2-(Trimethylsilyl)ethanethiol in nucleoside chemistry. A short route for preparing thionucleosides and their methyl disulfides

Full text of DOI:10.1021/jo9908492

J . Org. Chem. 2000, 65, 249-253
249
6-thioguanosine used for the treatment of leukemias,7
and, more recently, 2′-deoxy-2′-mercaptocytidine5 were
incorporated in oligonucleotides.
2-(Tr im eth ylsilyl)eth a n eth iol in Nu cleosid e
Ch em istr y. A Sh or t Rou te for P r ep a r in g
Th ion u cleosid es a n d Th eir Meth yl
Disu lfid es
The development of new methods for incorporating a
thiol function in nucleosides, nucleotides, and oligonucleo-
tides and for preparing their stable useful precursors is
thus of interest in the search of bioactive compounds or
biological tools. In this regard, mixed disulfides can be
interesting precursors that should be reduced efficiently
and rapidly under mild conditions for generating the
active species. For example, the RDPR inactivator 2′-
deoxy-2′-mercaptouridine 5′-diphosphate was obtained in
vitro by spontaneous reduction of the corresponding
stable mixed propyl disulfide with dithiothreitol (DTT).3
We report here a short route for preparing thionucleo-
sides and their corresponding methyl disulfides using
2-(trimethylsilyl)ethanethiol (â-ethylsilyl thiol, BEST) as
a source of sulfur, a method previously developed by
Fuchs and co-workers for synthesizing acyl- and alkyl-
substituted thiols.8 These authors have reported that the
2-(trimethylsilyl)ethyl sulfide intermediate did not afford
the corresponding thiol by treatment with fluorides.
Reaction with dimethyl(methylthio)sulfonium tetrafluo-
roborate in the presence of methyl disulfide gave the
corresponding acyl- or alkyl-substituted methyl disulfide,
which can be reduced with tributylphosphine. Nucleo-
sides in which a 2′-(2-(trimethylsilyl)ethyl)thio group is
linked to the sugar were prepared, and their reaction
with dimethyl(methylthio)sulfonium tetrafluoroborate led
to the corresponding methyl disulfide in high yield. On
the contrary, we describe here the first example of direct
and quantitative elimination of this group in 2′-deoxy-
8-(2-(trimethylsilyl)ethyl)thioadenosine with tetrabutyl-
ammonium fluoride at room temperature for obtaining
the corresponding thione on the base. Such a deprotection
should be of interest in the preparation of modified
oligonucleotides.
Ste´phane Chambert,† Isabelle Gautier-Luneau,‡
Marc Fontecave,§ and J ean-Luc De´cout*,†
Laboratoire de Chimie Bio-organique, UMR CNRS 5063,
De´partement de Pharmacochimie Mole´culaire, UFR de
Pharmacie, Universite´ J oseph Fourier-Grenoble I,
Domaine de la Merci, F-38706 La Tronche Cedex, France,
LEDSS 2, UMR CNRS 5616, Universite´ J oseph Fourier,
BP 53, F-38041 Grenoble Cedex 9, France, and Laboratoire
de Chimie et Biochimie des Centres Re´dox Biologiques,
DBMS-CEA/ CNRS/ Universite´ J oseph Fourier, Bat. K,
17 avenue des Martyrs, F-38054 Grenoble Cedex 9, France
Received May 25, 1999
In tr od u ction
Incorporation of a thiol function in nucleosides, nucleo-
tides, or oligonucleotides has led to a number of ana-
logues possessing interesting biological properties.1 For
example, 2′,3′-dideoxy-3′-mercaptonucleoside 5′-triphos-
phates (T, C, A, G) irreversibly stopped DNA chain
elongation by AMV and HIV reverse transcriptase, and
the corresponding nucleosides display antiviral activi-
ties.2a,b Recently, we have demonstrated that another
nucleotide possessing a thiol function on the sugar, 2′-
deoxy-2′-mercaptouridine 5′-diphosphate strongly inac-
tivates in vitro E. coli ribonucleoside diphosphate reduc-
tase (RDPR).3 This enzyme catalyzes the reduction of the
four natural ribonucleotides in the corresponding 2′-
deoxyribonucleotides and thus is a key enzyme in the
synthesis of DNA.4 The thiol function of the modified
nucleotide interacts with a cysteine residue at the active
site to lead to a perthiyl radical formed on the enzyme.
Numerous modified oligonucleotides containing a sul-
fur atom on the base, the sugar, or the phosphoryl group
were synthesized and used as tools in studies of nucleic
acid structure and function, protein-nucleic acid interac-
tions, and antisense therapy.5 For examples, 4-thiouri-
dine, a natural constituent of t-RNA used as a photo-
chemical probe in the study of nucleic acids,6 2′-deoxy-
Resu lts a n d Discu ssion
2′-Thioalkyl or -aryl uridine derivatives were prepared
by Divakar and Reese by ring-opening of 2,2′-anhydro-
uridine (1) with alkyl or arene thiolates formed with
triethylamine or N1,N1,N3,N3-tetramethylguanidine as a
base.9 To prepare 2′-deoxy-2′-(2-(trimethylsilyl)ethyl)-
thiouridine (2) (Scheme 1), 2,2′-anhydrouridine was
heated with 2-(trimethylsilyl)ethanethiol at 120 °C in
DMF in the presence of potassium carbonate. After 3 h
of heating and purification by chromatography on silica
* To whom correspondence should be addressed. Fax: (33) 04 76
51 86 67. E-mail: jean-luc.decout@ujf-grenoble.fr.
† UMR CNRS 5063, Universite´ J oseph Fourier-Grenoble I.
‡ UMR CNRS 5616, Universite´ J oseph Fourier.
§ DBMS-CEA/CNRS/Universite´ J oseph Fourier.
1
gel, the 2′-sulfide 2 was obtained in 88% yield. In the H
(1) Wnuk, S. F. Tetrahedron 1993, 49, 9877-9936.
(2) (a) Yuzhakov, A. A.; Chidgeavadze, Z. G.; Beabealashvilli, R. S.
FEBS 1992, 306, 185-188. (b) Yuzhakov, A. A.; Chidgeavadze, Z. G.;
Beabealashvilli, R. Sh.; Kraevskii, A. A.; Galegov, G. A.; Korneeva,
M. N.; Nosik, D. N.; Kilesso, T. Y. Bioorg. Khim. 1991, 17, 504-509;
Chem. Abstr. 1991, 115, 84923g). (c) Dueholm, K. L.; Aly, Y. L.;
J oergensen, P. T.; El-Barbary, A. A.; Pedersen, E. B.; Nielsen, C.
Monatsch. Chem. 1993, 124, 37-53.
(3) Le Hir de Fallois, L.; De´cout, J .-L.; Fontecave, M. J . Chem. Soc.,
Perkin Trans. 1 1997, 2587-2595. Cove`s, J .; Le Hir de Fallois, L.;
Lepape, L.; De´cout, J .-L.; Fontecave, M. Biochemistry 1996, 35, 8595-
8602.
NMR spectrum (DMSO-d6) of compound 2, the charac-
teristic signals of the (trimethylsilyl)ethyl group were
observed at 2.58, 0.80, and -0.01 ppm, respectively. This
compound reacted very slowly and incompletely with
tetrabutylammonium fluoride in THF at room temper-
ature. Heating at 60 °C, under an argon atmosphere, in
the presence of a large excess of tetra-n-butylammonium
(4) Fontecave, M. Cell. Mol. Life Sci. 1 1998, 684-695.
(5) Hamm, M. L.; Piccirilli, J . A. J . Org. Chem. 1997, 62, 3415-
3420 and references therein.
(6) Fourrey, J .-L.; Gasche, J .; Fontaine, C.; Guittet, E.; Favre, A. J .
Chem. Soc., Chem. Commun. 1989, 1334-1336. Coleman, R. S.;
Siedlecki, J . M. J . Am. Chem. Soc. 1992, 114, 9229-9230.
(7) Christopherson, M. S.; Broom, A. D. Nucleic Acids Res. 1991,
19, 5719-5724.
(8) Anderson, M. B.; Ranasinghe, M. G.; Fuchs, P. L. J . Org. Chem.
1988, 53, 3125-3127.
(9) Divakar, K. J .; Reese, C. B. J . Chem. Soc., Perkin Trans. 1 1982,
1625-1628.
10.1021/jo9908492 CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/16/1999

250 J . Org. Chem., Vol. 65, No. 1, 2000
Notes
Sch em e 1. Sh or t P r ep a r a tion of
2′-Deoxyu r id in -2′-yl a n d 2′-Deoxycytid in -2′-yl
Meth yl Disu lfid es 3 a n d 5 Usin g
2-(Tr im eth ylsilyl)eth a n eth iola
a
Key: (a) 2-(trimethylsilyl)ethanethiol/K2CO3/DMF, 120 °C, 2
h; (b) dimethyl(methylthio)sulfonium tetrafluoroborate/THF, rt;
(c) Ac2O/Py; (d) 1,2,4-triazole/POCl3/Et3N/CH3CN; (e) ammonia/
dioxane; (f) concentrated aqueous ammonia/ethanol.
fluoride, converted nearly completely compound 2 in a
complex mixture of compounds in which the uracil base
was identified as a major component (TLC, HPLC, H
1
F igu r e 1. X-ray structure of 2′-deoxycytidin-2′-yl methyl
disulfide (5).
NMR). In this mixture, 2′-deoxy-2′-thiouridine could be
identified as a minor component by comparison with a
sample synthesized and characterized previously (TLC,
Ellman’s reagent; HPLC).3 This result can be explained
by the previously observed lability of the glycosidic
linkage in 2′-thioribonucleosides.10 Elimination of the
base can result from intramolecular reaction of the
thiolate generated on the sugar at the 2′-position.
Finally, the trimethylsilyl sulfide 2 was quantitatively
converted to methyl disulfide 3 after reaction with
dimethyl(methylthio)sulfonium tetrafluoroborate accord-
ing to Fuchs and co-workers (Scheme 1).8 The reaction
should be conducted in a large excess of methyl disulfide
at room temperature to avoid the formation of the
symmetrical disulfide. Compound 3 was obtained in 90%
yield after C18 reversed-phase chromatography. This
method allowed the methyl disulfide 3 to be prepared
from 2,2′-anhydrouridine in two steps in 80% yield more
efficiently and rapidly than the method previously used
for preparing 2′-deoxyuridin-2′-yl propyl disulfide (40%
yield).3 Compound 3 could be crystallized from water to
give nice colorless needles.
Compound 2 was converted to its cytosine analogue 4
in four steps using a classical procedure:11 (i) acetylation
of the hydroxyl groups with acetic anhydride in pyridine,
(ii) reaction of the crude product with phosphoryl trichlo-
ride, triethylamine, and 1,2,4-triazole, (iii) reaction of the
triazolo derivative with an excess of ammonia in dioxane,
and (iv) deprotection of the hydroxyl groups with am-
monia in ethanol. Compound 4 was obtained from
compound 2 in 85% yield after chromatography on silica
gel. The 2′-(2-(trimethylsilyl)ethyl)thio group is, thus,
stable under the conditions of conversion of the uracil
base in cytosine. As for compound 2, deprotection of the
thiol group cannot be achieved cleanly and completely
by treatment with fluorides. The trimethylsilyl sulfide 4
was quantitatively converted to its methyl disulfide 5 by
reaction with dimethyl(methylthio)sulfonium tetrafluo-
roborate in the presence of a large excess of methyl
disulfide at room temperature. It was isolated in 80%
yield after C18 reversed-phase chromatography. Thus, 2′-
deoxycytidin-2′-yl methyl disulfide (5) was prepared in
60% yield from 2,2′-anhydrouridine (1). The X-ray struc-
ture of compound 5 (Figure 1) was determined after
crystallization from water (Supporting Information).
3′-Thiothymidine 5′-triphosphate was reported as a
highly effective terminator of DNA synthesis that is
catalyzed by HIV reversed transcriptase.2b,c However, the
antiviral activity of the corresponding nucleoside was
controverted.2c To prepare 3′-deoxythymidin-3′-yl methyl
disulfide (9), which is a potent antiviral agent and a
precursor of 3′-thiothymidine,2b,c we used the approach
developed for the synthesis of methyl disulfides 3 and 5.
5′-Protected derivatives of 3′-thiothymidine were pre-
pared previously by reaction of 2,3′-anhydro-5′-tritylthy-
midine or 1-(2-deoxy-3-mesyl-5-(4-monomethoxytrityl)-â-
D-lyxosyl)thymine with potassium or sodium thioben-
zoate,12 and a very fast oxidation of 3′-thiothymidine in
the corresponding symmetrical disulfide was observed.12a
To prepare the methyl disulfide of this nucleoside, 2,3′-
anhydro-5′-(4,4′-dimethoxytrityl)thymidine (6) was pre-
pared from thymidine. Reaction of this anhydro deriva-
tive with 2-(trimethylsilyl)ethanethiol at 140 °C in DMF
in the presence of potassium carbonate resulted in the
formation of the trimethylsilyl derivative 7 in a low 42%
(10) J ohnson, R.; Reese, C. B.; Pei-Zhuo, Z. Tetrahedron 1995, 51,
5093-5098.
(11) Divakar, K. J .; Mottoh, A.; Reese, C. B. J . Chem. Soc., Perkin
Trans. 1 1990, 969-974.
(12) (a) Miller, N.; Fox, J . J . J . Org. Chem. 1964, 29, 1772-1776.
(b) Cosstick, R.; Vyle, J . S. J . Chem. Soc., Chem. Commun. 1988, 992-
993.

Notes
Sch em e 2. Syn th esis of 3′-Deoxyth ym id in -3′-yl
J . Org. Chem., Vol. 65, No. 1, 2000 251
Meth yl Disu lfid e Usin g
2-(Tr im eth ylsilyl)eth a n eth iola
a
Key: (g) 2-(trimethylsilyl)ethanethiol/NaH/DMF, 90 °C, 1 h;
(h) 2% DCA/CH2Cl2, aqueous NaHCO3; (b) dimethyl(methylthio)-
sulfonium tetrafluoroborate/THF, rt.
yield. Surprisingly, the detritylated analogue of 6, 2,3′-
anhydrothymidine, did not react with the thiol under
these conditions. To increase the yield of compound 7,
sodium 2-(trimethylsilyl)ethanethiolate was prepared in
DMF by treatment of the corresponding thiol with 1.05
equiv of sodium hydride, and then the mixture was
heated with the anhydro derivative 6 at 90 °C for 1 h.
Under these conditions, the (2-(trimethylsilyl)ethyl)thio
derivative 7 was formed as the major product, whereas
a minor compound formed was identified as 3′-deoxy-2′,3′-
didehydro-5′-(4,4′-dimethoxytrityl)thymidine (5′-tritylat-
ed D4T; RfD 0.53) by detritylation (1H NMR, mass
spectrometry; RfB 0.62). This elimination with synchro-
nous decyclization has been previously observed in high
yield at room temperature with 2,3′-anhydro-5′-tritylthy-
midine in the presence of potassium tert-butoxide in
dimethyl sulfoxide.13
F igu r e 2. X-ray structure of 3′-deoxythymidin-3′-yl methyl
disulfide (9).
Sch em e 3. Syn th esis of 2′-Deoxy-8-th ioa d en osin e
Usin g 2-(Tr im eth ylsilyl)eth a n eth iola
The crude mixture isolated after ring-opening of the
anhydro derivative 6 with sodium 2-(trimethylsilyl)-
ethanethiolate was detritylated with 2% dichloroacetic
acid in dichloromethane for obtaining the (2-(trimethyl-
silyl)ethyl)thio derivative 8 in 62% yield for the two steps.
3′-Deoxythymidin-3′-yl methyl disulfide (9) was finally
obtained from compound 8 in 90% yield after reaction at
room temperature with dimethyl(methylthio)sulfonium
tetrafluoroborate in the presence of a large excess of
methyl disulfide (Scheme 2). After crystallization of this
compound from water, the X-ray structure (Figure 2) of
its crystalline monohydrate was obtained (Supporting
Information).
a
Key: (i) 2-(trimethylsilyl)ethanethiol/K2CO3/DMF, 60 °C, 8 h;
(j) 1 M tetrabutylammonium fluoride/THF, rt, 3 h.
8-Bromo-2′-deoxyadenosine (10) was prepared by bro-
mination of 2′-deoxyadenosine at the 8-position.15 Reac-
tion of this nucleoside with 2-(trimethylsilyl)ethanethiol
at 60 °C in the presence of potassium carbonate led to
the corresponding sulfide 11 in 95% yield (Scheme 3).
Deprotection of the thione function was achieved quan-
titatively by treatment with 1 M tetrabutylammonium
fluoride in THF at room temperature for 3 h. After
precipitation in water, a mixture containing only the
thione and its tetrabutylammonium salt was obtained (1:
Addition of a nearly stoichiometric amount of DTT (1.1
equiv) to a methanolic solution of one of the three methyl
disulfides 3, 5, and 9 led immediately and quantitatively
to the corresponding thiol evidenced by TLC (RfB 0.31,
RfD 0.33, RfA 0.48, respectively; Ellman’s reagent) and H
1
1
1; H NMR and mass spectrometry; 92% yield). It could
NMR spectrometry after removal of methanethiol formed
by argon bubbling.
be transformed to the pure thione form by chromatog-
raphy on a C18 reversed-phase column eluting with
aqueous ammonium carbonate, acidification of the re-
sulting solution, and desalting. All the characteristics of
the isolated compound appeared identical to those of an
authentic sample of 2′-deoxy-8-thioadenosine (12) pre-
pared previously.16
To demonstrate the potential of 2-(trimethylsilyl)-
ethanethiol for incorporating a thione function on the
base of nucleosides, 2′-deoxy-8-thioadenosine (12) was
synthesized using this reagent. This nucleoside was
previously used as an intermediate in the synthesis of
modified oligonucleotides carrying an 8-thio-substituted
adenine base.14
This result constitutes the first example of mild depro-
tection with tetrabutylammonium fluoride of a thione
(13) Horwitz, J . P.; Chua, J .; Da Rooge, M. A.; Noel, M.; Klundt, I.
L. J . Org. Chem. 1966, 31, 205-211.
(14) Pieles, U.; Sproat, B. S.; Neuner, P.; Cramer, F. Nucleic Acids
Res. 1989, 17, 8967-8978. Laayoun, A.; De´cout, J .-L.; Defrancq, E.;
Lhomme, J . Tetrahedron Lett. 1994, 35, 4991-4994.
(15) Ikehara, M.; Kaneko, M. Tetrahedron 1970, 26, 4251-4259.
Guy, A.; Duplaa, A. M.; Harel, P.; Teoule, R. Helv. Chim. Acta 1988,
71, 1566-1572.
(16) Laayoun, A.; De´cout, J .-L.; Lhomme, J . Tetrahedron Lett. 1994,
35, 4989-4990.

252 J . Org. Chem., Vol. 65, No. 1, 2000
Notes
stirred for 15 min in an ice bath, and then triethylamine (20
mL, 177 mmol) was added dropwise under argon. After 5 min,
function protected in the form of 2-(trimethylsilyl)ethyl
sulfide. The easy â-elimination of the trimethylsilylethyl
group can be explained by resonance interaction of the
unshared electrons of the sulfur atom with the p-electron
system of the aromatic adenine ring. Thus, the (trimeth-
ylsilyl)ethyl group appears very attractive for protecting
an aromatic thione function. 2-(Trimethylsilyl)ethyl sul-
fide 11 was found to be stable under strong basic condi-
tions in a 50:50 mixture of concentrated ammonia-
ethanol for 24 h at 50 °C. Synthesis of oligonucleotides
containing such a thione function on the base should be
possible with this protecting group.
a
solution of the crude acetylated nucleoside 2 previously
prepared in CH3CN (40 mL) was added. The mixture was stirred
for 24 h under argon at room temperature. Water (3 mL) was
added, and the solvents were evaporated. The residue was
dissolved in CH2Cl2 (100 mL), and the solution was washed with
aqueous NaHCO3 (5%, 5 mL) and then with water. It was dried
over Na2SO4, evaporated to dryness, and then coevaporated with
toluene. The residue was dissolved in an ammonia solution in
dioxane (40 mL, 0.5 M). The solution was stirred for 24 h under
argon and then evaporated to dryness. To a solution of the
residue in ethanol (20 mL) was added concentrated aqueous
ammonia (30%, 30 mL). The resultant solution was stirred for
12 h and then evaporated and coevaporated with ethanol. The
residue was chromatographed on silica gel in CH2Cl2-MeOH
(95:5) and then CH2Cl2-MeOH (9:1) to yield compound 4 (1.62
g, 4.51 mmol, 85%; RfB 0.37): mp 112 °C; 1H NMR (300 MHz,
DMSO-d6) δ 7.86 (1 H, d, J ) 7.5 Hz), 7.20 (2H, d, J ) 6.4 Hz),
6.19 (1 H, d, J ) 8.6 Hz), 5.83 (1 H, d, J ) 7.4 Hz), 5.60 (1H, d,
J ) 5.3 Hz), 5.14 (1H, t, J ) 5.3), 4.23 (1 H, m), 3.91 (1 H, m),
3.63 (2H, m), 3.38 (1 H, dd, J ) 8.7 Hz, J ) 5.5 Hz), 2.39 (2 H,
m), 0.77 (2 H, m), -0.03 (9 H, s); 13C NMR (75 MHz, DMSO-d6)
δ 165.4, 155.3, 141.5, 94.6, 89.0, 86.2, 72.3, 61.6, 51.4, 25.9, 17.2,
-1.8; LRMS [FAB+, glycerol] m/z ) 360 [M + H]+, 248 [M -
cytosine]+, 112 [cytosine + H]+. Anal. Calcd for C14H25N3O4SSi‚
1.5H2O: C, 43.50; H, 7.30; N, 10.87; S, 8.29. Found: C, 43.49;
H, 6.99; N, 10.95; S, 8.32.
2,3′-An h yd r o-5′-(4,4′-d im eth oxytr ityl)th ym id in e (6). To
a solution of thymidine (5.01 g, 20.7 mmol) in dry pyridine (50
mL) was added 4,4′-dimethoxytrityl chloride (7.72 g, 22.8 mmol)
at 0 °C under argon. The resultant solution was stirred at room
temperature for 20 h. Methanesulfonyl chloride (1.91 mL, 24.7
mmol) was added, and the mixture was stirred for 4 h under
argon at room temperature. Water (5 mL) was added, and the
solvents were evaporated. The residue was dissolved in CH2Cl2,
and the solution was washed with water, dried over Na2SO4,
and then evaporated. The colorless foam obtained was dissolved
in dry CH3CN (100 mL), and potassium carbonate (10 g) was
added. The mixture was stirred at room temperature for 24 h
under argon, and then the mineral salts were removed by
filtration. The filtrate was evaporated, and the residue was
dissolved in dichloromethane (150 mL). The resultant solution
was washed with water, dried over Na2SO4, and filtered, and
the solvent was evaporated. The residue was chromatographed
on alumina gel in CH2Cl2-MeOH (95:5) to lead to compound 6
(8.34 g, 15.8 mmol, 75%; RfA 0.42): mp 134 °C; 1H NMR (300
MHz, DMSO-d6) δ 7.64-7.20 (9 H, m), 6.85 (4 H, m), 5.90 (1 H,
d, J ) 3.5 Hz), 5.30 (1 H, m), 4.41 (1 H, m), 3.30 (6 H, s), 3.17-
3.05 (3 H, m), 2.60-2.40 (2 H, m), 1.78 (3 H, s); 13C NMR (75
MHz, DMSO-d6) δ 170.8, 158.1, 153.3, 144.6, 135.3, 135.1, 129.7,
127.8, 127.7, 126.7, 116.2, 113.2, 86.8, 85.8, 83.5, 77.1, 62.3, 55.0,
32.7, 13.0; LRMS [DCI, NH3/isobutane] m/z ) 527 [M + H]+,
303 [DMTr]+, 225 [M - DMTr + H]+, 127 [thymine + H]+.
3′-Deoxy-3′-(2-(tr im eth ylsilyl)eth yl)th ioth ym id in e (8). To
a stirred suspension of sodium hydride (60%; 215 mg, 5.38 mmol)
in dry DMF (10 mL) was added a solution of 2-(trimethylsilyl)-
ethanethiol (0.69 g, 5.12 mmol) in dry DMF (10 mL). The solution
was stirred for 15 min under argon, and then 2,3′-anhydro-5′-
(4,4′-dimethoxytrityl)thymidine (6) (2.57 g, 4.88 mmol; RfE 0.54)
was added. After being heated at 90 °C for 1 h under argon, the
mixture was filtered and DMF was evaporated off. The residue
was washed with pentane and then dissolved in dichloromethane
(150 mL). The solution was washed with aqueous NaH2PO4
(10%, 10 mL) and then with water, dried over Na2SO4, and then
evaporated.
These results point to 2-(trimethylsilyl)ethanethiol as
a useful tool in the synthesis of thionucleosides and
potentially of thio-nucleotides and -oligonucleotides.
Exp er im en ta l Section
Gen er al P r ocedu r es. Melting points are uncorrected. Chemi-
cal shifts are reported in parts per million relative to the residual
signal of the solvent. Thin-layer chromatographic data (Rf
values) were obtained with Macherey Nagel Alugram SIL
G/UV254 analytical sheets (layer, 0.25 mm) developed with
dichloromethane-methanol (95:5 (RfA), 90:10 (RfB), 85:15 (RfC),
80:20 (RfD)) or dichloromethane-ethyl acetate (75:25 (RfE)).
Syn th esis. 2-(Trimethylsilyl)ethanethiol (commercially avail-
able) was synthesized from vinyltrimethylsilane (Aldrich) and
thiolacetic acid according to procedures described previously.8,17
Dimethyl(methylthio)sulfonium tetrafluoroborate (commercially
available) was prepared from trimethyloxonium tetrafluoro-
borate in acetonitrile.18 2,2′-Anhydrouridine (1) was obtained
using the method reported by Hampton and Nichol and improved
by Mofatt and co-workers.19 Bromination of 2′-deoxyadenosine
leading to 8-bromo-2′-deoxyadenosine (10) was performed ac-
cording to the procedure of Ikehara and Kaneko described for
bromination of adenosine.15
2′-Deoxy-2′-(2-(tr im eth ylsilyl)eth yl)th iou r id in e (2). To a
suspension of 2,2′-anhydrouridine (1) (5 g, 22 mmol; RfD 0.30)
and anhydrous K2CO3 (11 g, 79 mmol) in DMF (110 mL) was
added 2-(trimethylsilyl)ethanethiol (3.5 g, 26 mmol). The mixture
was stirred at 120 °C for 3 h under argon. After cooling and
filtration to remove the mineral salts, DMF was evaporated off.
The residue was washed with hexane and then chromatographed
on silica gel in dichloromethane-methanol (95:5) to yield
compound 2 (6.9 g, 19 mmol, 88%; RfB 0.53): mp 59 °C; 1H NMR
(300 MHz, CD3OD) δ 8.00 (1 H, d, J ) 8.0 Hz), 6.14 (1 H, d, J )
8.5 Hz), 5.75 (1 H, d, J ) 8.0 Hz), 4.32 (1 H, dd, J ) 5.4, 2.1 Hz),
4.03 (1 H, m), 3.77 (2 H, m), 3.51 (1 H, dd, J ) 8.4, 5.3 Hz), 2.58
(2 H, m), 0.80 (2 H, m), -0.04 (9 H, s); 13C NMR (75 MHz,
CD3OD) δ 165.8, 152.4, 142.5, 103.3, 90.3, 88.1, 73.8, 63.1, 54.2,
28.1, 18.7, -1.8; LRMS [FAB+, glycerol] m/z ) 361 [M + H]+,
249 [M - Uracil + H]+; [FAB-, glycerol] m/z ) 359 [M - H]-,
111 [uracil - H]-. Anal. Calcd for C14H24N2O5SSi: C, 46.64; H,
6.71; N, 7.77; S. 8.89. Found: C, 46.47; H, 6.66; N, 7.58; S, 8.62.
2′-Deoxy-2′-(2-(tr im eth ylsilyl)eth yl)th iocytid in e (4). To
an ice-cold solution of compound 2 (1.92 g, 5.32 mmol) in dry
pyridine (25 mL) was added acetic anhydride (10 mL). The
resultant solution was stirred at room temperature for 24 h, and
then ethanol (20 mL) was added at 0 °C. The solvents were
evaporated, and the residue was dissolved in dichloromethane
(150 mL). The resultant solution was washed with water (100
mL), and the aqueous layer was extracted with dichloromethane.
The combined extracts were dried over Na2SO4, evaporated, and
coevaporated with toluene.
The residue was dissolved in 2% dichloroacetic acid solution
in dichloromethane (100 mL). The resultant orange solution was
stirred for 30 min under nitrogen at room temperature, and
aqueous NaHCO3 (5%, 80 mL) was added. The aqueous solution
was extracted with dichloromethane (2 × 50 mL), and then the
combined organic extracts were dried over Na2SO4 and evapo-
rated. Compound 8 was purified by chromatography on silica
gel in CH2Cl2-AcOEt (6:4) (1.08 g, 3.02 mmol; 62%; RfA 0.48):
mp 49 °C; 1H NMR (300 MHz, CDCl3) δ 8.48 (1 H, s), 7.52 (1H,
s), 6.07 (1 H, dd, J ) 7.0, J ) 4.1 Hz), 4.05 (1 H, m), 3.85 (2 H,
m), 3.47 (1 H, m), 2.64-2.50 (3 H, m), 2.41 (1H, m), 2.07 (1H, s),
1,2,4-Triazole (5 g, 72 mmol) was added to a POCl3 solution
(1.35 mL, 17.7 mmol) in CH3CN (20 mL). The mixture was
(17) Gornowicz, G. A.; Ryan, J . W.; Speier, J . L. J . Org. Chem. 1968,
33, 2918-2924.
(18) Helmkamp, G. K.; Cassey, H. N.; Olsen, B. A.; Pettitt, D. J . J .
Org. Chem. 1965, 30, 933-935. Smallcombe, S. H.; Caserio, M. C. J .
Am. Chem. Soc. 1971, 93, 5826-5832.
(19) Hampton, A.; Nichol, A. W. Biochemistry 1966, 5, 2076-2082.
Verheyden, J . P. H.; Wagner, D.; Moffatt, J . G. J . Org. Chem. 1971,
36, 250-254.

Notes
J . Org. Chem., Vol. 65, No. 1, 2000 253
1.90 (3 H, s), 0.84 (2 H, m), -0.02 (9 H, s); 13C NMR (75 MHz,
CDCl3) δ 164.3, 150.4, 136.8, 110.6, 85.9, 85.6, 61.2, 40.4, 40.2,
27, 17.3, 12.4, -1.8; LRMS [FAB+, glycerol] m/z ) 359 [M +
H]+, 233 [M - thymine]+, 127 [thymine + H]+, [FAB-, glycerol]
m/z ) 357 [M - H]-. Anal. Calcd for C15H26N2O4SSi‚0.25H2O:
C, 49.63; H, 7.36; N, 7.72; S, 8.83. Found: C, 49.47; H, 7.15; N,
7.48; S, 8.91.
Gen er a l P r oced u r e for P r ep a r in g th e Meth yl Disu lfid es
3, 5, a n d 9. To a solution of nucleoside 2, 4, or 8 (200 mg; 2,
0.55 mmol; 4, 0.55 mmol; 8, 0.56 mmol) and methyl disulfide
(2, 1.4 mL, 16 mmol; 4, 2.5 mL, 28 mmol; 8, 1.4 mL, 16 mmol)
in dry THF (10 mL) was added dropwise a solution of dimeth-
yl(methylthio)sulfonium tetrafluoroborate (2, 271 mg, 1.4 mmol;
4, 368 mg, 1.9 mmol; 8, 223 mg, 1.2 mmol) in THF (30 mL). The
mixture was stirred for 15 h under argon, and then it was
neutralized by addition of aqueous NaHCO3 (10%, 1 mL). After
evaporation, the residue dissolved in a minimal volume of water
was chromatographed on a Sep-Pak C18 cartridge (1 g) eluting
with H2O and then H2O-MeOH (95:5-90:10) to afford the
corresponding methyl disulfide 3 (154 mg, 0.50 mmol, 90%; RfB
0.43), 5 (134 mg, 0.44 mmol, 80%; RfC 0.53), or 9 (152 mg, 0.50
mmol, 90%; RfA 0.43). These compounds were crystallized from
water.
filtration, the solid was washed with water to obtain compound
11 (2.8 g, 7.3 mmol, 95%; RfB 0.59): mp 170 °C; H NMR (300
1
MHz, DMSO-d6) δ 8.04 (1 H, s), 7.22 (2 H, s), 6.23 (1 H, dd, J )
8.3, 6.3 Hz), 5.47 (1 H, dd, J ) 8.1, 4.0 Hz), 5.33 (1 H, d, J ) 4.0
Hz), 4.43 (1 H, m), 3.87 (1 H, m), 3.67 (1 H, m), 3.49 (1 H, m),
3.30 (2 H, m), 3.10 (1 H, m), 2.09 (1 H, m), 1.00 (2 H, m), 0.04 (9
H, s); 13C NMR (75 MHz, DMSO-d6) δ 154.5, 151.3, 150.4, 148.2,
119.7, 88.3, 84.9, 71.4, 62.3, 37.4, 29.1, 16.74, -1.7; LRMS
[FAB+, glycerol] m/z ) 384 [M + H]+, [FAB-, glycerol] m/z )
382 [M - H]-, 282 [M - 2H - C2H4SiMe3]-. Anal. Calcd for
C
15H25N5O3SSi: C, 46.97; H, 6.57; N, 18.26; S, 8.36. Found: C,
46.57; H, 6.68; N, 18.18; S, 8.27.
2′-Deoxy-8-th ioa d en osin e (12). To a solution of compound
11 (200 mg, 0.52 mmol) in anhydrous THF (2 mL) was added a
solution of tetrabutylammonium fluoride in THF (1 M, 2 mL).
The resultant solution was stirred for 3 h at room temperature
and then was evaporated. To the oily residue was added ether,
and the white solid obtained was filtered off and washed with
ether and then with water (5 mL at 0 °C). NMR analysis of this
dried solid revealed the presence of a 1:1 mixture of thione 12
and its tetrabutylammonium salt (194 mg, 92%; RfB 0.24): .1H
NMR (300 MHz, DMSO-d6) δ 7.90 (1 H, s), 6.84 (1 H, dd, J )
8.9, 6.0 Hz), 6.66 (2 H, s), 5.98 (1 H, s), 5.18 (1 H, d), 4.40 (1 H,
m), 3.85 (1 H, m), 3.65 (1 H, m), 3.52 (1 H, m), 3.15 (4 H, m),
2.84 (1 H, m), 1.95 (1 H, m), 1.55 (4 H, m), 1.32 (4 H, m), 0.92 (6
H, t, J ) 7.3 Hz); LRMS [FAB+, glycerol] m/z ) 284 [M + H]+,
[FAB-, glycerol] m/z ) 282 [M - H]-.
Isola tion of th e P u r e Th ion e (12). The obtained mixture
of thione 12 and the corresponding tetrabutylammonium salt
dissolved in aqueous NH4HCO3 (5%, 25 mL) was chromato-
graphed on a Sep-Pak C18 cartridge (1 g) with a mixture of 2%
aqueous NH4HCO3-MeOH (9:1). Fractions containing compound
12 were evaporated, giving a white residue which was dissolved
in aqueous HCl (0.2 M, 5 mL). The solution was chromato-
graphed on a Sep-Pak C18 cartridge (1 g) with H2O and then
H2O-MeOH (8:2) to lead to the thione 12: mp 141 °C; 1H NMR
(400 MHz, DMSO-d6) δ 12.55 (1 H, s), 8.18 (1 H, s), 6.95 (2 H,
s), 6.28 (1 H, t, J ) 6.4 Hz), 5.35 (1 H, s), 5.23 (1 H, s), 4.46 (1
H, m), 3.68 (1H, m), 3.53 (1H, m), 2.95 (1H, m), 2.07 (1 H, m);
13C NMR (75 MHz, DMSO-d6) δ 167.1, 151.9, 148.1, 107.5, 88.1,
85.1, 71.5, 62.4, 39.9, 36.8; IR (KBr) 3200, 2920, 1650, 1580,
1450, 1100 (CdS) cm-1; LRMS [FAB+, glycerol] m/z ) 284 [M +
H]+, [FAB-, glycerol] m/z ) 282 [M - H]-. Anal. Calcd for
C10H13N5O3S: C, 39.86; H, 5.02; N, 23.24; S, 10.64. Found: C,
40.09; H, 5.05; N, 22.90; S, 10.58.
Da ta for 2′-Deoxyu r id in -2′-yl Meth yl Disu lfid e (3): mp
175 °C dec; 1H NMR (300 MHz, CD3OD) δ 8.01 (1 H, d, J ) 8.1
Hz), 6.28 (1 H, d, J ) 8.7 Hz), 5.75 (1 H, d, J ) 8.1 Hz), 4.39 (1
H, m), 4.00 (1 H, m), 3.74 (2 H, m), 3.69 (1 H, dd, J ) 8.7, J )
5.5 Hz), 2.35 (3 H, s); 13C NMR (75 MHz, CD3OD) δ 165.9, 152.6,
142.7, 103.4, 90.2, 88.3, 74.1, 63.1, 60.0, 23.6; LRMS [FAB+,
glycerol] m/z ) 307 [M + H]+, [FAB-, glycerol] m/z ) 305 [M -
H]-. Anal. Calcd for C10H14N2O5S2: C, 39.21; H, 4.61; N, 9.14;
S, 20.93. Found: C, 39.30; H, 4.74; N, 9.01; S, 21.14.
Da ta for 2′-Deoxycytid in -2′-yl Meth yl Disu lfid e (5): mp
1
145 °C dec; H NMR (300 MHz, DMSO-d6) δ 7.79 (1 H, d, J )
7.5 Hz), 7.26 (2 H, d, J ) 9.6 Hz), 6.21 (1 H, d, J ) 8.9 Hz), 5.82
(1 H, d, J ) 5.3), 5.77 (1 H, d, J ) 7.5 Hz), 5.07 (1 H, t, J ) 5.2
Hz), 4.24 (1 H, m), 3.83 (1 H, m), 3.63 (1 H, dd, J ) 8.9, 5.4 Hz),
3.52 (2H, m), 2.24 (3H, s); 13C NMR (75 MHz, DMSO-d6) δ 165.4,
155.4, 141.7, 94.8, 88.3, 86.2, 72.3, 61.5, 58.8, 23.1; LRMS [FAB+,
glycerol] m/z ) 306 [M + H]+. Anal. Calcd for C10H15N3O4S2: C,
39.33; H, 4.95; N, 13.76; S, 21.00. Found: C, 39.23; H, 4.96; N,
13.65; S, 21.19.
Da ta for 3′-Deoxyth ym id in -3′-yl Meth yl Disu lfid e (9):
mp 83 °C; 1H NMR (300 MHz, CDCl3) δ 8.46 (1 H, s), 7.51 (1 H,
d, J ) 1.0 Hz), 6.09 (1 H, dd, J ) 7.0, 4.5 Hz), 4.10-3.84 (3 H,
m), 3.61 (1 H, m), 2.54 (2 H, m), 2.43 (3 H, s), 2.35 (1 H, t, J )
6.0 Hz), 1.89 (3H, d, J ) 1.0 Hz); 13C NMR (75 MHz, CDCl3) δ
163.4, 150.1, 136.5, 110.9, 85.8, 85.3, 61.3, 44.8, 38.1, 24.4, 12.6;
LRMS [FAB+, glycerol] m/z ) 305 [M + H]+, 179 [M - thymine
+ H]+, 127 [thymine + H]+, [FAB-, glycerol] m/z ) 303 [M -
H]-, 125 [thymine - H]-. Anal. Calcd for C11H16N2O4S2‚H2O:
C, 40.98; H, 5.63; N, 8.69; S, 19.89. Found: C, 41.15; H, 5.62; N,
8.51; S, 20.08.
2′-Deoxy-8-(2-(t r im et h ylsilyl)et h yl)t h ioa d en osin e (11).
To a suspension of 8-bromo-2′-deoxyadenosine (10) (2.5 g, 7.6
mmol; RfB 0.43) and anhydrous K2CO3 (3 g, 21.7 mmol) in DMF
(25 mL) was added 2-(trimethylsilyl)ethanethiol (1.2 g, 8.9
mmol). The mixture was stirred at 60 °C for 8 h under argon.
After cooling and filtration, DMF was evaporated off. The residue
was washed with hexane and dried. After addition of water and
Ack n ow led gm en t. This work was supported by a
grant from the Ligue Nationale contre le Cancer, Comite´
de la Droˆme.
Su p p or tin g In for m a tion Ava ila ble: Crystallographic
data for 2′-deoxycytidin-2′-yl methyl disulfide (5) and 3′-
deoxythymidin-3′-yl methyl disulfide (9) (tables of crystallo-
graphics details, positional parameters, B(eq), intramolecular
distances, intramolecular bond angles, anisotropic displace-
ment parameters, and least-squares planes). This material is
available free of charge via the Internet at http://pubs.acs.org.
J O9908492