COMMUNICATIONS TO THE EDITOR
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Vol. 77
have made theoretical calculations of the l / R 3 force modifications of the spectrum to be expected for myristamide (using both of the angles given it1 Fig. 1). These calculations support the interpretation of the 2120-2250 A. absorption as a crystal transition, in that the long-wave-length allowed component is predicted to occur in the a axis direction, split off by ca. 100 to 150 A., just as observed (and regardless of which angle is chosen). We have also made quantum-theoretical calculations of the direction of the transition moment for an amide model. In the model an amide is regarded as a perturbed allyl anion5with the nitrogen represented by a deepening of the coulomb potential on one of the end carbon atoms and the oxygen considered as being the same as carbon. The result is that the direction should be close to the nitrogen-oxygen line, but inclined toward the carbon nitrogen line. Of the two values fouiid in our experiments the one with an angle of 9.1' from the nitrogen-oxygen line is thus favored. Both the experimental and theoretical aspects of this investigation will be reported in detail in a forthcoming paper.
sive confirmation obtained through an 1I-methoxyalloyohimbane (IIa, R = R' = H ) (reserpane) synthesis which is stereochemically unambiguous insofar as the CI6-Cznasymmetric centers are con~erned.~ 6-Methoxytryptaminee (obtained through the route : 6-methoxyindole -t 6-methoxygramine methosulfate -t 6-methoxyindoleacetonitrile -+ 6-methoxytryptamine) was alkylated in boiling dimethylformamide by ethyl dl-cis-2-bromomethylcyclohexaneacetate,'~~ affording the lactam of dZ-cis-N-(P-3'indolylethyl) -2-aminomethylcyclohexaneacetic acid (111) [benzene solvate), m.p. 72.5-74.0" (Calcd. for CnoHa6Sz02.C6H6:C, 77.19; H, 7.97. Found: C, 77.02; H, 7.93). Heating I11 with phosphorus oxychloride in benzene, followed by platinum-catalyzed reduction of the unisolated A"ring-closed product, yielded the desired til-all0 base IIa, which melted a t 209-210" after crystallization from methanol. Infrared spectral comparison of chloroforni solutions of IIa and a reserpane derived by reduction from reserpone (IIb, IC, R' = 0)9 showed the two substances to be, apart from the racemic nature of the former, identical.'" DEPARTMEKT OF CHEMISTRY D. L. PE-I.ERSOX~ Publication of our views on the nature of the remaining asymmetric centers in reserpine and deUNIVERSITY OF WASHISGTOS TI., T. Srhwsos SEATTLE, WASHINGTOS serpidine, including evidence relating thereto, is RECEIVED J U T E 1.3, 1955 :i 11 ticipatcd. 15) Calculation waF facilitated by use of full conlixuration interacAcknowledgment.--The authors wish to express tion A.S.IVf.0. calculations on allyl anion (H. D H u n t , D L Peterson their gratitude t o the Department of Health, Weland Tir. T. Simpson, t o be published) fare and Education for financial support (Grant NO. (6) National Science Foundation Predoctoral Fellow 19.54-195.5. G-3S92'! and t o S. R.Penick and Co. for a gift of reserpine. ( 5 ) T h e matter of t h e stereochemistry a t C Ain o m synthetic prodiict
T H E D/E CIS RING JUNCTURE O F RESERPINE
Sir: Reserpine, the potent hypotensive agent obtained from various species of Rauwolfia, has been Because of its proxiassigned the structure I
is deferred for t h e present. (0) S.Akabori and K. Saito, B e y . , 6SB, 2245 (1930).
( 7 ) G . Stork and R . Hill, THISJOURNAL, 76, 949 (1954). ( 8 ) Unpiiblished results obtained in this Laboratory. (9) C . P. Iluehner, H . B. hIacPhillamy, A . F. S t . Snrlr6 and E. Schlittler, Tins J O ~ J R N A I . ,7 7 , 473 (195.5). ( I O ) n ' c should like t u thank Dr. Schlittler and Dr. St. Andr6 for their n;siitance in establishing t h e identity of t h e two specimens. Tn addition, o u r base Ita was shown t o be identical with material obtained b y them via a different synthetic route.
DEPARTMEXT OF CHEMISTRY EUGENE E. VAS TAMELEN USIVERSITY OF WISCOXSIS PAULD. HANCE MADISON, WISCOSSIS KENNETH V. SIEBRASSE PAULE. ALDRICH RECEIVED MAY3, 1955 OCH3
I
R
R'
TIa, b
POLY-GLUTAMYL PTERIDINE COENZYMES
.%: It was recently demonstrated that the conversion mate relation to deserpidine (formula I with the 11methoxyl replaced by - H ) , Schlittler, et al., sur- of serine to glycine by a bacterial extract is demised that reserpine too possesses a D/E cis (allo) pendent upon DPX, Mn++, pyridoxal phosphate, ring juncture3; and Wintersteiner, et u Z . , ~ on the orthophosphate and catalytic levels of a new coenbasis of the intramolecular N-4 quaternization of zyme, Co C.1,2 Co C is isolated from Clostridium methyl reserpate tosylate, inferred the same rela- cylindrosporum, and substitutes for but is not identionship. We wish to report for this view conclu- tical with known folic acid derivatives. By means of fractional acetone precipitation, chromatography (1) L. Dorfman, A . Furlenmeier, C. F. Huebner, R . Lncas, H . B. on cellulose columns, and repeated paper chromaMacPhillamy, J. hl. Muller, E. Schlittler, R. Schwyzer and A. F. tography in various solvent systems,3five groups of St. Andre, H d v . Chim. Acta, 37,59 (1954). (2) N. Neuss, H . E. Boaz and J. W. Forbes, THISJOURNAL,76, pteridine derivatives with Co C activity have been 2463 (1954). separated in relatively pure form from extracts of
(3) H . B. MacPhillamy, L. Dorfman, C. F. Huebner, E. Schlittler and A. F. St. Andre, ibid.,77,1071 (1955). (4) P. A. Diassi, F. I.. Weisenborn, C . M. Dylion and 0. Wintersteiner, ibid., 77, 2028 (1955).
(1) B. E. Wright, Biochini el Biophys. Acta, 16, 165 (1955) ( 2 ) B. E . Wright, F e d . PTOC., 14, 308 (1955). (3) B. E ITright and E. R. Stadtman, unpublished data.
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COMMUNICATIONS TO THE EDITOR
July 20, 1955
TABLE I Abbreviations: PatablerPtoC.l- P,o plus P i a b i i P ; Plu,phosphate liberated after 10 minutes of boiling in 1.0 K HC1, minus Pl,,bile, phosphate fully liberated after 20 minutes under conditions of orthophosphate determination by the FiskeSubbaRow method. This phosphate is stable in the Lowry-Lopez method. S o inorganic phosphate was present in any sample. Piabile.
Relative coenzymatic activity” C o C Type I Xhr. type = 10070 mp
I
I11 11.
100 80 50 40
V
40
I1
286 268
265 280 265
Moles pentose
Ptotai!6
Petable
PIO
27,900 S o n e 25,000 1 . 0 24,000 0.85 27,900 S o n e 2F,000O.85
Sone 1.28 1.0 None 4.05
Sone 1.0 1.0 Kone 2.0
Sone 0.25 Xone None 1.05
ehtar,a
riabile
Sone Sone None
Kone 1.05
.Imino acids present C 11oles t o t a l Moles other amino glutamica acidsa
2.6 2.7 >1.3” 6.1 5.95
0,37 0.50 >O .19 2.0 3,$1
Other amino acid in Order of Cuncn.
Glycine, serine Glycine, alanine, serine Glycine, serine Glycine, “x,” serine Alanine, “x,” glycine, serine
a The molecular extinction coeflicient of the unphosphorylated types I and I V is assumed to be t h a t for C.F. ( = 27,900) ; the others are calculated on stable phosphate content. 17alues for labile phosphate, pentose and amino acid content per Part of this sample was lost, CAmino acids were determined mole are based on these molecular extinction coefficients. quantitatively after acid hydrolysis by conversion to their D K P derivatives and subsequent c h r ~ m a t o g r a p h y ’ ~pentose ; was determined according t o the procedure of Pvlejbaurn.
C. cylindrosporum. These fractions are considered to be polyglutamyl pteridine compounds which contain other amino acids as parts of the molecule. Chemical data characterizing various types of Co C are given in Table I, which includes a triglutamyl derivative ( I ) , a triglutamyl and a diglutamyl form with one pentose and one stable phosphate (11) and (111), a hexa- or heptaglutamate derivative (IV), and a hexa- or heptaglutarnate with one pentose, two stable and two labile phosphates (V) . All types have a blue fluorescence except V, which is yellow in color and fluorescence. The fluorescence is enhanced under alkaline conditions. Co C types I-V are listed in Table I in order of decreasing coenzymatic activity on a molar basis. Compound I is the most fully characterized. It has the ultraviolet spectrum of C.F. (with a maximum a t %Gj a t neutral pH and shows the isoleucovorin characteristics (maximum a t 3531 in 0.1 LV HCl.5 The infrared spectrum is practically superimposable upon that for C.F.,6suggesting that I is closely related to triglutamyl C.F., some species of which contain amino acids other than glutamic Microbial assay of the Co C derivatives gave additional evidence for a pteridine structure.* Compounds 11, I11 and V keep the same ratio of pentose to ultraviolet absorption after chromatog( 4 ) I t is now clear t h a t t h e forms of Co C described pre\-iouslyl correspond t o compounds I and I V , Table I . T h e forms with 280 absorption arise a t least in part from t h e 260 absorbing forms during purification. ( 5 ) D. B. Cosiilich, B. Roth, J . M . Smith, Jr., RI. E . Hultquist and K. P . Parker, THISJ O U R N A I . , 74, 3232 (1952). (6) Infrared records of C o C I and C.F. were generously run by Dr. D . Anderson of Eastman Kodak Companp.7 (7) D. H. Anderson and N . B. Woodall, A n a l . Chem., 25, 1906 (1953). (7a) I t is possible t h a t this compound is similar t o one described by Ericson,’b in spite of differences in activity for Streptococcus faecalis
raphy in two solvent systems (pyridine-propanolHzO, 1: 1: 1 and EtOH-M/1 NH40H, 2 : 1). Preliminary evidence that pentose and phosphate are an integral part of these coenzymes was obtained by showing that when compound I11 (Rf = 0.32 in EtOH solvent system) is treated with phosphomonoesterase, about 50% can be recovered as a new compound (Rf = 0.57) which retains one pentose per mole. This compound has an emax = 260 mw and is not coenzymatically active. No pentose was recovered from the chromatogram of the incubated sample a t Rf = 0.32 nor from the control sample a t Rr = 0.57. These cofactors containing varying amounts of glycine and serine, and in some cases alanine and an unidentified amino acid, “x,” which is not identical with any amino acid normally found in protein hydrolysates.12 Chromatographic evidence shows that besides glycine, alanine and “x” are also formed from serine in the presence of Dowextreated, dialyzed enzyme. The enzymatic conversion of serine to glycine, alanine and compound “x” are all dependent on the presence of Co C. It is clear that the Co C types listed in Table I represent mixtures of species with respect to the amino acids other than glutamic acid. This is not surprising in view of the fact that only in the last stage of purification did the tri- and hexa-glutamates separate. Quantitative ninhydrin determinations on I, I1 and I V before hydrolysis revealed one free amino group
(12) An unidentified dicarboxylic acid was described by Ratner, Blanchard and Green,ld in a PAB conjugate containing 10-11 glutamyl residues. (13) S. Ratner , M . Blanchard a n d D . E. Green, J . B i d . C h o n . , 164, 691 (1946). (14) A. L. Levy, Nature, 174, 129 (1954). (15) W. Mejbaum, 2. physiol. Chem., 258, 117 (1939). (16) The stable phosphate on compound I1 and one phosphate of V R. (not Pisbile, which is liberated during orthophosphate determination) (7b) L. E . Ericson, Avkiv..for Keiiii, 6 , 503 ( l Y 5 3 ) . is removed by partially purified human semen phosphomonoesterase. (8) When various forms of Co C were assayed for microbiological Kornberg’s purified potato pyrophosphatase17 releases one phosphate activity using Streptococcus .fueculis R and I.rncurmstoc citrovorum,~ from V suggesting a free pyrophosphate linkage. Combining t h e activity was found only for t h e latter organism. and t h e growth curve pyrophosphatase and monoesterase removes phosphate quantitatively was unlike t h a t for C.F 10 Digestion with 7-ylutamic acid carhoxyfrom V. peptidase“ gave significant increases of C.1;. activity. (17) A . Kornherg and W. E. Pricer, J r , J . B i d . Chem., 182, 763 ( 9 ) h1 Silverman and R . E. Wright, unlnrblished data. (1950). (IO) hf. 13. Swendseid, P. D. Wright, I;. 11. Rethcll, PI.OG.SOL. (18) % activity = &if. glycine produced per hour X 1000 wlleu Expcr. B i d and Mcd., 80, 089 (1952) 0.003 ph1. of Co C is incubated with 4 units of eiizynie iiurler Pi-e(11) H , Kvzenkoand A I . Laskowski, J . Ziiul. C h i n , , 173,217 (1948). viuiisly defined assay conditions.1
BOOKREVIEWS
on I V but none on I and 11. _Ifter hydrolysis, total ninhydrin values corresponded to those obtained for the DNP derivatives. Acknowledgments.-I would like to thank Dr. AI. Kornberg for the purified nucleotide pyrophosphatase, Dr. L. Heppel for the phosphomonoester-
Vol. 77
ase, and Dr. K. Redfield for assistance in chroiriatographic techniques. LABORATORY OF CELLULAR PHYSIOLOCV BARBARA I< \I.RIC,H I
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BETHESDA14, MARYLAKD RECEIVED APRIL 16, I%>
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Advances in Carbohydrate Chemistry. Volume 9. By The Chemistry of Lipids of Biochemical Significance. BY MELVILLE L. WOLFROM,Editor, R . STUART TIPSON, J. A . LOVERX. John Wiley and Sons, Inc., 440 Fourth Assistant Editor, and E . L . HIRST, Associate Editor for Avenue, S e w York 16, S. Y . 1935. xiii 132 pp. the British Isles. Academic Press, Inc., Publishers, 123 11 X 17 em. Price, $1.73. East 23rd Street, S e w York 10, N. Y. 1954. xviii 426 ‘This relatively short monograph is intended to give a i l pp. 16 X 23 cm. Price, $10.50. over-all general picture of lipid chemistry and therefore I t is most appropriate t h a t this, the latest volume of a does not fully cover either subject matter or bibliography. distinguished series, should begin with a biographical sketch In general only major points are presented with a minimum of the late C. S. Hudson, written by the present editor, amount of experimental detail. The monograph brings to hI. L . Wolfrom. S o t only did Professor Hudson play a focus many important aspects of the lipids in a manner major part in the development of carbohydrate chemistry that the reader is not bothered with less important minutia. in this country but he was also closely associated in various Although the text covers lipid structure, preparation and capacities with this series of reviews from its inception until analysis, a considerable portion is devoted to the distribuhis death in 1952. tion, dynamic state and biochemical functions of the lipids. The present volume, like its predecessors, reminds the Emphasis is placed on the phosphatides. The carotenoids writer of the medieval Speculum Alchemiae, for it is a kind and fat-soluble vitamins are not included in the monograph. of mirror, a mirror of the state of carbohydrate chemistry, I t is the opinion of the reviewer that the title of the hook and affords even the most casual reader a n opportunity to might have been more appropiatel>- chosen since a l x g c see what areas of this fertile field are being tilled most part of the monograph deals with topics other than lipid intensively. I n this light two aspects of the latest volume chemistrj-. seem particularly worthy of note. First, several of the The author gives constructive comments on lipid nomciicontributions illustrate the gratifying extent t o which modern clature and classification and indicates the need for mor? theories of the mechanism of organic reactions have been uniform and discrete terminology. The interrelationships applied in the carbohydrate field and, second, the volume, of the various lipid classes are stressed and a n attempt i i taken as a whole, caters to a surprisingly broad spectrum of made to integrate the entire lipid subject. interests. The organic chemist concerned with reaction The reader will find the personal comments of the author mechanisms will find a chapter by R . U. Lemieux (“Some interesting, helpful and provocative. An excellent evaluaImplications in Carbohydrate Chemistry of Theories R r - tion of the various methods of lipid preparation arid analysi\ lating t o the Mechanisms of Replacement Reactions” 1 , is presented including a n informative discussion on lipid one by Clinton E . Ballou (“Alkali Sensitive Glycosides”) extraction and purification. In addition, therc is a stimuarid a third by Mary Grace Blair (“The 2-Hydroxyglpcnls”~. latiug discourse on the limitation5 of radioactive isotoi)cs The worker in natural products will turn especially to techniques for the study of lipid metabolism. Topics sclch Ballou’s chapter as well as to one by Dexter French entitled as lipid complexes with proteins and carbohgdrates, and lipicl “The Raffinose Family of Oligosaccharides.” If he i i digestion, absorption and biosynthesis are briefly covered. among the increasing number who deal with the uronic The subject matter of the book is well presented ai1d should have particular appeal to those interegted iri 01)acids, G. 0.Aspinall’s chapter on “The Methyl Ethers of Hexuronic Acids” will prove a useful compilation of imtainirig the essential highlights of the biochemistry of the portant data which have heretofore been widely dispersed major lipids. hIoreover, the fine integration of the m i t e in the literature. H e will also find a contribution by rial should make the monograph especially useful to thc l x Robert S. Teague entitled “The Conjugates of D-Glucuronic ginner in the lipid field. Acid of Animal Origin” which mill, of course, appeal as DEPARTMEXT OF BIOCHEMISTRY well to the biochemist and physiologist. One interest (>, 1..> I A K I \ li I I I THEUNIVERSITY OF ROCHESTER of the sugar technologist is represented by “Color and MEDICAL CESTER Turbidity of Sugar Products” written by R . \\:. Liggett ROCHESTER 2 0 , S E ~YORR V and Victor R . Deitz. The chemist interested in the indus-~ trial utilization of carbohydrates will find a contribution by T. V. Karabinos and Marjorie Hindert on “Carboxymethylcellulose” while laboratory workers in several fields will note The Vitamins. Chemistry, Physiology, Pathology. 1.01much of practical value in a review on the “Paper Chromaunie 111. Edited by \V, H. SEBRELL,J R . , Director, tography of Carbohydrates arid Related Compounds” bjSational Institutes of Health, Bethesda, Maryland, a i d George X . Kowkabany. The present volume is, incidenROBERTS.HARRIS,Professor of Biochemistry of S u t r i tally, the first of the series in which a n attempt has been tion, Massachusetts Institute of Technology, Cambridge, made t o use the new carbohydrate nomenclature [ Chem. Massachusetts. Academic Press, Inc., Publishers, 12.5 East 23rd Street, S e w York 10, S . Y . 1954. xi 663 Eng. -Vews, 31, 1776 (1953)]throughout. Taken as a whole, this volume, together with the precedpp. 16.5 X 23.5 cm. Price, $15.00. ing ones, forms a set which is of great utility to chemi5t. ’I‘liia i-, tlir third a11d last volulilr UT a w r k . I t i-, of griirrdly arld invalualjlt- t u tho>? >pc4aliyiiig i l l I I l r (~arli,i I Y ~ I I I X , ditficult L O rrvirw a ~ l e q i i ~ ~ toitr.tliird cl~. \jitliout Ii>,clr:ttvfirlii. rcfereucr t o t l i r otlirr t i 1 1 1 t l r i r i l ~ . l o 1 1 1 c . \ \ l i o I t ~ \c.r-iv, t.lii, S A I . I O ? ’ A I . ~ Y S l ’ I I ~ J l RORF I I F . I I . I ~ I T vit:iiiiiri\ urr lir-?,,erl1v(l i i i a l p I ~ ~ l ) r ~order i c ~ ~ i~i il t i l ? hioI , I ~ I . F ~ l c ‘ l i F KI ,R k.lleiiiiit, c.iieinist 2nd cliriiciLn, to \ \ 1 l i 1 1 n the wrirs i., dirrctr(1,
+
+
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