Cyclohexene nucleic acids (CeNA) form stable duplexes with RNA and induce RNAse H activity

Article abstract of DOI:10.1081/NCN-100002430

Cyclohexene nucleic acids (CeNA) were synthesized using classical phosporamidite chemistry. Incorporation of a cyclohexene nucleo-side in a DNA chain leads to an increase in stability of the DNA/RNA duplex. CeNA is stable against degradation in serum. A C

Full text of DOI:10.1081/NCN-100002430

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CYCLOHEXENE NUCLEIC ACIDS (CeNA) FORM STABLE
DUPLEXES WITH RNA AND INDUCE RNASE H ACTIVITY
J. Wang a , B. Verbeure a , I. Luyten a , M. Froeyen a , C. Hendrix a , H. Rosemeyer b , F.
Seela b , A. Van Aerschot a & P. Herdewijn c
a Laboratory for Medicinal Chemistry , Rega Institute, Katholieke Universiteit Leuven ,
Minderbroedersstraat 10, Leuven, B-3000, Belgium
b University Osnabrück , Laboratorium für Organische and Bioorganische Chemie,
Barbarastrasse 7, Osnabrück, D-49069, Germany
c Laboratory for Medicinal Chemistry , Rega Institute, Katholieke Universiteit Leuven ,
Minderbroedersstraat 10, Leuven, B-3000, Belgium
Published online: 07 Feb 2007.
To cite this article: J. Wang , B. Verbeure , I. Luyten , M. Froeyen , C. Hendrix , H. Rosemeyer , F. Seela , A. Van Aerschot
& P. Herdewijn (2001) CYCLOHEXENE NUCLEIC ACIDS (CeNA) FORM STABLE DUPLEXES WITH RNA AND INDUCE RNASE H
ACTIVITY, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 785-788, DOI: 10.1081/NCN-100002430
To link to this article: http://dx.doi.org/10.1081/NCN-100002430
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 785–788 (2001)
CYCLOHEXENE NUCLEIC ACIDS (CeNA) FORM
STABLE DUPLEXES WITH RNA AND INDUCE
RNASE H ACTIVITY
J. Wang,1 B. Verbeure,1 I. Luyten,1 M. Froeyen,1 C. Hendrix,1
H. Rosemeyer,2 F. Seela,2 A. Van Aerschot, 1 and P. Herdewijn1,∗
1Laboratory for Medicinal Chemistry, Rega Institute, Katholieke
Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
2Laboratorium fu¨r Organische and Bioorganische Chemie, University
Osnabru¨ck, Barbarastrasse 7, D-49069 Osnabru¨ck, Germany
ABSTRACT
Cyclohexene nucleic acids (CeNA) were synthesized using classical phospo-
ramidite chemistry. Incorporation of a cyclohexene nucleo-side in a DNA chain
leads to an increase in stability of the DNA/RNA duplex. CeNA is stable against
degradation in serum. A CeNA/RNA hybrid is able to activate E. Coli RNase
H, resulting in cleavage of the RNA strand.
Cyclohexene nucleic acids (CeNA) are a new class of nucleic acids in which
the furanose sugar moiety is replaced by a cyclohexene ring. It is general knowl-
edge that replacement of the five-membered ring of a natural DNA and RNA by a
six-membered ring increases the conformational rigidity of the oligomer. Several
types of six-membered nucleic acids have been studied. The pyranose nucleic acids
have an equatorially oriented base moiety at the 1ꢀ-position. These oligomers form
strong self-complementary duplexes, but don’t hybridize with natural nucleic acids.
On the other hand, the hexitol nucleic acids (HNA) have the base moiety located at
the 2ꢀ-position. They have axially oriented nucleobases and hybridize with natural
nucleic acids and themselves. The RNA selective hybridization of HNA is explained
∗Corresponding author.
785
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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WANG ET AL.
by the pre-organization of the hexitol ring in an N-type furanose conformer (3ꢀ-
endo conformation with an axially oriented base moiety). The cyclohexane nucleic
acids (CNA), however, have an equatorially oriented nucleobase at the monomeric
level, but at the oligomeric level the base moiety is axially oriented. The axial
orientation is required for hybridixation of CNA with natural nucleic acids. The
chair-chair conformation interconversion of the cyclohexane ring is energetically
highly demanding. This energy barrier is expected to be significantly reduced by
the introduction of a double bond in the cyclohexane ring, resulting in cyclohexene
nucleic acids (CeNA).
Cyclohexene nucleic acids were prepared by classical phosphoramidite chem-
istry. The cyclohexenyl adenine phosphoramidite building block was synthesized as
shown in Scheme 1. The adenine base moiety of 1 was protected as a benzoate and
the primary hydroxyl group as a monomethoxytrityl ether. The secondary hydroxyl
group was reacted with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidate to
yield the nucleotide building block. Assembly of the monomers into oligonu-
cleotides was accomplished using the standard phosphoramidite method. The cy-
clohexenyl analogue was used at a 0.12 M concentration and the coupling was
allowed to proceed for 3 min (>95% coupling efficiency).
When incorporating the cyclohexenyl-A (A1) in a mixed purine-pyrimidine
DNA sequence (5ꢀ-CCAGTGATATGC-3ꢀ) and in the corresponding RNA sequence
Scheme 1.

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CYCLOHEXENE NUCLEIC ACIDS
787
Figure 1.
(5ꢀ-CCAGUGAUAUGC-3ꢀ), a small decrease in stability of dsDNA (−0.5◦C
ꢀ Tm/mod) was observed. A similar drop in stability is observed after incorpora-
tion of anhydrohexitol-A (A2). The influence of A1 and A2 on the duplex stability of
dsDNA doesn’t seem to be fundamentally different. Of more interest is the influence
of A1 on the stability of a DNA/RNA duplex. Incorporation of 1, 2 or 3 cyclohex-
enyl nucleosides in the DNA strand increases duplex stability with 1.1, 1.6, and
5.2◦C, respectively, corresponding to a ꢀTm/mod of + 1.1, + 0.8 and + 1.7◦C.
As expectd, the influence on duplex stability is dependent on the incorporation site
of A1. The stabilization effect of A2 on the DNA/RNA duplex is twice as high as of
A1. However, the sequence selective effect is the same. An overlay of the melting
curves for DNA/RNA duplexes with a triple substitution of A1 and A2 is depicted
in Figure 1. Incorporation of three A1 in a dsRNA sequence leads to an increase in
duplex stability of + 0.6◦C. The increase in duplex stability after incorporation of
A1, using RNA as the complementary strand, suggests conformational preorgani-
zation of A1 (3H2, mimicking the 3ꢀ-endo conformation of the ribofuranose sugar).
The 3H2 conformation is indeed the most stable conformation of cyclohexenyl-A.
The complex formed between (A1)13 and poly(U) is of similar stability as
between (A2)13 and poly(U). However, the (A1)13-poly(dT) complex is much more
stable (ꢀTm = +10◦C) than the corresponding (A2)13-poly(dT). The titration
experiment showed the nature of (A1)13-poly(dT) complex is of triplex origin
((dT)13.(A1)13.(dT)13). The complex formed between oligo(A1) and its DNA or
RNA complement is of similar stability.
CD spectral analysis showed that the spectrum of the A1-DNA/DNA com-
plex (three A1 were incorporated in the above mentioned DNA mixing sequence)
is intermediate between that of a DNA/DNA and a RNA/DNA complex, while
the spectrum of the corresponding A2-DNA/DNA complex is more similar to the
DNA/RNA complex. The CD spectrum of all RNA containing complexes are sim-
ilar and suggest that they adopt similar conformations. This result suggests that the
incorporation of three A2 in a DNA strand is able to induce a conformational change

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of a dsDNA duplex in the direction of a DNA/RNA type structure. However, three
A1 residues don’t seem to have this effect and the DNA/A1-DNA conformation
stays close to the typical dsDNA form.
The complex formed between the A1 oligomer and its RNA complement is
able to activate E. Coli RNase H, resulting in the cleavage of the RNA strand, while
the HNA containing complex is devoid of this ability. CeNA has serum stability
similar to HNA. Both modified oligonucleotides are much more stable against
enzymatic degradation (no degradation after incubation for 3 h in serum at 37◦C)
than DNA (almost half degraded after 3 h incubation).
In conclusion, cyclohexene nucleic acids are much more flexible than other
six-membered nucleic acids and are good mimicks of natural furanose nucleic
acids. They combine the advantage of duplex stabilization and serum stability
with the potential to activate RNase H. They are potential candidates for antisense
application.
REFERENCES
1. Wang, J.; Verbeure, B., Luyten, I., Froeyen, M.; Hendrix, C.; Rosemeyer, H.; Seela, F.;
Van Aerschot, A.; Herdewijn, P. J. Am. Chem. Soc. 2000, in press.
2. Herdewijn, P. Liebigs Annalen 1996, 1337–1348.
3. Eschenmoser, A. Pure Applied Chem. 1993, 65, 11790–1188.
4. Hendrix, C.; Rosenmeyer, H.; De Bouvere, B.; Van Aerschot, A.; Seela, F.; Herdewijn,
P. Chem. Eur. J. 1997, 3, 1513–1520.
5. Maurinsh, Y.; Rosenmeyer, H.; Esnouf, R.; Medvedovici, A.; Wang, J.; Ceulemans,
G.; Lescrinier, E.; Hendrix, C.; Busson, R.; Sandra, P.; Seela, F.; Van Aerschot, A.;
Herdewijn, P. Chem. Eur. J. 1999, 5, 2139–2150.

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