Synthesis and duplex-forming ability of oligonucleotides containing 4′-carboxythymidine analogs

Article abstract of DOI:10.1039/c2ob26712h

Oligonucleotides containing 4′-carboxy-, 4′-methoxycarbonyl-, 4′-carbamoyl-, and 4′-methylcarbamoyl-thymidines, and their 2′-methoxy, 2′-amino or 2′-acetamido analogs were prepared. Their duplex-forming ability with DNA and RNA complements was evaluated by UV melting experiments. Interestingly, 4′-carboxythymidine existing in the S-type sugar conformation was found to lead to an increase in the stability of the duplex formed with RNA complements compared to natural thymidine. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob26712h

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Organic &
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Chemistry
PAPER
Synthesis and duplex-forming ability of
oligonucleotides containing 4’-carboxythymidine
analogs†
Cite this: Org. Biomol. Chem., 2012, 10,
9639
Yoshiyuki Hari, Takashi Osawa and Satoshi Obika*
Oligonucleotides containing 4’-carboxy-, 4’-methoxycarbonyl-, 4’-carbamoyl-, and 4’-methylcarbamoyl-
thymidines, and their 2’-methoxy, 2’-amino or 2’-acetamido analogs were prepared. Their duplex-
forming ability with DNA and RNA complements was evaluated by UV melting experiments. Interestingly,
4’-carboxythymidine existing in the S-type sugar conformation was found to lead to an increase in the
stability of the duplex formed with RNA complements compared to natural thymidine.
Received 30th August 2012,
Accepted 23rd October 2012
DOI: 10.1039/c2ob26712h
www.rsc.org/obc
Introduction
Modified oligonucleotides with high binding affinity to comp-
lementary single-stranded nucleic acids are promising
materials applicable to various nucleic acid-based technol-
ogies. In particular, those with high affinity for RNA comp-
lements are essential for applications in RNA-targeted
therapies like antisense methodology and siRNA therapy.1,2 To
date, many artificial oligonucleotides have been developed
towards practical use and to acquire increased RNA-binding
affinity by introduction of substituents at the 2′-position like
2′-alkoxy groups and bridged chains like 2′,4′-BNA/LNA.1 This
increased binding affinity is due to preorganization of the
sugar conformation to that in RNA duplexes, namely the
N-type sugar conformation. However, in some cases, oligonucleo-
tides, including several modified nucleotides, likely existing in
S-type conformations, were also observed to increase the stab-
ility of duplexes formed with RNA complements. For instance,
4′-methoxymethyl- or 4′-aminomethyl-thymidine units could
increase the melting temperature (Tm) value by up to 0.6 °C or
0.5 °C per modification, respectively, depending on
the number and position of the introduction.3 In contrast,
4′-hydroxymethylthymidine4 and 4′-methylthymidine,5 an analog
without any functional group on the 4′-methyl group, seem to
lead to a decrease in the stability. Given this background, we
were interested in the effect of thymidines with highly functio-
nalized methyl units at the 4′-position on the thermal stability
Fig. 1 Structures of 4’-carboxythymidine analogs used in this study.
of the formed duplexes. Here, the synthesis of oligonucleotides
bearing 4′-carboxy-, 4′-methoxycarbonyl-, 4′-carbamoyl- and
4′-methylcarbamoyl-thymidines and their 2′-substituted
analogs as shown in Fig. 1 was performed and their duplex
formation with DNA and RNA complements was studied.
Results and discussion
Synthesis
We planned to convert 4′-methoxycarbonylthymidines into
analogs with the desired 4′-carboxylic acid equivalents by base
treatment after oligonucleotide synthesis. Thus, the phosphor-
amidites 1–4 of 4′-methoxycarbonylthymidines were syn-
thesized as shown in Scheme 1. The diol 56 previously
reported by Wengel’s group was oxidized with a catalytic
amount of TEMPO and PhI(OAc)2, and the resulting carboxylic
acid was reacted with TMSCHN2 in MeOH to give the methyl
ester 6 in 72% over 2 steps. Deoxygenation of 6 afforded the
2′-deoxy compound 8 via thiocarbonate 7. By treatment of 8
with cyclohexene in the presence of Pd(OH)2-C, 4′-methoxycar-
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita 565-0871, Japan.
E-mail: obika@phs.osaka-u.ac.jp; Fax: +81 6 6879 8204; Tel: +81 6 6879 8200
†Electronic supplementary information (ESI) available: Additional experimental
procedures, NMR spectra of all new compounds, and HPLC and MALDI-TOF
mass spectra of the synthesized oligonucleotides. See DOI: 10.1039/c2ob26712h
bonylthymidine
97
was
obtained
in
45%
yield.
This journal is © The Royal Society of Chemistry 2012
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Organic & Biomolecular Chemistry
Scheme 1 Reagents and conditions: (i) TEMPO, PhI(OAc)2, MeCN–H2O (1 : 1), rt, 12 h, then TMSCHN2, MeOH, rt, 0.5 h, 52–72% for 2 steps; (ii) PhOCSCl, DMAP,
CH2Cl2, rt, 0.5 h, 79%; (iii) AIBN, n-Bu3SnH, toluene, reflux, 2 h, 55%; (iv) 20% Pd(OH)2-C, cyclohexene, MeOH, reflux, 10–12 h, 45%-quant.; (v) DMTrCl, pyridine, rt,
1–4 h, 83%-quant.; (vi) Pri2NP(Cl)OCH2CH2CN, DIPEA, CH2Cl2, rt, 2–4 h, 56–95%; (vii) BOMCl, DBU, THF, rt, 0.5 h, 86%; (viii) NaH, MeI, DMF, rt, 1 h, 58%; (ix) TBAF,
THF, rt, 11 h, quant.; (x) TMSCHN2, MeOH, rt, 0.5 h, 89%; (xi) NaBH4, NiCl2, MeOH–THF (1 : 1), rt, 0.5 h, 89%; (xii) CF3CO2Et, Et3N, MeOH, rt, 1 h, 92%; (xiii) Ac2O,
pyridine, rt, 1.5 h, quant.
Dimethoxytritylation of the 5′-hydroxyl group in 9 followed by phosphoramidite 2 was prepared from 16 via compound 17.
phosphitylation of 107 gave the desired phosphoramidite 1, a 2′-Amino and 2′-acetamido analogs were synthesized from 189
building block for oligonucleotide synthesis. The synthesis of prepared from 11 according to our previous report. Methyl
the 2′-methoxy analog 2 was achieved from 118 prepared esterification of 18 using TMSCHN2 yielded 19, which was
according to our previous report. After BOM-protection of the reduced by a combination of NiCl2 and NaBH4 to produce 20
nitrogen at the 3-position of 11, the resulting 12 was methyl- in 89% yield. Trifluoroacetylation and acetylation of 20 gave 21
ated to afford 13 by alkoxide formation, followed by treatment and 22 in high yields, respectively. These compounds 21 and
with MeI. Desilylation of 13 with TBAF produced 14, which 22 were hydrogenolyzed to afford monomers 23, protected as
was converted into methyl ester 15 as before. Removal the 2′-amino analog, and 24 as the 2′-acetamido one.
of benzyl and BOM groups by hydrogenolysis of 15 led to Dimethoxytritylated 25 and 26, and subsequently phosphora-
2′-methoxy monomer 16 quantitatively. Analogous to 1, the midites 3 and 4, were prepared as before. As expected, it was
9640 | Org. Biomol. Chem., 2012, 10, 9639–9649
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Scheme 2 Reagents and conditions: (i) oligonucleotide synthesis on an automated DNA synthesizer; (ii) 50 mM NaOH aq., rt, 1.5 h then 55 °C, 12 h (for X = OH),
50 mM K2CO3 in MeOH, rt, 2 h (for X = OMe), 28% NH3 aq., rt, 1.5 h then 55 °C, 12 h (for X = NH2) or 40% MeNH aq., rt, 1.5 h then 55 °C, 12 h (for X = NHMe).
1
Table 1 Sequence of the synthesized oligonucleotidesa
found from large J1′2′ values (6.5–9.0 Hz) obtained by H NMR
measurements that the monomers 9, 16, 23 and 24 existed in
Oligonucleotide
Sequence
S-type conformations.10
Next, introduction of the phosphoramidites 1–4 into oligo-
nucleotides was performed on an automated DNA synthesizer
using standard phosphoramidite chemistry (Scheme 2). For
the synthesis of 4′-methoxycarbonyl-modified oligonucleo-
tides, commercially available ultra-mild phosphoramidites‡ as
other phosphoramidites were used. After completion of the
synthesis on the DNA synthesizer, four base treatments
(50 mM NaOH aq., 50 mM K2CO3 in MeOH, 28% NH3 aq. and
40% MeNH2 aq.) afforded cleavage from the resin, deprotec-
tion of nucleobases and phosphates, and conversion of
4′-methoxycarbonyl moieties to give the corresponding oligo-
nucleotides containing 4′-carboxy, 4′-methoxycarbonyl, 4′-car-
bamoyl and 4′-methylcarbamoyl derivatives, respectively. The
removal of the standard isobutyryl protecting group of the
2-amino moiety in G was completed by treatment with 50 mM
NaOH aq. at 55 °C for 12 h. The list of the synthesized oligonu-
cleotides is shown in Table 1. In all cases, they were obtained
in moderate to good yields with high purities and their mo-
lecular weights were confirmed by MALDI-TOF-MS.
ON1a
ON1b
ON1c
ON1d
ON2a
ON2b
ON2c
ON2d
ON3a
ON3b
ON3c
ON3d
ON4a
ON4b
ON4c
ON4d
ON5a
ON6a
5′-GCGTTTTOHTTGCT-3′
5′-GCGTTTTOMTTGCT-3′
5′-GCGTTTTNHTTGCT-3′
5′-GCGTTTTNMTTGCT-3′
5′-GCGTTTTOH
5′-GCGTTTTOM
5′-GCGTTTTNH
5′-GCGTTTTNM
5′-GCGTTTTOHTTGCT-3′
5′-GCGTTTTOMTTGCT-3′
5′-GCGTTTTNHTTGCT-3′
5′-GCGTTTTNMTTGCT-3′
̲
TTGCT-3′
TTGCT-3′
TTGCT-3′
TTGCT-3′
̲
̲
̲
5′-GCGTTTTOH
5′-GCGTTTTOM
5′-GCGTTTTNH
5′-GCGTTTTNM
5′-GCGTTTOH
̲
TTGCT-3′
TTGCT-3′
TTGCT-3′
TTGCT-3′
̲
̲
̲
T
OH
TOHTGCT-3′
5′-GCGTTOHTTOHTTOHGCT-3′
a Bold letters: Modified thymidines (see Scheme 2).
out and the results are summarized in Table 2. In general, the
binding affinity of singly modified ON1–4 against complemen-
tary DNA and RNA was greatly affected by the 2′-substituent
and almost no effect by the 4′-modification was observed. The
order of stabilization was 2′-deoxy (ON1a–d), 2′-methoxy
(ON2a–d), 2′-amino (ON3a–d) and 2′-acetamido (ON4a–d)
groups. 2′-Amino and 2′-acetamido modifications led to an
especially large decrease in the duplex stability compared to
that of the unmodified DNA/DNA duplex (Tm = 51 °C). It is
Evaluation of duplex-forming ability
UV melting experiments of the synthesized ON1–4 with single
modification and DNA or RNA complement, 5′-d-
(AGCAAAAAACGC)-3′ or 5′-r(AGCAAAAAACGC)-3′, were carried
‡Purchased from Glen Research.
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Table 2 UV melting experiments with DNA and RNA complementsa,b
DNA complement
RNA complement
Tm
ΔTm/mod.c,d
(°C)
Tm
(°C)
ΔTm/mod.c,d
(°C)
Oligonucleotide (°C)
ON1a
ON1b
ON1c
ON1d
ON2a
ON2b
ON2c
ON2d
ON3a
ON3b
ON3c
ON3d
ON4a
ON4b
ON4c
ON4d
51
51
51
51
48
48
48
48
38
37
38
38
35
35
35
35
0
47
46
46
46
44
43
43
43
41
37
38
37
35
35
35
34
+1
0
0
0
0
0
0
−3
−2
−3
−3
−3
−3
Fig. 2 CD spectra of duplexes with RNA complements by DNA, ON1a, ON1b,
ON3a and ON3b.
−3
−3
−13 (−2)
−14 (−3)
−13 (−3)
−13 (−3)
−16
−5 (+3)
−9 (−1)
−8 (0)
−9 (−1)
−11
−11
−11
−12
−16
−16
−16
a Conditions: 10 mM sodium phosphate buffer (pH 7.2) and
100 mM NaCl. The final concentration of each oligonucleotide
used was 4 μM. The sequences of DNA and RNA complements
were 5′-d(AGCTTTTTTCGC)-3′ and 5′-r(AGCTTTTTTCGC)-3′,
respectively. b Tm values of unmodified DNA/DNA and DNA/RNA
duplexes were 51 °C and 46 °C, respectively. c The changes in Tm
per modification compared with the unmodified duplexes are
shown. d In parentheses, the changes in Tm per modification
Fig. 3 UV melting profiles of duplexes with RNA complements by ON1a,
ON5a and ON6a.
compared with the duplexes by 5′-GCGTTTTTTGCT-3′ (T
=
2′-aminothymidine) are shown. The Tm values of the duplexes with
DNA and RNA complements were 40 °C and 38 °C, respectively.
formed with RNA.12 However, in our case, only thymidine and
2′-aminothymidine with modifications by 4′-carboxy groups
known that 2′-amino modification drastically destabilizes stabilized the duplex with RNA compared to their 4′-unmodi-
duplexes11 and a UV melting experiment of 2′-aminothymi- fied ones. To study global conformational changes in duplexes
dine-modified oligonucleotide, 5′-GCGTTTTTTGCT-3′ (T
=
of ON1a, ON1b, ON3a and ON3b with RNA complements, cir-
2′-aminothymidine), under the same conditions showed cular dichroism (CD) spectroscopy was measured (Fig. 2). As a
decreased Tm values of 40 °C and 38 °C by 11 °C and 8 °C com- result, no major change of the global conformations was
pared to those of unmodified DNA/DNA and DNA/RNA observed although in the case of ON1b containing 4′-methoxy-
duplexes, respectively (see ESI† for the preparation of 2′-ami- carbonylthymidine the positive band at around 260–280 nm
nothymidine-modified oligonucleotide). From these points, was red-shifted.
the decreased binding affinity of 2′-amino-modified ON3a–d
Duplex-forming ability of oligonucleotides ON5a and ON6a
was considered to be reasonable. In the case of 2′-acetamido including three 4′-carboxythymidine units consecutively and
modification (ON4a–d), the destabilization of the formed alternately led to the highest stabilization of the duplex with
duplex was likely caused by the steric bulkiness of the 2′-aceta- an RNA complement. Under the same conditions shown in
mido group.
Table 2, the ability was compared to singly modified ON1a and
It was very interesting that 4′-carboxy modifications of thy- the UV melting profiles are displayed in Fig. 3. The Tm value of
midine and 2′-aminothymidine (ON1a and ON3a) had some ON5a with consecutive modifications was comparable to that
apparent effect on the duplex stability with RNA complements. of ON1a with a single modification. This indicated that con-
The Tm values of ON1a and ON3a were increased by 1 °C and secutive modifications led to a significant decrease in ΔTm/
3 °C compared to those of the 4′-unmodified oligonucleotides, mod. (+1 °C → +0.3 °C), perhaps due to slight electrostatic
46 °C for a natural DNA/RNA duplex and 37 °C for the duplex repulsion by contiguous carboxylate ions. On the other hand,
between 2′-amino-modified oligonucleotide and RNA comp- the Tm value of ON6a with alternate modifications was 49 °C.
lement, respectively, though the Tm value of ON3a was much Its ΔTm/mod. was 1 °C, almost the same as that of ON1a, and
lower than that of ON1a due to destabilization by the 2′-amino it formed a stable duplex with an RNA complement. The 1H
modification.11 Very recently, Leumann’s group reported that NMR spectrum of 4′-carboxythymidine13 exhibited a J1′2′ value
the existence of a carboxy group at the C6′-position of tricyclo- of 6.7 Hz, meaning that it preferentially adopted the S-type
DNA did not perturb the pairing affinity of tricyclo-DNA with conformation.10 These results could indicate that 4′-carboxy-
an RNA complement and in their case, carboxy and carbamoyl thymidine significantly stabilized the duplex with an RNA
modifications apparently increased stability of the duplexes complement compared to 4′-methoxymethylthymidine (ΔTm/
9642 | Org. Biomol. Chem., 2012, 10, 9639–9649
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mod. = up to 0.6 °C) or 4′-aminomethylthymidine (ΔTm/mod. combined organic layer was washed with water and brine,
= up to 0.5 °C), which also adopted S-type conformations.
dried over Na2SO4, and concentrated in vacuo. The residue
(1.52 g) was purified by column chromatography (silica gel,
30 g, n-hexane–EtOAc = 1 : 1) to give compound 7 (1.01 g, 79%)
as a white foam.
Experimental
General
Mp: 58–61 °C. [α]D23 −47.6 (c 1.00, CHCl3). IR: νmax (KBr):
3182, 3033, 2952, 2868, 2614, 2484, 1954, 1867, 1715, 1591,
All moisture-sensitive reactions were carried out in well-dried 1489, 1470, 1361, 1277 cm−1 1H NMR (400 MHz, CDCl3) δ:
.
glassware under a N2 atmosphere. 1H, 13C and 31P spectra were 1.57 (d, J = 1.0 Hz, 3H), 3.70 (s, 3H), 3.86 (d, J = 10.0 Hz, 1H),
recorded on JEOL JNM-EX300 and JEOL JNM-EX400 spec- 4.10 (d, J = 10.0 Hz, 1H), 4.59 (d, J = 11.5 Hz, 1H), 4.61 (d, J =
trometers. Chemical shifts are reported in parts per million 10.5 Hz, 1H), 4.68 (d, J = 10.5 Hz, 1H), 4.68 (d, J = 11.0 Hz, 1H),
referenced to tetramethylsilane (δ = 0.00 ppm) for 1H NMR 4.80 (d, J = 6.0 Hz, 1H), 5.79 (dd, J = 6.0, 7.5 Hz, 1H), 6.66 (d, J
spectra, CDCl3 (δ = 77.0 ppm) and CD3OD (δ = 49.0 ppm) for = 7.5 Hz, 1H), 6.96–7.40 (m, 16H), 8.80 (brs, 1H). 13C NMR
13C NMR spectra, and phosphoric acid (δ = 0.00 ppm) for 31P (100 MHz, CDCl3) δ: 12.06, 52.69, 71.83, 73.91, 75.47, 78.70,
NMR spectra. IR spectra were recorded on a JASCO FT/IR-4200 81.50, 86.38, 89.67, 111.84, 115.28, 121.55, 126.79, 127.71,
spectrometer. Optical rotations were recorded on a JASCO 127.85, 128.05, 128.25, 128.38, 128.66, 129.57, 135.75, 136.74,
P-2200 polarimeter. FAB mass spectra were measured on JEOL 136.85, 150.26, 153.28, 163.47, 168.82, 194.42. MS (FAB): m/z =
JMS-600 or JEOL JMS-700 mass spectrometers. MALDI-TOF 633 [M + H]+. HRMS (FAB): calcd for C33H33N2O9S [M + H]+,
mass spectra were recorded on a Bruker Daltonics Autoflex II 633.1907, found, 633.1920.
TOF/TOF mass spectrometer. Fuji Silysia silica gel PSQ-60B
3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYLTHYMIDINE
(8). Under
a
(0.060 mm) and FL-60D (0.060 mm) were used for flash nitrogen atmosphere, n-Bu3SnH (0.81 mL, 3.00 mmol) and
column chromatography. For HPLC, SHIMADZU LC-10ATVP, AIBN (33.1 mg, 0.202 mmol) were added to a solution of com-
SHIMADZU SPD-10AVP and SHIMADZU CTO-10VP instruments pound 7 (1.00 g, 1.58 mmol) in anhydrous toluene (20 mL) at
were used.
room temperature. The reaction mixture was refluxed for 2 h.
3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYL-5-METHYLURIDINE (6). PhI- The resulting mixture was concentrated in vacuo and the
(OAc)2 (3.86 g, 12.0 mmol) and TEMPO (170 mg, 1.09 mmol) residue (2.00 g) was purified by column chromatography (silica
were added to a solution of compound 56 (2.55 g, 5.45 mmol) gel, 60 g, n-hexane–EtOAc = 1 : 1) to give compound 8 (424 mg,
in MeCN–H2O (1 : 1, 30 mL) at room temperature. The reaction 55%) as a white foam.
mixture was stirred for 12 h at room temperature. The result-
ing mixture was concentrated in vacuo, and the residue (5.98 g) 3183, 3065, 3034, 2952, 2920, 2871, 1695, 1496, 1454, 1434,
was co-evaporated with anhydrous MeCN three times, and dis- 1399, 1365, 1281, 1207 cm−1 1H NMR (400 MHz, CDCl3) δ:
Mp: 72–74 °C. [α]D23 +24.6 (c 1.00, CHCl3). IR: νmax (KBr):
.
solved in anhydrous MeOH (30 mL). TMSCHN2 (2 M in 1.56 (d, J = 1.0 Hz, 3H), 2.13–2.22 (m, 1H), 2.53–2.61 (m, 1H),
n-hexane, 3.0 mL, 6.0 mmol) was added to the solution at room 3.75 (s, 3H), 3.88 (d, J = 10.0 Hz, 1H), 4.08 (d, J = 10.0 Hz, 1H),
temperature. The reaction mixture was stirred for 0.5 h at 4.48–4.61 (m, 5H), 6.58 (t, J = 6.5 Hz, 1H), 6.96–7.40 (m, 10H),
room temperature. The resulting mixture was concentrated in 7.52 (d, J = 1.0 Hz, 1H), 9.27 (brs, 1H). 13C NMR (75 MHz,
vacuo and the residue (6.11 g) was purified by column chrom- CDCl3) δ: 12.03, 37.71, 52.42, 70.98, 72.42, 73.74, 79.59, 85.70,
atography (silica gel, 150 g, n-hexane–EtOAc = 2 : 3) to give 89.90, 111.08, 127.41, 127.48, 127.62, 127.87, 128.11,
compound 6 (1.94 g, 72% for 2 steps from 5) as a white foam.
128.39, 128.48, 128.58, 135.81, 136.98, 137.06, 150.24, 163.84,
Mp: 62–64 °C. [α]D23 −35.4 (c 1.00, CHCl3). IR: νmax (KBr): 169.80. MS (EI): m/z = 480 (M+, 14.3), 355 (11.8), 261 (13.6), 248
3449, 3177, 3065, 3033, 2952, 2870, 1713, 1472, 1455, 1373, (14.2), 217 (85.5), 181 (29.5), 157 (13.8), 127 (16.3), 91 (100).
1
1279, 1240 cm−1. H NMR (300 MHz, CDCl3) δ: 1.59 (d, J = 1.0 HRMS (EI): calcd for C26H28N2O7 [M+], 480.1897, found,
Hz, 3H), 3.72 (s, 3H), 3.76–3.82 (m, 2H), 4.05 (d, J = 10.0 Hz, 480.1900.
1H), 4.55–4.74 (m, 5H), 6.21 (d, J = 6.5 Hz, 1H), 7.25–7.41 (m,
4′-METHOXYCARBONYLTHYMIDINE (9).7 Under a nitrogen atmos-
11H), 9.05 (brs, 1H). 13C NMR (75 MHz, CDCl3) δ: 12.09, 52.74, phere, a solution of compound 8 (420 mg, 0.874 mmol) in
71.83, 73.86, 74.36, 75.28, 80.24, 89.08, 89.20, 111.51, 127.61, MeOH (15 mL) and cyclohexene (8.8 mL, 87 mmol) were
128.02, 128.25, 128.26, 128.53, 128.69, 135.59, 136.59, 136.82, added to a suspension of 20% Pd(OH)2 on carbon (307 mg,
150.76, 163.54, 169.58. MS (FAB): m/z = 497 [M + H]+. HRMS 0.437 mmol) in MeOH (5.0 mL) at room temperature. The reac-
(FAB): calcd for C26H29N2O8 [M + H]+, 497.1924, found, tion mixture was refluxed for 12 h. The resulting mixture was
497.1923.
3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYL-2′-O-PHENOXYTHIOCARBONYL- (360 mg) was purified by column chromatography (silica gel,
5-METHYLURIDINE (7). Under nitrogen atmosphere, DMAP 10 g, CHCl3–MeOH = 20 : 1 to 7 : 1) to give compound 9
(493 mg, 4.04 mmol) and PhOCSCl (0.33 mL, 2.42 mmol) were (118 mg, 45%) as a white foam.
filtered and the filtrate was concentrated in vacuo. The residue
a
added to a solution of compound 6 (1.00 g, 2.02 mmol) in
1H NMR (400 MHz, CD3OD) δ: 1.87 (d, J = 1.0 Hz, 3H),
anhydrous CH2Cl2 (20 mL) at 0 °C. The reaction mixture was 2.32–2.36 (m, 2H), 3.75 (s, 3H), 3.92 (d, J = 12.0 Hz, 1H), 3.96
stirred at room temperature for 0.5 h. After addition of sat. (d, J = 12.0 Hz, 1H), 4.57 (dd, J = 5.5, 6.5 Hz, 1H), 6.48 (t, J = 6.5
NaHCO3 at 0 °C, the mixture was extracted with CH2Cl2. The Hz, 1H), 7.74 (d, J = 1.0 Hz, 1H). 13C NMR (75 MHz, CD3OD)
This journal is © The Royal Society of Chemistry 2012
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δ: 12.47, 40.56, 52.55, 64.25, 73.33, 87.12, 93.37, 111.70,
Mp: 40–42 °C. [α]D24 −6.8 (c 1.00, CHCl3). IR: νmax (KBr):
138.43, 152.26, 166.40, 172.39.
5′-O-(4,4′-DIMETHOXYTRITYL)-4′-METHOXYCARBONYLTHYMIDINE
3409, 3065, 3031, 2930, 2858, 1711, 1668, 1496, 1455, 1428,
1
(10).7 1362, 1265, 1209 cm−1. H NMR (300 MHz, CDCl3) δ: 1.06 (s,
Under a nitrogen atmosphere, DMTrCl (120 mg, 0.355 mmol) 9H), 1.62 (d, J = 1.0 Hz, 3H), 3.76–3.82 (m, 4H), 4.64 (d, J =
was added to solution of compound (71.0 mg, 11.0 Hz, 1H), 4.68 (s, 2H), 4.76 (d, J = 11.0 Hz, 1H), 5.45 (d, J =
a
9
0.236 mmol) in anhydrous pyridine (2.0 mL) at 0 °C. The reac- 9.5 Hz, 1H), 5.49 (d, J = 9.5 Hz, 1H), 6.00 (d, J = 2.0 Hz, 1H),
tion mixture was stirred at room temperature for 1 h. After 7.19–7.68 (m, 26H). 13C NMR (75 MHz, CDCl3) δ: 12.76, 19.02,
addition of sat. NaHCO3 at 0 °C, the mixture was extracted 26.75, 64.11, 70.47, 71.83, 72.10, 73.66, 73.86, 74.50, 78.06,
with EtOAc. The combined organic layer was washed with 88.20, 92.17, 110.90, 127.53, 127.60, 127.67, 127.80, 127.87,
water and brine, dried over Na2SO4, and concentrated in vacuo. 127.96, 128.03, 128.09, 128.22, 128.52, 128.55, 129.92, 129.95,
The residue (210 mg) was purified by column chromatography 132.06, 132.09, 135.24, 135.62, 137.18, 137.25, 137.97, 151.18,
(silica gel, 10 g, CHCl3–MeOH = 15 : 1) to give compound 10 163.46. MS (FAB): m/z = 827 [M + H]+. HRMS (FAB): calcd for
(118 mg, 83%) as a white foam.
C49H55N2O8Si [M + H]+, 827.3728, found, 827.3726.
3-N-BENZYLOXYMETHYL-3′,5′-DI-O-BENZYL-4′-TERT-BUTYLDIPHENYLSILOXY-
1H NMR (300 MHz, CDCl3) δ: 1.38 (d, J = 1.0 Hz, 3H),
2.37–2.56 (m, 2H), 3.58 (d, J = 10.0 Hz, 1H), 3.70 (d, J = METHYL-2′-O-METHYL-5-METHYLURIDINE (13). Under
a
nitrogen
10.0 Hz, 1H), 3.73 (s, 3H), 3.78 (s, 6H), 4.77 (dd, J = 3.0, 6.5 Hz, atmosphere, NaH (127 mg, 3.17 mmol) was added to a solu-
1H), 6.65 (dd, J = 6.0, 8.0 Hz, 1H), 6.81–7.40 (m, 13H), 7.55 (d, tion of compound 12 (2.50 g, 3.02 mmol) in anhydrous DMF
J = 1.0 Hz, 1H), 9.51 (brs, 1H). 13C NMR (75 MHz, CDCl3) (15 mL) at 0 °C. The reaction mixture was stirred at 0 °C for
δ: 11.60, 39.99, 52.59, 55.17, 65.03, 73.62, 85.66, 87.17, 91.55, 1 h. MeI (0.20 mL, 3.2 mmol) was added to the above mixture
111.36, 113.19, 127.15, 127.95, 128.06, 130.08, 134.77, 134.89, at 0 °C, and the mixture was further stirred at room tempera-
136.29, 143.91, 150.34, 158.61, 158.63, 163.93, 170.71.
3′-O-[2-CYANOETHOXY(DIISOPROPYLAMINO)PHOSPHINO]-5′-O-(4,4′-DIMETH- extracted with Et2O. The combined organic layer was washed
OXYTRITYL)-4′-METHOXYCARBONYLTHYMIDINE (1). Under nitrogen with brine, dried over Na2SO4, and concentrated in vacuo. The
ture for 1 h. After the addition of H2O at 0 °C, the mixture was
a
atmosphere, DIPEA (0.17 mL, 0.97 mmol) and i-Pr2NP(Cl)- residue (3.21 g) was purified by column chromatography (silica
OCH2CH2CN (52 μL, 0.23 mmol) were added to a solution of gel, 50 g, n-hexane–EtOAc = 3 : 1) to give compound 13 (1.48 g,
compound 10 (117 mg, 0.194 mmol) in anhydrous CH2Cl2 58%) as a colorless oil.
(2.0 mL) at 0 °C. The reaction mixture was stirred at room
[α]2D4 +15.4 (c 1.00, CHCl3). IR: νmax (KBr): 3068, 3031, 2955,
temperature for 3 h. After addition of sat. NaHCO3 at 0 °C, the 2930, 2858, 1707, 1668, 1463, 1428, 1389, 1362, 1298, 1254,
mixture was extracted with CH2Cl2. The combined organic 1211 cm−1. 1H NMR (300 MHz, CDCl3) δ: 1.05 (s, 9H), 1.45 (d, J
layer was washed with sat. NaHCO3, water and brine, dried = 1.0 Hz, 3H), 3.36 (s, 3H), 3.68 (d, J = 11.0 Hz, 1H), 3.71 (dd, J
over Na2SO4, and concentrated in vacuo. The residue (166 mg) = 3.5, 5.5 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 4.02 (d, J = 11.0 Hz,
was chromatographed (silica gel, 10 g, n-hexane–EtOAc = 2 : 3) 1H), 4.11 (d, J = 11.0 Hz, 1H), 4.27 (d, J = 5.5 Hz, 1H), 4.45 (d, J
to give 1 with a small amount of impurity (99.8 mg), which = 12.0 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.58 (d, J = 12.0 Hz,
was reprecipitated from n-hexane–CHCl3 to give compound 1 1H), 4.66 (d, J = 12.0 Hz, 1H), 4.69 (s, 2H), 5.46 (d, J = 9.5 Hz,
(86.7 mg, 56%) as a white powder.
1H), 5.50 (d, J = 9.5 Hz, 1H), 5.82 (d, J = 3.5 Hz, 1H), 7.20–7.71
Mp: 88–90 °C. H NMR (400 MHz, CDCl3) δ: 1.05–1.19 (m, (m, 26H). 13C NMR (75 MHz, CDCl3) δ: 12.52, 19.24, 26.88,
12H), 1.32 (d, J = 1.5 Hz, 1.5H), 1.33 (d, J = 1.5 Hz, 1.5H), 58.61, 64.47, 70.36, 70.67, 72.06, 73.97, 73.53, 75.55, 83.23,
2.41–2.65 (m, 4H), 3.50–3.80 (m, 15H), 4.84–4.91 (m, 0.5H), 87.75, 87.88, 109.66, 127.53, 127.59, 127.61, 127.64, 127.70,
4.98–5.06 (m, 0.5H), 6.61 (t, J = 8.0 Hz, 0.5H), 6.65 (t, J = 127.87, 127.98, 128.20, 128.34, 128.47, 128.53, 129.56, 129.69,
8.0 Hz, 0.5H), 6.82–7.41 (m, 13H), 7.59 (d, J = 1.5 Hz, 0.5H), 132.94, 133.40, 134.73, 135.56, 135.80, 137.40, 137.51, 138.00,
7.62 (d, J = 1.5 Hz, 0.5H), 9.26 (brs, 1H). 31P NMR (161 MHz, 150.72, 163.46. MS (FAB): m/z = 841 [M + H]+. HRMS (FAB):
CDCl3) δ: 149.89, 150.02. MS (FAB): m/z = 803 [M + H]+. HRMS calcd for C50H57N2O8Si [M + H]+, 841.3884, found, 841.3882.
1
(FAB): calcd for C42H52N4O10P [M + H]+, 803.3421, found,
803.3430.
3-N-BENZYLOXYMETHYL-3′,5′-DI-O-BENZYL-4′-HYDROXYMETHYL-2′-O-
METHYL-5-METHYLURIDINE (14). TBAF (1
M
in THF, 1.8 mL,
3-N-BENZYLOXYMETHYL-3′,5′-DI-O-BENZYL-4′-TERT-BUTYLDIPHENYLSILOXY- 1.8 mmol) were added to a solution of compound 13 (1.40 g,
METHYL-5-METHYLURIDINE (12). Under nitrogen atmosphere, 1.66 mmol) in THF (20 mL) at room temperature. The reaction
a
DBU (0.71 mL, 4.8 mmol) and BOMCl (0.61 mL, 4.4 mmol) mixture was stirred at room temperature for 11 h. The reaction
were added to a solution of compound 118 (2.60 g, 3.67 mmol) was concentrated in vacuo and the residue (2.54 g) was purified
in anhydrous THF (15 mL) at 0 °C. The reaction mixture was by column chromatography (silica gel, 50 g, n-hexane–EtOAc =
stirred at room temperature for 0.5 h. After addition of sat. 3 : 1 to 1 : 1) to give compound 14 (1.07 g, quant.) as a colorless
NaHCO3 at 0 °C, the mixture was extracted with EtOAc. The oil.
combined organic layer was washed with water and brine,
[α]1D9 +78.3 (c 1.10, CHCl3). IR: νmax (KBr): 3499, 3064, 3030,
1
dried over Na2SO4, and concentrated in vacuo. The residue 2941, 2867, 1708, 1668, 1496, 1455, 1362, 1274, 1209 cm−1. H
(3.40 g) was purified by column chromatography (silica gel, NMR (400 MHz, CDCl3) δ: 1.48 (d, J = 1.0 Hz, 3H), 2.75 (t, J =
60 g, n-hexane–EtOAc = 3 : 1) to give compound 12 (2.62 g, 7.0 Hz, 1H), 3.59 (s, 3H), 3.68 (d, J = 10.5 Hz, 1H), 3.75–3.87
86%) as a white foam.
(m, 4H), 4.40 (d, J = 6.0 Hz, 1H), 4.47 (d, J = 11.0 Hz, 1H), 4.48
9644 | Org. Biomol. Chem., 2012, 10, 9639–9649
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
(d, J = 11.5 Hz, 1H), 4.53 (d, J = 11.5 Hz, 1H), 4.68 (s, 2H), 4.75 J = 12.0 Hz, 1H), 4.05 (dd, J = 5.0, 7.5 Hz, 1H), 4.45 (d, J =
(d, J = 11.0 Hz, 1H), 5.44 (d, J = 10.0 Hz, 1H), 5.47 (d, J = 5.0 Hz, 1H), 6.20 (d, J = 7.5 Hz, 1H), 7.75 (d, J = 1.0 Hz, 1H).
10.0 Hz, 1H), 6.08 (d, J = 2.5 Hz, 1H), 7.19–7.38 (m, 15H), 7.63 13C NMR (100 MHz, CD3OD) δ: 12.45, 52.72, 58.50, 65.14,
(d, J = 1.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 12.48, 59.14, 72.05, 83.28, 88.44, 92.48, 112.13, 138.22, 152.41, 166.17,
63.59, 70.30, 70.33, 72.04, 73.00, 73.48, 75.86, 83.28, 87.21, 171.68. MS (FAB): m/z = 331 [M + H]+. HRMS (FAB): calcd for
89.28, 109.82, 127.50, 127.56, 127.60, 127.66, 127.99, 128.15, C13H19N2O8 [M + H]+, 331.1141, found, 331.1149.
128.48, 128.51, 134.52, 137.08, 137.14, 137.86, 150.59, 163.30.
5′-O-(4,4′-DIMETHOXYTRITYL)-4′-METHOXYCARBONYL-2′-O-METHYL-5-
MS (EI): m/z = 602 (M+, 14.0), 511 (16.3), 496 (96.4), 249 (10.2), METHYLURIDINE (17). Under a nitrogen atmosphere, DMTrCl
235 (20.5), 181 (34.0), 140 (23.5), 111 (24.1), 99 (37.4), 91 (100). (380 mg, 1.12 mmol) was added to a solution of compound 16
HRMS (EI): calcd for C34H38N2O8 [M+], 602.2628, found, (285 mg, 0.863 mmol) in anhydrous pyridine (5.0 mL) at 0 °C.
602.2623.
The reaction mixture was stirred at room temperature for 3 h.
3-N-BENZYLOXYMETHYL-3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYL-2′-O- After addition of sat. NaHCO3 at 0 °C, the mixture was
METHYL-5-METHYLURIDINE (15). PhI(OAc)2 (1.23 g, 3.80 mmol) and extracted with EtOAc. The combined organic layer was washed
TEMPO (53.9 mg, 0.345 mmol) were added to a solution of with water and brine, dried over Na2SO4, and concentrated
compound 14 (1.04 g, 1.73 mmol) in MeCN–H2O (1 : 1, 20 mL) in vacuo. The residue (710 mg) was purified by column chrom-
at room temperature. The reaction mixture was stirred for 12 h atography (silica gel, 15 g, CHCl3–MeOH = 30 : 1) to give com-
at room temperature. The resulting mixture was concentrated pound 17 (464 mg, 85%) as a white foam.
in vacuo, and the residue (1.88 g) was co-evaporated with anhy-
Mp: 81–91 °C. [α]D22 +4.8 (c 1.00, CHCl3). IR: νmax (KBr):
drous MeCN three times, and dissolved in anhydrous MeOH 3469, 3224, 3065, 2953, 2936, 2837, 1697, 1607, 1580, 1509,
1
(10 mL). TMSCHN2 (2M in n-hexane, 0.95 mL, 1.9 mmol) was 1464, 1387, 1252 cm−1. H NMR (400 MHz, CDCl3) δ: 1.40 (d, J
added to this solution at room temperature. The reaction = 1.0 Hz, 3H), 3.10 (d, J = 5.0 Hz, 1H), 3.54 (s, 3H), 3.56 (d, J =
mixture was stirred for 0.5 h at room temperature. The result- 12.0 Hz, 1H), 3.73 (d, J = 12.0 Hz, 1H), 3.74 (s, 3H), 3.78 (s,
ing mixture was concentrated in vacuo and the residue (1.26 g) 6H), 4.05 (t, J = 5.0 Hz, 1H), 4.60 (t, J = 5.0 Hz, 1H), 6.26 (d, J =
was purified by column chromatography (silica gel, 30 g, 5.0 Hz, 1H), 6.83–7.40 (m, 13H), 7.48 (d, J = 1.0 Hz, 1H), 9.96
n-hexane–EtOAc = 3 : 1 to 2 : 1) to give compound 15 (566 mg, (brs, 1H). 13C NMR (100 MHz, CDCl3) δ: 11.61, 52.52, 55.11,
52% for 2 steps from 14) as a colorless oil.
58.67, 64.76, 71.39, 82.33, 87.11, 87.20, 88.47, 111.44, 113.20,
[α]2D1 +9.7 (c 1.00, CHCl3). IR: νmax (KBr): 3064, 3031, 2952, 123.67, 127.12, 127.92, 128.00, 130.02, 134.62, 134.83, 135.19,
2927, 2867, 1762, 1713, 1673, 1497, 1454, 1363, 1278, 1239, 136.07, 143.85, 149.41, 150.37, 158.64, 163.96, 169.91. MS
1
1210 cm−1. H NMR (400 MHz, CDCl3) δ: 1.62 (d, J = 1.0 Hz, (FAB): m/z
= 655 [M +
Na]+. HRMS (FAB): calcd for
3H), 3.40 (s, 3H), 3.69 (s, 3H), 3.80 (d, J = 10.5 Hz, 1H), 3.96 C34H36N2NaO10 [M + Na]+, 655.2268, found, 655.2286.
(dd, J = 5.5, 6.0 Hz, 1H), 4.05 (d, J = 10.5 Hz, 1H), 4.35 (d, J =
5.5 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.60 (d, J = 11.5 Hz, 1H), OXYTRITYL)-4′-METHOXYCARBONYL-2′-O-METHYL-5-METHYLURIDINE
3′-O-[2-CYANOETHOXY(DIISOPROPYLAMINO)PHOSPHINO]-5′-O-(4,4′-DIMETH-
(2).
4.62 (d, J = 11.5 Hz, 1H), 4.68 (s, 2H), 4.79 (d, J = 11.5 Hz, 1H), Under a nitrogen atmosphere, DIPEA (0.63 mL, 3.5 mmol) and
5.46 (d, J = 9.5 Hz, 1H), 5.49 (d, J = 9.5 Hz, 1H), 6.40 (d, J = i-Pr2NP(Cl)OCH2CH2CN (0.18 mL, 0.84 mmol) were added to a
6.0 Hz, 1H), 7.23–7.38 (m, 15H), 7.42 (d, J = 1.0 Hz, 1H). 13C solution of compound 17 (444 mg, 0.702 mmol) in anhydrous
NMR (100 MHz, CDCl3) δ: 12.78, 52.52, 58.87, 70.47, 71.48, CH2Cl2 (5.0 mL) at 0 °C. The reaction mixture was stirred at
72.03, 73.77, 74.22, 78.38, 82.81, 87.96, 88.78, 110.63, 127.50, room temperature for 3 h. After addition of sat. NaHCO3 at
127.53, 127.56, 127.65, 127.67, 127.82, 128.18, 128.26, 128.54, 0 °C, the mixture was extracted with CH2Cl2. The combined
128.62, 134.44, 136.87, 137.30, 137.83, 151.01, 163.24, 169.33. organic layer was washed with sat. NaHCO3, water and brine,
MS (EI): m/z = 630 (M+, 3.6), 539 (10.8), 524 (52.8), 247 (8.3), 91 dried over Na2SO4, and concentrated in vacuo. The residue
(100). HRMS (EI): calcd for C35H38N2O9 [M+], 630.2577, found, (639 mg) was chromatographed (silica gel, 10 g, n-hexane–
630.2559.
EtOAc = 1 : 1) to give 2 with a small amount of impurity
4′-METHOXYCARBONYL-2′-O-METHYL-5-METHYLURIDINE (16). Under a (511 mg), which was reprecipitated from n-hexane–CHCl3 to
nitrogen atmosphere, a solution of compound 15 (550 mg, give compound 2 (450 mg, 77%) as a white powder.
1
0.872 mmol) in MeOH (8.0 mL) and cyclohexene (4.4 mL,
Mp: 86–88 °C. H NMR (400 MHz, CDCl3) δ: 1.04–1.19 (m,
44 mmol) were added to a suspension of 20% Pd(OH)2 on 12H), 1.37 (d, J = 1.0 Hz, 2.1H), 1.38 (d, J = 1.0 Hz, 0.9H),
carbon (306 mg, 0.436 mmol) in MeOH (2.0 mL) at room temp- 2.42–2.47 (m, 1.4H) 2.61–2.65 (m, 0.6H), 3.51–3.80 (m, 18H),
erature. The reaction mixture was refluxed for 12 h. The result- 4.04–4.13 (m, 1H), 4.65–4.72 (m, 1H), 6.29 (d, J = 5.5 Hz, 0.7H),
ing mixture was filtered and the filtrate was concentrated 6.32 (d, J = 6.0 Hz, 0.3H), 6.81–7.41 (m, 13H), 7.45 (d, J =
in vacuo. The residue (350 mg) was purified by column chrom- 1.0 Hz, 0.3H), 7.51 (d, J = 1.0 Hz, 0.7H), 8.15–8.21 (m, 1H). 31P
atography (silica gel, 10 g, CHCl3–MeOH = 15 : 1 to 7 : 1) to give NMR (161 MHz, CDCl3) δ: 151.02, 151.56. MS (FAB): m/z = 833
compound 16 (295 mg, quant.) as a white foam.
[M + H]+. HRMS (FAB): calcd for C43H54N4O11P [M + H]+,
Mp: 112–114 °C. [α]2D1 −16.4 (c 1.00, CH3OH). IR: νmax (KBr): 833.3527, found, 833.3551.
3506, 3285, 3050, 2955, 2833, 1702, 1471, 1378, 1277,
(2′R)-2′-AZIDO-3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYLTHYMIDINE (19).
1
1218 cm−1. H NMR (400 MHz, CD3OD) δ: 1.88 (d, J = 1.0 Hz, Under a nitrogen atmosphere, TMSCHN2 (2.0 M in n-hexane,
3H), 3.39 (s, 3H), 3.75 (s, 3H), 3.81 (d, J = 12.0 Hz, 1H), 4.00 (d, 0.13 mL, 0.26 mmol) was added to a solution of compound
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9639–9649 | 9645

Paper
Organic & Biomolecular Chemistry
189 (107 mg, 0.211 mmol) in anhydrous THF–MeOH (1 : 1, 6.5 Hz, 1H), 4.51 (d, J = 11.0 Hz, 1H), 4.57 (d, J = 11.0 Hz, 1H),
2.0 mL) at room temperature. The reaction mixture was stirred 4.59 (d, J = 11.5 Hz, 1H), 4.64 (d, J = 11.5 Hz, 1H), 4.71 (dt, J =
at ambient temperature for 0.5 h. The resulting mixture was 6.5, 8.0 Hz, 1H), 6.43 (d, J = 8.0 Hz, 1H), 7.25–7.41 (m, 11H),
concentrated in vacuo and the residue (113 mg) was purified 7.68 (d, J = 8.0 Hz, 1H), 9.44 (brs, 1H). 13C NMR (100 MHz,
by column chromatography (silica gel, 5 g, n-hexane–EtOAc = CDCl3) δ: 12.16, 52.87, 55.66, 71.21, 73.96, 75.66, 79.59, 87.18,
2 : 1) to give compound 19 (97.5 mg, 89%) as a white foam.
90.06, 112.26, 115.50 (q, J = 286 Hz), 127.82, 127.86, 128.35,
Mp: 159–161 °C. [α]D31 −50.9 (c 1.00, CHCl3). IR: νmax (KBr): 128.40, 128.58, 128.69, 128.84, 128.95, 134.99, 136.19, 136.75,
3179, 3066, 3031, 2954, 2927, 2878, 2110, 1755, 1691, 1664, 150.84, 157.75 (q, J = 38 Hz), 163.63, 169.38. MS (FAB): m/z =
1
1455, 1376, 1274 cm−1. H NMR (300 MHz, CDCl3) δ: 1.67 (d, J 592 [M + H]+. HRMS (FAB): calcd for C28H29F3N3O8 [M + H]+,
= 1.0 Hz, 3H), 3.67 (s, 3H), 3.77 (dd, J = 6.0, 8.5 Hz, 1H), 3.79 592.1908, found, 592.1886.
(d, J = 10.0 Hz, 1H), 4.06 (d, J = 10.0 Hz, 1H), 4.41 (d, J =
(2′R)-4′-METHOXYCARBONYL-2′-TRIFLUOROACETAMIDOTHYMIDINE
(23).
6.0 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.62 (d, J = 11.5 Hz, 1H), Under a nitrogen atmosphere, a solution of compound 21
4.62 (d, J = 11.0 Hz, 1H), 4.81 (d, J = 11.0 Hz, 1H), 6.49 (d, J = (95.5 mg, 0.162 mmol) in MeOH (4.0 mL) and cyclohexene
8.5 Hz, 1H), 7.28–7.39 (m, 11H), 8.31 (brs, 1H). 13C NMR (0.80 mL, 8.0 mmol) were added to a suspension of 20% Pd-
(75 MHz, CDCl3) δ: 12.29, 52.78, 63.82, 71.81, 74.04, 75.40, (OH)2 on carbon (57.0 mg, 0.0810 mmol) in MeOH (1.0 mL) at
81.50, 86.14, 89.81, 111.96, 127.75, 127.76, 128.22, 128.24, room temperature. The reaction mixture was refluxed for 10 h.
128.27, 128.46, 128.48, 128.82, 128.84, 134.93, 136.45, 136.69, The resulting mixture was filtered and the filtrate was concen-
150.39, 163.59, 168.95. MS (FAB): m/z = 522 [M + H]+. HRMS trated in vacuo. The residue (55.6 mg) was purified by column
(FAB): calcd for C26H28N5O7 [M + H]+, 522.1998, found, chromatography (silica gel, 10 g, CHCl3–MeOH = 15 : 1) to give
522.1973.
compound 23 (40.8 mg, 62%) as a white foam.
(2′R)-2′-AMINO-3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYLTHYMIDINE (20).
Mp: 108–110 °C. [α]D22 −5.7 (c 1.00, MeOH). IR: νmax (KBr):
Under a nitrogen atmosphere, NiCl2 (9.2 mg, 0.0709 mmol) 3481, 3261, 3068, 2957, 2841, 1710, 1552, 1471, 1388,
1
and NaBH4 (80.5 mg, 2.13 mmol) were added to a solution of 1276 cm−1. H NMR (400 MHz, CD3OD) δ: 1.90 (d, J = 1.0 Hz,
compound 19 (370 mg, 0.709 mmol) in anhydrous THF–MeOH 3H), 3.77 (s, 3H), 3.84 (d, J = 12.0 Hz, 1H), 4.08 (d, J = 12.0 Hz,
(1 : 1, 2.0 mL) at 0 °C. The reaction mixture was stirred at room 1H), 4.45 (d, J = 6.0 Hz, 1H), 4.75 (dd, J = 6.0, 8.5 Hz, 1H), 6.32
temperature for 0.5 h. The resulting mixture was filtrated and (d, J = 8.5 Hz, 1H), 7.81 (d, J = 1.0 Hz, 1H). 13C NMR (100 MHz,
the filtrate was concentrated in vacuo. The residue (550 mg) CD3OD) δ: 12.48, 52.78, 56.83, 65.09, 72.89, 87.99, 93.60,
was purified by column chromatography (silica gel, 20 g, 112.52, 117.20 (q, J = 285 Hz), 137.77, 152.68, 159.21 (q, J =
CHCl3–MeOH = 40 : 1) to give compound 20 (313 mg, 89%) as 38 Hz), 166.17, 171.46. MS (FAB): m/z = 412 [M + H]+. HRMS
a white powder.
(FAB): calcd for C14H17F3N3O8 [M + H]+, 412.0969, found,
Mp: 75–78 °C. [α]2D8 −27.0 (c 1.00, CHCl3). IR: νmax (KBr): 412.0966.
3372, 3178, 3066, 3031, 2948, 2923, 2867, 1759, 1703, 1496,
(2′R)-5′-O-(4,4′-DIMETHOXYTRITYL)-4′-METHOXYCARBONYL-2′-TRIFLUORO-
1
1472, 1454, 1394, 1364, 1278 cm−1. H NMR (300 MHz, CDCl3) ACETAMIDOTHYMIDINE (25). Under a nitrogen atmosphere, DMTrCl
δ: 1.60 (d, J = 1.0 Hz, 3H), 3.58 (dd, J = 6.0, 8.5 Hz, 1H), 3.69 (s, (80.0 mg, 0.238 mmol) was added to a solution of compound
3H), 3.80 (d, J = 10.0 Hz, 1H), 4.09 (d, J = 10.0 Hz, 1H), 4.21 (d, 23 (61.1 mg, 0.149 mmol) in anhydrous pyridine (2.0 mL) at
J = 6.0 Hz, 1H), 4.58 (d, J = 11.5 Hz, 1H), 4.63 (d, J = 11.0 Hz, 0 °C. The reaction mixture was stirred at room temperature for
1H), 4.66 (d, J = 11.5 Hz, 1H), 4.68 (d, J = 11.0 Hz, 1H), 6.14 (d, 3 h. After addition of sat. NaHCO3 at 0 °C, the mixture was
J = 8.5 Hz, 1H), 7.26–7.41 (m, 12H). 13C NMR (75 MHz, CDCl3) extracted with EtOAc. The combined organic layer was washed
δ: 12.21, 52.63, 58.75, 72.46, 73.96, 75.56, 82.39, 89.12, 89.83, with water and brine, dried over Na2SO4, and concentrated in
111.31, 127.76, 128.08, 128.23, 128.27, 128.53, 128.59, 128.81, vacuo. The residue (152 mg) was purified by column chromato-
130.79, 135.58, 137.01, 137.03, 151.00, 163.80, 169.85. MS graphy (silica gel, 10 g, CHCl3–MeOH = 50 : 1) to give com-
(FAB): m/z = 496 [M + H]+. HRMS (FAB): calcd for C26H30N3O7 pound 25 (95.7 mg, 90%) as a white foam.
[M + H]+, 496.2086, found, 496.2070.
(2′R)-3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYL-2′-TRIFLUOROACETAMIDO- 3414, 3271, 3066, 2955, 2839, 1703, 1608, 1509, 1466, 1386,
THYMIDINE (21). Under a nitrogen atmosphere, Et3N (12 μL, 1293, 1255 cm−1 1H NMR (400 MHz, CDCl3) δ: 1.40 (d, J =
Mp: 127–130 °C. [α]D22 −3.4 (c 0.66, CHCl3). IR: νmax (KBr):
.
0.085 mmol) and CF3CO2Et (24 μL, 0.19 mmol) were added to 1.0 Hz, 1H), 3.08 (d, J = 4.0 Hz, 1H), 3.58 (d, J = 10.0 Hz, 1H),
a solution of compound 20 (84.4 mg, 0.170 mmol) in anhy- 3.69, (d, J = 10.0 Hz, 1H), 3.78 (s, 3H), 3.80 (s, 6H), 4.64 (dd, J =
drous MeOH (2.0 mL) at 0 °C. The reaction mixture was stirred 4.0, 5.0 Hz, 1H), 4.39 (dt, J = 5.0, 8.5 Hz, 1H), 6.39 (d, J =
at room temperature for 1 h. The resulting mixture was con- 8.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.85–7.38 (m, 13H), 7.53
centrated in vacuo and the residue (134 mg) was purified by (d, J = 1.0 Hz, 1H), 8.06 (brs, 1H). 13C NMR (100 MHz, CDCl3)
column chromatography (silica gel, 5 g, n-hexane–EtOAc = δ: 11.49, 52.97, 55.30, 55.72, 65.43, 72.78, 85.91, 88.01, 91.04,
1 : 1) to give compound 21 (92.3 mg, 92%) as a white foam.
112.44, 113.49, 115.57 (q, J = 286 Hz), 127.50, 128.27, 130.28,
Mp: 75–77 °C. [α]D22 −19.8 (c 1.00, CHCl3). IR: νmax (KBr): 134.49, 134.69, 135.63, 135.65, 143.58, 151.36, 157.38, 157.95
3235, 3070, 2928, 2863, 1709, 1556, 1469, 1386, 1276 cm−1. H (q, J = 38 Hz), 158.94, 158.97, 164.39, 169.85. MS (FAB): m/z =
1
NMR (400 MHz, CDCl3) δ: 1.89 (d, J = 0.5 Hz, 3H), 3.71 (s, 3H), 714 [M + H]+, HRMS (FAB): calcd for C35H35F3N3O10 [M + H]+,
3.83 (d, J = 10.0 Hz, 1H), 4.03 (d, J = 10.0 Hz, 1H), 4.48 (d, J = 714.2276, found, 714.2247.
9646 | Org. Biomol. Chem., 2012, 10, 9639–9649
This journal is © The Royal Society of Chemistry 2012

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Paper
(2′R)-3′-O-[2-CYANOETHOXY(DIISOPROPYLAMINO)PHOSPHINO]-5′-O-(4,4′- = 7 : 1 → 4 : 1) to give compound 24 (181 mg, 72%) as a white
DIMETHOXYTRITYL)-4′-METHOXYCARBONYL-2′-TRIFLUOROACETAMIDOTHYMI- powder.
DINE (3). Under a nitrogen atmosphere, DIPEA (0.11 mL,
0.62 mmol) and i-Pr2NP(Cl)OCH2CH2CN (30 μL, 0.14 mmol) 3483, 3288, 3066, 2961, 2833, 1682, 1530, 1471, 1439, 1377,
were added to
solution of compound 25 (88.7 mg, 1291 cm−1. 1H NMR (300 MHz, CD3OD) δ: 1.89 (s, 3H), 1.94 (s,
Mp: 162–163 °C. [α]D27 −15.2 (c 0.85, MeOH). IR: νmax (KBr):
a
0.124 mmol) in anhydrous CH2Cl2 (2.0 mL) at 0 °C. The reac- 3H), 3.75 (s, 3H), 3.81 (d, J = 11.5 Hz, 1H), 4.05 (d, J = 11.5 Hz,
tion mixture was stirred at room temperature for 2 h. After 1H), 4.34 (d, J = 6.0 Hz, 1H), 4.70 (dd, J = 6.0, 9.0 Hz, 1H), 6.20
addition of sat. NaHCO3 at 0 °C, the mixture was extracted (d, J = 9.0 Hz, 1H), 7.81 (s, 1H). 13C NMR (75 MHz, CD3OD) δ:
with CH2Cl2. The combined organic layer was washed with sat. 12.48, 22.37, 52.68, 56.35, 65.30, 73.23, 88.12, 93.46, 112.11,
NaHCO3, water and brine, dried over Na2SO4, and concen- 138.04, 152.82, 166.22, 171.67, 173.82. MS (FAB): m/z = 358
trated in vacuo. The residue (122 mg) was chromatographed [M + H]+. HRMS (FAB): calcd for C14H20N3O8 [M + H]+,
(silica gel, 10 g, CHCl3–MeOH = 50 : 1) to give 6 with a small 358.1252, found, 358.1252.
amount of impurity (119 mg), which was reprecipitated from
n-hexane–CHCl3 to give compound 3 (102 mg, 95%) as a white NYLTHYMIDINE (26). Under
powder. (186 mg, 0.550 mmol) was added to a solution of compound
(2′R)-2′-ACETAMIDO-5′-O-(4,4′-DIMETHOXYTRITYL)-4′-METHOXYCARBO-
a
nitrogen atmosphere, DMTrCl
1
Mp: 85–88 °C. H NMR (400 MHz, CDCl3) δ: 1.07–1.16 (m, 24 (65.5 mg, 0.183 mmol) in anhydrous pyridine (2.0 mL) at
12H), 1.40 (d, J = 1.0 Hz, 1.5H), 1.43 (d, J = 1.0 Hz, 1.5H), 0 °C. The reaction mixture was stirred at room temperature for
2.36–2.80 (m, 2H), 3.45–3.80 (m, 15H), 4.86–5.08 (m, 2H), 6.35 3 h. After addition of sat. NaHCO3 at 0 °C, the mixture was
(d, J = 6.0 Hz, 0.5 H), 6.40 (d, J = 6.0 Hz, 0.5H), 6.86–7.52 (m, extracted with EtOAc. The combined organic layer was washed
15H), 7.95–7.99 (m, 1H). 31P NMR (160 MHz, CDCl3) δ: 151.96, with water and brine, dried over Na2SO4, and concentrated in
153.04. MS (FAB): m/z = 914 [M + H]+. HRMS (FAB): calcd for vacuo. The residue (315 mg) was purified by column chromato-
C44H52F3N5O11P [M + H]+, 914.3355, found, 914.3348.
(2′R)-2′-ACETAMIDO-3′,5′-DI-O-BENZYL-4′-METHOXYCARBONYLTHYMIDINE pound 26 (123 mg, quant.) as a white foam.
(22). Under a nitrogen atmosphere, Ac2O (80 μL, 0.85 mmol)
Mp: 142–144 °C. [α]D27 −19.5 (c 1.00, CHCl3). IR: νmax (KBr):
was added to solution of compound 20 (352 mg, 3489, 3323, 3188, 3063, 3017, 2953, 2835, 1694, 1607, 1579,
0.710 mmol) in anhydrous pyridine (2.0 mL) at 0 °C. The reac- 1509, 1465, 1378, 1291 cm−1 1H NMR (400 MHz, CDCl3) δ:
graphy (silica gel, 10 g, CHCl3–MeOH = 20 : 1) to give com-
a
.
tion mixture was stirred at room temperature for 1.5 h. After 1.34 (d, J = 1.0 Hz, 3H), 1.99 (s, 3H), 3.51 (d, J = 10.0 Hz, 1H),
addition of sat. NaHCO3 at 0 °C, the mixture was extracted 3.69 (d, J = 10.0 Hz, 1H), 3.64 (d, J = 4.5 Hz, 1H), 3.73 (s, 3H),
with EtOAc. The combined organic layer was washed with 3.78 (s, 6H), 4.57 (dd, J = 4.5, 5.5 Hz, 1H), 5.02 (dt, J = 5.5,
water and brine, dried over Na2SO4, and concentrated in vacuo. 8.5 Hz, 1H), 6.36 (d, J = 8.5 Hz, 1H), 6.64 (d, J = 8.5 Hz, 1H),
The residue (438 mg) was purified by column chromatography 6.84–7.42 (m, 13H), 7.55 (d, J = 1.0 Hz, 1H), 9.16 (brs, 1H). 13C
(silica gel, 15 g, CHCl3–MeOH = 30 : 1) to give compound 22 NMR (100 MHz, CDCl3) δ: 11.56, 23.00, 52.84, 54.95, 55.26,
(408 mg, quant.) as a white foam.
65.56, 72.71, 85.99, 87.74, 90.75, 112.05, 113.40, 127.33,
Mp: 85–88 °C. [α]D24 −31.4 (c 1.10, CHCl3). IR: νmax (KBr): 128.14, 128.25, 130.23, 130.25, 134.55, 134.77, 135.29, 143.63,
3317, 3204, 3067, 3033, 2952, 2927, 2867, 1762, 1683, 1543, 151.26, 158.85, 163.63, 170.21, 171.11. MS (FAB): m/z = 660
1
1455, 1374, 1283 cm−1. H NMR (300 MHz, CDCl3) δ: 1.56 (d, [M + H]+. HRMS (FAB): calcd for C35H38N3O10 [M + H]+,
J = 1.0 Hz, 3H), 1.84 (s, 3H), 3.67 (s, 3H), 3.81 (d, J = 10.0 Hz, 660.2559, found, 660.2538.
1H), 4.03 (d, J = 10.0 Hz, 1H), 4.39 (d, J = 6.0 Hz, 1H), 4.53 (d,
(2′R)-2′-ACETAMIDO-3′-O-[2-CYANOETHOXY(DIISOPROPYLAMINO)PHOSPHINO]-
J = 11.5 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.60 (d, J = 12.0 Hz, 5′-O-(4,4′-DIMETHOXYTRITYL)-4′-METHOXYCARBONYLTHYMIDINE (4). Under
1H), 4.68 (d, J = 12.0 Hz, 1H), 4.77 (dt, J = 6.0, 8.5 Hz), 6.41 (d, a nitrogen atmosphere, DIPEA (0.15 mL, 0.86 mmol) and
J = 8.5 Hz, 1H), 6.98 (d, J = 8.5 Hz), 7.26–7.39 (m, 10H), 7.51 (d, i-Pr2NP(Cl)OCH2CH2CN (45 μL, 0.21 mmol) were added to a
J = 1.0 Hz, 1H), 9.84 (brs, 1H). 13C NMR (75 MHz, CDCl3) δ: solution of compound 26 (113 mg, 0.127 mmol) in anhydrous
12.16, 22.76, 52.62, 55.45, 71.72, 73.85, 75.48, 80.68, 86.61, CH2Cl2 (2.0 mL) at 0 °C. The reaction mixture was stirred at
90.07, 111.83, 127.75, 128.11, 128.14, 128.18, 128.21, 128.45, room temperature for 4 h. After addition of sat. NaHCO3 at
128.50, 128.75, 135.46, 136.89, 137.00, 151.15, 163.83, 169.42, 0 °C, the mixture was extracted with CH2Cl2. The combined
171.02. MS (FAB): m/z = 538 [M + H]+. HRMS (FAB): calcd for organic layer was washed with sat. NaHCO3, water and brine,
C28H32N3O8 [M + H]+, 538.2191, found, 538.2194.
(2′R)-2′-ACETAMIDO-4′-METHOXYCARBONYLTHYMIDINE (24). Under
dried over Na2SO4, and concentrated in vacuo. The residue
(210 mg) was chromatographed (silica gel, 10 g, CHCl3–MeOH
a
nitrogen atmosphere, a solution of compound 22 (380 mg, = 20 : 1) to give 4 with a small amount of impurity (113 mg),
0.707 mmol) in THF–MeOH (1 : 1, 8.0 mL) and cyclohexene which was reprecipitated from n-hexane–CHCl3 to give com-
(3.6 mL, 35 mmol) were added to a suspension of 20% Pd- pound 4 (103 mg, 68%) as a white powder.
(OH)2 on carbon (199 mg, 0.283 mmol) in THF–MeOH (1 : 1,
Mp: 92–95 °C. 1H NMR (400 MHz, CDCl3) δ: 1.08–1.18
2.0 mL) at room temperature. The reaction mixture was (m, 12H), 1.35 (s, 1.8H), 1.37 (s, 1.2H), 2.00 (s, 1.8H), 2.03
refluxed for 10 h. The resulting mixture was filtered and the fil- (s, 1.2H), 2.40–2.64 (m, 2H), 3.51–3.80 (m, 15H), 4.68–4.76
trate was concentrated in vacuo. The residue (330 mg) was puri- (m, 1H), 4.99–5.07 (m, 1H), 6.23–6.41 (m, 2H), 6.83–7.43
fied by column chromatography (silica gel, 10 g, CHCl3–MeOH (m, 13H), 7.53 (m, 1H), 8.15–8.21 (m, 1H). 31P NMR (160 MHz,
This journal is © The Royal Society of Chemistry 2012
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Organic & Biomolecular Chemistry
CDCl3) δ: 152.39, 152.99. MS (FAB): m/z = 860 [M + H]+. HRMS CD measurements
(FAB): calcd for C44H55N5O11P [M + H]+, 860.3637, found,
860.3608.
Under the same conditions as those of UV melting exper-
iments, CD measurements were carried out from 350 nm to
200 nm at 4 °C on a JASCO J-720W spectropolarimeter.
Synthesis of oligonucleotides
Phosphoramidites 1–4 were used and the 0.2 μmol scale syn-
thesis of oligonucleotides was performed on an automated
DNA synthesizer (Applied Biosystems ExpediteTM 8909) using a
standard phosphoramidite protocol (DMTr-ON mode). ON1–6
were prepared by cleavage from the CPG supports, deprotec-
tion of nucleobase and phosphate moieties, and conversion of
4′-carboxyl units by treatment with the corresponding base
[50 mM NaOH aq., rt, 1.5 h then 55 °C, 12 h (for ON1a–6a);
50 mM K2CO3 in MeOH, rt, 2 h (for ON1b–4b); 28% NH3 aq.,
rt, 1.5 h then 55 °C, 12 h (for ON1c–4c); or 40% MeNH aq., rt,
1.5 h then 55 °C, 12 h (for ON1d–4d)]. After neutralization
with 1% HCl aq. for ON1a–6a and ON1b–4b, the solvent was
concentrated in vacuo. For ON1a–6a and ON1b–4b, removal of
ammonia or methylamine was carried out in vacuo. The crude
ON1–6 were purified with Sep-Pak® Plus C18 cartridges
(Waters) followed by reversed-phase HPLC (Waters XBridge®
MS C18 2.5 μm, 10 × 50 mm). The composition of the ON1–6
was confirmed by MALDI-TOF mass analysis. Yields and MAL-
DI-TOF-MS data ([M − H]−) for ON1–6; ON1a, 43% yield, found
3676.23 (calcd 3676.37); ON1b, 30% yield, found 3690.24
(calcd 3690.40); ON1c, 30% yield, found 3674.55 (calcd
3675.39); ON1d, 28% yield, found 3689.01 (calcd 3689.51);
ON2a, 38% yield, found 3706.23 (calcd 3706.40); ON2b, 33%
yield, found 3720.98 (calcd 3720.42); ON2c, 32% yield, found
3705.91 (calcd 3705.41); ON2d, 29% yield, found 3719.61
(calcd 3719.44); ON3a, 43% yield, found 3690.69 (calcd
3691.39); ON3b, 51% yield, found 3706.06 (calcd 3705.41);
ON3c, 40% yield, found 3689.73 (calcd 3690.40); ON3d, 61%
yield, found 3704.78 (calcd 3704.43); ON4a, 36% yield, found
3732.43 (calcd 3733.42); ON4b, 50% yield, found 3746.94
(calcd 3747.45); ON4c, 52% yield, found 3731.93 (calcd
3732.44); ON4d, 28% yield, found 3746.03 (calcd 3746.46);
ON5a, 29% yield, found 3765.21 (calcd 3764.39); ON6a, 49%
yield, found 3764.75 (calcd 3764.39).
Conclusions
The synthesis of oligonucleotides including 16 kinds of 4′-car-
boxythymidines was achieved by base treatment after oligonu-
cleotide synthesis. Evaluation of their duplex-forming ability
with DNA or RNA complements found that 4′-carboxythymi-
dine led to an increase in stability of the duplex formed with
RNA. In the recent report by Leumann’s group, tc-DNA with a
hexadodecyloxycarbonyl unit enhanced cellular uptake.12
Therefore, the carboxyl group of 4′-carboxythymidine that we
synthesized in this work might be practically useful as a
prodrug functional group. Thus, 4′-carboxythymidine was con-
sidered to be a potent candidate for targeting RNA.
Acknowledgements
This work was supported in part by the Program to Dissemi-
nate Tenure Tracking System of the Ministry of Education,
Culture, Sports, Science and Technology, Japan (MEXT), a
Grant-in-Aid for Scientific Research on Innovative Areas (no.
22136006) from MEXT, and the Program for Promotion of Fun-
damental Studies in Health Sciences of the National Institute
of Biomedical Innovation (NIBIO).
Notes and references
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UV melting experiments were carried out on SHIMADZU
UV-1650 and SHIMADZU UV-1800 spectrophotometers
equipped with Tm analysis accessory. ON1–6 and ssDNA or
ssRNA were dissolved in 10 mM sodium phosphate buffer (pH
7.2) containing 100 mM NaCl to give a final concentration of
each strand of 4 μM. The samples were annealed by heating at
100 °C followed by slow cooling to 15 °C. The melting profiles
were recorded at 260 nm from 15 °C to 85 °C at a scan rate of
0.5 °C min−1. The two-point average method was employed to
obtain the Tm values and the final ones were determined by
averaging three independent measurements which were accu-
rate to within 1 °C.
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