Chimeric peptide nucleic acids incorporating (2S,5R)-aminoethyl pipecolyl units: Synthesis and DNA binding studies

Article abstract of DOI:10.1016/j.tetlet.2004.02.099

The design and facile synthesis of a novel chiral six-membered PNA analogue (2S,5R )-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic acid, aepipPNA, that upon incorporation into PNA sequences effected stabilization of complexes with target complementary DNA

Full text of DOI:10.1016/j.tetlet.2004.02.099

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 3085–3088
Chimeric peptide nucleic acids incorporating (2S,5R)-aminoethyl
pipecolyl units: synthesis and DNA binding studiesq
Pravin S. Shirude, Vaijayanti A. Kumar* and Krishna N. Ganesh
Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411008, India
Received 23 December 2003;revised 12 February 2004;accepted 19 February 2004
Abstract—The design and facile synthesis of a novel chiral six-membered PNA analogue (2S,5R )-1-(N-Boc-aminoethyl)-5-(thymin-
1-yl)pipecolic acid, aepipPNA, that upon incorporation into PNA sequences effected stabilization of complexes with target com-
plementary DNA. This is the first example where a six membered-PNA is shown to be capable of forming stable complexes with
DNA and further expands the repertoire of cyclic PNA analogues.
Ó 2004 Elsevier Ltd. All rights reserved.
Peptide nucleic acids (PNA II), a new class of DNA (I)
mimics invented a decade ago, is emerging rapidly as
novel, potential antigene, and antisense agents, in the
field of medicinal chemistry. In PNA, a neutral and
achiral polyamide backbone consisting of N-(2-amino-
ethyl)glycyl, aeg units1 replaces the charged sugar-
phosphate backbone of DNA. The nucleobases are
attached to the backbone through a rigid tertiary acet-
amide linker and PNA binding to the target DNA/RNA
sequences occurs with high sequence specificity and
affinity.2 In spite of the resistance of PNAs to cellular
enzymes such as nucleases and proteases, the major
limitations complicating the therapeutic application of
PNAs are ambiguity in orientational selectivity of
binding, poor solubility in aqueous media, and ineffi-
cient cellular uptake.3;4 Our efforts5 and those of others6
to improve the properties of aegPNA and achieve fine-
tuning of an aminoethylglycyl, aegPNA backbone to
bind to the complementary nucleic acids through a
pre-organization strategy, has led to a number of five-
membered pyrrolidinyl PNA analogues. We have pre-
viously reported chiral aminoethylprolyl, aepPNA(III),
designed by linking the glycyl a0-carbon in the aegPNA
to the acetamido b0-carbon via a methylene bridge.5 In
aepPNA, the nucleobase is directly attached to a pyr-
rolidine ring instead of being linked to the backbone via
an acetamido moiety. This PNA analogue with posi-
tively charged tertiary amine in the backbone is con-
formationally constrained due to the five-membered
pyrrolidine ring and has significantly improved solubil-
ity accompanied by affinity and selectivity in DNA:PNA
binding, without compromising the base-pairing speci-
ficity.5 The stability of the duplexes was found to be
dependent upon the stereochemistry of the pyrrolidine
ring and also the nucleobase.5
~
~
~
~
2
3
1
HN
HN
HN
1
O
O
B
O
α
B
B
2
3
B
N
N
4
N
β'
4
5
α'
6
~
O
5
O
O
O
O
P
6
O
~
~
~
aepip PNA (IV)
aep PNA (III)
aeg PNA (II)
DNA (I)
In this communication, we report the synthesis
and preliminary evaluation of a six-membered homo-
logue of aepPNA, the chiral aminoethylpipecolylPNA,
aepipPNA (IV). This is a result of bridging the a0-C
atom of the glycyl unit and the b0-C atom of linker to a
nucleobase in PNA with an ethylene bridge instead of a
methylene bridge of aepPNA to generate a six-mem-
bered piperidine ring IV. Although the ethylene bridge
might be expected to be more flexible compared to the
methylene bridge, chair/boat conformations in six-
membered rings are rigidly locked structures in contrast
to the relatively flexible five-membered rings. It will be
Keywords: Six-membered PNA;Antisense therapeutics.
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2004.02.099
* Corresponding author. Tel./fax: +91-20-5893153;e-mail: vakumar@
dalton.ncl.res.in
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.02.099

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P. S. Shirude et al. / Tetrahedron Letters 45 (2004) 3085–3088
Table 1. UV–Tm (°C) of DNA:PNA2 complexes
interesting to study the DNA binding ability of the
resulting PNA sequences in order to understand the
contributing structural factors to assist evolution of
analogues with optimum properties. Herein, we report
the synthesis of one of the four possible stereoisomers,
(2S,5R)-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic
acid 4, its site-specific incorporation into various PNA
oligomers by solid-phase synthesis and hybridization
properties with complementary DNA sequences.
PNA
PNA2:DNA UV–Tm °C
5, H–t T T T T T T T–(b-Ala)–OH
5:14
5:16
6:14
6:16
7:14
8:14
9:14
43 (23.4)
19 (8.9)
6, H–T T T T T T T t–(b-Ala)–OH
48 (19.2)
24 (3.8)
7, H–T T T T t T T T–(b-Ala)–OH
8, H–T T T T T t T t–(b-Ala)–OH
9, H–T T T t T T T t–(b-Ala)–OH
44 (10.9)
52 (26.7)
51 (25.2)
49 (14.3)
60 (26.7)
43 (18.3)
51 (18.5)
10, H–T t T T T T T t–(b-Ala)–OH 10:14
11, H–t T C T C T T T–(b-Ala)–OH 11:15
12, H–T T T T T T T T–(b-Ala)–OH 12:14
13, H–T T C T C T T T–(b-Ala)–OH 13:15
Synthesis of aepipPNA monomer and oligomers. The
synthesis of cis-5(S)-hydroxy-2(S)-N1-benzyloxycar-
bonyl pipecolic acid methyl ester 1 from naturally
T ¼ aegPNA; t ¼ aepipPNA;DNA: 14, 50–GCAAAAAAAACG–30;
15, 50–AAAGAGAA–30; 16, 50–GCAAAATAAACG–30;buffer:
10 mM sodium phosphate, pH 7.30. The Tm values are accurate to ( )
0.5 °C. Experiments were repeated at least thrice and the Tm values
were obtained from the peaks in the first derivative plots. Values in
parentheses represents % hyperchromicity.
occurring L-glutamic acid was accomplished according
to the procedure of Bailey et al.7;8 The deprotection of
N1 in 1 followed by alkylation of the piperidine ring
nitrogen with N-Boc-aminoethyl mesylate6 afforded 1-
(N-Boc-aminoethyl)pipecolyl methyl ester derivative 2.
Displacement of the C5-(S)-hydroxyl function with N3-
benzoylthymine (BzT) under Mitsunobu reaction con-
ditions yielded the (2S,5R)-5-(N3-benzoylthymin-1-yl)
pipecolic ester derivative 3 with inversion of configura-
tion at C-5. The product, even after repeated purifica-
tion by column chromatography, was contaminated
with a small amount of diisopropyl hydrazide. The
hydrolysis of the ester function and N3-benzoyl depro-
tection of thymine in 3 was simultaneously effected, by
treatment with sodium hydroxide in aqueous methanol
to obtain (2S,5R)-1-(N-Boc-aminoethyl)-5-(thymin-1-
yl)pipecolic acid 4, the desired aepipPNA monomer for
PNA synthesis (Scheme 1).
NHCH2CH2–COOH 12 and the mixed homopyrimidine
H–TTCTCTTT–NHCH2CH2–COOH 13 were also
synthesized for control studies. The oligomers were
cleaved from the solid support by treatment with TFA-
TFMSA9 to yield the corresponding PNAs carrying
b-alanine at the carboxy terminus. All PNAs (5–13)
were purified by FPLC on a PepRPC column. The
purity of the oligomers was rechecked by HPLC on an
RPC-18 column and these were characterized by
MALDI-TOF mass spectrometry.10 The complementary
DNA oligomers 14 having a CG/GC lock at the ends to
prevent slippage, the mixed homopyrimidine sequence
15 and a mismatched sequence 16 were synthesized on
an automated DNA synthesizer using standard phos-
phoramidite chemistry11 and purified by C18 RPHPLC.
The pKa of the piperidine ring nitrogen of the aepipPNA
monomer as determined by pH-titration was found to
be 6.76, similar to that of pyrrolidine nitrogen in
aepPNA5 and may be partially protonated under the
experimental conditions. No precipitation was observed
in samples of aepipPNA even after prolonged storage.
The structural integrity of all the new compounds was
1
confirmed by H,13C NMR spectroscopic analysis and
mass spectrometry.
PNA oligomers 5–11 containing aepipPNA units were
assembled by solid-phase peptide synthesis on Merrifield
resin derivatized with N-Boc-b-alanine. The aepipPNA
monomer 4 was incorporated into the PNA octamer
sequence H–T8–NHCH2CH2COOH at predefined posi-
tions to yield the modified aegPNAs 5–11 (Table 1). The
chimeric PNA oligomers containing aepipPNA were
designed to test the effect of modifications at C/N ter-
minals and the distance between two modifications
on the stability of the derived PNA:DNA complexes.
UV–Tm studies on DNA:PNA2 triplexes. The PNA
sequences used here are homopyrimidine sequences that
are known to form DNA:PNA2 triplexes.12 The
DNA:PNA stoichiometry in these complexes was found
to be 1:2 as studied by mixing curves (JobÕs plot) gen-
erated from both UV absorbance at 260 nm and CD
ellipticity data.12 The annealed PNA2:DNA complexes
were subjected to temperature dependent UV absor-
bance measurements. The percent hyperchromicity ver-
sus temperature plots derived from these experiments
exhibited a single sigmoidal transition as observed in the
first derivative plots (Fig. 1). The Tm results (Table 1)
indicate that compared to unmodified aegPNA T8 oli-
gomer 12, the modified homothymine chimeric PNA
oligomers containing aepipPNA (5–10) stabilized cor-
responding DNA(14):PNA2 triplexes. Modification at
C-terminus in sequence 6 stabilized the complex whereas
with a single modification at the N-terminus and inter-
nal positions, as in 5 and 7, respectively, stability
remained unaffected. Increasing the number of modifi-
cations from the C-terminus end further enhanced the
The
unmodified
aegPNA
sequences
H–T8–
HO
i,ii
5
HO
iii
2
1
N
COOMe
N
z
COOMe
NHBoc
2
1
T
BzT
iv
N
COOH
NHBoc
N
COOMe
NHBoc
4
3
Scheme 1. Synthesis of aepipPNA monomer (i) H2/Pd–C, 60 psi (92%);
(ii) Boc-NH–(CH2)2OMs, DIPEA, acetonitrile–DMF (37%);(iii) BzT,
DIAD, PPh3, THF (32%);(iv) NaOH, MeOH–water (95%).

P. S. Shirude et al. / Tetrahedron Letters 45 (2004) 3085–3088
3087
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
a
h
0.3
0.2
0.1
b
c
i
0 10 20 30 40 5060708090
Temperature (°C)
Figure 2. PNA:DNA complexation by gel shift assay: lane 1, ssPNA
12 (aeg-T8);lane 2, ssDNA 14;lane 3, ssPNA 6;lane 4, PNA 6+ DNA
14;lane 5, PNA 12 (aeg-T8)+DNA 14;lane 6, PNA 8+ DNA 14.
d
e
is interesting since from an earlier study13 based on
piperidines with a different substitution pattern, it was
suggested that six-membered rings are unlikely to sta-
bilize the derived PNA structures for complex formation
with DNA. In the present aepipPNA analogues the
stereochemical dispositions of substituents seem to lead
to a favorable pre-organization of PNA for the forma-
tion of stable DNA triplexes. In this context, work is in
progress in our laboratory to examine the implications
on the duplex and triplex stability of aepipPNAs con-
stituted from other diastereomers and nucleobases.
f
g
0
10
20
30
40
50
60
70
80
90
Temperature (°C)
Figure 1. UV–Tm first derivative curves of DNA:PNA2 complexes. (a)
14:5;(b) 14:8, (c) 15:11, (d) 14:9, (e) 15:13, (f) 14:12, (g) 14:10. Inset: (h)
14:6, (i) 14:7.
Tm. Sequences 8, 9, and 10 were constructed with 2 aepip
units, one at the C-terminus and the second aepip unit at
third, fifth, and seventh base positions, respectively, to
study the positional effect of the modified units with
respect to each other. A synergistic stabilizing effect was
observed with the second modified aepip unit in all the
cases (8:14, 9:14, and 10:14). The maximum benefit per
additional unit was observed (DTm þ 4 °C) when the
second aepipPNA unit was placed nearer to the C-ter-
minal unit (8:14). The complexes of the modified PNAs
(5, 6) with DNA 16 (Table 1) having a single mismatch
in the center of the sequence, were destabilized with a
DTm ꢀ ꢁ24 °C (data not shown). However, the complex
of control PNA 12 with mismatch DNA 16, only shows
linear increase in absorbance and does not show a sig-
moidal transition. The stability of the mixed C/T base
aepip-PNA:DNA complexes containing N-terminus
substitution (11:15) was higher by 9 °C compared to that
of the control complex 13:15 when the orientation of the
DNA strand was antiparallel with respect to both PNA
strands. This result contrasts with the homothymine
oligomers where the relative orientation of the DNA
strand remains ambiguous.5c The percent hyperchrom-
icity accompanying the melting of DNA:aepip-PNA
complexes was also enhanced compared to the control
complex suggesting a better stacking of bases induced by
the incorporation of the aepipPNA modification. Only
in the case of the complex 7:14 where the aepip modifi-
cation is in the center of the sequence, was the percent
hyperchromicity accompanying the melting found to be
lower than the control. The formation of stable com-
plexes with the aepipPNA was further confirmed by a
nondenaturing gel shift assay (Fig. 2) in which the DNA
band exhibited characteristic retardation upon forma-
tion of the complex with PNAs. The ssPNAs alone
moved much more slowly compared to the complexes.
Acknowledgements
P.S.S. thanks DST and CSIR, New Delhi for a research
fellowship. V.A.K. thanks DST, New Delhi, for research
grants.
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In summary, the substitution of the six-membered
aepipPNA monomer into the aegPNA backbone in-
creased the Tm of the derived complexes with DNA. This

3088
P. S. Shirude et al. / Tetrahedron Letters 45 (2004) 3085–3088
10. PNA 5: Mobsd ¼ 2232; Mcalcd ¼ 2231:17, PNA 6: Mobsd
2232; Mcalcd ¼ 2231:17, PNA 7: Mobsd ¼ 2232; Mcalcd
¼
¼
11. Gait, M. J. Oligonucleotide Synthesis,
Approach;IRL Limited: Oxford, 1984.
12. Kim, S. H.;Nielsen, P. E.;Egholm, M.;Buchardt, O.
a Practical
2231:17, PNA 8: Mobsd ¼ 2243; Mcalcd ¼ 2244:23, PNA 9:
Mobsd ¼ 2243; Mcalcd ¼ 2244:23;PNA 10: Mobsd ¼ 2243;
Mcalcd ¼ 2244:23;PNA 11: Mobsd ¼ 2233; Mcalcd ¼ 2201:14,
PNA 12: Mobsd ¼ 2220; Mcalcd ¼ 2219:11, PNA 13:
Mobsd ¼ 2189; Mcalcd ¼ 2189:10.
J. Am. Chem. Soc. 1993, 115, 6477–6489.
€
13. Puschl, A.;Boesen, T.;Tedeschi, T.;Dahl, O.;Nielsen,
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