Introduction of chirality into PNA by replacement of the achiral methylene carbonyl linkage to the nucleobase

Article abstract of DOI:10.1080/15257770701527794

A novel approach to the introduction of chirality into peptide nucleic acid (PNA) by replacement of the methylene carbonyl linker by an alpha-amino acid derived moiety is described. A monomer compatible with Fmoc-based oligomerization chemistry possessing an L-serine derived linker has been synthesized and incorporated into PNA oligomers. A single, central substitution in a hexathymine PNA strongly destabilized triple helix formation whereas a central substitution in a mixed sequence is much better tolerated. We have investigated the influence of this substitution on the selectivity for strand composition (DNA versus RNA complement) and strand orientation (antiparallel versus parallel) in the context of duplex formation. A PNA 11-mer with a single substitution demonstrates a preference for an antiparallel RNA complement, as judged by thermal denaturation analysis of the complexes. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701527794

This article was downloaded by: [Memorial University of Newfoundland]
On: 07 August 2013, At: 00:21
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
Introduction of Chirality into PNA By
Replacement of the Achiral Methylene
Carbonyl Linkage to the Nucleobase
Filip Wojciechowski a & Robert H. E. Hudson a
a Department of Chemistry, The University of Western Ontario,
London, Ontario, Canada
Published online: 10 Dec 2007.
To cite this article: Filip Wojciechowski & Robert H. E. Hudson (2007) Introduction of Chirality into
PNA By Replacement of the Achiral Methylene Carbonyl Linkage to the Nucleobase, Nucleosides,
Nucleotides and Nucleic Acids, 26:8-9, 1195-1198, DOI: 10.1080/15257770701527794
To link to this article: http://dx.doi.org/10.1080/15257770701527794
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, 26:1195–1198, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701527794
INTRODUCTION OF CHIRALITY INTO PNA BY REPLACEMENT OF THE
ACHIRAL METHYLENE CARBONYL LINKAGE TO THE NUCLEOBASE
Filip Wojciechowski and Robert H. E. Hudson
University of Western Ontario, London, Ontario, Canada
Department of Chemistry, The
2
A novel approach to the introduction of chirality into peptide nucleic acid (PNA) by replacement
2
of the methylene carbonyl linker by an alpha-amino acid derived moiety is described. A monomer
compatible with Fmoc-based oligomerization chemistry possessing an L-serine derived linker has been
synthesized and incorporated into PNA oligomers. A single, central substitution in a hexathymine
PNA strongly destabilized triple helix formation whereas a central substitution in a mixed sequence
is much better tolerated. We have investigated the influence of this substitution on the selectivity
for strand composition (DNA versus RNA complement) and strand orientation (antiparallel versus
parallel) in the context of duplex formation. A PNA 11-mer with a single substitution demonstrates
a preference for an antiparallel RNA complement, as judged by thermal denaturation analysis of
the complexes.
Keywords Peptide nucleic acid; PNA; chirality; L-serine; PNA oligomers
INTRODUCTION
Peptide nucleic acid (PNA) is a nucleic acid analogue constructed with
a neutral polyamide backbone composed of N-(2-aminoethyl)glycine units
and a methylene carbonyl linkage between the alpha-amino group and a
nucleobase. Most modifications to PNA, including the introduction of chi-
rality, have been achieved by modifying the backbone structure. PNA being
acyclic has a significant conformational freedom in the nucleobase linker
and the methylene segments of the backbone. Thus, the formation of a
PNA-DNA or PNA-RNA complex results in decreased entropy and confor-
mational freezing of PNA.[1] PNA is also achiral and as a result may bind
to DNA/RNA in the parallel or antiparallel binding mode with almost
equal ability.[2] These issues have been addressed by introducing chirality
and cyclic conformational constraints into the PNA backbone. However,
most of these modifications are synthetically challenging. To our knowledge
The authors wish to thank NSERC (Canada), the Canadian Foundation for Innovation, and the
University of Western Ontario through the Academic Development Fund for support of this work.
Address correspondence to Robert H. E. Hudson, Department of Chemistry, The University of
Western Ontario, London, Ontario N6A 5B7, Canada. E-mail: rhhudson@uwo.ca
1195

1196
F. Wojciechowski and R. H. E. Hudson
introduction of chirality via the methylene carbonyl linker has not been re-
ported. Therefore we were motivated to replace the achiral methylene car-
bonyl linker by an alpha amino acid derived moiety. From a minimalist’s
point of view this is the simplest modification to the methylene carbonyl
linker that introduces both aqueous solubilizing groups and chirality while
preserving the original spacing of repeating units of 6 bonds along the back-
bone and 3 bonds linking the nucleobase, as first reported by Nielsen and
coworkers.[3]
RESULTS AND DISCUSSION
The synthesis of the nucleobase linker 3 was achieved in three steps,
starting with (S)-serine, as described by Scheme 1. Conversion of serine into
the α-halo acid 1 was readily done on a multi-gram scale in good yield.[4]
In order to alkylate thymine efficiently both the hydroxyl group and the
carboxylic acid required protection. The latter was protected under Fischer
esterification conditions to yield the ethyl ester 2, whereas the t-butyl ether
was conveniently prepared by treatment of 2 with t-butanol in the presence
of acid catalyst.[5]
SCHEME 1
The chiral-linker containing monomer was accessed using standard
chemistry as detailed in Scheme 2. Of note, slightly more forcing condi-
tions were required for the alkylation of thymine and the product was re-
covered in marginally lower yield as compared to the use of primary α-
bromoacetates. After ester hydrolysis, 4 was condesed with the Fmoc methyl
ester backbone to give the monomer ester 5a. Removal of the methyl ester
from 5a proved to be difficult in the presence of the base labile Fmoc group
but was accomplished in 52% yield to give the monomer 5b (T∗).
SCHEME 2

Chirality into PNA
1197
TABLE 1 Thermal denaturation dependence on strand composition and orientation
Tm, ◦C (ꢀ Tm)∗
Target Strand
PNA sequence
DNAAP
62.0
49.1 (−12.9)
DNAP
47.0
RNAAP
66.0
RNAP
52.0
49.1 (−2.9)
Ac-TCC-AGC-GTA-AC-K-NH2
Ac-TCC-AGC-GT∗A-AC-K-NH2
41.6 (−5.4) 58.2 (−7.8)
Tm values were estimated by the first derivative method. DNAAP
=
antiparal-
lel oligodeoxynucleotide target: 5ꢁ-GTT-ACG-CTGGA-3ꢁ; DNAP = parallel oligodeoxynu-
cleotide target: 5ꢁ-AGG-TCG-CAT-TG-3ꢁ; RNAAP = antiparallel oligoribonucleotide target: 5ꢁ-
GTT-ACG-CTGGA-3ꢁ; RNAP = parallel oligoribonucleotide target: 5ꢁ-AGG-TCG-CAT-TG-3ꢁ.
ꢀ Tm = difference in the Tm value of the unmodified PNA and the PNA containing T∗.
Incorporation of (T∗) into a PNA oligomer was done on NovaSyn TGR
resin using FastMoc chemistry module on an Applied Biosystems 433A
synthesizer. UV-Tm measurements of triplex (PNA2:NA), and duplex sys-
tems (PNA:DNAAP, PNA:DNAP, PNA:RNAAP, PNA:RNAP) were performed
at 2 µM in 100 mM NaCl, 10 mM PO34−, 0.1 mM EDTA at pH 7.0.
All compounds exhibited monophasic cooperative transitions. A PNA
hexamer bearing a single modification, at an internal position (Ac-TTT
T∗TT-K-NH2) was used to evaluate the effect of this substitution in the con-
text of triple helix formation with poly(rA). A single internal substitution
was found to strongly destabilize the complex (ꢀTm = −37◦C). However,
the substitution is better tolerated in duplex formation as shown in Table 1.
Although the chiral T∗ monomer is overall destabilizing when compared to
the unmodified PNA, it does exhibit some interesting hybridization proper-
ties. When incorporated near the C-terminus of an 11-mer mixed sequence
PNA the modified monomer (T∗) shows a 9◦C preference in Tm for hy-
bridization to an antiparallel RNA target (Tm = 58.2◦C) versus the DNA
(Tm = 49.1◦C), whereas the unmodified PNA only demonstrates a 4◦C pref-
erence for an antiparallel RNA target versus an antiparallel DNA target. The
chiral monomer also has a 9◦C discrimination for hybridization to the an-
tiparallel RNA target (Tm = 58.2◦C) versus the parallel RNA (Tm = 49.1◦C).
In summary, the introduction of a serine-derived linker into a
PNA monomer proceeded readily. An oligomer containing the modified
monomer exhibits preference for RNA binding in the antiparallel orienta-
tion. We currently are pursuing the synthesis of PNA monomer starting with
(R)-serine and will investigate the behaviour of the opposite enantiomer
on RNA/DNA binding, and the effect of multiple substitutions and posi-
tion/sequence effects. Substitution of the α-amino group of other amino
acids such as glutamic acid and lysine also will be investigated in order to
gain insight into why there is a preference for RNA binding and how the
stability of the complex may be increased. Additionally, it has not escaped
our attention that after reduction of the carboxyl group in compound 4,
we would be left with an acyclic nucleoside analog, similar to that used for

1198
F. Wojciechowski and R. H. E. Hudson
glycol nucleic acid.[6] Transformation of this compound into a monomer
for DNA analog synthesis using classical phosphoramidite chemistry is also
being pursued.
REFERENCES
1. Kumar, V.A.; Ganesh, K.N. Conformationally constrained PNA analogues: Structural evolution to-
ward DNA/RNA binding selectivity. Acc. Chem. Res. 2005, 38, 404–412.
2. Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D.W. PNA: synthetic polyamide nucleic acids with
unusual binding properties. Angew. Chem. Int. Ed. 1998, 37, 2796–2823.
3. Nielsen, P.E.; Egholm, M.; Berg, R.H.; Buchardt, O. Sequence-selective recognition of DNA
by strand displacement with
1500.
a thymine-substituted polyamide. Science 1991, 254, 1497–
4. Glabe, A.R.; Sturgeon, K.L.; Ghizzoni, S.B.; Musker, W.K.; Takahashi J.N. Novel Functionalized
acylphosphonates as phosphonoformate analogs. J. Org. Chem. 1996, 61, 7212–7216.
5. Wright, S.W.; Hageman, D.L.; Wright, A.S.; McClure, L.D. Convenient preparations of t-butyl esters
and ethers from t-butanol. Tetrahedron Lett. 1997, 38, 7345–7348.
6. Zhang, L.; Peritz, A.; Meggers, E. A simple glycol nucleic acid. J. Am. Chem. Soc. 2005, 127, 4174–
4175.