Novel synthesis of 2′-O-methylguanosine

Article abstract of DOI:10.1016/S0960-894X(03)00291-9

An efficient and chemoselective synthesis of 2′-O-methylguanosine (6) has been accomplished in high yield without protection of the guanine base. The salient feature of the synthesis of 6 lies in the application of methylene-bis-(diisopropylsilyl chloride), (MDPSCl₂, 2) as a new 3′,5′-O-protecting group for nucleosides. Use of CH₃Cl as a weak electrophile and NaHMDS as a mild base was crucial to the success of the 2′-O-methylation of 3′,5′-O-protected guanosine.

Full text of DOI:10.1016/S0960-894X(03)00291-9

Bioorganic& Medicinal Chemistry Letters 13 (2003) 1631–1634
Novel Synthesis of 20-O-methylguanosine
Suetying Chow,a Ke Wen,a Yogesh S. Sanghvib and Emmanuel A. Theodorakisa,*
aDepartment of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
bISIS Pharmaceuticals, Inc., Carlsbad Research Center, 2292 Faraday Avenue, Carlsbad, CA 92008, USA
Received 25 January 2003; accepted 19 March 2003
Abstract—An efficient and chemoselective synthesis of 20-O-methylguanosine (6) has been accomplished in high yield without pro-
tection of the guanine base. The salient feature of the synthesis of 6 lies in the application of methylene-bis-(diisopropylsilyl chlor-
ide), (MDPSCl2, 2) as a new 30,50-O-protecting group for nucleosides. Use of CH3Cl as a weak electrophile and NaHMDS as a mild
base was crucial to the success of the 20-O-methylation of 30,50-O-protected guanosine.
# 2003 Elsevier Science Ltd. All rights reserved.
In the last decade, several oligonucleotide-based drugs
have entered human clinical trials for the treatment of a
variety of viral, infectious or cancer-related diseases.1
Among these, 20-O-methyl ribonucleotides (Me-RN)
have been extensively utilized as a second-generation
oligonucleotide constructs due to their favorable in-
vitro and in-vivo properties.2,3 Additionally, Me-RN
have found recent applications in studying pre-mRNA
splicing, examining the structures of spliceosomes and
preparing nuclease-resistant hammerhead ribozymes.4
Me-RN have been found as minor components in RNA
of various origins.
and their incompatibility with any large-scale applica-
tions. Moreover, the low yields of product obtained
under these conditions and the concomitant formation
of the undesired 30-O-methyl isomer further complicates
purification.5 Alternatively, the use methyl iodide in
combination with sodium hydride9 or silver oxide10 has
been explored on nucleobase-protected ribonucleosides.
The problems of regioselectivity of methylation have led
to efforts to simultaneously protect the 30- and 50-OH
groups using tetraisopropyl dichlorodisilyloxane
(TIPDSCl2, 1),11 or TBSCl2.7,12 Since these protecting
groups were found to be labile under strong alkaline
conditions they require the use of sterically hindered
organicbases, such as BDDDP or BEMP, 13 whose high
cost renders this method unsuitable for any large-scale
nucleoside synthesis.14
The exceptional value of Me-RN has stimulated a con-
siderable effort towards synthesis of 20-O-methylated
nucleosides as key building blocks using a variety of
syntheticapproaches. Although some of these strategies
have been successfully implemented to the production
of pyrimidine containing 20-O-methyl nucleosides, they
are much less efficient in the case of the purine ana-
logues.5 In the latter case, the problems derive from the
inherent reactivity of the purine bases towards alkyl-
ation (chemoselectivity) and the need for selective pro-
tection of the 30- and 50-OH groups (regioselectivity).6,7
We have recently reported on the synthesis of a novel
silicon-based protecting group, referred to as methyl-
ene-bis-(diisopropylsilyl chloride), (MDPSCl2, 2) (Fig.
1).15 The design of this reagent was based on the
hypothesis that the fragility of TIPDSCl2, (1) was due to
the inductive effect of the oxygen atom that inter-
connects the two silicon groups. We envisioned that this
effect could favor the formation of a pentacoordinated
silicon intermediate during the basic conditions required
for alkylation, thereby accelerating its deprotection and
giving rise to side products formed by a non-selective
alkylation.16 Based on this hypothesis, we developed
reagent 2 in which a methylene unit is used to bridge the
two silicon atoms. Being isosteric to 1, we predicted that
2 would exhibit a similar reactivity and selectivity pro-
file as 1, but display an extended stability under basic
Initially methylation procedures were developed using
diazomethane or trimethylsilyldiazomethane on par-
tially protected nucleosides.8 However, these approa-
ches suffer from the toxicity of the methylating reagents
*Corresponding author. Tel.:+1-858-822-456; fax:+1-858-822-0386;
e-mail: etheodor@ucsd.edu
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00291-9

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S. Chow et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1631–1634
during methylation of 4 with other bases, such as
KOtBu and BEMP.
Further search for an ideal base resulted in a promising
lead with NaHMDS that in combination with MeI gave
rise to a mixture of monoalkylated adducts 5 and 7 in a
ratio of 4:6. Use of Li and K as alternative cations did
not provide further improvements over NaHMDS. The
encouraging initial results obtained with NaHMDS
together with its overall advantages versus other bases,
including low cost, good solubility and safer handling,
led us to attempt further improvements of the alkylation
reaction by tuning the nature of the electrophile. To this
end, we explored the use of (CH3O)2SO2, (CH3O)2POH,
CF3SO3CH3 and CH3Cl as potential electrophiles in
combination with NaHMDS. Among them, use of
CH3Cl furnished the desired O-alkylated product 5
together with dimethylated material 8 in 9:1 ratio and
excellent overall yield.18 The major adduct 5 was
obtained in 83% isolated yield by a simple extraction
and precipitation from the crude reaction mixture.19
This compound was easily crystallized and its structure
was further confirmed via a single crystal X-ray analysis
(Scheme 1). It is important to note that the methylation
was performed at lower temperature (À40 to 25 ꢀC)
because CH3Cl is a gas at higher temperatures. The
combination of NaHMDS as the base and CH3Cl as an
electrophile at low temperature was critical to the suc-
cess of this reaction.20,21 It is also worth mentioning that
guanosine protected with disilane 1 underwent depro-
tection under the above alkylation conditions, support-
ing our hypothesis that the fragility of 1 is derived from
the presence of the oxygen atom.
Figure 1. Structures of TIPDSCl2 (1) and MDPSCl2 (2).
conditions. Herein, we describe an application of 2 to a
new regio- and chemo-selective synthesis of 20-O-
methylguanosine (6).
The synthesis of 20-O-methylguanosine (6) is described
in Scheme 1. Protection of guanosine (3) proceeded
using a slight excess of disilane 2 in the presence of imi-
dazole and produced the desired 30,50-protected nucleo-
side 4 in 79% yield.17 The excellent regioselectivity of
the protection is attributed to the steric effects of the
isopropyl groups that favor initially silylation of the 50-
hydroxyl group and subsequently cyclization at the
neighboring 30-hydroxyl group.
To minimize the synthesis steps and maximize the over-
all efficiency of our strategy, we decided to examine the
methylation of 4 without prior protection of the
nucleobase. Some of our efforts to develop such a
chemoselective alkylation are summarized in Table 1.
Our initial studies, with traditionally used NaH and
methyl iodide, resulted in formation of substantial
amounts of N-alkylated product 7 together with dialky-
lated adduct 8 (Fig. 2). Although the decrease of tem-
perature slowed the formation of dialkylated adduct 8,
it did not improve upon the formation of desired pro-
duct 5. Undesirable N-alkylation was also predominant
The desilylation of 5 was achieved with both TBAF and
.
3HF Et3N as the fluoride sources. In our hands, use of
.
3HF Et3N proved to be advantageous, since it produced
compound 6, free of any residual ammonium salts, in
91% yield without column chromatography.
Table 1. Effect of base during methylation of 4
Base (3.0 equiv)
Temp (ꢀC)
R-X (equiv)
Product ratio
5:7:8
NaH
NaH
NaH
BEMP
KOtBu
NaHMDS
NaHMDS
NaHMDS
0
0
MeI (1.4)
MeI (5.0)
MeI (5.0)
MeI (5.0)
MeI (5.0)
MeI (5.0)
MeI (5.0)
MeCl (excess)
1:8:1
1:1:8
1:6:3
1:7:2
1:8:1
1:7:2
4:6:0
9:0:1
À30
0
0
0
À20
À40
Scheme 1. Reagents and conditions: (a) 1.15 equiv 2, 5.0 equiv imid,
DMF, 0–25ꢀꢀC, 5 h, 79%; (b) 3.0 equiv NaHMDS, MeCl(g), DMF,
91%.
ꢀ
.
À40 to À27 C, 5 h, 83%; (c) 1.0 equiv 3HF Et3N, THF, 35 C, 14 h,
Figure 2. Structures of mono- and di-alkylated adducts 7 and 8.

S. Chow et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1631–1634
1633
In conclusion, we describe herein an efficient chemo-
selective procedure for the synthesis of 20-O-methyl-
guanosine (6). Crucial to the success of our strategy
was the recognition that the limitations of the pre-
viously reported methods are due to the fragility of
the silicon-based protecting groups, such as 1, under
the basicocnditions required for alkylation. The
isostericsilane 2 was found to possess the necessary
stability to withstand the alkylation conditions,
thereby addressing the issue of regioselectivity of the
methylation. The chemoselectivity of this reaction was
achieved in favor of the O-alkylation by performing the
reaction in presence of NaHMDS as the base and MeCl
as the electrophile. This strategy allows the synthesis of
compound 6 in three steps and 61% overall yield. To
the best of our knowledge, this is the first report
describing a chemoselectivity for O-alkylation over
N-alkylation during methylation of guanosine with
unprotected base. An additional advantage of silane 2
over 1 is that it produces compounds that are highly
crystalline and easily isolated without the need of col-
umn chromatography.
7. Grotli, M.; Douglas, M.; Beijer, B.; Garcia, R. G.; Eritja,
R.; Sproat, B. J. Chem. Soc., Perkin Trans. 1 1997, 2779.
Gundlach, C. W., IV; Ryder, T. R.; Glick, G. D. Tetrahedron
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J. Chem. 1981, 59, 3360. Cramer, H.; Pfeiderer, W. Helv.
Chim. Acta 1996, 79, 2114. Leonard, T. E.; Bhan, P.; Miller,
P. S. Nucleosides Nucleotides 1992, 11, 1201.
9. Wagner, E.; Oberhauser, B.; Holzner, A.; Brunar, H.; Issa-
kides, G.; Schaffner, G.; Cotton, M.; Knollmueller, M.; Noe,
C. R. Nucleic Acids Res. 1991, 19, 5965. Yano, J.; Kan, L. S.;
Ts’o, P. O. P. Biochem. Biophys. Acta 1980, 629, 178.
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14. 20-O-Alkyl purine nucleosides can be prepared by cou-
pling a protected purine base to an alkylated sugar, but these
procedures add to the number of steps in a synthesis and can
result in undesirable isomers.
15. Wen, K.; Chow, S.; Sanghvi, Y. S.; Theodorakis, E. A. J.
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Acknowledgements
16. For selected recent references in the area of reactivity of
pentacoordinated silicon, see: Holmes, R. R. Chem. Rev. 1990,
90, 17. Corriu, R. J. P.; Guerin, C.; Henner, B. J. L.; Wong
Chi Man, W. W. C. Organomet. 1988, 7, 237. Schinzer, D.
Org. Synth. Highlights II 1995, 123. Corriu, R. J. P. Organo-
silicon Chem. 1994, 157. Chuit, C.; Corriu, R. J. P.; Reye, C.;
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Financial support from the BioSTAR (#00-10094) and
ISIS Pharmaceuticals, Inc. is gratefully acknowledged.
We thank Dr. P. Gantzel (UCSD, X-Ray Facility) for
his assistance with the crystallographic analysis of 5.
References and Notes
17. The effects of different bases (pyridine, imidazole,
triethylamine and lutidine) and temperature (0–70 ꢀC) for the
protection of 3 were investigated. Among them, imidazole was
found to produce the best results.
18. LC/MS analysis of the crude reaction mixture indi-
cated the presence of starting material 4 in 2% yield but
showed complete absence of any mono N1-alkylated pro-
duct 7.
1. For selected recent reviews in this area, see: Sohail, M. Drug
Disc. Today 2001, 6, 1260. Dean, N. M. Curr. Opin. Biotechnol.
2001, 12, 622. Crooke, S. T. Oncogene 2000, 19, 6651. Green,
D. W.; Roh, H.; Pippin, J.; Drebin, J. A. J. Am. Coll. Surg.
2000, 191, 93. Koller, E.; Gaarde, W. A.; Monia, B. P. Trends
Pharmacol. Sci. 2000, 21, 142. Sanghvi, Y. S. Comprehensive
Natural Products Chemistry. Editor-in-chief: Barton, D. H.
R.; Nakanishi, K. In DNA and Aspects of Molecular Biology;
Kool, E. T., Ed.; Academic: 1999; Vol.7, p 258.
2. For a recent monograph on this topic, see: Crooke, S. T.,
Ed. Antisense Drug Technology Principles, Strategies and
Applications; Marcel Dekker: New York, 2001.
3. The first generation of antisense nucleosides refers to modifi-
cations of the phosphodiester backbone by replacing the equa-
torial oxygen with a sulfur, whereas second generation refers to
the modification of sugar in addition to the backbone.
4. Beigelman, L.; McSwiggen, J. A.; Draper, K. G.; Gonzalez,
C.; Jensen, K.; Karpeisky, A. M.; Modak, A. S.; Matulic-
Admic, J.; DiRenzo, A. B.; Haeberli, P.; Sweedler, D.; Tracz,
D.; Grimm, S.; Wincott, F. E.; Thackray, V. G.; Usman, N. J.
Biol. Chem. 1995, 270, 25702.
5. For a recent account on 20-O-methylation of guanosine and
adenosine, see: Beigelman, L.; Haeberli, P.; Sweedler, D.;
Karpeisky, A. Tetrahedron 2000, 56, 1047.
6. Direct methylation of purine nucleosides has been con-
sidered but its syntheticuse is limited, due to the low regio-
selectivity of alkylation. For selected references on this topic,
see: Yamauchi, K.; Nakagima, T.; Kinoshita, M. J. Chem.
Soc., Perkin Trans 1 1980, 2787. Yamauchi, K.; Nakagima, T.;
Kinoshita, M. J. Org. Chem. 1980, 45, 3865. Pettit, G. R.;
Yamauchi, K.; Einck, J. L. Synth. Commun. 1980, 10, 25. For
a comprehensive analysis of this issue, see ref 5.
19. To a solution of compound 4 (5.0 g, 9.6 mmol) in 200 mL
DMF at À40 ꢀC was added MeCl (bubbled into the reaction
mixture for 3–5 min, approximately 5 g), followed by sodium
bis(trimethylsilyl)amide (NaHMDS 1.0 M in THF, 28.7 mL,
28.7 mmol). The reaction mixture was stirred for 5 h under
argon and subsequently quenched with methanol (5 mL).
After evaporation of the THF under reduced pressure the
remainder solution was poured into ice to provide compound
5 as a white solid (4.27 g, 7.8 mmol, 83% yield). X-ray quality
material was obtained after crystallization from CH2Cl2/
MeOH. 5: Rf=0.39 (silica, 10% methanol in dichloro-
25
methane); mp: 230–232 ꢀC (dec); ½aꢁD : +11.8 (c 0.13, CH2Cl2);
1
IR (film) nmax 1265, 1683, 3054; H NMR (400MHz, DMSO-
d6) d 10.64 (s, 1H), 7.73 (s, 1H), 6.49 (bs, 2H), 5.73 (s, 1H),
4.35 (dd, J=4.8, 9.2 Hz, 1H), 4.05 (t, J=6.4 Hz, 1H), 3.90 (dd,
J=7.0, 15.4 Hz, 2H), 3.78 (dd, J=2.2, 13.0 Hz, 1H), 3.53 (s,
3H, OCH3), 0.97–1.08 (m, 28H), 0.04 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d 157.3, 154.5, 151.0, 134.7, 117.4, 87,1,
83.9, 81.2, 71.2, 61.3, 59.5, 18.6, 18.4, 18.3, 18.3, 18.2, 18.2,
14.6, 14.5, 14.5, À9.04; HRMS, calcd for C24H43N5O5Si2
(M+Na+) 560.2695, found 560.2688.
20. The excellent regioselectivity of alkylation using HMDS
may be explained by considering that this base reacts tran-
siently with the guanine base to produce the corresponding O6-
TMS ether. The latter compound is known to be hydrolytically

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S. Chow et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1631–1634
ustable and upon extraction gives rise to the free nucleobase.
See also refs 7 and 13 on this topic.
21. For relevant references on this topic, see: Zhang, Z.-H.;
Li, T.-S.; Yang, F.; Fu, C.-G. Synth. Commun. 1998, 28, 3105.
Langer, S. H.; Connell, S.; Wender, I. J. Org. Chem. 1958, 23,
50. Sweeley, C. C.; Bentley, R.; Makita, M.; Wells, W. W. J.
Am. Chem. Soc. 1963, 85, 2497. Bruynes, C. A.; Jurriens, T. K.
J. Org. Chem. 1982, 47, 3966.