Synthesis and properties of pyrrolidine-based negatively charged dna mimics

Article abstract of DOI:10.1080/15257770701548345

A set of novel chiral pyrrolidine-based nucleotide mimics, in which nucleobase, hydroxyl group and phosphonic acid residue were attached to different carbon atoms of the pyrrolidine ring, was synthesized. These monomers were used for the synthesis of the

Full text of DOI:10.1080/15257770701548345

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Synthesis and Properties of Pyrrolidine-
Based Negatively Charged Dna Mimics
Vladimir A. Efimov a , Valeryi N. Klykov a & Oksana G.
Chakhmakhcheva a
a Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry , Moscow,
Russia
Published online: 05 Dec 2007.
To cite this article: Vladimir A. Efimov , Valeryi N. Klykov & Oksana G. Chakhmakhcheva (2007)
Synthesis and Properties of Pyrrolidine-Based Negatively Charged Dna Mimics, Nucleosides,
Nucleotides and Nucleic Acids, 26:10-12, 1595-1599, DOI: 10.1080/15257770701548345
To link to this article: http://dx.doi.org/10.1080/15257770701548345
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1595–1599, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701548345
SYNTHESIS AND PROPERTIES OF PYRROLIDINE-BASED
NEGATIVELY CHARGED DNA MIMICS
Vladimir A. Efimov, Valeryi N. Klykov, and Oksana G. Chakhmakhcheva
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
2
A set of novel chiral pyrrolidine-based nucleotide mimics, in which nucleobase, hydroxyl group
2
and phosphonic acid residue were attached to different carbon atoms of the pyrrolidine ring, was
synthesized. These monomers were used for the synthesis of the corresponding oligomers, and their
physico-chemical properties were evaluated.
Keywords DNA mimics; pyrrolidine; hybridization; antisense reagents
INTRODUCTION
In the last years, a search for modified oligonucleotides and their ana-
logues with improved physico-chemical and biological properties attracted
our attention in connection with their application in molecular biology
and medicine as diagnostic probes and antisense agents. With this aim
in view, we have undertaken the investigations on the synthesis of sev-
eral types of negatively charged oligonucleotide mimics constructed from
pyrrolidine-based chiral monomer units connected with phosphonate es-
ter bonds. Particularly, we reported pHypNA mimics containing 1-acetyl-4-
hydroxypyrrolidine-2-phosphonic acid backbone[1] (Figure 1). Trans-L type
of pHypNAs, similarly to HypNA-pPNA hetero-oligomers developed by us
earlier,[2] have demonstrated high hybridization and discrimination charac-
teristics as well as the ability to hybridize to complementary nucleic acids
in low salt concentrations.[1−3] Analysis of biological properties of pHypNA
oligomers has shown that this type of DNA mimics combined high biological
stability with the ability to penetrate cell membranes. They do not activate
The authors thank Russian Foundation of Basic Research for financial support.
Address correspondence to V. A. Efimov, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia.
E-mail: eva@mail.ibch.ru
1595

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V. A. Efimov et al.
FIGURE 1 General chemical structures of HypNA-pPNA and pHypNA mimic oligomers and four chiral
pHypNA monomers.
RNase H and inhibit mRNA translation by hybridization arrest. Other prop-
erties of pHypNA mimics include ability to bind RNA targets stronger than
DNA, good water solubility and cellular uptake with distribution in cytosol
and preferentially in nucleus as well as a long shelf-life at room temperature.
Thus, these compounds can be evaluated as useful tools for application in
molecular biology, particularly in nucleic acid based diagnostics, isolation
and analysis of nucleic acids and in the study of gene expression.[3−5] Gene
knockdown experiments in vivo revealed that their effect lasts over a pe-
riod of several days. So, the use of these mimics can represent a potent
technology for administering antisense-based drugs for future therapeutic
applications.[3,4] To continue these investigations, we obtained several novel
types of monomers on the base of pyrrolidine (PyrrNAs).
RESULTS AND DISCUSSION
In pyrrolidine-based monomers obtained, nucleobase, hydroxyl group
and phosphonic acid residue were attached to different carbon atoms of
the pyrrolidine ring (Figure 2). As examples, the synthetic routes to ob-
tain trans-L PyrrNA-2 and PyrrNA-3 monomers are depicted in Scheme 1.
The derivatives of trans-4-OH-N -acetyl-L-prolinol bearing heterocyclic bases,

Synthesis and Properties of Pyrrolidine
1597
FIGURE 2 General chemical structures of chiral monomer units synthesized for the construction of the
corresponding PyrrNA mimic oligomers.
which were used as starting compounds, were obtained as described.[6]
Schemes for the synthesis of other PyrrNA monomers will be published else-
where. The synthesis of the corresponding PyrrNA oligomers was carried out
by the modified solid-phase phosphotriester method with O-nucleophilic in-
tramolecular catalysis similar to the synthesis of pHypNA and HypNA-pPNA
oligomers.[2,4] Preliminary results obtained in the study of the hybridization
properties of PyrrNA-2 and PyrrNA-3 mimics revealed that oligomers con-
structed from monomers with cis-L configuration are not able to form sta-
ble complexes with the complementary oligonucleotides, whereas oligomers
constructed from trans-L monomers are able to form complexes with the
complementary targets, but the stability of these complexes is lower than

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V. A. Efimov et al.

Synthesis and Properties of Pyrrolidine
1599
TABLE 1 Stability of complexes formed by homo-Thy15 PyrrNA DNA mimics
with the complementary DNA, or RNA, targets in 5-10 µM concentration in
10 mM Tris-HCl (pH 7.5)/0.5 M NaCl/10 mM MgCl2
Tm,
◦C
Oligomer type
DNA target
RNA target
DNA
pHypNA
47
76
35
64
trans-L
trans-D
trans-L
cis-L
trans-D
cis-D
trans-L
cis-L
trans-L
cis-L
trans-L
cis-L
trans-D
cis-D
trans-L
cis-D
53
47
PyrrNA-1
NDa
<20
NDa
<20
35
NDa
<20
NDa
<20
30
PyrrNA-2
PyrrNA-3
PyrrNA-4
<20
31
<20
27
<20
NDa
<20
<20
NDa
NDa
NDa
<20
NDa
<20
<20
NDa
NDa
NDa
PyrrNA-5
aExamination is currently in progress.
the stability of natural DNA/DNA and DNA/RNA duplexes and significantly
lower than the stability of complexes between trans-L pHypNA oligomers
and DNA (or RNA) targets (Table 1). Also, cis-L and cis-D PyrrNA-1
oligomers as well as cis-L and trans-D PyrrNA-4 oligomers were not able to
form complexes with the complementary targets. The investigations on the
properties of other types of PyrrNA mimics are currently in progress.
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1. Efimov, V.; Klykov, V.; Chakhmakhcheva, O. Phosphono peptide nucleic acids with a constrained
hydroxyproline-based backbone, Nucleosides, Nucleotides Nucleic Acids 2003, 22, 593–599.
2. Efimov, V.; Buryakova, A.; Chakhmakhcheva, O. Synthesis of polyacrylamides N-substituted with
PNA-like oligonucleotide mimics for molecular diagnostic applications. Nucleic Acids Res. 1999, 27,
4416–4426.
3. Efimov, V.; Chakhmakhcheva, O.; Wickstrom, E. Synthesis and application of negatively charged
PNA analogues. Nucleosides, Nucleotides, Nucleic Acids 2005, 24, 1853–1874.
4. Efimov, V.; Birikh, K.; Staroverov, D.; Lukyanov, S.; Tereshina, M.; Zaraisky, A.; Chakhmakhcheva, O.
Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo. Nucleic
Acids Res. 2006, 34, 2247–2257.
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properties. Collect. Czech. Chem. Commun. 2006, 71, 929–955.
6. Ceulemans, G.; van Aerschot, A.; Wroblowski, B.; Rozenski, J.; Hendrix, C.; Herdewijn, P. Oligonu-
cleotide analogues with 4-hydroxy-N-acetylprolinol as sugar substitute. Chem. Eur. J. 1997, 3, 1997–
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