme 6 fulvene (13) avoids the problem of isomerization of monosubstituted cyclo-pentadienes and Prins reaction with n~rbornadiene~~ provides a four-pot reaction sequence to (14). Six-membered Rings.-Application of Robinson annelation procedures has been limited by failure to obtain products from a single enolate anion.Last year the important advance of regiospecific annelation -by preventing equilibration of enolate anions was reported [Ann. Reports (B) 1974 70 5811. This method has been further developed and will become established as a principal annelation technique. The annelations in Scheme 7 show that in ether43 or gl~rne,~~ equilibration of the lithium enolates is slow. Hence generation of a single enolate anion by reduction of (1 5). trapping as a silyl ether. and subsequent formation of the lithium enolate with methyl-lithium permits efficient annelation with the silylated vinyl ketone. An efficient synthesis of D-homotestosterone is described.43 42 R. Peel and J. K. Sutherland J.C.S. Chem. Comm. 1974 15 1 43 R.K. Boeckman J. Amer. Chem. SOC.,1974 90 6179. G. Stork and J. Singh J. Amer. Chem. SOC.,1974. 96 6181. 44 Alicyclic Chemistry 367 1iii Refs. 43 44 iv. v 0p-Liom H a2 -Ref.45 LiO H H 0 CH,OH Bu'0,C LiO H0,C -1xi xii Ref. 46 SiMe Reagents i Li NH, Bu'OH; ii Me,SiCl; iii MeLi; iv =(COMe; v NaOMe MeOH; vi CH,O -78°C; vii NaOEt EtOH CH,COCH,CO,Et; viii aq. KOH; ix THF; x H +;xi ClCO,Me Et,N; xii NaN,; xiii MeOH 40 "C;xiv 2 % KOH MeOH H,O Scheme 7 An alternative method ofannelati~n~~ proceeds by condensation of formaldehyde with a regiospecifically generated enolate anion to give an intermediate p-hydroxy-ketone. Elimination and Michael addition of ethyl acetoacetate com- pletes the annelation procedure in high yield.A further modification of the 45 G. Stork and J. d'Angelo J. Amer. Chem. SOC.,1974 96,71 14. 368 J. M. Mellor Robinson annelation is the use of y-hal~genotiglates,~~ which although it requires a multi-stage sequence proceeds under mild conditions. It has been recognized that equilibration of a kinetically generated enolate anion is much more facile with a potassium or sodium counter-ion but it has now been possible to alkylate at -70 "C with methyl iodide47 to give products from the kinetically generated potassium enolate without substantial formation of products from the thermo- dynamically stable enolate. The above use of silylated vinyl ketones and the use of silylallyl halides in alkylation procedures establishes the growing importance of silicon chemistry in the armoury of synthetic organic chemists.By suitable choice of substitution silylallyl halides can be used4* in the development of both 1,5-and lP-diketones and lead to new efficient methods of formation of cyclohexenones and cyclo- pentenones (Scheme 8). Me,% cr+ LiOni. Me,Si 1ii iii + 0.2 0-II Reagents i THF; ii m-ClC,H,CO,H; iii KOH; iv H' Scheme 8 Formation of cyclobutanes rather than cyclopentanes by anionic attack upon epoxides2' has been noted. The method may be used in a novel synthesis49 of six-membered rings. Thus (16)is converted into (17) in 70 yield. However 46 P. L. Stotter and K. A. Hill J. Amer. Chem. Soc. 1974 % 6524. L. Nedelec J. C. Gasc and R.Bucourt Tetrahedron 1974 30 3263. G. Stork and M. E. Jung J. Amer. Chem. SOC.,1974 % 3682. 49 G. Stork L. D. Cama and D. R. Coulson J. Amer. Chem. SOC.,1974 96 5268. Alicyclic Chemistry CN the critical steric constraints which determine the size of ring formed are illus- trated by conversion of (18) and (19)into (20)and (21) respectively. Addition” of enolate anions to phosphonium bromide (22) affords cyclohex- adienes. Yields are moderate but the reaction has considerable generality and complements the alternative cyclohexadiene synthesis of reaction of ally1 ylides with afl-unsaturated ketones. No new methods of effecting Johnson cationic cyclizations have been reported but full details of the synthesis of cis-51 and trans-5* fused tetracycles and preliminary communications of the synthesis of triterpenes ~erratenediol~ and hio on one^^ have appeared.Although 7r4s + 7r2s Diels-Alder cycloadditions by intermolecular reaction have been used in synthesis of nootkatone and ~t-vetivone~~ and [2,2]paracyclo- phane~~~ from 1,2,4,5-hexatetraene (Scheme 9) of greater interest has been the Scheme 9 P. L. Fuchs Tetrahedron Letters 1974 4055. ” K. E. Harding E. J. Leopold A. M. Hudrlik and W. S. Johnson J. Amer. Chem SOC. 1974 96 2540; R. L. Carney and W. S. Johnson ibid. p.. 2549 K. A. Parker and W. S. Johnson ibid. p. 2556. 52 W. S. Johnson K. Wiedhaup S. F. Brady and G. L. Olson J. Amer. Chem. SOC. 1974 96 3979. 53 G. D. Prestwich and J. N. Labovitz J. Amer. Chem. SOC.,1974 96 7103.54 R. E. Ireland C. A. Lipinski C. J. Kowalski J. W. Tilley and D. M. Walba J. Amer. Chem. SOC.,1974 96,3333. 55 K. P. Dastur J. Amer. Chem. SOC.,1974 96,2605. 56 H. Hopf and F. T. Lenich Chem. Ber. 1974 107 1891. 370 J.M. Mellor 1200"C Ref. 58 Ref. 59 Ref. 61 A licyclic Chemistry 371 +y-JL 'OH -280 "C 60 % 1 Ref. 62 Reagents Wolff-Kishner; ii HBr AcOH; iii LiAlH iv MeLi; v NaBH,MeOH. NaOH; vi MeO,CCrCCO,Me; vii Scheme 10 clever way in which intramolecular additions have been put to syntheticadvantage (Scheme An amusing footnote makes clear that the synthesis of pat- chouli alcohol (23) was not computer-inspired. Indeed with the Diels-Alder addition only proceeding at 280°C in the presence of potassium t-butoxide there is still a place for the traditional experimental skills.Seven-membered Rings.-No radically new syntheses have been reported but important extensions of the addition of ally1 cations to dienes have been made (Scheme 1163-66). The use of carbonyl-stabilized sulphoxonium ylides has been extended6 to the synthesis of hydroazulenes (Scheme 12). Cyclopropyl ketones 5' C. A. Cupas and L. Hodakowski J. Amer. Chem. SOC.,1974 96,4668. 58 A. Pryde J. Zsindely and H. Schmid Helv. Chim. Acta 1974 57 1598. 59 G. W. Klumpp and R. F. Schmitz Tetrahedron Letters 1974 291 1. '"H. Greuter and H. Schmid Helv. Chim. Acta 1974 57 1204. '' L. A. Paquette and M. J. Wyvratt J. Amer. Chem. SOC.,1974 96 4671; D. McNeil B. R.Vogt J. J. Sudol S. Theodoropulos and E. Hedaya ibid. p. 4673. 62 F. Naf and G. Ohloff Helv Chim. Acta 1974,57 1868. 63 R. Schmid and H. Schmid Helo. Chim. Acta 1974 57 1883. 64 H. M. R. Hoffmann and A. E. Hill Angew. Chem. Internat. Edn. 1974 13 136; A. E. Hill and H. M. R. Hoffmann J. Amer. Chem. SOC.,1974 % 4597. 65 J. G. Vinter and H. M. R. Hoffmann J. Amer. Chem. SOC.,1974 % 5466. '' R. Noyori Y. Baba and Y. Hayakawa J. Amer. Chem. SOC.,1974 % 3336. '' J. P. Marino and T. Kaneko J. Org. Chem. 1974 39 3175. 372 J. M. Mellor Ref. 63 Ref. 64 Ref. 66 Br Reagents i C,H6 CH,Cl, AgBF, -60 "C; ii MeOH NaOH; iii AgO,CCF, Na,CO, isopentane; iv H'; v Zn-Cu; vi Br,CHCOCHBr, FeJCO), CeH6 Scheme 11 0 0 CI CH=SOMe,1ii Ref.67 C0,Et t CHO \ Reagents i CH,SOMe, THF; il CH,=CHCHO; iii EtO,CCHLiPO(OEt) Scheme I2 A1icyclic. Chemistry 373 (24) (25) can undergo efficient ring expansion on thermolysis. Hence (24) gives6* (25) in 70% yield. Adamantans.-The increased commercial availability of adamantane and simple derivatives has led to great activity in simple transformations (described in ref. 1). Now a number of important advances are reported. Schleyer and his report with full experimental detail the procedure whereby diamantane (26) (26) may be obtained in better than 50% overall yield in three steps from norborn- adiene. This should make diamantane as readily available as adamantane and already the Princeton group have publi~hed’~ extensive details of the functional- ization of diamantanes.Derivatives of 1-aminoadamantane have attracted interest because of their anti-viral activity. Electrochemical oxidation of adamantane in acetonitrile7 ’ introduces functionality directly to give N-(1-adamanty1)acetamide in 75 x) yield. The use of empirical force-field calculations in understanding the chemistry of polycyclic systems has greatly advanced. An extensive review by M~Kervey~~ on adamantane rearrrangements gives a clear picture of the observed rearrange- ments and it is gratifying to find the reliability of strain calculations. Recent calculations rationalize the ob~ervation~~ that of the C12HI isomers ethano- S. A. Monti and T. W. McAninch Tetrahedron Letters 1974 3239.h9 T. M. Gund E. Osawa V. Z. Williams and P. von R. Schleyer J. Org. Chem. 1974 39 2979. ’O T. M. Gund M. Nomura and P. von R. Schleyer J. Org. Chem. 1974 39 2987; T. M. Gund P. von R. Schleyer G. D. Unruh and G. J. Gleicher ibid. p. 2995; I. Tabushi S. Kojo P. von R. Schleyer and T. M. Gund J.C.S. Chem. Comm. 1974 591. ” V. R. Koch and L. L. Miller Tetrahedron Letters 1973; 693; J. Amer. Chem. SOC. 1973 95 8631. l2 M. A. McKervey Chem. SOC.Rev. 1974 3 479. 73 D. Farcasiu E. Wiskott E. Osawa W. Thielecke E. M. Engler J. Slutsky P. von R. Schleyer and G. J. Kent J. Amer. Chem. SUC.,1974 96,4669. 374 3. M. Mellor adamantane (27) is the most stable. Ethanoadamantane can be prepared by rearrangement of (28) and it has been independently synthesized from adaman- tane-2-carboxylic acid.Calculations have also been used to predict bridgehead reactivities and rationalize the observations that manxane (29)very easily gives74 mono- and di-peroxides that 1-manxyl chloride is lo4 times more reactive74 in solvolysis than t-butyl chloride and that protoadamantane (30) preferentially brominates” to give (31). Spiro-compounds.-In addition to a number of specific syntheses of spirocyclic compounds (Scheme 1376-79),which are of some interest because of their physical Ph BF,-%Ph@ph ph$Lh Ref. 76 ___* MeOH Ph Ph Ph Ph Ph PI1 CH COPha~ COCl hv -7O”,C b Ref. 79 Ph Ph Ph Reagents i CH,=CCICOCl; ii NaN,; iii H’;iv TsNHNH,; v MeLi Scheme 13 74 W. Parker R.L. Tranter C. I. F. Watt L. W. K. Chang and P. von R. Schleyer J. Amer. Chem. SOC., 1974 96,7121. 75 A. Karim and M. A. McKervey J.C.S. Perkin I 1974 2475. 76 R. Weiss and S. Andrae Angew. Chem. Internat. Edn. 1974 13 271. 77 L. A. Hulshof and H. Wynberg J. Amer. Chem. SOC., 1974 96 2191; L. A. Hulshof M. A. McKervey and H. Wynberg ibid. p. 3906. A. de Meijere and L.-U. Meyer Tetrahedron Letters 1974 2051. 79 H. Durr M. Kausch and H. Kober Angew. Chem. Internat. Edn. 1974 13 670. A/icyclic Chemistrjq Ref. 80 Reagents i THF; ii SnCI, CH,Cl Scheme 14 properties some general methods (Scheme 1480,81)and a reviewg2 of intra-molecular alkylations leading to spiro-compounds have appeared. Of par-ticular interest is the high stereoselectivity observed in the Trost synthesis' ' and the further demonstration of the relatively complex systems which may be obtainedg0 by pyrolysis of ketones uiu their enols.Miscellaneous Syntheses.-Further approaches to the synthesis of trans-cyclic olefins are reported (Scheme 1 5"3-85) synthesis of trrrns-cyclo-octene ria a 0"" '0H OSPh Ref. 84 Reagents i Ph,PLi THF; AcOH H,O,; iii NaH DMF; iv NaBH,S,; v LiAIH,; vi PhCHO H'; vii LiNR,; viii KOBu' CHI,; ix AgCIO, MeOH; x MeLi Scheme 15 F. Leyendecker J. Drouin and J. M. Conia Tetruhedron Letters 1974 2931. n1 B. M. Trost and D. E. Keeley J. Amer. Chem. SOC.,1974 96,1252. B2 A. P. Krapcho Synthesis 1974 383. n3 A. J. Bridges and G. H. Whitham J.C.S. Chem. Comm. 1974 142. 13' M. Jones P. Temple E.J. Thomas and G. H. Whitham J.C.S. Perkin I 1974 433. B5 M. S. Baird J.C.S. Chem. Comm. 1974 196. 376 I:) J. M. Mellor /-P-hydroxyphosphine oxideg3 is particularly efficient. In improvements to the synthesis of large rings muscone (32) has been obtained via isocyanide insertion into a bis-77-allylnickel complexs6 and cembrene (33)via a nickel-tetracarbonyl- induced couplings7 of terminal allylic bromides. Efficient methods of ring ex- pansion by one carbon proceeding by carbenoid intermediates have been developed (Scheme 16g8,89). 1 li Ref. 88 Reagents i CH,Br, LiNR,; ii BuLi; iii CCI,; iv MeLi v H + ; vi H Scheme 16 n6 R. Baker R. C. Cookson and J. R. Vinson J.C.S. Chem. Comm. 1974 5 15. W. G. Dauben G. H. Beasley M.D.Broadhurst B. Muller D. J. Peppard P. Pesnelle and C. Suter J. Amer. Chem. SOC.,1974 96,4724. '' H. Taguchi H. Yamamoto and H. Nozaki J. Amer. Chem. Soc. 1974 96,3010,6510. 89 T. Hiyama T. Mishima K. Kitatani and H. Nozaki Tetrahedron Letters 1974 3297. 377 A licyclic Chemistry Ref. 90 Ref. 91 Ref 92 Reagents i electrochemical reduction; ii NCS RC0,H; iii KOBu'; iv hv; v CH,N,; vi CHBr, KOBu'; vii Bu,SnH Scheme 17 The continued interest in propellanes has stimulated further synthetic work (Scheme 1790-93).The [2,2,2]propellane (34) has not been isolated but electro- chemical reduction" of 1,4-dibromobicyclo[2,2,2]octane (35) at ca. -20 "C followed by addition of chlorine led to formation of 1,4-dichlorobicyclo[2,2,2]-octane.Reasonably it was concluded that the only molecular species capable of acting as a precursor of this dichloride was (34). In a similar analysis formation of (35) following irradiation of ~.4-dimethylene~yclohexane~~ and subsequent 90 K. B. Wiberg G. A. Epling and M. Jason J. Amer. Chem. SOC.,1974 96,912. 9' K. Weinges and K. Klessing Chem. Ber. 1974 107 1915. " D. H. Aue and R. N. Reynolds J. Org Chem. 1974,39 2315. y3 P. Warner R. LaRose and T. Schleis Tetrahedron Letters 1974,1409. y4 J. J. Dannenberg T. M. Prociv and C. Hutt J. Amer. Chem. Soc. 1974 96 913. 378 J. M. Mellor CI CI ~-~o+Br ~ 1 o-Br Ref. 95 Ref. 96 Ref. 98 Ref. 99 1xii. xiii Reagents i NBS aq. DMSO; ii CrO,; iii Et,N; iv hv;v NBS; vi Zn; vii AlCl,; viii CO; ix Base;-x 40% KOH; xi Ce4+;xii H,; xiii NaH Me1 Scheme 18 Alicyclic Chemistry 379 addition of bromine established the photoconversion in low yield of the diene into (34).Further novel transformations are reported in Scheme 18.95-99 3 Stereochemistry Conformational Aspects.-The commemorative issues of Tetrahedron celebrating the centenary of the development of the early stereochemical concepts of Van? Hoff and Le Be1 provide a wealth of interesting reviews. They appear at an im- portant time in the development of ideas concerning conformational analysis. The traditional basis of analysis of carbocyclic systems has concerned repulsive steric interactions dipolar interactions and hydrogen bonding. Various ex- perimental observations have for some time suggested the incompleteness of the earlier concepts.Some recent advances have concerned the &perimental observation of and the theoretical rationalization of additional types of inter- action. The importance of orbital interactions has been confirmed by photoelectron spectroscopy. Recognition of their importance permits an understanding of the anomeric effect the preferred ground states of simple acyclic molecules such as fluoroacetaldehyde and the greater stability of axial 2-halogenocyclohexanones over equatorial 2-halogenocyclohexanones. Ab initio calculations now pre-diet 100,101 conformer populations in good agreement with experiment for simple systems. However there remain areas of uncertainty concerning the very basic concepts of conformational analysis.A destabilizing gauche interaction (as in n-butane) has traditionally been interpreted to stem from a repulsive methyl- methyl interaction. Hence the greater stability of an equatorially substituted cyclohexane was attributed to the absence of a destabilizing gauche interaction. Now Wertz and Allinger,'02 using their strain calculations conclude that this analysis is probably incorrect. Instead they attribute the instability of a gauche conformation (as in n-butane) to a repulsive gauche hydrogen-hydrogen inter- action. The greater stability of an equatorial cyclohexane might then be at- tributed to the preference of the hydrogen for an axial site to minimize thegauche hydrogen-hydrogen interactions.Experimental methods for analysis of conformational phenomena continue to be perfected. Low-resolution microwave spectroscopy can now be used for molecules as complex as the ionones;lo3 the value of shift reagents in confor- " R. Breslow M. Oda and T. Sugimoto J. Amer. Chem. SOC.,1974 96 1639. 96 H.-P. Loffler Tetrahedron Letters 1974 787. '' V. Heil B. F. G. Johnson J. Lewis and D. J. Thompson J.C.S. Chem. Comm. 1974 270. J. M. Harless and S. A. Monti J. Amer. Chem. SOC.,1974 96 4714. 99 J. Meinwald and J. Mioduski Tetrahedron Letters 1974 4137. loo 0. Eisenstein N. T. Anh Y.Jean A. Devaquet J. Cantacuzene and L. Salem Tetra-hedron 1974 30 171 7. lo' J. A. Pople Tetrahedron 1974 30 1605 W. A. Lathan L. A. Curtis W. J. Hehre J. B. Lisle and J.A. Pople Progr. Phys. Urg. Chem. 1974 11 175. lo' D. H. Wertz and N. L. Allinger Tetrahedron 1974 30,1579. lo3 W. E. Steinmetz J. Amer. Chem. SOC.,1974. 96.685. 380 J. M. Mellor mational analysis has been reviewed,'04 and by a combination of deuteriation use of very low temperatures and a new generation of n.m.r. spectrometers great advances have been possible. Thus by ob~ervation'~~ of spectral changes at -183 "C,a AG* value of 4.2 kcal mol- ' was determined for cyclohexanone. Further AG * values are reported for substituted cyclohexanones and methylene- cyclohexanes,' O6 and reviews concern the preferred conformers of medium-ring ketones,'07 cyclobutanes,'08 and five-membered rings. 'O9 An important advance has been the unequivocal demonstration of the dis- tinctive photochemistry of individual conformers.As photochemical reactions of cyclohexyl phenyl ketones are more rapid than ring inversion product com- positions are determined' lo by populations of ground-state conformers (the axial phenyl ketone gives Norrish Type I1 products and the equatorial phenyl ketone gives Norrish Type I products). In contrast in cyclopentyl phenyl ketones conformational processes are fast with respect to photoreaction. Factors controlling Selectivity.-Last year theoretical studies were reported [Ann. Reports (B) 1974,70 5961 which concluded that stereoselectivity might be controlled by homoconjugative and hyperconjugative interactions. Further experimental evidence is now reported which in certain cases shows the dominant control of such interactions and suggests in other cases that it is an important factor which until recently had been neglected.Through-bond effects have been reviewed by Gleiter,' ' and the previously unexplained distinctive photochemistry of cyclobutanones which can undergo ring expansion to give oxacarbenes is now attributed to through-bond effects controlling the reaction pathway as a-cleavage proceeds.' ' The control of stereospecificity by 'remote' substituents is clearly observed in additions to 7-substituted-norbornadienes. Results shown in Scheme 19' '3,1 l4 indicate that both endo-em-and syn-anti-specificity is determined by the 'remote' substituent which in these examples would not be expected to exercise a steric influence.Although steric effects'I5 have been considered to be operative in the addition of hexachlorocyclopentadieneto 7-substituted norbornadienes where the endo mode is increasingly favoured through the series 7-H 7-Me 7-OAc and 7-OBut the present Reporter considers that these and the results in Scheme 19 may be attributed to orbital interactions. Products in each series will J. D. Roberts G. E. Hawkes J. Husar A. W. Koberts and D. W. Roberts J. Amer. Chem. SOC.,1974 96,1833. lo5 F. A. L. Anet G. N. Chmurny and J. Krane J. Amer. Chem. SOC.,1973 95,4423. Io6 M. Bernard L. Canuel and M. St. Jacques J. Amer. Chem. SOC.,1974 96 2929; J. B. Lambert R. R. Clikeman and E. S. Magyar ibid. p. 2265. lo' F. A. L. Anet M. St. Jacques P. M. Henrichs A.K. Cheng J. Krane and L. Wong Tetrahedron 1974 30 1629. '" F. A. Cotton and B. A. Frenz Tetrahedron 1974 30 1587. Io9 J. B. Lambert J. J. Papay S. A. Khan K. A. Kappauf and E. S. Magyar J. Amer. Chem. SOC.,1974 96 61 12. 'lo F. D. Lewis R. W. Johnson and D. E. Johnson J. Amer. Chem. SOC.,1974,96 6090. 'IL R. Gleiter Angew. Chem. internat. Edn. 1974 13 696. 'I2 W. D. Stohrer G. Wiech and G. Quinkert Angew. Chem. Internat. Edn. 1974,13 199. M. Franck-Neumann and M. Sedrati Angew. Chem. internat. Edn. 1974 13 606. ' '' K. Mackenzie Tetrahedron Letters 1974 1203. I L. T. Byrne A. K. Rye. and D. Wege Austral. J. Chem. 1974 27 1961. 38 1 Aliqvclic Chemistry Ref. 113 0Bu' c1 CI Me0 OMe -17 o( OMe -3Oo,/0 Ref. 114 CI + Me0 OMe -53% OBu' Scheme 19 382 J.M. Mellor be determined by the relative HOMO/LUMO interactions. Substituent-double- bond a-n interactions modify reactivity' 13,' l4 in the expected manner. It has been suggested that a major factor determining the direction of attack on a cyclohexanone or methylenecyclohexane is the interaction of the n-orbital with the symmetrical orbital of the fi C-C a-bonds which results in different electron densities on the two faces of the double bond. As the electron density is expected to be higher on the equatorial side of the double bond for the HOMO electrophilic attack should proceed from the equatorial side. Conversely nucleophilic attack should proceed from the axial face. Experimental support for these views challenging the importance of steric effects is obtained' ' from the preferred quenching of anions (36) and (37) from the equatorial face with methyl bromide the well-established preference for reduction of unhindered cyclo- hexanones from the axial face and the recently proven preference for electro- philic attack on thians [e.g.(38)] from the equatorial face.''' In the absence of substantial steric effects n-a interactions afford a good explanation of observed selectivity. However there are sufficient anomalies that one echoes the recent cri de coeur"* 'The origin of the marked stereochemical control in the reduction of cyclohexanones with metal-hydride reducing agents such as sodium boro- hydride is a fascinating but persisting puzzle in organic chemistry.' With highly hindered boranes axial cyclohexanols are obtained' with very high selectivity.A valuable approach to the problem of stereoselectivity is the computation of the ground-state congestion at the two faces of a carbonyl group. Calculations'20 agree reasonably with observed stereoselectivity. Recent studies of Grignard additionsI2' and the reaction of 'ate' complexes,'22 the products of the reactions of metal alkyls with Lewis bases with carbonyl com- pounds indicate that although high stereoselectivity may be observed the basis for such selectivity is not understood. '16 J. Klein Tetrahedron 1974 30,3349. 'I7 J. Klein and H. Stollar Tetrahedron 1974 30 2541. '18 D. C. Wigfield and D. J. Phelps J. Amer. Chem. SOC. 1974 96 543.'I9 J. Hooz S. Akiyama F. J. Cedar M. J. Bennett and R. M. Tuggle J. Amer. Chem. SOC.,1974 96 274. W. T. Wipke and P. Gund J. Amer. Chem. SOC.,1974,96 299. ''I E. C. Ashby J. Laemmie and H. M. Neumann Accounts Chem. Res. 1974 7 272. E. C. Ashby L.-C. Chao and J. Laemmie J. Org. Chem. 1974 39 3258.

ISSN:0069-3030    

DOI:10.1039/OC9747100359

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

14 Nucleic Acids By R. J. H. DAVIES Biochemistry Department Medical Biology Centre The Queen‘s University of Belfast Belfast BT9 7BL 1 Introduction The advent of a new journal is usually regarded ambivalently by those reviewing the literature! However the appearance of Nucleic Acids Research in 1974 is to be welcomed for concentrating many of the contributions in this field into a single source. The first two volumes in a new series entitled ‘Basic Principles in Nucleic Acid Chemistry’ contain a number of advanced reviews on physico- chemical aspects of nucleic acids.’ The latest additions to the series ‘Methods in Enzymology’ give practical details for many of the established procedures used in the study of nucleic acid and protein synthesis.2 A much-needed author- itative textbook on the physical chemistry of nucleic acids has been published which affords an excellent comprehensive treatment of this subject3 On a more relaxing note ‘The Path to the Double Helix’4 provides an interesting and readable historical perspective of the great discovery.It is particularly fitting that the year which marked the twenty-first anniversary of Watson and Crick’s announcement of the structure of DNA should see another triumph for X-ray crystallographers in the determination of the three-dimensional structure of a tRNA molecule. The honours for the first correct structure at 3 A resolution appeared to have been won by Rich and his group,’ at MIT when they published their results for yeast tRNAPhe in March. However in August Robertus and co-workers6 at the MRC Laboratory in Cambridge who had been working on another crystal form of the same tRNA proposed a model differing significant- ly from that of the MIT group.The outstanding feature of the MRC model was ‘ ‘Basic Principles in Nucleic Acid Chemistry’ ed. P. 0. P. Ts’O Academic Press London 1974 vols. 1 and 2. * ‘Methods in Enzymology’ ed. L. Grossman and K. Moldave Academic Press London 1974 vols. 29 and 30. V. A. Bloomfield D. M. Crothers and I. Tinoco ‘Physical Chemistry of Nucleic Acids’ Harper and Row London 1974. R. Olby ‘The Path to the Double Helix’ Macmillan London 1974. F. L. Suddath G. J. Quigley A. McPherson D. Sneden J. J. Kim S. H. Kim and A. Rich Nature 1974 248 20. J. D. Robertus J. E.Ladner J. T. Finch D. Rhodes R. S. Brown B. F. C. Clark and A. Klug Nature 1974 250 546; A. Klug J. D. Robertus J. E. Ladner R. S. Brown and J. T. Finch Proc. Nut. Acad. Sci. U.S.A. 1974 71 371 I. 383 R.J. H. Darks that it identified in addition to the base pairs of the familiar cloverleaf a number of base pairs and base triplets -mostly not of the Watson-Crick type which ~ help to maintain the tertiary structure of the molecule. Most of these additional interactions can be related to constant features of tRNA sequences and the structure as a whole accords well with the chemical reactivity of various tRNAs. The MIT workers have since revised their original structure7 so that it is now in close agreement with that of the MRC group apart from some minor differences in interpretation.This new model for tRNA structure. which is discussed further in the section on tRNA will no doubt inspire detailed investigations by n.m.r. spectroscopy. Thanks to recent advances in instrumentation this technique can now be effec- tively applied to conformational studies of tRNA molecules in solution.8 It should be possible to compare the crystal structure of yeast tRNAPhe with that in solution and to determine which features of the secondary and tertiary structure are common to other tRNAs. Perhaps another twenty one years will see the unveiling of a three-dimensional map of the ribosome. This year has seen some more elegant experiments involving affinity-labelling probes (see p. 400),which have begun to define the location of the ribosomal proteins and RNA molecules around the peptidyl-transferase centre.Some indication of the progress made in the sequence analysis of nucleic acids over the past few years can be gained from a recent handbook' containing all the sequences published up to March 1974. This year alone many more tRNA sequences have been reported together with substantial sequence data on a variety of mRNAs and viral RNAs. While the methodology for sequencing small RNA molecules is now well established that for DNA is still being actively explored and refined. The available methods for DNA sequencing have been reviewed," and a number of DNA sequences have appeared during the year. The longest of these is a tract of 89 nucleotides in phage fl DNA." It seems clear that the routine sequencing of DNA molecules containing up to several hundred nucleotides should be achieved in the near future.This will generally be sufficient to analyse the individual cleavage fragments produced from larger DNA molecules by the action of restriction endonucleases. Already the cleavage fragments produced by different restriction enzymes have been ordered for DNA molecules containing up to tens of thousands of base pairs and the complete sequencing of such molecules may now be anticipated. As will be discussed in the relevant sections the available sequences of RNA and DNA frequently reveal homologies and elements of symmetry which have profound implications for the evolution and function of the nucleic acid molecules.' S. H. Kim F. L. Suddath G. J. Quigley A. McPherson J. L. Sussman A. H. J. Wang N. C. Seeman and A. Rich Science 1974 185 435. D. R. Kearns and R. G. Shulman Accounts Chem. Res. 1974 7 33. B. G. Barrel1 and B. F. C. Clark 'Handbook of Nucleic Acid Sequences' Joynson- Bruvvers Oxford 1974. lo K. Murray and R. W. Old Progr. Nucleic Acid Res. Mol. Biol. 1974 14 117; W. A. Salser Ann. Reo. Biochem. 1974 43 923. '*F. Sanger J. E. Donalson A. R. Coulson H. Kossel and D. Fischer J. Mol. Biol. 1974 90 315. Nucleic Acids 385 Last year's Annual Report12 alluded to growing concern over 'plasmid engineering' experiments whereby restriction cleavage fragments of DNAs from different sources can be joined by ligase to produce biologically functional hybrid DNA molecules.Earlier this year the National Academy of Sciences in the United States took the unique step of calling for a worldwide moratorium on certain types of experiments in this area of research because of their potential danger to human health.13 In particular it was suggested that all experiments involving the introduction into bacterial plasmids or viral DNA of (i) genetic determinants for antibiotic resistance or bacterial toxin formation and (ii) of segments of DNA from oncogenic or other animal viruses be deferred until their hazards can be reliably assessed. Although this step undoubtedly excludes many experiments of great intrinsic interest and potential benefit it can only be a wise and responsible decision in our present state of knowledge.In the mean- time there is plenty of scope for 'safer' endeavour in this field. For example fragments of X. laeuis DNA coding for the 18s and 28s ribosomal RNAs and generated by EcoRI restriction endonuclease have been linked in vitro to a bacterial plasmid. l4 When introduced into E. coli by transformation the recom- binant plasmid can replicate stably and act as a template for the synthesis of RNA complementary to the X. lae~isribosomal DNA. Much of the DNA in the 3 phage genome is not essential for the viabiIity of the phage. However if too much of this non-essential DNA is deleted infection of non-restricting E. coli cells will not result in plaque formation because the phage DNA molecules are too short to be packaged into virus particles.The incorporation of restriction cleavage fragments from foreign DNA sequences can restore the infectivity of such heavily deleted A genomes and this system offers an effective assay for the production of viable hybrid DNA molecules. It has been used15 to produce a large number of functional hybrid iphages which contain DNA sequences derived from either prokaryotic (E. coli) or eukaryotic (D.mehogaster) sources. Instead of inserting DNA into plasmid and viral genomes the action of restric- tion nucleases may be exploited to construct defined deletion mutants lacking one or more restriction cleavage fragments from their DNA. This procedure constitutes a powerful new method of genetic mapping and it has been applied to the production of deletion mutants of SV40I6 and phage 2 Bases and Nucleosides A new oxidizing agent for nucleic acid bases has been investigated :I8 potassium peroxodisulphate K,S,O,.Reaction occurs only with the anionic forms of the '' R. T. Walker Ann. Reports (B),1973 70 623. l3 See Nature 1974 250 175. I4 J. F. Morrow S. N. Cohen A. C. Y. Chang H. W. Boyer H. M. Goodman and ' R.B. Helling Proc. Nar. Acad. Sci. U.S.A.,1974 71 1743. M. Thomas J. R. Cameron and R. W. Davis Proc. Nat. Acad. Sci. U.S.A.,1974 71 * 4579. l6 C.-J. Lai and D. Nathans J. Mol. Biol. 1974 89 179. N. E. Murray and K. Murray Nature 1974 251 476. R. C. Moschel and E. J. Behrman J. Org. Chem. 1974 39 1983 2699. R. J. H. Davies bases and guanine is oxidized much more rapidly than adenine thymine uracil or cytosine.In 1 M-potassium carbonate at 40 “C,guanosine and deoxyguano- sine are at least 500 times more reactive than any other nucleoside and are converted mainly into guanidine urea and ribosyl- or deoxyribosyl-urea. The reagent may therefore be useful for the selective alteration and cleavage of polydeoxyribonucleotides at guanine sites. 1,N6-ethenoadenosine (1) is defor- mylated at C-2 by the action of O.05N-alkali. l9 The product (2)reacts with nitrous acid to give the fluorescent 1,N6-etheno-2-aza-adenosine(3). Instead of carbon dioxide and oxides of nitrogen treatment of adenine with sulphuric acid potas- sium bromide potassium permanganate and hydrogen peroxide yields 8,8’-dioxo-6,6’-azapurine (4). This bright orange compound” is an extremely potent noncompetitive inhibitor of xanthine oxidase (Ki = 2.8 x mol I-’) which may prove useful in the therapy of gout.(4) The synthesis of a number of purine nucleoside-6-sulphonates has been des- cribed.21 This provides a simple route to the ribonucleoside and 5’-nucleotide derived from 6-aziridinylpurine (5) which are potential affinity-labelling analogues of adenosine and 5’-AMP. The synthesis of some tricyclic nucleoside analogues of the ‘Y’base found in tRNA has been reported.22 Stemming from work on synthetic analogues of CAMP a useful new procedure for purine ring closure has been developedz3 which permits introduction of alkyl and aryl sub- stituents into the 2-position. CAMP can be easily converted24 in four steps via l9 K.C. Tsou K. F. Yip E. E. Millar and K. W. Lo Nucleic Acids Res. 1974 1 531. 2o J. R. Davis A. L. Jadhav and J. Fareed J. Medicin. Chem. 1974 17 639. ” H.-R. Rackwitz and K.-H. Scheit Chem. Ber. 1974 107 2284. ’’ G. L. Anderson B. H. Rizkalla and A. D. Broom J. Org. Chem. 1974 39 937. ” R. B. Meyer jun. D. A. Shuman and R. K. Robins J. Amer. Chem. SOC.,1974 96 4962. 24 R. B. Meyer jun. D. A. Shuman R. K. Robins J. P. Miller and L. N. Simon J. Medicin. Chem. 1973. 16 1319. Nucleic Acids 387 the l-N-oxide into the 5-amino-4-carboxamidine-imidazolenucleotide (6). This reacts with aldehydes under mild oxidative conditions to give a variety of 2-substituted derivatives (7). The reaction sequence should be quite generally applicable to adenine-containing compounds.A detailed of the mechan- ism of the intramolecular rearrangement of 06-allylic guanines to 8-allylic guanines indicates that a double [3,3] sigmatropic shift via C-5 is involved. R1= Ribosyl 3’,5‘-CyclicPhosphate The acetone-photosensitized addition of vinylene carbonate to uracil26 gives a mixture of the cis-and trans-[2 + 21 photoadducts (8) which on treatment with triethylamine in DMF is converted into 5-(formylmethyl)uracil (9) in 30 7; overall yield. The latter compound is a useful intermediate for the introduction of extended carbon chains into the 5-position of uracil and its versatility has been demonstrated in the synthesis of (f)-5-(4’,5’-dihydroxypentyl)uracil.The ribo- nucleoside2’ and deoxyribonucleoside28 of ‘uracil anhydride’ (10) have both 0 0 .i’&x 0 N H (9) been synthesized.These analogues are of interest because due to their reactive anhydride function they may be effective as active-site-directed irreversible inhibitors of enzymes involved in pyrimidine metabolism. The first examples of 1’,2’-unsaturated pyrimidine29 (of uracil) and purine3’ (of adenine) nucleosides (11) have been synthesized. The stannic chloride-catalysed reaction of silylated purines with fully acetylated sugars3 is claimed to be equivalent if not superior to other procedures for the preparation of adenine nucleosides. 25 N. J. Leonard and C. R. Frihart J. Amer. Chern. SOC.,1974,96 5894. 2b D. E. Bergstrom and W. C. Agosta Tetrahedron Letters 1974 1087.21 T. Ling Chwang and C. Heidelberger Tetrahedron Letters 1974 95. M. Bobek A. Bloch and S. Kuhar Tetrahedron Letters 1973 3493. 29 M. J. Robbins and E. M. Trip Tetrahedron Letters 1974 3369. ’O M. J. Robbins and R. A. Jones J. Org. Chem. 1974 39 115. 31 F. W. Lichtenthaler P. Voss and A. Heerd Tetrahedron Letters 1974 2141. R. J. H. Davies II Bu9, Bu B = U. C. A. or Hypoxanthine (12) The reaction of ribonucleosides with dibutyltin oxide32 gives crystalline 2‘,3’-O-(dibutylstannylene)nucleosides (12) in high yield. The dibutylstannylene function serves as an activating group for the 2‘-and 3’-oxygen functions treatment of (12) with phosphorus oxychloride leads to the formation of mixed 2’(3‘)-phosphates acyl chlorides and anhydrides give 3’-O-acyl derivatives while reaction with tosyl chloride selectively yields the 2’-tosyl-nucleosides.This method has been used to prepare 2’-0-nitrobenzyl-uridine.~~ The o-nitro- benzyl group constitutes a photolabile protecting group (removable by irradiation at 320 nm) and it has been utilized in the synthesis of UpU and UpA. The use of alkysilyl groups appears to represent a major breakthrough in the development of protecting groups for the hydroxyl functions of ribon~cleosides~~ and deoxyribon~cleosides.~~ The crystalline alkylsilyl derivatives are readily prepared in good yield by the reaction of the nucleoside with the alkylsilyl chloride and imidazole in DMF. For ribonucleosides the most useful reagents are t-butyldimethylsilyl chloride and tri-isopropylsilyl chloride.With uridine it is a simple matter to prepare the 2-and 2‘,5’-protected nucleosides which have been used in a synthesis of UpU. Removal of the silyl groups is achieved by the action of Bu”,NF in THF which in general does not affect other acid- or base- labile protecting groups. The authors promise to describe the synthesis of some important oligoribonucleotides using only alkylsilyl groups for the protection of sugar hydroxyl functions. For the deoxyribonucleoside deoxythymidine specific protection of the 5’-hydroxy-group is possible with t-butyldimethylsilyl- imidazole in pyridine. Alternatively the 3’,5’-disilylated derivative (13) may be prepared which because of the difference in the rate of hydrolysis of the two silyl groups can be converted in 50% yield into the 3’-protected deoxyribo- nucleoside (14) by treatment with acetic acid.ROQ’ ---+ HOA~ HOGT Me I R = Me3C-Si-I OR OR Me (13) (14) 32 D. Wagner J. P. H. Verheyden and J. G. Moffatt J. Org. Chem. 1974 39 24. 33 E. Ohtsuka S. Tanaka and M. Ikehara Nucleic Acids Res. 1974 1 1351. 34 K. K. Ogilvie K. L. Sadana E. A. Thompson M. A. Quilliam and J. B. Westmore Tetrahedron Letters 1974 2861. 35 K. K. Ogilvie E. A. Thompson M. A. Quilliam and J. B. Westmore Tetrahedron Letters 1974 2865. Nucleic Acids 389 A high-pressure liquid chromatography system has been described36 which is capable of resolving each of the common purine and pyrimidine bases and their ri bonucleosides.Spectroscopic studies have shown that in aqueous solution in~sine~~ is at least 85 ”/ in the keto-form while 4-thiouracil 4-thiouridine and 5,6-dihydro-4- thiouracil all exist in the 2-keto-4-thione form.38 N.m.r. measurements have shown that for Nh-methyladenine and N4-methylcytosine there is restricted rotation about the nitrogen-ring carbon bond.39 The two possible rotamers in each case lie in the plane of the ring and show a 20 1 preference for the syn-position in which the methyl group prevents normal Watson-Crick hydrogen bonding. It is estimated that methylation of adenine or cytosine should destabilize a Watsonxrick double helix by approximately 1.4 kcal (mol of methyl sub- stituent)-‘. Rotation about the C-2-N-10 bond in methyl- and dimethyl- guanine does not appear to be restricted.A simple new method for determining the anomeric configuration of ~-ribofuranonucleosides~~ is based on the n.m.r. spectra of their 2’,3’-isopropylidene derivatives. For /I-anomers the chemical shifts of the two isopropylidene methyl groups differ by more than 0.18 p.p.m. while for a-anomers the difference is less than 0.10 p.p.m. In recent years a number of bivalent metal ions have been reported to complex with guanosine in DMSO. These conclusions were based mainly on observations of changes in the chemical shifts of the guanosine base protons in the presence of the metallic chlorides. Strong evidence has now been produced41 that the species being complexed in these cases is not the metal cation but the chloride anion! It is believed that the charge-reversed chelate complex (15) is formed in which two positive centres chelate the negative ion.This finding may be Ribosyl (15) relevant to the well-known destabilizing effect of many anions on nucleic acid secondary structure. A further cautionary note derives from the observation4’ that co-ordination of the methylmercury(I1) cation at N-7 of inosine or of 5’-GMP catalyses very rapid exchange of the proton at C-8 with solvent water leading to broadening or disappearance of this proton resonance in the n.m.r. spectrum. Binding sites of paramagnetic cations have often been assigned on the 36 P. R. Brown S. Bobick and F. L. Hanley J. Chromatog. 1974 99 587. 37 F. E. Evans and R. H.Sarma J. Mol. Biol. 1974 89 249. 38 A. Psoda Z. Kazimierczuk and D. Shugar J. Amer. Chem. Soc. 1974,96 6832. 39 J. D. Engel and P. H. von Hippel Biochemistry 1974 13 4143. 40 J.-L. Imbach J.-L. Barascut B. L. Kam and C. Tapiero Tetrahedron Letters 1974 129. 41 Chien-Hsing Chan and L. G. Marzilli J. Amer. Chem. Soc. 1974 96 3656. 42 S. Mansy and R. S. Tobias J.C.S. Chem. Comm. 1974 957. R.J. H. Davies basis of the broadening of proton resonances caused by the cation-assuming it to arise solely from dipolar spin-lattice relaxation. Metal-ion-catalysed exchange with solvent must be ruled out before this assumption is justified. The structure of coformycin (16) an unusual antibiotic from Streptomyces has been established by X-ray crystallography and confirmed by an elegant synthesis from neb~larine.~~ Another research isolated the correspond- ing deoxyribonucleoside and again determined its structure by X-ray diffraction.Both compounds are very powerful inhibitors of adenosine deaminase because it is believed they may be structural analogues of the transition-state intermediate in the deamination of adenosine. The crystal structures of at least twenty other bases base analogues and assorted nucleosides have been published this year.4s Of particular interest are 6-aza-cytidine and 8-aza-adenosine both of which adopt a ‘high-anti’ conf~rmation,~~ i.e. the torsional angle between the base plane and the sugar lies outside the ranges conventionally ascribed to either the syn-or anti-conformation.This probably results from removal of the rotational barrier caused by interactions between the sugar C-2’ and H-2’ with H-6 of normal pyrimidine bases and H-8 of normal purines. 3 Nucleotides and Oligonucleotides A simple acetoxymercuration reaction4’ has been used to prepare the 5-mer- curiacetate derivatives of UTP (17) CTP dUTP and dCTP as well as the 7-mercuriacetate of 7-deaza-ATP. These compounds are mostly potent in- hibitors of polymerase enzymes. However on conversion into the corresponding mercurithio-compounds (18) by the action of thiols they become excellent substrates for all the polymerases tested. Potential applications of these mer- curinucleotides include their use as (i) heavy-atom reagents for crystallographic purposes (ii) affinity probes for enzymes containing active-site sulphydryl groups and (iii) reagents for forming protein-polynucleotide complexes.The 43 H. Nakamura G. Koyama Y. Iitaka M. Ohno N. Yagisawa S. Kondo K. Maeda and H. Umezawa J. Amer. Chem. SOC. 1974 96 4327; M. Ohno N. Yagisawa S. Shibara S. Kondo K. Maeda and H. Umezawa ibid.,p. 4326. 44 P. W. K. Woo H. W. Dion S. M. Lange L. F. Dahl and L. J. Durham J. Heterocyclic Chem. 1974 11 641. 45 Mainly in Acja Cryst. 1974. VOl. B30. 46 P. Singh and D. J. Hodgson J. Amer. Chem. SOC. 1974 96 1239 5276. 4’ R. M. K. Dale D. C. Livingston and D. C. Ward Proc. Nar. Acad. Sci. U.S.A. 1974 70 2238. Nucleic Acids 391 0 0 II HIAyHgX Adenosine-S-O-P=O/o-pTo-ON \ /O 0-P I I1\ 0-(17)(17) XX == OACOAC 0 ‘0-(18)(18) XX = SR (1% = long-suspected formation of monoadenosine 5‘-trimetaphosphate(19) when ATP salts are treated with DCC in anhydrous pyridine has been confirmed by 31P n.m.r.measurement^.^^ The molecule is highly reactive and rapidly reverts to ATP on contact with water. For nearly forty years it has been recognized that NADH (20) is unstable in dilute acid and slowly rearranges to a modified pyr-idine coenzyme known as the primary acid product. By a combination of 220 MHz n.m.r. spectroscopy c.d. measurements chemical modifications and specific deuterium labelling Oppenheimer and Kaplan4’ have now successfully resolved the identity of the product (21). The rearrangement reaction is shown to be stereospecific and its mechanism has possible implications for the function of the coenzymes in dehydrogenases.HO OH (20) A method has been described” for the rapid and complete separation of the mono- di- and tri-phosphates of A G U and C on a conventional anion-exchange column operating at low pressure. Nucleotides can be efficiently separated by anion-exclusion chromatography ;5 its effectiveness has been critically compared with that of anion-exchange chromatography. A promising new chromatographic material having a combination of reversed-phase and ion-exchange characteristics has been useds2 for the high-speed resolution and analysis of mono-and oligo-nucleotides at the nanomole level. 48 T. Glonek R. A. Kleps and T. C. Myers Science 1974 185 352.49 N. J. Oppenheimer and N. 0. Kaplan Biochemistry 1974 13 4675. 50 J. X. Khym J. Chromatog. 1974,97 277. R. P. Singhal European J. Biochem. 1974 43 245. ’’ R. A. Holton D. M. Spatz E. E. van Tamelen and W. Wierenga Biochem. Biophys. Res. Comm. 1974 58 605. R.J. H. Davies Cyclic Nuc1eotides.-The central importance of the 3'.5'-cyclic purine ribo- nucleotides CAMP and cGMP in the regulation of metabolic processes is now well recognised. This year cCMP has been reported53 to regulate the initiation of growth of leukaemia cells in culture. Furthermore cCMP has been detected in extracts from these cells though it remains to be established that the compound is not an artefact of the isolation procedure. A number of ribonucleoside 3'3'- cyclic phosphates including CAMP have been prepared in good yield by the action of base on the corresponding 2'-protected 3'-diphenyl phosphate^.'^ Adenosine 3',5'-cyclic phosphorothioate (22) has been synthesized;55 both diastereomers are hydrolysed by diesterases much more slowly than CAMP.I I 0-0-(22) (23) The analogue 3'-amido-3'-deoxyadenosine3',5'-cyclic phosphate (23) has also been prepared. 56 The 2',5'-cyclic phosphate derived from cordycepin (3'-deoxyadenosine) contains a seven-membered cyclic phosphate ring." Synthesis and Sequencing of Oligonucleotides.-It has been pointed out5* that when phenyl is used as the protecting group for internucleotide linkages in oligonucleotide synthesis by the phosphotriester approach a substantial amount of internucleotide cleavage may occur during unblocking.However this problem appears to be surmountable by the introduction of chloro-substituents into the phenyl residue and the use of milder conditions for phosphotriester hydrolysis. Arylsulphonyl triazolides (24) have been proposed59 as alternatives to aryl- R = Me or Me,CH (24) 53 A. Bloch Biochem. Biophys. Res. Comm. 1974,58,652; A. Bloch G. Dutschman and R. Maue ibid. 1974 59 955. 54 J. H. van Boom P. M. J. Burgers P. van Deursen and C. B. Reese J.C.S. Chem. Comm. 1974 618. 55 F. Eckstein L. P. Simonson and H.-P. Bar Biochemistry 1974 13 4675. 5b M. Morr M.-R. Kula G. Roesler and B. Jastorff Angew. Chem. Internat. Edn. 1974 13 280. " M. Ikehara and J. Yano Nucleic Acids Res.1974 1 1783. J. H. van Boom P. M. J. Burgers P. H. van Deursen R. Arentzen and C. B. Reese Tetrahedron Letters 1974 3785. 59 N. Katagiri K. Itakura and S. A. Narang J.C.S. Chem. Comm. 1974 325. Nucleic A cids 393 sulphonyl chlorides as condensing agents. Although the rate of reaction is slower the yields are considerably higher. Further studies6' on the synthesis of oligodeoxyribonucleotides on polymer supports using modified polystyrene resins have been described. The pentamer and hexamer of 2'-O-methylinosine 3'-phosphate have been complexed with poly(C) as template and then poly- merized" with a water-soluble carbodi-imide. The chain length of the product depends upon the stability of the polymer-oligomer complex but a 157; yield of the 30-mer has been realized.Khorana" has devised an effective method for linking oligodeoxyribo- nucleotides to cellulose through a poly(dT) spacer attached to the 3'-OH ter- minus (25). Replication of the oligonucleotide by DNA polymerase I is then Cellulose-p(dT),,,-dCCACCCC (5') (25) possible when poly(dA) or oligo(rA) as primer is hybridized to the poly(dT) region. The cellulose-linked template is easily separated from the product of replication by denaturation and may then be used for further cycles of replication. So far the method has been investigated only for the oligonucleotide dCCCCACC -a sequence from the E. coli tRNATy' gene. The results are very promising and the method should be equally applicable to the linkage and replication of poly- deoxyribonucleotides.If so it should greatly simplify the problem of efficiently replicating synthetic DNA molecules. Polynucleotide phosphorylase has been used previously to add a single protected nucleotide residue to an existing oligo- ribonucleotide. A disadvantage of the method is that partial phosphorolysis of the acceptor molecule normally occurs. This has now been overcome63 by the expedient of removing (enzymatically) inorganic phosphate from the reaction mixture as it is formed. The terminal addition of nucleotides to (PA) has been achieved in 90 "/ yield with essentially no side-product formation. Poly- nucleotide phosphorylase also appears to be capable of catalysing the addition of a single deoxyribonucleotide to oligonucleotide primers.64 The unique sequence from the T4 lysozyme gene which was determined by Streisinger from the genetic analysis of frameshift mutants continues to inspire oligonucleotide synthesis.A nona- and a dodeca-deoxyribonucleotide corres-ponding to sections of the gene ~equence,~' and a tetradecadeoxyribonucleotide segment from the complementary strand,66 have been synthesized ; the latter " H. Koster and F. Cramer Annalen 1974 946; H. Koster A. Pollak and F. Cramer ibid. 1974 959; H. Sommer and F. Cramer Chem. Ber. 1974 107 24. 6' S. Uesugi and P. 0.P. Ts'o Biochemistry 1974 13 3142. '' A. Panet and H. G. Khorana J. Biol. Chem. 1974 249 5213. " J. J. Sninsky G. N. Bennett and P. T. Gilham Nucleic Acids Res. 1974 1 1665. 64 S. Gillam K. Waterman M.Doel and M. Smith Nucleic Acids Res. 1974 I 1649. 65 S. A. Narang K. Itakura C. P. Bahl and N. Katagiri J. Amer. Chem. Soc. 1974 96 7074. '' R. Padmanabhan E. Jay and R. Wu Proc. Nut. Acad. Sci. U.S.A. 1974 71 2510. 394 R.J. H. Dauies has been investigated as a primer for DNA sequencing purposes. A dodeca-nucleotide complementary to a defined region of the endolysin gene of phage A has also been prepared and ~haracterized.~~ The enzymatic synthesis of the double-stranded block polymer d(C 5A15).d(T1 5G1 5)-which corresponds to three double-helical turns -has been reported.68 Oligoribonucleotides may be sequentially degraded69 by the action of either alkaline phosphatase or snake-venom phosphodiesterase in Combination with periodate to give a series of oligonucleotide dialdehydes which can be reduced with tritiated sodium borohydride to give 3’-terminal trialcohols.Subsequent separation of these oligonucleotide trialcohols according to chain length and identification of the labelled end-groups provides a very sensitive post-labelling technique for sequencing oligoribonucleotides. As little as 0.01 Az6* units of a non-radioactive decanucleotide are sufficient for its sequence determination. Spectroscopic and Structural Studies.-Details have been published” of the sophisticated method for determining the conformation of nucleotides in solution which relies on the use of lanthanide ions as paramagnetic n.m.r. probes. The geometry of nucleotide-lanthanide complexes is deduced from perturbations of the nucleotide n.m.r.spectrum caused by the complexing of a combination of relaxation (e.g.Gd3+) and shift (e.g.Eu3+) probes. The procedure for selecting molecular conformations which fit the n.m.r. data is entirely computerized and for AMP a small family of closely related conformations -very similar to the crystal structure-is obtained. A study of CAMP has also been made and the results have been independently ~onfirmed.~ A more conventional approach to the conformational analysis of nucleotides in solution is based on a com- prehensive knowledge of the chemical shifts and coupling constants of all the protons in the molecule. This method has been widely applied; the systems studied include all the common 5’-ribo- and -deoxyribo-nu~leotides,~~ and the dinucleoside phosphates ~TPT,’~ N6-methyladenylyl-uridine,and APU.~~ The low-field resonances of the N-3-H proton of thymine and the N-1-H proton of guanine in base-paired oligodeoxyribonucleotides have been identified and the perturbations of their chemical shifts due to nearest-neighbour base pairs have been eval~ated.~ ” R.Wu C. D. Tu and R. Padmanabhan Biochem. Biophys. Res. Comm. 1974,55 1092. J. F. Burd and R. D. Wells J. Biof. Chem. 1974 249 7094. b9 K. Randerath E. Randerath L. S. Y. Chia R. C. Gupta and M. Sivarajan Nucfeic Acids Res. 1974 1 1121 1329. 70 C. D. Barry J. A. Glasel R. J. P. Williams and A. V. Xavier f. Mof.Biol. 1974 84 471. 71 C. D. Barry D. R. Martin R. J. P. Williams and A. V. Xavier J. Mof.Biof.,1974 84,491 ;D.K. Lavallee and A. H. Zeltmann f. Amer. Chem. Soc. 1974 96 5552. 72 D. B. Davies and S. S. Danyluk Biochemistry 1974 13 4417. 73 D. J. Wood F. E. Hruska and K. K. Ogilvie Canad. J. Chem. 1974,52 3353. ” C. C. Altona J. H. van Boom J. de Jager H. J. Koerners and G. van Binst Nature 1974 247 558; Rec. Trau. chim. 1974 93 169. ” D. J. Pate1 and A. E. Tonelli Biopofymers 1974 13 1943. Nucleic Acids 395 The helix-coil transition has been monitored for nineteen double-helical oligoribonu~leotides~~ containing between six and fourteen base pairs. Helix stability is strikingly dependent on sequence and thermodynamic parameters have been derived to predict the T of any RNA double helix of known sequence. Two have independently published the crystal structure of the di- sodium salt of 5'-dGMP.The conformation about the C-5'-C-4 bond is gauchr-trans in contrast to gauche-gauche for all the other nucleotides whose crystal structures are known. The structure of the sodium salt of cGMP~~ has been determined. X-Ray diffraction photographs of crystalline dTpdA 79 indicate that the molecules are base-paired and stacked on top of each other to produce an extended helical structure resembling the A-form of DNA. 4 Polynucleotides Poly(2-methyl-N6-methyladenylic acid)" is strongly stacked in neutral solution does not form a regular helix in acid solution but forms a 1 :1 complex with poly(U) by Hoogsteen or reverse Hoogsteen base-pairing. The effects of 0-alkylation on the stacking of single-stranded poly(A) the stability of its double- helical acid form and its complexes with complementary polynucleotides have been investigated by two groups.81 In contrast to poly(A) the fluorescent analogue poly( 1,N6-etheno-2-aza-adenylicacid) appears to have a random structure in solution.82 Both poly(5-fluorocytidylic acid)83 and poly(5-ethyl- cytidylic have been synthesized and shown to form 1 :1 complexes with POlY(1).DNA polymerase I has been used to prepare d(TCC),.d(GGA) and d(TTG),. d(CAA),.8s A persistent problem in such syntheses has been that the enzyme frequently introduces covalent links between the two complementary strands. A protein factor has now been identified whose presence prevents the formation of these interstrand linkages.The self-complementary alternating copolymer of guanosine and 2-thio-cytosine poly[r(G-s2C)] has exceptional thermal stability,86 in common with other helical polynucleotides incorporating 2-thioketo-pyrimidine bases. In " P. N. Borer B. Dengler I. Tinoco jun.+ and 0.C. Uhlenbeck J. Mol. Biof.,1974 86 843. " D. W. Young P. Tollin and H. R. Wilson Acta Cryst. 1974 B30 2012; M. A. Viswamitra and T. P. Seshadri Nature 1974 252 176. '8 A. K. Chwang and M. Sundaralingam Acta Cryst. 1974 B30 1233. 79 H. R. Wilson Nature 1974 251 735. M. Hattori M. Ikehara and H. T. Miles Biochemistry 1974 13 2754. *' J. L. Alderfer 1. Tazawa S. Tazawa and P. 0. P. Ts'o Biochemistry 1974 13 1615; F. Rottman K. Friderici P. Comstock and M. K. Khan ibid. 1974 13 2762.n2 K. C. Tsou and K. F. Yip Biopolymers 1974 13 987. J. 0. Folayan and D. W. Hutchinson Biochim. Biophys. Acta 1974 340 194. 84 T. Kulikowski and D. Shugar Biochim. Biophys. Acta 1974 374 164. n5 A. R. Morgan M. B. Coulter W. F. Flintoff and V. H. Paetkau Biochemistry 1974 13 1596; M. Coulter W. Flintoff V. Paetkau D. Pulleyblank and A. R. Morgan ibid. p. 1603; W. F. Flintoff and V. H. Paetkau ibid. p. 1610. 86 P. Faerber. F.E.B.S. Letters 1974 44 11 I. 396 R.J. H. Davies lop3M-sodium cacodylate buffer it remains undenatured even at 98 "C! The secondary structure formed by poly(2-thiouridylic acid) (T = 68 "C) is considerably more stable than that of poly(U) (T = 8 "C under the same con- ditions). X-Ray diffraction8' has revealed that it comprises a double helix with non-equivalent polynucleotide chains and an unusual base-pairing scheme N-3-H--0-4 and S-2..H-N-3. Double-stranded polydeoxyribonucleotides containing alternating purine and pyrimidine bases such as poly(dA-dT).poly(dA-dT),have a distinctive double- helical structure.88 There are only eight base pairs per helical turn and the bases themselves are highly tilted with respect to the helix axis. This unusual molecular geometry may have implications for the biological function of some satellite DNAs. The structure of poly(dA).(dT) closely resembles that of B-DNA with C-3-exo-furanose ring pucker while poly(dG).poly(dC) preferentially adopts the A-conformation with C-3-endo-furanose rings.89 In poly(dT). poly(dA).poly(dT) each of the chains is in a twelve-fold helix of the A-t~pe.'~ A new molecular model" has been proposed for the secondary structure of poly(1).It consists of four identical poly(1) chains related to one another by a four-fold rotation axis. Each hypoxanthine base is linked to two others by hydrogen bonds involving 0-6 and N-1. An isogeometrical structure can be built for poly(G) with additional hydrogen bonds between every N-2 and N-7. 5 tRNA The proposed model' for the tertiary structure of yeast tRNAPhe based on X-ray crystallographic analysis at 3A resolution is shown in Figure 1. The ribose- phosphate backbone has been traced through the whole molecule with the excep- tion of a small corner and the positions of most of the bases have been determined.The model confirms the presence of the base pairs incorporated in the convention- al cloverleaf formula as well as identifying a number of extra interactions respon- sible for maintaining the tertiary structure of the molecule. These additional interactions involve base pairs and base triplets which are stacked or intercalated so as to make their hydrogen bonds inaccessible to water molecules. They are especially prominent in the central region of the molecule where four ribose- phosphate chains are in close proximity. Consideration of the tRNA sequences known at the present time shows that these interactions which are vital to the integrity of the unique tertiary structure are highly conserved." For example a trans-bonded base pair between G15 and C48 occurs in thirty-nine tRNAs.The importance of this interaction is emphasized by the fact that in eleven other tRNAs there have been co-ordinated base changes to A15.U48 so as to conserve complementarity at these points. A base triplet between the invariant bases *'-S. K. Mszumdar W. Saenger and K. H. Scheit J. Mol. Biol. 1974,85 213. S. Arnott R. Chandrasekaran D. W. L. Hukins P. J. C. Smith and L. Watts J. Mol. Biol. 1974 88 523. 89 S. Arnott and E. Selsing J. Mol. Biol. 1974 88 509,551. 90 S. Arnott R. Chandrasekaran and C. M. Marttila Biochem. J. 1974 141 537. 9' A. Klug J. Ladner and J. D. Robertus J. Mol. Biol. 1974 89 51 1. Nucleic Acids 397 -a c stem '-plus18a 19 Figure 1. A schematic diagram of the tertiary structure of yeast tRNAPhe.The ribose- phosphate backbone is represented by a continuous dark line except where there is ambiguity when it is shown dashed. Base pairs in the double-helical stem are repre- sented by long light lines and non-paired bases by shorter lines. Many of the latter stack as indicated those which do not are drawn at an angle to the backbone. Base pairs additional to those in the cloverleaf formula are indicated by dotted lines. f denotes the region containing residues 16 17 and 20. (Reproduced by permission from J. Mol. Biol. 1974,89 51 1) U8 (or s4U8),A14 and A21 is expected to occur in all tRNA species. Two further base triplets C1 3.G22.m7G46 and U12.A23.A9 are replaced in a co-ordinated fashion in other tRNAs. The insight into the structure of tRNA afforded by this model will no doubt provide the inspiration for much future research work and the explanation for much that is past.Already n.m.r. evidence has been presented92 for the ter- tiary base-pairing of the 4-thiouracil base at position 8 in many E. coli tRNAs with the adenine at position 14. The location of the heavy-metal-atom binding sites in isomorphous derivatives of tRNA crystals is vital to the successful interpretation of X-ray diffraction data. K. L. Wong and D. R.Kearns Nature 1974 252 738. 398 R.J. H. Davies Conventional oligonucleotide-fractionation procedures have been used to iden- tify C38 as the site of osmium attachment93 in a crystalline isomorphous derivative of yeast tRNA,M" and to show that when trans-dichlorodiammineplatinum(1r) reacts with yeast tRNAPhe the platinum is bound to the oligonucleotide G,AAYAl//p which contains the anticodon When the lanthanide ions Tb3+ and Eu3+ are complexed with E.coli tRNA their fluorescence is enhanced several hundred-fold9' due to energy transfer from 4-thiouridine residues to bound lanthanide ions. The binding of lanthanide ions to tRNA also has important applications in n.m.r. studies. From changes in the n.m.r. spectrum induced by Eu3+ it has been deduced96 that the folding of yeast tRNAPhe in solution brings the phosphate backbone of the CCA ter- minus and the dihydrouridine stem into close contact -a result in agreement with the crystal structure. The thermal melting of the cloverleaf arms of E.coli tRNA:"' has been followed by a combination of temperature-jump relaxation measurements and high- resolution n.m.r. spectro~copy.~' The results indicate that the dihydrouridine helix melts first followed by the Tl//Chelix the anticodon helix and finally the acceptor-stem helix. A similar involving only n.m.r. has been carried out for E. coli tRNAG'". This approach which allows the individual resonances of all the hydrogen-bonded ring NH protons in the tRNA molecule to be assigned has been further exploited in an e~amination~~ of yeast tRNAk'". This tRNA is particularly interesting because it can exist in a biologically active (native conformer) and an inactive state (denatured conformer). An analysis of the tem- perature dependence of the n.m.r.spectra of the two conformers has confirmed the cloverleaf structure for the native conformer and led to a structural model for the denatured conformer. In this model only the acceptor stem the T$C stem and the minor stem from the cloverleaf are retained and a new helix is formed by pairing bases from the anticodon loop with bases in the T$C loop. These conclusions are supported O0 by the equilibrium binding patterns of complemen- tary oligonucleotides to the denatured conformer. A very rapid (and economical) procedure"' for isolating a specific tRNA from a crude preparation involves firstly amino-acylation with the specific amino-acid followed by treatment with p-chloromercuribenzenesulphonyl chloride to form the sulphonamide of the amino-acyl group.This derivative reacts covalently with a sulphydryl-resin. After removal of the unreacted t-RNA the specific tRNA is released from the resin by deacylation. 93 J. J. Rosa and P. B. Sigler Biochemistry 1974 13 5102. 94 D. Rhodes P. W. Piper and B. F. C. Clark J. Mol. Biol. 1974 89 469. 95 M. S. Kayne and M. Cohn Biochemistry 1974 13 4159. 96 C. R. Jones and D. R. Kearns Proc. Nat. Acad. Sci. U.S.A. 1974 71 4237. 97 D. M. Crothers P. E. Cole C. W. Hilbers and R. G. Shulman J. Mol. Biol. 1974,87 63. 98 C. W. Hilbers and R. G. Shulman Proc. Nat. Acad. Sci. U.S.A. 1974 71 3239. 99 D. R. Kearns Y. P. Wong S. H. Chang and E. Hawkins Biochemistry 1974,13,4736. loo 0.C. Uhlenbeck. J. G. Chirikjian and J. R. Fresco J. Mol. Biol. 1974 89 495.* ' D. J. Goss and L. J. Parkhurst Biochem. Biophys. Res. Comm. 1974 59 181. Nucleic Acids 399 Two laboratorieslo2 have identified the modified nucleoside in E. coli tRNAPhe that is responsible for its reaction with phenoxyacetoxysuccinimide as 3-(3- amino-3car boxypropy1)uridine. Streptococcusfuecalis grown in the absence of folate initiates protein synthesis with non-formylated tRNA,M"' which differs from the normal formylated tRNA,M"' only in the T$C loop where uridine replaces thymidine.lo3 The tRNA from an extremely thermophilic and aptly named bacterium -Thermus thermophilus HB8 -which can grow at 85"C contains 2-thio-thymidine in place of thy- midine' O4 in the GT$CG sequence. Introduction of the 2-thiopyrimidine base should considerably stabilize the interaction between this sequence and the complementary CGAAC sequence of 5s RNA in the ribosomal A-site for which there is very good evidence.lo5 In fact binding of the tRNA fragment T$CGp to a ribosome-mRNA complex is sufficient to induce the synthesis of ppGpp and pppG~p.'~~ Only a selection of the tRNA sequences published this year can be mentioned.The sequence of one of the dimeric tRNA precursor molecules coded for by bacteriophage T4 has been determined. lo' Besides the nucleotide sequences for tRNASe' and tRNAPr0 it contains at least thirteen precursor-specific nucleotides located at the 5' and 3' termini and in the interstitial region. All the minor bases present in the mature tRNAs are found except for a 2'-O-methylguanosine residue from tRNAS".The 3'-CCA, termini of both tRNAs are absent so these nucleotides must be added enzymatically at a later stage of maturation. The complete sequences of the mammalian cytoplasmic initiator tRNAs (tRNA?'') from rabbit liver sheep mammary gland and mouse myeloma P3 cells are iden- tical.'o8 A cytidine residue replaces the uridine that has hitherto always been found to precede the 5'-end of the anticodon in the tRNA sequences. The unusual loop IV (normally T$CG loop) sequence AUCGm'AAA may be responsible for some of the distinctive functional properties of mammalian initiator tRNAs. The sequences of tRNA?"' and the major tRNAVa' from mouse myeloma cells have also been determined. lo9 The tRNAp' functions only in protein elongation in contrast to the tRNAF'' whose sole function is initiation.Comparison of the sequences of these two methionyl tRNAs shows no striking homologies. The Io2 Z. Ohashi M. Maeda. J. A. McCloskey and S. Nishimura. Biochemistry 1974 13. 2620; S. Friedman H. J. Li K. Nakanishi and G. Van Lear ibid. p. 2932. lo' A. S. Delk and J. C. Rabinowitz Nature 1974. 252 106. lo* K. Watanabe. T. Oshima M. Saneyoshi and S. Nishimura F.E.B.S. Letters 1974,43. 59. lo' F. Grummt I. Grummt H. J. Goss M. Sprinzl D. Richter and V. A. Erdmann F.E.B.S. Letters 1974 42 15. D. Richter V. A. Erdmann and M. Sprinzl Proc. Nar. Acad. Sci. U.S.A. 1974 71 3226. lo' B. G. Barrell J. G. Seidman C. Guthrie and W. H. McClain Proc. Nat. Acad. Sci. U.S.A. 1974 71 413.P. W. Piper and B. F. C. Clark Nature 1974 247 516; Errropeun J. Biochem. 1974 45 589; M. Simsek U. L. Rajbhandary M. Boisnard and (3. Petrissant Nature 1974 247 518. Io9 P. W. Piper and B. F. C. Clark F.E.B.S. Letters 1974 47 56. R. J. H. Dauies tRNAp' contains fifteen modified nucleosides and is unique in having only four base pairs in the anticodon stem. Another highly modified eukaryotic tRNA (tRNAys from yeast) contains sixteen modified nucleosides,' lo including a 5'-terminal pseudouridine and 2-thio-5-carboxymethyluridine methyl ester in the first position of the anticodon. It is reported' that the nucleoside composition of the tRNA from mice and mosquitoes does not vary with their age. Hopefully this extends to other or- ganisms too! 6 Arrinity Labelling of Ribosomes Affinity labelling with reactive substrate analogues has proved a powerful method for identifying amino-acid residues adjacent to or forming part of the active sites of enzymes.There has recently been growing interest in this technique as a means of investigating the structure of ribosomes and it has been applied successfully in a number of instances. Most studies have employed alkylating acylating or photoalkylating derivatives of the wamino-group of Phe-tRNAPhe. Cantor and his colleagues' ' have proved that N-bromoacetyl-phenylalanyl-tRNAPhe can bind normally to the peptidyl (P) site on the E. coli ribosome and react specifically with proteins L2 and L26-L27 of the large (L) subunit. Under different experimental conditions,' ' it binds preferentially to the amino-acyl (A) site and then labels protein L16 in addition to L2 and L26-L27.The protein L16 is thus identified exclusively as an A-site protein and it is concluded that all three proteins are located close to one another in the peptidyl-transferase centre. This reagent has been modified by the introduction of up to sixteen glycyl residues to give a series of peptidyl tRNA analogues' l4 with varying peptide chain lengths. These all react covalently with certain ribosomal proteins and the extent of reaction depends on the peptide chain length. The results favour the existence of a ribosomal binding site for the growing polypeptide chain and indicate that the proteins in this site are ordered with L2 closest to the 3'-end of the tRNA followed by L26-L27 L32-L33 and then L24.It has been inferred' ' from affinity-labelling experiments with p-nitrophenylcarbamyl-phenylalanyl-tRNAPhe that the proteins L27 L15 L2 L16 and L14 are all at or near tRNA binding sites on the E. coli 50s subunit. Other investigators utilizing reactive N-bromoacetyl analogues of puromycin which is a substrate for the ribosomal 'lo J. T. Madison S. J. Boguslawski and G. H. Teetor Biochemistry 1974 13 518; J. T. Madison and S. J. Boguslawski ibid.. p. 524. J. L. Hoffman and M. T. McCoy Nature 1974 249 558. M. Pellegrini H. Oen D. Eilat and C. R. Cantor J. Mol. Bid. 1974 88 809. lI3 D. Eilat M. Pellegrini H. Oen N. De Groot Y. Lapidot and C. R. Cantor Nature 1974,250 514. l4 D.Eilat M. Pellegrini H. Oen Y.Lapidot and C. R. Cantor J. Mol. Biol. 1974 88 831. A. P. Czernilofsky E. E. Collatz G. Stoffler and E. Kuechler Proc. Nut. Acad. Sci. U.S.A. 1974 71 230. Nucleic Acids 40 1 A-site have observed affinity labelling of the 23s RNA molecule in E. coli ribosomes," and proteins L27 and L29 of rat-liver ribosomes.' The photoaffinity reagent N-(2-nitro-4-azidophenyl)glycyl-phenylalanyl-t-RNAPhe is bound specifically to the P-site of E. coli ribosomes,"* in the presence of poly(U). Upon irradiation it reacts covalently with proteins L11 and L18 which are therefore presumed to be part of or adjacent to the peptidyl-trans- ferase centre. These proteins have not been labelled by a-halogenocarbonyl affinity probes possibly because they lack easily accessible nucleophilic groups.The phenacyl-p-azide of 4-thiouridine in E. coli tRNAVa' has been prepared.' l9 Photolysis of p-azidophenacyl-valyl-tRNAval,bound to the ribosomal P-site causes covalent linking exclusively to the 16s RNA of the 30s subunit. Poly- (4-thiouridylic acid) which can act as an mRNA for polyphenylalanine synthesis in vim becomes covalently bound'20 to the 30s subunit protein S1 when a complex of E. coli ribosomes poly(4-thiouridylic acid) and phenylalanyl-tRNAPhe is irradiated at 30WOO nm. This finding supports current evidence that protein S1 is an important constituent of the mRNA ribosomal binding site. Affinity- labelling with an azidophenyl derivative of GDP strongly implicates proteins L5 L11 L18 and L30 in the elongation-factor G-dependent binding of GDP to E.coli ribosomes.121 7 RNA General.-The radioiodination of RNA with labelled sodium iodide in the presence of thallic trichloride can give activities in excess of lo7c.p.m. pg- '. A systematic evaluation of this reaction has been carried out122 and has led to a satisfactory procedure for the iodination of very small samples of RNA. An improved high-resolution fractionation procedure for the preparative isolation of RNA by polyacrylamide gel electrophoresis has been developed.'23 When RNA is attached to a solid support for affinity chromatography or hybridization studies a stable covalent linkage is desirable. This may be achieved124 by coupling the RNA to phosphocellulose using carbonyldi-imidazole.Treatment of per- iodate-oxidized RNA with several newly described hydrazide compounds' introduces a fluorescent label at the 3'-terminus. The 3'-terminal fragments of radioactively labelled RNA molecules produced by nuclease digestion may be selectively isolated in excellent yield on columns of cellulose derivatives containing dihydroxyboryl groups.' 26 Using partial digests overlapping terminal fragments containing up to sixty nucleotides have P. Greenwell R. J. Harris and R. H. Symons European J. Biochem. 1974 49 539. 'I7 J. Stahl K. Dressler and H. Bielka F.E.B.S. Letters 1974 47 167. 'Is N. Hsiung and C. R. Cantor Nucleic Acids Res. 1974,1 1753. ' I. Schwartz and J. Ofengand Proc. Nut. Acad. Sci. U.S.A. 1974 71 3951. 1. Fiser K.H. Scheit G. Stoffler and E. Kuechler Biochent. Biophys. Res. Comm. 1974,60 1112. J. A. Maassen and W. Moller Proc. Nut. Acad. Sci. U.S.A. 1974 71 1277. 122 N. H. Scherburg and S. Refetoff J. Biol. Chem. 1974 249 2143. 123 F. S. Hagen and E. T. Young Biochemistry 1974 13 3394. 24 T. Y.Shih and M. A. Martin Biochemistry 1974 13 341 1. ''' S. A. Reines and C. R. Cantor Nucleic Acids Res. 1974 1 767. 126 M. Rosenburg Nucleic Acids Res. 1974 1 653. 402 R.J.H. Davies been successfully isolated and sequenced. In the presence of manganese ions RNA polymerase can incorporate deoxyribonucleotides into an RNA transcript of either single-stranded or double-stranded DNA. 12' In a normal transcription reaction it is possible to replace one of the ribonucleoside triphosphate precursors entirely by the corresponding deoxyribonucleoside triphosphate.This finding opens up exciting new possibilities for nucleic acid sequencing. For instance by using U,-RNase with dG-RNA and pancreatic RNase with dC-RNA or dU-RNA base-specific cleavage is now possible at any of the four nucleotide residues (TI-RNase for G residues). In this way overlaps of at least six residues can be obtained for any site in the RNA. Some preliminary and promising applications of this method are reported. A new method for the preparative stepwise degradation of polyribonucleotides128 is based on the periodate oxidation-fl elimination+nzymatic dephosphorylation reaction sequence. The conditions for quantitative reaction in each step have been perfected and up to nine degradative cycles have been carried out successfully on tRNA fragments.Meanwhile the established RNA sequencing procedures have been used to determine the primary structure of the U-1 RNA of Novikoff hepatoma ascites cells which contains 171 nucleotide residues. 129 Ribosomal RNA.-A new model for the secondary structure of 5s RNA has been proposed on the basis of high-resolution n.m.r. ~pectroscopy.~ The room- 30 temperature structure contains five helical regions with a total of 28 base pairs of which 17 are GC. 32 The 3'-terminal sequence of E.coIi 16SrRNA which has been determined as -AUCACCUCCUUA,H suggests that it may play a direct part in the ter- mination and initiation of protein ~ynthesis.'~ The terminal trinucleotide UUA, is complementary to the terminator codon UAA while the sequence ACCUCC complements a conserved sequence found in the ribosome-binding sites of coliphage mRNAs.In eukaryotes,' 33 the corresponding 3'-sequence of the 18s rRNA is -GAUCAUUA, and it appears to be invariant. The terminal hexanucleotide is complementary to two termination codons and could recognize a third (UAG) if 'wobble' occurred. As no tRNA species appear to bind termination triplets it is possible that 18s RNA fulfils this function. The distribution of the polypyrimidine sequences in 28s rRNA from rat hepatoma cells has been studied.134 Of the 118 fragments identified with chain lengths between 5 and 21 nucleotides 94 (comprising a total of 667 nucleotides) have been sequenced.In addition a sequence of 99 nucleotides has been e~tablished'?~ 12' A. Van de Voorde R. Rogiers J. Van Herreweghe H. Van Heuverswyn G. Volckaert and W. Fiers Nucleic Acids Res. 1974 1 1059; G. V. Paddock H. C. Heindell and W. Salser. Proc. Nat. Acad. Sci. U.S.A. 1974 71 5017. 12* G. Keith and P. T. Gilham Biochemistry 1974 13 3601. Iz9 R. Reddy T. S. Ro-Choi D. Henning and H. Busch. J. Biol. Chem. 1974 249 6486. D. R. Kearns and Y. P. Wong J. Mol. Biol. 1974 87 755. ' 31 J. Shine and L. Dalgarno Proc. Nat. Acad. Sci. U.S.A. 1974 71 1342. C. Ehresmann P. Stiegler and J.-P. Ebel F.E.B.S. Letters 1974 49 47. 133 J. Shine and L. Dalgarno Biochem. J. 1974 141 609. 134 R. N. Nazar and H. Busch J. Biol. Chem. 1974 249 919. 135 R.Kanamaru Y. C. Choi and H. Busch J. Biol. Chem. 1974 249 2453. Nucleic Acids 403 which is remarkable in having just a single adenylic acid residue -located at its 5'-end. The methylation pattern of HeLa cell rRNA and its precursors has been in- ~estigated.'~~ The 18s RNA and 28s RNA contain approximately 46 and 70 methyl groups respectively whereas the 5.8s RNA contains only one. The transcribed spacer regions of the precursor molecules are unmethylated. When precursor and mature ribosomal RNA molecules are examined by electron microscopy,' 'a reproducible arrangement of hairpin loops reflecting discrete areas of secondary structure may be observed. Maps based on this secondary structure for X. laevis and mouse L-cell ribosomal precursors have revealed the arrangement of the mature RNA sequences within the precursors and have been used to follow the steps in the maturation process.mRNA and Viral RNA.-A brief review' 38 dealing primarily with the isolation and structure of eukaryotic mRNA has appeared. Despite intensive research and speculation the function(s) of the 3'-terminal poly(A) sequence found in eukaryotic mRNA molecules remains a mystery. Several reports this year have complicated rather than clarified the issue. Contrary to earlier opinion it now seems that a substantial proportion of cytoplasmic mRNA molecules at least from HeLa cells139 and from sea-urchin embryo^,'^' may lack poly(A). Although in mammalian cells poly(A) is added to the mRNA precursor (HnRNA) in the nucleus cytoplasmic polyadenylation of mRNA has now been demonstrated in sea-urchin embryos.14' The relative rates of translation (in Xenopus oocytes) of rabbit globin mRNA with and without the terminal poly(A) sequence have been measured.'42 Initially the rates of translation are comparable but at longer incubation times the poly(A)-free mRNA is translated much less rapidly. This finding suggests that the presence of the poiy(A) increases the functional stability of the mRNA. A similar study utilizing cell-free protein-synthesizing systems supports this view.'43 Perhaps this explains why the poly(A) sequence is necessary for the infectivity of poliovirus RNA. 144 Other representatives of the picornavirus group namely the cardioviruses and foot-and-mouth disease viruses contain poly(C)-rich tracts up to 100 nucleotides long in their RNA.14' It is not yet established whether these poly(C) sequences are terminally situated.136 B. E. H. Maden and M. Salim J. Mol. Biol. 1974 88 133. 13' P. K. Wellauer and I. B. Dawid J. Mol. Biof.,1974,89,379; P. K. Wellauer I. B. Dawid D. E. Kelley and R. P. Perry ibid. p. 397. 38 G. Brawermann Ann. Rev. Biochem. 1974 43 621. L39 C. Milcarek R. Price and S. Penman Cell 1974 3 1. I4O M. Nemer M. Graham and L. M. Dubroff J. Mof. Biol. 1974,89,435. 14' I. Slater and D. W. Slater Proc. Nur. Acad. Sci. U.S.A. 1974 71 1103. 142 G. Huez G. Marbaix E. Hubert M. Leclercq U. Nudel H. Soreq R. Salomon B. Lebleu M. Revel and U. Z. Littauer Proc. Nar. Acud. Sci. U.S.A. 1974 71 3143.143 A. E. Sippel J. G. Stavrianopoulos G. Schutz and P. Feigelson Proc. Nut. Acad. Sci. U.S.A. 1974 71 4635. L44 D. H. Spector and D. Baltimore Proc. Nut. Acad. Sci. U.S.A. 1944 71 2983. '45 F. Brown. J. Newman J. Stott A. Porter D. Frisby C. Newton N. Carey and P. Fellner Nature 1974 251 342. 404 R. J. H. Davies Methylated nucleosides have been identified in the mRNA from rat hep- at~rna'~~ and mouse L-~ells.'~' In the rat hepatoma mRNA all four 2'-0-methyl-nucleosides occur but base methylation is mainly confined to N6-methyladenosine. As with rRNA methylation may be important in the matura- tion of eukaryotic mRNA from its precursors. Bacteriophage T 7 early mRNAs are generated from a large precursor molecule by RNase I11 cleavage at specific base sequences.48 The sequencing of mRNA has been greatly advanced by methods involving the use of reverse transcriptase enzymes to produce faithful cDNA transcripts. A number of oligonucleotide sequences in human globin mRNA thus established have been shown to match unique amino-acid sequences in the a-or B-globin chains. 149 Sequence analysis of immunoglobulin light-chain mRNA has proved that a single mRNA molecule codes for both the variable and constant regions of the antibody light chain.'" In addition an untranslated sequence of 52 nucleotides adjacent to the poly(A) segment of this mRNA has been determined. This shows striking homologies with the corresponding sequence of 52 nucleo-tides from rabbit P-globin mRNA.' 51 In both cases identically positioned double-hairpin-loop secondary structures can be drawn which may have an important bearing on the biological function of these and other mRNA molecules.5'-Terminal sequences exceeding 60 nucleotides and including the initiation codons have been determined for the mRNAs from the lactose'52 and galactose'53 operons of E. coli. An examination of the sequences at the ribosome-binding sites of a number of RNA molecules' 54 has revealed some remarkable symmetries which may have implications for the evolution of the genetic code. True palindromic sequences (reading the same forwards and backwards) frequently occur overlapping with or immediately following the initiation codon. In addition sequences from this region often contain one or more out-of-phase termination codons.Sequences of 83 nucleotides from the replicase cistron'55 of phage Qp and of 54 nucleotides from the A protein ci~tron'~~ of phage R17 have been reported. The in oitro preparation of QP RNA with a G to A transition 16 bases from the 3'-terminus has been described.' 57 Further studies involving 'evolution in a 146 R. Desrosiers K. Friderici and F. Rottman Proc. Nut. Acad. Sci. U.S.A. 1974 71 397 1. R. P. Perry and D. E. Kelley Cell 1974 1 37. 148 R. A. Kramer M. Rosenburg and J. A. Steitz J. Mot. Biol. 1974 89 767 777. 149 C. A. Marotta B. G. Forget S. M. Weissman I. M. Verma R. P. McCaffrey and D. Baltimore Proc. Nut. Acad. Sci. U.S.A. 1974 71 2300. lSo C. Milstein G. G. Brownlee E. M. Cartwright J.M. Jarvis and N. J. Proudfoot Nature 1974. 252 354. lS1 N. J. Proudfoot and G. G. Brownlee Narure 1974 252 359. 152 N. M. Maizels Proc. Nut. Acad. Sci. U.S.A. 1973 70 3585. 153 R. E. Musso B. DeCrombrugghe I. Pastan J. Sklar P. Yot and S.Weissman Proc. Nat. Acad. Sci. U.S.A. 1974 71 4940. L54 G. Pieczenik P. Model and H. D. Robertson J. Mol. Biol.,1974 90 191. Is' A. G. Porter J. Hindley and M. A. Billeter European J. Biochem. 1974 41 413. IS' U. F. E. Rensing A. Coulson and J. G. G. Schoenmakers European J. Biochem. 1974 41 431. R. A. Flavell D. L. Sabo E. F. Bandle and C. Weissman J. Mol. Biol. 1974,89 255. Nucleic Acids 405 test tube' have been carried on the MDV-1 RNA mentioned in last year's Report." The sequence of a mutant RNA whose replication is resistant to ethidium bromide inhibition and which evolved during a serial transfer experi- ment differs from that of wild-type MDV-1 RNA at only three positions out of 218.The secondary structure of a 59-nucleotide fragment from phage R 17 mRNA has been e~tablished"~ by a combination of different physical techniques. mRNA is expected to form stable secondary structures computer-generated random base sequences can be arranged into thermodynamically stable conformations with about 50'1 of the bases paired.16' 8 DNA General.-Methods of gene isolation have been reviewed. lhl Solid supports bearing DNA sequences for specific genes can be preparedLh2 by hybridizing mRNA to oligo(dT)-cellulose by means of its poly(A) tail and then using reverse transcriptase to synthesize cDNA which is covalently attached to the cellulose.This provides a highly specific chromatographic medium for the isolation and purification of complementary nucleic acid sequences. The transformation of cells in uitro can be effected by a segment of human adenovirus type-5 DNA which contains about 1500 base pairs and which is located close to one end of the DNA molecule. 63 Excluded-volume interactions of DNA with other macromolecules such as polyethylene oxide in solution cause it to collapse into a compact relatively dense state known as $ DNA. Production of $ DNA in viw may be relevant to chromosome structure and the packaging of DNA in viruses. X-Ray scattering studies have now shown164 that $ DNA adopts a folded-chain structure com- parable to that of many crystalline linear polymers.Despite its highly anomalous c.d. spectrum the secondary structure of DNA differs little from that of the B-form of DNA to which it reverts on heating.'65 Lnteraction with Small Molecules.-Supercoiled DNA is twice as twisted as was thought! A careful study'66 of the unwinding of helical DNA by ethidium based on a new theoretical and experimental approach has shown that each bound ethidium molecule unwinds the helix by 26 _+ 3 degrees instead of the previously accepted value of 12". The antibiotic echinomycin which comprises two quinoxaline rings linked by a cyclic octapeptide is an extremely potent 15* F. R. Kramer.D. R. Mills P. E. Cole T. Nishihara and S. Spiegelman J.Mo/. Biol. 1974 89 719. J. Gralla J. A. Steitz and D. M. Crothers Nature 1974 248 204; C. W. Hilbers R. G. Shulman T. Yamane and J. A. Steitz ibid. p. 225. '(I" J. Gralla and C. DeLisi Nature 1974 248 330. Ih' D. D. Brown and R. Stern Ann. Rev. Biochem. 1974 43 667. 16' P. Venetianer and P. Leder Proc. Nut. Acad. Sci. U.S.A. 1974 71 3892. F. L. Graham A. J. van der Eb and H. L. Heijneker Nature 1974 251 687. T. Maniatis J. H. Venable jun. and L. S. Lerman J. Mol. Biol. 1974 84 37. 165 S.-M. Cheng and S. C. Mohr F.E.B.S. Letrers 1974 49 37. lh6 J. C. Wang J. Mol. Biol. 1974 89 783. R.J. H. Davies inhibitor of RNA synthesis. On binding to DNA both the quinoxaline rings of the antibiotic are intercalated into the helix.'67 Intercalative binding to DNA is also exhibited by a cationic terpyridyl platinum complexL68 and by the anti- tumour agent 9-hydroxyellipticene.'69 The ability of acridine dyes to cause frame- shift mutations appears to correlate with the quenching of their fluorescence when intercalated close to a GC base pair.'70 A phenazinium dye with a high specificity for complexing GC base pairs elutes DNA molecules from hydroxy- apatite according to their base comp~sition.'~ ' Preferential binding to GC-rich DNA is shown' 72 by the antitumour compound C~S-P~(NH,)~CI,. Tritium-displacement studies' 73 indicate that the binding of benzpyrene to DNA does not involve the K-region but probably occurs by covalent substitution at position 1 or 6. Sequence Studies.-A detailed account has now appeared' 74 of the methodology used in the direct determination of the sequence (48 bases) of a fragment from phage 4x174 DNA.DNA polymerase I repair synthesis with ribosubstitution and primed by appropriate oligodeoxyribonucleotides,has been used in estab- lishing the sequences complementary to a stretch of 81 nucleotides in phage fl DNA," and of 29 nucle~tides'~~ from the 1-strand of phage $8Opsu,,,+ DNA. The latter sequence (26) immediately precedes the starting point of transcription of the E. coli tRNATyr gene. It shows remarkable elements of two-fold symmetry (boxed in bases) about its central base pair and can loop out from the regular DNA helix to form cruciform arms in which 18 out of 25 residues are base- paired.RNA transcripts complementary to both the strands of SV40 DNA in the region immediately preceding the preferred site of initiation by E. coli RNA polymerase have been isolated and sequenced.' 76 The DNA template in this promotor region contains a palindromic sequence of 17 bases. '67 M. J. Waring and L. P. G.Wakelin Nature. 1974 252 653. 16' K. W. Jennette S. J. Lippard G. A. Vassiliades and W. R. Bauer Proc. Nar. Acad. Sci. U.S.A. 1974 71 3839. 169 J.-B. Le Pecq N.-D. Xuong C. Gosse and C. Paoletti Proc. Nat. Acad. Sci. U.S.A. 1974 71 5078. "* J. P. Schreiber and M. P. Daune. J. Mol. Biol. 1974 83 487. W. Pakroppa and W. Miiller Proc. Nat. Acad. Sci. U.S.A. 1974 71 699. 17' P. J. Stone A. D. Kelman and F. M. Sinex Nature 1974 251 736. 173 G.M. Blackburn P. E. Taussig and J. P. Will J.C.S. Chem. Comm. 1974 907. 17' F. Galibert J. Sedat and E. Ziff J. Mol. Bid 1974 87 377. 17' T. Sekiya and H. G. Khorana Proc. Nut. Acad. Sci. U.S.A. 1974 71 2978. B. s. Zain S. M. Weissman. R. Dhar and J. Pan Nucleic Acids Res. 1974 1 577; R.Dhar S. M. Weissman B. S. Zain J. Pan and A. M. Lewis jun. ibid. p. 595. Nucleic Acids 407 The specific cleavage of DNA molecules by restriction endonucleases and the ordering of the resulting fragments into physical maps has found wide application during the year. Several such studie~'~~-'~~ have been made on SV40 DNA and the results have proved very helpful in understanding certain aspects of the expression of this genome.' 79 Restriction-cleavage maps have also been reported for the replicative form of 6x174 DNA,'80 polyoma virus DNA,'" and mito- chondrial DNA.'82 1 1 (5') N-G-T-T-A-A-C-N (3') (5') N-C-C-G-G-N (3') (3')-N-C-A-A-J-T-G-N (5') (3') N-G-G-C-C-N (5') The symmetrical sequences at the cleavage sites of two restriction endo- nucleases from H.parainjluenzae Hpa I (27) and Hpa I1 (28) have been deter- mined.'83 The phage P1 modification enzyme methylates the central adenine in the sequence pAGATCT.la4 The duplex formed by this hexanucleotide with its complementary sequence resembles the recognition sites for several restriction enzymes in having a two-fold axis of symmetry. A review of DNA modification and restriction has been published.'8s Satellite DNA.-Restriction endonucleases have proved very useful in the identification and analysis of satellite DNAs.Because of their repetitive base sequence many satellite DNAs are resistant to the action of restriction enzymes and this may be used as a criterion for their identification and subsequent isolation.'86 In other cases such as some rodent satellite DNAs,lg7 a highly regular arrangement of restriction-endonuclease-sensitivesites has been ob-served. By its susceptibility to different restriction enzymes bovine satellite I DNA has been proved'88 to consist of direct tandem repeats 1400 base pairs in length. Renaturation kinetics indicate that this repeated sequence is itself internally repetitious. Three satellite DNAs from Drosophila virilis have repeating heptanucleotide sequences la9 which are related to one another by simple base- P.Lebowitz W. Siegel and J. Sklar J. Mol. Biol. 1974 88 105. K. N. Subramanian J. Pan S. Zain and S. M. Weissman Nucleic Acids Res. 1974 1 727. D. Nathans S. P. Adler. W. W. Brockman K. J. Danna T. N. H. Lee and G. H. Sack jiin. Fed. Pror. 1974 33 1135. A. S. Lee and R. L. Sinsheimer Proc. Nut. Acad. Sci. U.S.A. 1974 71 2882. B. E. Griffin M. Fried and A. Cowie Proc. Nar. Acad. Sci. U.S.A. 1974 71 2077. W. M. Brown and J. Vinograd Proc. Nar. Acad. Sci. U.S.A. 1974 71 4617. D. E. Garfin and H. M. Goodman Biochem. Biophys. Res. Comm. 1974,59 108. J. P. Brockes P. R. Brown and K. Murray J. Mol. Biol. 1974 88 437 W. Arber Progr. Nucleic Acid Res Mol. Biol. 1974 14 1. G. Roizes Nucleic Acids Res.1974 1 1099. W. Horz I. Hess and H. G. Zachau European J. Biochem. 1974 45 501. M. R. Botchan Nature 1974 251. 288. IB9 J. G. Gall and D. Atherton J. Mol. Biol. 1974 85 633 408 R.J. H. Ducks pair changes satellite I poly(dACAAACT).poly(dAGTTTGT) ; satellite 11 poly(dATAAACT).poly(dAGTTTAT);satellite 111 poly(dACAAATT).poly-(dAATTTGT). Hermit-crab satellite I DNA is p~ly(dTAGG).poly(dCCTA).'~~ Chromosome Structure.-Investigations of the organization of the DNA sequences in eukaryotic genomes have concentrated on the readily available ribosomal DNA from Xenopus. The 5s ribosomal DNA consists of repeated units of 750 base pairs of which 120 base pairs represent the actual gene for 5s RNA while the remainder is termed spacer DNA.Twelve closely related oligomers about 15 nucleotides long have now been identified'" from the spacer region and their tandem arrangement has been demonstrated. In the DNA containing the genes for Xenopus 18s and 28s rRNA the repeating units are arranged in head-to-tail fashion'92 and the spacer lengths are hetero-geneous.193 The individual strands of eukaryotic DNAs contain large numbers of self-complementary sequences which are capable of forming hairpin loops containing thousands of base pairs.'94 A new model for the structure of chromatin'95 is based on a repeating unit of two of each of the four main types of histone and about 200 base pairs of DNA. It is supported by endonuclease digestion studies.'96 A comprehensive survey of the structure and function of chromosomes has appeared.I9' Enzymes.-Many of the latest developments in the field of DNA replication are described in a collection of papers from a recent syrnp~siurn.'~~ The role of multi-enzyme systems in DNA replication has been reviewed.'99 At high tem- peratures cytosine bases in single-stranded DNA are deaminated to uracil at an appreciable rate whereas in native DNA they are well protected.200 An N-glycosidase activity has been detected,20' in extracts from E.coli which releases free uracil from single- and double-stranded DNA containing deaminated cytosine residues. An exonuclease from calf thymus has been characterized202 which introduces single-strand breaks into double-stranded DNA specifically Iyo D. M. Skinner W. G. Beattie F.R. Blattner B. P. Stark and J. E. Dahlberg Bio-chrmistrj,. 1974 13 3930. 19' G. G. Brownlee E. M. Cartwright and D. D. Brown J. Mol. Biol. 1974 89 703. Iy2 S. Henikoff J. Heywood and M. Meselson J. Mol. Biol. 1974 85 445. 19' P. K. Wellauer R. H. Reeder D. Carroll D. D. Brown A. Deutch.T. Higashinakagawa and 1. B. Dawid Proc. Nut. Acad. Sci. U.S.A. 1974,71. 2823. '94 D. A. Wilson and C. A. Thomas jun. J. Mol. Biol. 1974 84 115. Iy5 R. D. Kornberg Science 1974 184 868. '96 M. Noll Nature 1974 251 249. 19' Cold Spring Harbor Symp. Quant. Biol.,1974 vol. 38. 19' 'DNA Synthesis in Vitro' ed. R. D. Wells and R. B. Inman. Medical and Technical '" Publishing Lancaster 1973. R. Schekman A. Weiner and A. Kornberg Science 1974 186 987. 2oo T. Lindahl and B.Nyberg Biochemistry 1974 13 3405. T. Lindahl Proc. Nut. Acad. Sci. U.S.A. 1974 71 3649. 202 S. Ljundquist and T. Lindahl J. Biol. Chem. 1974 249 1530; S. Ljundquist A. Andersson and T. Lindahl ibid. p. 1536. Nucleic Acids 409 at apurinic sites. The covalent linking of polyribonucleotides to polydeoxyribo- nucleotides by DNA ligase has been dem~nstrated.”~ Microwave-induced emission spectrometry which can detect as little as 10-l4 (g atom) of metal in a 1 p1 sample has been used to prove that reverse transcriptase from avian myeloblastosis virus contains two atoms of zinc per enzyme molecule.204 This observation may account for demonstrated dif- ferences in zinc metabolism between normal and leukaemic leukocytes. Con-ditions have been estab1ished2O5 for using E.coli DNA polymerase I as a reverse transcriptase to produce cDNA from an RNA template; the molar ratio of enzyme to template is critical. The reconstitution of the RNA polymerase core and holoenzymes from their respective subunits has been achieved.206 A new form of RNA polymerase RNA polymerase 111 has been discovered207 which has an extra subunit of low molecular weight bound to the holoenzyme RNA polymerase I. lo3 K. Nath and J. Hurwitz J. Biol. Chem. 1974 249 3680. ‘04 D. S. Auld H. Kawaguchi D. M. Livingston and B. Vallee Proc. Nut. Acud. Sci. U.S.A..1974 71 2091. ’*’S. C. Gulati D. L. Kacian and S. Spiegelman Proc. Nut. Acud. Sci. U.S.A. 1974 71 1035; E. C. Travaglini and L. A. Loeb Biochemistry 1974 13 3010. ’04 L.R. Yarborough and J. Hurwitz J. Biol. Chem. 1974 249 5394. ‘07 W. Wickner and A. Kornberg Proc. Nat. Acad. Sci. U.S.A. 1974 71 4425.

ISSN:0069-3030    

DOI:10.1039/OC9747100383

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

15 Synthetic Methods By W. B. MOTHERWELL AND J. S. ROBERTS Department of Chemistry University of Stirling Stirling FK9 4LA 1 Alkanes The development of synthetically useful copper-derived reducing agents con- tinues to claim the attention of several groups. Masamune and his co-workers’ have prepared their second reagent by the addition of butyl-lithium to a stirred suspension of copper hydride in ether and have shown that primary secondary and tertiary halides mesylates and tosylates are smoothly reduced to the hydro- carbon at room temperature. The reduction of cyclohex-3-enyl tosylate to cyclohexene without elimination is particularly noteworthy. Although ester groups are inert aa-unsaturated ketones are reduced to the corresponding carbonyl compounds in very high yield.By way of contrast the ‘copper hydride’ derived by treatment of cuprous iodide with two equivalents of potassium tri-s- butyl borohydride is capable of reducing both sp3-and sp2-hybridized organic halides in addition to alkynes and ketones2 The direct conversion of esters and epoxides into alkanes is without precedent in the chemical literature. However van Tamelen and Gladysz3 have now devised an extraordinary reagent for this purpose from the reaction of titanocene di- chloride with sodium sand. Mechanistic studies have established that the most probable pathway involves deoxygenation to give an olefin complex of titanium which is subsequently reduced. The conversion of a formyl group into a methyl group is also possible thus avoiding the severity of Clemmensen or Wolff-Kishner reduction conditions.Full details are now available on the ‘exhaustive methylation’ of tertiary and alkyl-aryl carbinols by trimethylaluminium at elevated temperatures4 This excellent method for the replacement of the hydroxy-group by a methyl group can also be applied to various aldehydes and ketones to yield gem-dimethylated hydrocarbons in moderate yield. The reductive removal of phenolic hydroxy-groups in the form of their isourea or carbonimidic ester derivatives by catalytic hydrogenation at room temperature S. Masamune G. S. Bates and P. Georghiou J. Amer. Chem. SOC.,1974 96 3686. T. Yoshida and E.-I. Negishi J.C.S. Chem. Comm. 1974 762. ’ E. E. van Tamelen and J. A. Gladysz J. Amer. Chem. SOC.,1974 96 5290.D. W. Harney A. Meisters andT. Mole Austral. J. Chem. 1974 27 1639; A. Meisters andT. Mole ibid. p. 1665. 411 W.B. Motherwell and J. S. Roberts has been described. Aliphatic alcohols are also converted into alkanes in good yield oiu the isourea deri~ative.~ A convenient route to many electron-rich biaryl compounds involves the reaction of aryl-lead(1v) tricarboxylates with aromatic hydrocarbons.6 The Ullmann synthesis of biaryls has been re~iewed.~ A new method for the reduction of organomercurials by exchange with lithium powder followed by treatment with methanol has been described.8 A detailed investigation of the borohydride-reduced palladium catalyst reveals it to be highly selective for hydrogenation of the olefinic linkage.' Trityl tri- fluoroacetate generated in situ is an effective hydride abstractor and is particular- ly useful for the dehydrogenation of hydroaromatic compounds thus providing a good route to arenes." 2 Alkenes The Wittig olefination with non-stabilized ylides has been performed in a benzene- aqueous alkaline solution where the phosphonium salt functions not only to generate the phosphorane but also as a phase-transfer catalyst.' ' It is interesting to note that ketones are inert under these conditions although aliphatic and aromatic aldehydes give good yields of alkene.A timely review on olefin syn- thesis with organic phosphonate carbanions has been published. l2 An excellent alternative to the Wittig method for the methylenation of carbonyl compounds involves reaction with methylene iodide md magnesium amalgam.' Deuteriated terminal olefins have been prepared in this way without scrambling or loss of label. The synthesis of dienes continues to be an important area. Thus the reaction of enolates with readily prepared trans-1-butadienyltriphenylphosphonium bromide (1) results in a one-step synthesis of conjugated cyclohexadienes in moderate yield l4 (Scheme 1). The addition of ap-unsaturated aldehydes ketones 0-M' R' A Reagents i Ph,P; ii Na,CO Scheme 1 E. Vowinkel and C. Wolff Chem. Ber. 1974,107,907 1379; E. Vowinkel and I. Buthe ibid. p. 1353. ' H. C. Bell J. R. Kalman J. T. Pinhey and S. Sternhell Tetrahedron Letters 1974 857. ' P. E. Fanta Synthesis 1974 9. * V.G. Aranda J. Barluenga A. Ara and G. Asensio Synthesis 1974 135. T. W. Russell and D. M. Duncan J. Org. Chem. 1974 39 3050. lo P. P. Fu and R. G. Harvey Tetrahedron Letters 1974 3217. " W. Tagaki I. Inoue Y. Yano and T. Okonogi Tetrahedron Letters 1974 2587. l2 J. Boutagy and R. Thomas Chem. Rec. 1974 74 87. D. Hasselmann Chem. Ber. 1974 107 3486. l4 P. L. Fuchs Tetrahedron Letrers. 1974 4055. Synt he tic Methods 413 esters and nitriles to the hexacarbonyldicobalt complex of phenylacetylene occurs regiospecifically at the terminal end of the alkyne to afford good yields OF highly functionalized conjugated dienes. Vinylalanes prepared by the cis-monohydroalumination of acetylenes couple with allylic bromides in the presence of cuprous chloride to produce trans-1,4-dienes in a stereoselective manner.l6 An exceptionally mild and stereospecific diene synthesis relies on the extremely facile ,4s + ,2s cycloreversion of a dihydro-oxathiin 2-oxide (2)’ ’ (Scheme 2). x so2 CH,OH 1111. I\ I Ph Ph Reagents i m-chloroperbenzoic acid; ii N-chlorosuccinimide; iii CrO ; iv PhMgBr Scheme 2 Continuing interest is apparent in the preparation of olefins from precursors containing the sulphonyl grouping. The monoanion of a sulphone yields an olefin on treatment with lithium aluminium hydride,” as does the dianion on oxidation with cupric chloride.” Grieco and Masaki” have developed a method for the stereospecific formation of 1,5-dienes that involves the coupling of allylic sulphones with allyl halides followed by reductive cleavage.Three new procedures for the synthesis of symmetrical diallylic sulphones from allyl alcohols and sulphur-transfer reagents have been reported by Buchi and Freidinger.’ These authors have also developed a modified Ramberg-Backlund reaction for the subsequent formation of the triene by oxidation of the r,a’-dianion of the sulphone with a halogen. Julia and his colleagues22 have prepared a remarkable palladium 15 I. U. Khand and P. L. Pauson J.C.S. Chem. Comm. 1974 379. 16 R. A. Lynd and G. Zweifel Synthesis 1974 658. 17 F. Jung M. Molin R. Van Den Elzen andT. Durst J. Amer. Chem. Soc. 1974,96,935. In J. M. Photis and L. A. Paquette J. Amer. Chem. Soc. 1974 96 4715. 19 J. S. Grossert E.W. H. Asveld J. Buter and R. M. Kellogg Tetrahedron Letters 1974 2805. 20 P. A. Grieco and Y. Masaki J. Org. Chem. 1974 39 2135. 21 G. Buchi and R. M. Freidinger J. Amer. Chem. Soc. 1974 96 3332. ?> M. Julia and L. Saussine Terrahedron Letrers 1974 3443. W. B. Motherwell and J. S. Roberts complex (3) which has an unusual propensity for substituting into isolated (Scheme 3).6 R' R2 - R1 R2 + R1CH=CR2R3 -P \ /c=c I \ /c=c olefins. Methylated olefins are obtained from the allylic sulphones on reduction PhS0,CH /\R3 /\Me R3 /pd \CH,S02Ph (3) Reagents i Na-Hg Scheme 3 A full report on the synthesis of alkenes from carbonyls and carbanions adjacent to silicon is now available.23 An important observation in relation to stereospecific olefin synthesis has been made by Hudrlik and Peterson,24 who have achieved the selective reduction of a 8-ketosilane to one diastereoisomeric alcohol and have evolved elimination conditions which lead stereospecifically to either the cis-or the trans-olefin.New and improved methods are available for the stereospecific inversion of the carbon-carbon double bond via deoxygenation of epoxides. Reaction of UP-epoxy-esters with octacarbonyldicobalt affords high yields of olefins with inversion under mild conditions thus filling a gap where the previously reported phosphide-phosphonium salt method of Vedejs and Fuchs fail's.2 The latter method has also been improved by Bridges and Whitham,26 who have shown that ring-opening of the epoxide with diphenylphosphide anion followed by oxidation with hydrogen peroxide yields crystalline P-hydroxydiphenylphosphineoxides.Stereospecific fragmentation to the olefin results on treatment with sodium hydride in DMF. This four-step sequence is successful with di- tri- and tetra- substituted olefins and is experimentally convenient since the sodium diphenyl- phosphinate produced in the elimination is easily removed. Sulphoximines of N-amino-oxazolidones (4),readily prepared from the corresponding epoxides 0 )( ,N=S(O)Me ON (4) undergo a very efficient thermal fragmentation to alkenes with overall inversion of stere~chemistry.~' 23 T. H. Chan and E. Chang J. Org. Chem. 1974,39 3264. 24 P. F. Hudrlik and D. Peterson Tetrahedron Letters 1974 1133.25 P. Dowd and K. Kang J.C.S. Chem. Comm. 1974 384. 26 A. J. Bridges and G. H. Whitham J.C.S. Chem. Comm. 1974 142 Cf. E. Vedejs and P. L. Fuchs J. Amer. Chem. SOC.,1973 95 822. 27 J. D. White and M. Kim Tetrahedron Letters 1974 3361. Synthetic Met hods Pursuing their studies on the chemistry of lower-valent titanium species McMurry and his co-workers2* have found that the addition of one equivalent of lithium aluminium hydride to two equivalents of titanium trichloride furnishes a powerful reagent for the intermolecular deoxygenation of aliphatic and aromatic aldehydes and ketones to olefins. Adamantylideneadamantane can thus be prepared from adamantanone in 85 yield by this 'retro-ozonolysis' reaction. Other aspects of the chemistry of lower-valent titanium species have been con- cisely re~iewed.'~ A novel reaction of trialkylalkynylborates with dihalogenomethanes in- volves two alkyl migrations.Good yields of highly branched terminal olefins are obtained on hydrolysis (Scheme 4).30 [R:B-C=C-Rz]Li' + CH,Br -+-+RiCH \ /C=CH2 R2 Scheme 4 The formation of alkenes by the thermolysis of p-lactones continues to emerge as a synthetically useful proce~s.~' Barton and his collaborator^^^ have con- solidated their studies on the two-fold extrusion process by the synthesis of some very highly hindered olefins (Scheme 5). Reagents i A Ph,P Scheme 5 A new method which shows promise as a useful alternative to the Hofmann degradation involves preparation of the hydrazinium salt of the tertiary amine followed by elimination with potassium t-b~toxide.~~ This aza-analogue of the Cope amine oxide reaction proceeds at lower temperatures to form the olefin in reasonable yield.28 J. E. McMurry and M. P. Fleming J. Amer. Chem. SOC.,1974 96 4708. l9 J. E. McMurry Accounts Chem. Res. 1974 7 281. 30 A. Pelter and C. R. Harrison J.C.S. Chem. Comm. 1974 828. 31 A. P. Krapcho and E. G. E. Jahngen jun. J. Org. Chem. 1974 39 1322 1650. 32 D. H. R. Barton F. S. Guziec jun. and I. Shahak J.C.S. Perkin I 1974 1794. 33 H. Posvic and D. Rogers J. Org. Chem. 1974 39 1588. 416 W.B. Motherwell and J. S. Roberts RZ R2 i ii I I Me P(O)(OEt) Me CH P(0)(OEt), \ / \/ 111 //C-H C=CH-CH -* H-C / /C=Cx I R' R' H C Me /I\ H R' Reagents i Bu"Li; ii R2X; iii LiAIH Scheme 6 A useful two-step stereoselective synthesis of olefins from ally1 halides has been described (Scheme 6).34 Even if the phosphonate is not stereochemically pure reductive fission still produces the trans-olefin.The alkylation of benzyl phenyl selenides with alkyl or aralkyl halides followed by oxidation to the selenoxide and elimination provides a mild method for the formation of trans-alkenes in high yield.35 An unusual reaction of some synthetic potential is the formation of a remote double bond by the ferrous sulphatexupric-acetate-promoted decomposition of alkyl hydro peroxide^.^^ The availability of the 6 hydrogen atom is an impor- tant factor in this process and a preference for the least substituted double bond in the product has been noted (Scheme 7).Reagents i Fe"-Cu2+ HOAc Scheme 7 A mild non-oxidative method for the disengagement of sensitive organic ligands from their iron tricarbonyl complexes involves treatment with amine oxides in aprotic ~olvents.~' The importance of these complexes as reagents for organic synthesis is the subject of an excellent review.38 3 Alkynes and Allenes Lithium trialkylalkynylborates (5; R2 = alkyl) were highlighted last year as versatile synthetic intermediates. Activity in this area continues unabated. Full Li + [R :B-C-CR2] 34 K. Kondo A. Negishi and D. Tunemoto Angew. Chem. Infernat. Edn. 1974 13 407. 35 R. H. Mitchell J.C.S. Chem. Comm. 1974 990. 36 Z. Cekovic and M.M. Green J. Amer. Chem. Soc. 1974 96 3000. 37 Y. Shvo and E. Hazum J.C.S. Chem. Comm. 1974 336. 38 M. Rosenblurn Accounts Chem. Res. 1974 7 122. Synthetic Methods 417 experimental details have been published of their reaction with methanesul- phinyl chloride which provided a new route to internal acetylene^.^^ H. C. Brown and his co-workers4' have now succeeded in preparing the parents of the series (5; R2 = H) by the reaction of trialkylboranes with commercially available lithium acetylide-ethylenediamine thus considerably extending the utility of these intermediates as exemplified by the synthesis of terminal acetylenes by the subsequent reaction with iodine. Corey and Wollenberg4' have also developed a new route to terminal alkynes based on the facile metallation of readily avail- able trans-l,2-bis(tri-n-butylstannyl)ethylene(Scheme 8).Virtually quantitative Reagents i Bu"Li; ii R'X; iii Pb(OAc),; iv n-propylethynylcopper; v 4 Scheme 8 alkylation occurs with a variety of alkyl halides and subsequent unmasking of the acetylene is easily accomplished by treatment with lead tetra-acetate. Alternatively the reaction of the metallated reagent with n-propylethynyl- copper leads to the formation of a novel mixed organocuprate which can function as a nucleophilic ethynyl-group equivalent. A simple one-pot conversion of ketones into internal acetylenes has been achieved by heating them with phos- phorus pentachloride and pyridine in anhydrous benzene.42 The year under review has witnessed the growth of vinylsilanes as reagents which show promise of impressive synthetic utility.A particularly elegant method for the conversion of a carbonyl compound into the corresponding allene using the Peterson reaction illustrates the usefulness of a-lithiovinyltriphenylsilane (Scheme 9).43 Grieco and his ~o-workers~~ have once again exploited the utility of the [2,3]sigmatropic rearrangement in organic synthesis by providing a convenient route to terminal and internal allenes from acetylenic sulphoniuni ylides. The 34 M. Naruse K. Utimoto and H. Nozaki Tetrahedron 1974 30 2159. 40 M. M. Midland J. A. Sinclair and H. C. Brown J. Org. Chem. 1974 39 731. 4' E. J. Corey and R. H. Wollenberg J. Amer. Chem. Soc. 1974 96 5581. 42 C. M.Wong and T.-L. Ho Synthetic Comm. 1974 4 25. 43 T. H. Chan and W. Mychajlowskij Tetrahedron Letters 1974 171. For further reactions of vinylsilanes consult refs. 58 7 I 1 13 158 254 255 and 257. 44 P. A. Grieco M. Meyers and R.S. Finkelhor J. Org. Chem. 1974 39 119. W. B. Motherwell and J. S. Roberts R' CI R' i.ii ,cySiPh3 % \ \ c=o -? Rt,/ /C=C=CHz R B R2 CH R2 Reagents i. Ph,Si'-,.(Li ; ii SOCl,; iii F- DMSO CH Scheme 9 hydrolysis of l-bromo-2,2-dial kyl-3-meth ylenecyclopropanes in buffered medium provides a good route to tertiary a-allenic alcohols.45 Treatment of the lithium salt of propargyl chloride with a trialkylborane followed by reaction with acetic acid is an operationally convenient and high-yield method for the addition of the allene moiety to olefinic double Organoboranes also feature in the first stereoselective synthesis of cumulene derivatives (Scheme Reagents i l-+-BH2 0.5 equiv.; ii NaOMe 2.0 equiv.; iii I Scheme 10 4 AlkylHalides Tertiary alkyl iodides can be prepared from their corresponding chlorides in excellent yield by treatment with sodium iodide in a non-polar solvent containing a trace of ferric chloride as catalyst.48 Since primary and secondary alkyl chlorides are unreactive under these conditions this method forms a useful complement to the sodium iodide in acetone exchange procedure.The conversion of an al- cohol into its salicylate ester followed by reaction with phosphorus penta- chloride has been resurrected by Pinkus and Lin49 as an operationally simple method for the preparation of alkyl chlorides in high yield.45 G. Leandri H. Monti and M. Bertrand Bull. SOC.chim. France 1974 1919. 46 T. Leung and G. Zweifel J. Amer. Chem. SOC.,1974 96 5620. 47 T. Yoshida R. M. Williams and E. Negishi J. Amer. Chem. SOC.,1974 96 3688. '* J. A. Miller and M. J. Nunn Tetrahedron Letters 1974 2691. 49 A. G. Pinkus and W. H. Lin Synthesis 1974 279. Syntheiic Methods 419 The fluoride ion is generally considered to be a poor nucleophile for carbon compounds because of solvation. However the crown ether complex of potassium fluoride in benzene allows 'naked fluoride' to function as a potent nucleophile in displacement reactions with alkyl bromides and as a strong base.50 An alternative approach to the synthesis of primary and secondary alkyl fluorides by displacement reactions employs hexadecyltributylphosphonium bromide as a phase-transfer catalyst.51 The same catalyst is also effective for the con- version of primary alcohols into chlorides using aqueous hydrochloric acid.52 Selenium tetrafluoride and its pyridine complex are fluorinating agents of general use particularly for the replacement of hydroxy-groups in carboxylic acids arid alcohols and for the formation of gem-difluorides from aldehydes and ketone^.^ Olah and Welch54 have explored the use of a polyhydrogen fluoride-pyridine solution for the preparation of a-fluorocarboxylic acids from amino-acids and alkyl halides from alcohols.Reviews on uses of sulphur tetrafluoride and methods for the preparation of monofluoro-aliphatic compounds are included in the latest volume of Organic Reactions.55 The increasingly important role of vinyl-lithium and copper reagents in organic synthesis has led to the development of novel approaches to vinyl halides.The reaction of anhydrous hydrogen bromide with terminal trimethylsilylacetylenes results in the elimination of the silyl group as the halide and the production of 2-bromo-1-alkenes in high yield.56 Normant and his co-workers5 have found that the reaction of a vinylcopper reagent with mercuric bromide yields an organo- mercurial. Subsequent treatment with bromine in pyridine affords the vinyl bromide with retention of configuration. The vinylsilane (6),obtained by hydro- boration-protonolysis of the corresponding acetylene is a useful precursor for the stereospecific construction of vinyl halides (Scheme 1l).58 (6) Reagents i X (X = C1 or Br); ii NaOMe; iii I,; iv I, CF,CO,Ag; v F- DMSO Scheme 11 Two new methods for the generation of dichlorocarbene offering significantly improved yields of gem-dichlorocyclopropanesby reaction with alkenes are the 50 C.L. Liotta and H. P. Harris J. Amer. Chem. SOC.,1974 96 2250. 5' D. Landini F. Montanari and F. Rolla Synthesis 1974 428. 52 D. Landini. F. Montanari and F. Rolla Synrhesis 1974 37. 53 G. A. Olah M. Nojima and I. Kerekes J. Amer. Chem. SOC.,1974 96 925. s4 ' G. A. Olah and J. Welch Synthesis 1974 652 653. G. A. Boswell jun. W. C. Ripka R. M. Scribner and C.W. Tullock Organic Reactions 1974 21 1 ; C. M. Sharts and W. A. Sheppard ibid. p. 125. s6 R. K. Boeckman jun. and D. M. Blum J. Org. Chem. 1974 39 3307. 57 J. F. Normant C. Chuit G. Cahiez and J. Williams Synthesis 1974 803. R. B. Miller and T. Reichenbach Tetrahedron Letters 1974 543. W.B. Motherwell and J. S. Roberts use of lithium triethylmethoxide as base with chlor~form~~ and the thermal decomposition of trifluoro(trichloromethy1)silanein the vapour phase.60 5 Alcohols Lithium dimesitylborohydride bis(dimeth0xyethane) is a novel crystalline reducing agent which displays unprecedented stereoselectivity for the reduction of carbonyl compounds.6 For example trans-3-methylcyclohexanol is obtained in quantitative yield from 3-methylcyclohexanone.In contrast to the bulky trisubstituted borohydrides this reagent is totally unreactive towards highly hindered ketones such as camphor thus paving the way for greater selectivity. An interesting biomimetic reduction of carboxylic acids to alcohols in very good yield has been achieved by formation of the enol ester derivative (7) upon reaction 0 /I with N-ethyl-5-phenylisoxazolium-3'-sulphonate.This intermediate which had previously been reported by Woodward affords the alcohol on reduction with sodium borohydride.62 Borane-methyl sulphide continues to be advocated as a highly concentrated and inexpensive alternative to borane-THF for the hydroboration-oxidation of olefins and the reduction of benzoic acids to benzyl alcohols.63 Organoboranes are also important intermediates in two carbinol syntheses.Successive treatment of a trialkylborane with a 1-lithio-1,l-bis(pheny1-thio)alkane mercuric chloride and hydrogen peroxide gives excellent yields of tertiary alcohols in a process which is synthetically equivalent to the reaction of Grignard reagents with ketones or esters.64 The low migratory aptitude of the 1 - RIC-CRZ . ... ~lc~z;st/~ R3RlCH2-~-Rz YR-Me I OH R" Reagents i RiBH; ii MeLi; iii HC1; iv NaOH H,O Scheme 12 '' R. H. Proger and H. C. Brown Synrhesis 1974 736. 6o J. M. Birchall G. N. Gilmore and R. N. Haszeldine J.C.S. Perkin i 1974. 2530. " J. Hooz S. Akiyama F. J. Cedar M. J. Bennett and R. M. Tuggle J. Amer. Chem. SOC.,1974 96 274. 62 P. L.Hall and R. B. Perfetti J. Org. Chrm. 1974 39 1 11. 63 C. F. Lane J. Org. Chem. 1974 39 1437; C. F. Lane H. L. Myatt J. Daniels and H. B. Hopps ibid. p. 3052. 64 R. J. Hughes. A. Pelter and K. Smith J.C.S. Chrm. Comm.. 1974 863. Sypt thetic Methods 421 methyl group in a protonated lithium dialkylmethylvinylboronateforms the basis for a new synthesis of secondary and tertiary alcohols in very good yield (Scheme 12).65 A study of the ferrous ion-hydrogen peroxide oxidation of cyclohexanol has revealed a pronounced tendency for the regio- and stereo-selective formation of cis-cyclohexane-1,3-dio1.66Two economical methods for the preparation of 1-deuteriated secondary alcohols have been described.67 Heavily substituted ally1 alcohol units occur in many natural products and synthetic endeavour in this area has therefore been actively promoted.Ap proaches employing organoselenium reagents for the construction of this grouping have proved to be particularly fruitful. Three groups have independently Reagents i PhSeX (X = Br OAc or CF,CO,); ii hydrolysis; iii H,O Scheme 13 reported on the facile electrophilic trans-addition of phenylselenyl halides and acetates to isolated olefins.68 Solvolysis of the adducts followed by selenoxide fragmentation produces allylic alcohols (Scheme 13). Although this approach Reagents i PhSeH H+;ii Bu"Li; iii R4R5CO;iv H,O,; v (PhS) Scheme 14 " G. Zweifel and R. P. Fisher Synthesis 1974 339. bb J. T. Groves and M. Van Der Puy J. Amer. Chem. SOC.,1974 96 5274.67 R. Shanker Chem. and Ind. 1974 76; S. L. Regen J. Org. Chem. 1974,39 260. 68 K. B. Sharpless and R. F. Lauer J. Org. Chem. 1974 39 429; H. J. Reich ibid. p. 429; D. L. J. Clive J.C.S. Chem. Comm. 1974 100. W.B. Motherwell and J. S. Roberts avoids the previously described opening of an epoxide ring by highly nucleo- philic phenylselenide ion it does suffer from a lack of regioselectivity with un- symmetrical substrates. Two groups have discovered that the metallation of selenoacetals by butyl-lithium is a very efficient process.6g The derived car- banions which can thus be classed as masked vinyl-lithium reagents add readily to aldehydes and ketones to give j?-hydroxyselenides and hence allyl alcohols in a completely regiospecific manner (Scheme 14).Seebach and his colleagues have also shown that the carbanions (8) undergo thiolation to afford synthetically valuable secondary and tertiary phenylthioalkyl-lithium reagents. Another stereospecific synthesis of functionalized trisubstituted allylic al- cohols involves the reaction of the vinyl-lithium reagent (9) with aldehydes and ketones followed by allylic transposition and reduction (Scheme 15).70 p-R’O R/’H,” iii H ii R Me R’O,cv~ii+OHC+~~ ->-( + H CHO H CH,OH (9) 0 ‘$ Reagents i RCHO; ii M~~~-NJ ci-; iii L~AIH 0 Scheme 15 Hydroxysilanes obtained by the addition of a-lithiotriphenylvinylsilaneto a carbonyl group undergo a hydroxyl-assisted cleavage of the silicon-vinyl bond by fluoride ion to give allyl alcohols in good yield.71 Readily prepared diethylaluminium 2,2,6,6-tetramethylpiperidide has been used for the highly regioselective isomerization of epoxides into allylic alcohols under very mild conditions (Scheme 16).72 Scheme 16 69 W.Dumont P. Bayet and A. Krief Angew. Chem. Internat. Edn. 1974 13 804; D. Seebach and A. K. Beck ibid. p. 806. 70 J.-C. Depezay and Y. Le Merrer Tetrahedron Letters 1974 2751. 71 T. H. Chan and W. Mychajlowskij Tetrahedron Letters 1974 3479. ’’ A. Yasudo S. Tanaka K. Oshima H. Yamamoto and H. Nozaki J. Amer. Chem. SOC.,1974 96 6513. Synthetic Methods 423 6 Ethers Sharpless and his co-~orkers~~ have now extended their studies on the transition- metal-catalysed epoxidations with t-butyl hydroperoxide to acyclic allylic alcohols.Once again the method is highly stereoselective. A polymeric peracid has been prepared and used to oxidize several olefins to their epoxides in good yield.74 The regiospecific epoxidation of conjugated dienones and. diene esters at the y,&double bond is a potentially useful synthetic operation. This has now been realized by treatment with molecular oxygen in a solvent which contains readily abstractable hydrogen atoms.75 In a continuing exploration of the syn- thetic potential of diphenyI(dialkoxy)sulphuranes Martin and his co-~orkers’~ have shown that reaction with vicinal diols gives excellent yields of epoxides. 1,n-Diols (n = 4 5 or 6)form cyclic ethers in lower yield. Reagents i base; ii H + Scheme 17 Spencer and Garst7’ have developed another new dihydrofuran synthesis this year which relies on the reaction of an acyloin with a p-alkoxyvinylphosphon- ium salt (Scheme 17).7 Amines The [3,3]sigmatropic rearrangement of readily prepared allylic trichloroacetim- idates e.g. (10)-(ll) which results in the 1,3 transposition of alcohol and amino-functions is particularly useful for the synthesis of highly hindered amine~.~~ This reaction is markedly catalysed by the addition of mercuric salts. when a two-step mechanism involving intermediate (12) is probably CCI I ’3 S. Tanaka H. Yamamoto H. Nozaki K. B. Sharpless R. C. Michaelson and J. D. Cutting J. Amer. Chem. SOC.,1974 96 5254. 74 C. R. Harrison and P.Hodge J.C.S. Chem. Comm.1974 1009. ” H. Hart and P. B. Lavrik J. Org. Chem. 1974 39 1793. lb J. C. Martin J. A. Franz and R. J. Arhart J. Amer. Chem. SOC.,1974 96 4604. 7’ M. E. Garst and T. A. Spencer J. Org. Chem. 1974,39 584. L. E. Overman J. Amer. Chem. SOC.,1974 96 597. 424 W.B. Motherwell and J. S. Roberts operative. An efficient process for the synthesis of secondary or tertiary amines which has certain advantages over reductive amination of aldehydes and ketones involves the reaction of an arylmethanol or ally1 alcohol with primary or secon- dary amines in the presence of a palladium catalyst." An improved method for the preparation of amines from olefins via hydroboration employs treatment of the organoborane with the more electrophilic aminating agent O-mesitylene- sulphonyl-hydroxylamine.80 The Curtius reaction is a classical method for the conversion of carboxylic acids into urethanes.The use of tetrabutylammonium azide (soluble in organic solvents) for the formation of the azide from the acid chloride is recommended since thermolysis to the isocyanate can be carried out in the same reaction vessel.8 However full experimental details on the use of diphenylphosphoraz- idate for the one-step conversion of aromatic aliphatic and heterocyclic acids into urethanes are now available.82 The alkylation and arylation of primary and secondary aliphatic and aromatic amines by aliphatic and aromatic aldehydes occur readily in the presence of the hydridotetracarbonylferrate anion.83 Gassman and his colleagues have modified improved and extended their studies on the alkylation of anilines and phenols uia azasulphonium ylides to encompass new methods for the ortho-formylation of aniliness4 and phenols,85 the ortho-alkylation of phenols,86 and the synthesis of 2,3-disubstituted in dole^.^' Earlier studies in this area have now been fully documented.88 Aryl iodides react with bis(trimethylsily1amido)copper to give silylated amines which yield aniline derivatives on methan~lysis.~~ Two very mild methods for the demethylation of tertiary amines in good yield are conversion into the N-nitroso-derivative with silver nitrite in DMF.90 or reaction with 2,2,2-trichloroethyl chloroformate and subsequent liberation of the free amine from the demethylated trichloroethylcarbamate by reductive cleavage with zinc in acetic acid or rnethan01.~~Lithium n-propylmercaptide was reported last year as a reagent for demethylation of aromatic and aliphatic quaternary ammonium salts.Kutney and co-worker~~~ have now found that 79 S.-I. Murahashi T. Shimamura and I. Moritani J.C.S. Chem. Comm. 1974 931. Y. Tamura J. Minamikawa S. Fujii and M. Ikeda Synthesis 1974 196. " A. Brandstrom 9. Lamm and I. Palmertz Acta Chem. Scand. (B) 1974 28 699. K. Ninomiya T. Shiori and S. Yamada Tetrahedron 1974 30 2151; T. Shiori and S. Yamada Chem. and Pharm. Bull. (Japan),1974 22 849. 83 G. P. Boldrini M. Panunzio and A. Umani-Ronchi Synthesis 1974 733. 84 P. G. Gassman and H. R. Drewes J. Amer. Chem. SOC.,1974 96 3002. 85 P. G.Gassman and D. R. Amick Tetrahedron Letters 1974 3463. 86 P. G. Gassman and D. R. Amick Tetrahedron Letters 1974 889. '' P. G. Gassman D. P. Gilbert and T. J. van Bergen J.C.S. Chem. Comm. 1974 201. P. G. Gassman and G. D. Gruetzmacher J. Amer. Chem. SOC.,1974 96 5487; P. G. Gassman T. J. van Bergen D. P. Gilbert and B. W. Cue jun. ibid. p. 5495; P. G. Gassman and T. J. van Bergen ibid. p. 5508; P. G. Gassman G. D. Gruetzmacher and T. J. van Bergen ibid. p. 5512. " F. D. king and D. R. M. Walton J.C.S. Chem. Comm. 1974 256. L. Bernardi and G. Basisio J.C.S. Chem. Comm. 1974 690. '' T. A. Montzka J. D. Matiskella and R. A. Partyka Tetrahedron Letters 1974 1325. 92 J. P. Kutney G. B. Fuller R. Greenhouse and I. Itoh Synthetic Comm. 1974 4 183; Cf R.0. Hutchins and F. J. Dux J. Org. Chem. 1973 38 1961. Synthetic Methods selective debenzylation of quaternary ammonium salts is also possible without detriment to other sensitive functionality. 8 Aldehydes and Ketones To add to the growing list of reagents for the oxidation of primary and secondary alcohols to aldehydes and ketones are the following two modifications. The first one involves sodium dichromate and sulphuric acid in DMSO (as solvent),93 while the second makes use of a number of sulphoxonium complexes generated from DMSO and for example methanesulphonic anhydride or cyanuric chloride in HMPA.94 For the specific oxidation of secondary hydroxy-groups in the presence of primary hydroxy-groups a mixture of chlorine and pyridine can be used.95 Conversely a primary alcohol can be converted into the correspond- ing bromide by triphenylphosphine dibromide in DMF under mild conditions96 (which affords the formate ester of a secondary alcohol) and the primary bromide can be converted into the corresponding aldehyde by reaction with silver tetra- fluoroborate and triethylamine in DMS0.97 Ozonolysis of nitronates is an alternative method for the conversion of primary and secondary nitro-compounds into aldehydes and ketones.98 H SPh ArkN ArCCN ArLCN I I I R R Reagents I Bu"Li;ii PhSCI; iii.Bu'Me,SiCl; iv I,; v Ag,O; vi NBS Scheme 18 The oxidative decyanation of monosubstituted aryla~etonitriles~~" can be accomplished in high yield by two routes (Scheme 18).99b Unfortunately this method cannot be applied to the preparation of dialkyl ketones.Nitriles can be converted into aldehydes by reduction of the corresponding N-alkylnitrilium ions with triethylsilane followed by hydrolysis of the resultant aldimines. O0 93 Y. S. Rao and R. Filler J. Org. Chem. 1974 39 3304. 94 J. D. Albright J. Org. Chem.. 1974 39 1977. 95 J. Wicha and A. Zarecki Tetrahedron Letters 1974 3059. 96 R. K. Boeckman jun. and B. Ganem Tetrahedron Letters 1974 913. '' B. Ganem and R. K. Boeckman jun. Tetrahedron Letters 1974 917. 98 J. E. McMurry J. Melton and H. Padgett J. Org. Chem. 1974 39 259. 99 (a)D. S. Watt Tetrahedron Letters 1974 707; (6)D. S. Watt J. Org. Chem. 1974 39 2799; S. J. Selikson and D. S. Watt Tetrahedron Letters 1974 3029. I00 J.L. Fry J.C.S. Chem. Comm. 1974 45. W. B. Motherwell and J. S. Roberts The reaction of acids (with POCl,) and acid chlorides with 3-methyl- 1,4-diphenyl- isothiosemicarbazide produces the triazolium salts (13) which on borohydride reduction and subsequent acid hydrolysis produce the corresponding al- dehydes.lo l Ph Anderson et af.lo2have made the interesting observation that lithium dialkyl- cuprates react with S-alkyl and S-aryl thioesters to give ketones. Unlike acid chlorides for which an excess of organometallic reagent is used the thioester reaction only requires 0.55 equivalent of the lithium dialkylcuprate (i.e. one equivalent of R). This suggests that the intermediate complex lithium alkylthio- (alkyl)cuprate also reacts with the thioester a result which is in accord with Posner's recent observation that acid chlorides also react with mixed cuprate complexes.lo3 In a reaction analogous to the hydroformylation process acid chlorides can be converted into ketones by addition of olefins in the presence of hydrido(dinitrogen)tris(triphenylphosphine)cobalt(r)(Scheme 19).O4 Two years R'CH=CH + R2COCI + HCo(N,)(PPh,) -* R2COCH2CH,R* Scheme 19 ago it was reported that manganic acetate catalyses the addition of olefins to ketones -a similar process has now been shown to occur with olefins and acetone in the presence of silver oxide with the formation of methyl ketones in relatively high yield.lo5 A new synthesis of unsymmetrical ketones involves the addition of dialkyl-chloroboranes to lithium aldimines followed by electrophile-induced alkyl migration and oxidation (Scheme 20).'06 A variant of the Wittig reaction for the homologation of aldehydes makes use of thiophenoxymethylenetriphenyl-,R2 ... RiBCl + Me,CN=C /R2 -+ Me,CN=C 2R~R~C=O \ \ Li BR; Reagents i HSCH,CO,H; ii H,O, OH- Scheme 20 lo ' G. Doleschall Tetrahedron Letters 1974. 2649. R. J. Anderson C. A. Henrick and L. D. Rosenblum J. Amer. Chem. SOC.,1974,96 3654. Io3 G. H. Posner C. E. Whitten and J. J. Sterling J. Amer. Chem. SOC.,1973 95 7788. J. Schwartz and J. B. Cannon J. Amer. Chem. SOC.,1974 96 4721. M. Hajek P. Silhavy and J. Malek Tetrahedron Letters 1974 3193. lob Y. Yamamoto K. Kondo and I. Moritani Tetrahedron Letters 1974 793. Synthetic Methods phosphorane followed by acetoxymercuration of the derived thio-enol ether and subsequent demercuration and basic hydrolysis.Io7 a-Halogeno-ketones can be dehalogenated by the one-pot sequence of addition of pyridine followed by dithionite reduction of the derived pyridinium salt."' Organic chemists have engineered a number of ingenious guises for the nucleo- philic acyl anion equivalent (14) and this year has been no exception. Seebach and Kolb"' have reviewed this very interesting area of synthetic organic chemis- try and have proposed the term 'umpolung' to define the reversed polarization of carbonyl reactivity. While it may be argued that this terminology has the merit of brevity and avoids confusion with alternative nomenclature one could also make a case for using the term 'lynobrac' if only because it is Seebach carbonyl ! Two closely related acyl anion equivalents are the dithiocarbamate 0 R' I1 \-CH R /-/ R2 [15; R' = SMe It2 = SC(S)NMe,]'" and the bis(dithi0carbamate) [15; R' = R2 = SC(S)NMe,],"' both of which can be readily alkylated with iodides and subsequently converted into aldehydes by hydrolysis in the presence of mercuric ions.Schill and Jones112 have found that the Ogura and Tsuchihashi sulphoxide anion [15; R' = SMe R2 = S(O)Me] can be dialkylated in the presence of excess sodium hydride to produce symmetrical dialkyl ketones after acidic hydrolysis. 1,l-Bis(trimethylsily1)ethene (16) and bis(trimethylsily1)methane (17) have been to be useful precursors of the vinylsilanes (18) (19) and (20) which can be converted into ketones by epoxidation and acid hydrolysis (Scheme 21).(Me,Si),C=CH (Me,Si),CH / \ Me,% Me,Si rlh)jiv>v (17) \Ii Me,Si \ \ C=CHR2 ,,C=CR3R4 R /C=CH2 / R'CH H (18) (19) (20) Reagents i Br,; ii Bu'Li; iii RX; iv R'Li; v R'HCO; vi RLi; vii R3R4C0 Scheme 21 lo' I. Vlattas and A. 0.Lee Tetrahedron Letters 1974 4451. IDS T. L. Ho and C. M. Wong J. Org. Chem. 1974 39 562. log D. Seebach and M. Kolb Chem. and Ind. 1974,687 ;see also ref. 244. ILo I. Hori T. Hayashi and H. Midorikawa Synthesis 1974 705. 'IL T. Nakai and M. Okawara Chem. Letters 1974 731. G. Schill and P. R. Jones Synthesis 1974 117. 'I3 B.-T. Grobel and D. Seebach Angew. Chem.Internat. Edn. 1974 13 83; see also ref. 43. W.B. Motherwell and J. S. Roberts Walborsky et ~1.l'~ have published full details of the use of lithium aldimines (21 ; R = Et Bun Bus or Ph) which react with a number of electrophiles to give a variety of carbonyl compounds. A rather different type of masked acyl anion equivalent is the lithium acyloin enediolate (23),which is obtained by the addition of methyl-lithium to the enediolbis(trimethylsi1yl) ether (22) which. in turn is derivable from the acyloin condensation of an ester.' l5 Alkylation of (23) produces an cr-hydroxy-ketone which on borohydride reduction and subsequent oxidative cleavage produces the ketone (Scheme 22). Finally in the context of R' R' \ /R' I\/ R' -R*R~C=O+ R*CHO -* /c=c\ Me,SiO /c=c\ OSiMe -0 0-2Li + (22) (23) Reagents i 2MeLi; ii R2X; iii NaBH,; iv Pb(OAc) Scheme 22 acyl anions it should be noted that Fraser and Hubert"' have succeeded in preparing the lithio-carbanion (24)from di-isopropylformamide.Although nucleophilic attack at the p-position of an ap-unsaturated enone is a common feature in organic synthesis the reverse of this process namely the creation of a homo-enolate anion equivalent (25) and its subsequent reaction 0 I1 C /\ R CH,-CH (25) with electrophiles i.e.umpolung at C-3,is rather rare."' A number of interesting papers on this subject have been published during the year under review. These 1 I4 G. E. Niznik W. H. Morrison tert. and H. M. Walborsky J. Org.Chem. 1974 39 600. 115 T. Wakamatsu K. Akasaka and Y. Ban Tetrahedron Letters 1974 3879 3883. 116 R. R. Fraser and P. R. Hubert Canad. J. Chem. 1974 52 185; cf G. K. Koch and J. M. M. Kop Tetrahedron Letters 1974 603. 117 H. Ahlbrecht and G. Rauchschwalbe Synthesis 1973 417; G. Rauchschwalbe and H. Ahlbrecht ibid. 1974 663; H. Ahlbrecht and J. Eichler ibid. 1974 672; H. W. Thompson and B. S. Huegi J.C.S. Chem. Comm. 1973 636; M. Julia A. Schouteeten and M. Baillarge Tetrahedron Letters 1974 3433. Synthetic Methods 429 have centred around the addition of electrophiles (e.g. alkyl halides aldehydes ketones and epoxides) to metallated allylic ethers and related species e.g. (26; X = OR M = Li+ or Zn2+),l18 (26; X = OSiEt, M = Li+),"' (26; X = OTHP M = Li+),l2' (26; X = SPh M = Li+),I2' and (26; X = S- M = 2Li+)122 (Scheme 23).The regioselectivity of electrophilic attack at C-1 E * '?+X?" XM X Scheme 23 and/or C-3 appears to depend on a number of factors e.g. the steric bulk of OR,''8*' the nature' and solvation' of the counterion the degree of hin- drance associated with the alkylating agent,' '' and the nature of the electrophile (alkyl halide or ketone)."8,121 -Lif CH,CCH=PPh, II 0 (27) Further examples of the y-alkylation of the lithio-ylide anion (27) have been reported. 123 After alkylation the derived P-ketophosphorane can be hydrolysed thereby introducing an acetonyl grouping. An alternative method for the in- corporation of this moiety is the reaction of 1~-(2-methoxyallyl)nickelbromide with various halides followed by hydrolysis of the resultant enol ether.' 24 Aldehydes and ketones are less reactive towards this reagent but with longer reaction times p-hydroxy-ketones can be obtained.Sisti et ~1.'~'have published full details of the ring expansion of the magnesium salts of the bromohydrins (28;R1= H or Me R2 = Me or Ph). Another efficient means of ring expansion involves P-oxido-carbenoids which are obtained by treatment of the dibromo- alcohols (28; R' = H R2 = Br) with n-butyl-lithium.'26 'I8 D. A. Evans G. C. Andrews and B. Buckwalter J. Amer. Chem. Sac. 1974,96 5560. '" W. C. Still and T. L. Macdonald J. Amer. Chem. Soc. 1974,96 5561. lZo J. Hartmann M. Stahle and M. Schlosser Synthesis 1974 888; J.Hartmann R. Muthukrishnan and M. Schlosser Helv. Chim. Acta 1974 57 2261. ''I P. M. Atlani J. F. Biellmann S. Dube and J. J. Vicens Tetrahedron Letters 1974,2665. lz2 K. Geiss B. Seuring R. Pieter and D. Seebach Angew. Chem. Internat. Edn. 1974 13 479; see also ref. 152. M. P. Cooke jun. J. Org. Chem. 1973 38 4082; cJ refs. 142 143. 124 L. S. Hegedus and R. K. Stiverson J. Amer. Chem. SOC.,1974,96 3250. 25 A. J. Sisti and M. Meyers J. Org. Chem. 1973 38 4431 ;A. J. Ski and G. M. Rusch ibid. 1974 39 1182. H. Taguchi H. Yamamoto and H. Nozaki J. Amer. Chem. SOC.,1974,96,3010,6510. W. B. Motherwell and J. S. Roberts Effective methods for the regeneration of carbonyl compounds include for- molysis of acetal~'~~ and deoxymation of oximes with a dioxygen complex of palladium.'28 Acetals (29) derived from o-nitrophenylethylene glycol can be efficiently cleaved by irradiation at 350 nm thus serving as a photosensitive protecting group.12' Functionalized Aldehydes and Ketones.-Further work by Corey and Kim' 30 on the oxidative properties of chlorine in the presence of methyl sulphide or DMSO has shown that vicinal secondary-tertiary diols can be selectively oxidized to a-hydroxy-ketones without the normal cleavage of the carbon-carbon bond. Another route to a-hydroxy-ketones involves peracid oxidation of trimethyl- silyl enol ethers followed by acidic or alkaline hydrolysis -non-aqueous work-up yields a-trimethylsiloxy-ketonesand not trimethylsiloxy-epoxides.' ' It should be noted that bromination of trimethylsilyl enol ethers gives a-bromo-aldehydes and -ketones directly,' 32 thus obviating the intermediate formation of the lithium enolate from the silyl enol ether.'33 Prior cyclopropanation of the silyl enol ether followed by bromination yields P-bromo-ketones.'34 Undoubtedly the neatest solution to the problem of a-hydroxylation of ketones (and esters) has been provided by the work of Vedejs.' 35 This involves the reaction of ketone and ester enolates with the stable molybdenum peroxide complex MoO,(py)HMPA.Baldwin et have enlarged upon the use of a-methoxyvinyl-lithium (30) as an acyl-anion equivalent. This potent nucleophile has obvious synthetic uses as demonstrated by its reactions with a variety of substrates (Scheme 24).In the presence of thallium(1) ethoxide ketones react with tosylmethyl isocyanide to form 4-ethoxy-2-oxazolines (3l) which can be converted into a-hydroxy- aldehydes by acid hydrolysis.' The Pummerer rearrangement of /3-hydroxy-27 A. Gorgues Bull. Sac. chim. France 1974 529. K. Maeda I. Moritani T. Hosokawa and S. Murahashi Tetrahedron Letters 1974 797. Izy J. Hdbert and D. Gravei Canad. J. Chem. 1974,52 187. I3O E. J. Corey and C. U. Kim Tetrahedron Letters 1974 287. G. M. Rubottom M. A. Vazquez and D. R. Pelegrina Tetrahedron Letters 1974,4319. 13' R. H. Reuss and A. Hassner J. Org. Chem. 1974,39 1785; B. M. Trost and S. Kuro-zumi Tetrahedron Letters 1974 1929. 133 P. L. Stotter and K. A. Hill J. Org. Chem. 1973 38 2576. 134 S. Murai Y.Seki and N.Sonoda J.C.S. Chem. Comm. 1974 1032. 135 E. Vedejs J. Amer. Chem. SOC.,1974 96 5944. 136 J. E. Baldwin G. A. Hofle and 0.W. Lever jun. J. Amer. Chem. SOC.,1974,96 7125. 13' 0.H. Oldenziel and A. M. van Leusen Tetrahedron Letters 1974 167. Synthetic Methods 0 0 II I1 111. II C Ph CH,=C /OMe --+ C R' /\/ C-R2(H) Me C II 0 (30) /\/ \ Li <AH 431 R3 OH R4 OH Me\CxC/Me Me \C&9 II II 0 0 Reagents i PhCN(C0,H); ii H,O'; iii R'R2(H)CO; iv R3C0,Me; v CH,=CHCOR4 Scheme 24 sulphoxides (32) to give diacetoxy-sulphides (33)can also be used to produce a-hydroxy-aldehydes.'38 OAc I RCHCHSAr RZ RJ?N I OEt OH a-Chloro-aldehydes can be prepared by treatment of the 2-chloromethyl- oxazine (34) with lithium hexamethyldisilazane followed by alkylation boro- hydride reduction and acidic hydrolysis.'39 In addition (34) can also be converted into the corresponding phosphonium salt and phosphonate which undergo Wittig reactions with aldehydes and ketones to form the vinyl-oxazines (35).14* These via their N-methyl quaternary salts can be transformed into a variety of ag-unsaturated carbonyl compounds (Scheme 25).In the same way that trimethylsilyl enol ethers react with aldehydes and ketones in the presence of TiCl to form j-hydroxy-ketones so it has been found that both acetals and S. Iriuchijima K. Maniwa and G. Tsuchihashi J. Amer. Chem. SOC.,1974 96 4280. G. R. Malone and A. I. Meyers J. Org. Chem. 1974 39 618. I4O G. R. Malone and A. I. Meyers J.Org. Chem. 1974 39 623. W.B. Motherwell and J. S. Roberts R' 1iv. ii Reagents i R'Li; ii H,O'; iii NaBH,; iv OH-Scheme 25 orthoformates also react to produce P-alkoxy-ketones and P-keto-acetals respectively.141 The dianion (36) from the corresponding P-keto-sulphoxide undergoes a similar specific alkylation at the y carbon atom as the analogous dianion from a P-keto-ph~sphonate.'~~ This permits the introduction of the acetonyl 143 group into a number of substrates after reductive cleavage of the C-S bond (Scheme 26). In the case of #/?-unsaturated esters 1,4-addition takes place 0 d-PhkHCOCH MeCOCH,R' (36) MeCOCH,qHR2 I OH Reagents i 0;ii R'X; iii CrO, H'; iv R2CH0 Scheme 26 exclusively as opposed to 1,Zaddition to ap-unsaturated ketones.143 y-Alkyla-tion of the a-methyl analogue of (36) followed by thermal elimination of benzene-sulphenic acid provides a neat synthesis of vinyl ketones.'44 Trost and Kan~'~~ have described a short and efficient synthesis of methyl 3-oxopent-4-enoate (37) 14' T. Mukaiyama and M. Hayashi Chem. Letters 1974 15. 14' P. A. Grieco and C. S. Pogonowski J. Org. Chem. 1974 39 732. L43 I. Kuwajima and H. Iwasawa Tetrahedron Letters 1974 107. P. A. Grieco D. Boxler and C. S. Pogonowski J.C.S. Chem. Comm. 1974. 497. B. M. Trost and R.A. Kunz J. Org. Chem. 1974 39 2648. Synthetic Methods Reagents i PhSCH,I; ii NaIO,; iii A Scheme 27 (Scheme 27) which is similar in principle to the route used by Reich and Renga,'46 who have synthesized vinyl ketones and acids by alkylation with benzyl bromomethyl sulphide followed by oxidation and thermolysis.Reich et ~1.'~'have noted one weak feature of the synthetic utility of selenoxide syn-elimination from fl-keto-selenoxides. This is apparent in compounds such as (38) in which the acidic a-hydrogen facilitates a Pummerer-type rearrangement to give the vinyl selenide (39)as an unwanted by-product. This problem can be circumvented by the simple expedient of converting the a-phenylseleno-ketone into its corresponding ethylene ketal prior to oxidation and thermolysis. In the same paper it has been noted that ozone can be used as the selenide oxidant (especially in the case of labile ketones such as cyclobutanones) and also that copper enolates (after lY4-addition of a lithium dialkylcuprate to an enone) and fl-dicarbonyl enolates react with benzeneselenyl bromide.Another route to enones involves an extension of the Kochi reaction uiz. treatment of a y-keto- acid with lead tetra-acetate in the presence of cupric ions.14* y-Chloroallyl sulphoxides (40; X = S) and amine oxides (40; X = NMe) undergo a rapid [2,3]sigmatropic rearrangement to give enones via the sulphenate (41; X = S) (39) and hydroxylamine (41;X = NMe) re~pectively.'~~ Floyd''' has reported that acid chlorides will react with vinyl-lithium to form vinyl ketones when DME or glyme are used as solvents in place of THF or diethyl ether. Adirect attempt to influence the regioselectivity of Mannich-base formation from unsymmetrical ketones has produced an interesting result (Scheme 28) L46 H.J. Reich and J. M. Renga J.C.S. Chem. Comm. 1974 135. 14' H. J. Reich J. M. Renga and I. L. Reich J. Org. Chem. 1974 39 2133. L48 J. E. McMurry and L. C. Blaszczak J. Org. Chem. 1974 39 2217; cf. ref. 207. L49 P. T. Lansbury and J. E. Rhodes J.C.S. Chem. Comm. 1974,21; cf. V. Radtenstrauch Helu. Chim. Acra 1973 56 2492. ' J. C. Floyd Tetrahedron Letters 1974 2877. 434 W.B. Motherwell and J. S. Roberts 0 0 0 II 11 ii II Me,CHCCH,CH,NPr\ d-Me,CHCMe -+ Me,NCH,CMe,CMe 100'x isomerically pure 85 isomerically pure Reagents i Pr\N=CH ClO,- MeCN; ii Me,N=CH CF3C02- CF,C02H Scheme 28 for which there is no immediate explanation. 51 Seebachet al.' 52 have now shown that in general keten thioacetals (42) can be metallated with n-butyl-lithium in the presence of HMPA and that subsequent alkylation occurs with high regioselectivity to produce a/?-unsaturated ketones after hydrolysis (Scheme 29).(42) Reagents i Bu"Li HMPA R3X; ii hydrolysis Scheme 29 @-Unsaturated aldehydes can be obtained in high yield by two routes which depend upon the [3,3]sigmatropic rearrangement of S-ally1 dithiocarbamates (Scheme 30).'53 Newman et al.'54 have found that rearrangement of l-alkyl- R' R' .. NMe 2 v 1 Route B Reagents Route A (R' = alkyl halide RZ= SMe); i LiNPri; ii R'X; iii A; iv Me,S,; v Hg2+; Route B (R' = SMe RZ = alkyl halide); i LiNPr:; ii Me,S,; iii A; iv RZX;v Hg'+ Scheme 30 Is' Y. Jasor M.-J.Luche M. Gaudry and A. Marquet J.C.S. Chem. Comm. 1974 253. D. Seebach M. Kolb and B.-T. Grobel Tetrahedron Letters 1974 3 171. 153 T. Nakai H. Shiono and M. Okawara Tetrahedron Letters 1974 3625; see also T. Hayashi ibid. p. 339. Is4 M. S. Newman and G. M. Fraunfelder J. Org. Chem. 1974,39 251; M. S. Newman and M. C. V. Zwan ibid. p. 1186. Synthetic Methods I (43) (44) (45) (46) R3 MeI RC-/OCHOEt \ (47) (48) CN (49) %Me3 Br (52) idene-2-alkoxycyclopropanes(43;R = H alkyl aryl or alkoxy) in the presence of mercuric acetate leads to the vinylmercury derivatives (44),which can be reduced by hydrogen sulphide to produce yy-disubstituted-py-unsaturated carbonyl compounds. Following on from their previous work Cazes and Julia'5s have shown that carbanions generated from O-ally1 nitrile ethers (45) undergo a facile low-temperature [2,3]sigmatropic rearrangement to produce By-un- saturated ketones (46).Enamines of aa-disubstituted aldehydes (47) have been prepared by the Wittig reaction of the anion (48) with ketones. Subsequent allylation of these enamines proceeds in the normal manner to produce aa-disu bs tituted- y6-unsaturated aldehydes. In continuing the search for acyl anion equivalents which will add regiospeci- fically to the conjugate position of ap-unsaturated compounds a number of ingenious solutions have been found. Thus Stork and Mald~nado'~' have discovered that whereas the lithium anion (49; R = Me) shows a slight pre- ponderance for conjugate addition the lithium anion (49; R = CH=CHMe) derived from crotonaldehyde adds exclusively by 1,4-addition to produce after suitable elaboration the diketone (50) from 3-methylcyclohexenone.Yet another solution to this problem is the addition of the divinylcuprates derived from (51; R = H or Pr) and (52; R = H or Pr) to cyclohexenone to B. Cazes and S. Julia Tetrahedron Letters 1974 2077. "'S. F. Martin and R. Gornpper J. Org. Chem. 1974 39 2814. "' G. Stork and L. Maldonado J. Amer. Chem. SOC.,1974,96 5272. 436 W.B. Motherwell and J. S. Roberts give (53) and (54) respectively.' 58 Using modified conditions for the vinylsilane- carbonyl conversion these two compounds can be converted into a 1,4-diketone and a 6-keto-aldehyde respectively.Alternatively the carbanion (55) can be alkylated with 2,3-dichloropropene to give after hydrolysis the 1,4-diketone (56).lS9 0 R \ C-N-C,H, /-CH2Li+ Me,Si H (55) RCOCH,CH,COMe (56) Further work by Stetter et al. has established the generality of the cyanide-ion- catalysed addition of aromatic and heterocyclic aldehydes to aa-unsaturated nitriles16'" and ketones.'60b Of greater overall utility is the fact that thiazolium salts (cf the anion of hydroxyethyl-thiamine pyrophosphate as the source of 'active acetaldehyde' in the formation of branched sugars16 la) in the presence of base reversibly add to both aliphatic and aromatic aldehydes to form the acyl anion equivalent (57) which can then undergo conjugate addition to a variety of aP-unsaturated carbonyl compounds to form 1,4-dicarbonyl products.' Ib The interesting observation that aldehyde ketone ester and lactone enolates react with 1-dimethylamino-2-nitroprop- 1-ene to form the aci-nitro-derivatives (57) (58 ;R' = H alkyl aryl or alkoxy R2= H alkyl or aryl; or R' and R2= lactone grouping) has permitted the synthesis of 1,6dicarbonyl compounds. 62 Thus R. K. Boeckman jun. and K. J. Bruza Tetrahedron Letters 1974 3365; cf ref. 41. 159 Th. Cuvigny M.Larcheveque and H. Normant Tetrahedron Letters 1974 1237. loo (a)H. Stetter and M. Schreckenberg Chem. Ber. 1974 107 210; (6) ibid. p. 2453. 16' (a)R. Breslow and E. McNelis J. Amer. Chem. SOC.,1959 81 3080; (b)H. Stetter and H. Kuhlmann Angew. Chem. Internat. Edn. 1974 13 539; Tetrahedron Letters 1974 4505.16' T. Severin and D. Konig Chem. Ber. 1974 107 1499; H. Lerche D. Konig and T. Severin ibid. p. 1509. S-ynthetic Methods the appropriate derivatives of (58) can be converted into enediones and p-alkoxycarbonyl-ap-unsaturatedketones by treatment with silica gel or ascorbic acid. In the presence of copper powder the latter reagent can be used to produce 1,4-diketones. In this context it should be noted that titanous chloride will also reduce enedicarbonyl compounds (except enediesters).' 63 Other routes to 1,4-diketones include photolysis of a-hydroxy-By-unsaturatedketones (by a 1,3-acyl shift),'64 Grignard addition to mixed y-keto-carboxylic anhydrides (59),165and the reaction of enol esters with ketones in the presence of manganic ions.166 0 II R/c-fo* 00 (59) A number of new or modified cyclobutanone syntheses have been reported.These include two independent observation^'^^^'^^ that 1,l-dibromocyclo-propanes can be metallated with n-butyl-lithium and that subsequent reaction with a ketone produces an oxaspiropentane (60) which as Trost and co-workers have already shown rearranges to a cyclobutanone under acidic conditions. On the other hand the reaction of the carbenoid with an aldehyde produces a bromohydrin (61) which yields a cyclopropyl ketone (62) on treatment with sodium hydride.16* The reaction of the carbenoid with an a/?-unsaturated aldehyde proceeds by 1,2-addition while the reaction with diethyl carbonate leads to a 1,l-diethoxycarbonylcyclopropane.In a related area bicyclic 1,l-dichloro-2-ethoxycyclopropanes(63) can be ring-expanded to /?-methyl ap-unsaturated ketones (64) by the addition of two equivalents of methyl-lithium R' followed by hydrolysis.' 69 Whereas the reaction of 1,3-dibromides with dithian in the presence of base is not a very practical synthesis of dithian-protected '63 L.C. Blaszczak and J. E. McMurry J. Org. Chem. 1974 39 258. K. G. Hancock J. T. Lau and P. L. Wylie Tetrahedron Letters 1974 4149. M. Araki andT. Mukaiyama Chem. Letters 1974 663; M. Araki S. Sakata H. Takei and T. Mukaiyama ibid. p. 687. '66 R. M. Dessau and E. I. Heiba J. Org. Chem. 1974,39 3457. M. Braun and D. Seebach Angew. Chem. Internat. Edn. 1974 13 277. T. Hiyama S.Takehara K. Kitatani and H. Nozaki Tetrahedron Lerters 1974 3295. T. Hiyama T. Mishima K. Kitatani and H. Nozaki Tetrahedron Letters 1974 3297; cf. J. T. Groves and K. W. Ma ibid. p. 909. W. B. Motherwell and J. S. Roberts Scheme 31 cyclobutanones Tsuchihashi et al.' 70 have now shown that methyl methylthio- methyl sulphoxide is much superior (Scheme 31). In a continuation of their work on cyclobutanone synthesis Conia et al.' 71 have found that monocyclopropana- tion of silyl enol ethers of cisoid enones leads to trimethylsilyloxy-vinylcyclo-propanes e.g. (65) which on acid treatment ring expand to cyclobutanones. As shown previously thermolysis of compounds such as (65) leads to cyclo- pentanones via the corresponding trimethylsilyl enol ethers (Scheme 32).0 Scheme 32 Two interesting syntheses of substituted cyclopentenones have been reported by Hiyama et ~ (Scheme 33). It is probable that both these routes involve 1.~~~9~~~ a substituted pentadienyl cation (66; X = OCH,CHpOH and C1 respectively). X R~HR~OH c1 c1 ii -R~CHR~OH 2 R'CH H R3CH H Reagents i H,PO, HC0,H; ii :CC1,; iii 47 % HBr Scheme 33 'lo K. Ogura M. Yamashita M. Suzuki and G. Tsuchihashi Tetrahedron Letters 1974 3653. C. Girard P. Amice J. P. Barnier and J. M. Conia Tetrahedron Letters 1974 3329; see also B. M. Trost and S. Kurozumi Tetrahedron Letters 1974 1929. 172 S. Hirano T. Hiyama and H. Nozaki Tetrahedron Letters 1974 1429. T. Hiyama M. Tsukanaka and H. Nozaki J. Amer. Gem. SOC.,1974,96 3713.Synthetic Methods R' \+ /TPPh3 R2 C0,-(67) 9 Acids Alkylidenetriphenylphosphoranes react with carbon dioxide to give the salts (67) which lose triphenylphosphine oxide on alkaline hydrolysis to produce carboxylic acids.' 74 A considerable improvement in the Darzens condensation of ketones with chloroacetonitrile has been noted whereby the derived glycido- nitrile (68) can then be converted into the homologated asid (Scheme 34).17' CN AcO CO,H 04 -+ ICN5 I. ir R 'AR2 R' R2 R' R2 (68) Reagents i HCI gas; ii Ac,O py NEt,; iii OH-Scheme 34 Meyers et have achieved an asymmetric synthesis of dialkylacetic acids of high optical purity (-70%) from a single chiral oxazoline (69). This work OMe (69) resulted from a careful study of the sequential alkylation steps and the reaction conditions.A number of reagents have been recommended for their ability to remove protecting groups from carboxylic acid derivatives. These include boron tribromide (for a variety of esters),' 77 trithiocarbonate ion (for 2-halo- genoethyl esters),' 78 and sodium thiomethoxide (for 9-anthrylmethyl esters). 79 17* H. J. Bestmann Th. Denzel and H. Salbaum Tetrahedron Letters 1974 1275. 175 D. R. White and D. K. Wu J.C.S. Chem. Comm. 1974 988. 76 A. I. Meyers and G. Knaus J. Amer. Chem. SOC.,1974 96 6508; A. I. Meyers G. Knaus and K. Kamata J. Amer. Chem. SOC.,1974 96 268; see also A. I. Meyers, G. Knaus and P. M. Kendall Tetrahedron Letters 1974 3495; A. 1. Meyers and G. Knaus ibid.p 1333. 17'.A. M. Felix J. Org. Chem. 1974 39 1427. T. L. Ho Synthesis 1974 715. N. Kornblum and A. Scott J. Amer. Chem. SOC.,1974 96 590. W.B. Motherwell and J. S. Roberts Functionalized Acids.-A new synthesis of a-amino-acids has been reported which is based upon the reactivity of the carbanion of methyl methylthiomethyl sulphoxide with nitriles (Scheme 35).lgo An alternative route to certain a-amino-acids involves the electroreductive coupling of the Schiff bases (70)with . .. ... NHAc NHAc I 1. II H2N\ -CH -+ 2RCCOSMe 3 R(!HCO,H \ /c=c\ I SMe R SMe SMe Reagents i RCN; ii H,O; iii Ac,O; iv MeOH H,O K,CO Scheme 35 .~~~ alkyl halides (R2X)to give (71).18' Meyers et ~1 have published the full details of the reactions of the carbanions derived from 2-alkyl-4,4-dimethyl- A2-oxazolines (72) with various electrophiles to produce functionalized carboxylic acids.The dianion (73) reacts with aldehydes and ketones in a Wadsworth- Emmons reaction to produce ap-unsaturated acids directly. Me I PhCH,NHCC02R ' I RZ 0 II 2Li' -O,CCHP(OCH,Ph) The high nucleophilicity of silyl enol ethers is again manifest in their reaction with ozone to produce carboxylic acids and a carbonyl fragment.184 Since a high degree of control can be exercised over silyl enol ether formation from un- symmetrical ketones this method provides an opportunity for selective oxidative cleavage. a-Carboxylation of ketones can be achieved in reasonable yields by formation of the enolate with either 4-methyl-2,6-di-t-butylphenoxide ion lg5 86 or 1,8-diazabicyclo[5,4,0]undec-7-ene1 in the presence of carbon dioxide.K. Ogura and G. Tsuchihashi J. Amer. Chem. SOC.,1974,96 1960. T. Iwasaki and K. Harada J.C.S. Chem. Comm. 1974 338. Ia2 A. I. Meyers D. L. Temple R. L. Nolen and E. D. Mihelich J. Org. Chem. 1974 39 2778. Ia3 G. A. Koppel and M. D. Kinnick Tetrahedron Letters 1974 71 1. IS4 R. D. Clark and C. H. Heathcock Tetrahedron Letters 1974 2027. Is5 E. J. Corey and R. H. K. Chen J. Org. Chem. 1973 38 4086. lS6 E. Haruki M. Arakawa N. Matsumura Y.Otsuji and E. Imoto Chem. Lerters 1974 427. Synthetic Methods 441 In the same way that a-anions of esters can be carboxylated so it has been found that a-anions from carboxylic acids react with ethyl chloroformate or diethyl carbonate to form malonic acid half-esters.' '7 10 Esters and FunctionalizedEsters The use of boron trifluoride etherate and an alcohol for esterification has been reviewed."' An alternative route for the homologation of esters has been recor- ded (Scheme 36).lB9 OH SOMe .' I / RC0,Et RCOCF -!!+ RCHCF \ \ SMe SMe\111 LV ,SOMe ' RCH=C RCH,CO,Et \ SMe Reagents i MeSCHSOMe; ii NaBH,; iii Ac,O py; iv Et,N; v HCl EtOH Scheme 36 A simple continuous-flow reactor has been designed for the large-scale produc- tion of b-hydroxy-esters by the Reformatsky reaction.'" It has been found that a-phenylsulphinylacetate reacts with one mole of ethylmagnesium bromide to form the Grignard reagent (74) which reacts with aldehydes and ketones to form the adducts (75) which can be desulphurized to produce P-hydroxy-e~ters.'~~ MgBr C02Et I PhSOCHC0,Et PhSO &HCR1 R2 OH Continuing their work on ester enolates Rathke et have shown that lithio t-butyl trimethylsilylacetate reacts in high yield with ketones and aldehydes to produce ab-unsaturated t-butyl esters.A similar observation has been made using lithio ethyl trimethylsilylacetate and it is noted that much better yields are obtained by this method than by the Wittig-type process.'93 Carboalkoxy- lations of various vinyl bromides and iodides can be carried out using carbon 18' A. P. Krapcho E. G. E. Jahngen jun. and D. S. Kashdan Tetrahedron Letters 1974 272 1. Is' P. K. Kadaba Synthetic Comm.1974 4 167. 189 K. Ogura S. Furukawa and G. Tsuchihashi Chem. Letters 1974 659. I9O J. F. Ruppert and J. D. White J. Org. Chem. 1974 39 269. 19' N. Kunieda J. Nokami and M. Kinoshita Tetrahedron Letters 1974 3997. L92 S. L. Hartzell D. F. Sullivan and M. W. Rathke Tetrahedron Letters 1974 1403. 193 K. Shimoji H. Taguchi K. Oshima H. Yamamoto and H. Nozaki J. Amer. Chem. Soc. 1974 96 1620. W.B. Motherwell and J. S. Roberts R \ /H c=c H /\ C0,Et R Br R \ /CO& /c=c\H H Reagents i Br,; ii A; iii NaOEt Scheme 37 monoxide and an alcohol in the presence of a tertiary amine and a palladium-triphenylphosphine complex. The degree of stereoselectivity of these reactions however is rather variable.Better stereoselective syntheses of (E)-and (2)-afl-unsaturated esters can be achieved by decomposition of the dibromo-borane (76) under appropriate conditions (Scheme 37),Ig5 or by addition of lithium dialkylcuprates to (E)-and (2)-fl-acyloxy-ap-unsaturatedesters.196 Whereas photochemical decomposition of the fly-unsaturated diazomethyl ketone (77) in the presence of copper sulphate and methanol yields the normal Wolff re- arrangement product (78) the thermal process leads to the isomeric yd-unsatura- ted ester (79).’” Last year it was reported that sodium chloride in wet DMSO was very effective for decarboalkoxylations of geminal diesters fl-keto-esters and a-cyano-esters. While this is still true it has now been demonstrated that for the majority of cases wet DMSO alone will suffice.”* The necessity for added sodium chloride only occurs in the case of certain substituted malonates when it has an accelera- ting effect.1,4-Diazabicyclo[2,2,2]octanein refluxing xylene will also decarbo- alkoxylate 8-keto-esters. lg9 As anticipated ester enolates react with ethyl 194 A. Schoenberg I. Bartoletti and R. F. Heck J. Org. Chem. 1974 39 3318; see also A. Schoenberg and R. F. Heck ibid. p. 3327. ‘95 E. Negishi G. Lew andT. Yoshida J. Org. Chem. 1974 39 2321. 196 C. P. Casey and D. F. Marten Tetrahedron Letters 1974 925. L97 A. B. Smith tert. J.C.S. Chem. Comm. 1974 695. 19’ A. P. Krapcho E. G. E. Jahngen jun. A. J. Lovey and F. W. Short Tetrahedron Letters 1974 1091 ; C. L. Liotta and F. L. Cook ibid. p. 1095.IY9 B. Huang E. J. Parish and D. H. Miles J. Org. Chem. 1974 39 2647. Synthetic Methods chloroformate and in certain cases with chlorophosphates to produce substituted malonates and phosphonoacetates respectively.200 Lactones.-Once again the search for effective means of a-methylenation of y-butyrolactones has continued. For the trans-fused lactone (80)the best method for the introduction of the a-methylene group is methylation phenylsulphenyla- tion oxidation and pyrolysis while for the cis-fused isomer (81) the order of the first two steps is reversed.201 This stereospecific sequence ensures the correct stereochemical relationship in the syn-elimination step. Precisely the same methodology can be used where selenium replaces sulphur.202 The Michael- type addition of the phenylselenium anion to these a-methylene lactones serves as a method of protection.203 The reaction of the triphenylphosphorane (82) with paraformaldehyde is also an effective method of a-methylenati~n.~'~ PPh (80) (81) (82) Based upon results obtained last year,'" solvolysis of the tricyclic mesylate (83) produces the two lactones (84) and (85).206 Oxidative decarboxylation of a lactone bearing an a-acetic acid side-chain also produces an a-methylene la~tone.~'~ The dianion (86) reacts with epoxides to produce a-phenylsulphenyl y-lactones (87) which on oxidation and pyrolysis yield a/?-unsaturated-y- lactones.208 For the synthesis of saturated y-lactones routes involving anionic R' R' SPh -+ PhSt'CO -2Li R200 (86) (87) T.J. Brocksom N. Petragnani and R. Roadrigues J. Org. Chem. 1974,39 21 14. 201 P. A. Grieco and J. J. Reap Tetrahedron Letters 1974 1097; see also ref. 236. lo' P. A. Grieco and M. Miyashita J. Org. Chem. 1974 39 120. 203 P. A. Grieco and M. Miyashita Tetrahedron Letters 1974 1869. 204 P. A. Grieco and C. S. Pogonowski J. Org. Chem. 1974 39 1958. lo' P. F. Hudrlik L. R. Rudnick and S. €3. Korzeniowski J. Amer. Chem. SOC.,1973,95 6848. 206 F. E. Ziegler A. F. Marino 0. A. C. Petroff and W. L. Studt Terrahedron Letters 1974 2035; see also P. A. Grieco and K. Hiroi ibid. p. 3467. 2"7 K. J. Divaker P. P. Sane and A. S. Rao Tetrahedron Letters 1974 399; T. L. HOand C. M. Wong Synthetic Comm. 1974,4 133. 208 K. Iwai M. Kawai H.Kosugi and H. Uda Chem. Letters 1974 385. W.B. Motherwell and J. S. Roberts opening of epoxides with the lithium salt of 2,4,4-trimethyl-A2-oxazoline,20g and further examples with metallated carboxylic acids2" have been described. Undoubtedly one of the most significant advances in lactone chemistry is the report by Corey and Nicolaou,211 which describes the efficient cyclization of w-hydroxypyridine-2-thiol carboxylic esters (88). Except for n = 7 the yields of macrolides (up to n = 14) are consistently high and this has been attributed to a combination of dipolar attraction in the proton-transferred species (89) and the ease of elimination of pyridine-2-thione. This mild lactonization method augers well for syntheses of naturally occurring macrolides.a-Allyl-substituted y-and S-lactones can be prepared by a Claisen rearrangement involving an ally1 alcohol and a cyclic orthoester.212 11 Amides and Nitriles In the presence of boron tribromide and an amine esters can be converted directly into amide~.~'~ Transesterification takes place if an alcohol is added. Nitriles can be converted into amides in high yield using the nitrile-complexing reagent chloropenta-ammineruthenium(II1) chloride in trifluoroacetic acid and an oxidation-reduction system consisting of silver oxide and zinc amalgam.2 l4 Aldoximes can be rearranged to primary amides by refluxing in xylene over activated silica gel.* Better yields in the Hofmann rearrangement of amides to carbamates can be achieved by generating methyl hypobromite (as the source of positive bromine) at low temperature.216 Buchi et have found that allylic alcohols (but not those with yy-disubstitution) when treated with NN-dimethylformamide acetals rearrange smoothly to &unsaturated NN-dimethyl-amides.Evidence has been presented that the major reaction pathway is a [2,3 Jsigmatropic rearrangement ofthe carbene (90)(Scheme 38). Aliphatic aldehydes can be converted into nitriles by reaction with hydroxyl- amine-0-sulphonic acid at room temperature -for aromatic aldehydes the intermediate oximino-sulphonic acid requires additional heating or treatment '09 A. I. Meyers E. D. Mihelich and R. L. Nolen J. Org. Chem. 1974 39 2783. 2Lo T. Fujita S. Watanabe and K. Suga Austral. J. Chem. 1974 27 2205."' E. J. Corey and K. C. Nicolaou J. Amer. Chem. SOC.,1974,96 5614. C. B. Chapleo P. Hallett B. Lythgoe and P. W. Wright Tetrahedron Letters 1974 847. 'I3 H. Yazawa K. Tanaka and K. Kariyone Tetrahedron Letters 1974 3995. S. E. Diamond B. Grant G. M. Tom and H. Taube Tetrahedron Letters 1974,4025. J. B. Chattopadhyaya and A. V. Rama Rao Tetrahedron 1974 30 2899. P. Radlick and L. R. Brown Synthesis 1974 290. 217 G. Biichi M. Cushman and H. Wuest J. Amer. Chem. SOC., 1974 96 5563. Synthetic Met hods \/ \/ +I I N N hl I Scheme 38 with sodium hydroxide.2 l8 Aldoximes can be efficiently transformed into nitriles by an acid-catalysed reaction with orthoesters.2 l9 Hydrosilylation of afl-unsaturated nitriles’” in the presence of tris(triphenylphosphine)rhodium(I) chloride produces or-silylpropionitriles which on formation of anions with lithium di-isopropylamide followed by reaction with an aldehyde or ketone produce a new ap-unsaturated nitrile (Scheme 39).22 R’CH=CHCN R’CH,CHCN 5 R2R3C=CCH,R’ I I SiPhMe CN Reagents i PhMe,SiH (Ph,P),RhC1; ii PriNLi R2R3C=0 Scheme 39 12 Alkylation The generation and alkylation of enolate anions is beset by many ill-understood subtleties such as the role of the solvent and the nature of the counter-ion and alkylating agent.In consequence rational prediction of the outcome of such reactions is very difficult to achieve and experimental activity flourishes. The reaction of ketones with potassium hydride continues to be studied by C.A. Brown. Highly reactive potassium enolates are formed quantitatively within minutes at room temperature even from carbonyl compounds that are labile towards self condensation.222 Stork and his co-worker~~~~ have shown that the aldol condensation can be carried out regiospeclfically at the methyl group of a methyl ketone by addition of the aldehyde to the kinetically generated lithium enolate of the ketone. The condensation of formaldehyde with regiospecifically generated lithium enolates has also been shown by the same group to provide a convenient entry into or-(hydroxymethyl)ketone~.~~~ The addition of potassium t-butoxide to a cooled solution of a conjugated enone and methyl iodide results This reaction can be in methylation at the a’-position via the kinetic en~late.~~’ C.Fizet and J. Streith Tetrahedron Letters 1974 3187. ” M. M. Rogic J. F. Van Peppen K. P. Klein and T. R. Demmin J. Org. Chem. 1974 39 3424. “O D. N. Brattesani and C. H. Heathcock Tetrahedron Letters 1974 2279. 221 I. Ojima M. Kumagai and Y. Nagai Tetrahedron Letters 1974 4005. ‘12 C. A. Brown Synthesis 1974,427; C. A. Brown J. Org. Chem. 1974,39 1324. 223 G. Stork G. A. Krauss and G. A. Garcia J. Org. Chem. 1974 39 3459. 224 G. Stork and J. d’Angelo J. Amer. Chem. SOC.,1974 96 71 14. 22s L. Nedelec J. C. Gasc and R. Bucourt Tetrahedron 1974 30. 3263. W.B. Motherwell and J. S. Roberts controlled to give either the mono- or gem-di-alkylated product. By way of contrast however the carbanion alkylation of vinylogous amides esters and nitriles leads to the exclusive formation of a new carbon-carbon bond in the y-position.226 The magnesium enolates generated by the conjugate addition of Grignard reagents to enones have been successfully acylated alkylated and allowed to react in the aldol conden~ation.~’~ The alkylation of regiospecifically- generated kinetically stable organocopper enolates has received the attention of two groups.In the initial study Boeckman228 demonstrated the feasibility of the method but was unable to alkylate relatively hindered enones. Coates and Sandef~r~’~ have subsequently shown that the copper enolates while quite unreactive in ether become very receptive to alkylation in 1,2-dimethoxyethane thus permitting the formation of 2,2,3,3-tetramethylcyclohexanone in 86 % yield from 2,3-dimethylcyclohexenone.An alternative method for the regio- specific geminal dialkylation of ketones has also emerged from Coates’ laboratory involving alkylation of an a-phenylthioketone followed by reductive alkylation with lithium-ammonia and an alkyl halide (Scheme 40).230An attractive feature Reagents i NaH; ii R’X; iii Li-NH, RZX Scheme 40 of this sequence lies in the fact that the two alkyl groups can be introduced in either order thus allowing for the possibility of stereochemical control in a sterically biased substrate.Girard and C~nia~~’ have extended their work on the modified Simmons- Smith cyclopropanation sequence by demonstrating that 2-trimethylsiloxy- cycloalka-1,3-dienes undergo highly site-selective monocyclopropanation in the 1,2 position ;methanolysis then gives the a’-methylated cycloalkenone in excellent yield.Weiler and his collaborators have now published an extended study on the generation of the dianions of @-keto-esters by sequential treatment with one equivalent of sodium hydride followed by one equivalent of butyl-lithium. These dianions react with a wide range of alkylating agents to produce y-alkylated products in good yield. Acylations and aldol condensation have also been succe~sful.~ 32 226 T. A. Bryson and R. B. Gammill Tetrahedron Letters 1974 3963. 27 F. Naf and R. Decorzant Helv. Chim. Acta 1974 57 13 17. 228 R. K. Boeckman,jun. J. Org. Chem. 1973 38 4450. 229 R. M. Coates and L.0.Sandefur J. Org. Chem. 1974 39 275. 230 R. M. Coates H. D. Pigott and J. Ollinger Tetrahedron Letters 1974 3955. zJL C. Girard and J. M. Conia Tetrahedron Letters 1974 3327. 232 S. N. Huckin and L. Weiler Canad. J. Chem. 1974,52 1343 1379 2157; S. N. Huckin and L. Weiler J. Amer. Chem. SOC., 1974 96 1082. Synthetic Methods Hendrickson and his co-worker~’~~ correctly claim that the trifluoromethyl- sulphonyl group will prove most useful in many synthetic schemes. As one of the most powerful neutral electron-withdrawing groups known it can activate a neighbouring methylene towards alkylation; it can also function as a leaving group (Scheme 41). Reagents i K,CO,; ii PhCH,Br; iii OEt-Scheme 41 The steadily growing importance of mixed organocuprates has been reflected in a useful survey of their relative selectivities in typical reaction^.'^^ An ex- perimentally convenient preparation of lithium (phenylthio)alkylcuprates which are selective for the transfer of primary secondary and even tertiary alkyl groups has been reported.z35 The mixed a-ethoxycarbonylvinylcuprate(9l) readily prepared from ethyl a-bromoacrylate is highly specific for alkylations with ally1 halides (Scheme 42).236The reduced nucleophilicity of this species R = H or Br Scheme42 which is possibly due to the combined inductive effects of the ester moiety and the acetylenic ligand means that alkyl iodides and highly reactive benzyl bromide are totally inert.A study of the regio- and stereo-specific addition of organocopper reagents to terminal acetylenes has been made by Normant and his colleagues.237 The derived vinylcopper reagents are extremely versatile intermediates and can be transformed with retention of configuration into various ethylenic structures such as 1-deuterioalk- 1-enes symmetrical cis-dienes vinyl iodides di- or tri- substituted olefins and primary or secondary allylic alcohols.’38 Trost has reviewed his earlier work on new synthetic methods for the construc- tion of carbon-carbon bonds especially via sulphur ylide~.’~’ Although this 233 J.B. Hendrickson A. Giga and J. Wareing J. Amer. Chem. SOC.,1974 96 2275. 234 W. H. Mandeville and G. M. Whitesides J. Org. Chem. 1974 39 400. 235 G. H. Posner D. J. Brunelle and L. Sinoway Synthesis 1974 662.236 J. P. Marino and D. M. Floyd J. Amer. Chem. SOC.,1974 96 7138. 237 J. F. Normant G. Cahiez M. Bourgain C. Chuit and J. Villieras Bull. SOC.chim. France 1974 1656. 238 J. F. Normant G. Cahiez C. Chuit and J. Villieras J. Organometallic Chem. 1974 77 269 28 1. 239 B. M. Trost Accounts Chem. Res. 1974 7 85 W. B. Motherwell and J. S. Roberts approach has already led to a veritable host of usefully functionalized structural units further reports continue to be published with unfailing regularity. The complementary behaviour of 1-lithiocyclopropyl phenyl sulphide and di-phenylsulphonium cyclopropylide both in terms of stereoselectivity and chemo- specificity in reaction with conjugated enones considerably augments the syn- thetic potential of the spiroannelation sequence (Scheme 43).240 Reagents i PgPh ; ii LiClO,; iii ; iv SnCI Scheme 43 A further report on the 'allylic alkylation' sequence has demonstrated that the carbon-carbon double bond can be activated towards alkylation at the a-position even in the presence of its historically more familiar rival the carbonyl group (Scheme 44).241 Reagents i PdCI,; ii [MeS02CHC02Me]Na'; iii LiI-NaCN A; iv ketalize; v Li EtNH,; vi H' Scheme 44 240 B.M. Trost and D. E. Keeley J. Amer. Chem. Soc. 1974 96 1252. 241 B. M. Trost T. J. Dietsche and T. J. Fullerton J. Org. Chem. 1974 39 737. Synthetic Methods Among the new synthetic reactions reported this year ‘alkylative elimination’ represents a successful fusion of earlier interests.The anion of methyl phenyl- sulphinyl acetate can react with alkyl halides or Ic-allylpalladium complexes and the resultant adducts can be thermolysed to yield the aP-unsaturated product (Scheme 45).242 I ic $. 1‘Ii eCO,Me R PdCli2 ’ Reagents i NaH; ii RCH,X; iii A; iv R-R A Scheme 45 A series of articles on the topic of acylation has been provided by Chemistry and Industry as a companion set to the alkylation reviews published last year.243 Evans244 has published an account of his work on the potential of allylic sulph- oxides in organic synthesis. 13 Annelation New general methods for the construction of rings have burgeoned within the past year. Accordingly a separate section has been assigned in this Report.The geometric constraints imposed upon the base-catalysed isomerization of suitably constituted epoxynitriles lead to a reversal in the normal preference for ease of ring formation in carbanion-electrophile cycli~ations.~~~ The ‘epoxy- nitrile.cyclization’ has thus been used to construct the decalin (92)from (93),and even more remarkably the cyclobutane (94)from (95). The utility of this highly (95) (93) (94) 242 B. M. Trost. W. P. Conway P. E. Strege and T. J. Dietsche J. Amer. Chem. SOC.,1974 96 7165. 243 D. P. N. Satchell Chem. and Ind. 1974 683; D. Seebach and M. Kolb ibid. p. 687; J. H. Jones ibid. p. 723; P. H. Gore ibid. p. 727 P. W. Hickmott ibid. p. 731. 244 D. A. Evans and G. C. Andrews Accounts Chem. Res. 1974 7 147.245 G. Stork L. D. Cama and D. R. Coulson J. Amer. Chem. SOC.,1974 96 5268; G. Stork and J. F. Cohen ibid.. p. 5270. 450 W. B. Motherwell and J. S. Roberts stereoselective and non-photochemical approach to four-membered rings was demonstrated by an elegant synthesis of grandisol. The pyrethrins jasmones and prostaglandins which all contain highly functionalized cyclopentane rings have acted as a springboard for synthetic methodology. An intramolecular Wittig reaction has been employed for the crucial ring-closure step of two cyclopentane syntheses which differ in their ensuing pattern of functionality (Schemes 46246and 47247). 0 R1/ICCO,Et 1.11 -E102cn R2 R2' R' C02Et Reagents i NaH ii P<;h,"'' Scheme 46 0 R2 R3 ...11~., Iv ,R '93 C __* RO OR ,s-/ R3 Reagents i Li+ -ii H + ; iii NaH; iv -+ PPh X-Scheme 47 + iv 9'5 Meos+ SMe HO 0 Reagents i NaH; ii MeS SMe .c 0 Scheme 48 The one-step conjugate addition-alkylation of keten thioacetal monoxides has been used in a simple entry to rethrolones (Scheme 48).248 A direct method 246 P. L. Fuchs J. Amer. Chem. SOC.,1974 96 1607. 24' I. Kawamoto S. Muramatsu and Y. Yura Tetrahedron Letters 1974 4223. 248 R. F. Romanet and R. H. Schlessinger J. Amer. Chem. Soc. 1974 96 3701. Synthetic Methods 45 1 for the construction of five-membered rings which would rival the generality of the Diels-Alder reaction is the theoretically allowed 1,3-anionic cycloaddition of an ally1 anion to an olefin to produce a cyclopentane anion.Marino and Mes- bergen have implemented this approach by a judicious choice of substrate and developed a method for the further elaboration of the adducts to cyclopentanones (Scheme 49).249 liii.iv Reagents i LiNPr;; ii H + ; iii m-chloroperbenzoic acid; iv CF3C03H;v KOH-CHCI Scheme 49 An unusual reaction which also involves the direct addition of a three-carbon fragment to an unsaturated ester consists of heating a mixture of 1,3-di-iodo- propane diethyl fumarate cyclohexyl isocyanide and metallic copper in toluene under nitrogen. trans-1,2-Diethoxycarbonylcyclopentane is produced in high yield.250 Danishefsky and his colleagues have developed a fascinating double- cyclization technique through the transposition of activated cyclopropanes (Scheme 50).251This approach has also permitted an expeditious entry into heterocyclic systems.' 52 C0,Me Reagents i Na+[CH,S(O)Me]-; ii H+ Scheme 50 249 J.P. Marino and W. B. Mesbergen J. Amer. Chem. SOC.,1974,96; 4050. Y. Ito K. Nakayama K. Yonezawa and T. Saegusa J. Org. Chem. 1974,39 3273. "' S. Danishefsky J. Dynak E. Hatch and M. Yamamoto J. Amer. Chem. SOC.,1974 * 96 1256. ''' S. Danishefsky J. Dynak and P. McCurry J. Org. Chem. 1974 39 2658; S. Danishefsky and J. Dynak ibid. p. 1979. 452 W.B. Motherwelt and J. S. Roberts Preformed lithium enolates react rapidly and site-specifically with ally1 halides. This fact has been used by two groups for the introduction of a latent 3-keto-alkyl side-chain prior to cyclization.Stotter and Hill253 have employed 7-iodo-tiglates for the alkylation and a modified Curtius reaction of the derived azide for release of the carbonyl group. In a conceptually identical process Stork and his co-~orkers~~* have shown that the vinylsilane moiety can be un- masked under exceptionally mild conditions due to nucleophilic participation by the carbonyl in the epoxide ring-opening step. lP-Diketones and hence cyclopentanes can also be prepared from the appropriate vinylsilane. Further reports on the use of a-silylated vinyl ketones in the Robinson annelation have indicated that even readily equilibrated lithium enolates can be successfully trapped under aprotic conditions.255 These three approaches to the construction of the decalin system are summarized in Scheme 51.OLi SiM e Reagents I. ii m-chloroperbenzoic acid; iii base; iv SiMe vi. ClC0,Me-Et,N; vii NaN,; viii MeOH A Scheme 51 253 P. L. Stotter and K. A. Hill J. Amer. Chem. SOC.,1974 96 6524. 254 G. Stork M. E. Jung E. Colvin and Y. Noel J. Amer. Chem. SOC.,1974 96 3684; G. Stork and M. E. Jung ibid.,p. 3682. 255 R. K. Boeckman jun. J. Amer. Chem. SOC. 1974 96 6179; G. Stork and J. Singh ihid. p. 6181. Synthetic Methods 453 SIR 5ir3 Ph,yk-,Cl PhFCl 5Ph+ H H H 0-0 kPh 11 + PhA -Phd iii 0 Reagents i m-chloroperbenzoic acid; ii F- DMSO; iii. Q Scheme 52 Intramolecular alkylation of a regiospecifically generated copper enolate has been used in a synthesis of the sesquiterpene valerane.256 The nucleophilic affinity of fluoride anion for silicon has been employed in a very efficient entry to the allene oxide+yclopropanone system thus rendering possible the cycloaddition of an oxoallyl cation to a diene in preparatively useful yield (Scheme 52).257 Reagents i AgBF,; ii ; iii H' Scheme 53 The silver-fluoroborate-induced reaction of 3-chloro-2-pyrrolidinocyclo-hexane with 1,3-dienes also shows great promise as a method for the construction of even-membered rings (Scheme 53).258 S S 256 G.H. Posnet C. E. Whitten D. J. Sterling and D. J. Brunelle Tetrahedron Letters 1974,2591. 25' T.H. Chan M. P. Li W. Mychajlowskij and D. N. Harpp Tetrahedron Letters 1974 3511.2Je R. Schmid and H. Schmid Helu. Chim. Acta 1974,57 1883.

ISSN:0069-3030    

DOI:10.1039/OC9747100411

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

16 Biosynthesis By R. 6. BOAR Department of Chemistry Chelsea College London SW3 6LX and D. A. WIDDOWSON Department of Chemistry Imperial College London SW7 2A Y 1 Introduction The most notable trend this year has undoubtedly been a predictable increase in the application of 3C n.m.r. spectroscopy to biosynthetic studies. Successful application of this technique requires (a) that the 13C n.m.r. spectrum of the substance under study can be unambiguously interpreted (b)that incorporation is relatively high (in terms of that which is tolerable for methods using radioactive isotopes) and (c) that dilution by a pool of unlabelled endogenous material is not such as to obscure the benefits of a good incorporation. The last two con- ditions are normally satisfied in experiments using partially purified enzyme systems or micro-organisms.Most studies have been with such systems but this year has also seen the first successful applications of I3Cn.m.r. spectroscopy to problems of biosynthesis in intact higher plants. The use of precursors doubly labelled with 3C also offers intriguing (if expensive!) possibilities. This year has also seen the first application of 3H n.m.r. spectroscopy to a biosynthetic problem. The technique has the advantage as with 13C n.m.r. of not requiring degradation of the end product but the same metabolic limita- tions apply. The results complement carbon label studies and the method has obvious potential for the examination of biosynthetic hydrogen migrations. 2 Terpenoids Useful biosynthetic information is to be found in the proceedings of a symposium on phytosterols.‘ Sesterterpenes have been reviewed.2 Verrucarinic acid (l),a product of acid hydrolysis of the macrocyclic anti- biotic verrucarin A has been shown to be derived from (3R)-mevalonic acid (MVA) with loss of the 2-pro-S-hydrogen and migration of the 2-pro-R-hydrogen to C-3.3 Further support for the suggested pathway (Scheme 1)comes from the ‘Phytosterols’ AOCS Spring Meeting New Orleans April 1973 in Lipids 1974 9 pp.567-639. G. A. Cordell Phytochemistry 1974 13 2343. R. Achini B. Muller and C. Tamm Helu. Chim. Acta 1974 57 1442. 455 R.B. Bour und D.A. Widdowson HO HO/J J t CO H HO HO Scheme 1 isolation from Myrothecivrn species of other metabolites which incorporate the intermediates.Interconversion of trans,trans-farnesol and cis,trans-farnesol by a cell-free system from Andrographis paniculata occurs via the corresponding farnesols which are formed with loss of the 1-pro-S-hydrogen from trans,trans-farnesol but of the 1 -pro-A-hydrogenfrom cis,trans-farne~ol.~ CH,-COY -B . .H *Q-I-*+*P-.-,. \ Scheme 2 4'K. H. Overton and F. M. Roberts J.C.S. Chem. Comm. 1974 385; Biochem. J. 1974 144 585. Biosynthesis 457 The advent of techniques based on 13C n.m.r. spectroscopy has proved to be a great benefit to the field of sesquiterpenoid biosynthesis. A means now exists for unravelling the intricate rearrangements so characteristic of this area without the need for recourse to complicated degradative sequences.Scheme 2 summarizes the results obtained from a single feeding to cultures of Pseudeurotium oualis of a mixture of 90%enriched [1,2-'3C,]acetate and unlabelled a~etate.~ Carbon atoms joined by a heavy bond are those originally bonded together in acetate. Each of these twelve carbons appeared in the 13C n.m.r. spectrum of the derived ovalicin (2) as a triplet by virtue of spin-spin coupling with its partner. The effect of dilution by added and endogenous unlabelled acetate is such that there will only be a high probability of I3Cat two adjacent positions if these atoms have remained bonded together. The remaining three carbon atoms (a),which corres- pond to C-2 of MVA therefore appeared as singlets.Thus from a single experi- ment it is possible to locate unambiguously the biosynthetic origin of all fifteen carbon atoms of ovalicin. Examination by 13C n.m.r. spectroscopy of the hirsutic acid (3) formed during parallel feedings of [l-'3C]acetate (0)and [2-I3C]acetate (A)to cultures of Stereum complicatuln indicated the labelling pattern shown.6 Complementary results were obtained from a feeding of [1,2-'3C,]acetate to the fungus Coriolus Scheme 3 M. Tanabe and K. T. Suzuki,Tetrahedron Letters 1974 4417. ' T. C. Feline G. Mellows R. B. Jones and L. Phillips J.C.S. Chem. Comm. 1974 63. R. B. Boar and D. A. Widdowson consors which produces the related coriolins e.g. (4).' Modification of a humulene-type precursor in the manner indicated in Scheme 3 would account for these results.The incorporation of [2-' 3C]MVA into trichothecolone (5) by Trichothecium roseurn and location of the labelled atoms (e)by 13C n.m.r. spectroscopy in- dicated that the farnesyl pyrophosphate precursor must be folded as in (6)' qj& 0 - \ The precursor also contained a small amount of [2-14C]MVA. The incorporation judged from the enrichment of the 13C n.m.r. signals (0.35%) agreed well with that obtained by the normal radiochemical method (0.3 "/,). The presence of presqualene pyrophosphate in intact rat liver and in a micro- soma1 preparation from yeast has been demonstrated.' This compound is not therefore simply an artefact of NADPH deprivation. Related studies of pre-phytoene pyrophosphate have been reported.O The optically pure enantiomers of the important precursor squalene 2,3- epoxide are now readily available.' ' As would be expected for a cyclization that is a concerted process the (3S)-isomer (7) is the exclusive precursor of 3P-hydroxy- triterpenes [as (S)] and derived compounds (lanosterol in pig liver lanosterol H (7) and ergosterol in yeast and fi-amyrin lupeol and cycloartenol in pea seedlings). l2 Various suggestions have been made concerning the real nature/fate of the C-20 carbonium ion that is the immediate product of the cyclization of squalene 2,3- epoxide. Fusidic acid (9) that was biosynthesized from (3RS,2R)- and (3RS,2S)- [2-3H,2-'4C]MVA retained tritium in the 22-pro-R- and 22-pro-S-positions M.Tanabe K. T. Suzuki and W. C. Jankowski Tetrahedron Letters 1974,2271. J. R. Hanson T. Marten and M. Siverns J.C.S. Perkin I 1974 1033; E. McDonald Ann. Reports (B) 1972 69 471. F. Muscio J. P. Carlson L. Kuehl and H. C. Rilling J. Biol. Chem. 1974 249 3746; see also C. D. Poulter 0.J. Muscio and R. J. Goodfellow. Biochemistry 1974 13 1530. lo D. E. Gregonis and H. C. Rilling Biochemistry 1974 13 1538. " R. B. Boar and K. Damps Tetrahedron Letters 1974 3731. '' D. H. R. Barton T. R. Jarman K. G. Watson D. A. Widdowson R. B. Boar and K. Damps J.C.S. Chem. Comm. 1974 861. Biosyn thesis 459 HO-' respectively thus effectively precluding the intervention of a stable intermediate with a 20(22)-double bond.13 Examination of the 13C n.m.r.spectrum of fusidic acid derived from [l-'3C]acetate allowed the ready identification of those carbon atoms which are derived from C-1 of this precursor but only after considerable effort had been expended in the unambiguous identification of each resonance in the spectrum of unenriched rnaterial.I4 The enzyme system in plants which mediates the cleavage of the cyclopropane ring of 9p,l9-cyclo-triterpenes operates efficiently only on 4a-monomethyl or 4-desmethyl substrate^.'^ A similar system is not present in rat liver15 or yeast.I6 The sterol content of four single mutant strains of Saccharornyces cerevisiae clearly indicates that these have metabolic blocks at C -24-methylation at A' to A' isomerization at 5,6-dehydrogenation and at 22,23-dehydrogenation respectively.Each of these processes (and by implication 24,28-reduction) is thus controlled by a single enzyme system irrespective of substrate. As a result of this work the latter stages of ergosterol biosynthesis should now be viewed solely in terms of a permutation of these five independent unit transfor- mations rather than by any single sequence. This method of studying the bio- synthesis of complex systems has much to offer over methods based on the feeding of potential precursors. In another somewhat novel approach yeast which had been grown under anaerobic conditions (accumulation of squalene) was aerated in the presence of labelled methionine. The incorporations observed into various sterols suggested that C-24-methylation occurs principally at the 4-desmethyl stage.la Many insects have the ability to dealkylate dietary Cz8 and CZ9sterols to cholesterol.Tritium originally at C-25 is retained despite the apparent inter- l3 R. C. Ebersole W. 0. Godtfredsen s. Vangedal and E. Caspi J. Amer. Chem. SOC. 1974,96 6499. l4 T. Riisom H. J. Jakobsen N. Rastrup-Andersen and H. Lorck Tetrahedron Letters 1974 2247. l5 R. Heintz and P. Benveniste J. Biol. Chem. 1974 249 4267. l6 C. Anding L. W. Parks and G. Ourisson European J. Biochem. 1974 43 459. ' D. H. R. Barton J. E. T. Corrie D. A. Widdowson M. Bard and R. A. Woods J.C.S. Perkin I. 1974 1326. L. W. Parks C. Anding and G. Ourisson European J. Biochem. 1974 43 451. R. B. Boar and D. A. Widdowson mediacy of desrnosterol.it has now been established that the insect Tenebrio molitor converts [25-3H]clionasterol (10) into cholesterol (1 1) with migration of tritium to position 24 (Scheme 4).l9 / HO/+:I Scheme 4 Synthetic 2~-hydroxy-3,17-dioxoandrost-4-en-19-al (12) readily formed oes- trone (13) in aqueous solution at pH 2 7.20Since the 2a-hydroxy-isomer was relatively stable under the same conditions and the 2P-hydrogen is known to be lost it seems very likely that formation of (12)is the last enzymically controlled step in the biosynthesis of oestrone. (12) (13) Reduction of the 24,25-double bond of lanosterol occurs *ith overall cis stereochemistry in the rat,21 but the same process in the biosynthesis of tigogenin a (25R)-sapogenin in Digitalis lanata involves a trans-addition.22 The latter mode is also indicated for biosynthesis of (25S)-~apogenins.~~ l9 P. J. Pettler W. J. S. Lockley H. H. Rees and T. W. Goodwin J.C.S. Chem. Comm. 1974 844. H. Hosoda and J. Fishman J. Amer. Chem. SOC.,1974,96 7325. B. Yagen J. S. O'Grodnick E. Caspi and C. Tamm J.C.S. Perkin I 1974 1994. 22 L. Canonica F. Ronchetti and G. Russo J.C.S. Perkin I 1974 1670. " F. Ronchetti and G. Russo J.C.S. Chem. Cornm. 1974 785; A. G. Gonzalez C. G. Francisco R. Freire R. Hernandez J. A. Salazar and E. Suarez ibid. p. 905. Biosynthesis 461 3 Prostaglandins The question of the oxygenated intermediates between arachidonic acid and the prostaglandins has been resolved.24 Inhibition of the sheep vesicular gland prostaglandin synthetase with p-mercuribenzoate during incubation with [l-'4C]arachidonic acid (14)caused a build-up of peroxides and allowed the isolation and characterization of the 15-hydroperoxy-endoperoxidePGG (15) and the 15-hydroxy-endoperoxide PGH (16) (Scheme 5).These were HO / \yco2, Ho-r CO,H HO-\ HO Scheme 5 24 M. Hamberg J. Svensson T. Wakabayashi and B. Samuelsson Proc. Nut. Acad. Sci. W.S.A.,1974 71 345. R. B. Boar and D. A. Widdowson CO,H 0 (22) converted into PGF2cr(17) chemically (with stannous chloride) and enzymatically. In a parallel study it was fo~nd~~,~~ that the bulk of arachidonic acid was trans- formed in human platelets by a lipoxygenase into three metabolites (is),(191 and (20) which are not prostaglandin intermediates via a presumed hydro- peroxide (21).The cis-14,15-epoxyeicosa-cis-8,1l-dienoic prostaglandin intermediate.2 ' acid (22) is also not a 4 Polyketides Lynen as a part of his detailed study of patulin (23) biosynthesis has shown2* that its formation results from a minor hydroxylation process on m-cresol to form rn-hydroxymethylphenol. The major metabolite is 2-methylhydroquinone7 which is not a patulin intermediate. A new intermediate in stipitatonic acid (24) biosynthesis has been isolated from a feeding of [forrnyl-'4C]-3-methylorcylaldehyde (25) to Penicilliurn stipit~turn.~~ The aldehydo-acid structure (26) was assigned to the compound (Scheme 6). HOOYe -OQOH -oo \ /CO,H Me' CHO \/ OH HO * 0 HO (25) 0 (24) (26) Scheme 6 M..Hamberg and B.Samuelsson Proc. Nut. Acad. Sci. U.S.A.,1974 71 3400. M. Hamberg J. Svensson and B. Samuelsson Proc. Nut. Acad. Sci. U.S.A. 1974 71 3824. 21 R. Sood,M. Nagasawa and C. J. Sih Tetrahedron Letters 1974 423. 28 G. Murphy G. Vogel G. Krippahl and F. Lynen European J. Biochem. 1974,49,443. 29 R. W. Bryant and R. J. Light Biochemistry 1974 13 1516. Biosyn thesis 463 3H N.m.r. spectroscopy has been used for the first time for the solution of a biosynthetic problem.30 Carbon-14 work had shown the polyketide origin of penicillic acid (27) and the intermediacy of 6-methylsalicylic acid (28).31,32 By feeding [3H]acetate of high specific activity to Penicillium cyclopium samples of penicillic acid of sufficient tritium content for n.m.r.analysis were obtained. From the assigned tritium positions and notably the stereospecific (but undeter- mined) labelling of the vinyl group in (27) Scheme 7 was tentatively proposed for the biosynthesis. T (28) OMe HgGC:2T -+ H+ Scheme 7 Tetronic acids can arise either via cleavage of aromatic intermediates or from Krebs-cycle intermediates. Multicolic (29) and multicolosic (30) acids have been shown,33 by 13C n.m.r. spectroscopy to arise in Penicillium multicolor from acetate via cleavage of the aromatic acid (31) (see Scheme 8). From [1,2-' 3C,]acetate feedings (see above) the acetate residues C-2-C-5 C-6-C-7 C-8-C-9 and C-4-C-10 were shown to be incorporated intact. The fungal pigment tenellin (32) is derived from phenylalanine acetate and C units.34 Incorporation of the 13C-labelled precursors enabled the origin of all 30 J.M. A. Al-Rawi J. A. Elvidge D. K. Jaiswal J. R. Jones and R. Thomas J.C.S. Chem. Comm. 1974 220. 31 K. Mosbach Acta Chem. Scand. 1960 14 457. 32 R. Bentley and J. G. Kiel J. Biol. Chem. 1962 237 867. 33 J. A. Gudgeon J. S. E. Holker and T. J. Simpson J.C.S. Chem. Comm. 1974 636. 34 A. G. McInnes D. G. Smith J. A. Walter L. C. Vining and J. L. C. Wright J.C.S. Chem. Comm. 1974 282. R. B. Boar and D. A. Widdowson H0,CCH + Hrco2H (30) Scheme 8 carbon atoms to be determined (Scheme 9) and showed that the phenylalanine residue undergoes a carboxyl migration. [1,2-' 3C,]Acetate feedings showed that C-2-C-3 and the alkyl side-chain are derived from intact acetate units.(?H3-(?02-A 0 PhCH CH(NH 2)C0 H --* 0 CH,S(CH2),CH(NH2)C0,H Scheme 9 The cytochalasans e.g. cytochalasin D (33) also mixed phenylalanine-polyketides have been studied using both 14C and label^.^'.^^ The 14C work with Zygosporiurn rnasoni established two possible modes for joining the basic units [(34) or (3511. The 13C work with [1-'3C]- and [2-13C]-acetate could only confirm the earlier results without resolving the ambiguity. The macrocyclic polyketides have been receiving an increasing amount of attention. [Me-'3C]Methionine [l-'3C]propionate [l-'4C]acetate [1-i4C]-maionate ['4C]formate [Me-'4C]methionine and [1-' 4C]propionate have all been incorporated into geldamycin (36) by Streptomyces hygroscopicus var.geldanus var. 12o~u.~~ The o-methoxy-groups were derived from methionine. 35 C. R. Lebet and C. Tamm Heh. Chim. Ada 1974 57 1785. 36 W. Graf J. L. Robert J. C. Vederas C. Tamm P. H. Solomon I. Muera and K. Nakanishi Helv. Chim. Acta 1974 57 1801. 37 R. D. Johnson A. Haber and K. L. Rinehart J. Amer. Chem. SOC.,1974 96 3316. Biosynthesis 465 A A Methionine methyl group ;-m Acetate starter ; -3Malonate-derived C unit pPropionate As with streptovaricin and the rifamycins (see below) the compound is derived from a C7-N unit and a mixed propionate/acetate (malonate) chain (37). The Me0 Me -+Acetate/malonate ;pPropionate R.B. Boar and D.A. Widdowson origin of the C,-N unit was not determined.The antibiotics rifamycin W (38) and rifamycin B (39) have been examined in a continuing programme of studies of ansamycin bio~ynthesis.~'~~~ A m utant strain of Nocardia mediterranei which accumulates rifamycin W (38) was used for 3C-labelled acetate and propionate incorporations. The biosynthesis was shown to involve the C7 -N HOHZC HO MeOAc MeOH MeOH Me OH -o Acetate/malonate ;14Propionate/methylmalonate unit which was assumed to initiate a polyketide chain of mixed acetate and propionate units (40). The parent strain of the organism transformed the rifa- mycin W into rifamycin B a process which involves cleavage of the C-12-42-29 double bond More extensive studies39 on rifamycin B in N. mediterranei involving both 13C- and '4C-labelled precursors have shown that the propionate units are incorporated via carboxylation to methylmalonate.The origin of the C,-unit was not determined with any certainty. It is derived from glucose and glycerate but not shikimic acid. The authors suggested that an intermediate in shikimic acid metabolism possibly 5-dehydroquinic acid may be involved a conclusion which was corroborated by some work on mitomycin biosynthe~is.~~ 38 A. Karlsson G. Santori and R. J. White European J. Biochem. 1974 47 251. 39 R. J. White E. Martelli and G. Lancini Proc. Nat. Acad. Sci. U.S.A. 1974 71 3260. 40 U. Hornemann J. P. Kehrer and J. H. Eggert J.C.S. Chem. Cornm.. 1974,.1045. Biosynthesis 467 5 Alkaloids The conditions necessary for the successful application of 3C n.m.r.spectroscopy to studies of biosynthesis in higher plants have been clearly defined and illustrated by experiments which substantiate the established pathway for the biosynthesis of colchicine in Colchicum a~tumnale.~~ A comprehensive review of the biosynthesis of indole alkaloids has appeared.42 The (3S)-lactam (41) itself derived from isovincoside acted as a specific precursor of the interesting indole alkaloid camptothecin (42).43 Rather than attempt to dYo OGlu locate by degradation the labels of camptothecin derived from [14-3H2,5-14C]-(41) the authors fed [5-I3C]-(41) and showed by 13Cn.m.r. spectroscopy that the signal for C-5 of camptothecin was specifically enhanced (ca. 55 "/,). Previously vincoside but not isovincoside had been shown to be incorporated into a whole array of indole alkaloids.44 In uitro experiments showed that the indole a,&bond of lactam (41) is cleaved much more rapidly by either sodium periodate or oxygen-potassium t-butoxide than the corresponding bond in the (3R)-lactam but the suggestion that such differences in chemical reactivity rather than enzyme specificity could determine in uivo pathways seems unlikely.45 In an interesting application of stable isotopes a mixture of [4-'H2]- and [2-13C]-mevalonic acids was fed to the ergot fungus Cfauiceps,strain SD 58.Examination by mass spectrometry of the elymoclavine (43) formed showed that about 8% of the molecules contained both carbon-13 and deuterium.46 This together with other evidence requires that the conversion of chanoclavine-I (44)into elymoclavine involves the possibility for intermolecular hydrogen exchange at C-9 during isomerization of the 8,9-double bond.Dihydroergosine (45) that had been biosynthesized from [5(R,S)-3H,2-14C]MVA by the fungus 41 A. R. Battersby P. W. Sheldrake and J. A. Milner Tetrahedron Letters 1974 3315. 42 G. A. Cordell Lloydia 1974 37 219. 43 C. R. Hutchinson A. H. Heckendorf P. E. Daddona E. Hagaman and E. Wenkert J. Amer. Chem. SOC.,1974 96 5609. 44 See H. F. Hodson Ann. Reports (B) 1971,68 502. " C. R. Hutchinson G. J. O'Loughlin R. T. Brown and S. B. Fraser J.C.S. Chem. Comm. 1974,928. 46 H. G. Floss M. Tcheng-Lin C. Chang B. Naidoo G. E. Blair C. I.Abou-Chaar and J. M. Cassady J. Amer. Chem. SOC.,1974 96 1898. R.B. Boar and D.A. Widdowson OH OH OH 0 II H.' H H v '" (43) H Sphacelia sorghi retained tritium at C-10 thus discounting the possibility that ergosine (45 ;9,10-dehydro) is an intermediate." Interest continues in the synthesis of compounds containing a stereospecifically labelled methylene group and in the application of such materials to stereo- chemical aspects of biosynthesis. It has now been shown that the conversion of dopamine (46) into (-)-noradrenaline (47),48 and of (S)-amphetamine into (46) (47) (1 S,2R)-ephedrine4' by dopamine /I-hydroxylase both proceed with replacement of a benzylic pro-R-hydrogen by hydroxyl with retention of configuration.In the former case the crucial synthetic step was generation of the chiral benzylic methylene by asymmetric reduction of a substituted benzaldehyde by liver alcohol dehydrogenase. Kirby's group have continued to apply their ingenious and more strictly chemical solution to such syntheses. The indolylacetic acid (48) derived as indicated was hydrogenated to yield the racemate (49) + (50) which was then resolved using 1-phenylethylamine (Scheme 10). Further transformations then afforded (3R)-and (3S)-[3-*H]-~~-tryptophan.~' When admixtures of the corres- ponding tritiated compounds with 14C-labelled tryptophan were fed to the fungus Pithomyces chartarum clear evidence was obtained that hydroxylation at C-3 in the biosynthesis of sporidesmin A (51) occurs by replacement of a 3-pro-R-hydrogen of tryptophan with retention of configuration.When (2R)-[Z3H l-'4C]-O-methylnorbelladine (52) was fed to narcissi haemanthamine (53) and narcissidine (54) were formed with 5 and 91 % retention 47 K. D. Barrow P. G. Mantle and F. R. Quigley Tetrahedron Letters 1974 1557. 48 A. R. Battersby P. W. Sheldrake J. Staunton and D. C. Williams J.C.S. Chem. Comm. 1974 566. 49 K. B. Taylor J. Biol. Chem. 1974 249 454. G. W. Kirby and M. J. Varley J.C.S. Chem. Comm. 1974 833; see also ref. 66. Biosynthesis 469 Scheme 10 of tritium respectively. Complementary results were obtained using (2S)-(52) thus confirming that in the former case it is the pro-R- and in the latter case the pro-S-hydrogen that is lost.51 Galanthine (55) (5.2 incorporation) is evidently the immediate precursor of narcissidine.Me0 \ Meom (55) (54) C. Fuganti D. Ghiringhelli and P. Grasselli J.C.S. Chem. Comm. 1974 350. R.B. Boar and D.A. Widdowsoq The biosynthesis of the Erythrina alkaloids has proved to be a rewarding area of study. In uitro phenol oxidations completely belied the in uiuo origin of these compounds which was shown (Scheme 11) to involve (S)-N-norprotosinomenine (56).52 The stereochemistry of this precursor requires that the subsequent pathway involves either racemization or inversion of configuration. The resolu- tion of erysodienone has now been reported and as expected the (5s)-isomer (57)acts as a specific precursor of the Erythrina alkaloid^.'^ Reduction of resolved erysodienone with Ti”’ gives the dibenzazonine (58) which racemizes rapidly with f+ -1.2min at 20°C.It is likely that this is also the stage at which in uiuo racemization occurs. Erythrina ~ M::CC-& alkaloids Me0 0 (57) Scheme 11 It has been established that the furan ring of furoquinoline alkaloids and of furocoumarins arises by oxidative cyclization of an isoprenyl derivative and subsequent loss of an isopropyl fragment. Thus platydesmine (60)is an efficient precursor of dictamnine (62) (18.8 ”/ inc~rporation).’~ An intermediate of type (61) would be consistent with the observation that the [l’-(R,S)-3H 1‘-14C]-quinolone (59) afforded skimmianine (63) in which approximately half of the tritium had been lost.” ” D.H. R. Barton C. J. Potter and D. A. Widdowson J.C.S. Perkin J 1974 345 and earlier references. ” D. H. R. Barton R. D. Bracho C. J. Potter and D. A. Widdowson J.C.S. Perkin I 1974,2278. 54 J. F. Collins W. J. Donnelly M. F. Grundon and K. J. James J.C.S. Perkin I 1974 2177; M. F. Grundon D. M. Harrison and C. G. Spyropoulos ibid. p. 2181. ’’ M. F. Grundon D. M. Harrison and C. G.Spyropoulos J.C.S. Chem. Comm. 1974 5 1. Biosynthesis 47 1 (62) R = H (63) R = OMe H (44) Preliminary investigations of the biosynthesis of securinine (64)in Securinegu sufruticosa indicate that the piperidine ring derives from lysine whilst [2-’4C]-tyrosine specifically labelled the remaining C fragment at the position in- di~ated.~~ All incorporations were however extremely low.6 Amino-acid Metabolites Azetidine-2-carboxylic acid (65) arises from S-adenosylmethionine in Con-uullariu mujulis not by a direct displacement as might have been expected but by amination to y-aminohomoalanine (66)as shown (Scheme 12).57 d,,”,a;ozH --+ (65) Scheme 12 56 R. J. Parry Tetrahedron Letters 1974 307; U. Sankawa K. Yamasaki and Y. Ebizuka ibid. p. 1867. 57 E. Leete G. E. Davis C. R. Hutchinson K. W. Woo and M. R. Chedekel Phyro-chemistry 1974 13 427. R. B. Boar and D. A. Widdowson I CO H CO H (67) R = PhOCH,CO (68) (69) R = O,CCH(&H,)(CH,),CO The fate of the methyl groups of valine in penicillin biogenesis has been resolved.Firstly [Me,-’H,]valine was transformed by Penicillium chrysogenum into penicillin V (67),with retention of all the deuterium atoms.58 The ene reaction involving the species (68) suggested by Baldwins9 for penicillin biosynthesis is thus disproved. Two groups have tackled the problem of the stereochemical fate of the enantiotopic methyl groups of valine. Aberhart using (2RS,3S)- [4-’3C]valine showed that the chirality at C-3 was retained in the formation of penicillin V by P. chrysogenum.60 In a more detailed study,61 Sih and Abraham synthesized (2S,3S)-[4-’H3]- and (2S,3R)-[4-’H3]-va1ine. Each was incorporated into penicillin N (69) and cephalosporin C (70) in Cephalosporium acrernonium mutant C91. There was no scrambling of the [’H,]methyl groups.The (3s)-isomer of [4-’H,Jvaline gave (2R)-[Me-’H3]penici1lin N and cephalosporin C deuteriated only in the 3-acetoxymethylene group. The (3R)-isomer gave (2s)-[Me-’H ]penicillin N and [2,2-’H Jcephalosporin C. The overall retention of configuration of C-3 of valine in penicillin biosynthesis is thus confirmed and the involvement of a A’-cephem intermediate in cephalosporin biosynthesis can be excluded. The antitumour antibiotic anthramycin (71) produced by Streptomyces refuineus var. thermotolerans is derived from tryptophan and tyrosine (Scheme 13)uia the presumed intermediates 3-hydroxyanthranilic acid (72)and the proline derivative (73).62 The additional carbon atoms arise from methionine. Metacycloprodigiosin (74) and undecylprodigiosin (75) the tripyrrolic pig- ments from Streptomyces longisporus var.ruber are derived63 in an analogous 58 D. J. Aberhart J. Y.-R. Chu N. News C. H. Nash J. Occolowitz L. L. Huckstep and N. de la Higuera J.C.S. Chem. Comm. 1974 546. 59 J. E. Baldwin S. B. Haber and J. Kitchin J.C.S. Chem. Comm. 1973 790. 6o D. J. Aberhart and L. J. Lin J.C.S. Perkin I 1974 2320. 61 H. Kluender F. C. Huang A. Fritzberg H. Schnoes C. J..Sih P. Fawcett and A. P. Abraham J. Amer. Chem. SOC.,1974,96 4054. 62 L. H. Hurley and M. Zmijewsky J.C.S. Chem. Comm. 1974 337. 63 H. H. Wassermann C. K. Shaw and R.J. Sykes Tetrahedron Lerters 1974 2787. Biosynthesis 473 ,co,-(75) R = n-C,H, EH,~o,H (76) R = H HO~H,CH(kH,)CO +o H,NCH,CO,-Scheme 14 manner to prodigiosin (76)64in Serratia murcescens.Using 3Cn.m.r. techniques the 2,2-bipyrrolic moiety was shown to be generated from proline acetate glycine and serine (Scheme 14). It is coupled to a C,,-polyacetate chain which 64 H. H. Wassermann R. J. Sykes P. Peverada C. K. Shaw R. J. Cushley and S. R. Lipsky J. Amer. Chem. SOC.,1973 95 6874. R. B. Boar and D. A. Widdowson is cyclized to form the third pyrrolic ring and in the case of (74),further cyclized to the tvelve-membered cyclophane unit. Some scrambling of amino-acid label was evident from the labelling of the methine-bridge carbon by both glycine and serine. 7 Shikimic Acid Metabolites Aureothin (77) is formed in Streptomyces thiol~theus~~ from both threo-and erythro-p-aminophenylserine but not from p-aminocinnamic acid p-amino- benzoic acid or p-nitrophenylalanine.The formation of the p-nitrophenyl unit must therefore involve benzylic hydroxylation at the p-aminophenylalanine stage and formation of the nitro-group by oxidation at a late stage. (77) Floss has synthesized (2S,3R)-[3-3H]serine by an enzymatic procedure and used it to show that the stereochemistry of C-3 of serine is retained during tryptophan formation by tryptophan synthetase.66 HO ‘0H HO 65 R. Cardillo C. Fuganti D. Ghiringhelli D. Giangrasso P. Grasselli and A. Santopietro-Amisano Tetrahedron 1974 30 459. 66 G. E. Skye R. Potts and H.-G. Floss J. Amer. Chem. SOC.,1974,96 1593. Biosynthesis 475 In the biosynthesis of the proanthocyanidins by Aesculus carrea and Rubus SP.,~~ each half of proanthocyanidin A2 (78) and proanthocyanidin B2 (79) comes from ( -)-epicatechin (80) but the labelling levels (derived from [3-3H]-cinnamic acid) are different.The epicatechin units must therefore come from different pools and a simple oxidation of (80) is not involved. 67 D. Jacques and E. Haslam J.C.S. Chem. Comm. 1974 231.

ISSN:0069-3030    

DOI:10.1039/OC9747100455

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

17 Histaminic and Cholinergic Agonists and Antagonists By A. F. CASY School of Pharmacy Liverpool Polytechnic Byrom Street Liverpool L3 3AF 1 Introduction Ligands which bind to pharmacological receptors are of two chief types ; those which induce a physiological response are termed agonists while those which block the receptor and make it insensitive to an agonist are termed antagonists. Agonists and antagonists in the histaminic and cholinergic fields form the theme of this review. These areas concern the naturally endogenous agonists histamine and acetylcholine (ACh) respectively; the former is released as the result of various forms of bodily maltreatment and is responsible for allergic diseases in man while the chief role of the latter is the transmission of nerve impulses across the gaps (synapses) that link neuron to neuron neuron to muscle cell and neuron to secretory cell.Emphasis in the review is placed on the literature of the past 5 years. With certain exceptions e.g. dexetimide (73) and pancuronium (80) few novel clinical agents have been introduced as a result of the work described although substances of potential value in medicine have been discovered notably the selective H,-receptor histamine antagonists. Most of the work reviewed has been undertaken in a search for clues about the nature of histaminic and cholin- ergic receptors and modes of ligand-receptor interactions. 2 Histamine Agonists and Antagonists Interest in the naturally occurring amine histamine (1) [4(5)-(2-aminoethyl)-imidazole] has been stimulated by the need to define at least two types of histamine CH,CH,NH fi (and tautomer) HN N v receptor.Receptors blocked by mepyramine and other compounds clinically recognized as antihistaminics are termed H while mepyramine-insensitive types are designated H2.1 Thus the actions of histamine in stimulating secretion of ' A. S. F. Ash and H. 0.Schild Brit. J. Pharmacoi. 1966 27 427. 477 478 A. F. Cusy acid by the stomach,2 accelerating the heart,3 and inhibiting contractions of rat uterus4 are not antagonized by mepyramine and related drugs and are thus considered to be mediated at H receptors. This sophistication of histamine receptor theory has its counterparts in the catecholamine (a- P-receptors) and cholinergic (muscarinic nicotinic receptors) fields and has resulted in a search for specific agonists and antagonists.Kier's proposal5 that the drug-receptor interactions of histamine involve two distinct conformations [H trans-+NH3/Ar (2); H gauche (3)] of histamine monocation (population =-96% at Ar (2) (3) physiological pH) has prompted several conformational studies by M0,697uand 'H n.m.r. technique^.^ In general the data concur in showing that histamine has only a small preference for the trans-rotamer (2). In contrast crystalline histamine acid phosphate (dication) exists entirely in the truns-conformation.8 From differences between the pK values of N"-and N'-methyl(and -benzyl)- histamines [see (4)] it is calculated that the tautomeric ratio for histamine mono- cation in water is 4(N'-H) to l(N"-H).9 A similar value is reported for histidine monocation based on a I3C n.m.r.study." hCH ,6H2 N : 2CH I4H3 4d:H 'NUN" HN N' Y (4) (5) All possible monomethylhistamines (5) have been prepared and tested as H and H agonists.",'2 Full details of synthesis are not available but a convenient E. R. Loew and 0.Chickering Proc. SOC.Exp. Biol. Med. 1941 48 65. ' U. Trendelenburg J. Pharmacol. Exp. Therap. 1960 130,450. ' P. B. Dews and J. D. P. Graham Brit. J. Pharmacol. 1946 1,278. L. B. Kier J. Medicin. Chem. 1968 11 441. P. F. Periti Pharmacol. Res. Comm. 1970 2 309; J.-L. Coubeils P. Courriere and B. Pullman Compt. rend. 1971 272 D 1813; S. Margolis S. Kang and J. P. Green Internat.J. Clin. Pharmacol. 1971,5 279. (a) C. R. Ganellin E. S. Pepper G. N. J. Port and W. G. Richards J. Medicin. Chem. 1973 16 610; (6)A F. Casy R. R. Ison and N. S. Ham Chem. Comm. 1970 1343; (c) N. S. Ham A. F. Casy and R. R. Ison J. Medicin. Chem. 1973 16,470. * M. V. Veidis and G. J. Palenik Chem. Comm. 1969 196; M. V. Veidis G. J. Palenik R. Schaffrin and J. Trotter J. Chem. SOC.(A) 1969 2659. C. R. Ganellin J. Pharm. Pharmacol. 1973 25 787. lo W. F. Reynolds I. R. Peat M. H. Freedman and J. R.Lyerla J. Amer. Chem. SOC. 1973,95 328. J. W. Black W. A. M. Duncan C. J. Durant C. R. Ganellin and E. M. Parsons Nature 1972 236 385. ' la K. Kowalewski and A. Kolodeg Pharmacology 1974 11 207. C. R. Ganellin G. N. J. Port and W.G. Richards J. Medicin. Chem. 1973 16 616; C. R. Ganellin ibid. p. 620. Histaminic and Cholinergic Agonists and Antagonists 479 CH,CH(CH,OH)N H CH,CH( Me)NH Reagents i HBr-AcOH; ii H,-Pd/C AcOH,H,O Scheme 1 conversion of L-histidinol(6) into a-methylhistamine (7) (Scheme 1)is reported.' While methyl substituents generally depress the stimulant activity of histamine the 4-Me derivative is noteworthy in being a good H (43"/ of parent) but a feeble H agonist (H1/H2 ratio = 0.005),12a result confirmed by others.I4 The con- formationally restrained analogue of histamine 2-(4-imidazoyl)cyclopropylamine (lo) in which imidazoyl and amino functions are approximately anticlinal is a feeble agonist at both H and H sites;" the cyclopropane ring was constructed by the reaction of the trans-urocanic acid derivative (8) with dimethylsulphoxo- nium methylide and conversion of the alkoxycarbonyl group of (9)into an amino- group by a Curtius degradation (Scheme 2).H \ ,co2Bus ,dNH2 \ " * -+II IV II m hc==Lr-7do2Bus TrN N TrN N HN wN --& (81 (9) (10) Tr = CPh Reagents i Me,S(O)=CH,; ii KOH; iii HCI; iv Et,N-CIC0,Et-NaN Scheme 2 Most small-ring heterocycles with a P-aminoethyl side-chain are histamine agonists.' The anomalous report' that the claimed 2-positional isomer (1l) isohistamine is without histamine activity has been studied ;in reinvestigations of Jones' synthesis,' * the first step (reaction between 2-chloromethylimidazole and cyanide) was shown to involve nuclear rather than side-chain substitution giving l3 R.R. Ison and A. F. Casy J. Medicin. Chem. 1970 13 1027. l4 G. Bertaccini M. Impicciatore T. Vitali and V. Plazzi Farmaco Ed. Sci.,1972 27 680 l5 A. Burger M. Bernabe and P. W. Collins J. Medicin. Chem. 1970 13 33. l6 R. G. Jones in 'Handbook of Experimental Pharmacology' Vol. XVIII/I Springer- Verlag Berlin 1966 Ch. 1. " R. G. Jones J. Amer. Chem. Soc. 1949,71 383. '* (a) G. J. Durant M. E. Foottit C. R. Ganellin J. M. Loynes E. S. Pepper and A. M. Roe Chem. Comm. 1968 108; (b) E. C. Kornfeld L. Wolf T. M. Lin and I. H. Slater J. Medicin. Chem. 1968 11 1028. 480 A. F. Casy H,NCH k f=l HN HNYNCfi% HNyN 3 -HNYN (CH2)2NH CHACI Me Me (11) (12) (13) (12)('H n.m.r. evidence) hence Jones' product is an aminomethyl derivative (13).Reaction between 1-benzyl-2-chloromethylimidazoleand cyanide in aqueous ethanol (as Jones' ') gave a mixture of the two isomeric cyanides but reaction in Fl /=I / H,NCH,CH(OEt) HNyN HNYNcH2ph OEt PhCONHCH,CH,C ______+ CH,CN CH2CHz NHCOPh 1HCI (14) (15) (1 1) DMSO gave the 2-cyanomethyl compound (14) exclusively.18' Authentic iso-histamine obtained from (14) and also from the ethoxyimine (1 5) and 2-amino- acetaldehyde diethylacetal proved to be a weak histamine-like agonist. Several compounds are reported that are active in inhibiting gastric acid secretion e.g.(16),' (17),20and 2,2'-bipyridyl,' but only N-(4-imidazol-4-ylbutyl)-Ph S \ 4 CH-C\ NH PhCH,CMe,NHCH,COMe CN \ (17) (16) S ICH,.).NHC// CH,.SCH.CH .NHC \ NHMe \-/ HN VN "-methylthiourea (18) (burimamide) and the 4-methylimidazole derivative (19) (metiamide) prevent gastric secretion stimulated by histamine thereby being defined as specific H,-receptor antagonists.' '*'In Burimamide behaves as a competitive antagonist at H sites (PA 5 rat uterus and guinea pig atria)* and l9 R.G. Bianchi and D. L. Cook Fed. Proc. 1968,27 1331. 'O W. Lippmann Experientia 1968 24 1153. ' D. E. Butler P. Bass I. C. Nordin F. P. Hauck and Y. J. L'ltalien J.Medicin. Chem. 1971 14 575. * The parameter PA is defined as the negative logarithm ofthe concentration ofantagon- 1st which reduces the effects of a double dose of agonist to those of a single dose and it is a convenient means of comparing the potencies of different antagonists.It is also equivalent to the logarithm of the affinity constant of the antagonist in the equilibrium ligand + receptor complex. Histaminic and Cholinergic Agonists and Antagonists 481 does not interact significantly with H receptors as present in guinea pig ileum; its crystal and molecular structures have been reported,22 and it may be synthe- sized (Scheme 3) from lysine ethyl ester (19a).22a,b Metiamide2,' (cJ work on 4-methylhistamine described above) also inhibits bethanechol-chloride-induced (cholinergic) secretion of hydrochloric acid.' lo 2)4NH Et02CCH(CH2),NH2 HN/__((CH2)4NH2 % HN wN % (18) I YNH NH2 S (194 Reagents i Na-Hg; ii KCNS; iii FeCI,; iv MeNCS Scheme 3 CH(OH)CH,NHPr' (20) Since pronethal (20) a bicyclic analogue of isoprenaline is a /?-adrenergic antagonist aminoethylimidazo-[ 1,2-a]-and-[ 1,5-a]-pyridines have been examined as histamine antagonist^.^^ The derivative (22) was made (see Scheme 4) from + BrCH2COCH2CH2N=Phth2 vccH2CH2NH2 ONH2 (21) N=Phth = phthalimido Reagents i DMF HCO,-; ii 6M-HCI Scheme 4 2-aminopyridine and the a-halogeno-ketone (21) and the isomer (23) by a route starting with a Friedel-Crafts acylation of imidaz0[1,5-a]pyridine.~~ Neither (22) nor (23) had H agonist or antagonist activities but (22) was a moderately active H agonist.'' B. Kamenar K. Prout and C. R. Ganellin J.C.S. Perkin 11 1973 1734. 220G. J. Durant J. C. Emmett C. R. Ganellin and G.R. White B.P. 1 307 539 21 Feb. 1973. '"S. Akabori and T. Kaneko Bull. Chem. SOC. Japan 1936 11 208. '*'G.J. Durant J. C. Emmett and C. R. Ganellin B.P. 1 338 169 21 Nov. 1973. 23 G. J. Durant J. M. Loynes and S. H. B. Wright J. Medicin. Chem. 1973 16 1272. J. D. Bower and G. R. Ramage J. Chem. SOC.,1955,2834. 482 A. F. Casy Recent developments in conventional (H') histamine antagonists have empha- sized the importance of stereochemical relationships. Dextrorotatory isomers of the pheniramines all have the same configuration [S,(24)] since reduction of H (24) a; Ar = Ph b; Ar = C,H,-p-CI c; Ar = C,H,-p-Br ( +)-bromo- and (+)-chloro-pheniramine catalysed by palladium gives ( +)-pheniramine (24a).25 [( +)-(24b) is a more effective antihistaminic than the laevo-i~omer].~~ The configuration of dextrochlorpheniramine was correlated with that of the amide (25) of known stereochemistry by the sequence shown in Scheme 5.The more potent laevo-isomer of carbinoxamine tartrate (26) was qph -$ (+) -(24b) -+ +i ii CH,CH,NMe iii iv CH=CH Ph COPh COPh ' (isomers separated) 1v. vi OCH,CH,NMe H H Ph I H (26) (25) Reagents i H2-PtOz; ii PhCOCI; iii Mel; iv Ag,O-D,O on solid benzamide; v 0,; vi 12M-HC1 Scheme 5 shown to be sterically related to dextropheniramine by similar chemical inter- conversion^.^ ' Further studies of antihistamines that exhibit geometrical isomerism have been made. All four isomeric butenes (E- and Z-but-1-ene and but-2-ene pairs) that result when the tertiary alcohol (27) is dehydrated have been isolated and characterized by U.V.and 'H n.m.r. data;28 the most potent was the E-but-2-ene (28) (PA = 10.3),29which corresponds with the base of pyrrobutamine diphos- phate (Pyronil) a clinical agent of high potency. The more potent forms of 25 A. Shafi'ee and G. Hite J. Medicin. Chem. 1969 12 266. 26 R. T. Brittain P. F. D'Arcy and J. H. Hunt Nature 1959 183 734. 27 V. Barouh H. Dall D. Patel and G. Hite J. Medicin. Chem. 1971 14 834. 20 A. F. Casy and R. R. Ison J. Pharm. Pharmacol. 1970 22 270. 29 R. R. Ison F. M. Franks and K. S. Soh J. Pharm. Pharmacol. 1973 25 887 Histaminic and Cholinergic Agonists and Antagonists 483 OH Ph I \ /H PhCCH,CH /c=c\ Na I CH2 (27) Further related but-2-ene pairs all have an E(Ar/CH,N') config~ration.~~.~~ E-2 pairs related to triprolidine (29)have been obtained by the route (30)-(31):' v11 QH / c,x OH Y /H ArCO(CH,),NR 5 Ark.H,CH,NR c=c /\/---I Mannirh Mannich base (30) IJ-JI / Me (29) Ar \ C=CHCH,NR (a) R = Ph; (b) R = a; (c)R = CHPh -3 AcNHICqzH AcO RNH + NHR C 0 IIR or S 0 (32) AcNHGNR EtNHcNR 01LIAIH \ / (33) In all cases the E(2-py/CH2NR2)isomer had the higher potency with receptor affinity ratios near 10except for triprolidine and its isomer (> 1000).29Structural requirements for HI-receptor blockade have been discussed in the light of these data.28-31 30 A.F. Casy and A. P. Parulkar Canad.J. Chem. 1969,47 423. R. R. Ison and A. F. Casy J. Pharm. Pharmacol. 1971 23 848. 484 A. F. casy Several conformational studies of antihistaminics have now been made both in the solid32,33 and solute state;34 one of these33" confirms the S-configuration of ( + )-chlorpheniramine. The R-and S-3-ethylaminopyrrolidineunit [as in (33)] has been employed as a probe for the study of the stereoselective behaviour of a variety of pharmaco- logical receptors including those of histamine. Potential antihistaminics were made by treating enantiomorphic forms of N-acetylaspartic anhydride with various amines as shown [(32) +(33)]. The N-phenyl derivatives (33a) were weak antagonists of histamine at guinea-pig ileum sites with the S-ten times more active than the R-f~rm.~" Attachment of larger aromatic substituents to ring nitrogen led to compounds of far higher potency e.g.(33b) (molar ED,* -7 x lo-*) and (33c) (molar ED, -9 x lop9),but little difference in activity was found between isomeric pairs.35b The lack of stereoselectivity found is in accord with other studies employing isomers that have the asymmetric centres close to the basic nitrogen rather than an aromatic feature of the molecule.36 Analogues of (33a) with an additional phenyl substituent in the ring (34) are n-NMe CNh (34) (35) also much more potent than the parent and may be regarded as cyclic forms of phenbenzamine (35).3' The trans-isomer prevented histamine-induced contrac- tions of guinea-pig ileum at a concentration as low as 2 x lo-' mol kg-' and its action could not be readily reversed while the cis-form acted competitively Ph -+ cis-(34) Ph Reagents i NH,OH; ii Ac,O; iii B,H,; iv NaBH,; v TsCl; vi NaN,- ;vii Mel-LiAlH Scheme 6 32 G.R. Clarke and G. J. Palenik J. Amer. Chem. Soc. 1972,94 4005. 33 M. N. G. James and G. J. B. Williams (a)J. Medicin.Chem. 1971 14 670; (b)Cunud. J. Chem. 1974 52 1872; (c)ibid. p. 1880. 34 N. S. Ham J. Pharm. Sci. 1971,60 1764. 35 (a) D. T. Witiak Z. Muhi-Eldeen N. Mahishi 0. P. Sethi and M. C. Gerald J. Medicin.Chem. 1971,14,24; (6)D. T. Witiak S. Y. Hsu J. E. Ollmann R. K. Griffith S. K. Seth and M. C. Gerald ibid. 1974 17 690. 36 M. J. Jarrousse and M. T. Regnier 1951 Ann. pharm. franc. 1951 9 321 ; A. P. Roszkowski and W.M. Govier Pharmacologist 1959 1 60; F. E. Roth Chemo-therapiu 1961 3 120. 37 P. E. Hanna and A. E. Ahmed J. Medicin. Chem. 1973 16,963. Histaminic and Cholinergic Agonists and Antagonists 485 (PA 7.97). Both isomers were obtained from 1,5-diphenyl-3-pyrrolidone(36) (Scheme 6). and configurational assignments were made from ‘H n.m.r. data. Novel antihistaminics reported recently are mostly variants of known structural types (reviewed by Witiak).3B Thus the indole (37) is related to ~lernizole,~~ the phenothiazine (38) to pr~methazine,~’ and azatadine (39) to ~yproheptadine.~~ The last named a potent antihistaminic in animals and also effective ~linically,~~ is made by dehydration of the tertiary alcohol formed from the ketone (40)43 and Grignard reagent (41).The 4-benzamidopiperidine (42) (PA 9.6 cJ:chlor pheniramine 8.6)44 is of greater structural interest since it does not possess the geminal aromatic feature linked through a linear chain to basic nitrogen (Ar-Ar’X . . N) common to most other classes of antihistaminic. 3 Acetylcholine (ACh) Agonists and Antagonists Much of the work reviewed here concerns efforts to define the pharmacophoric or ‘active’ conformation of cholinergic molecules in the search for clues about the molecular nature of cholinergic receptors and the ways in which ligand- receptor combinations are achieved. There is promise from work on electric 38 D. T. Witiak in ‘Medicinal Chemistry’ ed. A. Burger 3rd edn. Wiley-Interscience New York,1970. 39 R. N. Schut F.E. Ward 0.J. Lorenzetti and E. Hong J. Medicin. Chem. 1970,13 394. 40 C. Kaiser D. H. Tedeschi P. J. Fowler A. M. Pavloff B. M. Lester and C. L. Zirkle J. Medicin. Chem. 1971 14 179. 41 F. J. Villani P. J. L. Daniels C. A. Ellis T. A. Mann K.-C. Wang and E. A. Wefer J. Medicin. Chem. 1972 15 750. 42 A. Sabbah La Vie Mkdicale 1969 41 5401; B. Sigal and M. Herblot Gar. Med. Fr. 1970 77 364. 43 F. J. Villani P. J. L. Daniels C. A. Ellis T. A. Mann and K.-C. Wang J. Heterocyclic Chem. 1971 8 73. 44 J. L. Archibald P.Fairbrother and J. L. Jackson J. Medicin. Chem. 1974 17 739. 486 A.F. casy organ tissues (electric eel and ray)45 and brain46 that direct study of the receptors themselves may eventually be possible but at present any receptor information must largely be inferred from ligand characteristics.Several groups notably that of Pa~ling,~~ have provided information about the solid-state conformation of a wide range of ACh agonists and antagonists. Torsion angles 72 and 73 are of most value in defining the conformations of ACh and its congeners (43). In most cases 73 values fall in the range 180’ & 36’ (44) which means that the acetyl portion of the molecule is set well away from the quaternary head. The torsion angle relating ‘NMe to OCOMe (72) commonly has a value in the range 73-94’ so the N and 0 functions are more or less synclinal (44). It turns out that most compounds with the molecular feature 0-C-C-N+ where the charged group is quaternary nitrogen or a protonated base and the oxygen function is OH or acyloxy prefer the synclinal N/O arrange- ment in the solid and evidence for a stabilizing electrostatic 0-N+ interaction has been ad~anced.~’ Antiplanar-anticlinal conformations are preferred by the potent agonists carbarnoylcholine (stabilized by hydrogen bonds)” and (+)-trans ACTM” (72 fixed by ring rigidity) and the weakly active thio- and seleno-analogues of AChS2 in which the ether oxygen is replaced by a bulkier and less electronegative atom.N/O conformations are also synclinal for the cyclic muscarinic agents L-( +kmuscarine iodide (45),’ the cis-dioxolan (46),54 and 5-methylfurmethide (47)47 but antiplanar for L-( +)-muscarone (48).” The crystal structures of nicotine and other nicotinic agonists e.g.a-methyl-ACh ” R. D. O’Brien M. E. Eldefrawi and A. T. Eldefrawi Ann. Rev. Pharmacol. 1972,12 19; G. Biesecker Biochemistry 1973 12 4403. 46 H. B. Bosmann J. Biol. Chem. 1972,247 130. *’ R. W. Baker C. H. Chotia P. Pauling and T. J. Petcher Nature 1971,230,439. ‘* M. Sundaralingam Nature 1968 217 85; C. Chotia and P. Pauling ibid. 1968 219 1156. 49 (a) F. G. Canepa and E. F. Mooney Nature 1965,207 78; (b)A. F. Casy M. M. A. Hassan and E. C. Wu J. Phurm. Sci. 1971,60 67. 50 Y.Barrans and J. Clastre 1970 Compt. rend. 1970 270 C 306. ’ C. Chotia and P. Pauling Nature 1970 226 541. 52 E. Shefter and H. G. Mautner Proc. Nut. Acad. Sci. U.S.A. 1969 63 1253. 53 F. Jellinek Acta Cryst. 1957 10 277. ’* P. Pauling and T. J. Petcher Chem. Comm.1969 1258. ” P. Pauling and T. J. Petcher Nature New Biol. 1972 236 112. Histaminic and Cholinergic Agonists and Antagonists + 487 NMe IVLt: M'e (45) (46) (47) (48) lactoylcholine and 1,l-dimethyl-4phenylpiprazine,have torsion-angle features similar to those of most muscarinic agentss6 Conformational studies by n.m.r. (chiefly 1H)49b*s7 and MO theory" are also extensive and by and large complement the results of X-ray diffraction. Thus n.m.r. analyses establish strong preferences for synclinal +N/O conformers in the cases of ACh B-methyl ACh and also carbamoylcholine (see above) as solutes in D,O ;acetylthio-and acetylseleno-choline are chiefly antiplanar in solution while a-methyl ACh (which exists in 2 crystalline forms)'' displays little conformational preference.It is clear that the preferred conformations of ACh and many of its active analogues are those with synclinal N and 0 functions but there is no guarantee that such forms represent the conformation adopted by the agonist at the cholin-ergic receptor especially as barriers to rotation in molecules such as ACh are low. Data on conformationally restrained analogues with onium and acetate functions relatively 'frozen' may thus provide clearer information about the active conformation. This approach has been followed but has the drawback of requiring molecules larger than the parent with reduced receptor affinity a likely consequence. If meaningful conclusions are to be made the mode of action of the analogue must be established (muscarinic nicotinic direct or indirect influ-ence of AChE) and regrettably evidence on this point is often in~omplete.~~" Smissman and co-workers* used trans-decalin as the restraining framework and prepared all four RS-isomers of (49);low orders of muscarinic activity were OCOMe hoCoMe +I -&Mel &Me3 NMe3 H (TIocoMe H (49) (50) (49) 56 C.Chotia and P. Pauling Proc. Nut. Acad. Sci. U.S.A. 1970 65 477 and references there cited. 57 C. C. J. Culvenor and N. S. Ham Chem. Comm. 1966 537; ibid. 1970 1242; R. J. . Cushley and H. G. Mautner Tetrahedron 1970 26 2151 ;P. Partington J. Feeney, and A. S. V. Burgen Mol. Pharmacol. 1972 8 269; T. D. Inch R. A. Chittenden and C. Dean J. Pharm. Pharmacol. 1970,22,956.58 L. B. Kier Mol. Pharmacol. 1967,3,487; B. Pullman,Ph. Courritre,and J. L. Coubeils ibid. 1971,7,397; M. Froimowitzand P. J. Gans J. Amer. Chem. SOC.,1972,94,8020; R. J. Radna D. L. Bevetidge and A. L. Bender ibid. 1973,95 3831. 59 C. Chotia and P. Pauling Chem. Comm. 1969,626. 59 (a) A. F. Casy Progr. Med. Chem. 1975 11 1 and references cited therein. *The untimely death of Professor Smissman in Summer 1974 has deprived medicinal chemistry of one of its leading exponents. 488 A. F. Casy shown by the NaOa(50) and NaOeisomers the former being the more potent.60 There is evidence that (50) maintains an anticlinal-antiplanar conformation in both solute ('H n.m.r.)60 and solid (X-ray)61 states. Analogues of (50) with methyl substituents at C-2and/or C-3 all had significant ACh-like activity.62 An example of the synthetic procedures used is shown (Scheme 7).The 4-t-butyl derivatives +M~I e &:ye meM OCOh rriins opening Me N3 NMe3 Reagents i NaN,; ii H,-PtO,; iii CH,O-H,; iv MeCOCl; v Me1 Scheme 7 (51) represent a quartet similar to (49),and of these the NaOaisomer again proved the more potent (but weak) muscarinic agent.63 More satisfying results in terms of'the potency were obtained with the cyclopropyl analogues (52).64 The (+)-OCOMe Bu' (51) trans-isomer (ACTM) equalled or surpassed ACh itself in two test systems while the (-)-enantiomer and RS-cis-form were feeble or inactive. From an X-ray crystallographic study ( +)-trans-ACTM has a 1S,2S configuration [as ( +)/3-methyl ACh and ( +)-muscarinel and an NCCO torsion angle of 137"(anticlinal)?l trans-ACTM was made from the trans-amide (54) itself derived from the ester (53) formed along with some cis-isomer during the copper-catalysed reaction between ethyl diazoacetate and 2-vinyloxytetrahydropyran (THP used as a protective 6o E.E. Smissman W. L. Nelson J. B. Lapidus and J. L. Day J. Medicin. Chem. 1966 9 458. 61 E. Shefter and E. E. Smissman J. Pharm. Sci. 1971,60 1364. 62 E. E. Smissman and G. R. Parker J. Medicin. Chem. 1973 16 23. 63 A. F. Casy E. S. C. Wu and B. D. Whelton Canad. J. Chem. 1972 50 3998; D. F. Biggs I. Chu E. S. C. Wu and A. F. Casy J. Pharm. Pharmacol. 1973,25 153P. 6* P. D. Armstrong J. G. Cannon and J. P. Long Nature 1968 220 65; C.Y. Chiou J. P. Long J. G. Cannon and P. D. Armstrong J. Pharmacol. Exp. Ther. 1969 166 243. 65 P. D. Armstrong and J. G. Cannon J. Medicin. Chem. 1970 13 1037. Histaminic and Cholinergic Agonists and Antagonists Other frameworks used to prepare rigid ACh analogues include bicyclo[2,2,2]- octane [anticlinal form (55) has significant muscarinic activity]66 and 2- and 3-aminobornanes (56).67Much chemical detail on the bornanes has been reported but little pharmacology. A A = OCOMe B = +NMe and vice versa OCOMe (55) (56) Analogues of muscarine (45)also provide evidence about the active conforma- tion of ACh. The difficult access of (+)-muscarine from natural sources (Fly Agaric) has been overcome by a convenient synthesis from 2-acetamido-4,5- dihydroxyhexanoic acid (from N-acetylcrotonylglycine and performic acid).68 The diol(57) was resolved enzymatically at the or-carbon and the resultant a-L(+)-amino-acid diol (58) was deaminated with nitrous acid-methanol to give the tetrahydrofuran (59) with retention of configuration at C-2.The reaction of (59) MeCH-CH-CH2eHC0,H MeCH-CHCH,CHC02 -II I II I OH OH NHCOMe OH OH +NH3 (57) selectively de-acetylated by hog-kidney acylase (59) (60) with dimethylamine gave two amides which were separated and transformed into L-( +)-muscarine (45) and L( +)-allomuscarine (60) respectively. The RS-deoxy- analogue of muscarine (45; cyclic 0 replaced by CH,) is reported69 to be 5-10 times more potent a spasmogen on guinea-pig ileum than the parent racemic mixture.Other analogues of (45) include 7-oxabicyclo[2,3,l]heptanes (61)" and of special importance the 1,3-dioxolan (62) which is closely related sterically to b6 W. L. Nelson and R. S. Wilson J. Medicin. Chem. 1971 14 169; A. H. Beckett and R. E. Reid Tetrahedron 1972 28 5555. 67 R. A. Chittenden and G. H. Cooper J. Chem. SOC.(0,1970 49; T. Ahmad M. N. Anwar M. Martin-Smith R. T. Parfitt and G. A. Smail ibid. 1971 847; A. H. Beckett N. T. Lan and A. Q. Khoklar J. Pharm. Pharmacol. 1971 23 528; G. H. Cooper D. M. Green R. L. Rickard and P. B. Thompson ibid. 1971 23 662. '* J. Whiting Y.-K. Au-Young and B. Belleau Cunad. J. Chem. 1972 50 3322. 69 K. G. R. Sundelin R. A. Wiley R. S. Givens and D. R. Rademacher J. Medicin. Chem.1973 16 235. 70 W. L. Nelson D. R. Allen and F. F. Vincenzi J. Medicin. Chem. 1971 14 698. 490 A. F. Cusy the natural agent.71 Variants of (62) restricted about the C-4-C-6 bond have been made e.g. (63)-(66).’* The spiran (66),with an anticlinal NCCO torsion angle was the most potent [about 0.1 x potency of parent (62)]; in contrast the open form (67) in which the 4-methyl group opposes an anticlinal22 was a feeble agonist. (66) (67) Several proposals about the conformational requirements of cholinergic receptors based on the one hand on preferred agonist conformation^^^.'^ and on the other on multiple modes of ligand binding,74 have been advanced and the. matter remains controversial. Turning to anticholinergic agents and especially the question of whether or not muscarinic agonists and antagonists occupy common receptor~,~~.’’ more evidence has appeared that underplays the influence of the nitrogen moiety in antagonist molecules.Earlier it was found that whereas the agonist properties of P-methyl ACh depend critically on the configuration of the p-centre enantio- mers of analogues that are antagonists show only modest differences in potency [see (68)]. In contrast the configuration of the acyloxy centre (when chiral) has a pronounced influence on blocking activity [see (69)].’6 Analogues of the ” B. Belleau and J. Puranen J. Medicin. Chem. 1963 6 325. 72 M. May and D. J. Triggle J. Pharm. Sci. 1968 57 51 1 ; D. R. Garrison M. May H. F. Ridley and D. J. Triggle J. Medicin.Chem. 1969 12 130; H. F. Ridley S. S. Chatterjee J. F. Moran and D. J. Triggle ibid. p. 931. 73 L. B. Kier in ‘Fundamental Concepts in Drug-Receptor Interactions’ ed. J. F. Danielli J. F. Moran and D. J. Triggle Academic Press New York 1970 p. 15; L. B. Kier ‘Molecular Orbital Theory in Drug Research’ Academic Press New York 1971. 74 D. J. Triggle ‘Neurotransmitter-Receptor Interactions’ Academic Press London 1971; J. F. Moran and D. J. Triggle in ‘Cholinergic Ligand Interactions’ ed. D. J. Triggle J. F. Moran and E. A. Barnard Academic Press New York 1971 p. 119. 7s E. J. Ariens and A. M. Simonis Ann. New York Acad. Sci.,1967 144 842; R.W. Brimblecombe D. Green and T. D. Inch J. Pharm. Pharmacol. 1970 22 951. 76 B. W. J. Ellenbroek R. J. F. Nivard J.M. Van Rossum and E. J. Ariens J. Pharm. Pharrnacol. 1965 17 393. Histaminic and Chotinergic Agonists and Antagonists 49 1 X Affinity ratio SIR + Me,NCH,CHMeOCOX Me 320 Ph,CH 0.2 (48) Ph,C(OH) 0.8 X AfJinity ratio ( -)/( +) + Me,NCH,CH20COX Ph(Me)C(CH,OH) 300 Ph(C,Hll)CH 100 (69) 1,3-dioxolan(62)with antagonistic properties have now been made with 2-methyl replaced by a bulky aromatic group. In especially painstaking work," all eight isomers of the dioxolan (71) were isolated (see Scheme 8) and tested ;configura-/-TcH2NMe3 Ph CH,OTs I I I .. . I1 C,H,,C-CHO + CHOH + rCCH20Ts I I OH CHzOH "Yo -"YO R or S R or S Ph/C\OH Ph/\\OH (70) I I (separate c and t) (71) Reagents i HNMe,; ii Me1 Scheme 8 tional priorities for activity were in the order (i) an R-benzylic centre ;(ii) a 2-S-centre (adjacent to benzylic carbon); and (iii) a 4-R-centre (adjacent to CH,&Me,).Similar conclusions of stereochemical influence were reached from studies on the dioxolans (72);thus R- and S-(72; Ar = Ph) were almost equally p~tent.~' rCCHzkMe3 00 PhCHZ, Y Ar Ph (72) Ar = PhorC,H, (73) Benzetimide (73),an anticholinergic agent as active as atropine,79 also contains an asymmetric benzylic centre and its dextro-isomer (dexetimide) is over 1000 times as active as the laevo-form from PA, values (guinea-pig ileum)." " R. W. Brimblecombe T. D. Inch J. Wetherell and N. Williams J. Pharm. Pharmacol. 1971 23 649. R. W. Brimblecombe and T. D. Inch J.Pharm. Pharmacol. 1970,22,881; K. J. Chang R. C. Deth and D. J. Triggle J. Medicin. Chem. 1972 15 243. 79 B. Hermans P. Van Daele C. Van de Westeringh C. Van der Eycken J. Boey J. Dockx and P. A. J. Janssen J. Medicin. Chem. 1968 11 797. P. A. J. Janssen C. J. E. Niemegeers K. H. L. Schellekens P. Demoen F.M. Lenaerts J. M. van Nueten I. van Wijngaarden and J. Brugmans Arzneim.-Forsch. 1971 21 1365. 492 A. F. Casy Dexetimide has a configuration (S from X-ray study)81 that is comparable with that of hexahydrobenzilates such as (74) if the sequence of aromatic hydrogen- + bonding donor and C..-Nfeatures be compared. The binding of R-and S-[3H]benzetimide to subcellular fractions of rat brain rich in membranes is stereospecific (R-isomer displaced by atropine but not by S-benzetimide or tubocurarine) and appears to involve muscarinic receptors.82 This use of dexetimide provides a further example of the use of optically active ligands for receptor localization (cf.work on opiate receptor^).^^ log K, MeND OCOCPh(C6H,,)OH HCI 10.92 Me1 11.08 R-(74) Bar10w~~ has emphasized the importance of optical purity in assessing the significance of enantiomeric potency ratios in anticholinergic drugs (considera- tions which apply equally well to other areas of medicinal chemistry). If isomers are only 95 % resolved the highest observed stereospecificity theoretically possible would be 19 while values of 100 and 1000 correspond to optical purities of 99.01 and 99.90 respectively. Indeed biological assays are capable of assessing purity more precisely than any physical technique (e.g.n.m.r.) presently available.R- and S-Hexahydrobenzilic acids may be obtained in high states of optical purity from arabin~se,~~ and a study of potency ratios of their esters with 2-dimethyl- aminoethanol and 1-methyl-4-piperidinol has been made by whole-animal and isolated-tissue methods.86 A divergence between in viuo and in uitro ratios was noted which became greater the higher the affinity constant of the ester of R-configuration and it was proposed that there is a minimum dose below which maximum anticholinergic effects cannot be obtained in uiuo. Thus with excep- tionally potent agents such as (74) the advantages of a high affinity for the receptor are offset by the need to administer enough drug to allow for its uptake at sites of loss.mocox N (75) a; X = CHPh b; X = C(OH)Ph c; X = Me Anticholinergics based on 3-quinuclidinol(75a,b) are unusual in having poten- cies that are markedly dependent on the configuration of the amino-alcohol 81 A. L. Spek and A. F. Peerdeman Nature 1971 232 575. 82 w. Soudijn I. van Wijngaarden and E. J. Ariens European J. Pharmacol. 1973 24 43; A. J. Beld and E. J. Ariens ibid. 1974 25 203. 83 C. B. Pert and S. H. Snyder Proc. Nut. Acad. Sci. U.S.A. 1973 70 2243. 84 R. B. Barlow J. Pharm. Pharmacol. 1971 23 90; R. B. Barlow F. M. Franks and J. D. M. Pearson ibid. 1972 24 753. 85 T. D. Inch R. V. Ley and P. Rich J. Chem. Soc. (0,1968 1693. 86 R. W. Brimblecombe D.M. Green T. D. Inch and P. B. J. Thompson J. Pharm. Pharmacol. 1971 23 745; T. D. Inch D. M. Green and P. B. J. Thompson ibid. 1973 25 359. Histaminic and Cholinergic Agonists and Antagonists centre.87 It is strange as well that the protonated base rather than the metho- salt form of the agonist (7%) is distinctly the more active form,** a fact that may be related to the rigid nature of the quaternary head compared with the situation in ACh and its close relatives. The more potent enantiomer of (75c) methiodide [R-(-); configuration by X-ray analysis]89 has an inverse steric relationship to agonists such as S-P-methyl ACh and (+)-muscarine. With hydrochlorides (75c) however receptor selectivity is reversed with the S-isomer the more potent agent (S :R ratio at least 13 :1); N-methylation drastically reduces the activity of the S-isomer (75c) (1100-fold) but has little effect on that of the R-isomer.” Finally a few developments in neuromuscular blocking agents are mentioned.In 1970 (+)-tubocurarine long accepted as a bisquaternary compound was shown by a ‘H n.m.r. study to possess in fact one quaternary and one protonated tertiary nitrogen centre ;” only three N-methyl resonance signals could be assigned one of which moved upfield when the pH was raised (typical of de- protonation of an +NHMe gro~p).~’ Cleavage experiments established the location of these centres and the structure (76) has now been confirmed by an X-ray diffraction The NN-dimethyl quaternary salt corresponding with (76) is actually (+)-chondrocurarine chloride earlier held to differ from tubocurarine in the location of a methyl ester residue.A solute conformation of (76) has been proposed based on a variable-temperature ‘H n.m.r. The neuromuscular blocking potency of ( +)-isotubocurarine (76; quaternary and tertiary N features interchanged) is almost twice that of (+)-tubocurarine in the cat.94a (76) 87 L. H. Sternbach and S. Kaiser J. Amer. Chem. SOC.,1952,74 2215 2219; A. Meyer- hoffer J. Medicin. Chem. 1972 15 994. 88 M. D. Mashkovsky in Proc. First International Pharmacological Meeting Stockholm 1961 Vol. 7 Pergamon Oxford 1962 p. 359; M. D. Mashkovsky and C. A. Zaitseva Arzneim.-Forsch. 1968 18 320. 89 R. W. Baker and P. Pauling J.C.S.Perkin IZ 1972 2340. 90 R. B. Barlow and A. F. Casy Mol. Pharmacol. in press. 91 A. J. Everett L. A. Lowe and S. Wilkinson Chem. Comm. 1970 1020. 92 A. F. Casy ‘PMR Spectroscopy in Medicinal and Biological Chemistry’ Academic Press London 1971 p. 58. 93 P. W. Codding and M. N. G. James J.C.S. Chem. Comm. 1972 1174; Acta Cryst. 1973 B29 935. 94 R. S. Egan R. S. Stanaszek and D. E. Williamson J.C.S. Perkin 11 1973 716. 940 T. 0.Soine and J. Naghaway J. Pharm. Sci. 1974,63 1643. 494 A. F. Casy Recent developments in novel curare-like agents have centred on steroid derivatives prompted perhaps by disclosure of the neuromuscular blocking activity of alkaloids related to connesine and of mal~uktine.~~.~~ The steroid skeleton also allows the synthesis of agents with varying but in each instance fixed +N to +N distance.In a series of Sa-and SP-androstanes (77) potency was most sensitive to the configuration of the 3-substituent (p-gave optimal a~tivity).~’ in Sa-derivatives a 3/I-quaternary nitrogen atom was essential but a /I-quaternary nitrogen atom could be replaced by a P-tertiary nitrogen at C-17 without change in potency.98 No critical relationship between inter-onium distance and potency was found-the +N to +N distance was close to 10% for both potent and weakly active derivatives. In the solid state the +N to +N distance for tubocurarine dichloride is 8.97 A,93 significantly shorter than that generally accepted as essential for neuromuscular blockade.99 The distance increases to 10.7 A when both nitrogens are quaternary as in 00”-trimethyltubocurarine di-iodide.loo Combination of knowledge about the monoquaternary compound (78) (1/16 x Br 0 (78) tubocurarine) lo’ which includes an ACh-like moiety in ring A and diquaternary salts like malouktine led to the synthesis of pancuronium (80)(+Nto +Ndistance 11.08 A) now established as a potent clinical agent of medium duration of acti~n.’~**’~~ For high potency two nitrogen atoms are needed at least one of which should be quaternary [the dihydrochloride form of (80) is inactive] but there are no data on the 2-N9 16-N‘ type of salt.Potency falls when the size of the quaternary groups is reduced and the diol corresponding with (80) is only about a tenth as active as the parent.With monoacetates deacetylation at 95 A. Quevauviller and F. Laine Ann. pharm. franc. 1960 18 678. 96 D Busfield K. J. Child A. J. Clarke B. Davies and M. G. Dodds Brit. J. Pharmacof. 1968 33 609. 97 D. G. Bamford D. F. Briggs M. Davis and E. W. Parnell Brit. J. Pharmacol, 1967 30,194. 98 D. G. Bamford D. F. Briggs M. Davis and E. W. Parnell J. Pharm. Pharmacof. 1971 23 595. 99 M. Martin-Smith in ‘Drug Design’ ed. E. J. Ariens Academic Press New York and London 1971 Vol. 11 p. 503. loo H. M. Sobell T. D. Sakore S. S. Tavale F. G. Canepa P. Pauling and T. J. Petcher Proc. Nat. Acad. Sci. U.S.A. 1972 69 2212. J. J. Lewis M. Martin-Smith T. C. Muir and H. H. Ross J. Pharm. Pharmacol. 1964 19 502. lo* W. R. Buckett C. E.B. Marjoribanks F. A. Marwick and M. B. Morton Brit. J. Pharmacof. 1968 32 67 1. ‘03 W. R. Buckett C. L. Hewett and D. S.Savage J. Medicin. Chem. 1973 16 1116. Histaminic and Cholinergic Agonists and Antagonists C-17 (dacuronium) causes the greater potency fall. In the solid state ring A of pancuronium adopts a skew-boat conformation (avoiding the 1,3-diaxial inter- actions of the chair),Io4 and there is 'H n.m.r. evidence that similar conformations obtain for the Pancuronium is made from the diepoxyandrostane (79)lo5 as shown in Scheme 9. (79) cN@,As 2 J.+ some 1b-epimer Me AcO" H (80) Reagents i NH, H,O; ii NaBH,; iii Ac,O-py; iv Me1 Scheme 9 Bisquaternary steroids of type (81) with an inter-onium separation of 5.8 A had their main action at an autonomic ganglion rather than the neuromuscular function,' O6 a result supporting experience with or,m-bisquaternary alkanes.lo7 (82) Y ED, (paralysing activity in mice) 0.16mg kg-' +NR3 (81) (+)-tubocurarine 0.05mg kg-' Some short-acting competitive neuromuscular blocking agents with the quaternary nitrogens linked by a nitrogen-rich chain have been described e.g.(82) I04 D. S. Savage A. F. Cameron G. Ferguson C. Hannaway and I. R. Mackay J. Chem. SOC.(B) 1971,410. lo' C. L. Hewett and D. S. Savage J. Chem. SOC.(0,1968 1134; A. Hassner and P. Catsoulacos J. Org. Chem. 1967 32 549. '06 I. G. Marshall and M. Martin-Smith European J. Pharmacol. 1972 17 39. lo' R. B. Barlow 'Introduction to Chemical Pharmacology' Methuen London 2nd end.1964 p. 165. 496 A. F. casy with an inter-onium distance of 7.5~.'08*'09The brief action of (82) may be due to its rapid metabolism to 3-methyl-2-phenylimidazo[ 1,2-a]pyridine. Reasonably potent monoquaternary neuromuscular blockers that lack a second basic nitrogen atom are rare and recent examples are carbonyl-functional- ized derivatives of alkyl and cycloalkyl aryl ketones e.g. (83),which is as active as suxamethonium in some species."' The hydrazones (84) and (85) also ap- (83) (84) (85) proach suxamethonium in potency and activity is lost when the position of the trimethylammonium substituent is changed. lo* L. Bolger R. T. Brittain D. Jack M. R. Jackson L. E. Martin J. Mills D. Poynter and M. B. Tyers Nature 1972 238 354.lo9 D. J. Pointer J. B. Wilford and D. C. Bishop Nature 1972 239 332. ' lo D. G. Bamford D. F. Biggs P. Chaplen M. Davis and J. Maconochie Experientia ' '' 1972 28 1069. I. Chu Ph.D. Thesis University of Alberta 1973.

ISSN:0069-3030    

DOI:10.1039/OC9747100477

出版商:RSC    

年代:1974

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18 Peptides and Proteins By P. M. HARDY Department of Chemistry University of Exeter Stocker Road Exeter EX4 400 1 Introduction Peptides were last covered in Annual Reports for 1969 as part of a chapter entitled 'Amino-acids and Peptides'. The aim of the present Report is to outline the advances made in the subsequent five years in the field of peptides with a very selective discussion of some aspects of proteins. Emphasis is laid principally on methods of structure determination and synthesis rather than on the sequences elucidated or the syntheses achieved. The space available has precluded any coverage of conformational studies cyclic peptides or peptides containing non-protein amino-acids. 2 Structure Elucidation Methods.-Enzymic Cleavage. Several novel enzymes have been characterized whose specificities are of interest to the chemist involved in sequencing work.Two of these pyrrolidonyl peptidase' and dipeptidyl aminopeptidase,2 have begun to be used in structural work. The former removes pyrrolidonecarboxylic acid (pyroglutamic acid) from the N-terminus of peptides thereby revealing a free amino-group and unblocking this end of the molecule for subsequent Edman degradation.' The latter cleaves peptides in a stepwise fashion from the N-terminus liberating dipeptides. If the amino-terminal amino-acid residue is removed by one cycle of the Edman degradation subsequent treatment with this enzyme gives a series of overlapping dipeptides. Comparison of the two series of dipeptide sequences should enable the original sequence to be established.The N-terminal pentapeptide segment of promelittin was established in this way.3 Ambiguities however can obviously arise with certain sequences. Application to the A-chain of insulin (21 residues) gave two possible primary ~tructures,~ indicating that this interesting approach may be limited to peptides of very moderate size. ' A. Szewczuk and J. Kwiatowska European J. Biochem. 1970 15 92. R. A. Valyulis and V. M. Stepanov Biokhimiya 1971 36 866. ' G. Kreil European J. Biochem. 1973 33 558. R. J. Rowlands and H. Lindley Biochem. J. 1972 126 685. 497 498 P.M. Hardy Two proteases as yet untested in sequencing applications have great potential in this field. Myxobacter AL-1 protease I1 attacks peptides or proteins only at the amino-side of lysine residues.It can thus provide peptide fragments which overlap those produced by trypsin which if the lysine side-chains are appro- priately masked are cleaved only at arginine residues.' A protease from Staphylo-coccus aureus specifically cleaves at the C-terminal side of aspartyl and glutamyl peptide bonds. Under certain conditions fission can be limited to glutamyl bonds. This enzyme may incidentally assist in the assignment of glutamine and asparagine residues to which it is not sensitive.6 A peptidoglutaminase from B. circulans has been isolated which attacks only the y-amide bonds of peptide- bound glutamine;' this may prove useful in combination with the S. aureus protease. Edman Degradation.Peptide sequencing by the automated Edman method first described in 1967,8 has rapidly gained ground at the expense of the manual technique since commercial machines for this purpose 'sequenators' became available in 1969. However the chemist cannot be entirely replaced by the technician. In general only the first thirty to fifty N-terminal amino-acid residues are directly accessible using this approach. In favourable cases more can be achieved e.g. the first 66 of the 88 residues of bovine parathyroid hormone could be identified.' The complete sequence of P-lactoglobulin has been deter- mined by sequence analysis of the intact protein and its tryptic peptides but this work involved modification of the classical Edman routine." The chief limitation of the automated method is its reliance on the insolubility of the residual peptide during the extraction of the liberated thiazolinones; short peptides are too soluble to be successfully retained during this procedure.Several ways of overcoming this limitation have been found to be useful. The use of buffers based on dimethylbenzylamine enables the less polar solvent benzene to replace ethyl acetate in the extraction step as its salts with organic acids are now sufficiently soluble in the hydrocarbon.' Alternatively the peptide itself can be rendered more hydrophilic. Tryptic peptides which remain the fragments most widely used in sequencing have either arginine or lysine at their C-terminus. The polarity of the arginine side-chain confers more resis- tance to extraction than does that of lysine.However if the first step of the degradation is carried out using a highly sulphonated isothiocyanate such as (1) or (2) the E-amino-group of the lysine can be modified to confer suitable ex- traction resistance even on a peptide as short as five amino-acid residues.I2 M. Wingard G. Matsueda and R. S. Wolfe J. Bacteriol. 1972 112 940. J. Houmard and G. R. Drapeau Proc. Nut. Acad. Sci. U.S.A. 1972 69 3506. ' M. Kikuchi H. Hayashida E. Nakano and K. Sakaguchi Biochemistry 1971,10 1222. P. Edman and C. Begg European J. Biochem. 1967 1 80. M. Tanaka M. Haniu G. Matsueda K. T. Yasunobu S. Mayhew and V. Massey Biochemisrry 197 1 10 3041. lo G. Braunitzer R. Chen B. Schrank and A. Stangl 2.physiol. Chem. 1972,353 832.'I M. A. Hermodson L. H. Ericsson K. Titani H. Neurath and K. A. Walsh Bio-chemistry 1972 11 4493. G. Braunitzer B. Schrank A. Ruhfus S. Peterson and U. Peterson 2.physiol. Chem. 1971 352 1730; G. Braunitzer B. Schrank S. Peterson and U. Peterson ibid. 1973 354 1563. Peptides and Proteins Aminoethylation of peptides with C-terminal cysteine residues effects a similar change in properties,' while peptides which lack suitably placed side-chain functional groups can be after their initial conversion into an N-phenylthio- carbamyl derivative coupled through their C-terminal carboxy-group to amino- methanesulphonic acid ' or 2-aminonaphthalene- 1,5-disulphonic acid. ' If a water-soluble carbodi-imide is used to effect reaction at pH 4 amino-groups other than that of the reagent are protected from reaction by protonation.Side-chain carboxy-groups however will always be tagged by additional solubilizing groups. Another successful strategy involves total insolubilization of the peptide by attaching its C-terminus to a polymer. An automated apparatus using this principle has been described.16 However while attaching the C-terminus to the resin amino-groups in side-chains can also form links to the polymer. Thus on degradation the thiazolinones corresponding to aspartic and glutamic acid residues may be missing. A more serious problem which occurs with aspartic acid is formation of the cyclic imide after activation of the P-carboxy-group during coupling to the resin. Edman degradation is then completely blocked when the first aspartimide residue is reached.16 This can be avoided either by coupling a C-terminal lysine residue to the resin through its &-amino-group using p-phenylene di-isothiocyanate' or by use of N-ethyl-N'-(3-dimethyl- aminopropy1)carbodi-imide'* as the coupling reagent.The C-terminal carboxy- group forms an oxazolinone which is susceptible to aminolysis by a resin-bound amino-group but the side-chain carboxy-groups are deactivated by isomerization to N-acyl-ureas. This method has been successfully tested on 20-cycle runs; the modified derivatives of aspartic and glutamic acids liberated can be hydro- . lysed to the parent phenylthiohydantoin for recognition.'8 The only specific chemical cleavage method widely used in protein-fragmenta- tion studies remains the cyanogen-bromide-induced fission at methionine C- terminal homoserine being generated.Homoserine lactone readily prepared from the parent acid by treatment with trifluoroacetic acid has been used to attach cyanogen-bromide-generated peptides to solid-phase supports as it will l3 J. K. Inman J. E. Hannon and E. Appella Biochim. Biophys. Acta 1972 46 2075. G. Crombie J. A. Foster and C. Franzblau Biochem. Biophys. Res. Comm. 1973,52 1228. J. A. Foster E. Bruenger C. L. Hu K. Albertson and C. Franzblau Biochem. Biophys. Res. Comm. 1973 53 70. l6 R. A. Laurson European J. Biochem. 1971 20 89. R. A. Laurson M. J. Horn and A. G. Bonner F.E.B.S. Letters 1972 21 67. I8 A. Previero J. Derancourt and M. A.Coletti-Previero F.E.B.S. Letters 1973 33 135. 500 P. M. Hardy acylate resin amino-groups. The peptide amino-groups need not be blocked if the resin amino-groups are present in large excess. A fragment of rabbit- muscle actin containing 13 residues which had resisted conventional methods of primary structure determination was successfully sequenced using this method. l9 The second and largest protein to have been totally sequenced by the automated Edman method is calf-skin collagen al-CB7. Correct placing of the 268 residues required studies on 19 peptides prepared by tryptic chymotryptic and thermo- lytic cleavage as well as on the intact protein.” It is clear that with large proteins although the use of sequenators may speed up realization of the desired objective the sequencing of the amino-acids is still in general dependent on the attainment of suitable peptides which provide overlaps of breakage points.Mass Spectrometry. Although the sequencing of peptides by mass-spectrometric means is in general only successful on compounds containing up to ten amino- acid residues and offers no advantages in economy of material over conventional sequencing methods work over the past few years has succeeded in considerably refining it as an analytical tool. It offers a single operation (as opposed to a repetitive subtractive method like the Edman degradation) although in general raw peptides must be derivatized prior to insertion into the instrument for analysis. End-group protection provides few problems but permethylation of the amide NH groups (to destroy their tendency to reduce volatility by formation of hydrogen bonds) can also attack a-CH groups and more seriously the side- chains of cysteine methionine and histidine.This opposes the effects of N-methylation because salts are formed and it also complicates the spectra. These side-reactions can be circumvented by conversion of the susceptible amino-acids into less sensitive structures. Methionine can be protected as its sulphoxide,21 and cysteine desulphurized with Raney nickel to alanine (normally using deuterium-saturated catalyst to enable the product to be distinguished from other alanine residues).22 Histidine may be cleaved with diethyl pyro- carbonate to a 1,2-bis(ethoxycarbonylamido)ethylenederivative (Scheme l) I 1 OH C0,Et C0,Et Reagents i diethyl pyrocarbonate; ii H,O Scheme 1 19 M.J. Horn and R. A. Laurson F.E.B.S. Letters 1973 36 285. 20 P. P. Fietzek F. W. Rexrodt K. E. Hopper and K. Kiihn European J. Biochem. 1973 38 396. 21 P. Roepstorff K. Norris S. Severinson and K. Brunfeldt F.E.B.S. Letters 1970 9 235. 21 Yu. A. Ovchinnikov A. A. Kiryushin V. A. Gorlenko and B. V. Rozynov Zhur. obshchei Khim. 1971,41 660. Peprides und Proteins 50 1 which does not form a quaternary salt.23 Arginine residues must also be modified otherwise the mass spectrum contains sequence information only from the N-terminus to the amino-acid preceding the first arginine residue. Hydrazine may be used to convert arginine into ~rnithine,'~ or else P-diketones to incorporate the guanido-group into an N6-pyrimidylornithine derivative." However proper treatment of any peptide presupposes knowledge of its amino-acid content and detracts from the simplicity of the method.A re-evaluation of permethylation in which peptides were treated with CD,I for a short period (in the presence of methylsulphinyl carbanion) and then CH,I for a longer period showed that the optimum time for methylation has in general been over-estimated as only deuteriomethyl groups were incorporated. Exposure for one minute is sufficient in most cases to give complete substitution of amide NH groups and under these conditions side-chains are largely unaffe~ted.~~~~~ If this short treatment is used arginine is the only residue requiring prior modi- ficat ion.Alternative methods to conventional electron-impact ionization (El) show promise for use in structure determination. Application of a field desorption (FD) technique gives a great reduction in fragmentation due to thermal de- composition and unprotected peptides show a molecular or a quasi-molecular (M + 1) ion. Arginine does not need to be protected ; even a nonapeptide con- taining an arginine residue at each end gives an intense molecular ion. Spectra can be obtained with as little as 10 ng adsorbed on the field anode.27 The use of chemical ionization (CI) allows the mass spectra of free oligopeptides to be obtained with sample temperatures at least 150 "Cbelow those required for EL The tetrapeptide Leu-Leu-Val-Tyr for instance gives an abundant (M + 1)' peak using CI at 150°C but none below 340°C with EI.The mass spectra obtained by CI also contain abundant sequencing peaks ; FD seems less satis- factory in this respect.28 The sequencing peaks produced by CI are also of relatively uniform intensity allowing the use of a smaller sample ;penta-alanine for example needs a 200 nmol sample for EI but only 2 nmol for CI.29 One facet of mass spectrometric sequencing that is being developed is the analysis of peptide mixtures. Here this technique may have clear advantages over stepwise degradation. Experiments with model peptides have established the applicability of this approach to sequencing enzymic digests of proteins which have simply been subjected to gel filtration to ensure that the material loaded into the instrument contains only peptides with ten residues or less.23 J. F. G. Vliegenthart and L. Dorland Biochem. J. 1970 117 31P. '' H. R. Morris R. J. Dickinson and D. H. Williams Biochem. Biophys. Res. Comm. 1973 51 247. 25 P. A. Leclercq L. C. Smith and M. M. Desiderio Biochem. Biophys. Res. Comm. 1971 45 937. 26 H. R. Morris F.E.B.S. Letters 1972 22 257. 27 H. U. Winkler and H.-D. Beckey Org. Muss. Spectrometry 1972 6 655; Biochern. Biophys. Res. Comm. 1972 46 391. 2B M. A. Baldwin and F. W. McLafferty Org. Mass. Spectrometry 1973 7 1353. 29 W. R. Gray L. H. Wojcik and J. H. Futrell Biochem. Biophys. Res. Comm. 1970 41 1111. 502 P.M. Hardy Larger peptides must be digested further with other enzymes before attempting ~equencing.~' Using a computer-assisted high-resolution instrument a mixture of four components (Gly Gly-Ala-Leu Val-Gly-Gly and MePhe-Gly) was correctly ~equenced.~ However such expensive and sophisticated hardware is not essential.A mixture of three rather larger peptides from a tryptic digest of pepsin (Val-Gly-Leu-Ala-Pro-Val-Ala, Ala-Asn- Asn-Lys and Gln-Tyr-Tyr-Thr- Val-Phe-Asp-Arg) was apart from the Asp-Arg terminus correctly sequenced using a relatively low-resolution in~trument.~' Without prior knowledge of the sequence as determined by classical methodology eighty per cent of the sequence of the 249-residue enzyme ribitol dehydrogenase from Klebsiellu uerogenes has been determined by m.s.of partially fractionated peptide mixtures.32 Partial- vaporization experiments are essential for mixture work whatever the instrument the change in relative abundance of peaks at varying temperatures serving to distinguish which peaks arise from the same oligopeptide. Sequence ambiguities may arise however due to the formation of 'non-sequence' ion peaks; these may usually be resolved by comparing the spectra of the acetylated and per- methylated peptides with those of the deuterio-acetylated and perdeuterio- methylated mixture. 33 The amide bonds of peptides are not essential to m.s. sequencing. The amines produced by reduction of the carbonyl groups of the peptide bonds show suitable sequencing ions and can be readily separated by g.1.c.Such an approach has been utilized in a computer-assisted g.1.c.-m.s. determination of the structure of a 20-residue fragment from the C-terminus of actin which was liberated by cleavage with cyanogen bromide. A partial acid hydrolysate of the peptide was esterified acetylated and then reduced with lithium aluminium hydride. The resulting polyamino-alcohols were 0-trimethylsilylated separated by g.l.c. and analysed by m.s. Twenty-seven peptides none larger than a tetrapeptide were identified. The single histidine-containing peptide required direct insertion into the ion source before a suitable spectrum could be obtained. The fragment peptides could be fitted together in two ways but identification of tryptophan as being N-terminal resolved the ambiguity.Digestion of the intact peptide with trypsin cleaved it at the single lysine residue exposing glutamine as the new N-terminus and hence establishing the position of the sole amidated side-chain carboxy- The same structure was deduced independently from a conventional sequencing Structures Determined.-The past few years have proved a particularly fruitful period for the isolation and determination of the primary structure of a variety 30 H. R. Morris D. H. Williams and R. P. Ambler Biochem. J. 1971 125 189. 31 F. W. McLafferty R. Venkataraghavan and P. Irving Biochem. Biophys. Res. Comm. 1970 39 274. 32 H. R. Morris D. H. Williams G. G. Midwinter and B. S. Hartley Biochem. J. 1974 141 701. 33 H. W. Wipf P. Irving M. McCamish R. Venkataraghavan and F.W. McLafferty J. Amer. Chem. SOC.,1973,95 3369. 34 H. Nau J. A. Kelley and K. Biemann J. Amer. Chem. SOC.,1973,95 7162. 35 M. Elzinga and J. H. Collins Cold Spring Harbor Symp. Quant. Biol. 1973 37 1. Peptides and Proteins 503 of peptide and protein hormones particularly those secreted by the pituitary gland. Among the proteins of medical interest so characterized are human growth hormone (HGH),36,38 ovine pr~lactin,~’ human placental lactogen (HPL),38 and ox parathyroid hormone.39 HGH and HPL are identical over 85% of their sequences. Porcine hypothalami have yielded a decapeptide (3) that controls the secretion of growth hormone from the anterior pituitary and the same source Val-His-Leu-Ser- Ala-Gly-Glu-Lys-Glu- Ala 12 3 4 5 6 7 8 9 10 (3) Glp-His-Trp-Ser-Tyr-GI y-Leu-Arg-Pro-Gly-NH 12 3 4 5 6 7 8 9 10 has yielded another peptide (4)of this size which stimulates the release of both luteinizing hormone (LH) and follicle-stimulating hormone.40 Curiously enough a tetrapeptide analogue of this latter peptide (Glp-Tyr-Arg-Trp-NH,) in which the amino-acids are scrambled from their natural order shows some LH-releasing power whereas its isomer Glp-Trp-Tyr-Arg-NH in which the residues are in the same relative order as in the decapeptide parent is inactive.41 The elusive substance P first described in brain extracts as long ago as 1931 has at last been isolated in a pure form from bovine hypothalami and its sequence (5)determined.42 Four of its five C-terminal amino-acids are identical with those Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 1 2 3 4 5 6 7 8 9 10 11 (5) of physalaemin (from amphibian skin) and eledoisin (from cephalopod salivary glands) ;all three compounds stimulate smooth muscle and exert a hypotensive effect.Two peptides which inhibit the release ofgrowth hormone and melanocyte- stimulating hormone from the pituitary have been identified as the pentapeptide 36 C. H. Li and J. S. Dixon Arch. Biochem. Biophys. 1971 146 233. 37 C. H. Li J. S. Dixon T.-B. Lo K. D. Schmidt and Y. A. Pankov Arch. Biochem. Biophys. 1970 141 705. 38 H. D. Niall Nature New Bid 1971 230 90. 39 H. B. Brewer and R. Ronan Proc. Nut. Acad. Sci. U.S.A. 1970,67 1862; H. D. Niall H. D. Keutmann R. Sauer M. Hogan B.Dawson G. Aurbach and J. Potts Z. physiol. Chem. 1971 351 1506. 40 A. B. Schally Y. Baba R. M. G. Nair and C. D. Bennett J. Bid. Cfiem. 1971 246 6647. 4’ J.-K. Chang H. Sievertsson C. Bogentoft B. L. Currie K. Folkers and C. Y. Bowers Biochem. Biophys. Res. Comm. 1971,44 409. 42 M. M. Chang S. E. Leeman and H. D. Niall Nurure New Biol. 1971 232 86. 504 P.M. Hardy Y 'i' Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-S~r-Cys 12 3 4 5 6 7 8 91011121314 (6) Pro-His-Phe-Arg-Gly-NH 43 and the disulphide-bridged tetradecapeptide (6) re~pectively.~~ The latter has been named somatostatin. The sinus gland near the eye of crustaceans resembles in function the hypo- thalamus of vertebrates. The blanching hormone of the shrimp Pandalus borealis which concentrates the red-pigmented areas has been isolated from the eyestalks in sufficient quantity (1OOpg) to be established as an octapeptide (7).4s The controversial compound scotophobin the first compound involved in the Glp-Leu-Asn- Phe-Ser-Pro-Gly-Trp-NH 12345678 Ser-Asp-Asn-Asn-Gln-Gln-Gly-Lys-Ser-Ala-Gln-Gln-Gly-Gly-Tyr-NH~ 1 2 3 4 5 6 7 8 9101112131415 chemical transfer of memory to which a structure has been assigned is thought to be a pentadecapeptide (8).This substance was isolated from the brains of rats trained to avoid the dark and is said to produce the same effect if injected into untrained animals. Material of this sequence synthesized by the solid-phase method gave a product apparently active in the behavioural bi~assay.~' How- ever this work has been criticized in almost all its aspects.47 A hexapeptide (9) 5-oxoPro-Ala-Gly -Tyr-Ser-Ly s 123456 (9) ameletin also isolated from the brains of rats is claimed similarly to transfer a lack of fright-response to noises.48 Hormones controlling the gastrointestinal tract continue to be isolated.That with the rather ungainly title 'gastric inhibitory polypeptide' contains 43 residues and as its name suggests it is a potent inhibitor of gastric acid secretion. It shows some homology to glucagon and rather less to secretin in its 43 R. M. G. Nair A. J. Kostin and A. V. Schally Biochem. Biophys. Res. Comm. 1972 47 1420. 44 R. Burgus N. Ling M. Butcher and R. Guillemin Proc. Nat. Acad. Sci. U.S.A. 1973 70 684; Biochem.Biophys. Res. Comm. 1973,50 127. A5 P. Fernlund Biochem. Biophys. Acta 1974 371 304. 46 G. Ungar M. M. Desiderio and W. Parr Nature 1972 238 198. 47 W. W. Stewart Nature 1972 238 202. 48 H. Lackner and N. Tiemann Naturwiss. 1974 61 217. Peptides and Proteins 505 N-terminal27 residue^."^ The rather smaller peptide motilin (22 residues) which stimulates gastric motor activity has been obtained from hog intestinal mucosa. Its structure is quite distinct from all previously described gastrointestinal pep tide^.^ Since the discovery of the insulin single-peptide-chain precursor proinsulin in 1968 there has been considerable work devoted to the characterization of other prohormones. Precursors of this type are not limited to disulphide-bridged materials.Proglucagon for example contains an extra eight amino-acid residues at its C-terminus,’ while promelittin contains its additional octapeptide unit at the N-termin~s.~ The ingenious use of ‘SC-labelled amino-acids allowed sequencing experiments on the latter compound to be carried out on very small amounts (-50 pg) of material3 Evidence has been obtained for pr~gastrin,~’ proparathyroid hormone,’ and pro~alcitonin,~~ and it seems likely that the formation of prohormones may be a general phenomenon. The most active growth area in primary structure determination over the past five years has been that of the snake-venom toxins; only in the cytochromes has there been a comparable number of individual compounds being sequenced annually.The most lethal of the various components are the ‘long’ toxins which exert a curariform effect. They contain 71-74 amino-acid residues and have four disulphide bridges. The ‘short’ toxins contain 60-62 residues but the cystine cross-links occur in the same positions as the deleted fragment is at the C-terminus. With a couple of dozen sequences now established only two residues besides the half-cystine residues are common to all toxins although considerable homology exists between those of similar length and physiological The structures of four scorpion neurotoxins have also been elucidated ; these resemble the ‘short’ snake toxins both in size and in their possession of four disulphide bridge^.'^ 3 Peptide Synthesis Protecting Groups.-cc-Group Protection.Attempts to extend the range of amino-protecting groups available which can be selectively removed continue to centre largely on the urethane type. The aa-dimethyl-3,s-dimethoxybenzyloxy-carbonyl group (10) is somewhat more labile to acid than the widely used t-butoxycarbonyl group but it can also be removed photochemically. Quantitative 49 J. C. Brown and J. A. Dryburgh Cunad. J. Chem. 1971,49 867. J. C. Brown M. A. Cook and J. A. Dryburgh Canad. J. Chem. 1973 51 533; €3. Schubert and J. C. Brown Cunad. J. Biochem. 1974 52 7. 5’ H. S. Tager and D. F. Steiner Proc. Nut. Acad. Sci. U.S.A. 1973 70 2321. 52 R. A. Gregory and H. J. Tracy Lancet 1972 ii 797. ’’ J. F. Habener B. Kemper J. T. Potts and A. Rich Endocrinology 1973 92 219.54 B. A. Roos K. Okama and L. J. Deftos Biochem. Biophys. Res. Comm. 1974 60 1 134. See ‘Amino-acids Peptides and Proteins’ ed. G. T. Young (Vol. 4) and R. C. Sheppard (Vols. 5 6) (Specialist Periodical Reports) The Chemical Society London 1973 1974 1975 Vols. 4-6 Ch. 2 under ‘Toxins’. 56 D. R. Babin D. D. Watts S. M. Goos and R.V. Mlejnek Arch. Biochem. Biophys. 1974 164. 694. 506 P.M. Hardy (10) deprotection occurs if 6-millimolar solutions are passed through a spiral made of one metre of quartz tubing irradiated with a one kilowatt high-pressure mercury lamp; trytophan seems to survive this treatment.57 The 1,l-dimethyl-2- propynyloxycarbonyl group (1 1)can be removed by hydrogenolysis in the presence Me 0 I II HCEC-C-0-C-I Me (1 1) of sulphur-containing amino-acid~.~ * Two base-sensitive groups the 9-fluorenyl- methoxycarbonyls9 and the cyano-t-butoxycarbony160 moieties have been examined.The former undergoes solvolysis in liquid ammonia (Scheme 2) Polymer +- A CO + RNH CH -0,CNHR Reagents i NH,; ii H,O Scheme 2 and the latter 8-elimination at pH 10 in aqueous solution. The 4-picolyloxy- carbonyl group (12) is stable in strong acids owing to protonation but it can be removed by zinc dust in 50 ”/ acetic acid or catalytic hydrogenation.61 The basic ‘handle’ in this group may aid purification of peptide derivatives. 57 C. Birr W. Lochinger G. Stahnke and P. Lang Annalen 1972 763 162. 58 G. L. Southard B. R. Zabarowsky and J. M..Pettee J. Amer.Chem. SOC.,1971 93 3302. ’’ L. A. Carpino and G. Y.Han J. Org. Chem. 1972 37 3404. 6o E. Wunsch and R.Soangenberg Chem. Ber. 1971 104 2427. 61 D. F. Veber S.F. Brady and R. Hirschmann Proceedings of the Third American Peptide Symposium held at Boston Massachusetts 1972 ‘Chemistry and Biology of Peptides’ ed. J. Meienhafer Ann Arbor Science Publishers Inc. Ann Arbor Michigan 1972 p. 315. Peptides and Proteins 0 NT CH2-0-C-II At the present time the most utilized amino-protecting groups as far as published syntheses of natural peptides and their analogues are concerned are those removable by acidic conditions or hydrogenolysis ;base-sensitive OT photolabile groups have not in general proved so applicable. Protecting groups cleavable by electrolysis and by solvolysis with organic solvents are now being developed although they have yet to be widely used.Two groups of the latter type have been reported. The 2-bromo-l,l-dimethylethoxycarbonylcan be removed simply by heating in ethanol (Scheme 3);62 the vinyloxycarbonyl group must be treated with bromine in dichloromethane to potentiate it. Subsequent treatment in methanol causes cleavage presumably by a mechanism similar to that outlined in Scheme 3.63 A comparative study of the electrolysis of 2- CH2cBr .q//I H02CCHRNH-C.\ -+ H0,CCHRAH <'&Me I 0-CMe ,O Me 'H Br-Reagents i EtOH Scheme 3 2,2-,and 2,2,2-halogenoethoxycarbonylprotecting-groups has established in simple models that if the half-wave potentials of two members of the series differ by at least 0.3 V selective reduction should be p~ssible.~~,~~ The use of metal complexes in connection with amino-protection is a further area beginning to be explored.The allyloxycarbonyl group can be cleaved from amino-acids with nickel carbonyl in the presence of tetramethylethylenediamine but peptides have yet to be examined.66 Treatment of pentacarbonyl[methoxy- (phenyl)carbene]chromium with amino-esters gives the corresponding [amino- (pheny1)carbenelpentacarbonylchromium complexes (13) which are stable to alkali enabling the ester group to be saponified and the peptide chain built up. The group can be removed from the yellow complex by trifluoroacetic The only other method of note using a non-urethane protecting group involves 62 T.Ohnishi H. Sugano and M. Miyoshi Bull. Chem. SOC.Japan 1972,45,2603. 63 R. A. Olofson and Y. S. Yamamoto U.S.P.3 711 458 (CI. 260/112.5; CO7cg COSh) (Chem. Abs. 1973,78 98 024). 64 E. Kasafirek Tetrahedron Letrers 1972 202 I. 65 M. F. Semmelhack and G. E. Heinsohn J. Amer. Chem. SOC.,1972 94 5139. 66 E. J. Corey and J. W. Suggs J. Org. Chem. 1973,38 3223. " K. Weiss and E. 0.Fischer Chem. Ber. 1973 106 1277. 508 P.M. Hardy (13) E and Z forms incorporation of the amino-group into a 4,S-diphenyl-A4-oxazolin-2-one ring system by successive reaction with benzoin cyclic carbonate and trifluoroacetic acid. Catalytic reduction or peracid oxidation afford derivatives sensitive to hydrolysis (Scheme 4).68 Ph Ph Ph Ph Ho0 +H,N- H -+ ~ O H 't(0 2 OKN-O Ph Ph Ph ph)=( II HOWOH 0 N-Ph Ph PhCOCOPh PhCH,CH,Ph + CO, HffH * +co + + + H,N-OK" + H,N-0 Reagents i CF,CO,H; ii m-CIC,H,CO3H; iii H +; iv Pd-H Scheme 4 Protecting groups which have fallen into disuse or which never fulfilled their initial promise can ofcourse take on new leases of life if novel methods of removal are found.Electrolytic cleavage of the tosyl group for example now enables it to be cleaved without affecting N-benzyloxycarbonyl groups or S-benzyl N-Trifluoroacetyl groups have been found to be sensitive to reduction by b~rohydride,~' and in contrast to earlier reports the o-nitro- phenylsulphenyl group can be cleaved with ammonium thiocyanate ;the secret here lies in the use of 2-methylindole as a scavenger for the o-nitrophenylsul- phenyl thiocyanate produced.' The N-chloroacetyl group can now be removed by thioureas under mild neutral conditions.It is preferable to use l-piperidine- thiocarboxamide (14) to avoid the further condensation of the 2-iminothiazolidi- J. C. Sheehan and F. S. Guziec J. Amer. Chem. SOC.,1972 94 6561. 69 K. Okamura T. Iwasaki M. Matsuoka and K. Matsumoto Chem.and Ind. 1971,929. 70 F. Weygand and E. Frauendorfer Chem. Ber. 1970 103 2437. 7' E. Wunsch and R. Spangenberg Chem. Ber. 1972 105 740. Peptides and Proteins none liberated if free thiourea is added (Scheme 5).72 The cleavage can also be effected after treatment with 3-nitropyridine-2-thione but liberation of the amino- group in this case requires the use of trifluoroacetic acid (Scheme 6).73 ,NHz CICH,CONH-+ S=C \ 1 - + NH2 l;>Yli +C1 H,N- H 1 Scheme 5 + 0 + CF,C02- H,N- Reagents i aq.NaHCO, 40°C; ii CF,CO,H Scheme 6 Two carboxy-protecting groups have similarly become more practical propo- sitions. The phthalimidomethyl group can be removed with zinc and acetic acid which are conditions leaving benzyl- and t-butyl-based protecting groups undi~turbed.’~ The resurrection of the use of phenyl esters is based on their rapid hydrolysis at pH 10.5 in the presence of hydrogen peroxide in aqueous dioxan or DMF. The apparently racemization-free reaction no doubt involves initial formation of the peptide pere~ter.~~ Several photolabile C-protecting groups ’’ W.Steglich and H. G. Batz Angew. Chem. Internat. Edn. 1971 10 75. 73 K. Undheim and P. E. Fjeldstat J.C.S. Perkin I 1973 829. 74 D. L. Turner and E. Baczynski Chem. and Ind. 1970 1204. ’’ G. W. Kenner and J. H. Seely J. Amer. Chem. SOC.,1972,94 3259. 510 P.M. Hardy -co-o Me -CO-0-CH-CO ..Omph OMe (15) HC -0-COW (15)-(17) have been de~cribed,~~-~~ but they have not so far been seriously evaluated in the synthesis of any sizeable peptide. Another group with potential is the NN'-di-isopropylhydrazide which is stable under both acidic and basic conditions but removable by mild oxidation with for example lead tetra-acetate in pyridine. The reaction is particularly clean as the carboxylic acid is the only non-volatile Side-chain Protection.The protection of side-chain functional groups has made some useful progress over the past few years. Conversion of the guanido-group of arginine into a bis-l-adamantyloxycarbonylderivative (18) provides such a hydrophobic shell for the side-chain that intermediates protected in this way readily dissolve in organic solvents. The urethane is like t-butoxycarbonyl derivatives stable to hydrogenolysis but cleaved by trifluoroacetic acid. Arginine protected in this way was used in the first synthesis of porcine proinsulin- connecting peptide.80 A new protecting group developed for histidine the 1,1,1,3,3,3-hexafluoro-2-(p-chlorophenoxymethoxy)propyl (HF-PA) group (19) is stable to base and hydrogenolysis but it can be removed with acid at room l6 A.Patchornik B. Amit and R. B. Woodward J. Amer. Chem. SOC.,1970,92 6333. 77 J. C. Sheeman and K. Umezawa J. Org. Chem. 1973 38 3771. la J. C. Sheeman R. M. Wilson and A. W. Oxford J. Amer. Chem. SOC.,1971,93 7222. 7g D. H. R. Barton M. Girijavallabhan and P. G. Sammes J.C.S. Perkin I 1972 929. G. Jager and R. Geiger Chem. Ber. 1970 103 1727. Peptides and Proteins 511 7F3 C-0-CH2-0 /I Nim I CF3 I -His-temperature. It can be introduced into N-benzyloxycarbonyl-histidine methyl ester by condensation with hexafluoroacetone followed by acylation with a,p-dichloroanisole.** Solid-phase peptide synthesis requires side-chain-protecting groups to be more stable to the acidic reagents used in the a-N-deprotection cycles than in ordinary peptide synthesis.The sheer number of repetitions of this treatment in a synthesis means that a level of fission acceptable in conventional synthesis may lead to considerable impurities in the polymer-linked stepwise process. It has been suggested that no more than 0.05% cleavage of a side-chain group should occur during the removal of more than 99.95%of the N-a-protecting group if it is to be acceptable for solid-phase work. The use of E-N-benzyloxy-carbonyl protection for lysine leads to a loss of 0.8% of protection per hour the free E-amino-group then being available for chain branching. Replacement with 2- 2,4-,or 3,4-chlorobenzyloxycarbonylprotection however leads to essentially linear products ;final deprotection now requires hydrogen fluoride treatment.82 In a similar way the use of S-4-methylben~yl~~ has advantages over.the S-p-methoxybenzyl group.84 For tryptophan the utility of the N'-formyl group removable by hydrazine in DMF to protect the indole nucleus has been proven in several ~yntheses.~~ Formation of the Peptide Bond.-The four most widely used methods for the synthesis of the peptide bond remain the active ester the azide the mixed anhy-dride and the dicyclohexylcarbodi-imide-mediatedreactions. As far as the latter is concerned the use of additives which suppress racemization caused by the reagent has made it more suitable for fragment condensations. The most efficient additive is 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (20),but '' H.H. Selztmann and T. M. Chapman Tetrahedron Letters 1974 2637. 82 B. W. Erickson and R. B. Merrifield J. Amer. Chem. SOC.,1973,95 3757. 83 B. W. Erickson and R. B. Merrifield J. Amer. Chem. SOC.,1973 95 3750. 84 M. Ohno S. Tsukamoto S. Sato and N. Izumiya Bull. Chem. SOC.Japan. 1973 46 3280. D. Yamashiro and C. H. Li J. Org. Chem. 1973,38 2594. 512 P. M. Hardy this can itself undergo a side-reaction through conversion into an acylating agent (21).86 I-Hydroxybenzotriazole (22) has become the most widely used promoter of this type.86 Other additives which have been recommended include ethyl 2-hydroximino-2-cyanoacetate(23),875,7-dichloro-8-hydroxyquinoline,88 and p-chlorobenzenesulphohydroxamic acid (24).89 N-Hydroxy-compounds FN HON=C \ C0,Et 0 have also been found to catalyse the aminolysis of certain active esters notably of the p-nitrophenyl and 2,4,5-trichlorophenyI types in polar solvents such as DMF.Reactions can be complete in a few minutes. Additives with a pK close to that of acetic acid are the most effective although the nature of the solvent plays a crucial role; in THF for example additives actually inhibit aminolysis.’* The assessment of novel types of active ester shows no sign of diminishing in interest. Perhaps the ones most likely to find use are those of 2-hydroxypyridine and 2-mer~aptopyridine.”.~~ These are more reactive than the standard p-nitrophenyl esters but in contrast to these the rate of coupling increases as the solvent polarity decreases.This seems to be characteristic of aminolyses which proceed by intramolecular general base catalysis (and hence are racemization- resistant) and renders esters like these useful in solid-phase synthesis.” An unusual type of active ester is that derived from 5-amino-3-methyl-4-nitroso-1-phenylpyrazole (25). In the presence of dicyclohexylcarbodi-imide it can be mono- R2 I MerlNo MerlNo R1 N-0-CO-CHNHZ N I ‘N NH ’\N NHCOCHNHZ R’ Ph Ph I N\N NCOCHNHZ Reagents i ZNHCHR’C0,H-dicyclohexylcatbodi-imide; ii ZNHCHR’C0,H-dicyclohexylcarbodi-imide Scheme 7 acylated (26)or diacylated. The diacyl-product (27) is a stable crystalline material which undergoes rapid aminolysis on treating with amino-esters (Scheme 7) n6 W.Koenig and R. Geiger Chem. Ber. 1970 103 788 2024 2034. ‘’ M. Itoh Bull. Chem. SOC.Japan 1973 46 2219; 1974,47 471. H. Yajima M. Kurobe and K. Koyama Chem. and Pharm. Bull. (Japan) 1973 21 1612. 89 H. Yajima K. Kitagawa and M. Kutobe Chem. and Pharm. Bull. (Japan) 1973 21 2566. 90 W. Koenig and R. Geiger Chem. Ber. 1973 106 3626. 91 K. Lloyd and G. T. Young J. Chem. SOC.(0,1971 2890. Peptides and Proteins C-CH-NHZ II I 0 R’ presumably by intramolecular catalysis (28) since it is racemization-free. The co-product (26) can be removed by extraction with aqueous sodium carbonate and recovered by a~idification.~~ A further active ester with a ‘safety catch’ has been described. N-Protected amino acids can be esterified with 2,2-diphenyl- 2-hydroxyethanol to give compounds (29) which can be N-deprotected and elaborated at their N-terminus before activating the ester by treatment with trifluoroacetic acid to generate the corresponding vinyl ester (30).94 0 II -C-0-CH,-C-OH Ph I I Ph 0 I1-c-0-CH=C /Ph \ Ph (29) (30) That original cornerstone of racemization-free synthesis the azide coupling has been shown to require careful control if it is in fact to live up to its reputation.Slight racemization may occur even if the added base is not present in excess especially if triethylamine is used. N-Methylmorpholine is more satisfactory but di-isopropylethylamine gives the best result^.'^ The use of the stable non- explosive diphenylphosphorazidate (31)as a coupling reagent involves formation of the acyl azide of the carboxyl component as an intermediate rather than a mixed anhydride ;this racemization-free method is a convenient complement to the classical azide procedure.96 The application of various reagents containing ’’ A.S. Dutta and J. S. Morley J. Chem. SOC.(C),1971 2896. q3 M. Guarneri P. Giori and C. A. Benassi Tetrahedron Letters 1971 665. 94 T. Wieland J. Lewalter and C. Birr Annalen 1970 740,31. 95 P. Sieber M. Brugger and W. Rittel Proceedings of the Tenth European Peptide Symposium held at Abano Terne Italy 1969 ‘Peptides 1969’ ed. E. Scoffone North- Holland Publishing Co. Amsterdam 1971 p. 60. 96 T. Shiori and S. Yamada. Chem. and Pharm. Bull. (Japan) 1974 22 849 855 859. 514 P.M. Hardy phosphorus to peptide synthesis is a considerable feature of the past few years.Diethylphosphoryl cyanide prepared by the action of cyanogen bromide on triethyl phosphite is able also to mediate racemization-free coupling.97 Both the 'oxidation-reduction' and the toluene-p-sulphonic anhydride-hexa- methylphosphortriamide methods of peptide synthesis [see Annual Reports (B) 1969,66 503-5041 involve acyloxyphosphonium salts as the effective acylating agent; a third method which generates this species in a different way is that involving triphenylphosphine and carbon tetrachloride (Scheme 8).98 Racemiza-0 Ph,P + CCI -+ Ph,&CCI Ph,&O-C-R1 II c1-/Cl-+ CHCI R'CONHR2 + Ph,P=O + HhEt C1-Reagents i R'C0,H; ii R2NH2 NEt Scheme 8 tion may occur on linking peptide component^,^^ but if the temperature is kept low and tris(N-methy1piperazino)phosphine is used instead of triphenylphos- phine this can be prevented.loo Amide bonds may also be formed by the action of diphenyl phosphite and pyridine presumably by the route outlined in Scheme H-P R~CONHR~ H-P-OCOR' HO/\OPh Reagents i RlCO,H-pyridine; ii R'NH Scheme 9 9,1°1 and by the use of hexachlorophosphatriazine. Half an equivalent of the latter reagent in the presence of one equivalent of triethylamine or N-methyl- morpholine is effective (Scheme 10).'O2 A particularly interesting novel synthetic method involves conversion of a urethane-protected amino-acid into an aziridin-2-one ; phosgene is the preferred reagent for this although thionyl chloride or phosphoryl chloride may be used " S.Yamada Y.Kasai and T. Shiori Tetrahedron Letters 1973 1595. 98 L. E. Barstow and V. J. Hruby J. Org. Chem. 1971 36 1305. 99 T. Wieland and A. Seeliger Chem. Ber. 1971 104 3992. loo Y. Takeuchi and S.-I. Yamada Chem. and Pharm. Bull. (Japan),1974 22 32 41. lo' N. Yamazaki and F. Higashi Terrahedron Letters 1972 5047. Y. Martinez and F. Winternitz Bull. SOC.chim. France 1972 4707. Peptides and Proteins c1 Cl N C1 \/\/ \p/N\p/C1 0 Cl'i CA IPCl --L I\O-!!-R1 \/ N \p/N C<p\C1 2R'NHCOR' 0/\ c1 I c=o I R' Reagents i 2R1C0,H-base; ii R'NH Scheme 10 0 Reagents i COC1,-2NEt3 -20 "C; ii R2NH Scheme 11 (Scheme11). In the presence of an amino-component the strained three-membered ring is opened to give a peptide without change of configuration.Formation of an aziridinone is markedly dependent on the N-protecting group ;they have been prepared so far only from N-benzyloxycarbonyl-amino-acidsor their para-substituted derivatives. N-Benzyloxycarbonylaziridinoneitself is too unstable to be isolated and proline of course cannot form such a derivative. The coupling of these compounds appears to be particularly effective with hindered com- ponent~.''~ Lactones involving seven-membered rings are also active towards aminolysis. Treatment of the Schiff base derived from an amino-acid and 5-nitrosalicylaldehyde with dicyclohexylcarbodi-imidegenerates such a species ; leucine for example forms 3-isobutyl-4-nitro-2,3-dihydrobenzo[f] [1,4]oxa-zepin-2-one (32).Other hydroxycarbonyl compounds may be used e.g. pentane-2,4-dione and a-formyl-N-hydroxysuccinimide.104Most work in the area of activation through cyclic derivatives of carboxyl components however still centres on the N-carboxy-anhydrides. Methods of preparing N-carboxy- anhydrides from amino-acids with functional groups in their side-chains either free or suitably protected have now been detailed in connection with the use of such compounds in the controlled stepwise synthesis of peptides in aqueous media without isolation of intermediate^."^ A two-phase solvent system Io3 M. Miyoshi Bull. Chem. SOC.Japan 1973 46 212 1489. lo' M. Bodanszky U.S.P. 3 704 246 (Cl. 260-333; C07d) (Chem. Abs. 1973,78 58 801).lo' R. Hirschmann H. Schwam R. G. Strachan E. F. Schoenewaldt H. Barkemeyer S. M. Miller J. B. Conn V. Gorsky D. F. Weber and R. G. Denkewalter J. Amer. Chem. Soc. 1971,93 2746. 516 P.M. Hardy (acetonitrile-water 60 50 v/v) containing sodium carbonate at an apparent pH of 11.5 can be used for this purpose at -15 "C;this mixture has the advantage ofnot requiring such careful control of pH as the original method in homogeneous solution and over-reaction tends to be less because the resulting carbamates are more stable under these conditions. O7 N-Thiocarboxy-anhydrides have been explored as alternative reagents but although in general they tend to give higher yields significant amounts of epimer are formed. In contrast to histidine N-carboxy-anhydride which tends to form an imidazo-tetrahydropyrimidinone (Scheme 12) the corresponding sulphur analogue can be successfully used in '0 1 0 Scheme 12 repetitive peptide synthesis.lo* N-Carboxy-anhydrides can be acylated with o-nitrophenylsulphenyl chloride in anhydrous solvents in the presence of tri- ethylamine to give the relatively stable crjstalline N-o-nitrophenylsulphenyl-N-carboxy-anhydrides which do not tend to polymerize. However these derivatives are unsuitable for use in aqueous media. Good yields of dipeptides occur on treatment with N-trimethylsilylamino-acid trimethylsilyl esters.log N-4,4'-Dimethoxybenzhydryl-N-carboxy-anhydrides can be prepared from N-4,4-dimethoxybenzhydryl-amino-acidsin 30-40 "/ yield using phosgene in THF and have been used in solid-phase synthesis.' lo '06 Y.Iwakura K. Uno M. Oya and R. Katakai Biopolymers 1971 9 1419. lo' R. Katakai M.Oya K. Uno and Y. Iwakura Biopofymers 1971 10 2199; J. Org. Chem. 1972 37 327. lo* R. S. Dewey E. F. Schoenewaldt H. Joshua W. J. Paleveda H. Schwam H. Barkemeyer B. H. Arison D. F. Weber R. G. Strachan J. Milkowski R. G. Denkewalter and R. Hirschmann J. Org. Chem. 1971 36 49. lo9 H. R. Kricheldorf Angew. Chem. Internat. Edn. 1973 12 73 H. R. Kricheldorf and M. Fehrle Chem. Ber. 1974 107 3533. lo J. Halstr~rm and K. Kovacs Proceedings of the Twelfth European Peptide Symposium held at Schloss Reinhardsbrun German Democratic Republic 1972 'Peptides 1972' ed. H. Hanson and H.-D. Jakubke North-Holland Publishing Co.Amsterdam and London 1973 p. 173. Peptides and Proteins Z-Gly-L-Ala + L-Leu-OBzl 1' Z-G~Y-D-and -L-Ala-L-Leu-OBzl 1ii G~Y-D-and -L-Ala-L-Leu Reagents i coupling agent; ii H,-Pd Scheme 13 The degree of racemization of amino-acids can be determined conveniently by their reaction with an optically active N-carboxy-anhydride and separation of the diastereoisomeric products on an amino-acid analyser.' ' A racemization test involving separation in a similar way of glycyl-L- and D-alanyl-L-leucines (Scheme 13) has become a popular method of monitoring coupling methods; 0.1 "/ D-L-isomer can be detected in 5 pmol L-L-peptide.lI2 The use of isotopic dilution for determination of racemate content has extended the range of applica-tion of methods based on oligopeptide diastereoisomers down to the 1.0-0.001 level much below that accessible by other methods.' 'I1 A.V. Barooshian M. J. Lautenschlager J. M. Greenwood and W. G. Harris Analyt. Biochem. 1972,49 602. ' l2 N. Izumiya H. Muraoka and H. Aoyagi Bull. Chem. Soc. Japan 1971,44 3391. 'I3 D. S. Kemp Z. Bernstein and J. Rebek J. Amer. Chem. SOC.,1970 92 4756.

ISSN:0069-3030    

DOI:10.1039/OC9747100497

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

19 Porphyrins and Related Compounds By A. H. JACKSON Department of Chemistry University College Cardiff In the present article emphasis will be placed on recent developments but some reference will also be made to other significant developments during the past few years since this topic was last reviewed (briefly) in Annual Reports'. 1 Porphyrins Structure and Synthesis.-Following the appearance of several new approaches* to porphyrin synthesis during the 1960's recent developments have been con- cerned more with their application. Earlier work describing the synthesis of various rhodoporphyrin derivatives e.g. (la) and (lb) related to chlorophyll (by the a-and b-oxobilane routes) has now been detailed;3 the 2,4-divinyl- rhodoporphyrin derivative (lc) has also been ~repared.~ Two general methods for the synthesis of porphyrin /3-keto-esters (2) from rhodoporphyrins have been PMe= CH2CH2C02Me (a) R' = R2 = Et (1) R = OMe (b) R' = V R2 = Et (2) R = CH2C02Me (c) R' = R2 = V V = vinyl developed (a) via the acid chloride and sodiomalonate esters and (b) more efficiently through mixed anhydrides or imidazolides and magnesium malonate~.~ B.C. Uff Ann. Reports (B) 1970 67,440. CJ A. H. Jackson and K. M. Smith in 'The Total Synthesis of Natural Products' ed. J. W. ApSimon Wiley New York 1973 p. 144. M. T. Cox T. T. Howarth A. H. Jackson and G. W. Kenner J. Amer. Chem. SOC. 1969 91 1232; T. T. Howarth A. H. Jackson and G. W. Kenner J.C.S. Perkin I 1974 502. M. T. Cox A. H. Jackson G.W. Kenner S. W. McCombie and K. M. Smith J.C.S. Perkin I 1974 5 16. 5 19 520 A. H. Jackson (3) (a) R' = H R2 = V (b) R' = V R2 = H M NHeN n 2 (4) (a) R' = CHO R2 = V (b) R' = V R2 = CHO (5) (a) R' = V R2 = PMe (b) R' = PMe,R2 = V (6) (a) R' = CH=CHCO,Me R2 = PMe (b) R' = PMe,R2 = CH=CHCO,Me A full account of the use of oxobilane routes2 in the synthesis of the methyl esters of pemptoporphyrin (3a) isopemptoporphyrin (?b) and chlorocruoro- porphyrin (4a) has recently appeared ;5 pemptoporphyrin and other porphyrins related to protoporphyrin-IX have also been prepared by the b-bilene method and from ac-biladienes.6 New syntheses of the trimethyl ester (5a) of hardero- porphyrin (one of the porphyrins found in the harderian gland of the rat) and its isomer (5b) have also been described using the MacDonald route.' The b-oxo- bilane approaches have recently been extended to the synthesis' of the 5411' porphyrin isolated some years ago' from meconium ;this porphyrin (so-called because of the position of its Soret band in the near;u.v.) had been assigned the structure (6a) or (6b) largely on the basis of analytical and spectroscopic evidence and biosynthetic reasoning.The former was synthesized via the b-oxobilane (7) and the latter by the MacDonald method from the pyrromethane intermediates (8) and (9) shown ; introduction of the acrylic side-chains was accomplished at the porphyrin stage by formylation followed by condensation with monomethyl malonate. Me\ NH HN / CO H PhCH,02C /-OHC CHO PNH HNA Interest in the synthesis of porphyrin-a," the iron complex of which is the prosthetic group of cytochrome oxidase has revived.For example a model A. H. Jackson G. W. Kenner and J. Wass J.C.S. Perkin I 1974 480. P. S. Clezy A. J. Liepa and N. W. Webb Austral. J. Chem. 1972 25 1991 ; G. V. Pomonarev S. M. Nasralla A. G. Bubnova and R. P. Evstigneeva Khim. geterorsikl. Soedinenii 1973 2027. J. A. S. Cavaleiro G. W. Kenner and K. M. Smith J.C.S. Chem. Comm. 1973 184. * CJ A. H. Jackson D. E. Games P. Couch J. R. Jackson R. V. Belcher and S. G. Smith Enzyme 1974 17 81. J. French D. C. Nicholson and C. Rirnington Biochem. J. 1970 120 393. lo G. A. Srnythe and W. S. Caughey J.C.S. Chem. Comm. 1970 809. Porphyrins and Related Compounds 521 porphyrincarboxylic ester was synthesized' by the ac-biladiene route3 and converted into a porphyrin heptyl ketone as shown schematically below; the porC0,Me + porCOCl -+ porCOCHN -+ porCO(CH,),Me -+ porCHOH(CH,),Me related alcohol was obtained by borohydride reduction and this opens up a possible route for the introduction of a hydroxyalkyl substituent such as that in a currently favoured structure" for porphyrin-a.A range of porphyrins with electron-withdrawing groups (e.g. formyl acetyl ethoxycarbonyl and vinyl) in opposite rings has also been synthesized12 by the b-bilene route,3 and one of these (with formyl and vinyl groups) has a very similar chromophore to that of porphyrin-a. The tendency of b-bilenes to rearrange to a-bilenes is a potential limitation of this route.' 2,1 Unsymmetrical formyl porphyrins have also been synthesized by oxidative cyclization of biladienes prepared by condensation of a dipyrromethane with two different pyrrole aldehydes.l4 A group of four related tetracarboxylic porphyrins was recently isolated from the faeces of patients suffering from symptomatic cutaneous hepatic por- phyria and also from rats poisoned with hexachlorobenzene. Analytical and spectroscopic evidence showed the presence of three methyl three propionic acid and one acetic acid group in each compound the eighth substituent being ethyl vinyl hydroxyethyl or hydrogen respectively cf (10). The precise order of these substituents around the ring has now been determined by studying the effects of the europium shift reagent Eu(fod) on the n.m.r.spectra of the tetra- methyl ester (10a) of 'isocoproporphyrin' and by degradation of the macrocycle to monopyrroles.16 The shift reagent chelates to pairs of neighbouring ester groups and as two of the rneso-bridge protons experience a very marked down- field shift only two structures were possible (on biosynthetic grounds). The alternative to (10a) was eliminated by reductive degradation to alkylpyrroles or chromic acid oxidation to maleimides ; the monopyrrolic products were characterized by gas chromatographic-mass spectrometric comparisons with authentic samples. The latter can be used with as little as 1Opg porphyrin in the case of the chromic acid oxidations,' 'and is thus a promising technique for other unknown porphyrins obtainable in only minute quantities.Isomers of isocoproporphyrin and its cogeners have been synthesized by the b-bilene route. l8 ' R. V. H. Jones G. W. Kenner and K. M. Smith. J.C.S. Perkin I 1974 531. P. S. Clezy and C. J. R. Fookes Austral. J. Chem. 1974 27 371. l3 J. M. Conlon J. A. Elix G. I. Feutrill A. W. Johnson M. W. Roomi and J. Whelan J.C.S. Perkin I 1974 713. l4 A. F. Mironov L. I. Fleiderman and R. P. Evstigneeva Zhur. ohshchei Khim. 1974 44 1165; A. F. Mironov M. A. Kulish V. V. Kobak B. V. Rozynov and R. P. Evstigneeva ibid. p. 1407. Is G. H. Elder Biochem. J. 1972 126 877. l6 M. S. Stoll G. H. Elder D. E. Games P. J. O'Hanlon D. S. Millington and A. H. Jackson Biochem.J. 1973 131 429. I' C' A. H. Jackson D. S. Millington and D. E. Games in Proceedings of the 6th International Congress on Mass Spectrometry Edinburgh 1973 p. 21 5. P. S. Clezy and V. Diakin Austral. J. Chem. 1973 26 2697. 522 A. H. Jackson \MIMe AMNH aN1 epMe AM' 'N R\ HN \ PM' PM' PMe AM' = CH,CO,Me (10) (a) R = Et (b) R = CH=CH (c) R = CH(0H)Me (d) R = H (11) R = AM" (12) R = Me [a,y-'4C,]Uroporphyrin-III octamethyl ester (11) has been synthesized by the MacDonald route the labels being introduced by Vilsmeier-Haack formyla- tion of intermediate pyrromethanes. A heptacarboxylic acid porphyrin recently isolated from avian erythrocytes is probably identical with a similar compound found in porphyrin faeces ; it is derived from uroporphyrinogen-I11 (an octa- carboxylic intermediate in porphyrin biosynthesis) by loss of one carboxylic acid group.Syntheses of the methyl esters of three of the isomers have been achieved;20*21 the fourth isomer (12) (not accessible by the MacDonald route) has now been prepared by the b-oxobilane method2'' and shown to be identical with the natural product by 'mixed' n.m.r. spectra and titration with europium shift reagent (cf. refs. 16 and 20). Full details have appeared of the synthesis of various deuteriated derivatives of protoporphyrin-IX by a variation of the MacDonald approach from pyrro- methane dialdehydes and pyrromethanedicarboxylic acids as well as by the b-oxobilane route.22 These were required for n.m.r. studies of haemoproteins and deuteriomethyl groups were introduced at the pyrrole synthesis stage by use of deuteriated 1,3-diketones.The meso-deuterium atoms were introduced by a novel exchange procedure which occurs under acidic conditions at the bridge methylene groups of pyrromethanes and intermediates in the MacDonald synthesis. In connection with biosynthetic studies the p- y- and 6-'3C-labelled proto- porphyrin-IX dimethyl esters have been synthesized by them-biladienemethod ;' l9 B. Franck D. Gantz and F. Huper Angew. Chem. Internat. Edn. 1972 11 420. 2o (a)A. R. Battersby E. Hunt M. Thara E. McDonald J. B. Paine tert. and J. Saunders J.C.S. Chem. Comm. 1974,994;(6)A. H. Jackson A. M. Ferramola H. A. Sancovich N. Evans D. E. Games S. A. Math G.K. Elder and S. G. Smith Phil. Trans. Roy. Soc. 1975 in the press. P. S. Clezy personal communication. 22 J. A. S. Cavaleiro A. M. Da Rocha Gonsalves G. W. Kenner and K. M. Smith J.C.S. Perkin I 1974 I77 1 ; cf. J. A. S. Cavaleiro A. M. Da Rocha Gonsalves G. W. Kenner K. M. Smith R. G. Shulman A. Mayer and T. Yamane J.C.S. Chem. Comm. 1974 392; see also G. W. Kenner K. M. Smith and M. J. Sutton Tetrahedron Letters 1973 1303. " A. R. Battersby G. L. Hodgson M. Ihara E. McDonald and J. Saunders J.C.S. Perkin I. 1973. 2923. Porphyrins and Related Compounds in the case of the labelled compound the label was introduced by ring closure with [’3C]formaldehyde. These experiments enabled the rigorous assignment of all four meso ’3C-resonances in protoporphyrin-IX.Porphyrin B-keto-esters (2) have been cyclized to the corresponding phaeo- porphyrins (13) in good yields by treatment of the former with two equivalents Me C0,Me (13) R = Et or V of thallium trifluoroacetate followed by photolysis and demetallation ;24 this accomplished the first formal total syntheses of the phaeoporphyrin dimethyl ester and of its 2-vinyl analogue. Use of thioacetals as protecting groups for formyl-pyrroles has enabled certain previously inaccessible pyrromethanes to be obtained ;25 MacDonald has described26 the use of Girard reagents for protecting formyl groups and has synthesized di- tri- and tetra-pyrroles related to porphobilinogen for biosynthetic studies. A new synthesis of porphobilinogen (PBG) (15) (the monopyrrolic PMeR CO H \ N Et0,COMe c\H2 /CO,H H (14) (a) R = COMe ‘Xi2NH2 N (b) R = CH,CO,Me H precursor of the porphyrin ring system) has also been described2’ in which the key step was the thallic nitrate oxidative rearrangement of an acetyl-pyrrole (14a) to the corresponding methoxycarbonylmethyl-pyrrole(14b).Enzymic catalysis of the condensation of 8-aminolaevulinic acid (8-ALA) by Propioni-bacterium shermanii affords PBG in 54% yield by a batch-wise process2* A continuous process for the synthesis of PBG has now been achieved by allowing 24 G. W. Kenner S. W. McCombie and K. M. Smith J.C.S. Perkin I 1974 527. L5 P. S. Clezy C. J. R. Fookes D. Y. K. Lau A. W. Nichol and G. A. Smythe Ausfral. J. Chem. 1974 27 357.26 J. M. Osgerby J. Plusec Y. C. Kim F. Boyer N. Stojanac H. D. Mah and S. F. MacDonald Canad. J. Chem. 1972,50 2652. ’’ G. W. Kenner K. M. Smith and J. F. Unsworth J.C.S. Chem. Comm. 1973 43. 2B G. Muller Z. Nururfursch. 1972 27b,473. 524 A. H. Jackson 8-ALA to percolate through a column of 6-ALA dehydratase linked to Sepharose ; up to 200mg per day has been obtained and the process is capable of being scaled up.29 Chemical and Physical Properties.-Proceedings of a recent ~onference,~ O which provided a valuable survey of the present state of the field have been published. They include articles on structural synthetic spectroscopic and physico- chemical studies. Detailed X-ray crystallographic studies3 of porphine itself showed that the free base has essentially two-fold symmetry with the internal hydrogen atoms fixed on opposite nitrogen atoms rather than equally distributed between all four nitrogens as had previously been suggested.N.m.r. studies32 of a number of porphyrins and chlorins also showed that at low temperatures the inner hydrogens become localized usually on opposite nitrogens although in some unsymmetri- cally substituted compounds adjacent nitrogens may be favoured ; this has also been studied by 13C n.m.r. spectroscopy and evidence has been presented that tautomerism occurs by successive prototropic shifts rather than by simul- taneous shifts.32b Porphyrins are converted into their monocations in toluene-acetic acid confirming earlier observations whereas in strong acids only the dication is observed.33 Monoazaporphyrins will add a third proton in strong sulphuric acid solution,33 and whilst such trications have not been observed with por- phyrins they are clearly transient intermediates in the well-known acid-catalysed exchange reactions which occur at the rneso-positions of porphyrins and chlorins.The crystal structure of octaethylporphyrin monocation tri-iodide has been reported.34 Several studies of the N-alkylation of p~rphyrins,~~,~~ corroles and related macrocycles by alkyl iodides and methyl fluorosulphonate have recently been described and mono-N-alkyl- NN-dimethyl- and NNN-trimethyl-octa-aIkyl-porphyrins and chlorins have been prepared. Three different types of NN-dimethylporphyrins were discovered i.e.the N,N,-trans- N,N,-trans- and N,N,-cis-dimethyl derivatives ;the trans relationship of the two methyl groups in the first of these three compounds was assigned on the basis of their n.m.r. spectra and their partial resolution as the D-camphorsulphonates. Detailed studies of their electronic spectra and of their basicity have been interpreted 29 D. Gurne and D. Shemin Science 1973 180 1188. 30 'The Chemical and Physical Behaviour of Porphyrin Compounds and Related Struc- tures' ed. A. D. Adler Ann. New Yurk Acad. Sci.,1973 206 pp. 1-761 ;see also J.-H. Fuhrhop Angew. Chew. Internat. Edn. 1974 13 321. 31 A. Tubinsky ref. 30 p. 47. 32 (a)C. B. Storm Y. Teklu and E. A. Stokolski ref. 30 p. 631 ;(b)R. J. Abraham G. E. Hawkes and K.M. Smith Tetrahedron Letters 1974 1483. 33 R. Grigg R. J. Hamilton M. L. Jozefowicz C. H. Rochester R. J. Terrell and H. Wickwar J.C.S. Perkin II 1973 407. 34 N. Hirayama A. Takenaka Y. Sasada E.-I. Watanabe H. Ogoshi and Z.-I. Yoshida J.C.S. Chem. Comm. 1974 330. 35 A. H. Jackson and G. R. Dearden ref. 30 p. 151. 36 R. Grigg G. R. Shelton A. Sweeney and A. W. Johnson J.C.S. Perkin I 1972 1789. Porphyrins and Related Compounds [cj ref. (35)]in terms of a high degree of sp3 character for one or more of the nitrogen atoms leading to considerable distortion of the macrocyclic ring ; X-ray crystallographic studies have shown that the N-substituted pyrrole rings of a cobalt N-methylcorrole is twisted 23" out of the plane.37 These and other studies with meso-tetraphenylporphyrinsand related compounds3* show that the porphyrin ring system can retain its aromatic character in spite of a considerable degree of distortion.N.m.r. studies show that N-methyloctaethyl- porphin monocation and free base undergo slow proton exchange ;35 similar slow processes occur between the free base and the dication of rneso-tetraphenyl- p~rphyrin.~~ These interesting results may be due to facilitation of the exchange process by the distortion of the macrocycle. In basic media N-alkylporphyrins are only converted into their monoanions whereas octaethylporphyrins and a monoazaporphyrin are converted into their dianion~.~' The reactions of di- azoacetic esters with porphyrins have also been studied ; rneso-tetraphenyl-porphyrin gives peripheral mono-and bis-cyclopropane adducts as well as N-substituted acetic ester derivatives with the zinc and copper complexes.41 70,Et Cobalt octaethylporphyin affords a novel bridged CO-CH-N adduct which is rearranged by chromous chloride to the chloro-cobalt(I1) complex of N-ethoxycarbonylmethyloctaethylporphin; with ethanolic hydrogen chloride the free "'-bridged derivative (16) is formed.42 The nickel rneso-tetraphenyl porphin adduct with diazoacetic ester undergoes a thermal rearrangement to a h~moporphin~~ (17) ;this product is reminiscent of the ring-expansion product of Et 37 R.Grigg T. J. King and G. Shelton J.C.S. Chem. Comm. 1970 56. 38 Cf.E. B. Fleischer Accounts Chem. Res. 1970 3 105; S. S. Eaton and G. R.Eaton J.C.S. Chem. Comm. 1974 576. 39 R. J. Abraham G. E. Hawkes and K. M. Smith Tetrahedron Letters 1974 71. 40 J. A. Clarke P. J. Dawson R. Grigg and C. H. Rochester J.C.S. Perkin 11 1973,414. 4' H. J. Callot Tetrahedron Letters 1972 1011; Bull. Soc. chim. France 1972 4381; H. J. Callot and T. Tschamber ibid. 1973 3192. 42 P. Batten A. Hamilton A. W. Johnson G. Shelton and D. Ward J.C.S. Chem. Comm. 1974 550. 43 H. J. Callot and T. Tschamber Tetrahedron Letters 1974 3155 3159. 526 A. H. Jackson octaethylporphin with etho~ycarbonylnitrene?~ X-Ray structures of N-ethoxy- carbonylmethyl~ctaethylporphin~~ have been and of the nickel h~moporphin~~ described. 2 Metalloporphyrins Chelation of porphyrins with metals is of great biological interest and a possible mechanism for metal-ion insertion involves distortion of the macrocycle by bonding of the ‘imino’ nitrogens of the free base to a metal ion aboue the molecule followed by insertion of a second metal ion from beneath after dissociation of the NH-pr~tons.~~ The high basicity of N-substituted porphyrins (mentioned above) and the formation of novel sandwich-type complexes of mercury [e.g.with three porphyrins and two mercury(@ ions] is of interest in this respect.48 Much interest has centred on the meso-reactivity of metalloporphyrins in relation to haem metabolism which involves initial oxidation at the a-meso- position. A range of deuteriation formylation and nitration reactions of metalloporphyrins has been described,36 and octaethylhaemin forms the meso-tetranitromethyl derivative by reaction with N,04 in dichl~romethane.’~ Magnesium porphyrins undergo a particularly facile deuterium- or tritium-exchange reaction 22 at the meso-positions which is of use for biosynthetic studies.Photo-oxygenation of magnesium octaethylporphyrin afforded a formyl-oxoporphyrin-magnesium complex.49 meso-Oxygenated metallopor- phyrins have been prepared’ by hydrogen peroxide-pyridine oxidation of several bivalent metal chelates (e.g. Fe Co Ni Cu Zn or Mn). Oxidation of a zinc or magnesium porphyrin with thallium trifluoroacetate5* affords the meso-trifluoroacetoxy-derivative in high yield ; acidic hydrolysis and demetallation then yields the free oxophlorin. The initial reaction with the thallium(1Ir) reagent was interpreted as an oxidation to the metalloporphyrin-n-cation radical ;52 such species are of considerable importance in oxidation reactions of metallopor- phyrins and in relation to the mode of action of cytochromes and chlorophyll^?^^'^ Both mono- anddi-cation radicals may be obtained by electrochemical oxidation and the dications react readily with nucleophiles to give an isoporphyrin e.g.(18). Two different ground states of the n-cation radicals were observed in some cases depending on the nature of the additional ligands attached to the metal. Further- more it was suggested that in the dication radicals derived from the iron(rI1) *‘ R. Grigg J. Chem. SOC.(0,1971 3664. 45 G. M. McLaughlin J.C.S. Perkin II 1974 136. 46 B.Chewier and R. Weiss J. C.S. Chem. Comm. 1974 884. *’ P. Hambright and P. B. Chock J. Amer. Chem. SOC., 1974,96 3123. M. F. Hudson and K. M. Smith Tetrahedron Letters 1974 2223 2227. 49 J. H. Fuhrhop and D. Mauzerall fhotochem. and Photobioi. 1971 13 453. 50 J. C. Fanning T. L. Gray and N. Dattagupta J.C.S. Chem. Comm. 1974 23. R. Bonnett and M. J. Dinsdale J.C.S. Perkin I ‘1972 2540. 52 G. H. Barnett M. F. Hudson S. W. McCombie and K. M. Smith J.C.S. Perkin I, 1973 691. ” D. Dolphin and R. H. Felton Accounts Chem. Res. 1974 7 26 and refs. therein; R. H. Felton G. S. Owen D. Dolphin A. Forman D. C. Borg,and J. Fajer ref. 30 p. 504; J. Fajer Proc. Nut. Acad. Sci. U.S.A. 1974 71 994. Porphyrins and Related Compounds Ph Me0 ’Ph (18) porphyrin one electron is removed from the ring and the other from the metal leading to the corresponding iron(1v) porphyrin cation radical.Reduction of metalloporphyrins affords the a,?-dihydro-derivatives ; inter-mediate radical dianions can be intercepted by methylation to form a,y-dimethyl- dihydr~porphyrins.~~ Bis-trimethylsilyloxy-silicon porphyrins can be subjected to gas chroma- tography-mass spectrometry in the case of porphyrins with only alkyl substi- tuents.’ ’ Germanium porphyrins can be coupled with other organic compounds through oxygen ligands and used as n.m.r. shift reagent^.'^ Very recently europium has also been introduced into tetraphenylporphyrin and other por- phyrins and preliminary studies with this new n.m.r. shift reagent show con- siderable p~tential.~’ Syntheses of a number of haemoglobin models and studies of their complexing with oxygen have recently been described ; these are of two types based either on coupling an imidazole-containing side-chain to an iron porphyrinS8 or on the synthesis of iron and cobalt complexes in which access to the metal atom is 54 J.W. Buchler Tetrahedron Letters 1972 3803; J. W. Buchler and L. Puppe Annalen 1974 1046. 55 D. E. Games A. H. Jackson and D. S. Millington in ‘Mass Spectrometry in Bio- chemistry and Medicine’ ed. A. Frigerio and N. Castagnoli Raven Press New York 1974 p. 251. 56 J. E. Maskasky and M. E. Kenney J. Amer. Chem. SOC. 1973,95 1443. 57 C. P. Wong. R. F. Venteich and W. D. Horrocks J. Amer. Chem. SOC. 1974,96 7149.58 (a) A. Vanderheijden H. G. Peer and A. H. A. Vandenoord J.C.S. Chem. Comm. 1971 369; (6) G. A. Vasileva A. F. Mironov and R. P. Evstigneeva Zhur. obshchei Khim. 1972,42,1402; (c)C. K. Chang and T. G.Traylor Proc. Nut. Acad. Sci. U.S.A. 1973,70 2647; (d)W. S. Brinigar C. K. Chang J. Geibel and T. G. Traylor .I. Amer. Chem. SOC. 1974,96 5597; (e)J. Almog J. E. Baldwin R. L. Dyer J. Huff and C. J Wilkinson ibid. 1974,96 5600. A. H. Jackson ar,a,r,a-H,TpivPP (20) hindered by bulky hydrophobic groups.’’ Examples of the latter include a and the non-porphyric iron complex (19),59b cyclophanep~rphyrin,~~~ and the so-called picket-fence porphyrin (20),59d an X-ray structure of which has recently been described.60 3 Chlorins and Chlorophylls Structure and Synthesis.-The structure of bacteriochlorophyll-b one of the pigments occurring in the purple photosynthetic bacteria has now been revised6’ in favour of the 4-ethylidene formulation (21).Whereas phytol is the normal esterifying group for the propionate side-chain of chlorophylls-a and -b and of the bacteriochlorophylls reports have appeared that in some bacteria the esterifying alcohol is geranylgeraniol or farnesol.62 As chlorophylls-c are 59 (a) H. Dieckmann C. K. Chang and T. G. Traylor J. Amer. Chem. Soc. 1971 93 4068; (h) J. E. Baldwin and J. Huff ibid. 1973 95 5757; (c) L. Vaska A. R. Ammundsen R. Brady B. R. Flynn and H. Nakai Finn. Chem. Lerrers 1974 66; (d)J. P. Collman R. R. Gagne T. R. Halbert J. C. Marchon and C.A. Reed J. Amer. Chem. SOC.,1973,95 7868; (e) J. P. Collman R. R. Gagne J. Kouba and H. Ljusbergwahren ibid. 1974 96 6800. 6o J. P. Collman R. R. Gagne C. A. Reed W. T. Robinson and G. A. Rodly Proc. Nat. Acad. Sci. U.S.A. 1974 71 1326. 61 H. Schaer W. A. Svec B. T. Cope M. H. Studier R. G. Scott and J. J. Katz J. Amer. Chem. SOC. 1974,96 3714. 62 J. J. Katz H. H. Strain A. L. Harkness M. H. Studier W. A. Svec T. R. Janson and B. T. Cope J. Amer. Chem. Soc. 1972 94 7938; H. Brockmann and G. Knoblock Arch. Mikrobiol. 1972 85 123; A. Gloe and N. Phenning ibid. 1974 96 93. Porphyrins and Related Compounds CH=CH Me H H ‘Me Me-\‘ pMe CO R (21) (22) (a) M =2H R =Me (b) M=Mg R =Et esterified with farnesol it seems likely that a variety of polyprenol esters of chlorophylls may exist.The absolute configuration of chlorophyll-a has been confirmedb3 by intro- duction of an additional chiral centre into the isocyclic ring by reduction of the C-9 carbonyl group of methyl phaeophorbide-a (22a). The resulting epimeric alcohols were separated and esterified with a-phenylbutyric anhydride and the observed stereoselectivity allowed deduction of the chirality at C-10 and hence the absolute configuration of chlorophyll-a. A convenient large-scale method for the separation of methyl phaeophorbides-a and -b by using the Girard-T reagent has been described.64 X-ray structure determinations of the chlorophylls themselves has so far proved impossible probably owing to association with traces of water or other ligands but those of methyl phaeophorbide-a (22a) and ethyl chlorophyllide (22b) have recently been rep~rted.~’ It has been suggested that minor components a’ and b’ in some preparations of chlorophylls-a and -b previously thought to be epimers at C-10 should now be re-assigned as chelated enol forms.h6 Chemical and Physical Properties.-Improved procedures for the degradation of phaeophytin-a to rhodoporphyrin-XV methyl ester (la) and the vinyl analogue (1 b) have been described as well as other novel oxidative reactions of the isocyclic ring.64 The photoreduction of chlorophyll-a by hydrogen sulphide affords the [Wdihydro-product as shown by 220 MHz n.1n.r.st~dies.~’ Extensive 3Cn.m.r. studies of chlorophylls and their derivatives have recently been reported68 using enriched material from algae grown in 13C02,and all the 63 H.Brockmann and J. Bode Annalen 1974 1017. 64 G. W. Kenner S. W. McCombie and K. M. Smith J.C.S. Perkin I 1973 2517. 65 (a)J. Gassman I. Strell F. Brandl M. Sturm and W. Hoppe Tetrahedron Letters 1971,4609; M.S. Fischer D. H. Templeton A. Zalkin and M. Calvin J. Amer. Chem. SOC.,1972,94 3613; (b)C. E. Strouse Proc. Nut. Acad. Sci. U.S.A. 1974,71 325. 66 P. H. Hynninen Actu Chem. Scand. 1973 27 1487 1771. h7 H. Scheer and J. J. Katz Proc. Nut. Acad. Sci. U.S.A. 1974 71 1626. 68 J. J. Katz and T. R. Janson ref. 30 p. 579; N. A. Matwiyoff and B. F. Burnham ref. 30 p. 365. 530 A. H. Jackson carbon resonances of the phytyl ester side-chain have also been assigned.69 As with the 'H n.m.r.spectra the 13Cn.m.r. spectra are also profoundly affected by intermolecular aggregation in non-polar solvents and the spectra change on addition of Lewis bases such as THF which bring about disaggregation. This work and related i.r. studies68a are of fundamental importance in relation to the organization of chlorophyll in living cells and the mechanisms of photosynthesis. Dimers and oligomers may be formed by co-ordination of the magnesium of one ring with the C-9 carbonyl group of another molecule in non-polar media whereas in the presence of water large polymers of a different structure (Chl- H,O) may be formed the water acting as a cross-linking agent by electron donation to the magnesium and by hydrogen-bonding of the carbonyl group.A number of other slightly differing pictures for the chlorophyll-water oligomers have been deduced from X-ray studies of methyl phaeophorbide-a and ethyl ~hlorophyllide.~~ The role of n-cation radicals in the photosynthetic process has received in- creasing attention; these can be readily produced by chemical oxidation or polarographic processes and their e.s.r. and optical spectra can be studied and compared with those observed for in uiuo systems.53 The observations were consistent with the interpretation that e.s.r. signal I (associated with the primary step of photosynthesis) is due to spin delocalization over a pair of chlorophyll molecules and this suggestion has recently received support from ENDOR studies.70 It has thus been suggested that the bulk of the chlorophyll in the synthetic unit is present as an oligomer (Chl,) ('antenna' or light-gathering chlorophyll) and the active centre is Chl-H20-Ch1.68b*70 Other chlorophylls also form dimers and oligomers and ENDOR and e.s.r.studies of bacteriochlorophyll have recently been reported ;these are consistent with the suggestion that the e.s.r. signal associated with photosynthesis in purple bacteria is due to delocalization of the unpaired electron over two active-centre molecules. Octaethylchlorin undergoes methylation on nitrogen and forms a mono-N- methyl two NN-dimethyl and NNN-trimethyl derivatives ; n.m.r. studies showed that the nitrogen of the partially reduced ring was not meth~lated.~~ Octaethylchlorin or its zinc complexes can be converted into bile-pigment-like products by treatment with thallium(1rI) trifl~oroacetate.~~ Cobalt corroles [e.g.(23)]are complexes of Co"' and the product from reaction with phenyl-lithium is probably the N-phenyl derivative.They can be obtained by a direct '2 + 2' type synthesis and oxidation with a high-potential quinone 69 A. Goodman E. Oldfield and A. Alkerhand J. Amer. Chem. Soc. 1973,95,7553. 70 J. R. Norris H. Scheer M. E. Druyan and J. J. Katz Proc. Nut. Acud. Sci. U.S.A. 1974,71,4897. 71 J. R. Norris M. F. Druyan and J. J. Katz J. Amer. Chem. Soc. 1973,95 1680; J. Fajer D. C. Borg A. Forman R. H. Felton and D. Dolphin Proc. Nut. Acud. Sci. U.S.A. 1974,71 994. 72 J. A. S.Cavaleiro and K. M. Smith J.C.S. Perkin I 1973 2149. Porphyrins and Related Compounds 531 CH=CH c10,-EtO CH = (24) M = Ni or Co Me clod-Me (28) can effect conversion of certain ring methyl groups into formyl groups.73 The bis-imino-ether of bilirubin diethyl ester can be cyclized to cobalt and nickel 1,19-diethoxytetradehydrocorrins(24); a variety of other metal tetradehydro- corrins with peripheral hydroxy-groups were also prepared by established The dicyano-cobalt(II1) tetradehydrocorrin (25) undergoes thermo- lysis to afford 5-cyano- (26) and 5,15-dicyano-c0mplexes.~ Hydrogenation of nickel and cobalt tetradehydrocorrins is markedly affected by the peripheral alkyl-substitution pattern and a variety of products can be obtained including corrin and dihydrocorrin salts.76 Nickel and cobalt tetradehydrocorrins (27) bearing acetate and/or propionate groups in rings A and D have been prepared by known methods and their 73 M.Conlon A. W. Johnson W. R. Overend D. Rajapaksa and C. M. Elson J.C.S. Perkin I 1973 2281. 74 H. H. Inoffen H. Maschler and A. Gossauer Annalen 1973 141; H. H. Inoffen N. Schwarz and K. P. Heise ibid. p. 146. 75 C. M. Elson A. L. Hamilton. and A. W. Johnson J.C.S. Perkin I 1973 775. 76 A. W. Johnson W. R. Overend and A. L. Hamilton J.C.S. Perkin I 1973,991. A. H. Jackson oxidation by osmium tetroxide to diols such as (28) and pinacol rearrangement of the latter to ketones have been st~died.~’ Certain tetra- tri- and di-hydro- corrin-nickel complexes are cleaved by reducing agents at the direct pyrrole- pyrrole link; the nickel in the resulting bilatrienes is then easily replaced by cobalt and recyclization affords the corresponding cobalt oorrinoids.’’ Palladium(i1) tetradehydrocorrins (30)can be prepared by oxidative cyclization of 1,19-ethoxycarbonyIbiladienes(29).79 CI--. Me ’Me Me Me (31) (32) Much of the work described above has been directed to studying the possibility of synthesizing corrins and ultimately Vitamin B1z by reduction and alkylation of the more unsaturated macrocycles; this has not been diminished even by the completion of total syntheses of Vitamin B by Woodward and Eschenmoser.*’ Variations of the sulphide-contraction method for the synthesis of corrinoid systems have since been described and a 1-hydroxy-zinc corrin (32) has been prepared by photocyclization of an A/D-seco-corrin (3 A metal-free ‘corphin’ (34) has been synthesized by cyclization of the metal complex (33) followed by demetallation.82 ” H.H. Inoffen F. Fattinger and N. Schwarz Annalen 1974 412. ’13 H. H. Inoffen and H. Maschler Annalen 1974 1269. 79 A. Gossauer H. Maschler and H. H. Inoffen Tetrahedron Letters 1974 1277. R. B. Woodward Pure Appl. Chem. 1973,33 145. 8’ E. Gotschi W. Hunkeler H.-J. Wild P. Schneider W. Fuhrer J. Gleason and A. Eschenmoser Angew. Chem. Internat. Edn. 1973,12 910; E. Gotschi and A. Eschen-moser ibid. p. 912. P. M. Miiller S. Farooq B.Hardegger W. S. Salmond and A. Eschenmoser Angew. Chem.Internat. Edn. 1973 12 914. Porphyrins and Related Compounds 533 Me Me Me Me hMe 0 --) Me 3:; H2C / \ Me N Me -N HA-Me / Me ’ I. Me‘ ’Me Me Me (33) ( 34) A radically different approach to corrin synthesis was suggested some years ago by Cornforth involving reductive cleavage of a system containing several isoxazole nuclei (cf ref. 2). This idea has recently been taken up by two other groups of workers and model ‘semi-corrins‘ (36) have been prepared from isoxazoles (35).83 Me Me R’Y o-! H2cve ’$.. R2 Me 0 Me (35) (a) R’ = CN R2 = COMe (36) (b) R’ = CMe RZ = C0,Me /\ 0? Interest in the reactioiis of Vitamin B, (cobalamin) and the mechanisms of its involvement in enzymically controlled reactions continues unabated e.g.diolde-hydrase and ethanolamine ammonia lya~e.~~ Reports have also appeared of the reactions of vinyl ethers with cobalamins and cobaloximes (simple analogues of cobalamin) and the synthesis of formylmethylcobalamin a postulated inter- mediate in the enzymic conversion of ethylene glycol into a~etaldehyde.~ Photolysis of 5’-deoxyadenosylcobalaminunder anaerobic conditions affords 8,5’-cyclic adenine.86 A series of analogues of adenosylcobalamin has also been prepared by allowing cobalamin to react with 5’-deoxy-5’-chloro-nucleotides and their properties have been studied.87 The 13Cn.m.r. spectra of a number of 83 C. Traverso G. P. Pollini A. Barco and G. Deguili Gazzetra 1972 102 243; R. V. Stevens L. E. Dupree W. L. Edmondson L.L. Magid and M. P. Wentland J. Amer. Chem. Soc. 1971,93 6637. 84 P. Y. Law and J. M. Wood Biochim. Biophys. Acta 1973 321 382. 85 G. N. Schrauzer W. J. Michaely and R. J. Holland J. Amer. Chem. Soc. 1973 95 2024; W. J. Michaely and G. N. Schrauzer ibid. p. 5771 ; R. B. Silverman and D. Dolphin ibid. 1974,96 7094; R. B. Silverman D. Dolphin T. J. Carty. E. K. Krodel and R. H. Abeles ibid. p. 7096. n6 P. Y. Law and J. M. Wood Biochim. Biophys. Acra 1973,331 451. 87 H. P. C. Hogencamp Biochemistry 1974 13 2736. 534 A. H. Jackson cobalamins and cobalamides selectively enriched with 3Cin the ligands attached to cobalt have been described.’’ 4 Bile Pigments Structure and Synthesis.-The absolute configuration of natural ( -)-stercobilin (37) has been defined through chromic acid degradation studies.89 Conflicting results have been obtained as to whether or not d-urobilin (a bacterial reduction product from bilirubin) contains a vinyl group in one of its terminal rings.” The structure of mesobilirhodin (38) one of the rearrangement products of e M H-NH HN U t -N HN \Me Me \ P (37) P = CH2CH2C02H urobilin-IXa has been defined as a result of n.m.r.and mass spectral studies;9’ its synthesis has since been achieved by the route indicated below involving the novel synthesis of the dipyrrolic intermediate (39) through coupling of a diazo- Me Me Me cHN2 + HN \Me Me,CO,C PMe (39) ketone with a pyrr~le.’~The total synthesis of phycocyanobilin (40) has also been achieved.93 T.E. Needham N. A. Matwiyoff T. E. Walker and H. P. C. Hogencamp J. Amer. Chem. Soc. 1973,95 5019; T. E. Walker H. P. C. Hogencamp T. E. Needham and N. A. Matwiyoff J.C.S. Chem. Comm. 1974 85; Biochemistry 1974 13 2650. 89 H. Brockmann G. Knobloch H. Pleininger K. Ehl and J. Ruppert Proc. Nat. Acad. Sci. U.S.A. 1971,68 2141. 90 S. D. Killilea andP. O’Carra Biochem. J. 1972,129,1179; A. R. Brewster Z. J. Petryka, M. Weimer C. J. Watson A. Moscowitz and D. A. Lightner Proc. Nat. Acad.Sci. U.S.A. 1974 71 1599. 91 D. J. Chapman H. Budzikiewicz and H. W. Siegelmann Experientia 1972 28 876. 92 A. Gossauer and D. Miehe Annaien 1974 352. 93 A. Gossauer and W. Hirsch Tetrahedron Letters 1973 1451. Porphyrins and Related Compounds 535 MePo NH HN H oy Me M e W M c PMe PMe PMe P Me (41) CHO M = Mg or Zn Bilirubin-IXa (41) labelled with 14C at the central methylene group has been synthesized through Vilsmeier-Haack formylation of an intermediate dipyrrole with [14C]dimethylformamide reduction to the Mannich base and coupling with an a-free dipyrrole; the XI11 a-isomer was also formed as a by-prod~ct.~~ Metal complexes of formyl-biliverdins (42) can be obtained by photo-oxidation of metall~porphyrins,~~ whilst the four isomeric biliverdins have been obtained and separated following oxidative cleavage of haemin in aqueous pyridine in the presence of ascorbic acid or hydra~ine.~~ Chemical and Physical Properties.-Ferric chloride oxidation of bilirubin-IXa dimethyl ester affords biliverdin-IXa dimethyl ester and the IIIa isomer and hydration products of the vinyl side-chains.” Isomerization of bilirubin also occurs reversibly under alkaline conditions to give a mixture of IXa- XIIIa- and IIIa-is~mers.~~ Photo-oxidation of bilirubin has been of considerable interest in recent years because of the use of phototherapy in the treatment of neonatal jaundice.Photo- oxidation in vitro occurs through singlet oxygen and a variety of products have been obtained depending on the solvent etc. including maleic imides 94 H. Plieninger F. El-Barkawi K. Ehl R. Kohler and A. F. McDonagh Annalen 1972,758 195. 95 F. H. Fuhrhop P. K. W. Wasser J. Subramanian and U. Schrader Annulen 1974 1450. 96 R. Bonnett and A.F. McDonagh J.C.S. Perkin I 1973 881. 9’ P. Mannitto and D. Monti Gazzetta 1974 104 513. 98 A. F. McDonagh and F. Assisi Biochem. J. 1972 129 797. A. H. Jackson 0 0 (43)R = H or Me dipyrrylmethenes and propentdyopents (43).99 Intermediate dioxygenated tetrapyrrolic pigments have also been tentatively identified. O0 Biliverdin may also be formed in small amounts but it is a singlet oxygen quencher and is degraded more slowly than bilirubin. Biosynthesis.-As this topic was reviewed in last year's Annual Reports only the more recent developments will be discussed. A recent conferencelo2 has dealt mainly with the more biochemical and clinical aspects of the subject including porphyrias (disorders of porphyrin metabolism). In the biosynthesis of 6-aminolaevulinic acid the pro-2R-hydrogen of glycine is removed and double-labelling experiments show that the pro-2s-hydrogen of glycine (44) occupies the S-configuration in the &position of 6-ALA (45); in the condensation of succinylcoenzyme-A with glycine and subsequent de- carboxylation one step occurs with retention of configuration and the other with inversion.lo3 (47) I 1 (49) P = CH2CH2C02H A = CH2C02H 99 R.Bonnett and J. C. M. Stewart Biochern. J. 1972 130 895; A. D. Lightner and D. C. Crandall Tetrahedron Lerrers 1973 953; A. F. McDonagh Biochern. Biophys. Res. Comm. 1972 48 408. loo C. S. Berry J. E. Zarembo and J. D. Ostrow Biochem. Biophys. Res. Comm. 1972 49 1366. lo' E. MacDonald Ann. Reports (B),1973 69 619.E. M. Doss 'Regulation of Porphyrin and Heme Biosynthesis' Part I Enzyme 1973 16 pp. 1-372; Part 11 ibid. 1974 17 pp. 1-136. Io3 M. M. Aboud P. M. Jordan and M. Akhtar J.C.S. Chem. Comm. 1974. 643. Porphyrins and Related Compounds The synthesis of four aminomethyl-pyrromethanes related to porphobilinogen (PBG) (46) and bearing acetic and propionic acid side-chains was described last year ;lo' other syntheses have also been described,lo4 including the direct use of PBG (46)cs an alkylating agent in reactions with its lactam (47)to form the pyrromethane lactam (48). Hydrolysis of the latter condensation with PBG lactam and hydrolysis then gave the aminomethyltripyrrane (49). This has also been synthesized by another similar route but details are not yet available;lo5 it did not appear to be a substrate for the deaminase-synthetase system obtained from wheatgerm or human erythrocytes and it appeared to inhibit the formation of uroporphyrinogen-III(50).'p5 No definitive indication of which pyrromethane is an intermediate in the formation of uroporphyrinogen-111 has yet appeared but an experimental solution to this outstanding problem cannot now be far away.Further theoretical speculations have also appeared. lo6 Doubly labelled coproporphyrinogen-I11 (51) ( 14C in the 2-propionic acid side-chain and 3H at the meso-positions) is converted into protoporphyrin-IX (52) by incubation (50) R = A (51) R = Me with a haemolysate from chicken erythrocytes with loss of 50% of the tritium label showing that the final oxidation of porphyrinogen to porphyrin is essentially a stereospecific process.* It has previously been accepted that a single enzyme ('coproporphyrinogen oxidative decarboxylase') catalyses the overall conversion of coproporphyrinogen-111 into protoporphyrin-IX but evidence for two separate enzymes has now been presented.'" It is suggested that the repression of haem biosynthesis observed in aerobic cultures of the yeast Saccharornyces cerezjisiae by high concentrations of glucose is due to inhibition of the second enzyme (responsible for oxidation of the protoporphyrinogen-IX).Confirmation that the pro-R-methyl group at C-12 of Vitamin B, is derived from methionine"' has now been provided by I3C- and 3H-labelling experiments using Propionibacteriurn shermanii; the methyl groups at both C-7 and C-12 *04 A.Valasinas E. S. Levy and B. Frydman J. Org. Chem. 1974 39 2872; J. Bausch A. Eberle and G. Muller 2. Naturforsch. 1974 29c 479. '05 R. B. Frydman A. Valasinas S. Levy and B. Frydman F.E.B.S. Letters 1974 38 134. lo' C. S. Russell J. Theor. Biol. 1974,47 145. lo' R. Poulson and W. J. Polglase F.E.B.S.Letters 1974 40 258. A. H. Jackson were shown to be incorporated intact."* A review of corrin biosynthesis has also appeared"'; full details of the Yale group's work confirming the inter- mediacy of uroporphyrinogen-111 the incorporation of seven methyl groups from methionine and 3Cstudies of the absolute stereochemistry of the methyla- tion process at C-12 have also been reported."' O8 A.R. Battersby M. Ihara E. McDonald J. R. Stephenson and B. T. Golding J.C.S. Chem. Comm. 1974,458. lo9 A. I. Scott Bioorg. Chem. 1974 3 229. 'lo A. I. Scott C. A. Townsend K. Okada and M. Kajiwara J. Amer. Chem. Soc. 1974 96,8054; A. I. Scott C. A. Towasend K. Okada M. Kajiwara R. J. Cushley and P. J. Whitman ibid. p. 8069.

ISSN:0069-3030    

DOI:10.1039/OC9747100519

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

20 Enzyme Mechanisms By A. D. B. MALCOLM and J. R. COGGINS Department of Biochemistry University of Glasgow Glasgow GI2 800 1 Introduction Many of the recent significant advances in enzymology have been the result of improvements in resolution and sensitivity of analytical techniques such as X-ray crystallography and n.m.r. The resulting knowledge is yielding an even more detailed picture of both the static and dynamic aspects of enzyme function. Enzymes whose three-dimensional structures were published during the year (although some had been publicized earlier) are given in Table 1. The successful design of specific and powerful enzyme inhibitors (with their obvious medical applications) is a direct consequence both of this detailed knowledge of the structure of enzymes and their active centres and substrate-binding sites and of increasingly accurate predictions about the structures of transition states in enzyme-catalysed reactions.Table 1 Recent three-dimensional structures of enzymes Enzyme Source Resolut ion/A Reference Glyceraldehyde phosphate deh ydrogenase lobster 3.0 a Alcohol dehydrogenase Hexokinase Phosphoglycerate kinase horse liver yeast yeast horse muscle 2.9 7.0 3.0 3.5 b d e c Adenylate kinase Trypsin Trypsin + inhibitor Ribonuclease A Phosphorylase b pig muscle cow pancreas cow pancreas rabbit muscle cow 3.0 2.7 1.9 2.5 6.0 gh f I j a M. Buehner G. C. Ford D. Moras K. W. Olsen and M. G. Rossmann J. Mol. Biol. 1974 90 25; * H. Eklund B. Nordstrom E. Zeppezauer G. Soderlund I. ohlsson T.Boiwe and C.-I. Branden F.E.B.S. Letters 1974,44.200; W. F. Anderson. R. J. Fletterick and T. A. Steitz J. Mol. Biol.,1974,%6,261 ; * C. C. F. Blake and P. R. Evans J. Mol. Biol. 1974 84 585; "T. N. Bryant H. C. Watson and P. C. Wendell. Nature 1974 247 14; f G. E. Schulz M. Elzinga F. Marx and R. H. Schirmer Nature 1974 250 120; 8 R. M. Stroud L. M. Kay and R.E. Dickerson J. Mol. Biol.,1974,83 185; R. Huber D. Kukla W. Bode P. Schwager K. Bartels J. Deisenhofer and W. Steigemann J. Mol. Biol. 1974 89 73; C. H. Carlisle R. A. Palmer S. K. Mazumdar B. A. Gormisky and D. G. R. Yeates J. Mol. Biol. 1974 85 1 ; j L. N. Johnson N. B. Madsen J. Morley and K. S. Wilson J. Mol. Biol. 1974 90 703. 539 A. D.B. Malcolm and J. R.Coggins 2 Hydrolases Chymotrypsin.-On the basis of the three-dimensional structure of chymo-trypsin'" and the known involvement in catalysis of Ser-195 and His-57 two mechanisms have been proposedlb which are assumed to apply to other mammalian and bacterial serine proteases (Scheme 1).Since protein X-ray crystallography cannot at present locate protons it has been impossible to estab- lish which of these two suggestions is correct. In fact some reviewers' have suggested that the two mechanisms may be canonical forms of each other and therefore identical. That this is not the case was shown in two similar paper^,^ which used 13Cn.m.r. to observe the state of ionization of the essential histidine in one particular serine protease uiz. a-lytic protease from Myxobacter 495.Since there is only one histidine in this enzyme there is no interference from other residues. The authors observed that over the pH range 5-9 where the enzymes pass from being inactive to active there was no change in the ionization state of the histidine residue as monitored by the C-H coupling constant of C-2. This implies that the observed pK of 6.7 in the enzyme's activity must be attributed to the aspartate residue and suggests that it is mechanism (a) in Scheme 1 which is operative. This has the attractive a posteriorijustification that the intermediate does not have to sustain a charge separation between the aspartate and the histidine since this would be expected to be energetically unfavourable in a non-polar environment. The unusually high pK of 6.7 for the asparate side- chain which this work indicates may be rationalized by noting that the histidine insulates it from the solvent and so destabilizes the carboxylate anion relative to the undissociated acid.Unfortunately this conclusion is at variance with that based on a 'H n.m.r. study (at up to 300 MHz)~of chymotrypsin. A single proton observed at about 15 p.p.m. downfield from dimethylsilapentane sulphonate is identified as being the one hydrogen-bonded between Asp-102 and His-57 on the basis of its chemical shift and its disappearance either on the use of 2H20as solvent or on specific alkylation of His-57. Furthermore a similar proton is seen in chymotrypsinogen trypsin trypsinogen subtilisin and a-lytic protease. However on varying the pH it is found that this proton titrates with a pK of 7.5 in chymotrypsin and at 6.5 in the acylated enzyme [N6-(N-acetylalanyl)-N '-benzoylcarbazoylchymo-trypsin] and therefore it is His-57 which is the residue to lose a proton on raising the pH to form the active enzyme.Further information can be gained about the electronic environment at the active site from the difference in chemical shift between high and low pH for the native and acyl enzymes. At low pH the proton resonance is seen in either enzyme at the low-field position of -18p.p.m. but ' (a) B. W. Matthews P. 9. Sigler R. Henderson. and D. M. Blow Nature 1967 214 652; (b)D. M.Blow J. J. Birktoft and B. S. Hartley ibid. 1969 221 337. S. Doonan C. A. Vernon and B. E. C. Banks Progr.Biophys. Mol. Biol. 1970,20,249. M. W. Hunkapiller S. H. Smallcombe D. R. Whitaker and J. H. Richards (a) Bio-chemistry 1973 12 4732; (b)J. Biol. Chem. 1973 248 8306. G. Robillard and R. G. Shulman J. Mol. Biol. 1974 86 519. Enzyme Mechanisms ??- I =F E 01 0=0\2 / 01 /O=U \El? /O /O N z N 3 O ?-X x I /\ 0IT II z I 0 / T 01 / z 1 0 O=U \g/ / A. D.B. Malcolm and J. R.Coggins at high pH it occurs at -15 p.p.m. in the native enzyme and at -14.4p.p.m. in the acyl enzyme suggesting the structures shown in Scheme 2. ASP-102 His-57 Ser-195 0 / II CH \ C \/\ ,CH* CH 0. .o I c=o I R' / 0 CH / II C CH \/\ h \* CH 0..H-N,,+,N /O c=o \ R' Scheme 2 Since protonation of His-57 shifts the resonance by 3.6 p.p.m.but the hydrogen- bonding to Ser-195 only shifts it by 0.6 p.p.m. it can be concluded that the serine proton is only slightly shifted towards the histidine. The differences between these authors will doubtless be resolved eventually. It is however very encouraging that n.m.r. is now sensitive enough to detect a single nucleus in a large protein molecule and that it can also provide information about the electronic distribution around that nucleus. The availability of such information means that the time when quantum mechanics can be used to predict the rates of enzyme-catalysed reactions has been brought a step nearer. It will be noticed that in both mechanisms (a) and (b) (Scheme l),a tetrahedral intermediate (1) has been drawn (rather than direct displacement by the serine hydroxy-group of the alkoxide moiety).Although there is still some dispute about this it is generally agreed to be the most likely mechanism. Considerable effort has been expended in attempting to delineate a precise structure for the tetrahedral intermediate. One fruitful approach to this problem is the use of various analogues of the proposed structure and in particular derivatives of boronic acid have been shown to bind extremely tightly to chymotrypsin.' For example 2-phenylethaneboronic acid (2)and trans-pdyreneboronic acid (3) form complexes with chymotrypsin having dissociation constants of 50 pmol 1-'and 26 pmol 1-',respectively. This is considerably tighter binding than is shown by amide and ester substrates of similar structure and it suggests that the oxygen atom of Ser-195 may occupy the vacant fourth co-ordination position on the boron atom generating tetrahedral co-ordination.The tight binding of J. D. Rawn and G. E. Lienhard. Biochemistry 1974 13 3124. Enzyme Mechanisms OH / these species is of course consistent with the hypothesis that the active sites of enzymes are complementary to the transition states of the reactions they cata- ly~e.~.’ 220MHz ‘H n.m.r. studies’ on various borate derivatives bound to chymo- trypsin show that the ionization of His-57 between pH 6 and 9.5 is prevented. (This is shown by the chemical shift of the proton in the hydrogen bond between Asp-102 and His-57).Also the increased downfield shift of this proton suggests that the histidine is hydrogen-bonded to the borate as shown in Scheme 3. ASP-102 His-57 Ser-195 n R /\ OH Scheme 3 There is still some doubt whether zymogens such as chymotrypsinogen possess any catalytic properties and if not why not.9a This problem is exemplified by chymotrypsinogen where the charge-relay triad is also present.’” The pK,‘s and reactivities of the two histidines (His-40 and His-57) have been determined by the competitive-labelling technique,’ * using l-fluoro-2,4-dinitrobenzene.’ For His-40 the relative rate us. pH profiles are qualitatively similar for the enzyme and zymogen. However the plot for His-57 in chymotrypsin shows a sharp decrease in reactivity at pH8 which is not found in chymotrypsinogen.The authors suggest that if the role of His-57 is firstly to activate Ser-195 by hydrogen bonding and secondly to catalyse the departure of the amino-moiety from the tetrahedral intermediate a rotation of His-57 will be necessary between these two L. Pauling Chem. Eng. News 1946 24 1375. J. B. S. Haldane ‘Enzymes’ Longmans Green London 1930. G. Robillard and R.G. Shulman J. Mol. Biol. 1974 86 541. (a) B. Kassell and J. Kay Science 1973 180 1022; (b) S. T. Freer J. Kraut J. D. Robertus H. T. Wright and Ng. H. Xuong Biochemistry 1970 9 1997. 10 H. Kaplan K. J. Stevenson and B. S. Hartley Biochem. J. 1971 124 289. I’ W. H. Cruickshank and H. Kaplan J. Mol. Biol. 1974. 83 267. 544 A. D. B. Malcolm and J.R. Coggins steps. Such a conformational change would require the breakage of the hydrogen bond between Asp-102 and His-57 causing the observed break in the reactivity us. pH curve. In the zymogen this conformational change cannot readily occur and so there is little catalysis and no discontinuity in the reactivity towards FDNB. Enzyme Subsites.-It has been recognized for some time that a substantial contribution to the rate enhancement in enzyme-catalysed reactions is made by raising the entropy of activation (in some cases this may be even more important than the lowering of the enthalpy of activationI2) and biochemists have used at least a dozen different names for this phenomenon. It was therefore encouraging to see a paper13 in which the authors volunteer to withdraw one of these terms -~ ‘togetherness’-which they had introduced.This increase in the activation entropy is achieved because in the uncatalysed rgaction there is a substantial loss of translational and rotational entropy in reaching the transition state but in the enzyme’s reaction some of this entropy has already been lost in the binding step and is therefore no longer involved in the rate-limiting step of the catalysed reaction. The free energy required to permit this entropy loss on binding is of course supplied by the free energy of binding of the various parts of the substrate. The realization of this has stimulated a study of the so called ‘subsites’ on the enzyme i.e. the area which is not involved in catalysis per se but is involved in substrate binding and which therefore influences the specificity of the enzyme and can also help in orienting the susceptible bonds at the active site.This topic was also reviewed four years ago.14 A study of the papain-catalysed hydrolysis of various oligomers of D-and L-alanine’ showed that the enzyme possessed binding sites for seven residues four (S,-S,) on the N-terminal side of the bond to be cleaved and three (S;-S;) on the C-terminal side (Scheme 4). Substrate [-{{I J-Enzyme Scheme 4 T. C. Bruice. in ‘The Enzymes’ ed. P. D. Boyer Academic Press New York 1970 Vol. 2 p. 217. l3 W. P. Jencks and M. 1. Page Biochern. Biophys. Res. Comm. 1974,57 887. l4 M. Akhtar and D. C. Wilton Ann. Reports (B),1971 68 167. Is I.Schechter and A.Berger Biochem. Biophys. Res. Cornm.. 1967 27 157. Enzyme Mechanisms A consideration of the three-dimensional structure of papainI6 led Lowe to suggest that the Sl site might contain Trp-177 and so might bind hydrophobic side-chains.l7 The hydrolysis of some carbobenzoxyglycyl-L-amino-acid amides by papain was studied on the basis that the known preference of the S site for L-Phe should ensure that these molecules are bound as indicated in Scheme 5. R ~CH,-O-CO-NH-CH2-CO-NH-CH-CONH2 I Scheme 5 S; was found to show a preference for the residues Val Leu and Trp and this preference occurs both in the formation of the initial Michaelis complex and also in the subsequent acylation of the enzyme. Additionally oxidation of the tryptophan residues of papain using N-bromosuccinimide'8 followed by a determination of the kinetic parameters of the enzyme modified at Trp-69 and also at both Trp-69 and Trp-177 showed that Trp-177 was indeed part of the S; site and furthermore that the S subsite (the one which shows a preference for L-Phe) contains Trp-69.The nature of some of the other subsites (S, S, S,) has been studied by using the fluorescence properties of the mansyl (Mns) group (4).19 In the series of (4) substrates X-Val-Glu-Leu-Gly the only bond to be cleaved is that between Glu and Leu. The introduction of the Mns group into X had the effect of diminishing K and decreasing k,,,. Papain does not significantly alter the fluorescence properties of free mansic acid but when the mansyl group is incorporated into a substrate for papain the addition of the enzyme results in an increase in the fluorescence showing that the Mns group is embedded in a hydrophobic part of the protein from which the polar solvent is excluded.It is interesting that this occurs even when the active-site sulphydryl (Cys-25)has been inactivated with l6 J. Drenth J. N. Jansonius R. Koekoek H. M. Swen and B. G. Wolthers Narure 1968,218,929. I' M. R. Alecio M. L. Dann and G. Lowe Biochem. J. 1974 141,495. G. Lowe and A. S. Whitworth Biochem. J. 1974,141 503. l9 J. Lowbridge and J. S. Fruton J. Biol. Chem. 1974 249 6754. 546 A. D. B. Malcolm and J. R.Coggins mercury mercaptoethanol iodoacetate or N-ethylmaleimide. This shows that the subsites of the enzymes are capable of binding substrate even when the active site has been blocked (or even in the absence of an intact site).The existence of such subsites has been neatly exploited in designing yet more specific active-site-directed inhibitors. For example kallikrein and thrombin both display trypsin-like specificity towards substrates. However the chloro- methyl ketone Ala-Phe-Lys-CH2C1 inactivates kallikrein but not thrombin,20 and this specificity must reflect differences in the subsites of the two enzymes. E1astase.-A discussion of the subsites of elastase will be found in last year's Report." A search for better substrates for elastase22 revealed that if a dicar- boxylic acid was used to provide the N-acyl group on the Ala-Ala-Ala-p-nitro- anilide substrate (instead of the usual acetyl or t-butyloxycarbonyl) there was a decrease of K, an increase in k,,, and the substrate was also more soluble.Succinyl and glutaryl groups were particularly effective suggesting that the S site may have a preference for binding anions. Alkaline Phosphatase.4f all the enzymes catalysing 'simple' reactions perhaps none has aroused as much controversy as alkaline phosphatase from E. coli. A short review of this enzyme will be found in a recent Report.23 Alkaline phos- phatase is known to consist of two identical subunits of molecular weight 40 OOO but incorporation of 32Pfrom labelled substrate reveals only one active site. This together with other data led Lazdunski to propose the so called 'flip-flop' mechani~m.~~ This involves anti-co-operativity of a very extreme type.The assumption is that each subunit may adopt one of three different conformations F in the free enzyme; R,when the active serine is phosphorylated; and T when the substrate is bound non-covalently. The catalytic cycle is then as shown in Scheme 6. In the flip-flop step the dephosphorylation of one subunit occurs simultaneously with phosphorylation of the other both subunits undergoing a conformational change. Only in this way could one explain the finding that p-chloroanilide phosphonate a competitive inhibitor which was presumed to bind at the active site accelerates the dephosphorylation of the monophosphorylated enzyme. Lazdunski's group have removed the Zn2+ by dialysis against edta and then added Zn2+ Co2+,CdZ+ Mn2+ or Cu2+.All the reconstituted metallo-enzymes are active. The use of a stopped-flow apparatus to study the pre- steady-state kinetics of the hydrolysis of 2,4-dinitrophenyl phosphate revealed a biphasic burst in all cases except fur the Mn2+ enzyme. The phorphorylation *' J. R. Coggins W. Kray and E. Shaw Biochem. J. 1974 138 579. M. Akhtar and D. C. Wilton Ann. Reports (B) 1974 70 98. 22 E. Kasafirek P. Fric and F. Malis F.E.B.S. Letters 1974 40 353. 23 M. Akhtar and D. C. Wilton Ann Reports (B) 1972,69 140. 24 M. Lazdunski C. Petitclerc D. Chappelet and C. Lazdunski European J. Biochem. 1971 20 124. 25 (a)D. Chappelet-Tordo M. Iwatsubo and M. Lazdunski Biochemistry 1974,13 3754; (b)D. Chappelet-Tordo M.Fosset M. Iwatsubo C. Gache and M. Lazdunski ibid. 1974,13 1788. Enzyme Mechanisms F-F cpw :“r ROH ‘Flip Flop’ P P R-T Scheme 6 of the first site was shown to be -lo3 times faster than phosphorylation of the second site. This extreme difference in reactivity between the two sites is supported by observation of the change in extinction at 640 nm of the Coz+ phospnatase on reaction with inorganic phosphate or P-glycerophosphate. This reaches a limit when the concentrations of substrate and dimeric enzyme are equal showing that at any time only one of the two active sites will bind substrate. These results are not in agreement with those of an earlier paper,26 where stopped-flow studies using a different substrate (p-nitrophenyl phosphate) suggest that the active sites are equivalent and independent.It is difficult if not impossible to reconcile these data although it may be noted that the stoicheio- metry of the metal in the enzyme and also of non-covalently bound inorganic phosphate is known to vary with the conditions used for isolation and storage of the enzyme. The stoicheiometry of 32Pincorporation is an important piece of evidence in favour of the flip-flop mechanism. It was therefore important to establish that neither phosphorylation nor dephosphorylation could occur during the quench- ing (by 10M-HC1 in 8M-urea) of the phosphatase reaction. The various controls have now been reported (but carried out on the enzyme from calf and pig intes- tine)25b and it has been confirmed that [32P]AMPwill only label half of the total available active sites.Stopped-flow studies show that the half-of-the-sites reactivity of this enzyme is so similar to that of the E. coli one that a flip-flop mechanism is again suggested. W. Blochand M. J. Schlesinger. J. Bioi. Chem. 1973 248 5794. A. D.B. Malcolm and J. R.Coggins At low concentrations pyrophosphate methylene diphosphonate and imido- phosphate stimulate alkaline phosphatase,” whereas at higher concentrations they behave as competitive inhibitors. The authors interpret this in terms of positive co-operativity between the active sites such that at low substrate con- centrations the inhibitors become activators in the way that maleate stimu- lates aspartate transcarbamylase at low aspartate concentrations.28 Since these inhibitors cannot phosphorylate the enzyme the question of whether or not a flip-flop mechanism exists here is not strictly relevant.However the authors feel that a flip-flop mechanism implies that stimulation of hydrolysis of p-nitro- phenyl phosphate requires association and subsequent dissociation between the activator and the other site. They dislike the idea that inserting an extra associa- tion-dissociation equilibrium could lead to an increase in the overall rate and they therefore maintain that their data are not consistent with a flip-flop mechan- ism for alkaline phosphatase. Whether or not such an activation is possible depends on the relative magnitudes of the rate constants involved.After all enzymes do catalyse reactions in spite of there being extra association4issocia- tion reactions. The above data therefore can most fairly be said to provide little information about this problem. With enzymes which display subunit interactions an interesting approach has been to prepare hybrids of the enzyme in which subunits from different species or chemically inactivated subunits are combined with ‘normal’ subunits and the resultant interactions have been studied. A hybrid of alkaline phos- phatase comprising a wild-type active subunit and an inactive mutant subunit has been made2’ and found to have properties identical with the wild-type dimer (after allowing for there being twice the number of active sites in the latter). The authors correctly point out that this is not consistent with the type of inter- action implied in the flip-flop mechanism.Of course until the reasons for the lack of activity of the mutant enzyme are known it would be dangerous to jump to conclusions about the native enzyme. 3 Pyridine-nucleotide-linked Dehydrogenases Crystallographers have continued to amass structural data on dehydrogenases and so although this area was reviewed last year,2’ it is useful to review some of the results obtained since then. Glyceraldehyde-3-phosphateDehydrogenase.-The most important progress has been made with lobster glyceraldehyde-3-phosphatedehydrogenase (GAPDH) which catalyses the oxidation of glyceraldehyde phosphate and the subsequent phosphorylation of the thioester intermediate (Scheme 7).Rossmann and his colleagues have determined an electron-density map at 3.0 8 resolution of an *’ S. J. Kelly J. W. Sperow and L. G. Butler Biochemistry 1974,13 3503. J. C. Gerhart and A. B. Pardee Cold Spring Harbor Symp. Quanr. Biol. 1963 28 491. 29 W. Bloch and M. J. Schlesinger J. Biol. Chem. 1974 249 1760. Enzyme Mechanisms 549 0\c/H 0\C/S-Enz I CH-OHI CH,-O-PO;- + Enz-SH + NAD' S CH-OH I CH,-O-PO$ + NADH + Hf - Scheme 7 averaged ~ubunit,~'.~' which shows that like other dehydrogena~es,~' the GADPH subunit is made up of two domains. One (residues 1-149) is the co- enzyme-binding fold while the other (residues 150-334) provides residues for catalysis specificity and subunit-subunit interactions.The coenzyme-binding domain is similar to that found in other dehydrogenases indeed some of the important residues involved in NAD+ binding are the same in GAPDH lactate dehydrogenase (LDH),33 and alcohol dehydrogenase (ADH). 34 For example the 0-2' of the adenine ribose in GAPDH is bound by Asp-32 and by the homolo- gous residues Asp-53 in LDH and Asp-223 in ADH while in all three cases there is a nearby glycine (Gly-7 in GAPDH Gly-28 in LDH and Gly-199 in ADH). It has long been known that there are stereochemically two classes of pyridine- nucleotide-linked dehydrogenases class A type dehydrogenases exemplified by LDH transfer hydrogen to the A-side of the nicotinamide ring (Scheme 8) Me CONH + D-C'~C02H + MeCOC0,H + H+ OCONH2 / " OH I I ADPR ADPR Scheme 8 30 M.Buehner G. C. Ford D. Moras K. W. Olsen and M. G. Rossmann J. Mu/. Biol. 1974 82 563. 31 M. Buehner G. C. Ford D. Moras K. W. Olsen and M. G. Rossmann J. Mol. Biol. 1974 90 25. 32 M. Buehner G. C. Ford D. Moras K. W. Olsen and M. G. Rossmann Proc. Nut. Acad. Sci. U.S.A. 1973 70 3052. 33 M. J. Adams M. Buehner K. Chandrasekhar G. C. Ford M. C. Hackert A. Liljas M. G. Rossmann I. E. Smiley W. S. Allison J. Everse N. 0.Kaplan and S. S. Taylor Proc. Nut. Acad. Sci.U.S.A. 1973 70 1968. 34 (a) H. Eklund B. Nordstrom E. Zeppezauer G. Soderlund I. Ohlsson T. Boiwe and C.-I. Brandtn F.E.B.S. Letters 1974 44 200; (b) R. Einarsson H. Eklund E. Zeppezauer T. Boiwe and C.-I. Brandtn European J. Biochem. 1974 49 41. A.D.B. Malcolm and J.R.Coggins and class B type dehydrogenases e.g. GAPDH transfer hydrogen to the B-side of the ring.35 A comparison of the GAPDH structure3' with the published LDH structure33 reveals the molecular basis of this stereochemical difference In LDH the A-side of the ring is exposed to substrate and the B-side is in a hydrophobic environment with access blocked by Val-32. However in GAPDH there is steric hindrance between the carboxyamide group of the coenzyme and residues Ala-120 and Pro-121. As a result the NAD+ conformation is changed in one respect :there is a 180" rotation about the ribose-nicotinamide glycosidic bond which leads to the exposure of the B-side of the nicotinamide ring to His-195 0 ,C-H HO R N LYS-183 Asn-3 13 LDH GAPDH (A-type dehydrogenase) (B-type dehydrogenase) Scheme 9 substrate (Scheme 9).This conformation of the coenzyme is stabilized by a hydrogen bond between the carbonyl group of the nicotinamide and Asn-313. The NAD+-binding site in GAPDH is shown diagrammatically in Figure 1. There appear to be two anion-binding sites in the active centre. One which is shown occupied by a sulphate is the correct distance from the essential Cys-149 to be the site which the phosphate group of glyceraldehyde phosphate occupies during formation of the thioester intermediate (see Scheme 7). The second anion- binding site (not shown in Figure 1) is 4 A from the first and it probably involves Lys-191 and Arg-231. This site is further from Cys-149 and is well situated to be the binding site for the inorganic phosphate required for phosphorylation of the thioester (Scheme 7).Figure 1 shows clearly the involvement of amino-acid side-chains from two different subunits in the active centre of GAPDH. Lys-183 which binds to the pyrophosphate of the NAD' is contributed by one subunit and most of the rest of the active site including the essential thiol group by a second subunit. GAPDH is only the second subunit protein for which sequence data from a number of different species as well as a three-dimensional structure are available. 35 M. Dixon and E. C. Webb 'Enzymes' Longmans London 2nd edn. 1964 p. 267. Enzyme Mechanisms 55 I Figure 1 The NAD+-binding site of GAPDH (Reproduced by permission from J.Mol. Biol. 1974,90 25) It has been pointed out that as with the haemoglobins the residues involved in subunit contacts are highly c~nserved.~’ Mammalian GAPDH’s show negative co-operativity in their binding of co-enzyme NAD+ and ‘half-of-the-sites’ reactivity towards substrate a~ylation.~~,~’ These molecular properties are important control mechanisms which are common to many regulatory systems.38 During the past year an understanding of the structural basis of these properties of GAPDH has begun to emerge. A detailed spectroscopic study of the interaction of rabbit muscle GAPDH with NAD’ and with a fluorescent analogue of NAD+ (5)39 has established Rib-a,-Rib-Nic (5) 36 A. Conway and D. E. Koshland Biochemistry 1968,7,4011. 37 0.P.Malhotra and S. A. Bernhard J. Biol. Chem. 1968 243 1243. 38 A. Levitzki W. B. Stallcup and D. E. Koshland Biochemistry 1971 10 3371. j9 J. Schlessinger and A. Levitzki J. Mu/. Biol. 1974 82 547. 552 A. D.B. Malcolm and J. R.Coggins that the greatest structural change in the protein tetramer occurs upon binding the first molecule ofcoenzyme. Progressive structural changes occur at the adenine subsite of the NAD+-binding site but the nicotinamide subsite is not significantly changed with the binding of successive molecules of coenzyme. The negative co-operativity must be due to these progressive conformational changes at the adenine subsite. The lack of change at the nicotinamide site accounts for the lack of change in the catalytic power of the whole NAD' site as a function of coenzyme ~aturation.~' It has been known for some time that acylation of GAPDH with substrate- like acyl phosphates leads to the rapid incorporation of two moles of acylating reagent per mole of tet~amer.~' There are four potential sites of acylation the active-centre thiol groups of Cys-149 in each of the four chemically identical subunit^,^' but only half of the sites react suggesting that in the tetramer there are two non-equivalent pairs of active-centre thiol groups.Some alkylating reagents (6)--(9) also inactivate GAPDH by reacting with half of the active-site CH,I CHzI I 1 F c=o c=o I I ONoz o=s=o ON". NO2 ON" OH CO,H thiols per tetramer.42 In contrast all four thiols are readily alkylated by iodo- acetate and iodoacetamide$2 and there is a linear dependence of enzyme inactiva- tion on the extent of alkylation.The spectroscopic results offered an explanation of the difference between half-of-the-sites acylating and alkylating reagents and the all-of-the-sites reagents. 39 The half-of-the-sites reagents must cause an interaction with the adenine subsite that can be transmitted to the other subunits whereas the all-of-the-sites reagents must affect only the nicotinamide region and no conformational changes are transmitted to the other subunits. In another paper this explanation of half-of-the-sites reactivity is developed further by taking account of the structural data available for GAPDH. The crystallo- graphic results show that GAPDH has tetrahedral symmetry to better than 0.5 The NAD' is bound close to a subunit contact; most of the binding site is on one subunit but a loop involving Lys-183 of a second subunit is also involved (see Figure 1).It is proposed that all-of-the-sites alkylating reagents result in non-co-operativity i.e. modify Cys-149 in all four subunits equally well because 40 J. Teipel and D. E. Koshland Biochim. Biophys. Acta 1970 198 183. 4' J. I. Harris and R.N. Perham J. Mol. Biol. 1965 13 876. 42 A. Levitzki J. MoI. Biol. 1974 90 451. Enzyme Mechanisms 553 they are too small to interact with both Cys-149 and the Lys-183 loop contributed by the other subunit. In contrast the usually larger alkylating reagents which show half-of-the-sites reactivity (negative co-operativity) modify Cys-149 and at the same time interact with the Lys-183 loop inducing a conformational change in that subunit which renders its Cys-149 inactive towards thiol reagents.The exact nature of the perturbations in conformation which half-of-the-sites reagents impose remains to be established. Careful model building or crystallo- graphy should soon provide an answer. Alcohol Dehydrogenase.-Liver alcohol dehydrogenase is a dimer of iden tical subunits each being a single polypeptide chain of 374 amino-a~ids.~~ There are two zinc atoms bound firmly to each subunit.44 Crystallography has shown that each subunit is divided into two domains separated by an active-site cleft.45 One of these domains (residues 176-318) binds the coenzyme and is structurally similar to the corresponding domains found in LDH,33 malate dehydrogenase (MDH),46 and GAPDH31 (see below).The other domain referred to as the catalytic domain consists of residues 1-175 and 319-374. The structure of the complex between the enzyme and adenosine diphosphate ribose (ADPR) (an NAD' analogue lacking the nicotinamide ring) has been studied.34 This shows that the adenine moiety binds in a hydrophobic pocket with its amino-group pointing away from the enzyme. The C-2' hydroxy-group of the adenosine ribose is hydrogen-bonded to Asp-223 which is one of the four invariant residues in the coenzyme-binding sites of LDH GAPDH and ADH4' (the other invariant residues using the ADH numbering system are Gly-199 Gly-203 and Gly-271).The invariance of Asp-223 suggests that this hydrogen bond is a very important feature of NAD' binding and it may even account for the fact that all of these dehydrogenases have a marked preference for NAD+ over NADP'. NADP' is phosphorylated on the C-2' hydroxy-group and it could not therefore be bound to Asp-223 in the same manner as NAD'. Hydrophobic coenzyme-competitive inhibitors e.g. salicylate and 8-anilino- naphthalenesulphonate (Ans) bind to the hydrophobic adenosine-binding pocket.34b The terminal ribose of ADPR is hydrogen-bonded through its 0-3' and 0-2' hydroxy-groups to the main-chain carbonyl oxygen atoms of Ile-269 and Gly-293. Very similar interactions with equivalent residues have been found for NAD' binding to LDH (the residues involved are Glu-140 and Ala-100).33 The guanidinium group of Arg-47 is the positively charged group involved in ionic interaction with the pyrophosphate group.It plays an analogous role to Lys-183 in GAPDH and Arg-101 in LDH although no structural homology is involved. A recent chemical study4* has shown that under mild conditions J3 H. Jornvall European J. Biochem. 1970 16 25. 44 A. Akeson Biochem. Biophys. Res. Comm. 1964 17 21 1. 45 C.-I Branden H. Eklund B. Nordstrom T. Boiwe G. Soderlund E. Zeppezauer I. Ohlsson and A. Akeson. Proc. Nut. Acad. Sci. U.S.A. 1973 70 2439. 46 L. E. Webb E. J. Hill and L. J. Banaszak Biochemistry 1973 12 5101. " I. Ohlsson B. Nordstrom and C.-1. Branden J. Moi. Biol. 1974 89 339. 48 L. G. Lange J.F. Riordan and B. L. Vallee Biochemistry 1974 13 4361. 554 A. D.B. Malcolm and J. R.Coggins butanedione and phenylglyoxal modify two arginine residues in ADH and there is a concomitant loss of NADH-binding ability. NADH protects these residues against modification. The modification of the two arginines affects the carboxy- methylation of the 'functional' Cys-46. Normally this cysteine is rapidly and preferentially modified by iodoacetate but not by i~doacetamide.~~ It is likely that one of the modified arginines is Arg-47 whose modification would block coenzyme binding. The preferential modification of Cys-46 may be explained as an affinity-labelling effect the reagent first binds to Arg-47 which has been shown to be a general anion-binding and then rapidly modifies the adjacent CYS-46.The two zinc atoms are bound in the catalytic domain. Only one of the zinc atoms is involved in ~atalysis,~*~~~ and this is situated at the bottom of a deep pocket between the two domains.45 It is tetrahedrally co-ordinated to three protein ligands (two sulphur atoms from Cys-46 and Cys-174 and a nitrogen atom from His-67) and to a water molecule or a hydroxyl ion (depending on pH). The other non-catalytic zinc is held in a distorted tetrahedral structure by four sulphur atoms from cysteines 97 100 103 and 11 1. The structure of the NAD+-enzyme complex is not yet available but it is possible by assuming that the whole coenzyme conformation is the same as in CYS-46 Zn ?-0H NAD' Zn!?-OH2 His-67 __-.cys-174 'ONH? L T I -NADY R AD'PR Scheme 10 49 T. K. Li and B. L. Vallee Biochemistry 1965 4 1195. H. Theorell The Haruey Lecfures 1965 61 17. 51 H. Jornvall Proc. Nat. Acad. Sci. U.S.A. 1973 70 2295. Enzyrne Mechanisms LDH (which like ADH is an A-type dehydrogenase) to deduce what it is like.34" The resulting model places the C-4 atom of the nicotinamide ring 4.5 A from the catalytic zinc with the A-side of the ring facing the water ligand on the zinc. A substrate alcohol can be built into this model (without distortion) with the alcohol hydroxy-group replacing the water hydroxy-group in the zinc co-ordination and the hydrogen atom that is to be transferred suitably placed for direct hydride shift to the nicotinamide.The active-site pocket is lined almost completely with hydrophobic residues and there are no histidines tryptophans cysteines aspartates nor glutamates which could participate directly in the catalytic action (cf.GAPDH where His-176 probably acts as the base for cata- lysisY3 and LDH where His-195 is e~sential~~). These structural results suggest that the only plausible mechanism for alcohol oxidation is that of electrophilic catalysis mediated by the active-site zinc through a bound water molecule as originally proposed by The~rell.~~ Binding of NAD' perturbs the pK of this water molecule leading to proton release. Alcohol then binds to the zinc as the alkoxide ion displacing hydroxyl ion. The formation of the alkoxide is mediated by the zinc-bound hydroxyl which acts as a base combining with the proton of the alcohol hydroxy-group.The zinc atom polarizes the bound alkox- ide so that direct hydride transfer and subsequent rearrangement to aldehyde occurs (Scheme 10). Common Structural Features in Dehydrogenases.-From the above sections on GAPDH and ADH it will have become clear that there are considerable structural similarities between the various dehydrogenases. Primary sequence homologies are well known between dehydrogenases of identical function from different species,51 but there is some disagreement in the literature about whether there are significant similarities between dehydrogenases with different functions. How- ever the three-dimensional structural similarities of the coenzyme-binding domains of LDH malate dehydrogenase (MDH) GAPDH and ADH3* are generally recognized.This homology has recently been put on a more quantitative footing by Ohlsson et using a technique first developed by Rossmann and R~o.'~ The nucleotide-binding domains of the three enzymes were super- imposed two at a time and the distances between the alpha-carbon atoms in the polypeptide chains and equivalent atoms in the coenzyme molecules were calculated. The detailed structure of the coenzyme-binding domains which have the well-defined secondary structure shown schematically in Figure 2 was strikingly conserved. There are six parallel strands of pleated sheet (PA to PF) and four helices (aB,aC aE,and alF). Not only do all four dehydrogenases contain the same structural elements but the connections between them are the same in all three cases (#?A-aB-/3B-aC-/3C-BD-aE-/?E-alF-/3F).The amino-acid sequence of an E. coli dihydrofolate reductase which is an NADPH-requiring enzyme has been deter~nined,~~ and there appear to be s2 S. T. Rao and M. G. Rossmann J. Mol. Biol. 1973 76,241 '' C. D. Bennett Narure 1974 248 67. A. D.B. Malcolm and J. R.Coggins Figure 2 Structure of coenzyme-binding domains in LDH MDH GAPDH and ADH (Reproduced by permission from J. Mol. Biol. 1974 89 339) similarities between part of this sequence and the sequences of the nucleotide- binding regions of pig GAPDH dogfish LDH horse ADH and cow glutamate dehydrogenase. It is attractive to speculate from these one-dimensional sequence data that the three-dimensional structures of the coenzyme-binding sites of all five enzymes are also similar particularly as this has already been established crystallographically for GAPDH LDH and ADH.47 4 Kinases Phosphoglycerate Kinase.-Crystallographic studies on horse muscle phospho- glycerate kinase (PGK) at 3 ,454have shown that this enzyme is made up of two domains which are clearly structurally independent and physically joined by a small ‘neck’ region.Each domain is organized around a central core of P-sheet. The binding site for Mg-ADP and Mg-ATP has been located on domain A and consists of a slot for the adenine ring at the edge of the p-sbeet. The position of the metal in the ADP complex has been defined partly from a difference map calculated between PGK-Mg-ADP and PGK-Mn-ADP and it appears to be linked to both the a-B-phosphates and an enzyme side-chain.54 C. C. F. Blake and P. R.Evans J. Mol. Biol. 1974 84 585. Enzyme Mechanisms 557 Partly because of the lack of primary sequence information for PGK it was difficult to trace the polypeptide chain through the structure and so although the regions of secondary structure (or-helix and fl-sheet) were obvious enough the connections between the various secondary structural elements were some- times ambiguous. However it was clear that domain A had the six-stranded parallel P-sheet that is a characteristic feature of the nucleotide-binding domain of the dehydrogenases (see above) and one of the possible solutions for connecting the polypeptide chain leads to the same connectivity as that observed for the four dehydrogenases.Furthermore the ADP/ATP binding site on domain A of PGK is in an equivalent position to the pyridine-nucleotide-binding site of the dehydrogenases. This possible identity of the B-sheet connectivity together with the clear similarity in the spatial organization of the secondary structural elements and the common position of the cofactor-binding site provide compelling evidence for the suggestion that the nucleotide-binding sites of PGK and the dehydrogenases are alike. This kind of structural similarity led Rossman and his colleagues32 to propose that these enzymes had evolved from a primordial nucleotide-binding unit. The specificity of this unit is for the adenosine group of the cofactor which occurs not only in NAD+ and ATP but also in flavin adenine dinucleotide and as the 3’-phosphate in coenzyme A.54 The presence of adenosine in these four important cofactors may be a consequence of a conservative evolu- tionary history from an ancestral nucleotide-binding protein.The structure of yeast phosphoglycerate kinase which was reported earlier is essentially the same as that of the horse muscle enzyme.55 Adenylate Kinme.-The structure of muscle adenylate kinase (myokinase) has also been reported at 3 A re~olution.~~ The interpretation of the electron- density map was greatly helped by the availability of the chemical ~equence,~’ and as a result the chain tracing was unambiguous.This enzyme (mol. wt. 22 OOO) although much smaller than other kinases and dehydrogenases still has a two-domain structure. One of the domains contains a five-strand parallel pleated sheet. The AMP-binding site has been located by n.m.r. studies58 and species sequence hornologie~.~~ The relation between this site and the five- strand pleated sheet is such that the topology of the nucleotide-binding site is very similar to that of the corresponding sites in dehydrogenases and PGK. Hexokinase.-Yeast hexokinase is a dimer of chemically identical subunits (subunit mol. wt. 51 OOO).59 Low-resolution (7 A) crystallographic studies have been reported on two crystal forms of the enzyme.60*61 The overall tertiary 55 T. N. Bryant H. C. Watson and P. L.Wendell Nature 1974 247 14. 56 G. E. Schulz M. Elzinga F. Marx and R. H. Schirmer Nature 1974 250 120. ’’ A. Heil G. Muller L. H. Noda T. Pinder I. Schirmer R. H. Schirmer and I. von Zabern European J. Biochem. 1974 43 13 1. 58 M. Cohn J. S. Leigh and G. H. Reed Cold Spring Harbor Symp. Quanr. Biol. 1970 36 533. 59 Y. M. Rustum E. J. Massaro and E. A. Barnard Biochemistry 1971 10 3509. 6o W. F. Anderson R. J. Fletterick and T. A. Steitz J. Mol. Biol. 1974 86 261. 6’ T. A. Steitz R. J. Fletterick and K. J. Kwang J. Mol. Biol. 1973 78 551. A. D.B. Malcolm and J. R.Coggins structure of each subunit is largely the same in both crystal forms. A deep cleft divides each subunit into two domains of roughly equal size. Helical regions account for between 40 and 50% of the polypeptide chain; much of the helical structure is concentrated in one of the domains which consists of between 70 and 80% helix.The molecule does not seem to be homologous to PGK or adenylate kinase. The quaternary structure of the dimer which differs in detail in the two crystal forms is very interesting because it is heterologous.60.6’ This means that at the interface between the two subunits a particular residue on one subunit may find itself in a different local environment from that of the same residue on the other subunit. Thus it is possible to have two non-identical binding sites for a given ligand in a dimer consisting of identical subunits. Pre- liminary X-ray studies on the binding of sugars and nucleotides have shown that some ligands do indeed bind very unequally to the two subunits.60 Further studies on hexokinase will hopefully lead to the first detailed structure of an oligomeric protein built from the heterologous association of subunits.

ISSN:0069-3030    

DOI:10.1039/OC9747100539

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

Abdulla R. F. 345 Abe E. 39 Abe N. 315 Abeles R. H. 533 Aberhart D.J. 32 472 Abou-Chaar C. I. 467 Aboud M. M. 536 Abraham A. P. 472 Abraham R. J. 18. 28 524 525 Abrahamsson S. 7 Abram T. S. 247 Abramova N. M.. 207 Abramova Z. A. 212 Abramovitch R. A. 153 155 157 159 166 289 342,343 Acheson R. M. 354 Achini R. 455 Ackerman B. K. 153 Adam W. 241 270 297. 358 Adams B. L. 359 Adams M. A. 23 Adams M. J. 549 Adams V. D. 346 Adcock W. 19 Ader. R. 24 Adler A. D. 150 Adler S. P. 407 Advena J. 341 Agami C. 269 Agosta W. C. 387 Agranat I. 317 Ahlbrecht H.. 428 Ahmad T. 489 Ahmed A. E. 484 Akabori S.. 481 Akasaka K. 428 Akasaki Y.. 325. 332 ,$kermark B.185 191 Akerstrom. S.. 57 Akeson A, 69 553 Akhmedov V. M. 172 Akhtar M. 536 544 546 Akhtar M. H. 108 Akiyama S.. 382,420 Akiyama T. 32 1 Akkerman J. M. 167 Akl N. S. 179 Akopyan M. E. 285 Albagli A. 290 Albaiges J. 192 Albertson. K. 499 Author Index Albin P. 177 Albright J. D.,425 Alderfer J. L. 395 Alecio M. R. 545 Aleksandrova E. K. 295 Alessi. P. 53 Alexander M. 19,277 Al-Kathumi K. M. 191 Alkins T. J. 357 Allan K. A. 202 Allen D. R. 489 Allen N. 52 Allen S. D. 43 Allenmark S. 173 278 Allerhand. A.. 32. 530 Allinger N. L. 166,272,302 379 Allison W. S. 549 Almlof J. 83 Almog J. 527 Alper H.. 178 184 Alpha S. R. 42 Al-Rawi. J.M. A,. 24 25 463 Alscher A, 307 Alston P. V. 105 Altenbach H.-J. 283 323 Altman J. A. 165 Altnau G. 13 Aitona C. C. 394 Alunni S. 124 Alwood J. L. 307 Aly N. F. 201 Amar F. 126 Ambler R. P. 502 Ames D. L.. 86 Amice P. 362,438 Amick D. R. 288,424 Amiet R. G. 194 Amit B. 510 Ammundsen. A. R. 528 Amy J. W. 8 Anand N. 104 Anastassiou A. G. 307,308 356 Andersen K. K. 153 Andersen N. H. 40 Anderson A. G. jun. 315 Anderson D. J. 320. 352 353 Anderson G. L. 386 Anderson J. E.. 282 358 Anderson P. H. 43. 303 Anderson R. A. 199 559 Anderson R. C. 346 Anderson R.J. 174 426 Anderson R.L. 173 Anderson W. F.. 539 557 Anderson W. K. 338 Andersson. A, 408 Anderson L.69 Anderton B. H. 68 Anding C. 459 Ando I. 25 Ando W. 169 Andose J. D.. 282 Andrae S. 374 Andreeta A. 175 Andrewes A. G. 42 Andrews G. C. 107 258 359,429,449 Andrieux C. P.,227 Andrieux J. 349 Anet F. A. L. 18 109 380 Anfinsen C. B. 73 Angele E. 30 Angeles R. M. 49 Anh N. T. 37Y Anricchio S. 334 Anselme J.-P. 157 Anthony M. T. 172 Anwar M. N. 489 Aoki K. 181 248 Aoyagi H. 517 Appel R. 321 Appella E. 499 Appleton D. C. 168 Appleton T. G. 183 Ara A. 204,412 Arai H.. 354 Arai M. 183 Arai S. 147 Arakawa M. 440 Araki M. 200 344 437 Araki Y. 169 Aranda V. G. 204,209,4 12 Arase A.. 207 Arber W. 407 Arbuzov. B. A.. 358 Archibald J.L. 485 Arens J. F. 255 Arentzen R. 392 Arhart. R. J.. 423 Ariel M. 220 Ariens E. J.. 490 492 Arison B. H. 516 Armitage I. M.,23 Armstrong P. D.. 488 Armstrong S. E. 330 Arnold B. J. 100 300 Arnold D. R.,162 Arnold J. C. 301 312 Arnold R.T. 110,259 Arnott S. 396 Arpino P. J. 54 Arsenio G. 204,412 Asao T. 306 Ash A. S. F. 477 Ashby. E. C.. 200,201,382 Ashby J. 357 Ashe A. J. 82 Ashley-Smith J. 190 Ashworth. P.. 144 Asmus P. 85 Aspberg K. 56 Asshauer J. 46.48 Assisi F. 535 Asveld E. W. H. 413 Atherton D. 407 Atkinson A.. 197 Atkinson R.S. 157 Atlani P. 273 Atlani P. M. 198,429 Attig T. G. 190 Au A. 240 Aue D. H. 377 Auer H.E. 41 Auld D. S. 409 Aumann. R.. 190 Aurbach G. 503 Au-Young Y.-K. 489 Avnir D. 3 17 Awang D. V. C. 276 Axen R.,56 Ayediran D. 289 Ayscough P. B. 137 Azarnoff D. L. 49 Azerbaev I. N. 212 Aznar F. 209 Baba Y. 101,371,503 Babiak A. 29 Babin D. R.,505 Baca B. 66 Baciocchi E. 124 Baczynski E. 509 Badder F. G. 201 Baddley W. H. 181 Backvall J. E. 185 191 Bar H.-P. 392 Bahl C. P. 393 Bailar J. C. jun. 17 1 Bailey T. D. 157 289 Bailey W. F. 351 Baillarge M.,330,428 Baird M. C. 185 Baird M. S. 162 375 Baitinger W. E. 8 Baker. M.M. 142 228 Baker A. D. 75 79 288 Baker D. R.,53.54 Baker R.,376 Baker R.W. 486,493 Baker V.J. 351 Bakke J. 329 Balakrishnan M.270 Baldock R.W. 149 358 Baldwin J. E. 106 108 198. 430,472,527,528 Baldwin J. G. 265 Baldwin M. A. 54 501 Bales S. E. 199 Ballester M. 294 Baltimore D. 403 404 Bal’yan Kh. V. 295 Barnford D. G. 494,496 Bamkole. T. O. 289 Ban Y . 428 Banaszak L. J. 553 Banda F. M. 217 Bandaev. S. G. 202 Bandle E. F. 404 Banks B. E. C. 540 Banks R.L. 188 Bannwarth W. 103 Ban-Oganowska H. 342 Bansal K. M. 143 Bansch J. 537 Barascut J.-L.. 389 Barbet J. 327 Barbey G. 220 Barclay L. R.C. 292 Barco A. 533 Bard A. J. 148 Bard M. 459 Barefoot A. C. 293 Barfield M. 27 Barford R.A. 47 Bargon J. 294 Barkemeyer H.. 515.516 Barlow M.G. 247 Barlow. R. B.. 492 493 495 Barluenga.J. 204 209 342. 412 Barmetler A. 98 Barnard E. A. 557 Barnes R.A.. 303 Barnett B. L. 194 Barnett G. H.,526 Barnett K.W. 180 187 Barnick J. W. F. K. 357 Barnier J. P. 362,438 Barofsky D. F. 16 Barofsky E. 16 Barondes S. H. 66 Barooshian A. V. 5 17 Barouh. V. 482 Barr J. J. 348 Barrall E. M. 47 Barrans Y. 486 Barrell B. G. 384 399 Author Index Barron L. D. 35 Barrow K. D. 468 Barry C. D. 20 394 Barry J. E. 221 Barry S. 60 Barstow L. E. 514 Bartels K. 539 Barth G. 35 Bartlett A. J. 296 Bartlett L. 41 Bartlett N. 282 Bartnik R.,321 Bartoletti I. 442 Barton D. H. R. 148 346 4 15,458,459,470,5 10 Barton T. J. 170 210 353 Bartsch R.A.123 124 Basilier E. 92 Basisio G. 424 Bass P. 480 Basselier J. J. 26 Basset J. M.,194 Bastide J. 327 Basu S. 234 Batchelor J. G. 27 Bates G. S. 184 41 1 Bates R.B. 197 Batich C. 79 80 82 Batten P.. 525 Battersby A. R. 31 467 468,522,538 Batz H. G. 509 Bauer L. 279 Bauer W. R. 406 Bauld. N. L. 140 312 Baumann R.,29 Bayet P. 198,422 Beach D. L. 187 Beach R.T. 192 Beak P. 281 Beames D. J. 185 Beasley G. H. 376 Beasley T. H. 49 Beattie W. G. 408 Beauchamp J. L. 13 15 Beavers W. A. 197 Bechgaard K. 218 Beck A. K.. 198,422 Beck J. R.,291 Beck W. 179 Becker A. 113 Becker D. 252 Becker J. V. 215,224,276 Beckett A. H. 489 Beckey H.-D..9,501 Beeby P.J. 309,310,356 Beez M. 84 Begg C. 498 Begtrup M.,327 Behforouz M.,338 Behrman E. J. 385 Beilan. H. S.. 13 Author Index Beisiegel E. 336 Bertrand M.,11 256 258 Bekeffy O. 47 418 Bektesh S. L. 48 Berwin H. J. 121 Belcher R.,15 Bestmann H. J.. 439 Belcher R.V. 520 Bethell D. 164 168 169 Beld A. J. 492 Betteridge D. 75 Bell H. C. 212 289,412 Betz W. 100 Bell R. A. 191 Beumel 0.F. 250 Bellamy A. J. 95 Beveridge D. L. 487 Belleau B. 489 490 Beynon. J. H. 7,8 11 Belloli R. C. 95 157 Bhacca N. S. 27 Belluco U. 180 184 Bhaduri S. 174 Bellus D. 104 Bhatia K. 143 Bellut H.,207 Bianchi R.G. 480 Belopotapova T. S. 184 Bichan D. 234,394 Belsharn M.G. 282 Bickart P. 102 Belue G. P.49 Bickelhaupt F. 356 Belyaev E. Yu. 297 Bicker U. 342 Benassi C. A. 5 13 Biehl E. R. 291 Benati L. 150 Bielka H. 401 Bencze L. 194 Biellmann J. F. 198,429 Bender A. L. 487 Biemann. K. 502 Benfield F. W. S. 172 Bierangel H. 167 Benjamins H.,282 Bieri G. 84 85 Bennett C. D. 503,505 Biesecker G. 486 Bennett G. N. 393 Biggi G. 351 Bennett J. E. 137 Biggs D. F. 488,496 Bennett J. T. 346 . Billeter M.A. 404 Bennett M.J. 382; 420 Billups W. E. 164 284 Benoit F. 16 Binger P. 180,181,206,360 Bentley R.,463 Bingharn. R.C. 120,138 Bentley. T. W. 16 116 Binh. S. K. 228 I8 Birch A. J. 292 346 Benveniste P. 459 Birchall J. M. 164,420 Berchtold G. A. 323 Birktoft J. J. 540 Berger A. 544 Birnberg G. H. 102 Berger J. G. 330 Birr C.506,513 Berger M..273 Bischof P. 79 Berger S. 22 Biserte G.. 74 Bergman R.G. 163 181 Bishop D. C. 496 Bergmann F. 2 1 Black D. St. C. 195,324 Bergstrom D. E. 325 387 Black J. W. 478 Bergstrom R. G. 291 Blackburn G. M.,270,406 Berk P. D. 64 Blackburne I. D. 19 351 Berkosky J. L. 93 Blackmore T. 184 Berkowitz J. 80 Blackwell D. S. L.. 326 BernaM M.,479 Bliiha K.. 38 Bernard M.,380 Blair G. E. 467 Bernardi L. 424 Blake C. C. F. 539 556 Bernasconi C. F. 291 Blake D. M.,179 Berndt A. 292 Blanchard M.,194 Bernhard. S. A. 551 Blankespoor R. 141 Bernstein Z. 5 17 Blasznak L. C. 433,437 Berridge J. 242 295 337 Blattner. F. R.,408 Berry C. N. 131 Blenkinsopp J. 40 Berry C. S. 536 Bloch A. 387,392 Berry R.S.344 Bloch W. 547,548 Berson J. A. 97 102 108 Block A. M.,145 Bertaccini G. 479 Bloomer J. L. 271 Bertini F. 140 Bloomfield V.A. 383 Bertram J. 2 15 Bloor J. 99 Bertrand. H. 108 Blount J. F. 39 56 1 Blout E. R. 32 Blow D. M.,540 Bloxsidge J. P. 25 Blu G. 55 Blum D. M.,21 1,254,419 Blum J. 177 188 Boar R.B. 458 Bobek M.,387 Bobick S. 389 Bobilliart F. 215 Bock H..75 79 82 84 Boekelheide V.. 303 Bodanszky M.,515 Bode J. 529 Bode W.,539 Bodner G. M. 30 Boeckman R.K. jun. 174 185 211 254 366 419 425,436,446,452 Boeckmann J. 356 Beg-Hansen T. C. 69 Bonnemann H. 182,341 Boerma J. A. 350 Boey J. 491 Bogaard M.P. 35 Bogel M.E. 19 277 Bogentoft C.. 503 Bognlr R.,39 Boguslawski S.J. 400 Bohlmann F. 11 12 13 14 256 Bohm A. 225 Bohm S. 328 Bohn B.. 276 Boicelli A. C. 288 Boido V. 297 Boisnard M.,399 Boiwe T. 539 549 553 Boldrini G. P. 424 Bolger L. 496 Boll P. M.,271 Bolleter W. T. 49 Bolton J. R.,162 Bolton P. H. 201 Bolton R.,135 Bornse D. S. 17 Bonati F. 173 Bond A. J. 136 Bonivento M.,185 192 Bonner A. G. 499 Bonnett R. 42 526 535 536 Bonte B. 348 Bontempelli G. 222 Boop D. C. 155 Boor J. E. 300 Borch G. 42 Borchardt J. K. 124 Bordens D. B. 323 Bordwell F.G. 112 Borer P. N. 395 Borg D. C. 150,526,530 Borgen G. 270 Borina E. L. 297 Borisov. A. E. 164 212 Boschi R. 82 Boschung A.F. 100 Bose A. K. 33 324 Bose S. 73 Bosmann H. B. 486 Bosshardt H. 12 Boswell G. A. jun. 419 Bosyakov Yu G. 212 Botchan M.R. 407 Botter R. 80 Bottini. A. T.. 98 Bottomley R. C. 68 Bouchet P. 327 Bouquant J. 19 Bourgain M.,447 Bourgeois P. 345 Boutagy J. 258,4 12 Bouvier P. 349 Bowden W. L. 288 Bowen D. V. 16 Bower J. D. 481 Bowers C. W. 357 Bowers C. Y. 503 Bo*ie J. H.,9 15 Bowne A. T. 154 Boxer S. G.,28 Boxler D. 432 Boyd G. V.. 335 Boyd R. J. 85 Boyer F. 523 Boyer H. W. 385 Boyer J. H. 169 Boyle F. T. 343 Bracho R.D. 470 Bradley J. S.. 178 Bradsher C. K. 104,358 Brady A. H. 42 Brady R. 528 Brady S. F. 369 506 Branden C.-I. 539,549,553 Brand! F.529 Brandsma L. 255 Brandstrom A. 424 Branton G. R. 88 Brassard P. 302 Brattesani D. N. 445 Brauman J. L. 359 Braun H. 258 Braun M.,198,437 Braun T. 47 Braunitzer G. 498 Brawerman G. 403 Brehm B. 87 Breslow R. 151 161 225 230,234,379,436 Bretde R. 216 217 Brewer H. B. 503 Brewster. A. R. 534 Bridges A. F. 277 Bridges A. J. 375 414 Brieger. G. 171 Brien J. F. 52 Brienne M.J. 38 Brigs D. F. 494 Brimblecombe R. W. 490 491,492 Brinigar W. S. 527 Brinkmann R. 182 341 Brintzinger H. H.,172 Brion C. E. 88 Brisk M.,79 Bristow T. H. C. 330 Brittain R.T. 482,496 Broadhurst M.D.. 376 Brockes J. P. 407 Brockman W. W. 407 Brockmann H.,528 529 534 Brocksom T.J. 443 Brockway N. M.,162 Brodelius P. 68 Brodersen R. 64 Brogli F. 82,84,85,93 Brook A. G. 2 11 Brook P. R. 106 Broom A. D. 386 Brossi A. 39 Brostrom K. 57 Brothers D. F. 8 Brown C. A. 445 Brown D. D. 405,408 Brown E. D. 365 Brown F. 403 Brown F. J. 115 Brown G. G. 191 Brown H. C. 120,204,205. 206,207,4 17,420 Brown J. C. 505 Brown J. M.,310 356 Brown K. J. 339 Brown L. R. 444 Brown M.P.. 178 Brown N. D. 52 Brown P. R. 44 389,407 Brown R. F. C. 165 253 324 Brown R. S. 12 1,383 Brown R. T. 467 Brown W. M.,407 Browne A. T. 299 Browning J. 180 Brownlee G. G. 404,408 Brownridge J. R. 115 Broxton T. L. 289 Bruce M.I. 184 Bruckmann E.M.,168 Bruenger E. 499 Brugger M.,513 Brugidou J. 275 Brugmans J. 491 Bruice T. C.. 131 544 Author Index Bruice P. Y. 131 Bruins A. P. 11 Brunee C. 8 Brunelle D.J. 174,185,447 453 Brunfeldt K. 500 Brust 0.E. 48 Bruza K. J. 436 Bryant R. W. 462 Bryant T. N.,539,557 Bryce T. 39 Bryce-Smith D. 242 243 295,337 Bryson T. A. 446 Bubb W. A. 346 Bubnov Yu. N. 207 Bubnova A. G. 520 Buchanan D. N. 106,237 Buchanan G. L. 359 Buchanan G. W. 26,33 Buchardt O. 336 Buchecker R.. 42 Buchler J. W. 527 Buchman O. 188 Buckett W. R. 494 Buckingham A. D. 35 Buckwaiter B. 429 Bucourt R. 368,445 Budzikiewin H. 534 Buchi G. 107,413,444 Buehner M.,539,549 Buenker R.J. 98 Bunzli J. C. 85 Burgi H.B. 274 Biissemeirer B. 181 248 Buglass. A. J. 28 Buhs R. P. 52 Bull D. C. 168 Bullpitt M.,212 Bunce N.J. 289 Buncel E. 290 297 Bundel Yu. G. 202 Bunina-Knvornkova L. I. 295 Bunker D. L. 112 Bunnenberg E. 35 Bunnett J. F. 289 291 Bunting J. W. 346 Burd J. F. 394 Burdon J. 290 312 Burtitt I. 27 212 Burgen A. S. V. 487 Burger A, 479 Burger K.,330 Burger U. 100 167 Burgers P. M.J. 392 Burgus R. 504 Burke S. S. 333 Burlingame A. L. 7 16 Burnham B. F. 529 Burns P.A. 324 Burroughs A. E. 19,277 Author Index Burrows E. P. 41 Bursey M.M.,7 Busch H. 402 Busch M.A. 194 Buschmann E. 342 Busfield D.494 Bushek J. M.,79 Rtttcher M.,504 Buter. J. 413 Buthe. I. 412 Butler D. E. 480 Butler L. G. 548 Buttrill S. E. 15 Byrd L. R.,224,276 Byrne. L. T. 380 Byme M.P. 357 Bystrek R.,190 Bywater R.,74 Cabrino R.,351 Caccamese S. 19 Cadogan J. I. G. 153 156 163,299,323,358 Caddy D. E. 25 Cahiez G. 185 203 419 Cain E. N. 277 Cairns M.A. 180 Cairns T. L. 167 Calas R.,210 Caldararn H. I47 Calder A. 148 Caldwell R. A. 243. 312 Callaway J. O. 208 Callot. H. J. 100 525 Calmus C. E. 241 Calvin M.,529 Calzaferri G. 334 Cama L. D. 368,449 Camerman N. 38 Cameron A. F. 495 Cameron J. R.,385 Camin. D. L. 44 Camp M.R.,300 Campbell J A. 292 Camps F. 192 Candlin J.P. 193 Canepa F. G. 486.494 Canet D. 28 Cannon J. B. 188,426 Cannon J. G. 488 Canonica L. 460 Canonne P. 201 Cantacuzene,J. 379 Cantello. B. C. C. 104 Cantor C. R. 400,401 Cantrell T. S. 242 325 Canuel L. 380 Capka M. 176 192 Capparella G. 175 Caprioli R.M.,7 Cardaci G. 184 Carde R.N. 158,346 Cardillo R.,474 Cardin D. J. 195 Carey N. 403 Cargill R.L. 359 Carless H. A. J. 147 Carlier P. 84 Carlisle C. H. 539 Carlson. J. P.,458 Carlson R. G. 105,236 Carlson R. M.,271 Carison,T. A. 90 Carmack M.,39 Carnahan. J. C. 248 Carney R. L. 369 Carnso T. C. 357 Carpenter B. K.,101 Carpenter P. D. 316 Carpino L. A. 506 Carr P. J. 54 Carre C. 300 Carrie R.,327 Carroll D.408 Carroll D. I. 54 Carroll F. A. 293 Carroll F. I. 31 Carsky P. 76 Carter R.,281 Carter W. P. L. 138 Carturan G. 184 Cartwright E. M.,404,408 Carty T. J. 533 Cary L. W. 31 Casanova J. 203,225 Case R.S.. 96 187 Caserio M.C.. 15 274 Caserio M. J. 15 Casey C. P. 173,442 Cash G. G. 190 Caspi E. 459 460 Cassady J. M.,271,467 Cassar L. 178 Cassidy R.M.,48,49 Castaiier J. 294 Castelli F. 201 Casy A. F. 478 479 482 483,486,487,488,493 Cato V.. 285 Catsoulacos P. 495 Catteral G. 301 Catuna S. 345 Caubere P. 153 155.300 Caughey W. S. 520 Caullet C. 219 220 226 Cauquis G. 222 Cava M.P. 306,338 Cavaleiro J. A. S. 520 522 530 Cazes B.107,435 Ceasar G. P.. 84 Cedar F.J. 382,420 Cekovic Z. 140,416 Cerfontain H. 287 312 Cernia E. 172 192 Cervinka O. 41 Chaband B. 222 Chadwick D. J. 19 197 327 Chalk A. J.. 341 Challis B. C. 131 273 Chalmers A. A. 34 Chambers J. Q. 222,232 Chambers R. D. 290,347 Chan A. S. K. 184 Chan R. P. K. 157 Chan T. H. 257 413 417 422,453 Chandler C. D. 45,49 Chandler W. D. 282 Chandrasekaran F. 396 Chandrasekhar K.,549 Chandross E. A. 282 Chang A. C. Y. 385 Chang C. 467 Chang C. K.,527,528 Chang E. 413 Chang H.-M., 294 Chang J.-K. 503 Chang K. J. 491 Chang K.Y. 141 Chang L. W. K. 128,375 Chang M.M.,503 Chang S. H. 398 Chang T. C. 37 Chanon F. 333 Chanon M.,333 Chao L.-C.201 207 382 Chaplen P. 496 Chapleo C. B. 444 Chapman D. J. 534 Chapman J. W. 286 Chapman 0.L. 245,283 Chapman T. M.,360,511 Chappelet D. 546 Chappelet-Tordo D. 546 Charles R.L. 49 Chase T. jun. 69 Chatterjee S. S. 490 Chattopadhyaya J. B. 444 Chauviere G. 209 Chedeket M.R.,471 Chen A. 315 Chen H.H. 209 Chen K.S. 137 Chen L. S. 184 Chen M.-C. 241 Chen R.,498 Chen R. H. K. 440 Chen S. L. 276 Cheng A. K.,109,380 Cheng J.-D. 297 Cheng S.-M.,259 405 Cheng Y. M.,169,173 Chernov A. 2..46 564 Chevrier B. 287 526 Chia L. S. Y.,394 Chiang Y.,273 Chiba T. 349 Chickering O. 478 Chickos J. S. 287 Chidgey R. 101 Chien-Hsing Chan 389 Chilcote D.D. 44 55 Child K. J. 494 Childs M. E. 170 Childs R. F. 108 Chimiak A. 39 Chini P. 29 Chint C. 203 Chiou C. Y. 488 Chira R. 345 Chirikjian J. G. 398 Chisholm M.H. 183 191 Chittenden R. A. 487,489 Chiusoli G. P. 185 Chiut C. 185 Chivers P. J. 19 351 Chizhov 0.S. 13 Chloupek F. J. 120 Chmurny G. N. 380 Cho H. 312 Chock P. B. 526 Choi Y. C. 402 Chotia C. 486,487 Chow S. T. 32 Chow W. Y. 164,284 Chow Y. L. 19 147,274 Christensen,J. J. 356 357 Christensen K. A. 23 Christensen,P. A. 240 Christie W. H. 44 Christol H. 275 Chu C.-H. 46 Chu I. 488 496 Chu J. Y.-R.,472 Chua C. 19,277 Chuihe J. 19 Chuikova T. V. 283 Chuit C. 419 447 Chujo R.25 Chung C. S. C. 141 Chung D. 57 Chung H. M. 314 ChursEek J. 44 55 Church D. F. 301 Churche J. 322 Chwang A. K. 395 Cinquini M.,278 Ciudaru C. 345 Clar E. 82 314 Clardy J. 38 187 Clark A. C. 188 Clark B. F. C.,383,384,398 399 Clark D. T. 82 Clark H. C. 30. 183 Clark J. 347 Clark M.,347 Clark P. A. 82 84 Clark R. D. 440 Clark R. T. 139 Clarke A. J. 494 Clarke D. E. 26 Clarke G. R. 484 Clarke J. A. 525 Clarke W. C. 57 Clarkson R. 365 Clary D. C. 82 Clastre J. 486 Cleary J. J. 350 Cleghorn H. P. 357 Clementi S 327 Clerc J. T. 16 Clerici A. 147 292 Clevenger J. V. 120 Clezy P. S. 14 520 521 522,523 Cliff G. R. 159 346 Clikeman R.R. 380 Clinging R. 338,355 Clinton N. A. 121 Clive D. L. J. 421 Closs G. L. 97 167 Clough S. C. 324 Clutter D. R. 19 277 Coates G. E. 199 Coates R. M.,269.446 Cobley U.T. 82 Coburn T. T. 164 284 Codding P. W. 493 Coffin R. L. 105,236 Coggins J. R. 546 Cogoni G. 222 Cohen J. F. 363,449 Cohen J. S. 28 Cohen M.,28 Cohen S. N.. 385 Cohen T. 174 360 Cohn M. 398,557 Cole B. J. 184 Cole P. E. 398,405 Coleman J. 219 Coleman J. P. 142,215,224 225 Coletti-Previero,M.A. 499 Collatz E. E. 400 Collier P. D. 39 Collins C. J. 115 Collins D. J. 350 Collins E. 295 Collins J. F. 470 Collins J. H. 502 Collins P. M.,236 Collins P. W. 479 Collman J. P.. 179 528 Colomer E.177 200 Colon M. 145 Colonna S. 278 Author Index Colton R. J. 93 Colvin E. 452 Combrisson S. 327 Comstock P. 395 Conan J. Y.,275 Concepcion. J. G. 145 146 Conde S. 353 Conia J. M. 259 362 375 438,446 Conlon J. M..521 531 Conover W. W. 321 Conn J. B. 515 Considine J. L. 208 2 12 Conway A. 55 1 Conway W. P. 192,449 Cook C. C. 357 Cook D. 123 Cook D. L. 480 Cook F. L. 357,442 Cook M.A. 505 Cook M.J. 358 Cook M. M. 37 Cook R. S. 286 Cooke B. J. A. 99 Cooke M.P. jun. 429 Cooke R. 198 Cooks R. G. 7 8 11 Cookson R. C. 237 376 Coombes R. G. 284 285 342 Cooper A. R.,47 Cooper G. H. 489 Cooper M.J. 357 Cope B. T. 528 Copenhafer W.C. 274 Coquelet C. 327 Corbon P. 220 Corcoran R. J. 151 234 Cordell G. A. 455,467 Corey E. J. 185 212 417 430,440,444,507 Corley L. 73 Cornish. D. W. 44 Corral C. 353 Corrie J. E. T. 459 Corriu R. J. P. 177 185 186. 200. 209 Corwin L. R. 97 Costa R. L. 303 Costin C. R. 40 Cotton F. A. 178,380 Cotton J. D. 184 209 Coubeils J.-L. 478 487 Couch P. 520 Coucouvanis D. 306 Coudurier G. 194 Coubon A. R. 384 404 Coulson D. R. 368,449 Coulter M. B. 395 Courriere P. 478,487 Courtois G. 198 259 Author Index Courtot. P. 240 Coutts R. S. P. 172 Covitz. F. H. 232 Cowie A. 407 Cowling S. A. 79 Cox J. A. 226 Cox M. T. 519 Cox O. 236 Cox R. E. 7 Crabb T.A. 19,351 Craig J. C. 157 Craig L. C. 32 Cram D. J. 272,357 Cramer F. 393 Crandall D. C. 536 Crandall J. K.,84,321 Craven D. B. 68 Crawford H. T. 284 Creed D. 243 Cremaschi P. 162 Cresp T. M. 309 31 1 356 Cripe K.,198 Crist D. k.,30 274 CFktOl s.J. 312 Criswell T. R.,312 Crombie G. 499 Cross H.S. 291 Crothers D. M. 383 398 405 Crouch R. 33 Cruickshank W. H. 543 Csumadia I. G. 165,274 Csontos G. 175 Cuatrecasas,P. 56 Cue B. W. jun. 329,424 Cukor C. 69 Cullen W. R. 213 Cullison D. A. 85 Culvenor C. C. J. 487 Cundy C. S. 204 Cunino C. M. 288 Cunningham W.D.,20 Cupas C. A. 371 Cupper Th. J. M.,313 Curphey M.,347 Currie B. L. SO3 Curtiss L.A. 379 Cushley R. J. 27 31 32 473,487,538 Cushman M. 107,444 Cutting J. D. 423 Cuvigny T. 363,436 Czernilofsky A. P. 400 Czuba. W.. 347 Dabrooski J. 17 Daddona P. E. 467 Dadok J. 20 Dagonneau M.. 201 Dahl L. F. 390 Dahlberg J. E. 408 Dais P. J. 114 Dale J. A. 234 Dale R. M. K.,390 Dalgarno L. 402 Dall H.,482 Dalton A. I. 138 Dalton D. R. 126 Damps K.,458 Danby C. J.. 87 Daneli R.,284 Daoeshralab M.,349 d’Anplo J. 367,445 Danieli R.,288 Daniels. J. 266 420 Daniels P.J. L. 485 bmilova T.A. 296 Danishefsky S. 363,451 Dann M.L. 545 Danna K.J. 407 Dannenberg J. J.. 377 Danyluk S. S. 394 Darby N. 310 D’Arcy P. F. 482 Daren W. C. 147 Dark W.A. 47 Da Rocha Gansalves A. M. 522 Dart E. C. 287 Darwish D. 122 Dastur K.P. 369 Dattagupta N. 526 Daub J. 100 Dauben W. G. 102,376 Daune M.P. 406 Dautrevaux M.,62 Dave V.,346 Davidson P. J. 197 Davies A. G. 136 207 Davies B. 494 Davies D. B. 394 Davis D. L. 48 Davis D. W. 86 Davis G. E. 471 Davis J. H. 97 Davis J. R.,386 Davis M. 336,494 496 Davis. R. E. 194 Davis R.W. 385 Davis W. 342 Davison A. 172 186 Dawid I. B. 403,408 Dawkins B. G. 54 Dawson B. 503 Dawson. P. J. 525 Day J. C. 147. 267 Day J. L. 488 Day V. M.,178 Dayal B. 324 Deacon G. B.,203 Deady L. W. 289,336 de Aguirre I. 176 Dean C. 487 Dean F. M. 338.355 5 65 Dean P.D.G. 56,62,68,74 Dearden G. R.,524 Debies T. P.,90,93 De Camp M.R. 109 154 Decorzant R.,446 De Crombrugghe B. 404 Dedieu M.,177 Deeming A. J. 184 Defay N. 313 De Flora A. 69 Deftos L. J. 505 De Graaf C. 200 257 De Oroot N. 400 Deguili G. 533 de Haan R.,180 Dehmer J. L. 80 Dehmlow E. V. 163 Deisenhder J. 539 Deits T. 64 de Jager J. 394 de Jong A. J. 255 Dekker J. 180 de la Higuera N. 472 de la Mare P.B. 285 Deleris G. 210 De Lisi C. 405 Delk A. S.,399 Delpuech J.4.. 28 de Marinis R. M.,323 de Mayo P. 110,245 de Meijere A. 186 374 Demerseman P.,26 Demidovich. G. V. 188 Demmin T. R. 445 Demoen P. 491 Demortier Y.,176 derr Elzen R. V. 279 Denes V.345 Dengler B. 395 den Hertog H. J. 347 Denis J. M. 248 Denkewaltex,R.G. 515,516 Denney D. B. 138 Denney D. Z. 190 Dennis N. 101,343 Deilnis R.W. ‘136 Denny R. W. 258 Denzei Th. 439 Depezay J.-C. 422 de Poortere M.,324 Derancourt J. 49 Derocque J.-L. 201 Derr H. 208 Derrick P.J. 7 16 de Santis. F. 327 de Saqui-Sannes G. 296 Desiderio M. M. 501 SO4 Deslausiers R.,22 Deslongchamps P. 273 Desrosiers R.,404 Dessau R.M.. 437 DeStefano J. J. 45 566 De Tar M.B. 99 Deth R. C.,491 Deutch A, 408 Devaquet. A. J. P. 107 379 Devaux P. 54 Devdar R. S. 326 de Vellis J. 64 Devillez C. 55 Devolder P. 162 Dewar M. J. S. 95 96 97 111,154,296,298,310 Dewar P.S. 79 Dewey R. S. 516 Dewhurst B. B. 272 de Wit J. 34 Dews P. B.,478 Deyrup J. A. 324 Dhar R. 406 Diab Y.,321 Diakin V. 521 Diamond S. E. 444 Dickason W.C. 120 Dickerson R. E. 539 Dickeson J. E. 346 Dickinson R. J. 501 Dickson R. S. 247 Dieck H. A. 183 Dieckmann H.,528 Diehl P. 24 Diels O. 334 Dietsche T. J. 185,192,448 449 Dietz A. G. jun. 174 Dietz K. P.,342 Dietzmann I. 192 Dijkink J. 345 Dillinger H. J.. 250 Dillon J.. 276 Dillon P. W. 258 Dinsdale M. J. 526 Dinsmore S. R.,44 53 Di Nunno L. 153,334 Dion H.W.,390 Dittrich. B. 167 Divaker K. J. 443 Diversi P. 194 Dixon J. S, 503 Dixon M.,550 Dixon W.T.,140 143,144 Djerassi C.8,35,36,37 Dobson C. M.,20 Dockx J. 491 Dodd J. R. 99 141 Doddrell D. 18 19 27 212 Dodds H.L. H. 270 Dodds M.G. 494 Doef M.,393 Doering W. von E. 97 Doherty R. F. 72 Dolcetti G. 171 Dolenko A. 297 Doleschall G. 426 Dolphin D. 526,530,533 Dolphin R. J. 54 Dolzine T. W.,208 209 Domanus J. 336 Dominguez X.,33 Do Minh T. 163 Donalson J. E.. 384 Done J. N. 48.53 Donley W.,199 Donneily W.J. 470 Doonan S. 540 Dorland L. 501 Dorn H. C. 26 Dorrer B. 173 Doss E. M, 536 Doty J. C.. 243 Dougherty R. C. 7 Dowd P. 185,414 Dowd W.,116 Drake A. F.,40 Drakenberg,T. 27 Drapeau G. R. 498 Dreilick R. W. 148 Drenth J. 545 Dressler K. 401 Drevin H.56 Drew M.G. B. 184 Drewes H. R. 288,424 Drouin J. 259 375 Drozd V.N. 290 Drury R. F.,-230 Druyan M. E. 530 Dryburgh J. A. 505 Dryhurst G. 220 D’Silva T. D. J. 188 Dube S. 198,429 DuBeshter B. 274 Dubini R. 107 Dubois J. 84 Dubois J. E. 139 Dubroff L. M. 403 Diinges W. 49 Diirr H.,167 Duffaut N. 345 Duflot C. 20 Dugas H. 25 Duldre J. P. 201 Dumont W.,198,422 Dunbar R.C. 76 Duncan C. D. 97 Duncan D. M.,412 Duncan W. A. M.,478 Dunkin I. R. 162 Dunnill P. 72 Dunogues J. 210 Dunstan B. T. 324 Dunstan D. R. 42 Duprk A. 220 Dupree L.E. 533 Durant C. J. 478,479,481 Durham L. J. 390 Durr H. 330,374 Author Index Durst,T. 102,260,279,324 350,413 Dutschman G.392 Dutta A. S.,513 Dux F. J. 424 Dvinin V. A. 188 Dyer R. L. 527 Dyke J. M.,86 162 Dymerski P. P. 76 Dynak J. 363,451 Dzhamatova G. 296 Dzidic I. 54 Eaborn C.. 2 12,289,294 Eadon G. 277 Eaton G. R. 525 Eaton S. S. 525 Eatough D. J. 357 Ebel J.-P. 402 Eberbach W.,85 Eberhardt M.K. 143 Eberle A. 537 Eberle G. 186 Ebersole R. C. 459 Eberson L. 217,218,225 Ebetiqo F. F.,358 Ebizuka Y.,471 Echols R. E. 22 Eckelman W.C.,.I69 Eckert C. A. 258 Eckert R. C. 294 Eckert-Maksic M.,283 Eckes L. 248 Eckhard I. F. 346 Eckstein F. 392 Edman P.,498 Edmonds A C. F. 40 Edmondson W. L..533 Edwards J. 294 Edwards R. 190 Eeles M.F. 164 Efner H.F. 199 Efraty A. 190 Egan R.S. 493 Ege G.,336 Eggelte H. J. 356 Egger K.W. 110 Eggerding D. 306 Eggert J. H. 466 Egorov Y.P.,13 Eguchi S. 163 Ehl K.534,535 Ehrenson S. 162 Ehresmann C. 402 Eibler E. 157 Eicher T. 328 Eichler J. 428 Eidenschink R. 103 Eidus Ya T. 189 Eikenberry,J. N.. 279 Eilat D. 400 Einarsson R. 549 Author Index Eisch J. J. 207,208 Eisenstein O. 379 Eisman G.. 126 Eizember R. F. 236 Eklund H. 539,549,553 Ekman S. 57 El-Abadelah M. M. 39 Eland J. H.D. 75.87 El-Barkawi F. 535 Eldefrawi A. T. 486 Eldefrawi M. E. 486 Elder G. H.,521 Elguero J. 327 Elia G. H.,349 Eliel E. L. 35,345,35 1 Elix J. A. 521 Ellenbogen L.73 Ellenbroek B.W. J. 490 Ellermann,J. 213 Elliott R. L. 308 356 Ellis C. A. 485 Ellis S. R. 54 Ellison F. O. 93 Elmitt K.,184 Elson C. M. 53 1 Elson I. H. 139 Elvidge J. A. 24 25 358 Elwood T. A. 286 Elzinga M. 502,539,557 Embree D. J. 11 Emch R. 19,277 Emmett J. C. 481 Emptoz G. 202 Endele R. 46 Endo K.,183 Endo M.,346 Engel J. D. 389 Engel P. S. 138,236 Engel R. 288 Engelhardt H.,46 Engler E. M. 340,373 Englert G. 13 Eon C. 55 Epiotis N. D. 95 106 359 Epling G. A. 225 377 Erdmann V. A. 399 Erickson B. W. 51 1 Erickson R. 226 Ericsson L. H. 498 Ermakov Yu. I. 193 Ermer O. 80 Erni F. 16 Ernst L. 18 Emst R. R. 23 31 Ernstbrunner E. 79 Ershov V.V. 167 Eschbach C. 173 Eschenmoser A. 532 Eshkami A. 69 Esperin W. G. 32 Etienne A. 348 Ette S. I. 290 Eugster C. H.,42 Evans D. A. 107 258,359 429,449 Evans D. H. 224,227 Evans E. A. 25,358 Evans F. E. 21,389 Evans G. O. 192 Evans J. 30 190 Evans P. R. 539,556 Everett A. J.. 493 Everse J. 549 Evstigneeva R. P. 520,521 527 Ewers U. 25 Exner 0..279 Eyndels Ch 3 13 Eye J. A. 137 Fachinetti,G. 175 Faerber P. 395 Fagan J. F. 113 Fahey M. R. 147 Fahey R. C. 125 127 Fahmy A. F. M.,201 Fairbrother P. 485 Fajer J. 150 526,530 Falck J. R. 218 Falk A. J. 49 Faller J. W. 23 Fan D. M. 203 Fanning J. C. 526 Fanta P. E.. 412 Farado S. 532 Farcasiu D.373 Fareed J. 386 Farid S. 243 Farina E. 291 Farquharson G. J. 203 Farr F. R. 140 Fasold H.,68 Fattinger F. 532 Faulkner T. R. 35 Favre E. 206 Fawcett P. 472 Fayat C. 157 Fayos J. 38,187 Fedarko M.-C. 281 Fedoronko M. 232 Feeney J. 28 271,487 Fehrle. M. 516 Feigelson P. 403 Feil D. 85 Feiring A. 161 Felice L. J. 53 Feline T. C. 31,457 Felis R. F. 193 Felix A. M..439 Fellner P.,403 Felsen-Rhengold D. 288 Felton R. H.,221 526 530 Fender G. 250 Fenton D. M. 189 Fend W. 207 Feoktishov V. M. 295 Ferguson G. 495 Fermandjian S. 28 Fernlund P.,504 Ferramola A. M. 522 Ferrari G. F. 175 Ferree W. I. jun. 243,244 Fessenden R. W. 143 Fetizon M.40 Feutrill G. I. 521 Fichter K.C. 207 Ficini J. 251 Field F. H.,16 Fielden. R.. 346 Fields E. K. 136 Fields T. R. 239 Fiers W. 402 Fierz G. 101 Fietzek P. P. 500 Filby G. 143 Filipescu N. 162 233 Filler R. 425 Finch J. T.. 383 Findlay. D.M. 238,300 Finkelhor R. S. 257,417 Finkelstein M. 221 Finocchiaro P. 26 281 Firl J. 359 Fironzabadi H.. 306 Fisch. H.-U. 57 Fischer A. 285 Fischer. A. J. 285 Fischer C. M. 226 Fischer D. 384 Fischer E. O. 173 507 Fischer M. S. 529 Fiser I. 401 Fisher R. D. 116 Fisher R. P. 421 Fishman J. 460 Fitton A. O. 346 Fitzgedd P. H.,273 Fizet C. 445 Fjeldstat P. E. 509 Flack W. R. 286 Flavell R. A. 404 Fleiderman L.I. 521 Fleischer E. B. 525 Fleischer J. 55 Fleischmann. M. 215 Fleming M. P. 415 Fleming R. H.,108,233 Fleming W. C. 241 Fletterick. R. J. 539,557 Flintoff W. 395 Fliszar S.,25 Flitsch W. 307,356 Floriani C. 175 Florio S. 153 334 Floss H.-G., 467,474 568 Flowers W. T.. 345 Floyd D. M. 447 Fbyd J. C. 433 Flygare W. H. 281 Flyua B. R. 528 Fiynn C. R. 299,338 Foi M.,178 Foffani A. 189 Folayan J. 0.. 395 Folkers K. 503 Fmbert C. 325 Fong,F. K.,121 Font J. 168 Foazaine M. 226 Fookes C. J. R. 521,523 Fq J. S. 79 Foote C. S. 324 Foottit M.E. 479 Forchioni A. 20 Ford G. C. 539 549 Ford W. T.. 98 123 Fmder. R. A. 184 Forget B.G.,404 Forman A. 150,526,530 Forrester A. R. 148 Forsen S.,27 F’orster A. 29 Forsythe P. P.,327 Fossel E. T. 32 Fosset M. 546 Foster C. H. 323 Foster 0.F. 259 Foster H. E. 330 Foster J. A. 499 Foster W. E. J. 144 Foocaud A. 157 Foutgar N.J. 299 Fourrey. J. L. 325 Fowler P.J. 485 Fox D. P.,164 FOX.M.-A. 241 Eoxal4 J. 140 Fsmcis R. F. 342 Francis W.,13 Francisco C. G. 460 Franck B. 275,522 Franck-Neurnann M. 380 Frangopol M.,162 Franwpol P.T. 162 Frank G. 356 Franklin J. L. 81 Franks. F. M.,482,492 Franz J. A. 423 Franz J. E.,335 Fraazbbu C. 499 Fraser P.J. 247 Fraser R.R. 197,428 Eraser S. B. 467 Frauendorfer E.. 508 FraunfeBder G. M.,434 Fray G.I. 243 Freedman M. H. 28,478 Freeman D. H. 49,55 Freeman R.,22 Freer S. T. 543 Frkhel D. 273 Frei R. W. 46,48.49 Freidinger R.M..107 413 Freidlin L. Kh. 188 Freire R. 460 Freiser. B. S.,13 French J. 520 Frenz B. A. 380 Fresco J. R.,398 Freudenberger,J. 69 Frey R.,87 Fric P. 546 Friderici K. 395 404 Fried M. 407 Fried W. 69 Friedman. H. S. 138. 140 Friedman L. 244 Friedman S. 399 Fries R.W. 179 183 Frihart C. R.,387 Friis. P. 271 Fringnelli F. 327 Frisby D. 403 Fritz. H. 33,353 Fritz H. P. 215 Fritz J. S. 46,49 Fritzberg A. 472 Fritzxhe U. 254,343 Froimowitz M.,487 FTO~OV, 1. I. 46 Fromageot P. 28 Frost D. C. 85 86 Frost D. J.39 Frost J. R. 346 Fruton J. S. 545 Fry J. L. 425 Frydman B. 537 Frydman R. 8..537 Fu E. 76 Fu P. P.,412 Fuchs P. L. 363 369 412 414,450 Fiisk W. 341 Fuganti C. 469,474 Fuhrer W. 532 Fuhrhnp J. H.,524,526,535 Fuji K.,38 Fujii S. 424 Fujimoto H. 167 Fujita K. 18 Fujita T.,314,444 Fujiwara F. 24 Fujiiara Y. 181 200 Fukata G. 33 1 Fukazawa Y. 307 Fukumoto K.,345 Fukuzumi K.,177 Futler G. B.,424 Fullerton,T. 4.. 185.448 Author Index Fu-Ming Chen 41 Furin G. C. 287 Furukawa J. 180 182 189 202 Furukawa S. 44 1 Furnse T. 330 Fuss W. 79 Futrell,J. H. 501 Gabriel,T. F. 49 Gacek M. 41 Gache C. 546 Gagne R.R. 528 Gajewski J. J. 109 Gakis N.,328 Galibert.F. 406 Gall J. G. 407 Galle J. E. 159 Galley M.W. 287 Gallup G. A. 281 Galuszko K. 303 Games D. E. 520,521,522 527 Gammill R. B. 446 Gan L. H..290 Gandhi S. S. 299 Ganellin C. R.,478,479,481 Gam B. 174,425 Gankin V. Yu. 188 Gans P.J. 487 Ganu D. 522 Garcia G. A. 445 Gafin D. E. 407 Garin D. L. 187 Garneau F. X.,163 Garnett J. L. 178 Garratt D. G. 266 Garratt P.J. 252 307 310 356 Garrison D. R. 490 Ganv R.,349 Garsky V. 110,259 Garst. M.E. 423 Garwood R. F. 224 Gasc J. C. 368,445 Gaspar P. P. 169 170 210 Gassman J. 529 Gassrnan P. G.,288 329 342,424 Gaudemar M. 206 Gaudty M..434 Gawronski J. 39 Gay I. D. 28 Geckle M.,19 Gee R.D.156 Geibel J. 527 Geiger R.,510,512 Geiss K.,429 Gelius U. 90 92 Gellender M.,79 Germ N.A. 178 Genies M.,222 Author Index Gennaro G. P.. 169 Genoni F.,175 Georghiou P. E.. 184,4 1I Gerald M.C. 484 Gerhart F. 97 Clerhart J. C. 548 Gerlock J. L. 141 Germeraad. P. 159,160,302 Germerhausen R.L.. 155 Gestrelius. S.,73 Geurtsen (3.. 347 Ghirardelli. R.G. 43 Ghiriaghelji,D.,469,474 Ghosez L. 106 261 324 362 Giangtasso D. 474 Giannini D. D..27 Giarrusso A. 178 Gibbons W.A.. 32 Gibson G. R.,49 Gibson T. P.. 52 Gieren A. 330 Giering W.P. 184 Gierke T. D. 281 Giga A. 447 Gil G. 108 Gil-Av E.,46 Gilbert A. 242,295,337 Gilbert B.C. 136 138 Gilbert D. P. 329,424 Gilbert J. C. 148 Gilbert R. 77 Gilbert S.G.! hY Gilbert T. W.,45 46 Gilchrist,T. L. 331,348,357 Gilde H.G. 225 Gil’denberg E.Z.. 189 Giles H.G. 110 Gilgen P. 328 Gilham P. T.,393,402 Gill G. B. 95 311 Gill G. N..65 Gillam S.,333 Gilman H.,196 Gilmore G:W. 164,420 Gilmore J. R. 79 Gilow H. M.,284 Gilpin R.K.,48 Ginsburg D. 225 Giori P. 513 Gipstein E. 49 Girard C. 362,438,446 Girijavallabhan,M.,5 10 Giuliano F. 69 Givens R.S. 105 236,489 Gladysz J. A. 41 1 Glasel J. A. 394 Gleason J. 532 Gleicher G. J. 301,312,373 Gleiter R. 85,95. 165,334 380 Gloe A. 528 Glonek T. 391 Gioor J. 233 Gloss G. L. 28 Gluckson J. p..20 Godbille E.54 Godfrey J. M.,42 Godfrey M.,79 Godtfredsen W.0.. 459 Goeke G. L. 183 Goerdeler J. 336 349 Goering H. L. 120 279 Goettert E. 141 Goetz H.,79 Gogte V. N..326 Goh S.H. 167 Gokel 0.W.,272,357 Golay M.J. E. 55 Goldberg I. R.,136 Goldberg S.Z. 307 Golding B. T. 538 Goldman N.L. 288 Goldstein,M.J. 85,109,307 Goldstein S.,288 Golob L. 86 162 Gompper R.,435 Goncharov A. V.,351 Gonzalez. A. G. 460 Goodbrand H. B.,261,338 Goodfellow R.J. 458 Goodin R.D. 148 Goodman A.. 530 Goodman. H.M.,385,407 Goodwin T. W.,460 Goos S. M..505 Gordon A. W.,277 Gordon J. K.,64 Gordon J. T. 241 Gore J. 201 Gore P. H. 31 1,444 Gore S.T. 339 Gorenstein D.G. 291 Gargues A. 430 Gorlenko V. A. 500 Gorinsky. B. A.. 53Y Gornosfaev,L. M..297 Gotsky V. 515 Gosney I. 156 Goss. D. J. 398 Goss H.J. 349 Gossauer A. 53 1,532,534 Gosse C. 406 Gator V. 342 Gotschi E. 532 Gottarelli G. 39 Gotthardt H. 245,325 Gotzler H.,258 Goudmand P. 162 Gould K. J. 206 342 Gounelle Y.,80 GOWC~. J.-G. 222,226 Goursot A. 25 Govier W.U. 484 Gowenlock B. G. 202 Graf W.,31. 464 Graham F. L. 405 Graham J. D. P. 478 Graham M.,403 Oralla J. 405 Grandberg I. I. 358 Grandolini G.. 327 Granoth I. 287 Grant B. 8,444 Grant D. M..23 Grant D. W.,46 Gras J. L. 108 258 Grasselli. P.,469,474 Gravei D. 430 Gravitz N.,272 Gray T. L.526 Gray W. R. 501 Graziani M. 180 184 185 192 Cream G. E. 224 Greaves E. 0.. 190 Oree R. 327 Green D. 490 Green D. M..489,492 Green J. P. 478 Green M.,180 Green M.K. 140 Green M.L. H. 172 184 Green M.M.,12,416 Greene R. L. 26 Greenhill J. V.,271 Greenhouse R.,424 Greenwell P.,401 Gteenwood J. M..517 Gregges A. R.,49 Gregonis D. E. 458 Gregoriou G. A. 114 Gregory R.A. 505 Greig C. C. 285 Grennberg B.,77 Greuter H. 371 Grey R.A. 191,249,306 Gribble G. W.,14 Gribnau T. C. J. 57 Grieco P. A. 257,413,417 432,443 Griffin A. C.,95 11 1 Grifin B. E. 407 Griffin T. 60 GrifTith R.C. 278 Griffith R.K. 484 Griffiths J. 312 Grigg R.,524,525,526 Grigoryan M.Kh.184 Griller D. 136 137 139 292 Grimm F. A. 90 Grimshaw J. 294 Grivet J. P. 162 Grobel B.-T. 2 1 1,427,434 Groenewege M.P. 38 570 Grohmann. K.,48 Grosjean 0.. 84 Grossert J. S.,413 Grover A. K.,69 Grover S. H. 25 Groves J. T.,421,437 Grubb H. M. 10 Grubbs R. H. 191,225,249 306 Gruetzmacher G. D. 288 424 Grutzmacher H.-F. 12 Gruetzmacher R. R. 120 Grummt F. 399 Grummt I. 399 Grundon M.F. 470 Grushka E. 48,55 Grutzner J. B,,27,33 Guameri M.,513 Gudgeon J. A. 3 1,463 Gudriniece E. 358 Gunther H. 25.33 283 Guerrera F. 301 Guerrieri F. 185 Guilford H. 56.66 Guillemin R. 504 Guinot F.. 275 Guiochon G. 55 Gulati S.C. 409 Gulyaev N.N. 65 Gund P. 382 Gund T. M.,373 Gunkel E. 84 Gunther K.,143 Gupta R. C. 394 Gupta S. K.,326 Gurari-Rotman D. 73 Guritz D. M.,124 Gurne D. 524 Gurvich L. G. 203 Gusel'nikov L. E. 210 Gusev A. O. 184 Gust D. 205,281 Guthrie C. 399 Gutpa B. D. 19 Guziec F. S.,jun. 4 15,508 Guzinski J. A. 221 Gymer G. E.,357 Haag J. 336 Haak P.,164 Haak W. J. 32 Haar W. 33 Habener J. F.,505 Haber A. 464 Haber S. B. 472 Habfest K.,8 Hach V.,266 Hackert M. C. 549 Haddad Y.M.Y.,177 Haddon R.C. 283,310 Haegele K.D. 54 Haenei M. W. 304 Hagaman E. 467 Hagaman E. W. 28 Hagen F. S.,401 Hagishita S.,38 39 Hagiwara T. 169 Hahn R. C. 287 Haiby W. A. 109 Hbjek M.,426 Haklits I.47 Halbsz I. 46,48 Halbert T. R.,528 Haldane J. B. S.,543 Halevi E. A. 115 Hall A. J. 273 Hall C. R. 239 Hall H. K.,361 Hall H. T. 191 Hall L. D. 22 Hall P. L. 420 Hall R. E. 115 Hallett P. 444 Halpern A. 289 Halpern J. 183 Halstr~m,J. 516 Halton B. 305 Ham N.S.,478,484,487 Hamada Y.,348 Hamana M.,342,343 Hamashima Y.,358 Hamberg M.,461,462 Hambright P.,526 Hamilton A. 525 Hamilton A. L. 53 1 Hamilton J. B. 244 Hamilton R. J. 524 Hamm P. 42 Hammer E. 198 Hammerich 0.. 217 219 Hammes G. G. 69 Hammett L.P.,112 Hammond G. S.,233 Hamner E. R.,180 Hamnett A. 75 Hamon D. P. G. 169 Hampton K.G. 275 Han G. Y.,506 Hanack M.,248 Hancock K.G. 437 Hancock R.D. 192 Hand R. L. 222 Haney W. G. jun. 53 Haniu M.,498 Hanley F. L. 389 Hanna. P. E. 484 Hannaway C. 495 Hannon J. E. 499 Hansen H. J. 328 Hansen L. C.,45,46,47 Hansen P. E. 271 Hansen R. S.,34 Hanson J. R. 32,458 Author Index Hanson P. E. 28 Hanstein W. 121 Hanzawa Y.,345 Hara K.,239 Hara O. 313 Harada H. 358 Harada K.,227,440 Harada N.,40,41,276 Harbison K.G. 291 Hardegger B. 532 Harding K.E. 369 Hardy M. 188 Hardy P.M. 20 Harger M. J. P. 161 Harkness A. L. 528 Harless J. M. 379 Harmon C. A. 307 Harmon T. E. 197 Harney. D. W. 208,411 Harpp D. N.,453 Harrington K,J. 165,253 Harris C. J. 331 Harris C.M.,275,350 Harris D. C. 29 Harris D. H. 209 Harris D. J. 352 Harris H. P. 357,419 Harris J. 348 Harris J. I. 552 Harris J. M.,112 113 115 119 Harris R.J. 401 Harris R. L. 148 Harris T. M.,275,350 Harris W. G. 517 Harrison A. G. 14 Harrison C. R. 206 269 415,423 Harrison D. M.,322,470 Harrison J. F. 162 Harrit N. 336 Harshbarger,W. R. 77 Hart H. 237,423 Hart H. I. 106 Hart S.G. 9 Hartley 8.S. 502 540 543 Hartley F. R. 195 Hartmann J. 198,429 Hartmann W. 147 Hartshorn S. R. 112 Hartzell S.L. 441 Haruki E. 440 Harvey M. J. 62,68,74 Harvey R. G. 312,412 Hasan F. 132 Hasegawa H. 354 Hasegawa M.,238 Haselbach E. 75 79 85 103 Hashimoto H. 202 Hashimoto K.,353 Haslam E..475 Author Index Hassan M.M.A. 486 Hasselmann D. 266,412 Hassloch M.A. 302 Hassner A. 159 320 333 352,353,430,495 Hasso S. 184 Hastings J. S. 110 Hastings 1. W.,63 Haszeldine R. N. 164 169 345,420 Hata K.,305 Hatanaka !%-I. 271 Hatano M.,38 Hatch E. 363,45 1 Hatch G. F. 148 Hatcher B. G. 106 Hattori M.,395 Hauck F. P.,480 Haugh M.J. 126 Hauptmann H. 338,341 Havel J. J.. 258 Hawkes G. E. 19 28 380 524,525 Hawkins C. 312 Hawkins E. 398 Hawkins S.,3I 1 Hawthorne M.F. 175 Hay J. V.,292 Hayakawa Y.,101,371 Hayashi M.,432 Hayashi S. 345 Hayashi T. 186,427,434 Hayashida H. 498 Hayes J. 168 Hayez E. 169 Haymore B.L. 174 Hazelton H. R. 48 Hazum E. 416 Hearn M.T. W. 28 Heathcock C. H. 440,445 Heather J. B. 271 Heaton B.T. 29 Htbert J. 430 Heck R. F. 171 183,442 Heckendorf,A. H. 467 Hedaya E. 104,250,371 Hedden G. 147,274 Heerd A. 387 Heeschen. J. P.,288 Hegedus L. S. 185.19 1,265 Hehre W.J.. 97 107 108 277,379 Heiba E.I. 437 Heidelberger C. 387 Heijneker H. L. 405 Heikman H. 329 Heil A.. 557 Heil B. 171 Heil V.,379 ffeilbronner. E. 75 76 79 80,82,84,85,93 Heilmann S.M.,224,228 Heimgartner H. 328 Heindell H.C. 402 Heine H. G. 147 Heine H. W. 322 Heinsohn G. E. 507 Heintz R. 459 Heintzelman R. W.,158 Heise K.P.,531 Heitz E. 227 Heitz L. 322 Helboe P.,271 Heldeweg R.F. 339 HelgCe B. 218 Helgeson R.C. 272 Heller C. 85 103 Hetler H. G. 110 Hellgren E.B. 329 Helling J. F. 190 Helling R. B. 385 Helmchen G. 53,276 Helmy E. 40 Helrre W. J. 283 Henbest H. B. 177 Henderson G. N. 99 Henderson R.,540 Hendrickson J. B. 95 359 447 Henglein A. 143 HenikoR S. 408 Henning D. 402 Henrichs P.M.,380 Henrici-OlivC G. 176 Henrick C. A. 174,426 Henrie R. 322 Henriquez R. 293 Henri-Rousseau,O. 327 Henry D. W.,241 Henry R. A. 54 Henschel R.,356 Hentrich G. 84 Herber R. H. 190 Herberhold M.,172 Herblot M.,485 Hercules D. M.,241 293 Herlem M.,215 Herman F. 161 Herman G. 360 Hennans B. 491 Hermodson M.A. 498 Hernandez R.,84,460 Herrig W.,33 Herrmann W.A.163 Herzberg G. 76 Hess I. 407 Hess J. jun. 54 Hesse Ch. 49 Hesse M.,12 Hesse R. H. 148 346 Hetflejs J. 176 192 Hewett C.L. 494,495 Hewitt. R. C. 24 571 Heyman M.L. 85 Heywood J. 408 Hezemans A. M.F.,38 Hibberty P.C.. 277 Hibino K.,223 Hickmott P.W. 449 Hierowski M.,64 Higashi F. 5 14 Higashinakagawa T.,408 Hiiragi M.,358 Hilbers C. W. 398,405 Hill A. E. 101 297 371 Hill C. L. 204 Hill E. J. 553 Hill H. D. W. 22 Hill K. A. 368,430,452 Hill R. K. 85 110. 259 Hillebrand G. 13 Hills D. J. 294 Hilton S. E. 158 Hinata S. 193 Hindley J. 404 Hindley K.B. 275 Hintz P.J. 147 Hipwell M.C. 62 Hirai H.192 Hirai K. 39 358 Hirano S. 304,438 Hirao K.,295,346 Hirao T. 173 Hirayama N.,524 Hirobe. M..347 Hiroi K.,443 Hirose Y.,345 Hirsch W.,534 Hirschmann R. 506 515 5 16 Hirsekorn F. J. 175 Hirst J. 289 290 Hisano T.,343 Hishida S. 16 Hite G. 482 Hixson S. S. 234 Hiyama T. 304 333 363 376,437,438 Ho T. L. 417. 427 439 443 Hochster H.S.,293 Hocks H. L.,287 Hocks L. 194 Hockswender,T. R. jun. 126 Hodakowski L. 371 Hodge P.,269,423 Hodgson D.J.. 390 Hodgson G. L. 522 Hodson H. F. 467 Hofle G.. 272,334 Hofle G. A, 198,265,430 Hohener A. 23 Hon W. 407 Hofbauer P.,335 Hoffman D. H. 272 572 Hoffman H. M.R.,101.297 371 Hoffman J.L. 400 Hoffman L. K.,109 Hoffmarln,N. W. 171 Hoffmann P. H. 43,302 Hoffmarln M. K.,11 Hoffmann R. 85 109 167 Hoffmadn R.W. 167 Hoffmadn-Ostenhof,0.. 69 Hogan M.,503 Hogenkamp H. P. C. 32 533,534 Hogeveen H. 186.339 Hohener A. 3 1 Hoiness C. M.,167 Holden C. M. 18 Holker J. S.E. 31,463 Holland G. W. 141 Holland R. J. 533 Hollander F. J. 306 Holliman F.G. 95 Hollis M.G. 46 Hollitzer O. 342 Hollyhead W. B. 290 Holm A. 336 Holm T. 199 Holman R.J. 229 Holmes J. L. 9 10 Holmfeld E. 336 Holness N. J. 122 Holt G. 287 Holton R.A. 391 Holy N. L. 199 Holzwarth G. 35 Hong E. 485 Hoobler J. A. 147,274 Hooper M.,330 Hooz J. 382,420 Hope J. 49 Hopf H.369 Hopkins B. J. 54 Hopkinson A. C. 197,274 Hoppach D. 31 Hoppe. W.. 529 Hoppen V. 26 Hopper K. E. 500 Hopper S. P. 168 Hopps H. B. 266,420 Horejsi V. 71 Hori I. 427 Horino H. 183 Horn M.J. 499,500 Hornback J. M.. 238 Hornemilnn U.,466 Horning E. C. 54 Horning M.G. 54 Hornung V. 82 85 Horrocks W. D. 527 Horvath C. 44 Hosaka K.,302 Hoshi T. 244 Hoshino M. 147 Hosoda H. 460 Hosokawa T. 430 Hosozawa. S. 41 Houghton L. E. 355 Houmard J. 498 Houng-min Shih 321 House H. O. 146,255 Houser J. J. 241 Hovland A. K. 209 Howard J. A. K. 192 Howarth T. T. 519 Howe D. V.,190 Howe R.K.,335 Howell 1. V. 192 Howell J. A. S. 190 Howells R.D. 196 Howie G.A. 271 Hoyana M.,343 Hoyle C. E. 147 Hoyle R.M.. 138 Hrbek J. 39 Hruban L. 39 Hruby V. J. 514 Hrucir D. C. 307 Hruska F. E. 394 Hsieh D. P. H. 49 Hsieh Z.-H. 30 274 Hsiung N. 401 Hsu E.C. 35 Hsu S. Y.,484 Hu C. L. 499 Huang B. 442 Huang C. T. 342 hang F. 165 183 Huang F. C. 472 Huang J.-T. J. 93 Huang Y. Y. 240 Hub L. 41 Huber H. 23 Huber R.,539 Hubert A. J. 169 194,287 Hubert E. 403 Hubert P. ft.,197,428 Huckin S. N. 446 Huckstep L. L. 472 Hudec J. 36 Hudrlik A. M.,369 Hudrlik P. F. 414 443 Hudson A. 209 Hudson D. R. 54 Hudson M.F. 526 Hudson P. 149,358 Huegi B. S. 428 Hunig S. 254 343 Huet. F.,202 Huez G. 403 Huff J. 527 528 Hugel H.M. 305 Hughes L. R. 252 Hughes M. T. 36 Hughes R.J. 206,420 Author Index Hughes W. B. 194 Huisgen R 103 Hukins D. W. L. 396 Hull R.,343 357 Hull V. J. 150 312 Hull W. E. 22 Hulla F. W. 68 Hulshof L. A. 374 Hummel J. P. 205 Humphreys D. J. 316 Humski K. 120 122 Hung P. L. K. 173 Hunkapiller M. W. 540 Hunkeler W. 532 Hunt E. 522 Hunt J. H. 482 Hunt K.,106 Huot R.,302 Huper F.,522 Hurley L. H. 472 Hurwitz J. 409 Husar J. 19 380 Husband S. 136 Husbands J. 177 Hussain S.A. M.T. 270 Hutchings M. G. 205 206 Hutchings R. O. 424 Hutchinson C. R.,467,471 Hutchinson D.W. 395 Hutchinson R.E. J. 123 Hutt C. 377 Hutton R.S. 162 282 Hutton W.C.32 Hutzinger O.,7 Hvistendahl G. 9,98 Hwang R. J. 169,170,210 Hyatt. J. A. 348 Hylton T. A. 303 Hynninen P. H. 529 1bers J. A. 174 178 1bii N. 309 I brahim B. 101 Ichikawa K.. 203 1chikawa M.,343 Iddon B.. 158.352 IBeta H. 354 1hara M.,522 538 I ida H. 346 1itaka Y.,390 I izuka. T. 20 I keda M.,346,424 1kegame M. 295 I kehara M. 388,392,395 Imaizumi S. 18 Imamura M.,147 Imanaka T. 181 Imbach J.-L. 389 Imhoff.M.A. 116 Impicciatore M. 479 Imoto E. 440 Inaba S.4 176 Author Index Inaba T. 52 Inch T. D. 487 490 491 492 Ingold K. U. 136 137 139 147,292 Ingrarn A. S. 335 Ingram G. 15 Inman J. K.,499 Inman K.J. 286 Inoffen H.H.. 531,532 Inoue H. 244 Inoue I. 412 Inoue N..183 Inouc. S. 185 Inoue T. 244 305 Ioffe B. V. 157 Ireland R.E. 191 348 369 Iriuchijima S. 431 Itngartinger H. 361 Irving P.. 502 Isaacs N. S.,9 106 Isaksson G. 336 Ishibe. N. 353 Ishiguro Y.,104 Ishihara S. 346 Ishii H. 330 Ishikawa M.,147 169 Ishikura. K.,147 Isida T. 321 Ison R. R. 478 479 482 483 Itakura K.,392 393 Ito O. 38 Itb S.,307 Ito Y.,173 185 300 451 Itoh I.. 424 Itoh M.,512 Itoh. O. 203 Ivanov. B. E.. 192 Iversen P. E. 227,23 1 Iwai K. 443 Iwakura Y.,516 Iwamura H. 164 244 298 Iwasaki T.. 227,440,508 Iwasawa H. 432 Iwata; S. 90 Iwatsubo M.,546 Iyoda M.,308 Izatt R. M.,357 Izmailov R.I.192 Izumi T. 183 Izumiya N. 5 11 5 17 Jablonski C. R.,183 Jack D. 496 Jackman D. E. 37 Jackowski G. 234 Jackson A. H. 346 519 520,521,522,524,527 Jackson J. L. 485 Jackson J. R.,520 Jackson M.R.,496 Jackson R.,294 Jackson T. E. 359 Jackson W. G. 29 Jacob T. A. 52 Jacobs P. 107 163 235 Jacques D. 475 Jacques J. 38 Jadhar A. L. 386 Jaeger D. A. 241 Jager G. 510 Jaenicke L. 25 Jahngen E. G. E. jun. 415 44 1,442 Jain P.C. 104 Jakobsen H. J. 34,459 James B. R.,175 James D. R.,102 James K.J. 470 James M.N. G. 484,493 James P.M. 49 Jamieson W.D. 11 Jandera P.,44 55 Janicki C. A. 48 Jankowski W.C. 458 Janson T. R.,528 529 Jansonius J.N. 545 Janssen P. A. J. 491 Jaouen G. 186.201 Jardetzky O. 2 1 Jarman T. R.,458 Jarrell H. 290 Jarrousse M.J. 484 Jarvis A. C. 191 Jarvis J. M.,404 Jasiwal D. K. 463 Jason M.,225,377 Jasor Y.,434 Jastod B. 392 Jaunin A. 333 Jay E. 393 Jean Y.,370 Jefferson I. 184 Jefford C. W.,100 167 Jeffrey A. M.,3 11,323 Jellinek F. 486 Jeminet G. 222 226 Jencks W. P.,131 272,544 Jenkins I. D. 212 289 Jenner G. 102 Jennette K.W. 406 Jennings E. C. 49 Jensen F. R.,121 Jensen N. J. 225 Jergil B.,66 Jerina D. M.,245 283 31 1 323 Jerkunica I. 64 Jesson J. P. 176 Jikeli G. 33 283 Jindal S. P.,120 Jirgensons. B. 35 Jochims J. C. 18 Jornvall H. 69 $53,554 Johansen H.,83 Johanssan A.274 Johnson A W.. 521 524 525,531 Johnson B. F. G. 29 174 179,189 190,363,379 Johnson C. D. 327 Johnson D. C. 53 Johnson D. E. 380 Johnson J. C. 172 Johnson J. F.,47 Johnson L. N.,539 Johnson R.,190 Johnson R. D. 464 Johnson R. W. 146,380 Johnson W. F. 54 Johnson W. S. 369 Johnstone R.A. W. 16,79 Jolly P.W.,172 181 248 Jonas A. E. 90 Jonathan N.. 86 162 Jones C. R..398 Jones D. N. 40 Jones D. W. 301 Jones G. 53 108 158 159 346 Jones J. B.,276 Jones J . D. 174 179 Jones J. H. 449 Jones J. R.,24,25,290,358 463 Jones L.B. 291 Jones M.,154,266,375 Jones M.,jun. 300,302 Jones P. R. 427 Jones R. 192 Jones R.A. 387 Jones R.A. Y.,351 Jones R.B. 26 31,457 Jones R.G. 479 Jones R.V.H. 521 Jones S. P. 131,273 Jones W. M.,101 164 165 167,284,294,295 Jongejan H. 347 Jordan G. J. 30 274 Jordan P. M.,536 Jordon J. E. 144 Joshua H.,516 Jouin P.,325 Jozefowicz M.L. 524 Juhlke T. 11 5 Jula T. F. 171 Julia M.,330,413 428 Julia S. 107 257 435 Jung F. 102. 260. 350 413 Jung M.E. 21 1,368,452 Junghaus K.,229 Jurand J. 45,49 Juvet R.S. jun. 54 5 74 Kaba R.A. 140 Kabengele A. N.. 167 Kacian D. L. 409 Kadaba P. K. 44 1 Kadar K. 47 Kaempfe L. A. 187 KAgerdal L. 57 Kahnus C. E.,293 Kainosho M. 26 Kaiser C. 485 Kaiser E. M.,197 Kaiser K. H. 107 235 Kaiser S. 493 Kaiser U.227 Kajiwara M. 32 538 Kalab P.. 53 Kalaus G.,329 Kalechits I. V. 192 Kalicky P. 234 Kalinowski H.-O. 25 275 Kalman J. R.,212 289,412 Kalo J. 225 Kam B. L. 389 Kamata K. 334,439 Kamenar B. 481 Kametani T. 345 358 KamiCnska-Frela K. 17 Kamm K. S. 233 Kan G. Y.-P. 352 Kanamaru. R.,402 Kane V. V. 302 Kaneda K. 181 Kaneda T.. 244,305 Kaneko C. 345 Kaneko S. 271 Kaneko T. 371,481 Kane-Maguire L. A. P. 191 Kanematsu K. 163 Kang K. 185,414 Kang S. 478 Kano S. 155 Kapecki. S. 106 Kaplan H. 543 Kaplan L. 272 Kaplan M.L. 144 Kaplan N. 0..391 549 Kapoor S. K. 202 Kapovits I. 222 Kappauf K. A. 380 Kappe T. 279,358 Kappler F. E. 271 Kappus G. 8 Kapur J.C. 324 Karchesy J. J.. 16 Kardos J. 292 Karger B. L. 49 55 Karim A. 374 Kariyone K. 444 Karlsson A.. 466 Karplus M. 162 Kartsova L. A. 157 Karup-Nielsen 1.. 153 Kasafirek. E. 507. 546 Kasahara A.. 183 Kasai P. H. 138 142 143 292 Kasai Y.. 514 Kashdan D.S. 441 Kaslow H. R.,65 Kasparek S. 357 Kassell B. 543 Kastening B. 227 Kasuga K. 347 Katagiri N. 392 393 Katakai R.,516 Katayama C. 38 Kato H. 309,349 Kato. K. 185 Kato. N.,41 Kato S. 220 231 Kato T. 325,343 349 358 Katrib A. 93 Katritzky A. R. 19 101 149,327,343,351. 358 Katsuhara Y. 239 Katz J. J. 28. 528. 529 530 Katz M. 217 Katz S. 44,53 Katzer E. 224 Kauffmann T. 95 103,247 358,359 Kaufman L.G. 304 Kaun L. C. 289 Kaupp G. 243 Kausch M.. 374 Kawabata N. 202 Kawaguchi H. 409 Kawai M.. 277.443 Kawamoto F.. 181 Kawamoto I. 363 450 Kawanisi M.,32 1 Kawata N. 193 Kawazoe Y. 331 Kay J. 543 Kay L. M.. 539 Kayane Y. 317 Kayne M. S. 398 Kayser R.H. 123 Kazansky B. A. 207 Kazimierczuk Z. 389 Kazimirchik 1. V. 358 Kearns D. R.,384,397,398 402 Kebarle P.,269 Keeley. D. E. 360 375,448 Kees F. 199 324 Kehrer. J. P. 466 Keith G. 402 Keller L. S. 154 Keller M. 41 Kelley D. E. 403 404 Kelley J. A. 502 Kellie G. M. 358 Kellogg R.M.. 413 Author Index Kelly D. P. 305 Kelly S. J. 548 Kelly W. J. 120 Kelman A. D. 406 Kemmitt R.D.W.. 180,191 Kemp D. S. 5 17 Kemper B. 505 Kendall P. M. 334,439 Kennepohl G. J. A. 163 Kenner G. W. 509 519 520,521,522,523,529 Kenney. M. E. 527 Kent G. J.. 373 Kenyon R.S.. 178 Kerekes I. 419 Kern R.,47 Kerr G. H. 328 Keske. R.G. 141 Kessar S. V. 299 Kessler H. 25 275 Keutmann H. D. 503 Khaddar. M. R.,28 Khairullina R.Z. 192 Khan M. K. 395 Khan S. A. 380 Khand 1. U. 189 191,413 Khandelwal G. D. 328 Khmel’nitskii,R.A. 358 Khoklar A. Q. 489 Khorana H. G. 393,406 Khusid B. L. 188 Khym J. X.,391 Kiel J. G. 463 Kier L. B.. 478,487,490 Kiewiet A. 34 Kigasawa K. 358 Kiguchi T. 99 Kiji J. 180 182 189 Kijima 1.. 212 Kikic I. 53 Kikta E. J. jun.48 Kikuchi M.,498 Kilcast D. 82 Kilgour J. A. 170. 210 Killilea S. D. 534 Kim. B. 163 Kim C. U.. 430 Kim C. W. 236 Kim J. H.. 155 Kim J. J. 383 Kim J. K. 15 274 K,im. K. 150 312 Kim K. H. 320 Kim M. 414 Kim S. H. 383,384 Kim Y. C. 523 Kimura B. Y..191 Kindsvater J. H. 53 King F. D.. 424 King R.B. 182 King T. J. 525 Kinloch E. F.. 146 Author Index Kinloch G. F. 255 Kinnick M. D. 440 Kinns M. 282 Kinoshita F. 271 Kinoshita M.,441 Kinoshita T. 337 Kinsinger J. A. 89 Kippenhan R.C. 353 Kirby G. W. 468 Kirk D. N.. 36 37 41 42 Kirkiacharian B. S. 349 Kirkland J. J. 44 46. 55 Kirschner S. 95.96 111 Kiryushin A. A. 500 Kiselev V. G. 207 Kishida Y.358 Kiso K. 186 Kissinger P. T. 53 Kitagawa K. 512 Kitakani. K. 333 Kitarnura T. 321 Kitatani K.. 376,437 Kitchin J. 472 Kitching. W. 18 19 212 Kizim N. G. 164 Klabunde K. J. 199 Kianderman B. 3 I2 Klarner F.-G. 100 109 Klaus P. K. 335 Klein J.. 382 Klein K. P. 445 Kleinstuck R.,321 Kleps R.A. 391 Kleschick W. A. 26 Klessing. K. 377 Klessinger M.,84 85 Kliment M.,110 Klimov A. A. 290 Klinge D. E. 347 Klingen T. J. 53 Klingsberg E. 352 Klopfenstein C. E. 147,274 Kloster-Jensen E. 82 84 85,93 Kluender H. 472 Klug A. 383. 396 Klumpp G. W. 37 1 Klyne W. 36 37 39,359 Kmel'nitskii R.,7 Knapp S. 245 326 Knauer K.-H. 334 Knaus G. 197,439 Knaus G.N. 153 157 289 Knauss. G. 334 Kneidl F. 35 1 Knight E. jun. 73 Knights E. F. 205 Knights J. R.,3 12 Knittel D. 227 Knobloch G.. 528,534 Knox G. R.,189 190 Knox,J. H.,44,45.48,49,53 Knox S. A. R.,192 Knust E. J. 289 Knyazev V. N.,290 Kobak V.V.,521 Kobayashi G. 336,344 Kobayashi T. 79 277 Kobayashi Y. 169,345 Kober H. 374 Koblicova Z.. 38 Kobrina L. S.. 293 357 Koch. D. 216 Koch G. K. 428 Koch K. F. 28 Koch P. 189 Koch R.W. 292 Koch V. R..223 373 Kocheshkov K. A. 202,203 Kochetkov S. N. 65 Kochi J. K. 137 139 140 171,218 Kochman M.,39 Kocourek J. 71 Koeberg-Telder A. 287 Kobrich. G. 198 267 Koekoek R.,545 Konig D. 436 Koenig T.. 147 274 Koenig W.5 I2 Konigshofen H. 308 315 Koppel C. 11 12 14 Koermer G. S. 279 Koerners H. J. 394 Kossel H. 384 Koster H. 393 Koster R.,206,207 Kogure T. 186 Kohler R.,535 Koizurni T. 325 Kojo S. 373 Kokensgard J. 144 Kokke W. C. M.C. 43 Kolb M. 197,427,434,449 Kolc J. 316 Kolinski R.A.. 351 Kollrnan P. A.. 27 274 Kollmar H. W. 96 Kolodeg A. 478 Kolomnikov I. S. 171 184 Komatsu K. 146 Komoroski R.A. 22,32 Kondo K. 206 337 416 426 Kondo M.,25,35 Kondo S. 390 Konishi A. 167 Konishi H.. 180 Konishi K.. 169 Kop. J. M.M.,428 Koppel. G. A. 440 Kopylova L. I. 176 Kopyttsev Yu. A.. 188 Koreeda M. 40.4 1,49,276 Koren R.,118 Kornberg A. 408,409 Kornberg R.D.408 Kornblum. N. 439 Kornfefd E. C. 479 Korpi J. A. 48 Kort C. W. F. 7 Korte W. D. 198 Korver O. 39.41 Kory D. R.,146,147 Korzeniowski S. H.. 443 Koshland D. E. 551,552 Kosikowski A. P. 102 Kossmehl. G. 276 Kost A. N. 358 Koster. D. F. 110 Kostin. A. J. 504 Kosugi H. 443 Kosugi Y. 141 Koto S. 18 Kotowycz G. 23 32 Kouba J. 528 Koudjis A. 347 Kouwenhoven C. G. 251 353 Kovacic P.,359 Kovacs. J. 100 167 Kovan K. 516 Kovar R.A. 208 Kovvali S. R.,322 Kowalewski. K. 478 Kowalski C. J. 369 Koyama G. 390 Koyama K. 5 12 Kozerski L. 17 Kpoton A. 209 Krabbenhoft H. O. 359 Kraft M.,8 Kramer A. V. 178 Krarner F. R. 405 Kramer C. W. 204 Krarner J.M. 344 Krarner. R. A.. 404 Krane J. 380 Krantz A. 245 Krapcho A. P. 375 415. 441,442 Krasnoshchek A. P.. 13 Kraus. J.. 18 Kraus M.,192 Krauss G. A. 445 Kraut .I.,543 Kray W. 546 Krebs A. 85,361 Krebs K.-F. 46.47 Kreiger M. K. 306 Kreil G.,497 Kreissl F. R.,22 Kresge A. J. 273 Krichefdorf H. R.,516 576 Kricka L. I. 259 357 Krief A.. 198 321,422 Krippahl G. 462 Kristinsson H. 346 Krodel E. K. 533 Kroll 3. O. 184 KrOOn A. P. 347 Kropp P.J.. 234. 239 Krow G. R. 203 Krusell W. C. 186 Krikstc P. J. 138 Ku T. 141 Kucera P. 46,55 Kucherov V. F. 253,363 Kuchler R. J. 69 Kuck. V. J. 282 Kuebrer N. A. 77 Kuechler E. 400 401 Kuehl L.458 Kiihn K. 500 Kustler A. 87 Kuhar S. 387 Kuhlmann H. 436 Kuhn D.. 360 Kuivila H. G. 212 Kukla. D. 539 Kukolev V. P.,171 Kula M.-R. 392 Kulikowski,T. 395 Kulish M. A.. 521 Kumada M. 169 186 Kumadaki. I. 345 Kumagai M. 176,210,445 Kumagai Y. 185 Kumar N. G. 20 Kunieda N. 441 Kuntz G. P.P. 23 Kunz. R.A. 432 Kunzru D. 46 Kuramitsu T. 182 Kurita J. 354 Kuriyama K. 38. 39 270 Kurkutova E. N. 351 Kurobe M.,512 Kurozurni S. 430,438 Kurtz A. N. 353 Kurz W. 107 261 Kuschel H. 12 Kutnevich A. M.,282 Kutney J. P. 38,424 Kuwajima I. 432 Kuznetsov B. N.. 193 Kwang K. J. 557 Kwart H. 132,263,296 Kwiatowska J. 497 Kyba E. P. 159 Laarhoven W. H. 313,314 La Bahn V.A. 157 La Bar R. A. 295 L'abbe G. 159 Labinger J. A, 178 Labovitz J. N. 369 Labrum J. Fvl. 316 Lackner H. 504 La Cour. T. 178 Ladner J. E. 383 396 Ladwig C. C. 233,311 Laemmle J. 200 201 382 Lai. C.-J. 385 Caini F. 494 Laird G. R. 45 Laird T. 296 Lakhan R.,357 Lakshinikantham M.V. 338 La] D. 136 Lalloz L. 153 Lam Y.-F.. 23 La Mar G. N. 23 Lamai-line R.,285 Larnaty G. 275 Lambert J. p.,380 Lamm B. 226,424 Lan N. T. 489 Lancaster J. E. 323 Lancini G. 466 Landberg B. E. 328 Landgraf W. C. 49 Landini D. 419 Landis R.T. jun.. 147 Lane C. F. 205,266,420 Lang P. 506 Lange L. G.,553 Lange S. M. 390 Langer E. 38,302 Lankin D. C. 346 Lanneau G.209 Lansbury P.T. 107,433 Lanyiova Z. 79 85 103 Lapidot Y. 400 Lapidus A. L. 189 Lapidus J. B.. 488 Lapierre Arrnande J. C. 167 Lapis S. 291 Lapper R.D. 31 Lappert M.F. 195 209 Larchevique M.,363,436 La Rose,R. 377 Larose R. H. 53 Larrahondo J. 109 264 Larsen J. W. 283 Larsen P. O. 271 Larsson P.-0.. 67,68 Lassettre E. N. 77 Lathan W. A. 370 Lattes A. 296 Lattimer C. J. 279 Lau D. Y. K. 523 Lau J. T. 437 Lau K. S. Y.,179 Lauer G. 361 Lauer M. L. 16 Lauer R. F. 421 Laurent A.. 223 321 Author Index Laurent E. 223 Laurent J. 245 Laurson R. A. 499,500 Lautenschlager M.J. 517 Lavallee D. K. 20 394 La Voie E. J. 338 Lavrik P. B. 423 Law P.Y. 73.533 Lawesson S. O. 362 Lawrence J. F. 49 Lawrence J. G. 49 Lawson D. F. 141 Layton A. J. 286 Lazdunski C. 546 Lazdunski M. 546 Leandri G. 4 18 Leavell S. 81 Lebedev B. L. 188 Le Belle M.J. 279 Lebet C. R.,464 Le Bigot Y. 40 Lebleu B.. 403 Lebowitz P. 407 Lebreux C. 273 Leclercq M.,403 Leclercq P. A. 501 Leder P. 405 Lednor P. W. 209 Ledwith A. 259 357 Lee A. 0..427 Lee A. S. 407 Lee C.-H. 21 212 Lee C. K. 234 Lee D. G. 115,274 Lee D.-J. 127 Lee G. A. 240 Lee J. H. 331,332 Lee M. S. 160 Lee T. H. 93 Lee T. N. ti. 407 Lee W. M. 241 Lee Y.C. 69. Lee Y.S. 147 Leeman S. E.,503 Leeney T. J. 365 Lee-Ruff E. 197 Leete E. 47 1 Lefferts J.L. 212 Le Gaillard F. 62 Legautt R.,324 Le Gay D. S. 48 Le Goffic F.. 66 Le Guen J. 3 13 Lehman P. G. 339 Lehn J. M. 272,274 Lehner. H. 38,302 Leibfritz D. 32 Leigh J. S. 557 Leitch L. C. 23 Leitch R. E. 45 Lemal D. M. 85,241 Le Merrer Y.,422 Author Index Lemieux R. U. 18 Lenaerts F. M. 491 Lenarda M. 180 Lenich F. T. 369 le Noble W. J. 102,154,298 Lenoir D. 119 359 Lenz G. R.,336 Leonard N. J.. 325,387 Leopotd E.J. 369 Le Pecq. J.-B. 406 Lepeska B. 125 Lepschy J. 272 Lerch E. 12 Lerche H.. 436 Lerman L. S. 405 Lerner. H. 307 356 Lester B. M. 485 Lester G. R.,7 Leung H. W. 290 Leung T. 206,256,418 Lever 0.W.. jun. 198 265 Levi A, 274 Levin R.H.154,299 300 Levitzki A. 55 1,552 Levsen K. 9 Levy A. B. 159 Levy. E. S. 537 Levy. G. C. 22 Lew G. 442 Lewalter J. 513 Lewis A. A. 82 Lewis A. M. jun. 406 Lewis D. W. 279 Lewis F. D. 146 147,380 Lewis J. 29 179 189 190 363,370 Lewis J. J. 494 Ley R. V. 492 Leyendecker. F. 259 375 Leznoff C. C. 338 Leznoff C. J. 241 Li C. H. 57,503.5 1 1 Li H. J. 399 Li M.P. 453 Li T. K. 554 Li W.-K. 154 298 Liaaen-Jensen S. I3,42 Liang G. 29 120 283 Liao C. S. 222 Libert M. 2 19 Librando V. 19 Lichtenberg D. 21 Lichtenthaler F. W. 387 Lien E. L. 73 Lienhard G. E. 542 Liepa. A. J. 520 Liggero S. H.. 116 Light R. J. 462 Lightner D. A. 37,534,536 Liler M.130 273 Lilie W. 338 Lilienbtum W. 167 Liljas A. 549 Liljefors T. 302 Lilly M.D. 72 Lirn C. L. 14 Limpert R.J. 47 Lin G. W. 346 Lin H.C. 284 Lin L. J. 32. 472 Lin L. P. 164 Lin T. M.,479 Lin W. H. 418 Lin Y. Y. 276 Linda P. 358 Lindahl T. 408 Linder R.E. 35 Lindley H.,497 Lindman B. 24 Lindner C. 336 Lindner D. L. 176 Lindsay D. 136 Lindsell W. E. 202 Lindstrom M. J. 147 267 Line L. L. 222 Lines R.,142 224 Ling N. 504 Ling Chwang T. 387 Linstrurnelle G. 257 Liotta C. L. 357,419,442 Liotta D. 288 Lipinski C. A. 369 Lipman A. L. jun. 293 Lippard S. J. 184,406 Lippke W. 244 Lipprnann W. 480 Lipsky S. R.,473 Lisle J. B.379 Listl M. 325 L’ltalien Y.J. 480 Littauer U. Z. 403 Little R. D. 298 Little W. F. 288 Liu M.S.-H. 14 Liu R. S. H. 233 31 1 Liu S. 234 Live D. H. 23 Livingston D. C. 390 Livingston D. M.,409 Ljundquist S. 408 Ljusbergwahren H. 528 Lloyd D. 357 Lloyd K. 5 12 Lloyd R. V. 137. 144. 277 Lo K. W. 386 Lo T. B. 503 Loaris G. 208 Lochinger W. 506 Locke D. C. 47 Lockley W. J. S. 460 Lodochnikova V. I. 203 Loeb L. A.. 409 Loew E. R.,478 Loewenstein A. 24 Lofberg R.T. 52 Lomer H.-P. 379 Loftus P. 18 Loginova N.F. 226 Loheac J. 48 Lomas J. S. 139 hbardo L. 154 Long J. P.,488 Long M. A. 178 Longone D. T. 304 Loosli H. R.,333 Lopez L. 285 Lorck H.459 Lorenzetti 0.J. 485 Lornitzo F. A. 62 Losch R.,336 Loucheux-Lefebvre M.-H. 20 Lovat M. M.,314 Lovey A. J. 442 Lovins R. E. 54 Lowbridge. J. 545 Lowe C. 56 Lowe C. R. 68 69.74 Lowe G. 545 Lowe L. A. 493 Lown J. W. 108,328 Loynes J. M. 479,481 Luche M.-J. 434 Lucherini A. 194 Ludwikow M.,163 Lukashenko A. P. 282 Lunassi L. 147 Lund H. 225,226,227 Lusch M.J. 185 Luskovits I. 292 Luton P. 113 Luz Z. 24 Lyerla J. R.,28 478 Lyn D. 290,297 Lynd R.A. 208,413 Lynen F. 462 Lysyak T. V. 184 Lythgoe H. 444 Ma K. W.. 437 Maassen J. A. 401 MacAlpine G. A. 27 1 McAninch T. W. 373 McBay H. C. 138 MacBride J. A. H. 344 McBride J. M. 293 McCabe J.R. 258 McCaffery A. J. 35 McCafferty D. J. A. 29 McCaffrey R. P. 404 McCamish M.,502 McCamrnon J. A. 22 Maccarone E. 288 McCarthy. A. R.,335 578 McClain W. H. 399 McClelland R. A. 27 273 McCloskey J. A. 399 McClure J. D. 180 McCombie S. W. 5 19 523 526,529 McConnachie G. 148 McCormack J. D. 289 McCoy M. T. 400 McCrearty M. D. 279 McCurry. P. 45 1 McDaniel R.S. 108 McDermott J. X..189,363 McDonagh A. F. 535,536 McDonald E. 458,522,536 538 McDonald K. 169 McDonald R.N. 288,314 MacDonald S. F. 523 Macdonald T. L. 429 McEntire E. E.. 159 McEwan R.,53 McFadden W. 7 McGillivray D. 9 McGillivray G. 324 McGinnis J. F. 64 McGrew J. G. 12 Mach M.H. 289 Machin. A. F.,49 Maciel G. E. 26 28 McInnes A. G. 31,463 McIntosh C. L. 245 283 McIntosh J. M. 261 338 Maciorowski C. A. 30 274 McIver R.T. 277 Mackay I. R.,495 McKelvey J. 274 McKelvey R.D. 105,239 McKenna J. 168 McKenna. J. M. 168 McKennis J. S.,187 194 Mackenzie K. 104,380 Mackenzie R.,172 McKenzie T. C.. 19 1 McKervey M. A. 373 374 Mackie R. K. 339,358 McKillop A. 209 McKinney C. R.,54 Mackor A. 140 147 McLafferty F. W. 7 9 10 12 14,54,282,501,502 McLaughlin G. M. 526 MacLaury M. R.,179 MacLean W. W. 49 McLennan D. J. 114 McLeod D. jun. 138 142 143,292 McLeod J. K. 123 McMahon. T. B. 15,269 McManus S. P. 119,126 McMeeking,J. 180,181,360 McMillan R.,192 McMullen G.L. 165,253 McMurry J. E. 415 425. 433,437 McMurry P. M. 363 McNair H. M. 45 McNeil D. 104,250,371 McNelis E.. 436 Maconochie J. 496 McPherson A. 383 384 McPherson A. M. 208 McPherson C. A. 125 McPherson E. 175 McQuillin F. J. 171 McVey J. K. 147 McWilliam D. 184 Maden B. E. H. 403 Madhavan S. 109 167,264 Madison J. T. 400 Madronero R.,353 Madsen J. @. 23 1 362 Madsen N. B. 539 Maeda. K. 390,430 Maeda M. 399 Markl. D. 350 Markl G. 225,341,351 Magatti C. V. 184 Magid L. L. 533 Magidman P.,47 Magno F. 222 Magyar E. S. 380 Magyar J. G. 147 Mah H. D. 523 Mahishi N. 484 Mahnke H. 30 Mahuzier G. 349 Maier G. 306 361 Maier J.P. 75 79 82 84 85,86 Mairanovski, V. G. 226 Maizels N. M. 404 Majer J. R.,15 Majerski Z. 169 Majoros G.. 47 Majors R.E.. 45.48 Maki T. 207 Makisumi Y.,338 Makosza M. 163 Malatesta V. 136 141 Malaval A. 273 Maldonado L. 197 435 Milek J. 426 Maletina I. I. 288 Malhotra 0.P. 551 Malik J. M. 32 Malis F. 546 Malkus H. L. 141 Mallory C. W. 281 Mallory F. B. 241 281 Mallory T. P. 298 Mallows R.,39 Malone G. R.,43 1 Mamer 0.A. 164 Author Index Mandeville W. H. 174,447 Mangini A. 288 Mangum M. C. 164 Manhas M. S. 324 Maniatis T.,405 Manion. C. V. 49 Manion M. L. 162 Maniwa K. 43 1 Mann M.,308 Mann T. A. 485 Manne R.,82 Mannik M.. 57 Manning C.241,338 Mannitto P. 535 Manocha A. S. 93 Mansfield C. A. 191 Mansy S. 389 Mantle P. G. 468 Mantovani A. 183 Mantsch H. H. 23 Mantzaris J. 182 Manus M. M. 281 Manyik R. W. 188 Manzer L. E. 30 Maravigna P. 19 Marbaix G. 403 March S. C. 56 Marchand A. P. 162 Marchand-Brynaert J. 106 262,324,362 Marchese S. 49 Marchon J. C. 528 Marcuzzi F. 127 254 Margaretha P. 233 Margolis S. 478 Marino A. F.. 443 Marino G. 327 Marino. J. P. 103 264 363 371,447,451 Marion M. L. 144 Marjoribanks C. E. B. 494 Mark V. 104 Markham L. D. 175 Marko L. 171 194 Marlinson P. 281 Marotta C. A. 404 Marquarding D. 186 Marquet A. 434 Marschner F. 79 Marsh R. E. 281 Marshall D.R.,357 Marshall G. R.,20 Marshall 1. G. 495 Marshall J. L. 305 Marshall R.A. G. 302 Martelli E. 466 Marten D. F. 442 Marten T. 32,458 Martin A. R.,351 Martin D. R.,394 Martin H.-D. 85 103 334 Martin J. 335 Author Index Martin J. C. 423 Martin J. S. 28 Martin L. E. 496 Martin M. 55 Martin M. A. 401 Martin R. 157 Martin R. B. 32 Martin R. H. 240. 313 Martin S. F. 435 Martinengo S. 29 Martinez Y.,514 Martin-Smith M. 489 494 Marttila C. M. 396 Marty R. A. 110 Maruya. K. 193 Maruyama K. 102 Marwick F. A. 494 Marx F. 539 557 Marxer A. 200 Maryanoff B. E. 350 Marziano N. C.. 286,288 Marzilli. L. G. 389 Marzin C.. 327 Masai N.18 Masaki Y.,413 Masamune S. 184,360,411 Maschler H. 53 1,532 Masclet P.. 84 Masda G. M. 315 Maseda C. 344 Mashkovsky M. D..493 Maskasky J. E. 527 Maslakiewicz J. R. 347 Mason S. F. 38.40 Massaro E. J. 557 Masse G. M. 261,338 Massei A.. 172 Massey. V. 498 Mastropaolo. M. 190 Masuda S. 286 Masuda Y.,207 Mataka S. 157 Mateescu G. D. 120 Math V. B. 42 Matheson T. W. 29 190 Mathews B. W. 303 Mathias R. 163 Mathieu J. 95 Mathis D. E. 53 Mathys G. 159 Matin S. B. 157 Matiskella J. D. 424 Matsuda Y.,336,344 Matsueda G.. 498 Matsukubo H. 349 Matsumoto A. 331 332 Matsumoto H. 86 169 Matsumoto K. 155 508 Matsumoto M. 337 Matsumura N. 440 Matsumura. Y.,223 Matsuo K.165 Matsuo T.,101,343 Matsuoka M. 508 Matsuoka T. 343 Matsuura N. 35 Matsuura T. 243 Matteson D. S.,205 Ma!thes. D. 350 Matthews B. W. 540 Mattox J.. 141 Matwiyoff N. A, 32 529 534 Maue R. 392 Mauer K. H. 8 Maujean A.. 19 Maurer W. 33 Mautner H. G. 486,487 Mauzerell D. 526 Maxim T. E. 52 May M. 490 Mayeda E. A. 221 Mayer A.. 522 Mayhew S. 498 Mayne-D'Haultfoeuille M. 74 Mayo P. D. 326 Mayr A. 225 Mazumdar S. K.,396,539 Mazzocchin G. A. 222 Meakin P. Z. 176 Meakin R. 138 Meakins G. D. 327 Mechlinski W. 49 Meese C. O. 273 Meesters A. 240 Mehta,G. 154 155.202 298 Mehta H. P. 15 Meiboom S. 24 Meier H. 181 Meijer J.200 257 Meiler W. 30 31 Meinwald. J. 109 245 249 326,379 Meiris R. B. 46 Meisters A. 208 41 1 Mejstrikova M. 192 Mellon F. A. 16 Melloni G. 127 254 Mellor J. M.,79 104 Mellows G. 3 1,457 Mellows S. M. 100. 300 Melton J. 425 Me'ndenhall G.D.,136 Menes F. 80 Menheere P. 55 Menon C. S. 166 Merault G. 345 Merk W.,194 Merlet P.,98 Mermet-Bouvier R. 28 Merrifield R. B.. 5 11 Merrill R. E. 199 207 Merritt V.Y.,242 Merzhauser J. 49 Mesbergen W. B. 103 264 363,451 Meselson M. 408 Meth-Cohn O. 328 335 339 Metz E. C. 23 Metzger J. 333 Meunier B. 185 Meyer A. 186,201 Meyer G. R. 125 Meyer H. 271 Meyer L.-U. 186,374 Meyer R. B.. jun. 386 Meyer T. J. 288 Meyerhoffer.A.. 493 Meyers A. I. 197 334.348 358.43 1,439.440.444 Meyers M. 257.4 17,429 Meyerson S, 10 136 Mez H. C. 104 Michaelis A. F. 44 Michaelson R. C. 186,423 Michaely W. J. 533 Michalewsky J. E. 49 Michalska 2. M. 171 Michel D. 30 31 Michel M.-A. 226 Michelot D. 257 Michl. J. 299 316 338 Midland M. M. 206,417 Midorikawa H. 427 Midwinter G. G. 502 Miehe D. 534 Miginiac L. 198 259 Migita T. 169 Mihelich E. D. 440,444 Mikes. F.. 46 Mikhailov B. M.,205 207 Milcarek C. 403 Miles D,H. 442 Miles H. T.. 395 Milkowski J. 516 Millar E. E. 386 Millefori A. 301 Millefori S. 301 Miller B. 108 Miller E.M. 179 Miller J. A. 418 Miller. J. G. 107 261 Miller J.P. 386 Miller J. S. 277 Miller L. L. 215 218 223 224,276,373 Miller R.B. 211 419 Miller S. M.,515 Millington D. S. 521 527 Mills D,R.. 405 Mills J. 496 Milner J. A, 31,467 580 Milstein C. 404 Milstein D. 188 Minamikawa J. 424 Minghetti G. 173 Minisci F. 147 292 358 Minn F. L. 233 Mintz E. A. 123 Mioduski J. 109 249,379 Mironov A. F. 521 527 Mironova I. V.,46 Mishima T. 376,437 Mislow K. 205 281 282 350 Mison P. 32 1 Mistra S. P. 294 Misumi S. 244 304 305 338 Mitani S.4 180 Mitchell G. H. 338 Mitchell H. L. 200 Mitchell L. W. 33 Mitchell R.H. 303 416 Mitchell T. R.B. 177 Mitsudo T. 186 188 Mitsuhashi T. 101 167 Mitsuyasu T.. 182 Mittal R.S. D. 271 Miura I.31 33 Miwa T. 337 Miya'ima G. 25 Miyahe. N. 186 Miyano S. 202 Miyashi T. 106 108,237 Miyashita M. 443 Miyawaki S. 223 Miyoshi M. 507 515 Mizimo K. 312 Muogami S. 304,338 Mizon J. 74 Muon-Capron C. 74 Mizoroki T. 193 Muuno K.,243,337 Mizuyama K.. 336 Mlejnek R. V. 505 Mochalov S. S. 202 Model P. 404 Modena G. 127,254,274 Modro A, 266 Meller J. 164 Moller W. 401 Moffatt J. G.,388 Moffitt W. 36 Moghimi M. 143 Mohr. S. C. 405 Mok K.-L. 101 262,344 Moldowan J. M. 12 Mole T. 208 41 1 Molenaar-Langeveld T. A. 9 Molho D. 349 Molin M. 102,260,350,413 Mollere P. D. 85 Monlaudo G. 26 Montanari F. 419 Montaudo G. 19 Monteiro H. J. 328 Montevecchi P. C. 150 Montgomery F.C. 96 Monti D. 535 Monti H. 418 Mooti S. A. 373,379 Montzka T. A. 424 Moodie R.B. 284 Moody C. J. 331 Mookerjee P. K. 298 Moon R. B. 32 Mooney E. F.. 486 Moore H. W. 159 160,302 Moran J. F. 490 Moran M. 147 Moras D. 539 549 Moreau J. J. E. 186 Moreau N. 66 Moreland C. G.. 31 Morelli A. hY Moreto J. M.. 192 Morgan A. R.,293,395 Morgan J. W. 110 259 Mori S. 47 Moriarty R.M. 358 Morigaki M. 308 Morishima I. 20 86 Morita N. 306 Morita T. 314 315 Moritani I. 178 205 206 424,426,430 Moriya H. 154 Morland. D. 82 Morley J. S. 513 Morocchi S.,334 Moron J. 325 Morozowich W. 47 Morr. M. 392 Morris A. 86 162 Morris H. R..501 502 Morrison H. 244 Morrison W. H. tert. 198 428 Morrow J.F. 385 Morton M. B. 494 Morton T. H. 17 Mortreux A. 194 Mosbach K,. 66,68,69,73 463 Moschel. R.C. 385 Moscowitz A. 35,36,534 Mow W. P. 42 Moses P. R. 232 Mosher H. S. 35,43 Mosley J. 539 Moss R. A. 163,360 Mossman A. B. 269 Mostad A. 92 Mourad. M. S. 155 Mouvier G. 84 Author Index Mowat W. 193 Mowery R.A. 54 Mrochek J. E. 44,53 Miillen K. 308 315 Miiller B. 11 Miiller G. 523 557 Miiller W. 406 Muera I. 464 Muetterties E. L. 175 194 Muhi-Eldeen Z. 484 Muir A. R..282 Muir T. C. 494 Mukai T. 101 Mukaiyama T. 200 206 344,432,437 Mukerjce Y. N.. 104 Mukherjee S. K. 132 Mukhtar R. 102 154,298 Mulholland P. 293 Muller B. 376 455 Muller C.. 361 Muller G. 537 Muller J.-F.84 Muller P. M. 532 Muller R.J. 205 Mullock E. B. 328 Mulvey D. 148 Munakata K.,41 Munroe M. 54 Murahashi S. 430 Murahashi S.4 178 225 424 Murai S. 430 Murakami Y. 330 Muramatsu S. 363,450 Muraoka H. 517 Murata,I.,313,317,353,354 Murata M. 166 Muratova R. G. 192 Murayama D. R. 225. 230 Murofushi K. 244 Murphy D.. 143. 144 Murphy G. 462 Murphy G. J. 198 Murphy J. R.,63 Murphy R. M.. 2~0 MUK,B. L. 205 Murray C. D. 153,299 Murray K. 384,385,407 Murray N. E. 385 Murray R. K. 29 Murrell J. N. 82 84 Muscio F. 458 Muscio 0.J. 458 Musgrave W. K. R.,290,347 Musso R. E. 404 Mustafa A. 357 Muthukrishnan R.,429 Myatt H. L. 266,420 Mychajlowskij W. 257,417 422,453 Myers.T. C.. 391 Author Index Mykytka J. P. 346 Mynott R. J. 212 Nabeck J. M.,281 Nadamuni G. 292 Nadir U.K.,299 Naf F. 104,263 371,446 Nagai T. 35 Nagai U. 39,277 Nagai Y.,169 176 186,445 Nagakura S. 79,90 Nagarajan K.N. 42 Nagarajan R.,39 Nagasawa M. 462 Naghaway. J. 493 Nahlovsky B. 296 Naidoo B. 467 Nair R. M. G. 503 504 Nair V. 320 Naito T. 99 Nakadaira Y.,169 Nakagawa M.,38,308 Nakagawa Y.,223 Nakai H. 528 Nakai T. 427,434 Nakajima T. 286 Nakamura A. 167 Nakamura H. 390 Nakamura Y.,192 Nakanishi K.,31 33.40.41 49,276,399,464 Nakano E. 498 Nakano T. 169 Nakashima T. T. 28 Nakaya J. 23 1 Nakayarna J. 155,299,340 Nakayarna K.,451 Nametkin N. S. 210 Naoi M.6Y Narang S. A. 392 393 Narayanau P. 330 Narula S. 299 Naruse M.,417 Narwid T. A. 348 Naser-uddin 224,225 Nash C. H.,472 Nasralla S. M.,520 Nasse B. J. 186 Natat A. 275 Nath K.,409 Nathans D. 385,407 Natowsky S. 85 Natsuki R.,344 Nau H. 502 Naundorf G. 49 Naylor R.,302 Nazar R. N. 402 Nazarova N. M.,188 Nazer M.Z.. 39 Nedelec L. 368,445 Needham T. E. 32,534 Neely J. W. 148 Neergaard J. R. 41 Neeter R. 7 Nef J. W.,333 Nefedov 0.M. 288 Negishi A. 416 Negishi E. 199 205 207 418,442 Negishi E.-I. 41 1 Neiman Z. 21 Nelsen S. F. 18 79 147 Nelson A. J. 353 Nelson D. J. 21 Nelson J. J. 52 Nelson R. B. 14 Nelson R.F. 222 Nelson W.L. 488,489 Nemer M. 403 Nesrneyanov A.N. 164,212 Nesterova M.V. 65 Nestrich T. J. 171 Neta P. 143 Neubert L. A. 39 Neubold H. B. 102 Neue U.. 46 Neuenschwander M. 84 Neumann H. M.,200,382 Nuenhoffer H. 342 Neurath H. 498 Neuss N. 472 Newall A. R. 168 Newcomb M.. 98,272 Newkome G. R.. 342 Newman J. 403 Newman M. S. 167 314 434 Newton C. 403 Newton M.D. 27,281 Newton M. G. 166,302 Ng H. Y.,245 Niall H. D. 503 Nibbering N. M. M. 9 11 14,282 Nichol A. W. 523 Nichols W. C. jun. 241 Nicholson D. C. 520 Nickle J. H.,132 Nickon A.. 165,258 Nicolaou K.C. 185,444 Nieberl S. 325 Niederberger W.,24 Niernegeers C. J. E. 491 Niirnura Y. 271 Niitsurna T. 343 Nikanorov V. A. 202 Nikiforov G. A. 167 Nilsson B. 281 Nilsson N.H. 350 Nilsson R. 92 Ninomiya I. 99 Ninorniya K. 424 Ninou M. C. 47 Nishida S. 147 162 Nishiguchi I. 293 Nishiguchi T. 177 Nishihara T. 405 Nishimoto K.,25 Nishimura J. 286 Nishimwa S.,399 Nishioka A. 25 Nishishita T. 14 282 Nishiyama K.,305 Nivard R. J. F.,313,490 Nixon. J. F.. 180 188 Niznik G. E.,198,428 Noda H. 343 Noda L.. 557 Noe E.A,. 270 Noel Y. 452 Nohara A, 349 Nojima. M.,419 Nokami J. 441 Nolen R. L. 440,444 Noll M.,408 Nomura M.,373 Nomura Y.,18 Nonhebel D. C. 293 Norden A. G.W. 66 Nordgren J. 77 Nordin. I. C. 480 Nordlander J. E.,120 Nordling C. 77 92 Nordstrom B. 539,549,553 NorgArd S.,13 Norman R. 0.C. 136 138. 191 Normant H.,363,436 Normant J. F.185,203,419 447 Norris A. R.,290 Norris J. R. 530 Norris K.,500 Norris T. 335 Norton J. R. 30 Novik J. J. K.,41 Novosel B. 55 Novotny M.,48 Noyori. R. 101 18$ 371 Nozaki H. 164 166 304 323 333 363 376 417. 422 423 429 437 438 44 1 Nozaki S.,185 Nozoe S. 39 nTanda Kabengele 100 Nudel U. 403 Nugent. M.J. 43,302 Nugent W. A. 140 Nunn M. J. 418 Nyberg B.. 408 Nyberg K.,217 Nye M. J. 101 262,344 NylTenegger L.,349 Nystrom R. S. 32 Nyu K.,358 582 Oagawa H.,309 Oberlin R.,327 O’Brien C. 25 358 O’Brien J. S. 66 O’Brien R.D. 486 O’Carra. P.. 58,60 534 Occolowitz J. 472 Ochiai S.. 18 Oda M.. 379 Odaira Y. 239 Ohlsson I. 539,553 Ohm Y.,165 294 Oen H. 400 Ofengand J.401 Ogasawara K. 345 Ogata Y.,300 Ogawa H.,309 Ogawa S.. 20 Ogawa T.. 163 Ogden J. S. 172 Ogilvie K. K. 388 394 Ogliaruso M. A. 292 Ogoshi H. 524 O’Grodnick J. S. 460 Ogura F. 38 Ogura K. 362,438,440,44 1 O’Hanlon P. J. 521 Ohashi M. 40 Ohashi Z. 399 Ohloff G. 40 104 263 371 Ohlsson I. 549 Ohnishi T. 20 33.507 Ohno. A. 325,332 Ohno M. 390,51 I Ohta Y.,40 Ohtsuka E. 388 Ojima I. 176 186,210,445 Okada K. 32,538 Okama K. 505 Okamoto K. 146 Okamoto T. 224,347 Okamura K. 508 Okawara M. 340,427,434 Okonogi T. 412 Oku M. 104 Okuda M. 86 162 Okuno Y.,346,355 Olah G. A. 29. 120 126 248,283,284,286,419 Olby R.,383 Old R. W. 384 Oldenziel 0.H. 333 430 Oldfield E. 530 Olin G.R. 35 Olin S.S. 102 165 Olive S. 176 Oliver J. P.. 208. 209 Olivetta S.,294 Ollinger J. 446 Ollis W. D. 107 270 296 335 Ollmann J. E. 484 Olofson R. A. 507 O’Loughlin G. J. 467 Olp D. 147 Olsen H. 102 Olsen K. W. 539 549 Olsen R. E. 98 Olson G. L. 369 Olson M. C. 49 Olson P. E. 305 Olsson K. 281 Olszewski L. T. 47 O’Neal H. E. 96 Ong E. C.,43,302 Ookita M.,183 Oosterhoff L. J. 43 Oostreen E. A. 347 Oparaeche N. N.. 236 Opella. S. J.. 21 Oppenheirner N. J. 391 Oppolzer W. 104,345 Orchard A. F.. 75 Orchin M. 188 Orda V.V. 288 Oretskaya T. S. 202 Orger B. 242 295 Osaka N.,304,338 Osawa E. 373 Osborn J. A. 178 Osgerby J. M.. 523 Oshirna K. 323,422,441 Oshima T.35,399 Ostercarnp D. L. 351 Ostojit. N.. 55 Ostrow J. D. 536 Ota K.. 244 Oth J. M. F. 308,315,356 Otsubo T. 304,338 Otsuji Y.,440 Otsuka S. 167 Otsuko T. 304 Otto E.,40 Ouano A. C.. 49 Ouelette R. J. 35 1 Ourisson G. 459 Ovchinnikov Yu. A. 32,500 Overend W. R. 531 Overman L. E. 263,423 Overton K. H.,456 Owen C. R.,345 Owen G. S.,526 Oxford A. W. 510 Oya M.,516 Oyama K. 266 Oyler A. R. 271 Ozaki A. 193 Ozaki Y.,304 Ozment C.L. 226 Ozubka R. S. 33 Pac C. 243 312 337 Pachla L. A. 53 Pachler K.G. R.. 34 Author Index Packer J. E. 135,294 Paddock G. V.,402 Paddon-Row M.N. 105 Padgett H. 425 Padmanabhan R.,393,394 Padwa A. 159,240,319 Paetkau V.,395 Pagani G. A. 349 Page M.I. 544 Paglietti G. 354 Pagni. R.M. 99 300 Paine J. B.,tert. 522 Pakroppa W. 406 Palacios F.. 342 Paladini J.-C. 322 Paleeva 1. E.,202 Palenik G. J. 478,484 Paleveda W. J. 516 Palladino N. 192 Palmer D. N. 276 Palmer G. E. 162 Palmer R.A. 539 Palmertz I. 424 Palmquist U. 219 Palsingh B. 298 Pan J.. 406 407 Panchenkov G. M.,188 Pancoast T. A. 191 249 306 Pandit. U.K.,167 Pandler W. W. 307 Panek E. J. 199 Panet. A. 393 Pankov. Y.A. 503 Pankstehs J. V.,314 Panov E. M. 203 Pant B. C. 21 1 Panunzio M. 424 Paoletti C. 406 Papay J. J. 380 Paquette L. A.,85 102 104 187.236.250.371,413 Pardee A. B. 548 Parfitt R.T. 489 Parham W. E. 305 Parikh I. 56 Parish E. J. 442 Park M.-G.166 Parker A. J. 123 Parker G. R.,488 Parker K. A. 369 Parker V. D. 156 217,218 219,294,296 Parker W. 128,374 Parkhurst L. J. 398 Parks L. W. 459 Parlman R. M. 123 Parnell E. W. 494 Parnell R. D. 136 Parr W. 504 Parr W. J. E. 191 Parrington B. D. 108 Author Index Parris N. A. 54 Parry R.J. 471 Parshall G. W. 178 Parsons E. M.,478 Parsons I. W. 3 12 Partington P. 28. 487 Partis R.A. 178 Parton S. K.. 101 343 Partyka R.A. 424 Parulkar. A. P. 483 Pascal Y.-L. 177 Passerini R.G.. 288 Pastan I. 404 Pasto D. J. 125 Patchornik A. 510 Patel D. 482 Patel D. J. 20 394 Patel. V. V. 340 Patrick T. B. 163 Patton E. 287 Pau. J. K.,15,274 Pauling L. 543 Pauling. P. 486 487 493 494 Paulissen R.169 Pauson P. L. 189 190 191 413 Pavlik J. W. 162 Pavloff A. M.,485 Paxson T. E. 175 Payne M.T. 127 Pearce D. S. 160 Pearce R. 174 195 294 Pearson H. 23 Pearson J. D. M.,492 Pearson R.G. 178 Peat I. R.,28,478 Pechet M.M.,148,346 Pechine J. M.,80 Pedersen C. Th. 164 Peek M.E.. 160 31 1 Peel R.,366 Peer H. G. 527 Peerdeman A. F. 492 Peereboom R.,347 Peet N. P. 359 Pelegrina D. R.,430 Pellegrini M.,400 Pelter A. 205 206 207 342,415,420 Pendarvis R.O. 275 Penman S. 403 Pennella F. 176 188 Peppard D. J. 376 Pepper E. S. 478,479 Perchinunno M.,147 Perfetti R.B. 420 Perham R.N. 552 Periti P. F. 478 Perkins M.J. 148 Perrin. C. L. 130 274 Perrin R.,285 Perrotti E.189 Perry J. S. 312 Perry R.A.. 19 Perry R.P. 403,404 Perry S. V. 63 Person H. 157 Persson B.-A. 49 Persson N. O. 24 Pert C. B.. 492 Perutz M.F. 20 Pew G. 285 Pesheck C. V. 15 Pesnelle P. 376 Petcher T. J. 486,494 Peter M. G. 42 Peters A. T. 15 Peters E. N. 120 Peterson D. 414 Peterson K.B. 178 Peterson M.R.,26 Peterson S. 498 Peterson U. 498 Petitclerc C. 546 Petragnani N. 443 Petrillo E. W. 102 Petrissant G. 399 Petroff 0.A. C.. 443 Petrova T. D. 357 Petryka Z. J. 534 Pettee J. M. 506 Petterson R.C. 346 Pettit R. 96 187 194 Pettler P. J. 460 Petty H. E. 314 Peuker H. 330 Peverada P. 473 Peyerimhoff S. D. 98 Peynircioglu N.B. 153 Pfadenhauer E. H. 49 Ptleiderer W.18 Phelps D. J. 382 Phenning N. 528 Phillips B. 72 Phillips L. 26 31 282 457 Phillips R.,286 Photis J. M.,187 413 Piccolo D. E. 323 Pickering M.W. 158 352 Pickholtz Y.,188 Pieaenik G. 404 Pierrard C. 169 Pieter. R.,429 Pietra F. 291 306 351 Pietrzyk D. J. 46 Pigott H.D. 446 Pilkiewia F. G. 163 360 Pincombe C. F. 224 Pinder T. 557 Pinhey J. T. 212,289,412 Pinion J. P. 233 Pinkus A. G. 284,418 Pinna. F..192 Pinson J. 226 Piper P. W. 398 399 Pirazzini G. 288 Pirisiono G. 297 Pitkethly R.C.,192 Pitt W. W.,jun. 44,53,54 Pittman C. U. jun. 192 193 Pittner F. 69 Plabst D. 173 Plachky M.,110 Platbrood G. 175 Platzer. N. 26 Plaui. V.,479 Pleininger H. 534 Pletcher D. 215 Plieninger H.,535 Plotz P.H.,64 Plummer B. J. 243 Plusec J. 523 Pogonowski C. S. 432,443 Pohjala E. 344 Poindexter G. S. 234 Pointer D. J.. 496 Polglase W. J. 537 Poliakoff M.,162 Poling M.,220 Pollak A. 393 Pollini G. P. 533 Polonski T. 39 Pomonarev G. V.,520 Pond D. M.,359 Ponomarev A. M.,226,285 Pople. J. A. 93,379 Poradowska H.,347 Porath J. 56. 57 Porri L. 194 Port G. N. J. 478 Porta 0..147,292,358 Porter A. 403 Porter A. G. 404 Porter J. W. 62 Porter R.S. 47 Porter W. A. 104 Posner G. H.,174,185,426 447,453 PospiSil J. 53 Posvic H. 415 Potenza J.. 190 Potter C. J. 470 Potts A. W. 92 Potts. J. 503 Potts J. T. 505 Potts K.T. 28 Potts R.,474 Poulson. R.,537 Poulter C.D. 458 Powell G. P. 36 Poynter D. 496 Praeger D. 18 Pragst F. 220 Praliaud. H. 194 Pratt A. C..238 299 584 Pratt D. W. 144 Pregaglia Ci. F.,175 Prestegard. J. H.,27 Preston C.M.,22 Preston P. N..357 Prestwich. G.D.,369 Pretorius V.,54 Previero. A. 499 Prk J. T.. 213 Price R.. 403 Prince. R.H. 179 Privett 0.S.,49 Prociv T. M.,377 Proctor G. R. 316 Proger R.H. 420 Pros A.. 118 Protiuava J. 53 Proudfoot N. J. 404 Prout K..184.481 Proverb; R.J. 109 167,264 Pryde A. 45,371 Psoda A. 389 Puddephatt R. J. 178 Pukharevich V. B. 176 Pulleyblank D. 395 Pullman. B. 478,487 Puppe L..527 Puranen J. 490 Quast H. 98. 199,324 Querauviller A. 494 Qutdey F.R. 468 Quidey G.J.383 384 Qullliam M.A. 388 Quina. F.H. 233 Quinkert G.. 107 163 235 380 Quinn C. B. 359 Quirt. A. R. 28 Qureshi. A. A. 62 Raatn V.F.. 115 Rabai. J. 222 Rabalais. J. W.,93 Raban N.,270 Raber D.J. 112 Rabideau P. W. 286 Rabinawitz J. C. 399 Racadot. A. 62 Racadot-Leroy N. 62 Rach J. F. 325 Rackwitz H.-R. 386 Rademacher D. R.. 489 Rademacher P. 79 Radlick P.,284 444 Radna R. J,. 487 Radom L. 93 Rajapaksa D. 531 Rajaram J.. 178 Rajbhandary U. L. 399 Rhkosi M.,39 Ramachandran V.. 2 15 Ramage E. M.,357 Ramage. G. R. 481 Ramakrishnan V.T. 169 Ramasseul R. 42 Rambeck B. 256 Ramsay J. N.. 285 Ramsden C. A. 97,335 Ramsey B. G.,75,84 Ramseyer J. 65 Rance M.J. 348 Randall G.L. P. 174 Randerath E.. 394 Randerath K.,394 Randic M.,31 1 Rao A. S.,443 Rao A. V. R.,444 Rao G. V. 270 Rao S. T. 555 Rao Y.S. 425 Raphael R. A. 252,271 Rasadkina E.N. 192 Rassat A. 42 95 Rastrup-Andersen N. 459 Rathke M.W. 441 Rauchschwalbe G. 428 Rauh R. 330 Rautenstrauch V. 40 107 198,433 Ravindran N. 205 Ravindranathan M.,120 Rawn J. D. 542 Raymond A. J. 44 Raymond K.N.,307 Readio J. D.. 69 Reap J. J. 443 Rebek J. 296,5 17 Reddy K.R. 305 Reddy R. 402 Redey A.. 194 Reed C. A. 528 Reed (3. H. 557 Reeder R. H. 408 Rees C. W. 160 311 331 348 Rees H.H. 460 Rees J. H.,286 Rees Y.,293 Reese C. B. 392 Reetz M.T. 110 Reeves L. W. 24 Reeves P.C. 291 Refetoff S.401 Regan M.T.,183 Regen S. L. 177.421 Regnier M.T. 484 Rei M.-H.,120 Reich H. J. 421 433 Reich I. L. 433 Reichardt C. 276 Reichenbach T.. 21 1.419 Reichmans E. 307. 356 Reid D. H. 335 Author Index Reid R. E. 489 Reiff. K. 155 Reines S. A. 401 Reinhoudt. D. N.. 251,353 Reitherman R. W..66 Rempel G. L. 177 Renaud R.. 225 Renaud R. N. 3 12 Renga. J. M..433 Rens J. 40 Rensing U. F. E. 404 Rericha R. 192 Resnick B. M. 105 Rettig W. 165 Reuss R. H. 344,430 Reutov 0.A. 202 Revel M.,403 Revillon A. 53 Rexrodt F. W. 500 Rey P. 42 Reyes-Zamora. C. 26 Reynolds R. 222 Reynolds R. N. 377 Reynolds W. F. 27,28,273 478 Rhine W. E. 208 Rhoades J. A. 28 Rhodes D. 383 398 Rhodes J.E. 107,433 Ricci A. 284,288,334 Rich A. 383 384 505 Rich P. 492 Richards A. C. 351 Richards E. E. 327 Richards J. H. 32,540 Richatds W.G.. 478 Richardson F. S. 39,41 Richardson R. K.. 135.294 Richardson T. J. 282 Richardson W. H. 96 Richman J. E. 357 Richmond J. M.,288 Richter D. 399 Rickard R. L. 489 Ridd J. H-. 284 286 Riddell F. G. 358 Ridley H. F. 490 Ridley T. Y.. 8 Rieder W.. 335 Riedmuller S. 173 Rieke R. D. 199,207 Riemenschneider J. L. 120 Riera V. 192 Rifi M.R. 232 Rigassi N. 13 Riggin R. M.,53 Rihs G. 104 Riisom T. 459 Riley R. G. 276 Rilling H. C. 458 Rimbault C. G. 100 Rimington C. 520 Author Index Rimmelin J. 102 Rirnther J. 348 Rinthart. K. L. 32 464 Rinte.P. V. 180 Rioidan J. F.. 553 Ripka. W. C.. 419 Ris; c.,287 Ritchie B.. 90 Rittel W. 513 Rivat. C. 226 Riviere. bl.,296 Rizkalla B. H.,386 Roddrigues R. 443 Rotibins. J. D.,23Y Ro6bins. M.J.. 387 Rotkrl J. L. 31 464 Robert J. R. 281 Roberts A. W. 19 380 Rotierts B. P.. 136 148. 207 Roberts D. R.. 38 Roberts D. W.. 19.380 Roberts F. M.. 456 Roberts G. C. K. 28 271 Roberts J. D.,19 22 23 29 278,281 327,380 Robertson. H. D.. 404 Robertus. J. D.. 368. 396. 543 Robillard G.. 540 543 Robin. M. B.. 77 Robjqs. J. D.. 105 Robins R. K. 386 Robinson B. H. 29 Rdbinson. G. E. 365 Robinson J. M. 342 Robinson P. J. 192 Robinson R. E. 54 Robinson S. R. 31 1 Robinson W. T. 528 Rotpon.P. 14 R&k J. 132 Rochester. C. H.,524. 525 Ro-Choi T. S.,402 Rdewald. H. 361 R~dly,G.A.. 528 Roe A. M. 479 Roder E. 49 Roepstorff. P.. 500 Roesler. G. 392 Rosrier M.. 267 Rottele H.,356 Rogers D..4 15 Rogers H. R.. 203 225 Rogers M.T.. 137 277 Rogers. N. R.. 237 Rogers R. B. 342 Rogers R. J. 200 291 Rogic M. M.. 445 Rogiers. R.. 402 Roitburd G. V. 253 363 Roizes G..407 Rolla. F. 4 19 Rolls J. P.,32 Romanet. R.F. 363 450 Rommel E. 79 Ronan R. 503 Ronchetti F. 460 Ronlan. A. 218. 219 Rood,J. I.. 59 Roomi M. W.. 521 Roos B. 83 Roos. B. A,. 505 Root W.G. 184 Ropartt. C.. 226 Roques €3. P. 327 Ros. R. 180. 185 Rosa J. J. 398 Rosen S. D..66 Rosenberg E.. 29 Rosenblum.L. D. 174,426 Rosenblum M. 171,416 Rosenburg M..401 404 Roskowski. A. P. 484 Ross. H. H.,494 Ross. K. J.. 86 Ross. S. D.. 221 Rossi. M.. 79 Rossi. R. 194 Rossi. R. A. 291 Rossmann M. G. 539 549 555 Roth F. E. 484 Roth H.D. 144 162 Roth. J. A, 186 Rothbart H. L. 47 Rothenberg S.. 274 Rottman F. 39s. 404 Roulan A. 294 Roulet R. 188 Roumestant. M. L. 201 Rowan R. tert. 22. 33 Rowe M. D. 35 Rowlands R. J. 497 Rowley A. G.. 156 Rowley. L. E. 350 Roy R. B. 328 Rozenberg V. I.,202 Rozhdestrenskaya 1. D. 192 Rozynov B. V.. 500. 521 Rubini P. 28 Rutmttom G. M.,430 Rudnik L. R.. 443 Ruchardt C. 98 Ruff B. A.. 32 Ruge B. I67 Ruhfus A.. 498 Rumin R.. 240 Runsink J. 34 Ruppert.J.. 534 Ruppert. J. F. 441 Rusch. G.M.. 429 Russell B. R. 147 274 Russell C. S.. 537 Russell. D. R.. I91 Russe1l.G. A. 140 141 144 147 Russell K.E. 136 290 Russell L. W. 284.285.342 Russell. T. *.,412 Russo G..450 Rustum Y. M.,557 Ruterjans. H.,33 Rutherford. K. G. 164 Ruuiconi R. 124 Ryan M.D.,227 Ryback. G.. 40 Rybakuv. V. A,. 188 Ryder; I. E. 190 Rydon H. N. 20 Rye A. R.. 380 Rylander P.N. 10 Sabbah A.. 485 Sabin. J. R. 165. 294 Sab. D. L.,404 Sachdev K. 97 Sack. G. H..jun.. 407 Sadana K. L. 388 Sadar M.H.. 274 Sadler. I. 23 Sadownick. J. A.. 184 Saegusa. T.. 173 185. 300. 45 I Saenger. W.. 396 Saeva F. D..35 Safe S. 7 1 I Saga S.. 286 Saika D. 13 Saikachi H.,309 St.Jacques. M.,380 Sairam M. R.. 57 Saitb H..23 Saito I. 243 Saito K. 101. 185 Sakaguchi K.. 498 Sakaguchi 0..146 Sakai. M.. 108 Sakamoto T..349 Sakata. S.. 200. 344 437 Sakata Y.. 304. 308. 338 Sakore T. D.,4Y4 Sakura. H. 337 Sakuragi. M.,238 Sakurai. H. 169. 243 312 Sala E. 28 Salazar. J. A.. 460 Salbaum H..439 Salem L.. 97 162,370 Sales R. 275 Salim M.,403 Salmond W. S..532 Salomon. R.. 403 Salser W. 402 Salser. W. A,. 384 Sammes P. G.. 100,300,510 Samori. B. 39 Samuel C. J. 105,239 Schaffrin R. 478 Samuelsson B.. 461,462 Schally A. V. 503 504 Sancovich H.A. 522 Scharf H.-D. 279.358 Sandall. J. P. B.. 135 Scharf V. 275 Sandefur. L. O. 446 Scharschmidt. B. F. 64 Sanders G.M.,347 Schaumann E. 199,273 Sandhu S. S. jun. 190 Schechter I. 544 Sandorfy C. 77 Scheer H. 529,530 Sandstrom J. 336 Schegolev A. A. 253,363 Sane P.P.,443 Scheit K.-H. 386 396,401 Saneyoshi M.,399 Schekman R..408 Sanger F. 384 Schellekens K. H. L. 491 Sanjoh H. 302 Schenkluhn H. 341 Sankawa U. 471 Scherburg. N.H. 401 Sanno Y.,349 Scherowsky G. 164,340 Sano H. 189 Schexnayder M.A. 236 Sano R. 39 Schiavon G. 222 SantaCy F. 39 Schickaneder M.,330 Santelli M.,256 Schick-Kalb J. 46 Santopietro-Amisano A. Schieke J. D. 54 474 Schild H. O. 477 Santori G.. 466 Schill G. 356,427 Saran. M.S. 182 Schinco F. P. 30 274 Sargent G. D.. 120 162 Schirmer. I. 557 Sargent M.V.,309,311,356 Schirmer R.H. 539,557 Sarma R.H. 21,212,389 Schleis T.377 Sasada Y.,524 Schlesinger M.J. 547 548 Sasaki T. 163 Schlessinger J. 55 1 Sasse W. H. F. 243 Schlessinger R. H. 363.450 Sasson S. 106 Schleyer P.von R.,112,115 Sasson Y.,177 188,237 116 118 120 128 373 Satchell D. P.N. 273,449 374 Sajek L. 199 Schlogl K. 38 Sato H. 358 Schloman W.W. jun. 243 Sato K. 185 Schlosser M. 198,429 Sato M.,325 Schmatz T. G.. 281 Sato S. 511 Schmelzer A. 79 85 93 Sato T. 26 154 305 Schmid G. 354 Satterthwait A. C. 131 Schmid G. H.,266 Sauer J. 157 Schmid,H.,42,328,371.453 Sauer J. D. 342 Schmid R.,37 1,453 Sauer. R. 503 Schmid U.. 328 Saunders D. H. 47 Schmidbauer E. 283 323 Saunders D. S. 234 Schmidt A. 198 Saunders J. 522 Schmidt E. 334 Saunders W. H. jun. 124 Schmidt H. 85 102 Saussine.L. 413 Schmidt J. A. 53 Sauter H. 33,104 Schmidt K.D. 503 Sautiere P.,74 Schmidt W. 82,330 Sauvageau P.,77 Schmitt K.. 141 Savage D. S.. 494,495 Schmitz E. 342 Saveant J.-M. 226,227,228 Schmitz H. 371 Savory C. J. 174 Schneider H.-J. 26 Scalan I. 82 Schneider M. 184 Schaasberg-Nienhus Z. R. Schneider P.,532 H.,287 Schnepp O.,43 Schadt F. L. 118 Schnoes H. 472 Schafer H. 216 Schoenberg A. 442 Schaer H. 528 Schoenewaldt,E. F.,515.516 Schaublin S. 23 31 Schoenmakers J. G. G. 404 Schaffner. c.P.,49 Schossner H.,2 13 Schaffner K..233 Schofield K..284 Author Index Schonteeten. A, 330,428 Schorpp K. 179 Schrader U. 535 Schrameyer M.,275 Schrank. B. 498 Schrauzer G. N.,533 Schreckenberg M.,436 Schreiber J. P.,406 Schreiner K.292 Schreurs H. 257 Schroder U. 8 Schriider G. 356 Schroeder M.A. 175. 176 209 Schroppel. F. 157 Schroth G. 181,360 Schubert H.,505 Schug R.,103 Schuler R.H. 143 Schulman E. M.,23 Schulman J. M.,27 281 Schulte K.-W. 361 Schultz A. G. 99 Schultz F. A.. 53 Schulz G. E.. 539 557 Schulze A. 349 Schulze P. 8 Schumacher U. 155 Schumann D. 250 Schurig V. 46 Schuster D. I. 105 236 Schut R.N.,485 Schutz. G. 403 Schwager P.,539 Schwam H. 5 15,516 Schwarcz R.,h9 Schwartz. I. 401 Schwartz J. 188.426 Schwartz J. L.. 263 296 Schwarz H. 11 12 13 14 Schwarz N.,531 532 Schweig. A. 82 85,93 102 361 Schweitzer. G. K. 90 Schwieter U.. 13 Schwyzer R..57 Scopes P.M. 39,4 1,42 Scorrano.G. 274 Scott A. 439 Scott A. I. 32 38,538 Scott C. D. 44 54 Scott C. G. 54 Scott C. J. 224 Scott R.G. 528 Scott R.J. 153 156 Scott R.P..162 Scott R.P.W. 46.54,55 Scouten C. G. 205 Scribner R..M.,419 Scriven E. F. V. 158 Scudder P.H. 102 Seal R. H. 38 Author Index Sealy R.C. 136 137 Seamans L. 35 Sebestian I. 48 Sedai J. 406 Sedrati M.,380 Seebach D. 174. 197 198 211 271 422 427 429 434,437,449 Seeber R.,222 Seela F. 64 Seelig A. 24 Seelig J. 24 Seeliger A. 514 Seely J. H.. 509 Seeman. J. I. 236 Seeman N. C. 384 Segal J. A,. 174 Seguchi K. 102 Seiber J. N. 49 Seidewand R. J. 164 Seidman J. G. 399 Seifert K.-G. 294 Seiler N. 49 Seiwell L. P. 178 Seki Y.430 Sekiguchi A. 169 Sekiya T. 406 Selikson S. J. 425 Selsing. E. 396 Selztmann H. H. 51 1 Semmelhack. M.F. 79 191 Senda Y.. 18 Sendijarevit V. 122 Senkler. G. H. jun. 350 Senning. A. 350 Seno S. 46 Sera A. 102 Sergeev Yu. L. 285 Sergio R.,330 Serratosa F. 168 Seshadri T. P..395 Seth S. K. 484 Sethi. 0.P. 484 Seto H. 31 Seuring. B. 429 Severin. E. S. 65 Severin T. 436 Severinson S. 500 Seyfreth D. 164 168 169 173. 198,204,212 321 Seyferth K. 194 Shabarov Yu. S. 202 Shafi’ee A. 482 Shafran R. N. 288 Shahak I. 415 Shalaev V.K. 153 Shamshurina S. A.. 13 Shanker R. 421 Shannon,J. S. 14 Shannon P..295 Shannon. P. V. R.,346 Sharp J. T. 53 153 163 299,323 Sharpe P.E.,35 Sharpless K.B. 42 1 423 Sharrocks,D. N. 207 Sharts C. M.,419 Shaw A. 271 Shaw B. L. 179,256 Shaw C. K. 3 1,472,473 Shaw E. 546 Shaw J. R.,40 Sheals. J. E. 291 Sheehan J. C.. 508,5 10 Shefter E. 486,488 Sheldrake P. W. 31 467 468 Sheldrick G. M.,179 Sheline R.K. 30 Shelkov A. V. 188 Shelton G.. 524. 525 Shelton J. R.,293 Shemin D. 524 Shen L. 138 Sheppard H. C. 358 Sheppard W. A. 419 Sheifinski J. S.. 281 Sheridan P.,290 Sherma J. 44 Sherrad S. A. 303 Shetty R.V. 110 259 Sheverdina N. I. 202 Shibara S. 390 Shibuya S. 155 Shiekh M.Y. 277 Shields S. 179 Shields T. C. 353 Shigemitsu Y. 239 Shih C. N. 109 Shih H. 168 Shih. S. 98 Shih T. Y. 401 Shillady D. D.. 105 Shimada K.,80 Shimamura T.424 Shimizu K. 154 Shimizu N. 147 162 Shimoji K. 441 Shiwojo N. 309 Shine H. J. 150 297 312 Shine J. 402 Shiner V. J. jun. 115 116 122 Shinkai I. 155. 343 Shiono H. 434 Shiono M.,206 Shiori T.. 424 513,514 Shiraishi T. 349 Shirley D. A.. 86. 197 Shoeman D. W. 49 Shono T. 223.293 Shoppee C. W. 99 Short F. W. 442 Shriver D. F. 180 Shtark A. A. 283 Shteingarts,V. D. 283 Shugar D. 389,395 Shulman R. G. 384 398 405,522,540.543 Shulyndin S. V.,192 Shuman D. A. 386 Shvo Y. 416 Sidani A. 106,262 362 Sieber P.,5 13 Siefke B. 220 Siegbahn K.. 77 90 92 Siegel A. 11 Siegel T. 215 Siegel W. 407 Siegelmann H. W. 534 Siegrist M.,200 Siehl H.-U.,248 Sierra Escudero.A. 321 Sieveking M.F.,273 Sievers R.E. 47 Sievertsson H. 503 Sigal B. 485 Sigler P.B. 398 540 Sih C. J. 271,462,472 Siirala-Hansen,K. 185 191 Silhav9 P.,426 Silverman R.B. 533 Silverstein R. M.,276 Silvon M.P. 172 265 Sim G. A. 190 Simamura O. 33 1 332 SimBnek V. 39 Simm I. G. 87 Simmons H. E. 167,357 Simon H. 256 Simon. L. N. 386 Simon S. R. 20 Simonet J. 222 226 Simonetta M.,162 Simonis A. M.,490 Simonson L. P.,392 Simonyi M.,292 Simpson T. J. 31,463 Simpson W. 314 Simsek M.,399 Sinclair J. A. 206 4 17 Sinex F. M..406 Singer G. M.,342 Singh B. P.,154 155 Singh J. 366 452 Singh P. 390 Singh R.K.. 363 Singhal R.P.,391 Sinoway L. 174 447 Sinsheimer. R.L.. 407 Sippel A.E. 403 Sisti A. J. 429 Sivarajan M.,394 Siverns M.,32,458 Sjoberg. K.. 185. 191 588 Skare D. 169 Skell P. S. 147 163 172. 265,267 Skinner. D. M.,408 Skinner K.J. 293 Sklar J. 404 407 Skolik. J. 39 Skorcz J. A. 155 Skrzelewski A. 18 Skvarchenko V.R.,153 Skyc G.E. 474 Slsck D. 185 Slater D. W.9 403 Slater I. 403 Slater I. H. 479 Slaven R.W. 187 Slegeir W. 96 187 Slifstein C. 220 Sloan K.B.,305 Slutsky J. 120 373 Smail G. A. 489 Small D.A. P. 68 Smallcombe S. H. 540 Smalley R.K. 352 Smart B. E,,121 138 Smi!ey 1. E.,549 Smirnov V. N. 205 Smissman E. E. 488 Smit W. A, 253,363 Smith A. B. tert. 166 442 Smith C. 270 Smith D. G. 31,463 Smith D. J. 86 Smith D.J. H. 239 Smith D. W. 49 Smith E. M.,166 Smith G. G.,293 Smith H. E.. 41 277 Smith I. C. P. 22.23 33 Smith J. 193 Smith, J. D. 195 Smith K.,110,204,205,206 420 Smith K. M.,28 519 520 521 522 523 524. 525 526,529,530 Smith L. 312 Smith L. C. 501 Smith M.,393 Smith M.A. R. 180 Smith M.B.,208 Smith N. G. 344 Smith P.,140 Smith P. A. S. 168 Smith P. J. C. 396 Smith R.A.. 125 Smith S. G. 520 Smith W. B.,95 Smith W. E.. 168 Smith W. N. 250 Smolanoff J. 159 319 Smythe G. A. 520 523 Snatzke F. 39,41,42 Snatzke,G. 39,40,4 1,42 Sneden D. 383 Snider B. B. 260 Snider B. R.,15 1 Snieckus V. 352 Sninsky J. J. 393 Snyder J. P.. 85 102 Snyder L. R.,44,55 Snyder S. H. 492 Sobczak R.L.291 Sobell H. M.,494 Soderlund,G. 539,549,553 Sop J. A. 32 Soh K.S. 482 Sojka S. A. 84 Solgadi D. 80 Solly R.K.,277 Solomon P. H. 31 464 Soma G. 271 Somanathan,R.,107,296 Sombreck J. 307 Sommer H. 328,393 Sornmer J. M.,27 Sondheimer F. 230 252 306 307 309 310 356 357 Sone M.,344 Song B.-H., 305 Sonne T. O. 493 Sonoda N. 430 Sood R. 462 Soole P. J. 135 Sorensen S. 34 Sorensen. T. S. 240 Soreq H. 403 Sorokin V. I. 358 Sosinsky B. A. 192 Soti F. 149 358 Soudijn W. 492 Souma Y.,189 Sousa L. R.,105,239,272 Southard G. L. 506 Southon 1. W. 347 Sowinski F. 346 Sow-mei Lai Chen 40 Spagnolo P. 348 Spangenberg R. 506,508 Spanget-Larsen J. 84 Spangler R.J. 155 Sparatoge F.297 Spatz D. M.,391 Speakman P. R. H. 14 Spear R.J. 248 Spears D. P. 90 Speck D. H. 357 Speckamp W. N. 345 Spector D. H. 403 Spek A. L. 492 Spencer J. L. 180 Spencer T. A. 423 Sperow J. W. 548 Author Index Spiegelman S. 405,409 Spiess H.W. 30 Spiteller,G. 7 8 Spiteller M.,7 Spoole P. J. 294 Sprague J. T. 302 Sprinzl M.,399 Spyropoulos C. G. 470 Srinivasan K.G.,169 Srinivasan R.,242 Srinvasan P.R. 33 Stack D. L. 287 Stackhouse J. 350 Stahle M.,198 429 Stage D. F. 57 Stahl,J. 401 Stahnke G. 506 Staley R. H. 15 Stallcup W. B. 551 Stanaszek R.S. 493 Stanford R.H. 191 Stang P. J. 164 Stangl A. 498 Stanislowski A. G. 178 Stanley K.,141 Staral J. 29 283 Stark B.P.,408 Startsev A.N. 193 Staunton,J. 468 Stavrianopoulos J. G.,403 Steckhan E. 216 Steevensz R. S. 261 338 Stegel F.,327 Steglich W. 272 342 509 Steichen J. 81 Steichen J. C. 53 Steigemann W. 539 Stein H.. 334 Steiner D. F. 505 Steiner G. 103 Steinmetz W. E.. 37Y Steinwand P. J. 189 Steitz,J. A. 404,405 Steitz,T. A.. 539.557 Stenhagen E. 7 Stepanov V. M.,497 Stephens R.D. 141 Stephenson B. 43 Stephenson J. R.,538 Stephenson L.M.,359 Stephenson R. W. 348 Stepovska G. 184 Sterling D. J. 453 Sterling J. J. 185 426 Stermitz F. R.,218 Stern R.,405 Sternbach L. H. 493 Sternerup H. 216,217 Sternhell S. 212 324 412 Sternhell,S. S. 289 Stetter H.,436 Stevens J. C. 312 Author Index Stevens R.V.533 Stevenson G. R.,145 146 Stevenson K.J. 543 Stewart P.G. 14 Stewart J. C. M.,536 Stewart K. K. 72 Stewart. R.,290 Stewzrt W. W. 504 Stiegier P.,402 Still W.C. 429 Stille J. K.,179 183 Stilwell R. N. 54 Stiverson R. K. 185 265 Stock L. M.,138 Stockbauer R.,88 Stocklin G. 289 Stockton G. W.,28 Stoddart J. F. 270 Stoffler G. 400,401 Stohrer W.-D. 107 163 235,380 Stojanac N. 523 Stokolski E. A. 524 Stoll M.S. 521 Stollar H. 382 Stolyhwo A. 49 Stone,F.G.A. 180,184,192 Stone P. J. 406 Storey H. T. 20 St0rk.G.. 107 197,211,261 363 366 367 368 435 445,449 Stork G. A.. 452 Storm C. B. 524 Storr R. C. 160 31 1 348 Story J. N. 49 Stothers J. B. 25 26 Stott J. 403 Stotter D.A. 179 Stotter P. L. 368,430,452 Strachan A. N.,286 Strachan R.G.. 515,516 Strachan W. A. 352 Strain H. H. 528 Strakov A. Y.,358 Straub H. 100,253,306 Straus M.J. 290 Strausz 0.P. 163 Streets D. G. 84.92 Strege P..E. 173 192,449 Streith J. 445 Streitwieser A. jun. 120 Strell I. 529 Strickland R.W. 39 41 Stridsberg B. 278 Stringer A. J. 256 Strobl G. 255 Strohmeier W. 175 Strope D. 180 Stroshane R. M.,32 Stroud R.M.,539 Strouse C. E. 529 Strubert W. 53 Struchkov Yu. T. 184 Strukul G,,185 192 Stubenrauch G. 155 Stucky G. D.,208 Student P.J. 310 Studier M.H. 528 Studt \IJ. L. 443 Sturm M.,529 su,s. c.,49 Su S. R.,184 Su Y.Y.,169 Suarez. E.,460 Suan R.,245,326 Subrahmanyam G.242 Subramanian,J. 535 Subramanian,#. N..407 Suda M.,360 Suddath F. L. 383,384 Sudol J. J. 104,250 371 Suffolk R..I., 84 85 Suga K.,444 Sugano H. 507 Suggs J. W.,507 Sugihara Y.,353,354 Sugimoto T. 379 Sugita T. 203 Suh J. T. 155 Suhr R.G. 291 Sullivan D.F.,441 Sullivan D.R.,298 Sullivan E. A. 131 Sullivan F. R..291 Sullivan J. M.,351 Summers L. A. 346 Summers R.,137 Summerville €2. H. 128 Sunami M.,353 Sundaralingam M.,21 395 486 Sundberg L. 57 Sundberg R.J. 158 Sundelin K.G. R..489 Sunderman,F. 147,274 Sunderman. F.-B. 201 Suschitzky H. 158,328,335 352 Sussman J. L. 384 Sustmann,R.,98 138 Sustmann S.,tr8 Suter C. 376 Sutherland I. O. 107 296 Sutherland J.K.,258 366 Sutton J. R.,216 Suzuki A. 207 Suzuki K.T. 31,457,458 Suzuki M.,302 362,438 Svanholm U. 156,294,296 Svec W. A. 528 Svedberg D. P. 40 Svendsen A.. 271 Svensson f. 461,462 Svenwn S.,92 Sviridov B. D.,167 Svoboda P. 176 Swain J. R.,188 Swan C.G. 291 Swan G. A, 328 Swan J. M.,195,350 Sweeney A.. 524 Swen H. M.,545 Swenton f. S.,348 Swern. D.,13 Swisher J. V. 209 Sychkova L.D. 202 Sykes B.D..22,33 Sykes R.J. 31,472,473 Symalla D.,141 Symon J. D.,335 Symons M. C. R. 150,294 Symons k.H. 401 Synerholm M.E..110,259 Syntkina 0.P.,203 Syrdal D. D. 40 Syska H. 63 Szabo L. 329 Szakasits. J. J. 54 Szantary C.,329 Szendrey t.hf..270 Szeto K.S.,303 Szewczuk A. 497 6zwarc M.,80 Taarit Y.P.,194 Tabei K.,325 Tabis M.S. 297 Tabushi I. 373 Tagaki. W. 412 Tager H. S.,505 Taguchi H. 164 166+ 376 429,441 Taillefer R.!273 Takada S. 338 Takagi K.,300 Takahagi H..46 Takaharhi K.,25 Takahashi. T. 345 Takaishi K.,25 Takamizawa A. 358 Takano T.,141 Takase K.,314,315 Takaya H. 185 Takaya T. 157,289 Takeda A. 104 Takeda H. 330 Takeda T.,. 183 Takegami Y. 186,188 Takehara. S.,437 Takei H. 200 344,437 Takemura T. 26 Takenaka A. 524 Takenaka S.,35 Takeuchi I. 348 Takeuchi Y. 18 19 101 343,351,514 Takhistov V.V.,285 Talik T.,342 Talik Z. 342 Tam 1.N. S. 147,274 Tarnao K.,186 Tamir I. 21 Tamm C. 3 1,455,460.464 Tamura Y.,424 Tan C.T. 26 Tanabe M.,3 1,457,458 Tanaka H.,224 Tanaka J. 38 Tanaka K.,444 Tanaka M. 186 188,498 Tanaka S. 323 388 422 423 Tanaka Y.,231 Tanba Y. 178 Tang D.Y.,352 Tang Y.-N.,169 Tanger C.. 141 Tang Wong K.L.,172 Taniguchi M.,32 Tapiero C. 389 Tardivel. R.,223 Tardy D. C. 138 Tasker F. A. 354 Tatemitsu H. 38 Taticchi A. 327 Tatlow J. C. 290,312 Tatsuno Y.,167 Tatsuoka S.,40 Tatsuoka T. 353,354 Taube H. 444 Taube R.,194 Taussig P.E. 406 Tavale S. S. 494 Tayim H. A. 179 Taylor A. W.,271 Taylor D.R.,247 Taylor D.S. 158 Taylor E.C. 209,346 Taylor J. W. 89 Taylor K.A.. 179 Taylor K.B. 468 Taylor L. R.,53 Taylor R.,179,313 Taylor R.J. K.,40 Taylor S.S. 549 Tazawa I. 395 Tazawa S.395 Tcheng-Lin M.,467 Tchir M. F.,238,300 Tedder. J. M.,339 Tedeschi D. H.,485 Tee 0.S. 346 Teetor G. H.,400 Teipel J. 552 Teitei T. 243 Teitel S.,39 Teklu Y.,524 Telder. A.-K. 312 Telepchak M.J. 45 Telesheva. A. T.,192 Temple D.L.. 440 Temple P. 266 375 Templeton D. H. 307,529 Tennant G. 332 Teranishi S.,181 Terapane J. F.,188 Terein. B.,327 Terent'eva. T. V.,287 Ternai B.,357 Terrell R.J. 524 Tesarik K.. 53 Tesser G. I. 57 Tetum C. M.,162 Teyssie P.,169 194 287 Thara M.,522 Thayer J. S.,195 Thenn W. 330 Theodoropulos,S. 104 250. 37 1 Theorell H. 554 Thiebault A.. 215 Thiel W. 93 Thielecke W. 373 Thirase G.. 199 Thomas. A. 345 Thomas A. F. 107 Thomas C.A. jun.. 408 Thomas C. B. 191 Thomas D.B.,72 Thomas E. J. 266 375 Thomas,H.,193 Thomas H.G. 224 Thomas J. L. 172 Thomas M.,385 Thomas P. E. 39 Thomas R.,24,25,258,412 463 Thomas R.K.,81 Thomas R.N.,39,42 Thomas. W. R.. 197 Thompson D. J. 189 363 370 Thompson E.A. 388 Thompson G.L. 85 Thompson H.W. 175,428 Thompson P.B. J. 489,492 Thompson R.C. 20 Tichy M.,43 Tidwell T.T. 136 266 Tiemann N.,504 Tilak B. D. 326 Tilley J. W. 369 Tirnko J. M.,272 Timms. P.L. 172 Timpe H.J. 358 Tin K.-C. 279 Ting J.-S.,174 Tingoli M.,124 Author Index Tinnemans A. H.,314 Tinoco I. jun. 383 395 Tipping A. E. 169,330 Titani K.,498 Tobias R.S. 389 Tobin M.,244 Tochtermann,W. 155 Todd L. J.29,30 Todesco P.E. 153,285,334 Toh S. H. 289 Toi. H. 205 Tokis. A. L. 39 Tokita. S. 155 Tbkura N.,35 Tolbert G. D.. 54 Tollin P.,395 Tolman C. A. 176 Tom G. M.,444 Toma S. 190 Tomada S. 307 Tomanova D. 192 Tomassini T. 271 Torner K.B.. 8 Tominaga Y.,336,344 Tomita H. 182 Tomlinson B. L.,22 Tonelli A. E. 394 Tonnard F. 327 Toome V.,39 Torii. S. 224 Tounard F. 157 Touzin A. M.,251 Townsend C. A. 32,538 Tracey A. S. 24 Tracey B. M.,42 Tracey H. J.. 505 Traficante D.D. 28 Trahanovsky W. S. 166 Tran-Dinh. S. 28 Tranter R.L. 128 374 Trattles M.J. 163 323 Travaglini E. C.. 409 Traverso C. 533 Trayer H. R.,68 Trayer I. P.. 63,68 Traylor T. G. 121,527,528 Treasurywala A. M.,38 Tremper A.159,319 Trendelenburg,U.,478 Trenerry V. C. 169 Trenwith M. 138 Triggle D. J. 490,491 Trill H.,138 Trip E. M.,387 Tripathy P.B. 205 Trippett S. 195 Trocha-Grimshaw J. 177 294 Troendle T. G. 346 Trofirnov B. A. 176 Trojhnek J. 38 Tronich. W..168 321 Author Index Trost B. M.,102 173 185 192 360 375 430 432 438,447,448,449 Trotter J. 38 478 Trowitsch W. 18 Troxler F. 333 Truce W. E. 185,325 Trueman R. E. 234 Truitt S.T. 32 Trynham J. G. 147 Tschamber T.. 100,525 Ts’o P. 0.P. 393 395 Tson K. C. 386,395 Tsuboi S.. 104 Tsuchihashi G. 362 431 438,440,44 1 Tsuchiya T. 354 Tsui F. P. 161 Tsuji J. 182 284 Tsujii R. 239 Tsukamoto S.,5 1 1 Tsukanaka M.,363,438 Tu C.D.. 394 Tubinsky A. 524 Tucker H. 110 Tucker P. A. 191 Tucker W. P.. 294 Tuggle R. M. 382,420 Tullock C. W. 419 Tundo A. 150 Tunemoto D. 416 Tupper G. B. 181 Turner D. L. 509 Turner D. W. 79.86 Turro N. J. 244 Twanmoh L. M.,282 Twigg M.V. 190 Tyers M.B. 496 Tyner R. L. 165 294 Tyrlik S. 184 Tyrrell H. M..242 243 295 Tysee. D. A. 142,228 Tyulin V. I. 259 Ubik. K. 279 Uchida I. 270 Uda H. 443 Ueno Y.,340 Uesugi S. 393 Uff B. C. 519 Ugi I. 186 Ugo R. 175 Uhlenbeck 0.C. 395,398 Ukawa K. 349 Uma V. 157 Umani-Ronchi A.. 424 Umenoto T. 304 Umetani T. 349 Umezawa H. 390 Umezawa K. 5 10 Una V. 289 Underhill M. 184 Underwood G. R.. 138,140 141,258 Undheim. K. 41,509 Ungar G.504 Unger K. 46.47 Unger. P. L. 345 Uno. K. 516 Unruh. 1. D. 312. 373 Unsworth J. F. 523 Untch. K. G. 107,261 Upton C. E. E. 178 Urban F. J. 148 Urban P. G. 347 Urban R. 186 Urbanski. T. 358 Urry D. W. 20 33 Utimoto K.. 417 Utley J. H. P. 142,224,225 229 Valasinas A, 537 Valenty S.,163 Vallee B. 409 Vallee B. L. 553 554 Valls J.. 168 Valyulis R. A. 497 van Bergen T. J. 288 329 424 van Binst G. 394 van Boom J. H. 392,394 Van Daele. P. 491 Van Dam E. M.,172,265 Vandenborn H. W. 224 Van Den Elzen R. 102.260 324,350,413 van den Ham D. M.W. 85 Vandenoord A. H. A. 527 van der Eb A. J. 405 Van der Eycken C. 491 Vanderheijden A. 527 van der Helm D. 220 van der Meer D.85 van der Plas H.C. 347 Van Der Puy M.,421 Van der Sande C. C. 14,282 van der Wiel. M.J.. 88 Vander Zwan M. C. 167 van Deursen P. 392 Van de Voorde A. 402 Van de Westeringh C. 491 van Dijk M.,347 van Ende D. 321 Vangedal S. 459 van Gorkom M.,41 Van Herreweghe J. 402 Van Heuverswyn H. 402 Van Hoof W.,136 Van Lear G. 399 van Leusen A. M.,333,430 van Nueten J. M.,491 Van Peppen J. F. 445 Van Rossum J. M.,490 591 van Tamelen E. E. 391,411 van Wijngaarden I. 491,492 Varadi V. 150 Varley M.J. 468 Vasileva G. A. 527 Vaska L.,528 Vassiliades G. A. 406 Vasvari G. 48 Vaughan G. G.. 47 Vazquez M.A. 430 Vdovin G. P. 13 Vdovin V. M.,210 Veatch W. R. 32 Veber D.F.. 506 Vedejs E. 204,414,430 Vederas J.C. 3 1,464 Veidis M.V.. 478 Veinberg A. Y.,226 Venable R. M.,165 Venable J. H.,jun. 405 Venema A. 1I Venetianer P. 405 Venkataraghavan R. 502 Venkatasubramanian N. 270 Venteich R. F. 527 Veracini C. A. 291,306 Vergalla J. 64 Verheyden J. P. H.,388 Verhiilsdonk R. 98 Verma I. M.,404 Vermeer H. 82 Vermeer P. 200,257 Vernon C. A. 540 Vernon J. M. 357 Vessiere R. 349 Vetter W. 13 Vevers R. J. S. 332 Vialle J. 201 Vicens J. J. 198,429 Vickery B. 243 Vierhapper F. W. 345 Viktorova E. A. 296 Villani F. J. 485 Villarreal R. 33 Villieras. J. 185 203,447 Vincenzi F. F. 489 Vining L. C. 31,463 Vinograd J. 407 Vinson J. R. 376 Vinter J. G. 371 Viola A. 109 167 264 Viriot-Yillaume M.L.300 Viswamitra M.A. 395 Vitali T. 479 Vitins P.,110 Vivilecchia R.,44 Vladuchick. S. A. 167 Vlasova L. V. 293 Vlattas I. 427 Vliegenthart J. F. G. 501 Vogtle F. 357 592 Vogel E. 283,307,308.3 15. 323 Vogel G. 462 Vdgel P. 188 Vogel T. M.,161 Vogt B. R.. 104 250,371 Vdigt. H. W..186 Volckatrt G.,402 Vollering M. C. 347 Vollhardt K.P. C. 181 Vol’pin h4. E. 171 184 von Bredpw K.,104 von Hippel P. H. 389 von Voitkenkrg H. 18 Von Werner. K.. 183 von Zabern. I. 557 Vorobyqva R.G. 46 Voronkqu M.G. 176 voss P. 387 Vowinkel E. 412 Vretblad f‘. 57 Vrkot J. 279 Wade L. E. 96 Wagatsurqa N. 358 Wagenkqecht J. H. 225 Wagner. p.,388 Wagner F. 181 Wahl G.H. Jun.. 26 291 Wahlgreq U. 83 Wailes. P.C. 172 Wakabayashi T. 461 Wakarnatqu T.,428 Wakatsukj. Y.. 1x1. 182.248 Wakefield b.J. 196 299 Wakelin L. P. G. 406 Wakeshima I. 212 Wakisakq #;. 358 Walba. a. M. 369 Walborsky H. M. 198,428 Walden p. A, 113 Walker F.A.. 84 Walker 4.T. 385 Walker T. E. 32 534 Walker. W. E. 188 Wallace T. W. 100 300 Waller T. 1. 302 Walley. 4. R.,168 Walling F. 23 139 Wallis T. O.,104 Walsh. K. A. 498 Walsh T.O.,244 Walter J. A. 31 463 Walter R. 22 Walter R. k. 174 Walter T. J. 166 Walter Vy. 199 273 Walton D. R.M.,195. 212 289.424 Walton H:F. 44 Wang A. H.J.. 384 Wang D. k.W. 175 Wang G.L.. 272 Wang I. S. Y. 162 Wang. J. C. 405 Wang. K.-C. 485 Wang.S. S. 241 Wankat. P. C.,74 Wannberg B. 90 Ward D. 525 Ward D. C. 390 Ward F. E. 485 Ward J. E. H. 30 Ware D. W.. 126 hareing J. 447 Waring M.J. 406 Warner P.. 377 warnhoff E. W. 346 Warrener. R. N. 105 Wartski. L. 321 Wasielewski M.R.,138 Wass J.. 520 Wassen J. 3 15 Wasser P. K. W.. 535 Wasserman E. 162 282 Wasserman. H. H.. 31 154 336,472.473 Watanabe. E.-I.,524 Watanabe. H.. I69 Watanabe K. 399 Watanabe S. 444 Watanabe. Y. 186 188 Waterhouse J. S.. 290 Waterman K. 393 Waters C. A, 63 Waters. W. A. 148 Watson C. J. 534 Watson C. R.,jun. 99 300 Watson D. J. 113 Watson H. C. 539 557 Watson K. G. 458 Watson W. D. 288 Watt C. I. F. 128. 374 Watt D. S. 425 Watt F. 86 *atts.D. D. 505 Watts L.. 396 Watts. R. O’B. 169 Watts W. E. 189 247 Wawzonek S.. 224 228 Weavers R.T. 303 30Y Webb B. C. 31 1 Webb C. F. 104 Webb E. C. 550 Webb J. 39 41 Webb J. G. K. 290 Webb L. E. 553 Webb N. W. 520 Weber D. F. 915 516 Weber E. 357 Weber H. P. 333 Weber J. L. 328 Weber L. 354 Weber W. P. 170 Author titdex Yebster. D. E.. 171 Webster D. R.,135 294 beedon B. C. L. 142 224. za5 Wpeks P. D. 204 WeesF G. M..10 Wefer E. A,. 485 WegF D. 380 Wehrli F. W. 2 1 Wei C. C. 38 Weidenbruch. M..169 Weidner U. 82. 85 Weigand N. 46 Weigang. 0. E.. jun.. 33. 302 Weiget. L.. 175 Weigold. H. 172 Weil T. A,. 183 Weiland J. 164. 340 Weiler L.. 85 446 Weimer M. 534 Weiner A, 408 Weiner P.H. 53 Weinges. K. 377 Weis C. D. 104. 353 Weise-Velpz C. A, 98 Weisinan 6.R..18 Weisman G.R. 147 Weiss B. 167 Weiss E. 184 199 Weiss G. 40. 49 Weiss #. 507 Weiss 8..287 374. 526 Weissberger E. 37. 182 Weissqan. C.. 404 Weissman S. M..404 406 407 Wege D. 154 Weglein R.. 165 Welch J. 419 Wellauer P. K. 403. 408 Wells C. H. J.. 31 1 Wells D. 243 Wells R.D. 394 Wendell P. L. 557 Wendling L. A. 163 Wendt. H. 217 Wehger. J. 188 Wenke D. C. 53 Wenkert E. 28 467 Wentland. M.P. 533 Wentrup C.,164 165 319 Weringa W. D.. 34 Werve L. O. 77 Werndod F. 167 Werner G. 342 Wernick D. L. 279 Werthemann D. P. 233 Wertz D. H. 370 Wessels P. L. 34 West C. T. 147 West J.C. 178 Author Index West R.,287,306 Westerman P.W. 248 Westmore J. B.,388 Weston J. B.,284 Wetherell J. 491 Wetmore S.I. 319 Wennerstrom H.,24 Weyerstahl P.,11. 163 Weygand F.,508 Weyler W. 160 Whan D. A. 193 Wheals B.B.,47 Whelan J. 521 Whelton B.D.,488 Whitaker D.R..540 White D.R..439 White G.R.,481 White H.A. 68 White J. 324 White J. D.,414 441 White R.J. 466 White R.M.,90 Whitehead M.A. 157 Whitehouse M.J. 47 Whiteley. J. M.,64 Whitesides G.M.,174 189 200,204,279,363,447 Whitesides T.H.,187 Whitfield G.F.,13 Whitham G.H.,266 375 414 Whiting J. 489 Whiting M.C.,113 Whitman P.J. 32 538 Whittaker. D.,18 Whittaker G.,307 Whitten C.E.,185,426,453 Whittle P.R. 141 Whitton. B.R.,236 Whitworth A. S.,545 Wkyman R.,188 Wibe-rg K.B.,132,225,377 Wicha J. 425 Wickner W. 409 Wickwar H.,524 Widdowson D.A. 458,459 470 Wiech G. lO7,163,235,3BO Wiedhaup K.,369 Wiegers K.E.,124 Wieland T.,513 514 Wienstein-Lanse F.,288 Wierenga W. 391 Wieseman T. L. 201 Wiewiorowski M.,39 Wife €4. L.,301 Wig!ield D.C.,382 Wihus K.,307 Wilwk J. D.,328 Wild H.-J. 271 532 Wild R. 12 Wilde L.,97 Wiley R.A.. 489 Wilford J. B.,496 Wilke G.,172 181 248 Wilkins C.K.,39 Wilkins J. D.,184 Wilkinson C.J. 527 Wilkinson J. R.,29 Wilkinson R.G.,59 Wilkinson S.,493 Will J. P.,406 Willard A. K.,348 Willcott M.R.,97 Williams B.D.,15 Williams D.C.,468 Williams D.H.,9 19 98 501,502 Williams G.H. 135 140 173,293 Williams G. J. B. 484 Williams H.,290 297 Williams J. 419 Williams J. L.R.,243 Williams J. W.,269 Williams N.,491 Williams R.C.,53 54 Williams R.J. P.,20 394 Williams R.M.,207 418 Williams T.A.. 92 Williams V.Z.,373 Williamson D.E.,493 Williamson K.L.,19 277 Willis M.R.,95 Willis R.B.,46 Willmott F.W. 54 Willputte-Steihert L.. 175 Wilshowitz L.,272 Wilson D.A. 408 Wilson H.,290 Wilson H.R.,395 Wilson J. A. 297 Wilson J. M.,7 Wilson K.S.,539 Wilson L.A. 24 Wilson N.H.,153 Wilson N.K.,26 Wilson R. M.,237,510 Wilson. R.S.,489 Wilt J. W. 298 Wilton D. C. 544 546 Win L.P.,284 Wingard M.,498 Wingard R.E.. 85 Winkler H.U.,501 Winkler J.10 12 282 Winnik M. A. 234 304 Winstein S. 108 122 Winterfeldt E.,250 Winternitz F.,514 Winters L.J. 344 Winton R.F.,99 Wipf H.W. 502 Wipff G.,272,274 Wipke. W. T. 183,382 Win J. 85 Wiseman J. R..80,359 Wisener J. T.,342 Wiskott E.,373 Witiak D.T.,484,485 Witkop B.,245,283 Wittel K.,82 Wittig G.,328 Wittington S.G.,234 Wittmer D.,53 Wojcicki A. 184 190 Wojcik L.H.,501 Wolf A. D.,302 Wolf F.J. 52 Wolf L.,479 Wolf S.F.,269 Wolfe J. F.,292 Wolfe N.L.,314 Wol€e R.S.,498 Wolff c.,4 12 Wolff M.E.,27 Wolkoff A. W. 53 Wollenberg R. H. 95 157 212,417 Wolters A. P. 243 Wolthers B.G.,545 Wong C.,307 Wong C.M.,417,427,443 Wong C.P.,527 Wong J. Y.,241 Wong.H.N.C. 252,307 Wong K.L..397 Wong L.,380 Wong P.K.,179 Wong Y.P..398,402 Woo,K.W. 471 Woo P.W.K.,390 Wood C.S.,241 Wood D.E.,137 144,277 Wood D.J. 394 Wood J. 174 Wood J. M.,73,533 Wood P.B.,140 Woodhouse D.I. 190 Woodruff R. A. 164 198 204 Woods R.A.. 459 Woodward P.,192 Woodward R. B.,36 109 510,532 Woody R.W. 39 Woolhouse A. D.,305 Woollard J. Mck. 159,346 Wreford S.S. 172 Wright B.J. 25 Wright H.T.,543 Wright J. L. C. 31,463 Wright P.W. 444 Wright S.H.B. 481 Wrighton M. S. 175 176 209 594 Wu D. K.,439 Wu E. C. 486 Wu E. S. C. 488 Wu R. 393,394 Wuest H. 107 444 Wurminghausen T.. 2 15 Wurthwein E.-U. 276 Wuilmet M.,19 Wulf C. A. 290 Wunderly S.W. 237 Wunsch E, 506,508 Wuthrich K.,29 Wylie P. L. 437 Wyman L. 179 Wynberg H. 339,374 Wynne-Roberts C. R. 48 Wyvratt M.J. 104,250,371 Xavier A. V. 20 394 Xuong N.-D., 406 Xuong Ng. H. 543 Yagen B. 460 Yagi H. 165 Yagisawa N. 390 Yaguchi T. 331 Yagupol’skii L. M.,13,288 Yahner J. A. 291 Yajima H. 512 Yakobson G. G. 287 293 357 Yamada K.,346 Yamada N. 155 Yamada S.,424,513,514 Yamada S.-I. 514 Yamada Y. 302 Yamamoto H. 164 166 323 376 422 423 429 44 1 Yamamoto K. 180 182 186,310,313,317 Yamamoto M.,363,451 Yamamoto S. 206 Yamamoto Y. 184 205 206,207,426 Yamamoto Y. S. 507 Yamamura. M.,178 Yamanaka H. 349 Yamanaka T. 358 Yamane T. 405,522 Yamasaki K.,243,471 Yamasaki Y.203 Yamashiro D. 51 1 Yamashita M..362 438 Yamashita S. 202 Yamashita Y. 101 Yamazaki H. 181 182,184 248 Yamazaki N. 514 Yamdagni R. 269 Yanada Y. 286 Yano J. 392 Yano K.,236 Yano Y.. 412 Yarborough L. R. 409 Yarnitsky Ch. 225 Yaroslavsky C. 288 Yarrow D. J. 190 Yashin Ya. I. 46 Yasuda A. 323.422 Yasufuku K. 183 Yasumori Y. 186 Yasunami M.,315 Yasunobu K.T. 498 Yatabe M.,342 Yates B. L. 109 264 Yates K. 165 266 Yates R. L. 106 Yathindro N. 21 Yazawa H. 444 Yeager S. A. 153 Yeates D. G. R.,539 Yeh H. J. C. 311,323 Yelvington M.B.,96 Yip K.F. 386 395 Yip R. W. 147 274 Ykman P. 361 Yoder C. H. 274 Yokomatsu T. 155 Yokota T. 79 Yonemitsu O. 295,346,355 Yonezawa K.,185,300,451 Yonezawa T.86 Yoshida M.,331,332 Yoshidai T. 207 411 418 442 Yoshida Z.-I. 524 Yoshikawa K.,86 Yoshikawa S. 180 189 Yoshimura H. 202 Yoshimura. K.,164 298 Yoshimura Y. 244 Yot P. 404 Young D. 172 Young D. W. 395 Author Index Young E. T. 401 Young G. T. 512 Young J. D. 312 Yukimoto Y.. 163 Yura Y. 363,450 Zabarowsky B. R. 506 Zachau H. G. 407 Zahradnik. R.,76 Zain B. S..406 Zain S. 407 Zaitseva C. A. 493 Zalkin A. 529 Zamudio A. 33 Zanardi G. 150 Zanella R. 184 Zarecki A. 425 Zarembo J. E. 536 Zarubin M.Ya. 282 Zawadski S. 161 Zecchin S.,222 Zeck 0.F. 169 Zefirov N. S.,203 35 1 358 Zeisberg R.,256 Zeltmann A. H. 20,394 Zeppezauer. E. 539,549,553 Zetterberg K.185 191 Zeya M.,108 Zhorov Yu. M.,188 Zicane. D.,358 Ziegler E. 279 358 Zeigler F. E. 443 Ziegler H. W. 49 Ziff E. 406 Ziffer H. 236 Zikmund L. 53 Zimmerman H. E. 105,233 239,298 Zirkle C. L. 485 Zmijewsky M.,472 Zobova N. N. 358 Zollinger H. 291 Zolotarev B. M.,13 Zon G. 161 Zotora S. B. 207 Zsindely J. 371 Zuccarrello F. 301 Zuurdeeg B. 347 Zwan M.C. V.. 434 Zweifel G. 206. 2.08 247 256,413,418,421 Zweig G. E. 44 Zwierzak A.. 161

ISSN:0069-3030    

DOI:10.1039/OC9747100559

出版商:RSC    

年代:1974

数据来源: RSC