C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

Article abstract of DOI:10.1016/j.bmc.2013.05.015

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60-80, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.

Full text of DOI:10.1016/j.bmc.2013.05.015

Bioorganic & Medicinal Chemistry 21 (2013) 4092–4101
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
0
C3 -endo-puckered pyrrolidine containing PNA has favorable
geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)
⇑
Anjan Banerjee, Vaijayanti A. Kumar
Division of Organic Chemistry, National Chemical Laboratory, Pune 411008, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of
the aegPNA backbone so that the dihedral angle b is restricted within 60–80°, compatible to form
PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/gua-
nidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA
oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated.
A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger
binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived
duplexes of modified PNA with complementary RNA were observed.
Received 1 April 2013
Revised 8 May 2013
Accepted 9 May 2013
Available online 16 May 2013
Keywords:
Peptide nucleic acid (PNA)
Conformationally constrained PNA
Antisense therapeutics
Diagnostics
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
charged sugar–phosphate backbone of DNA and RNA. Because of
highly favorable hybridization properties and high chemical and
Nucleic acid mimics are currently gaining considerable impor-
tance due to the interest in their development in the oligonucleo-
tide (ON) based therapeutics1,2 and diagnostic strategies.3–6 This
application of synthetic nucleic acids is based on the sequence spe-
cific recognition of target RNA or DNA sequences by Watson–Crick
hydrogen bonding schemes. The effectiveness of the designed ON
mimics as lead molecules for such applications primarily depends
on their specific hybridization ability with the target sequences.
The desired additional attributes of the synthetic ON mimics are
aqueous solubility, enzymatic stability and also the ease of synthe-
sis. To achieve these goals, several backbone modifications are
being developed that include the 2nd and 3rd generation antisense
ONs such as 20-O-alkyl,7 20-O-MTE,8 LNA,9 HNA,10 morpholino-
NA,11–13 aminoethylglycyl PNA (aegPNA),14 or several other PNA
modifications reported so far.15,16 Peptide nucleic acids (aegPNA,
Fig. 1) are designed oligonucleotide mimics, derived by replace-
ment of the anionic sugar–phosphate backbone with repeating
N-(2-aminoethyl)glycyl units, carrying adenine, guanine, cytosine,
and thymine nucleobases via methylene carbonyl linkages.17 PNA
acts as an excellent structural mimic of natural DNA/RNA and
exhibits strong sequence specific binding with complementary oli-
gonucleotide sequences following Waston–Crick base-pairing
rules.18 The relatively high binding affinity of PNAs towards the
natural oligonucleotides is attributed to the lack of electrostatic
repulsions between the uncharged PNA backbones and negatively
bio-stability, PNA is regarded as a very promising lead for develop-
ing into efficient antisense agents1,2 and diagnostic markers.3–6 The
positive attributes of PNA are accompanied by some disadvantages
which hamper the vivo applications of PNA. The main drawbacks of
PNA are its relatively poor water solubility19 and poor cellular up-
take20 due to the uncharged polyamide backbone. The acyclic flex-
ible PNA backbone also binds to the complementary DNA and RNA
sequences without any discrimination to form structurally diverse
PNA:DNA and PNA:RNA duplexes. The NMR21 and X-ray crystal
structural studies22 pointed out some crucial differences in
PNA:DNA and PNA:RNA duplexes. The main difference was in the
preferred dihedral angle b in the ethylenediamine segment (N10–
C20–C30–N40, Fig. 1) of PNA units while binding with RNA and
DNA. The preferred dihedral angle b was found to be 60–70° in a
PNA:RNA duplex and the same was ꢀ140° in PNA:DNA duplex.
In the literature, a variety of conformationally constrained PNA
analogues are known,15,16 which show differential stability and
parallel/antiparallel preferences while binding with cDNA and
RNA. We have earlier used the information from the structural
studies to design RNA selective cis-cyclopentyl23,24 (Fig. 1, 2b)
and cis-cyclohexyl25–28 (Fig. 1, 2c) based PNA in which the dihedral
angle b was restricted to 60–70°. The trans-cyclohexyl29 and
trans-cyclopentyl30,31 (Fig. 1, 2a) based PNAs were constructed by
Nielsen and Appella’s groups, respectively. The rigid nature of
cyclohexyl ring was found to be highly successful in stabilizing
the duplexes with RNA in a stereoselective manner when the
geometry of substitution was cis and the dihedral angle b was
restricted to the preferred value (ꢀ60°) in RNA:PNA duplexes.
⇑
Corresponding author. Tel.: +91 2025902340.
E-mail address: va.kumar@ncl.res.in (V.A. Kumar).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.015

A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
4093
Boc
N
Boc
N
BrCH2COOEt,
TEA,
NH
1'
H
NH
HOOC
N
(i) Ref. 40
(ii) Ref. 38
2'
B
β
B
NH
COOEt
β
MeCN, 0°C, 2 h
( )n
NH2
4'
OH
3'
N
N
5
6
O
7
O
O
O
N
O
Boc
N
Boc
HN
ClCH2COCl,
NaHCO3
N
trans-cyclopentyl PNA 2a, n=1
cis-cyclopentyl PNA 2b, n=1
cis-cyclohexyl PNA 2c, n=2
Cl
O
O
Thymine, K2CO3
DMF, rt, 6 h
aegPNA
1
N
8
N
H2O:Dioxane (1:1)
0°C, 1 h
O
COOEt
COOR
R
9, R = Et
1N LiOH / MeOH
rt, 0.5 h
R
1'
N
10, R = H
1
NH
5
2
B
β
2'
4'
Scheme 1. Synthesis of H-ethano-PNA T monomer.
β
N
3
NH2
β
3'
O
4
O
cis-4-amino-L-proline,
R = COOH
DNA and RNA with equal affinity and sequence selectivity.37a The
3
substitution of the glycine in aegPNA by
D-lysine, instead, was
found to exert right handed PNA helices.37d We envisage to confer
the modified oligomers with an ability to preferentially bind with
RNA.
ethano-PNA 4
H-ethano-PNA R=H
Am-ethano-PNA R= Aminomethyl
Gu-ethano-PNA R=Guanidinomethyl
2. Results and discussion
Figure 1. aegPNA, related PNA modifications and the designed ethano-PNA
analogues.
2.1. Synthesis of Boc protected H-ethano-PNA T monomer
(Scheme 1)
The cis and trans-cyclopentyl based PNAs, however, were more
flexible and were able to access the range of dihedral angles that
accommodated both the PNA:DNA and PNA:RNA structures.
In this context, we propose to use 3-aminopyrrolidine core
structure towards the rational design of conformationally re-
stricted aegPNA. The pyrrolidine ring conformations in substituted
prolines are known to be dictated by the R/S stereochemistry of the
electronegative substitutions at C-4 center32,33 (Fig. 1). The gauche
interactions of the substituent amino group and the vicinal ring
nitrogen allow either C4-endo or C4-exo conformations, depending
upon the R/S stereochemistry at C4 center, restricting the N1–C5–
C4–N4 dihedral angle in the pyrrolidine ring around 60–80°
(Fig. 1). This preference has been used by several researchers to
achieve excellent breakthroughs in designing collagen mimics.34–36
In the present PNA design, this N1–C5–C4-N4 segment of the
pyrrolidine ring coincides with the aminoethyl segment (i.e.,
N10–C20–C30–N40, Fig. 1) of aegPNA and the gauche geometry of
the vicinal electronegative substituents would coincide with the
preferred dihedral angle b in aegPNA:RNA duplexes. Thus, we pres-
ent here, the design, synthesis and biophysical studies of a novel
ethano-locked PNA (ethano-PNA) that is envisaged to confer con-
formational constraint on the relatively flexible PNA backbone
for favorable RNA binding. The conformational restriction is intro-
duced in the form of an ethylene bridge between aminoethyl linker
of aegPNA, hence, termed as ethano-PNA. It was envisaged that the
easy synthetic methodology developed from naturally occurring
The synthesis of H-ethano-PNA monomer was accomplished as
follows, starting from naturally occurring (2S,4R) hydroxyproline 5.
The N-Boc-3-S-aminopyrrolidine 6 was obtained by decarboxyl-
ation using reported procedure,40 followed by conversion of the
hydroxy to amino group.38 The primary amine was monoalkylated
with ethylbromoacetate in acetonitrile to get 7. Further, 7 was
acylated with chloroacetyl chloride in 1,4-dioxane-water (1:1)
maintaining a pH 8.0 with NaHCO3 solution during addition of
chloroacetyl chloride to obtain 8. Thymine was derivatized via
nucleophilic substitution of chloro group by N1 nitrogen of thy-
mine to get the base attached product 9. The ester was hydrolysed
using 1 N LiOH in MeOH to give (S) H-ethano-PNA thymine mono-
mer 10 (Scheme 1).
2.2. Synthesis of Fmoc/Boc protected Am-ethano-PNA T
monomer (Scheme 2)
The synthesis of Am-ethano-PNA monomer was also achieved
from (2S,4R) hydroxyproline. (2S,4R) hydroxyproline 5 was con-
verted to methyl ester by treatment with methanol and thionyl
chloride, which on subsequent protection with Boc anhydride gave
N-Boc hydroxyproline methyl ester derivative 11. The hydroxyl
group was mesylated using mesyl chloride in pyridine 12 and re-
duced to 13 with LiBH4 generated in situ with NaBH4/LiCl. The me-
syl derivative was converted to azide 14 by NaN3 in DMF and the
free hydroxyl was protected as TBS ether by reaction with TBSCl
and imidazole in DMF to get 15. The azido group in 15 was reduced
to amine with H2/Pd–C in methanol. The resultant amine 16 was
monoalkylated with ethyl bromoacetate in acetonitrile, to give
compound 17. Acylation of 17 with chloroacetyl chloride in 1,4-
dioxane–water (1:1) was carried out, maintaining the pH of the
solution at ꢀ8.0 with saturated NaHCO3 solution during the addi-
tion of chloroacetyl chloride, to obtain 18. Thymine was deriva-
tized via nucleophilic substitution of chloro group by N1 nitrogen
of thymine gave 19. Further deprotection of TBS group of 19 gave
trans-4-hydroxy-L-proline would be useful compared to the cum-
bersome resolution steps involved in the synthesis of enantiomeri-
cally pure cyclopentyl/cyclohexyl23–28 based PNA analogues. The
ethano group is further amenable for functionalization with ami-
nomethyl (Am-ethano-PNA, Fig. 1) and guanidinomethyl (Gu-eth-
ano-PNA, Fig. 1) group for better aqueous solubility.37–39
Additionally, the
cGPNAs prepared earlier from L-lysine were
found to be preorganized into a right-handed helix when the chiral
center was present in aminoethyl segment of aegPNA and bind to

4094
A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
HO
R2O
Boc
N
Boc
N
MeOH / SOCl2
rt, 8 h
HO
RO
NaN3 / DMF,
60°C, 7 h
H2 / Pd-C
40 psi, rt, 2.5 h
NaBH4/ LiCl
EtOH-THF,
COOMe
COOH
Boc2O / TEA
H2O:Dioxane (1:1), rt, 10 h
N
H
N
Boc
0°C, rt, 8 h
OMs
R1
14 R = N , R = H
5
11 R = H
12 R = Ms
MsCl / Pyridine
0°C, 2 h
13
TBSCl, Imidazole,
DMF, rt, 8 h
1
1
3
3
2
2
15 R = N , R = TBS
O
N
O
R1
Boc
N
RO
HN
Boc
HN
TBSO
ClCH2COCl,
NaHCO3
TBSO
Boc
Boc
N
NaN3 / DMF
60°C, 7 h
Thymine, K2CO3
DMF, rt, 6 h
N
O
O
N
N
Cl
H2O:Dioxane (1:1)
0°C, 1 h
N
N
O
N
NHR
O
COOR2
O
COOEt
BrCH2COOEt,
TEA, MeCN,
0°C, 2 h
16 R = H
17 R = CH COOEt
COOEt
2
22 R = N , R = Et
1
3
2
H2 / Pd-C/40 psi, rt, 2.5 h
1N LiOH / MeOH, rt, 0.5 h
18
1 M TBAF-THF
rt, 0.5 h
MsCl / Pyridine
0°C, 2 h
23 R = NH , R = Et
24 R = NH , R = H
19 R = TBS
20 R = H
21 R = Ms
1
1
2
2
2
2
Fmoc-OSu / NaHCO 3
H2O:Dioxane (1:1), rt 6 h
25 R = NHFmoc,
= H
2
R
1
Scheme 2. Synthesis of substituted aminomethyl-ethano T monomer.
Table 1
PNA sequences, HPLC retention time MALDI-TOF mass analysis and UV-Tm studies
No
Code
Sequences
HPLC tR (min)
MALDI-ToF mass
Calcd/Obsd
UV-Tm (°C)
RNAa 10 mM
cDNAa 10 mM
Mismatch RNAa 10 mM
1
2
3
4
5
6
7
PNA 1
PNA 2
PNA 3
PNA 4
PNA 5
PNA 6
PNA 7
H-CATTGTCACACT-Lys-NH2
H-CATTGtHCACACT-Lys-NH2
H-CATTGtAmCACACT-Lys-NH2
H-CATTGtGuCACACT-Lys-NH2
H-CAtHTGTCACACT-Lys-NH2
H-CAtAmTGTCACACT-Lys-NH2
H-CAtGuTGTCACACT-Lys-NH2
11.8
11.7
11.8
13.0
11.9
11.7
12.5
3332.24/3330.76
3356.40/3355.14
3385.42/3385.43
3427.44/3426.41
3356.40/3354.98
3385.42/3408.36 (M+Na+)
3427.44/3427.39
64.6 (60.1)
49.7 (46.2)
51.8 (46.6)
53.7 (48.8)
55.8 (50.0)
57.1 (53.8)
60.6 (56.3)
66.1 (63.7)
59.4 (52.3)
60.4 (57.2)
62.7 (59.4)
65.5 (59.9)
66.3 (60.1)
68.1 (60.7)
55.1 (53.0)
45.4 (42.2)
46.7 (44.7)
47.1 (46.3)
50.8 (43.3)
51.8 (45.7)
53.6 (46.8)
tH, tAm and tGu are the modified ethano-PNA monomers with H, aminomethyl and guanidinomethyl substituents, respectively.
a
values in parenthesis correspond to 100 mM salt concentration, respectively cDNA sequence 50 TGGAGTGTGACAATGGTG; RNA sequence 50 UGG AGU GUG ACA AUG GUG;
mismatch RNA sequence 50 UGG AGU GAG ACA AUG GUG.
20 and eventually the hydroxyl group generated was converted to
the mesylate derivative 21. The mesyl group of 21 was displaced by
azide 22, which was subsequently reduced to amine by H2/Pd–C in
methanol to obtain 23. The compound 23 was used without puri-
fication for hydrolysis of ester to give the amino acid 24. The exo-
cyclic amine was protected with orthogonal Fmoc protecting group
to give the protected Am-ethano-PNA thymine monomer 25.
the PNA oligomers were cleaved from the solid support by using
a standard protocol employing TFA-TFMSA to yield PNAs carrying
lysine at the carboxyl terminus. The PNA oligomers were purified
by HPLC on a PepRP column and characterized by MALDI-TOF mass
spectrometry (Table 1).
2.4. Post synthetic conversion of amino to guanidino group on
solid phase
2.3. Solid phase synthesis of oligomers incorporating modified
PNA thymine monomers
The synthesized oligomers having Fmoc-protected Am-ethano-
PNA modification were converted to Gu-ethano-PNA oligomers,
when still bound to the solid support.38,41 The oligomers 3a and
6a were treated with piperidine–DMF to selectively deprotect the
NH–Fmoc group to yield a free aminomethyl at the ethano-C2
position (Fig. 1 and Scheme 3, PNA 3b and PNA 6b) These Am-
ethano-PNAs on the resin were subjected to guanidinylation by sus-
pending the resin in the solution of 1H-pyrazole-1-carboxamidine
hydrochloride/DIPEA in DMF-THF (1:2) mixture. The Gu-
ethano-PNAs (Scheme 3, PNA 4 and 7, Table 1) were cleaved from
the resin by treatment with TFA-TFMSA and purified by reverse
phase HPLC. The conversion of amino to guanidino functionality
on PNA was confirmed by MALDI-TOF mass spectrometry and HPLC
analysis.
PNA synthesis was undertaken from the C-terminal to N-termi-
nal. PNA oligomers containing modified monomer were assembled
by solid phase synthesis on MBHA resin functionalized with the
first residue L-lysine, using TBTU/HOBt activation strategy. The se-
quences are shown in Table 1. The unmodified PNA and the mod-
ified ethano-PNA oligomers were designed to test the effect of
ethano locked modification of PNA on the stability of the PNA:
cDNA and PNA:RNA duplexes or triplexes. The modified monomers
were incorporated in pre-defined positions by solid phase synthe-
sis to yield a backbone modification at the central position (Table 1,
entries 2–4) and near the N-terminal of the aegPNA (Table 1, en-
tries 5–7). The conversion of amino functionality to guanidine
functionality on the side chain was achieved on solid support. All

A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
4095
HN
NH2
NHFmoc
NH2
DMF : THF (1 : 2)
DIPEA
NH
20 : 80
Piperidine : DMF
t
H-CATTG CACACT-Lys-NH
H-CATTG t CACACT-Lys-NH
H-CATTG t CACACT-Lys-NH
NH
PNA 3a
PNA 3b
PNA 4
HCl HN
N
N
HN
NH2
NH2
H-CA t TGTCACACT-Lys-NH
NHFmoc
DMF : THF (1 : 2)
DIPEA
NH
20 : 80
Piperidine : DMF
t
H-CA TGTCACACT-Lys-NH
H-CA tTGTCACACT-Lys-NH
NH
PNA 6a
PNA 6b
PNA 7
HCl HN
N
N
Scheme 3. Conversion of amino to guanidine-functionality on solid support.
A
cDNA : PNA2
cDNA : PNA3
cDNA : PNA4
B
cDNA : PNA5
cDNA : PNA6
cDNA : PNA7
C
cRNA : PNA1
cRNA : PNA2
cRNA : PNA3
cRNA : PNA4
D
cRNA : PNA1
cRNA : PNA5
cRNA : PNA6
cRNA : PNA7
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
cDNA : PNA1
cDNA : PNA1
0
10 20 30 40 50 60 70 80 90
0
10 20 30 40 50 60 70 80 90
0
10 20 30 40 50 60 70 80 90
0
10 20 30 40 50 60 70 80 90
Temp (°C)
Temp (°C)
Temp (°C)
Temp (°C)
Figure 2. UV-thermal melting profiles. The experiments were performed at 1 lM concentrations. The complexes were prepared in 10 mM sodium phosphate buffer, pH 7.0
containing NaCl (10 mM). Absorbance versus temperature profiles were obtained by monitoring at 260 nm from 10 to 85 °C at a ramp rate of 0.5 °C per minute. Experiments
were repeated at least thrice and the data was derived from the first derivative curves and are accurate to within 0.3 °C. (A) cDNA:PNA1/PNA2/PNA3/PNA4; (B) cDNA:PNA1/
PNA5/PNA6/PNA7; (C) cRNA:PNA1/PNA2/PNA3/PNA4; (D) cRNA:PNA1/PNA5/PNA6/PNA7.
2.5. UV-Tm studies on ethano-PNA:cDNA and ethano-PNA:RNA
We then synthesized PNA oligomers PNA 5–7, in which the
modification site was shifted towards C-terminal end, rather
than in the middle of the sequence, to study the effect of change
in the site of modification.42 When subjected for thermal dena-
turation studies, the UV-Tm profiles indeed improved than when
the modification was in the middle of the oligomers while bind-
ing with both DNA and RNA. In the case of complexation with
The synthesized PNA sequences were examined for their bind-
ing affinity with complementary DNA/RNA sequences by employ-
ing UV-thermal denaturation studies. The sequence chosen for
study was a 12mer sequence. The modified oligomers were an-
nealed with the complementary DNA and RNA and were subjected
to temperature dependent UV studies at 260 nm. Unmodified PNA
1 was used as a control in these experiments. The UV-Tm plots
show a single sigmoidal transition, characteristic of PNA:cDNA/
RNA duplex melting. The Tm values were obtained by the first
derivative and the values corresponding to peaks from such plots
for various PNA:cDNA/RNA duplexes of modified and unmodified
oligomers are shown in Table 1. It is seen from the UV-melting data
that the introduction of single ethano-PNA-T modification in the
middle of the sequence (PNA 2) decreased the Tm of derived PNA
cDNA, H-ethano-PNA, PNA 5, the
ethano-PNA (PNA 6) and Gu-ethano-PNA (PNA 7) substitution
D
Tm was ꢁ8.8 °C. The Am-
showed higher stability when compared with H-ethano-PNA oli-
gomer (Fig. 2B), but still destabilization was observed (DTm
ꢁ7.5 °C and ꢁ4.0 °C, respectively). In contrast, the result for
the duplex stability with RNA showed that the thermal stability
of the modified duplexes was as good as the duplex with
unmodified PNA (
respectively). When the amino group was converted to guani-
dino functionality (PNA 7), the Tm was found to be positive
D
Tm ꢁ0.6 °C and +0.2 °C for PNA 5 and 6,
2:cDNA duplex (
D
Tm ꢁ14.9 °C) over unmodified PNA 1:cDNA.
D
However, the substitution of H-ethano-PNA to get Am-ethano-
PNA (PNA 3) and Gu-ethano-PNA (PNA 4) reduced this change in
Tm values (Table 1). The destabilization of the duplexes with ami-
no/guanidino substituted modified PNA oligomers PNA 3 and PNA
4 was less compared to the uncharged modified PNA2:cDNA du-
as the Tm of the duplex PNA 7:RNA was higher by +2.0 °C com-
pared with the unmodified PNA 1:RNA duplex. Thus, the modi-
fied PNA 7 was able bind strongly with RNA. The comparison
of the melting data of these modified oligomers while binding
with cDNA and RNA, clearly indicated that the difference in Tm
plexes (
D
Tm ꢁ12.8 °C for PNA 3,
D
Tm ꢁ10.9 °C for PNA 4)
(DTm-RNA–DNA) is about +9 to 10 °C in all the modified oligomers
(PNA 2–PNA 7). In the case of the unmodified achiral PNA 1
there was relatively much less discriminating for RNA binding
(Fig. 2A). The UV-Tm studies involving complexation with RNA also
showed decrease in the Tm values but to a lesser extent as
compared to the complexes with cDNA. (Fig. 2C). In the case of
(DTm-RNA–DNA +1.5 °C). This result is in accordance with the ratio-
H-ethano-PNA, the complex PNA 2:RNA, the
D
Tm was found to
nale of our design which was aimed to be RNA selective.21,22 The
30-amino substituted pyrrolidine ring in which the dihedral angle
corresponding to the aminoethyl segment of PNA is locked near
ꢀ60 to 80° showed favorable for binding to RNA. The PNA:DNA
duplexes are at the same time much more destabilized and the
difference in RNA:PNA and DNA:PNA was therefore pronounced.
be ꢁ6.7 °C compared with the unmodified PNA 1:RNA (Table 1).
This destabilization was less as compared to PNA 2:cDNA duplex.
When the pyrrolidine ring carried the amino and guanidino
substituents in PNA 3 and 4, respectively, the
and ꢁ3.4 °C, compared to the unmodified PNA 1:RNA complex.
D
Tm was ꢁ5.7 °C

4096
A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
cRNA : PNA1
cRNA : PNA5
cRNA : PNA6
cRNA : PNA7
8
6
cRNA : PNA1
cRNA : PNA2
cRNA : PNA3
cRNA : PNA4
cDNA : PNA1
cDNA : PNA2
cDNA : PNA3
cDNA : PNA4
A
B
C
D
8
6
cDNA : PNA1
cDNA : PNA5
cDNA : PNA6
cDNA : PNA7
8
6
8
6
4
2
4
4
2
4
2
2
0
0
0
0
-2
-2
-4
-2
-2
-4
200 220 240 260 280 300 320
200 220 240 260 280 300 320
200 220 240 260 280 300 320
200 220 240 260 280 300 320
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Figure 3. CD profiles. the experiments were performed at 1 lM concentrations. The complexes were prepared in 10 mM sodium phosphate buffer, pH 7.0 containing NaCl
(10 mM). All the CD data were recorded at room temperature. All spectra represent an average of at least 8 scans recorded from 320 nm to 210 nm at a rate of 100 nm per
minute in a 1 cm path length cuvette. All specta were baseline subtracted and smoothed using a 5 point adjacent averaging algorithm. (A) cDNA:PNA1/PNA2/PNA3/PNA4; (B)
cDNA:PNA1/PNA5/PNA6/PNA7; (C) RNA:PNA1/PNA2/PNA3/PNA4; (D) RNA:PNA1/PNA5/PNA6/PNA7.
Being charge-neutral, the strength unmodified PNA:DNA du-
plexes is known to be little affected by increasing salt concentra-
tions, in contrast to the highly salt concentration dependent
duplex strength of native duplexes. In the present studies, a po-
sitive charge was introduced in the backbone of PNA and there-
fore at higher salt concentration some destabilization of the
duplexes could be expected, depending upon the electrostatic
properties and interactions of the charged amino- and guani-
dino-substitutions in the backbone.37–39 Thermal melting studies
were therefore undertaken at higher salt concentration (100 mM,
NaCl). PNA 1 showed slight destabilization of the duplexes with
2.6. Conformational analysis of the ethano-PNA:cDNA and
ethano-PNA:RNA
In order to examine if the ethano-PNAs cause any significant
differences on the overall conformational features of the du-
plexes, the CD studies of PNA complexes with cDNA as well as
RNA were undertaken. A maxima at 260 nm and a minima at
240 nm typically is seen for right handed helical structures.37,43
Base stacking interactions are reflected in the amplitude of the
CD signal at 260 nm in PNA:DNA/RNA duplexes.37 The CD profile
of the derived PNA oligomers (PNA 2–7, Fig. 3A and B) with the
ethano-PNA modification when duplexed with cDNA showed sim-
ilar CD pattern as that of the unmodified PNA1:DNA duplex
(Fig. 3A and B), although the amplitude of the CD-signal was
comparatively less in each case. The CD profile of the unmodified
PNA 1 when complexed with RNA showed a positive maximum
DNA at higher salt concentrations (
Tm values for uncharged H-ethano-PNA (PNA 2 and 5) were
similar when modification was present either at the central po-
sition or towards C-terminus (
Tm = ꢁ3 to 5 °C). In the case of
D
Tm = ꢁ4.1 °C, Table 1). The
D
D
Am-ethano-PNA and Gu-ethano-PNA (PNA 3, 4 and PNA 6, 7),
the average destabilization of the duplexes was about 3–4 °C.
In the case of RNA binding at higher salt concentrations, the
destabilization was more when the guanidino-group was present
in the backbone as compared to amino- or unsubstituted pyrrol-
idine ring modification at pH 7. The salt dependent destabiliza-
tion of the duplexes was observed by the earlier researchers
signal at 260 nm and
a minimum at 240 nm and 290 nm.
(Fig. 3C and D). In this case the amplitude of the CD signal at
260 nm was drastically reduced when the ethano-bridged PNA
monomers were introduced (PNA 2–7, Fig. 3C and D) compared
with the unmodified PNA 1:RNA complex. The reduction ob-
served in the minimum at 240 nm was indicative of tightened
helical pitch of PNA:RNA complexes compared with the PNA:DNA
complexes.37,44 Thus, no significant change in CD patterns was
observed in either the DNA or RNA duplexes and the fact suggests
that the overall structural features of the duplexes with either
DNA or RNA remained unchanged. The reduced amplitude of
the CD signal at 260 nm could be because of reduced base stack-
ing interactions due to the additional constraint in the backbone,
in contrast to the open chain charged PNA anlogues.37 The re-
duced binding strength of these modified oligomers compared
to the unmodified PNA could be therefore because of less effec-
tive stacking interactions. The hydrogen bonding interactions
were retained as seen by nucleobase sequence fidelity of binding.
Nevertheless, the modified oligomers were capable of inducing
differential duplex strength while binding with RNA and DNA
backbones.
also, when
c
PNA (corresponding to 20-position in the present
study) was constructed from the lysine residue. In that case,
the positively charged amino groups were shown to interact
with solvent molecules.37 Involvement of nonspecific electro-
static interactions of amino-/guanidino-groups in the side chains
was ruled out in these studies, given the fact that PNA:DNA he-
lix parameters did not comply with such interactions.37b We
envisage that the nonspecific charge–charge interactions with
the negatively charged phosphate groups may not be possible
in the present studies also, due to additional constraint of the
ethano bridge in the backbone.
To further rule out the possibility of involvement of nonspecific
electrostatic interactions at the expense of sequence specificity of
binding, we conducted experiments with RNA sequence compris-
ing a single mismatch. Thermal melting experiments were carried
out with mismatch RNA sequence (Table 1), to assess the effect on
Tm values for H-ethano-PNA, Am-ethano-PNA and Gu-ethano-PNA.
It was found that the all the PNA sequences (PNA 2–7) showed
much lower melting values and the destabilization was slightly
more with the unmodified PNA 1:mismatch RNA. (Table 1,
3. Experimental
3.1. Ethyl-[-N-Boc-pyrrolidin-3-yl-]glycinate (7)
The amino compound 6 (2.7 g, 14.5 mmol) was dissolved in dry
CH3CN (40 mL) and triethylamine (4.1 mL, 29.0 mmol) was added
with stirring. The reaction mixture was cooled to 0 °C and ethyl
bromoacetate (1.9 mL, 17.4 mmol) diluted with CH3CN (20 mL)
was added dropwise. The reaction mixture was stirred for 2 h at
room temperature. After the completion of the reaction, CH3CN
was removed under reduced pressure and the residue was redis-
solved in ethyl acetate (150 mL). The organic layer was washed
D
Tm matched–mismatched ꢁ11.0 °C). All the modified duplexes formed
using PNA 2–7 with mismatched RNA were found to be highly
destabilized (Table 1, Tm
D
ꢀ12 to 14.0 °C) at
matched–mismatched
either salt concentration studied. These experiments indicate that
although the electrostatic properties of modified PNA may affect
their preorganized structure and consequently may contribute to
the stability of the duplexes, it is not at the expense of nucleobase
sequence fidelity.

A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
4097
with water (2 ꢂ 30 mL), satd NaHCO3 (2 ꢂ 20 mL) followed by
brine (2 ꢂ 10 mL) and dried over anhyd Na2SO4. It was then
evaporated in vacuo to give the crude product, which on column
chromatography purification (3% MeOH–CH2Cl2) gave ethyl-[-N-
Boc-pyrrolidin-3-yl-]glycinate 7 (2.8 g, 70.9%). 1H NMR (CDCl3) d:
1.29 (t, 3H), 1.46 (s, 9H, C(CH3)3), 1.77–2.00 (m, 2H), 1.86 (s, 1H,
D2O exch.), 3.10 (m, 1H), 3.34–3.49 (m, 6H), 4.18 (q, 2H). 13C
NMR (CDCl3) d: 14.12, 28.43, 31.86, 44.23, 49.08, 51.14, 56.33,
by evaporation in vacuo and dessication to obtain Ethyl-[-N-(N-
Boc-pyrrolidin-3-yl)-N-(N1-thyminylacetyl)]glycine 10 (1.2 g,
85.5%). 1H NMR (D2O) d: 1.46 (s, 9H, C(CH3)3), 1.84 (s, 3H), 2.04
(m, 2H), 3.31 (m, 2H), 3.49 (m, 2H), 4.03–4.11 (2s, 2H), 4.59 (qu,
1H), 4.84 (m, 2H), 7.34 (s, 1H). 13C NMR (D2O) d: 11.34, 27.76,
29.67, 43.84, 44.96, 46.82, 49.58, 55.11, 81.58, 110.82, 143.25,
152.24, 156.11, 166.94, 168.67, 174.87. IR (m
max, cmꢁ1) (CHCl3):
723, 1377, 1465, 1649, 1702, 2855, 2926, 3433. ½a D27
ꢁ 24 (c 1.0,
ꢃ
60.91, 79.13, 154.50, 172.17. IR (
m
max, cmꢁ1) (CHCl3): 732, 912,
CH3OH). HRMS (ESI) calcd for C18H26N4O7Na: 433.1694, found:
433.1693.
1170, 1416, 1683, 1739, 2248, 2979, 3433. ½a D27
þ 68 (c 1.0, CHCl3).
ꢃ
HRMS (ESI) calcd for C13H25N2O4: 273.1809, found: 273.1806.
3.5. Methyl-(N-Boc-4R-hydroxypyrrolidine)-2-carboxylate (11)
3.2. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl)-N-
(2-chloroacetyl)]glycinate (8)
(2S,4R)-Hydroxyproline (5) (10 g, 76.3 mmol) was suspended in
methanol (100 mL) and stirred for 15 min at 0 °C. To that added
SOCl2 (8.3 mL, 114.4 mmol) dropwise and the reaction mixture
was stirred for 8 h at room temperature. The solvent was removed
under reduced pressure and the residue was co-evaporated thrice
with CH2Cl2. The crude product obtained was dissolved in 1:1 solu-
tion of 1,4-dioxane-water (200 mL). Triethylamine (31.8 mL,
228.8 mmol) was added slowly to neutralize and to provide basic
medium. Boc anhydride (20.0 mL, 91.5 mmol) was added in two
portions and the reaction mixture was stirred for 10 h. The result-
ing solution was concentrated to 75 mL and was extracted with
ethyl acetate (100 mL). The organic layer was then washed with
satd NaHCO3 (2 ꢂ 25 mL) followed by brine (2 ꢂ 20 mL). It was
kept over anhyd Na2SO4 and evaporated in vaccuo. The crude prod-
uct obtained was purified by column chromatography (40% ethyl
acetate-petroleum ether) to obtain the desired product methyl-
(N-Boc-4R-hydroxypyrrolidine)-2-carboxylate 11 (17.4 g, 93.0%
over two steps). 1H NMR (CDCl3) d: 1.41 (s, 9H, C(CH3)3), 2.06 (m,
1H), 2.30 (br, m, 2H), 3.58–3.62 (m, 2H), 3.74 (s, 3H), 4.40–4.49
(t, m, 2H). 13C NMR (CDCl3) d: 28.22, 39.09, 52.04, 54.68, 57.87,
Compound 7 (2.6 g, 9.5 mmol) was dissolved in 1:1 solution of
1,4-dioxane-water (70 mL) and NaHCO3 (8 g, 95.5 mmol) dissolved
in water (50 mL) was added. The whole content was stirred for
15 min at 0 °C and chloroacetyl chloride (2.3 mL, 28.6 mmol) was
added in two portions maintaining a pH 8.0. The reaction mixture
was kept for 1 h with stirring and then concentrated to 50 mL. It
was extracted in CH2Cl2 (50 mL) and was washed with brine
(2 ꢂ 10 mL). The organic layer was dried over anhyd Na2SO4 fol-
lowed by evaporation in vacuo to give the crude product. The prod-
uct was purified by column chromatography (2% MeOH–CH2Cl2) to
give the desired product Ethyl-[-N-(N-Boc-pyrrolidin-3-yl)-N-(2-
chloroacetyl)]glycinate 8 (2.8 g, 84.1%). 1H NMR (CDCl3) d: 1.28
(t, 3H), 1.46 (s, 9H, C(CH3)3), 1.97–2.21 (m, 2H), 3.15–3.35 (m,
2H), 3.55–3.65 (m, 2H), 3.94–4.21 (m, s.q 6H), 4.55 (qu, 1H). 13C
NMR (CDCl3) d: 13.90, 28.24, 29.27, 40.79, 43.63, 45.61, 47.32,
56.54, 61.32, 79.77, 154.01, 166.73, 168.51. IR (
732, 914, 1130, 1203, 1416, 1668, 1695, 1747, 2250, 2981.
m
max, cmꢁ1) (CHCl3):
½
a 2D7
ꢃ
ꢁ 16 (c 1.0, CHCl3). HRMS (ESI) calcd for C15H25ClN2O5Na:
371.1347, found: 371.1396.
69.45, 80.37, 153.95, 173.63. IR (m
max, cmꢁ1) (CHCl3): 773, 1159,
1416, 1682, 1749, 2978, 3447. ½a D27
ꢁ 54 (c 1.0, CHCl3). HRMS
ꢃ
3.3. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl)-N-(N1-
(ESI) calcd for C11H19NO5Na: 268.1155, found: 268.1158
thyminylacetyl)]glycinate (9)
3.6. Methyl-(N-Boc-4R-O-mesyl-pyrrolidine)-2-carboxylate (12)
The chloroacetyl derivative 8 (2.0 g, 5.7 mmol), K2CO3 (0.8 g,
5.7 mmol) and thymine (0.7 g, 5.7 mmol) was suspended in dry
DMF (20 mL) and stirred at room temperature for 6 h. After that
DMF was removed under reduced presssure and the residue was
redissolved in ethyl acetate (70 mL). The organic layer was washed
with water (2 ꢂ 25 mL) followed by brine (2 ꢂ 10 mL) and dried
over anhyd. Na2 SO4. It was then evaporated in vacuo to give the
crude product which was purified by column chromatography
(4% MeOH–CH2Cl2) to give ethyl-[-N-(N-Boc-pyrrolidin-3-yl)-N-
(N1-thyminylacetyl)]glycinate 9 (1.8 g, 71.6%). 1H NMR (CDCl3) d:
1.26 (t, 3H), 1.46 (s, 9H, C(CH3)3), 1.93 (d, 3H), 2.01 (m, 2H),
3.29–3.37 (m, 2H), 3.52 (m, 1H), 3.73 (q, 1H), 4.00 (d, 1H), 4.14–
4.29 (m, 3H), 4.46 (m, 2H), 5.06 (m, 1H), 7.04 (s, 1H), 9.34 (br, s,
1H). 13C NMR (CDCl3) d: 12.23, 13.98, 28.32, 29.56, 43.75, 45.03,
47.44, 48.03, 55.24, 62.30, 79.68, 110.75, 140.90, 151.16, 154.20,
The pure compound 11 (17.1 g, 69.7 mmol) obtained in the pre-
vious step was dissolved in dry pyridine (60 mL) and stirred for 15
mins at 0 °C. Mesyl chloride (6.5 mL, 83.7 mmol) was added drop
by drop to this solution at 0 °C with stirring and the reaction mix-
ture was left for 2 h. After that pyridine was removed under re-
duced pressure and the residue was redissolved in ethyl acetate
(100 mL). Organic layer was washed with water (2 ꢂ 30 mL), satd
NaHCO3 (2 ꢂ 20 mL) followed by brine (2 ꢂ 10 mL) and dried over
anhyd Na2SO4. The organic layer was concentrated in vacuo to give,
the crude product which was purified by column chromatography
(40% ethyl acetate–petroleum ether) to give methyl-(N-Boc-4R-O-
mesyl-pyrrolidine)-2-carboxylate 12 (19.4 g, 86.1%). 1H NMR
(CDCl3) d: 1.42 (s, 9H, C(CH3)3), 2.19–2.28 (m, 1H), 2.61–2.65 (m,
1H), 3.06 (s, 3H, SCH3), 3.75 (s, 3H), 3.79 (m, 2H), 4.41–4.45 (m,
1H), 5.27 (qu, 1H). 13C NMR (CDCl3) d: 28.15, 36.26, 38.71, 52.13,
164.37, 167.79, 169.52. IR (m
max, cmꢁ1) (CHCl3): 667, 761, 1048,
1217, 1409, 1677, 1742, 2931, 3019, 3455. ½a D27
ꢃ
ꢁ 14 (c 1.0, CHCl3).
52.28, 57.37, 77.85, 80.90, 153.29, 172.69. IR (m
max, cmꢁ1) (CHCl3):
HRMS (ESI) calcd for C20H31N4O7: 439.2187, found: 439.2183.
783, 907, 1173, 1402, 1699, 1748, 2978. ½a D27
ꢁ 32 (c 1.0, CHCl3).
ꢃ
HRMS (ESI) calcd for C12H21NO7SNa: 346.0931, found: 346.0923.
3.4. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl)-N-(N1-
thyminylacetyl)]glycine (10)
3.7. N-Boc-4R-O-mesyl-prolinol (13)
The amino ester 9 (1.5 g, 4.3 mmol) was dissolved in methanol
(10 mL) and to that was added 1 N NaOH (10 mL). The reaction
mixture was stirred for 30 min and after the completion of reaction
methanol was evaporated under reduced pressure. The resulting
aqueous solution was neutralized by Dowex H+ ion exchange resin.
The aqueous layer was washed with ethyl acetate (5 mL) followed
NaBH4 (2.5 g) was suspended in dry THF (105 mL) and absolute
alcohol (140 mL) and stirred for 30 min in ice bath. At 0 °C LiCl
(2.5 g) was added in two portions and the content was stirred for
another 1 h in ice bath for in situ generation of LiBH4. The solution
turned milky due to formation of NaCl. To this mixture, the mesy-
late derivative 12 (6.3 g, 19.5 mmol) dissolved in absolute alcohol

4098
A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
(60 mL) was added at 0 °C dropwise. The reaction mixture was stir-
red for 8 h. NH4Cl solution was then added for maintaining pH 7.0
and the solvents were removed under reduced pressure. The resi-
due was dissolved in ethyl acetate (125 mL). Organic layer was
washed with water (2 ꢂ 30 mL) followed by brine (2 ꢂ 20 mL)
and dried over anhyd Na2SO4. It was evaporated under vacuo and
the crude product obtained was purified by column chromatogra-
phy (50% ethyl acetate-petroleum ether) to give N-Boc-4R-O-me-
syl-prolinol 13. The same procedure was repeated thrice with
same amount of compound 12 to get compound 13 (15.4 g,
89.2%). 1H NMR (CDCl3) d: 1.46 (s, 9H, C(CH3)3), 2.05 (br, m, 1H),
2.33–2.40 (m, 1H), 3.05 (s, 3H, SCH3), 3.53–3.59 (m, 2H), 3.77–
3.92 (m, 2H), 4.14 (m, 1H), 5.20 (qu, 1H). 13C NMR (CDCl3) d:
28.28, 35.17, 38.64, 53.46, 58.49, 65.73, 78.32, 81.04, 156.05. IR
0.84 (s, 9H), 1.40 (s, 9H, C(CH3)3), 2.14 (t, 2H), 3.16 (m, 1H),
3.54–3.75 (m, 4H), 4.00 (m, 1H). 13C NMR (CDCl3) d: ꢁ3.68,
ꢁ5.48, 25.76, 28.34, 31.87, 32.85, 51.73, 57.59, 58.91, 62.54,
79.79, 154.01. IR (m
max, cmꢁ1) (CHCl3): 775, 837, 1123, 1416,
1665, 2930, 3433. ½a D27
ꢁ 26 (c 1.0, CHCl3). HRMS (ESI) calcd for
ꢃ
C
16H35N2O3Si: 331.2411, found: 331.2412.
3.11. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl)-2S-O-TBS-
methyl]glycinate (17)
The amino compound 16 (5.5 g, 16.6 mmol) was dissolved in
dry CH3CN (60 mL) and triethylamine (4.6 mL, 33.3 mmol) was
added with stirring. The reaction mixture was cooled to 0 °C and
ethyl bromoacetate (2.3 mL, 20.0 mmol) diluted with CH3CN
(25 mL) was added dropwise. The reaction mixture was stirred
for 2 h at room temperature. CH3CN was removed under reduced
presssure and the residue was dissolved in ethyl acetate
(150 mL). The organic layer was washed with water (2 ꢂ 30 mL),
satd NaHCO3 (2 ꢂ 20 mL) followed by brine (2 ꢂ 10 mL) and was
dried over anhyd Na2SO4. It was then evaporated in vacuo to give
crude product, which on column chromatography purification
(30% ethyl acetate-petroleum ether) gave Ethyl-[-N-(N-Boc-pyrr-
olidin-3-yl)-2S-O-TBS-methyl]glycinate 17. (3.2 g, 69.3%). 1H NMR
(CDCl3) d: 0.05 (m, 6H), 0.84 (s, 9H), 1.24 (t, 3H), 1.41 (s, 9H,
C(CH3)3), 1.81–2.00 (m, 2H), 2.15 (br, m, 1H), 2.95 (dd, 1H), 3.18
(qu, 1H), 3.35 (s, 2H), 3.68 (m, 4H), 4.09–4.20 (q, 2H). 13C NMR
(CDCl3) d: ꢁ5.43, 14.12, 25.85, 28.43, 33.74, 34.96, 49.32, 52.37,
(
mmax
, ) (CHCl3): 903, 1169, 1412, 1676, 2978, 3430.
cmꢁ1
½
a 2D7
ꢃ
ꢁ 34 (c 1.0, CHCl3). HRMS (ESI) calcd for C11H21NO6SNa:
318.0982, found: 318.0983.
3.8. N-Boc-4S-azido-prolinol (14)
The compound 13 (5.0 g, 16.9 mmol) was dissolved in dry DMF
(30 mL) and NaN3 (5.5 g, 84.6 mmol) was added to it. The reaction
mixture was stirred at 60 °C for 7 h. After completion of reaction
DMF was removed under reduced pressure and the residue was
dissolved in ethyl acetate (100 mL). Organic layer was washed with
water (2 ꢂ 25 mL) followed by brine (2 ꢂ 20 mL). The organic layer
was kept over anhyd Na2SO4 and then concentrated in vacuo. The
crude compound obtained was purified by column chromatogra-
phy (30% ethyl acetate-petroleum ether) to obtain N-Boc-4S-azi-
do-prolinol 14. (3.3 g, 80.5%). 1H NMR (CDCl3) d: 1.47 (s, 9H,
C(CH3)3), 1.79–1.87 (br, 2H), 2.26–2.40 (m, 1H), 3.27–3.36 (dd,
1H), 3.68–3.83 (m, 3H), 4.07 (m, 2H). 13C NMR (CDCl3) d: 28.29,
55.64, 57.70, 60.78, 64.43, 79.17, 154.28, 172.14. IR (m )
max, cmꢁ1
(CHCl3): 733, 837, 1103, 1169, 1402, 1697, 1742, 2359, 2930,
3333. ½a 2D7
ꢁ 30 (c 1.0, CHCl3). HRMS (ESI) calcd for C20H41N2O5Si:
ꢃ
417.2779, found: 417.2789.
33.95, 52.03, 58.65, 59.29, 67.20, 80.97, 156.44. IR (
m
max, cmꢁ1
)
3.12. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-O-TBS-methyl)-N-(2-
(CHCl3): 756, 870, 1045, 1167, 1406, 1676, 2104, 2976, 3424.
chloroacetyl)]glycinate (18)
½
a 2D7
ꢃ
ꢁ 12 (c 1.0, CHCl3). HRMS (ESI) calcd for C10H19N4O3:
243.1452, found: 243.1454.
Compound 17 (4.6 g, 11.0 mmol) was dissolved in 1:1 solution
of 1,4-dioxane-water (80 mL) and NaHCO3 (9.3 g, 110.4 mmol) dis-
solved in water (60 mL) was added. The reaction mixture was stir-
red for 15 min at 0 °C and chloroacetyl chloride (4.4 mL,
55.2 mmol) was added in two portions maintaining a pH 8.0. The
reaction mixture was kept for 1 h with stirring and then concen-
trated to 50 mL. It was extracted in CH2Cl2 (50 mL) and was
washed with brine (2 ꢂ 10 mL). The organic layer was dried over
Na2SO4 followed by evaporation in vaccuo to give the crude prod-
uct. The product was purified by column chromatography (20%
ethyl acetate–petroleum ether) to give the desired product ethyl-
[-N-(N-Boc-pyrrolidin-3-yl-2S-O-TBS-methyl)-N-(2-chloroacetyl)]
glycinate 18. The same procedure was repeated twice with same
amount of compound 17 to get compound 18 (8.7 g, 79.9%).
1H NMR (CDCl3) d: 0.05 (s, 6H), 0.89 (s, 9H), 1.27 (t, 3H), 1.46
(s, 9H, C(CH3)3), 1.64 (s, 2H), 2.05–2.30 (m, 2H), 3.56 (m, 1H),
3.91–4.03 (m, s, 4H), 4.14–4.21 (m, q, 5H). 13C NMR (CDCl3) d:
ꢁ5.42, 14.05, 18.27, 25.87, 28.45, 29.65, 40.95, 43.56,
48.63, 51.28, 56.25, 61.37, 62.13, 79.96, 153.65, 166.92, 168.75 IR
3.9. N-Boc-4S-azido-2S-O-TBS-prolinol (15)
The pure compound 14 (9.5 g, 39.2 mmol) was dissolved in dry
DMF (40 mL). Imidazole (8.0 g, 117.6 mmol) followed by TBSCl
(8.3 g, 54.9 mmol) was added to it. The reaction mixture was stir-
red for 8 h after the completion of the reaction the solvent was re-
moved under reduced pressure and the residue was dissolved in
ethyl acetate (150 mL). The organic layer was then washed with
water (2 ꢂ 30 mL) followed by brine (2 ꢂ 20 mL). It was kept over
anhyd Na2SO4 and evaporated in vacuo. The crude product
obtained was purified by column chromatography (10% ethyl ace-
tate-petroleum ether) to obtain N-Boc-4S-azido-2S-O-TBS-prolinol
15 (12.9 g, 92.8%). 1H NMR (CDCl3) d: 0.06 (s, 6H), 0.84 (s, 9H), 1.40
(s, 9H, C(CH3)3), 2.14 (t, 2H), 3.17 (m, 1H), 3.54–3.75 (m, 4H), 4.00
(m, 1H). 13C NMR (CDCl3) d: ꢁ3.68, ꢁ5.48, 25.76, 28.34, 31.87,
32.85, 51.73, 57.59, 58.91, 62.54, 79.79, 154.01. IR (
m
max, cmꢁ1
ꢃ ꢁ 4 (c 1.0,
)
(CHCl3): 775, 837, 1119, 1395, 1699, 2102, 2930. ½a D27
CHCl3). HRMS (ESI) calcd for C16H33N4O3Si: 357.2316, found:
357.2315.
(m
max, cmꢁ1) (CHCl3): 777, 837, 1113, 1167, 1408, 1667, 1694,
1753, 2361, 2930. ½a D27
ꢁ 22 (c 1.0, CHCl3). HRMS (ESI) calcd for
ꢃ
C
22H42ClN2O6Si: 493.2495, found: 493.2500.
3.10. N-Boc-4S-amino-2S-O-TBS-prolinol (16)
3.13. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-O-TBS-methyl)-N-
The azido compound 15 (4.2 g, 11.8 mmol) was dissolved in
methanol (35 mL) and was hydrogenated over 10% Pd–C catalyst
with 40 psi pressure for 2.5 h under hydrogen atmosphere. The
content was then filtered through a bed of celite and the filtrate
collected was concentrated to obtain the desired compound N-
Boc-4S-amino-2S-O-TBS-prolinol 15. (3.8 g, 97.6%) and was used
further without purification. 1H NMR (CDCl3) d: 0.02 (m, 6H),
(N1-thyminylacetyl)]glycinate (19)
The chloroacetyl derivative 18 (5.0 g, 10.1 mmol), K2CO3 (1.4 g,
10.1 mmol) and thymine (1.3 g, 10.1 mmol) were suspended in dry
DMF (30 mL) and stirred at room temperature for 6 h. After that
DMF was removed under reduced presssure and the residue was
redissolved in ethyl acetate (80 mL). The organic layer was washed

A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
4099
with water (2 ꢂ 20 mL) followed by brine (2 ꢂ 10 mL) and dried
over anhyd Na2SO4. It was then evaporated in vaccuo to give crude
product. The crude product was purified by column chromatogra-
phy (3% MeOH–CH2Cl2) to give ethyl-[-N-(N-Boc-pyrrolidin-3-yl-
2S-O-TBS-methyl)-N-(N1-thyminylacetyl)]glycinate 19 (4.5 g,
76.2%). 1H NMR (CDCl3) d: 0.02 (s, 6H), 0.84 (s, 9H), 1.21 (t, 3H),
1.42 (s, 9H, C(CH3)3), 1.87 (d, 3H), 2.10–2.28 (m, 2H), 3.51 (m,
1H), 3.86 (m, 2H), 4.02–4.15 (s, m, 5H), 4.32 (q, 2H), 5.06 (m,
2H), 6.97 (d, 1H), 9.17 (d, 1H). 13C NMR (CDCl3) d: ꢁ5.58, 12.25,
13.98, 25.81, 28.38, 29.59, 31.55, 43.43, 44.28, 47.70, 51.11,
56.20, 61.44, 62.22, 80.03, 110.70, 140.85, 151.04, 153.57, 164.25,
The reaction mixture was heated to 60 °C for 7 h. After completion
of the reaction DMF was removed under reduced pressure and res-
idue was redissolved in ethyl acetate (80 mL). Organic layer was
washed with water (2 ꢂ 25 mL) followed by brine (2 ꢂ 20 mL).
The organic layer was kept over anhyd Na2SO4 and then
concentrated in vaccuo. The crude compound obtained was
purified by column chromatography (3% MeOH–CH2Cl2) to obtain
ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-azidomethyl)-N-(N1-thymi-
nylacetyl)]glycinate 22 (1.8 g, 73.8%). 1H NMR (CDCl3) d: 1.28–1.34
(t, 3H), 1.46 (s, 9H, C(CH3)3), 1.92 (d, 3H), 2.14–2.35 (m, 2H), 2.95–
3.10 (m, 1H), 3.26–3.31 (d, 1H), 3.80 (m, 1H), 3.95–4.05 (m, 2H),
4.21–4.31 (m, 3H), 4.44–4.77 (m, 3H), 4.95–5.04 (m, 1H), 7.03 (s,
1H), 9.57 (d, 1H). 13C NMR (CDCl3) d: 12.27, 13.97, 28.29, 43.45,
44.79, 47.68, 48.09, 51.35, 54.83, 61.68, 62.38, 80.46, 110.81,
166.72, 168.89. IR (m
max, cmꢁ1) (CHCl3): 760, 839, 1117, 1215,
1412, 1680, 1686, 1744, 2400, 2928, 3393. ½a D27
ꢁ 16 (c 1.0, CHCl3).
ꢃ
HRMS (ESI) calcd for C27H47N4O8Si: 583.3158, found: 583.3157.
140.82, 151.13, 153.72, 164.25, 167.96, 169.51. IR (m )
max, cmꢁ1
3.14. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-hydroxymethyl)-N-
(N1-thyminylacetyl)]glycinate (20)
(CHCl3): 731, 912, 1115, 1206, 1470, 1651, 1694, 1748, 2104,
2251, 2978, 3175. ½a D27
þ 12 (c 1.0, CHCl3). HRMS (ESI) calcd for
ꢃ
C
21H32N7O7: 494.2358, found: 494.2355
Compound 19 (4.2 g, 7.2 mmol) was dissolved in dry THF
(20 mL), 1 M TBAF in THF (6.0 mmol) was added to it. The reaction
mixture was stirred for 30 min. THF was then removed under re-
duced pressure and the residue was dissolved in CH2Cl2 (50 mL).
The organic layer was washed with water (2 ꢂ 20 mL) followed
by brine (2 ꢂ 10 mL) and dried over anhyd Na2SO4. After evapora-
tion of the solvent in vacuo, the crude product was purified by
column chromatography (6% MeOH–CH2Cl2) to ethyl-[-N-(N-Boc-
pyrrolidin-3-yl-2S-hydroxymethyl)-N-(N1-thyminylacetyl)]glycin-
ate 20 (3.2 g, 94.8%). 1H NMR (CDCl3) d: 1.22–1.32 (t, 3H), 1.44
(s, 9H, C(CH3)3), 1.69 (m, 4H) 1.90 (d, 2H), 2.77–3.20 (m, 1H),
3.31–3.39 (m, 4H), 3.68–3.77 (m, 2H), 3.91–4.15 (m, 2H), 4.29
(m, 1H), 4.58–4.92 (m, 2H), 7.19 (s, 1H). 13C NMR (CDCl3) d:
12.01, 13.40, 19.44, 23.79, 28.09, 45.16, 48.21, 51.91, 58.61,
61.16, 61.98, 77.20, 80.38, 109.98, 141.28, 150.97, 164.26, 166.88,
3.17. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-aminomethyl)-N-(N1-
thyminylacetyl)]glycinate (23)
The azido compound 22 (1.5 g, 3.0 mmol) was dissolved in
methanol (20 mL) and was hydrogenated over 10% Pd–C catalyst
with 40 psi pressure for 3 h under hygrogen atmosphere. The
content was then filtered through a bed of celite and the filtrate
collected was concentrated to obtain the desired compound
ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-aminomethyl)-N-(N1-thymi-
nylacetyl)] glycinate 23 (1.4 g, 95.0%) and was used further
without purification. HRMS (ESI) calcd for C21H34N5O7: 468.2453,
found: 468.2450.
3.18. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-aminomethyl)-N-(N1-
thyminylacetyl)]glycine (24)
168.80. IR (m
max, cmꢁ1) (CHCl3): 770, 1028, 1163, 1416, 1649,
1686, 1744, 2359, 2965, 3416. ½a D27
ꢁ 4 (c 1.0, CHCl3). HRMS (ESI)
ꢃ
calcd for C21H33N4O8: 469.2293, found: 469.2294.
The amino ester 23 (1.2 g, 2.6 mmol) formed above was dis-
solved in methanol (10 mL) and to that added 1 N NaOH (10 mL).
The content was stirred for 30 min and after the completion of
reaction, methanol was evaporated under reduced pressure. The
resulting aqueous solution was neutralized by Dowex H+ ion ex-
change resin. The aqueous layer was washed with ethyl acetate
(8 mL) followed by evaporation in vaccuo and dessication to obtain
ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-aminomethyl)-N-(N1-thymi-
nylacetyl)]glycine 24 (0.9 g, 83.0%). 1H NMR (D2O) d: 1.42 (s, 9H,
C(CH3)3), 1.85–1.88 (d, 3H), 3.23–3.31 (m, s 3H), 3.65 (m, 1H),
3.80–3.90 (m, 2H), 3.97 (s, 2H), 4.25 (s, 1H), 4.58 (s, 2H), 7.34 (d,
1H). 13C NMR (CDCl3) d: 11.34, 23.30, 27.60, 45.54, 46.89, 49.62,
52.97, 53.89, 55.50, 82.20, 110.88, 147.23, 152.59, 155.89, 167.31,
3.15. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-O-mesyl-methyl)-N-
(N1-thyminylacetyl)]glycinate (21)
The pure compound 20 (2.9 g, 6.2 mmol) obtained in the previ-
ous step was dissolved in dry pyridine (20 mL) and stirred for
15 min at 0 °C. Mesyl chloride (0.7 mL, 9.3 mmol) was added drop-
wise to this solution at 0 °C with stirring and the reaction mixture
was left for 2 h. Pyridine was removed under reduced pressure and
the residue was dissolved in ethyl acetate (70 mL). Organic layer
was washed with water (2 ꢂ 30 mL), satd NaHCO3 (2 ꢂ 20 mL) fol-
lowed by brine (2 ꢂ 10 mL) and dried over anhyd Na2SO4. The or-
ganic layer was concentrated in vacuo to give the crude product
which was purified by column chromatography (4% MeOH–
CH2Cl2) to give ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-O-mesyl-
methyl)-N-(N1-thyminylacetyl)]glycinate 21 (3.0 g, 88.7%). 1H
NMR (CDCl3) d: 1.27–1.34 (t, 3H), 1.45 (s, 9H, C(CH3)3), 1.92 (d,
3H), 2.28–2.49 (m, 2H), 3.04 (s, 3H), 3.80 (m, 1H), 4.04 (s, 2H),
4.18 (q, 2H), 4.27–4.33 (s, m, 3H), 4.45 (m, 2H), 4.66–4.91 (m,
2H), 7.03 (s, 1H), 9.5 (br, s, 1H). 13C NMR (CDCl3) d: 12.25, 13.96,
28.24, 37.07, 43.49, 47.81, 48.27, 54.17, 61.60, 62.38, 69.68,
77.20, 80.64, 110.70, 140.93, 151.15, 153.82, 164.35, 167.93,
169.66, 175.43. IR (
1404, 1667, 1682, 1692, 1711, 1721, 2400, 3019, 3374, 3451.
m
max, cmꢁ1) (CHCl3): 729, 904, 1067, 1266,
½
a 2D7
ꢃ
ꢁ 2 (c 1.0, CH3OH). HRMS (ESI) calcd for C19H30N5O7:
440.2140, found: 440.2141.
3.19. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-Fmoc-aminomethyl)-
N-(N1-thyminylacetyl)]glycine (25)
The amino acid obtained in the previous step 24 (0.7 g,
1.6 mmol) was dissolved in 1:1 solution of 1,4-dioxane-water
(30 mL) and NaHCO3 (0.8 g, 9.6 mmol) and Fmoc-succinimide
(0.6 g, 1.9 mmol) was added to it. The reaction mixture was stirred
for 6 h at room temperature. After completion of reaction the sol-
vent was removed under reduced pressure and aqueous layer was
washed with ethyl acetate until impurity of Fmoc-succinimide was
removed. Then aqueous layer was neutralized with Dowex H+ ion
exchange resin. The compound was extracted in 5% MeOH–ethyl
acetate (30 mL). Organic layer was dried over anhyd Na2SO4,
169.46. IR (m
max, cmꢁ1) (CHCl3): 731, 914, 1198, 1406, 1647,
1685, 1744, 2361, 2982, 3198. ½a D27
þ 6 (c 1.0, CHCl3). MS (ESI)
ꢃ
calcd for C22H35N4O10S: 547.20, found: 547.3760.
3.16. Ethyl-[-N-(N-Boc-pyrrolidin-3-yl-2S-azidomethyl)-N-(N1-
thyminylacetyl)]glycinate (22)
The mesylate derivative 21 (2.7 g, 4.9 mmol) was dissolved in
dry DMF (30 mL) and NaN3 (1.6 g, 24.7 mmol) was added to it.

4100
A. Banerjee, V. A. Kumar / Bioorg. Med. Chem. 21 (2013) 4092–4101
concentrated and the crude product was purified by column chro-
matography (10% MeOH–CH2Cl2) to give ethyl-[-N-(N-Boc-pyrroli-
din-3-yl-2S-Fmoc-aminomethyl)-N-(N1-thyminylacetyl)]glycine
25 (0.7 g, 66.5%). 1H NMR (CDCl3) d: 1.41 (s, 9H, C(CH3)3), 1.75 (m,
br, 3H), 2.87–3.49 (m, br, 9H), 3.71–4.03 (m, br, 4H), 4.18–4.36 (m,
br, 4H), 4.71 (br, 1H), 6.92, (s, br, 1H), 7.35 (m, 4H), 7.55–7.72 (d, d,
4H) 13C NMR (CDCl3) d: 12.12, 14.42, 21.59, 23.66, 28.68, 30.66,
45.17, 48.13, 50.14, 57.17, 69.58, 81.78, 108.36, 111.05, 120.66,
122.03, 128.22, 129.89, 139.23, 141.32, 143.95, 144.85, 153.24,
3.22. UV-Tm measurements
The concentrations were calculated on the basis of absorbance
from the molar extinction coefficients of the corresponding nucle-
obases of DNA/RNA/PNA. The experiments were performed at
1 lM concentrations of each strand. The complexes were prepared
in 10 mM sodium phosphate buffer, pH 7.0 containing NaCl (10
and 100 mM) and were annealed by keeping the samples at 90 °C
for 5 min followed by slow cooling to room temperature and then
to 0 °C. Absorbance versus temperature profiles were obtained by
monitoring at 260 nm with Varian Cary 300 spectrophotometer
scanning from 10 to 85 °C at a ramp rate of 0.5 °C per minute.
Experiments were repeated at least thrice and the data were pro-
cessed using Origin software 6.1. Tm (°C) values were derived from
the first derivative curves and are accurate to within 0.3 °C.
167.21, 170.47, 175.39. IR (m
max, cmꢁ1) (CHCl3): 760, 878, 1047,
1215, 1371, 1510, 1622, 1667, 1694, 1732, 1755, 2361, 3019,
3242, 3451. ½a 2D7
þ 20 (c 1.0, CH3OH). HRMS (ESI) calcd for
ꢃ
C
34H40N5O9: 662.2821, found: 662.2823
3.20. Solid phase oligomer synthesis
The PNA oligomers were synthesized by standard Boc-solid
phase peptide strategy on MBHA resin having initial loading value
1.75 mmol/g. The loading value was lowered to 0.3 mmol/g by cap-
ping with acetic anhydride in dry CH2Cl2 and pyridine as base. The
free amines of the resin was then functionalized by coupling with
3.23. CD analysis of the oligomers
The complexes were prepared in 10 mM sodium phosphate buf-
fer, pH 7.0 containing NaCl (10 mM) and were annealed by keeping
the samples at 90 °C for 5 min followed by slow cooling to room
N (
a
)-Boc-N (
e
)-2-Cl-Cbz-
L-lysine using TBTU/HOBt as coupling re-
temperature. The experiments were performed at 1 lM concentra-
agent and DIPEA as base in DMF. The lysine functionalized resin
was swollen in dry CH2Cl2 for 30 min and then treated with 50%
TFA in CH2Cl2 (3 ꢂ 2 mL, 15 min each). After that the resin was
washed with CH2Cl2 (3 ꢂ 2 mL) followed by DMF (3 ꢂ 2 mL) and
again by CH2Cl2 (3 ꢂ 2 mL). The neutralization of the TFA salt
formed was done by treating the resin with 5% DIPEA in CH2Cl2
(3 ꢂ 2 mL, 5 min each) and washed further with CH2Cl2
(3 ꢂ 2 mL). The unmodified and modified PNA monomers were
coupled to the resin using TBTU/HOBt as coupling reagent and DI-
PEA as base in DMF. The efficiency of the Boc deprotection for free
amine and the coupling of monomers were monitored by Kaiser’s
test. At the end of the oligomers synthesis, the oligomers were
cleaved from the resin with standard TFA-TFMSA protocol.
tions of each strand. All the CD spectra were recorded at room tem-
perature with Jasco J-715 spectopolarimeter. All spectra represent
an average of at least 8 scans recorded from 320 to 210 nm at a rate
of 100 nm per minute in a 1 cm path length cuvette. All specta
were processed using Origin software 6.1, baseline subtracted
and smoothed using a 5 point adjacent averaging algorithm.
4. Conclusions
A novel conformationally constrained PNA analogue was de-
signed which would exert restrictions in the aminoethyl segment
of PNA such that the dihedral angle b is restricted for RNA selective
binding. The modified oligomers containing ethano-PNA mono-
mers did show preferential binding towards RNA than cDNA, The
modification also showed positional dependence as well as substi-
tution dependence binding as only PNA 7 could stabilize the du-
plex better than the unmodified PNA. The destabilizing effect and
preferential binding towards RNA could be accounted by the re-
duced stacking interactions as observed decreased amplitudes of
the signals in the CD studies.
In a typical cleavage reaction, the resin bound oligomers (5 mg)
were treated with thioanisole (10
for 10 min in an ice bath followed by and TFA (120
(16 L) at 0 °C for 2 h. After that the resin was filtered using TFA
(400 L) followed by evaporation of the filtrate under vaccuo.
The oligomers were precipitated by adding cold ether. The precip-
L) and
l
L) and 1,2-ethanedithiol (8
lL)
l
L) and TFMSA
l
l
itated oligomers were washed with cold ether (2 ꢂ 300
l
were dissolved in deionised water. Purification of all the PNA olig-
omers was carried out by HPLC on RP-C18 column with water:
CH3CN-0.1% TFA system and were characterized by MALDI-TOF
mass spectrometry. The spectra were acquired in linear mode
Acknowledgments
A.B. acknowledges CSIR, New Delhi for the research fellowship.
V.A.K. thanks ICMR, New Delhi for research funding. We dedicate
this work to Professor Krishna N. Ganesh on the occasion of his
60th birthday.
and the matrix used for analysis was CHCA (
namic acid).
a-cyano-hydroxyl-cin-
3.21. Post synthetic conversion of Am-ethano-PNA to
Gu-ethano-PNA
Supplementary data
MBHA resin (5 mg) attached to the Fmoc protected Am-ethano-
PNA was suspended in dry DMF. Fmoc protected amino group was
deprotected by 20% piperidine–DMF solution (3 ꢂ 2 mL, 30 min
each) and resin was then subjected for guanidinylation by sus-
pending in the solution of 1H-pyrazole-1-carboxamidine hydro-
chloride (10 equiv)/DIPEA in DMF–THF (1:2) mixture. After 6–7 h,
the resin was washed with dry DMF and treated with the same
guanidinylation mixture once again to ensure completion conver-
sion from amino to guanidino functionality. The resin was washed
with dry DMF (3 ꢂ 2 mL) followed by dry CH2Cl2 (3 ꢂ 2 mL) and
was then dessicated. Gu-ethano-PNA was cleaved from the resin
with standard conditions employing TFMSA–TFA and purified by
reverse phase HPLC (yield of conversion of amino to guanidino is
approx. 95%).
Supplementary data (1H, 13C and HRMS of all the new com-
pounds. HPLC and MALDI-TOF spectra of the synthesized oligomers
PNA1–7. CD spectra of single stranded PNA. Thermal melting
graphs for different salt concentration with cDNA, RNA and mis-
match RNA) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.05.015.
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