ACETONE DERIVATIVES OF d-ribose. II.

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1 ACETONE DERIVATIVES OF d-ribose. II. BY P. A. LEVENE AND ERIC T. STILLER* (From the Laboratories of The Rockefeller Institute for Medical Research, New York) (Received for publication, June 14, 1934) The present work was undertaken with the purpose of preparing partially substituted ribose having one hydroxyl group, namely the one in position (2) or in position (4), unsubst.ituted. This substance was needed for the synthesis of the optically active 2- (or 4-) phosphoribitol. It was expected that acetonylation of methylribopyranoside would lead to the desired derivative. From the figure below, it can be seen that acetone could be expected to enter the molecule of methylribopyranoside by condensing either in positions (2) and (3) or in (3) and (4). CH I H-C-OH. OCH31 I 0 H.C.OH I I H.C.OH I Consequently, normal methylriboside was prepared by essentially the same method as that employed by Levene and Tipson. Polarimetric observations of the initial glycoside formation in the cold showed that a maximum specific rotation of +7.7 was reached in 82 minutes and the final rotation was (after boiling for 130 minutes). The difference in final specific rotation from that * Commonwealth Fund Fellow. 1 Levene, P. A., Harris, S. A., and Stiller, E. T., J. Biol. Chem., 106, 153 (1934). * Levene, P. A., and Tipson, R. S., J. Biol. Chem., 92, 109 (1931). 421

2 Acetone Derivatives of d-ribose quoted by Levene and Tipson is probably due to the fact that in the present investigations a specially purified sample of dry ribose was used, and that heating was continued longer. The methylribopyranoside was condensed with acetone by the method previously described by the present authors3 In order to ascertain the structure of the condensation product, it was methylated by means of Purdie s reagents, yielding a monomethyl monoacetone methylriboside. On attempting to remove the acetone residue by means of 0.02 N hydrochloric acid-a reagent which should not effect the hydrolysis of the glycosidic methyoxyl group of a pyranoside4-the product reduced Fehling s solution strongly and was shown to contain 46 per cent of reducing monomethyl pentose. Further light was thrown on this unexpected result by the complete methylation of the hydrolyzed product, which gave a trimethyl methylriboside. The product on treatment with 0.02 N hydrochloric acid at 100 showed a fall in rotation and, by means of Willstatter titrations6 an increasing production of reducing mat erial to a maximum of 52 per cent was demonstrated. It is thus evident that during condensation of methylribopyranoside with acetone, a shift in the position of the lactal ring occurs, similar to that which has been shown by Levene and Meyer6 to take place in the case of the condensation of methylmannopyranoside with acetone. Confirmatory evidence of this partial ring shift was found in an examination of the action of p-toluenesulfonyl chloride on the acetone methylriboside. The product was shown to be a mixture of 5-p-toluenesulfonyl monoacetone methylribofuranoside with 4-p-toluenesulfonyl monoacetone methylribopyranoside. The former was compared with a specimen of known structure prepared directly from monoacetone methylribofuranoside and found to be identical. The latter was shown to be a pyranose derivative by its non-reaction with sodium iodide under standard conditions7 and by the resistance of its glycosidic methoxyl group to hydrolysis under conditions which would cause hydrolysis of a furanoside. 3 Levene, P. A., and Stiller, E. T., J. Biol. Chem., 102, 187 (1933). 4 Bott, H. G., Hirst, E. L., and Smith, J. A. B., J. Chem. Xoc., 658 (1930). 5 Willstgtter, R., and Schudel, G., Ber. them. Ges., 61, 780 (1918). 6 Levene, P. A., and Meyer, G. M., J. Biol. Chem., 78, 363 (1928). 7 Oldham, J. W. H., and Rutherford, J. K., J. Am. Chem. Sot., 64, 366 (1932).

3 P. A. Levene and E. T. Stiller 423 By treatment of the 5-p-toluenesulfonyl monoacetone methylribofuranoside with sodium iodide, an iodo derivative was obtained, conclusively demonstrating that the p-toluenesulfonyl residue was attached to the primary alcoholic group at position (5) of the ribose chain. By hydrolysis of the 4-p-toluenesulfonyl monoacetone methylribopyranoside with aqueous methyl alcohol containing 5 per cent sulfuric acid in the cold, a 4-p-toluenesulfonyl methylribopyranoside was formed. Thus it was definitely proved that on acetonylation methylribopyranoside in part rearranges into the furanoside. Indeed, study of a model reveals the fact that there is less strain in the structure of the 2,3-isopropylidene methylribofuranoside than in either of the two possible monoacetone derivatives of the methylpyranoside.8 EXPERIMENTAL Preparation of Methylribopyranoside-The methylribopyranoside was prepared in essentially the same manner as described by Levene and Tipson. Owing to the use of very specially purified ribose, the final rotation differed somewhat from that recorded by the above authors and by Levene, Raymond, and Dillon,g so a description of the preparation will be given. 20 gm. of finely powdered ribose (which had been twice recrystallized from absolute alcohol and dried to constant weight) were dissolved in 100 gm. of absolute methyl alcohol and 100 gm. of a.bsolute methyl alcohol containing 3 gm. of dry hydrogen chloride were added. The mixture was allowed to stand at room temperature while the specific rotation was followed polarimetrically. The specific rotation changed rapidly from (6 minutes after admixture) and reached a minimum of +7.7 in 113 minutes, after which the rotation commenced to increase very slowly. After 113 minutes the solution was boiled gently in a water bath at SO, polarimetric observations (see Table I) being made after the solution had been 8 The final purification of the tosyl derivatives was accomplished by Dr. R. S. Tipson. For this help as well as for his help in the preparation of the manuscript, we wish to express our thanks. 9 Levene, P. A., Raymond, A. L., and Dillon, R. T., J. Biol. Chem., 96, 699 (1932).

4 424 Acetone Derivatives of d-ribose cooled and shaken with a little charcoal to remove the slight strawyellow color. After boiling for 130 minutes, the specific rotation remained constant at The solution was now rendered neutral by addition of silver carbonate and the silver salts removed by filtration and thoroughly washed with small quantities of methyl alcohol. The combined filtrate and washings were evaporated to dryness under diminished pressure at a temperature below 35, the last traces of solvent being removed under the high vacuum pump. The product (22.2 gm.) TABLE Condensation of d-ribose with Methyl Alcohol in Presence of Hydrogen Chloride Cold (27 ) Time 14, min. degrees $ f $ I Cold (27 ) Boiling (65 ) Time min. degrees f was a pale yellow, viscous syrup which did not reduce boiling Fehling s solution. [al; = (in water) In order to demonstrate that the amount of furanoside in the normal methylriboside was small, a weighed quantity was dissolved in 0.1 N hydrochloric acid and during 24 hours at 25 the specific rotation remained unchanged. The solution was then diluted with an equal volume of water, making the solution 0.05 N and samples

5 P. A. Levene and E. T. Stiller 425 were heated in sealed tubes in a boiling water bath. During the first half hour, the specific rotation remained constant ( ) and thereafter fell very slowly (60 minutes, ). Acetonylation of Methylribopyranoside-Methylribopyranoside (21 gm.) was dissolved in pure, dry acetone (400 cc.) containing 0.2 per cent sulfuric acid, and anhydrous copper sulfate (40 gm.) was added.3 The mixture was shaken at 37 during 20 hours. The copper sulfate was now removed by filtration and thoroughly washed with small quantities of acetone. The combined filtrates and washings were shaken with calcium hydroxide until neutral and the calcium salts then filtered off and washed with acetone. The filtrate was evaporated to dryness under diminished pressure, the temperature being kept below 35, and the final traces of solvent were removed under the high vacuum pump. The product was distilled under a high vacuum, giving a main fraction (19.1 gm.) boiling at at mm. This product was refractionated, giving 17.0 gm. of a pale yellow syrup which distilled at at 0.05 mm. and had the following composition mg. substance: mg. AgI C8H1304 (OCH&. Calculated. OCH, Found Preparation of Monomethyl Monoacetone Methylriboside-Monoacetone methylriboside (3.9 gm.) was methylated by means of Purdie s reagents (20 cc. of methyl iodide and 16 gm. of silver oxide) with vigorous stirring. The product was isolated in the usual way and was distilled under a high vacuum, giving 3.5 gm. of a colorless mobile syrup (b.p at 0.04 mm.) which had t,he following composition mg. substance: mg. AgI CJLOJ (OCH,),. Calculated. OCHS Found Hydrolysis of Monomethyl Monoacetone Methylriboside-The product (3.5 gm.) was dissolved in 44 cc. of 0.02 N hydrochloric acid and heated at 100. Polarimetric observations showed a rapid change in rotation, from an initial value of to a constant value of after 30 minutes. The solution was rendered neutral with barium carbonate and the water removed under

6 Acetone Derivatives of d-ribose diminished pressure at a temperature below 30. The product was mixed with benzene and the solvent distilled off under diminished pressure in order to remove the last traces of water. This treatment was repeated three times. The product, 2.5 gm. (theory 2.6 gm.), was a pale yellow viscous syrup which was extracted by means of absolute alcohol. It reduced warm Fehling s solution strongly. The amount of reducing material present was estimated by the method of Willstatter and Schude15 and shown to be 46.5 per cent. Exhaustive Methylation of Hydrolytic Product-The product from the acid hydrolysis (2.3 gm.) was dissolved in 25 cc. of methyl alcohol containing 1.5 per cent dry hydrogen chloride and allowed to stand at room temperature until the rotation became constant. In 1 hour a constant specific rotat,ion of was attained. The solution was rendered neut,ral with silver carbonate and the silver salts removed by filtration and thoroughly washed. The combined filtrate and washings were evaporated to dryness under diminished pressure at a temperature below 30. The product (2.4 gm.) was methylated by means of Purdie s reagent (30 cc. of MeI, 20 gm. of AgzO) with vigorous stirring. On isolation in the usual manner, the syrupy product was remethylated with half the above quantities of Purdie s reagent. The product was isolated and dist,illed at at 0.03 mm. The ni7 was , and the composition as follows: mg. substance: mg. CO, and mg. Hz : AgI GH60 (OCH,),. Calculated. C 52.4, H 8.8, OCH, Found. 52.3, 8.9, 60.2 Partial Hydrolysis of Mixed Trimethyl Methylribosides mg. of the fully methylated product were dissolved in 25 cc. of 0.02 N hydrochloric acid. 4 cc. samples were sealed in tubes and heated at 98. At various intervals of time, a tube was removed from the heating bath and quickly chilled in ice. The polarimetric observations were performed for mercury green light (X = 5781) and the amount of reducing sugar present in each sample was determined by means of a Willstatter-Schudel titration (see Table II). Preparation of 5-p-Toluenesulfonyl 2,SMonoacetone Methylribo-

7 P. A. Levene and E. T. Stiller 427 furanoside-2,3-monoacetone methylribofuranoside was prepared by the method previously described by Levene and StillerlO by treating ribose with acetone containing 5 per cent of methyl alcohol in the presence of 0.2 per cent sulfuric acid and anhydrous copper sulfate. 2.8 gm. of monoacetone methylribofuranoside were dissolved in the minimum quantity of dry pyridine, p-toluenesulfonyl chloride (3.0 gm.) was added, and the mixture was allowed to stand at room temperature overnight. 1 drop of water was added and the solution (cooled in ice) was allowed to stand for 30 minutes. Chloroform and water were now added and the aqueous layer was extracted three times with small quantities of chloroform. The combined extracts were washed with dilute sulfuric acid, dilute TABLE Hydrolysis of Mixed Trimethgl Methylribosides Time [4181 hrs. degrees II Reducing per cent sodium hydroxide, and finally, with water. After drying over anhydrous sodium sulfate and filtering, the filtrate was evaporated under reduced pressure, giving 5.0 gm. of pale yellow, viscous syrup which crystallized rapidly on scratching under a little dry ether. After recrystallization from absolute alcohol the fine white needles had a melting point of The substance had the following composition mg. substance: mg. CO2 and mg. Hz : BaSOa : AgI ClaH220~S. Calculated. C 53.6, H 6.2, S 8.9, OCHl Found. 53.8, 6.0, 8.7, 8.6 The specific rotation of the p-toluenesulfonyl derivative was [a]; = x x = (in absolute ethyl alcohol) mgzw 10 Levene, P. A., and Stiller, E. T., J. Biol. Chem., 104, 299 (1934).

8 428 Acetone Derivatives of d-ribose Action of Sodium Iodide on 5-p-Toluenesulfonyl Monoacetone Methylribofuranoside-The 5-p-toluenesulfonyl monoacetone methylribofuranoside (1 gm.) was treated in acetone with sodium iodide (1 gm.) in a sealed tube at 100 for 2 hours. The solution was diluted with water and extracted repeatedly with chloroform. The chloroform extracts were washed with sodium thiosulfate solution in order to remove any free iodine and then dried over anhydrous sodium sulfate. On removal of the solvent, a pale yellow syrup (1 gm.) (which resisted all attempts at crystallization) remained. It had the following composition mg. substance: mg. AgI : C9HlsOJ. Calculated. I 40.4, OCHl Found. 40.4, * 9.8 Preparation of 4-p-Toluenesulfonyl Monoacetone Methylribopyranoside-The mixture of monoacetone methylribosides described above (5 gm.) was treated with 1.1 moles of p-toluenesulfonyl chloride in dry pyridine and allowed to stand overnight. The product was isolated as described above and 11.2 gm. of substance were isolated, crystallizing in white needles from absolute alcohol. The product began to soften at 78 and had melted completely at 127. By fractional crystallization from absolute alcohol, two homogeneous fractions were isolated. The lower melting fraction was identical with the 5-p-toluenesulfonyl monoacetone methylribofuranoside described above, m.p , giving no depression on admixture and forming an iodo derivative which did not crystallize. The other pure fraction, m.p , had the following composition mg. substance : mg. COz and mg. H : BaSOd : AgI C,eH,,OS. Calculated. C 53.6, H 6.2, S 8.9, OCH Found. 53.8, 6.3, 8.9, 8.8 It showed the following specific rotation x 100 [a]~ = 2 X = (in absolute ethyl alcohol)

9 P. A. Levene and E. T. Stiller This fraction of higher melting point did not react with sodium iodide, the starting material being recovered unchanged after treatment under standard conditions for 2 hours. The main portion of the original mixture of p-toluenesulfonyl derivatives, when heated with sodium iodide in acetone at 100 in a sealed tube for 2 hours, gave a product which partly crystallized. The crystals, on separation from the syrupy iodo compound identical with that already described, proved to be 4-p-toluenesulfonyl monoacetone methylribofuranoside (m.p ). Hydrolysis of Acetone Residue from..j-p-toluenesulfonyl Monoacetone Methylribopyranoside-4-p-Toluenesulfonyl monoacetone methylribopyranoside (2.5 gm.) was dissolved in 100 cc. of methyl alcohol and 20 cc. of 5 per cent sulfuric acid were added. A crystalline precipitate formed and on allowing the mixture to stand at room temperature for 4 hours, the suspension disappeared. 20 cc. of 5 per cent acid were then added and, after the precipitate had dissolved (4 hours), further addition of acid produced no precipitate. The acid was neutralized with barium carbonate and the barium salts removed by filtration. On removing the solvent under reduced pressure, the product crystallized spontaneously in white needles. It was recrystallized from ether, the substance having a melting point of 124 and the following composition mg. substance : mg. COz and mg. Hz : BaSOl : AgI C&H180~S. Calculated. C 49.0, H 5.7, S 10.1, OCHJ Found. 49.2, 5.8, 10.1, 9.9 Its specific rotation was as follows: [al:: = x x = (in chloroform)

10 ACETONE DERIVATIVES OF d-ribose. II P. A. Levene and Eric T. Stiller J. Biol. Chem. 1934, 106: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1

THE RING STRUCTURE OF THYMIDINE

THE RING STRUCTURE OF THYMIDINE BY P. A. LEVENE AND R. STUART TIPSON (From the Laboratories of The Rockefeller Institute for Medical Research, New York) (Received for publication, March 13, 1935) The 2-desoxy-ribose