MDPSCL2: a new protecting group for chemoselective synthesis of 2'-O-alkylated guanosines.

Article abstract of DOI:10.1081/NCN-120021959

An improved strategy for the synthesis of 2'-O-methyl-guanosine (6) and 2'-MOE-guanosine (8) is reported. The regioselectivity of the alkylation was attained using a novel silicon-based protecting group, methylene-bis (diisopropyl-silylchloride) (MDPSCl2, 2). The alkylation proceeded in a chemoselective manner using NaHMDS as the base and MeCl or MOE-Br as the appropriate electrophiles.

Full text of DOI:10.1081/NCN-120021959

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 583–587, 2003
MDPSCL2: A New Protecting Group for Chemoselective
Synthesis of 20-O-Alkylated Guanosines
Suetying Chow,1 Ke Wen,1 Yogesh S. Sanghvi,2
and Emmanuel A. Theodorakis1,
*
1Department of Chemistry and Biochemistry, University of California,
San Diego, La Jolla, California, USA
2ISIS Pharmaceuticals Inc., Carlsbad Research Center, Carlsbad,
California, USA
ABSTRACT
An improved strategy for the synthesis of 20-O-methyl-guanosine (6) and 20-
MOE-guanosine (8) is reported. The regioselectivity of the alkylation was
attained using a novel silicon-based protecting group, methylene-bis (diiso-
propyl-silylchloride) (MDPSCl2, 2). The alkylation proceeded in a chemoselective
manner using NaHMDS as the base and MeCl or MOE-Br as the appropriate
electrophiles.
INTRODUCTION
Antisense oligonucleotides constitute a promising class of therapeutic agents with
potential applications against a variety of inflammatory, infectious, cardiovascular
and malignant diseases. (For selected recent reviews in this area, see Ref.[1].) These
*Correspondence: Emmanuel A. Theodorakis, Department of Chemistry and Biochemistry,
University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA;
E-mail: etheodor@ucsd.edu.
583
DOI: 10.1081/NCN-120021959
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Chow et al.
compounds consist of short sequences of partially modified nucleic acids that are
designed to bind in a selective way to their target mRNA, thereby modulating
the expression of pathogenic proteins. Among the different strategies pursued for
the structural modifications of nucleic acids,[2] alkylations of the 20-OH group offer
several advantages including an improved binding affinity to the target RNA,
enhanced nuclease resistance and chemical stability against depurination, improved
pharmacokinetics and decreased toxicity. (For a recent monograph on this topic,
see Ref.[3].) In addition, 20-O-alkylated nucleosides maintain unaltered the basic
scaffold of the parent nucleic acid, ensuring the overall retention of base-pair
recognition, which in turn is required for target selectivity.
RESULTS AND DISCUSSION
The remarkable value of 20-O-alkylated nucleosides has provoked a considerable
effort directed toward the development of efficient synthetic approaches. (For
selected synthetic routes to 20-O-methylated nucleosides, see Ref.[4]. For selected syn-
thetic routes to 20-O-MOE nucleosides, see Ref.[5].) Although some of these strategies
have been successfully applied to the synthesis of pyrimidine containing 20-O-modi-
fied nucleic acids, they are much less efficient for purine analogs. (For a recent
account on this topic, see Ref.[6].) In the latter case, the problems stem from concur-
rent side-alkylations at both the ribose unit and the purine nucleobase, often requir-
ing the use of multiple protecting groups. The most popular strategies for protection
of the ribose unit involve the use of 1,1,3,3-tetraisopropyl-1,3-dichlorodisilyloxane
(TIPDSCl2, 1),[7] or TBSCl2.[8] Although such silanes can protect in a selective man-
ner the 30- and 50-hydroxyl groups, they are labile under strong alkaline conditions
that are required for the subsequent 20-O-alkylation.
To overcome the problems stemming from the fragility of 1 under basic condi-
tions we developed a new silicon-based protecting group, referred to herein as
methylene-bis-(diisopropylsilyl chloride), (MDPSCl2, 2).[9] The design of this reagent
was based on the hypothesis that the fragility of TIPDSCl2, (1) was due to the induc-
tive effect of the oxygen atom that bridges the two silicon groups. This effect could be
overcome using a methylene unit, instead of oxygen, to bridge the two silicon atoms.
Being isosteric to 1, we theorized that 2 would exhibit a similar reactivity and selec-
tivity profile as 1 but also display an extended stability under basic conditions.
Herein we describe an application of 2 to a chemoselective synthesis of 20-O-methyl-
guanosine (6) and 20-O-methoxyethyl (MOE) guanosine (8) (Sch. 1).
Figure 1.

Synthesis of 20-O-Alkylated Guanosines
585
Treatment of guanosine (3) with a small excess of disilane 2 in DMF using imi-
dazole as the base afforded compound 4 in 79% yield. The rate of this reaction was
slower than the one using disilane 1 as the protecting group, which may be explained
by considering that the inductive effect of the oxygen in compound 1 activates the
silicon toward nucleophilic substitution. Nonetheless, the regioselectivity during
the protection of 3 with 2 was comparable to that obtained with 1, since in both cases
the silicon atoms are hindered in a similar fashion by the presence of the isopropyl
groups. Compound 4 was easily crystallized from methanol=dichloromethane and its
structure was confirmed by a single crystal X-Ray analysis (Sch. 1).
In order to maximize the overall efficiency of this strategy we decided to study
the 20-O-methylation of 4 without protecting the nucleobase. Among the different
bases examined, use of NaHMDS in combination with MeI at ꢀ20ꢁC was found
to produce a mixture of 20-O-methylation along with N0-alkylation in a 4:6 ratio.
Further optimization of this reaction led us to employ MeCl as a less reactive elec-
trophile. In fact, best results for 20-O-methylation were obtained when this reaction
was performed in DMF at ꢀ40ꢁC using 3 equivalents of NaHMDS and excess of
Scheme 1. Reagents and Conditions: (a) 1.15 equiv 2, 5.0 equiv imid, DMF, 0ꢁC to 25ꢁC,
5 h, 79%; (b) 3.0 equiv NaHMDS, MeCl(g), DMF, ꢀ40ꢁC to ꢀ27ꢁC, 5 h, 83%; (c) 1.0 equiv
3HF-Et3N, THF, 35ꢁC, 14 h, 91%; (d) 3.0 equiv NaHMDS, 0.3 equiv TBAI, 3.0 equiv
MeOCH2CH2Br, DMF, ꢀ20ꢁC, 3 h, 85%; (e) 1.0 equiv TBAF (1.0M in THF (1.0 M in
THF), THF, 35ꢁC, 5 h, 97%.

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Chow et al.
MeCl allowing the isolation of 5 in 83% yield. The structure of 5 was unambiguously
established by a single crystal X-Ray study (Sch. 1). In a similar manner the synthesis
of 20-O-MOE guanosine (7) was pursued using NaHMDS in combination with
MOE-Br and TBAI. In this case compound 7 was obtained in 85% yield.
The choice of NaHMDS as the base proved to be essential for the chemo-
selectivity observed during the alkylation. This selectivity could be explained by
considering an initial reaction of HMDS with the nucleobase to produce the corre-
sponding O6-TMS ether. This may serve as a temporary protecting group guarding
the nucleobase against alkylation. Such compound is hydrolytically unstable and will
regenerate the free guanine upon aqueous extraction.
Deprotection of 5 and 7 was achieved with both TBAF and 3HFꢂEt3N as the
fluoride sources. This reaction was found to be considerable slower than the one
performed in identical substrates protected with TIPDSCl2 (1). Nonetheless, treat-
ing compound 7 with 1 equivalent of TBAF at 35ꢁC afforded 20-O-MOE guano-
sine (8) in 97% yield. Under similar conditions 20-O-methylguanosine (6) was
isolated together with a small amount of residual tetrabutyl ammonium salts in
95% overall yield. In our hands use of 3HFꢂEt3N was advantageous since it pro-
duced compound 6 free of any residual contaminations in 91% yield without
column chromatography.
CONCLUSION
In conclusion, we present herein an efficient and chemoselective protocol for the
synthesis of 2-O-alkylated guanosine. Essential to this strategy is the use of
MDPSCl2 (2), an isostere of TIPDSCl2 (1), that serves to protect the 50 and 30
hydroxy groups of the ribose moiety. Being more robust than 1, silane 2 withstands
the basic condition that are required during alkylation, thereby addressing the issues
of regioselectivity. Use of NaHMDS as the base is also essential for the chemo-
selectivity of alkylation. An additional advantage of silane 2 is that it produces
compounds that are highly crystalline and can be isolated without the need of
column chromatography.
REFERENCES
1. Sohail, M. Drug Disc. Today 2001, 6, 1260–1261; Dean, N.M. Curr. Opin. Bio-
technol. 2001, 12, 622–625; Crooke, S.T. Oncogene 2000, 19, 6651–6659; Green,
D.W.; Roh, H.; Pippin, J.; Drebin, J.A. J. Am. Coll. Surg. 2000, 191, 93–105;
Koller, E.; Gaarde, W.A.; Monia, B.P. Trends Pharmacol. Sci. 2000, 21, 142–
148; Sanghvi, Y.S. DNA and aspects of molecular biology. Kool, E.T., Ed.;
In Comprehensive Natural Products Chemistry; Barton, D.H.R., Nakanishi, K.,
Eds.; Academic Press, 1999; 258–311.
2. Herdewijin, P. Antisense & Nucl. Acid Drug Develop. 2000, 10, 297–310;
An, H.; Wang, T.; Maier, M.A.; Manoharan, M.; Ross, B.S.; Cook, P.D.

Synthesis of 20-O-Alkylated Guanosines
587
J. Org. Chem. 2001, 66, 2789–2801; Andrade, M.; Scozzari, A.S.; Cole, D.L.;
Ravikumar, V.T. Nucleosides Nucleotides 1997, 16, 1617–1620.
3. Antisense Drug Technology Principles, Strategies and Applications; Crooke, S.T.
Ed.; Marcel Dekker: New York, 2001.
4. Beigelman, L.; Haeberli, P.; Sweedler, D.; Karpeisky, A. Tetrahedron 2000, 56,
1047–1056; Ross, B.S.; Springer, R.H.; Tortorice, Z.; Dimock, S. Nucleosides
Nucleotides 1997, 16, 1641–1643; Chanteloup, L.; Thuong, N.T. Tetrahedron
Lett. 1994, 35, 877–880; Roy, S.K.; Tang, J.-Y. Org. Process Res. & Devel.
2000, 4, 170–171; Von Matt, P.; Lochmann, T.; Kesselring, R.; Altmann,
K.-H. Tetrahedron Lett. 1999, 40, 1873–1876; Beigelman, L.; Sweedler, D.;
Haeberli, P.; Karpeisky, A. US Patent 5,962,575, 1999.
5. Martin, P. Helv. Chim. Acta 1995, 78, 486–504; Legorburu, U.; Reese, C.B.;
Song, Q. Tetrahedron 1999, 55, 5635–5640; Altmann, K.-H.; Bevierre, M.-O.;
Mesmaeker, A.D.; Moser, H.E. Bioorg. & Med. Chem. Lett. 1995, 5, 431–436;
Cook, P.D.; Springer, R.H.; Sprankle, K.G.; Ross, B.S. US Patent 5,861,493,
1999.
6. Beigelman, L.; Haeberli, P.; Sweedler, D.; Karpeisky A. Tetrahedron 2000, 56,
1047–1056.
7. Markiewicz, W.T. J. Chem. Res. (S) 1979, 24–25; Beijer, B.; Grotli, M.; Douglas,
M.E.; Sproat, B.S. Nucleosides Nucleotides 1994, 13, 1905–1927.
8. Wada, T.; Tobe, M.; Nagayama, T.; Furusawa, K.; Sekine, M. Tetrahedron
Lett. 1995, 36, 1683–1684.
9. Wen, K.; Chow, S.; Sanghvi, Y.S.; Theodorakis, E.A. J. Org. Chem. 2002, 67,
7887–7889.