Design and synthesis of conformationally frozen peptide nucleic acid backbone: Chiral piperidine PNA as a hexitol nucleic acid surrogate

Article abstract of DOI:10.1016/j.bmcl.2004.02.034

The design and facile synthesis of novel chiral piperidine PNA from naturally occurring 4-hydroxy-L-proline is reported. The stereospecific ring-expansion reaction to get six-membered piperidine derivative from 5-membered pyrrolidine derivative is exploit

Full text of DOI:10.1016/j.bmcl.2004.02.034

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2147–2149
Design and synthesis of conformationally frozen peptide nucleic acid
backbone: chiral piperidine PNA as a hexitol nucleic acid
surrogateq
Pallavi S. Lonkar and Vaijayanti A. Kumar*
Division of Organic Chemistry, Synthesis, National Chemical Laboratory, Pune 411008, India
Received 12 November 2003;revised 9 February 2004;accepted 9 February 2004
Abstract—The design and facile synthesis of novel chiral piperidine PNA from naturally occurring 4-hydroxy-L-proline is reported.
The stereospecific ring-expansion reaction to get six-membered piperidine derivative from 5-membered pyrrolidine derivative is
exploited for this synthesis. The resulting conformationally constrained PNA is utilized for the synthesis of PNA mixmers and the
concept is substantiated by UV-Tm studies of the resulting PNA2:DNA complexes.
Ó 2004 Elsevier Ltd. All rights reserved.
The remarkable medicinal importance of the achiral,
O
acyclic, and uncharged aminoethylglycyl peptide nucleic
acids1 aegPNAs as DNA/RNA mimics has challenged
chemists to circumvent the limitations of their in vivo
efficacy.2 The efforts are mainly directed towards further
refining the aegPNA properties such as water solubility,
cellular uptake, and discrimination between parallel
versus anti-parallel binding modes.2;3 The conforma-
tional freedom in the nucleobase linker arm and the
backbone aminoethyl and glycyl segments in the aeg-
PNA were found to be a cause of unfavorable entropic
loss during complex formation with complementary
DNA/RNA.4 Complexation with target complementary
RNA by the DNA analogs that assume A-type of sugar
conformations such as LNA,5 hexitol nucleic acids6 or
20-modified nucleic acids7 (Fig. 1) was found to be much
stronger than natural nucleic acids. The main reason for
this improved stability of the complexes is attributed to
the conformational pre-organization due to either
locking or freezing the sugar ring conformation that is
prevalent in complexes with RNA. The structural
information of the PNA:DNA/RNA complexes8 is not
as completely available as for the DNA:DNA/RNA
complexes and there are possibilities for modulating the
conformational features of PNA in a variety of ways
O
B
B
O
F
B
O
O
O
O
O
O
O
P O
O
O
P O
O P
O
2'-fluoro ANA
c. rigid O4'-endo
Hexitol NA
b. Frozen 3'-endo
LNA
a. Locked 3'-endo
Figure 1. Conformationally locked and frozen nucleic acid analogs.
considering the acyclic nature of aegPNA. Construction
of the five-membered pyrrolidine-based conformation-
ally restricted chiral PNAs as well has met with some
success in this direction.9 The introduction of the rigid
chair conformations of the six-membered rings that
determine the orientation of the ring substituents with
respect to each other will further add to the structural
diversity of PNA. The earlier reported six-membered
PNA analogs10 did not exhibit stabilization of the
complexes with DNA as compared to aegPNA,
although limited binding selectivity for RNA over DNA
was observed. In this communication, we introduce a
new six-membered, chiral piperidine PNA analog that is
a hexitol-NA surrogate. Hexitol NA has equatorial
(hydroxymethyl), equatorial (hydroxy group) and axial
(base moiety) substitutions.11 The design of the mono-
mer is configured in such a way that, to maintain the
preferred N1-equatorial alkyl substitution on piperidine
ring,12 and 1,3 cis diequatorial disposition of the back-
bone, the orientation of nucleobase needs to be axial. As
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2004.02.034
* Corresponding author. Tel./fax: +91-2025893153;e-mail: vakumar@
dalton.ncl.res.in
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.034

2148
P. S. Lonkar, V. A. Kumar / Bioorg. Med. Chem. Lett. 14 (2004) 2147–2149
(3S,5R) tertꢀbutyloxycarbonyl piperidine derivative 6.
B
Compound 6 was subjected to hydrogenation to remove
ring nitrogen benzyl protection. Alkylation of ring
nitrogen using ethyl bromoacetate followed by removal
of silyl protection of the 5-OH using TBAF gave
7. trans-5S-(N3-Benzoyl-thymin-1-yl)-3S-tertꢀbutyloxy-
carbonylaminomethyl piperidine derivative 8 was syn-
thesized under Mitsunobu conditions with N3-benzoyl
thymine. The product was purified by column chroma-
tography and the ester function was hydrolyzed using
aqueous methanolic sodium hydroxide to get the thy-
mine-monomer 9 that could be used for solid phase
synthesis of PNA-PiperidinePNA mixmers. All new
compounds were characterized using suitable spectro-
scopic analysis. The synthesis of the other enantiomer
(3R,5R) can be achieved by using appropriate starting
material trans-(2R,4S)-4-hydroxy-D-proline.15 The syn-
thesis of the cis isomer proved to be difficult as the
Mitsunobu reaction with trans-7 failed.
O
B
BocHN
N
N
HO
O
BocHN
HO
(3R,5R)
(3S,5S)
Figure 2. Proposed conformationally frozen peptide nucleic acid
analogs.
in the case of hexitol-NA, the axial orientation of the
nucleobase and equatorial disposition of the backbone
may lead to the formation of stable duplexes with nat-
ural nucleic acids in contrast to hexose nucleic acids that
assume an all equatorial hexose ring13 substitution
pattern. We report herein, the synthesis of the monomer
unit (Fig. 2) trans-(3S,5S)-3-(thymin-1-yl)-5-tertꢀbutyl-
oxycarbonylamino-piperidin-1-yl acetic acid from nat-
urally occurring trans-4-hydroxy-proline.14 The PNA
oligomer synthesis incorporating this designed mono-
meric unit at pre-defined positions and the DNA-bind-
ing properties of the resulting oligomers with
complementary DNA is reported.
PNA 10 is the unmodified aegPNA with aminoethyl-
glycyl backbone. PNAs 11–14 are the PNA oligomers
with the modified PNA units incorporated at the pre-
defined sites as represented in Table 1. The UV-Tm
studies of the resulting complexes (Fig. 3) indicate that
the modified PNA unit at the C-terminus in PNA 11
stabilized the complex with complementary DNA by
about 7 °C. The synergistic effect was observed with one
more unit in the center of the sequence PNA 12 and the
PNA2:DNA complex was further stabilized by about
4 °C. The presence of conformationally frozen six-
membered piperidine at C-terminus seems to induce
favorable pre-organization of PNA to interact with
target DNA, leading to more stable PNA2:DNA com-
plexes. A single modified unit in the center of the
sequence in PNA 13 seems to be accommodated in the
uniform aegPNA backbone causing only a minor effect.
Interestingly, modification at the N-terminus destabi-
lizes the complex although the transition is sharp with
higher percent hyperchromicity. These preliminary
results are encouraging and invoke the possibility of
development of designed antisense oligonucleotide
mimics with minimum structural modifications. The
3S,5S stereochemistry of the piperidine ring reported
here might prefer axial nucleobase orientation and
equatorial backbone orientation as in the case of hexitol
nucleic acids.
The suitably protected derivative of trans-(2S,4R)-4-
hydroxy-L-proline 1 (Scheme 1) was converted to the
trans-(2S,4R) pyrrolidine-2-methanol 2 by reduction of
the ester function.15 Treatment with trifluoracetic
anhydride followed by diisopropylethylamine gave the
six-membered rearranged product 3 (3R,5R) with
retention of configuration.16 Mesylation of the resulting
unprotected hydroxy group and reaction with excess
sodium azide in dry DMF at 65 °C gave cis azide 5
(3S,5R) with inversion of configuration at C3. Com-
pound 5 was then selectively hydrogenated using Ra–Ni
and the resulting amine was Boc-protected to get the
OR
OR
RO
OH
c
b
a
N
N
N
OH
COOMe
Ph
3
Ph
Ph
1
2
R=TBDMS
RO
NHBoc
RO
OMs
RO
N3
e
f
d
g
N
N
4
N
Ph
Ph
Ph
Table 1. UV-Tm studies of PNA2:DNA complexesa
6
5
Sequencesb
UV-Tm (°C) Hyperchrom-
icity (%)
TBz
T
NHBoc
NHBoc
HO
NHBoc
h
PNA 10
PNA 11
PNA 12
PNA 13
PNA 14
DNA 15
H-TTTTTTTT-b-ala-OH 43
H-TTTTTTTt-b-ala-OH 50.7
10
15
9
N
N
N
COOH
COOR
COOEt
H-TTTtTTTt-b-ala-OH
54.4
9
8
7
H-TTTtTTTT-b-ala-OH 41.4
H-tTTTTTTT-b-ala-OH 35
50-GC(A)8 CG-30
6
15
Scheme 1. Synthesis of the 3S,5S piperidine monomeric unit. (a)
LiBH4/THF;(b) (i) TFAA, (ii) DIPEA;(c) MsCl/Pyridine;(d) NaN
DMF;(e) Ra-Ni/H 2, Boc anhydride;(f) (i) H 2/Pd–C;(ii) BrCH
COOEt/DIPEA;(iii) TBAF/THF;(g) N-3-benzoylthymine, PPh
DIAD;(h) NaOH/water:MeOH.
/
3
a Buffer: 10 mM sodiumcacodylate, 100 mM NaCl, 0.01 mM EDTA,
pH 7.3.
b T represents aegPNA unit and t represents piperidine PNA unit.
2
/
3

P. S. Lonkar, V. A. Kumar / Bioorg. Med. Chem. Lett. 14 (2004) 2147–2149
2149
ship. We thank Dr. K. N. Ganesh for his continued
support.
18
16
14
12
10
8
PNA12
PNA11
PNA10
PNA13
PNA14
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advantage of the conformationally frozen six-membered
ring having substituents in definite preferred orientations
with respect to each other. DNA complementation
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Acknowledgements
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V.A.K. thanks DST, New Delhi for financial support.
P.S.L. thanks CSIR, New Delhi for research fellow-
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