hydrogenasel0 activity, which is absent from the rat liver preparations used here.
m.p. mainly 1S5-19Oo. There was not suflicient material for elemental analysis, so a detailed study of spectra was made. DEPARTMEKT OF PHYSIOLOGICAL CHEMISTRY The absorption spectrum of V seemed to elimiTHEJOHSS HOPKINS UNIVERSITY SCHOOL OF MEDICINE BALTIMORE 5 , MARYLAND ROBERT G. L A N G D O Nnate '~ purines, pyrimidines, pyridines and alkyl.benzimidazoles and indicated an hydroxybenzirnid(10) N. 0. Kaplan, S . P. Colowick and E . F. Neufeld, J . Bioi. azole structure. The previously undescribed 5Chein., 205, 1 (1953). (11) This work was conducted during the tenure of a Lederle Medihydroxybenzimidazole was prepared by demethylacal Faculty Award. tion of 5-methoxyben~imidazole~with hydrobromic acid; m.p. 220-222'; Anal. Found: C , VITAMIN B12. XXVI. DEGRADATION OF FACTOR 111 62.26; H, 4.60; N, 20.90. The absorption spec2500 (485); 3050 (5733 : TO 5-HYDROXYBENZIMIDAZOLE AND DERIVATIVES trum AND BIOSYNTHESIS OF FACTOR I11 2100-2500 (shoulder); 2S70 (528)) was in good Sir: agreement with that of base V. The picrate and Factor 111 was isolated from fermented sewage.' hydrochloride of synthetic 5-hydroxybenzimidazole I t has been reported to have hematological activity had wide melting point ranges, but mixed melting similar to that of vitamin Biz, and appeared t o differ points with the isolated salts showed no depression. from vitamin B12 by having an unknown moiety in The infrared spectra of the hydrochlorides showed place of 5,G-dimethylbenzimidazole.' Friederich them to be identical. and Bernhauer3 have now reported S-hydroxyFactor B and synthetic 5-hydroxybenzimidazole benzimidazole as a degradation product of Factor were combined microbiologically using Escherichia 111. coli 113-3,8 and two red crystalline compounds Last year, through the generosity of Professor were isolated. One of these did not separate from Dr. K. Bernhauer, we received samples of Factor Factor I11 on mixed paper chrornatogratns. This 111. We have independently identified 5-hydroxy- result confirmed the chemical evidence that 3benzimidazole as a part of the molecule. We have hydroxybenzimidazole is present in Factor I11 and also prepared two crystalline cobalt complexes is not an artifact. The structure and synthesis from 5-hydroxybenzimidazole and Factor B by of the other degradation products of Factor I11 are biosynthesis. One of these appears to be Factor being studied further. I11 from comparison with the substance isolated (7) E . Ochiai and A I . Katagu, J . Phavln. SOL.J a p a ? i , 60, 543-550 from sewage. (1940); Chem. A h . , 35, 1785 (1931). Factor 111 was hydrolyzed with 6 N hydro(8) B. Davis and E. Mengioli, J . Bod., 60, 17 (1950). chloric acid for 20 hours a t room temperature, and RESEARCH LABORATORIES I?. M. ROBISSON the hydrolysate was subjected to paper electro- CHEMICAL DIVISIOS I. &I. MILLER J. F. h I C P H E R S O N phoresis in 0.5 N acetic acid containing a little MERCK& Co., INC. KARLFOLKERS cyanide. Material showing bright blue-white R A H W A Y , S E W JERSEY RECEIVED SEPTEMBER 12, 1955 fluorescence under ultraviolet light separated from the pigments present. On paper chromatography in a butanol-acetic acid-water ~ y s t e m ,it~ sep- ON T H E ORIGIN O F THE METHYL GROUPS OF arated into two spots (I and 11) of unequal inPHOSPHOLIPID CHOLINE IN THE RAT' tensity with Rfvalues of 0.16 and 0.22. Further Sir: hydrolysis of combined I and I1 with G N hydroWe previously reported that the extent of inchloric acid a t 95" for 24 hours gave a new sub- corporation of the carbon of the methyl group of stance (IV) with ail Rr value of 0.46. Hydrolysis methionine, labeled with C14, to phospholipid of Factor 111, or of the fluorescent materials I and choline is markedly reduced in the folic acid11, with G N hydrochloric acid a t 150" for 21 hours deficient rat, and as a tentative explanation of this gave another fluorescent substance (V) with an Rr observation we suggested that in the folic acidvalue of 0.63,and phosphate ion was detected in deficient rat the synthesis of the acceptor of the the hydrolysates. These same hydrolysis condi- methyl group of methionine for choline formation tions degrade vitamin Blz to isomers of ribazole is inhibited2. It is generally assumed that aminophosphate, ribazole and ~,G-dimethylbenzimid- ethanol is the acceptor of three methyl groups of azole.6 methionine in zivo, the formation of choline taking In the case of Factor 111, it was assumed that place via direct transmethylation from methionine. the substances obtained were isomers of a riboside However, existing evidence does not indicate that phosphate (I and 11), the riboside (IV) and the folic acid or its biological derivative is a co-factor base (V). The base was isolated as a crystalline in the enzymatic reactions involving transmethylapicrate, m.p. mainly 220-225", which was con- tions from methionine, neither is folic acid or its verted to a polymorphic crystalline hydrochloride, derivative involved in the i n vivo decarboxylation (1) (a) W. Friederich and K. Bernhauer, A n g e u . Chem., 65, 627 of serine to aminoethanol. It occurred to US, (1953); (b) K. Bernhauer and W. Friederich, ibid., 66, 776 (1954). therefore, that the acceptor of the methyl group (2) W, Friederich and K. Bernhauer, Z. Naluv.fovachi~ng.,Sb, 686 (1954). (3) W. Friederich and K. Bernhauer, A n g e v . Chem., in press. (4) J. E. Ford and J. W. G. Porter, Bvit. J. A'ulvition, 1, 326 (1953). ( 5 ) C. E. Carter, ibid., 72, 1466 (1950). (6) (a) N. G. Brink and K. Folkers, THISJ O U R K A L , 72, 412 (1950); (b) N. G. Brink and K. Folkers, ibid., 74,2856 (1952): (c) E . A. Kaczka and K. Folkers, ibid., 75, 6317 (1953).
(1) Aided b y grants from the National Cancer Institute, U. S . Public Health Service, from the U. S. Atomic Energy Commission, AT-30-1 (1552), and by an Institutional grant from the American Cancer Society. The isotopic material was obtained on allocation from the Atomic Energy Commission. (2) J. A. Stekol, S. Weiss, P. Smith and K. Weiss, J . B i d . Cheni., 201, 299 (1953).
Oct. 5 , 1955
COMMUNICATIONS TO THE EDITOR
of methionine by transmethylation in vivo is not ethanolamine, and that in the in vivo synthesis of this acceptor, folic acid or its derivative is a cofactor. The data summarized briefly in Table I show that the extent of incorporation of cholineCH3-CI4 into the phospholipid choline is not inhibited by folic acid deficiency, whereas the incorporation of the carbon of the methyl group of methionine or of aminoethanol into choline was markedly reduced. The administration of amino-
5193
The composition of the diets, the procedures for the isolation of choline, and the radio assay procedure employed were the same as previously reported. THELANKENAU HOSPITAL RESEARCH INSTITUTE JAKOB A. STEKOL SIDNEY U'EISS AND THE INSTITUTE FOR CANCER RESEARCH PHILADELPHIA 11, PA. ETHELI. ANDERSO?; RECEIVED AUGUST5, 1955 STEREOISOMERIC TETRA-0-TOLYLSILANES'
Sir: I n connection with an investigation of highlyTHEUTILIZATION O F AMINOETHANOL-1,2-C14 AND THE EFFECT OF AMINOETHANOL, MONOMETHYLAMINOETHANOL, DI- substituted aromatic organosilicon compounds, stereoisomers of a novel type have been isolated.3 METHYLAMINOETHANOL, AND Citrovorum FACTOR ON THE UTILIZATION O F METHIONINE-CH,yC14FOR THE SYNTHESIS The aromatic groups of tetra-o-tolylsilane are not OF PHOSPHOLIPID CHOLINE IN FOLICACID-DEFICIENT free to rotate about the carbon-silicon bonds and i t is possible to construct no fewer than eight RATSO models of the molecule, representing four meso Per cent. of total activity compounds and two racemic pairs. in phospholipid choline Using as starting materials six4 or ( O - C H ~ C ~ H ~ ) ~ Folic S i x (where -X is -C1, -0CH3, or -0CzHs) and acidSupplement injected Nordeo-CH3C6H4Li,four different compounds have been Isotope injected with the isotope mal ficient isolated which appear to be tetra-0-tolylsilane Choline-CH3-Cl4 None 73.0 76.0 stereoisomers. These melt a t 14j0, 228") 300" Methionine-CH8-C14 None 30.0 18.4 and 344'. A fifth material which melts a t 270" Methionine-CH3-C14 Aminoethanol 17.8 may also be a stereoi~omer.~JVe wish to draw Methi0nine-CH3-C~~ Monomethylaminoparticular attention to the 145' and 228" isomers. ethanol 20.0 145' Isomer.-o-Tolyllithium (1.16 moles) in Methionine-CHs-C14 Dimethylaminoethanol 38.8 ether was slowly added to methyl silicate (0.20 Methionine-CH3-C14 Citrovorum factor 29.8 34.8 mole). After 117 hours a t room temperature Aminoethanol- 1,2and 35 hours of reflux (all under nitrogen), the C'4 None 3.0 0.7 Crystalline Thirty-days old female rats were maintained for two reaction mixture was months on a folic acid-free diet or on the same diet supple- products could be isolated only after distillation of mented with folic acid. 1 X mM. of the isotope per the crude syrups. The fraction boiling 200-210" 100 g. wt. alone or together with 1 X 10-1 mM. of the non(1 mm.) (50% yield if tetra-o-tolylsilane) was exisotopic amines was injected intraperitoneally in a single dose. Citrooorum factor (Leucovorin, Lederle), 0.1 mg., was tracted repeatedly with hot methanol. The cooled injected two hours before the radiomethionine. All animals extracts slowly deposited solids. The solids from were sacrificed one hour after the injection of the isotopes. the first extracts melted as low as 45",but those Phospholipid choline was isolated from the entire pooled from later extracts 110-120°.6 Six recrystallizacarcasses (2-3 animals per pool). tions of the latter material raised the melting ethanol or of monomethylaminoethanol together point to 143-145') and five additional recrystalwith radiomethionine did not improve the utiliza- lizations to 144.8-145.7' (2 g., 3%). The retion of methionine methyl group for choline forma- crystallization solvents were methanol-benzene tion in the deficient animals. However, the ad- (9: l), ligroin, methanol, ethanol, propanol-2 and ministration of either the Citrovorum factor or di- acetic acid. Considerable amounts of low melting methylaminoethanol together with radiomethionine solids and syrups also were obtained. These appromptly enhanced the incorporation of the methyl peared to be mixtures of tetra-o-tolylsilanes. group of methionine into choline. Injection of the A n a l . Calcd. for CaHzsSi: C, 85.66; H, 7.19; Citrovorum factor into normal animals did not in- Si, 7.15. Found: C, 85.18; H, 7.05; Si, 7.15. crease the extent of transfer of the methyl group Ultraviolet data.6 Amax. in mp (and E) for cyclo(1) The authors gratefully acknowledge the financial support of the of methionine to choline. Increasing the period of the in Vivo reaction to 20 or 48 hours did not im- Research Corporation. H. Gilman and G. N. R. Smart, J. Ovg. Chem., 15, 720 (1950) ; prove the utilization of the methyl group of (b)(2)16,(a) 424 (1951); 19, 441 (1954). methionine on administration of aminoethanol or (3) The absorption spectra of triphenylmethyl radicals, Crystal monoaminoethanol in folic acid-deficient rats. Violet ions, and related types have been interpreted in terms of the These data strongly suggest that choline is syn- existence of stereoisomeric forms (G.N. Lewis, T. T. Magel, and D. Lipkin, T~IIS JOURNAL, 64, 1774 (1942)). T h e polymorphism of thesized in the rat not by transfer of three methyl certain tri-1-naphthylboron-amine addition compounds has been groups of methionine to aminoethanol, but by attributed t o the restricted rotation of the naphthyl groups (H. C . transfer of one methyl group of methionine to di- Brown and S. Sujishi, ibid., 7 0 , 2793 (1948)). (4) T h e principal position isomers have been prepared and are methylaminoethanol as the direct acceptor. The different from the tetra-o-tolylsilanes. data further indicate that the de novo synthesis of (5) Melting points are uncorrected. the two methyl groups of dimethylaminoethanol is ( 6 ) The absorption curves for the 145O and 228O isomers are exmediated by a folic acid derivative, and that folic perimentally indistinguishable in the region 290-212 mM. Above 290 acid or its derivatives are not involved in the ma, the solutions are transparent. Below 246 m r , there is a rapid rive absorption continuing into the vacuum ultraviolet (e a t 212 m r , transfer of the methyl group of methionine to in 60,000). These spectra are related t o those of tetraphenylsilane and dimethylaminoethanol. tetra-p-tolylsilane. TABLE I
