Exploring the synthesis of masked phosphoramido 6-vinylcytidine derivatives as building blocks for cross-linking oligonucleotides

Article abstract of DOI:10.3987/com-06-s(o)3

In the present work the synthesis of building blocks (2a,b), bearing a masked form of the reactive vinyl group bound to the C-6 position of a cytidine derivative, has been studied. The understanding of byproduct formation together with conformational considerations let us to plan a straightforward approach for the synthesis of the target compounds.

Full text of DOI:10.3987/com-06-s(o)3

HETEROCYCLES, Vol. 69, 2006
151
HETEROCYCLES, Vol. 69, 2006, pp. 151 - 166. © The Japan Institute of Heterocyclic Chemistry
Received, 13th January, 2006, Accepted, 28th February, 2006, Published online, 3rd March, 2006. COM-06-S(O)3
EXPLORING THE SYNTHESIS OF MASKED PHOSPHORAMIDO
6-VINYLCYTIDINE DERIVATIVES AS BUILDING BLOCKS FOR
CROSS-LINKING OLIGONUCLEOTIDES
Marco Radi, Raffaella Spinosa, Maria Cristina Parlato, Federico Corelli,
and Maurizio Botta*
Department of Medicinal Chemistry, University of Siena, Via Aldo Moro, 53100
Siena, Italy. *Corresponding author: botta@unisi.it
Abstract –In the present work the synthesis of building blocks (2a,b), bearing a
masked form of the reactive vinyl group bound to the C-6 position of a cytidine
derivative, has been studied. The understanding of byproduct formation together
with conformational considerations let us to plan a straightforward approach for the
synthesis of the target compounds.
INTRODUCTION
After the first example of oligonucleotides application as Rous Sarcoma Virus (RSV) inhibitors in 1978,1
a large number of antisense oligonucleotides are actually in preclinical and clinical trials mainly in the
cancer, cardiovascular and infectious disease fields.2 However, conventional, unmodified
oligonucleotides have drawbacks for their practical use, due to their unsatisfactory binding affinity to the
target sequence, the instability against cellular nucleases, the insufficient membrane penetration and the
low bioavailability.3 The function of oligonucleotides, either modified or unmodified, is passive, in that
they can dissociate from the complex and so their inhibitory effect is transient. For that reason, many
structural modifications have been brought so far to this class of compounds in order to improve their
binding affinity to the target sequence and consequently their pharmacological profile.4 In particular,
oligonucleotides bearing a reactive group have been regarded as promising candidates for irreversible
damage of the target sequence on account of their ability to cause an interstrand cross-linking reaction.5-7
On the basis of the interesting Michael acceptor properties showed by some 6-vinylpirimidine derivatives
synthesized in our group8 and on the work developed by Sasaki,9 the synthesis and evaluation of

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alkylating properties of cytidine derivative (1) (Fig. 1) has been recently reported.10 The vinyl group in
C-6 position constrains this molecule into a non-natural syn conformation11,12
:
NHBz
NHBz
N
N
O
N
O
N
SEt
DMTrO
NC
TBDMSO
O
O
O
O P
N
R
TBDMSO
O
2a; R = H
2b; R = OMOE
OMe
1
Figure 1
as a result a cross-linking reaction between the vinyl group of 1 and the amino group of a guanosine in the
target sequence may likely occur, thus enhancing the stability of the resulting duplex (Fig. 2). Moreover,
a masking strategy was developed in order to protect the reactive vinyl moiety of the monomer before its
introduction into the oligonucleotide sequence and to release subsequently this reactive
moiety.10Accordingly, the present work was focused on the synthesis of the active phosforamido
derivatives (2a,b) in order to subsequently introduce these two monomers into a specific oligonucleotide
sequence and to investigate the role played by the C-6 and C-2’ substituents in the duplex stabilization
and nuclease resistance.
Syn
conformation
Anti
conformation
N
O
NH2
O
O
N
O
N
HN
H2N
N
O
R
N
Target
sequence
Antisense
oligonucleotide
Figure 2
RESULTS AND DISCUSSION
In order to synthesize the target compound (2b), the reactive vinyl group in compound (1) was initially
masked as ethylthio derivative by reaction with ethanethiol in dichloromethane and the intermediate
obtained was then deprotected with tetrabutylammonium fluoride to give compound (3) in good yield
(Scheme 1). However, selective protection of the primary hydroxy group in 3 proved to be a difficult task.

HETEROCYCLES, Vol. 69, 2006
153
Reacting in fact compound (3) with dimethoxytrityl chloride either under classical, high temperature or
microwave-assisted conditions, the expected derivative (4) was never isolated.
NHBz
NHBz
NHBz
N
N
N
O
N
O
N
SEt
O
N
SEt
ii.
i.
TBDMSO
HO
DMTrO
O
O
O
TBDMS O
O
OH
O
OH
O
OMe
OMe
OMe
3
1
4
iii.
iv.
NHBz
N
NHBz
N
N
N
O
SEt
O
SEt
HO
TBDPSO
O
O
TBDPSO
O
HO
O
OMe
OMe
5
6
Scheme 1 Reagents and conditions: i. (a) EtSH, CH2Cl2, rt 2h; (b) TBAF, THF, rt 1h; ii. DMTrCl, pyridine, rt overnight; iii.
TBDPSCl, CH2Cl2, DMAP, Et3N, rt 48h; iv. TBDPSCl, CH2Cl2, DMAP, Et3N, MW, 60 °C, 1h.
The introduction of a different selective protecting group in 5' position was then investigated by reacting
compound (3) with tert-butyldiphenylsilyl chloride in the presence of dimethylaminopyridine and
triethylamine at room temperature, but the expected compound (5) was never obtained. However, running
the same reaction at 60°C under microwave assisted condition, the bulky tert-butyldiphenylsilyl moiety
was introduced on the secondary hydroxy group of 3 and compound (6) was obtained as the only product.
On the basis of the extensive literature reports12,13 we assumed that the unsuccessful tritylation of 3 and
the formation of compound (6) could be related to the formation of a strong hydrogen bond between the
carbonyl group of the nucleobase and the 5'-hydroxy group of the ribose moiety .As a consequence of this
hypothesis, we considered the formation of this strong hydrogen bond as an unconventional way to
protect the 5'-hydrox y group and the direct introduction of the phosphoramidite moiety on the secondary
hydroxy group was therefore investigated. Compound (3) was therefore directly reacted with
2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphoramidite in the presence of N,N-diisopropylammonium
tetrazole but no reaction occurred (Scheme 2). We tried therefore to perform the same reaction increasing
the temperature up to 70 °C under microwave irradiation obtaining a complex mixture with only traces of

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HETEROCYCLES, Vol. 69, 2006
the expected phosphoramidite (7) while, increasing the temperature up to 100 °C in a sealed tube, the
cyclic phosphite (8) was obtained as major reaction product.
NHBz
N
O
N
SEt
HO
(a)
O
(b)
NC
O
O
NHBz
N
OMe
O P
N
N
7
O
SEt
i
HO
O
NHBz
N
N
HO
O
OMe
3
O
SEt
O
P
(c)
O
NC
O
O
O
OMe
8
Scheme 2. Reagents and conditions: i. 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphoramidite, N,N-diisopropylammonium
tetrazole, CH3CN, (a: rt overnight; b: MW 70°C, 12 min; c: MW 100°C, sealed tube 12 min.)
It is well known how the complete definition of the conformation of a nucleoside is a complex subject. A
simple modification of the 2'-position with an electronegative substituent is in fact able to confer a north
conformation to the sugar moiety (3T2) while the 2'-deoxy derivatives possess a south conformation
(2T3).14 In order to investigate the relationship between the sugar conformation and the formation of the
intramolecular hydrogen bond, the 2'-deoxy analogue (13) of compound (3) was synthesized starting from
commercially available 2'-deoxycytidine (Scheme 3). Following the same synthetic approach used for the
synthesis of 3, compound (13) was easily obtained; however, the selective protection of the primary
hydroxy group proved again to be problematic. Since the failure of the 5'-hydroxy protection seems to be
exclusively related to the unnatural syn conformation imposed by the presence of a C-6 substituent, we
planned to introduce the 5'-protecting group first and to functionalize the C-6 position in a late stage. A
one pot procedure was used for the synthesis of the trityl derivative (16a) starting from 2’-deoxycytidine
(15a) which was first submitted to N4-benzoylation with benzoic anhydride in the presence of pyridine15
and after 1h at 90 ºC, treated with dimethoxytrityl chloride for 10 h to give 16a in good overall yield.
Compound (16a) was then protected with tert-butyldimethylsilyl chloride to give the fully protected

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155
derivative (17a) in quantitative yield. For the subsequent introduction of the 6-vinyl group, many
procedures were investigated
NHBz
N
NHBz
N
NHBz
NH2
N
N
N
N
N
O
O
O
N
CHO
O
TBDMSO
TBDMSO
TBDMSO
HO
iii.
ii.
i.
O
O
O
O
TBDMSO
TBDMSO
TBDMSO
OH
15a
10
9
11
NHBz
N
NHBz
N
NHBz
N
N
N
N
O
SEt
O
SEt
O
SEt
TBDMSO
DMTrO
HO
v.
iv.
vi.
O
O
O
TBDMSO
OH
OH
13
14
12
Scheme 3 Reagents and conditions: i. (a) TBDMSCl, imidazole, DMF, rt, 4h; (b) benzoic anhydride, DMF, MW, 160 °C. 15
min.; ii. (a) LDA, THF dry, -78 °C, 3h; (b) HCOOMe, -78 °C, 3h; iii. KHMDS, CH3P(C6H5)3Br, THF dry, from -78 to 0°C,
2h; iv. EtSH, CH2Cl2, rt, 2h; v. TBAF, THF dry, rt, 4h; vi. DMTrCl, Py, rt, overnight.
but, at last, the Wittig reaction on the 6-formyl derivative (18a) proved to give the best results.8
Accordingly, compound (17a) was first lithiated16 and then reacted with methyl formate to yield the
6-formyl derivative (18a) as a mixture (starting material still present) which was very difficult to purify
using common chromatographic techniques. Therefore, after a simple filtration of the crude on silica gel,
the 40:60 mixture of compounds (17a and 18a) was reacted with the suitable Wittig reagent to give the
6-vinyl derivative (19a). Unreacted 17a could be recovered at this stage from the reaction mixture and
reused in the formylation reaction to increase the overall yield of 19a. The vinyl group of compounds 19a
was then masked by reaction with ethanethiol in dichloromethane to give the corresponding
ethanethioethyl derivative which was subsequently deprotected with tetrabutylammonium fluoride to give
compound (20a). The latter compound was finally transformed into the target phosphoramidite monomer
(2a) by reaction with 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphoramidite at room temperature.
Following the same synthetic approach used for the synthesis of 2a, the 2'-MOE derivative (2b) was
obtained starting from 15b. The incorporation of these active monomers into a specific oligonucleotide
sequence is at present under study.
CONCLUSION

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HETEROCYCLES, Vol. 69, 2006
In the presentwork, thesynthesisofmasked phosphoramido 6-vinyl cytidine derivatives (2a,b) was studied.
It was shown how the failure of the 5'-hydroxy protection in compound (3) seems to originate from the
NHBz
N
NHBz
N
NH2
N
N
O
N
O
N
O
i.
ii.
DMTrO
DMTrO
HO
O
O
O
OH
R
TBDMSO
17 a,b
OH
15 a,b
R
R
16 a,b
NHBz
N
NHBz
N
NHBz
N
N
N
O
CHO
O
O
N
SEt
DMTrO
DMTrO
DMTrO
iii.
v.
iv.
O
O
O
TBDMSO
18 a,b
TBDMSO
R
R
OH
20 a,b
R
19 a,b
NHBz
N
N
O
SEt
DMTrO
a: R = H
b: R = OMOE
vi.
O
O
R
NC
O P
N
2 a,b
Scheme 4. i. (a) Benzoic anhydride, Pyr, 90°C, 1h; (b) DMTrCl, rt, 10h; ii. TBDMSCl, imidazole, DMF, rt, 4h; iii. (a) LDA,
THF dry, -78°C, 3h; (b) HCOOMe, -78°C, 3h; iv. KHMDS, CH3P(C6H5)3Br, THF dry, from -78 to 0°C, 2h; v. (a) EtSH, CH2Cl2,
rt, 2h; (b) TBAF, THF dry, rt, 1h; vi. 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphoramidite, diisopropylammonium tetrazolide,
CH3CN, rt, 4h.
formation of a strong intramolecular hydrogen bond as a consequence of the unnatural syn conformation
imposed by the presence of a C-6 substituent. In order to get round this stumbling block, the early
introduction of the trityl protecting group on the sugar moiety and the C-6 functionalization of the
nucleobase in a late stage allowed the obtainment of the activated monomers (2a,b) for the subsequent
introduction into a specific oligonucleotide sequence.

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ACKNOWLEDGEMENTS
This study was partially supported by grants from the European TRIoH Consortium
(LSHB-2003-503480).
EXPERIMENTAL
General Considerations. Reagents were obtained from commercial suppliers and used without further
1
purifications. Solvents were dried using standard procedures. H NMR spectra were measured at 200
MHz with a Bruker AC 200F spectrometer. Chemical shifts are reported relative to TMS at 0.00 ppm. EI
low-resolution MS spectra were recorded with an electron beam of 70 eV (GC-MS Varian Saturn 3 3400
CX). Elemental analyses were carried out on a Perkin-Elmer Elemental Analyzer 240L. Infrared spectra
were recorded on a Perkin-Elmer 1600 FT-IR spectrophotometer. Microwave reactions were conducted
using a CEM Discover Synthesis Unit (CEM Corp., Matthews, NC) consisting of a continuous focused
microwave power delivery system with operator-selectable power output from 0 to 300W. The reactions
were performed in 10 mL sealed vessel. The temperature of the contents of the vessel was monitored
using a calibrated infrared temperature control mounted under the reaction vessel. All experiments were
performed using a stirring option whereby the contents of the vessel were stirred by means of a rotating
magnetic plate located below the floor of the microwave cavity and a Teflon-coated magnetic stir bar in
the vessel.
N4-Benzoyl-2'-O-(2methoxyethyl)-6-{1-[2-(ethylthio)]ethyl}cytidine (3).
To a solution of N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-2'-O-(2methoxyethyl)-6-vinylcytidine
(1) (556 mg, 0.84 mmol) in CH2Cl2 (20 mL), ethanethiol ( 125 µL, 1.69 mmol) was added and the
mixture was stirred at rt for 1h. After evaporation of the solvent under reduced pressure, the resulting
residue was dried under high vacuum for 2 h, dissolved in anhydrous THF (20 mL) and treated with
TBAF (2.85 mL, 2.84 mmol) at rt. After stirring for 4 h, the solvent was removed in vacuo and the
residue was purified by flash chromatography (CHCl3:MeOH 98:2 v/v) to give pure compound (3) (345
1
mg, 83%). H-NMR (200 MHz, CDCl3): δ 1.28 (t, J = 7.7 Hz, 3H,), 2.60 (q, J = 7.7 Hz, 2H), 2.80-3.12
(m, 4H), 3.37 (s, 3H), 3.40-3.95 (m, 6H), 4.20 (m, 1H), 4.52 (m, 1H), 5.08 (t, J2'-3' = 5.4 Hz), 5.72 (d, J1'-2'
13
= 4.2 Hz), 7.40-7.70 (m, 4H), 7.82-7.97 (m, 2H); C-NMR (200 MHz, CDCl3): 14.7, 26.2, 34.8, 58.9,
63.2, 69.9, 70.3, 71.6, 78.2, 85.6, 89.8, 127.6, 129.1, 133.4; ESI-MS m/z: 516 (M+Na)+; Anal. Calcd for
C23H31N3O7S: C, 55.98; H, 6.29; N, 8.52. Found: C, 56.00; H, 6.20; N, 8.48.
N4-Benzoyl-2'-O-(2-methoxyethyl)-3'-O-(tert-butyldiphenylsilyl)-6-{1-[2-(ethylthio)]ethyl}cytidine
(6).

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N4-Benzoyl-2'-O-(2-methoxyethyl)-6-{1-[2-(ethylthio)]ethyl}cytidine (3) (124 mg, 0.25 mmol) was dried
over P2O5 in vacuo, dissolved in anhydrous CH2Cl2 (3 mL) and transferred in a microwave sealed tube.
DMAP (catalytic amount), triethylamine (241 µL, 1.5 mmol) and tert-butyldiphenylsilyl chloride (150 µL,
0.50 mmol) were added and the reaction was irradiated in the microwave at 60 °C for 10 min. The
mixture was then diluted with CH2Cl2 (10 mL) and washed successively with water, brine and dried over
anhydrous Na2SO4. Evaporation of the solvent gave a crude oil, which was purified on a silica gel column
(CHCl3:MeOH, 98:2 v/v) to give pure compound (6) (119 mg, 65%). 1H-NMR (200 MHz, CDCl3): δ 1.04
(m, 9H), 1.24 (t, J = 7.7 Hz, 3H), 2.59 (q, J = 7.7 Hz, 2H), 2.80-3.15 (m, 4H), 3.35 (s, 3H), 3.40-4.30 (m,
7H), 4.83 (m, 1H), 5.28 (m, 1H), 5.62 (d, J1'-2' = 3.2 Hz, 1H), 7.25-8.10 (m, 16H); ESI-MS m/z:
732(M+H)+; 754 (M+Na)+; Anal. Calcd for C39H49N3O7SiS: C, 64.02; H, 6.70; N, 5.75 Found: C, 64.05;
H, 6.81; N, 5.79.
N4-Benzoyl-2'-O-(2-methoxyethyl)-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-6-{1-[2-
(ethylthio)]ethyl}cytidine (7).
N4-Benzoyl-2'-O-(2-methoxyethyl)-6-{1-[2-(ethylthio)]ethyl}cytidine (3) (15.5 mg, 0.031 mmol) was
mixed with diisopropylamine tetrazolide (3.6 mg, 0.02 mmol) and dried over P2O5 in vacuo overnight.
The resulting mixture was dissolved in anhydrous CH3CN (5 mL) and 2-cyanoehtyl-N,N,N',N'-
tetraisopropylphosphorodiamidite (15 µL, 0.047 mmol) was added. The reaction mixture was irradiated
into the microwave (open vessel condition) under an argon atmosphere at 70 ºC for 12 min, followed by
removal of the solvent in a vacuum. EtOAc (10 mL) was added to the residue, and it was washed with 5%
aqueous NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The resulting residue was purified by preparative TLC
(CHCl3:MeOH, 9:1 v/v) to give trace of pure compound (7). 1H-NMR (200 MHz, CDCl3): δ 1.04-1.35 (m,
15H,), 2.58 (q, J = 7.7 Hz, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.85-3.18 (m, 4H), 3.32 (s, 3H), 3.43-3.98 (m,
10H), 4.20 (m, 1H), 4.51 (m, 1H), 5.08 (t, J2'-3' = 5.4 Hz, 1H), 5.72 (d, J1'-2' = 4.2 Hz, 1H), 7.40-7.70 (m,
4H), 7.82-7.97 (m, 2H); ESI-MS m/z: 732(M+K)+; Anal. Calcd for C32H48N5O8PS: C, 55.40; H, 6.97; N,
10.09 Found: C, 55.55; H, 6.90; N, 10.15.
N4-Benzoyl-2'-O-(2-methoxyethyl)-3',5'-O-(2-cyanoethyl phosphite)-6-{1-[2-(ethylthio)]ethyl}-
cytidine (8).
N4-Benzoyl-2'-O-(2-methoxyethyl)-6-{1-[2-(ethylthio)]ethyl}cytidine (3) (20 mg, 0.04 mmol) was mixed
with diisopropylamine tetrazolide (4.5 mg, 0.03 mmol) and dried over P2O5 in vacuo overnight. The
resulting mixture was dissolved in anhydrous CH3CN (5 mL) and 2-cyanoehtyl-N,N,N',N'-
tetraisopropylphosphorodiamidite (19 µL, 0.06 mmol) was added. The reaction mixture was irradiated

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159
into the microwave (sealed tube) under an argon atmosphere at 100 °C for 10 min, followed by removal
of the solvent in a vacuum. EtOAc (10 mL) was added to the residue, and it was washed with 5% aqueous
NaHCO3 (10mL) and brine (10mL). The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The resulting residue was purified by preparative TLC
(CHCl3:MeOH, 95:5 v/v) to give pure compound (8) (7 mg, 30%). 1H-NMR (200 MHz, CDCl3): δ 1.28 (t,
J = 7.7 Hz, 3H,), 2.60 (q, J = 7.7 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H), 2.82-3.12 (m, 4H), 3.30 (s, 3H),
3.40-4.55 (m, 9H), 4.72 (m, 1H), 5.08 (m, 1H), 5.50 (s, 1H), 7.40-7.70 (m, 4H), 7.82-7.97 (m, 2H);
ESI-MS m/z: 593 (M+H)+, 615 (M+Na)+; Anal. Calcd for C26H33N4O8PS: C, 52.70; H, 5.61; N, 9.45.
Found: C, 52.78; H, 5.71; N, 9.46.
N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-2'-deoxycytidine (9).
A suspension of 2'-deoxycytidine monohydrate (660 mg, 2.69 mmol), tert-buthylchlorodimethylsilane
(1.86 g, 12.37 mmol) and imidazole (842 mg, 12.37 mmol) in anhydrous DMF was stirred for 4 h at rt,
then diluted with EtOAc (100 mL).The separated organic layer was washed successively with H2O and
brine then dried over anhydrous Na2SO4. Evaporation of the solvent gave a crude oil, which was dried
under vacuum for 2 h and then dissolved in anhydrous DMF (10 mL). To this solution, benzoic anhydride
(1.22 g, 5.38 mmol) was added and the resulting mixture was irradiated into the microwave for 15 min at
160 °C, then diluted with EtOAc (10 mL) and washed successively with water, brine and finally dried
over anhydrous Na2SO4. Evaporation of the solvent gave a crude oil, which was purified on a silica gel
column (CHCl3:MeOH, 98:2 v/v) to give pure compound (9) (1.5 g, 99%). 1H-NMR (200 MHz, CDCl3):
δ 0.11, 0.13 (12H, each as s, 2×SiMe2), 0.87, 0.93 (18H, each as s, 2×SitBu), 2.02-2.25 (m, 1H), 2.42-2.68
(m, 1H), 3.65-4.10 (m, 3H), 4.31-4.50 (m, 1H), 6.25 (t, J = 5.6 Hz, 1H), 7.35-7.70 (m, 5H), 7.88 (d, J =
7.4 Hz, 1H), 8.41 (d, J = 7.4 Hz, 1H); 13C-NMR (200 MHz, CDCl3): -5.5, -5.4, -4.9, -4.6, 17.9, 18.4, 25.7,
25.9, 42.4, 61.8, 70.1, 86.9, 87.9, 96.3, 127.5, 129.0, 133.1, 144.8; HPLC reverse phase, zorbax C8
column, Eluent: CH3CN 95%, H2O 5%, flow 0.4 mL/min; retention time 9.29 min; ESI-MS m/z: 560
(M+ H)+; Anal. Calcd for C28H45N3O5Si2: C, 60.07; H, 8.10; N, 7.51. Found: C, 60.03; H, 8.11; N, 7.50.
N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-6-formyl-2'-deoxycytidine (10).
A solution of N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-2'-deoxycytidine (9) (1.03 g, 1.85 mmol) in
anhydrous THF (20 mL) was added dropwise to a -78 °C solution of freshly prepared LDA (11.10 mmol)
in anhydrous THF (20 mL) at -78 °C under a positive pressure of argon. After 4 h at -78 °C, HCOOMe
(1.14 mL, 18.50 mmol) was added, and the reaction mixture was kept under magnetic stirring at -78°C for
3 h. The mixture was then allowed to warm to rt, diluted with EtOAc and quenched by the addition of

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saturated NH4Cl solution. The organic phase was washed successively with water and brine then dried
over anhydrous Na2SO4. Evaporation of the solvent gave a crude white foam which was used in the next
Wittig reaction without further purification. HPLC reverse phase, zorbax C8 column, Eluent: CH3CN
95%, H2O 5%, flow 0.4 mL/min; retention time 6.65 min ESI-MS m/z 606 (M+H2O+H)+, 628
(M+H2O+Na)+; retention time 8.72 min ESI-MS m/z: 588 (M+ H)+, 610 (M+ Na)+.
N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-6-vinyl-2'-deoxycytidine (11).
A hexane solution of BuLi (1.6M, 5.7 mL, 9.11 mmol) was added to a suspension of
methyltriphenylphosphonium bromide (3.25g, 9.11 mmol) in THF (20 mL) at -78°C under a positive
pressure of argon. The mixture was stirred for 30 min at 0 °C, followed by addition of a solution of
N4-benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-6-formyl-2'-deoxycytidine (10) (1.07 g, 1.82 mmol) in
THF (30 mL). The reaction mixture was stirred for 1 h at rt. A saturated NH4Cl solution was added to the
mixture, which was extracted with EtOAc (30mL). The organic phase was washed successively with
water and brine then dried over anhydrous Na2SO4. Evaporation of the solvent gave a crude oil, which
was purified on a silica gel column (CHCl3:MeOH, 98:2 v/v) to give pure compound (11) (798mg, 75%).
1H-NMR (200 MHz, CDCl3): δ 0.05 and 0.07 (12H, each as s, 2×SiMe2), 0.87 and 0.90 (18H, each as s,
2×SitBu), 1.95-2.20 (m, 1H), 2.60-2.85 (m, 1H), 3.70-4.05 (m, 3H), 4.52-4.70 (m, 1H), 5.63 (d, J cis
=
11.2 Hz, 1H), 5.98 (d, J trans = 17.1 Hz, 1H), 6.51 (t, J = 7.3 Hz, 1H), 7.13 (dd, J cis = 11.2 Hz, J trans = 17.1
Hz, 1H), 7.32-7.70 (m, 5H), 7.82-8.00 (m, 1H); 13C-NMR (200 MHz, CDCl3): -5.4, -5.3, -4.8, -4.6, 17.9,
18.5, 25.7, 26.0, 38.7, 62.2, 70.9, 85.6, 79.8, 87.1, 124.3, 127.6, 129.0, 130.6, 133.1; HPLC reverse phase,
zorbax C8 column, Eluent: MeOH 95%, H2O 5%, flow 0.8 mL/min; retention time 4.67 min; ESI-MS
m/z 608 (M +Na)+; Anal. Calcd for C30H47N3O5Si2: C, 61.50; H, 8.09; N, 7.17. Found: C, 61.42; H, 8.15;
N, 7.20.
N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-6-{1-[2-(ethylthio)]ethyl}-2'-deoxycytidine (12).
To a solution of N4-Benzoyl-3',5'-bis-O-(tert-butyldimethylsilyl)-6-vinyl-2'deoxycytidine (11) (171 mg,
0.292 mmol) in CH2Cl2 (10 mL), ethanethiol (44 µL, 0.585 mmol) was added and the mixture was stirred
at rt for 1 h. After evaporation of the solvent under reduced pressure, the resulting residue was purified on
a silica gel column (CHCl3:MeOH, 98:2 v/v) to give pure compound (12) (161 mg, 85%). 1H-NMR (200
MHz, CDCl3): δ 0.03 and 0.06 (12H, each as s, 2×SiMe2), 0.87 (18H, each as s, 2×SitBu), 1.26 (t, J = 7.7
Hz, 3H), 1.92-2.25 (m, 2H), 2.57 (q, J = 7.7 Hz, 2H), 2.70-3.15 (m, 4H), 3.70-4.05 (m, 3H), 4.50-4.70 (m,
1H), 6.15 (t, J = 6.4 Hz, 1H), 7.38-7.70 (m, 5H), 7.80-7.95 (m, 1H); 13C-NMR (200 MHz, CDCl3): 14.6,
26.2, 34.4, 38.2, 62.0, 71.1, 86.8, 88.3, 127.8, 128.8, 132.8; ESI-MS m/z: 670 (M+Na)+; Anal. Calcd for

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161
C32H53N3O5SSi2: C, 59.31; H, 8.24; N, 6.48. Found: C, 59.37; H, 8.16; N, 6.39.
N4-Benzoyl-6-{1-[2-(ethylthio)]ethyl}-2'-deoxycytidine (13).
TBAF (3.84 mL, 3.84 mmol) was added to a solution of N4-benzoyl- 3',5'-bis-O-(tert-butyldimethylsilyl-
6-{1-[2-(ethylthio)]ethyl}-2'-deoxycytidine (12) (620 mg, 0.96 mmol) in THF (20 mL) at rt. After the
solution was stirred for 4 h, the solvent was removed in vacuo and the residue was purified by flash
1
chromatography (CHCl3:MeOH 98:2 v/v) to give pure compound (13) (318 mg, 79%). H-NMR (200
MHz, CDCl3): δ 1.24 (t, J = 7.7 Hz, 3H), 1.55-1.85 (m, 1H), 2.08-2.32 (m, 1H), 2.56 (q, J = 7.7 Hz, 2H),
2.68-3.20 (m, 4H), 3.72-4.20 (m, 3H), 4.85-5.10 (m, 1H), 6.13 (t, J = 6.9 Hz, 1H), 7.35-7.70 (m, 5H),
13
7.82-8.05 (m, 1H); C-NMR (200 MHz, CDCl3): 14.6, 26.2, 34.4, 38.2, 62.0, 71.1, 86.8, 88.3, 127.8,
128.8, 132.8; ESI-MS m/z: 442 (M+Na)+; Anal. Calcd for C20H25N3O5S: C, 57.26; H, 6.01; N, 10.02.
Found: C, 57.26; H, 6.07; N, 10.00.
General procedure for 16 a,b.
Benzoic anhydride (166 mg, 0.73 mmol) was added to a solution of 15a or 15b (0.66 mmol) in anhydrous
pyridine. The mixture was stirred at 90 °C; after 1 h, the reaction was cooled to rt and dimethoxytrityl
chloride (248 mg, 0.73 mmol) was added. The reaction was stirred at rt for 10 h then methanol (10 mL)
was added and the resulting solution evaporated to dryness under reduced. The residue was purified on a
silica gel column (CHCl3:MeOH, 97:3 v/v) to give pure compound (16a or 16b).
5’-Dimethoxytrityl-2’-deoxycytidine (16a).
1H-NMR spectrum was identical with that of an authentic sample of 16a.17
ESI-MS m/z: 634 (M+H)+; Yield: 70%; Anal. Calcd for C37H35N3O7: C, 70.13; H, 5.57; N, 6.63.
Found: C, 70.22; H, 5.64; N, 6.68.
5'-O-Dimetoxytrityl-2'-O-(2methoxyethyl)-cytidine (16b).
1H-NMR (200 MHz, CDCl3): δ 3.35 (s, 3H), 3.43-3.68 (m, 3H), 3.72-4.02 (m, 7H), 4.02-4.32 (m, 3H),
4.35-4.53 (m, 2H), 5.95 (m, 1H), 6.75-6.98 (m, 3H), 7.15-7.68 (m, 15H), 7.88 (d, J = 7.4 Hz, 1H), 8.55 (d,
13
J = 7.4 Hz, 1H); C-NMR (200 MHz, CDCl3): 55.19, 58.81, 60.91, 68.01, 70.28, 71.51, 83.11, 86.97,
89.42, 96.53, 113.2, 127.0, 127.5, 127.7, 128.0, 128.1, 128.8, 129.1, 129.9, 130.0, 132.9, 135.4, 135.6,
144.96; ESI-MS m/z: 730 (M+Na)+; 1437 (2M+Na)+; Yield: 77%; Anal. Calcd for C40H41N3O9: C, 67.89;
H, 5.80; N, 5.94 Found: C, 67.99; H, 5.98; N, 5.76.
General procedure for 17 a,b.

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A suspension of 16a or 16b (1,47 mmol), tert-butylchlorodimethylsilane (665 mg, 4.42 mmol) and
imidazole (600 mg, 8.83 mmol) in dry DMF (8 mL) was stirred for 4 h at rt, then diluted with EtOAc
(100 mL), washed successively with H2O and brine, then dried over anhydrous Na2SO4. Evaporation of
the solvent gave a crude oil, which was purified on a silica gel column (CHCl3:MeOH, 95:5 v/v) to give a
white solid. This intermediate was dissolved in anhydrous CH2Cl2 (10 mL), then benzoyl chloride (100
µL, 0.85 mmol) and triethylamine (140 µL, 1.01 mmol) were added, and the mixture was stirred
overnight at rt. After dilution with CH2Cl2, the mixture was washed successively with 0.01M HCl, H2O,
saturated solution of NaHCO3 and brine, dried over anhydrous Na2SO4. Evaporation of the solvent gave a
crude oil, which was purified on a silica gel column (CHCl3:MeOH, 95:5 v/v) to give pure compound
(17a or 17b).
N4-Benzoyl-3’-O-(tert-butyldimethylsilyl)-5’-dimethoxytrityl-2’-deoxycytidine (17a).
1H-NMR (200 MHz, CDCl3): δ 0.1 (s, 6H), 0.88 (s, 9H), 2.15-2.30 (m, 1H), 2.50-2.65 (m, 1H), 3.35 (m,
1H), 3.52 (m, 1H), 3.78 (s, 6H), 4.02 (m, 1H), 4.55 (q, J = 5.66 Hz, 1H,), 6.25 (t, J = 5.68 Hz, 1H), 6.85
(d, J = 8.17 Hz, 4H), 7.24-7.42 (m, 12H), 7.46 (d, J = 8.17 Hz, 1H), 7.96 (d, J = 7.4 Hz, 2H), 8.42 (d, J =
7.4 Hz, 1H); ESI-MS m/z: 749 (M+H)+, 771 (M+Na)+; Yield: 99%; Anal. Calcd for C43H49N3O7Si: C,
69.05; H, 6.60; N, 5.62 Found: C, 69.18; H, 6.76; N, 5.64.
N4-Benzoyl-2'-O-(2methoxyethyl)-3’-O-(tert-butyldimethylsilyl)-5'-O-dimetoxytritylcytidine (17b).
1H-NMR (200 MHz, CDCl3): δ 0.14 (s, 6H), 0.76 (s, 9H), 3.35 (s, 3H), 3.50-3.65 (m, 4H), 3.68-3.93 (m,
9H), 4.10-4.42 (m, 2H), 6.01 (s, 1H), 6.85 (d, J = 8.17 Hz, 4H), 7.24-7.42 (m, 12H), 7.46 (d, J = 8.17 Hz,
1H), 7.86 (d, J = 7.4 Hz, 2H), 8.73 (d, J = 7.4 Hz, 1H); ESI-MS m/z: 823 (M+H)+, 845 (M+Na)+; Yield:
99%; Anal. Calcd for C46H55N3O9Si: C, 67.21; H, 6.74; N, 5.11 Found: C, 67.11; H, 6.77; N, 5.11.
General procedure for 18 a,b.
A solution of 17a or 17b (0.16 mmol) in dry THF (3 mL) was added dropwise to a -78 °C solution of
freshly prepared LDA (2.1 mmol) in dry THF (3 mL) at -78 °C under a positive pressure of argon. After 4
h at -78 °C, methyl formate (60 µL, 0.96 mmol) was added, and the reaction mixture was kept under
magnetic stirring at -78 °C for 3 h. The mixture was then allowed to warm to rt, diluted with EtOAc and
quenched by the addition of saturated NH4Cl solution. The organic phase was washed successively with
water and brine then dried over anhydrous Na2SO4. Evaporation of the solvent gave a crude white foam
which was used in the next Wittig reaction without further purification.
N4-Benzoyl-3’-O-(tert-butyldimethylsilyl)-5’-dimethoxytrityl-2’-deoxy-6-formylcytidine (18a).

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163
HPLC reverse phase, zorbax C8 column, Eluent: CH3CN 95%, H2O 5%, flow 0.4 mL/min; retention time
4.39 min ESI-MS m/z 794 (M+ H2O +H)+; retention time 4.91 min ESI-MS m/z 808 (M+ CH3OH +H)+,
830 (M+ CH3OH +Na)+; retention time 6.74 min ESI-MS m/z 776 (M+H)+; retention time 7.98 min
N4-Benzoyl-2'-O-(2methoxyethyl)-3’-O-(tert-butyldimethylsilyl)-5’-dimethoxytrityl-6-formylcytidine
(18b).
HPLC reverse phase, zorbax C8 column, Eluent: CH3CN 95%, H2O 5%, flow 0.4 mL/min; retention time
4.71 min ESI-MS m/z 869 (M+ H2O +H)+; retention time 5.01 min ESI-MS m/z 883 (M+ CH3OH +H)+,
905 (M+ CH3OH +Na)+; retention time 6.74 min ESI-MS m/z 851 (M+H)+; retention time 8.51 min
General procedure for 19 a,b.
Methyltriphenylphosphonium bromide (140 mg, 0.390 mmol) and KHMDS 0.5 M (780 µL, 0.390 mmol)
were stirred together in dry THF (10 mL) at –78°C under a positive pressure of argon; after 15 min 18a or
18b (0.13 mmol) was added, and the mixture was stirred at –78 °C per 15 min and then at 0 °C for 2 h.
The mixture was allowed to warm to rt, diluted with EtOAc and quenched by the addition of saturated
NH4Cl solution. The organic phase was washed successively with water and brine then dried over
anhydrous Na2SO4. After evaporation of the solvent, the crude product was purified on a silica gel
column (CHCl3:MeOH, 98:2 v/v) to give pure compound (19a or 19b).
N4-Benzoyl-3’-O-(tert-butyldimethylsilyl)-5’-dimethoxytrityl-2’-deoxy 6-vinylcytidine (19a).
1H-NMR (200 MHz, CDCl3): δ 0.05 (s, 6H), 0.86 (s, 9H), 1.99-2.12 (m, 1H), 2.59-2.73 (m, 1H),
3.70-4.05 (m, 9H), 4.56 (m, 1H), 5.63 (d, J cis = 11.2 Hz, 1H), 5.98 (d, J trans = 17.1 Hz, 1H); 6.51 (t,
J = 7.3 Hz, 1H), 6.68-7.03 (m, 5H), 7.07-7.98 (m, 15H); ESI-MS m/z 775 (M+H)+; Yield: 45%
(from 17a); Anal. Calcd for C45H51N3O7Si: C, 69.83; H, 6.64; N, 5.43 Found: C, 69.68; H, 6.75; N,
5.40.
N4-Benzoyl-2'-O-(2methoxyethyl)-3’-O-(tert-butyldimethylsilyl)-5’-dimethoxytrityl-6-vinylcytidine
(19b).
1H-NMR (200 MHz, CDCl3): δ 0.05 (s, 6H), 0.85 (s, 9H), 3.26 (s, 3H), 3.37-4.05 (m, 13H),
4.48-4.68 (m, 2H), 5.69 (d, J cis = 11 Hz, 1H), 5.86 (d, J = 3 Hz, 1H), 6.02 (d, J trans = 17 Hz, 1H),
6.68-7.03 (m, 5H), 7.07-7.98 (m, 15H); ESI-MS m/z 849 (M+H)+; Yield: 40% (from 17b); Anal.
Calcd for C48H57N3O9Si: C, 67.98; H, 6.77; N, 4.95 Found: C, 67.99; H, 6.78; N, 4.90.
General procedure for 20 a,b.

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To a solution of 19 or 19b (0.84 mmol) in CH2Cl2 (20 mL), ethanethiol (125 µL, 1.69 mmol) was added
and the mixture was stirred at rt for 1h. After evaporation of the solvent under reduced pressure, the
resulting residue was dried under high vacuum for 2 h, dissolved in anhydrous THF (20 mL) and treated
with TBAF (2.85 mL, 2.84 mmol). After stirring at room temperature for 4 h, the solvent was removed in
vacuo and the residue was purified by flash chromatography (CHCl3:MeOH 98:2) to give pure compound
(20a or 20b).
N4-Benzoyl-5'-dimethoxytrityl-2'-deoxy-6-{1-[2-(ethylthio)]ethyl}cytidine (20a).
1H-NMR (200 MHz, CDCl3): δ 1.24 (t, J = 7.7 Hz, 3H), 1.55-1.85 (m, 1H), 2.08-2.32 (m, 1H), 2.56
(q, J = 7.7 Hz, 2H), 2.68-3.20 (m, 4H), 3.72-4.20 (m, 3H), 4.85-5.10 (m, 1H), 6.13 (t, J = 6.9 Hz,
13
1H), 6.75-6.98 (m, 3H), 7.15-7.68 (m, 16H); C-NMR (200 MHz, CDCl3): 14.6, 26.2, 34.4, 38.2,
62.0, 71.1, 86.8, 88.3, 113.2, 127.0, 127.5, 127.7, 128.0, 128.1, 128.8, 129.1, 129.9, 130.0, 132.8,
132.9, 135.4, 135.6, 145.0; ESI-MS m/z: 723 (M+H)+; Yield: 64%; Anal. Calcd for C41H43N3O7S:
C, 68.22; H, 6.00; N, 5.82 Found: C, 68.17; H, 6.08; N, 5.80.
N4-Benzoyl-5'-dimethoxytrityl-2'-O-(2methoxyethyl)-6-{1-[2-(ethylthio)]ethyl}cytidine
(20b).
1H-NMR (200 MHz, CDCl3): δ 1.27 (t, J = 7.7 Hz, 3H), 2.60 (q, J = 7.7 Hz, 2H), 2.78-3.12 (m, 4H), 3.27
(s, 3H), 3.33-3.62 (m, 4H), 3.65-3.84 (m, 8H), 3.84-4.01 (m, 1H), 4.54 (dd, J2'-3' = 5.4 Hz, J3'-4' = 4.3 Hz,
1H), 4.85 (dd, J1'-2' = 4.2 Hz, J2'-3' = 5.4 Hz, 1H), 5.67 (d, J1'-2' = 4.2 Hz, 1H), 6.74-7.03 (m, 3H), 7.15-7.66
13
(m, 16H); C-NMR (200 MHz, CDCl3): 14.7, 29.7, 29,8, 34.7, 58.8, 63.5, 70.4, 71.5, 72.0, 79.1, 85.7,
91.2, 113.0, 127.1, 127.5, 127.7, 129.8, 128.1, 128.7, 129.1, 129.9, 130.3, 133.0, 133.4, 135.5, 135.8,
144.9; ESI-MS m/z: 797 (M+H)+; Yield: 60%; Anal. Calcd for C44H49N3O9S: C, 66.40; H, 6.21; N, 5.28
Found: C, 66.41; H, 6.32; N, 5.20.
General procedure for 2 a,b.
Compound (20a or 20b (0.142 mmol) was mixed with diisopropylamine tetrazolide (25 mg, 0.142 mmol)
and dried over P2O5 in vacuo overnight. The resulting mixture was dissolved in anhydrous CH3CN (5
mL) and 2-cyanoehtyl N,N,N',N'-tetraisopropylphosphorodiamidite (180 µL, 0.568 mmol) was added.
The reaction mixture was stirred at rt for 4 h under an argon atmosphere, followed by removal of the
solvent in vacuo. EtOAc (10 mL) was added to the residue, and it was washed with 5% aqueous NaHCO3
(20mL) and brine (20 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The resulting residue was purified by flash silica gel column (CHCl3:MeOH, 98:2 v/v)
to give pure compound (2a or 2b).

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165
N4-Benzoyl-5'-dimethoxytrityl-2'-deoxy-6-{1-[2-(ethylthio)]ethyl}cytidine 3'-N,N-Diisopropyl
(cyanoethyl) phosphoramidite (2a).
1H-NMR (200 MHz, CDCl3): δ 1.06-1.40 (m, 15H), 1.55-1.85 (m, 1H), 2.08-2.32 (m, 1H), 2.43-3.20 (m,
8H), 3.70-4.20 (m, 12H), 4.35-4.48 (m, 2H), 6.09 (t, J = 6.9 Hz, 1H), 6.75-6.98 (m, 3H), 7.15-7.68 (m,
16H); 31P NMR (600 MHz, CDCl3): δ 151.8, 149.3; ESI-MS m/z: 923 (M+H)+; Yield: 53%; Anal. Calcd
for C50H60N5O8PS: C, 65.13; H, 6.56; N, 7.60 Found: C, 65.28; H, 6.50; N, 7.77.
N4-Benzoyl-5'-dimethoxytrityl-2'-O-(2methoxyethyl)-6-{1-[2-(ethylthio)]ethyl}cytidine
3'-N,N-Diisopropyl (cyanoethyl) phosphoramidite (2b).
1H-NMR (200 MHz, CDCl3): δ 1.04-1.39 (m, 15H), 1.55-1.80 (m, 1H), 2.05-2.29 (m, 1H), 2.42-3.25 (m,
8H), 3.33 (s, 3H), 3.65-4.22 (m, 16H), 4.35-4.48 (m, 2H), 6.10 (t, J = 6.9 Hz, 1H), 6.73-7.03 (m, 3H),
31
7.15-7.68 (m, 16H); P NMR (600 MHz, CDCl3): δ 150.5, 149.2; ESI-MS m/z: 997 (M+H)+. Yield:
35%; Anal. Calcd for C53H66N5O10PS: C, 63.90; H, 6.68; N, 7.03 Found: C, 63.95; H, 6.77; N, 7.05.
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