2′-O-hydroxyalkoxymethylribonucleosides and their incorporation into oligoribonucleotides

Article abstract of DOI:10.1080/15257770701543007

A simple and efficient method for the preparation of pyrimidine 2′-O-hydroxyethoxymethylribonucleosides and 2′-O- hydroxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were sh

Full text of DOI:10.1080/15257770701543007

This article was downloaded by: [Statsbiblioteket Tidsskriftafdeling]
On: 25 April 2014, At: 02:23
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
2′-O-
Hydroxyalkoxymethylribonucleosides
and their Incorporation into
Oligoribonucleotides
Sergey N. Mikhailov a , Georgii V. Bobkov a , Kyrill V. Brilliantov a
,
Jef Rozenski b , Arthur Van Aerschot b , Piet Herdewijn b , Michael H.
Fisher c & Rudolph L. Juliano c
a Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences , Moscow, Russia
b Laboratory of Medicinal Chemistry , Rega Institute for Medical
Research, Katholieke Universiteit Leuven , Leuven, Belgium
c Department of Pharmacology, School of Medicine , University of
North Carolina , Chapel Hill, North Carolina, USA
Published online: 05 Dec 2007.
To cite this article: Sergey N. Mikhailov , Georgii V. Bobkov , Kyrill V. Brilliantov , Jef Rozenski ,
Arthur Van Aerschot , Piet Herdewijn , Michael H. Fisher & Rudolph L. Juliano (2007) 2′-O-
Hydroxyalkoxymethylribonucleosides and their Incorporation into Oligoribonucleotides, Nucleosides,
Nucleotides and Nucleic Acids, 26:10-12, 1509-1512, DOI: 10.1080/15257770701543007
To link to this article: http://dx.doi.org/10.1080/15257770701543007
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Nucleosides, Nucleotides, and Nucleic Acids, 26:1509–1512, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701543007
2ꢀ-O-HYDROXYALKOXYMETHYLRIBONUCLEOSIDES AND THEIR
INCORPORATION INTO OLIGORIBONUCLEOTIDES
Sergey N. Mikhailov, Georgii V. Bobkov, and Kyrill V. Brilliantov
Engelhardt
2
Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Jef Rozenski, Arthur Van Aerschot, and Piet Herdewijn
Laboratory of
2
Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Leuven, Belgium
Michael H. Fisher and Rudolph L. Juliano
Department of Pharmacology, School
2
of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
A
simple and efficient method for the preparation of pyrimidine 2 ꢁ-O-
2
hydroxyethoxymethylribonucleosides and 2 ꢁ-O-hydroxypropoxymethylribonucleosides has been
developed. These modified nucleosides were incorporated into oligoribonucleotides, which were
shown to form stable RNA/RNA duplexes. The effect of 2 ꢁ-O-modification in the antisense and
sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells.
Keywords Oligoribonucleotides; ONs; RNA/RNA duplexes
Modified oligonucleotides (ONs) are widely used in molecular biology
and medicinal chemistry. Antisense agents are valuable tools to inhibit
the expression of a target gene in a sequence specific manner, and
may be used for functional genomics, target validation and therapeutic
purposes.[1] Research in this field increased in impact by the discovery of
RNA interference.[2,3]
Numerous attempts have been made to improve the properties of nat-
ural ONs. It is believed that the 2ꢁ-carbohydrate modifications are the
most universal and promising.[4−6] A typical strategy for the preparation
of such ONs is the synthesis of modified nucleoside followed by conver-
sion to the corresponding phosphoramidite suitable for automated ON
Financial support of the Russian Foundation for Basic Research, Programme “Molecular and Cellar
Biology,” INTAS and KUL Research Council is acknowledged.
Address correspondence to Piet Herdewijn, Laboratory of Medicinal Chemistry, Rega Institute for
Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, Leuven B 3000, Belgium.
E-mail: piet herdewijn@rega.kuleuven.ac.be
1509

1510
S. N. Mikhailov et al.
SCHEME
1 B
=
Ura, CytBz
;
Bꢁ
=
Ura, Cyt;
n
=
1,2. a) SnCl4/ClCH2CH2Cl,−12◦C;
b). Bu4NF/THF; c) NH3/MeOH, d) MMTrCl/Py; e) ClP(NiPr2)(OCH2CH2CN); f) oligoribonucleotide
synthesis.
synthesis. Most of these modifications were achieved via alkylation reac-
tions of partially protected ribonucleosides. The heterocyclic bases should
be protected in order to avoid their alkylation. For each nucleoside the
specific blocking groups are used,[7] which in most cases are not compat-
ible with standard automated ON synthesis. Very recently for the prepa-
ration of 2ꢁ-O-modified nucleosides we have developed another type of
chemistry based on O-glycosylation reactions, which have several important
advantages, such as increased yields and simplification.[8,9] A simple and
effective method for the preparation of 2ꢁ-O-(β-D-ribofuranosyl)nucleosides
starting from readily available 3ꢁ,5ꢁ-O-blocked N -acylribonucleosides and
1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose preactivated with tin tetra-
chloride in 1,2-dichloroethane at 0◦C, has been recently developed.[10−12]
RESULTS AND DISCUSSION
Here we present our recent results on the preparation of 2ꢁ-O-
hydroxyalkoxymethylribonucleosides and their incorporation into
oligoribonucleotides.[13] This scheme is nearly the same as for the
preparation of disaccharide nucleosides and their incorporation into
ONs.[14] The starting compounds 1(n = 1, 2)[15] were easily prepared from

2 ꢁ-O-Hydroxyalkoxymethylribonucleosides
1511
1,3-dioxolane and 1,3-dioxalane, respectively, and were condensed with
3ꢁ,5ꢁ-O-blocked ribonucleosides 2 (B = Ura, CytBz) in the presence of tin
tetrachloride in 1,2-dichloroethane at −12◦C for 20 minutes (Figure 1).
The yields (85–87%) of products 3 were even higher than in the case of
preparation of above mentioned disaccharide nucleosides.[10] The silyl
group was cleaved to yield partially protected 4 and subsequent deblocking
with ammonia in methanol gave free 2ꢁ-O-substituted nucleosides 5 in high
overall yield. Nucleosides 4 were converted using standard procedures to
the corresponding monomethoxytrityl derivatives 6 and their phospho-
ramidites 7. With these phosphoramidites, several oligoribonucleotides
with one or more modifications were assembled. It was shown that modified
ONs form stable RNA/RNA duplexes, with about 0.5◦C destabilization per
modification.
In addition several siRNAs comprising one or more of the new modi-
fications have been prepared and are currently under evaluation. The ef-
fect of 2ꢁ-O-modification in the antisense and sense strands of small inter-
fering RNA targeting the MDR1 gene was evaluated in multi-drug resistant
NIH 3T3 cells using previously described techniques.[16] Standard siRNA or
siRNAs with 2ꢁ-O-hydroxyalkoxymethylribonucleosides in the sense strand
effectively inhibited P-glycoprotein expression, whereas modifications in the
antisense strand were not tolerated.
CONCLUSIONS
The general method for the preparation of
2ꢁ-O-β-D-
ribofuranosylnucleosides was found to be applicable for the synthesis of
pyrimidine 2ꢁ-O-hydroxyalkoxymethylribonucleosides. The 2ꢁ-O-substitutent
was found to be stable during oligonucleotide synthesis. Additional work
on the preparation of other 2ꢁ-O-functionalized nucleosides and oligonu-
cleotides bearing amino groups is in progress and will be published
shortly.
REFERENCES
1. Kurrech, J. Antisence technologies. Improvements through novel chemical modifications. Eur. J.
Biochem. 2003, 270, 1628–1644.
2. Sioud, M.; Iversen, P.O. Ribozymes, DNAzymes and small interfering RNAs as therapeutics. Curr.
Drug Targets 2005, 6, 647–653.
3. Coppelli, F.M.; Grandis, J.R. Oligonucleotides as anticancer agents: From the benchside to the clin-
ical and beyond. Curr. Pharm. Des. 2005, 11, 2825–2840.
4. Manoharan, M. 2ꢁ-Carbohadrate modification in antisense oligonucleotide therapy: Importance of
conformation, configuration and conjugation. Biochim. Biophys. Acta 1999, 1489, 117–130.
5. Freier, S.M.; Altmann, K.-H. The ups and downs of nucleic acid duplex stability: Structure-stability
studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1997, 25, 4429–4443.
6. Jin, S.; Miduturu, C.V.; McKinney, D.C.; Silverman S.K. Synthesis of amine- and thiole-modified
nucleoside phosphoramidites for site-specific introduction of biophysical probes into RNA. J. Org.
Chem. 2005, 70, 4284–4299.

1512
S. N. Mikhailov et al.
7. Zatsepin, T.S.; Romanova, E.A.; Oretskaya, T.S. Synthesis of 2ꢁ-O-alkylnucleosides. Russ. Chem. Rev.
2002, 71, 513–534.
8. Efimtseva, E.V.; Mikhailov, S.N. Disaccharide nucleosides. Russ. Chem. Rev. 2004, 73, 401–414.
9. Nauwelaerts, K.; Efimtseva, E.V.; Mikhailov, S.N.; Herdewijn, P. Disaccharide nucleosides, an impor-
tant group of natural compounds. In Frontiers in Nucleosides and Nucleic Acids, Schinazi, R.F. Liotta,
D.C. eds. IHL Press, Tucker, GA, pp. 187–220 2004.
10. Mikhailov, S.N.; Efimtseva, E.V.; Gurskaya, G.V.; Zavodnik, V.E.; De Bruyn, A.; Janssen, G.; Rozenski,
J.; Herdewijn, P. An efficient synthesis and physico-chemical properties of 2ꢁ-O-β-D-ribofuranosyl-
nucleosides, minor tRNA components. J. Carbohydr. Chem. 1997, 16, 75–92.
11. Mikhailov, S.N.; Rodionov, A.A.; Efimtseva, E.V.; Ermolinsky, B.S.; Fomitcheva, M.V.; Padyukova
N.Sh.; Rothenbacher, K.; Lescrinier, E.; Herdewijn, P. Formation of Trisaccharide Nucleoside dur-
ing disaccharide nucleoside synthesis. Eur. J. Org. Chem. 1998, 2193–2199.
12. Mikhailov, S.N.; Efimtseva E.V. Disaccharide nucleosides and oligonucleotides on their basis. Collect.
Czech. Chem. Commun. Symp. Ser. 2002, 5, 181–194.
13. Bobkov, G.V.; Brilliantov, K.V.; Mikhailov, S.N.; Rozenski, J.; Van Aerschot, A.; Herdewijn, P. Syn-
thesis of oligoribonucleotides containing pyrimidine 2ꢁ-O-[(hydroxyalkoxy)methyl] ribonucleo-
sides.Collection Czech. Chem. Comm. 2006, 71, 804–819.
14. Efimtseva, E.V.; Bobkov, G.V.; Mikhailov, S.N.; Van Aerschot, A.; Schepers, G.; Busson, R.; Rozenski,
J.; Herdewijn, P. Oligonucleotides containing disaccharide nucleosides. Helv. Chim. Acta 2001, 84,
2387–2397.
15. Senkus, M. Reaction of some cyclic acetals with acetic anhydride. J. Am. Chem. Soc. 1946, 68, 734–736.
16. Xu, D.; Kang, H.; Fisher, M.; Juliano, R.L. strategies for the inhibition of MDR1 gene expression.
Mol. Pharmacol. 2004, 66, 268–275.