Nucleosides and nucleotides. 232. Synthesis of 2′-C-methyl-4′- thiocytidine: Unexpected anomerization of the 2′-keto-4′- thionucleoside precursor

Article abstract of DOI:10.1080/15257770500267204

□ The synthesis of 2′-C-methyl-4′-thiocytidine (16) is described. Since the 2′-keto-4′-thiocytidine derivative 2β unexpectedly isomerized to 2α and the methylation of 2β proceeded predominantly from the less hindered α-face to give 7, the desired product

Full text of DOI:10.1080/15257770500267204

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Nucleosides and Nucleotides. 232. Synthesis of 2′-C-
Methyl-4′-thiocytidine: Unexpected Anomerization of
the 2′-Keto-4′-thionucleoside Precursor
Daisuke Kaga a , Noriaki Minakawa a & Akira Matsuda a
a Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan
Published online: 16 Aug 2006.
To cite this article: Daisuke Kaga , Noriaki Minakawa & Akira Matsuda (2005): Nucleosides and Nucleotides. 232. Synthesis of
2′-C-Methyl-4′-thiocytidine: Unexpected Anomerization of the 2′-Keto-4′-thionucleoside Precursor, Nucleosides, Nucleotides
and Nucleic Acids, 24:10-12, 1789-1800
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1789–1800, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500267204
NUCLEOSIDES AND NUCLEOTIDES. 232. SYNTHESIS OF
2 -C-METHYL-4 -THIOCYTIDINE: Unexpected Anomerization
of the 2 -Keto-4 -thionucleoside Precursor
ᮀ
Daisuke Kaga, Noriaki Minakawa, and Akira Matsuda
Graduate School of
Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan
ᮀ
The synthesis of 2 -C-methyl-4 -thiocytidine (16) is described. Since the 2 -keto-4 -thiocytidine
derivative 2β unexpectedly isomerized to 2α and the methylation of 2β proceeded predominantly
from the less hindered -face to give 7, the desired product 16 was synthesized via the Pummerer
reaction of the sulfoxide 14 and N4-benzoylcytosine.
Keywords
Nucleoside; 4 -Thionucleoside; 2 -Keto-4 -thionucleoside; 2 -Branched-
chain-4 -thionucleoside; 2 -C-methyl-4 -thiocytidine
INTRODUCTION
Branched-chain sugar nucleosides are some of the most attractive
nucleoside derivatives for the development of antitumor and antiviral
agents. Consequently, much attention has been directed toward modifi-
cation of these derivatives, especially at the 2ꢁ-position, which has been
extensively investigated with a number of biologically active 2ꢁ-branched-
chain sugar nucleoside derivatives having been reported.[1,2] Our group
also has been engaged in the synthesis of 2ꢁ-branched-chain sugar pyrim-
idine nucleosides, and have found that 2ꢁ-C-methylcytidine,[3,4] 2ꢁ-deoxy-
2ꢁ(S)-methylcytidine,[5] 2ꢁ-deoxy-2ꢁ-methylidenecytidine (DMDC),[6,7] and
In honor and celebration of the life and career of John A. Montgomery.
Received 19 January 2005; accepted 18 April 2005.
This investigation was supported in part by a Grant-in-Aid for Scientific Research on Priority
Areas from the Ministry of Education, Science, Sports, and Culture of Japan. We would like to thank
Ms. H. Matsumoto and Ms. A. Maeda (Center for Instrumental Analysis, Hokkaido University) for
elemental analysis. We also would like to thank Ms. S. Oka (Center for Instrumental Analysis, Hokkaido
University) for measurement of mass spectra. This article constitutes Part 232 of Nucleosides and
Nucleotides (for part 231 in this series, see Takagi et al.[23]).
Address correspondence to Akira Matsuda, Graduate School of Pharmaceutical Sciences,
Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan. Fax: +81-11-706-4980; E-mail:
matuda@pharm.hokudai.ac.jp
1789

1790
D. Kaga et al.
FIGURE 1 The structures of 2 -branched-chain sugar pyrimidine nucleosides.
2ꢁ-deoxy-2ꢁ(S)-cyanocytidine (CNDAC)[8] possess potent antitumor activity
(Figure 1).
4ꢁ-Thionucleosides are nucleoside derivatives in which the furanose ring
oxygen is replaced by a sulfur atom. This series of nucleoside analogs
has long been recognized as a potential target of antimetabolites, and
much effort has been expended in the design and synthesis of 4ꢁ-thio-
nucleoside analogs.[9,10] Thus far, several groups have reported the synthesis
of 2ꢁ-branched-chain sugar 4ꢁ-thionucleosides,[11–13] and 4ꢁ-thio-2ꢁ-deoxy-2ꢁ-
methylidenecytidine (4ꢁ-thioDMDC) has been found to be a potent antineo-
plastic 4ꢁ-thionucleoside derivative.[14] Since the activity of 4ꢁ-thioDMDC was
higher than that of DMDC, the synthesis and biological evaluation of the 4ꢁ-
thio-congeners of 2ꢁ-branched-chain sugar pyrimidine nucleosides prepared
in our laboratory would be an ideal approach for developing new biologi-
cally active nucleosides. In this article, we describe the synthesis and antitu-
mor activity of 2ꢁ-C-methyl-4ꢁ-thiocytidine (16).
RESULTS AND DISCUSSION
The 2ꢁ-branched-chain sugar pyrimidine nucleosides reported by us have
been prepared from the corresponding 2ꢁ-ketonucleoside as the common
precursor. Therefore, it was thought that a 2ꢁ-keto-4ꢁ-thionucleoside would
be the ideal key compound for the preparation of the 4ꢁ-thio-congeners
of 2ꢁ-branched-chain sugar pyrimidine nucleosides, including the target
compound. To date, there have been no reports on a systematic study
of the synthesis and reactivity of the 2ꢁ-keto-4ꢁ-thionucleoside derivative.
Yoshimura et al. prepared the 2-keto-4-thiosugar derivative, a precursor
of 4ꢁ-thioDMDC, by oxidation of the hydroxyl group at the 2-position
with DMSO-Ac2O without affecting the sulfur atom of the 4-thiosugar.[14]
These conditions were examined as the first choice for the 2ꢁ-keto-4ꢁ-
thionucleoside derivative synthesis. When 1[15–17] was treated with DMSO-
Ac2O overnight according to the method reported by Yoshimura et al.
two inseparable compounds were obtained after column chromatography

Unexpected Precursor Anomerization
1791
(ca. 7:1 ratio). Although the ratio of the two compounds varied depending
on the oxidation reaction time (from 1:4 for 2.5 h to 7:1 for 24 h), neither
compound could be prepared as the sole product. To determine the struc-
tures of the oxidation product, the mixture, consisting of a 7:1 ratio, was suc-
cessively treated with sodium borohydride, ammonium fluoride, and meth-
ylamine. Consequently, the analytical data of the resulting major product
was identical with those of 4ꢁ- -thiocytidine (3) (Scheme 1).[18] Unexpected
anomerization of the 2ꢁ-keto-4ꢁ-thiocytidine derivative, i.e., from 2β to 2α,
occurred not only during oxidation but also during chromatographic pu-
rification. Such anomerization of 2ꢁ-ketonucleosides had not been observed
in the corresponding 4ꢁ-oxo-congener.[7] In addition, since the correspond-
ing 3ꢁ-keto-4ꢁ-thiocytidine derivative synthesized in our previous study was
also inactive for epimerization at the 4ꢁ-position,[15] we reasoned that this
equilibrium should be characteristic of the 2ꢁ-keto-4ꢁ-thiocytidine derivative.
The unexpected anomerization of the 2ꢁ-keto-4ꢁ-thiocytidine derivative can
be explained by the higher acidity of the -hydrogen (i.e., the H-1ꢁ proton
of 2β) of the thioether and the higher stability of the resulting carbanion
versus those of the corresponding ether.[19,20] Since other oxidation con-
ditions including SO3-pyridine complex and Dess-Martin periodinane, had
resulted in a diminished ratio and chemical yield of 2β, we decided to use
DMSO-Ac2O, which proved to be the best choice for obtaining 2β in a sat-
isfactory ratio.
In our study of the 2ꢁ-C-methylcytidine synthesis, we found that the stere-
oselectivity of the carbonyl methylation of the 2ꢁ-ketonucleoside derivative
SCHEME 1

1792
D. Kaga et al.
SCHEME 2
SCHEME 3
varied depending on the alkylating reagent (Scheme 2).[3] Thus, the reac-
tion of 4 with MeLi and AlMe3 afforded 5 in at least 80% yield as the sole
product arising from nucleophilic attack at the less hindered -face. While
the reaction with MeMgBr gave 5 and 6 in a 1:0.8 ratio (52% and 43%,
respectively) presumably due to a chelation-controlled process in which
the Grignard reagent would chelate between the 2-carbonyl oxygen of the
pyrimidine ring and the 2ꢁ-carbonyl oxygen of the sugar moiety. Accordingly,
we next examined the carbonyl methylation of the 2ꢁ-keto-4ꢁ-thiocytidine us-
ing MeLi, AlMe3, and MeMgBr (Scheme 3). Compound 1 was treated with
DMSO-Ac2O until the starting material had been consumed. The resulting
2β containing 2α (ca. 2α:2β = 1:4) was worked up in water and then sub-
jected to methylation under the conditions in Table 1. Although the reaction
with MeMgBr gave the best chemical yield, all reactions afforded 7 as the
sole product arising from nucleophilic attack on the less hindered -face.
TABLE 1 Reaction of Compound 2β with Organometallic
Reagents
Entry
Reagent
Conditions
Yield of 7
1
2
3
MeLi
Me3Al
MeMgBr
Et2O, −78 C
26
54
55
CH2Cl2, −78 to rt
Et2O, −78 C

Unexpected Precursor Anomerization
1793
Unlike the reaction with 4, the expected chelation-controlled process caus-
ing nucleophilic attack on the -face was not observed even in the reaction
of 2β with MeMgBr, probably due to the structural difference between 4 and
2β. Treatment of 7 with ammonium fluoride in MeOH, followed by meth-
ylamine, gave the free nucleoside 8 in 94% yield. The structure of 8 was
confirmed by NOE experiment. Thus, the expected NOEs were observed at
H-1ꢁ (4.6%) and H-4ꢁ (1.2%) upon irradiation of the methyl protons at the
2ꢁ-position.
Since we were unsuccessful in synthesizing 2ꢁ-C-methyl-4ꢁ-thiocytidine
from 2β, we decided to adopt an alternative route, in which the methyl
group would be introduced prior to the Pummerer reaction, as shown
in Scheme 4. Thus, 9 was converted to the ketone 10. The SO3-pyridine
complex proved to be superior to DMSO-Ac2O for use in this oxidation due
to the formation of the corresponding methylthiomethyl ether derivative
SCHEME 4

1794
D. Kaga et al.
under latter conditions. When the resulting 10 was treated with MeTiCl3, a
mixture of 11 and 12 was obtained in 72% yield (11:12 = 1:0.33). Although
other reagents such as MeLi, AlMe3, and MeMgBr were examined, none of
these increased the ratio of the desired 12. Since 11 and 12 were inseparable
by silica gel column chromatography, the mixture was subsequently treated
with dimethoxybenzoyl chloride (DMBzCl) in pyridine at 80◦C to isolate 13
in 19% yield. After conversion of 13 into the sulfoxide 14, the Pummerer
reaction of 14 was carried out in the presence of N4-benzoylcytosine, and
the 2ꢁ-C-methyl-4ꢁ-thiocytidine derivative 15 was obtained in 23% yield.
Although this reaction proceeded stereoselectively due to the effect of the
DMBz group, the chemical yield was insufficient because of steric hindrance
of the methyl substituent at the 2-position. Since no other coupling product
was detected, the thiocarbocation intermediate generated from 14 under
the Pummerer conditions would be decomposed prior to coupling with
N4-benzoylcytosine. Deprotection of 15 by tetrabutylammonium fluoride
(TBAF) in THF, followed by treatment with methylamine gave the desired
2ꢁ-C-methyl-4ꢁ-thiocytidine (16) in 97% yield. Although the antileukemic
activity of 8 and 16 was tested toward L1210 cells in vitro, neither compound
inhibited cell growth at a concentration of 100 g/mL, while the IC50 value
of 2ꢁ-C-methylcytidine was 12 g/mL.[3,4]
In conclusion, we have examined the synthesis of 2ꢁ-C-methyl-4ꢁ-thio-
cytidine (16) via the 2ꢁ-keto-4ꢁ-thiocytidine derivative 2β and found that
2β unexpectedly isomerized to its -anomer 2α under the oxidation con-
ditions, and that the methylation of 2β proceeded predominantly from the
less hindered -face to give 7 under all conditions. Accordingly, 16 was syn-
thesized via the Pummerer reaction between the 2-C-methyl-4-thiosugar 14
and N4-benzoylcytosine. The desired 16 showed no significant cytotoxic-
ity toward L1210 cells. In our previous paper, we reported the synthesis
of 1-(3-C-ethynyl-4-thio- -D-ribofuranosyl)cytosine, which had no significant
cytotoxicity.[15] From the results obtained in this and previous papers, it
may be concluded that 4ꢁ-thioribocytidine derivatives are less susceptible to
phosphorylation by cellular uridine-cytidine kinase. Since some 2ꢁ-deoxy-4ꢁ-
thiocytidine derivatives are suggested to phosphorylate by cellular deoxy-
cytidine kinase and show potent cytotoxicity,[21,22] further investigations of
the susceptibility of 4ꢁ-thioribocytidine derivatives to uridine-cytidine kinase
is needed.
EXPERIMENTAL SECTION
General Methods
Physical data were measured as follows: Melting points are uncorrected.
1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz instru-
ments in CDCl3 or DMSO-d6 as the solvent with tetramethylsilane as an

Unexpected Precursor Anomerization
1795
internal standard. Chemical shifts are reported in parts per million ( ), and
signals are expressed as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br (broad). All exchangeable protons were detected by addi-
tion of D2O. TLC was done on Merck Kieselgel F254 precoated plates. Silica
gel used for column chromatography was Merck silica gel 5715.
4 -α-Thiocytidine (3).[18] A mixture of 1[15] (244 mg, 0.40 mmol)
and Ac2O (2.0 mL) in DMSO (4.0 mL) was stirred at room temperature
overnight. The reaction was quenched by addition of saturated aqueous
NaHCO3, and the whole reaction mixture was stirred for 10 min. The
mixture was partitioned between AcOEt and H2O. The separated organic
layer was washed with saturated aqueous NaHCO3, followed by brine. The
organic layer was dried and concentrated in vacuo. The residue was purified
by a silica gel column, eluted with hexane/AcOEt (2:1–1:2), to give 2
(2α:2β = 7:1) as a mixture of diastereomers (227 mg, 93%). A solution
of the resulting 2 in MeOH (7 mL) was treated with NaBH4 (55 mg, 1.4
mmol) for 10 min at room temperature. The solvent was removed, and the
residue was purified by a silica gel column, eluted with hexane/AcOEt (2:1–
1:3), to give protected 4ꢁ- -thiocytidine derivative. The resulting compound
was then dissolved in MeOH (3 mL) and ammonium fluoride (122 mg, 3.3
mmol) was added to the solution. The whole reaction mixture was heated
under reflux for 12 h. The solvent was removed in vacuo, and the residue
was dissolved in methylamine in MeOH (40%, 5 mL). The reaction mixture
was kept for 2 h at room temperature, and the solvent was removed in vacuo.
The residue was purified by a silica gel column, eluted with 33% MeOH in
CHCl3, to give 3 (32 mg, 31% from 1).
N4-Benzoyl-1-[2-C-methyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-di-
yl)-4-thio-β-D-arabino-pentofuranosyl]cytosine (7). A mixture of 1 (302 mg,
0.50 mmol) and Ac2O (2.5 mL) in DMSO (5.0 mL) was stirred for 2.5 h
at room temperature. The reaction was quenched by addition of saturated
aqueous NaHCO3, and the whole reaction mixture was stirred for 10 min.
The mixture was partitioned between AcOEt and H2O. The separated
organic layer was washed with saturated aqueous NaHCO3, followed by
brine. The organic layer was dried and concentrated in vacuo to give crude
2β. The residue was dissolved in Et2O (5 mL), and MeMgBr (3.0 M in
Et2O, 0.83 mL, 2.5 mmol) was added dropwise to the solution at −78◦C.
The mixture was stirred for 4 h at the same temperature, and the reaction
was quenched by addition of saturated aqueous NH4Cl. After warming to
room temperature, the mixture was partitioned between AcOEt and H2O.
The separated organic layer was washed with H2O, followed by brine. The
organic layer was dried and concentrated in vacuo. The residue was purified
by a silica gel column, eluted with hexane/AcOEt (3:1–1:1), to give 7

1796
D. Kaga et al.
(174 mg, 56% as a white foam): FAB-LRMS m/z 620 (MH+, 25%); 1H NMR
(CDCl3) : 8.86 (d, 1H, J = 7.6 Hz), 8.72 (br s, 1H), 7.90–7.88 (m, 2H),
7.64–7.49 (m, 4H), 5.87 (s, 1H), 4.19 (dd, 1H, J = 2.9 and 12.6 Hz), 4.01
(d, 1H, J = 10.0 Hz), 3.98 (d, 1H, J = 12.6 Hz), 3.72 (dd, 1H, J = 2.9
and 10.0 Hz), 2.83 (br s, 1H), 1.57 (s, 3H), 1.20–0.92 (m, 28H); 13C NMR
(CDCl3) : 166.18. 161.78, 157.64, 147.09, 133.01, 132.86, 128.87, 128.43,
127.38, 127.17, 96.29, 80.21, 73.10, 66.48, 58.90, 47.57, 21.15, 17.70, 17.65,
17.57, 17.50, 17.40, 17.36, 16.99, 16.90, 13.73, 13.42, 13.27, 12.60. Anal. calcd
for C29H45N3O6SSi2: C, 56.19; H, 7.32; N, 6.78. Found: C, 56.19; H, 7.32;
N, 6.56.
1-(2-C-Methyl-4-thio-β-D-arabino-pentofuranosyl)cytosine (8). A mixture
of 7 (66 mg, 0.11 mmol) in MeOH (2 mL) containing ammonium fluoride
(79 mg, 2.1 mmol) was heated under reflux for 1 h. The solvent was re-
moved in vacuo, and the residue was dissolved in methylamine in MeOH
(40%, 2 mL). The reaction mixture was kept for 15 min at room tempera-
ture, and the solvent was removed in vacuo. The residue was purified by a
silica gel column, eluted with 33% MeOH in CHCl3, to give 8 (28 mg, 94%
1
as a white solid): FAB-LRMS m/z 274 (MH+, 17%); H NMR (DMSO-d6)
: 8.00 (d, 1H, J = 7.5 Hz), 7.12 and 7.03 (br s, each 1H), 6.14 (s, 1H),
5.67 (d, 1H, J = 7.5 Hz), 5.42 (d, 1H, J = 4.7 Hz), 5.18 (s, 1H), 5.09 (t,
1H, J = 5.3 Hz), 3.81–3.72 (m, 2H), 3.64–3.59 (m, 1H), 3.10 (m, 1H), 1.11
(s, 3H); 13C NMR (CDCl3) : 165.09, 155.90, 144.52, 93.04, 81.08, 78.83,
64.09, 63.32, 54.78, 21.03. Anal. calcd for C10H15N3O4S: C, 43.95; H, 5.53;
N, 15.37. Found: C, 43.72; H, 5.43; N, 15.47.
5,5,7,7-Tetraisopropyl-tetrahydro-4,6,8-trioxa-1-thia-5,7-disilacyclopen-
tacycloocten-3-one (10). To a solution of 9[17] (1.98 g, 5.1 mmol) in DMSO
(25 mL) was added SO3-pyridine (4.0 g, 25.2 mmol) and triethylamine (7.0
mL, 51.0 mmol), and the whole reaction mixture was stirred for 2.5 h at
room temperature. The reaction was quenched by addition of saturated
aqueous NaHCO3, and the mixture was stirred for 10 min. The mixture
was partitioned between AcOEt and H2O. The separated organic layer was
washed with saturated aqueous NaHCO3, followed by brine. The organic
layer was dried and concentrated in vacuo. The residue was purified by a
silica gel column, eluted with hexane/AcOEt (50:1), to give 10 (1.6 g, 81%
as a yellow oil): FAB-LRMS m/z 391 (MH+, 27%); 1H NMR (CDCl3) : 4.50
(d, 1H, J = 10.8 Hz), 4.18 (dd, 1H, J = 3.2 and 12.9 Hz), 3.94 (dd, 1H, J =
3.2 and 12.9 Hz), 3.40–3.29 (m, 3H), 1.12–1.03 (m, 28H); 13C NMR (CDCl3)
: 207.00, 76.21, 59.53, 48.09, 34.18, 17.51, 17.44, 17.40, 17.20, 17.11, 17.07,
17.01, 16.72, 16.65, 13.76, 13.39, 12.91. Anal. calcd for C17H34O4SSi2: C,
52.26; H, 8.77. Found: C, 52.16; H, 8.61.

Unexpected Precursor Anomerization
1797
1,4-Anhydro-2-O-(2,4-dimethoxybenzoyl)-2-C-methyl-3,5-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)-4-thio-D-ribitol (13). An Et2O solution (20
mL) containing TiCl4 (1.8 mL, 16.4 mmol) was cooled to −78◦C, and MeLi
(1.14 M in Et2O, 14.4 mL, 16.4 mmol) was added to the solution at the
same temperature. After being stirred for 10 min, the solution was warmed
to 0◦C, and a THF solution (20 mL) of 10 (1.6 g, 4.1 mmol) was added to the
resulting MeTiCl3 solution at the same temperature. The reaction mixture
was stirred for 8.5 h at room temperature, and the reaction was quenched by
addition of saturated aqueous NH4Cl. The mixture was partitioned between
AcOEt and H2O, and the separated organic layer was washed with H2O,
followed by brine. The organic layer was dried and concentrated in vacuo.
The residue was purified by a silica gel column, eluted with hexane/AcOEt
(50:1–20:1), to give 11 and 12 as a mixture of diastereomers (1.19 g, 72%;
11:12 = 1:0.33). To a mixture of 11 and 12 (1.19 g, 2.83 mmol) in pyridine
(14 mL) was added DMBzCl (1.7 g, 8.5 mmol), and the mixture was stirred
for 16 h at 80◦C. The reaction was quenched by addition of ice, and the
mixture was partitioned between AcOEt and H2O. The separated organic
layer was washed with saturated aqueous NaHCO3, followed by brine. The
organic layer was dried and concentrated in vacuo. The residue was purified
by a silica gel column, eluted with hexane/AcOEt (50:1–10:1), to give 13
1
(0.31 g, 19% as a colorless oil): FAB-LRMS m/z 593 (MNa+, 8%); H NMR
(CDCl3) : 7.94 (d, 1H, J = 8.9 Hz), 6.47–6.43 (m, 2H), 4.12 (dd, 1H,
J = 2.6 and 12.5 Hz), 3.96–3.83 (m, 9H), 3.66 (ddd, 1H, J = 2.6, 3.3, and
9.2 Hz), 2.91 (d, 1H, J = 12.5 Hz), 1.77 (s, 3H), 1.12–0.85 (m, 28H); 13C
NMR (CDCl3) : 163.99, 163.90, 161.60, 134.00, 112.87, 104.20, 98.72, 86.90,
80.65, 60.24, 55.82, 55.43, 49.82, 33.55, 21.56, 17.69, 17.45, 17.42, 17.38,
17.32, 14.16, 13.51, 13.41, 12.81. Anal. calcd for C27H46O7SSi2: C, 56.81; H,
8.12. Found: C, 56.83; H, 8.20.
1,4-Anhydro-2-O-(2,4-dimethoxybenzoyl)-2-C-methyl-3,5-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)-4-sulfinyl-D-ribitol (14). Ozone was bubbled
through a solution of 13 (306 mg, 0.54 mmol) in CH2Cl2 (5.0 mL) at −78◦C.
After 10 min, argon gas was bubbled through the solution to remove excess
ozone. The reaction mixture was allowed to warm to room temperature
and concentrated in vacuo. The residue was purified by a silica gel column,
eluted with hexane/AcOEt (1:1), to give 14 (251 mg, 80% as a colorless
oil): FAB-LRMS m/z 587 (MH+, 2%); FAB-HRMS calcd for C27H47O8SSi2
1
(MH+) 587.2530, found 587.2562; H NMR (CDCl3) : 7.93 (d, 1H, J =
9.2 Hz), 6.47–6.43 (m, 2H), 4.58 (d, 1H, J = 12.9 Hz), 4.26 (dd, 1H, J =
3.0 and 12.9 Hz), 3.93 (d, 1H, J = 15.8 Hz), 3.88–3.77 (m, 7H), 3.55 (dd,
1H, J = 3.0 and 11.5 Hz), 3.14 (d, 1H, J = 15.8 Hz), 1.79 (s, 3H), 1.13–
1.03 (m, 28H); 13C NMR (CDCl3) : 164.12, 163.90, 161.65, 134.03, 112.47,

1798
D. Kaga et al.
104.27, 98.71, 84.29, 74.09, 73.38, 56.50, 56.01, 55.82, 55.45, 22.12, 17.35,
17.26, 17.20, 17.16, 14.21, 13.26, 13.18, 12.62.
N4-Benzoyl-1-[2-O-(2,4-dimethoxybenzoyl)-2-C-methyl-3,5-O-(1,1,3,3-tet-
raisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribo-pentofuranosyl]cytosine (15).
To a suspension of N4-benzoylcytosine (130 mg, 0.6 mmol) in toluene
(4 mL) was added triethylamine (84 L, 0.6 mmol) and TMSOTf (0.47
mL, 2.4 mmol), and the mixture was stirred at room temperature until
giving two-phase clear solution. After being added CH2Cl2 (2 mL) to the
above solution, the whole solution was added to a solution of 14 (237
mg, 0.4 mmol) in CH2Cl2 (2 mL) dropwise over 15 min via a cannula.
An additional triethylamine (0.25 mL, 1.8 mmol) in toluene (2 mL) was
added dropwise to the reaction mixture. After being stirred for 5 min at
room temperature, the reaction was quenched by addition of ice, and the
mixture was partitioned between AcOEt and H2O. The separated organic
layer was washed with saturated aqueous NaHCO3, followed by brine. The
organic layer was dried and concentrated in vacuo. The residue was purified
by a silica gel column, eluted with hexane/AcOEt (5:1–1:1), to give 15
(72 mg, 23% as a white foam): FAB-LRMS m/z 806 (MNa+, 21%); FAB-
HRMS calcd for C38H53N3O9SSi2Na (MNa+) 806.2938, found 806.2942; 1H
NMR (CDCl3) : 11.40 (br s, 1H), 7.99 (d, 1H, J = 7.3 Hz), 7.77 (d, 2H,
J = 8.7 Hz), 7.62 (d, 1H, J = 7.3 Hz), 7.59–7.47 (m, 3H), 7.42 (d, 1H,
J = 7.3 Hz), 6.83 (s, 1H), 6.63–6.53 (m, 2H), 4.18–4.11 (m, 3H), 3.82 (s,
6H), 3.75 (m, 1H), 1.52 (s, 3H), 1.14–0.87(m, 28H); 13C NMR (CDCl3)
: 167.35, 164.23, 162.85, 162.63, 161.19, 154.74, 146.00, 133.45, 133.40,
132.96, 132.82, 128.47, 111.90, 105.32, 99.03, 96.57, 89.47, 76.81, 62.53,
59.38, 55.98, 55.78, 49.37, 17.62, 17.51, 17.40, 17.31, 17.17, 13.33, 13.02,
12.78, 12.73.
2 -C-Methyl-4 -thiocytidine (16). To a solution of 15 (24 mg, 0.03
mmol) in THF (0.5 mL) was added AcOH (3.5 L, 0.06 mmol) and TBAF
(1 M in THF, 60 L, 0.06 mmol) at 0◦C. After being stirred for 15 min at the
same temperature, the reaction mixture was partitioned between AcOEt and
H2O. The separated organic layer was washed with H2O, followed by brine.
The organic layer was dried and concentrated in vacuo, and the residue was
dissolved in methylamine in MeOH (40%, 1 mL). The reaction mixture
was kept for 2 h at room temperature, and the solvent was removed in
vacuo. The residue was purified by a silica gel column, eluted with 30%
MeOH in CHCl3, to give 16 (8 mg, 94% as a white solid): FAB-LRMS m/z
274 (MH+, 21%); FAB-HRMS calcd for C10H16N3O4S (MH+) 274.0862,
1
found 274.0864; H NMR (DMSO-d6) : 8.06 (d, 1H, J = 7.6 Hz), 7.19
and 7.11 (br s, each 1H), 5.96 (s, 1H), 5.73 (d, 1H, J = 7.6 Hz), 5.15–
5.05 (m, 3H), 3.84 (ddd, 1H, J = 2.9, 4.2, and 11.2 Hz), 3.71 (ddd, 1H,

Unexpected Precursor Anomerization
1799
J = 4.2, 5.9, and 11.2 Hz), 3.48 (m, 1H), 3.30 (m, 1H), 0.91 (s, 3H);
13C NMR (CDCl3) : 164.99, 155.76, 142.68, 94.15, 81.58, 76.08, 66.37,
61.28, 52.42, 20.72.
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