Efficient synthesis of N4-methyl-and N4- hydroxycytidine phosphoramidites

Article abstract of DOI:10.1055/s-0030-1258170

Nucleotide analogues empower chemical biologists to investigate the relationship between RNA structure and function on a level deeper than would be possible using only the four natural nucleotides. N⁴-Methylcytidine and N⁴-hydroxycytidine represent two cytidine analogues whose physicochemical properties enable biochemical tests for specific aspects of cytidine function within RNA. Here, we describe an efficient synthesis of N ⁴-methylcytidine and N⁴-hydroxycytidine derivatives, and their corresponding phosphoramidites in 40.7% and 25.2% yield, respectively, starting from uridine. These phosphoramidites couple efficiently during solid-phase synthesis, although N⁴-hydroxycytidine reverts partially to cytidine (5-10%) during oligonucleotide synthesis and deprotection. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0030-1258170

2708
PAPER
Efficient Synthesis of N4-Methyl- and N4-Hydroxycytidine Phosphoramidites
4
S
u
of
N
-Methy
n
l- and
N
-
Hydroxyc
L
ytidine Phosph
uoramidites , Nan-Sheng Li, Selene C. Koo, Joseph A. Piccirilli*
Department of Biochemistry and Molecular Biology and Department of Chemistry, University of Chicago, 929 East 57th Street, Chicago,
IL 60637, USA
Fax +1(773)7020271; E-mail: jpicciri@uchicago.edu
Received 22 February 2010; revised 1 May 2010
NH2
NH
N
Abstract: Nucleotide analogues empower chemical biologists to
investigate the relationship between RNA structure and function on
a level deeper than would be possible using only the four natural nu-
cleotides. N4-Methylcytidine and N4-hydroxycytidine represent two
cytidine analogues whose physicochemical properties enable bio-
chemical tests for specific aspects of cytidine function within RNA.
Here, we describe an efficient synthesis of N4-methylcytidine and
N4-hydroxycytidine derivatives, and their corresponding phosphor-
amidites in 40.7% and 25.2% yield, respectively, starting from uri-
dine. These phosphoramidites couple efficiently during solid-phase
synthesis, although N4-hydroxycytidine reverts partially to cytidine
(5–10%) during oligonucleotide synthesis and deprotection.
Ktaut = 10–5
N
NH
N
O
O
HO
HO
NH
N
N
N
Ktaut = 10
N
NH
O
O
Figure 1 Tautomeric forms of cytidine and N4-hydroxycytidine
Key words: N4-methylcytidine, N4-hydroxycytidine, nucleotides,
phosphoramidites, oligonucleotides
Several methods have allowed synthetic access to N4-
methyl- and N4-hydroxycytidine derivatives. Methods for
obtaining N4-methylcytidine derivatives include selective
oxidation of thiouridine with dimethyldioxirane,4 nucleo-
philic substitution of 4-triazolouridine,2 4-(tetrazol-1-
yl)uridine,5 or 4-(4-nitrophenoxy)uridine derivatives6
with methylamine, and reduction of 4-N-(dimethylform-
amidino)uridine with sodium borohydride.7 Methods for
obtaining N4-hydroxycytidine derivatives include hy-
droxyamination of 4-thiouridine,8 1,2,4-1H-triazolated 2¢-
deoxyuridine,9 or cytidine (via amine exchange).10 Grasby
and co-workers prepared the phosphoramidite derivative
of N4-methylcytidine via the fully deprotected parent nu-
cleoside.2 Bypassing complete deprotection to the parent
nucleoside, we develop here an alternate, more efficient
route to the N4-methylcytidine phosphoramidite and ex-
tend it to N4-hydroxycytosine phosphoramidite.
Triisopropylbenzenesulfonylation of uridine at the O4-po-
sition followed by treatment with concentrated ammoni-
um hydroxide has allowed efficient conversion of uridine
derivatives into the corresponding cytidine derivatives.11
We anticipated that this sulfonylation/displacement reac-
tion sequence could be used to prepare N4-methyl- and N4-
hydroxycytidine analogues by treatment with methyl-
amine or hydroxylamine, respectively (Scheme 1). Using
an established method,12,13 we obtained intermediate 1
from uridine in two steps with 73% yield. Reaction of si-
lyl-protected uridine 1 with 2,4,6-triisopropylbenzene-
sulfonyl chloride (TPSCl) in the presence of 4-
(dimethylamino)pyridine (DMAP) and triethylamine, fol-
lowed by treatment with methylamine or hydroxylamine
hydrochloride, gave the corresponding cytidine deriva-
tives 2a (80% yield) and 2b (75% yield), respectively.
Cytidine analogues bearing a C-4 exocyclic methylamino
or hydroxyamino group have distinct physicochemical
features that make them attractive reagents for studying
the relationship between RNA structure and function. N4-
Methylcytidine has distinct steric and hydrogen-bonding
properties compared to cytidine1 and thus can serve as a
specific biochemical probe to test the functional signifi-
cance of interactions involving the cytidine N4-amino
group. For example, Grasby and co-workers2 used N4-
methylcytidine to investigate the interaction of TAR and
RRE RNA elements in the HIV genome with their corre-
sponding regulatory proteins Tat and Rev. The other cyti-
dine analogue, N4-hydroxycytidine (N4-OH-C), favors
the 4-imino tautomeric state in aqueous solution, whereas
cytidine strongly favors the 4-amino tautomer (Figure 1).3
This distinct tautomeric preference directs nucleic acid
polymerases to incorporate adenosine as well as gua-
nosine during polymerization, accounting for the ob-
served mutagenic properties of N4-OH-C3e–g and
providing support for the rare tautomer hypothesis of sub-
stitution mutagenesis.3h Analogously, N4-hydroxycytidine
underpins a strategy to assess the functional contribution
of the 4-imino tautomer of cytidine in RNA-mediated bi-
ological processes. To allow the use of N4-methylcytidine
and N4-hydroxycytidine in structural and functional RNA
studies, we report here efficient synthetic access to the
corresponding phosphoramidites and their incorporation
into RNA oligonucleotides.
SYNTHESIS 2010, No. 16, pp 2708–2712
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x.
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1
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Advanced online publication: 12.07.2010
DOI: 10.1055/s-0030-1258170; Art ID: M01510SS
© Georg Thieme Verlag Stuttgart · New York
Acetylation of 2a using acetic anhydride in the presence
of DMAP and triethylamine generated 3a in 86% yield.

PAPER
Synthesis of N4-Methyl- and N4-Hydroxycytidine Phosphoramidites
2709
When compound 2b was treated with benzoic anhydride We incorporated the N4-methyl- and N4-hydroxycytidine
in dichloromethane in the presence of DMAP at room phosphoramidites into RNA oligonucleotides [5¢-
temperature for three hours, only monosubstituted hy- GGGCAUCUCCACCUCC*UCGC-3¢ (C*: 6a) and 5¢-
droxylamine derivative 3b was obtained in 89% yield.14 GGC*UA-3¢ (C*: 6b)] as described.17 The N4-methyl- and
Pyridinium poly(hydrogen fluoride) removed the di-tert- N4-hydroxycytidine phosphoramidites (~100 mg) were
butylsilylene group efficiently to afford 4a and 4b in dissolved in anhydrous acetonitrile (1 mL) and coupled
yields of 91% and 97%, respectively. No migration of the twice during the appropriate synthesis cycle. Based on tri-
2¢-O-tert-butyldimethylsilyl group to the 3¢-oxygen oc- tyl yields, coupling efficiencies throughout were excellent
curred under the reaction conditions.15,16 After 5¢-O- (data not shown). Following standard oligonucleotide
dimethoxytritylation of 4a and 4b to 5a and 5b, respec- deprotection conditions18 [NH4OH–EtOH (3:1), 55 °C, 2–
tively, phosphitylation of 5a using 2-cyanoethyl N,N-di- 4 h; Et3N·3HF, NMP, 65 °C, 1.5 h] and purification, we
isopropylchlorophosphoramidite in the presence of N,N- obtained MALDI mass spectra (see Supporting Informa-
diisopropylethylamine and 1-methyl-1H-imidazole gave tion,
Figures 1– 4)
for
5¢-GGGCAUCUCCAC-
phosphoramidite 6a in 94% yield. Under the same condi- CUCC*UCGC-3¢ (C*: N4-Me-C, 6a) and 5¢-GGC*UA-3¢
tions, we could not obtain phosphoramidite 6b from 5b, (C*: N4-OH-C, 6b). Each gave peaks consistent with the
possibly due to phosphitylation of the N4-hydroxy group. calculated mass (Table 1). The mass spectrum for the N4-
Compared to 2-cyanoethyl N,N-diisopropylchlorophos- hydroxycytidine oligonucleotide [5¢-GGC*UA-3¢ (C*:
phoramidite, 2-cyanoethyl N,N,N¢,N¢-tetraisopropylphos- N4-OH-C, 6b)] also gave a peak at mass 1569.6, consis-
phorodiamidite exhibits lower reactivity and higher tent with possible conversion of N4-hydroxycytidine into
selectivity. Using N,N,N¢,N¢-tetraisopropylphosphorodi- cytidine. Supporting this possibility, mutation of the HDV
amidite as the phosphitylating reagent, we obtained 6b ribozyme active site cytidine to C* (N4-hydroxycytidine)
from 5b in 82% yield, with no observed phosphitylation resulted in biphasic kinetic behavior. Approximately 10%
of the hydroxy group.
of the ribozyme reacted at the wild-type rate (as expected
if about 10% of C* was converted into C) while the re-
maining ~90% reacted at a diminished rate, attributed to
NHR1
O
N
N
NH
N
O
O
(1) TPSCl, DMAP, Et3N, MeCN
DMAP, Et3N, CH2Cl2
O
O
O
O
(2) MeNH2 in THF for 2a
NH2OH•HCl for 2b
Ac2O for 3a, Bz2O for 3b
(t-Bu)2Si
O
(t-Bu)2Si
OTBS
O
OTBS
1
2a R1 = Me, 80%
2b R1 = OH, 75%
NR1R2
N
NR1R2
NR1R2
N
N
N
O
N
O
DMTCl, py
N
O
HF-py, THF
DMTO
O
0 °C to r.t., 10 min
HO
O
O
O
(t-Bu)2Si
O
OH OTBS
OTBS
OH OTBS
3a R1 = Me, R2 = Ac, 86%
4a R1 = Me, R2 = Ac, 91%
5a R1 = Me, R2 = Ac, 95%
5b R1 = OH, R2 = Bz, 65%
3b R1 = OH, R2 = Bz, 89%
4b R1 = OH, R2 = Bz, 97%
NR1R2
N
i-Pr2NP(OCH2CH2CN)Cl, DIPEA
N
O
1-methyl-1H-imidazole, CH2Cl2 for 6a
DMTO
O
(i-Pr2N)2POCH2CH2CN
DCI, CH2Cl2 for 6b
O
OTBS
CN
P
(i-Pr)2N
O
6a: R1 = Me, R2 = Ac, 94%
6b: R1 = OH, R2 = Bz, 82%
Scheme 1 Synthesis of N4-methyl- and N4-hydroxycytidine phosphoramidites
Synthesis 2010, No. 16, 2708–2712 © Thieme Stuttgart · New York

2710
J. Lu et al.
PAPER
Table 1 Mass Data for 5¢-GGGCAUCUCCACCUCC*UCGC-3¢, 5¢-GGC*UA-3¢, and 5¢-C*A-3¢
5¢-GGGCAUCUCCACCUCC*UCGC-3¢ 5¢-GGC*UA-3¢
5¢-C*A-3¢
C(wt)
N4-Me-C
6264.9
C(wt)
N4-OH-C
1584.3
C(wt)
N4-OH-C
Calcd mass
6250.9
6250.5a
1568.3
1569.3a
571.1
587.1
MALDI mass
a Positive [M + H+].
6265.7a
1585.6a
571.3a
587.3a
13C NMR (100 MHz, CDCl3): d = 164.5, 156.2, 138.5, 95.6, 94.6,
76.0, 75.5, 74.5, 68.0, 28.0, 27.6, 27.1, 27.1, 22.9, 20.5, 18.4, –4.2,
–4.7.
HRMS–FAB: m/z [M + H+] calcd for C24H46N3O5Si2: 512.2976;
found: 512.2985.
the presence of C* (unpublished results). In order to con-
firm this observation, we synthesized a dinucleotide, 5¢-
C*A-3¢ (C*: N4-OH-C). Analysis after deprotection
showed that a small amount of C* was converted into C
(see Supporting Information). Deprotection of the oligo-
nucleotides with potassium carbonate in methanol gave
analogous results. These observations indicate that during
oligonucleotide preparation ~10% of the N4-hydroxycyti-
dine undergoes conversion into cytidine, possibly via
cleavage of the N–OH bond under the coupling conditions
during solid-phase synthesis19 or during the deprotection
of the oligonucleotide.4c,8b
2¢-O-[(tert-Butyl)dimethylsilyl]-3¢,5¢-O-(di-tert-butylsilylene)-
N4-hydroxycytidine (2b)
Compound 2b was prepared from uridine 1 (112 mg, 0.225 mmol),
TPSCl (204 mg, 0.675 mmol), Et3N (0.047 mL, 0.338 mmol),
DMAP (82.5 mg, 0.676 mmol) and NH2OH·HCl (78.2 mg, 1.125
mmol) as described for 2a. Silica gel chromatography (MeOH–
CH2Cl2, 3:97) was used to purify the residue, giving 2b as a white
foam; yield: 95 mg (75%).
In summary, we have described an efficient synthesis of
derivatives of N4-methylcytidine and N4-hydroxycytidine,
and their corresponding phosphoramidites with an overall
yield of 40.7% and 25.2%, respectively, starting from uri-
dine. These phosphoramidites couple efficiently during
solid-phase RNA synthesis, expanding the repertoire of
nucleotide analogues for investigation of cytidine func-
tion in RNA biology.
1H NMR (400 MHz, CDCl3): d = 6.60 (d, J = 8.2 Hz, 1 H), 5.67 (d,
J = 8.2 Hz, 1 H), 5.65 (s, 1 H), 4.47 (m, 1 H), 4.23 (d, J = 4.9 Hz, 1
H), 4.08 (m, 1 H), 3.95 (m, 1 H), 3.86 (m, 1 H), 1.91 (d, J = 1.8 Hz,
1 H), 1.05 (s, 9 H), 1.02 (s, 9 H), 0.92 (s, 9 H), 0.15 (s, 3 H), 0.12 (s,
3 H).
13C NMR (100 MHz, CDCl3): d = 148.8, 130.7, 98.3, 93.6, 76.3,
75.2, 74.1, 67.6, 27.4, 26.9, 25.8, 22.7, 20.3, 18.3, –4.4, –5.2.
HRMS–FAB: m/z [M + H+] calcd for C23H44N3O6Si2: 514.2769;
found: 514.2770.
All reactions were carried out under a positive pressure of argon in
anhydrous solvents. Commercially available reagents and anhy-
drous solvents were used as supplied without further purification.
1H, 13C, and 31P NMR spectra were recorded on a Bruker 400 MHz
NMR spectrometer; 1H chemical shifts are reported in d (ppm) rel-
ative to tetramethylsilane, 13C chemical shifts d (ppm) relative to
CDCl3, and 31P chemical shifts d (ppm) relative to 85% H3PO4.
MALDI-TOF spectra were obtained on an Applied Biosystems DE-
Pro MALDI-TOF instrument. High-resolution mass spectra were
measured at the Department of Chemistry, University of California
at Riverside, on a VG ZAB instrument.
N4-Acetyl-2¢-O-[(tert-butyl)dimethylsilyl]-3¢,5¢-O-(di-tert-butyl-
silylene)-N4-methylcytidine (3a)
To a soln of 2¢-O-[(tert-butyl)dimethylsilyl]-3¢,5¢-O-(di-tert-butyl-
silylene)-N4-methylcytidine (2a; 184 mg, 0.36 mmol), Et3N (145
mg, 1.44 mmol), and DMAP (9 mg, 0.08 mmol) in anhyd CH2Cl2
(15 mL) under argon was added Ac2O (73 mg, 0.72 mmol). The
mixture was stirred at r.t. for 24 h. TLC showed that the reaction
was complete. MeOH (1 mL) was added, and the solvent was re-
moved. The residue was purified by silica gel chromatography
(MeOH–CHCl3, 2:98), giving 3a as a white foam; yield: 172 mg
(86%).
1H NMR (400 MHz, CDCl3): d = 7.62 (d, J = 7.7 Hz, 1 H), 7.19 (d,
J = 7.7 Hz, 1 H), 5.68 (s, 1 H), 4.53 (m, 1 H), 4.33 (d, J = 4.2 Hz, 1
H), 4.27 (m, 1 H), 3.98 (m, 1 H), 3.78 (m, 1 H), 3.46 (s, 3 H), 2.39
(s, 3 H), 1.01 (s, 9 H), 1.00 (s, 9 H), 0.93 (s, 9 H), 0.25 (s, 3 H), 0.15
(s, 3 H).
13C NMR (100 MHz, CDCl3): d = 173.1, 164.6, 154.7, 141.2, 99.2,
94.3, 75.7, 75.1, 74.7, 67.7, 34.3, 27.4, 26.9, 25.9, 25.8, 22.7, 20.3,
18.1, –4.4, –4.9.
2¢-O-[(tert-Butyl)dimethylsilyl]-3¢,5¢-O-(di-tert-butylsilylene)-
N4-methylcytidine (2a)
2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl; 707 mg, 2.34
mmol) was added to a soln of 2¢-O-[(tert-butyl)dimethylsilyl]-3¢,5¢-
O-(di-tert-butylsilylene)uridine11 (1; 400 mg, 0.78 mmol), Et3N
(0.16 mL, 1.17 mmol) and DMAP (285 mg, 2.34 mmol) in anhyd
MeCN (20 mL). The mixture was stirred at r.t. for 24 h under argon,
at which time TLC showed that the reaction was complete. Then,
2.0 M MeNH2 in THF (15 mL, 30.0 mmol) was added to the mix-
ture, which was stirred at r.t. for 3 h. The solvent was removed, and
the residue was partitioned between CHCl3 and H2O. The organic
phase was washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by silica gel chromatography (2–4%
MeOH in CHCl3), giving 2a as a white foam; yield: 324 mg (80%).
1H NMR (400 MHz, CDCl3): d = 7.21 (d, J = 7.2 Hz, 1 H), 6.34 (d,
J = 4.4 Hz, 1 H), 5.82 (d, J = 7.2 Hz, 1 H), 5.69 (s, 1 H), 4.50 (m, 1
H), 4.33 (d, J = 4.0 Hz, 1 H), 4.20 (m, 1 H), 3.97 (m, 1 H), 3.86 (m,
1 H), 2.99 (d, J = 4.8 Hz, 3 H), 1.03 (s, 9 H), 1.02 (s, 9 H), 0.94 (s,
9 H), 0.23 (s, 3 H), 0.15 (s, 3 H).
HRMS–FAB: m/z [M + H+] calcd for C26H48N3O6Si2: 554.3082;
found: 554.3093.
N4-Benzoyl-2¢-O-[(tert-butyl)dimethylsilyl]-3¢,5¢-O-(di-tert-bu-
tylsilylene)-N4-hydroxycytidine (3b)
To a soln of 2b (125 mg, 0.243 mmol), Et3N (81.3 mg, 0.972 mmol)
and DMAP (59.4 mg, 0.486 mmol) in anhyd CH2Cl2 (15 mL) under
argon was added (PhCO)2O (109 mg, 0.486 mmol). The reaction
mixture was stirred at r.t. for 3 h. TLC showed that the reaction was
complete. MeOH (1 mL) was added, and the solvent was removed.
Synthesis 2010, No. 16, 2708–2712 © Thieme Stuttgart · New York

PAPER
Synthesis of N4-Methyl- and N4-Hydroxycytidine Phosphoramidites
2711
The residue was purified by silica gel chromatography (1% MeOH
in CH2Cl2) to give 3b as a white foam; yield: 133 mg (89%).
1H NMR (400 MHz, CDCl3): d = 8.42 (d, J = 7.6 Hz, 1 H), 7.42–
7.23 (m, 9 H), 6.85 (d, J = 8.8 Hz, 4 H), 6.64 (d, J = 7.5 Hz, 1 H),
5.86 (s, 1 H), 4.37 (m, 1 H), 4.28 (m, 1 H), 4.08 (m, 1 H), 3.80 (s, 6
H), 3.55 (m, 2 H), 3.42 (s, 3 H), 2.38 (s, 3 H), 0.93 (s, 9 H), 0.35 (s,
3 H), 0.21 (s, 3 H).
1H NMR (400 MHz, CDCl3): d = 8.07–7.46 (m, 5 H), 6.83 (d,
J = 8.2 Hz, 1 H), 5.92 (d, J = 8.2 Hz, 1 H), 5.65 (s, 1 H), 4.48 (m, 1
H), 4.22 (d, J = 4.9 Hz, 1 H), 4.04 (m, 1 H), 3.97 (m, 1 H), 3.84 (m,
1 H), 1.06 (s, 9 H), 1.03 (s, 9 H), 0.92 (s, 9 H), 0.15 (s, 3 H), 0.13 (s,
3 H).
13C NMR (100 MHz, CDCl3): d = 163.2, 149.0, 147.9, 133.4, 132.8,
129.6, 128.5, 128.4, 97.7, 93.2, 77.2, 76.7, 75.3, 74.1, 67.5, 27.4,
26.9, 25.7, 22.7, 20.3, 18.2, –4.4, –5.3.
13C NMR (100 MHz, CDCl3): d = 172.7, 164.9, 158.6, 155.0, 144.2,
142.9, 135.5, 135.2, 130.1, 128.2, 128.0, 127.1, 113.3, 113.1, 99.1,
90.9, 87.0, 83.0, 76.5, 68.8, 61.2, 55.2, 34.1, 25.9, 25.8, 18.1, –4.4,
–5.5.
HRMS–FAB: m/z [M + Na+] calcd for C39H49N3O8NaSi: 738.3187;
found: 738.3194.
HRMS–FAB: m/z [M + H+] calcd for C30H48N3O7Si2: 618.3031;
found: 618.3036.
N4-Benzoyl-2¢-O-[(tert-butyl)dimethylsilyl]-5¢-O-(4,4¢-dimeth-
oxytrityl)-N4-hydroxycytidine (5b)
Compound 5b was prepared from 4b (50 mg, 0.105 mmol) and
DMTCl (82 mg, 0.242 mmol) as described for 5a. Silica gel chro-
matography (1% MeOH in CH2Cl2 + 0.5% Et3N) was used to purify
the residue, giving 5b as a white foam; yield: 53 mg (65%).
1H NMR (400 MHz, CDCl3): d = 8.05 (m, 2 H), 7.61–7.25 (m, 13
H), 6.85 (d, J = 8.8 Hz, 4 H), 5.95 (d, J = 4.8 Hz, 1 H), 5.49 (d,
J = 8.2 Hz, 1 H), 4.38 (m, 1 H), 4.33 (m, 1 H), 4.12 (m, 1 H), 3.81
(s, 6 H), 3.47 (m, 2 H), 0.93 (s, 9 H), 0.17 (s, 3 H), 0.15 (s, 3 H).
13C NMR (100 MHz, CDCl3): d = 163.3, 158.7, 158.6, 149.2, 148.3,
144.2, 135.0, 134.8, 133.7, 133.5, 130.1, 130.0, 129.5, 128.6, 128.5,
128.0, 127.2, 113.2, 97.4, 87.9, 87.2, 83.5, 75.6, 71.1, 62.9, 55.2,
25.6, –4.8, –5.2.
N4-Acetyl-2¢-O-[(tert-butyl)dimethylsilyl]-N4-methylcytidine
(4a)
HF·pyridine (300 mL, 2.07 mmol) was carefully diluted with pyri-
dine (0.50 mL) and then added dropwise to a soln of 3a (229 mg,
0.41 mmol) in THF (6 mL) at 0 °C. The mixture was warmed to r.t.
and stirred for 10 min. TLC showed that the reaction was complete.
The mixture was diluted with pyridine (2 mL). This mixture was
partitioned between CH2Cl2 and H2O, and the organic layer was
washed with 5% NaHCO3 soln and brine, then dried (anhyd
Na2SO4). The solvent was removed, and the residue was purified by
silica gel chromatography (MeOH–CH2Cl2, 4:96), affording 4a as a
white foam; yield: 156 mg (91%).
1H NMR (400 MHz, CDCl3): d = 7.95 (d, J = 7.6 Hz, 1 H), 7.21 (d,
J = 7.6 Hz, 1 H), 5.51 (d, J = 4.0 Hz, 1 H), 4.74 (t, J = 4.6 Hz, 1 H),
4.21 (m, 1 H), 4.15 (m, 1 H), 4.00 (m, 1 H), 3.88 (m, 1 H), 3.80 (m,
1 H), 3.46 (s, 3 H), 2.68 (d, J = 5.5 Hz, 1 H), 2.40 (s, 3 H), 0.88 (s,
9 H), 0.10 (s, 3 H), 0.09 (s, 3 H).
HRMS–FAB: m/z [M + Na+] calcd for C43H49N3O9NaSi: 802.3136;
found: 802.3141.
N4-Acetyl-2¢-O-[(tert-butyl)dimethylsilyl]-5¢-O-(4,4¢-dimeth-
oxytrityl)-N4-methylcytidine-3¢-(2-cyanoethyl)-N,N-diisoprop-
ylphosphoramidite (6a)
13C NMR (100 MHz, CDCl3): d = 173.2, 164.8, 155.2, 145.0, 99.5,
94.5, 85.5, 74.1, 69.8, 61.3, 34.3, 25.9, 25.7, 17.9, –4.8, –5.4.
To a soln of 5a (171 mg, 0.24 mmol) in anhyd CH2Cl2 (5 mL) under
argon were added quickly DIPEA (208 mL, 1.2 mmol), 2-cyanoeth-
yl N,N-diisopropylchlorophosphoramidite (170 mg, 0.72 mmol),
and 1-methyl-1H-imidazole (19 mL, 0.24 mmol). The mixture was
stirred at r.t. for 1 h. TLC indicated that the reaction was complete.
The mixture was concentrated under reduced pressure, and the res-
idue was purified by silica gel chromatography (1% MeOH in
CH2Cl2 containing 0.5% Et3N), to give the corresponding phosphor-
amidite 6a; yield: 205 mg (94%).
HRMS–FAB: m/z [M + H+] calcd for C18H32N3O6Si: 414.2060;
found: 414.2064.
N4-Benzoyl-2¢-O-[(tert-butyl)dimethylsilyl]-N4-hydroxycyti-
dine (4b)
Compound 4b was prepared from 3b (427 mg, 0.691 mmol) and
HF·pyridine (500 mL, 3.45 mmol) as described for 4a. Silica gel
chromatography (MeOH–CH2Cl2, 2:98) was used to purify the res-
idue, giving 4b as a white foam; yield: 320 mg (97%).
1H NMR (400 MHz, CDCl3): d = 8.55 (br, 1 H), 8.08–7.48 (m, 6 H),
5.87 (d, J = 8.2 Hz, 1 H), 5.70 (d, J = 5.2 Hz, 1 H), 4.45 (t, J = 5.1
Hz, 1 H), 4.20 (m, 1 H), 4.11 (m, 1 H), 3.98 (m, 1 H), 3.86 (m, 1 H),
2.79 (br, 1 H), 0.89 (s, 9 H), 0.09 (s, 3 H), 0.08 (s, 3 H).
13C NMR (100 MHz, CDCl3): d = 163.5, 149.2, 148.5, 135.3, 133.6,
129.6, 128.6, 97.3, 90.6, 85.0, 74.7, 70.9, 61.9, 25.5, 17.9, –4.9,
–5.2.
31P NMR (162 MHz, CD3CN): d = 152.9, 151.8.
MS–FAB: m/z [M + H+] calcd for C48H67N5O9PSi: 916.5; found:
916.0.
N4-Benzoyl-2¢-O-[(tert-butyl)dimethylsilyl]-5¢-O-(4,4¢-dimeth-
oxytrityl)-N4-hydroxycytidine-3¢-(2-cyanoethyl)-N,N-diiso-
propylphosphoramidite (6b)
To a soln of 5b (185 mg, 0.24 mmol) in anhyd CH2Cl2 (5 mL) under
argon were added 4,5-dicyanoimidazole (DCI; 31 mg, 0.26 mmol)
and 2-cyanoethyl N,N,N¢,N¢-tetraisopropylphosphorodiamidite (86
mg, 0.29 mmol). The mixture was stirred at r.t. for 4 h. TLC indi-
cated that the reaction was complete. The mixture was partitioned
between CH2Cl2 and 5% aq NaHCO3. The organic layer was
washed with brine, then dried (anhyd Na2SO4). The solvent was re-
moved under reduced pressure, and the residue was purified by sil-
ica gel chromatography (1% MeOH in CH2Cl2 containing 0.5%
Et3N), to give the corresponding phosphoramidite 6b; yield: 190 mg
(82%).
HRMS–FAB: m/z [M + H+] calcd for C22H32N3O7Si: 478.2010;
found: 478.2005.
N4-Acetyl-2¢-O-[(tert-butyl)dimethylsilyl]-5¢-O-(4,4¢-dimeth-
oxytrityl)-N4-methylcytidine (5a)
To a soln of 4a (121 mg, 0.29 mmol) in anhyd pyridine (4 mL) un-
der argon was added 4,4¢-dimethoxytrityl chloride (DMTCl; 297
mg, 0.87 mmol), and the mixture was stirred at r.t. overnight. The
reaction was quenched with MeOH (1.0 mL), then the mixture was
partitioned between CH2Cl2 and H2O. The organic layer was
washed with 5% NaHCO3 soln and brine, then dried (anhyd
Na2SO4). The solvent was removed, and the residue was purified by
silica gel chromatography (2% MeOH in CH2Cl2 + 0.5% Et3N), af-
fording 5a as a pale yellow foam; yield: 199 mg (95%).
31P NMR (162 MHz, CD3CN): d = 152.5, 151.5.
MS–FAB: m/z [M – H+] calcd for C52H65N5O10PSi: 978.4; found:
978.2.
Synthesis 2010, No. 16, 2708–2712 © Thieme Stuttgart · New York

2712
J. Lu et al.
PAPER
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Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
Acknowledgment
This work was supported in part by the Howard Hughes Medical
Institute and an NIH grant (1R56AI081987-0109 and
1R01AI081987-01A1) to J.A.P. S.C.K. was supported in part by the
Medical Scientist Training Program (5 T32 GM07281). We thank
Q. Dai, E. Leung, K. Sundaram, N. Suslov, and N. Tuttle for helpful
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Synthesis 2010, No. 16, 2708–2712 © Thieme Stuttgart · New York