ORGANIC CHEMISTRYI. INTRODUCTIONAMONG the topics reviewed in the theoretical section of this Report are thechemistry of carbanions and carbenes, where valuable stereochemical in-formation has recently been obtained, and oxidation by ch-amic acid,permanganate, and other oxidants.There has been much progress in the assignment of absolute configur-ations. Conformational, mechanistic, and theoretical approaches to thisproblem are discussed. Much promise attaches to a procedure for the com-putation of molecular rotations from the polarisabilities of the componentsof asymmetric groups. A second example is recorded of a compound owingits optical inactivity to a four-fold alternating axis of symmetry. Theimportance of entropy in determining equilibria and rates of isomerisationhas been emphasised in a number of investigations.Among general synthetic methods, hydroboronation, followed by re-placement of boron by hydroxyl (H,O,) or hydrogen (H+) affords, respec-tively, means for anti-Markovnikov hydration and non-catalytic reductionof olefins.Serviceable procedures are described for stereospecific synthesisof trisubstituted ethylenes, and for repeated chain-lengthening by three-carbon units and by isoprene residues.Advances continue in the synthesis and identification of naturally occur-ring unsaturated acids. Much work has been done on acetylenes, on theone hand in exploiting the reactivity of halogeno- and alkoxy-acetylenes,and on the other in elucidating the structures of natural poly-ynes.Tri-merisation of hexa-1,5-diyne gives a partly conjugated cyclo-octadeca-hexayne, which by prototropic rearrangement followed by reduction givescyclo-octadecanonaene. This monocyclic olefin with 18 conjugated x-elec-trons would, according to Hiickel’s (4n+2) rule, be expected to have aromaticcharacter; it is at least not evidently unstable.Observations on non-benzenoid aromatic systems include niuch work oncyclopentadienyl anions and tropylium cations, the production of tetra-methylcyclobutadiene as its complex with nickel chloride, and the prepar-ation of new metallocenes. Several new boron-containing hetero-aromaticsystems have been described, as also has the first isothiazole (1-thia-2-aza-cyclopentadiene) .A new biosynthetic theory challenges the view that alkaloids arise in themain from amino-acids.Total syntheses of colchicine, cinchonamine, anddihydrocorynantheine are reported. The constitution and configuration ofaconitine have been elucidated.Recent developments in the chemistry of nitrogen-containing derivativesof sugars, of sugar anhydrides, and of deoxy- and branched-chain sugars arereviewed.New amino-acids have been isolated of the most diverse structures,including the first naturally occurring yyrazole. A process has been devise160 ORGANIC CHEMISTRY.which breaks a peptide chain at a tyrosine or tryptophan residue. Muchimportant work has been done on amino-acid sequences in normal andabnormal hanoglobin and on the structure and synthesis of biologicallyactive peptides.A total synthesis of coenzyme A has been effected.P. B. D. DE LA M.T. s. s.2. THEORETICAL ORGANIC CHEMISTRYEiectrophilic Aromatic Substitution.-This subject was not reviewed lastyear, and so the present report covers both 1955 and 1959.General theory. In recent years, the reactivity of aromatic compoundsto electrophilic reagents (e.g., X+) has generally been discussed in terms ofthe Wheland transition state (1) or a related unsymmetrical structure inwhich the C-X bond is only partly formed and in which the cyclic delocal-isation of the x-electrons is partly retained.l These approaches do notdistinguish between the effects of x-electrons in different molecular orbitals.At the same time there has been a growing body of evidence, supplied(1) (2)mainly by K.Fukui and his collaborators,2 indicating a relation betweenthe orientation of electrophilic substitution and the x-electron distributionin the highest filled molecular orbital of the aromatic compound. Relatedcorrelations have been made for nucleophilic and homolytic reactions, andthe whole has been termed the frontier orbital theory of aromatic sub-stitution.2 This approach has recently met considerable criticism,3 but itsimplication that the higher-energy molecular orbitals of the aromatic com-pound are particularly important in determining the reactivity has appar-ently influenced several new interpretations of the reaction path.435 Oneof these, by R. D. Brown,5 starts with the assumption that aromatic sub-stitution proceeds through two unsymmetrical charge-transfer complexes,e.g., (2) ; calculations suggest that in these complexes the charge is effectivelytransferred to the aromatic ring and the reagent is above one of the carbonatoms.For certain reactions, including nitration, it is assumed that thefirst step (a) is rate-determining, and calculations on this basis are in goodagreement with the relative reactivities in a series of polycyclic hydro-1 M. J. S. Dewar, Ann. Reports, 1956, 53, 133.3 K. Fukui, T. Yonezawa, and C. Nagata, J . Chem. Phys., 1957, 26, 831; K. Fukui,T. Yonezawa, C. Nagata, and H. Shingu, &id., 1954, 22, 1433, and references therequoted.K. Fukui, T. Yonezawa, and C. Nagata, J . Chem. Phys., 1959, 31, 550, and follow-ing letters.4 K.Fukui, T. Yonezawa, and C. Nagata, Bull. Chern. SOC. Japan, 1954, 27, 423;J . Cbem. Phys., 1957, 27, 1247; S. Nagakura and J. Tanaka, Bull. Chem. SOC. Japan,1959, 52, 734.6 R. D. Brown, J., 1959, 2224THEOKETICAL ORGANIC CHEMISTRY. 161carbons.6 However, rather similar results can be obtained by the calcul-ation of localisation energies (cf. ref. l), and methods for such calculationscontinue to be developed.'The equilibrium studies by Mackor and his co-workers * throw some lighton the possible intermediates in aromatic substitution. In ionising solvents(dimethyl sulphate, nitrobenzene, etc.) some aromatic compounds (ArH)react with Lewis acids (e.g., BF,, SO,) to form the corresponding aromaticcations (ArH+); in solvents of lower dielectric constant, addition canoccur to form compounds of type (3).In sulphuric acid alone, oxidationH H(3)to the cation is now known to occur,phatic character " of the CH, group(4)as well as protonationin compounds related(4). The " ali-to (4) has beenindicated by nuclear magnetic resonance measurement^.^Empirical rules related to the Hammett equation continue to be de-veloped. The " selectivity relationship " between the partial rate factorsfor substitution at the m- and the $-position of a monosubstituted benzenederivative has been re-examined for substitution in toluene lo and appliedwith less success to substitution in t-butylbenzene.ll Further considerationhas been given to the Hammett o-values12 and to their interpretation interms of separate contributions from the inductive and conjugative inter-action of substituents with the aromatic ring.ls The application of theo+-values to electrophilic substitution has been discussed in detail l4 anda critical analysis of the above approximations has been p~b1ished.l~At the recent Hyperconjugation Conference, de la Mare presentedevidence for the importance of O-H and N-H hyperconjugation in thereactions of phenols and amines,16 and in other papers the reactivity of thealkylbenzenes was discussed with special reference to the relative importanceof C-H and C-C hyperc~njugation.~~ Further work has been done on thesteric inhibition of mesomerism in substituted benzene derivatives.l*R.D. Brown, J., 1959, 2232.D.Peters, J., 1958, 1028.W. I. Aalbersberg, G. J. Hoijtink, E. L. Mackor, and W. P. Weijland, J., 1959,C. MacLean, J. H. van der Waals, and E. L. Mackor, Mot. Phys., 1958, 1, 247.lo L. M. Stock and H. C . Brown, J . Amer. Chem. SOC., 1959, 81, 3323.l1 L. M. Stock and H. C. Brown, J . Amer. Chem. SOC., 1959, 81, 5621.l2 M. M. Fickling, A. Fischer, B. R. Mann, J. Packer, and J. Vaughan, J . Amer.la R. W. Taft and I. C. Lewis, J . Amer. Chem. SOC., 1958, 80, 2436; 1959, 81, 5343,l4 H. C. Brown and Y . Okamoto, J . Amer. Chem. SOC., 1958, 80, 4979.l5 H. van Bekkum, P. E. Verkade, and B. hl. Wepster, Rec. Trm. chim., 1959, 78,l6 " Conference on Hyperconjugation," Pergamon Press, 1959, p. 126.l7 R. W. Taft, I. C. Lewis, ref. 16, p. 24; E. Berliner, 09.cit., p. 143.l8 T. C . van Hock, P. E. Verkade, and B. M. Wepster, Rec. Trav. chim., 1958, 77,559; H. Kafod, L. E. Sutton, P. E. Verkade, and B. M. Wepster, ibid., 1959, 79, 790.REP.-VOL. LVI F3049, 3055.Chem. SOC., 1959, 81, 4226.5352.815I 62 ORGAN I C CHEM I SllZY.Hydrogen-isotope exchange. Recent work is consistent with the theorythat hydrogen-isotope exchange in aromatic compounds can occur througha preliminary protonation, as suggested by Gold and Satchell and illustratedbelow. Thus the rate of detritiation of (~-~H)-p-cresol in D,O is greater( A 1 mechanism).than that in H,O by a factor of 1.6 (both media contained 4~-hydrochloricacid).l9 The variation of rate with composition in H,O-D,O mixtures isnot linear and is as expected for a preliminary protonation equi1ibri~m.l~The effect of zinc chloride and stannic chloride on the rate of hydrogen-isotope exchange of $-deuteroanisole in acetic acid containing hydrogenchloride parallels the effect of these salts on the protonation of anilinederivativesm However, there is also evidence that exchange can occur bya mechanism involving a Bronsted acid in the rate-determining step.Kresgeand Chiang 21 have shown that the detritiation of labelled 1,3,5-trimethoxy-benzene in acetate buffers is catalysed by molecular acetic acid, and thedetritiation of labelled 1,2,3-trimethoxybenzene in trifluoroacetic acid givessome evidence for the same effect.22 The importance of this mechanism(presumably A-SE2) (cf. ref. 23) relative to the A 1 mechanism mentionedabove is not yet understood, and it is possible that some of the facts usedin support of the A1 mechanism may permit other interpretation~.~3Primary hydrogen-isotope effects in these exchanges have been studiedby comparing the relative rates of displacement of deuterium and tritiumby h y d r ~ g e n .~ ~ , ~ ~ The values of kDlkT are all about 2, but their relativevalues show evidence of an increase with the reactivity of the positionsubstituted.Partial rate factors 25 have been obtained for the deuteration of alkyl-benzenes in trifluoroacetic acid containing D,O. Some work has beenreported on the orientation and rate of deuteration of benzene derivativesin the presence of platinum.26 Substituent effects have been reported forthe protodetritiation of a number of benzene derivatives in several acidicmedia, including aqueous sulphuric acid and trifluoroacetic a ~ i d .~ 7 Second-ary isotope effects (studied by comparing -CH3 and -CD, substituents) donot appear significant in aromatic hydrogen-isotope exchange.28Is V. Gold, R. W. Lambert, and D. P. N. Satchell, Chem. and Ind., 1959, 1312.2O D. P. N. Satchell, J., 1958, 1927, 3910.21 A. J. Icresge and Y . Chiang, J . Amer. Chem. SOC., 1959, 81, 5509.2z D. P. N. Satchell, J., 1958, 3904.29 F. A. Long and M. A. Paul, Chem. Rezi., 1957, 51, 935; cf. L. Melander and P. C.24 S . Olsson, Zrliiv Kemi, 1959, 14, 85.25 W. M. Lauer, G. W. Matson, and G. Stedman, J . Amer. Chem. SOC., 1958, 80,26 W. G. Brown and J.L. Garnett, J . Amer. Chem. SOC., 1958, 80, 8272.27 C. Eaborn and R. Taylor, Chem. and IBd., 1959, 949.28 A. J. Kresge and D. P. N. Satchell, Tetrahedron L e f f e r s , 1959, 13, 20; W. &I.Myhre, Arkiv Kemi, 1959, 13, 507.6433, 6437, 6439.Lauer and C. B. Koons, J . Org. Chem., 1959, 24, 1169THEOI<BTICAL ORGANIC CHEhIISTRY. 163'I'hie zeroth-order nitration obtained by E. I>. Hughes, SirChristopher Ingold, and others in nitromethane has now been extended tothe N-nitration of N-methyl-2,4,6-trinitroaniline 29 and to the O-nitrationof methyl alcohol, 4-nitrobenzyl alcohol, ethylene glycol, trimethyleneglycol, and glycerol.30 The zeroth-order rates are the same as for C-nitration,and the interpretation is also the same-a slow rate-determining formationof NO,+ followed by a fast reaction with the substrate.On the additionof sufficient water, the kinetic form of these reactions becomes first-orderwith respect to the substrate, and the relative reactivity of these substratesand of water towards NO2+ can then be obtained. Values on the scalebenzene = 1 are : H,O, 0.1--0*01; N-methyl-2,4,6-trinitroaniline, 1.4;methyl alcohol, 30.The comparison of the rates of nitration and l*O-exchange in aqueoussolutions containing nitric acid has now been extended to the nitration of2-mesitylethanesulphonic acid 31 (the most reactive substrate yet studiedin this way); the nitration still appears to involve NO2+. A kinetic studyof the nitration of benzene by nitric acid in acetic anhydride is inconsistentwith nitration by dinitrogen pentoxide and suggests that this reactionoccurs by the rapid formation of NO,+ in a pre-equilibrium step; thereaction is catalysed by added sulphuric acid and anticatalysed by nitrateions.32 The difference in isomer proportions sometimes observed betweennitrations in acetic anhydride and in sulphuric acid has been ascribed33to the increased electrostatic interaction between the electrophilic reagentand the molecular dipole of the substrate as a result of the low dielectricconstant of acetic anhydride.The orientation of nitration has been re-examined for substitution intoluene,M +-nitrotol~ene,3~ o-nitrotol~ene,~~ ethyl ben~oate,~' and benzo-nitrile.38 In the nitration of ethyl benzoate, the entropy of activation isthe same for substitution a t the o-, m-, and +-positions.37 In the nitrationof benzonitrile the o : p-ratio is 3-3; this is discussed with reference to themechanism by which -T substituents give high o :$-ratios (see also p.238).Partial rate factors have been published for the nitration of diphenyl-methane, fluorene, diphenyl ether, dibenzofuran, diphenylamine, carbazole,39and various nitro biphenyl^.^^ The partial rate factors for 4-nitrobiphenylNitration.hexanones besides the expected epoxides. Oxidation of cyclohex-2-enonewith trifluoroperoxyacetic acid 33 affords 2-hydroxyadipic acid, via theexpected hex-2-enolactone. Perhydrofluorenone is produced on thermalreaction of carbon monoxide and cy~lohexene,~4 and cyclopentene behavesanalogously.(29) (30) (31) (32)Pyrolysis of xanthates 35 or acetates 36 of tertiary alcohols of the type (30)yields both exo- and endo-cyclic olefins.The latter type of reaction involvesa cis-elimination.37 Pure methylenecycloalkanes are formed 38 whenhydroxy-acids (31; n = 1 4 ) are heated with copper in quinoline. 3,6-Dimethylenecyclohexene arises 39 from the pyrolysis of the acetate (32).1,2,4 and 1,3,5-Trimethylenecyclohexane and 1,3,5,7-tetramethylenecyclo-octane are produced by catalytic cyclopolymerisation of allene; and allenewith acetylene cyclopolymerises, according to the conditions, to 3,5- or3,6-dimethylenecyclohexene and 3,5,7-trimethylenecyclo-octene. The cyclo-hexene derivatives readily arornati~e.~~Hydration of cyclic olefins, via alkylborons, involves cisaddition of theelements of water.41 Cyclohexa-3,5-diene-l,2-diol (" benzene glycol ")has been prepared 42 from 3,4,5,6-tetrachlorocyclohexane-I,2-diol.l-Formylcyclo-hexene and -pentene have been togetherANSELL : ALICYCLIC COMPOUNDS. 223The action of zinc, in the presence of EDTA, on pentaerythritol tetra-bromide produces53 pure spiropentane.The spiran (42) is formed by theaddition of tetracyanoethylene to the diene (43; R = H), but with the diene(43; R = Me) addition occurs to the endocyclic double bond to form thebicyclo [2,2 ,O]hexane (44) .M Similar reactions are described by Blomquistand Mein~ald.~5 Syntheses of 6,9-methylenespiro[4,5]decane and spiro[5,6]-dodecane 66 are reported.Me.C(CHBr .CO,Et ) 2----.- ----_HOIC OZH (47)ICH,.CO,EtCOZH(46)The reported 57 formation of three isomeric bicyclobutanecarboxylicacids (45) from the ester (46) could not be sub~tantiated.~~ The structure(47) of the " cage compound " derived from one such acid is therefore ques-tionable. Ethyl bicyclo[l,l,0]butane-l-carboxylate, obtained from ethyl3-bromocyclobutane-l-carboxylate and triphenylmethylsodium, is the firstauthenticated member of this series.59The dione (48) is readily available 60 from methyl vinyl ketone and2-methylcyclopentane-lJ3-dione.Silver perchlorate with the chloride (49)forms the perchlorate of the ion (50).Ph. .(48) (49) (50)Claisen rearrangement of the ether (51) to the aldehyde (52;13 = CH,CHO) is a stereospecific method62 of introducing an angular sub-stituent. 9-Acetyldecalin and the ketone (52; R = COMe) are amongthe products from Friedel-Crafts acetylation of decalin.63224 ORGANIC CHEMISTRY.Norcaradienecarboxylic acid (and the derived nitrile) yield 64 phenyl-acetic acid when heated with sulphuric acid, and the dichloride (53) yields 65cycloheptatriene and toluene on pyrolysis.Dehydrative cyclisation of 2-3'-oxoalkylcyclohexanones yields bi-cycle [3,3,1] nonenones .66The free-radical halogenation of norbornane 67 gives mainly the 2-halide.Bromination of both norbornane-exo- and -endo-2-carboxylic acid yieldsem-2-bromobornane-l-carboxylic acid, whereas either acid chloride yieldsexo-2-bromonorbornane-endo-2-carboxylic acid.68 The novel rearrangementof 5-nitronorbornene to the oxazinone (54) has been des~ribed,~~ and amechanism proposed for it.The adamantane derivative (56) has been obtained '* by condensationof 4-dichloromethyl-4-methylcyclohexa-2,5-dienone and acetonedicarboxylicester, followed by ketonic hydrolysis to the diketone (55) and condensationwith nitromethane.The ketone formed by the action of boron trifluoride on endrin (57a) isconsidered'l to have structure (58), and not structure (57b) as suggestedprevio~sly.~~The sign and magnitude of optical rotation of some cyclic compoundsmay be estimated 73 by the use of the principles of conformational asym-metry and certain empirical constants. a-Axial and a-equatorial chloro-and bromo-ketones can be distinguished by measurement of their optical134 M, J.S. Dewar and C. R. Ganellin, Chem. and Ind., 1959, 458.65 €3. E. Winberg, J. Org. Chem., 1959, 24, 264.66 S. Julia and D. Varech, Bull. Soc. chim. France, 1959, 1127.67 E. C. Kooyman and G. C. Vegter, Tetrahedron, 1959, 4, 382,68 W. R. Boehme, J . Amer. Chem. SOC., 1959, 81. 2762; H. Kwart and G. Null,69 W. E. Noland, J. H. Cooley, and I?. A. McVeigh, J . Amer. Chem. Soc., 1959, 81,70 H. Stetter and J. Mayer, Angew. Chem., 1959, 71, 430.71 R. C. Cookson and E. Crundell, Chem. and Ind., 1959, 703.73 E. J . Skerrett and E. A. Baker, Chem. and Ind., 1959, 639.7 3 J . H. Brewster, J . Amer. Chem. SOL, 1959, 81, 6483, 5493.ibid., p. 2765.1209ANSELI. ALICYCLIC COMPOllNDS. 326rotatory disper~ion.'~ 2-Fluorocyclohexanone exists 75 in the theoreticallyless stable axial conformation. The absolute configurations of cis- and trans-( a ) x = 60( b ) X =(57)1,4dimethylcyclohexane have been established 75a by correlation with meso-and racemic-ad-dimethyladipic acid respectively.The cyclohexa-1 ,Pdiene molecule is considered 78 to be folded along the3,6-axis.The preferred conformations for cycloheptane (59) and (60) andcyclo-octane (61) and (62) have been predicted, and the last pair sub-stantiated by evidence based on the dipole moments of cyclo-octanonederivative^.^^ The conformation (59) has also been deduced on spectro-scopic grounds and those of cyclononane and cyclodecane have been dis-cussed.78 cis-Cyclodecene is less stable than its tra~~s-isomer.~~ The four-membered ring in pino- or isopino-camphone is consideredso to be planar,the strain of the system being accommodated in the six-membered ring.Further examples 81 of the stereospecificity of micro-organisms arepresented, e.g., A9-octalin-1,5-dione is reduced stereospecifically by CurvuZariafalcata and Rihizopus nigricans to (55)-5-hydroxy-As-octal-l-one.The stericeffect of 4-substituents in the mercuration of cyclohexene is reported; 82thus replacement of a l-hydroxy-l-methylethyl group by an isopropylgroup reverses the direction of addition. Norbornane-elzdo-2-carboxylicacid has been resolved and stereochemically correlated with the exo-isomerand with norborn-5-ene-exo- and -endo-2-carboxylic acid.On the basis of ultraviolet and infrared absorption 2-, 3-, and 4-acet-amido- and -benzamido-pyridine exist predominantly in the acylamino-form.84 o-, m-, and p-Aminophenyl 4-pyridyl ether (56) are rearranged byacids to N-4-pyridyl-o-, -m-, and -$-aminophenol (57). An essential stepin the proposed mechanism for the transformation is quaternisation of thepyridine-nitrogen atom.%With sodium hydrogen sulphite some pyridine quaternary salts yieldaddition compounds which are readily decomposed by alkali into alkyiaminesand diketones; the latter cyclise under acid conditions to phenoIs, asillustrated.s6+ NHzMe Me MeThe addition of ammonia to glutaronitrile yields glutarimidine (58).The imino-groups are reactive, undergoing displacement reactions with, forexample, water, hydroxylamine, and aniline.Glutarimidines are regardedas di-iminopiperidines or amino-imino-piperidehes, and not as diamino-dihydropyridines.87 Glutardialdehyde and ammonium cyanide react inaqueous solution, to yield 2,6-&~yanopiperidine.~82 Z. Arnold, Experientia, 1959, 15, 415.83 K. Mecklenborg and M. Orchin, J . Org. Chem., 1958, 25, 1591.84 R. A. Jones and A. R. Katritzky, J., 1959, 1317.85 D. Jerchel and L. Jakob, Chem. Ber., 1959, 92, 724.86 R. LukeS and J. Jizba, CoZZ. Czech.. Chem. Comm., 1959, 24, 1868.87 J. A. Elvidge, R. P. Liastead, and A. M. Salaman, J., 1959, 208.88 R. A. Henry, J . Org. Chem., 1959, 24, 1363PINDER: HETEROCYCLIC COMPOUNDS. 26 1Dieckmann cyclisation of the ester (59f yields, of the two possible isomers,A similar the p-keto-ester (60), the structure being proved by ozonolysis.cyclisation of the appropriate unsaturated diester affords the oxopiperideine(61).89The hydrochlorides of 1-methyl-3- and -4-oxopiperidine crystallise withone molecule of water. Infrared measurements show the water molecule ispresent as a hydrate of the carbonyl group, there being no carbonyl absorp-tion.w With the free bases, the carbonyl infrared absorption band appearsat a slightly higher frequency than that of cyclohexanone.A similar, butmore marked, difference is observed between 1-methyl-3-oxopyrrolidine andcyclopen tan~ne.~lActiphenol, a metabolic product of actinomycetes, is formulated as 4-(2-hydroxy-3,5-dimethylphenacyl)-2,6-dioxopiperidine (62), on the basis ofphysical and chemical properties and partial synthesis from the closelyrelated actidi0ne.9~Oxygen heterocycles. A synthesis of dkyl- and aryl-4-pyrones fromcarboxylic acids and anhydrides, by heating them at 220-300", is de-scribed.93 The photo-dimer of 2,6-dimethyl-4-pyrone has been assigned thecage-structure (63).The compound forms a bis-2,4-dinitrophenylhydrazone,and the other two oxygen atoms are ethereal, on infrared evidence.saturated, and reverts to the pyrone with dilute acid. Treatmentperbenzoic acid yields first a ketd-iactone, and finally two isomeric dilactones :hydrolysis of one of the latter affords isodehydroacetic acid (64) and thecyclobutanetetrol (65). The second dilactone yields, after several stages,the keto-lactone (66).The ultraviolet absorption of the dimer (Amx at233 mp, E 6600) is anomalous, probably having its origin in transannularIt iswithint e r a c t i ~ n s . ~ ~89 H. Plieninger and S. Leonhauser, Chem. Ber., 1959, 9Q, 157991 J , B. Jones and A. R. Pinder, J., 1959, 615.9% R. J. Highet and V. Prelog, Helv. Chim. Actu. 1959, &, 1523.93 J. D. von Mikusch, Angew. Chem., 1959, 11, 311.94 P. Yates and M. J. Jorgenson, J . Amar. Chem. SOL, 1958, 80, 6160.R. E. Lyle, R. E. Adel, and G . G. Lyle, J . Org. Chem., 1959, 24, 342263 ORGANIC CHEMISTRY.2- and 4-Pyrans can be prepared in good yield by the reaction of Grignardreagents with 2- and 4-pyrones respe~tively.~~Both kojic acid (67; X = OH) and a-deoxykojic acid (67; X = H)react with acrylonitrile, the former to yield a nitrogen-free productCl5Hl4O9,~ and the latter a compound formulated on chemical and physico-chemical grounds as (68) ,97The met hylat ion with diazome t hane of 6-substitut ed 3,4-dihydro-2,4-dioxo-2H-pyrans (69) leads to a mixture of the 2-methoxy-4-pyrone and the4-methoxy-2-pyrone , easily distinguished by their infrared spectra.Q8The ethylene ketal (70), derived from benzoin, undergoes an interestingmolecular rearrangement to 2,3-diphenyl-1,4-dioxen (71) with toluene-p-sulphonic acid.9QGeminal cyanonitrosoparaffins react readily with dienes to form dihydro-1,2-oxazines.100Diazines and triazines.Condensation of p-dialdehydes with guanidine,urea, and thiourea yields pyrimidines with a carbon-substituent at position 5,and an amino-, hydroxy-, or thiol group at position 2.The dialdehydes areprepared in situ from a-alkyl-p-chloroacrylaldehydes.101"XHCI.CH=CR'CHO - HO*CH=CR.CHO OHC.CHR.CHO &Reagent: I , (NH,),C=X (X = NH, 0, or S).The chlorinated enols of P-keto-aldehydes, obtained from cyclic ketoneswith the Vilsmeier reagent (H*CO*NMe,-POCl, or H*CO*NMe,-COCl,) , givewith formamide at 180-190" 4,5-disubstituted pyrimidines.lo22 Ha CO. N H2-95 R. Gompper and 0. Christmann, Angew. Chem., 1959, 71, 32.913 C. D. Hurd. R. T. Sims, and S. Trofimenko, .T. Amer. Chem. Soc., 1959, 82, 1684;cf. Ann. Reports, 1958: 55, 291.97 C. D. Hurd and S. Trofimenko, J . Amer. Chem. SOC., 1959, 81, 2430.98 D. Herbst, W. B. Mors, 0. R. Gottlieb, and C.Djerassi, J. Amer. Chem. SOC., 1959,81, 2427.99 R, K. Summerbell and D. R. Berger, J . Amer. Chem. Soc., 1959, 81, 633.100 0. Wichterle and V. Gregor, CoEZ. Czech. Ckem. Cowzm., 1959, 24, 1158.101 L. Rylski, F. Sorm, and 2. Arnold, Coll. Czech. Chem. Comm., 1959, 24, 1667.102 W. Ziegenbein and W. Franke, Angew. Chem., 1959, 71, 628PINDER HETEROCYCLIC COMPOUNDS. 263Malonamides condense with ethyl carbonate in liquid ammonia in thepresence of sodium hydroxide to give good yields of barbituric acids.lo3 Asynthesis of thymidine has been described.104Piperazine and 1-phenylpiperazine find use in the characterisation andidentification of perfluoro-organic acids as salts.lWSeveral 2-cyanopyridines have been trimerised in the presence of base to1,3,5-triazines of basic skeleton (72).A similar reaction with 2-cyano-pyrimidine yields the compound (73). Many of these compounds with6 (72)N A N u (73)ferrous ions yield deep blue chelate complexes.los The chlorine atoms inchloro-1,3,5-triazines can be replaced by fluorine by treatment of the tri-azines with antimony tri- or penta-fluoride; side-chain halogen atoms arcalso replaced.107 phcTo E t NHPh. CH I1f HS\ C=NH ‘“Cx:”’ !!&IEt Et-C I‘C02R NH20 0Sul’kur compounds. Thiourea reacts with trans-ac-ethylcinnamic acid orits ethyl ester to yield a 1,3-thiazan.l0*Butyl-lithium and methyl sulphate eliminate the sulphur atoms pro-gressively from 1,4-dithiins, yielding ultimately acetylenic hydrocarbons.SAXTON : ALK-iLOIDS. 279Uleine, the principal base of A .uulei, contains an exocyclic methylene groupconjugated with an indole ring. Two Hofmann degradations furnished3-ethyl-I-methyl-2-vinylcarbazole, which was degraded to the known3-ethyl-l-methylcarbazole. Since uleine contains C-ethyl and N-methylgroups, the only structure consistent with these results is (20).49 Twoalkaloids related biogenetically to uleine are ellipticine (21; R = Me,R' = H) 50 and olivacine (guatambuinine) (21; K = H, R' =which have been isolated from other Aspidosperma species; ellipticine alsooccurs in Ochrosia species, together with a methoxyellipticine,B and itsconstitution has been proved by a somewhat unconventional synthesis.501,2,3 ,4-Tetrahydro-N3-mcthylellip t kine (U-alkaloid R) 509 54 and ( + ) - 1,2,3,4-tetrahydro-N3-methylolivacine (guatambuine, U-alkaloid C) 52955 occur widelyin A spidosperma species.Ochrosia elliptica also contains elliptine (isoreser-piline) , which is structurally related to ajmali~ine.~~The bark of Hunteria eburnea, which has a marked hypotensive action,contains four alkaloids whose skeletal structures are clearly reminiscent ofaspidospermine. Eburnamine and isoeburnamine are stereoisomers (22) ,and they yield the same lactam, eburnamonine, on oxidation with chromicacid; dehydration of either stereoisomer gives the fourth alkaloid, eburna-menine. Wolff-Kishner reduction of eburnamine (22) , followed by racemis-ation at the asterisked carbon atom (via the tetradehydro-derivative), yieldsthe (&)-base (23), which was identified by total synthesis.56Curare groztp.The Proceedings of an International Symposium oncurare and curare-like agents have been published ; these contain discussionson the chemistry and pharmacology of all types of curare alkaloids.57 Anadmirable and comprehensive review of the calabash curare alkaloidssummarises this field up to the beginning of 1959.57 Diaboline, the tertiarybase which occurs in Strycktnos diaboli, is now known to be the N(a)-acetylderivative of the Wieland-Gumlich aldehyde.68 Most of the investigationsreported during this year have been concerned with establishing earlierstructural pr0posals,5~ and extending and amplifying interconversions inthis series. The structure of fluorocurarine (24) has been confirmed by tworoutes,GO one of which involves a direct synthesis from the Wieland-Gumlichaldehyde (24a; R = OH).Reduction of fluorocurarine with zinc and acid49 G. Biichi and E. W. Warnhoff, J . Atner. Chem. SOC., 1959, 81, 4433.50 R. B. Woodward, G. A. Iacobucci, and F. A. Hochstein, J . Amer. Chem. SOC.,1959, 81, 4435.51 J. Schmutz and F. Hunziker, Pharm. Acta Helv., 1958, 53, 341.52 M. A. Ondetti and V. Deulofeu, Tetrahedron Letters, 1969, No. 7, 1; G. B. Marini-Bettblo and J. Schmutz, Helv. Chim. Ada, 1959, 42, 2146; G. B. Marini-Bettblo andP. Carvalho-Ferreira, Ann. Chim. (Italy), 1959, 49, 869.53 S. Goodwin, A. F. Smith, and E. C . Homing, J . Amer. Chem. SOL, 1959, 81, 1903.54 J. Schmutz and F. Hunziker, Helv.Chim. Acta, 1958, 41, 288.55 P. C. Ferreira, G. R. Marini-Bettblo, and J. Schmutz, Exfierientia, 1959, 15, 179.56 F. Bartlett, W. I. Taylor, and R. Hamet, Comfit. rend., 1959, 249, 1259.57 " Curare and Curare-like Agents," ed. D. Bovet, F. Bovet-Nitti, and G. B.I\llarini-Bettblo, Elsevier, Amsterdam, 1959 ; K. Bernauer, Fortschr. Chem. org. Natur-sioffe, 1959, 17, 183.58 A. R. Battersby and H. I?. Hodson, Proc. Chem. SOC., 1959, 126.59 Cf. Ann. Reports, 1958, 55, 310.60 H. Fritz, E. Besch, and T. Wieland, Angew. Chem., 1959, 71, 126; N7. von Philips-Ilorn, K. Bernauer, H. Schmid, and P. Karrer, Nelzi. Chim. A d a , 1859, 42, 461280 ORGANIC CHEMISTRY.gives a dihydro-derivative by saturation of the 2,16-double bond; in bufferedsolution at pH 5 this intermediate dimerises to give dihydrotoxiferine (24b;R = R' = H).BO The latter has also been obtained from caracurine V,itself obtained by the dimerisation of the Wieland-Gumlich aldehyde.61The analogous dimerisation of the N(b)-metho-salts of this aldehyde yieldsR ' 0 CH * HC'CH.CH 2 R oc1Me, ,OHO\Me H OzC Me CI 0- coCOZH oc- /HOzC Me-$H-CTC!+- CHMeTC- Mea mixture of C-toxiferine I, diacetyl-C-toxiferine I, and N(b)-dimetho-caracurine V; this mixture can be converted without separation intoC-toxiferine I (24b; R = R' = OH) in an overall yield of 62y0.62 Theunsymmetrical bis-quaternary ion (24b; R = H, R' = OH) is the curare-alkaloid C-alkaloid H; this has been synthesised by a mixed condensationof heminordihydrotoxiferine (24a; R = H) with the N(b)-methochlorideof the aldehyde (24a; R = OH).Quaternisation of the tertiary basiccentre in the product then gives (24b; R = H, R' = OH), identical with61 I<. Bernauer, I?. Berlage, W. von Philipsborn, H. Schmid, and P. Karrer, Hetv.Chim. Acta, 1969, 42, 201.68 F. Berlage, K. Bernauer, W. von Philipsborn, P. Waser, H. Schmid, and P.Karrer, Helv. Chim. Ada, 1959, 42, 394SAXTON : ALKALOIDS. 281natural C-alkaloid H. Since photochemical oxidation of the latter givesC-alkaloid G and aerial oxidation in acetic acid-pyridine at elevated tem-peratures yields C-alkaloid F, these two “ dimeric ” alkaloids are alsoavailable by total synthesis.85The CM formulation of C-curarine I has received additional confirmation,since the Hofmann degradation to a ditertiary base can be achieved in twostages, via a monoquaternary ion containing a tertiary basic centre.64Pyrrolizidine Group.-The major alkaloid of Crutalaria anauruides hasbeen identified as l-methylenepyrrolizidine.a The evidence relating to thestructures of jacobine, jacoline, jaconine, and their hydrolysis products hasbeen re-examined and re-interpreted ; the new structures proposed removethe apparent anomalies in the chemistry of these substances.6s Jacobine isnow formulated as the epoxide (25), jacoline is the related 7,8-glycolJ andj aconine is the corresponding chlorohydrin containing the chlorine atom atposition 8.The two acids, jaconecic (26) and isojaconecic (27), are pre-sumed to be formed by nucleophilic attack on the oxide function in (25) bythe free 2-hydroxyl group and hydrolysis of both ester groups; the newformulations are confirmed by the results of the oxidative degradation ofthese two acids. The chlorodilactone, obtained from jacobine by the actionof hydrochloric acid, is the bis-&lactone (28) ; the infrared absorption at1781 cm.-l, earlier regarded as diagnostic of a y-lactone, is also exhibitedby a model bis-8-lactone.66The absolute configuration deduced earlier by Warren and Klemperer forposition 1 in (-)-heliotridane , and therefore in (-)-retronecanone, has beenconfirmed by the stereochemical correlation of the latter with (-)-methyl-succinic acid.67 Accordingly, the absolute configuration at Cc8) in (-)-retronecanone and the pyrrolizidine alkaloids, e.g., monocrotaline, shouldbe as shown in (29) and (30) respectively.This has been corroborated bythe degradation of monocrotaline (30) to the amino-alcohol (31), which wasidentical with that synthesised from ( -)-proline.68 Syntheses of heliotrineand supinine have been reported; heliotrine (32; R = OH, R’ = Me) wasobtained by preferential replacement of the allylic hydroxyl group in helio-tridine with chlorine, followed by reaction with the sodium salt of heliotricacid. Supinine (32; R = R’ = H) was synthesised in an exactly analogousmanner from supinidine and sodium trache1anthate.mPhenanthridine Group.-The constituents of further species belonging tothe family Amaryllidaceae have been examined.’* Of the new bases isolated,parkamine is the principal alkaloid of AmaryZZis parkeri.On the basis of itsconversion by sodium and pentyl alcohol into a mixture of caranine (33;63 F. Berlage, K. Bernauer, H. Schmid, and P. Karrer, Hetv. Chim. Acta, 1969, a,64 V. Boekelheide, 0. Ceder, M. Natsume, and A. Ziircher, J . Amer. Cheem. SOL,*ti C. C. J. Culvenor and L. W. Smith, Austral. J . Chem., 1959, 12, 255.T. A. Geissman, Austrul. J . Chem., 1959, 12, 247; R. B. Bradbury and S. Masa-67 R. Adams and D. Me& J. Amer. Chem. SOC., 1959, $1, 4946.R. Adams and D. Fle3, J . Amer. Chern. SOC., 1959, $1, 5803.6s C. C. J, Culvenor, A. T. Dann, and L. W. Smith, Chern. and Ind., 1959, 20.70 H.-G. Boit and W. Dopke, Naturwiss., 1969, 46, 228, 475; Chem. Ber., 1969, 92,2650.1959, 81, 2256.mune, J.Amer. Chem. SOL, 1959, 81, 5201.m a , 2582282 ORGANIC CHEMISTRY.R = OH, R’ = R” = H), dihydrocaranine, and deoxycaranine (33; R =R‘ = R” = H), and also the formation of anhydrofalcatine lactam by theaction of hot mineral acid, parkamine is formulated as (33; R = OH,R’ = R“ = OMe). Arnaryllidine, which occurs in A . behdonna, is prob-ably the corresponding glycol (33; R = R‘ = OH, R” = OMe).70 TheCH2*O-CO*$-CHMe H? ?R’ ,O & ’CHMe, o \(33)“Ci?3r-R”(32)position of the aromatic methoxyl group in these alkaloids is still sub judice.The position of the phenolic hydroxyl group in pseudolycorine (34; R = H,R’ = Me, R” = OH) has been established by ethylation and degradationto the phenanthridone (35; R = Et, R = Me), which was also obtainedby ~ynthesis.7~ Norpluviine (34; R = Me, R’ = R” = H) and demethyl-homolycorine (36), two new phenolic alkaloids from Lycoris radiata, have theH .OMeM e 0 / 6 0 @ Z e ’ H2Cb ,oa$;HO \ 0 H2C,o \ \(36) R (37) (3 8) 0o ; .- C H 2 -‘?HZ(3 9) HO2C-”‘Mefree phenolic group in the alternative position; the structures of both baseswere proved by degradation to the isomeric phenanthridone (35; R = Me,A comparatively abundant source of the hitherto rare alkaloid crinamine(37; R = H) has been found in several Crinum species, which also containa new alkaloid, 6-hydroxycrinamine (37; R = OH).72 The structure ofthe latter was confirmed by its ready conversion into apohaemanthidine,R’ = Et).7171 S.Uyeo and N. Yanaihara, J . , 1959, 172.72 H. M. Fales, D. H. S. Horn, and W. C. Wildman, Chew. aid Ind., 1959, 1416SAXTON ALKALOIDS. 283and by the rearrangement of its methiodide with alkali, which affordedcriwelline (38).72Diterpene Group.-Atisine has been degraded to a non-nitrogenous acid,which should have the structure and stereochemistry indicated in (39).This product is of interest in connection with the possible stereochemicalcorrelation of atisine with the diterpene~.~~The constitutions of hypognavine 74 and heti~ine,~~ which probablycontain the same ring system, have been discussed. The structure proposedearlier for hetisine 76 has been shown to be untenable, and a modified struc-ture has been suggested.75The combined efforts of two groups of investigators have resulted in theclarification of the structure of aconitine (40; R = Et, R’ = OH, R” =OH).77778 In another outstanding example of the application of X-raycrystallographic methods, Przybylska and Marion have deduced that thestructure and absolute configuration of (+)-demethanolaconinone are as@;: o@f% / *: * - --..f OH OHN E t N*o / 8OH HOOAc NR: I R’ 37OMe ’. OMe OMeCH OMe CH2*OMe CH2.0Me(40) (41) (4 2)given in (41).’8 Consequently, aconitine must be (40; R = Et, R’ = OH,R” = OH) or the isomer in which the benzoyl group is attached to theneighbouring tertiary alcohol function. This formulation explains satis-factorily the non-formation of a carbinolamine ether on oxidation (cf.delcosine and delsoline with OH at C,,), and also the significant reductionin basicity which accompanies the formation of aconitoline (the chromic acidoxidation product), which is an +unsaturated p‘-amino-ketone (ring A asin 41).Pyraconitine, the pyrolysis product of aconitine, behavesin the Wolff-Kishner reduction as an a-methoxy-ketone; hence thisprovides additional evidence for the presence in aconitine of the systemAcO-C-CH(OH) 1 -L The relationship of the nitrogen atom to theI Iacetoxy-group has been confirmed by the pyrolysis of aconitine N-oxide,which affords the nitrone (42); the latter can be reconverted into aconitineby reduction followed by ethylation. This behaviour is best explained bythe presence in aconitine of the system N-C(17)-C(7)-Cts)-OA~, which is73 0.E. Edwards and R. Howe, Proc. Chem. SOC., 1959, 62.74 S.-I. Sakai, Chem. Pharm. Bull. (Japan), 1959, 7 , 55.75 A. J. Solo and S. W. Pelletier, J. Amer. Chem. Soc., 1959, 81, 4439.76 K. Wiesner and 2. Valenta, Fortschr. Chem. wg. Naturstofle, 1958, 16, 62.77 I<. Wiesner, M. Gijtz, D. L. Simmons, L. R. Fowler, F. W. Bachelor, R. I?. C.78 H. Mayer and L. Marion, Canad. J. Chem., 1959, 37, 856; M. Przybylska andBrown, and G. Buchi, Tetrahedron Letters, 1959, No. 2 , 15.L. Marion, ibid., pp. 1116, 1843; D. J. McCaldin and L. Marion, ibid., p. 1071284 ORGANIC CHEMISTRY.sufficiently close to being planar to allow a concerted elimination duringpyrolysis.77 In detailed discussions on the structures of aconitine anddelphinine, Wiesner and his collaborators have established the substitutionpattern in rings c and D, and have deduced the constitution (40; R = Me,R = R" = H) for del~hinine,~~ in which C(3) is a possible alternative sitefor the methoxyl group of ring A.The complex Hofmann degradationwhich delphonine methiodide undergoes is at last given a rational explan-ati0n.7~In the lycoctonine series, the constituents of Imda roykana (fam.Compositae) have been investigated.*O Royline is identical with lycoctonine,and inuline is its anthranoyl derivative. This plant appears to be therichest known source of lycoctonine, and its occurrence here is the first to berecorded outside the Ranunculaceae.go Hydroxylycoctonine, the silveroxide oxidation product of lycoctonine, is now formulated as the semiacetal(43) ; its salts have an anhydronium structure, and contain a 7-keto-gro~p.~~~ OMe(4 4) (4 5 )The lycoctonine-type formulz for deltaline and delpheline have beenrejected, and have been replaced by perhydrophenanthrene structures.82Deltaline is formulated as (44; R = OAc, R' = OH) and delpheline as(44; R = OH, R' = H).The conversion of delpheline into deoxylycocton-ine involves, in the last stage, the acid-hydrolysis of the methylenedioxy-function; it is now suggested that this proceeds with simultaneous Wagner-Meerwein rearrangement of the 13,8-bond into the 13,9-positionJ to give thelycoctonine skeleton.82Miscellaneous.-Nitidine, the alkaloid of Zauzthoxylum nitidzcm (fam.Rutaceae) , is the quaternary benzophenanthridine derivative (45) .a Itoccurs in association with oxynitidine, the related benzophenanthridone,7s K.Wiesner, F. Bickelhaupt, and D. R. Babin, Experientia, 1959, 15, 93; I<.Wiesner, F. Bickelhaupt, D. R. Babin, and M. Gotz, Tetrahedron Letters, 1959, No. 3,11.80 0. E. Edwards and M. N. Rodger, Canad. J . Chem., 1959, 37, 1187; S. K. Tala-patra and A. Chatterjee, J . Indian Chsm. SOL, 1959, 86, 437.81 0. E. Edwards, M. Los, and L. IVfariorl, Proc. Cham. Soc., 1959, 192; Canad. J .Chem., 1959, 37, 1996; 2. Valenta, Chem. and Ind., 1959, 633.8% M. Carmack, D. W. Mayo, and J. P. Ferris, J . Amer. Chem. SOL, 1959, 81, 4110.8s H. R. Arthur, W. H. Hui, and Y . L. Ng, J., 1959, 1840SAXTON : ALKALOIDS. 285Purpurogall in A'0,4Me0 :93 r C02Me C02MeC I H2C0 94 (4 8)0Me0 \Me0 -OH(4 9) 0 OMe (50)Synthesis of colchicine by Eschenmoser and his colleagues.88Reagents: I, (a) Me$04-NaOH; (b) H2-Ni; (c) LiAIH,; ( d ) H3P04-k180.2, (a) HC-C.CO,Me-NEt,-C,Hll*OH, then C,Hll*OK; (b) MeI-K,CO,.3, Chlorornethylmaleic anhydride a t 175O.4, (a) MeOH-H,S04; (b) CH8N2.5, (a) C,HIl.0K-C6H,; (b) NaOH-MeOH.6, (a) OsO,; (b) NaHC0,-MeOH-0,; (c) NaOH; (d) Quartz powder a t 260'.7, (a) p-Me-C,ii,*SO,CI-pyridine; (b) NH,-EtOH a t 100"; (c) NaOH.8, (a) CH,N2; (b) separation of isomeric methyl ethers; (c) N-bromoruccinimide;9, NaOH.(d) NH3-EtOH at 1000.[Caption continued on p .286286 ORGANIC CHEMISTRY.Synthesis of colchicine by van Tarnelen etReagents: i, (a) CH,=CH*CN-KOBU~; (b) Zn-Br*CH,*CO,Me; (c) KOH; (d) dicyclohexycarbodi-imide; (e) CH2N2.ii, Na-NH,-ether.i i i , (a) Cu(OAc),-MeOH; (b) p-Me*C6H,*S0,H-C&t6; (c) N-bromosuccinimide.iv, (a) CH,N2; (b) N-bromosuccinimide; (c) NaN,; (d) H2-Pd; (e) H20-H+.Conversion of (49) into (-)-colchicine (50).*O10, (a) (+)-Camphor-I0-sulphonic acid; (b) Ac20-pyridine; (c) CH2N,.but the latter is probably an artifact.These are the first natural productsto contain this particular orientation of substituents ; the benzophen-anthridine alkaloids previously encountered have substituents in positions 7and 8 in ring A.Actinidia PoZygama contains two physiologically active substances , ofpresumably terpenoid origin. The basic component is actinidine (46) ;the other is a neutral lactone, matatabilactone.84The structure proposed for n~pharidine,~~ deduced from the results ofdegradation, has now been confirmed by the synthesis of (5)-deoxy-nupharidine.86 Nupharamine (47) , a companion alkaloid of the roots ofNuphar japonicum, possesses the same sesquiterpenoid skeleton.87Probably the most outstanding achievements in alkaloid synthesisduring the year under review are two total syntheses of (-)-colchicine,which were carried out according to the reactions outlined in the accompany-ing charL88 Both the Swiss and the American synthesis start from a deriv-ative of trihydroxybenzosuberone, and proceed, by different routes, todeacetylaminocolchiceine (48) , which was used as the relay compound, andthence to (&)-deacetylcolchiceine (49).Since the latter has already beenreconverted into (-)-colchicine (50),s9 the synthesis of the alkaloid iscomplete.J. E. S.10. CARBOHYDRATESAmino-sugars and Other Sugar Derivatives Containing Nitrogen.-The bio-logical importance of amino-sugars is becoming increasingly evident and sotheir chemistry is being rapidly developed.to have structure (1).Methanolysis of the substance resulted in scission at (a) to yield paromamineand an anomeric mixture of methyl paromobiosaminides.2 Paromamine wasshown to be a 2-amino-2-deoxyglucoside. The parent disaccharide (paromo-biosamine) of methyl paromobiosaminide is composed of a 2,6-diamino-2,6-84 T. Sakan:A. Fujino, F. Murai, Y . Butsugan, and A. Suzui, Bull. Chem. Soc.Japan,1959, 32, 315.85 M. Kotake, S. Kusumoto, and T. Ohara, Annalen, 1957, 606, 148.86 T. Kaneko, I. Kawasaki, and T. Okamoto, Chem. and Ind., 1959, 1191.Y. Arata and T. Ohashi, J . Pharm. Sac. Jafian, 1959, '79, 127.88 J. Schreiber, W. Leimgruber, M. Pesaro, P. Schudel, and A. Eschenmoser, Angew.Chem., 1959, 71, 637; E. E. van Tamelen, T. A. Spencer, D. S. Allen, and R. L. Orvis,J . Amer. Chem. SOC., 1959, 81, 6341.89 H. Corrodi and E. Hardegger, Helv. Chim. Actu, 1957, 40, 193.1 T. H. Haskell, J. C. French, and Q. R. Bartz, J . Amer. Chem. Soc., 1959,81, 3482.2 T. H. Haskell, J. C. French, and 9. R. Bartz, J . Amer. Clzem. Soc., 1959,81, 3480.The antibiotic paromomycin has been showOVEREND : CARBOHYDRATES. 287dideoxyhexosyl residue united to position 3 of a D-ribose unit.3 Synthetic2-amino-l,6-anhydro-2-deoxy-p-~-gulopyranose hydrochloride has beenshown to be identical with the amino-sugar derivative isolated from strepto-thricin and streptolin B.4 2-Acetamido-2-deoxy-~-galacturonic acid is acomponent of an antigenic polysaccharide isolated from Escherichia coZL5Much effort is concentrated on developing methods for the synthesis ofamino-sugars and derivatives.Conventional methods have been extendedand new routes devised. Enzymic conversions of N-acetylhexosamines into N-acetylhexosaminic acidsand of glucosamine into galactosamine have beenH,OH reported. The valuable reference compound (2) hasbeen prepared by Stoffyn and Jeanloz 8 who have also+NH, CI- reviewed the methyl ethers of 2-amino-2-deoxy-~ugars.~ The hydrochlorides of D-[ l-14C]glucosamineand ~-[l-~~C]galactosamine have been synthesised lofor use in metabolism experiments, and preparations of 2-acetamido-4,6-0-benzylidene-2-deoxy-glucose l1 and methyl 2-amino-4,6-0-benzylidene-2-deoxy- p-D-glucoside hydrochloride l2 have been described.The alkalineepimerisation of 2-acetamido-2-deoxy-~-glucose has been developed to yieldgram quantities of 2-acetamido-2-deoxy-~-mannose~~ Stereospecific addi-tion of ammonia to D-arabo-tetra-acetoxy-1-nitrohex-l-ene affords N-acetyl-l-deoxy-l-nitro-D-mannosamino1 which is convertible into 2-amino-2-deoxy-o-mann0~e.I~ The L-isomer was prepared similarly. Cramer et aZ.15 havedescribed syntheses of 6-amino-6-deoxy-~-glucose and derivatives thereof,and Muber et a2.le have converted methyl 3-amino-4,6-0-benzylidene-3-(2)T. H, Haskell, J.C . French, and Q. K. Bartz, J . Amer. Chem. Soc., 1959, 81, 3481.4 R. W. Jeanloz, J . Amer. Chem. SOC., 1959, 81, 1956.I<. Heyns, G. Kiessling, W. Lindenberg, H. Paulsen, and hi. E. Webster, Chent.6 L. I. Hochstein, J. B. Wolfe, and H. I. Nakada, J . Amer. Chent. SOC., 1950, 81,F. Maley and G. F. Maley, Biochim. Biophys. Acta, 1959, 31, 577.8 P. J. Stoffyn and R. W. Jeanloz, J . Anzer. Chem. SOC., 1958, 80, 5690.9 R. W. Jeanloz, Adv. Carbohydrate Chem., 1958, 13, 189.10 R. Kuhn, H. J. Leppelmann, and H. Fischer, Annalen, 1959, 620, 15.l1 H. Masamune, T. Okuyama, and H. Sinohara, TGhoku J . Exp. Med., 1958, 68,12 S.Akiya and T. Osawa, Chem. Pharm. Bull (Japan), 1959, 7, 277.l3 S. Roseman and D. G. Comb, J . Amer. Cham. SOL, 1958, 80, 3166; C . T. Spivak14 A. N. O'NeilI, Canad. J . Chem., 1959, 37, 1747.l6 G. Huber, 0. Schier, and J. Druey, HPIv. China. A d a , 1959, 42, 2447.Ber., 1959, 92, 2435,4111.181.and S . Roseman, ibid., 1959, 81, 2403.I?. Cramer, H. Otterbach, and H. Springmann, Chem. BEI., 1959, 92, 384288 ORGANIC CHEMISTRY.deoxy-a-D-altroside into 3-amino-3,6-dideoxy-~-altrose hydrochloride byconventional methods.Reduction of the hydrazino-compounds formed by replacement ofsulphonyloxy-groups in the sugar series provides a convenient approach tothe synthesis of amino-sugars.17 By this method methyl 3,4-0-isopropyl-idene-2-0-tosyl-p-~-arabinoside, lJ2-0-isopropylidene-3 ,5-di-O-tosyl-cc-~-xylofuranose, 1,2-O-isopropy~dene-5-0-tosy~-a-~-xy~ofuranose, methyl 3,5-O-isopropylidene-2-O-tosy~-or~-~-xylofuranoside , and methyl 3,4-O-iso-propylidene-6-0-tosy1-cr-~-galactoside have been converted via the hydr-azino-derivatives into amino-sugar derivatives.ls Crystalline 2-amino-2-deoxy-~-ribose and 2-amino-2-deoxy-~-~yxose (as hydrochlorides) wereprepared by this route.lB In 1938 Peat and Wiggins l9 showed that ammono-lysis of a tosyloxy-group proceeds through an epoxide intermediate when asuitably situated hydroxyl group is available; this results in retention ofconfiguration at the original site of the tosyloxy-group because of two suc-cessive inversions at this point.Using 1,2:5,6-di-O-isopropylidene-3-0-tosyl-D-glucofuranose (3) Lemieux and Chu 2o have now demonstrated that(3) MeCH2-0AcO AcHN Q I I (7)CH~*OACAcO 0 H,OAcAcHN(8)I(4) Me CH2.OH(5)JCH(SEt)ZCH (SO,Et),_2, H o g A c 4 OH HO Q HO NHAcCH2 *OH(9) (6)when the tosyloxy-group is not adjacent to free hydroxyl groups ammono-lysis or hydrazinolysis proceeds with Walden inversion to give a derivativeof 3-amino-3-deoxy-~-allose (4; R = H or NH, respectively).Oxidationwith peroxypropionic acid of 3-acetamido-3-deoxy-~-allose diethyl dithio-acetal(5) [obtained by treating compound (4; R = Ac) with ethanethiol andhydrochloric acid] yielded the cyclic disulphone (6) together with diethyl-17 M. L. Wolfrom, F. Shafizadeh, and R. K. Armstrong, J.Amer. Chem. Soc., 1958,80, 4885.18 M. L. Wolfrom, F. Shafizadeh, R. K. Armstrong, and T. M. Shen Han, J. Amer.Chema. SOC., 1969, 81, 3716.19 S. Peat and L. F. Wiggins, J., 1938, 1810.20 R. U. Leinieux and P. Chu, J . Amer. Chenz. SOL, 1958, 80, 4745OVIJHICNI) CAKHOHY DHA'lXS. 2 89sulplionylme thane and 2-acetamido-2-deoxy-D-ribose.21 The disulphone (6)could also be obtained by the sequence of reactions (7) + (8) ---;)- (9) --+c(6) (1, acetolysis; 2, de-0-acetylation and diethyl dithioacetal formation;3, oxidation with peroxypropionic acid). Both conversions (5) -+ (6)and (9) --t (6) proceed via the unsaturated intermediate (10). Theoxidation of the dithioacetal (9) also gave rise to 2-acetamido-2-deoxy-~-ribose. These results confirm the concIusion of Lemieux and Chu20 thatreplacement of the 3-tosyloxy-group in compound (3) by ammonia andhydrazine residues proceeds with inversion of configuration./SO,Et\SO,Et I HOCH, - 'i' 'i' 'i' CH=C I l lOH OH NHAc i (10)The epoxide route to amino-sugars has been further extended. Com-mencing with methyl 2,3-anhydro-4,6-0-benzylidene-a-~-alloside (1 1) Fosteret aZ.22 have carried out the sequence of reactions (11) ---t (14) to prepare2-amino-l,6-anhydro-2-deoxy-~-~-altropyranose hydrochloride.(14). The0- 0-Ph- CH-O I <&Me Ph . Cti-0 I <>MeLHO q.$oHe 3, Hoqy CI-HO (12)CH2.0H //;/ CH2-0( 1 1 ) O(13) HO HO (14)Reagents: I, MeOH-NH,, followed by N-acetylation. 2, Partial hydrolysis. 3, Total hydro-lysis.ammonolysis of methyl 2,3-anhydro-a- and -p-D-ribofuranoside has beeninve~tigated.~,~* With each anomer attack occurred almost exclusively atposition 3 and in this way methyl 3-amino-3-deoxy-CC- and -P-D-XylOfUran-oside were obtained. In the course of the synthesis of the P-epoxide (16)it was found that treatment of methyl 2-0-benzoyl-3-0-methanesulphonyl-Fi-O-trity~-~-~-xylofuranoside (15; Ms = MeSO,) with hot 80% acetic acid21 B.Coxon and L. Hough, Chem. alzd Id., 1969, 1249.22 A. B. Foster, M. Stacey, and S. V. Vardheim, Acta Chem. Scand., 1958, 12, 1605.23 C. D. Anderson, L. Goodman, and B. R. Baker, J . Amer. Chem. SOL, 1958, 80,24 R. E. Schaub and M. J. Weiss, J . ,4mer. Chem. SOC., 1958, 80, 4683.5247.REP.-VOL. LVI 2!)0 ORG.4NIC CHEMISTRY.resulted, not only in detritylation, but also in ring expansion of some of theproduct to the corresponding pyranoside which after oxide formation andammonolysis afforded methyl 3-amino-3-deoxy-P-~-xylopyranoside (17)The same results were obtained in the a-series.Thus by this novel ring-expansion with retention of configuration all four possible methyl 3-amino-3-deoxy-D-xylosides were obtained. Whitehouse and Kent 25 have preparedcrystalline methyl 2-acetamido-2-deoxy-p-D-glucofuranoside by treating2-acetamido-2-deoxy-D-glucose diethyl dithioacetal with mercuric oxide inmethanol. Aqueous mercuric chloride in the presence of mercuric oxidegives with 2-acetamido-2-deoxy-D-galactose diethyl dithioacetal a mixturefrom which by column chromatography on, successively, carbon, cellulose,and silicate it is possible to separate ethyl 2-acetamido-2-deoxy-l-thio-a-and -p-D-galactofuranoside and -/3-D-galactopyranoside.36 Ethyl 2-acet-CH1.OH (18) (19) (20) (2')Reagents: I, 10,.2, NaBH,. 3, HCI.amido-2-deoxy-l-thio-a-D-galactofuranoside (18) was converted into %amino-2-deoxy-L-arabinose (2 1) (isolated as the hydrochloride) by the annexedsequence of reactions.27Lindberg and Theander 28 have prepared an oxime (23) from the keto-glycoside (22) and this on hydrogenation in presence of Adams catalystCHl-OH2Lo?OPle Ho<o?OMeHOOH HO-N OHafforded methyl 3-amino-3-deoxy-~-~-allopyranoside (24) in 85% yield.Reduction of compound (23) with sodium amalgam yielded methyl 3-amino-3-deoxy-~-~-glucopyranoside (25) in 45% yield ; the presence of the amine(24) in the mother-liquors being demonstrated by paper chromatography andionophoresis.Baer and Fischer 29 have described a novel method for the synthesis of25 M.W. Whitehouse and P. W. Kent, Tetrahedron, 1958, 4, 425.26 34. L. Wolfrom and 2. Yosizawa, J . Arner. Chem. SOC., 1969, 81, 3474.27 M. L. Wolirom and 2. Yosizawa, J . Amer. Chem. SOL, 1959, 81, 3477.28 B. Lindberg and 0. Theander, Acin Chem. Scand., 1959, 13, 1226.28 H. H. Baer and H. 0. L. Fischer, Proc. Nat. Acad. Sri. C . S . A . , 1958, 44, 991;J . Amer. Chem. SOC., 1959, 81, 5185OVEKEND : CARBOHYDRATES. 29 I3-amino-3-deoxyribose. Cleavage with periodate of the methyl pento-pyranosides affords the dialdehydes (26) and (27) and it has been shown that10,- Meo-CHO 0 (26)Methyl /I-D- or a-L-pentopyranoside j-CHo i i CH2-'10,- CHO T H-C-OMeMethyl a-D- or p-L-pentopyranoside 0 (27) i"" 1 CH,-these can be cyclised with nitromethane and sodium methoxide, in yieldsup to 40%, to give intermediates which can be converted into amino-sugars.Thus the dialdehyde (26) is so converted into the sodium salt (28) of the3-acinitro-pyranoside which on dry acidification by grinding with solidpotassium hydrogen sulphate gives mainly methyl 3-deoxy-3-nitro-p-~-ribopyranoside (29) ; the configuration of this is shown by catalytic reduc-tion to a methyl 3-amino-3-deoxypentoside (30) from which crystalline3-amino-3-deoxy-~-~bose hydrochloride (31) can be obtained by hydrolysiswith hydrochloric acid. Reduction of the synip (29) remaining after < '?OMe O?OMe '?OMe '?OHHOOCN OH OIN OH HIN OH H,N+ OHCI' + 0- N3+(28) ( 2 9 ) (30) (31)crystallisation afforded up to 5% of methyl 3-amino-3-deoxy-p-~-xylo-pyranoside (32; X = NH,), but so far methyl 3-deoxy-3-nitro-p-~-xylo-pyranoside (32; X = NO,) has not been isolated in pure form from the dry-acidification mixture.The same sequence of reactions has been carried outwith the dialdehyde (27). In this series the amount of the xylose isomerwas greater (up to ll%), but the main final product was 3-amino-3-deoxy-a-L-ribose hydrochloride. Using thiourethane derivatives Baker et nZ.30developed a new synthesis of amino-sugars. Reaction of the sodio-derivative of methyl 4,6-O-benzylidene-cw-~-glucoside with phenyl iso-thiocyanate afforded an amorphous 2-phenylthiourethane (33) (53%) andthe crystalline %-isomer (34; R = €3) (21%).The 2-O-methanesulphonylderivative (34; R = Me-SO,), on treatment with methanolic sodium meth-oxide, underwent ring closure to give compound (35) whence alkaline hydro-lysis yielded methyl 2-anilino-4,6-O-benzylidene-2-deoxy-a-~-rnann0~ide30 B. R. Baker, K. Hewson, L. Goodman, and A. Renitez, J . Amer. Chem Soc.,1958, 80, 6577292 ORGANIC: CHIIMISTKY.(36). In view of Liebermann’s results 31 on the S-alkylation of the N-phenyl-thiourethane (37) to compound (38) it is surprising that the alkylation of0-Q”‘ 1 <aMe ]3c+) O-C*NHPhI {+ 1HO Ph-CH-0 Ph * C H - 0 OMe0 ORI(32) (33) SZC-NHPh (34)0- 0-Ph-CH-0 OMe Ph-CH -0(35) (36)the derivative (34; R = MeoSO,) proceeded by nitrogen-attack to lead tocompound (35) rather than by sulphur-attack to give compound (39).Onenoticeable difference in the two procedures is Liebermann’s use of the silversalt of the urethane (37) in contrast to the sodium salt used in the cyclisationof the 2-O-methanesulphonyl derivative (34; R = MeOSO,). This syntheticPhaNH*C.OEtII5(37)Ph.N=C*OEtISMe(38)pathway, whereby a 1 ,Z-frans-glycol system of a sugar can be convertedinto a 1,2-cis-amino-alcohol system, has considerable potentialities. Byuse of N-substituted thiourethane groups other than N-phenyl it should bepossible to prepare a variety of N-alkyl-, N-aryl-, and N-arylalkyl-amino-sugars.If an N-benzylthiourethane is employed it should be possible toremove the N-benzyl group by hydrogenolysis, thereby achieving thesynthesis of amino-sugars without a substituent on the nitrogen atom.Synthesis of the interesting disaccharide, 2-acetamido-6-0-(2-acetamido-2-deoxy-~-~-g~ucosy~)-2-deoxy-~-glucose has been described.32The order has been determined 33 in which amino-sugars are eluted withacid from columns of ion-exchange resin. These results coupled with aconsideration of the orders of separation of the N-acetylated sugars on paperchromatograms and pherograms, and of data on the estimation of thesesugars, make it possible to judge more accurately than by paper chromato-graphy alone the identity of certain sugars, without recourse to authenticmaterial as a standard €or comparison.Further investigations of theN-acetylation and estimation of hexosamines have been reported's andsome studies have been made of the effect of electronegative N-substituents81 C. Liebermann, Annalen, 1881, 207, 121.32 W. Yu and T. Hsing-I, Acta Chim. Sinica, 1959, 25, 50.33 M. J. Crumpton, Biochem. J , , 1958, 69, 25P; 1959, 72, 479.s4 G. A. Levvy and A. McAllan, Bioclzem. J., 1959, 71, 127OVEREND: CARBOHYDRATES. 293on the hydrolysis of 2-amino-2-deoxy-glucosides.35 By comparing theproducts of degradation of hexosamines by ninhydrin with those of briefoxidation of the corresponding hexoses by periodate it was concluded thatin both cases the 4-formic esters of the lower pentoses are produced and arehydrolysed to the pentoses.38 Some odd reactions have been noted.Period-ate oxidation of various 3-amino-3-deoxyribofuranosyl derivatives and ofone 3-amino-3-deoxyarabinofuranosyl compound resulted in the uptake oftwice the expected amount of oxidant .37 Two 5-amino-5-deoxyribo-furanosides each reacted with 4 molar equivalents of periodate, but various2- and 3-aminodeoxypyranosides behaved " normally." The bis-aldehydewhich is obtained on oxidation of the corresponding non-amino-furanosidesis neither an intermediate nor the final product of the oxidation of %amino-3-deoxypentofuranosides. Consequently the convenient periodate-molarrotation procedure 38 for the determination of anomeric configuration isinapplicable to nucleosides containing 3-amino-3-deoxypentofuranosylmoieties. Care must be exercised in the selection of the amine-protectinggroup in amino-sugar syntheses when the use of alkaline reagents is con-templated since it has been found 39 that aqueous-ethanolic sodium hydr-oxide converts methyl 2-benzyloxycarbonylamino-2-deoxy-6-O-tosyl-cr-~-glucopyranoside (40) into methyl 2-amino-3,6-anhydro-N2,0*-carbony1-2-deoxy-a-D-glucoside (41).2-Amho-2-deoxy-D-glucose has been transformed into oxazolone andoxazoline derivatives.@ Treatment of the N-benzoyl derivative withacetone and an acid catalyst produces a compound with structure (42).1,3,4,6-Tetra-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-~-glucose witheither titanium tetrachloride or aluminium trichloride-phosphorus penta-,CII 1 I N H-CO.0- CHzPhAcO 0CHI.OAC(43)CHI. OAC(44)35 W. Yu and H. Ching, A d a Chim. Sinica, 1958, 24, 413.36 P. J. Stoffyn, J . Org. Chein., 1959, 24, 1360.37 M. J. Weiss, J. P. Joseph, H. M. Kissman, A. M. Small, R. E. Schaub, and F. J.38 J. Davoll, B. Lythgoe, and A. R. Todd, J., 1946, 833.39 A. B. Foster, M. Stacey, and S. V. Vardheim, Acta Chem. Scand., 1959, 13, 281.40 S. Konstas, I. Photaki, and 1,. Zervas, Chem. Ber., 1959, 92, 1288.McEvoy, J . Amer. Chem. Soc., 1959, 81, 4050294 ORGANIC CHEMISTRY.chloride gives a l-chloro-derivative (43) from which benzyl chloride is elimin-ated to yield the oxazolone derivative (44). From 1,3,4,6-tetra-0-benzoyl-2-benzyloxycarbonylamino-2-deoxy-cc~-~-glucose and hydrogen bromide inacetic acid it is possible to obtain 2-amino-3,4,6-tri-0-benzoyl-l-bromo-2-deoxy-a-D-glucose hydr~bromide.~~ With alcohols both this salt and tbefree base give satisfactory yields of the hydrobromides of 2-amino-3,4,6-tri-0-benzoyl-2-deoxy-P-~-glucosides.The preparation of genuine 2-acet-amido-3,4,6-tr~-0-acetyl-l-bromo-2-deoxy-cc-~-glucose has been outlined; 42this substance rearranges in the presence of water to the known 1,3,4,6-tetra-O-acety~-2-amino-2-deoxy-a-~-gl~~0~e hydrobromide. The glycosyl-ation of acetohalogeno-derivatives of 2-amino-2-deoxy-~-glucose by Grignardreagents has been studied.43 The compound known in the literature as2-benzamido-l,3,4,6-tetra-0-benzoyl-2-deoxy-a-~-glucose is in fact an ano-meric mixture consisting mainly of the p-form; 44 with hydrogen bromidein acetic acid it affords 2-benzamido-3,4,6-tri-O-benzoyl-l-bromo-2-deoxy-a-D-g1UCOSe which will react readily with alcohols and thiols to give p-glucos-aminides or p-thioglucosaminides ; in moist ether it rearranges to 2-amino-1,3,4,6-tetra-O-benzoyl-2-deoxy-cr-~-glucose hydrobromide, the free base ofwhich can be smoothly benzoylated to give authentic 2-benzamido-I ,3,4,ci-tetra-0-benzoyl-2-deoxy-~e~-glucose.~~~~ By using only the first step, amperometric titration of liberated thiol groupspermits the determination of disulphide bonds in intact proteins.3' Oxid-ation of cysteine to cystine by Cu2+ ions,= iodosobenzoate, or tetra-thionate 38b during this fission, converts all cysteine-cystine residues intoS-sulphocysteine in the otherwise intact This sulphite fissionmay prove more useful than oxidation by performic acid for degradingproteins to single peptide chains for sequence determination, since otheramino-acids such as methionine and tryptophan are unaffected.Thebehaviour of cysteine in proteins has been of interest from other viewpoints.%The presence of a 2-thiazoline ring in bacitracin A has given credeme to thesuggestion 39 that these heterocyclic compounds, formed by condensationbetween the thiol group of cysteine and the carbonyl group of an adjacentamino-acid, may be normal features of protein structure. Although gluta-thione rearranges to the thiazoline in strongly acid solution, there is noevidence for cyclisation in solutions less acid than pH 1.40 This observation,as well as a detailed kinetic study of the interconversion of 2-methylthiazoline39 (a) L.Josefsson and P. Edman, Acta Chem. Scand., 1956,10, 148; ( b ) L. Josefsson,34 K. Narita, J . Amer. Chem. SOC., 1959, 81, 1751; J. L. Rabinowitz, Acta Chem.85 L. B. Smillie and H. Neurath, J . Biol. Chem., 1959, 234, 355.313 (a) D. T. Elmore and P. A. Toseland, J., 1957, 2460; ( b ) D. T. Elmore, J., 1959,3152.37 S, J. Leach, Biochim. Biophys. Acta, 1959, 33, 264; J. R. Carter, J. Biol. C h e w ,1959, 234, 1705.38 (a) J. M. Swan, Nature, 1957, 180, 643; (b) J. L. Bailey and R. D. Cole, J . Biol.Chem., 1959,234,1733; ( c ) J.-F. Pechhre, G. H. Dixon, R. H. Maybuky, and H. Neurath,J .Biol. Chem., 1958, 233, 1364; Biochim. Biophys. Acta, 1959, 31, 259; L. Weil andT. S. Seibles, Arch. Biochem. Biophys., 1959, 84, 244.39 K. Linderstram-Lang and C. F. Jacobsen, Compt. rend. Trav. Lab. Carlsberg,Ser. chim., 1940, 23, 289.40 R. B. Martin and J. T. Edsall, Bull. SOC. Chim. biol., 1958, 40, 1763.Arkiv Kemi, 1958, 12, 183.Scand., 1959, 13, 1463ELMOKE : AMINO-ACIDS, PEPTIDES, AND PROTEINS. :JOYand N-a~etyl-2-mercaptoethylamine,4~ has led to the conclusion that 2-thiazoline rings are not likely to be a normal feature of protein structure.Identification of C-terminal residues by fission with hydrazine has beenimproved. The reaction is accelerated by addition of hydrazine salts,42while separation of amino-acid hydrazides from amino-acids is more com-plete if polyacraldehyde, instead of a simple aliphatic or aromatic aldehyde,is added to the solution produced.&The unexpected formation ofcomplexes between N-benzyloxycarbonylamino-acids and their sodiumsalts 44 offers an explanation for discrepant melting points in the l i t e r a t ~ r e .~ ~Fortunately, formation of these complexes can be avoided by addition ofthe alkaline solution of N-benzyloxycarbonylamino-acid to an excess ofacid at 0". N-Benzyloxycarbonyl groups have been used to protect theguanidino-group of arginine 46 and the imidazole ring of histidineJ4' though ,for the latter, N-benzyl groups may be better as they are stable to bases.Removal of N-benzyloxycarbonyl groups from methionine peptides byhydrogen bromide or chloride has been troublesome owing to formation ofsulphonium C O ~ ~ O U ~ ~ S , ~ ~ ~ but addition of ethyl methyl sulphide preventsthis side-reacti~n.~~~ Boiling trifluoroacetic acid, a new reagent for remov-ing N-benzyloxycarbonyl groups, is claimed not to split peptide bonds orcause racemisation.M The chromophoric N-4-phenylazobenzyloxycarbonyland related groups, which can be removed by the usual methods, are usefulvariant^,^^^,^^ especially in syntheses where purification of intermediatesdemands chromatography or counter-current distribution. Although form-amido-acids tend to give racemic peptides, the carbodi-imide methodafforded optically pure products from which the formyl group was removedwith methanolic hydrogen chloride.62 N-Trityl groups may be used in theazide method under carefully controlled conditions,53u but N-tritylamino-acids, prepared by partial hydrogenolysis of their benzyl esters or directlyfrom amino-acids, gave only moderate yields of dipeptides in a variety of41 R.B. Martin, S. Lowey, E. L. Elson, and J. T. Edsall, J . Amer. Chem. Soc., 1959,81, 5089.4a J. H. Bradbury, Nature, 1956, 178, 912; Biochem. J., 1958, 68, 475, 482.43 T. Kauffmann and F.-P. Boettcher, An.nalen, 1959, 625, 123.44 E. P. Grommers and J. F. Arens, Rec. Trav. chim., 1959, 78, 558.46 W. Grassman and E. Wiinsch, Chem. Ber., 1958, 91, 462.46 L. Zervas, M. Winitz, and J. P. Greenstein, J. Org. Chem., 1957, 23, 1515; L.Zervas, T. Otani, M. Winitz, and J. P. Greenstein, Arch.Biochem. Biophys., 1958, 75,290; J. Amer. Chem. SOC., 1959, 81, 2878.47 A. Patchornik, A. Berger, and E. Katchalski, J . Amer. Chem. Soc., 1957, 79,6416; S. Akabori, K. Okawa, and F. Sakiyama, Nature, 1958, 181, 772; F. Sakiyama,K. Okawa, T. Yamakawa, and S. Akabori, Bull. Chem. SOC. .Japan, 1959, 31, 926.48 (a) D. Theodoropoulos and G. Folsch, Acta Chem. Scand., 1958, 12, 1955; D.Theodoropoulos, ibid., p. 2043; (b) R. Schwyzer and C . H. Li, Nature, 1958, 182,1669.49 (a) 0. Gawron and F. Draus, J. Org. Chem., 1958, 23, 1040; (b) R. A. Boissonnasand St. Guttmann, Helv. Chirn. Acta, 1959, 42, 1257.6o F. Weygand and W. Steglich, 2. Naturforsch., 1959, 14b, 472.61 (a) R. Schwyzer, P. Sieber, and K. Zatsk6, Helv. Chim. Acta, 1958, 41, 491;( b ) R.Schwyzer and F. Sieber, ibid., 1959, 42, 972.62 J. C. Sheehan and D.-D. H. Yang, J . Amer. Chem. SOC., 1958, 80, (a) 1164, (b)1158.68 (a) B. M. Iselin, Arch. Biochem. Biofihys., 1958, 78, 532; (b) G. C. Stelakatos,D. M. Theodoropoulos, and L. Zervas, J . Amer. Chem. Soc., 1959, 81, 2884.Peptide Synthesjs.-Protecthg grozlps310 ORGANIC CHEMISTRY.methods.53b A hydrazide may be covered by a N-trityl group when theazide method is to be used in a subsequent step.=t-Butyl esters of amino-acids, although less readily prepared in good yieldthan lower homologues, may occasionally be useful. The ester group can bepreferentially cleaved in presence of a N-benzyloxycarbonyl group byhydrogen chloride in benzene, or both groups may be removed by hydrogenbromide in acetic acid.55 4-Nitrobenzyl esters of amino-acids have beenrecommended in preference to the corresponding benzyl esters, since theirgreater stability to hydrogen bromide facilitates the selective removal ofN-benzyloxycarbonyl groups ; 51b356 hydrogenolysis of the ester group isstraightforward.Excess of alkali should be avoided in the hydrolysis ofN-benzyloxycarbonylpeptide esters, especially when glycine is adjacent tothe N-termin~s,~~ otherwise rearrangement to a hydantoin or urea derivativemay result (this reaction was employed by Wessely for the stepwise degrad-ation of peptides).Several methods have been reported for protecting the thiol group ofcysteine. N-Formyl-2,2-dimethylthiazolidine-4-carboxylic acid, obtainedfrom cysteine by successive condensation with acetone and formylation,gives peptide derivatives from which protecting groups are removed simul-taneously by aqueous-methanolic hydrogen The conventionalS-alkylcysteines have been made in good yield by a simplified method,58while S-(benzylthiomethy1)cysteine offers interesting possibilities, since theprotecting group is removed by mercuric chloride.59 S-(Tetrahydro-2-pyranyl)cysteine, from which the covering group is removed by silver ionsat 0", is unfortunately obtained in poor yield and has two asymmetriccentres.60Formation ofpeptide bond.The value of a method of peptide synthesisdepends chiefly on the yield and optical purity of the product.1a*2c Bodanszkyand du Vigneaud have critically discussed the various methods of formingpeptide linkages and favour the reaction of p-nitrophenyl esters of N-benzyl-oxycarbonylamino-acids with amino-esters.61 The tendency for racemis-ation may be assessed by the synthesis of Z.gly.-L-phe.gly.0Et,62 sincecrystallisation from ethanol at 0" can detect and separate the racemate evenwhen <1% is present.Success with the carbodi-imide method seems todepend on experimental conditions such as temperature and solvent.During the synthesis of the dipeptide derivatives (15; R = H,R' = H or [CHJ,=CO,Et), by the carboxylic-carbonic anhydride pro-cedure, reaction at the urethane-nitrogen atom led to by-products (15 ;R = CH,Ph*O*CO*NH-CH,-CO, R' = H or [CHJ,=CO,Et).@ In addition5* F. Weygand and W.Steglich, Chem. Ber., 1959, 92, 313.55 R. W. Roeske, Chem. and Ind., 1959, 1121.5G H. Schwarz and K. Arakawa, J. Amer. Chent. Soc., 1959, 81, 5691.57 J . A. Maclaren, Austral. J. Chem., 1958, 11, 360.58 D. Theodoropoulos, Acta Chent. Scand., 1959, 13, 383.59 P. J . E. Pimlott and G. T. Young, Proc. Chem. Soc., 1958, 257.Go G. F. Holland and L. A. Cohen, J. Amer. Chem. SOC., 1958, 80, 3765.62 G. W. Anderson and F. M. Callahan, J. Amer. Chem. Soc., 1958, 80, 2902.G3 K. D. Kopple and R. J . Renick, J . Org. Chenz., 1959, 23, 1565; P. SchellenbergM. Bodanszky and V. du Vigneaud, J , Amer. Chem. SOC., 1959, 81, 5688.and J . Ullrich, Chcnz. BPY., 1959, 92, 1256ELMORE : AIMINO-ACIDS, PEPTIDES, AND PROTEINS. 31 1the anhydrides RCO-O*CO,Et are prone to attack by secondary amines atboth carbonyl-carbon atoms, and the ratio of products depends on theamine and the group R, especially their steric effects.64 This may be im-portant in the synthesis of peptides of sarcosine, proline, and hydroxy-proline. The use of ethoxyacetylene in peptide synthesis has been furtherstudied,65 and experimental conditions have been improved.65b TheCH,Ph.OCO*N RCH,*CO*NHCH R’*CO,Et(15)R’*N HaR.CO,H + HCEC-OEt __+ CH,=C(OEt)*O*CO*R -+ R.CO*NHR’ 3- AcOEt(16)Anderson-Callahan test 62 was satisfactory, and the method deserves widerapplication.The mechanism does not necessarily involve formation of ananhydride; an O-acylketen hemiacetal (16) is a likely intermediate, and theO-(N-phthaloylglycyl) derivative [lS; R = o-C,H,(CO),N*CH,] has beenisolated.65u Related methods employ l-chlorovinyl ethyl ether or 1,l-dichloroethyl ethyl ether, and in these cases acyl chlorides are probablyint ermediat es.66It has been reported briefly that 1,l-carbonyldi-imidazole (17) gives agood yield of simple peptides with negligible racemisation at low temper-a t u r e ~ .~ ’ ~ The method depends on the formation of an N-acylimidazole (18)and its reaction with an amino-e~ter.~’~ In an ingenious, related method,reaction between N-acylamino-acid hydrazides and acetylacetone givescrystalline N-acylaminoacylpyrazoles (19 ; Tos = +-C,H,Me*SO,), whichgive peptides with amino-esters.68TO^ - NH-CH R - co .N H R‘EcO, FNP-NJ -EtO’EtO,HO’P 0 *NH.CHR. COzEtReagents: I , RC0,H; 2, R’*NH,; 3, CH,Ac,.Various methods which involve phosphorus compounds have receivedattention. Addition of imidazole during syntheses with tetraethyl pyro-phosphite accelerates the reaction and improves yields; it is suggested that64 N.*\. Leister and D. S. Tarbell, J . Org. Chem., 1958, 23, 1152.88 (a) J. C. Sheehan and J. J. Hlavka, J . 0t.g. Chem., 1958, 23, 635; (b) 13. J. Panne-man, A. F. Marx, and J. I;. Arens, Rec. Trav. chinz., 1969, 78, 487.O6 L. Heslinga and J. F. Arens, Rec. z’rav. chim., 1957, 76, 952.67 (a) G. W. Anderson and R. Paul, J . Anzer. Cheun. Soc., 1958, 80, 4423; (b) T.68 W. Reid and B. Schleimer, i I ~ i n ~ ! l c i ? , 19.58, 619, 43.lf-ielaiid anti G. Schneider, Annulen, 1063, 580, 1.59312 ORGANIC CHEMISTRY.N-(diethoxyphosphino)imidazole (20) is an intermediate.69 Bis-o-phenylenepyrophosphite has been recommended, since it is more readily prepared thantetraethyl pyroph~sphite.~~ Although syntheses of dipeptides were satis-factory, extensive racemisation occurred when N-acylpeptides were used asstarting materials.A thorough examination of the Goldschmidt andLautenschlager method 71 has revealed the same defect.72 A related methodinvolves reaction of phosphoric oxide with diethyl hydrogen phosphite at100” and then addition of an amino-ester. The mixture, which probablycontains a phosphoramidate (21), reacts with an N-acylamino-acid to give adipeptide.73 Advantage can be taken of the reactivity of enol phosphates(22) ZON H-CH WCO-NH R’Z-NHCH R.COIH RNHS(EtO),PO*O*C(OEt)=CH.CO,Et _____t Z*NH*CHR.CODO*PO(OEt)z -WCeH,,*N=C=N.CaH,,Z.NHCHR.CO2H + NH,’CHR’.CO,.C,H,.NO2-~ _______t(23) Z*NHCH R.CO.NH*CH R’CO2*C6H,*NO,-P(Z = CH2PhoO*CO-)to synthesise peptides.N-Acylamino-acids and (l-ethoxy-2-ethoxy-carbonylvinyl) diethyl phosphate (22) give unsymmetrical anhydrides,which react with esters or salts to give peptide derivative^.^^ Since carbodi-imide and unsymmetrical anhydride syntheses are considerably faster thanthose involving p-nitrophenyl esters, tripeptides can be obtained fromamino-acid derivatives without isolation of dipeptide interrnediate~.~~ AnN-acylamino-acid and a @-nitrophenyl ester of an amino-acid (23) arecoupled by one of the faster methods, and, without isolation, an alkyl esterof an amino-acid is added.Di- and tri-peptides of ory-diaminobutyric acidmay be synthesised easily 76 through the key intermediate L-S-amino-l-tosyl-pyrrolid-2-one (24). The aminoacyl insertion reaction has been reviewed :Tos*NHIRCO-NH N-Tos p 1 2R”O.CO.NH.CH.CO.NHR’ c Tos*NH I L4R - co -N H . c H - co - N H R ’ [y,04, R’-NH,.Reagents: I, HBr-AcOH-PhOH; 2, COCl2, then R”OH; 3, R.C02H, [(EtO),P]20;6g G. W. Anderson, A. C. McGregor, and R. W. Young, J . Org. Chem., 1958,23, 1236.70 P. C. Crofts, J. H. H. Markes, and H. N. Rydon, J., 1958, 4250; 1959, 3610.71 S. Goldschmidt and H. Lautenschlager, Annalen, 1953, 580, 68.7* W. Grassmann and E. Wiinsch, Chem. Ber., 1958, 91, 449; W. Grassmann, E.73 G. Schramm and H.Wissmann, Chem. Ber., 1958, 91, 1073.l4 F. Cramer and K.-G. Gartner, Chew. Ber., 1958, 91, 1562.75 M. Goodman and K. C . Stueben, J . Amer. Chem. Soc., 1959, 81, 3980.76 K. PoduSka and J. Rudinger, Coll. Czech. Chew. Comm., 1069, 24, 3449.Wunsch, and A. Riedel, ibid., p. 455ELMORE : AMINO-ACIDS, YEPTIDES, AND PROTEINS. 313more recent experiments have shown that the rearrangement is intra-molecular and proceeds without racemisation.''Cyclopeptides and Related Antibiotics.*-N-Cyclopeptides and antibioticshave been reviewed recently.2eJ Kenner and his have syn-thesised N-cyclopentapeptides from the diastereoisomers ofH .gly . leu. gl y . leu. gly . S . C6H,*N0,-pand correlated the yields with the lengths of the acyclic peptides as deter-mined by measurement of their dielectric increments.Synthesis of N-cyclo-peptides by procedures involving unsymmetrical anhydrides or phospho-amides 79 suffers from the risk of racemisation. One of the most interestingmethods of synthesis is the doubling reaction, in which a reactive ester of apeptide forms a N-cyclopeptide containing twice as many amino-acid residuesas the acyclic peptide. Schwyzer 8o has considered the factors which favourthis reaction. Thus, anti-parallel doubling assists the synthesis of grami-cidin S and of its homologue,81u in which lysine replaces ornithine, giving akind of anti-parallel pleated sheet. The configurations of the amino-acidsenable all their side-chains to be axially disposed. N-Cyclopeptides con-taining 2(2n + 1) residues (n = 1, 2, etc.) can be made by the doublingreaction. Bishomogramicidin S has an antibiotic activity comparable withthat of the parent compound.81 Various acyclic analogues of gramicidinSS2 and an acyclic pentapeptide sequence of tyrocidine A51b have beensyn thesised.A decapeptide corresponding to one of the possible structures for poly-myxin B, has been synthesised and is active against Brucella bronchisepti~a.~~This is a notable achievement, for the synthesis required three differentmethods of protecting amino-groups.The method of Podugka and Rudin-ger 76 may provide an alternative and simpler route to this compound.Rotatory dispersion studies have enabled Konigsberg and Craig@ todelineate partial structures for bacitracin A (25a-e), which may account forthe hitherto ill-defined linkage between phenylalanine and isoleucine as wellas their easy racemisation.Reduction of esterified bacitracin A with lithiumborohydride, followed by hydrolysis, revealed that both carboxyl groups ofL-aspartic acid were blocked, one with the c-amino-group of lysine, and thatthe 8- and y-carboxyl groups of D-aspartic acid and D-glutamic acid respec-tively were free.8577 Ref. 2e, p. 157; M. Brenner and J . P. Zimmermann, HeZv. Chim. Acta, 1958, 41,79 M. Rothe, I. Rothe, H. Briinig, and K.-D. Schwenke, Angew. Chem., 1959, '71, 700.81 R. Schwyzer and P. Sieber, HeZv. Chim. Ada, 1958, 41, (a) 2186, ( b ) 1582.82 B. F. Erlanger, W. V. Curran, and N. Kokowsky, J . Amer. Clzem. SOC., 1958, 80,8s K.Vogler, P. Lanz, and W. Lergier, Experientia, 1959, 15, 334.84 Ref. 2e, p. 226; W. Konigsberg and L. C. Craig, J . Amer. Chem. SOL, 1959, 81,85 D. L. Swallow and E. P. Abraham, Biochem. J., 1959, 72, 326.* Cyclic peptides, in which amino-acids are linked only through peptide bonds, areIf the ring contains an ester or disulphide linkage, the terms467; H. Dahn, R. Menass6, J. Rosenthaler, and M. Brenner, ibid., 1959, 42, 2249.G. W. Kenner, P. J. Thomson, and J. M. Turner, J., 1958, 4148.Ref. 2e, p. 171.1128; 1959, 81, 3051, 3055.3452.called N-cyclopeptides.0- or SS'-cyclopeytide are applied314 ORGANIC CHEMISTRY.Two antibiotics from different strains of Streptomyces are O-cyclo-peptides. Etamycin (26) contains allohydroxy-D-prolinc , a-phenyl-L-I i"' c p ,ssarcosine, and p ,N-dimethyl-L-leucine , which previously were not known tooccur naturally.86 Catalytic hydrogenation of the pyridine ring, followedby opening of the lactone, permitted sequence determination 86u by theQOH ~O-NH-CHB"~.CO-N CO-?Me/ CO-NH-CH CH2 ICHMe coI IO-CO-CHPh *NMe*CO - CHMe.NH*CO-CH*NMeIEtamycin (26) Me2CH-CHMe u:lco*NHEdmaii method. A penetrating degradative study led to the structure (27)for echinomycin, and the authors suggest that the unusual 1,4-dithian ringmay have been overlooked in other polypeptide^.^^Evolidine,ss from the leaves of Evodea xanthoxyloides, is an N-cyclo-85 (a) J.C. Sheehan, €1. G. Zachau, and W. B. Lawson, J . Amel.. Clzem. SOC., 1957,79, 3933; 1958, 80, 3349; ref.2e, p. 149; (b) R. B. Arnold, A. W. Johnson, and A . B.Mauger, J . , 1958, 4466.87 W. Keller-Schierlein, M. Lj. MilhailoviC, and V. Prelog, I€ch. Clzinz. i f cfa, 1959,42, 305ELMORE AMINO-ACIDS, PEPTIDES, AND PROTEINS. 315heptapeptide with the sequence asp.NH,.leu.ser.phe.leu.pro.val. In viewof its amino-acid composition, it would be interesting to lmow if it has anystrepogenin activity (see below).New Peptides.-In addition to ophthalmic and norophthalmic acid, bovinelens contains S-sulphogl~tathione.~~ y-Glutamyl-S-methylcysteine has beenisolated from lima and kidney beans; S-methylation of glutathione, fol-lowed by degradation with carboxypeptidase, gave an identical product andconfirmed the structure. Other new peptides of glutamic acid includey-glutamylvalylglutamic acid 91 from the rush, Junczis conglomeratus, andarginylglutamine 92 from the green alga Cladophora.A peptide from themycelium of Penicillium chrosogenuwz, which is probably a-aniinoadipyl-cysteicylvaline, is of interest because it is biogenetically related to cephalo-sporin N.93 Pencillin may arise by cyclisation of the corresponding cysteinetripeptide to cephalosporin N, followed by exchange of the side-chain for acarboxylic acid. Pyroglutamylglutaminylalanine has been synthesised andis identical with eisenin from the marine alga Eisenia b i c y ~ l i s . ~ ~ The re-lated pyroglutamylglutaminylglutamine,g4b~ gs however, apparently is differ-ent from fastigiatine from the brown alga Peluetia fasfigiata, which wasbelieved to have that s t r u ~ t u r e .~ ~Strepogenins.-A variety of peptides has been synthesised or isolatedfrom protein hydrolysates, having strepogenin activity. Merrifield andWoolley 97 have tentatively suggested that, for high activity, the peptideshould contain at least five amino-acid residues including either serine orcysteine, and the latter should preferably not be N-terminal. The presenceof amino-acids with lipophilic side-chains seems to be advantageous for highactivity. The sequence of amino-acids is not critical, since bothH.ser . his. leu.va1. glu. OH and H .Val. his.glu. ser .leu. OH were highly active .The related peptide, H.thr.his.leu.val.glu.OH was inhibitory to L. caseieg8From hydrolysed casein, a hexapeptide containing no serine or cysteine hasbeen isolated and was highly active.99 The relation between structure andactivity thus seems as elusive as ever.Mypertensins.-Full details have appeared of the syntheses of va15-hypertensins I and I1 (28; X = val), and the repeated and successful useof the carbodi-imide method is noteworthy.loO Tryptic degradation of a88 H.D. Law, I. T. Millar, H. D. Springall, and A. J. Birch, €'roc. Chenz. Soc., 1958,198.89 S. G. Waley, Biochem. J., 1959, 71, 132.I-I. Rinderknecht, D. Thomas, and S. A s h , Helu. Chiwz. Acta. 1968, 41, 1; 13. hl.91 A. I. Virtanen and T. Ettala, Actn Chenz. Sraizd., 1958, 12, 787.ga S. Makisumi, J . Biochem. (Japan), 1959, 46, 63.93 H. R. V. Arnstein, D. Morris, and E. J.Toms, Biochim. Bioplzys. A d a , 1959,35, 561.g4 (a) T. Kaneko, T. Shiba, S. Watarai, S. Imai, T. Shimada, and I<. Ueno, Cheni.upad I n d . , 1957, 986; (b) J. Rudinger and 2. Pravda, Coll. Cz~clz. Chem. Comm., 1958,23, 1947.g5 T. Shiba, S. Imai, and T. ICaneko, Bull. Clzem. SOC. Japan, 1958, 31, 244.96 C. A. Dekker, D. Stone, and J. S. Fruton, J . Eiol. Chem., 1949, 181, 719.97 R. B. hlerrifield and D. W. Xf-oolley, J . Ameu. Chem. SOC., 1958, 80, 6635.98 R. B. Merrifield, L. Biol. Chem., 1058, 232, 43.99 0. Mikeg, and F. Sorm, Coll. Czech. Chenz. Comm,, 1959, 24, 1897.loo R. Schwyzer, B. Iselin, €I. Kappelcr, B, Riniker, W. Rittel, and f l . Ziiher, ZIi~kv.I IZacharius, C. J. Morris, and J. F. Thompson, Arch. Biochem. Biophys., 1959, 80, 199.( ' h i m .Llctn, 1958, 41, 1273, 1287316 ORGANIC CHEMISTRY.plasma-protein fraction has furnished a tetradecapeptide (28; X = ileu) ,which is further hydrolysed by rennin to ileu5-hypertensin I. Degradativeevidence of structure of the rennin substrate has now been confirmed bysynthesis.lOlH.asp.arg.val.tyr.X.his.pro.phe.his.leu .leu.val.tyr.ser.OH/-Hypertensin I- ,-b4-,Hypertensin II+ TI 28; X = val o r ileu)Plasma RenninenzymeOxytocin and Vasopressin.-Improved methods for the isolation ofoxytocin and vasopressin have been described.lo2 Horse oxytocin andvasopressin are identical with the bovine horniones.lo3 New syntheses ofoxytocin have been announced; 61~104 one is of particular interest, since itinvolved starting with the C-terminal amino-acid and adding one residue ata time by reaction with a N-benzyloxycarbonylamino-acid 9-nitrophenylester.61 An analogue of oxytocin , in which phenylalanine replaced tyrosine,has been synthesised in two laboratories; lo5 bioassay revealed that thehydroxyl group of tyrosine is favourable, but not essential, for hormonalactivity.The isoasparagine isomer of oxytocin,lo6 like the isoglutamineisomer,lo7 is inactive, but unlike the latter, does not inhibit arginine-vasopressin.lo8 An improved synthesis of arginine-vasopressin , whichavoided exposure of peptide derivatives containing C-terminal asparagine totetraethyl pyrophosphite or carbodi-imide, obviated possible contaminationwith an anhydro-derivative, and gave a highly active product.log Ananalogue of vasopressin, in which histidine replaced lysine or arginine hadfeeble activity.ll0 The attenuation of pressor activity with decreasingpK,’ of the basic amino-acid is remarkable, and this relationship is furtherexemplified by the relatively high pressor activity of arginine-vasotocin,lllwhich is composed of the SS’-cyclopeptide fragment of oxytocin and thebasic side-chain of arginine-vasopressin.Pharmacological evidence has101 L. T. Skeggs, J. R. Kahn, K. E. Lentz, and N. P. Shumway, J . Exp. Med., 1957,106, 439; L. T. Skeggs, K. E. Lentz, J. R. Kahn, and N. P. Shumway, ibid., 1958, 108,283.102 R. Acher, A. Light, and V. du Vigneaud, J . Bid. Chem., 1958, 233, 116;A. Light, R. 0. Studer, and V. du Vigneaud, Arch.Biochern. Biophys., 1959, 83, 84;A. V. Schally, H. S. Lipscomb, and R. Guillemin, Biochim. Biophys. A d a , 1959,31, 252.10s R. Acher, J. Chauvet, and M.-T. Lenci, Bull. Soc. Chim. b i d , 1959, 40, 2005.104 &I. Bodanszky and V. du Vigneaud, J . Amev. Chem. Soc., 1959, 81, 2504.105 M. Bodanszky and V. du Vigneaud, J . Amer. Chern. Soc., 1959, 81, 1258, 6072;106 W. B. Lutz, C. Ressler, D. E. Nettleton, and V. du Vigneaud, J . Amer. Chem.107 C. Ressler and V. du Vigneaud, J , Amer. Chenz. SOC., 1957, 79, 4511.lo8 C. Ressler and J. R. Rachele, Pmc. SOL. Exptl. Bid. Med., 1958, 98, 170.lo9 P. G. Katsoyannis, D. T. Gish, G. P. Hess, and V. du Vigneaud, J . Amer. Chem.Soc., 1958, 80, 2558; V. du Vigneaud, D. T. Gish, P. G. Katsoyannis, and G. P.Hess,ibid., p. 3355.110 P. G. Katsoyannis and V. du Vigneaud, Arch. Bkochem. Biophys., 1958, 78,555.P.-A. Jacquenoud and R. A. Boissonnas, Helv. Chim. Acta, 1959,42, 788.SOC., 1959, 81, 167.P. G. Katsoyannis and V. du Vigneaud, J . BUioZ. Chem., 1958, 233, 13552I< T,MO KE : AM I NO --4CI DS , 1'K PTI DES , A N I) PHOTE I N S , 3 17subsequently indicated that arginine-vasotocin is a natural hormone of certaincold-blooded vertebrates.l12Melanocyte-stimulating Hormones (MSH) .-This subject has receivedconsiderable attention since it was last reviewed.wg Full details of sequentialstudies on the a- and p-porcine hormones have appeared.l13 In addition, thestructure of human p-MSH has been determined.l14 Three schoolshave synthesised biologically active peptides. The pentapeptideH.his.phe.arg,try.gly.OH had weak hormonal a ~ t i v i t y .~ ~ J ~ ~ A trideca-peptide derivative, differing from a-MSH in having a N-benzyloxycarbonylinstead of an N-acetyl group on the N-terminal serine residue, glutamine inplace of glutamic acid, and an N'-tosyl group on lysine, had about 1% ofthe activity of the natural hormone.l16 The corresponding N-acetyl deriv-ative, reported more recently, had 25% of the activity of the honnone.ll7ct-MSH itself has been brilliantly synthesised by Boissonnas, Guttmann, andtheir colleag~es.4~b N-Acetyl-~-seryl-~-tyrosyl-~-seryl-~-methionyl-~-glut-amic acid y-benzyl ester 118 and L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl- L - glycyl - (N" - benzyloxycarbonyl- L - lysyl) - L - prolyl- L - valineamide 118 were condensed by the carbodi-imide method; removal of protect-ing groups required very carefully controlled conditions.Finally,Schwyzer's group have synthesised a protected octadecapeptide with thecomplete amino-acid sequence of bovine p-MSH, which has about 1% ofthe biological activity of the natural horrnone.ll9 The N-terminal aspartylis replaced by a N-benz yloxycarbonylasparaginyl residue, lysine has anN'-toluene-9-sulphonyl substituent, glutamine replaces glutamic acid, andthe C-terminal aspartic acid is diesterified.I 2 3 4 5 6 7 8 9 1011 1213Ac.ser.tyr.ser.met.glu.his.phe.arg.try.gly,lys.pro.vai.~H2I 2 3 4 5 6 7 8 9 10 I 1 12 13 14 I 5 16 17 18 s (0x1 H.asp.ser.*gly.pro.tyr.lys.met.g lu.his.phe.arg.try.gly.ser.pro.pro.lys.asp.0HI 2 3 4 5 6 7 8 9 10 i I 12 13 14 15 16 I7 18 19 20 21 22a(human) H.ala.glu.lys.iys.asp.glu.gly.pro.tyr.arg,met.glu.his.phe.arg.try,gly.ser.pro.pro.lys.asp.OHa- and ~-Melanocyte-stimulating hormones. (* glu in porcine a-MSH.)112 B. T. Pickering and H. Heller, Nutwe, 1959, 184, 1463; W. H. Sawyer, R. A.113 J. I. Harris and P. Roos, Biochern. J., 1959, 71, 434, 445; J. I. Harris, ibid.,114 J. I. Harris, Nature, 1959, 184, 167.115 K. Hofmann, M. E. Woolner, G. Spuhler, and E. T. Schwartz, J . Awer. Chem.SOC., 1958, 80, 1486.116 K. Hofmann, M. E. Woolner, H. Yajima, G. Spiihler, T. A. Thompsoh, and E. T.Schwartz, J . Amer. Chem. SOC., 1955, 80, 6458.117 K. Hofmann, H. Yajima, and E. T. Schwartz, Biochim.Biophys. Acta, 1959, 38,252.l18 St. Guttmann and K. A. Boissonnas, Helv. Chim. A d a , 1958, 41, 1852; R. A.Boissonnas, St. Guttmann, R. L. Huguenin, P.-A. Jacquenoud, and E. Sandrin, ibid.,p. 1867.119 R. Schw!rzer, H. Kappeler, €3. Iselin, W. Rittel, and H. Zuber, Helv. Chim. Acta,1959, 42, 1702.Munsick, and H. B. van Dyke, ibid., p. 1464.p. 45131 s ORGANIC CHEMISTRY.Haemog1obins.-There are three recent reviews of hzemoglobins. 120 Thestructural comparison of normal adult (A) or normal foetal (F) with abnormalhaemoglobins (C, E, G, H, I, S, etc.), by electrophoretic and chromatographicexamination of enzyme digests of the proteins (" fingerprinting "),121 hasrecently provided results of profound significance to the study of the chemicalbasis of gene mutations.The cc- and the P-chains (two of each) 122 of humanhzmoglobin A may be separated chromatographically or electrophoretic-ally.123 The P-chains have the N-terminal sequenceH.val. hisleu. thr.pro.glu.glu.ly~,1~~~ 124whereas the a-chains begin with H.val.le~.I~~ Present evidence suggeststhat the C-terminal sequence of P-chains is tyr.his.0H.126 Haemoglobin Sand C are identical with hzmoglobin A except that glutamic acid at position 6of the N-terminal sequence of the two P-chains is replaced by valine andlysine ~ e v e r a l l y . l ~ ~ J ~ ~ Hzmoglobin G also differs from hamoglobin A a tone position; glycine replaces glutamic acid at position 7 of the N-terminalsequence of the p-chains.128 In hzmoglobin E, a further variation of asingle amino-acid has been demonstrated at a point remote from the N -termini of the p-chains.One of the peptides resulting from tryptic degrad-ation of haemoglobin A has the sequence:H.val.asp.val.asp.glu.val.gly.gly.gIu.ala.leu.gly.arg.0Hwhereas, in hzmoglobin E, lysine replaces glutamic acid at the ninth residuefrom the N-terminus of this p e ~ t i d e - l ~ ~ Normal foetal hzmoglobin F con-sists of 2a- and 2y-chains; the former are identical with the a-chains ofhzemoglobin A,130 while the latter have N-terminal g1y~ine.l~~ " Finger-printing " revealed many differences between the y-chains and the p-chainsof hzmoglobin A.130 An abnormal fatal haemoglobin (" Barts ") consistsof 4 y-cliains.132 Hzmoglobin H consists of 4 p-chains, identical with thoseof hzmoglobin A.lS A structural change in the a-chains is found in haemo-globin I ; one tryptic peptide contains tryptophan, unlike the correspondingpeptide from hzemoglobin A.lM120 H.A. Itano, A h . Protein Chem., 1957, 12, 215; H. K. Prins, J . Chromatog.,1959, 2, 445; W. A. Schroeder, Fortschr. Chem. org. Naturstoffe, 1959, 17, 322.121 V. M. Ingram, Biochim. Biophys. Acta, 1958, 28, 539.122 H. S. Rhinesmith, W. A. Schroeder, and N. Martin, J . Amer. Chem. SOC., 1958,80, 3358.123 S. Wilson and D. B. Smith, Canad. J . Biochem. Physiol., 1959, 37, 405; V. M.Ingram, Nature, 1059, 183, 1795.124 J. A. Hunt and V. &I. Ingram, Nature, 1959, 184, 640.125 H. S. Rhinesmith, W. A. Schroeder, and L. Pauling, J . Amer.Chem. SOC., 1957,79, 600.126 K. Hike and G. Braunitzer, 2. Naturforsch., 1959, 14b, 603; T. Kauffmann andF.-P. Boettcher, ibid., 1959, 13b, 467; Chem. Bey., 1959, 92, 2707.137 J. A. Hunt and V. M. Ingram, Nature, 1958, 181, 1062; Biochim. Biophys. Acta,1958, 28, 546; V. M. Ingram, ibid., 1959, 36, 402.128 R. L. Hill and H. C. Schwartz, Nature, 1959, 184, G41.1ZD J. A. Hunt and V. M. Ingram, Nature, 1959, 184, 870.130 J. A. Hunt, Nature, 1959, 183, 1373.131 W. A. Schroeder and G. Matsuda, J . Amer. Chem. SOC., 1958, 80, 1521.132 J. A. Hunt and H. Lehrnann, Nature, 1959, 184, 872.13* M. Murayama and V. M. Ingram, Nature, 1959, 183, 1798.R. T. Jones, W. A. Schroeder, J. E. Balog, and J. R. Vinograd, J . Amer. Chem.Soc., 1959, 81, 3161TSlMORE AM I N O-_L\C I 13s , I T P'TI I)E S , AND PH OlE I N S .319Phosphopeptides and Phosphopr0teins.-Phosphoproteins are increasinglybecoming a focus of attention. One reason is the elucidation of the amino-acid sequences at the " active centres " of a number of hydrolytic enzymes,which have been inhibited by phosphorylation with compounds such as di-isopropyl phosphorofluoridate (see Table). Some phosphate-transferringenzymes and phosphatases are phosphorylated at the hydroxyl group of aAmino-acid sequences in Phosllhorylated enzymes.Phosphoryl-EnzymeTrypsinChymotrypsinThrombinHorse-liver ali-esterase dHorse-serum pseudo-choline-esteraseElastaseRabbit-muscle phos-phoglucomutaseMuscle phosphorylaseBone or intestinal alk-aline phosphataseHexokinase JAcetylcholinesterasea (- b)atingreagentii, iiii11111 ...ivVviiStructure a t site of phosphorylationAsp.ser.cys.glu.gly.asp.se~.gly.pro.val.cys.ser.gl~.lys.gly.asp.ser.gly.gly.pro.leu.gl y .asp. ser . gl y . glu .ah.gly .asp. ser.gly .gl y . glu ser. g1 y . gl y . (glu , ser)phe.glp.glu.ser.ala.gly. (ala,,ser)*{gly.asp.ser.gly.asp.ser.gly.glu.ala.va1.lys .gluNH,.ileu. ser.val.arg.ser.ser.ser.* There is no satisfactory explanation for the different results.Reagents : i, (PrfO),PO*F; ii, (Pri0)MePO-F; iii, glucose 1- or 6-[32P]phosphate;iv, ATP + M P + " converting enzyme "; v, inorganic [32P]phosphate; vi, S2P-ATPor glucose 6-[32P]phosphate.References: a, G. H.Dixon, D. L. Kauffman, and H. Neurath, J . BioE. Chew., 1958,233, 1373. b, R. A. Oosterbaan, P. Kunst, J. van Rotterdam, and J. A. Cohen, Biochinz.Biophys. Acta, 1958, 27, 556; N. K. Schaffer, L. Simet, S. Harshman, R. R. Engle, andR. W. Drisko, J . Bid. Chem., 1957,225, 197; N. K. Schaffer and R. P. Lang, Fed. PYOC.,1959, 18, 317. c, J. A. Gladner and K. Laki, J . Amer. Clzem. Soc., 1958, 80, 1263.d , H. S. Jansz, C. H. Posthumus, and J, A, Cohen, Biochisn. Bioghys. Acta, 1959, 33,396. e, H. S. Jansz, D. Brons, and M. G. P. J. Warringa, Biochim. Biophys. Acta, 1959,34, 573. f, B. S. Hartley, M. A. Naughton, and F. Sanger, Biochim. Biophys. Acla,1959,34, 243. g , D. E. Koshland and M. J. Erwin, J . Amev. Chem. Soc., 1957, 79, 2657.h. E. H. Fischer, D.J. Graves, E. R. S. Crittenden, and E. G. Krebs, J . Bid. Chem.,1959, 234, 1698. i, G. Agren, 0. Zetterqvist, and M. Ojamae, Acta Ckem. Scand., 1959,13,1047; L. Engstrom and G. Agren, ibid., 1958,12,357. j , G. Agren and L. Engstrom,Acla Chew. Scand., 1056,10, 489. k , N. K. Schaffer, S. C. May, and W. H. Summerson,J . Bid. Chem., 1954, 206, 201.serine residue by incubation with substrate (see Table). O-Phosphoryl-serine and -threonine and several simple peptide derivatives have been syn-the~ised.l~~ Protected peptides have been synthesised and then phosphoryl-ated with diphenyl or dibenzyl phosphorochloridate. Hydrogenolysis isfavoured for the removal of protecting groups, since the phosphate grouptends to undergo p-elimination in alkali.Lombricine, isolated from earth-135 ( a ) D. Theodoropoulos, H. Bennich, G. Folsch, and 0. Mellander, Nature, 1959,184, 187; ( b ) G. Folsch, A c f a Chew. S c a d , 1055, 9, 1039; 1955, 12, 561; 1959, 13,1407, 1422; G. Riley, J. H. Turnbull, and If'. Wilson, J., 1957, 1373:I20 OKGANIC CHEMISTKY.w o r r ~ i s , ~ ~ ~ has been proved to be 0-(O’-(2-guanidinoethyl)phosplioryl)-u-serine by degradation and synthesis.137 N-Phosphoryl-lombricine, a naturalphosphagen of earthworm muscle, has also been synthesised.Evidence has accumulated, which weakens Perlmann’s 13* claim that, ofthe phosphorus present in a-casein, 40% is phosphoamide and 20% is pyro-phosphate. It is also unlikely that all the phosphorus of p-casein is in theform of phosphodiester linkages. Alkaline dephosphorylation of casein inH,180 gave unlabelled inorganic phosphate, and therefore represents ap-elimination ; N-P bonds were thereby ex~1uded.l~~ Degradation of CC-and p-casein with proteolytic enzymes, especially trypsin, produced phospho-peptones, in which the number of phosphate groups equalled the total numberof serine and threonine residues.lM titration of the phospho-peptones revealed that all phosphoric acid groups were monoesterified.Moreover, the phosphopeptide from p-casein is probably N-terminal in theprotein and contains all the phosphorus. The resistance of a-N-ethoxy-carbonyl-L-lysyl-(O-phosphoryl-L-sery1)glycine to trypsin is interesting inview of the limited attack on casein by this enzyme.135a It appears thatseveral consecutive residues of O-phosphorylserine occur in some phospho-proteins.141 Partial acid-hydrolysis of casein afforded HfP-ser],*OH(n = 1, 2, 3), while phosvitin furnished an even larger range (n = 1-6).Such accumulations of phosphoric acid groups may explain the slow hydro-lysis of casein by certain phosph~monoesterases,~~~ since similar observationshave been recorded with nucleosides bearing more than one phosphomono-esterRibonuc1ease.-Final elucidation of the complete amino-acid sequence isstill awaited. At least two differences between beef- and sheep-pancreaticribonucleases have been found; residues 3 and 37 are threonine and lysinein the former, and serine and glutamic acid in the latter.lM Attempts havebeen made to identify the “active centre’’ of the enzyme. Inhibition byiodoacetate at pH 5-5-6.0, followed by chromatographic purification andhydrolysis, gave one mol. of im-carb~xymethylhistidine.~~~ Subtilisincleaved the ala-ser linkage, but the two fragments remained stronglybound, so that chromatography and dialysis failed to separate them. Care-ful fractionation with trichloroacetic acid succeeded, however, and the twoIn onea0 21136 N. Van Thoai and Y . Robin, Biochiw. Biophys. Acta, 1954, 14, 76; R. Pant,137 I. M. Beatty, D. I. Magrath, and A. H. Ennor, Nature, 1959, 183, 591; I. M.138 G. Perlmann, Adv. Protein Chem., 1955, 10, 1.139 L. Anderson and J. J . Kelley, J . Amer. Chem. Soc., 1959, 81, 2275.140 (a) R. &kerberg, Arkiv Kemi, 1959, 13, 409; (6) R. F. Peterson, L. W. Nauman,and T. L. McMeekin, J . Amer. Chem. Soc., 1958, 80, 95; H. Bennich, B. Johansson, andK. Osterberg, Acta Chem. Scand., 1959, 13, 1171.141 J. Williams and F. Sanger, Biochim. Biophys. Acta, 1959, 33, 294; N. J . Hipp,M. L. Groves, and T. L. McMeekin, J. Amer. Chem. Soc.. 1957, ‘79, 2559.148 T. Hofmann, Biochem. J . , 1958, 69, 139.145 J: Baddiley, J . G. Buchanan, and R. Letters, J., 1958, 1000; C. A. Dekker,A, M. Michelson, and A. R. Todd, J . , 1953, 947.144 C. B. Anfinsen, S. E. G. Aqvist, J. P. Cooke, and B. Jonsson, J . Bid. Chem.,1959, 234, 1118.145 H. G. Gundlach, W. E. Stein, and S. Moore, J . Biol. Chem., 1959, 234, 1754.Biochern. J . , 1959, 73, 30.Beatty and D. I. Magrath, ibid., p . 591ELMOKE : AMINO-ACIDS, PEPTIDES, AND PROTEINS. 32 1fragments were separately inactive, but fully active when mixed.146 Sormhas made an interesting comparison of peptide sequences in ribonuclease,trypsinogen, chymotrypsinogen, and in~u1in.l~~D. T. E.M. F. ANSELL.C. A. BUNTON.A. G. DAVIES.P. D. B. DE LA MAKE.D. T. ELMORE.J. MCKENNA.W. G. OVEREND.A. R. PINDER.Y. POCKER.J. H. RIDD.J. E. SAXTON.T. S. STEVENS.J. TEDDER.G. H. WHITHAM.146 F. M. Richards, Proc. Nut. Acud. Sci. U.S.A., 1958, 44, 162; F. M. Richards,147 F. Sorm, Coll. Czech. Chem. Comm., 1959, 24, 3169.and P. J. Vithayathil, J . Bid. Chew., 1959, 234, 1459.