The effect of universal fluorinated nucleobases on the catalytic activity of ribozymes

Article abstract of DOI:10.1081/NCN-120022962

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2, containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribo

Full text of DOI:10.1081/NCN-120022962

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The Effect of Universal Fluorinated Nucleobases on the
Catalytic Activity of Ribozymes
A. E. Klöpffer a & J. W. Engels a b
a Institute for Organic Chemistry and Chemical Biology , Johann-Wolfgang-Goethe
University , Frankfurt am Main, Germany
b Institute for Organic Chemistry and Chemical Biology , Johann-Wolfgang-Goethe
University , Marie-Curie-Strasse 11, D-60439, Frankfurt a.M., Germany
Published online: 31 Aug 2006.
To cite this article: A. E. Klöpffer & J. W. Engels (2003) The Effect of Universal Fluorinated Nucleobases on the Catalytic
Activity of Ribozymes, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1347-1350, DOI: 10.1081/NCN-120022962
To link to this article: http://dx.doi.org/10.1081/NCN-120022962
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1347–1350, 2003
The Effect of Universal Fluorinated Nucleobases on the
Catalytic Activity of Ribozymes
*
¨
A. E. Klopffer and J. W. Engels
Institute for Organic Chemistry and Chemical Biology,
Johann-Wolfgang-Goethe University,
Frankfurt am Main, Germany
ABSTRACT
Four fluoro modified universal nucleobases have been synthesized. The universal
nucleobases 1 and 2, containing a 2,4-difluorobenzene as nucleobase and a 4,6-
difluorobenzimidazole, respectively, were chemically incorporated into a selected
hammerhead ribozyme sequence which has already been retrovirally expressed as
an anti-HIV ribozyme to investigate their effect on the catalytic activity of the
ribozymes. The substitution of the natural nucleosides with either 1 or 2 results
only in a small decrease of the catalytic activity. The Km value for the monosub-
stituted ribozyme with a 2,4-difluorobenzene is 309 nMꢀ1, the corresponding kcat
is 2.91 ꢁ 10ꢀ3 minꢀ1. A disubstituted hammerhead ribozyme carrying one of each
modification has also been synthesized. For a further stabilization of the ribo-
zyme=substrate complex 20-(b-aminoethoxy) modified fluorinated nucleosides 15
and 16 have been developed.
*Correspondence: J. W. Engels, Institute for Organic Chemistry and Chemical Biology,
Johann-Wolfgang-Goethe University, Marie-Curie-Strasse 11, D-60439 Frankfurt a.M.,
Germany; Fax: þ49 697 982 9148; E-mail: Joachim.engels@chemie.uni-frankfurt.de.
1347
DOI: 10.1081/NCN-120022962
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Klopffer and Engels
1348
INTRODUCTION
It has been shown that fluorinated nucleobase analogues 1 and 2 can act as uni-
versal bases and therefore do not discriminate between the four natural nucleo-
bases.[1] Furthermore the incorporation of the fluorine leads to a stabilization of
the investigated 12mer RNA duplexes compared to the oligoribonucleotides carrying
the benzene or the benzimidazole modification, respectively.[2] To determine whether
the nucleosides 1 and 2 effect the catalytic activity of ribozymes we incorporated
them into hammerhead ribozymes and determined the kinetic parameters.[3] To
further improve the catalytic cleavage reaction of the modified ribozymes through
electrostatic interactions we developed the nucleosides 15 and 16 and present an opti-
mized synthetic pathway for these compounds. Thus, it should be possible to direct
the ribozymes against the HIV polymerase gene containing point mutations (‘‘hot
spots’’) without the loss of activity due to mismatch base pairs.
THE EFFECT ON RIBOZYME CATALYSIS
The ribonucleosides with a 2,4-difluorobenzene 1 or a 4,6-difluorobenzimidazole
2 as base analogues for a pyrimidine or a purine scaffold, respectively, were synthe-
sized according to literature.[2] The corresponding phosphoramidites have been pre-
pared using the recently developed TOM chemistry.[4] The hydrolytic RNA cleavage
reactions were catalyzed by 10 mM MgCl2 and carried out at 37ꢂC under single turn-
over conditions. Cy5 labelling of the substrate allowed the fast and efficient kinetic
analysis on an ALF ExpressÕ DNA Sequencer.
The substitution of either 2.4U or 15.4A[5] with the nucleobases 1 or 2, respec-
tively, (Fig. 1) resulted only in a small reduction of the catalytic efficiency of the
hammerhead ribozyme compared to the unmodified one. The Km value obtained
for the ribozyme containing the 2,4-difluorobenzene modification is 309 nMꢀ1 with
an initial rate constant kcat of 2.9 ꢁ 10ꢀ3 minꢀ1. Comparable results were obtained
by the reaction with the ribozyme containing the 4,6-difluorobenzimidazole modifi-
cation on position 15.4. The doubly modified ribozyme showed decreased catalytic
activity.
IMPROVED SYNTHESIS OF 20-MODIFIED RIBONUCLEOSIDES
To enhance the rate of association between the ribozyme and the substrate we
further developed 20-modified fluorinated nucleobases. The 20-position was modified
with an b-aminoethoxy group[6] to yield the nucleosides 15 and 16 that are proto-
nated under physiological conditions. The electrostatic interaction between the pro-
tonated amino group and the negatively charged RNA phosphodiester backbone
should lead to an additional stabilization of helix I or III in the catalytically active
three dimensional conformation of the hammerhead ribozyme.
Starting from the universal nucleobases 3 and 4 we protected the 50- and the 30-
hydroxy group simultaneously with the Markiewicz reagent t-isopropyldisiloxane
dichloride (Fig. 2). After the reaction with methyl bromoacetate the nucleosides 7

Universal Fluorinated Nucleobases
1349
Figure 1. Secondary structure of the Cy5 labeled hammerhead ribozyme and modified
nucleosides.
and 8, respectively, were reduced with LiBH4 in THF=MeOH 8 : 2. The corresponding
alcohols 9 and 10 were tosylated with p-toluenesulfonyl chloride and afterwards con-
verted into the azides 13 and 14. In a final step the 20-(b-aminoethoxy) - substituted
nucleosides 15 and 16 were obtained by reduction of the azides with SnCl2 in methanol.
Figure 2. B1 ¼ 2,4-difluorobenzene, B2 ¼ 4,6-difluorobenzimidazole a) TiPDSiCl2, pyridine
b) BrCH2COOMe, NaH, THF c) LiBH4, THF=MeOH 8 : 2 d) r-TsCl, DMAP, NEt3, CH2Cl2
e) NaN3, DMF f) SnCl2, MeOH.

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Klopffer and Engels
1350
We optimized the synthesis of the 20- modified nucleosides by converting the
alcohol 9 directly into the azide via the MITSUNOBU reaction[7] using sodium
azide, DEAD (diethylazodicarboxylate) and triphenylphosphane.
ACKNOWLEDGMENTS
We want to thank the ‘‘Fonds der Chemischen Industrie’’ and the BMBF for a
Ph.D. fellowship and the SFB 579 for financial support. A.K. would also like to
thank Dr. U. Scheffer for the work on the ALFÕ Express System.
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2. Parsch, J.; Engels, J.W. Synthesis of fluorobenzene and benzimidazole nucleic-
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3. Klo¨pffer, A.E.; Engels, J.W. Universal fluorinated nucleobases: Their effect on
the catalytic activity of ribozymes. Coll. Symp. Series 2002, 5, 348–351.
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