Stabilization of RNA bulges by oligonucleotides containing 2′-naphthylmethyl-2′-deoxytubercidine

Article abstract of DOI:10.1081/NCN-120022948

A novel nucleoside analogue, 2′-naphthylmethyl-2′ -deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.

Full text of DOI:10.1081/NCN-120022948

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Stabilization of RNA Bulges by Oligonucleotides
Containing 2′-Naphthylmethyl-2′-deoxytubercidine
A. Madder a c , R. Ehrl a & R. Strömberg a b
a Division of Organic and Bioorganic Chemistry, MBB , Karolinska Institutet , Stockholm,
Sweden
b Division of Organic and Bioorganic Chemistry , MBB, Karolinska Institutet , Scheeles vägz, S
171 77, Stockholm, Sweden
c Department of Organic Chemistry , University of Gent , Gent, Belgium
Published online: 31 Aug 2006.
To cite this article: A. Madder , R. Ehrl & R. Strömberg (2003) Stabilization of RNA Bulges by Oligonucleotides Containing
2′-Naphthylmethyl-2′-deoxytubercidine, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1289-1291, DOI: 10.1081/
NCN-120022948
To link to this article: http://dx.doi.org/10.1081/NCN-120022948
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1289–1291, 2003
Stabilization of RNA Bulges by Oligonucleotides Containing
20-Naphthylmethyl-20-deoxytubercidine
A. Madder,# R. Ehrl, and R. Stromberg
*
¨
Division of Organic and Bioorganic Chemistry, MBB, Karolinska Institutet,
Stockholm, Sweden
ABSTRACT
A novel nucleoside analogue, 20-naphthylmethyl-20-deoxytubercidine, is synthe-
sized and incorporated in oligonucleotides that stabilize bulges in partially
complementary RNA.
Key Words: Oligonucleotides; RNA recognition; Bulges; Nucleic acids.
In our research towards development of oligonucleotide based artificial nucleases
we are targeting RNA bulges. However, since bulge-containing structures are inhe-
rently less stable than fully complementary duplexes,[1] introduction of potential
bulge stabilizing modifications is an important issue. To our knowledge no oligonu-
cleotide modifications have been designed for stabilization of RNA bulges.
In a first approach we concentrated on the direct introduction of an aromatic
substituent on the 20-position using a carbon-carbon bond forming strategy. Mole-
cular modelling suggested that incorporation of a 1-naphtylmethylgroup in the
20-position could reduce the destabilizing effect of the occurrence of a bulge and
#Current affiliation: Department of Organic Chemistry, University of Gent, Gent, Belgium.
*Correspondence: R. Stro¨mberg, Division of Organic and Bioorganic Chemistry, MBB, Kar-
olinska Institutet, Scheeles va¨gz, S 171 77 Stockholm, Sweden; Fax: þ46-8-311052; E-mail:
roger.stromberg@mbb.ki.se.
1289
DOI: 10.1081/NCN-120022948
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

¨
Madder, Ehrl, and Stromberg
1290
Scheme 1. Synthesis of the 20-(1-naphthyl)methyl substituted H-phosphonate building block:
a) TIPDSCl2, pyridine, 95%; b) CrO3, Ac2O, pyridine, CH2Cl2, 86%; c) naphthylmethyltriphe-
nylphosphonium chloride, nBuLi (1.75 eq), THF, rt, 73%; d) NH3(l), dioxane, 4 days, 80ꢀC; e)
Bu4NþFꢁ, THF, 80% over 2 steps; f) H2, Pd=C, CH3OH, 5 h, 82%; g) i. (CH3)3SiCl, pyridine,
ꢁ
ii. butyric anhydride, iii. H2O, 60%; h) MMTþBF4 , Li2CO3, 2,6-lutidine, 0ꢀC to rt, 93%; i) i.
Imidazole, PCl3, Et3N, CH2Cl2, ꢁ20ꢀC to ꢁ78ꢀC, 80% ii. H2O.
increase specificity for bulged out RNA-target sequences. The synthetic route is out-
lined in Scheme 1. 6-Cl-Tubercidine 1 (2) was protected with the Markiewicz
reagent[3], oxidized and reacted with the phosphorylide derived from naphtyl-
methylchloride. This gave an isomeric E=Z mixture in good yield. It was then neces-
sary to convert the 6-Cl-substituent to 6-NH2. The conditions developed by Seela
and coworkers,[2] involving heating with methanolic ammonia to 50ꢀC during
24 h, led in this case to the predominant formation of MeO-substituted base. How-
ever, treatment of 3, dissolved in dioxane, with liquid ammonia followed by heating
to 80ꢀC in a pressure vessel for 4 days led to the desired compound that was subse-
quently desilylated using TBAF in THF yielding 4 as an isomeric E=Z mixture,
which upon hydrogenation gave 5. Base protection followed by reaction with mono-
methoxytrityl tetrafluoroborate=LiCO3=2,6-lutidine[4] and phosphonylation[5a] gave
6 which was used in synthesis of both oligodeoxyribo- and oligoribonucleotides by
the H-phosphonate approach.[5b]
UV-melting studies were then performed of complexes between oligos contai-
ning 5, and RNA or DNA fragments that upon binding formed 1-3 nt bulged out
regions.[6]
Incorporation of 20-deoxy-20-b-(1-naphthyl) methyltubercidine in either an oligo-
deoxyribonucleotide or all-20-O-methyloligoribonucleotide has little effect on the
Tm values for complexes with DNA-complements that form small bulges. How-
ever, when the target sequence is an RNA-fragment that in the complex forms

Stabilization of RNA Bulges
1291
a small bulge, the naphthylmethyltubercidine modified oligonucleotides give
stabilisations of up to 4ꢀC.[6] This kind of stabilisation is only found with the
RNA complements and not with the DNA targets. Thus, the naphthylmethyl
tubercidine containing oligonucleotides specifically stabilize bulged RNA.
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