Pyrrolidine PNA-DNA chimeric oligonucleotides with extended backbone

Article abstract of DOI:10.1081/NCN-120022746

Cis-D-2-hydroxy-4-thymin-1-yl-pyrrolidine propionic acid unit is used to make PNA-DNA dimer block that is incorporated in DNA sequences at selected positions. Since the amide linkage is shorter than phosphodiester linkage, insertion of an extra atom in th

Full text of DOI:10.1081/NCN-120022746

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Pyrrolidine PNA-DNA Chimeric Oligonucleotides with
Extended Backbone
Vaijayanti A. Kumar a b & Meena a
a Division of Organic Chemistry , National Chemical Laboratory , Pune, India
b Division of Organic Chemistry , National Chemical Laboratory , Synthesis, Dr. Homi Bhabha
Road, Pune, 411008, India
Published online: 31 Aug 2006.
To cite this article: Vaijayanti A. Kumar & Meena (2003) Pyrrolidine PNA-DNA Chimeric Oligonucleotides with Extended
Backbone, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1101-1104, DOI: 10.1081/NCN-120022746
To link to this article: http://dx.doi.org/10.1081/NCN-120022746
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1101–1104, 2003
Pyrrolidine PNA-DNA Chimeric Oligonucleotides with
Extended Backbone
*
Vaijayanti A. Kumar and Meena
Division of Organic Chemistry, National Chemical Laboratory, Pune, India
ABSTRACT
Cis-d-2-hydroxy-4-thymin-1-yl-pyrrolidine propionic acid unit is used to make
PNA-DNA dimer block that is incorporated in DNA sequences at selected posi-
tions. Since the amide linkage is shorter than phosphodiester linkage, insertion of
an extra atom in the backbone with amide linkage seems to be better accommo-
dated for internucleotide distance-complementarity.
Key Words: PNA-DNA chimera; PNA.
INTRODUCTION
The combination of PNA and DNA as PNA-DNA chimera has useful biolo-
gical application such as primer for DNA polymerases and as antisense oligo-
nucleotides in therapeutics. Introduction of positive charges might increase the
binding ability of such chimeric oligonucleotides due to reduction of the nega-
tively charged phosphate interactions. In this regard cis-d-2-hydroxy-4-thymin-1-
yl-pyrrolidine acetic acid unit was used to make PNA-DNA chimera and when
*Correspondence: Vaijayanti A. Kumar, Division of Organic Chemistry, National Chemical
Laboratory, Synthesis, Dr. Homi Bhabha Road, Pune 411008, India; Fax: þ91 20 5893355;
E-mail: vkumar@dna.ncl.res.in.
1101
DOI: 10.1081/NCN-120022746
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1102
Kumar and Meena
incorporated into DNA sequences at selected positions was found to destabilize
the derived DNA=DNA-PNA chimera duplexes and triplexes.[1] Since amide link-
age is shorter than phosphodiester linkage, replacement of the phosphodiester
linkage with amide linkage may necessiate the insertion of an extra atom in
the backbone[2] to retain internucleotide distance-complementarity. With this
aim, study of PNA-DNA chimera where pyrrolidine propionic acid unit having
one extra carbon atom in the backbone as compared to phosphodiester linkage
was attempted. We present here the synthesis of the dimer building block used
for chimera, synthesis of DNAs with chimeric unit at selected positions and their
binding studies with complementary DNA.
RESULTS AND DISCUSSION
The pyrrolidine derivative 3 was synthesized from trans-4-hydroxy-D-proline
according to the reported procedure.[1] After DMT protection of the primary
hydroxy group, the ring nitogen was deprotected by hydrogenation using Pd-C
and was then alkylated by Michael addition to ethyl acrylate to get compound 4.
The ester was then hydrolyzed and coupled with 50-amino-50-deoxy thymidine deri-
vative using TBTU=HOBT in dry DMF to get the amide dimer 5a. Phosphitylation
of 5a gave the desired protected phosphoramidite 5b for site specific incorporation
into oligonucleotides (Sch. 1). The modified and control DNA sequences synthesized
are listed in Table 1. All the new compounds synthesized were characterized by spec-
troscopic analysis.
The stability (Table 2) of the triplex 5:6ꢀ7 containing polypyrimidine ODN 7
carrying amide linked dimer block at the center is similar to the stability of the cor-
responding unmodified triplex 5:6ꢀ1. This suggests that the distance between the
neighboring base pairs is affected by the incorporation of this extended backbone
and is better accommodated in the triplex. The duplexes are also less destabilized
with this modification as compared to our earlier modification.[1]

Pyrrolidine PNA-DNA Chimeric Oligonucleotides
1103
Scheme 1.
Table 1. DNA sequences.
1
2
3
4
5
6
7
8
50 TTC TTC TTC TTT TCT TTT
50 AAA AGA AAA GAA GAA GAA
50 CTT GTA CTT TTC CGG TTT
50 AAA CCG GAA AAG TAC AAG
50 TCC AAG AAG AAG AAA AGA AAA TA
50 ATA TTT TCT TTT CTT CTT CTT GGA
50 TTC TTC TTC tt C TCT TTT
50 CTT GTA CT tt TC CGG TTT
tt represents pyrrolidine-DNA thymine dimer block.
Table 2. UV-Tm studies of DNA duplex and triplex structures.
Duplex
Tm(ꢁC)
DTm
Triplex
Tm(ꢁC)
DTm
1:2
7:2
3:4
3:8
46
41
51.8
46.2
5:6ꢀ1
5:6ꢀ7
23.5
23.5
ꢂ5(ꢂ7)
0(ꢂ4)
ꢂ5.6(ꢂ6)
Values in parenthesis indicate the UV-Tm of duplexes and triplexes with modified pyrrolidine
oligomers reported earlier by us.[1]
CONCLUSIONS
We report here the synthesis of a PNA-DNA dimer block with controlled
conformational flexibility. The selected stereochemistry in the pyrrolidine PNA
monomer is 2R, 4R which is similar to the ribose sugar stereochemistry. This
dimer block is incorporated at predetermined positions in DNA oligomers. DNA

1104
Kumar and Meena
complementation studies indicate that the extended amide backbone shows better
nucleobase distance complemetarity to target DNA as compared to the shorter
amide linkage. Further studies to exploit this understanding are in progress in our
laboratory.
ACKNOWLEDGMENTS
VAK thanks Department of Science and Technology, New Delhi, for financial
support. Meena thanks CSIR, New Delhi, for research fellowship.
REFERENCES
1. Kumar, V.A; Pallan, P.S; Meena; Ganesh, K.N. Pyrrolidine nucleic acids:
DNA=PNA oligomers with 2-hydroxy=aminomethyl-4-(thymin-1-yl)-pyrro-
lidine-N-acetic acid. Org. Lett. 2001, 3, 1269.
2. Minasov, G.; Jasenka, M.-A.; Haeberli, P.; Usman, N.; Beigelman, L.; Egli, M.
Studies of a chemically modified oligodeoxynucleotide containing a 5-atom
amide backbone which exhibits improved binding to RNA. Nucleosides,
Nucleotides and Nucleic Acids 2001, 20, 991.