Enzymatic resolution and base pairing properties of D- and L-cyclohexenyl nucleic acids (CeNA)

Article abstract of DOI:10.1081/NCN-200060326

An enzymatic transesterification reaction afforded large scale resolution of the cyclohexenol precursor needed for preparation of both series of CeNA building blocks. CeNA oligos of "D-like" chirality display a strong and selective interaction with RNA, w

Full text of DOI:10.1081/NCN-200060326

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ENZYMATIC RESOLUTION AND BASE PAIRING PROPERTIES
OF D- AND L-CYCLOHEXENYL NUCLEIC ACIDS (CeNA)
Ping Gu a , Guy Schepers a , Carsten Griebel b , Jef Rozenski a , Hans-Joachim Gais b , Piet
Herdewijn a & Arthur Van Aerschot a
a Laboratory for Medicinal Chemistry , Rega Institute for Medical Research , Leuven, Belgium
b Institut fur Organische Chemie, Rheinische-Westfalische Technische Hohschule Aachen ,
Aachen, Germany
Published online: 15 Nov 2011.
To cite this article: Ping Gu , Guy Schepers , Carsten Griebel , Jef Rozenski , Hans-Joachim Gais , Piet Herdewijn & Arthur
Van Aerschot (2005) ENZYMATIC RESOLUTION AND BASE PAIRING PROPERTIES OF D- AND L-CYCLOHEXENYL NUCLEIC ACIDS
(CeNA), Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 993-998, DOI: 10.1081/NCN-200060326
To link to this article: http://dx.doi.org/10.1081/NCN-200060326
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):993–998, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060326
ENZYMATIC RESOLUTION AND BASE PAIRING PROPERTIES OF D- AND
L-CYCLOHEXENYL NUCLEIC ACIDS (CeNA)
5
Ping Gu and Guy Schepers
Laboratory for Medicinal Chemistry, Rega Institute for
Medical Research, Leuven, Belgium
5
Carsten Griebel
Institut fu¨ r Organische Chemie, Rheinische-Westfa¨lische Technische
Hohschule Aachen, Aachen, Germany
5
Jef Rozenski
Laboratory for Medicinal Chemistry, Rega Institute for Medical Research,
Leuven, Belgium
5
Hans-Joachim Gais
Institut fu¨ r Organische Chemie, Rheinische-Westfa¨lische Techni-
sche Hohschule Aachen, Aachen, Germany
5
Piet Herdewijn and Arthur Van Aerschot
Laboratory for Medicinal Chemistry, Rega
Institute for Medical Research, Leuven, Belgium
5
An enzymatic transesterification reaction afforded large scale resolution of the cyclohexenol
precursor needed for preparation of both series of CeNA building blocks. CeNA oligos of ‘‘D-like’’
chirality display a strong and selective interaction with RNA, while preserving RNase H activity, and
therefore have potential as antisense constructs. CeNAs of opposite chirality form a self-pairing system
on their own.
INTRODUCTION
Although studied for over a decade, antisense oligonucleotides remain an
important tool for possible therapeutic intervention. Among the many modifica-
tions of oligonucleotides to date, only a few demonstrate an increase in duplex
stability and few of them allow RNase H to cleave the target RNA. In 1988,
we started a program with the intent to substitute a 6-membered ring for the
5-membered (deoxy)ribofuranose ring.[1] These 6-membered rings should confer
increased rigidity to antisense constructs and therefore have less entropy loss upon
Address correspondence to Arthur Van Aerschot, Laboratory for Medicinal Chemistry, Rega Institute for
Medical Research, Minderbroedersstraat 10, Leuven B-3000, Belgium; E-mail: Arthur.VanAerschot@rega.
kuleuvan.ac.be
993
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994
P. Gu et al.
formation of a double stranded complex. As a result these constructs should be
endowed with higher affinity for a partner provided the correct orientation of the
base moieties is present. This endeavour afforded us the 1,5-anhydrohexitol nucleic
acids (HNA), with a strong and selective recognition of RNA, yielding the desired A-
type double helix.[2–4] At the same time, however, the HNA stiffnes prohibits RNase
H recognition. It was reasoned that introduction of a double bond should increase
the flexibility allowing adaptation and enzymatic recognition, while hopefully
preserving the increased affinity for complementary targets. A project on
cyclohexenyl oligonucleotides (CeNA) was thus started.
Synthesis of the desired monomers as required for incorporation into
oligonucleotides (see Figure 1A) is not straightforward, however, with a lengthy
chiral synthesis hampering further development of this new antisense backbone.[5]
A neat strategy for preparation of the racemic mixture of the nucleosides was
reported, and these can be separated by tedious chromatography of the
diastereomeric esters with (R)-methyl-mandelic acid.[6] We now report an
enzyme-catalyzed resolution of the enantiomers of the intermediate cyclohexenol
derivatives. This strategy affords sufficient quantities to allow preparation of all four
building blocks of the cyclohexenyl analogues, for both the D- and L-like series.[7]
It was described already that CeNA may adopt two half-chair conformations
2
(3H2and H3). The former one with axially oriented base moieties is energetically
favoured. Being a good mimic of a furanose ring in its C3’-endo conformation, this
analogue is preferred for hybridization with RNA.[5,8] As of the presence of the
double bond, these CeNAs can undergo conformational changes similar to those of
natural nucleic acids[8,9] (Figure 1B). The constraint of the six-membered ring,
endows these nucleoside analogues a ribonucleoside-like conformation with strong
affinity for RNA. The presence of the double bond on the other hand allows
sufficient flexibility within a CeNA-RNA double helix, to be recognized by RNase
H. However, these preliminary results were obtained with only a single CeNA-unit
FIGURE 1 Cyclohexenyl nucleoside building blocks for DNA synthesis of the a-type or the D-like series. Pannel
B: conformational equilibrium within the CeNA series.

P. Gu et al.
995
incorporated in an otherwise regular DNA helix, and awaited confirmation. In
addition, the influence of chirality of nucleic acids on hybridization is studied here
in evaluating homochiral D-CeNA and L-CeNA for their hybridization capacity
with DNA and RNA, respectively, and with itself.
Racemic compound 5 is the key intermediate for the subsequent introduction
of nucleobases, and is obtained via a Diels-Alder reaction. Several methods for
resolving 5 could be envisaged, i.e., kinetic resolution using sharpless epoxidation,
enzymatic resolution, or formation of diastereomeric esters. At first, several
conditions for enzyme-catalyzed hydrolysis of benzylidene-protected cyclohexenyl
esters 6--8 were studied, but none showed the desired selectivity and activity
profiles. Best results were obtained with PLE-catalyzed hydrolysis of the butyryl
ester with a selectivity of E = 45 (Figure 2).
However, all hydrolysis reactions had a much lower rate then the following
transesterifications. It was found already that transesterification of the cyclohexenol
rac-5 with Lipase PS afforded only 33% of enantiomeric excess.[7] We looked for
improvement using different lipases, co-solvents and reagents, which afforded a
transesterification methodology with Novozyme1 435 and isopropenyl acetate.
Followed by chromatographic separation and a double crystallization, this
methodology afforded a clean separation in good yield. Vinyl buyrate afforded
the highest selectivity (E = 127), but with slow reaction rate (52% conversion in 39 h)
and is economically more expensive. (Figure 3). The final methodology uses 14%
(w/w) of Novozyme1 435 with 5 equiv. of isopropenyl acetate in 50 volumes of
CH2Cl2 at RT (about 52% conversion after 22 h, E = 50). The method developed
allows isolation of both optically pure cyclohexenol enantiomers on a preparative
scale. These were used for introducing the base moieties via a Mitsunobu protocol.
FIGURE 2 Hydrolysis of different racemic esters rac-6, rac-7, rac-8 with different lipases and esterases.

996
P. Gu et al.
FIGURE 3 Transesterification of rac-5 with different enzymes and substrates.
Further elaboration into phosphoramidites (Figure 1) allowed straightforward
assembly into oligonucleotides (Figure 4) in acceptable yield.
D-CeNAs resembling the natural configuration, upon incorporation into DNA
strands only slihtly decrease the affinity for DNA strands, while with RNA
complements there is a slight and sometimes even large increase in affinity. This
can be seen in the following Table 1, with examples for incorporation of A and T
CeNA building blocks (A1, T1), but several new examples have confirmed these
findings. With fully modified sequences, however, the picture is clear, with CeNA
displaying an increased affinity for RNA targets (DTm/mod = 0.6 to 1.2°C). This
increase per basepair is 1°C less in comparison with the HNA skeleton (entries 5, 6,
FIGURE 4 Elaboration of the racemic cyclohexenol into, respectively, D-like and L-like cyclohexenyl
oligonucleotides.

P. Gu et al.
997
TABLE 1 Influence of Incorporation of Single and Multiple CeNA Monomers in DNA Strands on the
Affinity for DNA and RNA Complementary Sequences
Entry
Sequence
DNA complement
RNA complement
1
2
3
4
5
6
7
8
5’-CCAGTGATATGC-3’
5’-CCAGTG A1TATGC-3’
5’-CCAGTG A1T A1TGC-3’
5’-CC A1GTG A1T A1TGC-3’
5’-CCAGTGhATATGC-3’
5’-CCAGTGhAThATGC-3’
5’-CChAGTGhAThATGC-3’
5’-GGTCACTATACG-3’
5’-GGTCACT1ATACG-3’
5’-GGTC1A1C1T1A1T1ACG-3’
5’-GCGTAGCG-3’
49.8
49.4
48.5
48.1
49.5
49.3
47.5
46.1
44.8
37.7
—
44.0
45.1
45.6
49.2
46.9
48.7
53.1
46.1
49.1
53.5
21.0
47.6
50.8
54.4
9
10
11
12
13
14
5’-r(GCGTAGCG)-3’
5’-G1C1G1T1A1G1C1G1-3’
5’-h(GCGTAGCG)-3’
—
—
—
7, and 14), but the CeNA modification preserves RNase H cleavage activity for the
target sequence. This cleavage however takes place at much higher enzyme
concentrations, resulting in a 600-fold lower Kcat in comparison with a DNA
antisense oligo (not shown). Where the affinity for DNA targets is reduced (DTm/
mod = 1 to 2°C), the selectivity of interaction remains unaffected, analogous to the
selectivity for RNA complements.
Table 2 gives an overview of the strength of different homopolymer interactions
within an A-T or A–U context, a duplex, however, with different overall shape
already within the natural sequences. One clearly notices the effect of constraint
comparing the interactions of A1 or hA with RNA in comparison with the DNA-
RNA duplex (lines 3 and 6 versus line 1, with a rise from 15 to 34°C). Especially
enlightning therefore is the strong HNA-HNA interaction with a Tm of 78°C versus
TABLE 2 Tm (°C) Determination of the Complexes Formed Between Homo-Modified Oligonucleotides and
Their DNA, RNA, HNA, and CeNA Complement, Respectively
Entry
Sequence
(dA)13
DNA
RNA
HNA
CeNA
1
2
3
4
5
6
7
34.0
34.0
33.5
15.2
32.0
34.2
26
37–43b
33
45/58d
21
37–43b
33.5
NDc
67
(dT)13
*
(A1
(A2
)
)
72.5
13
13
a
–
48.5/43e
37–43b
78
*
(T2
)
13
37–43b
21
67
*
(hA)13
(hT)13
37–43b
72.5
45/58d
50
78
aNo melting transition detected.
bShallow melting curve without pronounced inflection point (T2 containing duplex).
cNot determined (ND).
dDouble transition, respectively for triplex and duplex.
eDissociation and reassociation values, respectively, when determined at 0.2°C/min.

998
P. Gu et al.
72.5°C for the CeNA-HNA and 67°C for the CeNa-CeNA counterparts. The latter
duplex was studied with oligomers of opposite chirality (the L-series) in view of
insufficient supply of the required T1 monomer. Herewith, however, we uncovered
that the CeNAs of opposite chirality constitute a strong self-pairing system on their
own, resembling L-RNA sequences, but not pairing with natural DNA or RNA.
This is actually the first example of a ‘‘nonnatural-like’’ spiegelmer. The unknown
thymine-thymine interaction as seen before within the HNA series (entry 4, Tm
48.5°C), was noticed here in both CeNA series as well, and is responsible for the
shallow melting curves with inflection point at 37–43°C. Finally, an unexpected
interaction occus between the A2 homopolymer with polyuridine (Tm of 26°C),
which cannot be explained either.
In view of the strong and selective interaction of CeNAs with RNA
complements, and the preservation of RNaseH recognition of a CeNA-RNA
duplex, the CeNAs have a strong potential for antisense strategies.
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