Vitamin B12. XVIII. The Degradation of Vitamin B12 to 1-α-D

Vitamin B12. XVIII. The Degradation of Vitamin B12 to 1-α-D-Ribofuranosyl-5,6-dimethylbenzimidazole. Norman G. Brink, Karl Folkers. J. Am. Chem. Soc...
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NORXANG. BRINKAND KARLFOLKERS

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ingaldchyde ciricL 0.83 g. (0.004 mole) of 2-hydroxy-3methoxy-5-propylacetophenone, and 6 ml. of an aqueous potassium hydroxide solution (26 g. of pellets, 75 g. of water) added. The mixture was heated in a closed flask for 24 hours at 70°, with frequent shaking at first and occasional shaking during the remaining time. The reaction mixture was dissolved in water, acidified with dilute hydrochloric acid, shaken with petroleum ether to remove the bulk of the unreacted phenone, and the aqueous solution drdined from the precipitated orange resin. The resin was dissolved in ether, the ether solution shaken with several portions of 30% sodium bisulfite solution, and the ether iolution dried. The ether was evaporated and the residue tlissolved by boiling with several portions of petroleum naphtha. The combined naphtha extracts were cooled in a refrigerator and the solvent, containing some residual phenone, was decanted. The resinous residue was crystallized from 50% aqueous ethanol, yielding 0.20 g. of orange

[Cox I R I R C I I O X

Vitamin BI2. XVIII.

I RUM M E

Vol. 74

crystals sintering at 68" and fusing to a viscous resin a t about 7 3 " . The product, apparently a hydrate, was dried a t 105' and recrystallized from getroleurn naphtba t o give orange needles, m.p. sinters 110 , melts 114-116 Anal. Calcd. for C2lHg406: C, 67.73; H, 6.50. Found: C, 68.03; H, 6.58.

.

Acknowledgment.-The author wishes to thank Dr. F. E. Rrauns, Institute of Paper Chemistry, for the sample of spruce "native lignin," Dr. K. Freudenberg, Heidelberg University, for the sample of enzymatically polymerized coniferyl alcohol and hfr. A. W. Goos, Cliffs-Dow Chemical Company, for the distillate containing 2,6-dimethoxy-4-propylphenol. MADISON 3, U'ISCONSIU

RECEIVED OCTOBER12, 1951

RESEARCH LABORATORIES OF A ~ E R C K& C o . , I N C I

The Degradation of Vitamin B12 to l-a-D-Ribof~~ranosyl-5,6dimethylbenzimidazole BY NORMAN G. BRINKAND KARLFOLKERS RECEIVED FEBRUARY 7, 1932

Acid hydrolysis of vitamin BIZyielded a product which has been identified as l-~u-~-rihofuranosyl-5,6-dimethylbenzimidazole. It was characterized as a l-pentofuranosyl-5,6-dimethylbenzimidazole by periodate oxidation studies, which led to aldehyde derivative, and which the isolation of a crystalline ~u-(5,6-dimethylbenzimidazole-l)-oc'-hydroxymethyldiglycolic suggested the a-configuration at carbon atom one of the ribose moiety of the glycoside.

The preparations of l-a-D-ribofuranosyl-S,G-diinethylbenzimidazole (a-ribazole) (I) from vitamin BI? and by synthesis have been communicated.' Details of the isolation of the degradation product

presence of a different substituted benzimidazole. When a solution of the previously obtained 5,6-dimethylbenzimidazole was made alkaline, an absorption maximum appeared a t 2470 A,, the magnitude of the absorption of this new peak being less, o - ~. -. however, than tho,se of the two principal maxima I a t 2810 and 2880 A. With the basic fraction from CFI OEI ? € I the hydrolysis dons a t 120°, the new peak which I \ s / .---i'-c- C-C --cI-{?()I I appeared a t 2.500A. when the solution was made I I II €1 11 alkaline wits higher than the two original absorpi tion peaks. The crude product was only sparfrom an acid hydrolysate of vitamin BIZ and its ingly soluble in ether; and the ether-insoluble macharacterization as a l-pentofuranosyl-5,6-dimeth- terial was indicated to contain carbohydrate by the ylbenzirnidazole are described herein. The final color test involving dehydration to furfural or its characterization of the degradation product as a- derivatives.j This same material yielded a crystalribazole resulted from its identification with syn- line picrate which melted a t 212-214', and hence thetic cu-ribazole.2 The assignment of the cu-config- was not identical with 5,B-dimethylbenzirnidazole uration to the degradation product followed from a picrate (ni.p, 273-275'). The crystalline picrate comparison of the optical rotations of synthetic a- also gave a positive carbohydrate test.5 Subsequent hydrolyses of vitamin Blz done in G N and @-ribazoles.2 A direct correlation of P-ribazole hydrochloric acid a t 120' afforded mixtures of the with l-P-~-glucopyranosyl-5,6-dimethylbenzimidabenzimidazole glycoside picrate and 5,6-dimethylzole is described below. Hydrolysis of vitamin BIZ in 6 nT hydrochloric benzimidazole picrate. However, when the hyacid a t 150' for 20 hours gave 5,6-dimethylbenzimid- drolysis was carried out overnight a t a temperature azole,3 an observation which was shortly confirmed of loo", splitting of the glycoside was negligible, by 0the1-s.~ When the hydrolysis was carried out and the glycoside picrate could be readily isolated a t a somewhat lower temperature, 120') and the in pure form. The picrate was dextrorotatory and hydrolysate treated to yield a basic fraction isolated had an ultraviolet absorption spectrum ia acidic by continuous chloroform extraction, the absorp- ethanol solutio? with maxima a t 2760 4. ( E M tion spectrum of the crude product suggested the 10,950), 2530 A. ( E M 10,600) and 3590 A. ( E M 13,000). Analyses indicated that the composition (1) N G. Brink, F. W. Holly, C. H. Shunk, B. W. Peel, J. J Cahill of the picrate corresponded to the formula C14H18a n d K . Folkers, T H I S JOURNAL, 72, 1866 (1950). N z O ~ * C B H ~ NRemoval ~ O ~ . of picric acid by chloro(2) F. W. Ilolly. C. H. Shunk, E. W. Peel, J. J. &hill, J. B. Larigne and K. Folkers, ibid., i n press. form extraction of a dilute aqueous hydrochloric (3) N. G. Brink and K. Folkers, ibid., 72, 4442 (19.50). acid solution of the picrate yielded the amorphous ( 4 ) E. R. Holliday a n d V. Petrom, J . P/zni.r,i. Phni?~tacol.,1, 7 3 4

j

1

(1949); G. R. Beaven, E. R. Iiolliday, E. A. Johnson, B. Ellis, P. Mamalts, V. Petrow a n d B. Sturgeon, ibid., 1, 957 (1949).

(5) F. Feigl, "Qualitative Analysis b y Spot Tests," Third English Edition, Elsevier, h'ew York. N. Y.,1046, p. 410.

June 5, 1952

TO ~-Q-D-~IBOFURANOSYL-~,~-DIMETHYL,BENZIIVIIDAZOLE 2857

VITAMIN

hydrochloride. The free base was obtained by dissolving the hydrochloride in water, making the solution weakly alkaline, and extracting continuously with chloroform. The amorphous free base prepared from the picrate Via the hydrochloride reacted with approximately 0.7 mole of periodate per mole. The crystalline glycoside picrate consumed 0.92 mole of periodate per mole. This latter oxidation yielded a crystalline picrate of map. 180-185' and [ a I z 3 D + 2 1 The product was unstable, its melting point f 4'. decreased upon recrystallization, and satisfactory analytical results were not obtained. From the method of preparation, i t was formulated to be an CY-( 5,6-dimethylbenzimidazole-l)-a' - hydroxymethyldiglycolic aldehyde picrate (11,R = 5,gdimethylbenzimidazole-1) , Similar periodate oxidation of a synthetic model compound, 1-P-D-glucopyranosyl5,G-dimethylbenzimidazolepicrate2 with two equivalents of periodate led not to the above dialdehyde picrate, but to a presumably anomeric picrate (111) of m.p. 141-144' and specific rotation of [CU]"D -20 i 2'. This product too was unstable, and its melting point decreased upon recrystallization. H

R

R

CHO

CHO

0

YHO I

HC-

I

CHzOH

I1

H

v

v

0

YHO I

HC-

I

CHzOH I11

The consumption by the glycoside derived from vitamin Blz of one mole of periodate permitted the assignment of a furanoid ring structure to the pentose moiety. The lack of identity of the oxidation products I1 and 111 suggested an a-configuration about the glycosidic carbon atom,6 leading to a structure of the degradation product as a l-cu-pentofuranosyl-5,6-dimethylbenzimidazole. In order to determine which of the four possible pentoses was combined with 5,6-dimethylbenzimidazole in the glycoside, experiments to hydrolyze the glycosidic linkage and identify the sugar were done. The conditions used in the hydrolytic degradation of vitamin Blz to the benzimidazole glycoside demonstrated the stability of the N-glycosidic linkage, which was not noticeably cleaved by treatment with strong mineral acid for several hours a t loo', and which was only partially broken by similar hydrolysis a t 120'. Indeed, hydrolytic conditions which sufficed to cleave the glycosidic linkage in the degradation product caused such extensive decomposition of the pentose that extensive degradative experiments to identify the carbohydrate moiety were not undertaken. It is well known that purine nucleosides are readily hydrolyzable by acids to give a pentose and a base, whereas pyrimidine iiucleosides cannot be hydrolyzed with dilute acids. Hydrolysis of compounds of the latter class under more drastic conditions gives only the base, the su(6) Cf.B. Lythgoe and A. R. Todd, J . Chcm. SOL.,592 (1944).

gar being destroyed.' It is evident that the behavior of the glycosidic linkage of the degradation product toward acid hydrolysis is the opposite of what might have been expected from the reported properties of the purine and pyrimidine glycosides and the obvious structural resemblance between the degradation product and the purine nucleosides. The final identification of the vitamin Blz degradation product as l-a-~-ribofuranosyl-5,6-dimethylbenzimidazole was accomplished by comparing the picrate with a synthetic specimen.2 The synthetic picrate melted a t 212-214' and showed a rotation of [ a I z 3+9.1 ~ f l o , as compared with the corresponding values of 212-214' and [ a I z a ~ +9.9 f1.6' for the compound obtained from vitamin BIZ. A mixture of the materials melted without depression, and the absorption spectra were identical. The synthetic a-ribazole picrate consumed one mole of periodate and gave a crystalline CY- (5,6-dimethylbenzimidazole1)-a' - hydroxymethyldiglycolic aldehyde picrate which was identical with the corresponding derivative of the picrate of the degradation product on the basis of melting point, mixed melting point and specific rotation. It is of interest that synthetic P-ribazole picrate on periodate oxidation gave a product which appeared to be identical with the corresponding derivative from l-P-~-glycopyranosyl-5,6-dimethylbenzimidazole picrate described above. This confirms the assignments of configuration about the glycosidic carbon atoms of CY- and P-ribazoles, which were originally made on the basis of the specific rotations. Experimental Part Acid Hydrolysis of Vitamin BI~.-A solution of vitamin BIZin 4 ml. of 6 N hydrochloric acid was heated in a sealed tube a t 120' for eight hours. The solution was then filtered, concentrated to dryness, and the residue dissolved in 4 ml. of water containing 3 drops of 1 N hydrochlorir acid. This acidic solution was extracted continuously with chloroform for 36 hours. The aqueous solution was then adjusted to pH 10 by the addition of sodium hydroxide solution, and extracted continuously with chloroform for 12 hours. The chloroform extract of the basic solution was evaporated to dryness, leaving a colorless oil which weighed 3.5 mg. A portion of this oily basic fraction showed in 0.01 N ethanolic hydrochloric acid solution an absorption spectrum characterized by maxima a t 2775 A. 222) and 28.50 A. (E:?m, 222). When the solution was made 0.01 N in sodium hydroxide, the spectrum showed maxima ?t 210), 2825 k . (E:?m. 152) and 2890 A . 2500 k . (E:?m.154): The remainder of the basic fraction was leached with 2 ml. of ether, and the ether extract discarded. Part of the ether-insoluble material which remained was heated with oxalic and sulfuric acids and the vapors tested for furfural or a related aldehyde with o-dianisidine according to Feigl.5 The test was strongly positive. Another portion of the ether-insoluble basic fraction upon solution in water and treatment with saturated aqueous picric acid solution gave a crystalline picrate, m . p . 211-214'. After recrystallization from aqueous ethanol, the product melted a t 212-214O. The crystalline picrate also gave a positive Feigl carbohydrate test. Preparation of l-or-~-Ribofuranosy1-5,6-dimethylbenzimidazole from Vitamin B12.-A solution of 86 mg. of vitamin BI?in 5 ml. of 6 N hydrochloric acid was heated at 100" for 18 hours. The solution was then evaporated to dryness in (7) P. A. Levene a n d L. W. Bass, "Nucleic Acids," Chemical Catalog Co. (Reinhold Publ. Corp.), New York, N. Y.,1931, p

144-145.

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N ~ R ~ I G. A NBRINKAND KARLFOLKEKS

Val. 74

vacuo and the residue was dissolved in 8 ml. of water and 0.4

ture then consumed 0.550 ml. of 0.0100 N arsenite solution. ml. of 1 N hydrochloric acid. This solution was extracted This corresponds to a periodate consumption of 0.00139 continuously with chloroform for eight hours. The aqueous millimole, or 0.92 mole per mole of a-ribazole picrate. solution was then made alkaline (PH 9-10) with sodium In another experiment, 5.1 mg. (0.010 millimole) of ahydroxide solution and extracted with a fresh portion of ribazole picrate was dissolved in 4 ml. of methanol and the chloroform for 16 hours. The chloroform extract of the solution was treated with 0.27 ml. of 0.0447 M sodium perioalkaline aqueous solution was filtered and evaporated to date solution (0.012 millimole). After having stood overdryness. The resulting 10.2-mg residue was extracted with night, the solution was diluted with 2 ml. of water and confour 3-mi. portions of boiling ether and the combined ethe- centrated in vacuo to a volume of about 2 ml. Another 2 real extracts were concentiated t o a volume of 0.5 ml. The mi. of watei was added, and the solution concentrated to a white, amorphous precipitate which had formed during the volume of 1 ml. and allowed to stand a t 5’ overnight. The concentration was separated from the supernatant solution, yellow crystals which had separated were collected by cenh i e d and dissolved in 0.5 ml. of water. The addition of 1 trifugation, washed twice with water, and dried in vacuo. nil. of a saturated aqueous sclution of picric acid resulted in The product weighed 4.2 mg. and melted a t 180-185’ (dec.). the immediate formation of a crystalline piecipitate. After One recrystallization from aqueous ethanol failed to change two hours, the mixture was centrifuged, and the precipitate the melting point. The material showed a specific rotation was washed twice with water and dried ilt vucuo. The of [ a I z 3 D +24 4’ ( c 0.58 in pyridine). yield of l-a-~-ribofuranosyl-5,6-dimethylbenzimidazole picPeriodate Oxidation of Synthetic a-Ribazole Picrate.-A rate was 7.0 mg. I t melted a t 212-214’, and was recrystal- 31-mg. quantity of synthetic a-ribazole picrate2 was oxidized lized from aqueous ethanol to give a product of m.p. 213with periodate exactly as described above for the vitamin €312 214” degradation product. Recrystallization of the resulting Anal. Calcd. for ClaHl*N,O, C B H ~ N J O C, ~ : 47.34; H, compound from aqueous ethanol gave a picrate which 4.17; N , 13.80; picric acid, 45.3. Found: C, 47.52; H, melted a t 183-186’ and had a rotation of [ a ] 2 3 ~$20 f 4’ (c 5.5 in pyridine). Upon admixture with the peliodate 3 93; X, 14.07; picric acid, 15.9 (spectrophotometric). oxidation product (m.p 180-185’) of “noatural” a-ribazolc The picrate had a specific rotation [ a I z 3 D 4-9.9 i 1.6’ picrate, the material melted at 183-185.5 . (c 2.4 in pyridine). Its ultraviolet absorption spectpm in A n a l . Calcd. for C ~ O H I ~ S ~N, O ~13.86. I: Found: N, acidic ethanol solution showed maxima a t 2780 A. ( E x 13 08. 10,950), 2850 8. ( E X 10,600) and 3590 A. (E,, 13,000). Periodate Oxidation of 1-p-~-Glucopyranosyl-5,6-diA solution of 6 mg. of 1-a-~-ribofuranosyl-5,6-dimethylmethyl-benzimidazole Picrate.--A solution of 9.6 mg. of benzimidazole picrate in 4.5 ml. of water containing 0.2 ml. P-D-glucopyranosyl-5,fi-dimethylbenzimidazolepicrate2 in of 1 N hydrochloric acid was extracted for two hours with 5 ml. of methanol was treated with 0.77 ml. of 0.0533 &’ chloroform. The colorless aqueous solution was then lyo- sodium periodate solution (2.3 moles per mole of picrate). philized, yielding 3.5 mg. of oily a-ribazole hydrochloride. The reaction mixture stood a t room temperatui e overnight This &as in turn dissolved in water (3 ml.), and the solution and was then worked up by the addition of 1 ml. of water brought to pH 8 with sodium hydroxide, and extracted con- and concentration in vacuo to a volume of 1 ml. The crystinuously for six hours with chloroform. Evaporation of talline product which separated was collected, washed twice the chloroform extract gave 2.8 mg. of white, amorphous a- with watei and dried in VUCUQ. It weighed 6.4 mg., melted ribazole free base. ~ D f 2” a t 741-144’ and had a specific rotation of [ C Y ] ~ -20 Periodate Oxidations .-One and two-tenths milligrams of (c 1.0 in pyridine). Recrystallization of the picrateofrom ‘imorphous a-ribazole (0.0043 millimole) was dissolved in aqueous ethanol lowered the melting point to 136-141 . 2.00 ml. of water and 0.50 ml. of 0.0207 M sodium periodate Periodate Oxidation of I-fl-~-Ribofuranosyl-5,6-dimethylsolution was added. After 4.5 minutes, a 1.00-ml. aliquot benzimidazole Picrate.-Oxidation of 2 4 mg. of synthetic 8portion was withdrawn from the reaction solution and ana- ribazole picrate2 in the manner described above gave a lyzed for unieacted periodate by addition of sodium bicar- product in the form of yellow platelets. It melted a t 132bonate and potassium iodide and titration with 0.0100 N ar- 1 3 6 O , and no depression of melting point was observed with a senite solution. A volume of 0.600 ml. of reagent was re- mixture of this product and the oxidation product of 1-@-Dquired, corresponding to a consumption of periodate of 0.67 glycopyranosyl-5,6-dimethylbenzimidazolepicrate described mole per mole of a-ribazole. Subsequent titrations after above. 80 and 170 minutes gave values for periodate consumption of Acknowledgment.-The authors wish to thank 0.65 and 0.67 mole per mole, respectively. 30 change in the PH of the reaction solution could he detected during the Ilk. R. N. Boos and his associates for the microanalyses reported here. They are indebted t o Dr. oxidation. A 0.771-mg. (0.00152 millimole) sample of a-ribazole pic- N. R. Treiiner and co-workers for some of the deterrate was dissolved in 1.8 ml. of pure methanol, and 0.20 minations of specific rotation and ultraviolet abi d . of 0 0207 hl aqueous sodium periodate solution was added. After the solution had stood a t room temperature sorption. for one hour, hicarbonate and iodide n ere added; the mix- RAHWAY, KEWJERSEY

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1-