Aminoethylprolyl peptide nucleic acids (aepPNA): chiral PNA analogues that form highly stable DNA:aepPNA2 triplexes.

Article abstract of DOI:10.1021/ol990835i

[formula: see text] The replacement of the glycyl component in the peptide nucleic acid (PNA) backbone by a prolyl unit bearing a nucleobase leads to the aminoethylprolyl (aep) PNAs, which are chiral and cationic. The homooligomeric aepPNA binds to complementary DNA sequences with high affinity and sequence specificity, forming highly stable triplexes.

Full text of DOI:10.1021/ol990835i

ORGANIC
LETTERS
1999
Vol. 1, No. 10
1513-1516
Aminoethylprolyl Peptide Nucleic Acids
(aepPNA): Chiral PNA Analogues That
Form Highly Stable DNA:aepPNA2
Triplexes
Moneesha D’Costa, Vaijayanti A. Kumar,* and Krishna N. Ganesh*
DiVision of Organic Chemistry (Synthesis), National Chemical Laboratory,
Pune 411008, India
kng@ems.ncl.res.in
Received July 20, 1999
ABSTRACT
The replacement of the glycyl component in the peptide nucleic acid (PNA) backbone by a prolyl unit bearing a nucleobase leads to the
aminoethylprolyl (aep) PNAs, which are chiral and cationic. The homooligomeric aepPNA binds to complementary DNA sequences with high
affinity and sequence specificity, forming highly stable triplexes.
One of the most prominent outcomes of the search for DNA/
RNA (I) analogues as effective antigene/antisense agents is
the emergence of peptide nucleic acids (PNAs, II) in which
and affinity.2 In spite of its resistance to cellular enzymes
such as nucleases and proteases, the major limitations
confounding its application are ambiguity in orientational
selectivity of binding, poor solubility in aqueous media, and
inefficient cellular uptake.1d,3 Attempts to resolve the orien-
tational ambiguity focus on introduction of chirality to PNA
by linking chiral amino acids,4 peptides,5 and oligonucle-
(1) (a) Nielsen, P. E. Annu. ReV. Biophys. Biomol. Struct. 1995, 24, 167-
183. (b) Hyrup, B.; Nielsen, P. E. Bioorg. Med. Chem. Lett. 1996, 4, 5-23.
(c) Good, L.; Nielsen, P. E. Antisense Nucleic Acids Drug DeVelop. 1997,
7, 431-437 (d) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew.
Chem. Int. Ed. 1998, 37, 2796-2823.
(2) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S.
M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
Nature 1993, 365, 566-568.
(3) Hanvey, J. C.; Peffer, N. J.; Bisi, J. E.; Thomson, S. A.; Cadilla, R.;
Josey, J. A.; Ricca, D. J.; Hassman, F.; Bonham, M. A.; Au, K. G.; Carter,
S. G.; Bruckenstein, D. A.; Boyd, A. L.; Noble, S. A.; Babiss, L. E. Science
1992, 258, 1481-1485.
the charged sugar-phosphate backbone of DNA is replaced
by a neutral and achiral polyamide backbone consisting of
N-(2-aminoethyl)glycine units.1 In PNAs, the nucleobases
are attached to the backbone through a conformationally rigid
tertiary acetamide linker group and PNA binding to the target
DNA/RNA sequences occurs with high sequence specificity
(4) Kim, S. H.; Nielsen, P. E.; Egholm, M.; Buchardt, O. J. Am. Chem.
Soc. 1993, 115, 6477-6489.
(5) Koch, T.; Naesby, M.; Wittung, P.; Jørgensen, M.; Larsson, C.;
Buchardt, O.; Stanley, C. J.; Norde´n, B.; Nielsen, P. E.; Ørum, H.
Tetrahedron Lett. 1995, 36, 6933-6936.
(6) (a) Petersen, K. H.; Jensen, D. K.; Nielsen, P. E.; Egholm, M.;
Buchardt, O. Bioorg. Med. Chem. Lett. 1995, 5, 1119-1124. (b) Bergmann,
F.; Bannwarth, W.; Tam, S. Tetrahedron Lett. 1995, 36, 6823-6826.
10.1021/ol990835i CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/15/1999

Scheme 1
otides6 to the PNA or by using chiral amino acids in the
advantages over a similar modification reported earlier,11
which has the nitrogen of the pyrrolidine ring in the backbone
in the tertiary amide form and, hence, is structurally too rigid.
The nucleobase in III is directly attached to the pyrrolidine
ring at the C4 position, without changing the net number of
atoms connecting two successive nucleobases. Further, the
two chiral centers at C2 and C4 offer an opportunity to study
the stereochemical implications on the interactions of chiral
PNA with ss/ds DNA/RNA. The synthesis of two of the four
possible stereoisomers, (2S/R,4S)-1-(N-Boc-aminoethyl)-4-
(thymin-1-yl)proline, and their site-specific incorporation into
various PNA oligomers by solid-phase synthesis and hy-
bridization properties with complementary DNA is reported.
Chemical Syntheses of aepPNA Monomer and Oligo-
mers. The syntheses of the (2S,4S) and (2R,4S) isomers of
1-(N-Boc-aminoethyl)-4-(thymin-1-yl)proline were achieved
in four steps starting from the naturally occurring (2S,4R)-
4-hydroxyproline (1) as indicated in Scheme 1. The N-
alkylation of the pyrrolidine ring in trans-4-hydroxy-L-proline
methyl ester 2 by N-Boc-aminoethyl bromide afforded the
1-(N-Boc-aminoethyl)proline derivative 3. The replacement
of the C4 hydroxyl function with N3-benzoylthymine under
Mitsunobu reaction conditions12 then yielded the (2S,4S)-4-
(N3-benzoylthymin-1-yl)proline derivative 4 with inverted
stereochemistry at C4. The hydrolysis of the methyl ester
and N3-benzoyl protecting group of thymine was achieved
in a single step, by employing sodium hydroxide (1 N) in
aqueous methanol to provide (2S,4S)-1-(N-Boc-aminoethyl)-
4-(thymin-1-yl)proline (5) as the desired monomer. Starting
from (2R,4S)-4-hydroxyproline 68a and by following the same
backbone itself.7 Bridging the â-C of the ethylenediamine
moiety of PNAs and the glycyl R′-carbon with a methylene
group generates a five-membered pyrrolidine ring which
imparts structural rigidity in the PNA backbone with
simultaneous introduction of chirality.8 This has marginally
improved the affinity and selectivity in DNA:PNA2 binding
in a desired manner. Conjugation of spermine to the
C-terminus rendered the PNA cationic and improved its
aqueous solubility.9 Linking of the nucleobase to the poly-
amide backbone via a flexible ethylene linker instead of an
acetamide linker creates a positive charge in the PNA
backbone at the expense of the conformational rigidity of
the tertiary amide linkage.10 Although this improved the
aqueous solubility of the PNA, the detrimental effect on the
stability of DNA:PNA2 complexes stressed the importance
of rigid preorganization of the PNA structure for effective
binding to ss/ds DNA.
In this Letter, we introduce the new chiral aminoethyl-
prolyl (aep) PNA backbone (III), designed by linking the
glycyl R′-carbon of the PNA with the acetamido â′-carbon
via a methylene bridge. This modification is aimed at tuning
the rigidity and flexibility of the PNA backbone, with
concomitant introduction of a positive charge on the tertiary
nitrogen of proline. This has potential conformational
(7) Haaima, G.; Lohse, O.; Buchardt, O.; Nielsen, P. E. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1939-1941.
(8) (a) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetrahedron
1996, 52, 15017-15030. (b) Gangamani, B. P.; Kumar, V. A.; Ganesh, K.
N. Tetrahedron 1999, 55, 177-192. (c) Jordan, S.; Schwemler, C.; Winfried,
K.; Kretschmer, A.; Schwenner, E.; Stropp, U.; Mielke, B. Bioorg. Med.
Chem. Lett. 1997, 7, 681-686, 687-690.
(9) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Biochem. Biophys.
Res. Commun. 1997, 240, 778-782.
(10) Hyrup, B.; Egholm, M.; Buchardt, O.; Nielsen, P. E. Bioorg. Med.
Chem. Lett. 1996, 6, 1083-1088.
(11) Lowe, G.; Vilaivan, T.; Westwell, M. S. Bioorg. Chem. 1997, 25,
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(12) Lowe, G.; Vilaivan, T. J. Chem. Soc., Perkin Trans. 1 1997, 539-
546.
1514
Org. Lett., Vol. 1, No. 10, 1999

steps (i to iv), the (2R,4S)-1-(N-Boc-aminoethyl)-4-(thymin-
1-yl)proline isomer (7) was prepared as shown in Scheme
1. The structural integrity of the aepPNA monomers 5 and
7 was confirmed by spectral data (NMR and mass spectros-
copy).
PNA oligomers containing the (2S/R,4S)-1-(N-Boc-ami-
noethyl)-4-(thymin-1-yl)proline units were assembled by
solid-phase peptide synthesis on Merrifield resin derivatized
with N-Boc-â-alanine (0.29 mequiv/g of resin). The aepPNA
unit was incorporated into a PNA octamer, H-T8-NHCH2-
CH2COOH at predetermined positions to yield the aepPNAs
8-11. The unmodified aegPNA sequence H-T8-NHCH2CH2-
COOH 12 was also synthesized for control studies. The
oligomers were cleaved from the solid support by treatment
with trifluoroacetic acid-trifluoromethanesulfonic acid14 to
yield the corresponding sequences carrying â-alanine at their
carboxy termini. These were purified by FPLC on a PepRPC
column. No precipitation was observed in samples of
aepPNA even after prolonged storage. The purity of the
oligomers was rechecked by HPLC on a RPC-18 column
and characterized by MALDI-TOF mass spectrometry (PNA
8: Mobsd ) 2218; Mcalcd ) 2217.17. PNA 9: Mobsd ) 2219;
Mcalcd ) 2215.19. PNA 10: Mobsd ) 2212; Mcalcd ) 2212).
The DNA oligomers 13 and 14 were synthesized on a
Pharmacia Gene Assembler Plus synthesizer by employing
standard phosphoramidite chemistry,15 followed by ammonia
deprotection. These were purified by gel filtration and their
purities checked by HPLC.
Figure 1. Melting profiles of DNA:PNA2 complexes. A: for the
2S modification, (a) 13:12, (b) 13:8, (c) 13:9, (d) 13:10, (e) 13:11.
B: (a) 13:11, (b) 14:11, and for single strands, (c) 14, (d) 11.
The presence of a single 2S,4S/2R,4S aep unit at the
C-terminus of the oligomer 8 has no detrimental effect on
the stability of its complex with the complementary DNA
(Table 1, entries 1 and 7). Increasing the number of aep units
Table 1. UV-Tm (°C) of DNA:PNA2 Complexes
The PNA sequences used here are homopyrimidines that
are known to form DNA:PNA2 triplexes.4 UV mixing and
titration experiments indicated a 1:2 binding stoichiometry
(DNA:aepPNA2) for PNA oligomers of both alternating aep-
aeg units and homooligomers of aep units, as in the control
aegPNA. Hence, all complementation studies were performed
with 1:2 stoichiometry of DNA and aeg/aepPNA. The percent
hyperchromicity-temperature plots derived from the UV
melting data indicated a single transition (Figure 1A),
characteristic of DNA:PNA2 triplexes, in which both PNA
strands dissociate from DNA simultaneously, in a single step.
in the oligomer (two in the aep-aeg oligomer 9, four in the
alternating aep-aeg oligomer 10, and eight in the aep
homooligomer 11) leads to a progressive increase in the
stability of complexes with DNA (∆Tm ) +5-8 °C per aep
unit, Table 1, entries 2-4), with incomplete melting for
oligomers 10 and 11 even beyond 75 °C. When a single T-T
mismatch is present in the middle of the sequence as in 14:
10 and 14:11, the DNA:PNA2 complexes failed to show
sigmoidal transitions in UV-melting curves, resulting in only
a small linear increase in percent hyperchromicity (entries 5
and 6 and Figure 1B). Thus aepPNA units enormously
stabilize DNA:PNA2 triplexes but still retain the stringency
(13) (a) (2S,4S)-1-(N-Boc-aminoethyl)-4-(thymin-1-yl)proline 5: 1H
NMR (D2O) δ 7.4 (s, 1H), 4.1 (br m, 2H), 3.6 (br m, 4H), 3.4 (br m, 1H),
3.1 (br m, 2H), 2.4 (m, 1H), 1.8 (s, 3H), 1.4 (s, 9H). m/e ) 382. [R]25
D
-34.5° (c ) 0.12, CH3OH). (b) (2R,4S)-1-(N-Boc-aminoethyl)-4-(thymin-
1-yl)proline 7: 1H NMR (D2O) δ 7.4 (s, 1H), 4.4 (t, 1H), 4.2 (m, 1H),
3.7-3.3 (br m, 6H), 2.9 (m, 1H), 2.6 (m, 1H), 1.9 (s, 3H), 1.4 (s, 9H). m/e
) 382. [R]25 +43.6° (c ) 0.12, CH3OH).
D
(14) Fields, G. B.; Fields, C. G. J. Am. Chem. Soc. 1991, 113, 4202-
4207.
(15) Gait, M. J. Oligonucleotide Synthesis, a Practical Approach; IRL
Press Limited: Oxford, 1984.
Org. Lett., Vol. 1, No. 10, 1999
1515

in terms of base mismatches. PNA single strands, when
subjected to the same temperature program as the DNA:
PNA2 complexes, exhibited <3% change in absorbance
(Figure 1B), thus ruling out any contribution from single-
stranded ordering to the sigmoidal transition of the DNA:
PNA2 triplex. The tight binding of aepPNAs with comple-
mentary DNA is also seen in diagnostic gel mobility shift
experiments, where even a single modification effected
significant retardation.
case with control aegPNA. Both isomers studied here have
the same spatial disposition of the attachment of the
nucleobase to the proline ring at C4 equivalent to the
nucleobase attachment at C1′ in DNA. Changing the stereo-
chemistry at the other center, i.e., C2, located in the PNA
backbone does not appreciably affect the stability of DNA:
aepPNA2 complexes. The structural changes caused by the
difference in stereochemistry at C2 are probably accom-
modated due to the flexibility imparted to the backbone by
the aminoethyl moiety linking the proline ring. Such flex-
ibility is lost when the proline nitrogen is part of an amide
moiety.11 Molecular modeling studies are in progress to
further examine these effects.
To study the effect of pH on the stability of the DNA:
aepPNA2 complexes, the hybrids were constituted in 10 mM
sodium phosphate buffers of different pHs in the range 6.0-
7.4. No change in Tm (∆Tm ) (1°) was detected in the above
pH range. DNA:PNA2 complexes are known to be destabi-
lized in the presence of salts, and the stability of complexes
of DNA with positively charged ligands is strongly salt-
dependent.16 The presence of salt (100 mM) in the medium
destabilized the higher substituted DNA:aepPNA2 complexes
13:10 and 13:11 to a greater extent (∆Tm g 8°), compared
to the complexes of control aegPNA 13:12 or monosubsti-
tuted aepPNA 13:8 (∆Tm ) 4-5°). The higher salt depen-
dence of Tm of DNA complexes with aepPNAs, both in
alternating and homooligomers, is perhaps a consequence
of the protonated proline ring N in aepPNA units, leading
to electrostatic destabilization under increased ionic strengths.
The pH-titration curve of 4 indicated a pKa ≈ 6.5 for the
pyrrolidine nitrogen, substantiating its protonation status even
at neutral pH. The large stabilization observed for the
alternating aep-aeg oligomer (∆Tm per aep unit ≈ +7-8°;
entry 3) is at least partly due to electrostatic interaction
between the positively charged backbone of aepPNA and
the negatively charged sugar-phosphate DNA backbone, and
this is further amplified in aepPNA homooligomers, which
are polycationic. The main cause of DNA:aepPNA recogni-
tion still appears to be the specific hydrogen bonding between
the complementary nucleobases of PNA (aep/aeg) and DNA,
since even a single mismatch in the middle of the sequence
was found to inhibit DNA:aepPNA complexation, as is the
In summary, we have shown that new peptide nucleic acid
analogues aepPNAs offer one of the best tunings between
rigidity and flexibility of a PNA backbone with positive
charge and show high sequence specific binding to target
DNA sequences. The controlled flexibility in the backbone
accommodates both 2(S/R) stereocenters with equal ef-
ficiency. The introduction of a positive charge in PNA also
enhances its solubility in aqueous media. The synthesis of
mixed oligo aepPNA sequences is currently underway to
explore the potential for orientational selectivity of binding
and efficient DNA duplex strand invasion by these aepPNA
oligomers similar to that seen recently with other cationic
PNAs.17
Acknowledgment. M.D. thanks CSIR, New Delhi, for a
fellowship. V.A.K. and K.N.G. acknowledge the Department
of Biotechnology, New Delhi, for a financial grant for the
project. K.N.G. is an Honorary Senior Faculty of the
Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore.
Supporting Information Available: UV titration and
mixing profiles indicating PNA-DNA binding stoichiom-
1
etry, H and 13C NMR data for selected compounds 3-7,
MALDI-TOF mass spectrum of aepPNA oligomer 8-10,
and gel mobility shift data. This material is available free of
charge via the Internet at http://pubs.acs.org.
(16) Linkletter, B. A.; Szabo, I. E.; Bruice, T. C. J. Am. Chem. Soc.
1999, 121, 3888-3896.
(17) Ishihara, T.; Corey, D. R. J. Am. Chem. Soc. 1999, 121, 2012-
2020.
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