Aminoethylprolyl (aep) PNA: Mixed purine/pyrimidine oligomers and binding orientation preferences for PNA:DNA duplex formation

Article abstract of DOI:10.1021/ol015546k

(equation presented) The synthesis of (2S,4S)- and (2R,4S)-aepPNA monomers of adenine, guanine, and cytosine (3-5) and their incorporation at appropriate positions into aegPNA sequence 7 leads to mixed aeg-aep backbone/mixed nucleobase PNAs 8-11. The ther

Full text of DOI:10.1021/ol015546k

ORGANIC
LETTERS
2001
Vol. 3, No. 9
1281-1284
Aminoethylprolyl (aep) PNA: Mixed
Purine/Pyrimidine Oligomers and
Binding Orientation Preferences for
PNA:DNA Duplex Formation
Moneesha D’Costa, Vaijayanti Kumar,* and Krishna N. Ganesh*
DiVision of Organic Chemistry (Synthesis), National Chemical Laboratory,
Pune 411008, India
kng@ems.ncl.res.in
Received January 11, 2001
ABSTRACT
The synthesis of (2S,4S)- and (2R,4S)-aepPNA monomers of adenine, guanine, and cytosine (3−5) and their incorporation at appropriate
positions into aegPNA sequence 7 leads to mixed aeg-aep backbone/mixed nucleobase PNAs 8−11. The thermal stabilities of the derived
duplexes with DNA are found to be dependent on nucleobase and backbone stereochemistry.
Peptide nucleic acids wherein the nucleobases A, G, C, and
T are linked to acyclic, achiral N-(2-aminoethyl)glycyl
(aegPNA) backbone through a methylene carbonyl linker
have emerged as successful DNA mimics.1 Several analogues
and derivatives of aegPNA2 have been synthesized to
overcome the limitations of PNA such as poor aqueous
solubility and orientational ambiguity in complementary
DNA recognition by making them cationic and chiral. Recent
attempts toward designing newer PNA analogues are based
on constraining the flexibility of the acyclic backbone to pre-
organize the PNA structure for favorable and selective DNA/
RNA recognition. These have resulted in a novel class of
PNA analogues (Figure 1) derived from cyclic, chiral
monomers of either neutral prolyl residues3 or the cationic
pyrrolidine core.4,5 The two chiral centers of the five-
membered pyrrolidine ring along with the different possible
ring substitutions in such monomers lead to a combinatorial
library of regio- and stereoisomers with the derived PNA
oligomers possessing wide conformational diversity. The
foremost analogue in this series, the prolyl PNA,3 was
derived by the introduction of a methylene bridge between
the â-C of ethylenediamine unit with the R′-C of the glycyl
unit on the PNA backbone. These marginally improved the
affinity and selectivity in DNA:PNA binding, but were found
to be less soluble in aqueous medium. The replacement of
the glycyl segment of the PNA backbone by a prolyl unit to
which the nucleobase is directly linked led to the cationic
and chiral aminoethylprolyl PNAs (aepPNA).4 The mixed
backbone aeg-aepPNA T8 oligomers derived from either
(3) (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.
(4) D′Costa, M.; Kumar, V. A.; Ganesh, K. N. Org. Lett. 1999, 1, 1513-
1516
(5) (a) Hickman, D. T.; King, P. M.; Cooper, M. A.; Slater, J. M.;
Micklefield, J. Chem. Commun. 2000, 2251-2252. (b) Vilaivan, T.;
Khongdeesameor, C.; Harnyuttanakorn, P.; Westwell, M. S.; Lowe, G.
BioMed. Chem. Lett. 2000, 10, 2541-2545. (c) Puschl, A.; Tedeschi, T.;
Nielsen, P. E. Org. Lett. 2000, 2, 4161-4163.
* Authors to whom correspondence should be addressed. (V.K.) E-
mail: vkumar@dna.ncl.res.in.
(1) (a) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624-630. (b) Ulhmann,
E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998,
37, 2796-2823.
(2) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 4, 931-943.
10.1021/ol015546k CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/04/2001

Scheme 1a
a Reagent: (i) methanesulfonyl chloride, pyridine; (ii) B (Abz/
2-amino-6-chloropurine/Cz), K2CO3, DMF; (iii) NaOH, CH3OH,
H2O.
with the undesired N7 isomer in higher proportion. To
overcome this, 2 was alkylated with 2-amino-6-chloropurine,
which gave the N9 isomer 4a as a major product (90%).
The (2R,4S)-aepPNA monomers were likewise synthesized
from the diastereomer 1(2R,4R) with similar yields. The
hydrolysis of the methyl esters was effected with 1 N NaOH
in aqueous MeOH to get the desired PNA monomer synthons
3b-5b (2R/S,4S) in 95% yield. These and the corresponding
T monomer4 6 were individually incorporated into the PNA
sequences 8-11 by solid-phase synthesis using the standard
Boc-chemistry as previously reported.4 The unmodified PNA
7 and DNA 12, 13 sequences required for constituting the
duplexes were synthesized by standard procedures.
Figure 1. Strategies for the design of conformationally constrained
PNA analogues.
(2R,4S) or (2S,4S) backbone stereochemistry exhibited high
affinity and sequence specificity to form DNA:PNA2 tri-
plexes. Further, in such homopyrimidine aepPNA oligomers,
the nature of the backbone chirality was inconsequential in
differentiating the binding of complementary DNA in both
parallel (HG) and antiparallel (WC) orientations.6
In view of the above results, it was thought worthwhile
to examine the effect of aep backbone chirality on the
binding orientation in duplexes with mixed purine/pyrimidine
sequences. This and the combined characteristics of high
sequence specificity, affinity, improved aqueous solubility,
intrinsic positive charge, and the ease of synthesis of aepPNA
prompted us to construct aeg-aepPNA oligomers comprised
of pyrimidine-purine units and study preferences in duplex-
ation with DNA. We herein report the synthesis of (2S,4S)-
and (2R,4S)-aepPNA monomers of cytosine, adenine, and
guanine (3-5) and their incorporation, along with thymine4
6, in a control PNA sequence 7 at appropriate positions to
generate aeg-aepPNAs 8-11. Preliminary UV-Tm studies
of their binding with antiparallel and parallel complementary
DNA sequences (12, 13) reveal interesting stereochemical
and orientation preferences in PNA:DNA duplex formation.
Synthesis of aepPNA Monomers and aeg-aepPNAs. The
(2S,4S)-aep-PNA monomers 3-5 were synthesized (Scheme
1) by N-alkylation of protected nucleobases Abz, GiBu, and
CZ with the common intermediate 1-(N-Boc-aminoethyl)-
4(R)-O-mesyl-2(S)-proline methyl ester 2. This was obtained
from mesylation of the known (2S,4R) 4-hydroxyl com-
pound4 1 (45% yield). While Abz and CZ gave the expected
products 3a and 5a in 65% and 60% yield, respectively, GiBu
gave a mixture of N7:N9 alkylated products in a 5:1 ratio
All the modified PNA monomers were characterized by
1H and 13C NMR and mass spectrometry.7 The PNA
oligomers were purified by C18 reversed-phase HPLC and
(7) Characteristic signals. 3a (2S,4S): 1H NMR (CDCl3) δ 9.27 (br s,
1H, Benzamide-NH), 8.76 (s, 1H, A-H2), 8.70 (s, 1H, A-H8), 8.20 (m, 2H,
Bz, o-CH), 7.56 (m, 3H, Bz, p-CH, m-CH), 5.40 (m, 1H, H4), 5.15 (m,
1H, Boc-NH), 3.74 (s, 3H, OCH3), 3.60-2.60 (m, 9H, H5, H5′, H3, H3′,
H2, Boc-NH-(CH2)2), 1.45 (s, 9H, C(CH3)3); [R]25 ) +12° (c ) 0.1,
D
CH3OH). 3a (2R,4S): 1H NMR (CDCl3) δ 9.27 (br s, 1H, benzamide-NH),
8.76 (s, 1H, A-H2), 8.10 (s, 1H, A-H8), 8.00 (d, 2H, Bz, o-CH), 7.56 (t,
1H, Bz, p-CH), 7.45 (t, 2H, Bz, m-CH), 5.27 (m, 2H, H4, Boc-NH), 3.93
(m, 1H, H2), 3.74 (s, 3H, OCH3), 3.60 (m, 2H, H5, H5′), 3.20 (m, 2H, H3,
H3′), 2.83 (m, 2H, Boc-NH-CH2), 2.60 (m, 2H, Boc-NH-CH2CH2), 1.42
(s, 9H, C(CH3)3); [R]25D) +20.2° (c ) 0.1, CH3OH); MALDI-TOF M+)
509.6. 4a (2S,4S): 1H NMR (CDCl3) δ 8.35 (s, 1H, 2-amino-6-chloropurine,
H8), 5.20 (br s, 2H, N2H2), 5.10 (m, 2H, H4, Boc-NH), 3.75 (s, 3H, OCH3),
3.30 (m, 4H, H5, H5′, Boc-NH-CH2), 2.90 (m, 3H, H2, Boc-NHCH2CH2),
2.70 (m, 1H, H3), 2.15 (m, 1H, H3′), 1.45 (s, 9H, C(CH3)3); [R]25D ) -24°
(c ) 0.1, CH3OH). 4a (2R,4S): 1H NMR (CDCl3) δ 7.85 (s, 1H, 2-amino-
6-chloropurine, H8), 5.20 (m, 4H, N2H2, H4, NH), 3.92 (t, 1H, H2), 3.76
(s, 3H, OCH3), 3.60-3.00(br m, 4H, H5, H5′, H3, H3′), 2.84 (t, 2H, Boc-
NH-CH2), 2.56(t, 2H, Boc-NH-CH2CH2), 1.46 (s, 9H, C(CH3)3); [R]25
)
D
-4.8° (c ) 0.19, CH3OH); MALDI-TOF M + H ) 441.6. 5a (2S,4S): 1H
NMR (CDCl3) δ 8.45 (d, 1H, C-H6), 7.40 (s, 5H, Ph), 7.25 (d, 1H, C-H5),
5.35 (t, 1H, Boc-NH), 5.20 (s, 3H, Ph-CH2, H4), 3.75 (s, 3H, OCH3), 3.20
(br m, 4H, H5, H5′, Boc-NH-CH2), 2.80 (m, 2H, Boc-NH-CH2-CH2), 2.60
(m, 1H, H2), 1.90 (m, 2H, H3, H3′), 1.45 (s, 9H, C(CH3)3); [R]25D ) +9.6°
(c ) 0.3, CH3OH). 5a (2R,4S): 1H NMR (CDCl3) δ 7.90 (d, 1H, C-H6),
7.35 (s, 5H, Ph), 7.25 (d, 1H, C-H5), 5.40 (m, 1H, Boc-NH), 5.20 (s, 2H,
Ph-CH2), 4.90 (m, 1H, H4), 3.95 (dd, 1H, H5), 3.74 (s, 3H, OCH3), 3.40
(m, 1H, H5′), 3.25 (m, 2H, Boc-NH-CH2), 2.95 (m, 1H, H2), 2.77 (m, 2H,
Boc-NH-CH2CH2), 2.60 (m, 1H, H3), 2.15 (m, 1H, H3′), 1.45 (s, 9H,
C(CH3)3); [R]25 ) +24.2° (c ) 0.1, CH3OH); MALDI-TOF M + H )
D
517.7. PNA 8, 9, 10, 11: (MALDI-TOF) Mcalc)2796, Mobs)2796.
(8) Nielsen, P. E.; Egholm, M., Eds. Peptide Nucleic Acids: Protocols
and Applications; Horizon Scientific Press: Norfolk, England, 1999.
(6) parallel (p): N-end of PNA facing the 5′-end of DNA in the complex,
antiparallel (ap): C-end of PNA facing the 5′-end of DNA.
1282
Org. Lett., Vol. 3, No. 9, 2001

characterized by MALDI-TOF mass analysis. The mixed
PNA sequences 8-11 constituted by systematic replacement
of specific aeg units in control aegPNA 7 by appropriate
aep unit were synthesized to test the explicit effect of each
nucleobase in (2S/R) aep backbone on duplex formation
(Scheme 2).
To examine whether the observed higher stabilities of aeg-
aepPNAs were accompanied by any loss in base specificity,
UV-Tm’s were determined for hybrids having single base
mismatches on the antiparallel DNA sequences at the site
complementary to the aepPNA unit. While any single
mismatch in the control PNA lowered the duplex Tm by
4-8°, the mismatches in aep-PNA induced larger de-
stabilization in the range 9-30° (Table 2). The mismatch
Scheme 2. DNA and PNA Sequencesa
Table 2. UV-Tm (°C) of Mismatched PNA:DNA Duplexesa
DNA (5′f 3′)
aegPNA 7
aepPNA
14 AGT GAT CCA C
15 AGT GTT CTA C
16 AGT GAT ATA C
17 AGT TAT CTA C
a Buffer: 10 mM sodium phosphate, pH 7.4. The mismatch data reported
above are for the (2S,4S)-aepPNA units, a, t, g, and c respectively. Values
in parentheses indicate the ∆Tm values compared to the fully matched PNA:
DNA 12 complexes.
35.4 (-8.4)
36.8 (-7.0)
39.6 (-4.2)
36.8 (-7.0)
8 (a ), 44.4 (-9.4)
9 (t), 26.9 (-29.7)
10 (g), 24.7 (-18.5)
11 (c), 43.6 (-11.6)
a a, t, g, and c represent (2S/R,4S)-aepPNA units.
UV-Tm and CD Studies of PNA:DNA Duplexes. The
UV-Tm data of complexes of 8-11 with DNA in both
orientations (12, antiparallel; 13, parallel) obtained as a
measure of duplex stability are summarized in Table 1 and
effects followed the destabilization order t:T (9:15) > g:A
(10:16) > c:T (11:17) > a:C(8:14) and the results indicate
that the higher affinity of aeg-aepPNA for complementary
DNA was achieved without any loss of selectivity.
The magnitude of percent hyperchromicity changes in the
melting transitions was also base dependent. The purine
substitutions exhibited hyperchromicity changes (9-12%)
as good as the control aegPNAs (11-13%) and higher than
pyrimidines (6-9%). The parallel and antiparallel PNA:DNA
Table 1. UV-Tm (°C) of PNA:DNA Duplexesa
DNA 12
DNA 13
antiparallel
parallel
PNA
PNA 7
(2S,4S)
(2R,4S)
(2S,4S)
(2R,4S)
43.8
40.3
PNA 8 (a )
PNA 9 (t)
PNA 10 (g)
PNA 11 (c)
53.8
56.6
43.2
55.2b
53.1
50.2
50.2
33.0b
34.0b
26.2b
53.1
62.3b
58.3
31.2b
28.3
34.0b
a Buffer: 10 mM sodium phosphate, pH 7.4. Tm values are accurate to
((1 °C). Experiments were repeated at least three times, and the Tm values
were obtained from the peaks in the first derivative plots. b Low hyper-
chromicity (∼6%), for others 9-12%.
shown in Figure 2. In the unmodified aegPNA 7, the anti-
parallel duplex was more stable than the parallel duplex by
3.5°. The aeg-aepPNAs 8, 9, and 11 having (2S,4S)-aep-T,
A, and C units uniformly stabilized the antiparallel duplexes
(DNA 12) by 10-13°/unit compared to the control PNA 7.
The complex formed by aepPNA-G 10(2S,4S) was as good
as that of the control. The (2S,4S)-aep purine A/G units (8/
10) also stabilized the parallel PNA:DNA duplexes (DNA
13), whereas the corresponding PNAs with T/C units (9/11)
destabilized the parallel duplexes. In the other stereochem-
istry (2R,4S), the aepPNAs show base related variations in
both parallel and antiparallel orientations; the aep units with
A/G/C stabilized the antiparallel duplexes (PNA8/10/11:DNA
12) and T aepPNA 9 had detrimental effects in both parallel
and antiparallel orientations. The effect of stereochemistry
on stability was most prominent in case of guanine aepPNA
10, where the (2S,4S) isomer preferred a parallel orientation
(∆Tm(p-ap) +15°) and (2R,4S) isomer was more stable in the
antiparallel orientation (∆Tm(ap-p) +24°).
Figure 2. UV-Tm first derivative plots of aeg-aepPNA:DNA
duplexes. (2S,4S)-PNA (s); (2R,4S)-PNA (- - -). Melting experi-
ments were performed with a PNA:DNA strand concentration of
0.5 µM each. The concentrations were calculated on the basis of
the absorbance at 260 nm using the molar extinction coefficients
of A, T, G, and C as for DNA.8 Y-axis data is derived from change
in percent hyperchromicity with temperature.
Org. Lett., Vol. 3, No. 9, 2001
1283

duplexes are expected to differ in the base stacking patterns,
and this should be reflected in their circular dichroic (CD)
spectra. Figure 3 shows the CD profiles of control aegPNA
and selected aeg-aepPNAs in parallel and antiparallel
orientations.
complexes of achiral PNA and DNA. This is perhaps due to
the fact that CD of PNA:DNA complexes are dominated by
DNA contributions.
The overall results indicate that single aep modifications
are satisfactorily accommodated in the aeg PNA backbones
leading to significant thermal stabilization effects. The
stereochemistry at C2 has considerable effect on the stability
of hybrids since R isomers except 9 lowered the duplex
stability (with DNA 13). The fact that aep modifications in
mixed sequence duplexes exhibit stabilization effects de-
pending on the nucleobase and orientation suggests that the
positive charge on the pyrrolidine may not be entirely
responsible for the properties of aepPNA.
The pyrrolidine ring pucker and/or syn-anti orientation of
the nucleobases directly attached to the ring perhaps dictate
the individual parallel/antiparallel preferences seen for the
nucleobases on chiral aep units. It is known that the nature
of the 4-substituent plays an important role in defining the
pucker of the pyrrolidine ring in 4-substituted prolines.9 The
individual purines or pyrimidines present at the 4-position
in aepPNA monomers may cause differential pyrrolidine ring
puckers and consequent backbone conformational changes.
The stereochemistry at C4 of the proline ring (4S) to which
the nucleobase is attached is same as the spatial disposition
in natural DNA.4
In summary, the incorporation of single (2S,4S)-aep-A/
T/G/C units in 7 stabilize the antiparallel duplexes with DNA.
The individual nucleobases in (2R,4S)-aepPNA display
interesting binding preferences and affinity with comple-
mentary DNA. In view of these interesting results, it emerges
that mixed purine-pyrimidine sequences with a homogeneous
(2S,4S) aep backbone may be appropriate to study the
cumulative effects on the stability of PNA:DNA duplexes.
Such studies are currently in progress.
Figure 3. CD profiles of (A) PNA 10 single strands, (B) Selected
aeg-aepPNA:DNA hybrids, (C) aegPNA:DNA hybrids, and (D)
observed (s) and addition (- - -) spectra of PNA 10 and DNA 13.
Each strand was taken in a concentration of 1 µM, and each CD
spectrum was recorded as an accumulation of eight scans.
Considerable differences were seen in CD signals of the
parallel and antiparallel duplexes, such as the wavelengths
and amplitudes of the positive/negative bands in both 210-
230 nm and the 240-280 nm regions and the crossover
wavelength in 240-280 nm region. These differential
features were preserved in the parallel and antiparallel
duplexes from aeg-aepPNAs. The observed CD effects arise
from duplexation since the experimental CD profiles differ
from that of the sum of the components. The slightly
enhanced amplitudes of both positive and negative bands
suggests an improved base stacking in the modified PNAs,
but with overall retention of conformational integrity. The
backbone configuration of (2S,4S/2R,4S)aep units did not
have any dramatic effect on the geometry of either parallel
or antiparallel PNA:DNA complexes and the CD patterns
of the duplexes were similar to those of parallel/antiparallel
Acknowledgment. M.D. thanks CSIR, New Delhi, for a
research fellowship. V.A.K. and K.N.G. thank the Depart-
ment of Biotechnology, New Delhi, for a research grant.
Supporting Information Available: Mass spectra for key
compounds (2S,4S)-3a-5a, HPLC profiles, MALDI-TOF
mass spectra of (2S,4S)-PNAs 8-11 and UV-Tm plots of
aeg-aepPNA:DNA duplexes. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL015546K
(9) Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines,
R. T. Int. J. Pept. Protein Res. 1994, 44, 262-269.
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