Facile and efficient approach for the synthesis of N2-dimethylaminomethylene-2′-O-methylguanosine

Article abstract of DOI:10.1016/j.bmcl.2009.11.016

A convenient method for the synthesis of N²-dimethylaminomethylene-2′-O-methylguanosine (1), which is a useful intermediate for oligonucleotide construction, was developed. We chose the di-tert-butylsilyl group and the triisopropylbenzenesulfonyl group as sugar and base protecting groups, respectively. These protecting groups were stable during the 2′-O-methylation step with MeI and NaH. Our six-step synthesis of 1 is easy to perform using commercially available reagents, and requires only three chromatographic purifications. Compound 1 was obtained in 56% yield from guanosine.

Full text of DOI:10.1016/j.bmcl.2009.11.016

Bioorganic & Medicinal Chemistry Letters 20 (2010) 129–131
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile and efficient approach for the synthesis of
N2-dimethylaminomethylene-20-O-methylguanosine
*
Tsuyoshi Mukobata, Yosuke Ochi, Yuji Ito, Shun-ichi Wada, Hidehito Urata
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
A convenient method for the synthesis of N2-dimethylaminomethylene-20-O-methylguanosine (1), which
is a useful intermediate for oligonucleotide construction, was developed. We chose the di-tert-butylsilyl
group and the triisopropylbenzenesulfonyl group as sugar and base protecting groups, respectively. These
protecting groups were stable during the 20-O-methylation step with MeI and NaH. Our six-step synthesis
of 1 is easy to perform using commercially available reagents, and requires only three chromatographic
purifications. Compound 1 was obtained in 56% yield from guanosine.
Article history:
Received 8 October 2009
Revised 2 November 2009
Accepted 6 November 2009
Available online 12 November 2009
Keywords:
Guanosine
20-Hydroxyl group
Methylation
Ó 2009 Elsevier Ltd. All rights reserved.
Triisopropylbenzenesulfonyl group
20-O-Methylguanosine
Oligonucleotides containing 20-O-methylribonucleosides have
high resistance to nucleases and form hybrids with complementary
RNAs, the thermal stability of which is higher than that of unmodi-
fied oligoribonucleotide duplexes.1–4 Because of these characteris-
tics, 20-O-methyloligonucleotides have been employed to regulate
specific gene expression in antisense and RNAi methodologies.4–6
The usefulness of 20-O-methylribonucleosides increases the impor-
tance of developing efficient methods for their synthesis. Among
thefourribonucleosides, guanosineis themostdifficulttomethylate
selectively at the 20-hydroxyl group because of the acidic N1-proton
at the base moiety. Methylation procedures for non-protected or
partially protected ribonucleosides were developed using diazo-
methane or trimethylsilyldiazomethane.7 However, the yields were
oftenvery low andthe presenceof theundesired 30-O-methylisomer
complicated the purification, which was usually achieved by anion-
exchange chromatography on Dowex. Robins et al. reported that the
use of 1 equiv of diazomethane and stannous chloride as the catalyst
gave relatively high yields of 20-O-methylribonucleosides except for
guanosine that underwent base methylation.8 The use of bifunc-
tional sugar protecting groups, such as the tetraisopropyldisiloxane
(TIPDS) bridge, has resolved the isomer issue. However, the methyl-
ation of 30,50-O-protected guanosine using MeI/NaH or MeI/Ag2O
reagent was not successful due to the undesired base methylation.2,9
Therefore, protection of the O6-position of guanosine prior to the
methylation is necessary. Recently, Chow et al. reported that the
methylation of 30,50-O-methylene-bis-diisopropylsilyl (MDPS)-gua-
nosine in the presence of sodium bis(trimethylsilyl)amide as a base
proceeded in good yield with high selectivity without the base
protection.9,10 However, MDPS-Cl2 is not commercially available
and the use of MeClgas as the methylating reagent is inconvenient
because it is difficult to handle. Therefore, one general way to avoid
the methylation at N1 of guanosine is to use 2,6-diaminopurine or
2-amino-6-chloropurine analogues11–13 as the starting material.
However, these analogues are much more expensive than guanosine
and the transformation into guanine analogues is required after the
methylation step. Anotherway to avoidthemethylation at N1 of gua-
nosine is to use O6-protected guanosine derivatives, such as
O6-nitrophenyl,14 O6-nitrophenylethyl,15 and O6-diphenylcarba-
moyl (DPC).14 O6-Nitrophenylethyl and O6-nitrophenyl protections
have been reported to be useful for the methylation of guanosine,
while O6-DPC protection gave unsatisfactory results. Grotli et al.
reported the methylation of O6-tert-butyldiphenylsilyl (TBDPS)-
30,50-O-TIPDS-guanosine with MeI and expensive 2-tert-butylimi-
no-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine
(BEMP).14,16 They achieved the five-step synthesis of N2-dimethyl-
aminomethylene-20-O-methylguanosine (48% from guanosine).
However, O6-silyl protecting groups, such as tert-butyldimethylsilyl
(TBDMS) and TBDPS, are known to be unstable during chromatogra-
phy on silica gel.17,18 Zlatev et al. reported that O6-trimethylsilyleth-
yl was stable during silica gel column chromatography.17 However,
this eight-step synthesis of N2-isobutyryl-20-O-methylguanosine
gave low yield (28% from guanosine). Meanwhile, Saneyoshi et al.
reported a convenient method for cyanoethylation at the 20-hydro-
xyl group of O6-triisopropylbenzenesulfonyl (TPS)-protected guano-
sine derivative with acrylonitrile and Cs2CO3.19 Hence, we studied
* Corresponding author. Tel./fax: +81 72 690 1089.
E-mail address: urata@gly.oups.ac.jp (H. Urata).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.016

130
T. Mukobata et al. / Bioorg. Med. Chem. Lett. 20 (2010) 129–131
Table 1
O6-Arenesulfonylation of 30,50-O-protected guanosinesa
Entry
Starting material
Reagent
Reaction time (min)
Product
Yield (%)
Product ratio
1
2
3
4
5
2
2
3
3
3
Ts-Cl (3.0 equiv)
Mes-Cl (3.0 equiv)
Ts-Cl (3.0 equiv)
Mes-Cl (3.0 equiv)
TPS-Cl (3.0 equiv)
60
75
60
75
120
4a, 4b
4c, 4d
5a, 5b
5c, 5d
5e, 5f
82.1
99.8
Quant.
Quant.
Quant.
4a:4b = 70:30
4c:4d = 78:22
5a:5b = 90:10
5c:5d = 97:3
5e:5f = 100:0
a
Reactions were carried out using recrystallized starting materials in the presence of p-toluenesulfonyl chloride (Ts-Cl), 2-mesitylenesulfonyl chloride (Mes-Cl) or TPS-Cl,
triethylamine (6 equiv) and DMAP (0.2 equiv) in CH2Cl2 at room temperature.
whether or not arenesulfonyl groups could be used as an O6-protect-
ing group under conventional MeI/NaH methylation conditions.
Herein we describe an easy and efficient method to prepare
N2-dimethylaminomethylene-20-O-methylguanosine (1).
tivity is based on the steric effect of the alkyl chain of silyl protect-
ing groups and the rigidity of the sugar moiety. As a result, we
chose to use DTBS and TPS groups as sugar and base protecting
groups, as shown in Scheme 1.
First, we studied the selectivity of the arenesulfonylation at the
O6-position of 30,50-O-protected guanosine without 20-O-protection
by using 30,50-O-TIPDS-guanosine (2) and 30,50-O-di-tert-buty-
lsilanediyl (DTBS)-guanosine (3). The results are shown in Table 1.
The arenesulfonylation of 2 showed moderate selectivity for the
O6-position, while that of 3 showed high selectivity. Moreover,
the selectivity increased as the arenesulfonylating reagents be-
came bulkier. Indeed, the arenesulfonylation of 3 using TPS-Cl
was specific for the O6-position. A conformational search suggested
that 3 has more rigid conformational features than 2, and the dis-
tance between the 20-hydroxyl group and the silicon alkyl side
chain is longer in 2 than in 3 (Fig. 1).20 This suggests that the selec-
The 30,50-hydroxyl groups of guanosine were protected using
DTBS-ditriflate and 30,50-O-DTBS guanosine 3 was given in 89%
yield. Then, compound 3 was treated with TPS-Cl, triethylamine,
and DMAP to give O6-TPS-30,50-O-DTBS-guanosine (5e). Undesired
20-O-TPS product and 6,20-O-di-TPS product were not observed
on TLC. After column chromatography, compound 5e was obtained
in 97% yield. Subsequently, we attempted to perform the methyla-
tion of 5e with MeI and NaH. However, the yield of the 20-O-methyl
derivative was only 58% because of N2-methylation. Therefore, we
considered it necessary to introduce a protecting group of the 2-
NH2 group to avoid the undesired N2-methylation. Moreover, the
amino protection is necessary to synthesize the 30-O-phosphorami-
Figure 1. Low-energy conformers of 2 and 3 calculated with OPLS_2005 force field (MACROMODEL ver. 9.1). (A) TIPDS-protected guanosine (2), (B) DTBS-protected guanosine (3);
compound 2 has more flexible sugar and silicon rings than compound 3. The average distances between the 20-hydroxyl group and the alkyl chain of the silyl protecting group
of 2 and 3 are 4.79 Å and 4.17 Å, respectively.

T. Mukobata et al. / Bioorg. Med. Chem. Lett. 20 (2010) 129–131
131
Scheme 1. Reagents and conditions: (i) di-tert-butylsilyl ditriflate/DMF, 0 °C, 1.5 h, 89.3%; (ii) 2,4,6-triisopropylbenzenesulfonyl chloride, triethylamine, DMAP/CH2Cl2, 3 h,
96.8%; (iii) N,N-dimethylformamide dimethyl acetal/DMF, overnight, 99.9%; (iv) MeI, NaH/DMF, molecular sieve 3 Å, 0 °C, 30 min, 84.7%; (v) 2-nitrobenzaldoxime, N,N,N0,N0-
tetramethyguanidine/MeCN, 1 h; (vi) triethylamine trihydrofluoride, triethylamine/THF, 1 h, 77.0%.
dite derivative, which is a synthetic unit of oligonucleotides. Then,
O6-TPS-30,50-O-DTBS-guanosine (5e) was subjected to formamidin-
ation using N,N-dimethylformamide dimethyl acetal (DMFDA).21
This reaction proceeded almost quantitatively and the resulting
mixture (6) was sufficiently pure to be used in the next step. The
methylation reaction of 6 was performed with MeI and NaH in
dry DMF at 0 °C, and was completed within 30 min. After column
chromatography, compound 7 was obtained in 85% yield.21 Thus,
the O6-TPS protection was proven to be stable under these methyl-
ation conditions. Then, compound 7 was subjected to removal of
the O6-TPS group with 2-nitrobenzaldoxime and N,N,N0,N0-tetra-
methylguanidine.19 Finally removal of the silyl group using trieth-
ylamine trihydrofluoride and triethylamine afforded N2-
dimethylaminomethylene-20-O-methylguanosine (1).19 After chro-
matographic purification, compound 1 was obtained in 77% yield.22
Supplementary data
Supplementary data (general experimental procedures) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.11.016.
References and notes
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In conclusion, we have developed a novel six-step approach
for the synthesis of N2-dimethylaminomethylene-20-O-methylgu-
anosine (1). The use of DTBS protection and NaH is more eco-
nomical than the use of TIPDS protection and
a sterically
hindered organic base, such as BEMP. Moreover, the reactions
are easy to perform and the overall yield from guanosine is
56%. From the viewpoint of laborsaving, chromatographic isola-
tion in the methylation step is not necessarily required. In this
case, 1 was obtained in 62% yield from 5e, and the overall yield
from guanosine was 53% with two chromatographic purifica-
tions. The syntheses of N2-dimethylaminomethylene-20-O-
alkylguanosines are currently underway.
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Acknowledgment
This work was supported by a Grant-in-Aid for High Technology
Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.
22. The spectroscopic data were in good agreement with previously published
data; Ref. 14.