Study on disulfur-backboned nucleic acids: Part 3. Efficient synthesis of 3′,5′-dithio-2′-deoxyuridine and deoxycytidine

Article abstract of DOI:10.1080/15257770802520474

A general method is described for synthesizing 3′,5′-dithio- 2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′- deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl- 3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH- ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802520474

Nucleosides, Nucleotides and Nucleic Acids, 27:1272–1281, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802520474
STUDY ON DISULFUR-BACKBONED NUCLEIC ACIDS: PART 3.
EFFICIENT SYNTHESIS OF 3ꢀ,5ꢀ-DITHIO-2ꢀ-DEOXYURIDINE
AND DEOXYCYTIDINE
Peihua Shang, Hua Wang, Changmei Cheng, Hongchao Zheng, and
Yufen Zhao
Key Laboratory of Phosphorus and Chemical Biology, The Ministry of Education,
Department of Chemistry, Tsinghua University, Beijing, P. R. China
A general method is described for synthesizing 3ꢁ,5ꢁ-dithio-2ꢁ-deoxypyrimidine nucleosides 6 and
2
13 from normal 2ꢁ-deoxynucleosides. 2,3ꢁ-Anhydronucleosides 2 and 9 are applied as intermediates
in the process to reverse the conformation of 3ꢁ-position on sugar rings. The intramolecular rings
of 2,3ꢁ-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3ꢁ-S-acetyl-
3ꢁ-thio-2ꢁ-deoxynucleosides 3 or 5. To cytidine, OH− ion exchange resin was used to open the ring
and 2ꢁ-deoxycytidine 10 was abtained in which 3ꢁ-OH group is in threo- conformation. The 3ꢁ-OH
is activated by MsCl, and then substituted by potassium thioacetate to form the S,Sꢁ-diacetyl-3ꢁ,5ꢁ-
dithio-2ꢁ-deoxycytidine 12. The acetyl groups in 3ꢁ,5ꢁ position are removed rapidly by EtSNa in EtSH
solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine
and cytidine are also discusssed.
Keyword 3ꢁ; 5ꢁ-dithio-2ꢁ-deoxyuridine; 3ꢁ; 5ꢁ-dithio-2ꢁ-deoxycytidine; 2; 3ꢁ-anhydronucl-
eosides; synthesis
INTRODUCTION
Since the early 1960s, thiol group has been used to replace the hydroxyl
in the sugar ring of normal nucleosides.[1] But the work of both 3ꢁ,5ꢁ
hydroxyl groups were replaced by thiol was rarely mentioned before, not
only because of the hard conformation conversion of 3ꢁ-position on the
sugar ring, but also the easily formation of S-S linkage between bare thiol
groups. In 1996, Reese’s group developed an efficient route to synthesize the
3ꢁ,5ꢁ-dithio-2ꢁ-deoxythymidine through 2,3ꢁ-anhydrothymidine.[2] And this
Received 10 March 2008; accepted 21 September 2008.
Address correspondence to Changmei Cheng, Key Laboratory of Phosphorus and Chemical
Biology, The Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084,
P. R. China. E-mail: chengcm@mail.tsinghua.edu.cn
1272

Study on Disulfur-Backboned Nucleic Acids
1273
procedure was simplified by Mitsuhiko Shinoya’s group in 2002.[3] However,
the previous work mainly focused on the synthesis of 3ꢁ,5ꢁ-dithiothymidine,
and the other four kinds of dithionucleosides were almost ignored. Since
2004, our group has started a project to synthesize a series of novel disulfur-
backbones nucleic acids, and to apply them to the study of antisense and
the synthesis of the metal-mediated DNA strand. In the present research,
3ꢁ,5ꢁ-dithio-2ꢁ-deoxyadenosine[4] has been succeffully synthesized and a
novel efficient synthetic route toward 3ꢁ,5ꢁ-dithiothymidine[5] has also been
developed. In this article, we report the synthetic process of 3ꢁ,5ꢁ-dithio-2ꢁ-
deoxyuridine and deoxycytidine.
RESULTS AND DISCUSSION
As uridine has a very similar base moiety with thymidine, the similar syn-
thetic route with thymidine was applied in the research. The synthesis of 5ꢁ-
O-mesyl-2ꢁ-deoxy-2,3ꢁ-anhydrouridine was followed the procedure reported
by Reese.[2] Then there were two methods for synthesis of S,Sꢁ-diacetyl-
3ꢁ,5ꢁ-dithio-2ꢁ-deoxyuridine, the first one was to open the intramolecular
ring in 2 by treatment with thioacetic acid, which served as both solvent
and nucleophilic reagent. The product of 5ꢁ-O-mesyl-3ꢁ-S-acetylthiouridine
was then reacted with potassium thioacetate to form 5. The second method
was the reversed order of two steps.
In our previous report,[5] we found that 5ꢁ-O-tosyl-2,3ꢁ-anhydrothym-
idine had a completely different regioselectivity to nucleophilic reagent
between basic and acidic conditions. In acidic solution, the 3ꢁ-position had
a higher activity than 5ꢁ-position. When it was treated with nucleophilic
reagent, the bond of C3ꢁ-O2 prefered to break first, to form the 3ꢁ-
substituted product; while in basic solution, the activity of 5ꢁ-position was
higher than 3ꢁ, so it lead to the only 5ꢁ-substituted product, in which
the intramolecular ring was not opened. It seemd that the 5ꢁ-O-mesyl-2ꢁ-
deoxy-2,3ꢁ-anhydrouridine had the same property as thymidine. When 2 was
reacted with AcSH, it can only produce 3, the 3ꢁ-substitution; when reacted
with KSAc, it led to 4, the 5ꢁ-substituted product.
The acetyl groups of 5 can be easily removed by strong base, such
as NaOMe in MeOH, MeOH-NH3. But the bare thiol groups can be
easily oxidized to form disulfur bond in these conditions. Our group has
developed a facile method to deprotect the acetyl groups that can efficiently
prevent the oxidization between thiol groups. 5 was treated with EtSNa
in the solution of EtSH under N2 atmosphere. The acetyl groups were
completely removed in 2 to 3 minutes. Then the mixture was neutralized
with 0.1N HCl, and extracted with dichloromethane. After evaporating the
solution to dry, the residue was saturated with ethyl ether to precipitate
3ꢁ,5ꢁ-dithio-2ꢁ-deoxyuridine in 85% yield. Under N2 atmosphere and -20◦C
the solid state of 6 can be stored for three months.

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P. Shang et al.
However, the successful route for synthesizing dithiothymidine and
uridine is not suitable for the synthesis of dithiocytidine.
The N4-benzoyl-3ꢁ,5ꢁ-di-O-mesyl-2ꢁ-deoxycytidine was readily prepared
in two steps.[6] However, the 2,3ꢁ-anhydro product cannot be obtained
by heating with Et3N and EtOH. A stronger base DBU had to be used,
which has a weak nucleophilic property, to produce 9. When 9 was treated
with refluxing AcSH, the desired 3ꢁ-substituted product also cannot be
obtained as uridine and thymidine. When the reaction time was elongated
to 3 days, the solution became a black-viscous syrup. Some 5ꢁ-substituted
compound can be found in its ESI-MS detection, but it still had no trace
of the formation of 3ꢁ-substituted production. Apparently, under AcSH
acid condition, the 3ꢁ-position of anhydrocytidine had less activity than the
3ꢁ-position of anhydrothymidine and uridine, even than its 5ꢁ-position.
So the synthetic route had to be changed. Previous report showed that
the bond of C3ꢁ-O2 in anhydronucleosides can be breaked by OH− ion
exchange resin.[7] 9 was heated with Dowex 1×2, OH− ion exchange resin
on 50◦C in the solution of EtOH: H2O = 1:1 for 5 hours, and 10 was
obtained in 75% yield. The threo-conformation of 3ꢁ-hydroxyl group was
1
confirmed by NOE H-NMR spectra. 11 was obtained in a high yield by
treatment of 10 with 1.5 mol equiv. of MsCl. Then the 3ꢁ,5ꢁ-dimesyl groups
were readily substituted to thioacetyl groups by reacted with KSAc. With the
SN2 nucleophilic substitution, the 3ꢁ-S-acetyl group was reversed to a normal
conformation.
The deprotection step of 12 was quite similar with 5. Under N2 atmo-
sphere, the acetyl groups were rapidly removed by adding of the solution
of EtSNa in EtSH. However, the benozyl group was unexpectedly resisted
to the EtSNa/EtSH system without leaving. Amplified the usage amount of
EtSNa to 5 mol equiv., and also elongated the reaction time to 1hr. But he
benozyl group was still stable. So it was believed some stonger bases other
than EtSNa could remove the Benozyl group.
So many different properties of cytidine caused our strong curiosity.
First is the difference in preparation of 2ꢁ-deoxy-2,3ꢁ-anhydronucleoside.
The intramolecular ring of thymidine and uridine can be easily formed by
treating with Et3N/EtOH; while to cytidine, there was a stronger base DBU,
other than Et3N, can react. A proposed reaction mechanism (Figure 1)
accounts for it. In the process of the formation of 2,3ꢁ-anhydronucleosides,
base was used to deproton of –NH in the base group to form an enolate
intermediate. In thymidine and uridine, the –NH groups were connected
with two C = O groups, while in cytidine, the –NH group group was bonded
with a C = O and a C = N. So the proton’s acidity in cytidine was not so
strong as that in thymidine and uridine, which needed a stronger base such
as DBU to realize the deproton step.
Another difference has the reactive preference between 3ꢁ and 5ꢁ
position. It was clearly that anhydrothymidine and uridine had a significant

Study on Disulfur-Backboned Nucleic Acids
1275
FIGURE 1 The differences between 2ꢁ-deoxy-uridine and cytidine in the process of formating 2,3ꢁ-
anhydronnucleosides.
regioselectivity to nucleophilic reagents under acidic and basic conditions,
which was not seen to anhydrocytidine. Another mechanism was suggested
(Figure 2) to try to explain it. Firstly, it was believed that the 5ꢁ-position has
a stronger reactive activity than the 3ꢁ-position. So in basic condition, the
nucleophilic reagent prefered to choose the C5ꢁ atom to react. But when
in acidic condition, the nitrogen atom in base group prefered to accept
a proton to form an aminium cation. In anhydrothymidine and uridine,
it was the N3 atom to accept the proton. The positive charge on N3 can
be delocalized to O2 by resonance effection, and made a strong electron
attraction on C3ꢁ, by which the 3ꢁ-position was activated with a stronger
reactivity than 5ꢁ-position. While in anhydrocytidine, it was the N4 to accept
the proton. Because of the conjugation influence between N4 = C and C5
= C, the positive charge can be easily delocalized to the base ring, and
had less influence with the C3ꢁ. So the reactive activity of 3ꢁ-position in
anhydrocytidine still remained less than that of 5ꢁ-position.
FIGURE 2 The difference between 2,3ꢁ-anhydronuridine and cytidine in the process of protonation.

1276
P. Shang et al.
SCHEME 1 Synthetic route for 3ꢁ,5ꢁ-dithio-2ꢁ-deoxyuridine. a) MsCl, pyridine, 97%; b) Et3N, EtOH,
92%; c) AcSH, reflux, N2, 77%; d) KSAc, DMF, N2, 73%; e) KSAc, DMF, N2, 70%; f) AcSH, reflux, N2,
76%; g) EtSNa, EtSH, 85%.
SUMMARY AND CONCLUSIONS
Our work presented an efficient route to synthesize the 3ꢁ,5ꢁ-dithio-2ꢁ-
deoxyuridine (Scheme 1) and deoxycytidine (Scheme 2). The use of 2,3ꢁ-
anhydronucleosides as intermediate molecules provided a general method
for 3ꢁ conformation conversion for pyrimidine nucleosides’ modification.
The regioselectivity of 5ꢁ-mesyl-2,3ꢁ-anhydrothymidine and anhydrouridine
can be applied as a useful synthetic intermediate. By choosing basic or acidic
conditions, we can easily make selective substitution on 3ꢁ or 5ꢁ position
on sugar rings could easily made. These work should be an important
supplement to nucleoside modification. The further work to study the
properties of 3ꢁ,5ꢁ-dithio-2ꢁ-deoxynucleosides being carried on.
SCHEME 2 Synthetic route for 4-benzoyl-3ꢁ,5ꢁ-dithio-2ꢁ-deoxycytidine. a) 1) Me3SiCl, pyridine, BzCl; 2)
H2O, NH3•H2O, 96%; b) MsCl, pyridine, 92%; c) DBU, CH3CN, 87%; d) Dowex, 1×2, OH ion exchange
resin, EtOH/H2O, 75%; e) MsCl, pyridine, 91%; f) KSAc, DMF, N2, 70%; g) EtSNa, EtSH, N2, 80%.

Study on Disulfur-Backboned Nucleic Acids
1277
EXPERIMENTAL
General
All solvents were freshly distilled. DMF and pyridine were dried by
standard procedures. All solvents and reagents used can be obtained by
commercial sources. TLC analyses were carried out on silica gel F254
and the spots were examined with UV light or by exposure to vaporised
iodine. Column chromatography was carried out by using silica gel (100–200
mesh). Optical rotation data was obtained on Automatic Polarimeter
WZZ-2A. UVλmax and ε data was obtained on Ultrospec 4000 UV/Visible
Spectrophotometer. 1H NMR spectra were obtained on JOEL JNM-ECA600
and JOEL JNM-ECA300 spectrometers. Mass spectra were recorded with a
Bruker ESQUIRE-LC ion trap spectrometer equipped with a gas nebulizer
probe, capable of analyzing ions up to m/e 6000. The compound 2, 8 were
prepared by literature procedures.
5ꢁ-S-Acetyl-2,3ꢁ-anhydro-1-(2ꢁ-deoxy-β-D-xylofuranosyl) uracil 4
5ꢁ-O-mesyl-2,3ꢁ-anhydro-1-(2ꢁ-deoxy-β-D-xylofuranosyl) uracil 2 (576 mg,
2.00 mmol), potassium thioacetate (456 mg, 4 mmol) and dry DMF
(25 mL) were stirred together at 50◦C for 30 hours. The resulting solution
was evaporated in reduced pressure. The residual oil was dispersed by 50
mL CH2Cl2 and 50 mL H2O. The organic layer was then separated, washed
twice by two portions of 30 mL sodium bicarbonate, and then concentrated.
The residue was applied to column chromatography, which was eluted with
CH2Cl2-MeOH (98:2 to 90:10 v/v), yielding 390 mg (1.46 mmol, 73%) of
1
compound 4. H NMR (300 M Hz, DMSO-d6): δ 7.11 (d, J = 7.22 Hz, 1H,
6-H), 6.02 (d, J = 7.57 Hz, 1H, 5-H), 5.52 (d, J = 3.78 Hz, 1H, 1ꢁ-H), 5.19 (s,
1H, 3ꢁ-H), 4.33 (ddd, J = 2.06, 7.22, 7.22 Hz, 1H, 4ꢁ-H), 3.25 (dd, J = 7.22,
14.10 Hz, 1H, 5ꢁ-H), 3.03 (dd, J = 7.22, 14.10 Hz, 1H, 5ꢁ-H), 2.68 (d, J =
12.73 Hz, 1H, 2ꢁ-H), 2.49 (ddd, J = 3.44, 2.75, 12.73 Hz, 1H, 2ꢁ-H), 2.36 (s,
3H, Ac-Me). ESI-MS: m/e = 269 [M + H]+, 291 [M + Na]+, 307 [M + K]+.
S,Sꢁ-Diacetyl-3ꢁ,5ꢁ-dithio-2ꢁ-deoxyuridine 5
Method A
Compound 4 (270 mg, 1.00 mmol) was dissolved in 20 mL AcSH. The
reaction mixture was heated to reflux under nitrogen for 12 hours. The
solution was evaporated, extracted by 50 mL CH2Cl2 and 5 portions of
30 mL sodium bicarbonate. The organic layer was separated, dried, and
concentrated. The residual oil was applied to column chromatography
(eluted with petrol ether-EtOAc, firstly 80:20, then 30:70 v/v), to give 260
mg (0.76 mmol, 76%) of compound 5.

1278
Method B
P. Shang et al.
5ꢁ-O-mesyl-2,3ꢁ-anhydro-1-(2ꢁ-deoxy-β-D-xylofuranosyl) uracil 2 (576 mg,
2.00 mmol) was dissolved in 30 ml thioacetate acid under N2 atmosphere.
The reaction mixture was stirred, heated to refluxing temperature (100◦C)
for 15 hours. The extra AcSH was evaporated in reduced pressure. The
residue was added 50 mL CH2Cl2, and then washed 5 times by portions
of 30 mL sodium biocarbonate. The organic layer was seperated, dried
and concentrated. The residual oil was purified by a fast column chro-
matography, eluted with petrol ether-EtOAc (firstly 80:20, then 30:70 v/v).
Concentration of the appropriate fraction gave crude compound 3 as a
yellow solid. (560 mg, approximately in 77% yield). Compound 3 (360 mg,
1.00 mmol), potassium thioacetate (228 mg, 2 mmol) and 20 mL DMF was
stirred together at 50◦C for 32 hours. The extra solvent was removed in
reduced pressure. The residue was dissolved by 50 mL CH2Cl2 and washed
twice with 30 mL sodium bicarbonate. The CH2Cl2 layer was dried and
concentrated, purified by silica column chromatography, which was eluted
with CH2Cl2-methanol (98:2 to 95:5 v/v), yielding 239 mg (0.70 mmol, 70%)
of compound 5. [α]20 +74.5◦ (c 1.00, CH2Cl2). UV(CH2Cl2)λmax (ε) 313
D
1
nm (368). H NMR (300 MHz, CDCl3): δ 9.64 (s, 1H, -NH), 7.51 (d, J =
7.84 Hz, 1H, 6-H), 6.09 (s, 1H, 1ꢁ-H), 5.80 (d, J = 7.89 Hz, 1H, 5-H), 4.03 (s,
1H, 3ꢁ-H), 3.79 (m, 1H, 4ꢁ-H), 3.37 (dd, J = 3.44, 14.45 Hz, 1H, 5ꢁ-H), 3.24
(dd, J = 6.19, 14.45 Hz, 1H, 5ꢁ-H), 2.46 (m, 2H, 2ꢁ-H), 2.37 (s, 3H, Ac-Me),
2.36 (s, 3H, Ac-Me). ESI-MS: m/e = 367 [M + Na]+, 383 [M + K]+.
S,Sꢁ-3ꢁ,5ꢁ-Dithio-2ꢁ-deoxyuridine 6
The reaction system was operated under nitrogen protection. To a
stirred solution of compound 5 (103 mg, 0.30 mmol) in 15 mL EtSH was
added 50 mg (0.60 mmol) EtSNa. The reaction was taken place at room
temperature for 5 minutes. Then 0.1 N HCl solution was added to ajust the
pH to neutral. Evaporated the EtSH, extracted with 20 mL CH2Cl2 and 15
mL H2O. Concentrated the CH2Cl2 layer, the residual oil was poured into 20
mL cold ethyl ether. Collected the white precipitate, and washed with cold
ethyl ether, yielding 66 mg (0.254 mmol, 85%) compound 6. Compound 6
was soon kept in a case with nitrogen atomosphere at -20◦C. 1H NMR (300
MHz, DMSO-d6): δ 7.44 (d, J = 7.23 Hz, 1H, 6-H), δ 6.13 (d, J = 3.44 Hz,
1H, 1ꢁ-H), δ 5.83 (d, J = 7.22 Hz, 1H, 5-HH), δ 3.89 (s, 1H, 4ꢁ-H), δ 3.46
(m, 1H, 3ꢁ-H), δ 3.08 (m, 1H, 5ꢁ-H), 2.92 (m, 1H, 5ꢁ-H), δ 2.55–2.38 (m, 2H,
2ꢁH). ESI-MS: m/e = 283 [M + Na]+.
N4-Benzoyl-5ꢁ-O-mesyl-2,3ꢁ-anhydro-1-(2ꢁ-deoxy-β-D-xylofuranos-
yl) cytosine 9
To a stirred suspension of N4-benzoyl-3ꢁ,5ꢁ-di-O-dimesyl-2ꢁ-deoxycytidine
8 (2.85 g, 5.82 mmol) in dry CH3CN (50 mL) was added dropwise a solution

Study on Disulfur-Backboned Nucleic Acids
1279
of 1 mL (6.58 mmol) DBU in 5 mL CH3CN at room temperature Compound
8 was dissolved with adding of the DBU solution. The mixture was stirred for
2 hours. The solution was evaporated. The residual oil was extracted twice
with 50 mL sodium bicarbonate and 50 mL CH2Cl2. The organic layer was
dried and evaporated in reduced pressure to give 1.98 g (5.06 mmol, 87%)
crude product 9. A part of crude product was recrystallized in CH3CN to
1
give pure compound 9 for H NMR detectation. The main crude product
was directly used to prepare compound 10. 1H NMR (300 MHz, DMSO-d6):
δ 7.90 (d, J = 7.57 Hz, 2H, phenyl-H), 7.62 (d, J = 7.22 Hz, 1H, 6-H), 7.48 (t,
J = 7.57, 6.88 Hz, 1H, phenyl-H), 7.39 (t, J = 7.57, 7.22 Hz, 2H, phenyl-H),
6.41 (d, J = 7.22 Hz, 1H, 5-H), 5.94 (d, J = 3.44 Hz, 1H, 1ꢁ-H), 5.36 (s, 1H,
4ꢁ-H), 4.50–4.44 (m, 2H, 3ꢁ-H, 5ꢁ-H), 4.26–4.19 (dd, J = 7.57, 12.04 Hz, 1H,
5ꢁ-H), 3.14 (s, 3H, Ms-Me), 2.63 (d, J = 12.73 Hz, 1H, 2ꢁ-H), 2.53–2.46 (m,
1H, including the peak of DMSO, 2ꢁ-H). ESI-MS: m/e = 392 [M + H]+, 414
[M + Na]+, 430 [M + K]+.
N4-Benzoyl-1-(5ꢁ-O-mesyl-2ꢁ-deoxy-β-D-xylofuranosyl) cytosine 10
A solution of 9 (1.50 g, 3.84 mmol) in EtOH-H2O (1:1 v/v, 100 mL) was
heated to 50◦C. Ion exchange resin (Dowex 1×2, OH−) 20 g was added, and
the mixture was stirred at 50◦C for 5 hours. Then the resin was filtered off
an washed with H2O and EtOH, the combined filtrate was concentrated in
reduced pressure. The residual oil was applied to column chromatography,
eluted with CH2Cl2-MeOH (95:5 v/v) to give 1.18 g 10 (2.88 mmol, 75%).
1H NMR (600 MHz, DMSO-d6): δ 8.32 (d, J = 7.56 Hz, 1H, 6-H), 8.00 (d,
J = 7.56 Hz, 2H, phenyl-H), 7.63 (t, J = 7.56 Hz, 1H, phenyl-H), 7.52 (t, J
= 7.56 Hz, 2H, phenyl-H), 7.37 (d, J = 7.56 Hz, 1H, 5-H), 6.06 (d, J = 6.87
Hz, 1H, 1ꢁ-H), 4.62–4.60 (dd, J = 3.44, 11.00 Hz, 1H, 5ꢁ-H), 4.57–4.54 (dd,
J = 8.25, 11.00 Hz, 1H, 5ꢁ-H), 4.35 (t, J = 4.12 Hz, 1H, 3ꢁ-H), 4.29–4.27 (m,
J = 3.44, 6.87 Hz, 1H, 4ꢁ-H), 3.26 (s, 3H, Ms-Me), 2.65–2.61 (ddd, J = 4.81,
8.25, 14.43 Hz, 1H, 2ꢁ-H), 2.05 (d, J = 14.43 Hz, 1H, 2ꢁ-H). ESI-MS: m/e =
410 [M + H]+, 432 [M + Na]+.
N4-Benzoyl-1-(3ꢁ,5ꢁ-di-O-mesyl-2ꢁ-deoxy-β-D-xylofuranosyl)
cytosine 11
To a stirred suspension of 10 (1.23 g, 3.01 mmol) in dry pyridine (30
mL) was added a solution of 0.3 mL (3.90 mmol) MsCl in 3.0 mL pyridine
at 0◦C. When compound 10 was disappeared in TLC detection, 5 mL
ice-water was added to the mixture. Removed the solvent under reduced
pressure. The residual oil was poured into 100 mL ice-water. The precipitate
was collected, washed with ice water, yielding 1.32 g (2.74 mmol, 91%) of
1
compound 11. H NMR (300 MHz, CDCl3): δ 8.11 (d, J = 7.57 Hz, 1H,
6-H), 7.95 (d, J = 7.22 Hz, 2H, phenyl-H), 7.70–7.60 (m, 2H, phenyl-H),

1280
P. Shang et al.
7.51 (dd, J = 7.57, 7.22 Hz, 2H, phenyl-H), 6.24 (d, J = 5.85 Hz, 1H, 1ꢁ-H),
5.35 (m, 1H, 3ꢁ-H), 4.65–4.63 (m, 2H, 4ꢁ-H, 5ꢁ-H), 4.55–4.50 (m, 1H, 5ꢁ-H),
3.13 (s, 3H, Ms-Me), 3.03 (s, 3H, Ms-Me), 2.99–2.91 (m, 1H, 2ꢁ-H), 2.72 (d,
J = 15.82 Hz, 1H, 2ꢁ-H). ESI-MS: m/e = 488 [M + H]+, 510 [M + Na]+.
N4-Benzoyl-S,Sꢁ-diacetyl-3ꢁ,5ꢁ-dithio-2ꢁ-deoxycytidine 12
Compound 11 (1.0 g, 2.05 mmol), potassium thioacetate (935 mg, 8.2
mmol) and 30 mL DMF was stirred together at 50◦C under N2 atomosphere
for 24 hours. The potassium salt was removed by filtration. The filtrate was
evaporated to dry under reduced pressure. The residue was dissolved in
50 mL CH2Cl2, and was washed twice by 30 mL sodium bicarbonate. The
organic layer was dried and concentrated, then was applied to a column
chromatography, which was eluted with CH2Cl2-MeOH (98:2 to 95:5 v/v),
yielding 640 mg (1.43 mmol, 69.8%) compound 12. [α]20 +36.2◦ (c 1.00,
D
1
CH2Cl2). UV(CH2Cl2)λmax (ε) 341 (669), 322 nm (618). H NMR (300
MHz, CDCl3): δ 8.88 (s, 1H, -NH), 8.16 (d, J = 7.57 Hz, 1H, 6-H), 7.91 (d,
J = 7.57 Hz, 2H, phenyl-H), 7.61 (t, J = 7.22 Hz, 2H, phenyl-H), 7.54–7.49
(m, 2H, 5-H, phenyl-H), 6.08 (dd, J = 4.13, 6.54 Hz, 1H, 1ꢁ-H), 4.12 (ddd, J
= 4.13, 6.19, 8.26 Hz, 1H, 4ꢁ-H), 3.72 (d, J = 8.60 Hz, 1H, 3ꢁ-H), 3.41–3.27
(m, J = 4.13, 6.54, 14.79 Hz, 2H, 5ꢁ-H), 2.68–2.55 (m, 2H, 5ꢁ-H), 2.40 (s, 3
H, Ac-Me), 2.36 (s, 3 H, AC-Me). ESI-MS: m/e = 448 [M + H]+, 470 [M +
Na]+, 486 [M + K]+.
N4-Benzoyl-S,Sꢁ-3ꢁ,5ꢁ-dithio-2ꢁ-deoxycytidine 13
To a solution of compound 12 (100 mg, 0.224 mmol) in 15 mL
EtSH was added 68 mg (0.809 mmol) EtSNa under N2 atmosphere at
room temperature The compound 12 disappeared in 2 minutes. by TLC
detection. 0.1 N HCl was added dropwise to adjust the pH of the solution
to neutral. Evaporated the solvent, and extracted the residue with 15 mL
CH2Cl2 and 15 mL H2O rapidly. A concentration of the CH2Cl2 layer and
20 mL cold Et2O was added. The white sediment was collected to yield 65
mg (0.179 mmol, 80.0%) pure compound 13. Compound 13 was then kept
1
under nitrogen protection at −20◦C. H NMR (300 MHz, CDCl3): δ 8.98
(s, 1H, -NH), 8.26 (d, J = 7.57 Hz, 1H, 6-H), 7.90 (d, J = 7.56 Hz, 2H,
phenyl-H), 7.62–7.50 (m, 2H, 5-H, phenyl-H), 7.49 (t, J = 7.56 Hz, 2H,
phenyl-H), 6.13 (dd, J = 2.75, 6.54 Hz, 1H, 1ꢁ-H), 4.00–3.95 (m, 1H, 4ꢁ-H),
3.25–3.20 (m, 1H, 3ꢁ-H), 3.15–2.93 (m, 2H, 5ꢁ-H), 2.68–2.50 (m, 2H, 2ꢁ-H).
ESI-MS: m/e = 364 [M + H]+, 386 [M + Na]+.
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