摘要:

14 ALKALOIDS By J. A. Joule (Chemistry Department University of Manchester Manchester M13 9PL) THEvolume' of 'The Alkaloids' published this year is devoted to updating previous chapters on steroidal (Solanurnand Veratrurn) Erythrophleurn Taxus Lycopodiurn benzylisoquinoline (tricyclic-monomeric and cularine types) papaveraceae u-naphthaphenanthridine and simple indole alkaloids and on the alkaloids of the Calebar bean Picralirna nitida Mitragayna and Ouruparia species together with a compilation of alkaloids of as yet unknown structures. Two books2V3 deal with alkaloids and reviews have appeared devoted to bisc~claurine,~ ipecac,6S~larnander,~ Rau ~ol$a,~ calebash curare,8 ~trychnine,~ and proaporphine" alkaloids. Pyrrolizidine Alkaloids.-Two bases nilgirine (1 ; R = H) and axillarin (2) from Crotalaria rnuoonata' 'and C.axzllarisl2 respectively with new variations in the necic acid portions of their structures have been isolated and character- ised. Nilgirine lacks the one-carbon unit [R = Me or CH20H in (l)] which Me ,OH I C0,Me b' (3) R H. F. Manske 'The Alkaloids,' Academic Press New York 1968 vol. X. W. Dopke 'Einfuhrung in die Chemie der Alkaloide,' Akademie-Verlag Berlin 1968. G. A..Swan 'An Introduction to the Alkaloids,' Blackwell Oxford 1967. J. Sakakibara Nagoya Shiritsu Daigaku Yakugakubu Kenkyu Nempo 1966 14,l. S. C. Pakrashi and B. Achari J. Sci. Znd. Res. 1968,27,58. C. Szanthy Recent Developments Chem. Natural Carbon Compounds 1967,2,65. ' G. Habermehl Progr. Org. Chern.1968,7 35. H. Schmid Bull. Schweiz. Akad. Med. Wiss. 1967,22,415. R. N. Chakravarti J. Inst. Chemists (India) 1968,40 85. lo K. L. Stuart and M. P. Cava Chem. Reo. 1968,68,321. C. K. Atal R. S. Sawhney C. C. J. Culvenor and L. W. Smith Tetrahedron Letters 1968 5605. l2 D. H. G. Crout Chem. Comm. 1968,429. 490 J. A. Joule when present in other related bases it is suggested,' is methionine-derived. The necic acid portion of axillarin it is proposed," may be biogenetically derived from valine and isoleucine (with loss of carbon dioxide). The pyrrolizidine ester (3) obtained from Chysis bracte~cens'~displays an intriguing U.V. absorption at 290 nm. (E 40),the cause of which is not known. Neither the corresponding sodium salt nor the lithium aluminium hydride reduction product (identical with the alkaloid lindelofidine) show this pheno- menon.Pyridine and Piperidine Alkaloids.-A detailed e~amination'~ of hemlock (main alkaloids coniine and y-coniceine) has shown that the alkaloids present in the living plant occur in part in bound forms with the remainder as simple salts. The concentration and location within the plant of bound forms varies during the day and according to the stage of the plant's development. These findings have suggested that in this plant at least the alkaloids may be intimately involved in the metabolism perhaps in some oxidation-reduction sequence resembling the role of the pyridine nucleotides. It is intriguing to speculate whether other alkaloid-bearing plants utilise their alkaloids whether of greater or lesser complexity in a metabolically more fundamental way than has hitherto been believed.The trimer (4) has been isolated'' from Nicotiana tabacum and the dimer (9,named hystrine and isolated'6a from Genista hystrix has been obtained by partial synthesis16b from ammodendrin. RD Myosmine (6;R = 3-pyridyl) and apoferrorosamine (6;R = 2-pyridyl) have been neatly synthesisedl' by acid-catalysed isomerisation of a cyclopropyl imine (7; R = 2-or 3-pyridyl) derived in turn by addition of the appropriate pyridyl lithium to cyclopropanecarbonitrile. Two intriguing variations on the monoterpene-pyridine theme are jasminine (8) from Jasminum speciesI8 and gentiaflavine (9) from Gentiana species.'' The B.Liining and H. Tranker Actu Chem. Scud. 1968 22,2324. l4 J. W. Fairbairn and A. A. E. R. Ali Phytochemistry 1968,7 1593,1599. Is T. Kisaki S. Mizusaki and E. Tamaki Phytochemistry 1968,7,,323. l6 (a)E. Steinegger C. Moser and P. Weber Phytochemistry 1968 7,849; (b)E. Steinegger and P. Weber Helv. Chim. Actu. 1968 51 206. '' R. V. Stevens M. C. Ellis and M. P. Wentland J. Amer. Chem. SOC., 1968.90 5576. Alkaloids 491 isolation of jasminine did not involve the use of ammonia and so one may be sure that this base containing two nitrogen atoms occurs naturally. The yellow alkaloid gentiaflavine has been ascribed the novel 1,2-dihydropyridine structure (9) mainly on the basis of its n.m.r. spectrum. Of0 I 0 MeO,C 0HC.p CH ” N HH 0 (8) (9) (10) Lupin Alkaloids-Lamprolobine (10) from Lamprolobium fruticosum2 co-occurs with cytisine.A biogenesis from three moles of lysine is suggested for the new base. Structurally useful mass spectral studies,’ have been made on the cytisine and lupanine types. When deoxyangustifoline (11; R = H,) was treated with formaldehyde in the presence of acid a salt (13) was formed,22 from which a crystalline perchlo- rate was obtained. The salt was stable to hot water and cold sodium hydroxide and required treatment with hot alkali to hydroryse it back to starting material. When boiled in acetic acid it was converted into 13-epiacetoxylupanine (12; R = H,) [the analogous amide (1 1 ;R = 0)gave a cyclised product (12 ;R = 0) directly on reaction with formaldehyde and acid].An X-ray analysis of the perchlorate of (13) showed that the bonds N(1&C( 17) and C(17)-N( 12) were not equal in length. The former is ascribed a bond order of 0.86 and the latter 1.13. The suggestion which is to be tested is that the methylene group in this salt may behave biologically like the ‘active formaldehyde’ of hydroxymethyl- tetrahydrofolic acid. N. K. Hart S. R. Johns and J. A. Lamberton Austral. J. Chem. 1968 21,1321. l9 N. L.Marekov and S. S. Popov Tetrahedron 196% 24,1323. ’O N.K.Hart S. R. Johns and J. A. Lamberton Austral. J. Chem. 1968 21,1619. ” D.Schiimann N. Neuner-Jehle and G. Spiteller Monatsh. 1967,98,836; 1968,9!9,390; M. Silva M. V.Medina and P. G. Sammes Phytochemistry 1968,7,661. 22 G. I. Birnbaum K.K. Cheung M. Wiewiorowski and M. D. Bratek-Wiewiorowska J. Chem. Soc. (B),1967 1368. 492 J. A. Joule Neat synthesesz3 of lupinine continue to appear ;onez3' involved the reaction of methyl pyridine-2-acetate with carbon suboxide to give a methoxycarbonyl- quinolizinone system suitable for elaboration. Isoquinoline Alkaloids.-The powerful combination of g.1.c. and mass spectrometry has been applied to extracts of Peyote cactusz4 and of Lophophora wiEliamsii2 and Trichocereus pa~hanoi.~ In this way it was possible to demon- strate the presence of minute quantities of partially methylated and oxygenated intermediates2' on the biosynthetic pathway from tyrosine to mescaline and the isoquinoline alkaloids. The presence of small quantities of many compounds for example peyonine (14) derived by interaction of mescaline with acids of the Krebs cycle was dem~nstrated.~~ From Cryptostylis fulva a l-phenyl- 1,2,3,4-tetrahydroisoquinolinealkaloid has been isolated26 for the first time.::q -aq. HC1 PhCH C0,H PhCH 23 (a)Th. Kappe Monatsh. 1967,96,1853; (b)E. Wenkert K. G. Dave and R V. Stevens J. Amer. Chem. Soc. 1968,!W 6177. 24 G. J. Kapadia and H. M. Fales Chem. Comm. 1968 1688. 25 S. Agurell and J. Lundstrom Chem. Comm. 1968 1638. 26 K. Leander and B. Luning Tetrahedron Letters 1968 1393. Alkaloids 493 In support of a suggestion27 that the biosynthesis of isoquinoline alkaloids involves peptide chains-the head turning and biting its tail-a model sequence (15) -+ (19) designed to simulate this process has been in~estigated.~' The compound (15) was treated with the masked phenylpyruvate (16) to give the diamide (17) which cyclised easily to a tetrahydroisoquinoline derivative (18) hydrolysis of which gave (19).Presumably if nature does indeed take a course analogous to this laboratory model then carboxy-benzylisoquinoline corn-pounds like (19) may well exist in benzylisoquinoline-producingplants. As aids for the elucidation of structures mass spectrometric studies have been carried out on tetrahydroprotoberberine2* and cryptaustoline alkaloid^,^ an analysis3' of the effect of oxygen substitution on U.V. absorption in the benzylisoquinoline and tetrahydroprotoberberine alkaloids has been made and n.m.r. examinations have shown that because of proximity effects caused by the ring systems the chemical shifts of aromatic methoxy-groups on macro- cyclic bisbenzylisoquinoline alkaloids can aid in deciding the type of oxygen bridge present3' and that the stereochemistry at C-14 of the rh~eadine-type~~" of alkaloid can be deduced.33 The racemisation at C-13a of tetrahydroprotoberberine alkaloids like corexi- mine (20) when catalysed by metals involves exchange of the 13a-h~drogen~~ in contrast to the situation in the acid-catalysed epimerisation of the analagous C-3 position in tetrahydro-P-carboline alkaloids which can occur without exchange of the hydrogen.3s A certain degree of success36 has been achieved in attempts to make bisbenzyl- isoquinoline alkaloids by forming the ether links by oxidative coupling.27 G. E. Krejcarek B. W. Dominy and R. G. Lawton Chem. Comm. 1968 1450. 28 C.-Y. Chen and D. B. MacLean Canod. J. Chem. 1968,46,2501. 29 T.Kametani and K. Ogasawara Chem. and Pharm. Bull. (Japan) 1968 16,1498. 30 L. Hruban and F. SantavjS Coll. Czech. Chem. Comm. 1967 32 3414. 31 J. Baldas P. N. Porter I. R. C. Bick G. K. Douglas M. R. Falco J. X. De Vries and S. Yu. Yunosov Tetrahedron Letters 1968,6315. 32 Ann. Reports:(a)1965,378;(b)1966,506;(c) 1967,437; 1965,38O;(d)1956,247;(e)1960,290;(f) (9)1967,439;(h) 1966,511;(i)1965,384;(j)1962,355. 33 M. Shamma J. A. Weiss S. Pfeifer and H. Dohnert Chem. Comm. 1968 212. 34 T.Kametani and M. Ihara J. Chem. SOC. (C) 1968,191. 35 A.J. Gaskell and J. A.Joule Tetrahedron 1967,23,4053. 36 M. Tomita Y. Masaki K. Fujitani and Y. Sakatani Chem. and Pharm. Bull. (Japan),1968,16. 688; A. M. Choudhury I. G. C. Coutts A. K. Durbin K. Schofield and D. J. Humphreys Chem. Comm. 1968 1341 ;A. Rieker H. Kaufmann D. Briick R. Workman and E. Miiller Tetrahedron 1968 24 103. 494 J. A. Joule A neat synthesis37 of the erythrina skeleton has as its key step the enamine alkylation of (21) with methyl vinyl ketone. The first members of the homo- erythrina type3* have been obtained from Schelharnrnera pedunculata ; for example schelhammerine is (22). A homoerythrina skeleton has been synthe- ~ised~~ by oxidative coupling. investigation^^^ of the thebaine system continue to provide interesting chemistry for example the thebaine-iron tricarbonyl adduct gave4OU with acid a salt (23) which is the result of yet another new type of rearrangement in this series.Me0 / Me / Me0 / Fe (CO) (23) Morphinane derivatives have been obtained by use of oxidative coupling4' and by use of a Pschorr reaction42 to form the final bond. Thus reticuline (24) was converted4lU to isosalutaridine (25)by treatment with manganese dioxide- silica gel (a means of supplying the substrate to the oxidant at high dilution and thereby improving the yield by cutting down on competing polymerisation reactions). The amino-derivative (26)was converted42u into (27) by successive treatments with nitrous acid and heat. Me0 / $,NMe -Me0 MeO&,II NMe Me0 \ OH 0 37 R.V. Stevens and M. P. Wentland Chem. Comm. 1968 1104. 30 S. R. Johns C. Kowala J. A. Lamberton A. A. Sioumis and J. A. Wunderlich Chem. Comm. 1968,1102. '' T. Kametani and K. Fukumoto J. Chem. SOC.(C) 1968,2156. 40 (a)A. J. Birch H. Fitton M. McPartlin and R Mason Chem. Comm. 1968,531 ;(b)J. B. Taylor J. Chem. SOC.(C),1968,1506; Z. J. Barneis J. D. Carr R. J. Warnet and D. M. S. Wheeler Tetrahedron 196$,24,5053. 41 (a)B. Franck G. Dunkelmann and H. J. Lubs Angew. Chem. 1967,79,1066; (b)A. R. Battersby A. K. Bhatnagar P. Hackett C. W. Thornber and J. Staunton Chem. Comm. 1968,1214. 42 (a)T. Kametani K. Fukumoto andT. Sugahara Tetrahedron Letters 1968,5459;(b)T.Kametani K. Fukumoto F. Satoh and H. Yagi Chem. Comm. 1968 1398. A lkaloids 495 PO 0Me (26) (27) A morphinan skeleton has been converted43 into a hasubanan skeleton32b and a totally synthetic route44 to a simple hasubanan compound has been developed.Kreysiginine (28) is a homomorphine type4' containing the carbon skeleton of andr~cymbine~~" with the additional ether bridge. This new alkaloid has an absolute configuration opposite to that of morphine.46 The carbon skeleton of these alkaloids has been produced both by ferricyanide oxidative coupling4' and the use of a Pschorr reaction.48 OMe Rb Rb HO (28) (29) A group of bases (29; R1.= R2= Me R3 = H OH) (29; R1= Me R2= H R3 = H OH) and (29; R1+ R2 = CH, R3 = 0)from Furnariu officinalis have structures49 allied to that of ochotensimine (29; R1= R2 = Me R3 = CH,).( f)-Ochotensimine has been synthesised." " T. Ibuka and M. Kitano Chem. and Phann. Bull. (Japan),1967,15,1944. 44 M. Tomita M. Kitano and T. Ibuka Tetrahedron Letters 1968,3391. '' A. R. Battersby M. H. G. Munro R. B. Bradbury and F. Santav); Chem. Comm. 1968 695; N. K. Hart S.R Johns J. A. Lamberton and J. K. Saunders Tetrahedron Letters 1968 2891 ; J. Fridrichsons M. F. Mackay and A McL. Mathieson ibid. 1968 2887. 46 A. F. Beecham N. K. Hart S.R.Johns and J. A. Lamberton Austral. J. Chem. 1968 21,2829. 47 T. Kametani K. Fukumoto M. Koizumi and A. Kozuka Chem. Comm. 1968,1605. '* T. Kametani K. Fukumoto F. Satoh and H. Yagi J. Chem. SOC.(C),1968 3084. 49 J. K. Saunders R. A. Bell C.-Y. Chen D. B. McLean and R H. F. Manske Canad.J. Chem.1968 46,2873,2876. 'O H. Irie T. Kishimoto and S. Uyeo J. Chem. SOC.(C) 1968,3051;S. McLean Mei-Sie Lin and J. Whelan Tetrahedron Letters 1968 2425. 496 J. A. Joule Amaryllidaceae Alkaloids.-Quantitative analysis5 of the alkaloidal content of Amaryllidaceous plants can be carried out by g.1.c. of their tetramethylsilyl ethers. This technique makes easy a progressive analysis of the alkaloidal content of a plant throughout its growth cycle. Two new alkaloids isolateds2 from many varieties of daffodils are narciclasine (30) and narciprimine (31). Ta~ettine~~~ is in fact an artifact produceds3 from pretazettine (32) during work-up. Lycorine has been converted5 into hippea- trine.^^^ OMe (32) 0 0 MeoG7 N.CO CF CO * CF3 I Me0d \ OH OH OH (33) (34) A biogenetically patterned oxidative coupling route (33) + (34) has been employed to produces5 a crinine ring system.Indole Alkaloids.-Simpler alkaloids. Brevicolline (35) from Carex brevi- c01Iis~~" have been assigned the and nitrarine (36) from Nitraria ~choberi~~' novel structures shown. Elaeocarpidine (37) from Elaeocarpus archbo1dianuss7 is also clearly different in type to previously isolated indole bases. Dasycar- pidone (38 ;R = 0)58 and uleineS8" and their C-3 epimersS8 have been synthe- 51 S. Takagi T. Katagi and K. Takebayashi Chem. and Pharm. Bull. (Japan) 1968 16 1116 1121. 52 F. Piozzi C. Fuganti R. Mondelli and G. Ceriotti Tetrahedron 1968,24,1119. 53 W. C. Wildman and D. T. Bailey J. Amer.Chem. Soc. 1967,89,5514. 54 K. Kotera Y. Hamada and R. Nakane Tetrahedron 1968,24,759. s5 B. Franck and H. J. Lubs Angew. Chem. 1968,80,238. 56 (a) P. A. Vember I. V. Terent'eva and A. V. Ul'yanova Khim prirod. Soedinenii 1968 4 98 (b)M. Normatov and S. Yu Yunosov ibid. p. 139. " S. R. Johns J. A. Lamberton and A. A. Sioumis Chem. Comm. 1968,410. 58 (a)A. Jackson A. J. Gaskell N. D. V. Wilson and J. A. Joule Chem. Comm. 1968 364 584; (b)L. J. Dolby and H. Biere J. Amer. Chem. SOC.,1968,90 2699 Alkaloids 497 rNM. (35) NMe R (37) sised in two different ways. Both methods involved as a key step a formation of the C-ring ones8' by cyclisation on to the indole P-position and the other58b by cyclisation on to the indole a-position.YohirnbP and related alkaloids. This year has seen the isolation (or detection) of two compounds (39 epimers at C-3) vincoside and isovincoside (together with the N-acetyl derivative59c of vincoside) from Vinca and strichto- sidine [same overall structure as (39) and probably identical stereochemically with one of the vincosides] from Rhazia~tricta,~~~ which lie even closer than tryptamine ~ordifoline~~f to the biogentic pathway from loganin to the indole alkaloids. One of the isomers vincoside has been shownsgc to be a direct precursor of the three main indole alkaloid skeletal types and both are formed in vitro when secologanin (40)is treated with tr~ptamine.'~" 59 (0)A. R. Battersby,A. R Burnett. and P. G. Parsons,Chem Comm. 1968 1282; (b)G.N.Smith ibid. 1968 912; (c) A. R. Battersby A. R. Burnett E. S. Hall and P. G. Parsons ibid. 1968 1582. 498 J. A. Joule Adina cordqolia has now yielded another P-carboline alkaloid adifoline (41),60 which retains the carboxy-group derived biogenetically from tryptophan. The novel seven-membered ring in this alkaloid is formed by linkage of the terpenoid part of the molecule to the aromatic nitrogen instead of as is more usual to the aliphatic nitrogen atom. Quinine has been transformed6' via a series of degradations including a von Braun cleavage into indole alkaloids of the dihydroantirhine [42; R' = CH(Et)*CH,OH,R2 = H] and dihydrocorynantheol(42 ;R' = CH2*CH2*OH R2 = Et) types. The key steps involved the treatment of the requisite dihydro- quinolones e.g.(43) with dihydropyran in acetonitrile when the hexahydro- quinolizones (44) were formed in high yield. Standard procedures then allowed closure to the required indoloquinolizine systems and removal of the aromatic oxygen function. THP = tetrahydropyranyl Synthetic ( & )-akuammigine and ( f)-tetrahydroalstonine each having cis D/E ring junctions have been obtained62 via the product [45; R = CH(CO,Me),] ofkinetically controlled Michael addition of dimethyl malonate to (45; R = H 15,20-dehydro). More examples of the utility of the synthetic approach to indole alkaloids which uses partial hydrogenation of 3-acyl-pyridinium species have appeared.63 6o R. T. Brown K. V. J. Rao P. V. S. Rao and L. R. Row Chem. Comm. 1968,350. 61 Y.K. Sawa and H. Matsumura Chem. Comm. 1968,679. '* E. Winterfeldt H. Radunz and T. Korth Chem. Ber. 1968 101,3172. 63 E. Wenkert K. G. Dave C. T. Gnewuch and P. W. Sprague J. Amer. Chem. SOC. 1968 90 5251; E. Wenkert K G. Dave R G. Lewis and P. W. Sprague ibid. 1967,89,6741; for a review see E. Wenkert Accounts Chem Res. 1968 1 78. A lkaloids 499 (45) A study of the mass spectral fragmentati~n~~ type has been of the voba~ine~’~ carried out. Another alkaloid of the vobasine class is taberpsychine (46) from Tabernaemontana psychotri$olia.65 Strychnos and related alkaloids. A study66 has shown that care is needed in the interpretation of 0.r.d. curves of indolines and N-acylindolines of the Strychnos and Aspidosperm series if the molecule also contains an aliphatic carbonyl group.An analysis of the mass spectral fragmentation of the strychnine type67 helped the elucidation of the structure of rindline from Strychnos henningsii. 4-Hydroxystrychnine has been isolated from S. icaja6’ and brucine has been obtained6’ by a series of oxidative steps from strychnine. (47) ii-iv 1 0 Reagents:i (EtCHCl-CO),O ;ii NaOH ;iii MnO ;iv NaOC(Me),Et. 64 T. Shiori T. Nakashima and S. Yamada Tetrahedron 1968.24,4177. 65 P. R Benoin R. H. Burnell and J. D. Medina Tetrahedron Letters 1968 807. 66 W. Klyne R. J. Swan A. A+ Gorman A. Guggisberg and H. Schmid Helv. Chim. Acta 1968,51 1168. 67 M. Spiteller-Friedmann and G. Spiteller Annalen 1968,711 205. 68 F. Sandberg K. Roos K. J. Ryrberg and K.Kristiansson Tetrahedron Letters 1968,6217. 69 P. Rosenmund W. H. Haase and K. Kaiser Chem. Ber. 1968,101,2754; E. Tedeschi S. Dukler P. Pfeffer and D. Lavie Tetrahedron 1968,24,4573. 500 J. A. Joule The simple precursor (47)has been neatly utilised7' in a total synthesis of (+)-tubifoline (48;R1= a-H R2 = H R3 = Et) and (+)-condyfoline (48; R' = P-H R2 = Et R3 = H). The synthesis centred on the production of the medium sized ring intermediate (49) which after modification was oxidatively closed to give both alkaloids. Continuing concentration on the relative and absolute stereochemistries of the tetra-71 and penta-~yclic~~ oxindole alkaloids has clarified the use of the various spectral and chemical methods for such assignments.Even mass spectrometric fragmentation [the relative intensity of the peak at m/e 180 (50) derived from the pentacyclic series] can have stereochemical ~ignificance.~ Aspidosperma alkaloids. An X-ray analysis74 of (-)-kop~anone~~~ methio-dide shows it to have the absolute configuration opposite to that of (+)-aspidospermine. Two intriguing bases from Aspidosperma dispermum7' Me0,C-0 provisionally assigned structures (5 1;R = H) and (51 ;R = OH) are the first known alkaloids of this group not to have an ethyl side chain in these bases replaced by a hydroxy-group. Q~ebrachamine~~~ by a route in which the nine- has been ~ynthesised~~ membered ring was created by acid-catalysed intramolecular acylation3 2g of the indole a-position. Iboga alkaloids.Much has been achieved this year in the synthesis of this class of alkaloid. Two more syntheses77a* of (_+)-ib~gamine~~' have appeared together with two for (+)-epi-ib~gamine~~~*' and syntheses of ~oronaridine~~ and ~elbanamine.~'? 32j One of the appro ache^'^" to the ibogarnine system utilised the cis-enedione (52) as starting material. The seven-membered 6-ring of the alkaloids was produced by monoacetalisation oxime formation and Beckman rearrangement (53). Next the double bond was used to introduce a 70 B. A. Dadson J. Harley-Mason and G. H. Foster Chem. Comm. 1968 1233. '' W. F. Trager C. M. Lee J. D. Phillipson R. E. Haddock D. DwumaBady and A. H. Beckett Tetrahedron 1968,24 523. 72 A. F. Beecham N. K. Hart S. R. Johns and J. A. Lamberton Austral.J. Chem. 1968,21,491. 73 M. Shamma and K. F. Foley J. Org. Chem. 1967,32,4141. 74 B. M. Craven B. Gilbert and L. A. Paes Leme Chem. Comm. 1968,955. 75 M. Ikeda and C. Djerassi Tetrahedron Letters 1968 5837. 76 F. E. Ziegler and P. A. Zoretic Tetrahedron Letters 1968,2639. " (a)S. 1. Sallay J. Amer. Chem. SOC.,1967,89,6762; (b)W. Nagata S. Hirai T. Okumura,and K. Kawata ibid. 1968 90 1650; (c) Y. Ban T. Wakamatsu Y. Fujimoto and T. Oishi Tetrahedron Letters 1968 3383. S. Hirai K. Kawata and W. Nagata Chem. Comm. 1968,1016. 79 G. Biichi P. Kulsa and R. L. Rosati J. Amer. Chem. SOC.,1968 90 2448. Alkaloids 501 i-iii - vii-xiii - (55) Reagents i HOCH,.CH,OH; ii H,NOH; iii TsC1-pyridine; iv PhC0,H; v LiAlH,; vi CrO pyridine; vii Ph,PCH,; viii B,H,-H,O,; ix LiAlH,; x PhCH20COCl; xi TsCl ; xii HBr-HOAc ; xiii heat.carbonyl group at C-7 (54 ;R = 0),whence by Wittig reaction and hydrobora- tion the corresponding hydroxymethyl compound (54; R = CH,OH) was obtained. A series of standard steps culminating in the formation of the isoquinuclidine ring system by intramolecular N-alkylation gave (55) which when subjected to a Fischer indole synthesis gave (-t-)-ibogamine. The relationship between yohimbk/strychnos aspidosperma and iboga skeleta. Although it has been known for some time that all of the three main skeletal types of indole alkaloid are derived from a common precursor which in a pre-alkaloid stage would be represented by a secologanin (40)and at an alkaloid level by vincoside (39) the manner in which this yohimbk/strychnos skeleton becomes rearranged to the two other types and indeed which of the yohimbk and strychnos types if either comes first in the biogenetic sequence is not certain.However this year considerable evidence". 82a has appeared which ''9 suggests that rearrangement occurs at an alkaloid level and which gives an inkling as to the sort of mechanisms which may be involved. The secamines (56; together with 15,20- or 15',20'-dihydro- and 15,15',20,20- tetrahydro-; the alternative structures with C-2' and its accompanying ester attached to C-17' are much less likely) a series ofnovel dimeric alkaloids isolated from Rhazya stricta" and Rhazya orientalis and an alkaloid (57)from Taber-namontana cumminsii,*' may well represent the species which lie on the pathway D.A. Evans G. F. Smith G. N. Smith and K. S. J. Stapleford Chem. Comm. 1968,859. P. A. Crooks B. Robinson and G. F. Smith Chem. Comm. 1968 1210. 82 (a) A. A. Qureshi and A. I. Scott Chem. Comm.,1968,945,947; (b)cf E. Wenkert J. Amer. Chem. SOC. 1962,84,98 (c)Doubts have been expressed on the reproducibility of those experiments; Alka- loids Conference Manchester Univ. April 1969. 502 J. A. Joule dfiN\ H dfiN Meocw Q-Jy (57) between the alkaloid types in these cases removed from the interrelationship pathway by some other irreversible process. The structure of (57) followed simply from its mass spectrum and the base has been synthesised.8' The struc- tures of the secamines were elucidated" (stereochemistry as yet unspecified) by an elegant combination of mass spectrometry labelling and chemical studies.The most relevant results are as follows. Hydrolysis of tetrahydrosecamine led to a didemethoxycarbonylated product (58) which suggested the presence of two a-indolylacetic ester units and to the fission product (59),which was identi- fied by synthesis. The latter arises by a reverse Mannich reaction from the bottom half of the molecule. Reductive didemethoxycarbonylation gave (60) which was synthesised. The positions of the double bonds in the less reduced members were established by Hofmann degradation and identification with the known 3-ethyl-l-methyl-l,2,5,6-tetrahydropyridine. These alkaloids are the first recognised members of a new class with the (monomeric) skeleton of (61).82b Their existence is a strong indication that the alkaloid rearrangement could occur through an intermediate with the (61) type of structure.Thus for example a species of the form (61) could clearly be reversibly derivable (see Scheme) from any of the three types of skeleton and could thereby become the crucial go-between.82 A series of in uitro experimentss2" have provided striking evidencesZc in sup-port of these ideas. When (-)-tabersonine (62)(an aspidosperma alkaloid with A lkaloids 503 C0,Me C0,Me Aspidosperma series (62) 11 Stemmadenine 11 Strychnos series (63) CbzMe Iboga series SCHEME the appropriate oxidation level) was simply heated in acetic acid there were obtained as well as starting material ( +)-catharanthine (63)(12%) and pseudo- catharanthine (28%)(which is in any case formed from catharanthine under these conditions).Even more remarkable when ( +)-stemmadenine (64) (with double bond at 19,20) was heated under reflux in acetic acid ( f)-tabersonine (12 %) and consequently ( *)-catharanthine (9 %) and pseudocatharanthine OH 504 J. A. Joule (16 %) were formed. When the yohimbk alkaloid geissoschizine (65) were treated with hot acetic acid under nitrogen catharanthine (5 %) and pseudo- catharanthine (15 %) were formed. These rearrangements almost certainly proceed through a ring-opened intermediate of the (61) type and constitute strong support for the idea that the in vivo processes OCCUTby analogous routes.Steroidal Alkaloids.-The 0.r.d. of steroidal amines has been studied by use of the aliphatic amino-absorption as the asymmetric chromophore. The highly poisonous batrachotoxinine-A from the Columbian arrow poison Me \ MeCHOH n v (66) (67) frog has the novel structure (66).84 Spiropachysine (67) is yet another variant of the pachysandra alkaloid theme.85 Lycopodium Alkaloids.-Annopodine (68) another alkaloid from Lycopo-dium annotinum has a novel ring structure.86 Two elegant syntheses8’ of (-I-)-lycopodine (73; R = H,) have appeared. One of these87“ utilised the cyclo- hexanone (69) which was converted into the hexahydroquinoline derivative (70) (and its unwanted isomer methyl and benzyl interchanged) by reaction of the corresponding pyrrolidine enamine with acrylamide.Acid-catalysed intra- molecular alkylation of the aromatic ring gave (71). The aromatic ring was then destroyed by a sequence of reactions leaving only the desired functionalities (72). Removal of the N-protecting group then gave a keto-lactam (73; R = 0) which was reduced and re-oxidised to (-t )-lycopodine (73 ; R = H2). Miscellaneous Alkaloids.-Several macrocyclic polyamino-bases have figured in the literature this year. Palustrin from Equisetum palustre88probably has the structure (74) and two bases from Oncinotis nitZda8’ have been assigned the structures (75) oncinotine and (76) iso-oncinotine. Homaline from Homalium alni$oliumgOhas been given the working structure (77) and pithecolobine from 83 J.Parello and F. Picot Tetrahedron Letters 1968 5083. 84 T. Tokuyama J. Daly B. Witkop I. L. Karle and J. Karle J. Amer. Chern. SOC.,1968,90 1917. 85 T. Kikuchi T. Nishinaga M. Inagaki and M. Koyama Tetrahedron Letters 1968,2077. 86 W. A. Ayer G. G. Iverach,J. K. Jenkins and N. Masaki Tetrahedron Letters 1968,4597. 87 (a)G. Stork R. A. Kretchmer and R. H. Schlessinger,J. Amer. Chem. SOC.,1968 90 1647; (6) W. A. Ayer W. R. Bowman T. C. Joseph and P. Smith ibid. p. 1648. 88 C. Mayer W. Trueb J. M. Wilson and C. H. Eugster Helv. Chim. Act4 1968,51,661. 89 M. M. Badawi A. Guggisberg P. van den Broek M. Hesse and H. Schmid Helv. Chim. Acta 1968,51,1813. 90 M. Pais G. Rattle R. Sarfati and F.-X. Jarreau Compt. rend..1968 266 C 37. Alkaloids 505 OMe 70,Me CH,Ar i ii 0aMe -\/CO,Me ix x] Me -\/COz Me xi R' u (73) Reagents i C,H,N; ii CH = CH*CO.NH,; iii H,PO,-HC0,H; iv LiAlH,; v Li-NH,-Bu'OH; vi Bu'O- ; vii CCl,*CH,.O*COCl; viii 0,;ix Se0,-H,O,; x Me0 -;xi Zn. Pithecolobiurn sarnan91 seems to have the structure (78) despite the difficulty in definitely establishing the absence of a third oxygen atom suggested by earlier analyses. The novel imidazolium salt (79)has been isolated92 from Dendrobium anosmum and D. parishii. Novel indolizidines have been obtained from Elaeocarpus species (see also under simple indole alkaloids). Elaeocarpine (80; R = a-H) and isoelaeocarpine (80; R = P-H) occur in E. polyda~tylus,~~" and their dihydroaromatic counterparts elaeocarpiline (81; R = a-H)and isoelae-carpiline (81; R = p-H) in E.doli~hostilis.~~~ Borohydride reduction of iso- elaeocarpiline gave (82).93b Slaframine a fungal alkaloid has been reformulated as (83).94 91 K. Wiesner D. M. MacDonald C. Bankiewicz and D. E. Orr Cad.J. Chem. 1968,46,1881. 92 K. Leander and B. Liining Tetrahedron Letters 1968,905. 93 (a)S. R. Johns J. A. Lamberton A. A. Sioumis and J. A. Wunderlich Chem. Comm 1968,290; (b)S. R. Johns J. A. Lamberton and A. A. Sioumis ibid. 1968 1324. 506 J. A. Joule Et NH [dH] CO 1;JHY (74) (75) PhCH Me[CH,],-CH[CH,],. CO I CO-CH-NMe I I CCHzI3 'N' [CH2]4. N [CH,] 3 I I NH* [CH,I4. NH MeN-CH-CO I (79) PhCH (78) (77) OMe Ar 0ge Me Me (85) Alkaloids 507 Borohydride reduction of isoelaeocarpiline gave (82).93bSlaframine a fungal alkaloid has been reformulated as (83).94 Several different waysg5 of synthesising the mesembrine (84) system have come to light this year.Three groups have used the reaction of an arylpyrroline (85) as an enamine (see also under erythina alkaloids). Thus for exampleg5" the appropriate aryl cyclopropyl nitrile was converted into the corresponding imine which was rearranged (see also under pyridine alkaloids) to (85). Reaction of (85) with methyl vinyl ketone gave mesembrine. 94 R. A. Gardiner K. L. Rinehart J. J. Snyder and H. P. Broquist J. Amer. Chem. SOC. 1968,90 5639. 95 (a) S. L. Keely and F. C.Tahk J. Amer. Chern. SOC.,1968,90 5584; (b)R. V. Stevens and M. P. Wentland ibid.,p. 5580; T. J. Curphey and H. L. Kim Tetrahedron Letters 1968 1441 ;M. Shamma and H. R. Rodriguez Tetrahedron 1968 24 6583; T. Oh-Ishi and H. Kugita Tetrahedron Letters 1968. 5445; H. Taguchi T. Oh-Ishi and H. Kugita ibid. p. 5763.

ISSN:0069-3030    

DOI:10.1039/OC9686500489

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

15 AMINO-ACIDS AND PEPTIDES By P. M.Hardy (Department of Chemistry University of Exeter Stocker Road Exeter) Last year’s Report took stock of the methods currently in general use for amino- acid protection formation of the peptide bond and structural elucidation and outlined present ideas on racemisation and peptide conformations. Methods and ideas do not change rapidly enough to warrant such an approach more than once in about every three years. This year’s Report then concentrates on innovations in the field and shows the advances made during 1968to update last year’s more general review. Publications this year include an introductory monograph on peptides and proteins,’ the Proceedings of the Ninth European Peptide Symposium,2 and I.U.P.A.C. “Tentative Rules” for the abbreviated nomenclature of synthetic polypeptides3 and a one-letter notation for amino- acid sequence^.^ Amino-acids.-Several new unsaturated amino-acids have been isolated.From the seeds of Aesculus cali$ornica 2-amino-4-methylhex-4-enoicacid (1;R1 = H R2 = H R3 = Me) 2-amino-6-hydroxy-4-methylhex-4-enoic acid (1; R’ = OH R2 = H R3 = Me) the dipeptide y-glutamyl-2-amino-4- methylhex-4-enoic acid and 0-(methylenecyclopropy1)-P-methylalanine (2; R = Me) together with 2-amino-4-methylhexanoicacid have been character- ised.’ L-( -)-2-Amino-5-methylhex-4-enoic acid (1;R’ = H R2 = Me R3= H) D. T. Elmore ‘Peptides and Proteins’ Cambridge University Press 1968. Peptides 1968 Proceedings of the Ninth European Peptide Symposium Orsay France April 1968 ed.E. Bricas North Holland Publishing Co.;(a)P. Sieber and B. Iselin p. 85 ;(b) B. Rzeszotarska and S. Wiejak p. 86; (c) E. Schnabel H. Herzog P. Hoffman E. Klauke and I. Ugi p. 91; (d)P. G. Pietta F. Chillemi and A. Corbellini,p. 104;(e) L. Birkhofer and F. Miiller p. 151 ;(f)R. Hirschmann p. 139; (4) R. B. Merrifield and V. Littau p. 179; (h) A. Loffett and J. Close p. 189; (i) K. Brunfeldt J. Halstrom and P. Roepstorff p. 194; (j)G. W. H. A. Mansfeld H. Hindriks and H. C. Beyerman p. 197; (k) R. Garner D. J. Schafer W. B. Watkins and G. T. Young p. 145; (I) E. Bayer H. Hagen- maier G. Jung and W. Konig p. 162; (m) E. Wunsch G. Wendlberger,E. Jaeger and R. Scharf p. 229 ; (n)E. Scoffone F. Marchiori L. Moroder R Rocchi and A.Scatturin p. 325; F. M. Finn J. P. Visser and K. Hofmann p. 330; (0)A Anastasi L. Bernadi G. Bosiso R De Castiglione 0.Goffredo G. Bertaccini V. Erspamer and M. Impicciatore p. 247; (p)S. Bajusz Z Paulay Z Lhng K. Medzihadszky L. Kisfaludy and M. Low p. 237; (4)V. K. Antonov L. I. Andreeva and M. M.Shemyakin p. 78; (r) M. Bodansky J. Izdebski I. Muramatsu and k Bodansky p. 306; (s) R G. Hiskey R L. Smith A. M. Thomas J. T. Sparrow and W. C. Jones jun.,p. 209; (t) H. Zahn W. Danho G. Schmidt J. Dahlman A. Costopanagiotis and E. Engels p. 222; (u)G. Gawne G. W. Kenner N. H. Rogers R C. Sheppard and K. Titlestad p. 28. I.U.P.A.C. Combined Commission on Biochemical Nomenclature Tentative Rules for Abbreviated Nomenclature of Synthetic Polypeptides Biochemistry 1968 7 483 Arch.Biochem. Biophys. 1968,123,633; J. Bid. Chem 1968,243,2451. I.U.P.A.C. Combined Commission on Biochemical Nomenclature Tentative Rules for a One-letter Notation for Amino-acid Sequences Biochemistry 1968,7,2703 ; Arch. Biochem Biophys. 1968,125 i-v; J. Biol. Chem. 1968,243 3557. L. A. Fowden and k Smith Phytochemistry 1968,7,809; D. S Millington and R C. Sheppard ibid. p. 1027. 510 P.M. Hardy has been found in the mushroom Leucocortinurius bulbiger,6 while L-2-amino- 3-formylpent-3-enoic acid (3 ;R = CHO) and~-2-amino-3-hydroxymethylpent-3-enoic acid (3;R = CH,*OH) have been isolated from another mushroom Bankera fuligineoalba.’ Degradation of hypoglycin A (2; R = H) to (+)-3- methylpentanoic acid of known S configuration now establishes the stereo- chemistry as ( +)-(2S,4S)-2-amino-4,5-methanohex-5-enoic acid.’ Conversion of allenic aldehydes into allenic amino-acids has been found useful only for the synthesis of 2-amino-3,3-dimethylalka-4,5-dienoic acids but a more general route involving the reaction of 1-bromoalkadienes with diethyl formylamino- malonate has been established.’ In this way hypoglycin A has been synthesised in only three stages.8 R I ,NH2 MeCH=C-CH ‘CO,H A survey of the amino-acids present in the seeds of forty species of the genus Acacia has been made and L-P-N-acetyl-ap-diaminopropionicacid isolated for the first time.lo E-N-methyl- and E-NN-dimethyl-lysines have previously been shown to occur in histones; c-N-trimethyl-lysine has now been isolated from the same source.A hepatotoxic amino-acid L-a-amino-samidino- caproic acid (indospicine) has been identified in Indigofera spicata.l2 The first example of a naturally occurring pyridinium containing amino-acid has been found in tobacco leaves. The diester of this amino-acid N-(3-amino-3-carboxy- propy1)-P-carboxypyridinium betaine (4),was synthesised from methyl-L-a- amino-y-iodobutyrate hydrochloride (derived from homoserine) by heating with ethyl nicotinate ; acid hydrolysis liberated material identical with the natural product. Other new naturally occurring amino-acids include rn-hydr~xyphenylglycine,~~ 3,5-dihydroxyphenylglycine,’ and p-hydroxymethyl- L-phenylalanine.15 Fusarinines A and B have been found to contain two and three residues respectively of 6-N-(cis-5-hydroxy-3-methylpent-2-enoyl)-6-N-hydroxyornithine (5) previously found in ferrirhodin.Labile ester bonds join the carboxy-group of one unit with the 6-N-hydroxy-group of another. l6 The asparagine residue of bacitracin has been converted into a 2,4-diaminobutyric G. Dardenne and J. Casimir Phytochemistry 1968,7 1401. ’ R. P. Doyle and B. Levenberg Biochemistry 1968,7 2457. D. K. Black and S. R. Landor J. Chem. SOC. (C) 1968,281,283. D. K. Black and S. R. Landor J. Chem. SOC.(C),1968,288. lo A. S. Seneviratne and L. Fowden Phytochemistry 1968,7 1039. K. Hempel H. W. Lange and L. Birkhofer Naturwiss. 1968,55 37. M. P. Hegarty and A. W. Pound Nature 1968,217 354. l3 M. Noguchi H.Sakuma and E. Tamaki Arch. Biochem. Biophys. 1968,125,1017. l4 L. P. Miiller and H. R. Schiitte Z. Naturforsch. 1968,23b 659. l5 N. H. Sloane and S. C. Smith Biochim. Biophys. Acta 1968,158,394. l6 J. M. Sayer and T. F. Emery Biochemistry 1968,7 184. Amino-acid and Peptides 51 1 unit by dehydration of the side-chain amide to a nitrile and subsequent reduction. After acid hydrolysis of the modified antibiotic the liberated 2,4-diaminobutyric acid was found to have the L-configuration showing that it must be the aspartic acid residue of bacitracin which has the D-configuration. l7 N-Acetylcysta-thionine has been isolated from the urine of cases of congenital cystathioninuria,' and the rather unstable mixed disulphide of P-mercaptolactate and cysteine has been identified in the urine of mentally defective patients.lg OH I r r2 (5) HO*CH2*CH2* -CH.C0.N.CH2*CH2.CH2* H.CO2H A new general synthesis of amino-acids from glycine involves alkylation of the NN-bis(trimethylsily1) ethyl ester after conversion into the sodio-derivative with sodium bis(trimethylsily1)amide.All the Si-N bonds are then cleaved rapidly by dilute aqueous hydrochloric acid to liberate the new amino-acid.20 The glycine residues in protected dipeptides can be alkylated photochemically in an acetone-initiated reaction ; isobutene but-1-ene and toluene produce leucine norleucine and phenylalanine respectively. 21 An interesting asym- metric synthesis of L-aspartic acid has been described. L-erythro-Diphenyl-1 ,2-ethanolamine (6),prepared from L-glutamic acid gives (7) on condensation with (6) (7) (8) C1 H,N- -H I kH,CO,Me E.Ratti C. Lauinger and C. Ressler J. Org. Chem. 1968,33 1309. T. Perry S. Hansen D. Love and C. A. Finch Nature 1968,218,178. M. Ampola E M. Bixby M. F. Efron R Parker W. Seddon and E. P. Young,Biochem J. 1968 107,16P. K. Ruhlmann and G. Kuhrt Angew. Chem Internat. Edn. 1968,7,809. D. Elad and J. Sperling Chem. Comm. 1968 655. 512 P.M. Hardy dimethyl acetylenedicarboxylate. Catalytic hydrogenation using Raney nickel stereospecifically generates (8) with the ester group axial. Hydrogenolysis of (8)with palladium as the catalyst followed by the addition of hydrogen chloride gives the hydrochloride of optically pure a-methyl L-aspartate (9).22D-( -)-a-Aminophenylacetic acid has been prepared by the hydrolysis of D-( -)-a-aminophenylacetonitrile ( + )-hemitartrate with aqueous hydrochloric acid.This seems to be the first preparation of an optically active amino-acid from the corresponding optically active a-ami~~o-nitrile.~~ N-Acetyl-N-(3-amino-2,4,6-tri-iodophenyl)-~-aminoisobutyric acid has been synthesised and found to be an excellent oral cholecystographic agent of low cis-Glycolation of 3,4-dehydroproline is described in the first part of a project to synthesise all the isomers of the hitherto unknown 3,4-dihydroxy- prolines.25 Racemisation during the conversion of cystine dimethyl ester into a-benzyloxycarbonyldiaminopropionicacid frustrated the synthesis of optically active material.26 N(2)-Hydroxyasparagine was also found to racemise during preparati~n.~~ Recent synthetic studies on amino-acids include the preparation of 2,5-dihydr0phenylalanine,~y6 b’-trihydroxy-~-leucine,~~ hexafluoroleucine [2-amino-4,4-bis(trifluoromethyl)butyricacid],30 the leucine antagonists DL-cyclopent-2-enyl and cyclohex- 1-enyl-alanine~,~’ and tri-N-methylhi~tidine.~~ DL-Alanine benzenesulphonate may be resolved by seeding a supersaturated solution in 97 % aqueous acetone with one optical antipode. A 91 % yield of almost optically pure material can be obtained. The mother liquor yields the other enantiomorph. 33 Resolution by stereoselective interaction of basic amino-acids (including arginine) and metal complexes has been described.For example when DL-histidine is added to DL-[CO(EDTA)] -ion in acidic aqueous ethanol the optically pure diastereoisomer [~-hisH,]( +)s46 1 [Co(EDTA)], 2H20 crystallises first.34 Lysine can also be resolved by adding a solution of + dodecatungstophosphoric acid slowly to a concentrated solution of K ( -)546 [Co(EDTA)] -(1rnol.) and DL-lysine hydrochloride (2mol.). The initial precipi- tate is the tungstophosphate of D-lysine. If sufficient is added then the L-lysine salt eventually precipitate^.^^ In the cobalt complex of diglycine (10)the protons of the C-terminal residue (H;) exchange with deuterium in alkaline deuterium oxide but the (H;) protons do not. In the corresponding complex of glycyl-L- alanine no change occurs in the c.d.spectrum during exchange so the reaction ’’ J. P. Vigneron H. Kagan and A. Horeay Tetrahedron Letters 1968 5681. 23 J. Schawartz and G. Eibel Chem and Ind. 1968 1698. 24 J. A. Korver Rec. Trau. chim. 1968,87 308. ” C. B. Hudson A. V. Robertson and W. R J. Simpson,Austral. J. Chem. 1968,21 769 26 L. Benoiton Canad. J. Chem. 1968,46 1549. ’’ E. Falco and G. B. Brown J. Medicin Chem. 1968 11 142. M. L. Snow C. Lauinger and C. Ressler J. Org. Chem. 1968,33 1774. ’’ F. Weygand and F. Mayer Chem Ber. 1968,101 2065. ’O J. Lazar and W. A. Sheppard J. Medicin Chem. 1968,11,138. 31 T. H. Porter R M. Gipson and W. Shive J. Medicin Chem. 1968,11,263. ’’ V. N. Reinhold Y. Ishikawa and D. B. Melville J. Medicin Chem. 1968 11 258. ’’ I. Chibata S. Yamada M. Yamamoto and M.Wade Experientia 1968,24,638. 34 R D. Gillard P. R Mitchell and H. L. Roberts Nature 1968 217 949. Amino-acids and Peptides 513 seems to be stereospecific. 3s 0.r.d. studies on N-dimed~nyl~~ and N-(2-pyridyl N-o~ide)~amino-acids suggest correlations with absolute configuration. ' The optical activity of the disulphide bond in L-cystine and some of its deriva- tives has been studied in 0 I-\ I=/"CHR'Co2H (R = amino-acid side chain) (R = amino-acid side chain) (13) L-and ~-3,5-Dicklorotyrosines have been made by the chlorination of tyrosine in propionic acid at 0-5". The product can be salted out. L-Amino- acid oxidase and catalase will attack the L-derivative. 39 Benzyloxycarbonyl-L-asparagine reacts with phosgene to give benzyloxycarbonyl-P-cyano-L-alanine.L-Asparagine itself reacts with phosgene to give P-cyano-N-carboxy-L- alanine anhydride which forms poly-( P-cyano-L-alanine) on treatment with t~iethylamine.~~ N-a-Formyl-L-tryptophyl peptides have been found to cyclise in trifluoroacetic acid to 3,4-dihydro-~-carboline-3-carboxamides (11); this may be useful for the synthesis of alkaloids derived from P-~arboline.~' Amino-acids will react with S-methylisothiosemicarbazide hydriodide in aqueous ethanol to produce N-aminoguanyl amino-acids (12). These deriva- tives have been condensed into peptide analogues.42 a-Amino-acids or their esters also react with 2,5-diethoxytetrahydrofuran in acetic acid to give a-pyrrolo-acids and esters (13). Regeneration of the amino-acid is not possible 35 R.D. Gillard P. R Mitchell and N. c. Payne Chem Comm. 1968 1150. 3b P. Crabbe B. Halpern and E. Santos Tetrahedron 1968,4299,4315. 37 V.Tortorella and G. Bettani Cazzetta 1968 % 316. D. Coleman and E.R Blout J. Amer. Chem SOC. 1968,90 2405. 39 K. R Brady and R P. Spencer J. Org. Chem. 1968,33 1665. 40 M. Wilchek S. Ariely and k Patchornik,J. Org. Chem. 1968,33 1258. 41 k Previero,MA Coletti-Previero and Lb-G. Barry Canad. J. Chem. 1968,46,3404. 42 J. Gante Chem Ber. 1968,101 1195. 514 P.M. Hardy and optical homogeneity has not been e~tablished.~~ Progress has been made in g.1.c. techniques enabling all the protein amino-acids to be separated on a single run.44 Prebiotic Studies.-There is much interest in models of prebiological systems.The thermal synthesis of polyamino-acids (‘pr~teinoids.)~~ and their MSH46 and glutamic acid oxyreductase4’ activities have been examined. The high- temperature synthesis of amino-acids from acetylene carbon dioxide and ammonia has been shown not to be a Strecker reaction4* U.V. irradiation of ammonium thiocyanate has produced methionine ; the sulphur-containing amino-acids have previously resisted synthesis under possible prebiotic condition^.^^ Experiments with oligomers of hydrogen cyanide such as diaminomaleonitrile have shown that when heated with water alone they will give rise to peptides which contain up to eleven types of amino-a~id.~’ Peptides containing up to five types of amino-acid have been prepared by irradiation of hydroxylamine and ethylene glycol mixtures at 185-235 nm.5 Montmoril- lonite gel (a clay of silicon and aluminium oxide layers) in water at pH 5 will convert alanine adenylate into homopeptides of D.P.10-12 in 16% yield.52 Aspartic acid copper complex is partially resolved when a supersaturated solution is seeded with wool or cotton,53 while D-tyrosine decomposes in solution more quickly than L-tyrosine when bombarded with polarised p-particles from 90Sr;54 these observations are of interest in connection with the natural predominance of L-amino-acids. L-Amino-acids have also been shown to stabilise nucleic acid helices more effectively than D-aminoacids. Peptides :Structural Elucidation.-The Edman degradation has been carried out with a copolymer of acrylic acid and styryl isothiocyanate.’ Fluorescein isothiocyanate can also be used in the Edman method; its fluorescence enables the thiohydantoin to be detected easily.57 An isotope dilution technique has also been described in which phenyl [35Slisothiocyanate is used.58 2-Fluoro- 43 J.Gloede K. PoduSka H. Gross and J. Rudinger Coll. Czech. Chem. Comm. 1968,33 1307. 44 A. Darbre and A. Islam Biochem J.,1968,106,923;C. W. Gehrke R W. Zumwalt and L. L. Wall J. Chromatog. 1968,37 398; See also G. E. Pollack and A. H. Kawauchi Analyt. Chem. 1968 40,1356;B. A. Halpern V. A. Close A. Wegmann and T. W. Westley Tetrahedron Letters 1968 31 19; F.Shakrokhiv and C. W. Gehrke J. Chromatog.,1968,36,31; J. R Coulter and C. S. Hann ibid.p. 42. 45 S. W. Fox and T. V. Waekneldt Biochim Biophys. Acta. 1968,160 246. 46 S.W. Fox and W. Ching-Tso Science 1968,160 3827. 47 G. Krampitz W. Haas and S. Baars-Diehl,Nntunviss. 1968 55 345. 48 K.Samochocka,A. L. Kawezynski and M. Taube Angew. Chem Znternat. Edn. 1968,7 392. 49 G. Steinman k E. Smith and J. J. Silver Science 1968 159 1108. 50 R E.Moser and C. N. Matthews Experientia 1968,24,658;R E.Moser A. R Claggett and C. N. Matthews Tetrahedron Letters 1968 1599. 51 A. Zamoram C. Lanzarinii and A. Rura Gazzetta 1968,9?3,210 214. ” M. Paecht-Horowitz I.U.P.A.C. 5th International Symposium on the Chemistry of Natural Products London July 8-13tk 1968,Abstract D24,p. 232 53 K. Harada Nature 1968,218 199. 54 A S.Goray Nature 1968,219 338.” G. Manecke and G. Giinzel Naturwiss. 1968,55,84. ’‘ E.J. Gabbay R Kleinman and S. R Shimshak J. Amer. Chem SOC.,1968 90,927. 57 H. Maeda and H. Kawauchi Biochem Biophys. Rex Comm. 1968,31 188. G. L. Callewaert and C. A. Vernon Biochem J. 1968,107 728. Amino-acids and Peptides pyridine N-oxide has been suggested for stepwise N-terminal analysis but it can also be used for carboxy-group activation giving rise to N-acyloxy-2- pyridones.59 7-Chloro-4-nitro-2,1,3-benzo-oxa-diazole is a new fluorogenic reagent for amino-acids. Derivatives are excited by visible light and the reagent is more stable to moisture and more soluble in aqueous solutions than dansyl chloride.60 Reduction of 3,5-dinitro-2-pyridyl derivatives of peptides with sodium borohydride at pH 8-9 gives dihydro- or tetrahydro- pyridine products with enhanced nitrogen basicity.Cleavage of the N-terminal amino-acid from such reduced peptide derivatives occurs at pH 5-6 over 12 hr at 40-50" or in 5 min. at 100". Other amide bonds and disulphide bridges are untouched.61 Tetrafluorosuccinic anhydride can be used for the reversible protection of protein amino-groups ; the derivative is cleaved by trifluoro- acetic acid.62 A six-stage sequential degradation of amino-acids from the C-terminus of a peptide has been carried out in a favourable case by use of ammonium thiocyanate and acetic anhydride. The peptidyl thiohydantoins formed are cleaved with acetohydroxamic acid under mild condition^.^ A method for selective cleavage of peptides at serine and threonine residues has shown reasonable results in preliminary tests on dipeptide derivatives.The side-chain hydroxy-groups react with phosgene to give O-chlorocarbonyl derivatives (14) which cyclise to oxazolidones (15) when heated under reflux in xylene. Mild alkaline treatment then liberates the carboxylic acid derived from the N-terminal amino-acid and a 2-0x0-oxazolidine-4-carboxylic derivative (16).64 Lysine residues can be converted into NN-dimethyl-lysine by a reductive alkylation method under conditions where disulphide bridges are not attacked.65 Modification of tryptophan and cysteine residues with (14)-NH*CHR*CONH-CH*CO-(15)-NH*CHR.CON-CH.C0-I I I kH2 k0 CH ClCO~O/ 'O/ (16) -NH*CHR.CO,H + N-CH.CO-I I 59 D.Sarantakis J. K Sutherland C. Tortorella and D. Tortorella J. Chem Soc. (C) 1968 72. 6o P. B. Ghosh and M W. Whitehouse Biochem J. 1968,108,155. 61 A. Signor and E.Bordignon Tetrahedron 1968,24,6995. 62 G. Braunitzer K Bayreuther H. Fujiki and B. Schrank Z. physiol. Chem. 1968,349 265. 63 G. R Stark Biochemistry 1968,7 1796. 64 T. Kaneko S. Kusomoto T. Inui and T. Shiba Bull. Chem Soc.Japan 1968,41,2155; T. Kaneko I. Takeuchi and T. Inui ibid. p. 974. 65 G. E. Means and R E Feeney Biochemistry 1968,7 2192 Diacetyl 6-Deoxy talose 0 p I OMe CH,Ph CHMe Mo % I *CH*C04NH*CH.C0,NH*&-I*C0,NH*CH*CH,0 Tri-o-met hyl % Me[CH,],,CH==CH-CH.CH,*CO,NHI I rhamnose r; i Phe i AlloThr i AlA i Alaninol @ Amino-acids and Peptides 517 sulphenyl halides has been further explored,66 and azobenzene-2-sulphenyl bromide has been found to be a selective reagent for cysteine thiol modifica- tion being unreactive towards amino-groups and tryptophan.This reagent has the advantages of high solubility and great stability in water since its true structure is 2-phenyl-1,2,3-benzo-thia-diazoliumbromide (17).67 Amino-terminal tyrosine residues can be determined spectrophotometrically after conversion into 5,7-dibromo-6-hydroxyindole-2-carboxamide with N-bromo- succinimide.6 Mass spectrometry. Techniques using N-methylation of amide groups to increase the volatility of peptides continue to be explored. Methylation of peptides by silver oxide-methyl iodide-dimethylformamide mixtures is not straightforward for methionine glutamic acid and aspartic acid residue^.^' It has been found difficult in some cases to determine the C-terminal residues of N-methylated large peptides as they are too weak to be seen by mass spectro- metr~.~' The structure of mycoside, (18)has been determined by mass spectro- metry of a derivative prepared by Hakamori methylation.This method of methylation originally developed for use with glycolipids involves the reaction of the methylsulphinyl carbanion in dimethyl sulphoxide with the peptide prior to treatment with methyl iodide. After 12 hr. amide NH methylation was complete. The mass spectra of N-acyl-N-methyl oligopeptide methyl esters has been discussed,72 and the mass spectrometry of amino-acids and peptides in general has been reviewed.73 Extensive studies on the modes of fragmentation of peptides containing aromatic and heterocyclic residues have been rep~rted.'~ Peptide Synthesis-Protecting groups.A study has been made of a series of aralkyloxycarbonyl amino-protecting groups and the 1-@biphenyl)-1-methylethoxycarbonyl residue has been proposed as especially suitable for use in peptide synthesis. This group is cleaved with dilute acetic acid 3000 times faster than is the t-butoxycarbonyl group which permits its selective removal in the presence of the latter. No effects due to steric hindrance are observed,in contrast to the trityl re~idue.'~*~" An alternative route to t-butoxy- carbonyl and t-pentyloxycarbonyl amino-acids by use of t-butyl and t-pentyl 66 E.Scoffone A. Fontana and R Rocchi. Biochemistry. 1968,7.971. 67 A. Fontana F. M. Veronese and E. Scoffone Biochemistry 1968 7 3901; A. Buroway F. Liversedge and C. E. Vellins J. Chem SOC., 1954 4481. M. Wilchek T.Spande and B. Witkop Biochemistry 1968,7 1787. 69 K. L. Agarwal R A. W. Johnstone G.W. Kenner D. S. Millington and R C. Sheppard Nature 1968,219,498. 70 D. W. Thomas B. C. Das S. D. GCro and E. Lederer ref. 52 Abstract A24 p. 38. E. Vilkas and E Lederer Tetrahedron Letters 1968 3089. 72 B. C. Das S. D. GCro and E. Lederer Nature 1968,217 547. 73 J. H. Jones Quart. Rev. 1968,22 302. 74 E. 1. Vinogradova V. M. Lipkin Y. B. Alakhov M. Yu Feigina Yu. B. Schvetsov Zhur. obshchei Khim. 1968,38,777; E. I. Vinogradova V. M. Lipkin Y.B. Alakhov and Yu B.S'chvetsov ibid. p. 787; M. M. Shemyakin Yu A. Ovchinnikov A. A. Kiryushin,E.I. Vinogradova Yu R Alakhov V. M. Lipkin and B. V. Rozynov ibid. p. 798. '' P. Sieber and B. Iselin Helu. Chim Acra 1968 51 614. 518 P.M. Hardy quinolin-8-yl carbonates is now available.2b,76 t-Butyl fluoroformate has also been used to introduce the t-butoxycarbonyl group; in contrast to t-butyl chloroformate it is stable at 0” for several months but the required carbonyl chlorofluoride is not readily 77 The useful derivatives N(a)-t- butoxycarbonyl-N(o)-t-butoxycarbonyl-L-arginine, some of its active esters and the t-butyl ester of N(o)-t-butoxycarbonyl-L-argininehave now been prepared. Catalytic hydrogenation of benzyloxycarbonyl amino-acid and peptide p-nitrophenyl esters in the presence of hydrochloric acid (1 equiv.) is reported to remove the amino-protecting group without appreciable reduction of the nitro-group.When prehydrogenated solvent and catalyst are used reaction is complete in 5 min.79 It has also been found possible to hydrogenolyse benzyloxycarbonyl groups quantitatively from peptides containing methionine in the presence of boron trifluoride+ther in anhydrous methanol. Under these conditions free carboxy-groups are esterified. This can be avoided by use of t-butyl alcohol or acetic acid as solvent but reaction times are much prolonged.8o Schiff bases derived from benzoylacetone have been proposed for amino-protection. Yields are high and the protected amino-acids can be coupled through mixed anhydrides or uia cyanomethyl or phenacyl ester derivatives.Recrystallisation of cyanomethyl esters of this type is however accompanied by racemisation. 81 Hydroxylaminolysis can be used to remove the phthaloyl amino-protecting group from peptides under mild conditions. The sodium salt of N-hydroxyphthalimide can be filtered off from the methanolic solution after 15 min. and the product can then be isolated.82 p-Nitrophenyl acetate and other esters except formates have been found to acylate homolysine lysine and ornithine exclusively at the o-ami9o-group at pH 11. At less alkaline pH values the reaction is preferential but not selective. ap-Diamino-propionic and butyric acids are not attacked selectively at any pH.83 Pentamethylbenzyl ethers of a-hydroxy-acids have been used successfully in depsipeptide synthesis.These esters are crystalline when the corresponding benzyl and t-butyl esters are oils and can be removed with cold trifluoroacetic acid.84 An alternative procedure to treatment with methyl iodide for sensitising C-protecting p-methylthioethyl ester groups towards alkali has been developed. Oxidation of a relatively alkali-sensitive series of y-benzylglutamyl peptides with a-C- terminal P-methylthioethyl esters by use of hydrogen peroxide in the presence 76 B. Rzeszotarska and S. Wiejak Angew. Chem Internat. Edn 1968,7 379; Annalen 1968,716 216. 77 E.Schnabel H. Herzog P. Hoffman E Klauke and I. Ugi Angew. Chem Internat. Edn. 1968 7,380;Annalen 1968,716 165. ” H.Arold and S. Reissmann Z. Chem. 1968,3 107. 79 J. Kovacs and R L. Rodin J. Org. Chem. 1968,33 2418. H. Yajima K. Kamasaki Y. Kinomura T. Oshima S. Kimoto and M.Okamoto Chem and Pharm Bull (Japan) 1968,16,1342 D.Breazu A. Balog C Daicoviciu and E Varga ref. 52 Abstract D22 p. 228. 82 0.Neunhoeffer G. Lehmann D.Haberer and G.Steinle Annalen 1968,712 208. 83 J. LeClerc and L.Benoiton Cad J. Chem. 1968,46,1047. 84 F.H. C. Stewart Austral. J. Chem. 1968,21,1327. Amino-acids and Peptides 519 of ammonium molybdate produced the P-methylsulphonylethyl esters which lost no y-ester on fission of the a-ester with alkali8’ The similarly alkali- labile 2-p-tolylsulphonylethyl esters have also been advocated for C-protec- tion. 86 4-(Methy1thio)phenyl esters can be used for C-protection during peptide synthesis and converted when required into an active ester by oxidation to the 4-(methylsulphony1)phenyl ester ;87 this active ester was originally introduced for peptide cyclisation.88 A preliminary study indicates that NN-dimethylaminoethyl esters of N-protected amino-acids are rapidly hydrolysed by 2-4 % sodium hydrogen carbonate in aqueous dimethylform- amide but are resistant to hydrogenolysis or acid cleavage. 89 Two new S-protecting groups for cysteine are proposed. The l-phenyl- cyclohexyl group emerged as the most promising from a study of eleven types of S-derivative. S-( 1-Phenylcyclohexy1)cysteine can be prepared by a boron trifluoride-ther-catalysed condensation of cysteine hydrochloride with 1-phenylcyclohexanol.The group is cleaved by trifluoroacetic acid.” S-Acet-amidomethylcysteine can be prepared from cysteine and acetamidomethanol in strongly acid solution. This protecting group is stable both to acid and to alkali including trifluoroacetic acid and liquid hydrogen fluoride at O” but can be removed by Hg’ + at pH 4.91 Peptide synthesis using O-~arbamoyl~~ and 0-alkoxycarbonylg3 tyrosine protection has been explored. The 2,2,2- trifluoro-N-benzyloxycarbonylaminoethylresidue has been suggested for the protection of serine and threonine hydroxy-groups. It can be introduced with N-benzyloxycarbonyl- l-chloro-2,2,2-trifluoroethylamine(19 ; R’ = C1 R2= PhCH,) in the presence of triethylamine and removed when required either by catalytic hydrogenolysis or by fission with hydrogen fluoride.94 The closely similar 1-butoxycarbonylamino-2,2,2-trifluoroethylgroup has been used to protect the iminazole NH of histidine; it is inserted by the reaction of (19; R’ = OAc or OBz R2= CMe,) with N-t-butoxycarbonylhistidine methyl ester.” The piperidino-oxycarbonyl group has also been proposed for histidine imino-group prote~tion.~~ An improved synthesis of O-benzyl- threonine without concomitant racemisation has enabled O-benzyl-N-t-butoxycarbonyl-L-threonine to be conveniently ~repared.’~ N(a)-(2-Nitro- phenylsulpheny1)-0-t-butyl-L-threonine, serine hydroxyproline and tyrosine P.M. Hardy H. N. Rydon and R C. Thompson Tetrahedron Letters 1968 2525. 86 A. W. Miller and C. J. M. Stirling J.Chem SOC. (C) 1968 2612 13’ 3.J. Johnson and P. M. Jacobs Chem Comm. 1968 73. ‘I3 R Schwyzer and P. Sieber Helu. Chim Acta. 1958 41 2190. A. E. Greben V. F. Martynov and M. A. Titov Zhur. obschei Khim. 1968,38,664. W. Konig R Geiger and W. Siedel Chem Ber. 1968,101 681. 91 D. F. Veber J. D. Milkowski R. G. Denkewalter and R. Hirschmann Tetrahedron Letters 1968,3057. 92 G. Jager R Geiger and W. Siedel Chem. Ber. 1968,101,2762. 93 R Geiger G. Jager and W. Siedel Chem Ber. 196% 101 2189. 94 F. Weygand W. Steglich F. Fraunberger P. Pietta and J. Schmid Chem Ber. 1968 101,923. 95 F. Weygand W. Steglich A. Maienhofer and k Bauer Chem Ber. 1968 101 1894. 96 G. Jager R Geiger and W. Siedel Chem. Ber. 1968 101 3537. 91 T. Mizoguchi G. Levin P.M. Woolley. and J. M. Stewart. J. Org. Chem. 1968 33 903. 520 P. M. Hardy have been obtained as solid dicyclohexylammonium salts and several active esters of each have been prepared.98 The 2,4-dimethoxybenzyl and 2,4,6- trimethoxybenzyl groups show great promise for the protection of asparagine and glutamine amide groups. They improve the solubility in organic solvents of peptides containing these residues and prevent dehydration to the nitrile during some coupling reactions. The o-protected amides are prepared by coupling the appropriate methoxybenzylamine with dicyclohexylcarbodi-imide to the free p-and y-carboxy-groups of a-protected aspartic or glutamic acids. The groups are stable to hydrogenolysis and to hydrogen chloride in methanol but treatment with trifluoroacetic acid at room temperature over 25-30 hr.liberates the free o-amides.2d* 99 OEt (2 1) Formation of the peptide bond. Several ingenious new methods of coupling amino-acids have been described. 2-Ethoxy-M-ethoxycarbonyl-1,2-dihydro-quinoline (20) does not easily react with amines but with N-protected amino- acids mixed anhydrides are formed presumably uia (21) liberating quinoline. To prepare peptides the mixed anhydride is formed in the presence of an amino-acid or peptide ester. Under these conditions racemisation was shown to be absent by the Young test probably because the slow formation but relatively rapid consumption of the mixed anhydride minimises its accumula- tion. O0 A method of peptide synthesis involving oxidation-reduction con-densation has been developed.The cupric salt of an N-protected amino-acid couples with an o-nitrophenylsulphenyl amino-acid ethyl ester in the presence of triphenyl phosphine to give the N-protected dipeptide ethyl ester triphenyl- phosphine oxide and cupric o-nitrophenylsulphenate. A further example is illustrated in (22).lo1 N-Protected amino-acid or peptide active esters can be coupled with free amino-acids or peptides in the presence of N-trimethyl- silylacetamide and a trace of sulphuric acid under anhydrous conditions without apparent racemisation. Reaction goes presumably through the amino-acid trimethylsilyl ester ; the resulting peptide ester is hydrolysed by 98 E. Wiinsch and F. Angelo Chem. Ber. 1968,101 323.99 F. Weygand W. Steglich T. Bjornason R Aktar and N. Chytil Chem. Ber. 1968 101 3623 3642; P. Pietta F. Chillemi and k Corbellini ibid. p. 3649. loo k Belleau and G.Mnlek J. Amer. Chem Soc. 1968,90 1651. T. Makaiyuma M. Veki M. Maruyama and R Matsueda. J. Amer. Chem. SOC..1968,W.4490. Amino-acids and Peptides 521 the sodium hydrogen carbonate wash during work-up.2e Peptide bond forma- tion has been found to occur in surprisingly high yields on a column of Dowex 50 (H') ion-exchange resin on which an amino-acid is held when a solution of a second amino-acid is simply passed down the column. Yields of up to 50% of dipeptide have been obtained.lo2 Synthesis of peptides by oxidation of N-acyl-a-amino-acid phenylhydrazides has been studied. The phenyl- hydrazides were prepared by a papain-catalysed condensation.After oxidation with N-bromosuccinimide or lead tetra-acetate the unstable but often crystal- line phenyldi-imide can be isolated and stored at -70" until coupled with the amino-component. Some racemisation has been found to occur. lo3 Use of thiazolidine-2,5-diones instead of N-carboxy-anhydrides for the synthesis of peptides in aqueous medium by reaction with free amino-acids at carefully controlled pH values as developed by Hirschmann '04 gives higher yields but racemisation occurs. Glycine however has less tendency to undergo isocyanate formation and histidine less tendency to undergo other side reac- tions than if the N-carboxy-anhydrides were used.2f '05 Further details and studies on o-hydroxyphenyl 4,5-dichloro-2-hydroxyphenyl,'06and piperidino active ester^''^ are reported.Their resistance to racemisation during coupling is ascribed to their possession of a neighbouring group capable of hydrogen bonding to the incoming amine and accepting a proton from it thus accelerat- ing aminolysis. N-t-Butyl-5-methylisoxazoliumperchlorate will form enol esters with N-protected amino-acids. These esters are stable enough to be stored but so far only coupling with benzylamine has been reported.''* (22) Z-L-Phe-OH + H-Gly-OEt + HgCl + (O-NO~*C~H~*S)~ + 2NEt3 + Ph,P + Z-L-Phe-Gly-OEt (89%) + 2NEt3H'Cl-+ H~(o-NO~.C~H~*S)~ + Ph3PO Solid-phase methods of peptide synthesis continue to receive much attention both from the point of view of improvement of materials and yields on coupling and also in their increasing routine application.Autoradiographs of solid- phase resin beads bound to peptides containing tritium-labelled proline show clearly that the peptide chains are located quite uniformly throughout the polymeric support and not just near the surface.2g Two papers on polymers derived from phenol for use in solid-phase synthesis have appeared. After methylation of phenol-formaldehyde resin with diazomethane it can be S. Yamashita and N. Ishikawa Experientia 1968,24 1079. H. B. Milne and C. F. Most jun.,J. Org. Chem. 1968,33 169. lo4 R Hirschmann R G. Strachan H. Schwam E. F. Schoen~waldt,H. Joshua H. Barkemeyer D. F. Veber W. J. Paleveda T. A. Jacob T. E. Beesley and R. G. Denkewalter J.Org. Chem. 1967 32 3415. lo' R S. Dewey E. F. Schoenewaldt H. Joshua W. J. Paleveda jun. H. Schwan H. Barkemeyer B. H. Anson D. F. Veber R G. Denkewalter and R. Hirschmann,J. Amer. Chem SOC.,1968,W. 3254. '06 J. H. Jones and G. T. Young J. Chem. SOC.(C) 1968,436. lo' J. H. Jones and G. T. Young,J. Chem. SOC.(C) 1968,53. lo* R. B. Woodward and D. J. Woodman J. Amer. Chem. SOC.,1968,90,1371. 522 P.M. Hardy chloromethylated in the usual way. Its utility is demonstrated by the synthesis of a hexapeptide.Iog A polyphenol resin prepared from phenol and s-trioxan in bis-(2-ethoxyethyl) ether has been used for a synthesis of oxytocin and fragments of physalaemin. ' Two new groups for attaching amino-acids to polystyrene resins have been explored (23 ;R = benzene ring of polystyrene).These can be esterified by N-protected amino-acids by use of carbonyl(di- imidazole) and are compatible with N-benzyloxycarbonyl protection. '' (23) RCO. [CHZI3- OH and RCH,*NAc. [CH2In. OH Peptides can be removed from polymer supports if they are present as sub- stituted benzyl esters by transesterification with an anion exchange resin. a-Functions of aspartic and glutamic acids can also however be trans- esterified during this process. Attempted preparation by the Merrifield method of hexapeptides containing the sequence Asp-Gly gave predominantly the succinimido-derivatives. This occurred during conventional synthesis only when the P-carboxy-group of the aspartic acid was esterified.I13 Three more types of automated solid-phase apparatus have been described.2~-i Two new polymeric hydroxy-compounds for activating N-acyl amino-acids upon esterification have been applied. Poly-(8-hydroxy-5-vinylquinoline) is a copolymer prepared from 8-benzyloxy-5-vinylquinolineand divinyl- benzene. Condensation of copoly(ethy1ene-maleic anhydride) with hy- droxylamine gives copoly(ethy1ene-N-hydroxymaleimide) a polymeric analogue of N-hydroxysuccinimide. 11' A novel way of utilising polymers for peptide synthesis has been described. The 4-picolyl ester of an amino-acid is coupled to an N-protected amino-acid by dicyclohexylcarbodi-imide in solution. After filtration from urea the dipeptide is absorbed on Sulphoethyl Sephadex through its basic ester group and excess of acylating agent and the co-products can be washed away leaving the pure dipeptide held to the Sephadex.The peptide can be eluted from the polymer by 2% triethylamine in aqueous tetrahydrofuran the amino-protecting group split off and the cycle is repeated until the desired peptide is built 'I6 Esters of p-dimethyl- aminoazobenzyl alcohol can be used in the same way; they have the added advantage of being easily followed on a Sephadex column by their dark red colour. ' '09 G. Losse C. Madlung and P. Lorenz Chem. Ber. 1968,101 1257. N. Inukai K. Nakano and M. Murakami Bull. Chem. SOC. Japan 1968,41,182. ''I M. A. Tilak and C. S. Hollinden Tetrahedron Letters 1968 1297. B. Halpern L. Chew V. Close and W. Patton Tetrahedron Letters 1968,5163. '13 M.A. Ondetti A. Deer J. T. Sheehan J. Pluscec and D. Kocy Biochemistry 1968,7 4069. l4 Van G. Manecke and E. Haake Naturwiss. 1968,55,343. '15 D. Laufer T. M. Chapman D. I. Marlborough V. M. Naidya and E. R Blout J. Amer. Chem. Soc. 1968,90,2696. R. Camble R Gamer and G. T. Young Nature 1968,217,247. T. Wieland and W. Racky Chimia (Switz.) 1968,22 375. Amino-acih and Peptides Racemisation. A method for determining racemisation in the C-terminal amino-acid of a benzyloxycarbonyl dipeptide after coupling with an amino-acid t-butyl ester has been evolved. After removal of the protecting groups the N-terminal amino-acid is degraded by the Edman technique and any resulting diastereoisomeric dipeptides are separated by paper chromatography."* The extent of racemisation of an amino-acid can be estimated by conversion to a dipeptide with L-leucine N-carboxy-anhydride and running the product on an amino-acid analyser to separate the diastereoisomeric dipeptides.By use of pmole samples one part of D in 10oO parts of L can be detected. Basic amino-acids are treated with L-glutamic acid N-carboxyanhydride to give dipeptides with favourable elution positions.' Racemisation of amino-acids can also be detected by the g.1.c. of N-(-)-menthyloxycarbonyl'20 or ( +)-2-butyl esterI2' derivatives. Diastereoisomeric dioxopiperazines can also be separated by g.l.c.'22 Use of a I3C-H satellite peak of the methyl group doublet in the n.m.r. spectrum of alanine dipeptides is claimed to improve the sensitivity of detection of racemisation tenfold over a previously published method.72 The ready racemisation of S-benzyl-L-cysteine has received further study.The pentachlorophenyl ester of S-benzyl-N-benzyl- oxycarbonyl-L-cysteine on incubation with [35S]-a-toluenethiol in the presence of triethylamine racemised without incorporation of radioactivity ruling out a mechanism involving p-elimination and re-addition. Under these conditions the corresponding p-nitrophenyl ester does incorporate radioactivity but this is thought to be due to conversion into the thiobenzyl ester since the radioactivity is lost again on hydrazinolysis of the product. S-Benzyl-L- cysteine active esters also show no incorporation of deuterium after treatment with alkaline deuteriomethanol seemingly also ruling out racemisation by direct ionisation of an a-hydrogen atom.123 Synthesis of Natural Peptide.-The outstanding achievement of the year was the synthesis of a peptide with the amino-acid sequence of C.pasteurianum ferredoxin which contains fifty-five amino-acid residues by the solid-phase technique. Amino-acids were coupled as their t-butoxycarbonyl derivatives by use of dicyclohexylcarbodi-imide in dichloromethane. After fifty-four coupling steps 38 % of free amino-group (relative to the amino-group liberated by the first t-butoxycarbonyl splitting) was available. This corresponds to an average yield of 98.9 % for the two steps in each synthetic cycle. A 51"/,crude yield of S-benzyl peptide was obtained. After removal of the benzyl groups the peptide was purified as the S-sulphonate.The yield of product from the E. Taschner,L. Lubiewska M. Smulkowski and H. Wajciekowska Experimeniu 1968,24 521. J. M. Manning and S. Moore J. Biol. Chem. 1968 243 5591. J. W. Westley and B. Halpern J. Org. Chem. 1968,33 3978. G.E.Pollack and A. H. Kawauchi Analyt. Chem. 1968,40 1356. "'J. W. Westley V. A. Close D. E. Nitecki and B. Halpern Analyt. Chem. 1968,40 1888; A.B. Mauger J. Chromatog. 1968,37,315. J Kovacs G. L. Mayers R H. Johnson. and U. R. Ghatak. Chem Comm.. 1968 1066. 524 P.M. Hardy thiol peptide was similar to the 15% of S-sulphonate obtained from natural ferredoxin under these conditions.26*'24 Five groups of workers have been studying porcine thyrocalcitonin the thyroid hormone which lowers plasma calcium by direct inhibition of bone breakdown.The amino-acid sequence of this dotriacontapeptide (24a) has been completely established by three groups working independently,'25* and partially elucidated by a fourth group.'" Two syntheses of material possessing full biological activity have appeared.'28*lZ9 The CIBA group link protected sequences 10-24 and 25-32 and finally add sequence 1-9 by a dicyclohexylcarbodi-imide coupling in the presence of N-hydroxysuccinimide as the preformed heterodetic cyclic peptide.lz8 The Sandoz group link sequences 10-19 and 20-32 before similarly adding sequences 1-9 already containing the disulphide bridge. The sequence 10-32 differs in the two cases in the degree of side-chain protection; in one peptide there is none,lz9 and in the other five t-butyl groups are present.l2 * Human calcitonin has been isolated from thyroid tumour tissue as a mixture of monomer and dimer. The dimer reverts to the monomer on treatment with ammonium hydroxide. The molecule is the same size as the porcine variety and has a similar disulphide bridge but differs in sequence at eighteen posi- tions (24b).I3' Synthesis has also been reported. 13' The pharmacology of thyrocalcitonin has been reviewed. 13' s---S (24) I I [a) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-TrpArgAsn 12 3 4 5 6 7 8 9 101112131415 (b) GlY Met-Gly-Thr-Thr Gln-Asp. ~ Leu-Am-AspPhe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Phe-Lys-Thr -Pro Gln-Thr-Ala-Ile Val-GI y-Ala-~ 124 E.Bayer G. Jung and H. Hagenmaier Tetrahedron 1968,24,4853. It' F. W. Kahnt B. Riniker I. MacIntyre and R Neher Helu. Chim Act4 1968,51,214; R Neher B. Riniker H. Zuber W. Rittel and F. W. Kahnt ibid. p. 917; P. H. Bell W. F. Barg Jr. D. F. Colucci M. C. Davies C. Dziobnowski M. E. Englert E. Heyder R Paul and E. Snedeker,J.Amer. Chem SOC. 1968,W. 2704. Iz6 H. B. Brewer H. T. Keutmann J. T. Potts,jun. R. A. Rensfeld R. Schlueter and P. L. Munson J. Biol. Chem 1968,243,5739; J. T. Potts jun. H. D. Niall H. T. Keutmann H. B. Brewer jun. and L. J. Deftos Proc. Nat. Acad. Sci. U.S.A 1968,59 321. 127 T. E. Beesley R. E. Harmon,T. A. Jacob C. F.Hammick D. F. Veber F.J. Wolf R.Hirschmann and R. G. Denkewalter J. Amer. Chem. SOC.,1968,W 3255. 12' W. Rittel M. Brugger B. Kamber B. Riniker and P. Sieber Helu. Chim Acta 1968 51 924. 129 St Guttman J. Pless Ed Sandrin P-A Jacquenoud H. Bossert and H. Willems Helv. Chim. Acta 1968,51,1155. lJo B. Riniker R Neher R Maier F. W. Kahnt P. G. H. Byfield T. V. Gudmundson L. Galante and I. MacIntyre Helu. Chim Acta 1968 51 1738; R Neher B. Riniker R Maier P. G. H. Byfield T. V.Gudmundson and I. MacIntyre Nature 1968,220,984; R Neher B. Riniker W. Rittel and H. Zuber Helu. Chim Acta 1968 51 1900. P. Sieber M. Brugger B. Kamber B. Riniker and W. Rittel Helv. Chim Acta 1968,51 2057. A. Tenenhouse H. Rasmussen and C. D. Hawker Ann Rev. Pharmacol. 1968,8 319. Amino-acih and Peptides 525 A cyclic decapeptide antitoxin antamanide has been isolated from Amanita phalloides.If administered before or simultaneously with the toxins phalloidin or a-amanatine it counteracts their lethal action. The sequence (25) was determined by a combination of gas chromatography and mass spectrometry. (25) Pro -+ Phe - Phe - Val - Pro t 1 Pro tPhe tPhe tVal + Pro Antamanide has been synthesised from the linear decapeptide by a mixed anhydride cyclisation in 30% yield.'33 Further details of the synthesis of glucagon have appeared.2m- 134 The 0.r.d. of aqueous solutions of glucagon indicate that it is largely a random coil with perhaps one turn of an a-helix. There can be no steric objection to helix formation as it is largely helical in 2-chloroethanol.When the compound is left in acid solid possessing the antiparallel p-conformation is precipitated. 35 A second synthesis of secretin by use of a fragment condensation approach136 complements the original stepwise preparation. Peptides corresponding to two proposed sequences 42-46 of tobacco mosaic virus protein have been synthesised and the sequence Thr-Val-Val-Glu-Arg has been found to correspond to a tryptic peptide obtained from the protein. 38 The isolation structure and synthesis of the identical ovine and bovine gastrins (ValSAla'o-porcine gastrin I) has been reported. 39 The same group has prepared Leu" and Leu'.Le~'~ porcine gastrin I analogues.140 An exten- sive series of modifications of the C-terminal tetrapeptide sequence of gastrin has been detailed.I4' Bradykinin has been synthesised by a solid-phase method142 and also by an improved conventional method in which the N(o)-133 Tk Wieland G.Liiben H. Ottenheym J. Faesel J. Y. De Vries A. Prox and J. Schmid Angew. Chem Internat. Edn 1968,7 204. 134 E. Wunsch A. Zwick and A. Fontana Chem Ber. 1968,101 326; E. Wiinsch A. Zwick and E. Jaeger ibid. p. 336; E. Wunsch and G. Wendlberger ibid.,pp. 3418 and 3659; E. Wiinsch E. Jaeger and R Scharf ibid. p. 3664. 135 W. B. Gratzer C. H. Beavan H. W. E. Rattle and E M. Bradbury European J. Biochem. 1968 3 276. M. A. Ondetti V. L. Narayanan M. von Satza J. T. Sheehan F. F. Sabo and M. Bodansky J. Amer. Chem Soc. 1968,90,4711. 13' M. Bodansky M. A. Ondetti S. D. Levine and N. J.Williams J. Amer. Chem SOC.,1967 SS 6753. J. D. Young C. Y.Leung and W. A. Rombauts Biochemistry 1968,7,2475. 139 K. L. Agarwal J. Beacham P. H. Bentley R A. Gregory G. W. Kenner R C. Sheppard and H. J. Tracy Nature 1968,219 614. 140 G. W. Kenner J. J. Mendive and R C.Sheppard J. Chem SOC.(C) 1968,761. 14' K. L. Agarwal G. W. Kenner and R C. Sheppard J. Chem SOC.(C), 1968,1384; H. Gregory A. H. Laird J. S. Morley and J. M. Smith ibid. p. 522; H Gregory D. S. Jones and J. S. Morley ibid. p. 531; H. Gregory J. S.Morley J. M. Smith and M. J. Smithen ibid. p. 715; J. S. Morley and J. M. Smith ibid. p. 726; H. Gregory and J. S. Morley ibid. p. 910. 14' M. Fridkin A. Patchornik and E. Katchalski J. Amer. Chem SOC. 1968 90 2953. 526 P.M. Hardy tosyl group used for arginine protection is removed rapidly and cleanly by liquid hydrogen fluoride at despite a previous report of its stability to this reagent.144 N(o)-Nitro protecting groups can also be cleaved from bradykinin by hydrogen fluoride in the presence of anisole. Bradykinin and the hitherto unknown Val' Thr6 analogue have been found in the skin of Rana nigromaculata Hall0we11.~~~ Structure-function relationships in some partly synthetic modified ribonucleases continue to be explored. 2n* 14' Full details of the isolation and structural determination of caerulein have now appeared the synthesis of eighteen analogues is reported.20 Details of the synthesis of the sequence 1-20 of melittin are also available.'" ACTH and MSH. Monkey P-MSH (Arg6-bovine P-MSH) has been synthe- sised.The &-amino-group of the single lysine residue was protected with a formyl group which was eventually removed with hydrazine acetate. ' ' A synthetic stereoisomer of a-MSH containing four D residues has been prepared but is much less active than u-MSH.'~~ Human ACTH has now been synthe- sised,2r and a 6-aminovaleric acid analogue of the sequence 11-19 of ACTH has been reported. Three analogues of porcine corticotropin with high steroidogenic activity in uiuo have been isolated from pituitary sources. They are similar in size to ACTH but differ in amino-acid comp~sition.'~~ A new pituitary peptide H-Arg-Trp-Asp-Arg-Phe-Trp-OH has been identified but has no vasopressin- or corticotropin-releasing activity.Oxytocin and vasopressin. Isomeric dimers of oxytocin probably parallel and antiparallel dimers have been isolated as by-products in the oxidation of oxytoceine to oxytocin. They can also be prepared from oxytocin by di- sulphide interchange in the presence of triethylamine. Both compounds have low oxytocic activity. '56 Acetone inactivation of oxytocin has been found to be due to the formation of a substituted 2,2-dimethyl-imidazolidin-4-one derivative of the N-terminal Cys-Tyr sequence (26),in which the isopropylidene group from acetone forms a bridge between the N-terminal amino-group and the NH of the adjacent peptide bond. Treatment with 0.25% acetic acid at 143 R H. Mazur and G. Plume Experientia 1968,24 661. 144 S. Sakakibara Y. Shimonishi Y.Kishida M. Okada and H. Sugihara Bull. Chem SOC.Japan 1967,40,2167. 14' S. Sakakibara H. Nakamizy Y. Kishida and S. Yoshimura Bull. Chem SOC.Japan 1968,41 1477. 14' T. Nakajima Chem and Pharm Bull. (Japan) 1968,16 769. 14' R Rocchi L. Moroder F. Marchiori E. Ferranese and E. Scoffone J. Amer. Chem SOC., 1968 90,5885. 14' A. Anastasi V. Erspamer and R Endean Arch. Biochem Biophys. 1968,125 57. 149 A. Anastasi,L. Bernardi G. Bertaccini G. Bosiso R DeCastiglione,V. Erspamer 0.Goffredo and M. Impicciatore Experientia 1968,24 771. lSo E. Schroder Annalen 1968,711,227. H. Yajima Y. Okada Y. Kinomura and H. Minami J. Amer. Chem SOC., 1968,90 527. lS2 H. Yajima and K Kawasaki Chem and Phann Bull. (Japan) 1968,16 1387. lS3 W. Oelofson and C.H. Li J. Org. Chem. 1968,33 1581. 154 S. Lande M. Sribuey R K McDonald and W.Boxt Biochem Biophys Actu 1968,154,429. lS5 A. V. Schally and J. F. Barrett Biochem Biophys. Acta 1968,154 595. D. Yamashiro D. B. Hope and V. du Vigneaud J. Amer. Chem. SOC.,1968 90 3857. Amino-acids and Peptides 90" for 0-5 hr. regenerates the active hormone in good yield. '" Preparation of new analogues of oxytocin,15* vasopre~sin,'~~ and angiotensin16' are reported. Val'-angiotensin 11 containing an isosteric analogue of histidine P-(pyrazoly1)-L-alanine has high pressor and myotropic activities indicating that activity is independent of the acid-base properties of the irnidazole ring. OH Both swine kidneys and human urine contain a new angiotensinase which breaks the Pro-Phe bond of angiotensin.The resultant heptapeptide is bio- logically inactive.162 Thin-film dialysis and 0.r.d. studies have shown that a remarkable increase in the size of the angiotensin I1 molecule occurs which coincides with the ionisation of the tyrosine phenolic H-Leu-Leu-Val-Tyr-OMe and H-Leu-Leu-Val-Phe-OMe have been found to be competi-tive inhibitors of the renin-angiotensin system. Insulin. Pro-insulin the biosynthe tic precursor of insulin has been isolated from a porcine insulin preparation. The A and B chains are linked (27) with a loop of thirty-three amino-acids (28). Trypsin cleaves the bond between the A chain and the loop correctly but frees the B chain one amino-acid too far down liberating desalanine insulin. "'Cod insulin is found to differ in sequence from ox insulin at sixteen positions.The B chain is one amino-acid short at the C-terminus and has one extra residue at the N-terminus.166 Insulin 15' V. J. Hruby D. Yamashiro and V. du Vigneaud J. Amer. Chem SOC. 1968,90,7106; D. Yama-shiro and V. du Vigneaud. ibid. p. 487. H. Takashima V. du Vigneaud and R B. Merrifield J. Amer. Chem SOC. 1968 90 1323; M. Bodansky and R J. Bath Chem Comm,1968,766; M. Manning T. C. Wiu J. W. M. Baxter and W. H. Sawyer Experientiu 1968,24,660;E Klieger ibid. 1968,24,13;Zh.D. Bespalova,V. F. Martynov and M. k Titov Zhur. obshchei Khim. 1968,38 1684; 0.k Kaurov I. M. Lushchitskaya and V. F. Martynov ibid. pp. 720 and 724; H. Nesvadba K. JoSt J. Rudinger and F. Sorm Coll. Czech. Chem.Comm. 1968,33 2918; k Chimiak K Eisler K JoSt and J. Rudinger ibid. p. 2918. J. Meienhofer and Y. Sano J. Amer. Chem SOC.,1968,90,2996. 160 M. C. Khosla N. C. Chaturvedi R R Smeby and F. M. Bumpus Biochemistry 1968,7 3417. K. Hofmann R Andreatta and H.Bohm J. Amer. Chem SOC. 1968,90,6207. 16' H. Y.T.Yang E. G. Erdos and T. S. Chiang Nature 1968,218 1224. 163 M. T. Franze de Fernhdez A. E.Delius and A. C. Paladini Biochem Biophys. Actu 1968,154 223. 164 T. Kokaby E. Ueda S. Fujimoto K. Howada k Kato H. Akutsu and S. Yamamura Nature 1968,217,456. 16' R E. Chance R M. E Ellis and W. M. Bromer Science 1968,161 165. 166 K. B. M. Reid P. T. Grant and A. Youngson Biochem J. 1968 110 289. 528 P. M. Hardy containing cystathionine in place of the two cysteine residues involved in the intra-A chain disulphide bridge ('carba' insulin) has been synthesised.Hypo- glycaemic activity is of the same order as that of synthetic insulin. 167 Cyclic Peptides.-The use of mixed anhydrides for peptide cyclisation has been investigated,'68 and the method has been applied in the synthesis of antamanide (25). Two papers discuss improved dioxopiperazine syntheses. Free dipeptides or their hydrobromides cyclise in high yield when heated in phenol to just below its b.p.;16' dipeptide ester formate salts when boiled in neutral solvents such as toluene also give good yields of sterically pure dioxopiperazines.''O Cyclisation of dipeptide methyl esters by treatment with ammonia in methanol on the other hand has been shown to cause 5-40 % racemisation.''O In connection with studies on the antibiotic albo- mycin a series of cyclic hexapeptides containing only ornithine and serine residues have been prepared and their complexes with iron have been studied.The yields on cyclisation were found to be very dependent on the configuration of the amino-acids present. Cyclisations were carried out by the aide method. ' ' Studies on the transannular interactions of 1-(hydroxyalkanoyl)piperazine-2,5-diones172 and medium sized cyclodepsipeptides2q are reported. Trans- annular amide-amide interactions have been examined by mass spectro- metry. '73 Aromatic o-mercaptoacyl-lactams such as N-(0-mercaptobenzoy1)- 6-valerolactam have been found to form the thiacyclol or the cyclodepsipeptide (29) spontaneously.'74 The L-and D-forms of P-(0-t-butylsery1oxy)propionic acid cyclodimerise in a straightforward way on cyclisation but the DL-compound yields both the meso form and a racemic mixture of the D,D-and LA-forms. The rneso-form has been synthesised unambiguously. '' 16' K. JoSf J. Rudinger H. Klostermeyer and I€ Zahn 2.Naturjbrsch. 1968,23b 1059. Th. Wieland F. Jiirgen and H. Faulstich Annalen 1968,713,201. 16' K. D.Kopple and H. C. Ghorazian J. Org. Chem. 1968,33,862 D. E Nitecki B. Halpern and J. W. Westley J. Org. Chem. 1968,33,864. N. A.Poddubnaya and A. M. El-Haggar,Zhur. obshchei Khim. 1968,38,450;N. A. Poddubnaya A. M. El-Haggar I. N. Skvortsova and G. L Balandini ibid. p. 732 V. K. Antonov A. M Schkrob and M M.Shemyakin,Zhur. obshchei Khim. 1968,38,2225. 173 V. V. Denisov V. A. Ruchkov N. S. Vulfson T. E. Agadzhanyan V. K. Antonov and M. M. Shemyakin Zhur. obshchei Khim. 1968,38,770. 174 M. Rothe and R Steinberger Angew. Chem Internat. Edn 1968,7,884. C. H. Hassall and J. 0.Thomzs J. Chem SOC.(0,1968 1495. Ammo-acids and Peptides 529 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 (28) Leu-Gly-G1 y-Leu-Gln -Ala-Leu-Ala-Leu-Glu-Gl y-Pro-Pro-Gln-Lys-Arg 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Two groups of workers have independently concluded that the structure of the peptide alkaloid ceanothine B should be revised to (30).'76i177 Complete hydrolysis of dihydroceanothine B yields tyramine,177 and not o-aminoethyl- phenol as would be expected from the structure originally proposed.'78 Mass spectral evidence also points to (30).176Elucidation of the structure of viomycin first isolated in 1958 has proved difficult.Acid hydrolysis yields the amino- acids L-serine L-aP-diaminopropionic acid L-P-lysine and a new basic amino-acid viomycidine (ratio 2 :1 :1 :l) together with carbon dioxide ammonia urea and traces of glycine. The structure (31) is now proposed for viomycin although it is possible that the p-lysyl and one of the seryl units may be reversed in position. Viomycidine appears to be an artefact derived from the guanidine unit;'79 its structure (32) has been confirmed by mass spectrometry. The dehydroamino-acid unit proposed which breaks down on acid hydrolysis agrees with the spectral properties of viomycin and other degradative evidence.179 0 0 II 176 F. M. Klein and H. Rapaport J. Amer. Chem Soc. 1968,90 3576. 177 R E. Semis and A I. Kosak J. Amer. Chem Soc. 1968,90,4179. 17' E. W. Warnhoe S. K Pradham and J. C. N.Ma Canad. J. Chem. 1968,43,2594. B. W. Bycroft D. Cameron L. R Croft A Hassanali-Walji k W. Johnson,and T. Webb Tetrahedron Letters 1968. 5901 ;see also T. Kitagawa Y. Sawada and T. Miura ibid. p. 109. J. C. Floyd J. A. Bertrand and L R Dyer Chem Comm.,1968,998. 530 P.M. Hardy Gramicidin S18' and Gly5G1y1O-gramicidin S18* have been synthesised by a solid-phase method and retrogramicidin S' and all-L-gramicidin S'84 by conventional methods. The latter was cyclised with o-phenylene chlorophosphite and cyclopenta- and cyclodeca-peptides were obtained in a 2:3 ratio.The conformation adopted by gramicidin S in solution continues to attract attention. A preliminary n.m.r. study has been interpreted to support a compact model previously proposed on 0.r.d. evidence,' but more detailed work'86 is thought to show a conformation fairly similar to that adopted in the crystal,' 87 i.e. an all-trans-amide antiparallel pleated sheet The phthalimide group is a promising marker for signalling the proximity of the phenyl side- chains of phenylalanine residues by n.m.r. Absorption occurs in a 'window' region at ca. 8 p.p.m.188 The proximity of the D-phenylalanine residues in diphthaloyl gramicidin S as shown by this method agrees with the results of the detailed n.m.r.study reported above. 18' H The structure of telomycin first isolated in 1958 has now been elucidated (33). This antibiotic contains P-methyltryptophan cis-and trans-3-hydroxy- prolines and erythro-0-hydroxyleucine. A dehydrotryptophan (A-Trp) is postulated in order to account for the spectral properties. About half a residue of tryptophan is formed on alkaline hydrolysis. The threonine residue joining the peptide side chain to the ring is linked to cis-3-hydroxyproline through its hydroxy-group as a lactone.lgO A re-examination of the structure of circulin H. Klostermeyer Chem Ber. 1968,101 2823. lE2 J. Halstrom and H. Klostermeyer Annulen 1968,715 208. lE3 M.Waki and N. Izumiya Tetrahedron Letters 1968 3083; Bull. Chem SOC.Japan 1968 41 1909.lE4 M. Rothe and F. Eisenbeiss Angew. Chem Internut. Edn 1968,7 883. A. M. Liquori and F. Conti Nature 1968,217,635. lB6 A. Stern W. A. Gibbons and L. C. Craig Proc. Nut. Acad. Sci. U.S.A. 1968,61 134. IE7 D. C. Hodgkin and B. M. Oughton Biochem J. 1957 65 752; ct also R Schwyzer CIBA Foundation Symposium on Amino-acids and Peptida with Antimetabolic Acitivity 1958 p. 171. IE8 R Schwyzer and U. Ludescher Biochemistry 1968,7,2514. lE9 R Schwyzer and U. Ludescher Biochemistry 1968,7 2519. 190 J. C. Sheehan D. Mania S. Nakamura J. A Stock and K. Maeda J. Amer. Chem Soc. 1968 90.462 Amino-acids and Peptides 531 B shows it to be a cyclic heptapeptide and not a cyclic decapeptide as previously suggested. It now differs from circulin A only in having an N-terminal iso- octanoyl group in place of a (+)-6-methyloctanoyl residue.lgl all-^-and all-D-enniatin B stereoisomers have been synthesised together with enantio-enniatin B.lg2Although it has not yet been obtained homogeneous a tentative proposal of the structure of the antifungal antibiotic stendomycin has been put forward (34).It contains seven or eight D-residues and a new ‘cyclic arginine’ amino-acid (35).’‘9 lg3 Further extensive work on the synthesis of the actino- mycin series has been published.194 The fact that in peptide antibiotics all the isoleucine and all the threonine residues belong to the L-series while all the do-isomers of these belong to the D-series is thought to support the view that all the D-amino-acids of microbial peptides originate from L-amino- acids.lg5 (33) cis-3-Hypd-Trp-P-rvle-Trp-erythro-3-Hyleu / / / / Ser-Thr-Thr-Ala-Gl y-trans-3-Hyp H-AspOH (34)Fatty acid-Pr~N-Me-Thr-Gly-Val-ullo-Ile-d-But-allo-Thr-Val-Va1 ” t do-Ile-Ser-do-Thr B= ,CH2\ IHN ICH Me-N=C CH- CH-CO2H I Me NH (39 Important advances have been made this year in the successive selective linkage of cysteine residues via disulphide bridges.An insulin model has been synthesised by making three disulphide linkages in a stepwise and specific fashion by use of the sulphenyl thiocyanate coupling method. This method does not involve an intermediate thiol and avoids the possibility of thiol- disulphide interchange. Peptide (36) was prepared from the corresponding t-butyl ester by use of boron trifluoride in acetic acid to effect cleavage and 19’ K.Hayashi Y. Suketa and T.Suzuki Experientia 1968,24,657. 19’ G. Losse and R Hartmut Chem. Ber. 1968,101 1532 193 I. Muramatsu and M. Bodansky J. Antibiotics (Japan) 1968 21 68. 194 H. Brockmann and H. Lackner Chem Ber. 1968,101 1321 and 2231; H. Brockmann and P. Boldt ibid. p. 1940; H. Brockmann and F. Seela Tetrahedron Letters 1968 161 ; J. Meienhofer Experientia 1968,24 776. 19’ M. Bodansky and D. Perlman Nature 1968,218 291. 532 P.M. Hardy the resulting peptide acid was coupled with a tripeptide to yield (37). The S-trityl group of this model A chain (37) reacted with the sulphenyl thiocyanate of a model B chain to eve the 9-12 disulphide bridge (38).Treatment of (38) with trifluoroacetic acid in the presence of thiocyanogen removed the benzhy- dry1 groups and formed the final 2-16 third disulphide link. Partial cleavage of the t-butyl ester also occurred and fission was completed by treatment with f Z-Cys-Cys-Gly-Phe-Gly-ys-Phe-Gly-OH + H-Cys-Gly-Val-OBu' 1 1234 5 6 1 I SBzh (74 % yield) STr dicyclo hexylcarbodi-imide (37) cs I I Z-Cys-Cys-Gly-P he-Gly-C ys-Phe-Gly-Cys-Gly-Val-OBu' model A chain 1 (23 4 5 6 7 8 191011 SBzh STr ZISCN r (76 % yield) ys-Gly-Gly-Gly-ys-Gly-OBu' model B chain 12 13 14 15 16 17 I I HO-Gly-~y~-Gly-Gly-Gly-by~-Z model B chain 17 16 15 14 13 12 Amino-acids and Peptides boron trifluoride in acetic acid to give the insulin model (39).During this series of reactions no evidence of disulphide interchange was detected.2s* The B chain of insulin has been synthesised efficiently without using S-protec- tion by using polydisulphide intermediates and finally converting into the bis-S-sulphonate.2' Protected peptides containing S-trityl cysteine residues can also be converted directly into cystine peptides by treatment with iodine in methanol. This method has been used to prepare the 1-7 disulphide link in the synthesis of human calcitonin. lg7 Peptide Conformations.-0.r.d. studies on all the diastereoisomers of tri-and tetra-leucines show that with respect to the peptide chain and its immediate substituents the conformations adopted in solution closely resemble those of the corresponding alanine stereoisomers.* Theoretical calculations of the mean square dipole moments of polypeptide chains when applied to fourteen diastereoisomeric oligomers of ala~~ine'~~ show good agreement with the values previously obtained experimentally.200 'H N.m.r. spectra of oligo- glycines indicate that in aqueous solution they are not constrained to a small number of preferred conformations,20' while similar studies on DL and LL-phenylalanylvalines suggest that their conformations are determined primarily by nonbonded interactions and secondarily by some weighting of those conformations that bring the opposite charges of the dipolar form somewhat nearer.2o2 The i.r. spectra of the pentamer hexamer and octamer of t-pentyl- oxycarbonyl-L-proline are similar to those of polyproline-11 and it is concluded that these oligomers possess a left-handed three-fold screw axis helical struc- t~re.~'~ An n.m.r.study of cyclo(G1y-Phe) shows that in trifluoroacetic acid the phenyl group is positioned above the dioxopiperazine ring which is itself buckled into a boat form so that the phenyl group occupies a 'flagpole' type orientation.2" Similar conformations have been deduced for cyclo(G1y- Trp)2" and a series of dioxopiperazines containing one tyrosine residue.204 Experiments on N-methylated dioxopiperazines which are more soluble in less polar solvents show that this attractive interaction between an aromatic residue and the amide bonds persists in solvents of lower dielectric constant.'" Copolymers of P-benzyl and P-ethyl-L-aspartate prepared by the partial transesterification of poly( P-L-aspartate) have been found to undergo a transition from a right-handed to a left-handed a-helix when the temperature of a solution in chloroform is raised.Poly-( P-n-propyl-L-aspartate) undergoes a similar transition at 59°.205It is well known that poly(cr-amino-acids) con- ''' R G. Hiskey and R L. Smith J. Amer. Chem SOC. 1968,90,2677. B. Kamber and W. Rittel Helu. Chim Acta 1968 51 2061. D. R Dunstan and P. M. Scopes J. Chem SOC.(C) 1968 1585. P. J. Flory and P. R Schimmel J. Amer. Chem SOC.,1967,89,6807. 'O0 J. Beacham V. T. Ivanov G. W. Kenner and R C. Sheppard Chem Comm. 1965 386. A. Nakamura and 0.Jardetzky Biochemistry 1968,7 1226.'02 V. J. Morlino and R B. Martin J. Phys. Chem. 1968 72 2661. '03 H. Okabayashi T. Isemura and S. Sakakibara Biopolymers 1968,6 307 323. '04 K. D. Kopple and D. H Marr J. Amer. Chem SOC. 1967,89 6193. '05 E. M. Bradbury B. G. Carpenter and H. Goldman Biopolymers 1968,6 837. 534 P.M. Hardy taining branches at the P-carbon atom or oxygen or sulphur atoms in the side- chain at the y-position formP-structures preferentially. Theoretical calculations however now suggest that po1y-L-serine2O6 and poly-~-valine~~~ could exist in the left- and right-handed helical conformations respectively. To test this theory poly-L-valine has been prepared as a block copolymer using poly- D,L-lysine to increase its solubility. 0.r.d. and c.d. evidence showed that about half of the fifteen-residue valine block was in the right-handed a-helical conformation in 98 % aqueous methanol but in water only the intermolecular- p-structure was formed.208 The collagen model poly(L-Pro-L-Pro-Gly) has been synthesised by a solid-phase method with addition of successive tripep- tide units.Material containing twenty units was practically monodisperse and the temperature dependence of the optical rotation was closer to that of natural collagen than that of previously prepared polydisperse material. 209 lo6 K. P. Sarathy and G. N. Ramachandran Biopolymers 1968,6 461. '07 T. Ooi R A. Scott G. Vanderkooi and H. A. Scheraga J. Chem Phys. 1967,46 410. R F. Epand and H. A. Scheraga Biopolymers 1968.6 1551. '09 S. Sakakibara Y.Kishida. Y. Kikuchi R. Sakai and K. Kakiuchi Bull. Chem. SOC. Japan 1968,41 1273.

ISSN:0069-3030    

DOI:10.1039/OC9686500509

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

16 NUCLEIC ACIDS Part (i) By G. Michael Blackburn (Department of Chemistry University of Sheffield Sheffield S3 7HF) “THATwas Molecular Biology that was.” Gunther Stent’s essay’ provides the backcloth against which two of the year’s highlights can be seen in perspec- tive. The Nobel Prize awarded2 to Holley Khorana and Nirenberg signifies again the experimental success of the ‘Academic Phase’ of nucleic acid studies. Watson’s ‘The Double Helix’3 records the termination of the ‘Romantic Phase’ both with the solution of the structure of DNA4 and by the exposition of the Central Dogma; it also implies in diverse ways that there can be occupa- tions more profitable than bench experimentation. The book3 has provoked reviews,’ counter reviews,6 and reviews of reviews,’ none of which appears to have noted that its dust jacket depicts a left-handed golden helix! The year’s achievements have included Khorana’s synthesis of a DNA duplex of 30 nucleotide residues the increase to eleven of the number of known t-RNA sequences and the crystallisation of both homogeneous and hetero- geneous t-RNA’s.Topics reviewed elsewhere include RNA from animal cells,8 sense and non- sense in the Gmetic Code,g and the molecular biology of viruses.” In uiuo codon assignments have been summarised” and the physical chemistry of polynucleotides has been the subject of a group of papers. l2This and doubtless other literature searches has been assisted by SCAN a computer-controlled search profile for nucleic acids published by the Chemical Society.Prebiotic Chemistry.-Many components of the genetic system can be made from simple precursors under mild conditions. Among the amino-acids glycine alanine serine and aspartic acid are most easily produced and ribose is formed G. S. Stent Science 1968 160 390. ’ Chum iii Britain 1968,4 557; Cheni. adEng. Ne1i.q 1968 46,NO. 46 66. J. D. Watson ‘The Double Helix,’ Weidenfeld and Nicholson London 1968. Abbreviations DNA deoxyribonucleic acid; RNA ribonucleic acid; t-RNA transfer (soluble) RNA; m-RNA messenger RNA; r-RNA ribosomal RNA; 5s-RNA RNA with sedimenta- tion coefficient of 5s; A adenosine; C cytidine; G guanosine; H hypoxanthine; I inosine; Y pseudouridine; T thymidine; rT ribothymidine; U uridine; U* uridine hydrate; dA deoxy- adenosine etc.; AMP CMP PA pC etc.5‘-phosphates; Ap Cp etc. 3‘-phosphates; poly-A polyadenylic acid ;d-pTpC etc. deoxyoligonucleotides. ’ Lord Todd,Chem. in Britain 1968 4 268; J. Hollander Nature 1968 218 791. A. Klug Nature 1968 219 808 880; L. D. Hamilton Nature 1968 218 633. J. Donohue Chem. in Britain 1968 4 468. J. E. Darnell Buct. Rev. 1968 32 262. A. Garen Science 1968 160 149. lo L. V. Crawford and M. P. G. Stoker (ed.) ‘The Molecular Biology of Viruses,’ C.U.P. London 1968; Symp. SOC.Gen. Microbiol 1968 18 125. H. Berger W. J. Brammar and C. Yanofsky J. Mol. Biol. 1968 34 219. l2 J. chim. Phvs.. 1968 65 No. 1. G.Michael Blackburn more readily than deoxyribose. Adenine which can be made from ammonium cyanide in liquid ammonia,14 is more reasonably formed in aqueous solutions of 4-aminoimidazole-5-carbonitrileand cyanide under conditions which could produce significant quantities of adenine and 4-aminoimidazole-5- carboxamide in a few years at -20 to 0".' Pyrimidines are less readily con- jured out of a 'primordial soup,' but a restricted reasonable route16 has been demonstrated from cyanoacetylene (1) via cyanovinylurea (2) to cytosine (3) and thence by hydrolysis to uracil (4).2HCNO HO HC =C-CN---OCN-C-NH.CH=CH.CNAH,N.C.NH.CH=CH-CN I1 II (1) 0 0 (2) (4) (3) There is still no convincing nucleoside synthesis under prebiotic conditions" but skirting this hurdle experiments on the phosphorylation of nucleosides have produced good yields of IMP from isopropylideneinosine and tri-n- butylammonium phosphate in non-aqueous solvent by the action of U.V.radiation. '* In dilute aqueous solutions the stable enol phosphate formed by addition of phosphate to cyanoacetylene slowly converts uridine into UMP.' Solid-state phosphorylation isanother prebiotic possibility :uridine and uridine- 2'(3')-phosphate at 160" yield a mixture of products including a significant amount of UpUpU 35 % of which has natural (3' + 5') linkages2' Greater preference is observed in UpUpU than in UpU formed for the (3'-,5') linkage ; this may be a clue to its evolution. Given poly-U as a template (another step in the dark) it is possible to achieve nucleotide condensations in aqueous solution by use of water-soluble carbodi- imides2 or phosphorimidazolides22 as condensing reagents.Three features l3 C. Ponnamperuma and N. W. Gabel Space Life Sciences 1968,1 65. l4 Y. Yamada I. Kumashiro andT. Takenishi J. Org. Chem. 1968,33,642. l5 R. A. Sanchez J. P. Ferris and L. E. Orgel J. Mol. Biol. 1968,38 121. l6 J. P. Ferris R.A. Sanchez and L. E. Orgel J. Mol. Biol. 1968,33 693. '' C. Reid L. E. Orgel and C. Ponnamperuma Nature 1968,216,936. '* Y. Sanno and A. Nohara Chem. and Pharm. Bull. (Japan) 1968,16,2056. l9 J. P. Ferris Science 1968 161 53. 2o J. MorBvek J. Kopeck$ and J. Skoda CON.Czech. Chem. Comm. 1968,33,960. 21 J. Sulston R.Lohrmann L. E. Orgel and H. T. Miles Proc. Nar. Acad. Sci. U.S.A. 1968 60 409. 22 B. J. Weimann R.Lohrmann L. E. Orgel H. Schneider-Bernloehr and J. E. Sulston Science 1968 161 387.Nucleic Acids 537 are noteworthy first the process is catalytic23 and enhances the rate of phosphodiester formation by an order of magnitude; second the system is species selective-poly-U facilitates only the homocondensation of adenosine units but not heterocondensation with C G or U; and third the phosphate linkage formed between A and pA is 96% (2‘ - 5‘) and that between d-pA and dA is mainly (5’ - 5’).24 Why did evolution select for (3‘ - 5’) phosphate esters?25 This and other speculative matters are expertly and lengthily dis- cussed by Crick,26 Orge1,27 and Woese.28*29 The clear conclusion is that selection among tenable but conflicting hypotheses cannot proceed without more facts. The whole problem of translation of information into control involves the interaction between nucleic acid bits and amino-acids three aspects of which are under scrutiny.Ralph3’ argues that interaction of amino-acid and its anticodon should involve the recognition of the aminoacyladenylate by the appropriate t-RNA-hence an explanation for the invariant location of U next to the anticodon (pp. 554-559). Next the binding of nucleotides to poly- peptides shows3’ that poly-L-arginine binds pG more effectively than PA pC or pU. Lastly Gabba~~~ finds that double helices of poly-I poly-C are thermally stabilised by interaction with lysyl dipeptides or aminoacyldiamines. Dilysine is ten times more effective than lysine and L-amino-acids are superior to their D-enantiomers. Alongside this chiral specificity should be noted the partial resolution of DL-adenosine by template-controlled condensation with D-adenosine-5’-phosphorimidazole.33 These two developments appear to simplify the problem of chiral evolution in the biosphere.Chemistry of Bases.-Transfer RNA continues to be a good source of new minor bases. 2-Thiocytosine (5) and 5-methylaminomethyl-2-thiouracil(6) have been isolated34 as their 2’(3’)-nucleotides from t-RNAE. coli 5-carboxymethyl-(7) and 5(6)-carbomethoxymethyl-2-thiouraci136(8) nucleotides have been identified as products from t-RWAYeast and 2’-0N(4)-dimethylcytidine 23 J. Sulston R. Lohrmann L. E. Orgel and H. T. Miles Proc. Nut. Acad. Sci. U.S.A. 1968,59 726. 24 H. Schneider-Bernloehr R. Lohrmann J. Sulston B.J. Weimann L.E. Orgel and H. Todd Miles J. Mol. Biol. 1968 37 151. 25 Anon. Nature 1968 218 523. 26 F. H. C. Crick J. Mol. Biol. 1968,38 367. ” L. E. Orgel J. Mol. Biol. 1968 38 381. 28 C. R. Woese Proc. Nut. Acad. Sci. U.S.A. 1968 59 110. ” See also M. Nei Nature 1969 221,40; J. E. Edstrom ibid. 1968 220 1196; D. C. Reanney and R. K. Ralph J. Theoretical Biol. 1968 21,217. 30 R. K. Ralph Biochem. Biophys. Res. Comm. 1968,33,213. 31 K. G. Wagner and R. Arav Biochemistry 1968 7 1771. 32 E. Gabbay and R. Kleinman J. Amer. Chem. SOC.,1968,90,7123; E. J. Gabbay R. Kleinman and R. R. Shimshak ibid. p. 1927. 33 H. Schneider-Bernioehr R. Lohrmann L. E. Orgel J. Sulston and B. J. Weimann Science 1968,162,809. 34 J. Carbon H. David and M. H.Studier Science 1968,161 1146. 35 M. W. Gray and B. G. Lane Biochemistry 1968 7 3441. 36 L. Baczynskyj K. Biemann and R. H. Hall Science. 1968. 159. 1481. G. Michael Blackburn is found in 16s-RNA from E. ~oli.~’ N(6)-isopentenyl-2-The cyt~kinin,~~ methylthioadenosine (9) isolated from crude t-RNA and ~ynthesised,~~ originates in t-RNAz:o,i.40 Cultures of L. aciduphilus but not of yeast incor- porate [2-14C]mevaIonic acid into N(6)-isopentenyladenosine (lo) which is a minor base in several t-RNA species (pp. 554-559).41 Sfj H (9) R = MeS (7) (8 (10) R = H r = P-D-ribofuranosyl 4-Thiouracil residues in t-RNA are selectively modified by eth~leneimine~~ and by N-eth~lmaleimide,~~ and the reduction of 4-thiouridine dihydrouridine and N(4)-acetylcytidine with sodium r3H1borohydride gives characteristic products from each; this permits estimation of these bases in t-RNA.44 Such reduction of t-RNA,,,,, without further degradation produces changes in biological activity.45 In contrast to the random methylation of bases by dimethyl s~lphate,~~ a purified enzyme catalyses methylation of certain base sequences in methyl- 37 J.L. Nichols and B. G. Lane Biochim. Biophys. Acta 1968 166 605. 38 J. P. Hegelson Science 1968 161 974. 39 W. J. Burrows D. J. Armstrong F. Skoog S. M. Hecht J. T. A. Boyle N. J. Leonard and J. Occolowitz Science 1968 161 691. 40 F. Harada H. J. Gross F. Kimura S. H. Chang S. Nishimura and U. L. RajBhandary Biochem. Biophys. Res. Comm. 1968,33 299. 41 A.Peterkofsky Biochemistry 1968 7 472; F. Fittler L. K. Kline and R. H. Hall ibid. p. 940. 42 B. R. Reid Biochem. Biophys. Res. Comm. 1968,33,627. 43 J. Carbon and H. David Biochemistry 1968 7 3851. 44 P.Cerutti J. W. Holt and N. Miller J. Mol. Biol. 1968 34 505. 45 P. Cerutti Biochem. Biophys. Res. Comm. 1968 30,434; M. Molinaro L. B. Sheiner F. A. Neelon and G. L. Cantoni J. Biol.Chem. 1968 243 1277. 46 R.A. Zakharyan T. V. Venkstern and A. A. Bayev Biokhimiya 1967 32 1068. Nucleic Acids 539 deficient t-RNA and nucleotides Ap2MeGPCp Gp1MeApApUp and Apl MeApApUp have been ~haracterised.~~ Much effort in cancer research is currently focussed on the alkylation of DNA and RNA both in uitro and in uiuo. Carcinogens which effect such alkyla- tion include N-nitro~odimethylamine,~~’*~ N-methyl-N-rnethyl~rethane,~~ N-nitroso-toluene-p-sulphonamide,so N-methyl-N-nitroso-N’-nitroguani- dine,49-ethyleneiminium nitrogen mustards,53 sulphur mustards methyl methanesulphonate and dimethyl s~lphate,~~‘? and N-hydroxyfluoren- 2-ylacetamide.’’Although the carcinogenic polycyclic aromatic hydrocarbons are not recognised alkylating agents they bind to DNA after incubation and treatment with hydrogen peroxide.56 Similar binding has been observed to result under the influence of U.V. irradiati~n.~~ Since it is known that the K-region epoxides have little carcinogenic power,58 the possibility that aromatic peroxides are the intermediates in DNA alkylation by these hydrocarbons must be considered.With the exception of N-hydroxyfluoren-2-y1acetamide,’ all alkylations of hydrogen-bonded base pairs give predominantly 7-alkylguanosinet Bi-functional sulphur mustards produce bis-2-(7-guanyl)ethyl sulphide (1 1). 54a Lesser amounts of 1- and 3-alkyladenosines and 1-methylcytidine are found.48b Organ specificity is exhibited by certain reagents ;dimethyl sulphate acts mainly on brain cells and dimethylnitrosamine can alkylate up to 1% of G residues in rat liver RNA but is ten times less active on kidney nucleic Some of these results are consistent with the hypothesis that there is a casual relationship between alkylation of DNA and carcin~genesis,~~~ but firm con- clusions are as yet premature. However in one case at least there is good evi- dence for a direct relati~nship.~’ After treatment of the skin of a mouse with P-propiolactone the initiation of tumourigenesis correlates well with covalent binding of the p-lactone to skin DNA.Isolated DNA yields 7-(2-carboxyethyl)- guanine (12) on hydrolysis. Two pyrimidine ring substitution reactions have been investigated. Uracil 47 B. C. Baguley and M. Staehlin Biochemistry 1968 7. 45. 48 (a)P. D. Lawley P. Brooks P. N. Magee V. M. Craddock and P. F. Swann Biochim. Biophys. Actu 1968 157 648; (b)W. Lijinsky J. Loo,and A. E. Ross Nature 1968 218 1174. 49 P. D. Lawley Nature 1968 218 580. R. R. McCalla Biochim. Biophys. Actu 1968 155 114. P. Chandra A. Wacker R. Sussmuth and F. Lingens 2.Nuturforsch 1967 22b 512; V. M. Craddock Biochem. J. 1968 106,921 ;F.Lingens J. Rau and R. Sussmuth Z. Nuturforsch. 1968 23b 1565; B. Singer H. Frankel-Conrat J. Greenberg and A. M. Michelson Science 1968 160 1235. 52 C. C. Price G. M. Gaucher P. Koneru R. Shibakawa J. R. Sowa and M. Yamaguchi Biochim. Biophys. Acta 1968 166 327. s3 K. W. Kohn and C. L. Spears Biochim. Biophys. Actu 1967 145 734. 54 (a)P. D. Lawley and P. Brookes Biochem. J. 1968,109,433;(b)P. F. Swann and P. N. Magee ibid. 110 39. 55 C. M. King and B. Phillips Science 1968 159 1351. 56 C. E. Morreal T. L. Dao K. Eskins C. L. King and J. Dienstag Biochim. Biophys. Actu 1968,169,224. ” P. 0.P. Ts’o and P. Lu Proc. Nut. Acad. Sci. U.S.A. 1964 51,272. J. A. Miller and E. C. Miller Lab. Invest. 1966 15 226. 59 N. H. Colburn and R. K. Boutwell Cancer Res.1968 28 642. G.Michael Blackburn II?-.X JL ““Y2 Q (13) (14) and uridine rapidly exchange hydrogen at C-5 in acid6’ and in alkaline6‘ solu-tion. An examination of the mechanism for 2-hydroxyprimidine and its deri- vatives suggests that hydrogen exchange is consequent upon hydration of the 5,6-double bond.62 This is supported by hydrogen exchange in the photo- hydrate of UMP6 and of cytidine. 64 0(6),5’-Cyclonucleosidesare produced from 5-halogenopyrimidine nucleosides with strong base.65 Thus 5-iodouri- dine (13) readily obtained by iodination of uridine,66 is converted into 1-[0(6),5‘-cyclo]-p-D-ribofuranosylbarbituric acid (14) with potassium t-but- oxide.67 Both addition4imination and heteryne mechanisms have been con- sidered but the observed68 base-catalysed exchange of C-6 hydrogen in 5-fluoropyrimidine nucleosides seems best interpreted in favour of the latter mechanism.Nucleosides and Nucleotides-Much synthetic work continues to be directed at the modification of nucleosides in either the base or the sugar fragment. The 2’-C-methyL6’ and 3’-C-methyl-ribo~ides~~ and 3’-deoxy-3’- C-hydroxymethyl-erythro-furanoside”of adenine have been prepared and 5’-homothymidine has been made by extension of the sugar.72 An elegant syn- thesis of the carbon isostere of UMP 6’-deoxyhomouridine-6’-phosphonic “ K. Kusama J. Biochem. (Japan) 1968 63 561. S. R. Heller Biochem. Biophys. Res. Comm. 1968 32 998. A. R. Katritzky M. Kingsland and0. S. Tee J. Chem. SOC.(B),1968 1484.63 R. W. Chambers J. Amer. Chem. SOC.,1968,90,2192. 64 L. Grossman Photochem. and Photobiol. 1968 7,727. 65 B. A. Otter E. A. Falco and J. J. Fox Tetrahedron Letters 1968,2961. 66 H. Yoshida J. Duval and J.-P. Ebel Biochim. Biophys. Acta 1968,161 13. 67 D. Lipkin C. Cori and M. Sano Tetrahedron Letters 1968 5993. R. J. Cushley S. R. Lipsky and J. J. Fox Tetrahedron Letters 1968 5393. 69 S. R. Jenkins B. Arkon and E. Walton J. Org. Chem. 1968,33,2490. 70 R. F. Nutt M. J. Dickinson F. W. Holly and E. Walton J. Org. Chem. 1968,33 1789. 71 E. J. Reist D. F. Calkins and L. Goodman J. Amer. Chem. SOC.,1968,90,3852. ’I2 G. Etzold G. Kowollik and P. Langen Chem. Comm. 1968,422. Nucleic Acids 541 acid (16) has been accomplished in four stages from 5’-aldehydo-2,3’-0-iso- propylideneuridine (15).The AMP analogue can also be made by this route.73 0 Reagents:i Ph,P=CH*PO(OPh) ;ii Pd-BaS0,-H ;iii PhCH,.ONs; iv Pd-H,-H+ The three nucleotide antibiotics toyokamycin (17) sangivamycin (18) and tubercidin (19) have been ~ynthesised~~ and new pyrimidine nucleoside anti- biotics have been identified. Polyoxin A and B,75active components of agri-cultural antifungal agents have the unique structures (20a and 20b). Gougero- tin has the revised structure (21).76 \ (17) R = CN (18) R = CONH (19) R = H r = P-D-ribofuranosyl 0 CH,OH L &H OH (20b) R = €€ 73 G. H. Jones and J. G. Moffatt J. Amer. Chem. Soc. 1968 90 5337. 74 R. L. Tolman R. K. Robins and L.B. Townsend J. Amer. Chem. SOC.,1968,90 524. 75 K. Isono and S. Suzuki Tetrahedron Letters 1968 1133. 76 J. J. Fox,Y. Kuwada and K. A. Watanabe Tetrahedron Letters 1968 6029. 542 G. Michael Blackburn A synthesis of pseudouridine (22) and of 5-p-D-ribofuranosyluridine(23) has been described,77 though the latter product does not appear to be identical with the naturally occurring material.78 A related synthesis of 6-azapseudouridine failed when the ribityl fragment in (24) cyclised in an unexpected fashion given (25).79 A novel preparation of purine nucleosides provides a-anomers virtually free of the p-isomers :N(6)-octanoyladenine is heated with the boron trichloride complex of methyl ribofuranoside in chloroform. a-Adenosine is obtained after deacylation with sodium methoxide.80 (22) R = H (25) (23) R = P-D-ribofuranosyl The readily available nucleoside thiophosphates promise to be good probes for many enzyme reactions.Thus adenosine-5‘-thionophosphateis a poor substrate for enzymic deamination or phosphorolysis but as an activator for phosphorylase b it is more effective than AMP.81 The introduction of one thio- group into the valine codon GpUpU markedly lowers its capacity to bind t-RNAVa’ to ribosimes.82 Uridine-2’,3’-cyclothiophosphate(26) is hydrolysed in alkali to uridine-2’(3’)-thiophosphate,whereas acid hydrolysis results in significant loss of sulphur.83 The diastereoisomers of (26) can be separated by crystallisation one binds to ribonuclease as strongly as does uridine cyclic phosphate but both are hydrolysed by the enzyme more slowly than the natural substrate.The complete retention of sulphur in the product has strong implica- tions for the stereochemistry at phosphorus in the mechanism of action of ribon~clease.~~ Oligonucleotides.-Well established methods of synthesis leave some room for improvement. A third oligonucleotide synthesis using phosphotriesters has been reported phenyl phosphate triesters can be hydrolysed by alkali but are stable under milder conditions appropriate for the removal of 0-acetyl groups.85 One of the abiding problems in ribonucleotide synthesis is the pro- pensity of (3’ + 5‘) phosphate diesters to isomerise into (2’ + 5’) linked nucleo- 77 D. M. Brown M. G. Burdon and R.P. Slatcher J. Chem. SOC.(C) 1968 1051. A. W. Lis and E. W. Lis Fed. Proc. 1964 23 532. ’’ M. Bobek J. FarkaS and F. Sorm,Tetrahedron Letters 1968 1543. Y. Furukawa K. Imai and M. Honjo Tetrahedron Letters 1968 4655. A. W. Murray and M. R. Atkinson Biochemistry 1968 7 4023. V. LisL F. Eckstein and J. Skoda CON. Czech. Chem. Comm. 1968,33,2734. 83 F. Eckstein and H. Gindl Chem. Ber. 1968 101 1670. 84 F. Eckstein F.E.B.S. Letters 1968 2 85. 85 C. B. Reese and R. Saffhill Chem. Comm. 1968,767. Nucleic Acids 543 tides. This can be avoided successfully by careful selection of the conditions for removal of protecting groups. 86 Terminal phosphomonester functions also require special consideration 3’-phosphates are best generated from their benzyl esters87 and 5’-phosphates can be produced by selective cleavage of trichloroethyl esters88 or through removal of a terminal nucleoside protecting group by means of periodate oxidation and p-elimination.89 Nonetheless it is obvious that oligoribosides will continue to be made best by enzymic transcription of synthetic DNA. Khorana’s progress towards gene synthesis already appears irresistible. The methods of synthesis described in last year’s report” have led to the synthesis9 of the eicosadeoxynucleotide (27) by the block condensation of an appropriately protected oligonucleotide with a free 3’-hydroxy-group and 5’-phosphates of tri- and tetra-nucleotides as shown in the Scheme. Khorana considers this to be the maximum chain length that current methods of chemical synthesis and purification allow for deoxynu~leotides.~~ Similar methods are MMTrGpApA + pCpC -MMTrGpApApCpC PGPGPA I MMTrGpApApCpCpGpGpApGpApCpT-pGpApCpT MMTrGpApApCpCpGpGpA PCPTPAPC I MMTrGpApApCpCpGpGpApGpApCpTpCpTpApC PCPAPTPG I MMTrGpApApCpCpGpGpApGpApCpTpCpTpApCpCpApTpG (27) 86 B.E. Griffin M. Jarman and C. B. Reese Tetrahedron 1968,24,639; B. E. Griffin and C. B. Reese ibid. p. 2537; H. P. M. Fromageot C. B. Reese and J. E. Sulston ibid. p. 3533. 87 F. Cramer and G. Schneider Annalen 1968 717 193. A. Franke F. Eckstein K.-H. Scheit and F. Cramer Chem. Ber. 1968 101,944. 89 F. Kathawala and F. Cramer Annalen 1968 712 195. 90 G. M. Blackburn and M. J. Waring Annual Reports 1967 B 64,479. 91 N.K. Gupta E. Ohtsuka V. Sgaramella H. Biichi A. Kumar H. Weber and H. G. Khorana Proc. Nat. Acad. Sci. U.S.A. 1968 60,1338. 92 H. G. Khorana Biochem. J. 1968 109 709. 544 G. Michael Blackburn used to make the second eicosanucleotide which has ten bases complementary to the first and is of opposite polarity so that together (28) the two oligomers span the length of residues 21-50 of the gene for t-RNA$is (p. ). This molecular duplex has two 'sticky' ends which can complex with an added complementary oligonucleotide. Thus the nonanucleotide (29) com-bines with (28) to form a stable trimolecular complex which 'melts' at 42". This is effectively a segment of a DNA helix with one single-strand break and application of the ligase ('sealing' enzyme) from bacteriophage T4 joins together the eicosa- and nona-nucleotides to give a molecule of 29 nucleotide units.A similar operation after the addition of the heptanucleotide (30) gives the DNA duplex (31). The success of these operations is monitored first by the incorpora- tion of the 32P-label from (29) into the product second by nearest neighbour analysis and third by addition of the nucleotide residues complementary to the single-strand termini of (31) by use of DNA polymerase from E. coli. Thus the dC residue at the right-hand end of (31) and the dA dG and dC at the other are specifically added by the polymerase enzyme to complete the thirty-base double-stranded DNA molecule. Detailed experiments show that a 'cohesive' end of 5 or 6 deoxyribonucleo-tide residues provides sufficient overlap for the ligase extension procedure.93 Thus blocks of decanucleotides can be added step by step to elongate either end of the molecule and leave a new cohesive end the addition of a pentanuc- leotide d-CpTpApApG to (28)can be followed by the further extension of the left-end by use of decanucleotides corresponding to t-RNAA'" sequence 46-55 then 51-60 and so on to completion of the gene.Using very similar techniques a second group is intent on the synthesis of the gene corresponding to chain A of bovine insulin.94 Thus far the protected duodecanucleotide d-pTpTpApApTpTpApCpApApTpA has been prepared. Syntheses on polymer supportsg have been comparatively unsuccessful measured against the achievements in peptide synthesis.The best support for nucleotide studies appears to be a highly cross-linked rigid polystyrene- divinylbenzene copolymer. Even with this the efficiency of addition of monomers falls alarmingly by the third or fourth deoxynucleotide residue.96 Nevertheless cellulose-bound polynucleotides have opened some interesting opport~nities.~' Their use as solid-state primers for enzymic polymerisations includes the synthesis of a polymer-bound single-strand DNA complementary to a given soluble single-strand DNA.98 Details of sequence determination are described elsewhere [Part (ii)] but the potential application of mass spectrometry for this purpose is heralded by 93 N. K. Gupta E. Ohtsuka H. Weber S. H. Chang and H. G. Khorana Proc. Nut. Acad.Sci. U.S.A. 1968,60,285. 94 S.A.Narang S. K. Dheer and J. J. Michniewicz,J. Amer. Chem. SOC. 1968 90 2702. 95 T. Shimidzu and R. L. Letsinger J. Org. Chern. 1968,33 708. 96 F. Cramer and H. Koster Angew. Chem. Internut. Edn. 1968 7 473. 97 P. T. Gilham Biochemistry 1968 7,2809. 98 T. M. Jovin and A. Kornberg J. Biol. Chem. 1968 243 250. SCHEME G-T-A-C -C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G-OH 5’ IIIIIIIIII S’HO-G-C-T-C-C-C-T-T-A-G-C -A-T-G-G-G-A-G-A-G 3’HOG-G-G-A-A-T-C pT-C -T-C-C-G-G-T-T-OH 3‘ ‘i‘ (30) ligase (29) .................... t i C G A i G-G-G-A-A-T-C-G-T-A-C-C-C-T-C-T-C-A-G-A-G-G -C-C-A-A-G z-b .................... I I I I I I I I I I I I I I I I I I I I I I I I I I...... G~-T-C-C-~-T-T-A-G-C-A-T-G-G-G-A-G-A-G-T~-T-€-C-G-G-T-T i......C i (31) MMTr = 5’-monomethoxytrityl. Amino-groups are protected by acylation with anisoyl for C benzoyl for A and isobutryl for G respectively. The 3‘-OAc at the end of each new block is removed by hydrolysis before condensation of the next block. (The phos- phates are omitted from deoxynucleotides (28H31) for clarity.) G. Michael Blackburn studies on purines and pyrimidine^,^' nucleosides and nucleotides,'" and dinucleoside phosphates. lo' Photochemistry.-Sensitised photochemistry has clarified the mechanism of pyrimidine dimer formation. Cytosine uracil thymine and orotic acid all exhibit triplet state dimerisation in solution. '02 Dimethylthymine gives all four cyclobutane products on photosensitised dimeri~ation."~ A detailed study of the solvent effects on the relative yields of the four and the influence of triplet quenching additives suggests a complex pattern of behaviour in which all dimers are formed independently from both singlet and triplet states with different adducts showing a preference for the one or the other.'04 The singlet pathway becomes important at high concentration of monomer.'03 Thymine dimers frozen in a glass at 80°K can be monomerised by irradiation at 248 nm. Subsequent irradiation at 280 nm. reforms dimers with high quantum yield and with quenching of monomer fluorescence. This ingenious experi- ment105 suggests a mechanism for dimerisation in which a singlet excimer is formed involving two suitable oriented thymines some 2.8 A apart which collapses to a cyclobutane dimer before fluorescent emission.The properties of all four thymine dimers have been surveyed.'06 X-Ray diffraction structures are available for cis-syn dimers of dimethylthymine'07 and uracil.lo8 Whereas the major thymine photoproduct from native DNA is the cis-syn dimer," irradiation of denatured DNA yields lesser amounts of a second dimer identified as the trans-syn isomer (32) which is not cleaved by the photoreactivating enzyme. '09 Although the cyclobutane dimers have received 0 0 Y-NH 02% KNH 0 0 (3 2) (33) 99 M. H. Studier R. Hayatsu and K. Fuse Analyt. Biochem. 1968 26 320. loo J. A. McCloskey A. M. Lowson K. Tsuboyama P. Krueger and R. N. Stillwell J. Amer. Chem. Soc. 1968,90,4182. lo' D.F. Hunt C. E. Hignite and K. Biemann Biochem. Biophys. Res. Comm. 1968 33 378. lo' C. L. Greenstock and H. E. Johns Biochem. Biophys. Res. Comm. 1968,30 21. H. Morrison A. Feeley and R. Kleopfer Chem. Comm. 1968,358. lo4 H. Morrison and R. Kleopfer J. Amer. Chem. SOC.,1968,90,5037. lo5 A. A. Lamola and J. Eisinger Proc. Nat. Acad. Sci. U.S.A. 1968 59 46. lo6 M. A. Herbert J. C. LeBlanc D. Weinblum and H. E. Johns Photochem. and Photobiol. 1968 9 33. lo' N. Camerman and A. Camerman Science 1968 160 1451. Io8 E. Adman M. P. Gordon and L. H. Jensen Chem. Comm. 1968 1019. log E. Ben-Hur and B. Ben-Ishai Biochim. Biophys. Acta 1968 166 9. Nucleic Acids 547 most attention a minor photoproduct of thymine' lo is assigned structure (33) and on heating is dehydrated to give (34).The pyrimidine photohydrates whch are formed from singlet excited states,'" have had their structures confirmed both by n.m.r. analysis' l2 and by chemical degradation of both cytidine and uridine hydrates. '' The transient formation of unstable cytidine hydrates can be assayed by reaction with hydroxy- lamine to give N(4)-hydroxycytidine ;'l4 photoreduction of thymine and uracil dimers with sodium [3H]borohydride provides a radioisotope marker for photodimerisation. ' ' The mechanisms of photoreduction and hydrogenolysis of pyrimidine nucleosides and their photoproducts have been reviewed. 'l6 Much recent work on the molecular biology of U.V. lesions in DNA has been surveyed.'' While photohydrates are primarily mutagenic (p.548) pyrimidine dimers appear to be less lethal than was thought. In a population of E. coli cells which lack the enzyme for repairing U.V. damage a dose of U.V. radiation sufficient to create 50 pyrimidine dimers in each DNA molecule only kills 63% of the bacteria. Experiments indicate that the DNA of the survivors' progeny has gaps in one strand but that these defects slowly disappear during incubation. ''*Other studies demonstrate that thymine dimers in bacterio- phage DNA can be handed on efficiently from parent to progeny under non- repair conditions and suggest that fewer than one in five thymine dimers is lethal.' '' Pulse radiolysis of uracil'20 and thymine12' in aqueous solution causes the addition of hydroxyl radicals to the pyrimidine.Thymine forms the tertiary radical which in the presence of oxygen is converted into 5-hydroperoxy-6- hydroxydihydrothymine (35). The use of hydroxyl scavengers shows that while HO' and H' react destructively with thymine the solvated electron does not.'22 y-Radiolysis of solutions of AMP produces the unusual cyclonucleoside phos- phate (36).'23 Base Pairing and Stacking.-The specificity of base pairing through hydrogen bonds is most simply observed in non-aqueous solvents. Dimethyl sulphoxide appears to be an excellent choice and n.m.r. investigations of cytidine and guanosine give value of -6 kcal. M-' for the enthalpy of base pairing while showing no evidence of base stacking.'24 A similar value has been measured 'Io A. J. Varghese and S.Y. Wang Science 1968 160 186. '" I. H. Brown and H. E. Johns Photochem. and Photobiol. 1968,8 273. '" W. J. Wechter and K. C. Smith Biochemistry 1968 7 4064. 'I3 N. Miller and P. Cerutti Proc. Nat. Acad. Sci. U.S.A. 1968 59 34. G. D. Small and M. P. Gordon J. Mol. Biol. 1968,34 281. T. Kunieda L. Grossman and B. Witkop Biochem. Biophys. Res. Comm. 1968,33,453. '16 B. Witkop Photochem. and Photobiol. 1968 7 813. I" Photochem. and Photobiol. 1968 7 578 ff. ''13 W. D. Rupp and P. Howard-Flanders J. Mol. Biol. 1968 31 291. 'I9 W. Sauerbier and M. Hirsch-Kauffmann Biochem. Biophys. Res. Comm. 1968 33 32. R. M. Danziger E. Hayon and M. E. Langmuir J. Phys. Chem. 1968,72 3842. P. T. Emmerson and R. L. Willson J. Phys. Chem. 1968 72 3669. H. Looman and J.Blok Radiation Res. 1968 36 1. '" K. Keck 2.Naturforsch. 1968 23b 1034. R. A. Newmark and C. A. Cantro J. Amer. Chem. SOC. 1968 90 5010. G. Michael Blackburn ..XoH OL OH (35) for the interaction of 9-ethyladenine and 1-cyclohexyluracil in pure chloro- form.12' This pairing is weakened by the presence of ethanol in the solvent and base pairing between monomers appears to be excluded in aqueous solution.126 There is little evidence for complexing between di- tri- or tetra-nucleotides and their complements in water. The formation of a 2:l complex between GpGpC and GpCpC is possibly non-specific since GpGpC self-aggregates strongly.127 This is interpreted to indicate that codon :anticodon interactions must be augmented by ribosome and t-RNA interactions.The biological effects of barbiturates may relate to the observation that 9-ethyladenine forms specific hydrogen-bonded complexes with phenobar- bital barbital and thiopental which are much more stable than its complex with l-cyclohexyluracil.128 The X-ray structure of a 2 1 complex of 8-bromo- g-ethyladenine and phenobarbital shows Watson-Crick hydrogen-bonding between bases. 12' The tautomeric constant for N(4)-hydroxycytosine derivatives (37) favours the imino-form (38) for N(4)-aminocytosine the amino-form (39) is more stable than the imino-form (40). Thus the mutagenic action of hydroxylamine on DNA can be identified with the transformation of a cytosine residue (41) which base pairs with guanine into an N(4)-hydroxycytosine unit (38) which tautomerically resembles uracil and so will base-pair with adenine.On repli- cation this change will result in progeny DNA containing an A residue where the parent had a G.130 Since modification of (41) to (39) by hydrazine does not involve a tautomeric change hydrazine would be predicted to be an indifferent mutagen-as observed. Changes in base pairing consequent on the photohydration of pyrimidines are environment dependent. in vitro Polypeptide synthesis shows that uracil hydrate U* codes as cytosine when it is in the first position in a codon triplet but that in the centre of the triplet it codes as C with only 20 % efficiency. A striking example of this change in base pairing is provided by the reversal 12' J. S. Binford and D.M. Holloway J. Mol. Bol. 1968 'l 91. 126 T. N. Solie and J. A. Schellman J. Mol. Biol. 1968 33 61. S. R. Jaskunas C. R. Cantor and I. Tinoco Biochemistry 1968 7 3164. 12* Y. Kyogoku R. C. Lord and A. Rich Nature 1968,218 69. S.-H. Kim and A. Rich Proc. Nut. Acad. Sci.,U.S.A.,1968 60,402. 130 D. M. Brown M. J. E. Hewlins and P. Schell J. Chern. SOC.(0,1968 1925. F. P. Ottensmeyer and G. F. Whitmore J. Mol. Biol. 1968,38 1. Nucleic A cids 549 (37) R = OH (38) R = OH (39) R = NH (40)R = NH2 (41)R = H of a lethal mutation with U.V. irradiation. Bacteriophage h containing an amber mutation infect a host bacterium and produce m-RNA which contains a nonesense codon UAG. This has the effect of terminating protein synthesis at the amino-acid of the codon preceding UAG-with lethal consequences for the phage.A dose of U.V. radiation a short time after the phage has begun to make the defective m-RNA converts some of the nonesense triplet UAG into the hydrated form U*AG. This behaves in protein synthesis like CAG and codes for glutamine. Thus normal protein synthesis is restored and the bacteriophage multiply.' 32 An investigation of the kinetics of base pairing can determine whether the rate of certain biological processes is limited by this factor. T-Jump methods'33 show that the rates of formation of dimers U :A U :U and A :A are essentially diffusion controlled. The different stabilities of these three complexes are governed by different rates for their dissociation. The kinetics of base pairing and co-operative helix formation have been measured for oligomers from tri- to deca-nucleotides giving time constants from to sec.134 The rate of combination of poly-A and poly-U to give a double helix decreases with increasing temperature and reaches zero 0.2" below the 'melting' temperature of the complex.However the rate of dissociation of the duplex is complicated and exhibits a minimum value at T,.13' This behaviour requires a revision of the mathematical models for stacking and suggests that the requirement for helix growth is a stable nucleus of more than one base pair. Single-strand stacking in ribotrinucleotides shows that stability depends not only on the nature of the bases involved but also on their sequence.'36 Thus UpApGp stacks better than ApUpGp.Purines tend to stack better than pyrimidines; this fact has led to the prediction that hairpin turns in single- stranded RNA should beexpected in pyrimidine rather than in purine regions. 137 The interactions of polymers with monomers are not as simple as first appeared. Adenosine interacts with poly-U below 26" to give a rigid A:2U complex probably involving co-operative hydrogen-bonding and A-A F. P. Ottensmeyer and G. F. Whitmore J. Mol. Biol. 1968,38 17. 133 G. G. Hammes and A. C. Park J. Amer. Chem. Soc. 1968 90,4151. 134 M. Eigen J. chim. Phys. 1968 65 53. 13' R. D. Blake L. C. Klotz and J. R. Fresco J. Amer. Chem. Soc. 1968,90 3556. S. Aoyagi and Y. Inoue J. Biol.Chem. 1968 243 514. 137 R. C. Davis and I. Tinoco Biopolymers 1968 6 223.G.Michael Blackburn base stacking stabilisation. Above 26" there is non-co-operative base stacking with some intercalation of adenosine into the poly-U.'38 Intercalation is best seen in n.m.r. studies of poly-U and purine interactions-where no hydrogen- bond base pairing is p~ssible.'~' Other work shows that 3,4-benzpyrene intercalates between adjacent bases in the DNA helix. Finally the complexes formed at low temperature betwen poly-U and adenine nucleotides are in- soluble in water. 141 While this phase-transition contributes to the stability of the complex and provides a useful mechanism for prebiotic sequestration of AMP it impedes further study of these interesting complexes. 13* B. W. Bangerter and S. I. Chan Proc.Nat. Acad. Sci.U.S.A.,1968,650 1144. 139 B. W. Bangerter and S. I. Chan Biopolymers 1968 6,983. S. A. Lesko,A. Smith P. 0.P. Ts'o and R. S. Umans Biochemistry 1968 7 434. 141 P. 0.P. Ts'o and M. P. Schweizer,Biochemistry 1968,7,2963;P. 0.P. Ts'o and W. M. Huang ibid. p. 2954.

ISSN:0069-3030    

DOI:10.1039/OC9686500535

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

16 NUCLEIC ACIDS Part (ii) By M. J. Waring (Departmentof Pharmacology Downing Street Cambridge) RNA.-Double-helical complexes formed by mixtures of synthetic poly- nucleotides (poly-A plus poly-U ; poly-I plus poly-C ; poly-G plus poly-C) have been examined in detail by X-ray diffracti0n.l They reveal three distinct helical conformations of RNA the A-form with eleven base-pairs per turn; the A’-form with twelve ; and the A”-form which is non-integral and may be a family of closely related structures rather than a single molecular species. Transitions from one form to another are related to changes in salt concentra- tion. All three conformations resemble the A-form of DNA but no structure like the DNA B-form is seen. This could be explained by the T-hydroxy- group of ribose keeping the sugar ring in the C(3)-endo-conformation since when DNA takes up the B-helical form its sugar rings change to C(2)-endo.Such a restriction imposed by the ribose 2’-hydroxy-group could also explain the failure of a DNA-RNA hybrid to show an A - B transition.’r2 The new X-ray data favour an eleven-fold helix (rather than ten-fold) for the double- helical RNA of re0virus.l Electron microscopy of the replicative form (RF) and replicative intermediate (RI) of the RNA phage R17 reveals a translation per residue of 3.14 A which the authors3 consider more consistent with a ten- fold helix but would also be consistent with the twelve-fold A’-form. Ribosomal RNA.-Ribosomes from different sources yield r-RNAs of characteristically different size (see the 1967 Report).Occasionally the r-RNA shows evidence of in~tability,~ e.g. to heat,5*6 and in one instance (Euglena gracilis) more careful preparative techniques have revealed the occurrence as expected of a large and a small component where only a single species had previously been detected.6* A survey of r-RNA size in many different organisms has interesting implications for evolution procaryotic organisms have r-RNAs of molecular weight 1.09 and 0.56 million; all plants and animals have their smaller r-RNA of molecular weight 0.7 million ; and while the molecular weight of the larger r-RNA of plants is consistently 1.3 million that of the r-RNA of animals increases up the evolutionary scale from 1.4 million to 1.75 milli~n.~ ’ S.Arnott W. Fuller A. Hodgson and I. Prutton Nature 1968 220 561. * G. Milman R. Langridge and M. J. Chamberlin Proc. Nut. Acad. Sci. U.S.A.,1967,57 1804. ’ N. Granboulan and R. M. Franklin J. Virol. 1968 2 129. U. E. Loening J. Mol. Biol.. 1968 38 355. ’ J. J. Pene E. Knight jun. and J. E. Darnell jun. J. Mol. Biol.,1968 33 609. ’ C. Portier and V. Nigon Biochim. Biophys. Acta 1968 169,540. ’ J. R. Rawson and E. Stutz J. Mol. Biol. 1968 33 309; K. E. Schuit and D. E. Buetow Biochim. Biophys. Acta 1968 166. 702. 552 M. J. Waring An unexpectedly close correlation between the (G + C):(A + U) ratios of the two r-RNAs in various organisms has been described.' Sequence analysis however is still at an early stage. Partial sequence analysis has been performed on oligonucleotides in a pancreatic RNAse digest.' Bases modified by methyl- ation seem to occur in clusters; the major methylated oligonucleotides all occur twice in E.coli 23s-r-RNA. This could be the result of gene duplication during evolution or dimerization of precursor half-molecules.'O Alternatively the apparent duplication of sequences might represent the result of 'convergent' evolution" if for example the duplicated sequences were involved in the two t-RNA-binding sites' believed to occur on the ribosome. Infrared difference spectra indicate that the secondary structure of E. coli r-RNA involves 60% of the total bases in pairing; this agrees reasonably well with estimates from other methods. 5s-RNA.-Full details have been published describing the determination of the nucleotide sequences of E.c0liI4 and KB cell15 SS-RNAs. The KB cell SS-RNA exists in two forms; one has 120 nucleotides and the other is identical but for an additional U at the 3'-end (Figure 1). Since it is an axiom of molecular biology that primary sequence determines secondary and tertiary structure which in turn determine biological function it is a challenging (not to say paradoxical) situation for the molecular biologist to be confronted with mole- cules of accurately known sequence in search of a structure and a function. Yet this is still the case with SS-RNA. Various conformations have been pro- posed (see the 1967 Report) to which may be added a pair of structure^'^ which resemble the clover-leaf conformation suggested for t-RNA (see Figures 1 and 2).Optical data18 and sensitivity to monoperphthalic acid o~idation'~ indicate quite a high degree of base pairing and are perhaps most consistent with the model of Cantor,20 but the case is far from proved. Indeed it seems that E. coli 5s-RNA can exist in more than one structural form21*22 and can undergo a partially reversible transition to a 'denatured' state,22 as can some t-RNAs. F. Amaldi Nature 1969 221 95. F. Amaldi and G. Attardi J. Mol. Biol. 1968 33 737. lo P. Fellner and F. Sanger Nature 1968 219 236. C. R. Woese Nature 1968 220 923. M. Bretscher Cold Spring Harbor Symp. Quant. Biol. 1966 31 289. l3 R. 1. Cotter and W. B. Gratzer Nature 1969 221 154. l4 G. G. Brownlee F. Sanger and B.G. Barrell. J. Mol. Biol. 1968 34 379. l5 B. G. Forget and S. M. Weissmann J. Biol. Chem. 1968,243 5709. l6 B. G. Forget aad S. M. Weissmann Science 1967 158 1695. I. D. Raacke Biochem. Biophys. Res. Comm. 1968,31 528. C. R. Cantor Proc. Nat. Acad. Sci. U.S.A. 1968,59,478. l9 F. Cramer and V. A. Erdmann Nature 1968 218 92. 2o C. R. Cantor Nature 1967 216 513. M. E. Geroch E. G. Richards and G. A. Davies European J. Biochem. 1968 6,325. 22 M. Aubert J. F. Scott M. Reynier and R. Monier Proc. Nut. Acad. Sci. U.S.A. 1968,61,292. Nucleic Acids 553 uOH U U pG-C U-G C-G U-A CG c A A “AU c ‘I CCUGA --A,-u C-GG GU C G-C C C-G C-G C-G G-C A-U U-G C-G U-G CA G-C U-G CA U-A G-U AC OGA A GG 5s-RNA (KB cells) RGURE1 Nucleotide sequence of 5s-RNA from KB cells.The sequence is arranged in the clover leaf‘ formsuggestedby Raacke.l7 Another variant exists which lacks the third U at the 3’-end shown here. 554 M. J. Waring 2OH C A PC A G-C C-G G-C G-C G-C G-C *U CGG ccuAA G Ill II A CCGACGA,G GUC GGT4 U 111 I C C 7Me GG~AGCU uG \ U-A AG A C-G G-C G-C G-C 2’0MeC A UA CAU t-RNA Fet (E.Coli) RGURE 2 Nucleotide sequences of t-RNAs arranged in the clouer- leaf form. Bases in t-RNA A adenosine; AMe position of methyl group unknown ; A lMe 1-methyladenosine; A” N(6)-isopentenyl-adenosine ;A* N(6)-acylated adenosine with bulky substituent on the amino-group; At Nature of modification unknown; Y,fluorescent modified adenosine.G guanosine; GMe,position of methyl group unkown; GIMe 1-methylguanosine ;GZMe,2-methylguanosine ; G7Me, 7-methyl-guanosine ; WMe,N(2)-dimethylguanosine; 2’0MeG 2‘-0-guanosine ; G* nature of modification unknown. C cytidine; CMe position of methyl group unknown; C3Me, 3-methylcytidine ;CSMe, 5-methylcytidine;CAc N(4)-acetylcytidine ; 2’0MeC 2’-O-methylcytidine; Ct probably N(4)-acylated cytidine. U uridine ; 2’0MeU 2’-0-methyluridine ; U* 4-thiouridine. @,.pseudouridine;2’OMe@,2’-O-methylpseudouridine. I inosine; IMe,position of methyl group unknown. T thymidine. X,unknown. Nucleic Acids A OH C C A pG-C C-G G-C G-U A-U U-A U-A U GA A II DGACUC G2~e 5MeCU G 111 I cU G7Me G~AG~~ 'GDiMe C-G AG t-RNA (Yeast) A OH C C A pG-C G-C G-U C-G G-C uu G-C U AGGCC u'A MeG 11111 G CG UCCGG T4 cU,D AG C-G u-A C-G C-G C-G u9 u IMe IGC t-RNA :la (Yeast) 556 M.J. Waring gOH C G pG-C G-U C-G A-U A-U G C-G t U-A u CGUCC~~A DGAG AC G 1111I A*G 2'0MeG 5MeC 777 GCAGGTqC G GGC 2'OMe GC DDAA oiMeG u '/'CG A-U GG // u A-U G~ c A-U 'u G-C A-Y -YA U AiP IGA t-RNA 7'' (Yeast) t0" t-RNA2er differs in 3 bases as shown. c A pG-C C-G G-C G-C GA G-C A-U U CGCAC A 11111 DG A c ucG2~e GCGUG n 1111 G DG7Me GG A AG *-A - - C-G A-U G-C A-3, 2'0MeC A UY 2'0MeG A A t-RNA (Wheat germ) NucIeic Acids 557 OH C G pG-C U-A A-U G-C u-A C -G G-C u CGUCC~~A~M~ 11111 G FC CG GCAGGTqC I I I 5MeCm G-C v G-C A-92'OMe 3MeC A U AiP IGA t-RNA Ser (Rat liver) AOH C C A pC-G U-A C-G U-G C-G G-C G-C u CCCGC~~AM~ 11111 G A-U A-U G- C A-9 CA U AiP GWA t-RNA Tyr (Yeast) M.J. Wating OH C A pG-C G-U U-A U-A U-A C-G G-C U-A G-C C-G 3rc UA OH IAC C A PG-C t-RNA Va I (Baker's yeast) G-U-G-G-G-G-*U GCCcu I1II AGGGA G-C-A-G-A-C A u+ A+ GUA t C Species I,Sum as shown.Su+ . G * replaced by c . rn' Species II uc replaced by CA . Nucleic Acids 559 C A pG-C G-C C-G U-A A-U C-G D G-C (I *U UGCCC UAA A II II G G ACAGG TW I C A C-G A-u U-A C-G A-W CA U A* CtA U t-RNA :et (E.Coli) AO" C c A pG-C G-U U-A U- A U-A C-G -c GGG uc AIM^ 11111 G D DGAqC CCCAGTqC G I 5MeC G UGGC C DC A AA 9-A C-G U-A G-C C-G "C UA I AC t-RNA y"' (Torula yeast) 560 M. J. Waring (This may also be true of 28S-r-RNA.23) Clearly a primary requirement for formulating likely structural models for molecules of known sequence is some means of maximising base pairing; a mathematical approach to this problem has been de~cribed.’~ The function of 5S-RNA remains unknown :its location in the ribosome would suggest some role in protein synthesis for which there is limited e~idence,~’ but little more data are available other than studies on its removal from and association with ribosomal particles.’’9 26 Viral RNA.-Table 1 summarises data on terminal sequences of viral RNAs; the great majority of which were published during 1968. In most in- stances the methods involved specific labelling of one end of the RNA e.g. by attaching a [32P]phosphate group at the 5’-end by use of labelled ATP and polynucleotide kina~e,~~. 39 or selective periodate oxidation of the 3’-terminal nucleoside followed by reduction with [3H]borohydride ;29 then cleavage with an endonuclease isolation of the labelled oligonucleotide and sequence analysis.One new technique however identifies the oligonucleotide derived from a 3’-hydroxy-end on the basis of its unchanged electrophoretic mobility after phosphatase treatment.27 It is striking that all the bacteriophage RNAs begin with a G residue bearing a 5‘-triphosphate group; this invites comparison with t-RNAs most of which also begin with a G residue and all of which bear a 5‘-phosphate (see Figures 2 and 3). Perhaps more striking is the 3‘-terminal --CCA, grouping which occurs in all the viral RNAs (including the three plant viruses) and has long been recognised as characteristic of t-RNA. This similarity has prompted some workers to speculate that perhaps the --CCA, serves to protect both t-RNAs and viral RNAs from nuclease attack in the cell.Be that as it may it now appears that the terminal adenosine of viral RNAs is probably added by some host cell enzyme,41 42 perhaps the enzyme involved in the turnover of the --CCAoH end of t-RNAs since infectivity of R17 RNA 23 H. Singh and D. Keller Biochim. Biophys. Acta 1968 169 150. 24 V. G. Tumanyan L. E. Sotnikova and A. V. Kholopov Doklady Akad. Nauk S.S.S.R. 1966 166,1465. 25 D. M. W. Kirtikar and A. Kaji J. Biol. Chem. 1968 243 5345. 26 P. Morel1 and J. Marmur Biochemistry 1968 7,1141 ;M. A. Q.Siddiqui and K. Hosokawa Biochem. Biophys. Res. Comm. 1968,32 1. 27 J. E. Dahlberg Nature 1968 220 548. H. L. Weith and P.T. Gilham J. Amer. Chem. SOC.,1967,89 5473. 29 D. G. Glitz A. Bradley and H. Fraenkel-Conrat Biochim. Biophys. Acta 1968 161 1. 30 R. Roblin J. Mol. Biol. 1968 36 125; 1968,31 51. 31 D. G. Glitz Biochemistry 1968 7,927. 32 R. De Wachter J. P. Verhassel and W. Fiers F.E.B.S. Letters 1968,1,93. 33 R. De Wachter and W; Fiers J. Mol. Biol. 1967 30,507. 34 M. Watanabe and J. T. August Proc. Nut. Acad. Sci. U.S.A. 1968,59 513. 35 R. De Wachter and W. Fiers Nature 1969 221 233. 36 H. L. Weith G. T. Astenadis and P. T. Gilham Science 1968,160 1459. 37 S. Mandeles J. Biol. Chem. 1967 242 3103. 3a J. Suzuki and R. Haselkom J. Mol. Biol.. 1968,36 47. 39 E. Wimmer and M. E. Reichmann Science 1968,160 1452; E. Wimmer A. Y. Chang J. M. Clarke jun. and M. E. Reichmann J.Mol. Biol. 1968 38 59. 40 D. H. L. Bishop D. R. Mills and S. Spiegelman Biochemistry 1968 7 3744. 41 A. Vandenberghe B. Van Styvendaele and W. Fiers European J. Biochem. 1969,7,174. 42 R. Kamen Nature 1969 221,321. TABLE 1 Terminal sequences of viral RNAs Ref. RNA Sequence Y-end 3’-end f2 pppG ________________G U U A C C A C C C AOH 27 27-29 R17 p p p G pu py ____-_______ G U U A C C A C C C A 30 27 MS2 31 29,33 PPPGGU 1 GUUACCACCCA, 32 or p p p G G G U pppGGGGAAC G C C C U C C U C U C U C C C A, 27,34,35 27,36 QP pppGGGGGAAC $ b ‘Little’variant pppGGGGA A 5. 40 % ofQP 29,37 Turnip yellow A py______-_--_-_l-_-.l-l_-l_-__-38 mosaic virus Satellitetobacco p p A G U -39 necrosis virus 562 M.J. Waring Pu 1-3 Ant icodon t-RNA Homologies * indicates G-C pairing PU indicates a purine py indicates a pyrimidine. RGURE 3 Generalised clover-leafstructure for t-RNA. is retained after removal of the 3’-terminal adenylate (but not if the penultimate C is removed as well).42 Qp RNA provides a curiosity in that both the 5’-terminal sequences shown in Table 1 occur in the progeny of an infection initiated by a single parental phage particle suggesting that some sort of equi- librium between the two types is established during growth.35 On the other hand it seems unlikely3’ that a similar explanation can account for the different 5’-terminal sequences reported for MS2 RNA (Table 1).The apparent comple- mentarity of the terminal sequences of MS2 RNA and Qp RNA has prompted the suggestion that the ends of these molecules might associate by hydrogen bonding to form pseudo-circles.3’ 329 t-RNA.-The complete sequences of six more t-RNA species and variants of them are now known valine t-RNA from Torulopsis utiIis;43 serine t-RNA 43 S. Takernura T. Mizutani and M. Miyazaki J. Biochem. (Japan) 1968 63 277. Nucleic Acids 563 from rat liver ;44 phenylalanine t-RNA from wheat germ ;45 tyrosine t-RNA and the amber suppressor su& t-RNA derived from it from E. COIZ;~~ and the methionine-specific t-RNA and t-RNA from E. coli.47,4a Full details of the yeast phenylalanine t-RNA sequence have also been p~blished.~’ The new sequences are shown together with the five reported earlier (Reports for 1965-1967),in the conventional clover leaf form in Figure 2.The considerable degree of homology in the eleven sequences is clear. One can divide the anatomy of the clover leaf into five distinct sections the ‘amino-acid’ helix of seven base pairs with its unpaired --CCAoH end (top); the seven-nucleotide ‘TJIC’ loop with its helical stem of five base pairs (right); the seven-nucleotide ‘anti- codon’ loop with five base pairs in its stem (bottom; the anticodon itself consists of the three bases at the very bottom); the ‘dihydro-U’ loop with three or four base-pairs in its stem (left); and the ‘extra’ loop or ‘lump’ which varies widely in the different molecules and lies between the TJIC and anticodon arms. Much can be learnt from a detailed study of the apparent homologies.To facilitate comparison a generalised clover leaf structure has been compiled in Figure 3 which draws attention to the most striking regularities in the known sequences. Certain irregularities may also be noted for example the ‘dihydro-U’ loop shows substantial variation and indeed in the E. coli tyrosine t-RNAs does not even contain any dihydro-U. In some cases the base-pairing in the ‘amino- acid’ and ‘TJIC’ stems appears to be imperfect. Knowledge of regions shared in common between different t-RNAs is important for formulating detailed ter- tiary structure models (see later) and for identifying regions concerned with particular functions such as interactions with the ribosomea during protein synthesis.Special interest attaches to the t-RNA:“ and t-RNA:“ sequences in view of the peculiar role of the latter in the initiation of polypeptide synthesis (see the 1966 Report). The two sequences are surprisingly dissimilar (only forty- one nucleotides are held in common); this makes it difficult to determine recog- nition sites for aminoacyl t-RNA synthetases the transformylase or initiation factors for protein synthesis. It is hypothesised that the tertiary structures of the molecules must be of importance for these functions.47 In any event the unique ability of t-RNA to recognise both AUG and GUG codons in ribosome- binding experiments is not due to the fact that two forms differing in the 7-methyl-G or A replacement exist. 50N-formylmethionyl t-RNA has been found in mitochondria of yeast and liver but not in their cytoplasmic protein- synthesising systems.s’ 44 M. Staehelin H. Rogg B. C. Baguley T. Ginsberg and W. Wehrli Nature 1968,219 1363. 45 B. S. Dudock G. Katz E. K. Taylor and R. W. Holley Proc. Nut. Acad. Sci. U.S.A. 1969 in the press. 46 H. M. Goodman J. Abelson A. Landy S. Brenner and J. D. Smith Nature 1968 217 1019. 47 S. Cory K. A. Marcker S. K. Dube and B. F. C. Clark Nature 1968,220 1039. 48 S. K. Dube K. A. Marcker B. F. C. Clark and S. Cory Nature 1968 218,232. 49 U. L. Rajbhandary and S. H. Chang J. Bid. Chem. 1968 243 598. S. Cory S. K. Dube B. F. C. Clark and K. A. Marcker F.E.B.S.Letters 1968 1,259. 51 A. E. Smith and K. A. Marcker J. Mol. Bid. 1968 38. 241. 564 M.J. Waring The modified nucleotide Y which occurs next to the anticodon in t-RNAPhe of yeast and wheat germ is strongly fluorescent and occurs in t-RNAPhe of other organisms.52 The base is a rather hydrophobic adenine derivative of as yet unknown structure; it can be selectively removed by mild acid without breaking the chain and the treated t-RNA can still be charged with phenyl- alanine but it is unable to bind to ribosomes in the presence of poly-U or to transfer its Phe to growing peptides.53 Thus bases in the anticodon loop as well as the anticodon itself are needed for codon-anticodon interaction but the integrity of the anticodon loop cannot be necessary for recognition by the aminoacyl synthetase. Similar conclusions may be drawn from other instances where base changes in the presumed anticodon affect the coding properties of the t-RNA but still permit charging with the amino-a~id.~~ Especially eloquent is the production of tyrosine-specific su,’; amber suppressor t-RNA by a mutation which substitutes C for G* in the anticodon of a minor tyrosine t-RNA of E.(Figure 2). Codon-anticodon interaction also seems to require the helical stem of the anticodon loop? There is evidence that the ‘amino-acid’ helical stem and the unpaired --CCAoH end may be directly involved in recognition by the aminoacyl synthetaseS6 and ribosomal peptidyl transferase5’ respectively. Large fragments derived from t-RNA may help to identify regions which interact specifically and non-specifically with ribo- some~.~~ Although the susceptibility of t-RNA sequences to chemical and enzymic attack yields results in broad agreement with the clover leaf model (p.566) the most compelling reasons for believing that it is basically correct are to be found in Figures 2 and 3 in the high degree of homology between different sequences which becomes evident when they are written in the ‘standard’ clover-leaf form. The thermal denaturation pattern of E. coli methionine t-RNA shows its structure to be perceptibly more stable than that of t-RNA (ref 59) which is consistent with an unusually high proportion of GC pairs in helical regions (compare Figure 2) but definitive proof of the clover leaf will probably have to wait for detailed X-ray diffraction examination of t-RNA crystals.At the end of 1968 papers from six laboratories reported that crystallisation of t-RNA had been achieved. Crystals were obtained with formylmethionine 52 D. Yoshikami G. Katz E. B. Keller and B. S. Dudock Biochim. Biophys. Acta 1968 166 714; L. M. Fink T. Goto F. Frankel and I. B. Weinstein Biochem. Biophys. Res. Comm. 1968,32 963; B. S. Dudock G. Katz E. K. Taylor and R. W. Holley Fed. Proc. 1968 27 342. 53 R. Thiebe and H. G. Zachau European J. Biochem. 1968 5 546; Biochem. Biophys. Res Comm. 1968,33,260. 54 J. Carbon and J. B. Curry Proc. Nat. Acad. Sci. U.S.A. 1968 59 467; J. Mol. Biol. 1968 38,201 ;G. Sundharadas J. R. K&ze D. Soll W. Konigsberg and P. Lengyel Proc. Nat. Acad. Sci. U.S.A. 1968 61 693. 55 B. F. C. Clark S. K. Dube and K.A. Marcker Nature 1968 219 484. s6 L. H. Schulman and R. W. Chambers Proc. Nat. Acad. Sci. U.S.A. 1968,61 308. s7 R. E. Monro J. CernB and K. A. Marcker Proc. Nut. Acad. Sci. U.S.A. 1968,61 1042. 58 A. D. Mirzabekov D. Griinberger and A. A. Bayev Biochim. Biophys. Acta 1968,166,68. 59 T. Seno M. Kobayashi and S. Nishimura Biochim. Biophys. Acta. 1968 169,80. Nucleic Acids t-RNAc0 and phenylalanine t-RNA6' from E. coli ;serine t-RNA,62 phenylal- anine t-RNA,63 and formylmethionine t-RNA61 from yeast; and even with unfractionated mixtures of ~-RNAs.~~ Unit cell dimensions were given for formylmethionine t-RNAgO and phenylalanine t-RNA6' of E. coli but the data were clearly rudimentary. While the clover-leaf model provides an acceptable representation of the secondary structure of t-RNA there remains the possibility that the molecule may have an ordered tertiary structure.Low-angle X-ray scattering by t-RNA solutions6s and X-ray diffraction from oriented fibres66 both indicate that the arms of the clover leaf may be folded together possibly in pairs to form an RGURE 4 Two-dimensional projection of a proposed tertiary struc- ture for yeast phenylalanine t-RNA after Cramer et and personal communication from Professor F. Cramer. 6o B. F. C. Clark B. P. Doctor K. C. Holmes A. Klug K. A. Marcker S. J. Morris and H. H. Paradies Nature 1968,219,1222; S. H. Kim and A. Rich Science 1968 162 1381. 61 A. Hampel M. Labanauskas P. G. Connors L. Kirkegard U. L. RajBhandary P. B. Sigler and R.M.Bock Science 1968 162 1384.H. H. Paradies F.E.B.S. Letters 1968 2 112. 63 F. Cramer F. v. d. Haar W. Saenger and E. Schlimme Angew. Chem. 1968,80,969. 64 J. R.Fresco R.D. Blake and R.Langridge Nature 1968,220 1285. 65 J. A. Lake and W. W. Beeman J. Mol. Biol. 1968,31 115. 66 B. P. Doctor W. Fuller and N. L. Webb Nature 1969 221 58. 566 M. J. Waring H shape with the ‘amino-acid’ stem stacked on the T$C-containing arm and the anticodon arm stacked on the arm which bears the dihydro-U loop.66 A more detailed model proposing additional fixation of the arms has been based on measurements of aminoacylation rates and susceptibility of adenosines to monoperphthalic acid oxidation at various temperatures (Figure 4). In this model the helical regions of the dihydro-U-containing arm the amino-acid-bearing arm and the T$C-containing arm are packed to form a trigonal prism with hydrogen bonding between the $C in the T$C loop and the AG in the dihydro-U loop and between the CC at the --CCAoH end and the GG in the dihydro-U loop.These interactions are possible for all the known t-RNA sequences (Figure 3). The anticodon arm extends away from the trigonal prism in the opposite direction. For different species of t-RNA various addi- tional base pairs are feasible which would provide further stabilisation and might help to confer characteristic differences in tertiary structure.67 Such differences doubtless of importance for recognition would be largely deter- mined by the size of the dihydro-U loop and the central part of the molecule at the junction of the arms,including the ‘extra’loop.Weak ‘additional’ interactions like those proposed in this model could account for conformational changes in t-RNAs which might be of functional significance and might also include the reversible denaturation referred to in the 1967 Report. Recent evidence indicates that the native + denatured interconversion involves breakage and re-formation of hydrogen-bonded base pairs as well as changes in base stack- ing,68 and that the nature and concentration of cations (but not necessarily Mg+ +) are 69 Related to the phenomenon of denaturation is the occurrence of aggregates of t-RNA especially dimers frequently observed during purification procedures. It now appears that at least in some instances dimers are formed from monomers by intermolecular interactions resembling normal intramolecular interactions.70p 71 Dimers of yeast alanine t-RNA are interconvertible with monomers by the action of heat ;7 their hypochromism and denaturation spectra are so similar to those of the monomer that they must be presumed to contain the same extent of base pairing; they accept two mole- cules of alanine in the enzymic charging reaction but their anticodon sequences are much less readily accessible for specific cleavage by RNAse T,. These observations7’ suggest the sequence of events in Figure 5. DNA.-An entertaining recent discovery is the finding that certain mutants of E. coIi produce considerable amounts of DNA-less cells which can be separated from the normal (DNA-containing) cells.72* 73 Two such mutants have been 67 F.Cramer H. Doepner F. v. d. Haar E. Schlimme and H. Seidel Proc. Nat. Acad. Sci. U.S.A.,1968 61 1384. ‘* T. Ishida and N. Sueoka J. Biol. Chem. 1968 243 5329. 69 T. Ishida and N. Sueoka J. Mol. Biol. 1968 37 313. 70 A. Adams and H. G. Zachau European J. Biochem. 1968 5 556. 71 J. S. Loehr and E. B. Keller Proc. Nat. Acad. Sci. U.S.A. 1968 61 1115.. 72 H. I. Adler W. D. Fisher A. Cohen and A. A. Hardigree Proc. Nat. Acad. Sci. U.S.A. 1967 57 321. 73 Y.Hirota F. Jacob A. Ryter G. Buttin and T. Nakai J. Mol. Biol. 1968 35 175. unfolded MONOMERS by heat DIMER RGURE 5 Dimers of t-RNA possible structure and mechanism of interconversion with monomers.568 M. J. Waring studied ;in both cases the DNA-less cells arise because of a more or less normal septation process occurring to one side of the nuclear material but while one mutant produces DNA-less cells of relatively normal dimension^,^ in the other mutant septation occurs near one or both poles of the cell giving DNA- less ‘minicells’.72 Apart from their lack of DNA the cells contain protein and RNA and carry out several normal metabolic processes but not DNA-dependent syntheses.72*73 Minicells can be mated with F+ male strains of E. coli; after mating the minicells can be reisolated and extracted to yield the transferred DNA which very probably is the F epis~me.~~ Application of electron microscopy to the study of DNA continues to yield important results especially with circular DNA (see below).High-contrast staining techniques have been de~cribed,~ and a micro-procedure has been developed which requires as little as 0.01 pg of nucleic acid.76 The extreme sensitivity of electron microscope observations enabling single molecules of DNA to be seen has been employed to measure the diffusion coefficient of DNA at essentially ‘zero’ c~ncentration.~~ A by-product of ths work worth noting for its practical implications in work with very dilute DNA solutions is the finding that adsorption of DNA to glass was undetectable with detergent- cleaned glass but did occur with glass cleaned by chromic acid and water.77 An elegant method for studying deletion mutations at the level of the DNA molecule has been described:78 if DNAs from a deletion mutant and the wild- type organism are denatured and annealed together the hybrid molecules have a lowered contour length and a ‘bush’ (formed by the wild-type strand which is in effect looped out) visible at the position of the deletion (Figure 6).Thus the position and length of the deleted region can be mapped directly on the DNA molecule. Partial sequences have been determined for the cohesive ends of phage h DNA by use of the DNA polymerase-catalyzed ‘repair’ reaction in which the 3’-ended strands are lengthened by incorporation of nucleotides comple- mentary to those in the protruding 5’-ended strands7’ In this way thirteen residues of dG thirteen of dC seven of dA and seven of dT were added the complementarity implicit in these values is further evidence that the cohesive ends are indeed complementary and the sum shows that each protruding strand is twenty nucleotides long.79 The partial sequence data are shown in Figure 7.Ofthe other phage DNAs known to possess cohesive ends a h-related group will form mixed hybrid concatemers with h DNA and the phages from which these DNAs are derived will ‘help’ h DNA in infectivity assays; these effects are not shared by a h-unrelated group but within that group a similar relationship 74 A. Cohen W. D. Fisher R. Curtis tert. and H. I. Adler Proc. Nut. Acud. Sci. U.S.A. 1968 61 61. 75 C. N. Gordon and A. K. Kleinschmidt Biochim. Biophys. Actu 1968 155 305. 76 H. D. Mayor and L. E. Jordan Science 1968,161 1246.D. Lang and P. Coates J. Mol. Biol. 1968 36 137. 70 R. W. Davis and N. Davidson Proc. Nat. Acad. Sci. U.S.A. 1968,60,243. 79 R. Wu and A. D. Kaiser J. Mol. Biol. 1968 35 523. 6 FIGURE Electron micrograph of a renatured hybrid DNA molecule formed by annealing denatured wild-type A DNA with denatured DNA from a deletion mutant. A bush marking the position of the deletion mutation is visible about half-way along the length of the molecule. c.s.-~.P.5G8 Nucleic Acids 569 between cohesive end complementarity and helper function can be observed." This is the first evidence of biological function of cohesive ends. The h-related and h-unrelated DNAs also differ with respect to the stability of interaction of their cohesive ends.'l A genetic function Ter has been identified which generates the ends of mature h DNA ;it might control the production of a highly specific nuclease.82 (3') (r-strand) (5') PyAG Il A G GGGGGC GC GC,, AA I1 (5') (1-strand) (3') FIGURE 7 Partial nucleotide sequence of the cohesive ends of phage h DNA The structure of a native linear h DNA molecule is repre- sented by two lines representing the l-strand and r-strand with 3'-and 5'-terminal nucleotides identified.The nucleotides added in the presence of DNA polymerase which have been identified are written beyond the ends of the lines. An additional dG residue is located at one of the three caret marks." For many years it has been a puzzle to explain why denatured DNA always seems to retain a few per cent of the transforming activity of native DNA.Compelling evidence has now been obtained that this residual activity is associated with a small proportion of 'naturally occurring' cross-linked DNA molecules which resist the irreversible strand separation characteristie of normal DNA molecules upon denaturation. 83-'5 The cross-links appear to be located at or close to the ends of the molecules and it is suggested84 that they might arise during shear breakage encountered during DNA extraction and purification perhaps by a mechanism similar to that shown in Figure 8. A novel and rigorous approach to problems of helix-random coil transition in polydeoxyribonucleotides is suggested by results obtained with alternating dAT oligomers of known chain length.At low salt concentrations they form one-chain hairpin helices while at higher salt concentrations two-chain helices form which when heated rearrange into hairpins before 'melting' completely into random coils.86 If more than thirty-two nucleotides long they can be coil- verted into covalently closed circles by polynucleotide ligase ; the circles can 8o M. Mandel and A. Berg Proc. Nat. Acad. Sci. U.S.A.,1968,60 265. 81 M. Mandel and A. Berg J. Mol. Biol. 1968 38 137. 02 S. Mousset and R. Thomas Nature 1969 221 242. 03 R. Rownd D. M. Green R. Sternglanz and P. Doty J. Mol. Biol. 1968,32,369; B. M. Alberts and P. Doty ibid. p. 379; C. Mulder and P. Doty ibid.,p. 423. 84 B. M. Alberts J. Mol. Biol. 1968 32 405.'' M. R. Chevallier and G. Bernardi J. Mol. Biol. 1968 32 437. 86 I. E. Schemer E. L. Elson and R. L. Baldwin J. Mol. Biol. 1968,36,291. 570 M. J. Waring Reaction with solvent FIGURE 8 Possible mechanism for formation of cross-links during breakage by shear ofDNA. The percentages indicate the relative weighting of the alternative pathways as suggested by the degree of cross-linking observed in standard DNA preparations. The creation of ion pairs on breakage is assumed C+ indicating a carbonium ion. Free-radical mechanisms are also possible. It should be noted that breakage could require incipient solvolysis in which case no reactive intermediates of any type need be formed.84 form a base-paired helix (with a loop at each end) which is markedly more resistant to thermal denaturation than the helix formed by the equivalent linear oligomer.87 Circular DNA.-Closed circular duplex DNAs with their twisted super- helical structure or supercoils continue to attract attention.One of the chief sources is mitochondria1 DNA from higher organisms which seems always to occur in the form of 5 p twisted circles.88* 89 In lower organisms such as yeast however the situation appears to be different and as yet rather confused heterogeneous linear molecules are often found and sometimes circles.88 -It is suggested that the complexity of the picture might be due to fragmentation of larger perhaps circular molecules. 88r 89 Twisted circular intracellular forms of two more phage DNAs have been fo~nd,~~.~~ and also ‘minicircles’ (extremely small closed circular duplexes) in uninfected ba~teria.~ Bacterial ” B.M. Olivera I. E. Schemer and I. R. Lehman J. Mol. Biol. 1968,36,275. E. F. J. Van Bruggen C. M. Runner P. Borst G. J. C. M. Ruttenberg A. M. Kroon and F M. A. H. Schuurmans Stekhoven Biochim. Biophys. Acta 1968 161,402. 89 ‘Biochemical Aspects of the Biogenesis of Mitochondria,’ ed. E. C. Slater J. M. Tager S. Papa and E. Quagliariello Adriatica Editrice Bari Italy 1968. 90 L. Shapiro L. I. Grossman J. Marmur and A. K. Kleinschmidt J.Mol. Biol. 1968,33,907 C. J. Avers F. E. Billheimer H. P. Hoffmann and R. M. Pauli Proc. Nut. Acad. Sci. U.S.A. 1968 61 90; Y.Suyama and K. Miura ibid. 1968 60,235; G. E. Sonenshein and C. E. Holt Biochem.Biophys. Res. Comm. 1968 33 361 ; D. R. Wolstenholme and N. J. Gross Proc. Nut. Acad. Sci. U.S.A. 1968 61 245. 91 M. Rhoades and C. A. Thomas jun. J. Mol. Biol. 1968,37,41. 92 C. S. Lee N. Davidson and J. V. Scaletti Biochem. Biophys. Res. Comm. 1968 32 752. 93 N. R. Cozzarelli R. B. Kelly and A. Kornberg Proc. Nut. Acad. Sci. U.S.A. 1968,60,992; C. S. Lee and N. Davidson Biochem. Biophys. Res. Comm. 1968 32 151. Nucleic Acids 57 1 sex factors can be isolated in the form of twisted circles ;94 they have been used to study the rate of production of single-strand breaks in DNA by X-irradiation of cells,95 and the method has been calibrated to permit measurement of molecular weights of the DNA of several F’ element^.'^ Estimates of the number of superhelical turns in naturally occurring closed circular duplexes have been made by several methods The values obtained have on the whole been in very good agreement and lead to the conclusion that the number of turns is proportional to the molecular weight of the DNA (Table 2).The method based on untwisting caused by the intercalating drug ethidium bromide”. lo2(see the 1967 Report) remains the simplest and most straight- forward technique. Confidence in its fundamental assumption that intercala- tion of ethidium uncoils the double helix by 12”may be gained from the agree- ment with values derived by other methods (Table 2). Use of ethidium also enables the number and sense of supercoils to be varied over a continuous range at will a facility which can be expected to prove valuable in investigation of the hydrodynamic behaviour of closed circular DNAs.The alkaline denatura- tion method for estimating superhelical turns depends upon the disruption of a small percentage of the base pairs in the DNA which occurs at pH values just below the pH at which nicked or linear molecules are denatured ~ompletely.~~ In this condition both strands are still intact and topologically bonded but the supercoils have been lost.98 Disruption of more base pairs would be expected to lead to superhelix formation in the opposite (left-handed) sense before the molecule collapses into the double-stranded cyclic coil form. These changes in supercoiling have been observed by electron microscopic examination of p~lyoma~~’ and papillomalo6 DNAs after progressive denaturation by heating to various temperatures in the presence of formaldehyde.The initial stages of denaturation seem to occur preferentially in only a few regions the relative positions of which can be mapped.’” Direct electron microscopy of super- coiled DNA also provides an estimate of supercoiling turns since each turn should give rise to a visible ‘crossover’ of the double helix. In early work little confidence was placed in this type of measurement :it is often difficult to be sure 94 D. Freifelder J. Mol. Biol.. 1968 34 31. 95 D. Freifelder J. Mol. Biol. 1968 35 303. 96 D. Freifelder J Mol. Biol. 1968 35 95. 97 L. V. Crawford and M. J. Waring J. Mol. Biol. 1967 25 23. ’* J. Vinograd J. Lebowitz and R.Watson J. Mol. Biol. 1968,33 173. 99 W. Bauer and J. Vinograd J. Mol. Biol. 1968 33 141. loo M. J. Waring Nature 1968 219 1320. H. Bujard J. Mol. Biol. 1968 33 503. L. V. Crawford and M. J. Waring J. Gen. Virology,1967 1 387. lo’ G. J. C. M. Ruttenberg E. M. Smit P. Borst and E. F. J. Van Bruggen Biochim. Biophys. Acta 1968 157,429. lo4 V. C. Bode and L. A. MacHattie J. Mol. Biol. 1968 32 673. M. F. Bourguignon and P. Bourgaux Biochim. Biophys. Acta 1968 169,476. E. A. C. Follett and L. V. Crawford J. Mol. Biol. 1967 28,455. E. A. C. Follett and L. V. Crawford J. Mol. Biol. 1967 28 461; E. A. C. Follett and L. V. Crawford ibid. 1968 34 565; M. F. Bourguignon Biochim. Biophys. Acta 1968 166 242. T ul -4 N TABLE 2 Number of superhelical turns in closed circular duplex DNAs Mol.wt Number DNA Method Solvent (millions) of turns Polyoma 3-2 Ethidium O.OSM-~I%-HC~ -12 97 Alkaline denaturation Buoyant CsCl -15+1 98 SV-40 3-2 Ethidium 1M-NaC1 -16 f3.5 99 g Buoyant CsCl -12.7f1.5 4 Phage +X 174 3.4 Ethidium O.OSM-tris-HC1 -12 100 P B replicative form Crossover count O-~M-NH, acetate (-113 88 f' @Q Bovine papilloma 4.9 Ethidium O.OSM-tris-HC1 -18+3 101 Human papilloma 5.3 Ethidium -20 102 Shope papilloma 5.3 Et hidium -20 102 Chick liver Ethidium O.OSw-tris-HC1 -40 103 10-1 1 acetate (-)35 +6 88 mitochondria Crossover count O-~M-NH Phage h 31 Crossover count Ionic strength 0.06 (-)117 f11 104 intracellular form Crossover count Ionic strength 2.0 (-112 Nucleic Acids 573 of an accurate count partly because it is not always possible to distinguish left- handed from right-handed crossovers.Some workers have however claimed that the sense of crossovers can be seen,91* lo5and at least in two cases agree- ment with the ethidium intercalation method is good (Table 2). Examples are shown in Figure 9. The ratio of sedimentation coefficients of closed circular DNA and 'nicked' circles provides a crude estimate of supercoiling (crude because the ratio is rather insensitive to the number of turns if it is greater than about threeg9) but it has been used to show that the extent of super- coiling is influenced by temperaturelo8 and ionic strengthlo4* '08* log (com-pare also Figure 9). This leads to the important conclusion that the pitch of the DNA double helix varies with salt concentration and temperature.It also suggests a simple explanation for the origin of supercoils i.e. that they arise because the helix pitch increases in response to differences between the environ- ment in which closure was made and that used to study the DNA in uitro. This would be consistent with the finding that the number of supercoils is apparently a function of the length of the DNA (Table 2). It is not certain however that the ionic strength effect alone could account for the observed extent of super- coiling in natural DNAs.lo4 How a closed circular duplex DNA could replicate in uiuo poses an interesting topological problem but replicating circles have been seen.' ' O Catenated circular DNA molecules have now been reported in sea urchin eggs,'" phage h-infected E.c~li,~' and mitochondria from a number of mam- malian tissues. 'l2 The mammalian mitochondrial DNAs all showed one major difference from the leucocyte mitochondrial DNA of patients with chronic granulocytic leukaemia they contained no circular dimers which accounted for as much as 26 % of the complex mitochondrial DNA from leukatmic cells. '' Sedimentation velocity data have been published for various species of catenanes and circular oligomers of mitochondrial DNA; the most surprising result was the finding that catenated dimers of open (nicked) circles sediment slower than the free monomeric circles themselves. '' Binding of Drugs.-A number of drugs most of which are potent anti- tumour agents appear to bind to DNA both in uiuo and in uitro.It is generally believed that their biological effects and occasional therapeutic value may be accounted for by interference with the structure and function of DNA. This topic has been reviewed.'00* 'I4 The mitomycin antibiotics activated by reduc- tion act as powerful bifunctional alkylating agents and form cross-links be- tween the DNA strands :l ''their action is similar to but probably not identical coiled DNA also provides an estimate of supercoiling turns since each turn lo' J. C. Wang D. Baumgarten and B. M. Olivera Proc. Nut. Acud. Sci. U.S.A. 1967 58 1852. log J. A. Kiger jun. E. T. Young,jun. and R. L. Sinsheimer J. Mol. Biol. 1968,33 395.'lo T. Ogawa J.4.Tomizawa and M. Fuke Proc. Nat. Acad. Sci. U.S.A. 1968 60,861; R.H. Kirschner D. R. Wolstenholme and N. J. Gross ibid. p. 1466. 111 L. Pik6 D. G. Blair A. Tyler and J. Vinograd Proc. Nut. Acud. Sci. U.S.A. 1968,59 838. ''' D. A. Clayton C. A. Smith J. M. Jordan M. Teplitz and J. Vinograd,Nature 1968,220,976. 'I3 B. Hudson and J. Vinograd Nature 1969 221 332. G. Hartmann W. Behr K. A. Beissner K. Honikel and A. Sippel Angew. Chern.Internat. Edn.. 1968 7 693. FIGURE Electron micrographs of closed circular h DNA molecules. Examples of twisted 9 (1 28 crossings) and relatively untwisted (22 crossings) circular h DNA mole-cules the former prepared from low-salt solution and the latter prepared from high-salt solution.c.s.-Jp. 573 574 M. J. Waring with the action of nitrogen rnustards'l6 which are of great importance in cancer chemotherapy. Another group of drugs form reversible complexes with DNA by intercalation of their planar polycyclic ring systems between adja- cent base pairs of the double helix. ''' Among these proflavine is important for its role in the production of frame-shift mutants (see the 1966 Report) while ethidium has found valuable application in the study of closed circular duplex DNA (see previously and the 1967Report). Intercalation has recently become of interest in the interaction of nucleosides with polynucleotides (p. 549).A third mode of interaction with DNA is provided by the peptide-containing antibiotic actinomycin D. This drug (I) is much used by molecular biologists Sarcosine Sarcosine L-N-L-N-I \L-Thr/" \L-Thr/o \ / Actinomycin D.The 2-amino-4,6-dimethylphenoxazinone 3-ring system constitutes the chromophore of the molecule the 7-position is indicated. as a specific inhibitor of DNA-dependent RNA synthesis ;''* it is also a powerful inhibitor of certain tumours probably for the same reason. Its binding to DNA is specific for deoxyguanosine residues in double-helical (not denatured or single-stranded) DNA ;moreover it is the 2-amino-group of guanine which is particularly important since actinomycin will bind to a synthetic dAT copolymer containing occasional 2,6-diaminopurine-thyminepairs but not to ordinary dAT copolymer."g The binding of actinomycin to DNA requires the unsubstituted amino-group and quinoidal oxygen of the chromophore.' ' A molecular model which explains these results proposes that the drug molecule attaches itself in the narrow groove of the DNA and forms hydrogen bonds W. Szybalski and V. N. Iyer in 'Antibiotics 1 Mechanism of Action,' ed. D. Gottlieb and P. D. Shaw Springer-Verlag New York 1967 p. 21 1. I l6 P. D. Lawley Prog. Nucleic Acid. Res. and Mol. Biol. 1966 5 89. 'I' L. S. Lerman J. Mol. Biol. 1961,3 18 ;M. J. Waring in 'Biochemical Studies of Antimicrobial Drugs,' ed. B. A. Newton and P. E. Reynolds Symp. SOC. Gen. Microbiol. Cambridge University Press 1966 16 235; A. Blake and A. R. Peacocke Biopolymers 1968 6 1225. E. Reich and I. H. Goldberg Prog. Nucleic Acid. Res.and Mol. Biol. 1964 3 183. E. Reich A. Cerami and D. C. Ward in ref. 115 p. 714. Nucleic Acidr between the chromophore amino-group and quinoidal oxygen and the 2-amino-group N-3 and sugar ring oxygen of deoxyguanosine.''* This model has recently been challenged. The new data revealed hydrodynamic changes associated with actinomycin binding which have hitherto been regarded as characteristic of intercalation ; moreover it is shown that bulky substituents on the 7-position of the chromophore cause profound changes in the kinetics of association and dissociation,12' yet this side of the chromophore is away from the DNA in the narrow groove-binding model. Accordingly an inter- calation model is proposed with the specificity for deoxyguanosine explained by electronic interactions in the x-complex formed in an intercalated struc- ture.I2' It will take a good deal of work to apply crucial tests to distinguish which model is more correct ; preliminary studies indicate that actinomycin affects the supercoils of closed circular DNA in the same fashion as ethidiurn.lz2 This is more readily explicable by the intercalation model ;yet flow dichroism measurements indicate that the plane of the actinomycin chromophore is inclined at an angle of 23 & 5" to the perpendicular to the helix axis; this is more compatible with the narrow groove-binding model.' 23 Thank's are due to N.L. Webb for collating data on sequences of t-RNA and drawing diagrams of t-RNA molecules. L. D. Hamilton. W. Fuller and E.Reich Nature 1963 198 538. W. Miiller and D. M. Crothers J. Mol. Biol. 1968,35 251. 122 M. J. Waring Biochem. J. 1968,10!3,28P. lZ3 M.Gellert C. E. Smith D. Neville and G. Felsenfeld J. Mol. Biol. 1965 11 445.

ISSN:0069-3030    

DOI:10.1039/OC9686500551

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

17 BIOSYNTHESIS By R. Ramage (The Robert Robinson Laboratories The University Oj'Liverpool) THE major concentration of effort in this field during the last year was con- cerned with the genesis and interrelationships of the indole alkaloids together with studies of the mechanisms involved in higher terpenoid biosynthesis. Alkaloids.-There has been a rapid decrease in effort devoted to the bio- synthesis of compounds derived from amino-acids with the exception of the isoprenoid alkaloids which will be discussed separately. In an excellent review' it has been shown that the biosynthesis of aromatic amino-acids is not regulated in the same way by all organisms. Two groups have studied'. the biosynthesis of capsaicin (1)and have showed that phenyl- alanine is the source of the benzylamine residue.The aliphatic moiety was found to be derived from valine. [~-'~C]Valine was incorporated into the terminal isopentylene part of the molecule in accord with the suggestion4 that valine is a precursor of isobutyryl coenzyme A which serves as a starter unit for the production of even-numbered iso-fatty acids. The unusual amino-acid p-aminophenylalanine (2) has been shown to be a precursor of chloramphenicol(4; R = NO,) produced by cultures of Strepto-myces species. [c1-l 4C a-' 'Nlp-Aminophenylalanine (2) was incorporated into the p-nitrophenylserinol moiety with only a small change in the 14C ''N ratio. Specific incorporation of ~~-threo-p-aminophenyl[c~rboxy-'~C]serine (3) and the amino analogue of chloramphenicol (4; R = NH') delineate the late stages of the biosynthesis of chloramphenicol(4; R = NO,) in considerable detail.' A study6V7 of gliotoxin (5) biosynthesis in Trichoderma viride made use of 13C 14C and "N as labelling isotopes.Incorporation of [l-I4C ''Nlphenyl- alanine indicated that phenylalanine only labelled N-5 in gliotoxin (5) but that transamination must also occur. [1-' 3C 3-'4C]Phenylalanine was however incorporated with unchanged isotope dilution showing that the carbon skeleton of phenylalanine remained intact. ["NlGlycine labelled both nitrogen atoms to different extents. F. Lingens Angew Chcm. 1968,350. ' D. J. Bennett and G. W. Kirby J. Chem. SOC. 1968,442. E. Leete and M. C. L. Louden J. Amer. Chem. SOC.,1968,90,6837. P. E. Kolattukudy Science 1968 159 498.' R. McGrath L. C. Vining F. Scala and D. W. S. Westlake Canad. J. Biochem. 1968,46,587. A. K. Bose K. G. Das P. T. Funke I. Kugajevsky 0.P. Shukla K. S. Khanchandani and R. J. Suhadolnik J. Amer. Chern. SOC.,1968,90 1038. ' A. K. Bose K. S. Khanchandani T. Tavares and P. T. Funke J. Amer. Chem. SOC. 1968,90 3593. 578 R. Ramage DL[~-’~C; 3,5-3H,]Tyrosine was fed to ‘Twink’ and ‘Deanna Durbin’ daffodils in an attempt’ to elucidate the relative stereochemistry of in vim protonation and hydroxylation at C-2 in the biosynthesis of norpluviine (6) and lycorine (7; R = OH) respectively. As expected the isolated alkaloids had lost half of the tritium fed but more significantly the biosynthetic lycorine (7; R = OH) had the same tritium content as norpluviine (6).This suggests that both protonation and hydroxylation at C-2 are stereospecific and further that the hydrogen removed on hydroxylation at C-2 is the same as that intro- duced in the formation of norpluviine (6). An intermediate in the biosynthesis of lycorine (7; R = OH) could conceivably be (8) which should yield the alkaloid by allylic rearrangement. This would also explain the result of Wild- man’ who found that caranine (7; R = 3H) stereospecifically labelled with tritium at C-2 was incorporated into lycorine (7; R = OH) with retention of tritium. Further work” on the biosynthesis of mesembrine (9) which has structural NMe (7) (8) (9) I. T. Bruce and G. W. Kirby Chem. Comm. 1968,207. W. C.Wildman and N. E. Heimer J. Amer. Chem. Soc. 1967,89,5265. lo P. W. Jeffs 1.U.P.A.C.Meeting London 1968. Biosynthesis 5 79 similarities to the Amaryllidaceae alkaloids showed that o-methylnorbelladine (10; R' = OMe R2= OH) was not an intermediate. However the isomer (10; R2= OH R2= OMe) was incorporated well into mesembrine (9) in Scelatium stricturn implicating the intermediacy of the spiro-dienone (11) which would be expected to fragment in the manner shown. (10) (11) Investigations"* l2 into the biosynthesis of pellotine (12) in the peyote cactus Lophophoru williamsii have shown the difficulties involved in a detailed examination of more primitive alkaloid structures. It was readily shown that dopamine was incorporated into the tetrahydroisoquinoline ring system however the timing of the hydroxylation of the aromatic ring and the subsequent methylation still remains uncertain.Feeding of [2-14C]acetate led to equal labelling of C-1 and C-9 but [l-'4C]acetate was incorporated better into C-1. [14C]Formic acid was a better precursor and again this produced almost equal labelling at C-1 and C-9. The work failed to identify the source of the 2-carbon unit but indicated the problems involved in the early stages of a1 k alo id bi osyn t he sis . [Il-14C]Tryptophan was administered13 to Nicotiana tabucum and the 6-hydroxykynurenic acid (13) isolated was shown to have the label in the carboxy-group. The indolenine peroxide (14) was postulated as a likely inter- mediate. The biosynthesis of psilocybin (15) was studiedI4 in Psilocybecubensio and the results indicate that hydroxylation of NN-dimethyltryptamine is the penultimate step.N-Acetyltryptamine (16) has been shown" to be the pre- cursor of harman (17) in PassiJloru edulis. l1 A. R. Battersby R. Binks and R. Huxtable Tetrahcdron Letters 1968,6111. l2 J. Lundstrom and S. Agurell Tetrahedron Letters 1968,4437. l3 M. Slaytor L. Copeland and P. K. Mac Nicol Phytochernistry 1968 1779. l4 S. Agurell and J. L. G. Nilsson Acta Chem. Scad. 1968,22 1210. is M. Slaytor and I. J. McFarlane Phytochemistry 1968 605. 580 R.Ramage Anthranilic acid and phenylalanine have been provenI6 to be the structural units of aborine (18) in Glycosrnis artoreu. Further r3H]anthranilic acid was found to be the precursor of ring A in the acridine alkaloid aborinine (19).A study of quinazoline alkaloid formation in Peganurn harnala revealed" that vasicine (20) was synthesised in uiuo from anthranilic acid and putrescine (21; R = H). ''N Studies showed that both the CI and 6 amino-functions of ornithine (21; R = C02H) were incorporated to the same extent suggesting the symmetrical intermediate (21 ;R = H). N-Methylisopelletierine (22 ; R = Me) formation in Sedurn sarrnentosurn has been found" to involve lysine. [6-I4C; 4,5-3H2]Lysine was utilised with the 3H :14C ratio unchanged and specific incorporation of 14C into C-6 of the alkaloid. This indicates a nonsymmetrical intermediate in the biosynthesis of N-methylisopelletierine (22; R = Me) unlike the degradation of ornithine to putrescine discussed earlier.[l-14C]Acetate was found' to be incorporated into the 2-position of the side-chain in (22; R = Me). Dimerisation of pelle-tierine (22; R = H) has been implicated" in the biosynthesis of lycopodine l6 D. Groger and S. Johne 2.Naturforsch. 1968,236 1072. 17 D. Liljegren Phytochemistry 1968 1299. l8 R. N. Gupta and I. D. Spenser Chem. Comm. 1968,85. l9 D. G. O'Donnovan and M. F. Keogh Tetrahedron Letters 1968,265. R. N. Gupta M. Castillo D. B. MacLean I. D. Spenser and J. T. Wrobel J. Amrr. Chem. Soc. 1968,90 1360. Biosynthesis 581 (23) in Lycopodium flabelliforme. Incorporation of [2-14C]- and [6-14C]-lysine into lycopodine (23) disposed of the idea that the lycopodium alkaloids are polyketide in origin.If the lysine were transformed into a symmetrical inter- mediate the lycopodine would be labelled as shown i.e. the carbonyl carbon would contain 25 % of the 14C activity. This was found to be the case which is interesting in view of the specific incorporation of [6-'4C]lysine into N-methylisopelletierine(22;R = Me) discussed previously. Isoprenoid Alkaloids.-The discovery2'-' that loganin (24) is a progenitor of the indole and ipecacuanha alkaloids has stimulated much effort in the later stages of the biosynthesis of these classes of alkaloids. Loganin was however never considered to be the actual unit which combined with trypta- mine or dopamine. Further oxidative processes namely cleavage of ring A to give the aldehyde (25) would have to be involved before such a union could be achieved in uiuo or in vitro.Proof of this came as a result of both structural .O Gluc. C.0 Me d02Me (27) a-@Me (28) \N OAc Me HO C02Me (29) 21 A. R. Battersby R. S. Kapil J. A. Martin and Mrs. L. Mo Chem. Comm. 1968 133. 22 P. Loew and D. Arigoni Chem. Comm. 1968,137. 23 A. R. Battersby and B. Gregory Chem. Comm. 1968 134. 582 R. Ramage elucidation of trace alkaloids and ultimately tracer methods. The E.T.H. group showed24 the glycoside foliamenthin ex. Menyanthes trfoliata to have the structure (26a) easily recognisable as a derivative of the elusive aldehyde (25). In keeping with this structure [4-14C] geraniol was incorporated into foliamenthin (26a) in Menyanthes trijoliata.Another important glycoside menthiafolin was identified25 as (26b) and [2-14C]geraniol feeding produced menthiafolin (26b) in which the 14C label was incorporated into the ester and lactol moieties in the rxtio 3:l. Menthiafolin (26b) thus gave a source of synthetic and biosynthetic secologanin (25) which was exploited26 in an elegant synthesis of ipecoside (27). That secologanin (25) was indeed the building block of the indole alkaloids was shown by feeding [O-r~thyl-~Hl- secologanin (25) to Vinca rosea. This afforded the following alkaloids with the corresponding incorporations of 14C ajmalicine (28) 0.55 % ; vindoline (29) 0.12 % ; catharanthine (30) 016 % ; and perivine (31) 0.13 %. The incorpora- tion2' of sweroside (32) into vindoline (29) in Vinca rosea and also28 reserpine and quinine probably proceeds by oxidation to secologanin (25).However since the mechanism of the transformation of loganin (24) to secologanin (25) is as yet unknown this result may have some deeper significance. The indole counterpart of ipecoside (27) would be expected to have structure (33) which has been assigned without stereochemical detail to a new alkaloid strictosidine isolated29 from Rhazia stricta and R. orientalis. [O-methyL3H]-Loganin (24) was incorporated3' efficiently into strictosidine (33) showing it to be an important intermediate in indole alkaloid biosynthesis. Condensation of [O-methyl-3H]secologanin (25) with tryptamine afforded31 the expected p-carbolines (33) epimeric at position 5.This mixture was shown to be efficiently 24 P. Loew Ch. V. Szczepanski C. J. Coscia and D. Arigoni Chem. Comm. 1968 1276. " A. R. Battersby A. R. Burnett G. D. Knowles and P. G. Parsons Chem. Comm. 1968 1277. " A. R. Battersby A. R. Burnett and P. G. Parsons Chem. Comm. 1968 1280. "l H. Inouye S. Ueda and Y. Takeda Tetrahedron Letters 1968,3453. 28 H. Inouye S. Ueda and Y.Takeda Tetrahedron Letters 1969,407. 29 G.N. Smith Chem. Comm. 1968,912. 'O R. T. Brown G. N. Smith and K. S. J. Stapleford Tetrahedron Letters 1968,4349. A. R. Battersby A. R. Burnett and P. G. Parsons Chem. Comm.,1968 1282. Biosy n thesis 583 incorporated into a ajmalicine (28) vindoline (29) catharanthine (30) and perivine (31).With doubly labelled p-carbolines (33)prepared from [O-rnethyl-3H]secologanin (25) and [3H]tryptamine it was shown that biosynthesis occurred without a change in the 3H:14C ratio.By dilution techniques it was shown that both p-carbolines and secologanin are present in Vinca rosea. C0,Me Another area of indole alkaloid biosynthesis which received much attention was the genesis of the three structural types corynanthe (34) aspidosperma (35) and iboga (36). The ready isomerisation of the Aspidosperma alkaloid (-)-tabersonine (37) to the Iboga alkaloid (+)-catharanthine (38) is thought32 to go via the intermediate (39) which could also be a transformation product of the alkaloid stemmadenine (40). This led to stemmadenine (40) being postulated 32*33 as a key intermediate for Aspidosperma and Iboga alkaloids.It is interesting to note that the alkaloids named secamines (41) isolated34 from Rhazia stricta may be regarded as dimers of (39) or closely related struc- ture. There are plausible pathways for the formation of stemmadenine (40) from the Corynanthe alkaloid geissoschizine (42). Acid-catalysed rearrange- ment3’ of (42) afforded catharanthine (30) together with pseudocatharanthine (39) (40) 32 A. A. Qureshi and A. I. Scott Chem. Comm. 1968,945. 33 J. P. Kutney C. Ehret. V. R. Nelson and D. C. Wigfield J. Amer. Chem. SOC. 1968,90 5929. 34 D. A. Evans G. F. Smith G. N. Smith and K. S. J. Stapleford Chem. Comm. 1968,859. 35 A. A. Qureshi and A. I. Scott Chem. Comm. 1968,947. 584 R. Ramage Me (42) OH (43). The latter can be considered to be derived from (44),an intermediate of the Strychnos alkaloid type.A novel approach36 to the elucidation of the late stages of indole alkaloid biosynthesis employed short-term germination (43) (44) of Vinca rosea seeds. The sequence of alkaloid formation was found to be Corynanthe Aspidosperma and Iboga. [O-methyL3H]Sternmadenine was incorporated well into catharanthine (30) and vindoline (29); similarly [O-methyL3H]tabersonine was found to be efficiently transformed into catharan- thine and vindoline. However cathatanthine was not incorporated into vindoline thus showing the irreversible nature of the change from Aspido- sperma to Iboga alkaloids. It was also found that specific incorporations of Corynanthe precursors in Vinca rosea were much higher in germinating seedlings than in the whole plant.By usine 6-month old Vinca rosea plants K~tney~~ also found that labelled tabersonine (37) was incorporated into catharanthine (30) and vindoline (29). The incorporation of DL-[~-'~C]-tryptophan into vincadine (45) and vincadifformine (46) was studied33 over different time intervals and the results obtained indicate that there is no direct biosynthetic relationship between the two alkaloids in spite of the smooth in vitro conversion of (45) into (46). 36 A. A. Qureshi and A. I. Scott Chem. Comrn. 1968,948. 37 J. P. Kutney W. J. Cretney J. R. Hadfield E. S. Hall V. R. Nelson and D. C. Wigfield J. her. Chem. Soc. 1968,90,3566. Biosynthesis 585 1soprenoids.-Monoterpenes.A of the biosynthesis of (+)-and ( -)-camphor (47) by using [2-'4C]mevalonate produced the interesting result that all the 14C was incorporated into the 6 position of camphor. Normal union of two C-5 units would have led to additional labelling at positions 8 and 9. This result agrees with earlier work3' on thujone (48) biosynthesis where [2-14C]me~alonate was incorporated specifically at the position shown. 0 9e (48) Obviously more work. must be done on the initial stages of terpene for- mation in the higher plants. Isopentenyl pyrophosphate isomerase has been isolated from liver.40 It was found to be activated by Mn2+ ions and the equilibrium between isopentenyl pyrophosphate and dimethylallyl pyro- phosphate favoured the latter.[2- ''C]Mevalonate feeding4' to Santolina chamaecyparissus gave a very low incorporation into artemesia ketone (49). The biosynthesis of nepetalactone (50) in Nepeta cataria L. was studied42 by using [2-'4C]mevalonate and it (49) 38 D. V. Banthorpe and D. Baxendale Chem. Comm. 1968,1553. 39 D. V. Banthorpe and K. W. Turnbull Chem. Comm. 1966,177. 40 P. W. Holloway and G. Popjak Bochom. J. 1968,106 835. 41 G. R. Waller G. M. Frost D. Burleson D. Brennon and L. H. Zalkow Phytochemistry 1968 213. 42 F. E. Regnier G. R. Waller. E. Z. Elsenbraun. and H. Anda Phytochmistry 1968 7 221. 586 R. Ramage was found that positions 3 8 6 and 9 had 36 17,29 and 18 % of the activity respectively. This indicates considerable randomisation at the isopentenyl stage.A cell-free system has been prepared43 from Mentha piperita which can convert pulegone (51) to menthone (52) and isomenthone (53) in the presence of NADPH,. [l-'4C]Geranyl pyrophosphate was tran~formed~~ @ GoMe& into cineole (54) in Rosrnarunus oficinalis 2nd degradation gave the expected equal labelling so shown. Qo Me Me Me Me Me Me (54) (51) (52) (53) Sesquiterpenes. In vitro acid-catalysed transformations4' of the epoxides (55) and (56) wlll undoubtedly have relevance to future biosynthetic studies in the endesmane and guaiane series. Rearrangement of (55) afforded (57) and (58) which is explicable in terms of a strict application of the Markownikoff rule; the epoxide (56) however gave the guaiane-type (59) predicted on steric grounds.This delicate balance between steric and electronic effects can be expected to play at least as important a role in sesquiterpene biosynthesis as in the biological transformations of 2,3-oxidosqualene leading to triterpenoids. The biosynthesis of ~antonin~~ (60) in Arternesia rnaritirna gave very low incorporations of all precursors including mevalonate and [l-3H]farnesol showing the great difficulty of transporting the precursor to the site of synthesis. Incorporation of the lactones (61) and its double-bond isomer indicate that the introduction of oxygen into ring A is a late step. Diterpenes. [l-3H]Geraniol pyrophosphate was incorporated specific- 48 into rosenonolactone (62) in Tricothecium roseurn.Administration of 43 J.Battail A. J. Burbott and W. D. Loomis Phytochemisrry 1968 1159. 44 B. Achilladelis and J. R. Hanson Phytochemistry 1968. 1317. 45 E. D. Brown and J. K. Sutherland Chem. Comm. 1968. 1060. 46 D. H. R. Barton G. P. Moss,and J. A. Whittle J. Chem. SOC.(C) 1968,1813. 47 B. Achilladellis and J. R. Hanson Phytochemistry 1968 589. 48 B. Achilladellis and J. R. Hanson Tetrahedron Letters 1968,4397. Biosynthesis 587 Me 4R-[4-3H 2-'4C]mevalonolactone afforded (62) with an unchanged 3H :14C ratio. Degradation placed the 3H labels as shown thus verifying the postu- lated4' hydrogen migration from C-9 to C-8 during biosynthesis and also eliminated the lactone formation via a A59 rosadiene intermediate. A studys0 of the biosynthesis of viridin (63) showed that the furan carbon indicated was not labelled by [2-14C]mevalonate and presumably arose by oxidation of a 3a-methyl group.[7-3H]Kaur-16-en-19-oic acid (64; R = H) was founds1 to be an effective precursor of steviol (64; R = OH) in Stevia rebandiana suggesting bridgehead hydroxylation as the final biosynthetic step. In a further investigation5 into the biosynthesis of tetracyclic diterpenes the Sussex group fed 4R-[4-'HH 2-'4C]mevalonate to Gibberella fujikuroi and isolated labelled gibberellic acid (65) plus 4,18-dihydroxykaurenolide(66). The 3H labels in the biosynthetic diterpenes were shown to be at the positions indicated and the P-CH,OH grouping in (66) found to be derived from [2-14C]mevalo- nate. Since 4R-[13H]mevalonate should give 3H at C-2 of gibberellic acid (65) in the P-configuration it follows that hydroxylation at this centre must have occurred with inversion.Retention of mevalonate-derived hydrogen at C-4b and C-lOa in (65) excludes the formation of either 4a 4b- or 4a 10a-double- bonds during loss of the angular methyl group and lactone formation. Also retention of the 3H at C-9 in (66) rules out any intermediacy of pimara-8,9- diene (67) during the biosynthesis of tetracyclic diterpenes of the kaurane type. In order to determine whether pimara-7,8-diene was in fact involved [Z3H, 49 A. J. Birch R. W. Richards H. Smith A. Harris and N. B. Whalley Tetrahedron 1968 7,241. M. M.Blight J. J. W. Coppen and J. F. Grove Chem. Comm. 1968,1117. J. R. Hanson and A. F.White Phytochemistry 1968,595. '2 J. R. Hanson A. Hough and A. F. White Chem. Comm. 1968,467. 588 R. Ramage Me 10a HO 10 OH Me 98 C0,H (65) (66) (67) 2-'4C]mevalonate was fed5 to Gibberellafujikuroi which should label position 7 of kaurene (68) nonstereospecifically with 3H.No loss of 3H from this position was observed which eliminated pimara-7,g-diene as a precursor. Pimara- 8,14-diene was specifically incorporated into the tetracyclic diterpenes (65) and (66) although with low efficiency. In studiess4. 55 designed to determine the oxidative sequence leading from a tetracyclic gibberane skeleton to gib- berellic acid (65) structure (69) and gibberellin A14 (70) were found to be Me Me 8Me Me (68) 'OZR CHO effective precursors of gibberellic acid (65) and gibberellin A13 (71).The latter was found not to be an intermediate in the biosynthesis of gibberellic acid (65). These workers also synthesised (69) by base treatment of (72). Bearing in mind the oxygenation pattern of (66) which co-occurs with gibberellic acid (65) this transformation must be closely analogous to the biosynthetic pathway. 53 J. R.Hanson and A. F. White Chem. Comm. 1968 1689. 54 B.E. Cross R. H. B. Galt and K. Norton Tetrahedron 1968,24,231. 55 B.E. Cross K. Norton and J. C. Stewart J. Chem. SOC.(C),1968 1054. Biosynthesis 589 Steroids and Triterpenes. Investigations into the reductive dimerisation of two CI5 units in the biosynthesis of squalene have shown that thiamine pyrophosphate is implicateds6 in the formation of squalene.Stereoisomers having structure (73) assigned to an isolated intermediate in squalene bio- synthesiss7 were synthesised” and shown to be different from the natural material. H R (74) 0& RR (77) In order to elucidate details of substrate specificity of 2,3-epoxysqualene cyclase unnatural epoxides (74) were treated with 100,OOO g. supernatant preparation of rat liver microsomes.s9~60 Only the trans-oxide (74; R1= Me ” G. E. Risinger and H. D. Durst Tetrahedron Letters 1968,3133. ” H. C. Rilling J. Bid. Chem. 1966,241 3233. ’* E. J. Corey P. R. Ortiz de Montellano Tetrahedron Letters 1968 5113. ” R. B. Clayton E. E. van Tamelon and R. G. Nadean J. Amer. Chem. Soc. 1968,90 820. 6o E.J. Corey K. Lin and M. Jantelar J. Amer. Chem. Soc. 1968,90. 2724. 590 R.Ramage R2 = H) yielded the 4-desmethyllanosterol analogue (75 ;R' = Me R2 = H). [23-14C]29,30-Bisnor-2,3-epoxysqualene(76; R' = H R2= R3= Me) was treated with a particle-free solution of 2,3-epoxysqualene-amyrincyclase from pea seedlings6' and gave 29,30-bisnoramyrin (77; R = H). Although the ion (78 ;R = Me) has been proposed62 as an intermediate in p-amyrin (77 ;R = Me) biogenesis it would appear unlikely that the corresponding primary carbonium ion (78 ;R = H)would be involved in the bisnor series. In an attempt to separate the cyclisation and rearrangement processes involved in sterol biosynthesis the bisnor-2,3-epoxysqualene(76; R' = Me R2 = R3= H) was treated with the cyclase enzyme system.63 One ofthe important driving forces causing rearrange- ment of the primary cyclic intermediate (79; R' = R2 = Me) to lanosterol (75; R' = R2 = Me) is the relief of repulsive interactions in ring B (twist boat) R HO (79) (80) due to the methyl group at C-8.In the bisnor intermediate (79; R' = R2= H) no such interactions exist and not surprisingly the product of cyclisation was found to be (80) formed by deprotonation of (79; R' = R2 = H). Thus the important function of the enzyme is the cyclisation stage after which the relative stabilities of carbonium ions control the product formation. Another interesting example which showed the importance of the C-8 methyl group in (79) causing rearrangement to the lanostane skeleton was given by treatment of 15-nor-2,3-epoxysqualene (76; R2= H R' = R3= Me) with 2,3-epoxysqualene- lanosterol ~yclase.~~ The product (75; R' = H R2 = Me) was shown not to E.J. Corey and S. K. Gross J. Amer. Chem. SOC. 1968,90,5045. A. Eschenmoser L.Ruzicka 0.Jeger and D. Arigoni Helu. Chim. Acta 1955,38 1890. 63 E. J. Corey P. R. Ortiz de Montellano and H. Yamamoto J. Amer. Chem. SOC.,1968,90,6254. 64 E.E.van Tamlen R. P. Hanzlik K. B. Sharpless,R. B. Clayton W. J. Richter and A. L. Burlin-game J. Amer. Chem. SOC.,1968,90,3284. Biosynthesis 591 have incoporated 3H from the medium hence cyclisation and subsequent rearrangement of the 15-nor series is identical to that occurring in 2,3-epoxy- squalene. HO' In a study6' of the biosynthesis of the antibiotic fusidic acid (81),in Fusidiurn coccineum it was shown that 2,3-epoxysqualene was incorporated intact confirming the earlier work66 with [2-14C]mevalonate.The formation of helvolic acid (82) in Cephalosporiurn caerulens has been shown6' to involve 3~-hydroxy-4-~-hydroxymethylfusida-l7(20)[ 16,20-cis]24-diene (83). In the transformation of (83) to helvolic acid (82) the 4P-hydroxymethyl group is eliminated and thus gives a clue to the possible mechanism and sequence of demethylation of 4 :4-dimethyl sterols. An investigation6' designed to give more information on this problem revealed that 4P-methyl-4a-hydroxymethyl-cholestanol (84 ; R' = CH20H R2 = Me) and 4a-hydroxymethylcholestanol (84; R' = CH20H R2 = H) were converted efficiently into cholestanol (84; R' = R2 = H) by rat liver homogenates.However in contrast to the helvolic acid biosynthesis it was found that. 4a-methyl-4P-hydroxymethyl-cholestanol(84; R' = Me R2 = CH20H) was not transformed into cholestanol (84; R' = R2 = H). These results indicating initial removal of the 4a-methyl group should be compared with earlier on the biosynthesis of choleste- rol from [2-14C]mevalonate which suggested that the 4P-methyl group is the first to be eliminated as in the case of helvolic acid (82). The idea that cycloartenol (85) is the first product of cyclisation and re- arrangement of 2,3-epoxysqualene is supported by the incorporation of 2,3-epoxysqualene into cycloartenol (85) in a cell-free system from newly '' W.0.Godtfredsen H. Lorck E. E. van Tamelen J. D. Willett and C. B. Clayton J. Amer. Chem. SOC.,1968,90,208. 66 D. Arigoni Conference on the Biogenesis of Natural Products Academia Nzlzionale dei Lincei Rome 1964. '' I. Okuda Y. Sato T. Hattori and H. Igarashi Tetrahedron Letters 1968,4769. 68 K. B. Sharpless T. E. Snyder T. A. Spencer K. K. Maheshwari G. Guhn and R. B. Clayton J. Amer. Chem. SOC.,1968,90,6874. 69 J. L. Gaylor and C. V. Delwiche Steroids 1964 4 207. 592 R. Ramage HO developed bean leaves.” The biosynthesis of (85) was studied71 by using 4R[2-14C 4-3Hl]mevalonate and it was found that the 3H:14C ratio in cycloartenol (85) was the same as in the squalene isolated. This indicates a hydrogen migration from C-9 to C-8 with concomitant cyclopropane-forma- tion ; this eliminates lanosterol (86) as an intermediate in the biosynthesis of cycloartenol(85).2,3-Epoxysqualene was shown to be a precursor of lano- sterol (86)in yeast and thus the low turnover of lanostadiene (8,24) to lanosterol must be due to hydroxylation of an unnatural precursor.72 The remarkable triterpene tetrahymanol (87) which is a metabolite of the protozoan Tetrahymena pyricforrnis has been shown7 3*74 to be produced by acid-catalysed cyclisation of squalene terminated by nucleophilic attack at C-21 rather than from 2,3-epoxysqualene. Since deoxytetrahymanol has C2 symmetry both biosynthetic routes were possible. Elimination of the 14~-methyl group of lanosterol (86) in the biosynthesis of sterols is accompanied by stereospecific removal of the 15a-hydr0gen.~ 5-77 4,4-Dimethyl-5a-cholesta-8,14-dien-3~-ol(88) produced by such a transforma- tion has been shown7* to be an intermediate between lanosterol (86) and 70 H.H. Rees L. J. Goad and T. W. Goodwin Tetrahedron Letters 1968 723. 71 H. H. Rees L. J. Goad and T. W. Goodwin Bochem. J. 1968,107,417. 72 D. H. R. Barton A. F. Gosden G. Mellows and D. A. Widdowson Chem. Comm. 1968 1067. 73 E. Caspi J. B. Greig and J. M. Zander Bochem. J. 1968,109,931. l4 E. Caspi J. M. Zander J. B. Greig F. B. Mallory R. L. Couner and J. R. Landrey J. Amer. Chem. SOC. 1968.90.3563. 75 L. Canonica H. Fiecchi M. G. Kienle H. Scala G. Galli E. G. Paoletti and R. Paoletti J. Amer. Chem. SOC., 1968,90,3597. 76 G.F. Gibbons L. J. Goad and T. W. Goodwin Chem. Comm. 1968 1458. 77 E. Caspi J. B. Greig P. J. Ramm,and K. R. Varna Tetrahedron Lettcrs 1968,3829. 78 L. Canonica A. Fiecchi M. Gallikiente A. Scale G. Galli E. Grossi E. G. Paoletti and R. Paoletti J. Amer. Chem. SOC. 1968,90 6532. Biosyn thesis 593 (87) (88) HO cholesterol (89) using rat liver homogenates in the presence of oxygen. Under anaerobic conditions the conversion of (88) is stopped at C-29-A8-sterols (90; R = Me). The biological conversion of 5a-cholest-8-en-3P-01 to 5a-cholest-7-en-3P-01 during biosynthesis of cholesterol (89) has been shown to involve elimination of the 7fLhydrogen. No migration of hydrogen from C-7 to C-9 was observed ;799 8o the hydrogen attached to C-9 in (91) being acquired from the medium.The remaining steps between (91) and cholesterol (89) involve introduction of a A5-double bond to give (92) followed by reduction of the A'-double-bond. cis-Elimination of the 5a-and 6a-hydrogens have been sh~wn,~'-~~ to be involved in the formation of the As-double-bond. In agree- ment with this the formation of the phytosterol periferasterol (93) in Ochro-monas malhamensis has been shown,84 by feeding 3R-[2-14C (5R)-R-3Hl]-mevalonate to involve elimination of the 6a-hydrogen and also the 23-pro-R- hydrogen. A study of the biological reduction of (92) to cholesterol revealed that the 8P-hydrogen in cholesterol was obtained from the medium and the 7a-hydrogen from NADH. The removal of the C-19 methyl group in the biological conversion of androsterone (94; R = Me) to oestrone (95) has been showng5 to involve the 19-hydroxymethylandrosterone(94; R = CH,*OH) and the 19-formylandrosterone (94; R = CHO).Polyketide-derived Compounds.-Bohlmann has studied the biosynthetic relationships of many natural acetylenes. The biosynthesis of phenylhep- 79 L. Canonica A. Fiecchi M. Gallikienle A. Scala G. Galli E. Grossi E. G. Paoletti and R. Paoletti Steroids 1968 287. M. Akhtar and A. D. Rahimtala Chem. Comm. 1968,259. M. Akhtar and S. Marsh Biochem. J. 1967,102,462. S. Dewhurst and M. Akhtar Biochem. J. 1967,105 1187. 83 A. M. Paliokes and G. J. Schroepfer J. Biol. Chem. 1968,243,453. 84 A. R. H. Smith L. J. Goad and T. W. Goodwin Chem. Comm. 1968 1259. M. Akhtar and S.J. M. Skinner Biochem. J. 1968,109 318. 594 R.Ramage HO (94) (95) tatriyne (96) was investigated86 by feeding [2,3-3H2 14-14C]tetradec-5-en-8,10,12-triyn-l-ol (97) to Coreopsis lanceolata L. A possible intermediate (98) would result in the loss of all 3H at C-3 but this is not observed. The origin of the thiomethyl grouping in (99) and (100) was studied87 by using C3H 35S]-methionine which was administered to Anthemis tinctoria L. Isolated esters (99) and (100) had a different 3H:35Sratio from the methionine fed hence the sulphur and methyl group were donated independently to matricaris ester (101) which was also shown to be a precursor of (99) and (100) in Anthemis tinctoria. In order to investigategg the formation of the highly unsaturated hydrocarbons (102) (103) and (104) in Centaurea dilute L.the alcohol (105) was fed and found to be incorporated into the hydrocarbons. This gives some idea of the sequence of triple-bond formation and it was suggested that the terminal olefinic linkage is formed by breakdown of the P-hydroxy-acid intermediate (105). A startg9 has been made in the isolation of the important 86 F. Bohlmann H. Bonnet and K. Jente Chem. Ber. 1968,101,855. '' F. Bohlmann and T. Burkhardt Chem. Ber. 1968,101 861. F. Bohlmann M. Wolschokowsky J. Laser C. Zdero and K. D. Bach Chem. Ber. 1968,101 2056. Biosynthesis 595 enzymes concerned with the biosynthesis of naturally occurring acetylenes with the conversion of oleic acid (106)into (107)with a cell-free system.Linoleic acid (109) had earlierg0 been shown to be an intermediate in the biosynthesis of crepenynic acid (108).It was also proven that neither the diol nor epoxide was an intermediate in the conversion of (109) into (108). Ph[-],Me (96) Me[Cx] 3-CH *CHLCH. [CH,] .CH2 OH (97) (98) MeC=C.CHACH.C==CH-CH=kH*CO,Me $Me (99) 09 F. Bohlmann and H. Schulz Tetrahedron Letters 1968,4795. 90 F. Bohlmann and H. Schulz Tetrahedron Letters 1968 1801. 596 R.Ramage Me [kC] ,CH= w (107) Me[CH,] C=C* CH CH=CH. [CH2] CO,H (108) MeCCH,] .H&H CH,CH=CH * [CH,] * CO H (109) There has been a reviewg1 of the prostaglandins and much work on the biosynthesis of this important class of biologically active substances.The mechanism of the conversion of eicosa-8,11,14-trienoic acid (110) into prosta- glandin El (111) and prostaglandin F1a (112) was studiedg2 with [13D-3H 3-14C]- and [13L-3H 3-14C]-eicosca-8,1 1,14-trienoic acid. The hydrogen lost from C-13 was shown to have the L-configuration and (112) was shown not to have involved (111) as an intermediate. It is suggested that the loss of hydrogen from C-13 leads to the intermediate (113) which can form the cyclic peroxide (114) by a concerted process. Reductive fission of the peroxide would yield prostaglandin F a (112) and oxidative cleavage would afford prosta- glanding El (111). Additional evidence for the cyclic process was providedg3 by the biosynthesis of 12-hydroxyheptadeca-8,lO-dienoicacid (115) and malondialdehyde from eicosa-8,11,14-trienoic acid The fragmentation may be formally represented as a cyclic process.Biosynthesis of 8-isoprostaglandin El 94 (1 11 epimer at C-8) involves isomerisation of prostaglandin El (111). Earlier workgs on the biosynthesis of glauconic acid (116) had shown the pathway involved dimerisation of (117). Further studies96 strongly suggest the 91 S. Bergstrom and B. Sammelsson Science 1968 109. 92 M. Hamberg and B. Sammelsson. J. Biol. Chem. 1967,242,5336. 93 M. Hamberg and B. Sammelsson J. Biol. Chem. 1967,242 5344. 94 E. G. Daniels W. C. Krueger F. P. Kupiecki J. E. Pike and W. P. Schneider J. Amet. Chem. Soc. 1968,90 5894. " C. E. Moppett and J. K. Sutherland Chem. Comm. 1966,772. 96 J. L. Bloomer L. E. Moppett and J.K. Sutherland J. Chrm. SOC. (C),1968 588. Biosynthesis 597 COOH C,-dicarboxylic acid oxalacetic acid as the source of C-8 C-6 and C-7 in (1 17) the remainder being polyketide in origin. The aliphatic lichen acid proto- lichesterinic acid (118) has been shownQ7 to be derived from a C-16 polyketide unit together with a C-3 fragment from another source. An investigationg8 of the synthesis of triacetic acid lactone (119;R = H) by pigeon liver fatty acid synthetase showed that (119;R = H) was formed in the absence of TPNH but that palmitic acid was obtained in the presence of TPNH. Although methyltriacetic lactone (119; R = Me) is not incorporated into stripitatic acid (120) in P. Stipitatum it has been foundQ9 that [14C]-formate is a source of C-1 units for both structures at the positions indicated.These results indicate that the hydroxy-pyrones are free forms of enzyme-held polyketides which are true tropolone precursors and that addition of the C-1 unit occurs at the polyketide level. By using [2-' 3C] -,[1-' 3C]-acetate and [ 3C]formate to study the biosynthesis of sepedonin (121) it was found that formate labelled C-8 specifically in agreement with the previous example of stipitatic acid (120). loo Studies of the biosynthesis of terreic acid (122) indicatelo' that it is formed from 6-methylsalicyclic acid in Aspergillus terreus the epoxide oxygen being 97 J. L. Boomer W. R. Eder W. F. Hoffmann Chem. Comm. 1968,354. 98 J. E. Nixon G. R.Puty and J. W. Porter J. w'ol. Chem. 1968 5471.99 G. S. Man and S.W. Tananbaum J. Amer. Chem. SOC.,1968,90,5302. loo A. G. McInnes D. G. Smith L. C. Vining and J. L. C. Wright Chem. Comm. 1968 1669. G. Read and L. C. Vining Chem. Comm. 1968,935. 598 R. Ramage Me (121) derived from the atmosphere. The very remarkable natural product giorosein (123) which prefers not to assume an aromatic structure has been shownlo2 to be produced by Gliocladiurn roseurn viu (124) and the quinone (125). "'0"' 0 HOOMe 6:: 0 Me0 "//,Me HO\ HO ' 'Me 0 CHO 0 (123) (124) (125) N ~ OH zOH :OH OH H 2 Early worklo3 on the biosynthesis of tetracycline had shown that 6-methyl- pretetramid (126) was a precursor of 7-chlorotetracycline. Recent worklo4- lo6 in this field employed mutants in order to identify the intermediates.In this way the anthraquinone (127) was isolated however on treatment with a tetracycline producing organism it was shown to be a shunt-product from the main pathway. Another such metabolite was (128) in which the 6-methyl group was introduced at an early stage before cyclisation of the tetracene system was complete. It is interesting to note that the oxygen at C-8 is already missing even at this relatively early stage of the biosynthesis. The biosynthesi~'~~-'~~ of lo2 M. W. Steward and N. M. Packter Bochcm. J. 1968,109 1. lo3 J. R. D. McCormick S. Johnson and N. 0.Sjolander J. Amer. Chem. SOC.,1963,85,1692. lo4 J. R. D. McCormick and E. R. Jensen J. her. Chem. SOC.,1968,90,7126. lo5 J. R. D. McCormick E.R. Jensen N. H. Arnold M. S. Carey H. H. Joachim S. Johnson P. A. Miller and N. 0.Sjolander J. Amer. Chem. SOC.,1968,90 7127. Io6 J. R. D. McCormick E. R. Jensen S. Johnson and N. 0.Sjolander,J. Amer. Chem. SOC.,1968 90 2201. lo' M. Biollaz G. Buchi and G. Milne J. Amer. Chem. SOC.,1968,90 5017. Io8 M. Biollaz G. Buchi and G. Milne J. Amer. Chem. SOC.,1968,90,5019. lo9 J. A. Dunkerslott D. P. H. Hsieh and R. I. Matelas J. Amcr. Chem. SOC. 1968 90,5020. Biosynthesis 599 (127) 0 45 (129) aflatoxin B (129) has been studied with [1-14C]- and [2-14C]-acetate feedings to Aspergillusflauus. One fascinating result which emerged was that C-11 and C-14 are both derived from [2-'4C]acetate. A very interesting biogenetic route was proposed'08 involving a tetracene (130; R = H or OH) similar to that involved in tetracycline biosynthesis.The closely related metabolite of Asper-gillus versicolor sterigmatacystin (131; R = H) has also been shown''O to involve head-to-head coupling of two acetate units. A mutant of Aspergillus uersicolor produced by irradiation yielded' ' 5-methoxysterigmatacystin (131;R = OMe).The co-occurrence of versicolorin A (132) and sterigmatacystin in certain strains of the organism suggest that the xanthone may be derived from a related anthraquinone which in turn could be produced via a tetracycline intermediate as suggested by Buchifo8 for the aflatoxin B1 biosynthesis. -0 -70 R 0 OH OH OH (130) (U1) OH 'lo J. S. E. Holker and L. J. Mulheirn Chem.Comm. 1968 1576. J. S. E.Holker and S. A. Kagal Chem. Comm. 1968 1574. 600 R. Ramage The role of aren-oxide-oxepin systems in the metabolism of aromatic systems has assumed greater importance with the synthesis of (4-2H] 3,4-epoxytoluene (133) and its subsequent rearrangement to [3-2H] -p-cre~ol."~ From the meta- bolism of naphthalene' l by rat-liver microsomes 2,3-epoxynaphthalene (135) was isolated by dilution techniques and was found to be hydrated enzymatically to trans-1,2-dihydro-l,2-dihydroxynaphthalene (136). Acetylaranotin (137) is a naturally occurring substance recently isolated which contains the dihydro- oxepin system.'I4 Baldwin has discussed the cleavage of aromatic rings in terms of enzymic generation of a species equivalent in its powers to singlet oxygen.' It was shown that 1,4-peroxides formed from aromatic rings may be trans- formed by acid to cleavage products of the type found in biological systems.'12 D. M. Jerina J. W. Daly and B. Witkop J. Amer. Chem. SOC. 1968,90 6523. D. M. Jerina J. W. Daly B. Witkop P. Zaltzman-Nirenberg and S. Udenfriend J. Amer. Chem. SOC.,1968,90,6525. li4 R. Nagarajan N. Neuss and M. M. Marsh J. Amer. Chem. SOC. 1968,90,6519. '15 J. E. Baldwin H. H. Busson and H. Krauss Chem. Comm. 1968 984.

ISSN:0069-3030    

DOI:10.1039/OC9686500577

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

18 ENZYME MECHANISMS By M. R. Hollaway (Department of Biochemistry University College London W.C.1) Introduction.-There can be little doubt that the most exciting recent develop- ments relevant to an understanding of enzyme mechanisms have come from X-ray crystallographic studies. The structures of several enzymes and enzyme- inhibitor complexes have now been described at the 2-3 A level; this has made possible a fairly unambiguous identification of protein side-chains which interact with the substrate molecules during catalysis. A review of the structures published up to October 1967 has been given by Stryer’ and some subsequent developments will be referred to in this account. Such a complete knowledge of the three-dimensional structure of an enzyme cannot in itself inform us how the enzyme functions; as Gutfreund and Knowles2 pointed out :‘making a model of a horse from photographs does not necessarily tell us how fast it can run’.Nevertheless,any proposed mechanism must be compatible with the X-ray structures in terms of the geometry of the active site. Furthermore the proposed reactivity of catalytic groups invoked in the mechanism should be consistent with their position in the enzyme-substrate complex i.e. their microenvironment. Significant advances have also been made in the study of enzyme reactions by use of rapid reaction techniques. The approach to mechanism through observation of the nature of intermediates by their physical signals3 and measure- ment of their speed of interconversion is made difficult in enzyme reactions because if the very high rates involved.Thus most enzymes transform between 10 and 1000 substrate molecules per second per active site. This sets an upper limit to half-lives of intermediates to around 100 milliseconds so that special techniques are required for the elucidation of transient steps in enzymic catalysis. Such techniques including stopped-flow. temperature-jump and combined stopped-flow-T-jump are being continually refined so that now Chance et d4report time resolutions below 1millisecond with rapid mixing techniques and Eigen and his co-workers’ are able to study transients in perturbed equili- L. Stryer Ann Rev. Biochem. 1968,37,25. H. Gutfreund and J. R Knowles ‘Essays in Biochemistry’ vol. 3 ed. P.N. Campbell and G. D. Greville Academic Press 1967 p. 25. ’ M. L. Bender in ‘Rates and Mechanisms of Reactions’ pt. 11 ed A. Weissberger Interscience New York 1963 p. 1427. B. Chance D. DeVault V. Legallais L. Mela and T. Yonetani in Nobel Symposium 5 ‘Fast Reactions and Primary Processes in Chemical Kinetics’ ed S. Claesson Interscience New York 1967 p. 437. ’ M. Eigen Quart. Rev. Biophys. 1968 1 3 and refs. therein. M. R. Hollaway bria with half-lives below 1 p second using the temperature-jump technique. The recent development of the combined-flow-T-jump technique’ enables the study of enzyme transients by perturbation of an enzyme-substrate system in the steady state rather than at equilibrium. Excellent accounts of rapid reaction methods as applied to the study of enzymes are given in the books by Gutfreund6 and Bernhard7 and in the reviews by Chance* and Eigen.s*9 A number of very fine reviews have recently been published including the essay on ‘The Foundations of Enzyme Action’ by Gutfreund and Knowles;2 ‘New Looks and Outlooks in Physical Enzymology’ by Eigen ;’an account by Singer” on covalent labelling of the active site enzymes and a review by Kosh- land and Neet on ‘The Catalytic and Regulatory Properties of Enzymes’.Volume XI of the invaluable series ‘Methods in Enzymology”2 contains sections on specific modification reactions and the investigations of confor- mation changes. There is a book on ‘Design of Active-site Irreversible Enzyme Inhibitors’ by Baker.13 Some of the other excellent reviews which are more specific in nature will be referred to in the relevant section.In this report consideration will only be given to those enzymes for which X-ray crystallographic studies are relatively advanced. This limitation may give a somewhat one-sided view of enzymic mechanisms since the enzymes which fall into this category are low molecular-weight hydrolytic proteins com- prising a single-polypeptide chain. Furthermore the molecules usually contain intramolecular disulphide bonds; this seems to be a feature of extra- cellular enzymes whereas as far as the reviewer is aware enzyme molecules found in the interior of cells do not contain disulphide links and usually contain more than one polypeptide chain. (Hartley has pointed out that the disulphide bond is a characteristic structural feature of extracellular proteins e.g.ref. 14.) Nevertheless it is to be hoped that principles discovered for the single chain hydrolytic enzymes will be applicable in more complex systems. The Basis of Enzyme Catalysis.-A great deal of consideration has been given to factors which could explain why enzymes are such good catalysts. The book by Bruice and Benkovic,” the essay by Gutfreund and KnowlesY2 and the review by Koshland and Neet” all give a careful evaluation of the extent to which all or some of the following effects may contribute to enzymic catalysis (u)a proximity effect arising from an enhanced collision frequency for reactant molecules bound to the enzyme; (b) an orientation effect resulting from the H.Gutfreund ‘An Introduction to the Study of Enzymes,’ Blackwell Oxford 1965. ’ S. A. Bernhard ‘The Structure and Function of Enzymes,’ Benjamin New York 1968. B. Chance in ref. 3. M. Eigen ref. 4 p. 333. lo S. J. Singer Adv. Protein Chem. 1967,22 1. l1 D. E.Koshland and K. E. Neet Ann. Rev. Biochern. 1968,37,359. l2 ‘Methods in Enzymology,’ vol. XI ed. C. H. W. Hirs Academic Press New York 1967. l3 B. R Baker ‘Design of Active-Site Directed Irreversible Enzyme Inhibitors,’ Wiley New York 1967. l4 P. B. Sigler D. M. Blow B. W. Matthews and R Henderson J. Mol. Biol. 1968,35 143. T. C. Bruice and J. Benkovic ‘Bioorganic Mechanisms,’ vol. I Benjamin New York 1966. Enzyme Mechanisms optimal positioning of functional groups of the enzyme so as to assist in bond- forming bond-breaking processes ;(c) general base or general acid catalysis including bound metals acting as Lewis acids; (d)covalent catalysis whereby a group on the enzyme is a stronger nucleophile than the final acceptor and a better leaving group than the leaving group portion of the substrate; (e) a ‘strain’ effect in which the substrate molecule binds to the enzyme in a confor- mation that is part of the way towards the transition state of the catalysed reaction (an excellent discussion of this and other types of strain theory has been given by JencksI6); (f)a microenvironment effect whereby the local environ- ment of groups in the enzyme-substrate complex may be such as to facilitate reaction pathways which are energetically unfavourable in aqueous solution ; and (9)concerted catalysis a concept arising largely from the oft-quoted work of Swain and Brown.17 These last authors found that in benzene solution 2- hydroxypyridine was a 7000-fold more effective catalyst of the mutorotation of a-tetra-0-methyl-D-glucosethan a mixture of pyridine and phenol each at the same concentration as the 2-hydroxypyridine.This result cannot be accoun-ted for on the basis of increased acidity of the hydroxy-group or increased basicity of the ring nitrogen in the 2-hydroxypyridine. The proposed inter- pretation was that a concerted acid-base catalysis was operable in the rate- limiting ring-opening step. It may be argued that this system cannot represent a model for enzymic catalysis because the studies were carried out in a non- polar solvent whereas enzyme reactions proceed in aqueous solution.Such an objection may be invalidated by the consideration that the active sites of at least some enzymes contain regions of low polarity (see later). Koshland and Neet l1 have extrapolated from model systems in an attempt to discover what order of rate-enhancement would be expected if the effects (a)-(d) were to operate simultaneously. The results of their calculations showed that a factor of at least lo8 remains to be accounted for in enzymic catalysis by strain concerted catalysis or other effects. Snell Kwok and Kim18 have put forward some suggestions for enzymic mechanisms based on some work on the aminolysis of the methyl esters of salicyclic acid (1) and p-hydroxybenzoic acid (2) in dry dioxan solution.The l6 W. P. Jencks in ‘Current Aspects of Biochemical Energetics,’ ed. N. 0.Kaplan and E. P. Kennedy Academic Press New York 1966,273. C. G. Swain and I. F. Brown J. Amet. Chem. SOC.,1952,74,2538. R. L. Snell W-K. Kwok and Y. Kim J. Amer. Chem. SOC.,1967,89,6728. U M. R.Hollaway reaction of (1) with n-butylamine in dry dioxan at 50 and at 70" was found to be first-order in ester but second-order in amine. Under the same conditions (2) did not react with n-butylamine to any detectable extent. The suggested inter- pretation of the second-order component in amine was that one molecule combined with a molecule of ester to give an ion pair represented simply by (3 in which the butylammonium ion served as an acid catalyst during the nucleo- philic attack of a second amine molecule.This system is considered by the authors as a model for enzyme reactions involving the hydrolysis of esters or amides. A hypothetical enzyme is postulated to contain in its active site a carboxy-group and two amine groups one a stronger base than the other. On binding substrate it is proposed that all water is excluded from the active site and the substrate optimally orientated with respect to the catalytic groups ('microstereochemically oriented'). The carboxy-group and strong base form an ion pair which interacts with the substrate so as to facilitate nucleophilic attack by the weaker amine base. This work is predated by that of Menger,lg who studied the reactions of benzamidine or n-butylamine with p-nitrophenyl acetate in dry chlorobenzene solution.Benzamidine was found to react with the ester some 15,000 times faster than the butylamine monomer and only fourfold slower than hydroxide ions in water. As in the system studied by Smell Kwok and Kim'* the p-nitrophenyl acetate-butylamine reaction was second order in amine but the rate-law for the benzamidine reaction only contained a first-order component in amidine. Menger ' concluded that benzamidine acted as a bifunctional reagent in a manner depicted in (4). OAr ph-I H (4) Such a process would be feasible in chlorobenzene since there is no charge formation in the transition state. This model reaction was considered to support the suggestion that multifunctional catalysis by proteolytic enzymes may occur in a cyclic fashion in non-polar regions of the active sites.As indicated by Menger,Ig the model is reminiscent of the Swain-Brown system." WangZ0 has suggested that facilitated proton transfer along rigidly held hydrogen bonds in the enzyme-substrate complex might account for both the specificity of enzymes and their efficiency as catalysts. It is proposed that this factor enables enzyme systems to reach transition states faster often by a l9 F.M. Menger J. Amer. Chem. SOC. 1966,88,3081. 2o J. H. Wang Science 1968,161,328. Enzyme Mechanisms 605 process which is essentially a pre-transition-state protonation. Mechanisms were suggested for the reactions catalysed by carbonic anhydrase chymotryp- sin ribonuclease and some dehydrogenases.Another suggestion by Braunstein Ivanov and Karpeisky,’ is that enzyme reactions are distinguished from chemical transformations in solution by a high degree of conformational mobility in the enzyme-substrate complexes. This mobility is considered to provide the capacity to meet different sets of requirements in consecutive steps of the catalytic process. A consideration of the chemical and physical properties of metalloenzymes has led Vallee and Williamsz2 to propose that these enzymes are in an entatic state i.e. that they might be ‘poised for catalytic action in the absence of substrate.’ This concept is presented largely on the basis of the atypical spectral properties of metalloenzymes which suggest an unusual geometry in the arrangement of ligands about the bound metal ions.The active site is seen as an area which is ‘closer to a unimolecular transition state than to that of a conventional stable molecule thereby constituting an energetically poised domain’. This concept is reminiscent of the ‘strain’ theory16 but with the enzyme in the deformed state. It should be emphasized that the entatic-state theory cannot account for the specificity of enzymes. Thus if a metallopep- tidase catalyses the hydrolysis of a peptide bond between two amino-acid residues the theory would predict that the enzyme would also readily catalyse the hydrolysis of say acetamide. Such indiscriminate catalysis is not observed in the hydrolytic reactions of metallopeptidases.Nevertheless when taken in conjunction with other factors the unusual environment of bound metals at the active site of metalloenzymes may well be an important factor in catalysis. The role of ligand-induced conformation changes in enzymic reactions i.e. the induced fit theory of Koshland has been extensively discussed in the review by Koshland and Neet.” The evidence for such changes appears convincing especially in the case of carboxypeptidase A,z3 where binding of molecules of similar structure to substrates leads to the movement through about 14 A of one of the enzyme tyrosine hydroxy-groups. An important aspect of the induced-fit theory is its capacity to account for the specificity of enzyme reactions.Wagner and his co-workersz4 have carried out a series of interesting experi- ments on a model system designed to investigate the possible role of apolar bonding in enzymic catalysis. The rate of aqueous hydrolysis of the NN-dimethyl-N-(p-nitrophenyloxycarbonylethyl)dodecylammonium ion (5) was 21 A. E. Braunstein V. I. Ivanov and M. Ya Karpeisky in ‘Pyridoxal Catalysis Enzymes and Model Systems,’ ed E. E. Snell A. E. Braunstein E. S. Severin and Yu M. Torchinsky Interscience New York 1968,291. 22 B. L. Vallee and R J. P. Williams Proc. Nut. Acud. Sci. U.S.A.,1968,59,498. 23 G. N. Reeke J. A. Hartsuck M. L. Ludwig F. A. Quiocho T. A. Steitz and W. N. Lipscomb Proc. Nur. Acud. Sci. U.S.A.,1967 58 2220. 24 T. E. Wagner C-J. Hsu and C. S.Pratt J. Amer. Chem SOC. 1967,89,6366; R G.Shorenstein C.S. Pratt C-J. Hsy and T. E. Wagner ibid. 1968,90 6199. M. R.Holluwuy cNa N H fH2 -0 (6) OOC. CH. NHCO. C17 H35 1 O=C -[CH2I2-i (CH3I2. C12 H25 strongly catalysed by N-stearoylhistidine (6). The latter compound was a 2000-fold better catalyst than N-acetylhistidine. Moreover the reaction catalysed by (6) exhibited a hyperbolic relationship (the so-called Michaelis- Menten kinetics) between rate of hydrolysis and ester concentration at a fixed concentration of (6). It was concluded that the rate enhancement was due to apolar bonding between the lyophobic groups in (5) and (6). The reaction was subject to competitive inhibition by trimethyl(steary1)ammoniumbromide and was also inhibited by urea; these two types of effect are often observed in enzyme reactions.Lysozyme.-Hen eggwhite lysozyme is a small protein of molecular weight CU. 14,600 comprising a single linear polypeptide chain of 129 L-a-amino-acid residue^.^'-^^ There are four intra-chain disulphide bonds in the m~lecule.~’ The bacteriolytic action of lysozyme resides in its abilit~~*-~’ to catalyse the hydrolysis of the p-N-acetylmuraminyl (NAM) glycosidic link in polymers of alternately linked p-(1 4)-N-acetylmuraminyl and N-acetylglucosaminyl (NAG) residues (NAM-NAG),. Chitin,31 the p-(1 + 4)-linked N-acetyl- glucosamine polymer represented (NAG), and derived oligosa~charides~~ are also substrates for lysozyme action. The bond cleaved during catalysis is that between C-1 of the pyranoside ring and the glycosidic oxygen atom and the reaction proceeds with greater than 99 % retention of configuration about the glycosidic carbon atom.32 The elegant X-ray crystallographic studies of Blake et al.33-35and Phillips36 25 R E.Canfield and A. K. Liu J. Biol. Chem. 1963,239 2698. l6 J. Jolles J. Tauregui-Adell and P. Jollbs Biochim. Biophys. Acta 1963 78 668. ’’ R E. Canfield and A. K. Liy J. BioI. Chem. 1965,240 1997. ” M. R J. Salton Ann Rev. Biochem 1965,34,143. 29 J. A. Rupley and V. Gates Proc. Nat. Acad. Sci. U.S.A. 1967,57 496. 30 D. M. Chipman J. J. Pollock and N. Sharon J. Biol. Chem. 1968,243 487. ” L. R. Berger and R. S. Weiser Biochim. W’ophys. Acta 1957,26 517. 32 M. A. Rafferty and T. Rand-Meir Biochemistry 1968 7 3281.’’ C. C. F. Blake D. F. Koenig G.A. Mair A. C. T. North D. C. Phillips and V. R Sarma Nature 1965,206,757. 34 C. C. F. Blake G. A. Mair A. C. T. North D. C. Phillips and V. R. Sarma Proc. Roy. SOC. B 1967,167,365. ” C. C. F. Blake L. N. Johnson G. A. Mair A. C. T. North D. C. Phillips and V. R Sarma Proc. Roy. SOC. B 1967,167 378. 36 D. C. Phillips Proc. Nat. Acad. Sci. U.S.A. 1967,57 484. Enzyme Mechanisms have led to a detailed knowledge of the three-dimensional structure of the hen eggwhite lysozyme molecules and of several enzyme-inhibitor complexes. Key features of the structure are (i) that the shape of the molecule is slightly ellipsoid with a large cleft at its surface and (ii) that the interior of the molecule contains amino-acid residues with non-polar side chains (apart from Gln-57 and Ser-91) a feature in common with other globular proteins of known structure.Furthermore these studies have indicated that the cleft region is the active site of the enzyme. A substrate comprising six sugar residues e.g. (NAG) can be built into the cleft giving an arrangement of protein side-chain and substrate as represented in crude schematic form in (7). Detailed drawings of the active site region containing a bound (NAG) molecule are given in refs. 35 and 36 and an excellent three-dimensional representation produced by the Xograph technique has been published. It should be noted that the proposed conformations and interactions of the substrate residues A B and C are those found in the lysozyme-(NAG) crystal struct~re,~~,~~ whereas the arrangement of residues D.E and F is based on careful model building. Some noteworthy points regarding the lysozyme-(NAG) complex are as follows. s*36 (a) In order to build residue D into the active site it must be distorted towards a half-boat conformation. (b) For steric reasons a sugar with a 3-lactyl substituent as in NAM cannot be placed in position C. This fact in conjunction with the hydrolysis pat- tern2'-,' for (NAM-NAG) and the stability of the lysozyme-(NAG) complex in which the sugars are bound at A B and C suggested that cleavage occurs between residues D and E. (c) The carboxy-groups of Asp-52 and Glu-35 are located at either side of the D-E glycosidic link and are consequently regarded as potential catalytic groups.The environments of these residues are such as to suggest that Glu-35 may have a high pK value whereas Asp-52 being placed in a polar environ- ment may have a low pK,. The foregoing studies have led to a proposal of a mechanism for lysozyme action by the group at The Royal Instit~tion~~~~~ in discussion with Prof. C. A. Vernon.,' It is suggested that the substrate binds to the active site in such a way that sugar residue D is distorted towards the half-chair conforma- tion. In the subsequent bond-breaking step the protonated form of Glu-35 provides general acid catalysis and the ionised Asp-52 serves to stabilise the incipient carbonium ion as shown in (8).The latter postulate serves to explain the retention of configuration at the glycosidic carbon atom.It is significant that the solvolysis of 1,3,4,6-tetra-O-acety1-~~-glucopyranosyl chloride in 37 R A. Harte and J. A. Rupley J. Biol. Chem.,243 1663. C. A. Vernon Proc. Roy. SOC.1967 B 167 389. M.R. Hollaway '/ h c 3 In 3 2-U cy In Enzyme Mechanisms acetic acid is considered to proceed via a carbonium ion with the half-chair conf~rmation,~~ so that the distortion of the D ring towards this arrangement on binding of a substrate to lysozyme could represent an example of catalysis through induced ‘strain’ discussed in an earlier section.’ Lowe and his ~o-workers~~~~ have proposed an alternative mechanism for lysozyme action in which the 2-acetamido-group of the D sugar residue provides anchimeric assistance and Glu-35 acts as a general acid catalyst during the rate limiting bond-breaking step (9).In the most recent com- m~nication~~ no mechanistic role is ascribed to Asp-52. The implication of the 2-acetamido-group was based on the observation that the k, value for the di-N-acetylchitobioside (10) was at least 100 times that of the corresponding glycoside (11) although the CT values for OH ( +0-25) and NHAc ( +028) are (10) R’ = NHAc R2= p-nitrophenyl (11) R’ = OH R2= p-nitrophenyl (12) R’ = NHAc R2= aryl similar. Earlier studies by Lowe et aL41 had. implicated either concerted acid- base or acid-nucleophilic catalysis in the rate-limiting step for lysozyme hydrolysis of some P-aryldi-N-acetylchitobiosides(12) because KM values for these substrates were independent of the aglycone whereas the k,, values varied with the leaving group giving a Hammett p value of + 1-2.Some care must be exercised in extrapolating the mechanistic conclusions derived from these studies to lysozyme action on ‘normal’ substrates e.g. (NAG), since the largest k,, value reported4042 for the derivatives (lo) (ll),and (12) was such that one molecule of lysozyme catalysed the hydrolysis of one glycosidic bond every 40 min Normal substrates are probably hydrolysed at least 4 orders of magnitude faster.43 39 R. U. Lemieux and G. Huber Cad. 1.Res. 1955,33,128. 40 G. Lowe Proc. Roy. SOC.B 1967,167,431. 41 G. Lowe G.Sheppard M. L. Sinnott and A. Williams Biochem. J. 1967,104,893. 42 G. Lowe and G. Sheppard Chem. Comm. 1968,529. 43 J. A. Rupley L. Butler M. Gerring F. J. Hartdegen and R Pecoraro Proc. Nat. Acud. Sci. U.S.A. 1967,57 1088. 610 M. R. Hollaway Piszkiewicz and Br~ice~~ (see also ref. 49) have also discussed the possibility of anchimeric assistance by the 2-acetamido-group in lysozyme action. Raftery and Rand-Meir45 have recently considered several possible mecha- nisms for lysozyme-catalysed reactions. They exclude single displacement reactions on the basis of the greater than 99 % retention of configuration about the glycosidic carbon atom. Anchimeric assistance by the 2-acetamido- (or 2-hydroxy)-group was also shown to be unnecessary by the observation that lysozyme catalysed the rate of release of p-nitrophenol from 2-deoxy-P-~- glucosides (13) at about 16 times the rate for the corresponding glucoside (14).(13) R = H (14) R = OH The authors were careful to state,45 however that ‘. ..although anchimeric assistance is not necessary to explain catalysis by lysozyme the present findings do not of course exclude its occurrence in NAG substrates’. The remaining possibilities are either the carbonium ion intermediate rnechani~m,~ or a double-displacement mechanism involving a glycosyl-enzyme intermediate.46 Vernon38 considers the latter mechanism inoperable on steric grounds. The acid-catalysis carbonium ion mechanism3* is further supported by some work by Rupley Gates and Billbre~.~’ These authors measured the relative rates of hydrolysis and transglycosylation (krel)in the system :lysozyme-(NAG) plus an acceptor nucleophile.Firstly it was found that krelwas essenti- ally constant for a series of alcohols of similar steric requirements but with pK values ranging over 4 units. General-base catalysis by a group on the enzyme was invoked to explain this levelling effect. This group would act as a general acid in the first step of the hydrolytic reaction. Secondly strongly nucleophilic sulphur acceptors were less reactive than their oxygen analogues? a fact which is incompatible with a glycosyl-enzyme inte~mediate.~~ The low reactivity of the thiol analogues cannot be accounted for on steric grounds because the reaction centre is available to a sulphur atom as evidenced by the lysozyme-catalysed hydrolysis of phenylthioglu~oside.~~* 49 44 D.Piszkiewicz and T. C. Bruice J. Amer. Chem. SOC. 1967 89 6237. 45 M. A. Raftery and T. Rand-Meir Biochemistry 1868,7 3281. 46 D. E. Koshland jun. Bid. Rev. 1953,28,416. 47 J. A. Rupley V.Gates and R Billbrey J. Amer. Chem Soc. 1968,90 5633. 48 A. J. Rhind-Tutt and C. A. Vernon J. Chem SOC.,1960,4637. 49 D. Piszkiewicz and T. C. Bruice Biochemistry 1968 7 3037. Enzyme Mechanisms 61 1 Dahlquist and Rafteryso have described some interesting n.m.r. studies on lysozyme-inhibitor interactions. Thus both the glycosidic methyl protons and the acetamido-protons of methyl 2-acetamido-2-deoxy-~-~-glucopyranoside underwent a chemical shift in the enzyme-inhibitor complex.The glycosidic methyl proton shift was pH-independent whereas the extent of the acetamido- proton shift varied with the ionisation of the two groups of pK 4-7 0.1 and 7.0 0.5. Furthermore the pH-dependence of the dissociation constant for the enzyme inhibitor complex implicated a group of pK 6.1. Previous studies5’ on the binding of (NAG) to lysozyme involved two groups of pK values 4-2 and 5.8 in the free enzyme and 3.6 and 6.3 respectively in the complex. A consideration of the crystal structure of the lysozyme<NAG) complex3’ together with the foregoing observations has led Dalhquist and Rafteryso to make the following assignments of pK values Asp-101 pK 4.2(3-6 in the complex); Glu-35 6-1 (7.0 0.5 in the complex); and Asp-103 4-7.The bell- shaped pH profiles with inflections near 4and 6 for the action of lysozyme on low molecular weight substrate^^^*'^ may reflect two or more of these ionisa- tions. An exciting recent discovery is that the amino-acid sequence of bovine a-lactalbumin is largely homologous to that of hen eggwhite lyso~yrne.~~ Furthermore a-lactalbumin is one of the two protein components of lactose synthetase which catalyses the reaction uridine diphosphate galactose + D-glucose + lactose + uridine diphosphate The other protein component A protein catalyses the reaction a-uridine diphosphate galactose + N-acetylglucosamine -+ N-acetyl-lactosamine + uridine diphosphate However addition of a-lactalbumin (designated B protein) to the A protein results in a system which gains ability to catalyse (15) whilst losing the facilitys4 to catalyse (16).The similarity of a-lactalbumin to lysozyme is further accentuated by the fact that it has proved possibless to build a model of a-lactalbumin based upon the main chain backbone of ly~ozyme.~’ In this model the interior 50 F. W. Dahlquist and M. C. Raftery Biochemistry 1968,7 3269 3277; see also M. A. Raftery F. W. Dahlquist S. I. Chan and S. M. Parsons J. Biol. Chem. 1968,243,4175. 51 F. W. Dahlquist L. Jao and M. A. Raftery Proc. Nut. Acad. Sci. U.S.A.,1966,56 26. ’’ T. Osawa and Y.Nakazawa Biochim Biuphys. Acta 1966 130 56. 53 K. Brew T. C. Vanaman and R L. Hill J. Biol. Chern. 1967,242 3747. 54 K. Brew T. C. Vanaman and R L.Hill Proc. Nut. Acad. Sci. U.S.A.,1968,59,491. 55 A. C. T. North Abstracts of the Meeting of The British Biophysical Society Dec. 18th-l9th 1968 p. 6. 612 M. R. Hollaway retains the apolar character seen in the lysozyme molecule and it seems likely that the two proteins have very similar structures. Serine Proteases.-The excellent review by RyleS6on ‘The Endopeptidases of Vertebrates’ contains a description of the relevant literature up to 1966. Two reviews by Bender and KCzdy consider respectively the mechanisms of action of proteolytic enzymess7and of chymotryp~in.~~ Books containing discussionson this group of enzymesare by Gutfreund,6Bernhard,7and Bruice and Benkovic.’ CunninghamS9has also presented an excellent comprehen-sive account of the ‘Structure and Mechanism of Action of Proteolytic En-zymes’.The reader is also referred to the important review by Neurath Walsh and Winter6’ which gives consideration to the ‘Evolution of Structure and Function of Proteases’. The terms ‘analogous proteins’ and ‘homologous proteins’ are used here in the manner defined by these authors. Chyrnotrypsin (CT). There is a considerable body of evidence that the CT-catalysed hydrolysis of specific substrates involves a minimum of three steps (reviewed in refs. 15 and 56-62) k+l k+2 k+3 E+S====ES =EP,eE+P k-1 k-2 ++ P,P k-3 (17) Initial formation of an enzyme-substrate complex through non-covalent bonding is followed by catalytic steps involving acylation (k+ step) and deacylation (k+3 step) of a group on the enzyme.The role of the uniquely reactive Ser-195 residue as the group acylated and the involvement of His-57 in catalysis have been extensively reviewed.”*56-62 Specific substrates for chymotrypsinaction are typically N-acyl-substitutedesters,amides or peptides of L-a-amino-acids with acyl side chains. X-Ray crystallographic studies14* 63 combined with a knowledge of the amino-acid sequence of u-CT~~ have enabled Blow and his collaborators to determine the structure of bovine p-tolylsulphonyl-Ser-195-CTat 2 Aresolu-tion. A difference electron density map between tosylated and native enzyme has also been calculated.’ The a-CT molecule comprises three polypeptide chains designated A B 56 A. P. Ryle Ann Reports 1966,63 614.’’ M. L. Bender and F. J. KCzdy Ann Rev. Biochem. 1965,34 49. ’’ M. L. Bender and F. J. KCzdy J. Amer. Chem SOC.,1964,86 3704. 5g L. Cunningham in ‘Comprehensive Biochemistry,’ ed. M. Florkin and E. H. Stotz Elsevier Amsterdam 1965 VOL 16 p. 85. 6o H. Neurath K. A. Walsh and W. P. Winter Science 1967,158,1638. 61 A. Himoe P. C. Parks and G. P. Hess J. Biol. Chem. 1967,242,919. 62 M. L. Bender M. J. Gibian and D. J. Whelan Proc. Nut. Acad. Sci. U.S.A. 1966,56,833. 63 B. W. Matthews P. B. Sigler R Henderson and D. M. Blow Nature 1967,214 652. 64 B. S. Hartley in ‘The Structure and Activity of Enzymes,’ ed T. W. Goodwin J. 1. Hams and B. S. Hartley Academic Press London 1964 p. 47; B. S. Hartley and D.L. Kauffman,Biochem J.,1966 101 229; B.Meloun I. Kluh V. Kostka L. Moravek Z. Prusik J. VanBkk B. Keil and F. Sorm Biochim. Biophys. Acta 1966 130 543. Enzyme Mechanisms 613 and C; the B chain is covalently joined to A and C by disulphide bonds and the B chain has one and the C chain two intrachain disulphide bonds.64 Apart from eight residues at the N-terminus of the C chain in an a-helical arrangement the polypeptide chains are in an extended conformation and folded so as to form a compact approximately spherical structure with an interior comprised almost entirely of non-polar residues. There is a slight groove at the surface of the molecule in the region of the active site.I4 The most recent reveals that in the uninhibited enzyme the centre of the imidazole ring of His-57 is about 3 %i from the oxygen atom of Ser-195 a distance consistent with hydrogen-bonding between the imidazole 1-nitrogen atom of His-57 and the serine oxygen.However in the tosylated enzyme at low pH values these appears to be a hydrogen bond between imidazole 1-nitrogen of His-57 and the sulphonyl oxygen atom mediated by a water molecule. It is noteworthy that significant differences in the electron density map between native and tosyl-CT are only apparent in the active centre region and the largest side-chain movement that of Met-192 is less than 1A. An exciting recent de~elopment~~ is that residue 102 formerly designated as an asparagine is in fact aspartic acid. This group is located in an essentially non-polar ‘pocket’ generated by residues Ala-55 Ala-56 Cys-58 Tyr- 194 Ile-99 and Ser-214.The entrance to the pocket is sealed by His-57 whose imidazole 3-nitrogen atom is hydrogen-bonded to Asp-102 thereby rendering the latter residue inaccessible to solvent molecules (18). Blow Birktoft and Hartley6’ consider that the species (18) which is only one of several possible mesomeric and tautomeric representations is catalytically inactive but may lose a proton in a process of pK ca. 7 (the pK value observed in acylation and deacylation reactions see previously) to give an active species (19). It is worth mentioning a caueat entered by WallenfelP who emphasised that observed pK values for such a process may not be ascribed to any par- ticular group but are a characteristic of the system as a whole.This arrangement (19) has been christened by Blow Birktoft and Hartle~~~ as a ‘charge-relay system’ for relaying electrons from Asp-102 to the surface of the molecule via His-57 and has led these authors to suggest a mechanism for CT action (20) incorporating many of the features of a mechanism proposed by Wang.,’ This depicts the rate-limiting acylation step in the CT-catalysed hydrolysis of a peptide amide or anilide. Deacylation would involve re- placement of RNH by a water molecule and a reversal of the acylation process. Key features of the mechanism are that (a) The ‘charge relay system’ ensures a significant concentration of the Ser- 195-0 species at neutral pH values ; (b) the ‘charge relay system’ also serves to assist the removal of the leaving group by transfer of the Ser-195 proton; and ‘’ D.M.Blow J. J. Birktoft and 8. S. Hartley Nature 1969,221 337. “ K. Wallenfels personal communication 1964. M. R. Hollaway 614 In z L QrY p\T Enzyme Mechanisms ASP-102 1 95 A (c) The overall process is facilitated by electron transfer uiu pre-formed rigidly held hydrogen bonds.20 It is interesting that an electrophilic component in CT-catalysed reactions has been implicated by various observations. Thus Metzger and Wilson67 found that the rate constant for acylation of CT by diphenylcarbamyl fluoride (3790 M-’ sec-I) was eight times that for the corresponding chloride an inversion of the expected reactivity for these reagents. Such an inversion may occur in situations where electrophilic catalysis can operate e.g.in the reaction of benzoyl halides with Grignard reagents6* where kgCl may act as an acid catalyst. Secondly Inagami York and Patchornik6’ observed that k, (acylation rate constant) for the CT-catalysed hydrolysis of a series of substituted anilides of N-acetyl-L-tyrosine was subject to a large negative p value suggesting the involvement of general acid catalysis. This was supported by a kinetic isotope effect k+2(H20)/k+2(D20) = 3-4. These studies have been extended by Wang and Parker,70 who found that the increase in k, in H20 or D20 for a series of anilides paralleled an increase in the basicity of the substrated as measured by titration in protonated or deuteriated glacial acetic acid.However the kinetic isotope effect diminished with increasing basicity a finding which was interpreted as evidence for protonation of the ” H. P. Metzger and I. B.Wilson Biochemistry 1964,7,926. ‘* C. E. Entemann and J. R Johnson J. Amer. Chem SOC. 1933,55 2900. ‘’ T. Inagami S. S. York and A. Patchornik J. Amer. Chem SOC.,1965,87 126. 70 J. H. Wang and L. Parker Proc. Nut. Acad. Sci. U.S.A.,1967,58,2451; L. Parker and J. H. Wang J. Biol. Chem. 1968 243 3729. 616 M. R. Hollaway anilide nitrogen atom prior to the rate-limiting step in acylation i.e. specific acid catalysis. This argument is subject to the objection that the basicity of the anilide in glacial acetic acid might be associated with protonation of the carbonyl oxygen rather than the nitrogen atom.Nevertheless if this is correct then (17) must be expanded to give (21) k+1 k+2 k+3 k+4 E + S ES /ES' /EP -. . .E + Pz k-1 k-2 k-3 + p (21) It may be of significance in this context that Hess7' has recently described an extra intermediate in the CT hydrolysis of furoylacryloyltryptophanamide. Rapid reaction studies enabled the measurement of the various rate constants k+ = 6-2 x lo6 M-' sec-'; k- = 2.7 x lo3 sec-'; k, = 13 sec-'; k- = 30 sec-' ; and k+4 = 50 sec-'. However whether the k+z step involves a protonation of substrate tetrahedral intermediate or conformation change in the enzyme remains to be seen. Another attempt to account for the exceptional nucleophilicity of Ser-195 in CT is that by Epstein Michel and Mosher7 who consider that Arg-14 may be sufficiently close to Ser-195 to lower the pK value of its hydroxy-group to ca.8. In discussing the nucleophilicity of Ser-195 it is important to note that it is unreactive towards iodoacetic acid. Thus Botvinik and Novodarova7 have shown that reaction of CT with this reagent at pH values between 5 and 9 leads only to substitution of histidine and lysine residues. In view of the ready reaction of alkoxide ion with alkyl halides74 this suggests that in the absence of substrate or substrate analogues there is no significant concentration of the 4form of Ser-195 at neutral pH values. Hydrogen bonding of Ser-95 to His-57 in itself is also insufficient to account for the reactivity of Ser-195 since Piszkiewicz and Bruice7' have shown that the His-15 Thre-89 hydrogen- bonded system in hen eggwhite lysozyme does not possess abnormal esterolytic activity.In (18) an electrostatic interaction is represented between the carboxylate anion of Asp-194 and Ile-16. The importance of this interaction in maintaining an active conformation of CT has been established largely through the elegant work of Hess and his collaborators.61*76*77 Thus the deprotonation of a 71 G. P. Hess Proc. Roy. SOC. December 1968,in preparation; K.G. Brandt A. Himoe and G. P. Hess J. Biol. Chem. 1967,242 3973. 72 J. Epstein H. 0.Michel and W. A. Mosher J. Theor. BWL 1968,19,320. 73 M. M. Botvinik and G. N. Novodarova Biokhimiya 1968,33,296. 74 C. K.Ingold 'Structure and Mechanism in Organic Chemistry,' Bell London 1953.75 D. Piszkiewicz and T.C. Bruice Biochemistry 1968,7,3037. 76 H. L. Oppenheimer B. Labouesse and G. P. Hess J. Biol. Chem 1966 241 2720 and refs. therein. 77 B. H. Havsteen and G. P. Hess Biochem Biophys. Res. Comm. 1964,14,313;k Y.Moon J. M. Sturtevant and G.P. Hess J. Biol. Chem. 1965,240,4204. Enzyme Mechanisms group in CT pK ca. 8.5-9.0 has been shown to be associated with (i) the formation of enzyme unable to bind substrate in a mode leading to catalysis ;61 (ii) an alteration in the 0.r.d. spectrum of the enzyme and a change in the extent of hydrogen ion uptake on binding di-isopropyl phosphofluoridate (DFP).76*77 Evidence has been presented that the ionising group is the a-ammonium group of Ile-16.76* 78 This group cannot be titrated in di-isopropyl-phosphoryl-CT (DIP-CT) which has a pH-independent 0.r.d.spectrum similar to that of the active low-pH form of the enzyme.76 Furthermore chymotrypsinogen in which the a-amino-group of Ile-16 is involved in a peptide bond also has a pH-independent 0.r.d. spectrum but this is similar to the inactive high-pH form of the enzyme. The X-ray crystallographic 63 revealed that the protonated a-amino-group of Ile-16 forms a 'buried' ion-pair with the carboxylate anion of Asp-194 which has led Blow and his co-~orkers'~ to suggest an interpretation of the pH-dependent struc- tural transition. Loss of the proton from the a-ammonium group of Ile-16 is considered to lead to a movement of the carboxylate anion to the surface of the molecule away from the non-polar interior.The resulting catalytically inactive structure is thought to be similar to that of the zymogen and to be completely devoid of capacity to bind sub~trate.'~ In DIP-CT the bulky DIP group hinders the movement of the Asp-194 group thereby explaining the inability to titrate the a-ammonium group. Hess and his collaborator^'^ have observed a proton uptake by 6-CT on binding the specific substrate N-acetyl-L-tryptophanamide.The pH dependence of the number of protons taken up per molecule of CT was interpreted according to the complex ionisation scheme (22) ................................. KSA where E represents an inactive conformation of the enzyme and E* an active conformation.Hence a pK value (pK*,) was determined for the overall process involving ionisation and a conformation change of 9.0 in the free enzyme (Kg = =9.6 in the enzyme-substrate complex (K& = on the binding of proflavin (regarded as a substrate analogue) to a-CT seemingly support a scheme such as (22),since binding appears to proceed in two steps a rapid second-order process followed by a slower first-order isomerisation of the enzyme-'sub- strate' complex. 8o 78 C. Ghelis J. Labouesse and B. Labouesse Biochem Biophys. Res. Comm. 1967,29 101. 79 J. McConn E. Ky C. Odell G.Czerlinski and G. P. Hess Science 1968 161,274. B. H. Havsteen J. Bid. Chem. 1967 242 769. M. R.Hollaway Ligand-induced pK shifts in CT have also been noted by Bender and Wedler," who observed the raising of the pK value of a group (originally 8.8) by at least 2 units on binding the competitive inhibitor benzyl alcohol.Glick82 has investigated the perturbation of prototropic equilibria on ligand binding to CT over a wider pH range than other workers. In particular it was shown that binding of the CT inhibitor,83 chloromethyl L-1-tosylamidophenethyl ketone (23) gave proton release or uptake depending on the starting pH. The results were interpreted as a ligand-induced decrease in the pK value of a group of pK 6-7 and elevation of the pK value of a group with original pK 8-6. The downward shift in the pK value of the group of pK 67 was considered to result from the formation of a hydrogen-bond between His-57 and Ser-195 only on binding of ligand.(24) R' = H,R2 = C0,Me (23) (25) R' = CO,Me R2 = H Several attempts have been made to probe the topography of the active site of CT most often by use of substrates or inhibitors of fixed conformation. There seems to be general agreement (see later) with Hein and Niemann's postulate84 that substrates of the type R'CHR' COR3 bind at three loci p' p2 and p3,and that hydrogen bonding is important in R1-pl interaction and apolar bonding in R2-p2 interaction and that the R3-p3 interaction involves the catalytic groups i.e. Ser-195 and His-57. The original interesting observa- tion by Hein and Niemann84 that ~-3-methoxycarbonyl-3,4-dihydroiso-quinolin-1-one (24)is a 2oO-(kc,) to 4000-(kcaJKd times better CT substrate than the L-analogue (25) and of comparable reactivity to 'normal' substrates has stimulated several authors to consider whether the methoxycarbonyl group in this substrate is required axial or equatorial to the heterocyclic ring for CT-catalysis to occur.Awad Neurath and Hartley8' favoured the axial hypothesis on the grounds that the enzyme would by unable to discriminate between equatorial D-and M. L. Bender and F. C. Wedler J. Amer. Chem. SOC.,89 3052. D. M. Glick Biochemistry 1968,7 3391. G. Schoellman and E Shaw,Biochemistry 1963,2,252 84 G. Hein and C. Niemann Proc. Nat. Acad. Sci. U.S.A. 1961,47 1341. " E. S. Awad H. Neurath and B. S. Hartley. J. Bid. Chem. 1960,235 FT 35. Enzyme Mechanisms L-isomers because they contain the ester group in a similar spatial location.Recent studies by Lawsons6 on the CT-hydrolysis of substrates with highly restricted conformation have also supported the axial hypothesis and some work by Balleau and Chevalliers7 has added further substantiation. The latter authors found that the R-S isomer of the 2,2'-bridged biphenyl analogue of benzoylphenylalanine (26) was not susceptible to hydrolysis by CT but iso- merised in solution to give the conformer (27) which has an axial methoxy- carbonyl group and is a substrate for CT action. Conversely Erlangers8 concluded from studies employing sterically con- strained inhibitors of CT and reactivators of diethylphosphoryl-CT that the equatorial conformer of (24) is the active species. This conclusion is sup- ported by recent work by Silver and Sones9 on the stereospecificity of CT towards equatorial and axial p-nitrophenyl esters of 3-t-butylcyclohexane- carboxylic acids.Cohen and his co-workersgO* also favour the equatorial 91 hypothesis and like Hein and Niemann visualise the active site of CT as containing three binding sites (28). These authors consider the am interaction 86 W. B. Lawson J. Biol. Chem. 1967,242 3397. 87 B. Belleau and R Chevalier J. Amer. Chem SOC. 1968,90 6864. B. F.Erlanger Proc. Nat. Acad. Sci. U.S.A. 1967,s 703. a9 M.S.Silver and T. Sone J. Amer. Chem SOC. 1968,90 6193. S. G. Cohen L. H. Klee and S. Y. Weinstein J. Amer. Chem SOC. 1966,88,5302;S. G. Cohen Z Neuwirth and S. Y. Weinstein ibid. p. 5306;S. G.Cohen R M.Schultz and S. Y. Weinstein ibid. p. 5315. S. G. Cohen and R. M. Schultz Proc. Nat. Acad. Sci. U.S.A.,1967,57 243. 620 M. R. Hollaway unimportant in the CT-catalysed hydrolysis of (24) since ~-3,4-dihydroiso- coumarin-3-carboxylate the oxygen analogue of (24) is also a good substrate for CT.” Of especial significance is the observation by Cohen and Milovano- vic92 that the h site is unable to accommodate a methyl group. A different approach to stereospecificity in CT-reactions has been developed by Ingles and Kno~les.~~~ 94 The deacylation rates (k 3) of acyl-CT derivatives were found to decrease in the order N-acetyl-L-phenylalanyl > N-acetyl-L-tryptophanyl %-N-acetyl-L-leucy % N-acetylglycyl. However the k + values for the corresponding D-isomers were much smaller and the above order was reversed.The results were interpreted in terms of a three-site interaction as in (28). Ingles and Knowles emphasized the fact that specificity is expressed in the catalytic rather than in the binding steps. These authors have presented further evidenceg4 for the hydrogen bonding site (am) by comparing the ratio of deacylation rates for the L-and D-isomers of a series of substrates in which hydrogen-bonding capacity was systematically decreased. The free energy of formation of the hydrogen bond with the substrates employed was estimated at 4 kcal./mole and since this is not expressed as a difference in bonding of derivatives with and without hydrogen-bonding potential it was suggested that CT binds a high-energy conformation of the substrate (an aspect of the strain theory discussed by Jencks16).The X-ray crystallographic studies14*65 are rapidly approaching the refinement where it will be possible to discriminate between the various postulates for substrate binding. Thus studies on the CT-N-formyl-L-trypto- phan complex have revealedg5 that the tryptophan ring is located in a hydro- phobic pocket known as the ‘tosyl-hole’ (since this is the position occupied by the p-tolylsulphonyl group in tosyl-CT). This would correspond to the ar site in (28). Ser-189. at the bottom of the ‘tosyl hole’ is hydrogen bonded to the nitrogen atom of the tryptophan ring of the ligand and further interactions are with Tyr-146 which is C-terminal in the B chain Met-192 and the carbonyl group of Gly-193 which is hydrogen-bonded to the amido-nitrogen atom of the ligand and so may represent the am site.It seems likely that the non- specific hydrophobic site at the active centre of CT described by Canadyg6* 97 and his co-workers and the hydrophobic locus with which the p-nitrophenyl- sulphonyl group in the Ser-195 substituted enzyme interacts described by Kallos and A~atis,~~ are both identical to the ‘tosyl-hole’. McClure and Edelman” have observed that the non-competitive inhibitor 92 S. G. Cohen and A. Milovanovic J. Amer. Chem SOC. 1968,90,3495. 93 D. W. Ingles and J. R Knowles Biochem J. 1967,104,369. y4 D. W. Ingles and J. R Knowles Biochem J. 1968,108 568. 95 D. M. Blow Proc. Roy. SOC. December 1968 in preparation 96 R Wildnauer and W.J. Canady Biochemistry 1966,5,2885; A. J. Hymes D. A. Robinson and W. J. Canady J. Biol. Chem. 1965 240 134. 9’ G.Royer and W. J. Canady Arch. Biochem Biophys. 1968,124 530. 98 J. .Kallos and K. Avatis Biochemistry 1966,5 1979. 99 W. 0.McClure and G. E. Edelman Biochemistry 1967,6 559 567. Enzyme Mechanisms 62 1 of CT 2-p-toluidinylnaphthalene-6-sulphonate(TNS) fluoresces when bound to the enzyme. Since TNS is only fluorescent in non-polar solvents this finding represents evidence for a second hydrophobic regon in the active site of CT probably distinct from the tosyl hole. The fluorescent TNS peak was not observed with benzylsulphonyl-CT or with chymotrypsinogen. Another interesting study on the active site of CT using a chromophoric probe has been carried out by Hille and Koshland.'OO A catalytically active CT derivative was prepared in which Met-192 was substituted with a 2-aceta- mido-4-nitrophenol group.The spectrum of this derivative suggested that the chromophore was in a polar environment and the pH-dependence of the spectrum indicated that (i) the phenolic group of the chromophore was a stronger acid when com- bined with Met-192 (its pK shifts from 6.1 to 5-8); (ii) titration of a group in the enzyme of pK 7.6 alters the spectrum of the phenolate anion of the reporter group; and (iii) there is another group pK ca. 9 which also affects the spectrum. Also substrate binding or covalent substitution of Ser-195 abolishes the dependency of the spectrum on the group of pK 7.6.Hille and Koshland'" attributed the ionisation of pK 7.6 to the imidazolium group of a histidine residue presumably that of His-57 and suggested that the anomalously high value was due to the proximity of the phenolate anion of the reporter group. As indicated by these authors the finding of a polar region in the active site of CT is not incompatible with other studies which indicate non-polar areas but merely serves to illustrate the heterogeneous nature of the active site. The use of n.m.r. spectroscopy for the investigation of CT-substrate-analogue interactions has also proved informative. Thus Spotswood Evans and Richards' O' observed that the binding of N-acetyl-D-p-fluorophenylalanine to CT was attended by a downfield shift in the 19Fresonance consistent with the binding of this competitive inhibitor in a hydrophobic pocket.Gerig,lo2 in investigating the binding of tryptophan to CT by 'H n.m.r. observed that complex formation was accompanied by line-broadening in the alkyl and aromatic regions of the tryptophan spectrum. The extent of broadening suggested that binding was tight enough for tryptophan to assume the rota- tional characteristics of the enzyme. Line broadening of the tryptophan 'H n.m.r. spectrum was not apparent when di-isopropylphosphoryl-Ser-195or S-(N-3-trifluoromethylphenyl)carbamoylmethyl-Met-192 derivatives of CT were employed. Complementary to this study is the investigation of the binding of DL-N-trifluoroacetylphenylalanine to CT carried out by Zeffren and Rea~i1l.l'~ A downfield shift in the "F n.m.r.peak was considered compatible with the location of the trifluoroacetyl group in a polar environment when bound to the enzyme. loo M. B. Hille and D. E.Koshland,jun.,J. Amer. Chem SOC.,1967,89,5945. lo' T. McL. Spotswood J. M. Evans and J. H. Richards J. Amer. Chem SOC. 1967,89,5054. J. T.Gerig J. Amer. Chem SOC. 1968,90 2681. lo' E. Zeffren and R E. Reavill Biochem Biophys. Res. Comm. 1968,32 73. M. R. Hollaway An important contribution to the understanding of CT catalysis has recently been made by Bernhard and Rossi.lo4 These authors have discussed the im- portance of conformation differences between native and acyl-CT derivatives. Also some elegant experiments were described indicating that the rate-limiting step in the deacylation of indoleacryloyl-CT involves the formation of a tetrahedral intermediate.Direct observation of an acyl-enzyme intermediate in the CT hydrolysis of a specific substrate has been reported by Miller and Bender.'" Thus incuba- tion of N-(2-furyl)acryloyl-~-tryptophan (FAT) with CT at low pH gave a derivative considered to be acylated enzyme. The first-order breakdown of this derivative to give free enzyme and FAT was dependent on a group of pK 6.95 required in the base form. Furthermore k, values for the CT hy- drolysis of FAT methyl ester also exhibited a sigmoid pH dependence pK 6.95 and the absolute values of k, were only slightly less than those for the breakdown of the low pH CT-FAT intermediate.Miller and Bender concluded that the CT-catalysed hydrolysis of FAT methyl ester at neutral pH values involved a single kinetically important intermediate namely the acyl-enzyme. Elastase. Elastase another representative of the serine proteases exhibits the usual characteristics of these enzymes.56* 57 In particular the pig pancreatic enzyme contains a single reactive serine residue per molecule which is in identical amino-acid sequence to the reactive serines in chymotrypsin-A and trypsin.lo6Furthermore the remainder of the elastase sequence is so extensively homologous with the other pancreatic serine proteases that Hartley and his collaborator^^^^ have suggested that they have all evolved from a common ancestor. Elastase also shows some functional similarity to the other serine protease in that it catalyses a biphasic release of p-nitrophenol from p-nitrophenyl pivalate,'08 a finding consistent with the formation of an acyl-enzyme inter- mediate.As in the case of chymotrypsin-catalysed reactions both acylation and deacylation steps exhibit large deuterium isotope effects and are dependent on an ionising group of pK 6.7 required in the base form.'os However elastase is less specific in its action than chymotrypsin in that it readily catalyses the hydrolysis of bonds involving the carboxy-group of a side variety of neutral amino-acids including Leu Ile Val Ala Ser Gly Tyr and Gln.Io9 An extremely rapid X-ray crystallographic determination of the structure of pig pancreatic tosyl-elastase at 3.5 A resolution has recently been accom- plished by Watson and Shottonl'O (a concise account of this and other work lo4 S.A. Bernhard and G. L. Rossi in 'Structural Chemistry and Molecular Biology,' ed. A. Rich and N. Davidson W. H. Freeman and Co. San Francisco 1968,p. 98. lo5 C. G. Miller and M. L. Bender J. Amer. Chem SOC. 1968,90,6850. lo6 M.A.Naughton F. Sanger B. S. Hartley and D. C. Shaw Biochem. J. 1960,77 149. lo' L.R Smillie and B. S.Hartley Biochem. J. 1966,101,232;J. R Brown D. L. Kauffman and B. S. Hartley ibid. 1967,103 497. lo* M. L. Bender and T. H. Marshall J. Amer. Chem SOC. 1968,90,201. log M. k Naughton and F. Sanger Biochem J. 1961,78 156. 'lo H.C.Watson and D. M. Shotton Proc. Roy. SOC.,December 1968,in preparation.Enzyme Mechanisms presented at the British Biophysical Society meeting December 1968 on 'Structural Aspects of Enzymatic Activity' has been given by Johnson' '). This work shows that the high degree of homology between chymotrypsin-A and elastase is reflected in the close similarity of their crystallographic struc- tures. Especially significant is the fact that of the 72 residues in the interior of the elastase molecule 78 yi &is homologous with the interior chymotrypsin residues whereas the 169 external residues are oniy 200,; homologous. Also the arrangement of His-57 Asp-102 and Ser- 195 in tosyi-elastase although not identical to that in chymotrypsin could allow the operation of the type of 'charge-relay system' suggested for the functioning of the latter enzyme.110* In view of the many similarities in structure and function between chy- motrypsin and elastase it would be surprising were they to catalyse reactions by different mechanisms.Thus it becomes of interest to account for their differing specificities. This is almost certainly connected with the fact revealed by the X-ray structure"'* ' '' that the entrance to the 'tosyl hole' in elastase is blocked by the bulky side chain of Val-216. This does not occur in chymotryp- sin where residue 216 is a glycine so that the major difference between the enzymes is probably in the presence of an ar site in chymotrypsin (28) but not in elastase. Trypsin. The extensive homology between chymotrypsin and trypsin has been discussed by several authors.60* '12 'l3 In particular Smillie and his co-workers' l3 have pointed out that the amino-acid sequences of chymo- trypsinogen A chymotrypsinogen B and trypsin exhibit a common pattern of invariant non-polar residues which are almost certainly located in the interior of the molecules.This implies that these enzymes have very similar structures confirmed by the fact that it has proved possible to build a model of trypsin based on the crystallographic structure of a-chymotrypsin. l4 These considerations together with many functional ~imilarities,'~ strongly suggest that chymotrypsin and trypsin have a common catalytic mechanism. The chief difference between trypsin and chymotrypsin is in their speci- ficities chymotrypsin hydrolyses bonds involving the carboxy-groups of amino-acids with bulky non-polar side-chains whereas trypsin shows specificity for bonds involving amino-acids with positively charged side-chains (for a review see ref.59). This suggests the participation of a negatively charged group on the enzyme in substrate binding. Smillie et u!.''~ consider this group to be the carboxylate anion of Glu-188 (corresponding to Ser-186 in the chymotrypsinogen sequence) on the basis that this is the only substitution of a negatively charged group for a neutral one within a sequence of otherwise invariant non-polar residues. Furthermore the corresponding residue in L. N. Johnson F.E.B.S.Letters 1969,2 201. B. S. Hartley J. R Brown D. L. Kauffman and L. B. Smillie Nature 1965,207 1157.L. B. Smillie A. Furka N. Nagabhusan K. J. Stevenson and C. 0.Parkes Nature 1968,218 343. P. B. Sigler D. M. Blow B. W. Matthews and R Henderson J. Mol. Biol. 1968,35 143. M. R.Hollaway chymotrypsin lies close to the active centre. However in view of the uncer- tainties in discriminating between aspargine and aspartic acid residues,65 it would also seem that the replacement of Sex-189 in the chymotrypsinogen sequence with what has been considered to be an asparagine in trypsinogen could also represent the crucial amino-acid substitution. This replacement is particularly attractive since Ser-189 in chymotrypsin is located114 at the bottom of the 'tosyl-hole' which seem likely to be the region involved in the expression of chymotrypsin specificity.Some elegant experiments on the interaction of trypsin with the com- petitive inhibitor' l5 benzamidine (29) as measured by difference spectroscopy have been reported by East and Trowbridge.'16 The dependency of the dif- ference spectra on benzamidine concentration at different pH values was consistent with the competition between inhibitor and protons for a single site in the trypsin molecule. This site was characterised by a pK value of 4.6 (29) R' = H R2= H (30) R' = any1 or alkyl R2 = H (31) R' = H R2= phenoxyalkoxy consistent with that expected for the ionisation of a side-chain carboxy-group. East and Trowbridge' l6also made the interesting observation that benzamidine binds to di-isoprop ylphosphoryltrypsin albeit more weakly than to native enzyme but not to trypsinogen or to trypsin modified by reaction with p-tolylsulphonyl-L-lysyl chloromethyl ketone.Baker and Erickson' '' have studied the inhibition of trypsin action by a number of substituted benzami- dines (30) and (31) and shown that although substitution at the R2 position (31) did not greatly diminish binding substitution at R' (30) gave a large decrease in affinity. The latter result would be anticipated on steric grounds if the benzamidine derivatives were to bind in the modified 'tosyl-hole'. This would correspond to the hydrophobic slit suggested by Mares-Guia Shaw and C~hen.~~~*''~ 'ls M. Mares-Guia and E. Shaw J. Biol. Chem. 1965,240 1579. E. J. East and C. G. Trowbridge Arch.Biochem. Biophys. 1968 125 334. 11' B. R. Baker and E. H. Erickson J. Medicin Chem. 1967,10 1123. '18 M. Mares-Guia E. Shaw and W. Cohen J. Biol. Chem. 1967,242,5777; M. Mares-Guia and E. Shaw ibid. p. 5782. Enzyme Mechanisms 625 An important finding by Beeley and Neurath'lg is that the reaction of the active centre histidine residue of trypsin (His-46 in the trypsinogen sequence corresponding to His-57 in chymotrypsinogen) reacts with bromoacetone at a similar rate to that of the model compound a-N-benzoyl-L-histidine methyl ester. In the resulting substituted trypsin derivative Ser-183 had lost its unusual reactivity towards acylating reagents. However His46 was still reactive towards bromoacetone in di-isopropylphosphoryl-Ser-183-trypsin.These findings serve to emphasize that in the absence of substrate the catalytic residues of trypsin do not possess exceptional reactivity and that the type of 'charge-relay system' represented in (19) is unlikely to be present in the free enzyme. Elmore and Smyth12' have made the interesting observation that the rate of deacylation of a-N-p-tolysulphonyl-L-lysyl-trypsin is 260 times that of a-N-methyl-a-N-p-tolylsulphonyl-L-lysyl-trypsin. This finding illustrates the importance of the am site interaction (28) during catalytic steps in trypsin reactions and is thus reminiscent of chymotrypsin-catalysed reactions.93* 94 The elegant rapid-reaction studies by Bernhard and Gutfreund12' using chromophoric substrates and dye-displacement techniques have shown that the simple three-step mechanism (17) is probably insufficient to describe trypsin-catalysed reactions.Some of the evidence for this conclusion is given in the excellent book by Bernhard7 together with a discussion of possible mechanistic consequences. Subtilisin. The extracellular bacterial proteases subtilisin Carlsberg and subtilisin BPN' (probably identical to subtilisin Novo'~~) are similar to other serine proteases in their reaction with acylating agents'23-' 27 and involvement of a histidine residue in cataly~is.'~~*~~* In addition the specificity of the subtilisins resembles that of chymotrypsin in that they preferentially hydrolyse derivatives of N-acyl-L-amino-acids with aromatic side chains.' '27 Ho wever 39 although the amino-acid sequences of Carlsberg and BPN subtilisins are 70 % homologous neither exhibits any obvious homology with the chymo- trypsin or trypsin sequences.' 24 Wright Alden and Kraut'29 have recently determined the crystallographic structure of benzylsulphonyl-subtilisinBPN (PMS-subtilisin) at 23A resolu-tion.The roughly spherical molecule which has few structural features in li9 J. G. Beeley and H. Neurath Biochemistry 1968,7 1239. 120 D. T. Elmore and J. J. Smyth Biochern. J. 1968 107 97. 12' H. Gutfreund and S. A. Bernhard Proc. Roy. SOC.,December 1968 in preparation. lZ2 S. A. Olaitin R J. DeLange and E. L. Smith J. Biol. Chem. 1968,243,5296. 123 A. 0.Barel and A. N. Glazer J. BioZ. Chem 1968,243 1344. 124 E. L. Smith F. S. Markland C. B.Kasper R. J. DeLange M. Landon and W. H. Evans J. Bid. Chem. 1966,241 5974. lZ5 A. N. Glazer J. Biol. Chem. 1968,243 3639. 126 K. E. Neet and D. E. Koshland Proc. Nat. Acad. Sci. U.S.A.,1966,56 1606. 127 A. N. Glazer,J. Biol. Chem. 1967,242,433. 12* L. Polgar and M. L. Bender Biochemistry 1967,6,610. 129 C. S. Wright R. A. Alden and J. Kraut Nature 1969 221 235. 626 M. R. Hollaway common with chymotrypsin contains eight a-helical regions involving 3 1% of the residues and much of the hydrophobic interior is built up from the side-chains of residues arranged in the parallel pleated-sheet structure. The most striking feature however is that the reactive Ser-221 residue lies close to His-64 whose imidazole 3-nitrogen atom is within hydrogen-bonding dis- tance of Asp-35.This arrangement is reminiscent of the Ser-195 His-57 Asp-102 arrangement seen in the chymotrypsin structure (19) and its occurrence in the active sites of enzymes with otherwise widely different structures indicates that it plays a key role in the catalytic processes. Wright Alden and Kraut'29 have also calculated a difference electron density map between PMS-subtilisin BPN and the native enzyme. This map reveals that the imidazole ring of His-64 which is within 3.5 A of the oxygen atom of Ser-221 in the native enzyme is rotated through approximately 80"in the PMS-enzyme to a position 4 A away from its original location. It is also noteworthy that the side-chain of Met-222 moves by about 1 A on substitution of Ser-221 a similar movement to that of Met-192 in chymotrypsin on substitution of Ser-195 with a tosyl group.I4 The essential serine of subtilisin has been chemically converted into a cysteine residue by two groups of workers.12'*'27 The product thiolsubtilisin possessed less than 1 % of the esterolytic activity of the native enzyme when specific substrates were employed,'26 although exhibiting comparable activity if substrates with good leaving groups (such as p-nitrophenol) were used.These findings were contrary to those expected on the basis of the superior nucleo- philicity of the thiol group and so Koshland and Neet'26 attributed the loss of activity in the thiol-enzyme to the larger van der Waals radius and different bond angle of sulphur failing to conform to the stringent steric requirements of the subtilisin active site.A not unrelated interpretation has been offered by Wang,20 who has suggested that subtilisin functions uiaa catalytic mechanism involving facilitated proton transfer along rigid accurately-held hydrogen- bonds. The sulphur atom because of its larger size and bond angle is considered unable to take part in the correct steric arrangement. Thiol Proteas=.-The thiol proteases are low molecular weight proteolytic enzymes containing a single thiol group per molecule essential for catalytic activity. Reviews of earlier studies with these enzymes have been given by Cunningham" and by Kimmel and Smith.'30 The 2.8 A resolution X-ray crystallographic structure of papain the most extensively studied of the thiol proteases has recently been published13' and confirms many of the conclusions reached by chemical studies.Papain. An examination of the detailed X-ray structure of papain deter- mined by Drenth and his c~llaborators,'~~ has shown that it is necessary to 30 E. L. Smith and J. R Kimmel in 'The Enzymes,' vol. IV,ed. P. D. Boyer H. Lardy and K. Myrback Academic Press New York 1960 p. 133. 131 J. Drenth J. N. Jansonius R. Koekoek H. M. Swen and B. G. Wolthers Nature 1968,218 929; J. Drenth et al. Proc. Roy. Soc. December 1968 in preparation. Enzyme Mechanisms 627 amend the amino-acid sequence proposed by Light Frater Kimmel and Smith.132 In this report the revised numbering from the X-ray sequence is employed.' 31 Prior to the X-ray crystallographic studies a considerable body of evidence existed for the implication of a thiol group' 303 132*'33 and the imidazole group of a histidine residue' 34-' 36 in papain-catalysed reactions.Furthermore the active-centre thiol group has been identified as that of Cys-25 and convincing evidence has been presented that this group is acylated by substrate during the course of cataly~is.'~' The amino-acid sequence'32 in the vicinity of this group is given in (32) together with the strikingly similar sequences around the essential thiol groups of ficin'38 and bromelain.'39 25 Papain Lys-Asn-Gln-Gly-Ser-Cys-Gly-Ser-Cys* -Ficin Arg- Gly-Gln-Gly -Gln-Cys- Gly- Ser- Cy s*-Stem Bromelain -Am-Gln-Asp-Pro-Cys-Gly-Ala-Cys*-(32) It is noteworthy that Ser-24 in papain is replaced by alanine in bromelain and is thus unlikely to play a crucial role in catalysis emphasizing the value of comparative studies of enzymes which operate by similar catalytic mechanisms.The earlier somewhat indirect kinetic evidence' 34 for the involvement of a histidine residue in papain catalysis has recently been substantiated by the elegant chemical modification studies of Husain and LOW^.'^^^ 13' Th ese authors demonstrated that the reaction of papain with the bifunctional reagent 1,3-dibromoacetone led to the intramolecular cross-linking of the thiol group of a cysteine residue to N-1 of a histidine residue indicating that these groups are within 5 A of each other. Sequencing studies identified these residues as Cys-25 and His-158.A similar procedure was employed to determine the sequences around the active centre histidine and cysteine residues in brornelain.l3' The histidine sequences for the two enzymes are given in (33). -_--_ I----Papain -Val-Asp+ His*- Ala-Va$ Ala~Al~Val-IGly-Tyr-/ 1158 I I IIlll l I I I Ill I Stem Bromelain XiHis*-Ala-Val~-Thr-jAla+ L---IleJGly-Tyr-/ --- - - - -. __ (33) i32 A. Light R Frater J. R. Kimmel and E. L. Smith Proc. Nut. Acud. Sci. U.S.A. 1964,52 1276. 133 S. S. Husain and G. Lowe Chem Comm. 1965,345. lJ4G. Lowe and A Williams Biochem J. 1965,96,194; A W. Lake and G. Lowe ibid. 1966,101 402. 13' S. S. Husain and G. Lowe Chem Comm. 1968 310; S. S. Husain and G. Lowe Biochem. J. 1968,108,861. S.S. Husain and G. Lowe,Biochem J. 1968,108,855; G.Lowe,Proc. Roy. SOC. December 1968 in preparation. IJ7 G. Lowe and A. Williams Proc. Chem. SOC. 1964 140; G.Lowe and A. Williams Biochem. J. 1965,96 189; L. J. Brubacher and M. L. Bender J. Amer. Chem. SOC.,1966,88,5871. 13' R C. Wong and I. E. Liener Biochem. Biophys. Res. Comm. 1964,17,470. lJ9 S. S. Husain and G. Lowe Chem. Comm. 1968 1387; Li-Pen Chao and I. E. Liener Biochem. Biophys. Res. Comm. 1967,27 100. 628 M. R.Hollaway The group X is not an aspartic acid residue,'39 so that Lowe and Husain have concluded that Asp-157 is unlikely to play a crucial role in papain catalysis. Wallenfels and EiseleI4* have also suggested the presence of a histidine residue in the active site of papain largely on the basis of a bell-shaped pH profile for the rate of inhibition of the enzyme by (-)-L-a-iodopropionic acid The prototropic groups giving the bell-shaped curve had pK values of 4.0 and 7-82 and were designated as a carboxy-group and an imidazolium group respectively.It was proposed that electrostatic interaction between the latter group and the carboxylate anion of the inhibitor served to orientate the inhibitor favourably for reaction with the essential thiol group. Kinetic studies have also led Cohen and PetraI4' to suggest the involvement of a histidine residue in the deacylation of (a-N-benzoyl-L-citrulliny1)papain. The detailed X-ray structure of papain'3' confirms many of the findings from chemical investigations. The molecule is made up from a single polypep- tide chain of 21 1or 212 residues folded so as to form two 'wings' separated by a marked cleft.The active site lies at the surface of the cleft and contains the imidazole side-chain of His-158 at a distance of 4 A from the sulphur atom of Cys-25. Trp-176 is also in the active site close to His-158 an observation of particular interest since Shinitzky and G01dman'~~ have obtained evidence from fluorometric measurements for an indole-imidazolium charge-transfer interaction in papain. Examination of the structure of papain given in ref. 131 shows that His-81 the only other histidine residue in papain is not near to a tryptophan so that the charge-transfer interaction is almost certainly between His-158 and Trp-176. Shinitzky and G~ldman'~' have also calculated from the dependence of fluorescence on pH that the interacting imidazolium group has a pK value of 7.22.Other groups in the active site identified from the X-ray stru~ture'~' are Gln-19 Asp-64 and Asp-157. Furthermore His- 158 is hydrogen-bonded to residue 174 (122 in the sequence of Light et which has been identified as an a~paragine.'~~ However in view of uncer- tainties in distinguishing between aspartic acid and a~paragine.~' residue 174 could be an aspartic acid thereby providing an interesting parallel to the serine proteases (18). Drenth and his collaborators' 31 have also calculated a difference electron- density map between native papain and papain substituted at Cys-25 by reaction with tosyl-L-lysyl chloromethyl ketone (TLCK) an irreversible inhibitor with some substrate-like pr0~erties.I~~ In this derivative the lysyl side-chain is seen to point towards A~p-64,'~' suggesting a possible role for this group in orientating substrates with positively charged side-chains during catalysis.The presence of the large cleft in the papain in the active site region had been predicted by some earlier studies by Schechter and Berger.'44 140 K. Wallenfels and B. Eisele European J. Biochem. 1968,3,267. 14' W. Cohen and P. H. Petra Biochemistry 1967,6 1047. 142 M.Shinitzky and R Goldman European J. Biochem. 1967,3,139. 143 B. G. Wolthers F.E.B.S. Letters 1969,2,143. 144 I. Schechter and A. Berger Biochem Biophys. Res. Comm.,1967,27 157; 1968,32 898. Enzyme Mechanisms 629 From a comparison of the rates of papain-catalysed hydrolysis of a series of pairs of diastereoisomeric alanine peptides (up to six residues) it was concluded that the active site was able to accommodate seven amino-acid residues.'44 The binding subsites were designated S S3 S2 S, S; Si and Sl, where the bond cleaved is between S and S;.In the extension of these investigations to map the subsites Schechter and Berger'44 have demon- strated that the S2 site has a high affinity for a phenylalanyl side-chain. Different mechanisms for papain action have been proposed by Husain and L~we'~~ The mechanism proposed by Husain and and by S1~yterman.l~~ L~we,'~~ which is similar to that proposed by Wang2' for chymotrypsin action starts with enzyme in which the thiol group of Cys-25 is hydrogen bonded to the imidazole side-chain of His-158 (34).Acylation of the thiol group by substrate is then con~idered'~~ to take pkce by a process involving a four-membered transition state as represented schematically in (34). H I H i I His-158 I 'Cys -25 ' \ (34) \ (35) In the deacylation step the leaving group X is replaced by water or another nucleophile YH and the imidazole group functions as a general base. Evidence for His-158 acting as a general base in deacylation has been presented by L0~e.l~~ This is based on the observation that the pK value of a group required in the deprotonated form for deacylation to occur decreased from 4-65 in water to 4-15 in 20% dioxan a shift expected for a cationic rather than a neutral acid i.e.an imidazolium rather than a carboxylate ionisation. Sl~yterrnan'~~, however favours a mechanism (35) whereby the imidazolium form of His-158 functions as a general acid catalyst at the substrate carbonyl oxygen atom during the rate-limiting acylation of Cys-25. The key difference between this mechanism (35) and that of Lowe (36) is that His-158 is required in the acid form during acylation whereas in Lowe's mechanism it is required in the base form. Hence the discrimination between the mechanisms depends on 145 L. A. Ae. Sluyterman Proc. Roy. SOC.,December 1968 in preparation. M. R. Hollaway H2 Hh . the interpretation of pH profiles of rate constants for separate processes in catalysis.Such a discrimination is not possible at the moment because of the conflicting conclusions'46~ 147 about which step is rate-limiting in the papain- catalysed hydrolysis of benzoly-L-arginine ethyl ester probably the most in- tensively studied substrate for papain. As pointed out by Brocklehurst Crook and Whart~n,'~~~ non-productive binding of this substrate to papain could lead to the assignation of incorrect values of rate constants for acylation and deacylation. Further complications could also ensue from the suggestion by Henry and Kirsch14* that the three-step mechanism may be insufficient to describe some papain-catalysed reactions although this appears not to be the case149 with benzyloxycarbonylglycine aryl esters as substrates.Despite these considerations however it seems likely from the data of Whitaker and Bender that acylation of papain by substrate is dependent on two ionising groups of pKk values 4-3and 85 one required in the acid form and the other in the base form. It remains to identify the systems contributing these ionisations un- ambiguously and to establish their exact role in catalysis. Other thiol proteases. The other less extensively studied thiol proteases resemble papain in both structure and function.59* 13* The similarities of the amino-acid sequences in the vicinity of the active-site cysteine residues of fi~in,'~' br~melain,'~~ and papain'32* 1339 13* have already been indicated (32) and there is convincing evidence that the thiol groups of these residues are acylated during ~atalysis.'~' Furthermore a histidine residue is a component of the 146 J.R Whitaker and M. L. Bender. J. Amer. Chem SOC. 1965. 87.2728. 14' L. A. Ae. Sluyterman Biochim. Biophys. Acta 1968 151 178. 148 A. C. Henry and J. F. Kirsch Biochemistry 1967,6 3536. 148 'K. Brocklehurst E. M. Crook and C. M. Wharton F.E.B.S. Letters 1968 2 1969. C. D. Hubbard and J. F. Kirsch Biochemistry 1968,7 2569. 14' Enzyme Mechanisms 631 active site in all three enzymes'35. and the amino-acid sequences around these residues in papain and bromelain are similar.' 39 These considerations together with the overall similarities in amino-acid composition,' 50* 15' strongly suggest that these enzymes have a common catalytic mechanism.' 51 like papain,143- lS3are It is noteworthy that fi~in'~~.and br~melain,'~~ inhibited by the chloromethyl ketone derivatives of tosyl-L-phenylalanine (TPCK) and tosyl-L-lysine (TLCK) through reaction at the active centre thiol groups. Also each enzyme reacts faster with TLCK than with TPCK although these compounds react with cysteine at similar suggesting that TLCK binds to the enzymes so as to facilitate subsequent reaction with the essential thiol group. As with papain,'46 evidence has been presented'54 that the acylation of ficin is dependent on two ionising groups of pK values 4.4 and 8.5 at 25" and I = 01 one of which is required in the protonated form and the other in the deprotonated form. It is tempting to assign the ionisation of pK 8.5 to the active centre thiol residue which has been shown'55 to have a pK value of 8.55 (25" I = 0-1)but it is not possible to decide from available data whether this group is required in the acid or the base form for catalysis to occur.How- ever in view of the greater nucleophilicity of the ionised form of thiols it seems likely that the base form of the thiol group would be required in acylation which means that the group of pK 4.4 would be required as an acid. Another thiol protease known to contain a histidine residue in the active site,' 56 is the so-called streptococcal proteinase. Of particular interest was the finding by Gerwin157 that whereas the rate of inhibition of this enzyme by chloroacetamide gave a sigmoid pH-dependence a bell-shaped pH-profile was obtained with chloroacetate ion as inhibitor.Furthermore at pH 4.2 the rate with chloroacetate ion was at least one hundred times the rate with chloro- acetamide. These results which suggest' 57 the presence of an ionising cationic group in the active centre of the enzyme are essentially similar to those ob- tained in the reaction of papain with the (-)-L-antipodes of a-iodopropionate ion and a-iodopropionamide. 140 Carboxypeptidase A.-Reviews of earlier work on this enzyme are by ningham5' and Vallee.' 58 Carboxypeptidase A (CPA) specifically catalyses the hydrolysis of peptide bonds at the carboxy-terminus of polypeptide chains. It is a small extracellular enzyme of molecular weight 34,600 containing a single zine atom per molecule essential for catalysis.However the derivatives S. S. Husain and G. Lowe. Biochem. J.. 1968.110 53. P. T. Englund T. P. King L. C. Craig and k Walti Biochemistry 1968,7 163. 152 W. J. Stein and I. E. Liener Biochem Biophys. Res. Comm. 1967,26 376. 15' T. Murachi and K. Kato J. Biochem (Japan) 1967,62,627. M. R Holliaway European J. Biochem 1968,5 366. M. R Hollaway A P. Mathias and B. R Rabin Biochim. Biophys. Act4 1964,92,111. T-Y.Liu J. Biol. Chem 1967,242,4029. 15' B. I. Gerwin J. Biol. Chem. 1967,242,451. "* B. L. Vallee Fed. Proc. 1964,32 8. 632 M.R.Hollaway of CPA in which Zn2 + is replaced by Mn2 +,Co2+,or Ni2 + are still catalytic- ally active.158* lS9 The elegant X-ray crystallographic studies by Lipscomb and his co-workers'60 have resulted in the determination of the structure of bovine CPA at 2 A resolution.The molecule contains a single disulphide link and 30% of the 307 amino-acid residues are in an a-helical arrangement with a further 20% in a twisted P-pleated sheet conformation of either the parallel or the antiparallel type. As with some of the other hydrolytic enzymes there is a pronounced cleft in the molecule at the active site.'60 This observation confirms the prediction by Abramowitz Schechter and Berger16' from peptide 'mapping' studies that the active site of CPA would be able to accom- modate a substrate comprising 5 residues i.e. should be at least 18 A in length. The zinc atom is bound within the cleft through the side-chains of residues His-69 Glu-72 and Lys- 196 the fourth co-ordination position being occupied by a water molecule.Lipscomb and his collaborators' 6o have also calculated difference electron density maps for CPA and CPA containing bound glycyr-L-tyrosine or L-~YSY~-L-tyrosinamide and have made the following striking observations. (a) The tyrosine side-chain corresponding to the normal C-terminus of a substrate is located in a pocket sufficiently large to accommodate a tryptophan side-chain. This pocket is located at the end of the cleft. (b) The guanidinium side-chain of Arg-145 moves through about 2A to form an ionic bond with the carboxylate anion of the substrate. This movement is largely effected by rotation about the C(P)-C(y) bond of Arg-145. (c) The water molecule bound at the active centre zinc atom is displaced by the carbonyl oxygen of the substrate peptide bond.(d) In the glycl-L-tyrosine-CPA complex the carboxy-side-chain of Glu-270 is hydrogen-bonded to the amino-group of the glycyl residue. However model-building has shown that in substrates with more than two amino-acid residues this interaction may not occur and that Glu-270 may then approach the peptide bond which is bound at the zinc atom. The glycyl-Glu-270 inter- action could explain the stability of the CPA-glycyl-L-tyrosine complex. (e) On binding glycyl-L-tyrosine the phenolic oxygen atom of Tyr-248 moves through a distance of 12 A as a result of a shift in the peptide backbone and rotation about the C(ol)-C(P) bond. (f) The zinc atom moves through about 1 A.159 R. C. Davies J. F. Riordan D. S. Auld and B. L. Vallee Biochemistry 1968,7,1090. M. L. Ludwig J. A. Hartsuck T. A. Steitz H. Muirhead,J. C. Coppola G. N. Reeke and W. N. Lipscomb Proc. Nut. Acud. Sci. U.S.A. 1967,57,511;G.N. Reeke J. A. Hartsuck,M. L. Ludwig F. A. Quiocho T. A. Steitz and W. N. Lipscomb ibid. p. 2220; W. N. Lipscomb in 'Structural Chemistry and Molecular Biology,' ed. A. Rich and N. Davidson W. H. Freeman and Co. San Francisco 1968 p. 38; W. N. Lipscomb et al. Proc. Roy. SOC. December 1968 in preparation; W. N. Lipscomb J. A. Hartsuck G.N. Reeke F. A. Quiocho P. H. Bethge M. L. Ludwig T.A Steitz H. Muirhead and J. C. Coppola Brookhaven Symp. Biol. 1968,21 in the press 16' N. Abramowitz I. Schechter and A.Berger Biochem Biophys. Res. Comm. 1967,29,862 Enzyme Mechanisms (g) The conformation changes do not take place on the binding of poly- peptides in which the C-terminal carboxy-group is blocked e.g. with L-lysyl- L-tyrosinamide.'60 This suggests that the movements of CPA side-chains are triggered off by the substrate-carboxy-group-to-Arg-145ionic bond possibly by disruption of the hydrogen-bonding around this residue.' 6o A diagrammatic representation of the postulated interactions' 6o in a CPA-substrate complex is given in (36). The foregoing observations seem to provide one of the most clear-cut examplesof Koshland's 'induced-fit' theory 162 whereby the specific interaction between an enzyme and its substrate results in a conformation change in the enzyme protein so as to give a catalytically active structure.(37) R' \ 2+. Zn- / I Glu-270 On the basis of the structural work Lipscomb and his co-workers'60 have suggested two possible mechanisms. In the first (37) the carboxylate anion acts as a nucleophile and attacks the carbonyl carbon atom of the C-terminal peptide bond of the substrate. This is facilitated by the zinc atom acting as a Lewis acid at the carbonyl oxygen atom and removal of the leaving group by interaction with the phenolic hydroxy-group of Try-248 acting as an acid. This mechanism therefore involves an intermediate anhydride of Glu-270 and the acyl portion of the substrate. lci2 D. E. Koshland jun.,Fed. Proc. 1964,23 719. 634 M. 4.Holloway In the second mechanism involving a single displacement (38) the carboxy- late anion of Glu-270 functions as a general base in abstracting a proton from a water molecule as it adds to the substrate carbonyl carbon atom.The latter mechanism is favoured'60 on the grounds that CPA does not catalyse trans- peptidation reactions. Vallee and his co-workers163 have presented a schematic model based on multiple modes of substrate binding which attempts to explain the complex kinetic behaviour of CPA towards ester and peptide substrates. This model has correctly predicted a reciprocal effect of bound ligands on esterase and peptidase activities of CPA. Ribonuc1ease.-Bovine pancreatic ribonuclease A (RNAse A) molecular weight 13,600 comprising a single polypeptide chain of 124 residues catalyses the hydrolysis of internucleotide phosphodiester bonds in ribonucleic acids only if the phosphate of the susceptible P-0 bond is attached to the 3'-OH of a pyrimidine nucleotide.The reaction is known to proceed with the inter- mediate formation of 2'3'-cyclic phosphates which are themselves substrates for ribonuclease action. Reviews of earlier work are by Anfin~on'~~ and by Mathias Deavin and Rabin.' 65 Kinetic evidence has been presented'66 for the implication of two imidazole groups in RNAse-catalysed reactions and this has been substantiated by the results of chemical modification studies 67 which have identified the active- site imidazole residues as those of His-12 and His-119. A mechanism involving concerted acid-base catalysis by these groups has been suggested by Rabin and his c~llaborators.'~~~ 166 The detailed X-ray crystallographic structures of RNAse and RNAse Si70 have been published and confirm many of the chemical predictions.In the structure of RNAse A presented by Harker and his co-~orkers,~~~ the active site region which contains a bound phosphate ion is at the surface of a distinct cleft and contains the side-chains of residues His-12 His-1 19 Lys-7 Gln-11 Lys-41 and His-48. The 3.5 A resolution structure of RNAse S (a derivative of RNAse A with the Ala-20-Ser-21 peptide bond cleaved by treat- ment with subtilisin) published by Richards and his collaborators' 70 closely resembles the RNAse A structure apart from the region of Ala-20 and Ser-21 which are separated by 10-15 A in RNAse S.163 B. L. Vallee J. F. Riordan J. L. Bethune T.L. Coombs D. S. Auld and M. Sokolovsky Bio-chemistry 1968,7 3547. 164 C. B. Anfiison Brookhaven Symp. Biol. 196515 184. 165 A. P. Mathias A. Deavin and B. R. Rabin in 'Structure and Activity of Enzymes,' ed. T. W. Goodwin J. I. Harris and B. S. Hartley Academic Press New York 1964 p. 19. 166 D. Findlay D. G. Herries A. P. Mathias B. R Rabin and C. A. Ross Biochem J. 1962,85,152. 16' A. M. Crestfield W. H. Stein and S. Moore J. Biol Chem. 1963 US,2413. 16' A. Deavin A. P. Mathias and B. R Rabin Nature 1966,211,252; A. Deavin A. P. Mathias and B. R. Rabin Biochem J. 1966 101 14C. G. Kartha J. Bello and D. Harker Nature 1967,213,862; G. Kartha J.Bello and D. Harker in 'Structural Chemistry in Molecular Biology' ed A Rich and N. Davidson W. H. Freeman and Co. San Francisco 1968 p. 29. ''O H. Wyckoff K. D. Hardman N. M. Allewell T. Inagami L. N. Johnson and F. M. Richards J. Biol. Chem. 1967 242 3984 3749. 16' Enzyme Mechanisms 63 5 Electron-density maps for the complexes of RNAse S with the competitive inhibitors uridine 2'(3')-phosphate and 5-iodouridine 2' (3')-phosphate have also been calculated' 70 and the following locations of active-site residues established. (a)His-12 and His-119 are close to the nucleotide binding site. Furthermore His-119 is freely accessible to solvent whereas His-12 is partly buried with the N-1 atom inaccessible to solvent. This observation is consistent with the fact that iodoacetate ion reacts at the N-3 atom of this residue.'67 (b) The uracil portion of the inhibitors is located in a groove beneath the histidine residues bounded by residues Val-43 Phe-120 Thr-45 and Ser-123.Position 5 of the uracil ring points towards the liquid and is close to Ala-122. (c) Gln-11 and Asn-44 are above the histidine residues and Asp-121 within a slot to the left of His-119 (positions as depicted in ref. 170). (d) The E-amino-groups of Lys-7 and Lys-41 are in the active site region as predicted by chemical st~dies,'~' and could move close to the bound nucleo- tide. It is noteworthy that there are no marked conformation changes in the enzyme on binding the inhibitors."' More recent work by Richards Wyckoff and their collaborators has extended the crystallographic data for RNAse S and several RNAse S-sub- strate analogue complexes to the level of 2 A resolution (an account of this work was given by N.Allewell at the British Biophysical Society Meeting Dec. 1968 on 'Structural Aspects of Enzymatic Activity' which has been reported by Johnson'"). Thus the electron density map of the RNAse S-cytidine 3'-phosphate complex reveals that the 2-OH group of the ribose ring is hydrogen bonded to His-12 and the 2- and 6-positions of the cytosine ring are hydrogen-bonded to Thr-45. These interactions probably account for the specificity of the enzyme for pyrimidine nucleotides. His-119 appears to be displaced by the phosphate group of the inhibitor towards Asp-121.A schematic representation of these interactions is given in (39). An additional interaction shown in (39) is that of the E-NH group of Lys-7 with the N-7 atom of the guanine base in the guanosine nucleotide binding site. Such an interaction has been predicted on the basis of spectrophoto-metric investigation of the binding or guanosine nucleotides to RNAse.'" The crystallographic studies are thus consistent with the predicti~n'~~.'~~~ 168 that catalysis by RNAse involves concerted acid-base catalysis by the imidazole side chains of His-12 and His-119. A schematic representation of a possible mechanism,' ''which may involve the formation of a pentacovalent phosphate intermediate is given in (40). The formation of this intermediate (39) would be 17' C.H. W. Hirs Brookhaven Symp. Biol. 1962 15 154; P. S. Marfey M. Uziel and J. Little J. Biol. Chem 1965,240 3270. 17* A. Deavin R Fisher C. M. Kemp k P. Mathias and B. R Rabin European J. Biochem 1968,7 21. W M. R.Holloway facilitated by the interactions with His-12 and His-119 as indicated by the elegant studies of model systems by Westheimer and his collaborators.17 + Lys-7 -NH2 I H 0 I-r12 I His-12 173 F. H. Westheimer Accounts Chem. Rex 1968 1 70 Enzyme Mechanisms Some other interesting studies involving the histidine residues of RNAse are those by Jardetzky and his colleagues.'74 These authors have resolved the C(2r-H n.m.r. absorptions of the histidine residues and from the change in position of the peaks with pH determined the individual pK values of these groups.Furthermore the binding of cytidine 3'-phosphate to RNAse was shown to shift the pK value of His-119 from 5.8 to 7.4 and of His-12 from 6-2 to 8.0. These results have been discussed in terms of a model involving the binding of the dianion of the inhibitor to the active site of RNAse with both His-12 and His-1 19 in the protonated form. 174 D. H. Meadows and 0.Jardetzky Proc. Nut. Acad. Sci. U.S.A. 1968,61,406; D. H. Meadows J. L. Markley J. S. Cohen and 0.Jardetzky ibid. 1!?67,58,1307;D. H. Meadows 0.Jardetzky R M. Epand H. H. Ruterjans and H. A. Scheraga ibid. 1968.60 746.

ISSN:0069-3030    

DOI:10.1039/OC9686500601

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

ERRATA Vol64 Section B 1967 Page 163 Footnotes. Transfer asterisked footnote at bottom of page to page 187. Page 207. The transylidation reaction scheme should be CH,=CH-CN + Ph,P=CHCO,Et CH,=CH--CO,Et + Ph,P=CH-CN Page 207 Reference 70. For 1967 88 5654 read 1966 88 5654. Page 208 line 10. For diazophospholidines read diazaphospholidines. Page 210 line 13. For Dickmann read Dieckmann. Page 216 line 21. For 2,2,7-read 2,2,2-Page 494 line 14-15. For nicotinamide mononucleotide19~ 21 read nicotin-arnidel9.21 639

ISSN:0069-3030    

DOI:10.1039/OC9686500639

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Abakumov G. A. 183 Abe N. 418 Abegg V. P. 276 Abelson J. 563 Abenhaim D. 291 Abermark B. 332 Abraham M. H. 131,289,317 Abrahams S. C. 58 Abrahamson E. W. 143,188 Abramovitch R. A. 168 179 347 Abramowitz N. 632 Abramson H. N. 62 Achari B. 489 Achenbach H. 452 Acheson R. M. 465 Achilladelis B. 586 Acton E. M. 472 Adachi K. 37,391 Adachi T. 467 Adams A. 566 Adams D. G. 285 Adams F. H.. 233 Adams. G. E.. 224. 225 226. 229 Adams J. 71 Adams J. Q.,31,183 Adams R. F. 29 Adams R.N. 29,247,248,249 Adams R. W. 326 Adamson D. W. 146 Adesogan E. K. 427 Adickes H. W. 358,456 Adler H. I. 566 568 Adman E. 49 57 546 Adolph H. G. 134 Aeberli P. 220 Agadzhanyan T.E. 528 Agami C. 280 Agarwal K. L. 5 17 525 Agatsuma K. 417 Agosta W. C. 200 Agranat I. 362 Agurell S. 492 579 AH T. D. 60 Ahlbrecht H. 385 Ahmad M. S. 436 Ahmed F. R. 64 Ahmed N. A. K. 42 AUTHOR INDEX Ahoud A. 275 Aikens D. A. 244 Akabori S. 354 Akashi M. 402 Akchurina I. S. 315 Akermark B. 259 Akhmetova N. E. 24 Akhrem A. A. 398 Akhrem I. S. 103 Akhtar A. 286 Akhtar M. 316,593 Aksnes G. A, 83 Aktar R.,520 Akutsu H. 527 Alakhov Y. B. 517 Alazrak A. 240 Albert A. H. 257 Alberts B. M. 569 Albery W. J. 79 Albrecht J. 313 Alcais P. 97 Alcock N. W. 312 Alden C. K. 176 Alden R. A. 625 Alder R. W. 370 Aleksandrov Y. A, 318 Alexander E. 50 Alexander L.E. 66 Alexandrou A. U. 314 a1 Holly M. M. 204 Ali A. A. E. R. 490 Ah Y.,264 Alkaitis A, 467 Allen D. W. 350 Allen F. H. 41 60 Allen L. C. 115 Allendoerfer R. D. 20 Allenmark S. 80 127 Allewell N. M. 634 Alley E. G. 220 Allinger N. L.,380 Allmann R.,46 Allred A. L. 24 26 308 Allred E. L. 123 385 Almenningen A. 287 Almy J. 133 Al'perovich N. E. 297 Alt A. 308 Alt H. 26 641 Altman. L. J. 481 Altona C. 62 Aluotto P. F. 129 Alvarez F. S. 277 Alves H. M. 476 Amaldi F. 552 Ambady G. K. 43 Ambrose J. F. 249 Amiet R.G. 359 Amit A. 53 Ammon H.,L. 55 Amphlett C. B. 224 Ampola M. 511 Amy J. W. 16 Anastasi A. 509 526 Anastassiou A.G. 161 166. 167 Anbar M. 224 Anda H. 585 Andersen A. G. jun. 22 Andersen N. H. 273,406 Andersen P. 56 Anderson A. G. 375 Anderson C. B. 469 Anderson C. M. 13 Anderson D. F. 413 Anderson D. G. 110.308.309 310,311 Anderson J. D. 252 Anderson J. E. 280,478 Ando S. 316 Ando W. 221,268 Andreatta R. 527 Andreeva L. I. 509 Andrejevic V. 184 Andrew H. F. 94 Andrews K. J. M. 480 Andrews L. J. 126 127 128 271,349 Andrews S. D. 206 Andrews T. D. 296 Anet E. F. L. J. 471 Anet F. A. L. 194 380 Anfinson C. B. 634 Angelo F. 520 Angyal S.J. 469 Anh N. T. 131 Anisimov K. N. 313 Anjaneyulu B. 447 Anner G. 261,434 Annino R.,257 Anorova G. A, 297 Ansell M.F. 374 Anson F. C. 259 Anteunis M. 381 Anthoine G. 365 Antipin L. M. 312 Antonov V. K. 509 528 Aoki D. 330 Aoyagi S. 549 Applequist D. 118 Applequist D. E. 172 208 220,373 Appleton R. A. 114 382 Apt K. E. 380 Arai H. 215 Arai S. 225 Arapakos P. G. 265 Arav R.,537 Arcamone F. 37 Archibald T. G. 458 Arens J. F. 391 Aresta M.,.337 Arguelles M. 486 Ariely S. 513 Arigoni D. 581 582 590 59 1 Arihara S. 438 Arison B. 540 Armand J. 254 Armstrong D. J. 538 Armstrong D. R. 293 Armstrong V. C. 67,68 Amakasu T. 475 Amett E. M. 109 Arnold D. R. 187 442 460 Arnold N. H. 598 Amold R. A. 465 Amott R.C. 288 Arnott S. 551 Arold H. 518 Arotsky J.97 Arrington J. P. 201 Arthui P. 277 Asaki Y. 216 Asami R.,135 Asato A. E. 268 Ashby E. C. 262 283,288 Ashe A. J. 118 Ashida T. 45 Asinger F. 327 391 Asmus K. D. 224,225 Asselin A, 418 Asteriadis G. T. 560 Atal C. K. 489 Atherton J. H. 152 Atherton N. M. 29 171 Atkins G. M. 352 Atkins J. M. jun. 447 Atkins P. W. 33 Author Index Atkins R 456 Atkinson M. R. 542 Atkinson R. S. 169,441 Attardi G. 552 Atwell W. H. 310 Atwood J. L. 305 Aubert M. 552 August J. T. 560 Auld D. S. 632,634 Aurich H. G. 28 Austin J. D. 309 Avatis K. 620 Avers C. J. 570 Avery E. C. 33 Avigad G. 40 Avila N. V. 432 Avram M. 387 Avrutskaya I. A. 236 Awad E. S. 618 Axelrod H.D. 259 Axelrod M. 38 Axen U. 406 Axenrod T. 14 Ayer D. E. 198 Ayer W. A. 504 Aylett B. J. 283 Aylward J. B. 184 Ayscough P. B. 17 Azaroff L. V. 41 Aziz S. 79 Baars-Diehl S. 514 Baba Y. 307 Babad E. 197 395 Babb B. E. 293 Babler J. H. 303 Babu B. H. 91 Babushkina T. A, 314 Bach G. 266 Bach K. D. 594 Bacha J. D. 183,184,271 Baciocchi E. 96 Bacon J. 248 Baczynskyj L. 537 Badawi M. M. 504 Badger R. A, 402,416,419 Baenziger N. C. 108 Baer F. 28 Baer H. H. 474 Bafoz-Lambling J. 260 Baggaley A. J. 246 Bagli J. F. 218 Baguley B. C. 539 563 Bailar J. C. jun. 326 Bailey D. T. 496 Bailey G. C. 323 Bailey K. 263 Bailey P. S. jun. 450 Baillie k C.423 Baiocchi L. 343 Baird M. C. 283 329 Baird M. S.,394 Baird R.,348 Baird W. C. 404 Baird W. C. jun. 405 Baitis F. 214 Baizer M. M. 250 252 Bajer F. J. 318 Bajusz S. 509 Baker A. W. 136 Baker B. R.,602,624 Baker E. J. 349 Baker W. 372 Balaban A. T. 183 370 Balakrishran P. V. 401 Balandini G. L. 528 Balazs E. A. 230 Baldas J. 493 Baldock R.D. 463 Baldwin J. E. 115 168 176 205 270 278 279 376 398 411,431,451,461 6QO Baldwin R.L. 569 Balgir B. S. 96 Ballard D. 311 Ballard D. G. H. 330 Ballio A, 427 Balog A. 518 Balzer W. D. 38 Bamford C. H. 162 Ban Y. 500 Band S. J. 308 Bangerter B. W. 550 Bank R E. 23 Bankiewicz C. 505 Banks R L.323 Banthorpe D. V. 585 Barbeis G. 97 Barber L. 204 Barbieri G. 266 Barbieux M. F. 37 Barbour R.V. 176,437 Barclay L. R C. 344 346 Bard A. J. 249,256,259 Barel A. O. 625 Barg W. F. jun. 524 Barkemeyer H. 521 Barltrop J. A, 198 201 206 Barneis Z. J. 494 Barner R.,350 Barnes C. S. 13 Barnes D. 252,253 Barnes J. M. 283 Barnett C. 135 Bamett J. E. G. 471 Barney A. L. 330 Bamhardt R. G. jun. 274 Barrell B. G. 552 Barrett E. J. 379 Barrett G. C. 39 Barrett H. C. 418 Barrett J. F. 526 Barrett J H. 57 482 Barrow K. D. 427 Barry LCo-G. 513 Barstow L. E.,404 Bart J. C. J. 46 51 57 Bartell L. S.,293 Bartlett P. D. 111 148 149 173 174 206 379 387 Bartoli.G. 100 101 Barton D. H. R. 176,221,267 272 344 345 419 427 428 431 586,592 Barton J. W. 372 Barton T. J. 104 197,483 Bartsch R.A, 137 Bateman J. H. 202 Bates R B. 278,411,417 Bath R. J. 527 Batley G. E. 326 Battail J. 586 Battersby A. R. 494 495,491 579,581,582 Battiste M. A. 104 123 Bauer A. 519 Bauer F. 375 Bauer S. 470 Bauer S. H. 382 Bauer W. 361 571 Baukov Yu. 1.. 3 11 Bauld N. L. 237 Baum E. J. 32 Baum J. W. 200 Baumann N. 189,461 Baumgarten D. 573 Barry R H. 450 Bawn C. E. H. 153 Baxendale. D. 585 Baxendale J. H.. 224 226 Baxter J. W. M. 527 Bayer E. 509,524 Bayev A. A. 538,564 Bayless A. 318 Bayreuther K. 515 BaZant V. 262 284 Bazhin N.M. 24 Beach R L.,,271 Beacham J. 525 533 Beak P. 215,460 Beal D. A. 266 Bean G. P. 97 Beard. R. D. 280 286 Beauchamp k L. 319 Beavan C. H. 525 Beck B. H. 381 Beck F. 252 Beck G. 225 Beckett A. H. 500 Beckey H. D. 15 Beckwith,A. L. J.. 168 178,372 Bednowitz. A L. 48 Author Index Beecham,A. F.,38,39,495,500 Beeley. J. G.. 625 Beeman W. W. 565 Beer R J. S. 462 Beesley T. E. 521 524 Begland R. W. 124 Behar J. V. 175 Behn C. G. 137 Behr W. 573 Beissner K. A. 573 Belanger A. 391 Belavin I. Yu. 311 Beletskaya I. P. 283 292 Bell J. A. 161 Bell K. H. 351 Bell M. R. 448 Bell P. H. 524 Bell R. A. 495 Bell R.H. 221 265 473 Bell R. P. 141 Belleau A, 520 Belleau B.619 Bellin S. A. 415 Bellingham P. 97 Bello J. 634 Bellobono I. R. 101 BelluS D. 187 210 Beltrame P. 101 130 Beltrame P. L. 101 Bemis A, 109 184 Bender M. L. 601 612 618. 622 625,627,630 Ben-Efraim D. A. 323 392 Beng M. 93,344 Ben-Hur. E. 546 Benion P. R. 499 Ben-Ishai B. 546 Benkeser R. A. 250,251 285 289 310 Benkovic J. 602 Benkovic P. A, 86 Benkovic S. J. 86 Bennett D. J. 577 Bennett J. E. 30 Bennett M. A. 337 Bennett M. J. 319 Bennett R. P. 346 Benoit R. L. 260 Benoiton L. 512 518 Benson R. E. 196 Benson S. W. 161,379 Benthin I. 290 Bentley M. D. 124 348 Bentley P. H. 525 Bentley T. W. 13 Bentrude W. G. 178 Berchtold G. A. 221 Berdahl J.M. 267 Berg A. 569 Berger A. 632 Berger C. 418 Berger H. 535 Berger L. R. 606 Bergeson K. 83,85 Bergin R. 45 Bergman R. G. 116 117 125 Bergmann E. D.,329,361,362 379 Bergmark W. 449 Bergomi A. 207 Bergson G. 133 Bergstrom G. 414 Bergstrom S. 596 Beringer F. M. 204 Berkoz B. 432 Berliner E. 97 113 Berlman 1. B. 228 Berman H. M. 59 Bernard S. A. 622 Bernadi L. 509,526 Bernardi G. 569 Bernardi R. 178 Bernasconi C. F. 97 Bernauer K. 219 Bernett W. A. 118 Bernhard S. A,,602,625 Berntsson P. 339 Berse C. 437 Berseck L. 79 Berson J. A 125,145,398 Berson J. M. 116 117 Bertaccini G. 509 526 Bertele E. 415 Bertelli D. J. 135 364,418 Bertrand J.A, 529 Bertrand M. 124 Bertrand M. P. 176 Besolova E. A. 317 Bespalova Zh. D. 527 Bessler E. 311 Bestmann H. J. 370 391 Bethea T. W. 183 Bethell D. 79 Bethge P. H. 632 Bethune J. L. 634 Bettani G. 513 Bevan C. W. L. 89 Bevan P. L. T. 226 Beveridge A. D. 316 Beveridge D. L. 19 Beychok S. 35 Beyer H. 458 Beyer K.-H. 172 Beyerman H. C. 509 Beyrich W. 16 342 Bhacca N. S. 439 Bhakuni D. S.,414 Bhatnagar A. K. 494 Bhatnagar S. P. 476 Bhattacharya S. K. 447 Biale G. 138 Biallas M. J. 293 Bick I. R.C. 493 Bickart P. 38 Bickelhaupt F. 394 Bidzilya V. A. 171 Bielavsky J. 67 Biellmann J. F. 279 325 Bielski B. H. J. 24 Biemann K.,10 537 546 Bien A.S. 249 Biere H. 496 Bigam G. 196,395,399 Bigley D. B. 303 Bigotto A. 314 Billbrey R 610 Billheimer F. E. 570 Billig E. 331 Billups W. E. 394 Binch A. J. 337 Bindra A. P. 367 Binger P. 298 Bingham R. C. 113 Binkley R.W. 473 Binkley W. W. 473 Binks R 579 Binford J. S. 548 Binns T. D. 235,240 Binsch G. 148 Biollaz M. 598 Birch A. J. 414,431,494 587 Bird C. W. 219 Birkhofer L. 509 510 Birktoft J. J. 613 Birnbaum G. I. 61,491 Biryukov B. P. 313 Bishop C. E. 221 392 Bishop D. H. L. 560 Biswas A. B. 43 Biswas K. M. 264 Bittler K. 276 328 Bittner G. 295 Bixby E. M. 511 Bjamer K. 62 Bjornason T. 520 Bjorvatten T. 44 Black D. K. 510 Blackburn B.J. 33 183 Blackburn E. V. 208 Blackburn G. M. 78,278,279 357,411,543 Blackwell L. F. 140 Blackwood J. E. 268 Blaha K. 37 39 Blair D. G. 573 Blake A. 574 Blake C. C. F. 606 Blake R D. 549,565 Blanc-Guenee J. 280 Blank D. R.,286 Blank H. U. 479 Blankley C. J. 400 Blanton C. D.,jun. 455 Author Index Blass A. S. 117 Blatter H. M. 183 Blatz P. E. 104 Blejwas M. 343 Blewett C. W. 318 Blight M. M. 587 Blok J. 547 Blomberg C. 289 Bloomer J. L. 596 597 Bloomfield J. J. 262 387 Blosich G. J. 444 Blouri B. 139 Blout E. R.,38 513 522 Blow D. M. 602,612,613,620 624 Blum J. 329 Blumberg P. 83,128 Boag J. W. 224,226 Bobek M. 542 Boberg F. 105 Boche G.371 Bock C. M. 50 Bock H. 26,308 Bock L. A. 194 Bock R M. 565 Bokalski R 467 Bodansky A. 509 Bodansky M. 509 525 527 531 Bode V. C. 571 Bodea C. 418 Bohme E. 273 Boekelheide V. 353,457 Boelhouwer C. 323 Boll W. A. 214 Boer F. P. 87 309 Boerma J. A. 486 Boerwinkle F. 271 Boeyens J. C. A. 50 Bogatkov S. V. 89 Bogdanov V. S. 293 302 Bohlmann F. 150,594,595 Bohm C. G. 76 Bohm H. 527 Bobnert T. 373 Boll W. A. 365 385,405 Bollinger J. M. 105 106 114 Bolton J. R.,18,22 31 Bolton P. D. 89 Bolton R.,96 97 Bonacci A. C. 37 Bond A. C. 293 Bond F. T. 141,392,404 Bond G. C. 326 Bonnet H. 594 Bonner T. G. 92 Boocock D. G. B. 14.27 183 Booth H. 493 Boots S.G. 432 Boozer C. E. 82 Borch R. F. 107 Bordignan E. 515 Bordwell F. G. 133 381 446 456 Borisov A. E. 317 Bormann H. 309 Borsdorf R 382 Borst P. 570 571 Bos H. J. T. 391 Bosche W. L. 68 Boschung A. F. 112 Bose A. K. 182 211 447 577 Bosiso G. 509 526 Bosmans R.,./i? Bossenbroek B. 370 Bossert H. 524 Bott K. 277,405,445 Bott R.W. 283,284 Bottcher R. J. 394 Bottino F. 376 Botvinik M. M. 616 Bouchard R. 266 Bouchoule C. 286 Boue S. 318 Bouguerra M. L. 253 Bouillon C. 462 Boulton A. J. 360 372 Bouman T. D. 36 Bourene M. 223 Bourgaux P. 571 Bourguignon M. F. 571 Boutwell R K. 539 Bovey F. A. 35 Bowden K. 88,89 Bower J. D. 443 Bowie J. E.14 Bowie J. H. 13 14 Bowman P. S. 370 Bowman W. R.,504 Boxt W. 526 Boyd G. V. 458 Boyd M. R.,452 Boyle J. T. A. 538 Bozzato G.,418 Braams J. F. H. 391 Braams R.,229 Braatz J. A. 400 Bradbury E. M. 525 533 Bradbury R B. 495 Bradbury W. C. 74 Braddon D. V. 122 Bradley A. 560 Bradley E. le R.,179 Bradshaw J. S. 208,210 Brady K. R 513 Brady S. F. 268 391 409 Brady W. T. 150,447 Braga de Oliveira A 377 Brainard R. 188 Brakas A, 318 Bramlett C. L. 298 Brammar W. J. 535 Brandt K. G. 616 Bratek-Wiewiorowska M. D. 49 1 Brattsev V. A. 297,298 Brauman S. K. 139 Braumann J. I. 144 Braunitzer G. 515 Braunstein A. E. 605 Bravo P. 386 Breazu D. 518 Breen D.E. 188 Bregadze V. I. 284 Brelikre J.-C. 213 Brennan J. 314 Brennan M. E. 123 Brennan T.,311 Brennan T. M. 145,202,402 Brenner M. 218 Brenner S. 563 Brennon D. 585 Breslow D. S. 168 Breslow R. 103 134 176 179 342,379,410 Bretscher M. 552 Brettle R. 235 240 246 267 Brew K. 611 Brewer H. B. 524 Brewer J. P. N. 356,357 Briat B. 40,364 365 Brigs W. S. 36 140 Brimacombe J. S. 271,472 Brinen J. S. 32 Brinich J. 106 Brinkman F. E. 26 Bristow P. A. 87 Britton D. 54 Britton R. W. 418 Broadbent A. D. 198 Broaddus C. D. 133 141 Brock F.-X. 426 Brockman C. J. 255 Brockmann H. 37 531 Brockmann H. jun. 37 Brod L. H. 71 Brodherr N. 62 Brodsky A. E. 252 Broidy J.M. 81 Brois S. J. 441 Bromer W. M. 527 Brook A. G. 110 308 309 310 311 Brook P. R. 117,403 Brookes P. 539 Brookhart M. 119,120 Broquist H. P. 907 Broser W. 117 Broughton S. M. 339 Brouwer D. M. 107,108 341 Brown C. 120 Brown C. J. 45 Brown D. J. 479 Author Index Brown D. M. 542 548 Brown E. A. 243,246 Brown E. D. 416,586 Brown E. L. 189 Brown G. B. 512 Brown G. W. 353 Brown H. C. 121 122 137 262 263 265 273 274 276 280 299 300 301 348 391 Brown I. H. 547 Brown J. F. 603 Brown J. K. 23,24 Brown J. M. 135 Brown J. R. 622,623 Brown M. 421 Brown P. 12 16 156 373 Brown R. 48 260 Brown R. K. 471 Brown R. T. 498 582 Brown S. H. 65 Brown T.L. 284 291 Brown W. 432 Browne M. W. 173 Brownlee G. G. 552 Brownlee R. G. 412 Brubacher L. J. 129,627 Bruce I. T. 578 Bruce M. J. 284 Bruderer H. 10 Briick D. 493 Brufani M. 427 Brugger M. 524 Bruice T. C.,71,72,74,76,126 602,610,616 Brune H. A. 386 Brunee C. 15 Brunfeldt K. 509 Bryan J. G. H. 271 Bryan R. F. 312 313 326 Bryant C. P. 469 Bryce-Smith D. 157,208,210 211 346 Bryne W. E. 97,99 Bubnov N. N. 24 Bubnov Y. N. 2Y3,302 Buchachenko A. L. 32 Buchardt O. 216,467,481 Buchholtz A. C. 11,12 Buchi G. 598 Buckley A. 89 136 Buckley P. J. 463 Budesinky M. 430 Buding H. A. 316 Biiche M. 325 Buchi G.. 198 277 415. 418 500 Buchi H. 543 Buhler R. E.224 229 Burger H. 283 Buetow D. E. 551 Bugerenko E. F. 283 Buick A. R. 23 Bujard H. 571 Bukhari S. T.K. 470 Bukhtiyarov V. V. 24 Bull G. R 309 Bull J. R 435 Bulten E. J. 283 312 Bultsma T. 464 Bumpus F. M. 527 Buncel E. 171 342 Bunnell C. A. 400 Bunnenberg E. 40,364,365 Bunnett J. F. 99 100 348 Bunnett J. H. 137 Bunton C. A, 68 82. 101 137 Buntrock R. E. 342 Burbott A. J. 586 Burckhatter J. H. 62 Burdon M. G. 542 Burgert B. E. 241 242 244 246 Burgess E. M. 352 447.461 Burgess J. 22 Burgmaier G. 403 Burka L. T. 452 Burkhardt T. 594 Burleigh J. E. 290 Burleson D. 585 Burley D. R. 198 Burlinghame A. L. 12 311 427 590 Burlitch J. M. 164 Burnashova T.D. 310,315 Burnell R. H. 499 Burnett A. R. 497 582 Burnett M. G. 326 Burnham D. R. 23 Burow N. E. 43 Buroway A. 517 Burrows W. J. 538 Bursey M. M. 8 14 91 Burton C. S. 187 Burton F. J. 256 Busetti V. 318 Bush J. B. jun. 267 334 Bush L. W. 24 26,308 Bush R. P. 312 Bushweller C. H. 478 Buss V. 262 399 Busson H. H. 600 Butcher F. K. 36 Buter J. 455 Butler A. R. 97 Butler L. 609 Butler P. E. 271 Butler R S. 341 Butterworth R. F. 470 Buttin G. 566 Buxa M. 404 Buxton M. 228 Buys H. R. 380 By A. W. 435 Bycroft B. W. 529 Byfield P. G. H. 524 Bylina A. 188 Byrne W. E. 349 Cabaleiro M. C. 142 Cabell M. 363 Cadiot P. 305 315 Cadle S. H.255 Cadogan J. I. G. 101,178,265 342,346,468 Cahill R. W. 223 Cais M. 261 326 Calder I. C. 366 Calderon N. 322 323 Calin M. 103 107 Calkins D. F. 540 Callewaert G. L. 514 Cafo V. 371 Calvin M. 16 220,467 Calvo C. 207 Camaggi C. 207 Cambie R. C. 424,431 Camble R. 522 Camerman A. 57,546 Camerman N. 57,546 Cameron A. F. 52 Cameron D. 529 Cameron D. W. 213 Camp R B. 91 Campbell N. 94 Campbell N. C. G. 113 Campbell S. F. 268,415 Canady W. J. 620 Canceill J. 137 Candlin J. P. 329 Canfiefd R. E. 606 Canonica L. 592 Cantoni G. L. 538 Cantor C. R 548,552 Cantrell T. S. 214 364,461 Cantrill H. L. 11 Cantro C. A. 547 Canty A. J. 291 Capella P. 13 Capitaine J.266 Capka M. 262 Caple R. 404 Capon B. 165 171,178 187 Carbon J. 537 538 564 Carbonaro A. 343 Cardellina J. H. I 270 Carey F. A. 110,265 Carey M. S. 598 Carhart R.E. 50,381 Carithers R. 461 Carless H. A. J. 206 Carlon F. E. 265 Carlough K. H. 220 Carlson J. A, 277 Author Index Carlson R G. 202 Carlson R M. 406 Carlstrom D. 45 Carmack M. 38 Carman R M. 422 Carpenter B. G. 533 Carpenter J. H. 293 Carpenter W. 11 Carr J. D. 494 Carr R. P. 462 Card S. 321 Carrahar P. 258 Carraway R. 22 Carre F. H. 309 Carrell H. L. 51 382 Carroll B. L. 293 Carson M. S.,413 Carstensen-Oeser E. 48 Carter J. 292 Carter J. H. jun. 288 Carter 0.L. 48 Carter R E.339 Cartledge F. 308 Cartwright D. 462 Carty D. 236 Casanova J. 237 Crisenky B. 262 Caserio M. C. 360 Casimir J. 510 Casinovi C. G. 427 Caspi E. 36,430 592 Cassal J. M. 216,483 Cassinelli G. 37 Casson J. E. 162 Castellano J. A, 204 Castillo M. 580 Castle R. B. 293 Caticha-Ellis S. 58 Caton M. P. L. 94 Cattanach C. J. 314 Cattania M. G. 101 Cauquis G. 27 240,249 Causse J. 310 Cava M. P. 489 Cavanaugh R. 431 Cavk A, 275 Cazes J. 181 Cekovic Z. 184 Celini S. 333 Cerami A. 574 Cerceau C. 139 Cercek B. 224 225 Cerfontain H. 97 Ceriotti G. 496 Cernri J. 564 cerny M. 262 Cerrini S. 427 Cerutti P.,217,538 547 Cevidalli G. 207 Chain E. B. 427 Chait.E. M. 16 Chakravarti R. N. 489 Chakravorty K. 175 Chalfont G. R. 181,183 Chalk A. J. 285,324 Chalk R C. 473 Challand B. D. 157 205 Challis B. C. 95 344 Chalmers A. M. 414 Chalvet O. 219 Chamberlin M. J. 551 Chambers D. B. 284 Chambers 3. Q. 255 Chambers R. D.. 350,480 Chambers,R. W. 218,540,564 Chamot D. 348 385 Chance B. 601,602 Chance R E. 527 Chandhuri J. 135 Chandra P. 539 Chandross E. A. 339 Chan J. H. H. 381 Chan S. I. 550,611 Chan W. R. 427 Chang A. Y. 560 Chang F. C. 435 Chang K. Y. 30 Chang M. L. 438 Chang P. 257 Chang R. 25 Chang S. H. 538,545,563 Chang S. Y. 18 Chantooni M. K. 67 Chapman N. B. 89 136 Chapman 0. L. 13 189 202 204 211 212 213,453 Chapman T.M. 522 Chapman R. A, 200 Charbonneau G.-P. 46 Charles G. 428 Charlton J. L. 198 Chasar D. N. 477 Chatt J. 165 324 332 Chaturvedi N. C. 527 Chaturvedi R. K. 77 Chatwoerdi R K. 71 Chawdhury S. A. 47 Chekulaeva V.N. 292 Chen C. J. 175 Chen C. M. 93 Chen D. H. 30 Chen C.-Y. 493,495 Cheng C. C. 479 Cheng K. F. 261 Cherest M. 381 Cherkasova E. M. 89 Chernyshev E. A. 283 Cheung H.-C. 415 Cheung K. K. 491 Chevalier,M. R. 569 Chevalier R. 619 Chevalier,Y. 325 Chew L. 522 Chiang T. S. 527 Chiang J. F. 382 Chiang Y. 68 Chibata I. 512 Chickos J. 402 Childs R. F. 104 109 341 Chillemi F. 509 520 Chilton W. S. 39 Chlmiak.A, 527 Ching 0.A. 472 Ching-Tso W. 514 Chiovini J. 39 Chipman D. M. 606 Chippendale J. C. 31 Chitwood J. L. 113 Chiu K. K. 314 Chiusoli G. P. 328 Chivers T. 3 17 Chizhov 0.S. 470 Chlebowski,J. F. 97 Chloupek F. J. 348 Chmurny G. N. 380 Chodroff S. D. 172,405 Chorvat R J. 83 Chottard J. C. 178 Choudhury A. M. 493 Chow Y. L. 210,345 Christensen A. T. 62 Christl B. 459 Christl M. 152 457 Christobel W. 318 Chrysochoos J. 230 Chu N. W. K. 393 Chu S. S. C. 59 Chuit C. 270 Chutny B. 224 Chvalovsk9,V. 262 Chytil N. 520 Ciabattoni J. 103 358 363 Ciamician G. 199 Ciganek E. 213 Cimarusti C. M. 271 Cinquini M. 266 Claggett A. R. 514 Clapp R. C. 231 Clark B.C. jun. 392 Clark B. F. C. 563 564 565 Clark H. C. 307,316 Clark R. A. 442 Clark R. J. H. 314 318 Clark T. J. 345 395 Clark W. D. 307 Clarke G. M. 116 117 125 Clarke J. M. jun. 560 Clarke T. G. 267 Clark-Lewis,J. W. 37 Clauson-Kass N. 246 Claxton T. A.,'30 Clayton. D. A, 573 Author Index Clayton R. B. 427 589 590 59 1 Cleary J. C. 455 Cleary J. W. 290 Cleghorn J. T. 93 Clementi E. 342 Clerc J. T. 40 Clewell W. 90 Clifton E. C. 91 Close J. 509 Close V.A, 514 522 523 Closs,G. L. 160. 163. 214. 385 395 Clough S. C. 445 Clusius K. 231 Coates G. E. 283 287 288 Coates P. 568 Coates R. M. 417,418 Coburn J. E. jun. 379 Cochrane J. C. 131 Cocker W.413 Cockerill A. F. 79 Coffey R. S. 324 Cohen A, 568 Cohen J. I. 189 Cohen J. S. 637 Cohen K. F. 204 Cohen M. H. 351,456 Cohen S. C. 356 Cohen S. D. 209 Cohen S. G. 619,620 Cohen T. 179 Cohen W. 624,628 Coke J. L. 139 140 Colburn N. H. 539 Cole R. S. 189 Cole T. M. 212 359 Cole W. G. 433 Coleman A. E. 244 Coleman D. 513 Coleman D. L. 38 Coleman R. A. 273 Coles L. 430 Coles M. A. 290 Coletti-Previero M. A. 513 Colin G. 316 Collier J. R. 200 Collin P. J. 195 Collington D. 179 Collington D. M. 350 Collington E. W. 363 Collins J. C. 266 Collins J. H. 11 Collins P. M. 203 473 Collman J. P. 321,332 Colonna S. 266 Colpa. J. P.. 18 Colter A. K.109 Colucci D. F. 524 Comisarow M. B. 25 Commeyras. A.. 105 Conan J.-Y. 129 Conia J.-M. 118 379 391 Conlay C. 427 Connolly J. D. 427 Connolly J. S. 217 Connolly J. W. 311 Connors P. G. 565 Conolly P. J. 326 Considine J. L. 307 Consonni P. 448 Conti F. 333 530 Contrell T. S. 135 Conway B. E. 171 231 233 234 Cook C. C. 94 Cook C. D. 321,355 Cook D. 128 Cook J. D. 466 Cook R. D. 67 Cooke B. 262 Cooke M. P. 139,140 Cooke R. S. 189 Cooks R G. 8 9 10 11. 13 14 Cooksey C. J. 142 Cookson R. C. 156 199 373 376,383 Coombes R. G. 91 Coomber J. W. 201 Coombs R A. 36 Coombs T. L. 634 Coomes. R. M.. 65 Cooper A. 62 Cooper J. C. 466 Cooper J. T. 23 Cooper R.227,228 Cooper W. 158 Cope A. C. 146 191,336,339 340,382,403 Copeland L. 579 Coppen J. J. W. 587 Coppola J. C. 632 Corbellini A. 509 520 Corbett J. F. 262 Corbett R. E. 430 Cordes C. 382 Cordes E. H. 71 78 81 Corey E. J. 237,267 268 269 273 277 278 279 280 281 386 403 406 415 589 590 Corfield P. W. R. 381 Cori c. 540 Cornforth J. W. 268 Cornforth R H. 268 Corriu R. 309 311 Corriu R. J. P. 309 Corsano S. 428 Corse J. 213 Cory S. 563 Coscia C. J. 582 Coscia J. 414 Costain C. C; 463 648 Costapanogiotis A. 509 Costin C. R. 44 Cosyn J. P. 37 377 Cotter R. I. 552 Cotton F. A. 319 Cottrell W. R. T. 250 Coulter C. L. 57 Coulter J. M. 262 Coulter J.R. 514 Couner R. L. 592 Courtot-Coupez J. 260 Coutts I. G. C. 493 Cowley D. E. 422 Cowley D. J. 29 Cox A, 345 Cox G. B. 235 Cox J. D. 286 cox o.,201,212 Coxon J. M. 413,435 Coyle J. D. 198 Coyle J. J. 163 Coyle T. D. 294 Cozzarelli,N. R. 570 Cozzone P. 176 Crabbe P. 35 39 277 439 513 Crabtree J. H. 68 137,418 Craddock J. H. 327 Craddock V. M. 539 Cragg R. H. 284 Craig J. T. 94 Craig L. C. 530 631 Crain D. L. 392 Cram D. J. 95 118 125 131 133 282,353 Cramer F. 543 545 552 565 566 Cramer R. 321 Cramer R. E. 18 Crampton M. R 98 Crandall J. K. 201 205 400 Crashey R. 145 Craven B. M. 65,500 Crawford L. V. 571 Crawford R.J. 206 393 Crecely R.W. 341 Creemers,H. M. J. C.,314,318 Cremer S. E. 83 Crestfield A. M. 634 Cretney W. J. 584 Crews P. 135 Crews P. O. 364 Crick F. H. S. 537 Criegee R. 352 379 386 397 401 Crimmins T. F. 285 Crktol S. J. 176 437 Croft L. R 529 Cromarty A. 482 Crombie L. 277,426 Cromwell N. H. 443 Author Index Crooks P. A. 501 Cross A. D. 432 Cross B. E. 263 588 Cross M. L. 91 Cross P. E. 337 Cross R. J. 317 Crothers D. M. 575 Crouse D. 403 Crouse D. N. 280,285 Crout D. H. G. 489 Crow W. D. 166,469 Crowell T. I. 138 Cruickshank B. 325 Crumrine D. S. 202,402 Cryberg R. L. 463 Cserhegyi A, 135 Cubbon R. C. P. 154 Cuellar L. 432 Culbertson B. M. 445 Cullen W.R. 315 Culvenor C. C. J. 489 Cundall R. B. 68 207 227 Cunningham L. 612 Cupas C. A. 93,343 Cuppen T. J. H. M. 209 Curphey T. J. 907 Curran. C. 314 Curran W. V. 203 Curry J. B. 564 Curtiss R. tert. 568 Cushley R.J. 540 Czerlinski G. 617 Czochralska B. 258 Czuba L. J. 11 1 Dack M. R. J. 89 136 Dadson B. A. 500 Dahl K. 184 267 Dahlberg J. E. 560 Dahlman J. 509 Dahlquist F. W. 611 Dahn H. 79 Daicoviciu C. 518 Dainis I. 176,431 Dainton F. S. 226,227 Dallas G. 443 Dall’Asta G. 343 Dalle J.-P. 218 Dalton C. K. 353 Dalton D. R. 20 Dalton J. C. 206 Daly J. 65 504 Daly J. W. 600 Daly N. R. 16 Damiani A. 60 Damodaran N. P. 419 Damrauer R. 291 Danen W.C. 30 135 Danho W. 509 Daniel H. 319 Daniels E. G. 596 Danielsen J. 61 Daniker F. A,. 271 Danilova G. N. 297 Danishefsky S. 431,464 Danks L. J. 428 Danner J. C. 20 Danstead E. 413 Danziger R. M. 229,547 Das T. L. 539 Darbre A, 514 Darby A. C. 97 Darcy R. 183 Dardenne G.,510 Darling T. R.,156,389 Darnall K. R. 178 Darnell J. E. 535 Darnell J. E. jun. 551 Darragh K. V. 164,291 Darwish D. 128 Das B. C. 517 Das K. G. 577 Das P. K. 259 Daub J. 394 Dauben H. J. 184 Dauben H. J. jun. 21. 236 271 Dauben W. G. 113 188 197 203,211,270 Daubendiek R. L. 190 Daudel R. 219 Daudt W. H. 231 Dave;K. G. 262,492,498 Davey R 27 David B. 317 David H. 537 538 Davidsohn W.E. 315 Davidson A. 308,568 Davidson B. E. 470 Davidson E. R. 18 Davidson 1. M. T. 308 Davidson J. M. 336 Davidson N. 570 571 Davies A. 94 375 Davies A. G. 284 303 314 318 Davies D. G. 109 Davies D. I. 176 183 Davies D. W. 19 Davies G. A. 552 Davies G. T. 326 Davies J. S.,373 Davies J. V. 230 Davies J. W. 230 Davies M. C. 524 Davies M. T. 36 Davies N. R. 325 Davies R C. 632 Davies V. H. 373 Davis F. A 293 294 Davis G. T. 265 Davis M. M. 89 Davis R. A. 450 Davis R. C. 549 Davis R. W. 568 Davison B. E. 307 Dawber J. G. 67 Dawes K. 469 Day A. C. 187 206 209 214 221,389 Day J. 282 Day W. A. 348,385 Dayagi S. 285 Deacon G.B. 291 Deady L. W. 446 De-Alti G. 314 Dean R. R 285 Deavin A. 634,635 de Bie D. A. 456 De Boer A. 177 De Boer C. 176 De Boer C. D. 221 de Boer E. 23 33 De Boer F. P. 105 de Boer J. L. 59 60 de Boer Th. J. 11 28 183 De Bruin K. E. 69 141 De Bruyne C. K. 71 Debye N. W. G. 314 De Castiglione R. 509 526 Deer A. 522 Degani C. 471 Degtiarev L. S. 252 De Graff W. L. 394 de Groot. A. 57,270,486 de Groot M. S. 32 De Haan J. W. 398 Dehmlow E. V. 402 Dehnicke K. 304 Dekker H. 381 Dekker J. 14 Dekker M. 169 de la Mare P. B. D. 95 128 De Lange R J. 625 de Lavieter L. 381 Del Cima F. 100 Delhoste J. 130 Delius A. E. 527 Del Praa. A, 57,318 Delwiche C. V.591 De Mare. G. R 190 Demarne H. 305 de Mayo P. 157,189,198,205 DeMember J. R. 188 385 Dernisch W. H. 118 Demole E. 219,413 de More W. B. 161 den Hertog H. J. 466,467 Denisov V. V. 528 Denkewalter R. G. 519 521 5 24 Denney D. B. 182 Dennis E. A. 83 Author Index Dennis N. 422 Dennis W. H. jun. 265 326 Demon D. D. 392 de Oliveira A. B. 476 De Puy C. H. 143 177 379 386 Derieg M. E. 481 Derissen J. L. 44 de Rostolan J. 275 Dertouzos D. 316 de Santis P. 60 Desbarres J. 260 de Selms R. C. 93 de Silva L. B. 371 Deslongchamps P. 391,418 Dessau R M. 184,267,334 Destro R 46 DeTar D. F. 184 Dev S. 419 421,427 Dev V. 214,385 de Valk J. 486 Devaprabhakara D. 301 De Vault D.601 De Vries J. X.,493 De Vries J. Y.,525 De Vrieze J. D. 197 353 394 399 de Waal W. 418 Dewar M. J. S. 88 89 117 124,293,294,295 348 De Watcher R. 560 Dewhurst S. 593 Deyrup C. L. 123 Deyrup J. A. 445 Dheer S. K. 545 Diaz A. F. 112 di Bello C. 57 Dickerson J. E. 97 Dickerson R. E. 56 Dickinson C. 44 132 Dickinson M.J. 540 Dickinson T. 234 Diekman J. 12 311 Dienstag J. 539 Dierks H. 49 Dietl H. 331 332 343 401 Dietrich H. 49 163 220 Dietrichs H. H. 377 Dietz F. 187 Dietz S. 445 Dillard C. R. 314 Dillon M. A, 228 DiMichiel A. D. 325 Dimroth K. 26,104,150,204 343 Dines M. 14 Dingwall J. D. 462 Dingwall J. G. 462 Dinh-Nguyen N.14 Dinse K. P. 26 Dinulescu 1. G. 387 Di Nunno L. 100 Di Pasquo V. J. 392 Dirania M. K. M. 210 Dirlam J. 122 Dischler B. 340 Ditter J. F. 298 Dixon J. A. 287 Dixon J. R. 382 Djerassi C. 11 12 16 36 38 39 40 62 65 140 261 311 364 365 500 Doak G. O. 283,319 Dobis O. 175 Dobosh P. A. 19 Dobson J. E. 293 Doctor B. P. 565 Dodson C. L. 22 Dodson R. M. 450 Dohnert H. 493 Dtipke W. 489 Doepner H. 566 Dorfelt C. 313 Doering W. Von E. 160 201 379 Dorscheln W. 215 Dokro E. A 402 Dolak L. A, 409 Dolbier W. R jun. 387 Dolby L. J. 454,496 DolejS L. 417 Dolfini J. E. 463 Dolman D. 141 DoMinh T. 185 291 Dominy B. W. 493 Donaldson C. W. 89 Donaghue P.F. 14 Donaruma L. G. 48 Donninger C. 38 Donohue J. 41 45 51 382 535 Doorakian G. A, 144,397 Dorfman L. M. 224 225 Dorman D. 419 Dorn H. 459 Dorokhov V. A. 302 Dorsch H. L. 403 Dorsey E. D. 150,447 dos Santos Veiga J. 22 Dost F. 385 Dotsenko L. A. 220 Doty P. 569 Doty J. C. 293 DO&H. J.-M. 181 Douek M. 134,342 Dougherty R. C. 7 15 Douglas G. K. 493 Douthit C. E. 319 Dow A. W. 3 11 Dow J. 57 Dowd W. 140 Downing A. P. 36 361 377 Doyle R. P. 510 Drago R. S. 18 Drakesmith F. G. 258 31 1 Dreiding A. S. 399 Drenth J. 626 Drenth W. 316 Drew E. H. 175 Drew M. G. B. 392 Drews H. 14 Dreyer D. L. 37 427,475 Druckery 220,352,384 Dryhurst G.249 Dua S. S. 311 Dube S. K. 563,564 Dubini M. 328 Dubois J. E. 97 141 142 Ducep J. B. 279 Dudock B. S. 563 564 Dudukina 0.V. 31 1 Diirr H. 347 Duff J. M. 308,311 Duffield A. M. 11 Duffin B. 56 Duffy D. 305 Duholke W. K. 310 Dukler S.,499 Duncan J. H. 270 Duncan W. G. 412 Duncanson L. A. 165 Dunitz J. D. 51 52 Dunkelblum E. 380,381 Dunkelmann G. 494 Dunkersloot J. A. 598 Dunks G. B. 298 Dun G. E. 67 Dunn G. L. 392 Dunn J. T. 374 Dunny S. 310 Dunstan D. R. 40 533 Duphorn I. 426 Dupuy A. E. jun, 445 Duquette L. G. 464 Durand D. A. 446 Durbin A. K. 493 Durst H. D. 589 Durst T. 268 Dusenbery R. L. 198 Dusold L. R 14 Dutton G. G. S. 267 Duty R.C. 257 Duval J. 540 du Vigneaud V. 526 527 DwumaBadu D. 500 Dyall L. K. 97 Dyatkin B. L. 283 Dyatkina M. E. 171 Dyckes D. 348 Dyer 1. R. 529 Dygos J. 463 Dyumaev K. M. 93 Dzieciuch. M. 233 Author Index Dziobnowski C. 524 Eaborn C. 97,284,309 Eargle D. H. jun. 17 East E. J. 624 Eastmond R. 310 Easton D. B. J. 462 Easwaren C. V. 71 Eaton D. R. 325 Eaton P. E. 187 Ebel J.-P. 540 Eberbach W. 191,396,486 Ebert M. 224 229 230 Eberius W. 386 Ebersole R. C. 435 Eberson L. 231 233 234,244 245,246 348 Ebsworth E. A. V. 284 Echenmoser A. 453 Eckhard I. F. 356 Eckhardt G. 36 Eckstein F. 542 543 Edelman G. E. 620 Edelson S. S. 156 389 Eder W. R. 597 Edge G.214 Edman J. R. 189 191,387 Edmondson R. C. 318 Edstrom J. E. 537 Edwards E. I. 168 Edwards J. A, 269 273 415 438 Edwards J. M. 391 Edwards 0.E. 422 Eeckhart Z. 313 Efron M. F. 511 Egan C. 382 Ege G. 481 Ege S. N. 460 Egger K. W. 398 Eguchi S. 110,405 Ehmann W. J. 331,343 Ehrenberg A. 20 Ehrenberg M. 48 Ehret C. 583 Ehrhardt M. 290 Ehsan A. 136 Eian G. L. 213 453 Eibel G. 512 Eiben K. 29 Eigen M. 549 601 602 Eiglmeier K. 304 Ei-Ichi Negishi 391 Eilermann L. 46 Einstein F. W. B. 313 Eisch J. J. 283 306 307 Eisele B. 628 Eisenbeiss F. 530 Eisenbraum E. J. 267 277 Eisenhardt W. 443,444 Eisinger J. 217 546 Eider K. 527 Eisner T.,414,415 Eiss E.W. 287 Eistert B. 371 Eizember R. F. 201 Elad D. 217 511 El Ashry S. H. 475 El-Haggar A. M. 528 El-Haj M. J. A, 435 Eliel E. L. 262 380 381 478 Elix J. A, 367,455 El Khadem H. 475 Ellam R. M. 117 Elliason R. 68 Elliott C. S. 162 Ellis M. C. 451,490 Ellis R. M.E. 527 Elmore D. T. 509 625 Elphinoff-Felkir I. 266 El-Sayed M. A. 187 Elschenbroich C. 285 Elsenbraun E. Z. 585 Elson E. L. 569 Elvidge J. A. 478 Elving P. J. 249 254 Elwood T. A. 14 Emery E. 117 Emery T. F. 510 Emmerson P. T. 230 547 Endean R. 526 Endeman H. J. 44 Endres L. S. 392 Enemark J. H. 50,381 Engberts J. B. F. N. 78 Engel Ch. R. 266 Engel P. S. 174 206 Engelmann T. R. 285 Engels E.509 Enggist P. 219 413 Englard S. 40 Englert M. E. 524 Englhardt G. 309 Englund P. T. 631 Entemann C. E. 615 Epand R. F. 534 Epand R. M. 637 Eppley R. L. 287 Epstein J. 82 616 Epstein R. 293 Epstein W. W. 95 Eraker J. 49 Erdmann V. A. 552 Erdos E. G. 527 Erickson B. W. 269,415 Erickson E. H. 624 Erickson K. 415 Erickson K. L. 403 Erickson R. E. 257 Eriksson S. O. 78 Erlanger B. F. 619 Ermer O. 52 Ershov V. V. 32 Erspamer V. 509 526 Eschenmoser A. 441,445,590 Eskins K. 539 Espinosa F. G. 472 Etheredge S. J. 406 Etzold G. 540 Eucokpae T. A. 89 Eugster C. H. 424 504 Evans D. 324,327 Evans D. A. 501,583 Evans D. F. 290 Evans G.B. 227 Evans J. H. 254 Evans J. M. 382,621 Evans M. E. 472 Evans M. G. 157 Evans T. R. 188 190 Evans W. H. 625 Evenhuis B. 270 Evnin A. B. 109 286 460 Eyman D. P. 305 Eyring H. 40 Eyton W. B. 377 Fabey R. C. 141 Fadeeva T. M. 398 Faesel J. 525 Fairbairn J. W. 490 Fairless B. 197 395 Fairlie J. C. 114 Fajer J. 24 Falco E. 512 Falco E. A. 540 Falco M. R. 493 Fales H. M. 492 Falk H. 340 Fallis A. G. 397 Fang K.-N. 209 Fantazier R. M. 173 Fanucci R.,68 Farber S. J. 82 Farcasiu D. 370 Fargher J. M. 168 Farid S. 211 Farine J.-C. 221 392 Farkas E. 432 FarkaS J. 542 Farlow D. W. 68 Farnham W. B. 404 Farnum D. G. 420 Farquhar D. K. 465 Farrisey W.J. jun. 449 Fatiadi A. T. 21 Faubion B. D. 21 135 Faubl H. 392 Faulkner D. J. 268,415 Faulstich H. 528 Favirie G. 394 Fawcett J. K. 62 Fearon F. W. G. 311 Author Index Fedeli W. 51 427 Fedin E. I. 103 317 Fedoruk N. A. 457 Feeley A. 217 546 Feeney R E. 515 Fehlhaber H.-W. 427 Feigina M. Yu. 5 17 Feiler L. A. 148 Feinberg R S. 465 Feinleib S. 35 Feit E. D. 199 Feld D. 278,411 Feldman M. 464 Felix D. 441 445 Felkin H. 270 381 Fell B. 327 391 Feller L. W. 103 Fellner P. 552 Felsenfeld G. 575 Felton R. H. 24 Felton S. M. 76 Fendler E. J. 82,86,97,98,99 349 Fendler J. H. 86 97 98 99. 349 Fenical W. 398 Fenoglio R. A. 379 Ferbig A. E.350 Ferguson G. 42,48 51 52,53 56 61 62 318 Fergusson J. E. 332 Ferles M. 250 Fernandez J. M. 357 Ferranese E. 526 Ferraris M. 328 Ferrier R J. 471 Ferris 3. P. 536 Fersht A. R. 74,76 126 Fessenden R W. 29 Fetizon M. 267 Fetter N. R.,283 Feuer H. 275 Fichteman W. L. 325 Ficini J. 357 464 Fickes G. N. 125 Fiecchi A. 592 593 Fiecchi H. 592 Fielden E. M. 224 230 Fields E. K. 14 Fields R. 152 Fiers W. 560 Fieser L. F. 231 267 374 Fieser M. 267 Fife T. H. 70 71 77 82 Figeys H. P. 354 372 Fiiipescu N. 188 385 Filler R. 134 350 Finch C. A. 511 Findley J. W. A. 432 Fink G. 371 Fink L. M. 564 65 1 Finkbeiner H. 267,334 Finkelstein M. 240 246 249 Finn F.M. 509 Finnegan R.A. 210 Finseth G. A. 265 326 Fioshin M. Ya. 236 Firestone R. A. 152 457 Fischer A. 140 Fischer E. O. 165 Fischer G. 286 Fischer H. 131 Fischer H. P. 286 339 Fischer M. 201 221 434,449 Fischer M. S. 49 Fischer P. H. H. 24 32 Fischer R. H. 286 Fischer W. F. jun. 277 Fish R. H. 177 Fisher G. S. 425 Fisher L. P. 335 Fisher R. 635 Fisher T. H. 176 Fisher W. D. 566,568 Fishman J. 432 Fishwick A. H. 287 Fishwick S. E. 83 84 Fittler F. 538 Fitton H. 494 Fitzgerald R. 373 Fitzsimmons B. W. 314 Flammang R. 354 Flammang-Barbieux M. 377 Flamme W. 382 Flautt T. J. 413 Fleischmann M. 233 234 Fleming J. S. 291 Fletcher H. G. jun. 472,475 Fletcher R.453 Fleury M. B. 254 Flint J. A. 83 84 Flom M. S. 435 Flood T. C. 311 Florin R. E. 31 Flory P. J. 533 Floyd J. C . 529 Floyd M. B. 220 Flynn J. J. 87 Fochler M. 308 Fodor G. 471 Fogelsong W. D. 115 Foley K. F. 500 Follett E A. C. 571 Fomina 0.S 176 Font J. 185.291 Fontaine M. C. 190 Fontana A, 517 525 Foote C. S. 114 187 218 219 268,274 Foote R. S. 210 Forbes E. J. 212 219 357 364 Forbes W. F. 23 652 Author Index Ford B. F. E. 314 Friary R. J. 424 Ford P. W. 479 FriE I. 39 Ford W. T. 131 Frickey D. G. 356 Fordice M. W. 382 Fridkin M. 525 Forget B. G. 552 Fridrichsons J. 64 65 495 Forrester A. R. 28 171 181 Fried J. 406 Forschult S. 33 183 Fried J.H. 273,438 Fort R. C. 405 Friedemann D. 105 Foss V. L. 317 Friedman G. 220 Foster G. H. 454 500 Friedman L. 97,195,355 Foster R. 99 183 Friedrich E. C. 336 373 Fowden L. 128,509,510 Fritchie C. J.,jun. 56 450 Fowler F. W. 441,442 Fritsch J. M. 22 247 249 Fowler J. S. 345 Fritze P. 355,403 Fowler M. S. 177 311 Froemsdorf D. H. 140 Fox B. L. 238 Frolov S. I. 293 302 Fox,J. J. 479 540 541 Fromagest H. P. M. 543 Foxton M. W. 306 Frost G. M. 585 Fraenkel G. 285,466 Fry A. J. 254 257 404 Fraenkel G. K. 17 19,21,22 Fryer R. I. 481 Fraenkel-Conrat H. 539,560 Fuchs R.,452 Frainnet T. 310 Fueno T. 69 111 141 Frajerman C. 270 Fiirst A. 439 Frame R. R 133 Fuganti C. 496 Franceschi G. 37 Fujiki H. 515 Francis J.N. 297 Fujimoto H. 143 309 Franck B. 494,496 Fujimoto S. 527 Franck R. W. 357 Fujimoto Y. 500 Franck-Neumann. M. 385 Fujinaga T. 258 418 Fujita K. 112 Frangopol M. 183 Fujita S. 444 Frangopol P. T. 183 Fugitani K. 493 Frank F.,J. 266 Fujiwara T. 62 Franke A. 543 Fukazawa T. 200 Franke C. 191 Fuke M. 573 Frankel E. N. 261 326 Fukomoto K. 65 Frankel F. 564 Fukui K. 31 143 171 Frankevich E. L. 227 Fukui S. 129 Franklin R. E. 172 405 Fukumoto K. 495 Franklin R. M. 551 Fukushima K. 426 Franta E. 135 Fukuyama M. 206 Franzede Fernandez M. T. Fuller W. 551 565 575 527 Funke P. T. 577 Franzus B. 405 Funt B. L. 247 FrBter Gy. 350 Furaya Y. 97 Frater R. 627 Furberg S. 57 Fraunberger F. 519 Furey R L. 213,449 Fredlen R.A. 68 Furic I. 474 Freed J. H. 19 Furka A. 623 Freedman H. H. 132 144 Furlenmeier A, 439 397 Furukawa J. 69 111 141. Freedman L. D. 283 319 384 Freeman F. 465 Furukawa Y. 542 Freifelder D. 571 Furusaki A. 46 52 Freitag D. 342 Furutachi N. 218,439 Freon P. 290 291 Fuse K. 546 Fresco J. R. 549 565 Fyfe C. A. 350 Freudenberg B. 181 Fyffe C. A. 99 Frey D. W. 197 Frey H. M. 160 161 162 Gabard J. 137 163 Gabbay E. J. 40 514 537 Gabel N. W. 536 Gadsby B. 266,433 Gaertner V. R.,445 Gafield W. 37 Gait R. J. 462 Gal J. 109 Galante L. 524 Galasso V. 314 Galbraith M. N. 438,439 Gale D. M. 189 347 Gale L. H. 381 Gale 0.M. 384 Gall J. S. 128 Gallagher R. T. 424 Galli G. 592 593 Galli R.,178 Gallikiente M.592,593 Gallivan R. M. 301 Gallivan R. M. jun. 263 Gallo A. A. 380 Galt R. H. B. 588 Games M. L. 426 Gandhi R. P. 199 Ganem B. E. 267,415 Ganguly A. K. 344,471 Gante J. 5 13 Ganter C. 200,419 Ganz C. R. 191 Gaoni Y. 366 Gar T. K. 283 Garbisch E. W. 381 Garbisch E. W. jun. 404 Gardiner R. A. 907 Gardner J. N. 265 Gardner P. D. 196,486 Garen A. 535 Garland R. P. 435 Garnau F. X. 219 Garner A. Y. 160 Garner R. 509,522 Gamier F. 141 Garratt P. J. 196 366 368 Garratt S. 103 Garrett P. M. 296.297 Garrison A. W. 82 Garst R. H. 99 100 Garwood R. F. 246 Gasic M. 124 Gaskell A. J. 493 496 Gassman P. G. 120 122 128 237,238 386,400,401,437 463 Gates P.N. 104 Gates V. 610 Gaucher G. M. 539 Gaudemer A, 417 Gaudiano G. 386 Gault R. 465 Gavrilenko V. V. 306 Gawne G. 509 Gaylor J. L. 591 Gaylord N. G. 159 Geens A. 381 Gehrke C. W. 514 Geiger R. 519 Geiger W. E. jun. 29 Geise H. J. 62 Geissler H. 314 Gelbeke M. 37,377 Gellert M. 575 Gemies M. 27 Gendell J. 252 George T. A, 299,316 Gerhart F. 271 Gerig J. T. 364 381 621 Gerlock J. L. 24 255 GBro S. D. 517 Geroch M. E. 552 Gerrard A. F. 83 Gerring M. 609 Gerson F. 17,26 171 308 Gervais H. P. 36 Gerwin B. I. 631 Geske D H. 29 Gesner B. D. 383 Ghatak U. R ,523 Ghelis C. 617 Ghera E. 274 Ghisalberti E. L. 426 Ghorazian H.C. 528 Ghosez L. 386 Ghosh P. B. 515 Giacobbe T. J. 275 Giacometti G. 18 Giants T. W. 380 Gibbons C. S. 65 Gibbons G. F. 592 Gibbons W. A. 530 Gibian M. J. 612 Gibney K. B. 267 Gibson D. H. 177 Gibson T. W. 414 Gielen M. 318 Gieren A, 62 Giersch W. 413 Giesel M. 112 Giesler G. 389 Gifford W. A. 140 Giglio E. 60 Gil-Av E. 384 Gilbert A, 157 210 21 1 Gilbert B. 65 500 Gilbert B. C. 27 184 Gilbert E. C. 380 Gilchrist M. 78 Gilchrist T. L. 321 355 Gilde H. G. 23 1 236 Giles R. G. F. 213 Gilham P. T. 454 560 Gill G. B. 143 187 Gill P. S. 30 Gillard R. D. 512 513 Author Index Gilles J.-M. 365 383 Gillespie R. J. 105 Gillet I. E. 252 Gilliom R.D. 178 Gillis R.G. 345 Gilman H. 286 311 314 Gilman N. W. 267,269,415 Gilow H. M. 91 Gindl H. 542 Ginsberg R. 130 Ginsberg T. 563 Ginsburg D. 197,395 Giovannini E. 453 Gipson R. M. 512 Girijavallabhan V. M. 177 Gitier C. 81 Givens R. S. 18 194 Gladys C. L. 268 Glass C. A. 261 326 Glaze W. H. 283,285 Glazer A. N. 625 Gleicher G. J. 410 178 Gleifer R. 161 Glens K. 246 Glick D. M. 618 Glinski R. P. 469 Glitz D. G. 560 Glockling F. 284 312 Gloede J. 514 Glogowski M. E. 293 Glover D. J. 130 Glusker J. P. 43 57 Goad L. J. 592 593 Godfrey M. 90 Godinho L. S. 272 Godtfredsen W. O. 428 591 Goe G. L. 191,403 Goebel P. 379 Goering H. 120 Goering H.J. 125 Goering H. L. 120 Goth H. 215 Goffredo O. 509,526 Gohlke R. S. 15 Golborn P. 97 Gold H. 79 Gold V. 70 74 98 137 Goldanskii V. I. 314 Goldberg I. H. 574 Golden D. M. 144 Golden S.,275 Goldfinger P. 190 Goldman H. 533 Goldman R. 628 Goldman N. L. 208 Goldschmidt S. 233 Goldstein J. H. 341 Goldstein L. 30 Goldstein M. J. 125 Goldstein M. L. 38 Goldstein. P. 59 Golfier M. 267 Gollnick K. 187 218 Gomez G. 130 Gompper R. 355 359 327. 386 Gondo Y. 32 Goodall D. M. 69 Goodman H. M. 563 Goodman L. 471,472,540 Goodwin H. W. 380 Goodwin T. W. 592 593 Gopalakrishna E. M. 62 Goray A. S. 514 Gordienko L. L. 252 Gordon C. N. 568 Gordon M.E. 291 Gordon M. P. 57,546,547 Gofinkel M. I. 370 Gorlitz M. 382 Gorman A. A. 37,499 Gornowicz G. A. 31 1 Gorringe A. M. 95 Gosden A. F. 267 592 Gosselck J. 385 Gosser L. 131 Goto K. 64 Goto T. 564 Goto Y. 209 Gotter L. D. 152 Gotthardt H. 152,357,459 Gottlieb 0.R. 37 377,476 Gough A. 324 Gough T. E. 30 Gould E. S. 333 Gourley R. N. 370 Gouzerh M. 254 Gowenlock B. G. 308 Gowling E. W. 321 354 Govindachari T. R. 429 Goyert W. 286 Gozzo F. 207 Gnewuch C. T. 498 Gnoj O. 265 Graf B. 403 Graf R. 280 Graham S. H. 382 Gramaccioli C. M. 46 Gramaccioni P. 394 Granboulan N. 551 Grant D. M. 372 Grant M. W. 309 Grant P. T. 527 Grashey R. 152,457,459 Grassberger M.A. 298 Grastraminza A. 91 Gratzer W. B. 525 552 Graveling F. J. 321 355 Gray A. C. G. 374 Gray C. J. 357 Gray D. G. 247 Gray M. W. 537 Grayson D. H. 413 Gream G. E. 178,387 Greatbanks D. 271 Greben A. E. 519 Grebennikov A. V. 297 Greco A. 343 Greeley R. 212 359 371 Green D. M. 569 Green M. 16,336 Green M. B. 37 Green M. J. 38 Green M. L. H. 283 Green M. M. 38 Greenberg B. 50 Greenberg J. 539 Greene D. C. 3 17 Greene F. D. 173 373,446 Greene J. M. 264 Greenspan G. 266 Greenstock C. L. 217 229 Greenwald R. B. 465 Greenward R. B. 449 Gregersen N. 472 Gregor V. 297 Gregory B. 581 Gregory H. 525 Gregory M. J. 364 Gregory R. A, 525 Gregson M.377 Greig J. B. 592 Grezemkovsky R. 433 Gribble G. W. 454 Grief N. 26 Griffin B. E. 543 Gain C. E. 97,98,99,349 Griffin G. V. 220 Griffin G. W. 163,235 Griffin S. 364 Griffith D. B. 138 Griffith M. G. 381 Grimths J. 204,212 219 Griffiths P. A. 227 Griffiths W. E. 29 Grigg R. 169,453 Grigsby R. D. 14 Grimes R N. 298 Grimme W. 365,395 Grimshaw J. 254,256,370 Grinter R 207 Grisdale P. J. 190 293 Griswold A. A. 202 Grobb C. A 112 Grodski A. 68 Groen S. H. 455 Groger D. 580 Grohmann K. 367 Gronowitz S. 294 Groom T. 380 Gross D. E. 334 Gross E. 178 Author Index Gross H. 514 Gross H. J. 538 Gross M. L. 9 Gross N. J. 570 573 Gross S.K. 590 Grossi E. 592 593 Grossman L. 540,547 Grossman L. I. 570 Grossweiner L. I. 226 Grotewold J. 293 Groth J. L. 257 Groth P. 44 50,58 Grove J. F. 587 Groves J. T. 179,410 Grubb H. M. 11 Grubbs E. J. 373 Gruber G. W. 193 213 356 400 Gruber R. 449 Griinberger D. 564 Griiner H. 352 Grunbaam M. 178 Gruner H. 401 Grunwell J. R. 221 Grutzner J. B. 341,405 Guaraldi G. 79 Guarnaccia R 414 Gudmudson T. V.,524 Giinther H.,365 Giinzel G. 514 Guemeri F. 328 Giisten H. 209 Guggisberg A. 37,499,504 Guhn G. 591 Guillemonat A. 289 Gulden M. 295 Gulick W. M. jun. 29 Guliev A. M. 176 Gulyas E. 39 Gundel L. 201 Gunning H. E. 185 Gunsher J. 113 Gunthard Hs.H. 30 Gunther H. 382 Gupta D. N. 421 Gupta N. K. 543,545 Gupta R K. 20,25,29 Gupta R N. 580 Gupta S. K. 469 Gurbaxam S. 415 Gustak E. 256 Gutch C. J. W. 29 Gutfreund H. 601,602,625 Guthrie R D. 111,307,470 Gutowski G. E. 469 Gutsche C. D. 200 Guttman St,524 Haake E. 522 Haake M.,281 Haake P. 67 Haaland A. 287 Haas H. 233 Haas W. 514 Haase W. H. 499 Habermehl G. 48,489 Haberer D. 518 Habicht E. R 432 Hackett P. 494 Hackler R. E. 278 279,411 Hackney R J. 438,439 Haddadin M. J. 482 Haddock R E. 500 Hadfield J. R 584 Hafner K. 361 362 375 Hafner K.H. 362 Hafner-Schneider G. 375 Hagberg C.-E. 127 Hageman ,H. J. 210 Hagen E. L. 108 Hagen R 212,213 Hagenmaier H.509,524 Hagihara N. 329 Haglid F. 262 Hahn S. J. 40 Haiduc I. 286 31 1 Halgren T. A. 176 Hall A. M. 275 Hall D. W. 110 Hall E. S. 497 Hall F. M. 89 Hall J. H. 168 Hall R H. 537 538 Hall S. R 64,65 Hall T. K. 221 Haller I. 207 397 Haller W. S. 214 461 Hallman P. S. 324 Halls P. J. 183 Halmann M. 471 Halpern B. 39 513 522 523 528 Halpern B. A. 514 Halpern J. 141 321,333 Halsall T. G. 263 Halstram J. 509 530 Halton B. 376 383 Halton R 123 Haluska R. J. 57 482 483 Ham G. E. 154 Ham N. S. 305 Hamada Y.,37,496 Hamann H. C. 271 Hamberg M. 596 Hamer J. 187 Hamer N. K. 83 Hamilton J. B. 195 Hamilton J. B. A. 97 Hamilton L. D. 535 575 Hamilton W.C. 48 53 319 Hammen P. D. 450 Hammes G. G. 549 Hammick C. F. 524 Hammond,G. S. 187,188,189 195,486 Hammond W. B. 402 Hamon D. P. G. 386 Hamor T. A. 271 Iiampel A. 565 Hampson N. A. 267 Hamuro J. 172 Han J. 16 Hancock. K. G. 202,266 Hancock. R. A. 92 Hancock. R. I. 336 Handa K. L. 427 Haner B. A. 62 Hanic F. 48 Haniotis Z. 30 Hanisch G. 470 Hann C. S. 514 Hanna D. P. 413 Hannay A. R.. 326 Hansel R. 476 Hansen H.-J. 350 Hansen S.. 511 Hanson A. W. 47,48 57. 59 Hanson E. M. 511 Hanson J. C. 62 Hanson J. R. 265 426 586 5 Hanson M. J. 89 Hanze. A. R. 266 Hanzlik R. 427 Hanzlik R. P. 278 412 590 Happ G. P. 293 Harada F.538 Harada K. 514 Harbert C. .4. 409 Hardigree A. A. 566 Harding C. J. 128 Harding K. 406 Harding M. M. 44 Hardgrove G. L. 50 Hardman K. D. 634 Hardy J. P. 257 Hardy P. M. 519 Hardy W. B. 346 Harget A. J. 79 Hargreaves A. 47 Harker D. 634 Harkness A. L. 207 Harley-Mason J. 454 500 Harman M. 406 Harmon R. E. 524 Harper E. T. 97,456 Harrington M. J. 436 Harriman J. E. 19 Harris C. M. 455 Herris A, 587 Harris C. M., 455 Harris D. 462 Harris D. R. 65 Author Index Harris F L. 128 Harris J. M. 11 7 Harris T. M. 455 Harrison C. R. 372 Harrison H. R. 277 Harrison I. T. 65 426 Harrison R. 356 Harrison R. W. 308 Harrison S. 65,426 Harrod J. F. 324 Hart E.J. 224 Hart F. A. 290 Hart H. 12 104 197 203,204 344 353 360 374 390 Hart N. K. 491 495 500 Hartdegen F. J. 609 Harte R. A. 607 Harter D. A. 224 Hartlage J. A. 360 Hartley A. M. 259 Hartley B. S. 612 613 618 622,623 Hartman P. M.,397 Hartmann G. 573 Hartmut R. 531 Hartmuth R. 286 Hartter D. R. 69 139 Hartshorn M. 415 Hartshorn M. P. 413,435 Hartsuck J. A, 605 632 Haruki E. 458 Harvey G. R. 443 Hasegaws H. 31,33 Hasekamp C. D. E. 54 Haselkorn R. 560 Haseltine R. P. 205 Hassall C. H. 373 528 Hassanah-Walji A, 529 Hassel O. 60 Hassner A. 141 271,441,442 465 Hata G ,304 330 Hata K. 209 354 Hata T. 48 Hatada K. 305 Hatano H. 31 Hater G.337 Hattori T. 428 591 Hauck H. 152,457 Hauptmann H. 63,319 Hauptmann S. 452 Hauser C. R. 285 Hauser K. L. 406 Hausser J. W. 130 Hausser K. H. 32 Havanec J. W. 128 Havas J. 259 Havel M. 139 Haven A. C. 146 Havinga E. 380 Havsteen B. H. 616,617 Hawes W. 83,84,85 Hawker C. D. 524 Hawks G. H. 280 Hawley D. M.,56,318 Hawley M. D. 248 Haworth G. B. 473 Hawthorne M. F. 284 295 296 297 298,299 Hay J. M. 171 Hayamizu K. 304 Hayase Y. 273 Hayashi K. 531 Hayashi M. 418 Hayashi N. 417 Hayashi S. 209,412,417 Hayashi T. 316 Hayashi Y.,425 Hayatsu R. 546 Hsyberg C.-E. 80 Haynes W. M. 277 Hayon E. 229 547 Hayrac T. 481 Heaney H. 350 356 357,457 Heathcock C.H.. 262 397 402,416,419 Hecht J. K. 359 Hecht S. M. 538 Hecht S. S. 446 Heck R. F. 335,336 Heckert D. C. 413 Heckman R A. 415 Hedaya E. 166 Hedayatallah M. 280 Hedberg A. 20 Heep U. 481 Heeres G. J. 54 Heffron P. J. 37 Hegarty A. F. 142 Hegarty M. P. 510 Hege B. P. 415 Hegedus L. S. 278 Hegelson J. P. 538 Hehre W. J. 355 Heiba E. I. 184 267 334 Heidema J. H. 87 Heil B. 327 Heiibronner E. 212 213 Heim S. 406 Heimbach P. 329,343 Heimer N. E. 578 Hein. G. 618 Heindl. L.. 276 Heine H. W. 443,444 Heinzer J. 26 308 Heller S. R. 540 Hellner E. 46 Hellwinkel D. 350 Hemesley P. 277 Hemingwaf.,R. J. 417 Hemmerich P. 216 Hempel K. 510 656 Hems M.A, 208,346 Hemwall R W. 456 Hen J. 201 Henderson R. 602,612,624 Hendrickson J. B. 379 419 Hendrix J. P. 286 Henery-Logan K. R. 442,448 Henglein A. 224 225 Henner B. 3 11 Henning J. C. M. 18 Henold K. L. 307 Henrici-OlivC G. 332 Henrick C. A. 273 Henrickson C. H. 305 Henry A. C. 630 Henry M. C. 315 Henry P. M. 334,335 Henry-Basch E. 290 291 Henseke G. 470 Hensens 0. D. 429 Herak J. N. 217 Herbelin J. M. 364 Herbert C. A. 391 Herbert M. A. 546 Herbig K. 147 Herkstroeter W. G. 188 Heimanek S. 297 Herout V. 417 Herries D. G. 634 Herriott J. R 44 Herron D. K. 200 Hershman A. 327 Hertzberg G. 160 Hertzler D. V. 267 Herz W. 423 Herzog H. 509,518 Hess D.211 Hess G. P. 612,616,617 Hess W. W. 266 Hesse G. 295 Hesse M. 504 Hesse R. H. 176 272 344 Hesselmann I. A. M. 32 Heslop J. A. 288 HetflejS J. 31 1 Hetzer H. B. 89 Hetzheim A. 458 Heusler K. 171 Hewlins M. J. E. 548 Hey D. H. 171 178,179 181 350 Hey H. 15 Heyder E. 524 Heyn H. 214,385 Heyns K. 472 Hiatt J. E. 118 403 Hiatt R. R. 333 Hibbert F. 80 Hickman J. 263 301 Hidai M. 331,332 Higgins R. 268 Author Index Hignite C. E 546 Higo M. 455 Hikino H. 417 438 439 Hikino Y. 438 439 Hill D. T. 113 Hill E. A. 103 110 Hill H. A. O. 259 Hill H. C. 15 Hill J. 200 210 Hill J. W. 168 Hille M. B. 621 Hill M. L. 196,486 Hill R. E. E.309 Hill R. K. 145 202 402 465 Hill R. L. 61 1 Hill R. R. 113 377 Hill W. E. 297 Himizu J. I. 406 Himoe A. 612,616 Hinchcliffe A, 18 Hindle P. R. 22 Hindriks H. 509 Hine J. 134 Hines J. N. 270 Hinrichs H.-H. 365,382 Hinshaw J. C. 123,385 Hirai S.,500 Hirai Y.,458 Hiranuma H. 467 Hiraoka H. 21 5,455 Hirasawa R. 33 Hirata Y. 418 Hirota N. 22 Hirota T. 464 Hirota Y. 566 Hiroto K. 8 Hirsch J. A. 380 Hirsch-Kauffmann M. 547 Hirschmann R.. 509 519. 521 524 Hirs C. H. W. 635 Hirst J. 89 Hishida T. 91 Hiskey R. G. 509 533 Hjortaas K. E. 52 Ho S.K. 228 Hobbs W. E. 285 Hobson J. D. 204,463 Hochstetler A. R. 190 Hocks P. 439 Hodgkin D. C. 62 530 Hodgson A.551 Hodgson B. 172 Hodgson W. G. 255 Hoefnagel M. A. 381 Hohn E. G. 36 Hoppner K. 314 Hofmann K. 509,527 Hoffman A. K. 255 Hoffman H. 259 Hoffman. P. 509 518 Hoffman R.. 18 202 Hoffmann A. K. 160 161 Hoffmann E. G. 298 Hoffmann H. 314 Hoffmann H. M. R 145,157 158,388 Hoffmann H. P.. 570 Hoffmann R. 143 187 188 355 Hoffmann W. F. 597 Hogarth M. J. 289 Hoge R. 59 Hogeveen H. 114,397 Hogg D. R 345 Hohnstedt L. F. 297 Hoignt J. 210 Hojo M. 88 Holden J. R. 44 132 Holden K. G. 406 Holder N. L. 427 Holker J. S. E. 599 Holland J. M. 377 Hollander J. 535 Holley R. W. 563 564 Holliday A. K. 293 Hollinden C. S. 522 Hollins R. A. 218 Hollis M.L. 229 Holloway D. M. 548 Holloway M. R 631 Holloway P. W. 585. Holly F. W. 540 Holmes K. C. 565 Holovka J. M. 196,486 Holt A. 309 Holt C. E. 570 Holt J. W. 538 Holzapfel C. W. 39 Honda O. 425 Honeck H.,458 Honikel K. 573 Honjo M. 542 Hoogeboom T. J. 285 Hoover J. R. E. 392 Hooz,J.,274,276,301,391,409 Hope D. B. 526 Hope H. 62 Hoppe W. 53,62 Horder J. R. 307,317 Horeau A. 512 Horibe I. 417 Horman I. 126 128,349 Horn D. H. S. 438,439 Horn U. 445 Hornback J. M. 120 Hornby R. B. 481 Horner L. 258 309 325 Horsfield A. 183 Hortmann A. G. 416,442 Horton D. 221,265,274,473 Horton N. 363 Horwitz J. P. 71 Hosokawa K. 560 Hosomi A, 171,309 Hossain M.B. 418 Hostynek J. J. 275,433 Hota N. K. 297 Hough A. 587 Hough E. 427 Houlihan W. J. 220,272 Houlten P. R.,370 House H. O. 135 274 277 381 400 Housty J. 42 Hovance J. W. 83 Howada K. 527 Howard E. G. 177 Howard J. A. 184 185 Howard R. D. 79 Howard-Flanders P. 547 Howden M. E. H. 176 Howe C. A. 181 Howe D. V. 296 Howe I. 8 10 14 Howles J. R. 178 Howsam R. W. 268 Hoy T. G. 47 Hoya W. K. 94 Hoyda F. 463 Hoyer H. W. 379 Hoyt E. B. 446 Hranilovic J. 256 HrdloviE P. 187 Hruban L. 493 Hruby V. J. 527 Hsieh D. P. H. 598 Hsu F. M. 404 Hsu H. Y. 438 HSU,S.-J.,605 Huang B. 406 Huang C. W. 207 Huang H. H. 314 Huang S. J. 264 Huang W.H. 216 Huang W. M. 550 Hubbard A. T. 259 Hubbard C. D. 630 Huber C. P. 60,65 Huber G. 609 Huber H. 146 Huber J. E. 219 274 433 Huber R. 53 Huber U. A. 399 Hubner. H. 175 Huckstep L. L. 486 Hudrlick P. F. 279 31 1 Hudson A. 28 Hudson B. 573 Hudson C. B. 512 Hudson J. A. 348 Hueblin G. 141 Hiippi G. 438 Author Index 657 Huff R.K. 277 389 Ilatovskaya M. A. 332 Huffman R. W. 424 Iles D. H. 27 Hughes A. N. 162 Illuminati G. 99 Hughes E. S. 128 Ilton M. A. 268,409 Hughes E. W. 43 Ilyas M. 476 Hughes M. T. 398 Imai K. 542 Hughes N. A. 472 Imamura A. 355 Hui K. M. 314 Imamura H. 425 Huisgen R. 145 146 147 148. Impicciatore M. 509 526 151. 152 357 447 457 Imoto E. 458 459 Inaba T.475 Huisman H. O. 430 431 Inada H. 344 Hulscher J. B. 58 Inagaki M. 504 Hume K. 59 Inagami T. 615,634 Humphrey L. B. 172 Inamoto N. 182 Humphreys D. J. 493 Ingles D. W. 620 Humski K. 120 Ingold C. K. 128,616 Hunt D. F. 546 Ingold K. U. 184 185 Hunt G. W. 312 Inomata I. 331 Hunt J. W. 226 229 Inoue T. 244 Hunter F. D. 59 Inoue Y.,549 Hunter F. R. 21 Inouye H. 582 Hurd C. D. 146 Inui T. 515 Hurd R. M. 252 Inukai N. 522 Hursthouse M. B. 427 Ioffe S.T. 22 283 Husain S. S. 627 631 Ionin B. I. 314 Hussain H. A. 28 Ipaktschi J. 204 369 Husson H. 275 Iqbal K. 264 Hutchins J. E. C. 79 Irie H. 495 Hutchins R.O. 414,478 Irie T. 122 Hutchinson D. A. 16 Irwin K. C. 333 Hutchinson R E. J. 446 Isada T. 344 Hutley B.G. 130 Isaev I. S.,370 Hutson D. H. 38 Isele P. R.,113 Huxtable R. 579 Iselin B. 509 517 Huyser E. S. 178 Isemura T. 533 Hvoslef J. 58 Ishida T. 566 Hyde J. S. 176 Ishii H. 418 Hyde R. M. 79 Ishikawa F.,464 Hylton T. 353 Ishikawa M. 216 311 312 Hymans W. E. 437 468 Hymes A. J. 620 Ishikawa N. 521 Ishikawa Y. 512 Iam F. L. 141 Ishitobi H. 122 Iball J. 60 183 Ishiz~mi,K. 264 Ibers J. A. 326 Islam A. 514 Ibuka T. 495 Islam K. M. S.,61 Iburg W. J. 402 Ismail R M. 311 Ichet P. 260 Isomura K. 441 Ichihara A. 421 Isono K. 541 Ichikawa T. 43 Israel M. 484 Igaki H. 330 Issidorides C. H. 482 Igarashi H. 591 Itaya N. 426 Igeta H. 215 Ito I. 464 Igeta S. I. 182 Ito S.,36,425 Ihara M. 37,65 493 Itoh I.36 464 Iitaka Y. 43 55,65,426 Itoh K. 33 Iizuka T. 200 Itoh M. 301 Ikeda M. 500 Ivanov V. T. 533,605 Ikeda S. 329,332 Iverach G. G. 504 Ikemoto I. 60 Iwamoto H. 464 Iwamoto K. 96 Iwamoto M. 330 Iwamura H. 194 Iwamura M. 182 Iwanami M. 464 Iwasaki S. 200 Iwatani K. 256 Iyer V. N. 574 Izawa Y. 200 Izdebski J. 509 Izumiya N. 530 Jablonski J. M. 356 357 475 Jackovitz J. F. 293 Jackson A, 496 Jackson A. H. 264 Jackson G. F. 319 Jackson R. A. 284 Jackson W. R. 264 Jacob F. 566 Jacob T. A, 521,524 Jacobs P. M. 519 Jacobs T. L. 123 124 Jacquenoud P.-A. 524 Jacques J. 137 Jaeger E. 509 525 Jager G. 519 Jagur-Grodzinski J. 109 135 James B. R. 326 James L.L. 93 344 346 Janata J. 252 Janeck A. 313 Janes W. H. 153,329,330 Janiga E. R. 281 Jansonius J. N. 626 Jantelar M. 589 Janzen E. G. 24,33,183,255 Jao L. 611 Jao L. K. 70 Jarcho M. 375 Jardetzky O. 533,637 Jarman M. 543 Jarreau F.-X. 504 Jarvie A. P. 312 Jarvie A. W. P. 309 Jarvis B. B. 446 Jaskunas S. R. 548 Jauhal G. S. 321 355 Jautelat M. 281 Jefcoate C. R. E. 32 Jefferies P. R. 426 Jeffery E. A. 304 305 Jefford C. W. 113,384,494 Jeffrey G. A. 59 Jeffs P. W. 578 Jeger O. 200,437 590 Jencks W. P. 73 78 126,603 Jenevein R. M. 253 258 Jenkins C. J. 184 Jenkins J. K. 504 Jenkins S. R. 540 Author Index Jennings C. A. 285 Jennings J. R. 302 307 Jensen E.R. 598 Jensen F. R. 380 381 Jensen L. H. 41 57 546 Jensen N. P. 391,409 Jente K. 594 Jeremu D. 184 Jerina D. M. 600 Jernow J. L. 165 Jersley B. 61 Jervis G. J. 309 Jessop G. M. 293 Jewell J. S. 473 Joachim H. H. 598 Job B. E. 207 347 Joklik J. 284 Johne S. 580 Johns H. E. 217,546,547 Johns J. W. C. 160 Johns S. R. 491,494,495 496 500 505 Johnson A. P. 453 Johnson A. W. 453,529 Johnson B. 124 Johnson B. F. G. 284,337 Johnson B. J. 519 Johnson C. R. 281 Johnson C. D. 97 Johnson C. K. 43 177,413 Johnson C. S. jun. 20 25 Johnson D. A 447 Johnson F. 380,464 Johnson F. A. 293 Johnson J. R. 286,615 Johnson K. 227 Johnson K. L. 178 Johnson L. N.623,634 Johnson M. D. 95,142 Johnson M. G. 211 Johnson P. C. 421 Johnson R. H. 79,523 Johnson S. 598 Johnson S. M. 53,57,482 Johnson W. S. 268 391,409 415,432 Johnston T. E. 289 Johnstone R. A W. 8 9 13 91,462 517 Jolles J. 606 Jollks P. 606 Joly J. 265 Joly R. 265 Jones A. J. 372 Jones D. N. 38 Jones D. S. 525 Jones D. W. 377 Jones F. N. 389 Jones G. 277 363 Jones G. H. 541 Jones IT. 124 Jones J. B. 434 Jones J. H. 517,521 Jones J. K. N. 473 Jones J. R. 67 135 Jones L. B. 193 Jones L. C. 484 Jones L. H. 177 Jones M. 347,356 Jones M. jun. 194 197 221 384,386,395 Jones M. M. 321 Jones N. R.,403 Jones P. F. 308 Jones P. L. 285 Jones R. 293 294 295 Jones R.V. H. 417 Jones T. C. 129 Jones V. K. 193 Jones W. C. jun. 509 Jones W. J. 293 Jones W. M. 342 Jonkhoff T. 328 Jordan J. M. 573 Jordan L. E. 568 Jordan M. D. 141 Jorgenson M. J. 141,201,395 401 Joseph T. C. 210 419 504 Joshi G. C. 301 Joshua H. 521 JoSt K. 527 528 Jothan R. W. 293 Joule J. A. 493,496 Joullic M. M. 89 Jovin T. M. 545 Joy D. R.,157 i58,388 Judy W. A. 322,323 Juenge E. C. 266 Jurgen F. 528 Jugelt W. 79 247 Jula T. F. 316 317 Julia M. 171 178,403 Jung G. 509,524 Jung J. M. 325 Jungmann H. 209 Jura W. H. 255 Just G. 184,267 Kabachnik M. I. 22 Kabalka G.W. 273,276,300 30 1 Kagal S. A, 599 Kagan H. 512 Kagan H. B. 150,447 Kagawa S.421 Kahn P. C. 35 Kahnt F. W. 524 Kai Y.,307 Kaiser A. D. 568 Kaiser E. M. 250 280 285 286 Kaiser E. T. 17 25 86 87 Kaiser K. 499 Kaissi F. E. 317 318 Kaji A. 560 Kakihana T. 485 Kakisawa H. 423,424 Kakiuchi K. 534 Kakova K. 37 Kakudo M. 45,307,312 Kalfogloy N. 105 Kalinin V. N. 297 Kalinina G. S. 317 Kallos J. 620 Kalvoda J. 436 Kamasaki K. 518 Kamber B.,524,533 Kamen R 560 Kametani T. 37 65 469,493 494,495 Kamiet M. J. 134 Kamiya K. 64 Kampmeier J. A. 173 Kan R. O. 213,449 Kanaoka Y.,487 Kaneko. C. 216,468 Kaneko T. 515 Kan-Fan C. 275 Kang J. W. 332,401 Kang S. Z. 301 Kaniosho M. 354 Kankaanpera A, 71 Kanto S.410 Kano H. 213,468 Kanoya A. 182 Kanters J. A. 58 Kapadia G. J. 492 Kapecki J. A 451 Kapil R S. 414 581 Kaplan E. 286 Kaplan L. 103,207 Kaplan L. E. 172 Kaplan M. L. 218 Kappe Th. 492 Kappus G. 15 Karafiath E. 191 Karanatsios D. 424 Kardos A. M. 259 Karger M. H. 281 Karim A 417,424 Karle I. L. 63 65,487 504 Karle J. 63,65 504 Karliner J. 470 Karmann H. G. 105 Karpeisky M. Ya,605 Karplus M. 17 Karrer F. 453 Kartha G. 43,62,634 Kasahara A. 336 Kasai K. 458 Kasai N. 307 312 Author Zndex Kasper C. B. 625 Katagi T. 496 Katakami T. 368 Katchalski E. 525 Kathawala F. 543 Kato A. 527 Kato H. 109 355,442 Kato K. 631 Katb M. 202 205 Kato M. 487 Kato T.410,418,467 Katritzky A. R. 92 97 183 463,467,540 Katsumura S. 418 Katsurakawa K. 88 Katz G. 563,564 Katz T. J. 21 196 Katze J. R. 564 Katzenellenbogen J. A 269 415 Katzin L. I. 37 39 Kauer. J. C. 389 Kauffman D. L. 612 622 623 Kauffman K-D. 309 Kaufmann H. 493 Kaupp G. 30 Kaurov 0.A. 527 Kawabata N. 384 Kawamura T. 26 Kawasaki K. 526 Kawata K. 500 Kawauchi A. H. 514,523 Kawauchi H. 514 Kawazoe V. 463 Kawezynski A. L. 514 Kazamkova M. R. 316 Kazartsev A. V. 297 Kay H. F. 52 Kealy T. J. 330 Keana J. F. W. 214 Kearns D. R. 218 Keck K. 547 Keefer R. M 126 127 128 349 Keeley S. L. 907 Keene J. P. 227 Keeton R. 414 Keil B.,612 Keim W.337 Kei-Wei Shen 319 Keller D. 560 Keller E. B.,564 566 Keller L. S. 386 Keller R. A, 188 Kellogg R. M. 97 189 455 456 Kelly D. P. 278,376,411 Kelly J. F. 447 Kelly R. B. 570 Kemball C. 326 Kemp C. M. 635 Kemp T. J. 23 228 Kemp R. T. 138 Kemp W. 370 Kemppainen A. E. 198 Kemula W. 90 Kennard C. H. L. 51 Kennard O. 62 Kcnner G. W.. 509. 517. 525. 533 Kenner J. W. 146 Kent G. J. 141,271 Kent M. E. 166 Keogh M. F. 580 Kerb U. 439 Kern R. J. 332 Kerr K. A. 62 Kerst F. 83 Kerzomard A. 81 Kessar S. V. 439 Kesselaar F. H. 431 Kessler H. 339 352 384 Kessler H. 352 Kessler H. 384 Ketley A. D. 335,400 Kettle S. F. A. 321 354 Keutmann H.T. 524 Kevan L. 17 Keve E. T. 42 Keys R. T. 20 Kezdy F. J. 612 Keziere R. J. 417 Khachaturov A. S. 176 Khalil F. Y.,68 Khan A. A. 226 Khan A. U. 218 Khanchandani K. S. 577 Kharasch M. S. 242 Kharasch N. 171,187,342 Khiznyi V. A. 171 Kholopov A. V. 560 Khorana H. G. 543,545 Khotimskaya G. A. 262 Khrapov V. V. 314 Kice J. L. 79,80 Kido F. 421 Kiefer E. F. 268 Kiefer H. 175 Kiely D. E. 475 Kienle M. D. 592 Kier L. B. 433 Kiger J. A,jun. 573 Kikkawa S. 312 316 Kikuchi T. 64,504 Kikuchi Y.,534 Kim B.,221 Kim H. L. 907 Kim H. S. 59 Kim J. B. 97 Kim S.-H. 548,565 Kim Y.,603 Kimmel J. R. 626,627 Kimmer H. 314 Kimoto S. 518 Kimura F. 538 King c.L.539 King C. M. 539 King K. 342 King,R. C. 53 King R W. 202 King T. P. 631 King T. J. 371 Kingsland M. 92 540 Kingston D. G. I. 8 Kinomura Y.,518,526 Kinstle T. H. 13 14 277 Kiovsky T. E. 107 108 341 Kirby A. J. 74 76 82 126 Kirby G. W. 345,577,578 Kirk D. N. 439 Kirkegard L. 565 Kirmse W. 159 160 Kirchoff K. 105 Kirsch J. F. 630 Kirschner R.H. 573 Kirst H. A. 279 Kirtikar D. M. W. 560 Kiryushin A. A. 517 Kisaki T. 490 Kisfaludy L. 509 Kishida Y.,55,526,534 Kishimoto T. 495 Kisin A. V. 308 Kitagawa I. 429 Kitagawa T. 529 Kitahara Y.,392,410,418 Kitahonoki K. 443 Kitano M. 495 Kitazawa K. 429 Kitching W. 283 Kitzing R 452 Kivelson D.20 Kjaer A. 38 Klarner F.-G. 371 Klasine L. 209 Klauke E. 509 518 Klaus M. 396 Kleb K. G. 350 Klee L. H. 619 Klein F. M. 381 529 Klein H.-F. 304 Klein H. S. 332 Klein J. 380 381 Klein L. 371 Klein M. 375 Klein P. 189 Kleiner F. G. 315 Kleinman R. 514 537 Kleinschmidt A. K. 568,570 Kleinschmidt R. F. 392 Klem R 184,236,271 Klemchuk P. P. 182 Author Index Kleopfer R 217 546 Klewe B. 56 Klieger E. 527 Klimova T. V. 297 Kline L. K. 538 Klinman 3. P. 77 Klippert T. 227 Klok R. 406 Kloosterziel H. 398 Klostermeyer H. 528,530 Klotz L. C. 549 Klug A 535,565 Klug H. P. 53 Kluger R. 83 Kluh I. 612 Klumpp G. W. 394 Klundt I. L. 94 Klyne W. 37,39,439,499 Knight E.jun.,551 Knights E. F.. 263 299 230 301 Knoepfel H. P. 448 Knoppel H. 16,342 Knorr R 357,459 Knothe L. 363 Knowles G. D. 582 Knowles J. R. 601,621 Knowles W. S. 261 326 Knox,G. R 169,353 Knox L. H. 160 Knutson D. 210 Kobayashi K. 257 Kobayashi M. 418,564 Kobayshi S. 285 441,479 Kobelt D. 313 Koblikova Z. 37 Kobrich G. 160 387 Koch F. 182 Koch J. K 177 Koch K. 211 Koch T. H. 189 Kocheshkov K A. 290 Kochetkov N. K. 470 Kochi J. K. 109 183,184,271 Kochler S. 78 Kochloefl K. 262 Kocy D. 522 Kodama M. 425 Kodama T. 251 Kobrich G. 286 Koehl W. J. 184,238,239,267 3 34 Koehl W. J. jun. 267 Koekoek R 626 Konig C. 362 Koenig D. F.606 Konig W. 509 519 Koerner von Gustorff E. 412 Koster H. 545 Koster R. 298 Koga K. 264 Kohler H. J. 452 Kohll C. F. 328 334 Kohn K. W. 539 Koizumi M. 27 495 Kokabu T. 527 Kokoszka G. F. 26 Kolattukudy P. E. 577 Kollmeyer W. D. 131 Kolobova N. E. 313 Kolthoff 1. M. 67 Komae H. 421 Komari S. 271 Komarov N. V. 310,315 Komem A. 220 Komeno T. 38 Komori T. 182 Konaka R. 30 Kondon H. 261,326,337 Kondo K. 387 Kondo Y.,217,264 Koneru P. 539 Kong N. P. 173 Konig H. 282 Konigsberg W. 564 Konishi H. 33 355 Konno C. 417 Konstyanovsky R. G. 314 Kopchik R. M. 173 Kopecky J. 212 536 Kopp L. D. 478 Kopple K. D. 528,533 Koptyug V. 109 Koptyug V. A. 370 Koreeda M.438 Kornberg A. 545 570 Kornet M. J. 264 Kornev K. A. 310 Kornhauser A. 217 Korobeiinikova S. A. 302 Korshak K. V. 284 Korsloot I. G. 431 Kort C. W. F. 97 Korte S. 365 Korth T. 498 Koruncev D. 256 Korver J. A. 512 Korytko L. A. 314 Kosak A. I. 529 Kosaka T. 243 Koshland D. E. 602,625 Koshland D. E. jun. 621,633 Koshland T. E. jun. 610 Kossler I. 159 Koster R 263 Kostka V. 612 Kostyanovsky R. G. 314 Kosyakova L. V. 332 Kotera K. 37 443,496 Kothe W. 211 Kotz J. 302 Koudijs A. 480 Author Index 66 1 Kovac P. 470 Kovacic P. 182 Kovacik V. 470 Kovacs J. 518 523 Kovol 0.I. 68 Kowala C. 494 Kowaleszyk L. S. 71 Kowollik G. 540 Koyama G.55 Koyama H. 45 Koyama K. 241,244 Koyama M. 504 Koyama T. 464 Kozima S. 316 Kozuka A, 495 Kozyukov Y. P. 310 Krafft W. 343 Kraft M. 10 Krahn R. C. 39 Kraihanzel C. S. 310 Kralic I. 226 Kramarova E. N. 31 1 Kramer B. D. 148 387 Krampitz G. 514 Krapcho P. 118 Kraus M. 262 Kraus W. 402 Krauss H. 600 Kraut J. 625 Kravtsov D. N. 314 Krebs A. 382 Kreevoy M. M. 68,69 Krejcarek G. E. 493 Krepinsky J. 417 Kresge A. J. 68 Krespan C. G. 177 Kretchmer R. A. 398 504 Kreuder M. 362 Kreuz K. L. 271 Krief A. 357,464 Krieg G. 290 Kriegl V. 304 Kriegsmann H. 309,314 Krigbaum W. R. 44 Krisch J. F. 90 Kristiansson K. 499 Krizhanskii L. M. 314 Kronfeld L.R. 54 Kroon A. M. 570 Kropf R. 48 Kropp J. E. 141 271 Kropp P. J. 413 Kroto H. W. 166 Krow G. R. 114,197,352,401. Krueger P. 546 Krueger W. C. 596 Kruger G. 432 Kruger G. J. 50 Kruglaya 0. A. 283 317 Krukonis A. P. 48 Krumbiegel P. 175 KrupiEka J. 139 257 Krusic P. J. 183 Krygowski M. T. 90 Ku E. 617 Kubo R.,19 Kubota T. 45 256 Kudryavtsev R. V 265 322 355 Kudryuski J. 89 Kuhlien K. 312 Kuehne M. E. 275 Kugajevsky I. 182,577 Kugita H. 907 Kuhn A. T. 252 Kuhrt G. 511 Kuimova M. E. 303 Kuivila H. G. 131 177 284 316 317 Kulik S. 346 Kulish L. F. 68 Kuilenberg B. 414 Kulsa P. 500 Kumada M. 308 309 310 31 1 Kumar A, 543 Kumari D.428 Kumashiro I. 536 Kumazawa Z. 422 Kumler P. L. 216 467,481 Kumpfert H. 382 Kunieda T. 547 Kunkel W. 360 Kunstmann W. 360 Kuo C. H. 431 Kuo I. 229 Kupchan S. M. 417,424,435 Kupiecki F. P. 596 Kurabayashi K. 399 Kuran W. 307 Kurihara T. 418 Kurioka E. 326 Kurita M. 202 205 Kuriyama K. 36 38 Kurkov V. P. 333 Kurland R. J. 109 Kuroda H. 60 Kurosawi K. 37,377,476 Kursanov D. N. 262,265 Kurt R. N. 95 Kurts A. L. 292 Kuruma K. 30 Kurvajima I. 274 Kurz M. E. 182 Kusama K. 540 Kuse T. 332 Kusomoto S. 515 Kusumi T. 423 Kutikova G.A. 27 183 Kutney J. P. 429 475,583 584 Kutter E. 377 Kuwada Y. 541 Kuwana T. 259 Kuyama J. O. 69 Kuznetsov V.I. 283 Kuznetsova V. P. 310 Kuzuhara H. 472 Kvasov B. A. 103 Kwan T. 326 Kwart H. 342 351,456 Kwiatek J. 326 Kwiatowski G. T. 192,268 Kwitowski P. T. 105,362 Kwok W.-K. 603 Kyogoku Y. 548 Laarhoven W. H. 209 Labanauskas M. 565 Labeeuw B. 436 Labouesse B. 616 617 Labouesse J.. 617 Lachowicz D. R. 271 Lackner H. 531 Lacount R.B. 375 Ladenberger V. 140 Lader H. 276 LaFollette D. 103 Lagercrantz C. 27,28,29,33 Lagercrantz E. 183 Lahournkre J.-C. 312 Laing S. B. 431 437 Laird A. H. 525 Lajierowicz-Bonneteau J. 56 Lake A. W. 627 Lake J. A. 565 LaLancette E. A. 196 LaLonde R. T. 237 Lam A. Y. 109 Lamaty G. 130 Lamb R.C. 176 Lambert C. A. 308 Lambert F.L. 257 Lambert J. B. 50,319,381 Lambert R.F. 250 Lamberton J. A, 491 494 495,496 500 505 Lampe F. L. 313 Lampe F. W. 313 Lampman G. M. 380 Lamola A. A. 217 546 Lance D. G. 271,473 Land E. J. 244 225 226 227 Lande S. 526 Landgrebe J. A. 68 Landini D. 80 Landis W. R. 217 Landon M. 625 Landor S. R. 510 Landrey J. R. 592 Landsbury P. T. 477 Landy A. 563 Lane B. G. 537 538 Lane G. A, 435 Lanet J. C. 436 Lang D. 568 Lhg z. 509 Lange H. W. 510 Lange R M. 197 Langen P. 540 Langmuir M. E. 229,547 Langridge J. L. 67 Langridge R. 551,565 Lankamp H. 182,340 Lansbury P. T. 375 Lanzarinii C. 514 Lapkin I. I. 288 310 La Placa S. J. 53 319 Lappert M. F. 294 302 307 312,316,317 Larber M.E. 270 Larbi Bouguerra M. 252 Large G. B. 80 La Rochelle R. 278 411 Larsen 1. K. 44 Larsen J. W. 109 Larson J. K. 274 Larsson G. 42 Laser J. 594 Lassila J. D. 204 21 1 Lassmann G. 314 Lathan D. W. 235 Lauder I. 68 Lauer D. 339 Laufer D. 522 Lauinger C. 511 512 Laur P. 38 Lavie D. 499 Lavigne J. B. 333 Law D. C. F. 387 Lawesson S.-O. 13 14 Lawler J. M. 81 Lawler R. G. 21 372 Lawley P. D. 539 574 Lawrence R. V. 423 Lawson A. J. 95 344 Lawson A. M. 12 Lawson B. W. 619 Lawton R. G. 252,493 Lazar J. 512 Lazdins I. 112 Leander K. 492,505 Leard M. 309 Leaver D. 462 Lebedev Ya. S. 17 171 Lebel N. A. 177 Le Blanc J. C. 546 Lebowitz. J. 571 Le CalvC J.223 Le Clerc J. 518 Leclerc J. C. 8 Le Demezet M. 260 Lederer E. 517 Ledwith A. 27 146 154 159 Author Index Lee A, 213 Lee C. M. 500 Lee C. S. 570 571 Lee D. G. 83 Lee F. T.-H. 201 Lee L. T. C. 276 Lee J. B. 267 Lee S. L. 262 Lee w. s.,447 Leedy D. W. 249 Leeming M. R. G. 266,433 Leermakers P. A. 188 190 198,233 Leete E. 577 Lees P. 350 Leffek K. T. 130 Lemer J. E. 90 174 Leftin J. H. 384 Legallais V. 601 Le Goff E. 375 Legrand M. 37 Lehman I. R. 570 Lehmann C. 200,434 Lehmann G. 518 Lehmann H.-G. 439 Lehmann M. S. 57 Lehn J. M. 441 Leicher W. 233 Leitch L. C. 12 Leites L. A. 297 Leitich J. 412 Lemaire H. 27 Lemaire J. 187 Leme L. A. P.,65 Lemieux R. U.609 Lenard K. 479 Lengyel I. 445 Lengyel P. 564 Lenz H. 294 Leonard N. J. 442,446 538 Leone R 168 Leopold E. J. 391,409 Lepley A. R. 394 Le-Quanh-Minh 315,318 Lerman L. S. 574 Lesko P. M. 379 Lesko S. A. 550 Lester E. W. 342 Letsinger R L. 545 Leung C. 87 Leung C. Y.,525 Leusink A. J. 316 Leutzow A. E. 274 Lewin A. H. 179 Lewis A, 372 Lewis F. D. 221 Lewis I. C. 23 30 Lewis J. 284 337 Lewis J. W. 262 264 Lewis K. G. 429 Lewis R. G. 262,498 Levenberg B. 510 Levin G. 519 Levin R H. 356 386 Levine S. D. 525 Lezina V. P. 93 Lhomme J. 184,420 Li C. H. 526 Li G. S. 310 Li T. 268,415 Liang C. D. 380 Liang K. S. Y. 181 Licke G. C. 202,266 Liebman S. A. 20 Liehr J. G. 455 Liener I.E. 627 631 Liengme B. V. 314 Lier E. F. 428 Lieske C. N. 83 128 Light A. 627 Light J. R. C. 284 312 Lightner D. A, 38 Lijinsky W. 539 Lilie J. 225 Liljegren D. 580 Lillford P. J. 81 Lillicrap S. C. 230 Lim D. 172 Limborg F. 246 Liminga R. 42 Lin B. T.-S. 53 Lin C. H. 406 Lin J. W. 274 Lin K. 589 Lin Y. Y. 423 Lincoln F. H. 406 Linde H. 428 Linder E. 105 Lindow D. F. 355 Lindquist R. N. 81 Lindsay D. G. 394 Lindsey R. V. 231 Lindsey R.V. jun. 389 Ling D. 458 Lingens F. 539 577 Lini D. C. 381 Link H. 419 Link M. 97 Linke S. 166 274,276 301 Linn W. J. 156 Linschitz H. 217 Linstead R. P. 238 Linter M. A. 208 Liotta C. L. 89 134 Li-Pen Chao 627 Lipkin D. 540 Lipkin V.M. 517 Lippmaa E. 109 Lipscomb W. N. 65 605 632 Lipsky S. R 540 Liquori A. M. 60 530 Lis A. W. 542 Lis E. W. 542 Lishanskii I. S. 176 Lissi E. A. 293 Listowsky I. 40 Lis); V. 542 Litle R. L. 373 Littau V. 509 Little J. 635 Little W. F. 332 Littlehailes J. D. 207,250,347 Lui A. K. 606 Lui C. Y. 105 Liu R. S. H. 189 Liu T.-Y. 631 Liversedge F. 517 Livingston R.,32 Lloyd D. 95 Lloyd D. J. 138 Lloyd N. C. 312 Loader C. E. 208 Locke A. W. 68 Loder J. W. 38 Loehr J. S. 566 Loemker J. E. 341 Loening K. L. 268 Loening U. E. 551 Loeschen R. L. 189 Low M. 509 Loew P. 581,582 Loewensteing R. M. J. 362 Loffett A. 509 Logan R. H. 294,295 Lohrmann R. 536,537 Lohse C.468 Loman H. 229 Lomas D. 387 Lomas J. S. 95 Long F. A. 67 69 Long G. G. 319 Long H. A, 44 Long L. jun. 412 473 Long L. H. 293 Longevialle P.,11 Longster G. F. 29 Longuet-Higgins H. C. 143, fa Loo J. 539 Loo S. N. 344 Looman H. 547 Loomis W. D. 586 Lorand J. P. 172,405 Lorberth J. 313 314 Lorch H. 428 Lorck H. 591 Lord R. C. 548 Lorenz P.,522 Lorquet A. J. 8 Lorquet J. C. 8 Losse G. 522 531 Losse M. L. 310 Author Index 663 Lossing F. P.,166 McBride J. M. 174,206 Lott J. 262 McCaffery A. J. 40 Loudon A. G. 128 Maccagnani G. 132,133 Loudon M. C. L. 577 McCalla R.R.,539 Low M. J. D. 293 McCapra F. 143 Lowe G. 126,609,627,631 McCarthy A. R 459 Lowenstein A. 30 McCarthy E.D. 16 Lowers R 227 McCarthy J. P. 482 Lown J. W. 443 McCloskey J. A. 12,546 Lowrie G. B. tert. 444 McClure J. D. 331 Lowry N. N. 173 McClure W. O. 620 Lowson A. M. 546 Maccoll A. 128 Love D. 51 1 McConn J. 617 Love J. L. 332 McConnell H. M. 17 Love W. E. 44 57 MacConnell J. G. 62 Loveridge E. L. 210 McCormick A. 16 Lu C. T. 62 McCormick J. R.D. 598 Lu P. 539 McCreadie T. 422 Lubiewska L. 523 McCrindle R.,114,423,427 Lubs H. J. 494,496 McCullagh L. 461 Luche J. L. 150 447 MacCulloch W. J. 24 Lucken E. A. C. 105 McCullough J. J. 192 207 Luckhurst G. R. 28 33 McDaniel M. C. 205,398 Ludescher U. 530 MacDonald A. A. 22 375 Ludwig M. L. 605,632 Macdonald C. J. 339 Liiben G. 525 MacDonald, D. M. 505 Liining B. 490,492 505 Macdonald L.H. 39 Luetzow A. E. 473 MacDonald M. C. 346 Lui S. C. 146 McDonald R. K. 526 Luijten J. G. A, 283 McDonald R. N. 356 Lukas J. 105 McDonnell J. 31 Lukaszewski H. 183 McEwen W. E. 274 319 Lukina M. Yu. 262 McFarlane I. J. 579 Lumb J. T. 122 McFarlane W. 313 Lund H. 245,255 McGarvey B. R.,324 Lundin A. F. 275,433 McGeachin S. G. 309 Lundin R. E. 213 McGhie J. F. 428 Lundstrom J. 492 579 MacGillavry C. H. 51 Lunn W. H. W. 405,432 McGrath R.,577 Lupton E. C. 90 McGreer D. E. 393 Lupton E. C. jun. 144 MacGregor D. 95 Lushchitskaya I. M. 527 McGregor D. R 42 Lustgarten R. K. 119 120 Mach K. 159 Lutsenko I. F. 311 316 Machzek J. 262 317 MacHattie L. A. 571 Lutz R E. 138,444,445 Machlis L. 418 Luz Z. 17 18 Maciaszek S. 307 L’vov A.I. 297 Mclnnes A. G. 597 Lwowski W. 166 168 MacIntyre I. 524 Lyall J. 430 McIntyre T. W. 248 Lynch B. M. 93 Mack H. 369 Lynch J. 312 Mackay A. C. 219 Lynch P P. 262 McKay B. 257 Lynch T. R 393 Mackay M. F. 65,495 Lynden-Bell R. M. 36 McKechnie J. S. 53 56,62 Lyukas S. D. 310 McKendry L. H. 213 McKenna J. 130 Ma J. C. N. 529 McKenzie S. 462 Mabry T. J. 37,417,476 Mackie F. D. H. 128 McAllister T. 166 Mackie R. K. 468 MacBride J. A. H. 480 McKillop A, 277 280 664 McKinley S. V. 132 284 McKinnon D. M. 462 Mackor A, 28,183 McLachlan A. D. 20 McLafferly F. W. 8 9 10 16 91 McLauchlan. K. A, 33 McLay G. W. 277 MacLean C. 182,340 MacLean D. B. 493,495,580 McLennan D. J. 138,348 McLean S. 495 McLung R.21 McMaster P. D. 380 McMillan G. R. 190 MacMillan J. 439 McMurry J. E. 264 275 419 McNab C. A. 472 McNaughton G. S. 226 McNeil D. W. 166 McNew W. E. 176 MacNicol D. D. 51 MacNicol P. K. 579 McPartlin M. 494 McPhail A. T. 48 60,62,307 41 7,470 Macomber R.,123 124 McOmie J. F. W. 94 372 McQuillin F. J. 268 MacRae D. 309 MacRae D. M. 308 Macrosson W. D. K. 53,402 McVicker G. B. 287 Madlung C. 522 Madsen J. O. 14 Maeda H. 514 Maeda K. 55,530 Magalhaes Alves H. 37 Magee P. N. 539 Magid H. 190 Maginuson V. R. 304 Magos L. 283 Maheshwari K. K. 591 Mahone L. G. 134 Mahta G. 421 Maienhofer A, 519 Maier D. P. 293 Maier G. 481 Maier R. 524 Mairanovskii V. G. 259 Mair G.A. 606 Maire J. C. 314 Maisch H. 294 Maisey R. F. 277 Maitlis P. 332 Maitlis P. M. 331 343 401 Majerski Z. 262 351 399 Majeski E. J. 247 Makaiyuma T. 520 Makaminami G. 368 Makarova L. G. 283 Author Index Maki A. H. 20 32 Malek G. 520 Milek J. 262 Malhotra S. K. 275 380,433 Malina Yu. F. 89 Mallon B. J. 379 382 Mallory F. B. 592 Malpam J. R. 483 Malpass J. R. 197,204,483 Malrieu J.-P. 17 Malter M. Q. 113 Maltsev V. A, 314 Maltseva E. N. 316 Mamantov A. 384 Mammi M. 57,318 Mandanas B. Y. 277 Mandel M. 569 Mandelbaum A, 10 Mandeles S. 560 Mandolini L. 96 Manecke G. 152,514 Manecke Van G. 522 Mangini A. 25 Manhas M. S. 211,447 Mani I. 223 Mani J. C. 198,218 Mania D. 447 530 Mann C.K. 243,258 Manning A. R.,313 Manning J. M. 523 Manning M. 527 Manning T. D. R. 431 Mannschreck A. 339 ManojloviE L. 42 Manser G. E. 79 87 Mansfeld G. W. H. A. 509 Mansfield J. R. 233 234 Mansfield K. T. 400,401 Manske R. H. F. 489,495 Manuer J. A, 177 Manulkin S. M 318 Marchese G. 130 Marchiori F. 509 526 Marcker K. A. 563 564 565 Marcks C. 424 Marcoux L. S. 249 Marcus M. F. 248 Marcus N. L. 271 Marcus R. A. J. 229 Marekov N. L. 491 Mares F. 132 134 348 Mares-Guia M. 624 Maretina I. A, 316 Marfey P. S. 635 Margerison D. 287 Margulis T. N. 49 Mariani C. 394 Marino J. P. 165 Mark H. 211 Mark H. B. 252 Markby R. E. 251 Markham K. R. 37 Markl G. 319 Markland F. S. 625 Markley J.L. 637 Marko L. 327 Marlborough D. I. 522 Marmur J. 560 570 Marolewski T. A, 385 Marple L. W. 260 Marples B. A, 356,436 Marr D. H. 533 Marsh M. M. 600 Marsh S. 593 Marshall J. A. 190 303 392 402,418,419,421 Marshall J. L. 120. 128. 386 Marshall D. R. 95 Marshall T. H. 622 Martin A. 423 Martin E. J. 380 Martin H.-D. 396 Martin J. 402 Martin J. A. 581 Martin J. C. 175 176 177 263 Martin P. K. 458 Martin R. B. 533 Martin R. H. 37 339 354,377 Martin R. J. L. 380 Martinelli J. E. 442 Martinson P. 343 Mirton J. 343 Martuscelli E. 58 Martynov V. A. 519 Martynov V. F. 527 Maruishi T. 424 Marumoto R. 479 Maruyama K. 177,236 Maruyama M. 520 Maruyama T. 31 33 Marx G. S.597 Man M. 12 Masaki N. 504 Masaki Y. 493 Masamune S. 196 395 399 Maslen E. N. 61 65 Maslowsky E. 291 Mason J. 36 377 Mason R. 321,333,494 Mason S. F. 36 377 Masse J. 309 31 1 Massey A. G. 284,356 Massey V. 216 Massingill J. L. 231 Masson J. C. 315 Masthoff R 289,290 Masui M. 29 255 Mataga N. 33 Mateescu G. D. 341 387 Matelas R.I. 598 Mathew K. K. 268 Mathew M. 47 Mathews H. R.,458 Mathias A. P. 631 634 635 Mathieson A. M. 65 MathieSon A. McL. 47 65 495 Mathieu J. 265 Mathur P. B. 260 Matic R 184 Matsubara S. 454 Matsuda S. 312,316 Matsueda R. 520 Matsui M. 426 Matsui N. 316 Matsumoto J. 464 Matsumoto M. 468 Matsumoto S. 421 Matsumoto T. 421 Matsumura H. 498 Matsumura I.69 141 Matsumura Y. 242 Matsuno M. 311 Matsuoka K. 244 Matsuoka T. 244 Matsushima H. 220 Matsuura A. 443 Matsuura T. 187 220 412 417 Matt J. W. 176 Matteson D. S. 283 293 297 Matthews B. W. 602 612 624 Matthews C. N. 514 Mattison P. 36 Matveeva Z. A. 302 Matyska B. 159 Mauger A. B. 523 Maugh T. 260 Maumy M. 171,178 Maurer K.-H. 15 Maurey M. 249 Mayell J. S.,249 Mayer C. 504 Mayer F. 512 Mayer K. K. 214 Mayer R 15 Mayers G. L. 523 Mayers R. F. 42 Mayo F. R. 171 Mayor H. D. 568 Mazeline C. 105 Mazur R. H. 526 Mazur Y.,281,434 Meaburn G. M. 223 Mead W. L. 16 Meadows D. H. 637 MeaKins G. D. 381 Means G. E. 515 Mease A. D. 128,349 Medema D. 328 Medina J.D. 499 Author Index 665 Medina M. V. 491 Miginiac P. 286 Medinger T. 330 Mihailovic M. L. 184 Medof. M. E.. 176 Mijovic M. V.,209 Medzihradszky K. 509 Mikhailov B. M. 293 302 Meek J. S.,345 303 Mehta G. 420 Mikhailova L. N. 302 Meienhofer J. 527 531 Mile B. 30 Meinwald J. 200 270 392 Miles D. W. 40 414 415,435 Miles F. B. 69 120 139 Meinwald Y. C. 415 Miles H. T. 536 537 Mei-Sie Lin 495 Milewich L. 37 Meislich M. 343 Milkowski J. D. 519 Mela L. 601 Miller A. W. 519 Melchiar M.T. 271 Millar I. T. 350 Melillo J. T. 38 Miller B. 351 Melloni G. 358 Miller C. G. 622 Mellows G. 267 592 Miller D. B. 307 Meloun B. 612 Miller D. W. 14 Melville D. B. 512 Miller E. C. 539 Melville R.D. 62 Miller F. W. 110 Menapace M. R,323 Miller H.E. 417 Mendelsohn J.-C. 312 Miller J. A. 539 Mendelson W. L. 218 Miller J. J. 293 Mendive J. J. 525 Miller M. A, 380 Menger F. M. 604 Miller N. 538 547 Menon N. A. 326 Miller P. A. 598 Menzel H. 31 1 Miller R E. 312 Merenyi R.,394 Miller R. G. 330,400 Merk W. 104,341 Miller S. I. 109 143 171 451 Merrifield R.B. 509 527 Millington D. S. 509 517 Merritt F. 141 Mills D. R.,560 Merzoni S. 328 Mills 0.S. 48 165 Meske-Schuller J. 403 Milman G. 551 Messer W. R.,215,460 Milne G. 598 Messmer A, 97 Milne G. W. A, 14 Mesure A. D. 79,87 Milne H. B. 521 Metler T. 451 Milner D. L. 337 Metras F. 312 Milovanovic A. 620 Metzger H. P. 615 Milstien J. B. 77 Metzger J. 181 Minami H. 526 Metzner W. 198 Minas H. 371 Meyer H. 316 Minato H.417,418 Meyer J. D. 466 Mincione E. 428 Meyer W. C. 447 Mingaleva K. S. 314 Meyer W. L. 424 Minisci F. 178 Meyers A. I. 264 Minkin J. A. 43 57 Meyers T. J. 359 386 Miocque M. 280 Meyerson C. 12 Mironov V. A 398 Meyerson S. 11 14 133 Mironov V. F. 283 310 312 140 Mirrington R N. 423 Michael B. D. 224 225 226 Mirzabekov A. D. 564 Michaels R J. 349 Mishra A. 168,206 Michalovic J. 257 Mislow K. 38 Michejda C. J. 460 Misono A. 251 329,331,332 Michel H. O. 616 Mitani M. 271 Michelson A. M. 539 Mitchell E. D. 14 Michielli R.F. 254 Mitchell G. H. 377 Michniewicz J. J. 545 Mitchell P. R. 512 513 Mickewich D. 31 Mitchell R.H. 368 370 Middleton E. J. 439 Mitchell R.W. 402 Miesel J. L. 215,460 Mitscher L. A, 37 Mitsuhashi T.435 Mitsui R. 37 Miura K. 570 Miura T. 529 Miwa T. 454,487 Miyake A. 261,326 337 Miyashi T. 211 316 Miyazaki H. 256 Miyazaki M. 432 562 Mizoguchi T. 519 Mizuta E. 216 Mizuta T. 326 Mizusaki S. 490 Mizuiani T. 562 Mo F. 44 581 Mobius K. 22,26 Mochida I. 140 Mock W. L. 55 Modena G. 80,130 Modest E. J. 484 Modro T. A. 91 Moedritzer K. 284,312 Moffat J. 331 401 Moffatt J. G. 541 Mohilner D. H. 251 Moissieva S. 130 Mol J. C. 323 Mole T. 304,305 Molin Yu N. 27,183 Molinaro M. 538 Molloy B. B. 486 Molodtsov M. V. 470 Mondelli G. 328 Mondelli R. 37,496 Mongrain M. 418 Monier R 552 Monro R. E. 564 Monroe P. A. 345 Montaigne 386 Montana A. F. 22 375 Montanari F. 80 132 266 44 1 Montelatici S.325 Montgomery L. K. 148 149 176,387 Moodie R B. 67 68,91 93 Moon A. Y. 616 Mooney E.F. 314 Moore D. 356 Moore J. A. 483 Moore R. 206 Moore R H. 257 Moore S. 523 634 Moore W. R 191 Mootz D. 50 Moppett C. E. 277 389,596 Moran M. D. 392 Morand P. 430 MorBvek J. 536 Moravek L. 612 Morcom K. W. 260 Author Index Moreland C. G. 319 Morell P. 560 Moretti I. 441 Morgan C. H. 60 Morgan G. L. 287 Morgan K. J. 348 Mori H. 439 Mori K. 426 Moriconi E. J. 447 Morifuji K. 329 Morikawa M. 147 Morin R B. 406 Morisaki M. 65,426 Morita A. 97 Morita K. 216,479 Moritani I. 137 Moriyama H. 410,438 Morley J. R. 267 Morley J. S. 525 Morlino V. J. 533 Morocova I.D. 94 Moroder L. 509,526 Morozova I. D. 171 Morozowich W. 71 Morreal C. E. 539 Morrell M. L. 21 Morris D. G. 52 Morris J. W. 99 Morris M. D. 235 Morns P. J. 311 Morris R. A. N. 250 Morris S. J. 565 Morrison H. 188 202 217 540 Morrison R. T. 181 Morrison W. H. tert. 221 Morrow T. 227 Morse R L. 202 Moscowitz A. 36 39 Moskowitz M. 280 Moseley P. T. 288 Moser C. 490 Moser J.-F.,200 Moser P. 40 Moser R E. 514 Mosher H. S. 140,289 Mosher W. A. 616 Moshuk G. 21 Mosler W. A 82 Mosley K. 401 Moss G. P. 586 Moss R A. 117 164 384 Mosselman C. 381 Most C. F.,jun. 521 Moulijn J. A. 323 Mourning M. C. 139 140 Mousseron-Canet M.,218,436 Mousset S. 569 Mowery P. C. 372 Moyer C.E. 348 Moyer C. L. 445 Moyce D. S. 141 Mtol O. 417 Muchin G. 130 Muck D. L. 141 Mudd A. 439 Muhlstadt M. 187 Miiller E. 352 493 Miiller F. 509 Miiller J. 304 Miiller L. P. 510 Mueller R A. 406 Miiller W. 575 Mueller W. J. 103 Muensch H. 339 Muetterties E. L. 301 Muhlstadt M. 382 Mui J. Y.-P. 291 Muir D.M. 11 3 Muir K. W. 48 Muirhead H. 632 Muju N. L. 260 Mukai T. 211,399 Mukaibo T. 33 Mukaiyama T. 274,455 Mukki K. 71 Mulder C. 569 Mulheirn L. J. 599 Muller E. 384 Muller L. L. 187 Muller P. 384 Mulligan L.A. 95 Mulligan P. J. 364 Mullineaux R. D. 326 Mullins M. A. 314 Munday R. 374 Mundy D. 38 Muneyaki R 122 Munro M. H. G. 495 Munson P. L. 524 Murachi T. 631 Murai S.312 329 Murakami K. 177,236 Murakami M. 464,522 Muramatsu I. 509 531 Muramoto N. 174 Muraoka Y.,55 Murase K. 464 Murata I. 392 Murov S. 189 Murov S. L. 189 Murphy J. W. 438 Murphy T. J. 212 401 Murphy W. S. 482 Murr B. L. 103 Murray A. W. 542 Murray J. J. 377 Murray R. A. 374 Murray R D. H. 423 Murray R K. 344,374 Mutray R. K. jun. 203,204 Murray R. W. 218 Murrell J. N. 18 Murrell L. L. 291 Musgrave W. K. R. 480 Muskatirovic M. 140 Musso H. 37 Muth E. F. 442 Muto H. 255 Muto S. 354 Myatt I. 29 250 Myer Y. P. 39 Myers A. I. 464,468 Myers C. 31 Myers L. S. jun. 229 Myhre P. C. 93 344 346 Mylonakis S. G. 175 Mylori B. L. 176 Nabika K. 316 Nace H. R 133 Nadean R.G. 589 Naemura K. 37 391 Nagabhusan N. 623 Nagai T. 45 1 Nagarajan R. 486,600 Nagase S. 240 Nagata W. 273 500 Nagaura S. 240 Nahringbauer I. 42 Naidya V. M. 522 Nair V. 442 Naito T. 458 464 Nakabayashi N. 428 Nakadaira Y. 218,439 Nakagawa M. 368 Nakagawa S. 458 Nakai T. 566 Nakajima T. 526 Nakamaye K. L. 380 Nakamizu H. 526 Nakamoto K. 291 Nakamura A, 533 Nakamura H. 55 Nakamura S. 418,530 Nakane R. 496 Nakanishi K. 218 410 438 Nakanishi T. 62 Nakano K. 522 Nakano T. 65 221 345 Nakashima T. 499 Nakata T. 182 Nakatsuji H. 109 Nakatsuji T. 111 Nakatsuka N. 196 395 Nakazaki M. 37 391 Nakazawa Y. 611 Namstvedt J. 294 Naqvi S. M. A, 260 Narang S. A, 545 Narasimhan P.T. 19 20 29 Narayanan K. V 339 Author Index Narayanan V L.. 525 Naser-ud-din. 246 Nash R. 183 Nasielski J. 318 Naso F. 130 Nathan E. C. 103 Nathan E. C. tert. 402 Natatsuka N. 399 Nattaghe A. 129 Naughton M. A. 622 Nauto W. T. 182 340,464 Naya K. 418 Nayak V. R. 421 Neal G. T. 23 Neale R. S. 178 271 Neckers D. C. 176 Neelon F. A. 538 Neet K. E. 602 625 Negishi E. 301 Negoita N. 183 Neher R. 524 Nei M. 537 Neidle S. 41,427 Neilson D. G. 380 Nelson A. D. 108 Nelson L. E. 310 Nelson P. H. 438 Nelson P. J. 189 202 Nelson R F. 22 29 248 249 Nelson V. R. 583 584 Nenitzescu C. D. 341 Nerlekar P. G. 310 Nesmeyanov A. N. 314 317 Nesvadba H. 527 Neta P. 224 Neubauer D. 276,328 Neubert L.A. 38 Neuenschwander M. 386 Neuernschwander P. N. 286 Neugebauer F. A. 27 Neuman R. C. 175 Neumann. W. P. 312 315 Neumuller 0.-A, 187 Neuner-Jehle N. 491 Neunhoeffer O. 518 Neurath H. 612,618,625 Neuss N. 486 600 Neuwirth Z. 619 Neuwirth-Weiss Z. 21 1 Neville D. 575 Neville G. A, 421 Newberg J. H. 254 Newkome G. R. 465 Newlands M. J. 318 Newman B. A. 52 Newman G. A. 209 Newman H. 461 Newman M. S. 164 Newmark R. 547 Nibbering N. M. M. 11 Nichol K. J. 94 Nichol S. L. 256 Nichols J. L. 538 Nicholson H. 25,67 Nickon A. 218 Nicodem D. E. 216 Nicolas M. 253 Niederhauser A. 386 Niehaus A. 313 Nielsen A. T. 272,458 Nielsen N. B. 208 Niemann C. 618 Niemeyer J. 37 Nigam I.C. 421 Nigon V. 551 Niikolaeva N. A, 302 Niizuma S. 27 Nilsson A, 42 Nilsson B. 44 63 Nilsson J. E. 287 Nilsson J. L. G. 579 Nilsson S. 233 246 Nishida S. 421 Nishikawa M. 64 Nishikida K. 256 Nishimoto N. 439 Nishimura J. 384 Nishimura S. 538 564 Nishimura T. 429 Nishinaga T. 504 Nissen A, 369 Kitecki D. E. 523 528 Nitta I. 52 64 129 Nitta T. 392 Nivard R. J. F. 209 Niwa Y. 8 Nixon J. E. 597 Ng M. 229 Noack K. 327 Noda I. 26 Noel Y. 403 Noth H. 293 294. Noguchi M. 510 Nohara A, 536 Nolde C. 13 Nollett A. J. H. 431 Noltes J. G. 283 312 314 318 Nomoto K. 438 Nomura M. 316 Nomura T. 258 Nonaka T. 253 Nordio P. L. 18 Nordman C. E. 62 Norman R. 0.C. 27 32 184 224 Normant H.280 Normatov N. 496 Norris A. R. 171 342 Norten D. G. 129 North A. C. T. 606,611 North A. M. 153 Norton D A, 62 668 Norton K. 588 Nouls J. C. 339 Novikova 0.A. 310 Novkova N. Y. 317 Novodaro G. N. 616 Novotny L. 417 Noyce D. A. 69 Noyce D. S. 139 Noyes W. A. jun. 187 Noyori R. 202,205,444 Nozaki H. 205 387,444 Nozako H. 202 Nozoe S. 65,426 Nozoe T. 363 Nugteren D. H. 406 Nussim M. 434 Nutt R. F. 540 Nutting W. H. 418 Nyberg K. 244,245,246 Nykerk K. N. 305 Nyman C. J. 329 Nyss V. A. 287 Oakenfull D. G. 74 137 Oakes J. 29 30 Oberhansli W. E. 51 Obi K. 310 O’Brien M. H. 319 O’Brien R. E. 176 Occolowitz J. 538 Occolowitz J. L. 16 342 Occolowitz L.V. 135 Ochiai M. 216,479 Ochiai T. 174 Ochoa-Solama A. 81 O’Connel A. M. 42 O’Connor C. 325 Oda R. 112 172 356 386 402 Odan N. 33 Odell B. G. 125 Odell C. 617 O’Donovan D. G. 580 Oelofson W. 526 Oelschlager H. 259 Oesterlin R.,448 O’Ferrall R. A. M. 109 Ofstead E. A. 322 323 Ogasawara K. 469,493 Ogata M. 213,468 Ogata Y. 200 Ogawa S. 439 Ogawa T. 426 573 Ogihara Y.,65 Ogliaruso M. 21 Ogorodnikova N. A. 297 Oh Y. L. 61 Ohashi M. 424 Ohashi T. 271 Oh-Ishi T. 907 Author Index Ohki. E. 443 Ohloff G. 218 413 Ohmori K. 301 Ohnesorge W. E. 247 Ohnishi S. 31 Ohnishi Y. 206 Ohno A. 206 260 Ohno K. 276,328,329 Ohnsorge U. F. W. 427 Ohrt J. M. 62 Ohta M. 442 Ohtsuka E.543 545 Oida S. 443 Oishi T. 262 500 Oishi Y. 256 Ojima J. 368 Oka H. 243 Okabayashi H. 533 Okada M. 526 Okada Y. 526 Okahara M. 271 Okamoto K. 129 Okamoto M. 518 Okamoto Y. 64,346 Okamura W. H. 483 Okazaki K. 386 Okhlobystin 0.Y. 283 284 Okorie D. A, 427 Okorodudu A. 0.M. 164 Oksinoid 0.E. 308 Oktura Y. 464 Okuda I. 591 Okuda M. 27 Okuda S. 426 428 Okuda T. 418 Okumura J. 458 Okumura T. 500 Okuno Y. 487 Okuyama T. 111,141 Olah G. A. 25 103 105 106 107,108,114 341 343 Olah J. A. 343 Olaitin S. A. 625 Olin S. S. 179 410 Olive S. 332 Oliver J. H. 135 Oliver J. P. 304 305 307 Olivera B. M. 570 573 Ollis W. D. 36 37 278 279 357 Olmstead H. D. 381 Olofson R. A. 136 165 Olovsson I.42 olsen B. A, 133 Olson A. J. 62 Olson D. C. 260 Olson G. L. 391,409 Omae I. 312 Ona H. 425 Onak T. 298 Ondetti M. A. 522 525 O’Neal H. E. 379 Ong A. S. H. 178 Onoda R. 418 Ooi T. 534 Oosterhoff L. J. 188 Oppenheimer H. L. 616 Orchin M. 128 325 359 Orezzi P. 37 Orgel L. E. 536 537 Orlando C. M. jun. 21 1 Orloff M. K. 32 Orlova L. V. 24 Ormand K. L. 476 Orr D. E. 505 Ortiz de Montellano P. R. 589,590 Osa T. 251 Osaki K. 64 Osawa E. 392,402 Osawa T. 61 1 Osborn C. L. 210,404 Osborn J. A. 324 325 Oshika T. 323 Oshima T. 518 Osiecki J. H. 27 183 Ota E. 96 0th J. F. M. 365 379 383 394 Ottenheym H. 525 Ottensmeyer F. P. 548 549 Otter B. A, 540 Ottley R.P. 293 Otto A. 459 Otto P. 148,447 Ouaki R 314 Oughton B. M. 530 Ourisson G. 184 418 420 428 Ovchinnikov Yu. A, 517 Overchuk N. A. 343 Overend W. G. 470 Overton K. H. 422,427 Owen D. A, 296 Owen G. S. 346 Owens P. D. 178 Owens R. M. 21 135 Owyang R. 331,387 Oyeda M. 67 Pabon H. J. J. 406 Pacifici J. G. 176 Packter N. M. 598 Paddon-Row M. N. 479 Padwa A. 50,443,444,449 Paecht-Horowitz M. 514 Paes Leme L. A. 500 Paetsch J. 319 Paetzold P. I. 294 Pagni R. M. 194 Pals M. 504 Paknikar S. K. 421 Pakrashi S. C. 489 Paladini A. C. 527 Palazzo G. 343 Paleeva I. E. 290 Palei B. A. 306 Palenik G. J. 45 Palevada W. J. jun. 521 Paliokes A. M. 593 Pallaud R. 253 287 Palm J.H. 381 Palmberg P. F. 91 Palmer K. J. 52 Panetta C. A. 447 Pankova M. 139 Pannell K. H. 110 310 Pant B. C. 310 Paoletti E. G. 592 593 Paoletti R.,592 593 Papouchado L.. 247 Paquette L. A. 57 114 124 197 201 212 352 363 392 450,477,482,483,485 Paradies H. H. 565 Parello J. 504 Pares Z. N. 262 Parish J. H. 11 3 Park A. C. 549 Parker A. J. 128 138,277 Parker L. 615 Parker R. 5 11 Parker V. F. 136 139 241 242,244,246 Parker W. 114,402 Parkes C. O. 623 Parkham W. E. 11 1 Parkin D. C. 88 Parkin J. E. 463 Parkin J. G. 240 Parkins A. W. 337 Parks P. C. 612 Parky J. Z. 333 Parrish F. W. 472 473 Parrish R. V. 314 Parry D. 146 Parshall G.,337 Parsons. P. G. 497 582 Parsons S. M. 611 Partchanazad I.289 Partridge J. J. 419 Pasedach F. 214 Pashayan D. 449 Pashegorova V. S. 398 Pasquinelli E. A, 236 Passannanti S. 426 Pasto D. J. 263 301 381 Pasynkiewicz S. 307 Patchornik A. 513 525,615 Patel D. J. 203 Pattenden G. 277 Patterson A. L. 43 57 Author Index Patterson J. M. 452 Patterson J. W. 419 Pattison I. 302 307 Patton W. 522 Paul I. C. 53 56 57 62 293 451,482 Paul R. 524 Paulay Z. 509 Pauli R.M. 570 Paulson D. R. 400 Paulson H. 39 Paulsen H. 472 473 Paulus E. F. 313 Paust J. 406 Pavan M. V. 18 Pawson B. A,. 339 340 382 415 Payling D. W. 9 13 91 303 Payne N. C. 513 Pazdro K. M. 249 Peacocke A. R. 574 Peagram M. J. 270 Pearce C. A, 312 Pearce P. J. 89 Pearson J.M. 175 Pearson R.L. 93 343 Pecci G. 260 Pechet M. M. 176,344 Pecoraro R. 609 Peddle G. J. D. 309,314 Peddle G. J. G. 313 Pedersen C. 472 Pedone C. 58 Pedulli G. F. 25 Peerdeman A. F. 44 Peet J. H. 128 Pehk T. 109 Peiffer R. 188 Pelizza G. 448 Pelletier S. W. 421 Pellicciani R. 427 Pelter A, 476 Penczek S. 109 Pene J. J. 551 Penfold B. R. 313 Peng C. T. 227 Penkowski N. J. 130 Pennella F. 323 Penrose A. B. 402 Percy R. K. 381 Pereira J. L. C. 176 431 Pereyre M. 316 Perkampus H. 105 Perkins M. J. 171. 181 183 187 Perkins P. G. 293 Perlman D. 531 Perner D. 223 Perry T. 5 11 Perst H. 204 Pesaro M. 418 Peshet M. M. 272 Pete J.-P. 189 Peterkofsky A 538 Peters R.C. H. 118 Petersen H. jun. 18 Petersen R. C. 240 246 249 Peterson M. L. 231 Peterson P. E. 106 Petke J. D. 15 Petra P. H. 628 Petrellis P. 163 Petrie G. 247 Petrorca A. E. 268 Petrov A. A, 314 315 316 Petrovich J. P. 252 348 Petrowskii G. 21 Petrzilka T. 207 Pettersen R. C. 61 66 163 220 Pettit R. 104 341 359 Pfeffer P. 499 Pfeffer P. E. 395 399 Pfeifer S. 493 Pfeiffer J. G. 118 380 Pfenninger E. 437 Pfister J. 434 Pfoertner K. 219 Pfundt G. 21 1 Philbin E. M. 87 Philippossian G. 191 Philips. J. C. 485 Phillips B. 539 Phillips D. 187 Phillips D. C. 606 Phillips D. R. 82 Phillipson J. D. 500 Piatak D. M. 36 Pickard A. L. 307 333 Pickles V. A, 97 Picot F. 504 Pierce A.E. 284 Pierce J. B. 262 285 Pierre G.. 240 Picrs E. 261 117,118 Piers K. 445 Pietra F. 100 Pietta P. 519 520 Pietta P. G. 509 Pifferi G. 448 Pike J. E. 406 596 Pike M. T. 418 Piko L. 573 Pilar F. L. 141 Pilling R.L. 296 Pilloni G. 318 Pincock R. E. 172 173 Pines A. 384 Pinhey J. T. 204,210 437 Pinke P. A. 400 Pinkus A. J. 339 Piozzi F. 426,496 Pippert D. L. 104 Pirkle W. H. 14 213 348 385 Pistoia G. 260 Piszkiewicz D. 71 72 126 610,616 Pitea D. 130 Pitt C. G. 177 311 Pitts A. D. 296 Pitts J. N. jun. 198 201 Plackett J. D. 278 357 Plackett L. D. 411 Plank D. A. 210 Plate N. A. 314 Plato M. 22 Platt R. H. 314 Pleau J.-M. 287 Pleiss M. G. 483 PleSek J. 297 Pless J.524 Pletcher T. C. 78 Pliske T. E. 414 Ploss G. 362 Plume G. 526 Pluscec J. 522 Poddubnaya N. A, 528 Podmore W. D. 405 PoduSka K. 514 Podvisotskaya L. S. 297 Poel D. E. 437 Pointer D. J. 426 Poist J. E. 310 Pojarlieff I. 97 Pokhodenko V. D. 171 Polgar L. 625 Polgar N. 430 Pollack G. E. 514 523 Pollack R. M. 69 Pollak P. I. 461 Pollet R. 89 Pollitt F. J. 98 Pollock J. J. 606 Polston N. L. 271 301 Pomerantz M. 193 213 356 379 397,400 Ponce C. 219 Ponder B. W. 272 Ponnamperuma C. 536 Ponsold K. 39 Ponticello G. S. 486 Ponticello I. S. 477 Popjak G. 585 Pople J. A. 19 Popov S. S. 491 Popp F. D. 252,465 Popper T. L. 265 PoppIeton B. J. 47 Porter G. 355 Porter J.W. 597 Porter N. A. 174 206 Porter P. N.. 493 Author Index Porter Q. N ,438 Porter T. H. 512 Portier C. 551 Posler J. 424 Posner G. H. 277 Possinet G. 428 Post B. 50 Post E. W. 302 Post H. W. 318 Potier P. 275 Potts J. T. jun. 524 Potts K. T. 459 Poulter C. D. 113 Poulter S. R. 262 Pound A. W. 510 Poupart J. 391 Poupko R.. 30 Povarnitsyna T. N. 288 310 Powell J. 330 Powell J. T. 221 389 Powell P. 283 Powell R. E. 16 Powell-Wiffen J. W. 162 Prabhakar S. 421 Pradham S. K. 177 529 Prager R. H. 96 Pragst F. 247 Prakash D. 429 Prasad N. 471 Pratt A. C. 413 Pratt C. S. 605 Pratt D. G. 112 Pratt J. M. 259 Pratt R. F. 81 Prelog V. 35 382 PremuziC E. 265 Previero A. 513 Price C.C. 539 Price R. 356 Price S. J. W. 307 Prihar H. S. 473 Prince R. H. 309 Prince S. R. 294 Pring B. G. 350 Pritchett R. J. 28 Prinzbach H. 191 352 363 384 387. 396,452,486 Prochazka M. 172 Proctor G. R. 364 482 Prodayko L. A. 332 355 Prokai B. 317 Prokofiev A. K. 314 Protsenko N. P. 316 Prox A. 525 Pruett R L. 331 Priitz W. 226 Prusik Z. 612 Prutton I. 551 Pryor W. A. 171 175 Przybyla J. R. 384 Przybylska. M.. 62 65 Puddephatt R. J 314 318 Puddussery R. G. 461 Puliti R. 60 Pungor E. 259 Purmort J. I. 284 Puty G. R. 597 Pyl T. 458 Quane D. 312 Quiocho F. A. 605 632 Qureshi A. A, 501 583 584 Qureshi A. K. 461 Qureshi E. A. 93 Raacke I. D. 552 Rabideau P. W. 195 356 Rabiman W.476 Rabin B. R. 631 634 635 Rabinovitch B. S. 162 Rabinovitz M. 362 Racky W. 522 Radecka C. 421 Radlick P. 184 218 236 271 398 Radunz H. 498 Rafferty M. A. 606 610 611 Rahimtala A. D. 593 Rahman A, 360 Rahman M. 25 Rahn D. 11 7 Raj Bhandary U. L. 538 563 565 Rakita P. E. 308 Ralph R. K. 537 Ramachandran G. N. 534 Ramm P. J. 592 Ramp F. L. 146 Rampal A. L. 439 Ramsden C. A. 459 Ramsey J. S. 256 Ranby B. 31 Rand L. 238 Randall E. W. 313 RandiC M. 351 Rand-Meir T . 606,610 Rando R R. 201 Rao A. S.,37 Rao K. V. J. 498 Rao P. V. S. 498 Raphael R. A. 51 Rapoport H. 418,458,529 Rappe C. 135 Rappoport Z. 138 Rasmussen H. 524 Rasmussen S. E. 57 Rassat A. 27 Ratajczak A.118 Ratajczak E. 397 Ratcliffe B E ,397 Rathke M. W. 273 274 276 280 300 301 Rathousky J. 284 Ratti E. 51 1 Rattle. G. 504 Rattle H. W. E. 525 Rau J. 539 Rauh R. D. 188,198 Rautenstrauch V. 365 Ravet J. P. 27 Rawson J. R. 551 Ray G. J. 109 Ray N. K. 19,29 Rayner D. R. 38,282 Rayn-Jonsen E. J. 61 Razuvaev G. A. 283 Rea E. J. F. 254 Read G. 597 Read J. M. 341 Readhead M. J. 264 Readio P. D. 177 Reanney D. C. 537 Reavill R. E. 621 Records R. 40 364 365 Reddoch A. H. 22 Reddy T. B. 243 Redhouse A. D. 165 Redl G.. 3 13 Redman B. T. 37 377,476 Redmond J. W. 168 Redwood M. E. 288 Reece C. A. 412 Reed R. I. 15 Reeke G. N. 605,632 Reeke G. N. jun. 65 Rees,C. W.169 171 178 179 187,321,350,355 Rees D. P. 208 Rees H. H. 592 Reese C. B. 394 542 543 Reeve W. 129 Regan T. H. 293 Regnier F. E. 585 Rehberg R. 123 Rei M. 121 Reich E. 574 575 Reich H. J. 353 Reich H. T. 95 Reichardt. C. 150 Reichenbacher P. H .]lo,234 235,238 Reichmann M. E. 560 Reichstein T. 62 Reid B. R. 538 Reid C. 536 Reid. D H . 462 Reid D S. 260 Reid K. B. M. 527 Reid S. T. 187 Reiff H. 273 Reiff H. F. 310 Reimann H. 471 Reimlinger H. 442 Author Index Reinecke. M. G. 358,456 Reiner R. 448 Reinhardt H. G. 397 Reinheimer H. 331,401 Reinhold V. N. 512 Reintjes M. 296 Reis H. 328 Reissmann S. 518 Reist E. J. 540 Reitz R. L. 275 Rejoan A. 261 Rejoan A. J. 326 Remko J.R. 33 Rempp P. 171 Renard M. 81 Renfrew A. H. 364 Renfroe H. B. 354 Renfrow A. 372 Renfrow W. B. 372 Rensfeld R. A. 524 Renwick J. D. 39 Respess W. L. 288 Ressler C. 512 Reusch W. 36 177 197,413 Reutov 0.A. 283,292 Rewicki D. 131 Rezvukhin A, 109 Rezvukhin A. I. 370 Rey M. 399 Reynier M. 552 Reynolds W. F. 339 Rhind-Tutt A. J. 610 Rhoades M. 570 Rhodes C. A. 37 Rhodes Y.E. 117 Ricard M. 373 Ricciardi R J. 449 Rice S. N. 168 Rich A. 548 565 Richards E. E. 381 Richards E. G. 552 Richards F. M. 634 Richards J. H. 110,621 Richards K. E. 413 Richards R. L. 332 Richards R. W. 587 Richardson A. C. 264 Richardson D. 188 Richardson K. 453 Richey H. G. 391 Richmond G. D.400,401 Richmond R R. 265,326 Richter W. 10 Richter W. J. 12 311 590 Richtol H. H. 244 Rick E. A. 404 Rickborn B. 381 Ridd J. H. 91 Riddell W. D. 463 Ridley D. 288 Riebel H. J. 395 671 Ried W. 342. 360 Riedel A. 165 Rieke D. 14 Rieke R 21 Rieker A. 339,493 Hi,M. R 256 Rigau J. J. 177 Rigaudy J. 213,219,373 Riggs J. I. 339 Riley J. F. 223 Riley T. 74 137 Rilling H. C. 589 Rinehart Z. L. 907 Rinehart K. L. jun. It 12 Ring D. F. 162 Rinicker B. 524 Riordan J. F. 632,634 Ripamonti A. 60 Ripley R. A. 212 Ripoll J.-L. 435 Risinger G. E. 589 Ritchie A. B. 40 Ritchie C. D. 68 Ritscher J. S. 207 Rittel W. 533 Ritter A. 221 309 Ritter J. J. 294 Riva di Sanseverino L. 62 Rivero J.M. 28 Rivier J. 387 Rizvi S.H. 47 Ro K. 418 Roark D. N. 309 Robert-Lopes M. T. 15 Roberts B. P. 303 Roberts B. W. 51,382 Roberts D. L. 415 Roberts E. V. E. 472 Roberts H. L. 512 Roberts J. D.. 176. 360 380 478 Roberts J. P. 228 Roberts R J. 377 Roberts R M. G. 317,318 Roberts S.M. 467 Robertson A. V. 12 512 Robertson G. B. 333 Robertson J. M. 56 Robertson R E. 129 Robins M. J. 40 Robins R.K. 40,541 Robinson B. 501 Robinson C. H. 37 Robinson D. A. 620 Robinson D. J. 51 Robinson D. R. 78 Robinson L. 68 137 Robinson M. J. T. 462 Roblin R 560 Robson A, 179 Robson J. H. 165 Robson R. 472 Rocchi R 509,517,526 Rocek J. 384 Rochev V. Y. 314 Rockett B. W. 128 Rodda H.A. 14 Rodde A. F.,jun. 226 Rodgers J. E. 381 Rodgers M. A. J. 228 Rodgers M. G. 89,136 Rodgers P. G. 128 Rodin J. O. 412 Rodin R L. 518 Rodionov A. N. 290 Rodricks J. V. 448 Rodriguez H. R. 907 Roe R-J. 44 Roe D. K. 260 RoepstoB P. 509 Rogers D. 41,60 427 Rogers G. T. 348 Rogers I. H. 429 Rogers J. W. 24 Rogers N. H. 509 Rogg H. 563 Rogic M. M. 273 274 276 280 300 301 Rogozev B. I. 314 Rohrer D. 57 Rois H. 276 Rokhlina E. M. 314 Rolle F. R. 92 Rolston J. H. 173 Roman S. 415 Roman S. A. 269 Rombauts W. A. 525 Romers C. 54,62 Rsmming C. 49 Romo J. 419 Romo de Vivar A. 419 Romstead L. R. 81 Rona P. 277 Ronayne J. 10 Ronwin E. 478 Rony P. R. 81 Roos K. 499 Rosati R.L. 500 Rosen M. 450 Rosenberg E. 314 Rosenblatt D. H. 265 Rosenblatt P. H. 326 Rosenkranz H. J. 221,460 Rosenman H. 329 Rosenmund P. 499 Rosenstein R D. 59 Rosenthal I. 217 Rosich R S. 422 Rosik J. 470 Rosmus P. 15 Ross A. E. 539 Ross C. A. 634 Author Index Ross R. A, 128 Ross S. D. 101,240,246,249 Rossi G. L. 622 Roth W. R. 192 Rothberg I. 121 Rothe M. 528,530 Rothenwohrer W. 402 Rothman A. M. 391 Rothstein E. 112 Rottele H. 394 Rottenberg F. 97 Roussi G. 270 Row L. R. 498 Rowe J. J. M. 267 Rowe J. L. 242 Rowland F. S.,163 Rowley P. J. 176 Rownd R. 569 Roy D. N. 459 Roy J. 347 Roy N. 71 Roy S.B. 464 Royer G. 620 Royo G. 309 Rozantsev E. G. 33 Rozynov B.V. 5 17 Ruane M. 138 Rubin M. B. 197 211,395 Rubinchik G. F. 318 Ruby W. R. 260 Ruchkov V. A. 528 Ruden R A 208 Rudinger J. 514,527 528 Riichardt C. 181 Riihlmann K. 309,511 Rummel S. 175 Rumpf P. 139 Rundel W. 339 Runner C. M. 570 Rupilius W. 327 Rupley J. A. 606 607,609,610 Rupp W. D. 547 Ruppert D. 370,391 Rura A. 514 Rush J. E. 268 Russell G. A, 17 27 30 31 135 171 184 381,387 Russell G. B. 38 Russell J. D. 115 Russell K. E. 171 342 Ruterjans H. H ,637 Ruttenberg G. J. C. M. 570 571 Rutter A. W. 30 Ruzicka L. 590 Ryan K. J. 472 Ryba O. 172 Rychnovsky V.,54 Rydon H. N. 519 Ryle A. P. 612 Ryrberg K. J. 499 Ryter A. 566 Ryvolovh-Kejharova A, 252 Rzezotarska B. 509 518 Sacco A.337 Sachetto J. P. 471 Sachs W. H. 135 Sabacky M. J. 261,326 Sabo F. F. 525 Saboz J. A. 200 Sadek H. 68 Sadler J. L. 256 Saeed M. A. 38 Saeed S. A. 472 Saeki Y. 65 Saenger W. 565 Saeva F. D. 38 Saffhill R. 542 Sagdeev R Z. 27 183 Saito I. 220 Saito K. 455 Saito T. 329 Sajus L. 325 Sakabe N. 46,59 Sakai M. 109 Sakai R. 534 Sakakibara J. 489 Sakakibara S. 526,533,534 Sakamoto H. 220 Sakan F. 421 Sakan T. 425,454 Sakatani Y. 493 Sakuma H. 510 Sakuma R. 421 Sakurai H. 26 171 308 309 311 Sakurai T. 60 Salem L. 131 143 Sallay S. I. 500 Salmon G. A. 227 Salton M. R. J. 606 Samejima H. 326 Samkoff N. 181 Sammelsson B. 596 Sammes P. G. 221 345,491 Samochocka K.514 Samokhvalov G. I. 259 Sams J. R. 314 Sanchez R. A, 536 Sanchez del Olmo V. 95 Sanford E. 176,405 Sanford E. C. 344 Sandberg F.,499 Sandel V. R. 132 Sanders J. R jun. 288 Sandhagen H. J. 403 Sandner M. R. 209 Sando K. M. 19 Sandosham J. 360 Sandrin Ed. 524 Sands B. W. 31 1 Author Index Sandstrom J. 461 Sanger F. 552,622 S ankey G. H. 471 Sanno Y. 536 Sano H. 314 Sano M. 464,540 Sano Y. 527 Santavy F. 493,495 Santelli M. 124 Santhanakrishran T. S. 406 Santhanam K. S. V. 259 Santoro A. V. 379 Santos E. 39 513 Santry D. P. 19 Santurbano B. 427 Sarantakis D. 515 Sarathy K. P. 534 Sarel S. 379 Sarfati R. 504 Sargeant P. B. 177 Sargent G. D. 118 Sargent M. V.367 Sargent S. D. 173 Sarishvilli I. G. 284 Sarma V. R. 606 Sarre 0. Z. 471 Sasada Y. 45,48 Sasaki K. 240 Sasaki T. 110,405,481 Sass R. L. 54 Sasse W. H. F. 195 Satchell D. P. N. 80 81 Sato K. 475 Sato N. 233 Sato S. 55,441 442 Sato T.,209 354 Sato Y.:428 591 Satoh F. 494,495 Saucy G. 415 Sauer J. 145 147,214 Sauer M. C. jun. 223 Sauerbier W. 547 Sauers R. R.,192 399 Saunders B. C. 98 Saunders D. A, 304 Saunders J. K. 304 305,495 Saunders M. 20 108 Saunders M. D. 71 Saunders V. R. 36 Saunders W. H.,jun. 221 Saurborn E. 203 Savedoff,L. G. 128 Savina L. A, 297 Sawa Y.-K. 36,498 Sawada Y. 529 Sawhney R. S. 489 Sawai M. 439 Sawyer W. H. 527 Sax M. 41 Sayer J. M. 510 Sayigh A.A. R. 147,449 Sayo H. 29,255 Scala A. 592 593 Scala F. 577 Scaletti J. V. 570 Scamehorn R G. 133,381 Scanio C. J. V. 402 Scanlon B. 267 Scarpa J. S. 42q Scatturin A. 509 Scerbo L. 392 Schaags A. P. 97 Schaap A. P. 456 Schade G. 218 Schaefer J. P. 44 55 392 Schaefer T. 339 Schaeffer R. 293,294 Schafer D. J. 509 Schafer L. 381 Schaffer G. W. 203 Schaffner,K. 36 200,210 43 7 Schally A. V.,526 Schanzer W. 231 Schappele S. E. Scharf D. J. 477 Scharf R. 509,525 Schatz P. N. 40 Schawartz J. 512 Schechter I. 628 632 Scheer W. 146 Schemer I. E. 569 570 Scheiner P. 174 206,442 Scheinmann F. 350 Scheit K.-H.,543 Schell P. 548 Schellman J. A, 35 548 Schenck G. O. 187 Schenck H.U. 152 Schenk H. 385 Scheppele S. E. 175 Scheppers G. 347 Scheraga H. A, 534,637 Scherer H. 313 Scherer K. V. 359 Scherer K. V. jun. 386 Scherer 0.J. 283 Scheufele D. S. 230 Schexnayder D. A, 456 Schiavilli M. D. 69 Schifer H. 240 Schimmel P. R 533 Schimpf R. 329 343 Schindel W. G. 173 Schkrob A. M. 528 Schlessinger R H. 477. 486 504 Schleyer P.v R. 113 114 118 168 221 262 392 394 399 Schlimme E. 565 566 Schlogl K. 340 Schlosberg R. 107 Schlosberg R. H. 344 Schlosberg 8. B. 399 Schlosser M. 140 Schlueter R. 524 Schmeisser M. 312 Schmid G. 294 Schmid H. 37 215 221 350 351,460,489,499 504 Schmid J. 519,525 Schmidbaur H. 304 318 319 Schmidt D. 79 Schmidt G. 385 509 Schmidt M. 302 318 Schmieder W.P. 406 Schmiegel,W. W. 424 Schmir G. L. 77 Schmitz E. 163 Schmolke B. 355 Schnabel E. 509 518 Schneider C. 226 Schneider G. 543 Schneider H.-J. 141 Schneider J. J. 439 Schneider W. P. 266 596 Schneider-Bernloehr H. 537 Schonherr H.-J. 292 Schollkopf U. 271 Schoellman G. 618 Schoen L. J. 167 Schonberg A, 187 Schoenewaldt E. F. 521 Schofield K. 91,93,493 Scholer F. R. 298 Scholes F.,229 Scholl H.-J. 365 Schollkopf U. 379 Scholz K.-H.,211 Scholz M. 187 452 Schomburg G. 298 Schonberg A. 477 Schook W. 22 Schooley D. A. 38 40 364 365 Schowen R. L. 76 137 Schrank B. 515 Schrauzer G. N. 321 379 Schreiber J. 445 Schrock R. R. 201 Schroder E. 526 Schroeder G. 394 Schroeder P.G. 424 Schroek C. W. 281 Schroepfer G. J. 593 Schudel P. 415,418 Schiiler H. 290 Schueller K. 33 149 Schiimann D. 491 Schiitte H. R. 510 Schuit K. E. 551 Schulenberg J. W. 453 Schuler R. H. 29 Schuller W. H. 423 Schulman L. H. 564 Schulte Eke K. H. 218 Schulte-Frohlinde,D. 209 Schultz A. G. 221,477 Schultz H. P. 252 Schultz J. E. 177 Schultz R. G. 334 Schultz R. M. 619 Schultze G. R.,105 Schulz G. 361 362 Schulz H. 595 Schumacher H. 212,359 Schuster D. I. 201 203 Schuurmans Stekhoven F. M A. H. 570 Schvetsov Yu. B. 517 Schwab L. O. 194 Schwam H. 521 Schwartz M. A, 424 Schwartz M. M. 174 Schwarz H. A, 228 Schwarz J. C. P. 472 Schwarzenbach D. 53 Schweizer M.P. 550 Schwene C. B. 120 Schwenk R. 339 Schwerin S. G. 296 Schwiezer E. E. 455 Schwoerer M. 33 Schwyzer R. 519 530 Sckolnick B. R 201 Scoffone E. 509,517 526 Scoggins M. W. 14 Scopes P. M. 39 40 533 Scorrano G. 80 Scotney J. 428 Scott A. I. 470 501 583 584 Scott J. E. 298 Scott J. F. 552 Scott K. W. 322 Scott M. K. 265 Scott R. A. 534 Scrosati B. 260 Sczware M. 109 Searle R. 208 373 Sears D. J. 101 Sease J. W. 256,257 Sechser L. 318 Seddon D. 267 Seddon W. 5 11 Seden T. P. 271 Seebach D. 280,285,403 Seela F. 531 Seeley N. J. 314 Seff K. 46 59 Segal G. A. 19 Segre A, 376 Seiber R. P. 216 Seide W. 391 Seidel H. 566 Author Index Seidl H. 26 152 308 457 Seidner R. T.196 395 Seifert H. 350 Seiler P. 207 Sekine T. 233 Selinger B. 208 Selke E. 261 326 Selley D. B. 192 Selman C. M. 285 Selvarajan R. 339 Seltzer S. 175 Semin C. K. 314 Semmelhack M. F. 278 409. 48 1 Senda Y. 381 Seneviratne A. S. 510 Seno M. 91 Seno T. 564 Seo E. T. 22 249 Sepp D. T. 469 Servis K. L. 98 143 209 382 Servis R. E. 529 Seter F. 386 Sethi D. 301 Sevilla M. D. 20 30 Seybold G. 355,359 386 Seyferth D. 164 283 286.291 311,316 317 Seyler J. K. 326 Sgaramella V. 543 Shadoff L. A, 15 Schafir J. M. 309 Shafiullah 436 Shagova E. A, 303 Shakrokhiv F. 514 Shalek R. J. 230 Shamma M. 493 500,907 Shani A. 189 Shank N. E. 225 Shannon P. V. R. 413 Shannon T. W. 16 Shapiro J.128 Shapiro L. 570 Shapiro R. H. 101 270 Shapiro R. K. 207 Sharanina L. G. 314 315 Sharma B. D. 43 Sharma M. 471 Sharma R. K. 171 187,342 Sharma R.P. 436 Sharon N. 606 Sharp J. H. 247 Sharp J. T. 178 Sharp R. L. 263 301 Sharples G. M. 321 354 Sharpless K. B. 278 412 427 590 591 Shaw B. L. 325 330 Shaw D. C. 622 Shaw E. 618,624 Shaw J. E. 417 418 Shchegoleva L. N. 24 Shearer H. M. M. 288 Shechter H. 135 165 370 Sheehan J. C. 445 530 Sheehan J. T. 522 525 Shefter E. 50 Sheiner L. B. 538 Sheinson R. S. 20 Sheley C. F. 339 Sheller A. 197 Shelton G. 453 Sheludyakov V. D. 310 Shemyakin M. M. 509 517 528 Shepelavy J. N. 167 Shephard B. R. 238 Shepilov I. P. 297 Sheppard G.126 609 Sheppard R. C ,39 509 517 525 533 Sheppard W. A. 512 Sherer K. V. 113 Sherman E. O. 260 Sherman P. D. 126 Sheverdina N. I. 290 Shew D. C. 424 Sheyanov N. G. 318 Shiba T. 515 Shibakawa R. 539 Shibano T. 329 Shibata K. 439 Shibata S. 65 Shields J. E. 212 Shields T. C. 394 Shigorin D. N. 290 Shih S. 28 Shiina K. 314 Shilov A. E. 162 Shimanouchi H. 48,65 Shimazu H. 479 Shimidzu T. 545 Shimizu H. 346 Shimizu Y. 435 Shimonishi Y. 526 Shimshak R. R. 537 Shimshak S. R 514 Shin H. 421 Shina K. 31 1 Shine H. J. 25 Shiner V. J. 101 Shingu H. 129 Shinitzky M. 628 Shiori T. 499 Shiozaki M. 426 Shirabama H. 421 Shirahata K. 418 Shirley D. A, 286 Shiro M. 45 Shive W.512 Shono T. 242 243 271,402 Shorenstein R. G. 605 Shorter J. 89 136 Shostakovskii M. F. 310 315 Shotton D. M. 622 Shriver D. F. 293 Shronin G. P. 94 Shteingarts V. D. 24 Shteinman A. A. 162 Shukla 0.P. 577 Shulman F. C. 117 Shulman J. I. 386 Shur V. B. 332,355 Shurpik A. 192 Shushunov V. A. 318 Shvo Y. 275 Sicher J. 139 257 382 Sicilio F. 31 Siddiqui H. 436 Siddiqui N.A. Q. 560 Siddall J.B. 269,415 438 Siddall T. H. 339 Sieber P. 509 517 519 524 Siebert W. 302,315 Siedel W., 519 Siegel H. 325 Siegel S. 228 Siepmann T. 150 Sigler P. B. 565 602 612 624 Signor A, 3 18 515 Silber P. 199 Silberg I. 478 Silhankova A. 250 Sillescu. H.. 19. 20 Silver B L.,17,30 Silver J.J.514 Silver M. S. 619 Silverman J.,48 Silverstein R. M. 412 Silvestri N.H. 447 Sim G. A. 48 53 60,62,417 Simamura O. 173 174 182 Sime J. G. 61 42 Simeone J. F. 424 Simkins. R. J. J. 89 Simmie J.,309 Simmons H. E.,166 191 387 389 Simmons T. 38 Simpson A. F. 33 Simpson P. 309 Simpson P. G. 65 Simpson W. R. J. 512 Simon A, 90 Simon W. 40 Simonetta M. 46 101 130 394 Simmons B. K. 14 Sims J. J. 184 236 271 Sinclair J.,20 Sinclair R. W. 386 Author Index Singer B. 539 Singer H. 332 Singer L. A. 173 Singer L. S. 23 30 Singer R. J.,258 Singer S. J. 602 Singerman B. 138 Singh B. 215 216 460 481 Singh G. 318 Singh P. 468 Singh R. 560 Singh S. 464 Singleton D. M. 177,271 Sinnott M.L.609 Sinsheimer R. L. 573 Sioda R. E. 260 Sioumis A. A, 494,496 505 Sippel A, 573 Sircar J. C. 264 425 Sisido K. 316 Sisido S. 344 Sitzmann M.E. 134 Sjolander N.O. 598 Skapski A. C. 42 Skell P. S. 110,160 177 234. 235 238 Skinner S. J. M. 593 Sklarz B. 461 Skoda J.,536,542 Skoog F. 538 Skvortsova I. N., 528 Slatcher R. P. 542 Slater C.D. 129 Slater R A, 462 Slaugh L.H. 176 326 331 Slaytor M.?579 Sloane N.H. 510 Slocum D. W. 285 Sluyterman L. A, Ae. 629. 630 Smale T. V. 427 Small G. D. 547 Smaller B. 33 Smalley R.K. 443 Smeby R. R 527 Smedman L. 419 Smentowski F. J. 21 135 Smetankina N.P. 310 Smillie L.B. 623 Smillie L.R. 622 Smirnov L. D. 93 Smit E.M., 571 Smit P., 480 Smith A, 509 550 Smith A. B. tert. 443 Smith A. E. 514 563 Smith A. R. H. 593 Smith A. W., 314 Smith C. 278 357,411 Smith C. A. 573 Smith C. E. 575 Smith C. L. 466 Smith D. G. 597 Smith D. M. 101 Smith E. B. 176 Smith E. L.,625,626,627 Smith G. F. 501 583 Smith G. N.,497 501 582 583 Smith H. 266 587 Smith H. D. 297 Smith H. E. 37 Smith J. D. 563 Smith J.H. 345 Smith J.M. 525 Smith K. 175 Smith K. C. 547 Smith L.M. 178 Smith L.W., 489 Smith P.,26 504 Smith P. J. 243 Smith R. A. 168 Smith R. A. J. 430 Smith R. J.,204 Smith R. L. 509 533 Smith S. C. 510 Smith S. G. 128 Smith S. L. 211 Smith W.B. 231 232 Smithers M. J. 525 Smulkowski M.523 Smutny E. J. 329,360 Smyth J.J.,625 Snatzke G. 35,36 37 427 Snedeker E. 524 Snegur L. N.,318 Snell R. L. 603 Snider W.H. 483 Snow M. L. 512 Snyder J. J. 907 Snyder T. E. 591 Sobolev. E. S. 312 Sobolevskii M. V. 284 Sobti R. R.,427 Soedigdo. S. 452 Soll D. 564 Serrum H. 44 Sokolik R. A. 283 Sokoloski T. D. 37 Sokolov V. I. 292 Sokolovsky M. 634 Solie T. N., 548 Sollaide G. 140 Solodar A. J.,356 Solodar J.,386 Solodnikov S. P. 22 Someswara Rao C. 238 Sommer J. lG8 Sommer L. H. 309 Sommer R. 316 317 Sommermayer K. 226 Somsen G. 381 Sondheimer F. 353 364 366 367,368,370 Sone T. 619 Sonenshein G. E. 570 Sonntag F. I. 203 Sorm F. 527,542,612 Soshka S. A, 297 Sotnikova L.E. 560 Sotolova T. D. 89 Souchay P. 254 Southam R. M. 113 199 Southwick P. L. 48 Sowa J. R. 539 Sowinski F. 479 Spadafino L. P. 176 Spalding T. R. 131 317 Spande T. 517 Sparrow J. T. 509 Speakman J. C. 42 Spears C. L. 539 Speet A. 343 Spence G. G. 216,468 Spencer R. P. 513 Spencer T. A. 424,591 Spenser I. D. 580 Sperling J. 51 1 Spialter L. 309 Spicer E. K. 236 Spiegelman G. 447 560 Spielman J. R 298 Spillett R. E. 97 Spiro v.,39 Spiteller G. 10,491 499 Spiteller-Friedmann M. 499 Spitzer W. A. 197 Splitter J. S. 220 Spotswood T. McL. 621 Sprague P. W. 498 Spring D. J. 350 Spry D. O. 406 Spurlock S. 184 236,271 Squires T. G. 89 Sribuey M. 526 Srinivasan R 192 199 203 215,220,397,455 Srivastava S.N. 62 65 Staab H. A. 204,339,369,370 Stiide W. 26 Staehelin M. 539,563 Stiillberg-Stenhagen S. 414 Stafford C. 14 Stam,C. H. 46 Stam J. G. 14 Stanfield R. A. 48 Stanger H. 136 Stanko V. I. 297,298 Stanovnik B. 478 Stapleford K. S. J. 501 582 583 Stark G. R. 515 Starka L. 430 Author Index Starnes W. H. 184 Staunton J. 494 Steard M. W. 598 Stechelberg W. 37 Stedronsky E. R. 109 Steele D. 104 Steele W. R S. 370 Steelhammer J. C. 223 Steffan G. 21 1 Stegel F. 99 Steglich,W. 520 Steglich W. 519 Stein W. H. 634 Stein W. J. 631 Steinberg G. M. 83 128 Steinberger R. 528 Steinegger E. 490 Steiner G. 39 Steinfeld J. I. 355 Steinfelder K. 15 Steinhardt M.373 Steinle G. 518 Steinman G. 514 Steitz T. A. 605 632 Stenhager E. 414 Stent G. S. 535 Stepanov I. P. 220 Stepanovic R. D. 391 Stephens P. J. 40 Stephenson I. L. 308 Stephenson L. M. 189 Stermitz F. R. 65 216 Stem A. 530 Stem E. W. 334 Stern R. 325 Sternbach L. H. 481 Sternberg H. W. 251 Sternglanz R. 569 Stetter H. 392,403 Steuber F. W. 26 Steudel R. 141 Stevens C. L. 469 Stevens,I. D. R. 128,163 376 383 Stevens R V. 262 451 490 492,494,907 Stevenson G. R. 21 Stevenson J. 308 Stevenson K. J. 623 Steward D. W. 3 11 Stewart F. H. C. 518 Stewart J. C. 263 588 Stewart J. M. 55 519 Stewart 0.J. 31 1 Stewart W. E. 339 Steyn P. S. 39 Stille J. K. 152 Stiller K. 220 Stillwell R N.12 546 Stimson V.R. 128 Stipanovic R. D. 409 Stirling C. J. M. 519 St. Jacques M. 380 Stjernstrom N. E. 350 Stock J. A. 530 Stock L. M. 91 Stocker J. H. 253,258 Stoddart J. F. 486 Stockel K. 621 Stoeckler H. A. 314 Stocklin W. 62 Stohr G. 294 Stolka M. 159 Stolow R. D. 380 Stone F. G. A. 283,293 Stone T. J. 23 Stork G. 279 311 504 Story P. R. 221 392 Stott D. A. 226 Stout. G. H. 41 Stowe M. E. 342 Stoye D. 473 St. Pierre T. 73 126 Strachen R. G. 521 Strahs G. 56 Strating J. 405 Straub P. A. 212 213 Strauss M. J. 127 128 349 Strausz 0. P. 185 291,310 Streef J. W. 467 Streith J. 216,483 Streitwieser A. 132 134 348 372 Strojek J. W. 259 Strom E. T. 30 Strong J.G. 133 Strow C. B. 196,331,486 Struble D. L. 178 Struchkov U. T. 313 Stryer L. 601 Stuart J. D. 247 Stuart J. M. 130 Stuart K. L. 489 Stuart S. R. 325 Stucki H. 450 Stucky G. D. 304,305 Studier M. H. 537 546 Sturm E. 362 Sturtevant J. M. 616 Stutz E. 551 Subba Rao G. S. R. 431 Subba Rao H. N. 419 Subramaniam M. S. 337 Subramanian J. 20 29 Suchy M. 36 Suehiro T. 182 Sueoka N. 566 Siissmuth R. 539 Sugahara T. 494 Sugie M. 271 Sugihara H. 526 Sugihara Y. 410 Sugino K. 233 253 Sugiyama N. 206 Suhadolnik R. J. 577 Suketa Y.,531 Sullivan M. F. 332 Sullivan P. D. 23,25 31 Sullivan R. A. L. 47 Sulston J. 536 537 Sulston J. E. 543 Sultanbawa M. U. S. 269,409 Sumiki Y.426 Summerford C. 302 Sunagawa M. 425 Sundaralingam M. 57 Sundberg R. J. 468 Sunder S. 455 Sunder-Plassmann P. 406 Sundharadas G. 564 Sung M.. 13 189,461 Sung Moon 191 Surridge J. H. 405 Surzur J M. 176 Suschitzky H. 467 Susuki T. 241 Sutcliffe F. K. 94 Sutcliffe L. H. 29 Suter A. K. 157 388 Suter S. R. 392 Sutherland H. H. 47 Sutherland I. O. 36 37 278 357 361 374 377 411. 476 Sutherland J. K. 277,389,416 515,586 596 Sutherland M. D. 417 Sutter B. 350 Suyama Y. 570 Suzuki A. 301 Suzuki H. 95,96 Suzuki J. 560 Suzuki S. 541 Suzuki T. 91 531 Svoboda M. 139,382 Swain C. G. 90 260,603 Swain E. J. 463 Swallow A. J. 225,226 227 Swaminathan S. 339 Swan G. A. 464,485,489 Swan R.J. 37,499 Swann P. F. 539 Swedlund B. E. 142 Swen H. M. 626 Swenton. J. S. 148 203 387 Swern D. 271 Swierczewski,G. 270 Swinbourne E. S. 128 Swindell R T. 123 Symonds M. 184 Symons M. C. R. 22 29 30 206 Sykes P. J. 431,437 Author Index Szantay C. 489 Szarek W. A, 271,473,486 Szczepanski Ck V. 582 Szeimies,G. 115 395 Szepesvary E. 259 Szeytli J. 71 Sziman O. 97 szinai s. s.,405 Szmant H. H. 177,266 Szwarc M. 105 135 171 175 Szybalski W. 574 Tabuchi H. 323 Tabushi I. 112,172,356,386 Tada H. 36 Taddei F. 132 133 Tagliavini G. 318 Taguchi H. 907 Tahk F. C. 907 Tai W. T. 133 Tajima Y. 326 Takada S. 418 Takagi H. 454 Takagi I. 418 Takagi K. 200 Takagi S. 496 Takahashi H.336 Takahashi K. 135 274 458 464 Takahashi M. 392,48 1 Takahashi S. 329 Takahashi T. 425 Takakura K. 31 Takano Y. 443 Takaoka K. 258 Takashima H. 527 Takebayashi K. 496 Takeda H. 417,423 Takeda K. 38 Takeda T. 243 Takeda Y. 582 Takei H. 455 Takemoto T. 417,438,439 Takemura S. 562 Takenaka H. 301 Takenishi T. 536 Takeshita A. 211 Takeshita H. 200 Takeuchi I. 515 Takeuchi K. 122 Takino T. 117 Takita T. 55 Talaty E. R. 445 Talcott C. 21 Tallec A. 255 Tam S. W. 9 10 Tamaki E. 490 510 Tamano T. 487 Tamao K. 31 1 Tamazawa K. 464 Tamborski C. 288 Tamelen E. E. 590 591 Tamura C. 53 55,62 Tan C. C. 181 Tan H. W. 404 Tan S. I. 264 Tanaka J. 46 59 Tanaka M. 451 Tanaka N.45 Tanaka Y. 211 Tananbaum S. W. 597 Tani H. 307 Tani K. 330 Tanida H. 119 122 379 Taniguchi H. 31 441 Tannenbaum H. P. 8 Taraszka M. J. 71 Tarkhanova M. V. 32 Tarrant P. 311 387 Tarkakovskii V. A, 292 Taschner E. 523 Tateishi M. 423 Tatwawadi S. V. 259 Taub D. 431 Taub I. A,. 224,225 Taube M. 514 Tauregui- Adell J. 606 Tavares R. 577 Tavernier D. 381 Taylor D. A. H. 427 Taylor D. R. 427 Taylor E. C. 216 275 277 280. 342,449,464,468,479 Taylor E. K. 563 564 Taylor G. A. 202 Taylor G. R. 89 Taylor J. B. 494 Taylor J. W. 175 263 Taylor K. G. 285 Taylor R. 97 Taylor R L. 118 Tchir M. 189 Tebbe F. N. 296,297,301 Tedeschi E. 499 Tee 0.S. 540 Tefertiller B. A. 381 Temnikova T.I. 220 Temple R. D. 90 Templeton D. H. 50 52 Templeton L. K. 50 Temussi P. A, 376 Tenenhouse A, 524 Teplitz M. 573 Terabe S. 30 Terada A. 465 Teranishi R. 415 Teraoka M. 417 Terashima M. 262 Terataki S. 122 Terauchi K. 26 Terent’eva 1. V. 496 Ternat A. L. jun. 477 Ternay A. L. 38 Ter-Sakisyan G. S. 302 Testa E. 448 Teubner J. K. 372 Texier P. 260 Tezuka T. 202 Theard L. M. 229 Theilacker W. 172 182 Thiebe R. 564 Thiele K.-H. 290 Thrill R J. 62 Thio P. A. 264 Thirsk H. R. 234 Thomas A. 30 Thomas A. M.,509 Thomas C. A. jun. 570 Thomas C. B. 184 Thomas D. W. 517 Thomas. H. G. 392 Thomas H. T. 233 Thomas J. 290 Thomas J. K 226,227,228 Thomas J. O. 528 Thomas M.B.476 Thomas P. C. 293 Thomas P. J. 128 Thomas R 427,569 Thomassin R.B. 309 Thomasson J. E. 332 Thompson C. 171 Thompson D. T. 324 Thompson G. F. 227 Thompson J. A 125 Thompson J. E. 277 Thompson J. L. 406 Thompson R C. 519 Thompson R H. 171 Thompson T. W. 480 Thomson,A. F. 415 Thomson C. 17,24,32,187 Thomson,J. B. 12 311 Thomson R.H. 28,423 Thornber C. W. 494 Thornton E. R 77 Thyagarajan G. 458 Tie P.R 255 Tichy M.,139 Ticozzi C. 386 Tidwell T. T. 1 11 404 Tiecco M.,25 Tiefenthaler H. 215 Tikhomirov B. I. 302 Tilak M.A. 522 Tillett J. G. 67 79 87 Timberlake J. W. 177 Timell T. E. 71 Timmons C.J. 208 Timms P. L. 293 Timms R E. 312 Tincher. C. A. 250 251 Tinker H. B. 141 Author Index Tinoco I.548,549 Tisler M.,478 Tisue G. T. 166 Titlestad K. 509 Titov M. A. 519 527 Titova N. S. 297 Tiwari H. P. 109 Tjabin M.B. 162 Tjoa B. T. 429 Tobey S. W. 387 Tochtermann W. 191 Toda T. 363 Todd (Lord) 535 Todd A. R 179 Todd L. J. 298 Todd M. J. 109,346,468 Todd P. F. 29,250 Todesco P. E. 100 101 371 Tohyama T. 209 Tokumaru K. 173,174,182 Tokunaga Y.,454 Tokura N. 451 Tokuyama T. 65,504 Tollin P. 47 Tolman R L. 541 Tomalin D. A. 87 Tomer K 101,207,270 Tomiie Y. 64 Tomita K. 62,64,493,495 Tomizawa J. I. 573 Tong B. P. 480 Tong W.-H. 285,289 Tong Y. C. 87 Toporcer L. H. 310 Topping R. M.,126 Topson R. D. 446 Torii S. 243 Toromanoff E. 379 Torre G.441 Torssell K. 27,28 29 Tortorella C. 515 Tortorella D. 515 Tortorella V. 513 Toru T. 110,405 Toubiana R 417 Townsend L. B. 541 Toyne K. J. 136 Tozyo T. 418 Trachtenberg E. N. 142 Tracy H. J. 525 Tranker H. 490 Trager W. F. 500 Trapp H. 286 Trautwein W. P.,472 Travers N. F. 316 Traylor T. G.,175 404 Traynard J. C. 289 Trecker D. J. 202 209 210 404 Treindl L. 129 Tremmel C. G. 260 Tremper H. S. 110,265 Treves G. R.,136 Triggs C. 336 TrinajstiC N. 217 Trippett S.,83 84 85 Tritle G. L. 122 Trojanek J. 37 Tronich W. 319 Trost B. M.,278,411,456 Trotman- Dickenson A. F. 397 Trotter J. 46 59,65 308 Trowbridge C. G. 624 Troxler E. 382 Truce W. E. 450 Trueb W. 504 Trueblood K.N. 45,46,59 Trumbore C. N. 226 Trumbull E. R 146 Truter E. V. 428 Tsao F. H. C. 130 Tschesche R 426,429 Tsechesche R 62 Ts’o P. 0. P. 539 550 Tsuboi M.,43 Tsuboyama K. 546 Tsuchihashi G. 206 Tsuchiya T. 215 Tsuda M.,463 Tsugi T. 122 Tsuji J. 276 328 329 336 Tsukitani Y.,255 Tsuneda K. 439 Tsurata H. 211 Tsuruta H. 399 Tsushima T. 122 Tsutsumi S.,241 244,336 Tucci E. R.,327 Tucher P. M.,293 Tucker B. 147 Tucker L. C. N. 271 Tufariello,J. J. 276 Tuinman A 200 Tumanyan V. G. 560 Tummler R. 15 Turkevich J. 31 Turley J. W. 53 309 Turnbull K. W. 585 Turner A. B. 432,444,445 Turner R B. 379 382 Turner R W. 271 Turner S. 39 Turro N. J. 156 199,206,389 402 Tursch B. 62 Tutt D.E. 126 Tvorogov A. N. 311 Tyler A, 573 Tyminski I. J. 177 380 Ubersax R.W. 392 Uchida A, 451 Uchida Y. 329,331,332 Uchijuma T.,91 Uda H. 421 Udding A. C. 405 Udenfriend S. 600 Uebel J. J. 246 380 Ueda E. 527 Ueda H. 46 Ueda S. 387,582 Ueki I. 312 Ueno K. 464 Ugi I. 509,518 Ugo R. 321 333 Uhl,W. J. 311 Ulbright T. L. V. 348 Uliss D. B. 445 Ulland L. A. 309 Ullman E. F. 27 183 189 215,460,461 Ulrich H. 147 Ul’yanova A. V. 496 Uma V.. 168,347 Umani-Ronchi A. 386 Umans R. S. 550 Umemoto K. 252 Umezawa H. 55 Unchold R. E. 68 Underwood G. R 18,31,381 Uneyama K. 240 Unni M. K. 263 301 Uno F. 270 392 Uraneck C. A. 290 Urasaki I. 97 Urata H. 240 Urberg M.M. 25 Urry W. H. 242 Usher D. A. 76,77 Usmani J. N. 476 Ustynyuk Yu. A. 308 Utley J. H. P. 91 Uyeda R. T. 133 Uyeo S. 495 Uzan R. 97 Uziel M. 635 Vaciago A. 427 Vahrenkamp H. 293,313 Valade J. 312,316 Valentini F. 254 Vallee B. L. 605,631,632,634 Vanaman T. C. 61 1 Van Auken T. V. 196,404,486 van Bekkum H. 381 van Brockhoven J. A. M. 33 Van Bruggen E F. J. 570,571 van Bruijnsvoort A. 46 Van Catledge F. A, 380 Vance R. L. 298 Vandenberghe A, 560 van den Broek P. 504 Author Index vanden Eynde H. 89 van der Ent A 325 van der Goot H. 464 van der Helm D. 43,57,418 Van der Jagt D. L. 121 van der Kelen G. P. 3 13 van der Kerk G. J. M. 314,318 Vanderkooi G. 534 van der Lans H. N. M. 466 van der Lugt W.T. A. M. 188 van der Meer H. 46 van der Plas H. C. 456,480 van der Waals J. H. 32 Vanderwaart B. E. 403 Van Dine G. W. 118,160 Van Dorf D. A 406 Van Dorp D. A. 406 Van&ek J. 612 van Helden R. 328 334 van Heuvelen A. 30 Van Lear G. E. 12 Vanlear G. F. 11 Van Leusen A. M. 464 van Oyen J. W. L. 54 Vanstone A. E. 439 Van Styvendaele B. 560 van Tamelen E. E. 184 212 215 236 259 271 278 332 359 371,412,409,427 455 589 van Veen A 381 Van Vitnendaele F. 71 van Wazer J. R.,312 van Willigen H. 23 33 Varga E. 518 Varghese A. J. 547 Vargolis A. G. 82 Varna K. R. 592 Vaska L. 321 Vaughan J. 140,446 Vaughan T. A. 91 v.d. Haar F. 565 566 Veber D. F. 519,521,524 Vecera M. 67 Vedejs E.273 357,406 Vederas J. C. 481 Veinberg,.A. Ya. 259 Veki M. 520 Vellins C. E. 517 Vellturo A. F. 235 Vember P. A. 496 ven Donck L. 313 Venkstern T. V. 538 Venter D. P. 14 Verberg G. 328 Verbit L. 37 Verhassel J. P. 560 Verkade P. E. 381 Vernin G. 181 Vernon C. A 514,607,610 Veronese F. M. 517 Verrier M. 266 Verschoor G. C. 54 Verstraeten L. M. J. 471 Vetesinki P. 67 Vetter W. 10 Vialle J. 462 Vieroth C. 289 Viertler H. 238 Vietmeyer N. D. 203 270 Vigneron J. P. 512 Vijh A K. 171 231 233 234 Villa A. E. 293 Villatica R. M. 424 Vilkas E. 5 17 Vincent C. A 260 Vincent R. L. 65 Vincow G. 18,20,21,30 Vining L. C. 577,597 Vinograd J. 571,573 Vinogradova E. I. 517 Vipond P.W. 345 Viscontini M. 478 Visser G. J. 54 57 Visser H. D. 305 Visser J. P. 509 Viswanathan N. 429 Vit J. 262 Vitali D. 100 v. Kutepow N. 276,328 Vlatters I. 273 406 Vodehnal J. 159 Voelter W. 40 261 Vogel E. 365 371 395 Vogel K. 343 Vogel P. 191,486 Vogt B. R 392 Voisey M. A. 162 Volodarsky L. B. 27 183 Volger H. C. 114,334,397 Volke J. 259 Vol’kenau N. A. 283 Volland W. V. 21 Vollmer J. J. 51 143 382 Volod’khin A. A. 32 Vol’pin M. E. 103,332 355 von Ardenne M. 15 von Ardenne R 477 von Daehne W. 428 von den Berghe E. V. 313 Vonkeman H. 406 von Satza M. 525 von Schriltz D. M. 282 von Veh G. 396 Vopel K. H. 362 Vos A. 54 57 59 60 Voss A. J. R. 207 Vostrikova T. N. 297 Vulfson N.S. 528 Vyazankin N. S. 283,317 Vystrcil A, 430 Wachs T. J. 10 Wacker A, 539 Wada M. 512 Wade K. 283,284; 302,307 Waekneldt T. V. 514 Wagenkneckt J. H. 250,257 Wagner E. 387 Wagner H.-U. 377 Wagner J. 441 Wagner K. G. 537 Wagner P. J. 187 198 Wagner T. E. 605 Wagner W. J. 128 Waiss A. C. 37 Waiss A. C. jun. 213 Waight E. S. 14 Waight E. S. 427 Waits H. P. 175 Wajciekowska H. 523 Wajer Th. A. J. W. 28 183 Wakamatsu T. 500 Wakefield B. J. 284,286,466 Waki M. 530 Walborsky H. M. 175 262 285,373 Waldman H. 20 Waldman M. C. 315 Waldron N. M. 345 Walia J. S. 276 Walia P. S. 276 Walinsky S. W. 165 Walker D. R. 272 Walker J. 312 Walker J. A, 359 Walker R. L. 465 Walker W.H. 216 Wall A. A. 138 Wall E. N. 269,415 Wall H. M. 89 136 Wall L. A. 31 Wall L. L. 514 Wall R E. 373 Wallace R. W. 172 405 Wallbillich G. E. H. 148 387 Wallenfels K. 613 628 Waller G. R. 14 585 Walling C. 171 175 236 Wallis E. S. 233 Wallis S. R. 37 Wallwork S. C. 60 Walsh E. J. jun. 398 Walsh K. A. 612 Walti A. 631 Walton D. R. M. 310 Walton E. 540 Wang C. S. 134 Wang J. 323 Wang J. C. 573 Wang J. H. 604,615 Wang S. Y.,547 Wangen L. E. 380 Author Index Wanzlick H.-W. 292 Ward D. C. 574 Ward H. R. 126,191,207,351 Ward J. F. 229 Ward J. P. 322 Ward R. L. 29 Ward R. S. 8 10 Ward S. D. 8 Warhurst E. 30 31,157 Waring C. 183 Waring M. J. 543 571 574 575 Warkentin J.135 176,405 Warnant J. 265 Warnet R J. 494 Warnhoff E. W. 133 529 Warrell D. C. 364 Warren K. D. 79,94,375 Warren L. F. 296 Warren M. E. 37 Warren R 298 Warren S. G. 284 Warrener R. N. 13 Washecheck P. H. 418 Wasserman E. 33 218 323 392 Wasserman H. H. 220 356 357 386 Watanabe A. 202 Watanabe H. 211,285 Watanabe I. 65,426 Wataliabe K. A. 541 Watanabe M. 560 Watani N. 429 Watenpaugh K. 57 Waterman D. C. A. 70 Watkins W. B. 509 Watson D. G. 62 Watson H. C. 622 Watson J. D. 535 Watson K. J. 60 Watson R. 571 Watson R. A. 184 Watson W. H. 24 Watt A. N. 277 Watt D. S. 424 Watts P. 71 Watts P. H. jun. 55 Wawzonek S. 248,251,257 Wayne R. P. 162 Wearring D. 251 Wease J. C.274,473 Weaver T. D. 424 Webb N. L. 565 Webb R. G. 176 Webb T. 529 Weber H. 543 545 Weber H. P. 51 Weber P. 490 Webster D. E. 309 Webster J. R. 176 Wechter W. J. 547 Wedler F. C. 618 Weedon B. C. L. 231,238,246 277 Weeks D. P. 68 Wege D. 116 117 125 Wegmann A. 514 Wegner P. A. 296 Wehniger E. 369 370 Wehrli H. 200 434 437 Wehrli P. 453 Wehrli W. 563 Wei C. C. 216 Weidenbruch M. 312 Weidlein J. 304 Weidler A. 133 Weigang 0.E. 36 Weimann B. J. 536 537 Weinberg D. S. 14 Weinberg H. R. 171,240,342 Weinberg N. L. 171,240,243 246,342 Weinblum D. 546 Weingarten H. 247 Weinheimer A. J. 418 Weinshenker N. M. 373 Weinstein 1. B. 564 Weinstein S. Y. 619 Weinstock J. 406 Weinstock L.M. 461 Wei-Ping Lin J. 219 Weis L. D. 190 233 Weisbach J. A. 406 Weisenberger C. R. 15 Weiser R S. 606 Weisflog J. 452 Weiss D. S. 199 Weiss J. A. 493 Weiss M. J. 16 Weiss U. 35 391 Weiss W. 105 Weissenfels M. 452 Weissmann S. M. 552 Weith H. L. 560 Wells E. E. 342 Wells J. L. 56 450 Welzel P. 428 Wemmers A. 381 Wender I. 251 Wendlberger G. 509 525 Wendler N. L. 274,431 Wenkert E. 176,262,422,492 498,501 Wentland M. P. 490,494,907 Wentrup C. 166,469 Wentworth W. E. 223 Wepster B. M. 381 Werner D. 417 Werner E. M. 452 Werstiuk N. H. 419 Wessal N. 312 West P. 284 West R. 285 311 312 362 402 Westberg H. H. 184 236,271 Westen H. H. 382 Westheimer F. H. 82 83 636 Westlake D.W. S. 577 Westley J. W. 523 528 Westley T. W. 514 Wexler S. 268 Weyenberg D. R. 310 Weygand F. 512,519,520 Whaler D. 124 Whalley N. B. 587 Wharf I. 290 Wharton P. S. 398 Wheatley P. J. 55 Wheeler D M S. 494 Wheeler 0.H. 252 Whelan D. J. 612 Whelan J. 495 Whipple E. B. 197 Whitaker J. R. 630 Whitaker K. E. 372 White A. C. 154 White A. F. 426 587 588 White A. M. 107 White D. A. 337 White E. H. 109 White J. D. 383,421 White L. 210 Whitehouse M. W. 515 Whitehouse R. D. 38 Whiteside T. 236 Whitesides G. M. 191 291 331 340 343,403 Whitesides T. 184 271 Whitesides T. H. 215,455 Whitham G. H. 270 Whiting M. C. 113 214 Whitmore G. F. 548 549 Whitney C. C. 271 301 Whitten D.G. 189 Whitten J. L. 15 Whittingham A. 312 Whittle J. A. 586 Whittle P. R. 387 Wiberg K. B. 115 118 379 380 392 395,403 Wiberg K. 109 Wiberg K. W. 118 Wicha J. 430 Widdowson D. A, 267 592 Wie C. T. 455 Wiechert R. 434,439 Wiedhaup K. 391,409,431 Wiejak S. 509 518 Wieland P. 261,434 Wieland T. 522 525 528 Wiemann J. 252 253 Wiesel M. 196 395 399 Author Index Wiewiorowski M. 491 Wigfield,D. C. 434 583 584 Wigger A. 224 Wiggins D. E. 87 Wilairat P. 208 Wilchek M. 513 517 Wilcke S. 290 Wilcox C. F.,jun. 382 Wild S. B. 337 Wilde A. M. 30 Wildman W. C. 496 578 Wildnauer R. 620 Wiley D. W. 150 Wiley G. A. 122,404 Wilke G. 329 Wilke R. N. 356,400 Wilkinson G. 324 325 327 329 332 Willemsens L.C. 283 318 Willems H. 524 Willett J. D. 221 591 Willette R. E. 465 Williams A. 609 627 Williams D. H. 8 9 10 11,14 16,433 Williams D. M. 221 265 473 Williams F. J. 386 Williams G. H. 171 181 Williams J. G. 142 Williams J. L. R. 190 293 Williams J. M. 69 Williams J. M. jun. 446 Williams J. R. 156 201 389 Williams K. R. 406 Williams L. L. 451 Williams N. J. 525 Williams N. R. 470 Williams R. J. P. 259 605 Williams R. M. 60 Williams R. O. 398 Williams W. G. 24 Wills J. 201 Willson R L. 225 230 547 Wilsbach K. E. 207 Wilson C. F. 87 Wilson H. G. E. 234 Wilson H. R 60 Wilson I. B. 615 Wilson J. 504 Wilshire J. F. K. 339 Wilson J. M. 10 Wilson J. N. 43 Wilson N. D. V.496 Wilson R. 32 Wilt J. W. 128 276 Wiu T. C. 527 Wimmer E. 560 Wingrove A. S. 148 387 Winstein S. 21 104 109 112 119 120 122 124 128 138 237 341 348,405 68 1 Wiesner K.. 505 Winter R. E. K. 192,204 273 406 Winter W. P. 612 Winterfeldt E. 389,498 Winterman D. R 370 Winters R. E. 11 Wipf H. K. 40 Wise L. D. 477 Wise M. L. 392 Wiseall B. 226 Wiseman J. R. 392 Wishnok J. S. 207 351 Wisnosky D. E. 109 Wittig G. 273 355 356 357 403 Witkop B. 65 217 264 487 504,517,600 Witkop P. 547 Witte H. 295 Wladislaw B. 238 Woerner F. P. 442 Woese C. R. 537 552 Wohllebe J. 404 Wojcicki A. 332 Wojnarowski W. 384,404 Wojtkowski P. 276 Wold S. 29 Wolf A. P. 319 Wolf F. J. 524 Wolf H.C. 33 Wolfe E. S.,91 Wolfe S. 447 Wolff D. 331,401 Wolfrum R. 287 Wolinsky J. 271,403,414 Woller P. B. 443 Wollheim R. 371 Wolovsky R 323,392 Wolschokowsky N. 594 Wolstenholme D. R. 570 573 Wolthers B. G. 626 628 Wolters J. 54 Wong C. M. 133 Wong R. C. 627 Wong S. K. 128 Wood P. B. 26 Woodbury E. C. 254 Woodhall B. J. 250 Woodman T. J. 521 Woods J. D. 44 Woods M. C. 392,418 Woodward R B. 143 187 188,521 Woodworth C. W. 262,399 Woodworth R C. 160 Woolley P. M. 519 Work S. D. 310 Workman R. 493 Worm M. 309 Worthley S. 387 682 Wriede P. A. 206 Wright C. S. 625 Wright G. J. 446 Wright J. L. C. 597 Wright M. 270 Wrixon A. D. 470 Wrobel J. T. 249 580 Wszolek P.C. 427 Wu R 568 Wiiest H. 415 Wiinsch E. 509 520 525 Wiirsch P. 191 Wulff G. 62,429 Wunderlich J. A. 494 505 Wyatt B. K. 302 Wyatt P. A. 67 Wyatt P. A. H. 67 Wyckoff H. 634 Wyler H. 39 Wynberg H. 37 57 97 189 270,405,455,456,486 Wynne-Jones (Lord) 234 Wynne-Jones W. F. K. 233 234 Yager W. A. 33,218 Yagi H. 65,494,495 Yagupsky G. 327 Yajima H. 518 526 Yakobson G. G. 24 Yakubchik A. I. 302 Yek-Yung Wigfield 93 Yamabe T. 91 Yamada K. 206 Yamada L. K. 418 Yamada M. 215 Yamada S. 216,264,468,499 512 Yamada Y. 536 Yamagishi T. 251 Yamaguchi M. 539 Yamaguti T. 387 Yamakawa M. 45 Yamamaka T. 469 Yamamori H. 308,311 Yamamoto A. 329 332 Yamamoto H. 590 Yamamoto K. 310 Yamamoto M.512 Yamamoto O. 304 Yamamura S. 527 Yamanaka H. 467 Yamanouchi A. 363 Yamashiro D. 526 527 Yamashita S. 521 Yamato M. 464 Yamauchi T. 182 Yamazaki N. 329 Yanagi K. 357 Author Index Yandle J. R.,79 Yang H. Y. T. 527 Y ang M. T. 267 Yang N. C. 189 198,199,385 Yannoni N. F. 48 Yano K. 412 Yanofsky C. 535 Yasuda H. 307 Yasukouchi K. 255 Yasuoka N. 307,312 Yates P. 219 397,451 Yen Chien Liu M. 234 Yeo A. N. H. 11 16 Yergey A. L. 313 Yeboah S. K. 439 Yi-Noo Tang 163 Yoneda Y. 91 Yonemitsu O. 487 Yonetani T. 601 Yonezawa T. 26,355 Yoon N. M. 262,265 York S.S. 615 Yoshida H. 540 Yoshida M. 174 312 Yoshida T. 418 Yoshida Z. 88 Yoshikami D. 564 Yoshikawa K. 258 Yoshikoshi A.421 Yoshimura S. 526 Yoshioka H. 417 Yoshioka M. 206 Yoshizawa J. 432 Yosioka I. 429 Youeda Y. 140 Young A. E.. 87 Young D. C. 796 Young D. W. 47 Young E. P. 511 Young E. T. jun. 573 Young G. T. 509,521,522 Young H. 423,430 Young J. D. 525 Young J. F. 283 Young R. 42 Young R.C. 67 87 Young R.N. 381 Youngson A. 527 Yousefzadeh P. 258 Youzeawa T. 109 Yu Chen H. 322 Yuguchi S. 330 Yuh Y. H. 232 Yui H. 173 Yukawa T. 336 Yuki H. 305 Yunosov S. Yu. 493,496 Zabkiewicz J. A, 51 Zaborsky 0.R.,86 87 Zachau H. G. 564,566 Zahn H. 509,528 Zaidi N. A. 478 Zaitsev B. E. 93 Zak A. G. 176 Zaks Yu. B. 22 Zakharkin L. I. 297 306 Zakharyan R. A. 538 Zalar F. V. 237 Zalewski R.I. 67 Zalkow L. H. 585 Zaltzman-Nirenherg P. 600 Zamoram A, 514 Zander J. M. 434,592 Zaugg H. E. 349 Zavada J. 139,257 Zavarin E. 419 Zavgorodnii V. S. 314 315 316 Zdanovich V. I. 265 Zdero C. 594 Zee-Cheng K. V. 479 Zeeh B. 201 434 Zeffren E. 621 Zefirov N. S. 292 Zeiss G. D. 160 Zeldes H. 32 Zeller E. A, 91 Zeller J. 122 Zeller P. 448 Zenda H. 196,395,399 Zhigach A. F. 284 Zhigareva G. G. 297 Ziegler F. E. 500 Ziegler G. R. 195 372 486 Ziegler P. F. 128 Ziffer H. 37 201 Zimmer H. 318 Zimmerman H. 24 Zimmerman H. E. 188 194 198,202 266,402 Zirkle C. L. 465 Zoltewicz J. A, 466 Zoretic P. A. 500 Zorzut C. M. 13 Zoziara A. 271 Zsindley J. 351 Zuber H. 524 Zuckerman J. J.313,314 Zuech E. A, 323 392 Zuman P. 252 253 Zumwalt R. W. 514 Zunker D. 124 Zurfliih R 269,415 Zverev V. V. 94 Zwanenburg B. 78 Zweifel G. 271 301 Zwick A. 525 Zwierzak A, 271

ISSN:0069-3030    

DOI:10.1039/OC9686500641

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Acid-base catalysis 68 Acidity functions 67 Alicyclic compounds 379 Alkaloids 489 biosynthesis 577 Alkylation reactions 277 Aluminium compounds 304 Amino-acids 509 Amaryllidaceae alkaloids 496 Annulenes 364 Anode processes 231 Antimony compounds 318 Aromatic compounds 45,206,339 Arsenic compounds 318 Azepines 482 Azetidines 446 Azirines 441 Azulenes 363 Benzenes 342 Benzynes 354 Beryllium compounds 287 Biosynthesis 577 Bismuth compounds 3 18 Boron compounds 292 Bridged rings 382 Cadmium compounds 290 Calcium compounds 289 Carbanions 13 1 Carbenes 159 Carbohydrates 53,470 Carbonium ions 103 Carbonyl compounds 198,272 Carbonyl groups 136 Catalysis acid-base 68 enzymic 602 Cathode processes 249 Chromans 475 Circular dichroism 35 Conformational analysis 3 79 Crystallography 41 Cyclic peptides 528 Cycloaddition reactions 145 Cyclobutadienes 359 Cyclophanes 353 Disproportionation reactions 322 Diterpenes 421 DNA 566 Drugs binding to DNA 573 Electrochemistry 23 1 Electrophilic substitution 91 Elimination reactions 137 SUBJECT INDEX Enzyme mechanisms 601 Fragmentation processes 10 Free radicals 20 171 Fulvalenes 362 Fulvenes 361 Furans 455 Gallium compounds 304 Germanium compounds 308 Halogens e.s.r.23 Heterocycles 53 213 441 e.s.r. 25 Hydrocarbon radicals 20 Imidazoles 458 Indium compounds 304 Indole alkaloids 496 Indoles 453 Insertion reactions 323 Isoprenoid alkaloids biosynthesis 581 Isoquinoline alkaloids 492 Isothiazoles 458 Isoxazoles 457 Kolbk reaction 231 Lead compounds 3 18 Linewidths 19 Lithium compounds 284 Lupin alkaloids 491 Lycopodium alkaloids 504 Lysozyme 606 Magnesium compounds 288 Mass spectrometry 7 Medium rings 382 Mercury compounds 291 Molecular basicity 67 Molecular complexes 59 Monoterpemes 412 Natural products 60 Nitrenes 166 Non-classical carbonium ions 115 Nucleic acids 535 Nucleophilic aromatic substitution 97 Nucleosides 540 Nucleotides 540 Olefins 190,268,322 Optical rotatory dispersion 35 Orbital symmetry correlations 143 684 Subject Index Organometallic compounds 283 Reaction mechanisms 67 Oxidation methods 266 Reduction methods 261 Oxidations 333 Relaxation theory 19 Ribonuclease 634 Palladium complexes 335 Ring synthesis 383 Peptides 514 RNA 551 Phosphorus esters 81 Sesquiterpenes 414 Photochemistry 187 Sesterpenes 426 of nucleic acids 546 Silicon compounds 308 Photo-oxidation 218 Spectroscopy e.s.r.17 Piperidine alkaloids 490 Steroid alkaloids 504 Polar additions 141 Steroids,409 Polycycles 370 biosynthesis 589 Polyketides biosynthesis 593 Substituent effects 87 Porphyrins 452 Substitution reactions 128 Prebiotic studies 514 535 Sydnones 459 Prostaglandins 405 Symmetry rules 36 Proteases 612 Pulse radiolysis 223 Terpenes 409 Purines 537 biosynthesis 585 Pyrans 469 Thallium compounds 304 Pyrazoles,459 Thermolyses 392 Pyridine alkaloids 490 Thietans 450 Pyridines 462 Thiirans 446 Pyrimidines,478 537 Thiophens 455 Pyrroles 451 Tin compounds 312 Pyrrolizidine alkaloids 489 Transannular reactions 368 Triterpenes 427 Quinolines 464 Troponoids 211 Quinone methides 377 Quinones 210 1 Zinc compounds 290

ISSN:0069-3030    

DOI:10.1039/OC9686500683

出版商:RSC    

年代:1968

数据来源: RSC