Formation of 3,5,5-trimethyl derivatives of dihydrouracil nucleosides by reaction with methylsulfinyl carbanion and methyl iodide

Biochimicaet Biophysica Acta, 331 (1973) 147-153

Elsevier Scientific Publishing Company,Amsterdam- Printed in The Netherlands 8BA 97824

FORMATION OF 3,5,5-TRIMETHYL DERIVATIVES OF DIHYDROURACIL NUCLEOSIDES BY REACTION WITH METHYLSULFINYL CARBANION AND METHYL IODIDE RAYMOND P. PANZICA a, LEROY B. TOWNSEND ~, D. L. VON MINDEN b, M. S. WILSON b and JAMES A. McCLOSKEY b'*

aDepartments of Chemistry and Biopharmaceutical Sciences, University of Utah, Salt Lake City, Utah 84112 and bInstitute for Lipid Research and Department of Biochemistry, Baylor College o f Medicine, Houston, Texas 77025 (U.S.A.) (Received May 21st, 1973)

SUMMARY

1. 5,6-Dihydrouracil nucleosides undergo selective reaction with methylsulfinyl carbanion and methyl iodide to form derivatives which are O-methylated in the sugar and methylated at positions 3,5,5 in the base. This behavior is unique to the 5,6-dihydropyrimidine system and provides a sensitive mass spectrometric test for the 5,6-dihydro moiety. 2. Structures of reaction products were determined by electron- and chemicalionization mass spectrometry, and by proton magnetic resonance.

[NTRODUCTION

Previous work in this laboratory has involved the preparation of volatile derivatives of nucleosides 1-3 and nucleotides 4 for mass spectrometry. Of special interest are reactions which exhibit high selectivity toward modified bases, such as trimethylsilylation of 7-methylpurine nucleosides 5, because of their use for the mass spectrometric characterization of modified nucleosides from RNA (e.g. ref. 6). We have recently studied the mass spectra of N,O-permethyl derivatives of nucleosides7, which have been prepared using methylsulfinyl carbanion 8'9 and methyl iodide. Reactions of the 5,6-dihydrouracil nucleosides 1-3 were found by mass spectrometry to unexpectedly produce in each case a single permethylated product containing three methyl groups in the base. The behavior is apparently unique to the dihydrouracil moiety, and has no analogy in any of the forty other nucleosides we have examined. For example, uridine incorporates three methyl groups in the ribosyl moiety and only one group in the base, at N-3 (ref. 7). The present report provides evidence based on mass spectrometry and proton magnetic resonance spectroscopy that the Products 4 and 5 have the unique 3,5,5-trimethyl structures shown**. * Author to whom correspondence should be addressed. ** It is o f interest to note that reaction between a related reagent, dimethyloxosulfonium methylide, and the 5,6-double bond of uracil produces a 5,6-methylenepyrimidine derivative 1°.11.

148

R . P . PANZICA et al.

0 HN"~

0 RI

CH~N.,'~CH 3 CH3[

HO

R2

0 " ~ N- /

CH30

R

1

RI =R2=H

4

R=H

2

RI =CHs, R2=H

5

R=OCH3

3

RI=H, R2=OH

MATERIALS AND METHODS

Electron ionization mass spectra were obtained using an LKB 9000 instrument, with sample introduction by gas chromatograph (3 ft, 1% OV-1); ionizing energy 70 eV, ion source and separator temperatures 250 °C. Chemical ionization and high resolution mass spectra were recorded on a CEC 21-110B instrument a2, with sample introduction by direct probe; ion source and probe temperatures 140 °C; methane reagent gas pressure 0.4 torr during high pressure operation; electron energy 200 eV; repeller voltage 0-30 V/cm. Ultraviolet spectra were recorded on a Beckman DK-2 spectrophotometer. NMR spectra were obtained on a Varian XL-100-12 spectrometer using sodium 3-trimethylsilyl[2H4]propionate (Thompson-Packard) as an internal standard and [2Hr]dimethylsulfoxide as solvent. Thin-layer chromatography was run on glass plates coated (250/~m) with SilicAR 7 GF (Mallinckrodt) and the solvent system used was chloroform-methanol (32:1, v/v). Short-wave ultraviolet light (254 nm) was used to detect the spots.

M icroscale permethylation reactions Microscale permethylation was carried out by solution of Compounds 1-3 (20-50 #g) in dimethylsulfoxide (80 #1), followed by addition of methylsulfinyl carbanion solution in 10-fold excess (on a "reactive equivalent" basisT'13), prepared by addition of sodium hydride to dimethylsulfoxide 13. After 30 min an equimolar amount of methyl iodide (for Compounds 4 and 5) or [2Ha]methyl iodide (Compound 6) was added and allowed to stand 90 min. After addition of water (1 ml) the product was extracted by shaking with chloroform (1 ml). The chloroform layer was washed with three successive 1-ml portions of water and then transferred to the mass spectrometer. 2'-Deoxydihydrouridine (Compound 1), dihydrothymidine (Compound 2)and dihydrouridine (Compound 3) were obtained from Sigma Chemical Co.(St. Louis, Mo.). [2H3]Methyl iodide was purchased from Merck and Co., Teterboro, N.J. Preparation of 3,5,5,0-3',5'-Pentamethyl-2'-deoxydihydrouridine (4) To a solution of Compound I (69.4 rag, 0.3 mmole)in dimethylsulfoxide (50 ml) at room temperature was added methylsulfinyl carbanion solution 13 (15 ml, 15 mmoles), under N2. After 30 rain methyl iodide (0.94 ml, 15 mmoles) was added

149

T R I M E T H Y L D I H Y D R O U R A C I L NUCLEOSIDES

with stirring; 90 min later water (30 ml) was added and Compound 4 was extracted with chloroform (10 ml), then washed 4 times with 20-ml portions of water. Upon evaporation, Compound 4 was recovered as a syrup (55.4 mg, 61.4 % yield). Thinlayer chromatography (chloroform-methanol (32:1, v/v)) showed the material to be homogeneous; NMR showed two singlets at 6 1.13 and 6 1.15 (C-5 methyl groups), a singlet at 6 3.02 (N-3 methyl group), two singlets at 6 3.30 and 6 3.34 (C-3' and C-5' methoxyl groups) and a triplet centered 6 6.14 (pk. wd. 15 Hz) (anomeric proton). RESULTS A N D DISCUSSION

Mass spectrometry

The electron ionization mass spectrum of the methylation product (Compound 4) of Compound 1 is shown in Fig. 1. The molecular ion peak (m/e 300) is of low intensity, but the molecular weight and elemental composition are unambiguously established by the high resolution chemical ionization spectrum which is shown in Fig. 2. The protonated molecular ion (m/e 301) is characteristically a major ion species 14, t 5, which shows the molecular weight to be 300. Other ions in Fig. 2 conform to the usual behavior of nucleosides with methane reagent gas ~5. The molecular mass is

100.

~-5

-8.7

(M-2CH3OH)

71

(C3 H4OCH3)

236

Z

50 ~: _d 25"

-4

{B+2H)

i

(B+ C3H40)

157{8+ CH2)

50

I00

150

(M-CH~OH) M

211

200

250

300

m/e

Fig, 1. Mass spectrum of the permethylation product of 2'-deoxydihydrouridine (Compound 1).

,ooI

34.4

MH 301.1729

z

( M+C2H5)

LLI

(S)

(B* 2H)

(MH-CHsOH)

~45:°9°° /s~.po,

E: 25

I ,oo

,50

269.~468

,'/

17

529.2042

I (M+ c3H~) , 34, 2028

500

550 m/e

Fig. 2. Chemical ionization mass spectrum of the permethylation product of 2'-deoxydihydrouridine (Compound 1), obtained using methane reagent gas.

150

R . P . PANZICA et al.

corroborated in Fig. 1 by rn/e 268 and 236 arising from elimination of methanol, and by m/e 255 and its daughter role 223, associated with loss of the C-5' moiety7. o

•

O.~N' ~cH3 N CH3

CH3N

_J B 055)

223

jCH30H

45 . ' / F~ ;,~'~" "~,,,,,~.11 CH~O 4

The methylation reaction has therefore incorporated five methyl groups into Compound 1. The principal base-containing ions show that three groups are in the base. These are base (B)+2H (m/e 157) and B-LCH2 (C-I') (role 169) which often occur in nucleoside mass spectra 16, and in those of the permethyl derivatives7'17. Mass 211 includes the base plus three carbons of the ribose skeleton and 0-4' (ref. 7). These correlations require that the ribose portion of the molecule be dimethylated, which is independently confirmed by the sugar ion m/e 145 (also in Fig. 2) and its daughter ion m/e 113. All of these interpretations are supported by appropriate mass shifts in the spectrum of the hexa[2H3]methyl derivative of Compound 1, and in the spectrum of Compound 5. The spectrum shown in Fig. 1 is indistinguishable from that derived from Compound 2, thus requiring that at least one of the three methyl groups in the base be located at C-5. This is supported by the spectrum of Compound 6 (Fig. 3), and of the 0

CH3SOCH2-

C2H3N'~ cH3

l I -c'H3 C2H31 > 0$>--..N j C2H30C~

2

CZH30 6

TABLE I SELECTED IONS FROM THE H I G H RESOLUTION MASS SPECTRUM OF 6 Exact mass (error*)

Composition

Structure

277.1976 264.1875 242.1538 229.1439 217.1451 175.1337 163.1348 151.1236

C~31:[112H9N204 C 12H1 o2I-[9N20~.

M-C2I-[aOH M-CHzOCZH 3 M-2C2HaOH 264-CZHaOH

(-- 1.2) (3.5) (0.0) (--2.0) (--0.9) ( - 1.7) (--0.5) (--0.6)

C12Nlo2H9N203 C11H9ZH6N203

Cl oHo2H6N203 CsH72H6N202 C71-[72H6N202 C7H72I-[60 3

* Millimass units; found minus theoretical value.

B-~C 3H40 B~-CH2 B~-2H S

TRIMETI-IYL DII-IYDROURACIL NUCLEOSIDES

151

TRIMETFIYL DI14YDROURACIL NUCLEOSIDES

153

13 Polan, M. L., McMurray, W. J., Lipsky, S. R. and Lande, S. (1970) Biochem. Biophys. Res. Commun. 38, 1127-1133 14 Field, F. 14. (1968) Accounts Chem. Res. 1, 42--49 15 Wilson, M. S., Dzidic, I. and McCloskey, J. A.. (1971) Biochim. Biophys. Acta 240, 623-626 16 Shaw, S. J., Desiderio, D. M., Tsuboyama, K. and McCloskey, J. A. (1970) J. Am. Chem. Soc. 92, 2510-2522, and references cited therein 17 Dolhun, J. J. and Wiebers, J. L. (1970) Or#. Mass. Spectrom. 3, 669-681 18 House, H. O. (1965) Modern Synthetic Reactions, pp. 163-215, W. A. Benjamin, New York 19 Jackman, L. M. and Sternhell, S. (1969) Applications of Nuclear Maynetic Resonance Spectroscopy in Oryanic Chemistry, 2nd edn, p. 180, Pergamon Press, 20 Ma, J. C. N. and Warnhoff, E. W. (1965) Can. J. Chem. 43, 1849-1869 21 Robins, M. J. and Naik, S. R. (1971) Biochemistry 10, 3591-3597 22 Townsend, L. B. (1973) in Synthetic Procedures in Nucleic Acid Chemistry I (Zorbach, W. W. and Tipson, R. S., eds), Vol. II, pp. 267-398, Wiley-Interscience, New York