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dec; uvmax(HzO) 0.05 N HCl, 267 (e 8500); 0,05 N NaOH, 269 ( E 7000); mass spectrum of the trimethylsilyl d e r i ~ a t i v e ,found ~ for M - CH3, m j e 650.2400; calcd for C24H49N307Si5F, 650.2401. Principal fragment ions6 agree with structure 5 (see Chart 11) and
Chart I n
Chart I1
- - - - - -. ‘05 ( l6 I AHOSiMe3 __-____-____
t-
lo3 CHpOSiMe3
4
M=525( 0.3% )
M03Si0zC r------
548 (23)
Me3Si”Cy
-218 - - -(35),’ -_-
Me3SiO
____-__--_
201 t 2 H = 2 0 3 ( 4 . 8 ) 464 (2.5)
OSiMe3
5-(SiMe3)5, M.665 (0.3%)
correlate with the spectrum of 6, with all base-containing ions shifted 17.991 f 0.003 mass units higher due to fluorine. The pmr spectrum (100 MHz) of 5 (compared with 6) is shown in Table I. Close resemTable I. Nuclear Magnetic Resonance Spectral Data of 5-Fluorouracil Polyoxin C and Uracil Polyoxin C in 20 DCl in DzO
75-Fluorouracil polyoxin C (5)- -Uracil polyoxin C (6)-Assign- Chem Coupling Assign- Chem Coupling ment shift. constant* ment shifta constantb 1’-H 2’-H 3’-H 4’-H 5’-H 6-H
= 4 . 0 ~1’-H 5.81 (br d) J1,,2, 4.51 (9) Jz,,a,= 6.2 2’-H 4.76 (9) Ja,,4, 6 . 6 3’-H 4.51 (q) J4r,jr 3 . 0 4’-H 4.76(d) 5’-H JGF= 6.0d 5-H 7.75(d) JIC-F Id 6-H
5.82 (d) = 3.8” 4.54(q) Jz,,~, = 6.0 4.77 (4) Jar,a, = 6 . 8 4.52(q) J 4 P . 5 , = 2.7 4.75(d) 5.98(d) J j 6 = 8.1 7.61 (d)
Shifts are given in pprn from DSS as an internal standard. * J values are in Hz; read from first-order splittings; d, doublet; q, quartet; br, broad. Coupling was confirmed by the spindecoupling experiment. See ref for H6-F and H1,-F coupling of 5-fluorouracil nucleosides.
blance of chemical shifts and coupling constants of 1 ’, 2’, 3’, 4’, and 5’ protons strongly suggests the same configuration and conformation in 5 and 6. Moreover, typical coupling of H- l ’ and H-6 with 5-fluorine was observed.’ Compounds 7 and 8 were not crystallized. However, the Rr values of 7 and 8 were identical with authentic 7 and 8 (tlc; three solvents). Mass spectra (relative abundance) of the major ions shown below (Chart 111) from gas chromatography-mass spectrometry6 of the trimethylsilyl derivatives of 7 and 8 were the same as those derived from authentic 7 and 8 and clearly establish the overall structure as 1 and 2. Whereas the natural polyoxins only inhibit the fungi, 1 and 5 are extremely inhibitory to Streptococcus faecalis and Escherichia coZi ( 10-j-10-6 M ) . 5-Fluorouracil-2’-deoxyuridine 5‘-monophosphate inhibits thy(5) J. A. McCloskey, A. M. Lawson, K. Tsuboyama, P. M. Krueger, and R. N. Stillwell, J . Amer. Chem. Soc., 90, 4182 (1968). (6) Mass spectra were recorded with 70-eV ionizing energy using LKB 9000 (low resolution) or CEC 21-llOB (high resolution) instruments. (7) R. J. Cushley, I. Wempen, and J. J. Fox, J . Amer. Chem. Soc., 90, 709 (1968).
7-
(
SiMe3 ) 5
205 (26)
_----------
bHOSiMe3
1--
lo3 ( 3 ’ 3 CH20SiMe3
M.437 (4.0% 1 8- (SiMe3I 4
midylate synthetase.8 With partially purified thymidylate synthetase from E. coli, the fluoropolyoxins (1 and 5) were not inhibitory. Therefore, the mode of action of the fluoropolyoxins is unclear. This mode of action is under study. Finally, 1 and 3 had the same inhibitory properties against purified chitin synthetase from Saccaromyces cerevisiae 5233C; 5 was not inhibitory. Acknowledgment. This work was supported by National Institutes of Health Research Grant No. AI08932-12 and National Science Foundation Research Grant No. GB32288X.9 (8) (a) T. I. Kalman, Ann. N . Y . Acad. Sci., 186, 166 (1971); (b) R. J. Landenbach, P. V. Danenberg, and C. Heidelberger, Biochem. Biophys. Res. Commun., 48, 1565 (1972); (c) D. V. Santi and C. McHenry, Proc. Nut. Acad. Sci. U.S., 69, 1855 (1972). (9) This paper is part XVI of a series; the previous paper is T. Uematsu and R. J. Suhadolnik, J . Med. Chem., in press. (10) On leave from The Institute of Physical and Chemical Research, Wako-shi, Saitama, Japan. * Address correspondence to this author at Albert Einstein Medical Center.
K. Isono,Io P. F. Crain, T. J. Odiorne James A. McCloskey, Robert J. Suhadolnik* Department of Bioorganic Chemistry Albert Einstein Medical Center Philadelphia, Pennsylvania I914I Institute f o r Lipid Research and Department of Biochemistry Baylor College of Medicine, Houston, Texas 77025 Receioed M a y 21, 1973
5-Chlorocytosine. Occurrence in Salmon Sperm Deoxyribonucleic Acid Sir: Fractionation of a deoxyribonucleic acid (DNA) hydrolysate from 900 kg of salmon sperm resulted in the isolation of 5-chlorocytosine (I). While numerous halogen-containing compounds have been isolated from marine organisms,’ this is, to our knowledge, the first halogenated nucleic acid constituent to be isolated.*, ( 1 ) See, for example, J. J. Sims, W. Fenical, R. M. Wing, and P. Radlick, J. Amer. Chem. Soc., 95, 972 (1973); W. Fenical, J. J. Sims, and P. Radlick, Tetrahedron Lett., 313 (1973); G. E. VanLear, G. 0. Morton, and W. Fulmor, ibid., 299 (1973); W. B. T.Cruse, M. N. G. James, A. A. AI-Shamma, J. K. Beal, and R. W. Doskotch, Chem. Commun., 1278 (1971); J. J. Sims, W. Fenical, R. M. Wing, and P. Radlick, J. Amer. Chem. Soc., 93, 3774 (1971); S. Hunt and S. Breuer, Biochem. SOC.Trans., 1,215 (1973). (2) Halogenated nucleosides, e.g., 5-bromouridine, when administered to certain organisms are incorporated into nucleic acid chains. See P. Hackett and P. Hanawalt, Biochim. Biophys. Acra, 123,356 (1966). (3) Nucleocidin, an antibiotic nucleoside produced by Strepromyces calaus, has a fluoro substituent at C-4 of the ribosyl moiety. See G. 0. Morton, J. E. Lancaster, G. E. Van Lear, W. Fulmor, and W. E. Meyer, J . Amer. Chem. SOC.,91, 1535 (1969).
Communications to the Editor
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DNA was isolated from a 900 kg lot of salmon sperm by the method of Emanuel and Chaikoff4s5 and hydrolyzed to the nucleoside level using snake venom diesterase and intestinal phosphatase. From the resulting hydrolysate, fractions enriched in each of the four principal nucleosides were obtained by ion exchange chr~matography.~Each of these fractions was concentrated by distillation at 50’ under reduced pressure and followed by crystallization of the principal nucleoside present in the respective fractions. The crystalline nucleosides were removed and the resulting filtrates were concentrated further and analyzed6 by paper chromatography and electrophoresis.’ NH2
hibited intense ions at m/e 110, 145, and 14713 confirming the assignment of the base structure as 5chlorocytosine (I). However, no molecular ions corresponding to 5-chlorodeoxycytidine (11) were observed ; instead apparent molecular ions were present at m/e 225 and 22713 corresponding to loss of two molecules of water from the expected molecular ions (m/e 161 and 163). Although the complexities involved in processing 900 kg of salmon sperm to isolate very small samples (
