5410
Anal. Calcd for CeHuN307P.H20(323.20): C, 33.45; H,4.35; N, 13.01; P,9.60. Found: C, 33.47; H, 4.44; N, 12.90; P, 9.8 (titration). The uv spectrum exhibited the characteristic features of this system: A,, 231 ( E 9550), 262.5 mp ( e 10,640); Amin 243 mp ( E 6480) in water, pH 1-7. Characteristic ir frequencies were 1665, 1376, 1358, 1252, 1212, 1060, and 932 cm-'. The nmr spectrum taken at 100 MHz in D 2 0 at p D 7 featured the following resonances [6 ppm (J Hz) relative to TMS]: a doublet of H-6 at 8.61 (7.5); two unresolved doublets of H-5 and H-1' centered at 7.09 (7.5) and 7.15 (5.9, respectively; a doublet of H-2' at 6.24 (5.5). The clear separation of the H-2' signal is remarkable when compared with other nucleotide spectra,' and it must be due to the combined deshielding effect of the isourea and phosphate groups. The ORD characteristics in water ([MI at c M ) were: peak at 282 mp, +6200", trough at 239 mp, -20,800°; crossover at 268 mp. Several interesting reactions of 3 are currently under study. Its hydrolysis is pH dependent and general base catalyzed above its pKz (5.7).2 At pH 1 to 7 a partial conversion to cytidine 2 ',3 '-cyclic phosphate can take place, which can be followed by electrophoresis at pH 6. Treatment with alkali or bicarbonate gave aracytidine 3'-phosphateB (4) as the only product. Scheme I
+
1
2
4
3
The identification of 4 was carried out by comparison with all published data. Its 100-MHz nmr spectrum in DzO exhibited the following signals relative to acetone as internal standard: at pD 7: H-6, 6 5.65 (8); H-5, 3.83 (8); H-l', 4.03 ( 3 ) ; at pD 4: H-6, 5.87 (8); H-5, 4.02 (8); H-1 ', 3.99 (3). The nmr and uv spectra were in good agreement with those of Wechter.9 The O R D characteristics in 0.1 M Na2HP04, pH 7.8 ([MI at c 9.3 X M ) were: peak at 288 mp, +15,900°; broad trough centered at 240 mp, - 18,800"; crossover at 272 mp. We also obtained good elementary analysis from the crystalline free acid. Furthermore, alkaline phosphatase hydrolysis liberated l-fl-D-arabinosylcytosine which was identical with the commercial sample (Sigma) by all usual criteria. The concept of a cyclic phosphate derivative as an intramolecular leaving group has already been proposed lo as the mechanism of anhydronucleotide formation in polyphosphoric acid.3 Our work on the alkyl' and silyl esters of cytidine 2 ',3 '-cyclic phosphate represents a direct experimental proof and a further development of the same general concept. This novel reaction provides an extremely simple and economical way to the most suitable intermediate for a direct polymerization (7) 0. Jardetzky, Biopolymers, Symp., 1, 501 (1964); C. D. Jardetzky and 0. Jardetzky, J . Amer. Chem. Soc., 82, 222 (1960). (8) W. J. Wechter, J . Med. Chem., 10, 762 (1967). (9) W. J. Wechter, personal communication. (IO) See Ph.D. theses of E. R. Walwick and W. I
