THE RING STRUCTURE OF THYMIDINE

Transcription

1 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 nucleosides, first isolated by Levene and London by the hydrolysis of thymus nucleic acid, are characterized by their extreme ease of hydrolysis by very dilute mineral acids. Thus, guanine-2-desoxy-riboside is completely hydrolyzed by heating with 0.01 N hydrochloric acid during 5 minutes. Since the rate of hydrolysis is of the same order as that of the furanosides, it was considered possible that the 2-desoxy-ribonucleosides, similarly to the ribonucleosides, have the furanoside ring structure. When, however, it was discovered2 that methyl-2-desoxyglucopyranoside is almost as readily hydrolyzed, it appeared just as feasible that the natural derivatives might have the pyranoside ring structure. Hence the question of their ring structure was in need of further special investigation. We have now studied the ring structure of the sugar portion of thymidine (thymine-2-desoxy-riboside). This substance is found to react with triphenylmethyl chloride in pyridine to give a monotrityl derivative. Since it was shown by us3 that the analogous monotrityl uridine is the 5-substituted derivative, the formation of a monotrityl thymidine might, of itself, be considered a good indication that thymidine is, likewise, a furanoside. Definite proof of this conclusion was provided by substituting the remaining free hydroxyl group by a tosyl group and examining the behavior of the resulting monotosyl trityl thymidine on treat- Downloaded from by guest on August 25, Levene, P. A., and London, E. S., J. Biol. Chem., 81, 711 (1929); 83, 793 (1929). 2 Bergmann, M., and Breuers, W., Ann. Chem., 470, 51 (1929). Levene, P. A., and Mikeska, L. A., J. Biol. Chem., 88, 791 (1930). * Levene, P. A., and Tipson, R. S., J. Biol. Chem., 106,419 (1934). 623

2 624 Ring Structure of Thymidine ment with sodium iodide dissolved in acetone. It was found that t.he tosyloxy group in this case was much more stable than if attached at a primary hydroxyl but, presumably owing to the presence of the CH2 group in position (2), the tosyloxy group in position (3) was somewhat less stable than the same group at- CR3 I "'"-"=I: I H.N - c - B II, Thymidine 0 C,H502p I R.C I 1 % 1 0 I C"2"+%~ /c&5 > '%W2N2 B.C 1 CH2 I Trityl I /w5 CH,O.C-C,H5 bk lbymidine 3-T03yl 5-Trityl!l'hymidine tached at a secondary hydroxyl group in true sugars. In confirmation, it was found that the secondary tosyloxy group in monotosyl 5-iodothymidine had about the same stability towards the above reagent as had the secondary tosyloxy group in monotosyl trityl thymidine.

3 P. A. Levene and R. S. Tipson 625 Thus it is evident that in desoxy-ribose nucleic acid the positions of the phosphoric acid radicles are carbon atoms (3) and (5) of the desoxy-ribose,4 as in (I). This fact offers the hitherto missing explanation for the differences in behavior of the nucleic acids of the two types. The differences are the following. The ribose nucleic acid is less resistant towards the action of dilute alkali and -- _-- H, H H! (adenine) -_-- Jf----- h u (guanine) ---; -0-- '0' Desoxy-ribose nucleic acid (CSH~~OZ,NXP~) I furthermore, on hydrolysis with dilute mineral acids, yields pyrimidine nucleotides having one phosphoric acid radicle only and that attached in position (3), whereas desoxy-ribose nucleic acid under 4It is more likely that the phosphoryl residue connecting the thymineand adenine-desoxy-ribosides is situated at positions (5) of their sugar chains and that the union from the adenine- to the cytosine-desoxy-ribosides is through positions (3).

4 626 Ring Structure of Thymidine similar conditions yields pyrimidine nucleotides with two phosphoric acid radicles, which must be in positions (3) and (5). Inasmuch as the phosphoric acid radicle in ii-phospho-ribose is naturally quite resistant towards the hydrolytic action of dilute mineral acids, it is justifiable to assume that the hydroxyl in position (5) of ribose nucleic acid is not substituted and hence it is warranted to assign to this acid structure (II), in which the phosphoryl residues are attached at positions (2) and (3). This structure explains the behavior of this acid towards alkalies. Since substituents, even ether linkages, in position (2) are characterized by greater instability than in other positions, it is readily understood that nucleotides linked to one another through position Ribose nucleic acid (C~SOO~ON~'~) II (2) of the sugar should dissociate with greater velocity, thus yielding nucleotides with the phosphoric acid radicle in the more stable position on carbon atom (3) of the ribose chain. 0 EXPERIMENTAL The thymidine used in the following experiments had a melting point of 184 and the following specific rotation. [& = +o.w x 100 = i-30.6" (in water) 2 x It was soluble in cold absolute methyl alcohol, pyridine, glacial acetic acid, and water; fairly soluble in cold and soluble in hot absolute ethyl alcohol; very sparingly soluble in cold but fairly

5 P. A. Levene and R. S. Tipson 627 soluble in hot acetone or ethyl acetate; insoluble in cold but very sparingly soluble in hot chloroform. It crystallizes from ethyl acetate in rosettes of needles. Preparation of Monotrityl Thymidine-A mixture of dry, finely powdered thymidine (0.5 gm.) with pure, dry triphenylmethyl chloride (0.6 gm.) was dissolved in 15 cc. of dry, redistilled pyridine. The solution was allowed to stand, with the exclusion of atmospheric moisture, during 7 days at room temperature. The solution, which was still clear and very pale yellow in color, was then poured into 100 cc. of ice water, with vigorous stirring. The pale yellow gum which was precipitated was twice washed with 100 cc. portions of ice water and allowed to stand under ice water during 3 days in the refrigerator. The gum had now changed to a hard, friable mass which was pulverized, filtered off, washed with water until free from pyridine, and air-dried. The finely powdered product was now shaken with dry ether and the ether extract decanted. Pentane was added to the ether extract until no more precipitate formed. The precipitate was united with the ether-insoluble portion, dissolved in acetone, and the acetone solution evaporated to dryness under diminished pressure, giving a pale yellow glass (weight, 0.9 gm.). A further portion (0.1 gm.) was recovered from the ether-pentane filtrate in the following way. The solution was evaporated to dryness, giving a colorless crystalline mass which was dissolved in 5 cc. of dry ether. To this solution, 100 cc. of pentane were cautiously added, with shaking, and the flocculent precipitate filtered off. The material was first obtained crystalline in the following manner. The glassy substance (1 gm.) was dissolved in 2.5 cc. of acetone and 100 cc. of dry ether were cautiously added to the solution, producing a small precipitate. The mixture was now evaporated under diminished pressure without a water bath. When the volume of the solution had been decreased to about 10 cc., the trityl thymidine commenced crystallizing spontaneously. It was recrystallized by dissolving in a little acetone and adding dry ether. On nucleating and stirring it set to a solid mass of colorless crystals having a melting point of 125. It was insoluble in cold or hot water; insoluble in cold but very sparingly soluble in hot pentane or heptane; sparingly soluble in cold but

6 628 Ring Structure of Thymidine slightly soluble in hot carbon tetrachloride; fairly soluble in cold but quite soluble in hot benzene; and quite soluble in cold dry ether, absolute ethyl alcohol, absolute methyl alcohol, acetone, chloroform, pyridine, ethyl acetate, and glacial acetic acid. It crystallized in rosettes of needles from hot benzene. It had the following composition mg. substance: mg. CO2 and mg. Hz ( : cc. N, (762 mm. at 24 ) CzsHzsOeNz. Calculated. C 71.87, H 5.8, N 5.79 Found. I 71.80, 6.0, 5.78 It had the following specific rotation. [ I24 = X 100 ffll 2 x = $11.4 (in acetone) Preparation of Monotosyl Trityl Thymidine-Dry, recrystallized trityl thymidine (0.4 gm.) was dissolved in 2.5 cc. of dry pyridine contained in a glass-stoppered 10 cc. Erlenmeyer flask, tosyl chloride (0.17 gm.) was added, and the mixture was shaken until the chloride had dissolved. After standing overnight at room temperature, with the exclusion of atmospheric moisture, the pale brown solution was cooled in ice and 2 drops of ice water were added. The resulting solution was kept at room temperature during 30 minutes and then poured into 100 cc. of ice water with stirring. The white powdery precipitate was filtered off, repeatedly washed with ice water until free from pyridine, and dried overnight in the vacuum desiccator over phosphorus pentoxide and soda-lime. It was soluble in cold absolute ethyl alcohol, absolute methyl alcohol, acetone, chloroform, carbon tetrachloride, benzene, pyridine, ethyl acetate, and glacial acetic acid; fairly soluble in cold or hot dry ether; insoluble in cold and very sparingly soluble in hot pentane or heptane; and insoluble in cold or hot water. It had the following composition mg. substance: mg. CO2 and mg. H,O IL : cc. N2 (776 mm. at 25 ) : mg. BaSOd C&H3a0,N2S. Calculated. C 67.67, H 5.4, N 4.39, S 5.02 Found , 5.2, 4.50, 5.05, Cl 0.00

7 P. A. Levene and R. S. Tipson 629 It had the following specific rotation. [, s = fo.60 X 100 (YD 2 x = (in acetone) Action of #odium Iodide on Tosyl Trityl Thymicline-A mixture of 100 mg. of dry tosyl trityl thymidine with 100 mg. of dry sodium iodide was dissolved in 2 cc. of acetone and the solution heated in a sealed tube at 100 during 2 hours. The solution became very light brown in color and a small amount of flaky crystalline material was deposited. The crystals were filtered off, dried, and weighed. Weight, 10 mg. (about 30 per cent of what should have been formed, had the reaction proceeded to completion). The acetone solution and washings were united and evaporated to dryness and the product isolated in the usual way,3 giving a white amorphous powder having the following analysis mg. substance: mg. CO2 and mg. Hz0 C&H3407N2S. Calculated. C 67.67, H 5.4 CzsHzr04NJ , 4.6 Found , 5.4, ash 0.00 In a second experiment in which 180 mg. of tosyl trityl thymidine were treated with sodium iodide in acetone at room temperature (18 hours), then at 100 (2 hours), and finally at room temperature (18 hours), the yield of sodium p-toluenesulfonate was 27 mg. (about 50 per cent of the theoretical). The product from the acetone solution had the following analysis mg. substance: mg. CO, and mg. Hz : AgI C&,~H,TO~NJ. Calculated. C 58.57, H 4.6, I Found , I 5.5, Action of Tosyl Chloride on Thymidine-Dry thymidine (0.5 gm.) was dissolved in dry pyridine (3 cc.) and tosyl chloride (0.9 gm., approximately 2.2 moles) was added. After standing overnight at room temperature with the exclusion of atmospheric moisture, 0.1 cc. of water was added to the brown solution. The resulting solution was kept at room temperature during 30 minutes and the product isolated by pouring into 100 cc. of filtered ice water with stirring. The pale pink powdery precipitate was filtered off and

8 630 Ring Structure of Thymidine washed repeatedly with ice water until free from pyridine. It was then dissolved in acetone and evaporated to a hard glass (1.0 gm.) which could not be obtained crystalline as it consisted of a mixture of ditosyl thymidine with some monotosyl chloro- thymidine (about 25 per cent). Action of Sodium Iodide on Tosylated Thymidine-A mixture of 0.6 gm. of crude ditosyl thymidine (containing monotosyl chlorothymidine) with 0.6 gm. of dry sodium iodide was dissolved in 5 cc. of acetone and the solution heated in a sealed tube at 100 during 2 hours. The solution became brown in color and a large amount of crystalline material was deposited. The crystals were filtered off, dried, and weighed. Weight, mg. The crystals were dissolved in water and the solution, diluted to 25 cc., had the following analysis. 10 cc. required 1.35 cc. 0.1 N AgN03 (Volhard); found Cl 4.34 Hence the starting material contained about 25 per cent of monotosyl chlorouridine; and about 43 per cent of the secondary tosyloxy group was removed by sodium iodide in acetone. The reaction product was isolated in the usual manner, giving a yellow, glassy material which had the following analysis mg. substance: mg. AgI ClrHtsOeNJS. Calculated. I C1oHxt0aN& Found. I 42.17

9 THE RING STRUCTURE OF THYMIDINE P. A. Levene and R. Stuart Tipson J. Biol. Chem. 1935, 109: 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

10 CORRECTIONS On page 630, Vol. 109, No. 2, May, 1935, line 4 from foot of text, read chlorothymidine for chlorouridine. On page 675, Vol. 109, No. 2, May, 1935, line 13, last figure, read 6.0 for 60.0.