Backbone extended pyrrolidine PNA (bepPNA): A chiral PNA for selective RNA recognition

Article abstract of DOI:10.1016/j.tet.2005.12.002

Synthesis of cationic, chiral PNA analogues with an extra atom in the backbone (bepPNA) is reported. The (2S,4S) geometry of the pyrrolidine ring, and an additional carbon atom in the backbone of homopyrimidine-bepPNAs resulted in the optimization of the inter-nucleobase distance, such that selective binding to complementary RNA over DNA was observed in the triplex mode. It was evident from circular dichroism studies that oligomers with mixed aminoethylglycyl-bep (aeg-bep) repeating units, and also bepPNA with homogeneous backbone attained structures quite different from those of aegPNA₂:RNA/DNA complexes. The bepPNA, when incorporated in a duplex forming mixed purine-pyrimidine sequence, also showed a preference for binding complementary RNA over DNA.

Full text of DOI:10.1016/j.tet.2005.12.002

Tetrahedron 62 (2006) 2321–2330
Backbone extended pyrrolidine PNA (bepPNA): a chiral
PNA for selective RNA recognition
*
T. Govindaraju and Vaijayanti A. Kumar
Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411008, Maharashtra, India
Received 12 September 2005; revised 8 November 2005; accepted 1 December 2005
Available online 27 December 2005
Abstract—Synthesis of cationic, chiral PNA analogues with an extra atom in the backbone (bepPNA) is reported. The (2S,4S) geometry of
the pyrrolidine ring, and an additional carbon atom in the backbone of homopyrimidine-bepPNAs resulted in the optimization of the inter-
nucleobase distance, such that selective binding to complementary RNA over DNA was observed in the triplex mode. It was evident from
circular dichroism studies that oligomers with mixed aminoethylglycyl–bep (aeg–bep) repeating units, and also bepPNA with homogeneous
backbone attained structures quite different from those of aegPNA2:RNA/DNA complexes. The bepPNA, when incorporated in a duplex
forming mixed purine–pyrimidine sequence, also showed a preference for binding complementary RNA over DNA.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
HN
5
HN
5
HN
6
O
6
4
7
6
B
B
B
*
*
Synthetic oligonucleotides (Fig. 1, DNA/RNA I) have been
considered as potential gene targeted therapeutic agents
(antisense and antigene).1 However, the unmodified
oligonucleotides are rapidly identified and cleaved by the
action of nucleases that hydrolyze the inter-nucleoside
phosphodiester linkage of the backbone.1 Among the known
oligonucleotides analogues,2,3 acyclic N-(2-aminoethyl)-
glycyl peptide nucleic acids (Fig. 1, aegPNA II), are
found to be very good mimics of DNA/RNA.3 Because of
the higher thermal stability of PNA:DNA/RNA hybrids and
their stability toward proteases and nucleases, PNA has
generated interest in medicinal chemistry, having potential
for the development as gene targeted drugs and as reagents
in molecular biology and diagnostics.3 The ability of the
negatively charged DNA, as well as that of uncharged
aegPNA, to cross the cell membrane is poor. Additionally,
PNA suffers from drawbacks such as poor aqueous
solubility, cell permeability, and ambiguity in binding
complementary DNA/RNA in both parallel and antiparallel
orientations. To overcome these obstacles and to facilitate
their use as antisense therapeutic agents in biological
systems, several rational modifications have been reported
to-date.4 Editing at the molecular level of designed
backbones is translated in imparting selectivity of binding
with DNA or RNA, due to the intrinsic structural differences
at duplex/triplex level of the natural nucleic acids.5 Neutral
O
4
3
5
B
*
*
+
+
N
N
H
N
H
4
3
O
3
2
2
2
O
P
H(OH)
-
O
O
O
O
O
1
1
1
HN
HN
HN
O
B
B
B
*
*
*
*
+
+
O
B
N
HN
HN
O
(OH)
H
O
O
O
O
bepPNA IV
Pyrrolidine PNA/ (POM) III
DNA/RNAI
aegPNA II
Figure 1. Structure of DNA, PNA, and modified PNAs.
pyrrolidinone and cationic pyrrolidine PNAs (Fig. 1, III)
belong to this class of modifications. (3S,5R)-Pyrrolidinone
PNA destabilized both DNA and RNA complexes6 whereas
aepone-PNA (2S,4S) stabilized complex with DNA over
RNA.7 A pentameric (2R,4R) pyrrolidine-amide oligo-
nucleotide mimic (Fig. 1, POM III), showed kinetic binding
preference to RNA over DNA.8 Pyrrolidine PNA (Fig. 1, III)
having one (2R,4S)-modified unit, showed destabilization
with DNA and RNA, but bound strongly when fully
modified.9 (2S,4S)-Pyrrolidine PNA destabilized complexa-
tion with both DNA and RNA.10 Previous studies on DNA/
RNA analogues have shown that the length of the linkage in
the repetitive DNA/RNA backbone can be varied from five
to seven.11 An example of the five-bond contracted
backbone is TNA, an extraordinary oligonucleotide system
introduced by Eschenmoser et al. TNA cross-paired
Keywords: Peptide nucleic acids; bepPNA; RNA recognition.
*
Corresponding author.
e-mail: va.kumar@ncl.res.in
Tel./fax:
C91
20
25893153;
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.12.002

2322
T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
efficiently with complementary DNA and RNA, and showed
relatively high affinity to RNA over DNA.11 Matsuda et al.
studied oligonucleotides containing oxetanocin A,12 an
isomer of deoxyadenosine with an oxetane sugar moiety,
and a higher analogue. These analogues with extended
seven-bond backbones cross-pair with RNA with high
affinity compared to DNA. A carbocyclic analogue of the
oxetanocin A oligonucleotide system also showed a
tendency to form stronger complexes with RNA rather
than with deoxyribonucleotide.13 Many examples in the
literature suggest that a five-atom amide leading to a seven-
atom repeating backbone may be more useful, because of
the reduced conformational flexibility of the amide relative
to phosphodiester backbone.14 This postulate has been
supported by X-ray studies. X-ray data confirms that the
amide backbone has a trans conformation, and that the
distance between neighboring base pairs is not affected by
incorporation of a longer backbone.14a,e On the contrary,
Lowe et al. very effectively accomplished preferential DNA
binding by replacing glycine with one-atom extended
b-amino acids in his prolyl-glycyl PNA analogues.15 Our
preliminary results on the chimeric phosphate-amide
extended backbone revealed that 2R,4R pyrrolidine–amide
chimera were accommodated better in triplex forming
sequences.16
detailed studies on the hybridization properties of bepPNA
using UV–Tm measurements, gel electrophoretic shift assay,
and circular dichroism analysis of the bepPNA hybrids that
address binding properties in both triplex and duplex modes,
using either homopyrimidine or mixed purine–pyrimidine
base sequences, respectively.
1.1. Synthesis of bepPNA monomer [(2S,4S)-2-(tert-butyl-
oxycarbonylaminomethyl)-4-(thymin-1-yl) pyrolidin-
1-yl] propanoic acid
The ring nitrogen of the naturally occurring trans-4-
hydroxy-L-proline 1 was protected as the carbamate to
give compound 2, which was then converted into its methyl
ester 3 on treatment with MeOH/SOCl2.12 Methyl ester 3
was reduced using LiCl/NaBH4 to yield diol 4 (Scheme 1).
Selective tosylation of the primary hydroxy group by
controlled dropwise addition of p-TsCl (freshly crystallized
from chloroform and petroleum ether) in pyridine gave
monotosylate 5 (some ditosylate formed was removed by
column chromatography). The monotosylate 5 was treated
with NaN3 to obtain azido compound 6, and selective
reduction of azide functionality using Raney Ni in methanol
gave free amine 7. Protection of free amine 7 using BocN3 in
DMSO yielded compound 8. Compound 8 was subjected
to hydrogenation using Pd–C catalyst to remove the
benzyloxycarbonyl group to get free amine 9. The free
ring nitrogen was subsequently alkylated using ethyl acrylate
in methanol as solvent, giving alkylated product 10.
In view of the above reports, we recently presented
preliminary results on the backbone extended pyrrolidine
PNA (Fig. 1, bepPNA IV).17 In this paper, we present
HO
HO
HO
ii
i
iii
OMe
OH
OH
N
N
H
1
N
z
2
91%
88%
98%
O
O
O
z
3
trans-4-hydroxy-L-proline
(2S,4R )
HO
HO
HO
HO
iv
v
96%
vi
OH
N
OTs
N3
N
N
75%
95%
z
z
5
z
6
4
HO
HO
ix
viii
95%
vii
NHBoc
NHBoc
NH2
72%
N
N
N
76%
H
z
z
9
8
7
O
O
HO
BzN
N
H
N
N
NHBoc
O
O
N
NHBoc
NHBoc
x
xi
N
N
72%
98%
10
11
12
COOEt
COOH
COOEt
Scheme 1. Reagents and conditions: (i) Z–Cl (50% toluene soln), Et3N, NaHCO3, water, rt, 8 h; (ii) SOCl2, Et3N, MeOH, rt, 7 h; (iii) LiCl, NaBH4, ethanol–
THF (4/3), rt, 7 h; (iv) TsCl, pyridine, rt, 7 h; (v) NaN3, DMF, 70 8C, 8 h; (vi) Raney Ni, H2, MeOH, 35 psi, rt, 3 h; (vii) BocN3, DMSO, 50 8C, 5 h; (viii)
H2/Pd–C, MeOH, 60 psi, rt, 7 h; (ix) ethyl acrylate, MeOH, rt, 2.5 h; (x) N3-benzoylthymine, DEAD, PPh3, benzene, rt, 4 h; (xi) 2 M aqueous NaOH, rt, 5 h,
Dowex-HC
.

T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
2323
No external base was required, as the amine 8 itself acted as
a base, and the inclusion of an extra atom in the backbone
was easily achieved by using conjugate addition to ethyl
acrylate as compared to cumbersome methods in extended
sugar-phosphate backbone.14 Secondary alcohol 10 was
converted to protected monomer ethyl ester 11 on treating
with N3-benzoyl thymine under Mitsunobu conditions.18
Hydrolysis of ethyl ester 11 and simultaneous deprotection
of N3 of thymine was achieved using 2 M NaOH in aqueous
methanol. The aqueous layer was washed with DCM to
remove benzoic acid, and was then neutralized with a cation
exchange resin (Dowex-HC) by careful acidification of the
reaction mixture to pH 7.0. The neutral aqueous layer was
filtered and concentrated under reduced pressure to yield
required monomer [(2S,4S)-2-(tert-butoxycarbonylamino-
methyl)-4-(thymin-1-yl) pyrolidin-1-yl] propanoic acid (12)
in good yield (Scheme 1). All new compounds were
characterized by NMR, mass spectroscopy and elemental
analysis. One-step conversion of the 4-OH in 10 to the
corresponding thymin-1-yl derivative with an inversion of
configuration can be achieved under Mitsunobu conditions.
As the reactivity of N1 and N3 of thymine are comparable
towards alkylation, this reaction results in both N1-alkylated
and N1,N3-dialkylated products. Hence, N3 of the thymine
was first protected as its benzoyl derivative.19 The
aminoethylglycyl PNA (A/G/C/T) monomers were
synthesized following the literature procedure,20 and these
monomers were used for the synthesis of aegPNA T8
octamer (13) and aegPNA 19 mixed decamer for the control
studies and to synthesize bepPNA–aegPNA chimeras.
overlapped at w10. The pKa of the ring nitrogen was found
to be w7.7 (Fig. 2). The pyrrolidine ring nitrogen in the
monomer could partially be protonated under physiological
conditions (pH). This pKa (7.7) of the ring nitrogen is higher
than any other pyrrolidine PNAs, and can be attributed to
the fact that the additional methylene group in the backbone
increases the basic character of the ring nitrogen.
1.3. Synthesis of cationic backbone extended pyrrolidine
peptide nucleic acids (bepPNA) and UV-melting studies
of bepPNA:DNA/RNA complexes
The modified cis-(2S,4S)-bepPNA thymine monomer 12
was incorporated into PNA sequences using Boc-chemistry
on L-lysine-derivatized (4-methylbenzhydryl)amine
(MBHA) resin as reported before, using HBTU/HOBt/
DIEA in DMF as the coupling reagent. Various homo-
thymine PNA oligomers (13–18, Tables 1 and 2) incorpo-
rating modified monomers at the middle (15), N-terminus
(16), C-terminus (14), at alternative positions (17), and
through the entire sequence (homo-oligomer 18) were
synthesized in order to study their triplex formation and
stability with DNA/RNA. Octamer aegPNA T8 (13)
sequence was also synthesized, incorporating aegPNA
thymine monomer for the use of control studies. In order
to study the duplex formation potential, and in particular
DNA/RNA discrimination of the bepPNA monomeric units,
it was imperative to synthesize mixed sequences, incorpo-
rating both purines and pyrimidines. Mixed sequences
aegPNA 19 and bepPNA 20 were also synthesized by
incorporating aegPNA (A/G/C/T) monomers and bepPNA
(T) monomers (Table 3). The oligomers were cleaved from
the resin using a ‘low–high TFA–TFMSA’ procedure,21
followed by RP-HPLC purification and characterization by
mass spectrometry (LC-TOF-MS). The complementary
DNA oligonucleotides 21, 23, 24 were synthesized on
Applied Biosystems ABI 3900 High Throughput DNA
Synthesizer using standard b-cyanoethyl phosphoramidite
chemistry. The oligomers were synthesized in the 30–50
direction on polystyrene solid support, followed by
ammonia treatment.22 The oligonucleotides were desalted
by gel filtration, their purity ascertained by RP-HPLC on a
C18 column to be more than 98%, and were used without
further purification in the biophysical studies of PNAs.
The RNA oligonucleotides 22, 25, and 26 were obtained
commercially.
1.2. Determination of the pKa of the pyrrolidine ring
nitrogen (N1) of the bepPNA thymine monomer 12
The bepPNA monomer has a tertiary amino group in the
pyrrolidine ring that can be protonated. pH titration
experiment was carried out with NaOH to determine the
approximate pKa of this nitrogen atom. A plot of pH versus
volume of NaOH-added gave three transitions; the first one
corresponding to the carboxylic acid, second corresponding
to the tertiary ring nitrogen. The third transition could
correspond to the free amine group of the deprotected
monomer and the N3–H of thymine (Fig. 2). This transition
is not very well resolved under the present conditions. The
Boc protected monomer 12 was deprotected using trifluoro-
acetic acid, and the product titrated against 0.2 M NaOH to
find out the approximate pKa value of pyrrolidine ring
nitrogen. The pKa values for free –COOH was found to be in
the range of 3–4 and those of –NH2 and N3–H of thymine
The Tm values of homopyrimidine PNAs 13–18, hybridized
with complementary DNA and RNA were obtained from
14
0.14
b
d
pKa = ~ 10.3
0.12
0.10
0.08
0.06
0.04
0.02
0.00
12
10
T
c
NH2
a
8
N
b
pKa = ~ 7.7
6
4
2
c and d
COOH
a
pKa = ~ 3.76
0
50
100
150
200
Vol. of NaOH added (µL)
Figure 2. pH titration curve of (2S,4S)-bepPNA monomer (free amine) with NaOH (empty triangles) and change in pH with volume of NaOH (filled squares).

2324
T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
Table 1. HPLC and mass spectral analysis of synthesized PNAs
0.7
0.6
0.5
0.4
0.3
0.2
Entry PNA
HPLC
(RT, in min)
MW (calcd)
MW
(found)a
1
2
3
4
5
6
7
8
aegPNA 13
7.536
7.596
7.270
7.359
9.562
8.246
10.505
7.852
2274.00
(C96H132N35O32
2286.00
(C96H132N35O32
2286.00
(C96H132N35O32
2286.00
(C96H132N35O32
2323.00
(C102H144N35O29
2370.00
(C110H160N35O25
2852.00
(C114H147N60O31
2887.00
(C120H159N60O28
2275.90
2288.00
2288.00
2288.04
2326.06
2374.43
2853.00
2891.36
)
)
)
)
bepPNA 14
bepPNA 15
bepPNA 16
bepPNA 17
bepPNA 18
aegPNA 19
bepPNA 20
)
)
)
)
0
20
40
60
80
100
Mole fractions of bepPNA 18
a LC-TOF-MS, RTZretention time.
Figure 3. UV–Job’s plot for bepPNA 18 and the complementary RNA
(poly rA) mixtures in the molar ratios of 0:100, 10:90, 20:80, 30:70, 40:60,
50:50, 60:40, 70:30, 80:20, 90:10, 100:0 at 260 nm (buffer, 10 mM sodium
phosphate pH 7.0, 100 mM NaCl, 0.1 mM EDTA).
Table 2. Melting temperatures (Tm) of PNA2:DNA/RNA triplexesa
No.
Homopyrimidine PNA sequence DNA
RNA
1
2
3
4
5
6
13, H-TTTTTTTT-LysNH2
14, H-TTTTTTTt-LysNH2
15, H-TTTtTTTT-LysNH2
16, H-tTTTTTTT-LysNH2
17, H-TtTtTtTt-LysNH2
18, H-tttttttt-LysNH2
51.5
49.0
nd
53.0
nd
65.8
59.9
59.2
59.0
84.4
58.9
complex with DNA as shown in UV-melting curves. When
the complexation studies were performed with RNA,
chimeric PNAs with a single bepPNA unit were found to
bind with approximately same Tm, but slightly lower than
that of control aegPNA 13. bepPNA 17, with alternating
aeg–bep units exhibiting a very high binding affinity
(DTmZC4.5 8C/mod) (Fig. 4B, Table 2). The observed
transitions were very sharp with RNA compared to that
with DNA. The sequence 18 comprised only of a bepPNA
backbone also recognized only RNA, but with reduced
strength compared to the alternating aeg–bepPNA. These
results suggest that bepPNA monomer in chimeric and
homooligomeric PNAs introduced binding selectivity for
RNA over DNA. Incorporation of the modified units at the
terminals (C-/N-) seems to exert very weak effective
preorganized conformation, and allowed binding with
DNA as well as RNA. When in the center of the sequence,
the induced conformation allows recognition of RNA but
that of DNA is suppressed. The high affinity binding of
alternating aeg–bepPNA 17 with RNA suggests that the
alternating aeg–bep units are uniformly spaced, such that a
balanced optimum conformation may be reached for
recognition of RNA. The fully modified backbone in 18
binds to RNA but with reduced strength compared with the
alternating sequence 17. This could be because of over-
organization of single strand as suggested for fully modified
LNA,14b,d or high positive charge concentration of two
bep-homooligomers in 2:1 binding mode. The 2:1 binding
stoichiometry for:18:RNA was confirmed by UV–Job’s plot
(Fig. 3). The charge–charge repulsions could therefore be
the possible reason for the observed reduced Tm. To study
the RNA binding selectivity of bepPNA in duplex
formation, purine–pyrimidine-mix bepPNA decamer 20
was synthesized incorporating the bepPNA-T monomer at
three thymine positions in control aegPNA 19. The UV and
CD-thermal denaturation24 studies of bepPNA 20 with DNA
and RNA were carried out (Fig. 5 and Supplementary
information). Data in Table 3 shows that the reference
aegPNA 19 forms ap-duplexes with complementary DNA
23 and RNA 25 with equal stability. Mixed bepPNA 20 did
nd
a TmZmelting temperature (measured in buffer 10 mM sodium phosphate,
pH 7.0 with 100 mM NaCl and 0.1 mM EDTA). Measured from 10 to 90 8C
at ramp 0.2 8C/min. UV-absorbance measured at 260 nm. All values are an
average of three independent experiments and accurate to within G0.5 8C.
DNA 21ZdCGCA8CGC; RNA 22Zpoly rA; ndZnot detected.
temperature dependent UV-absorbance data (Fig. 4,
Table 2). The UV and CD Job’s plots23 suggest the
formation of 2:1 bepPNA2/DNA and bepPNA2/RNA
triplexes (Fig. 3) and hence all the complementation studies
were performed with 2:1 PNA/DNA stoichiometry. The
UV–Tm values were obtained from the first derivatives of
the normalized absorbance-temperature plots of the corres-
ponding PNA:DNA complexes (Fig. 4A, Table 2). The
C-terminal modified bepPNA 14 binds to DNA with
slight decrease in Tm (DTmZKl 8C) where as bepPNA 16
modified at N-terminal stabilizes the complex (DTmZC
2 8C) compare to the control aegPNA 13. Surprisingly,
bepPNA 15, with a modified unit at the center did not
show any complexation with DNA. Alternate and
homooligomeric bepPNAs (17 and 18) also did not form
Table 3. UV–Tm (8C) of PNA:DNA/RNA duplexesa
Mix PNA sequence
DNA 23
RNA 25
19, H-GTAGATCACT-LysNH2
20, H-GtAGAtCACt-LysNH2
55.0 (40.0)b
nd
55.4
81.0 (74)
a TmZmelting temperature (measured in buffer 10 mM sodium phosphate,
pH 7.0 with 100 mM NaCl and 0.1 mM EDTA). Measured from 10 to
90 8C at ramp 0.2 8C/min. UV-absorbance measured at 260 nm. DNA
23Z50AGTGATCTAC (ap); DNA 24, 50-CATCTAGTGA-30(p); RNA
25Z50 AGUGAUCUAC (ap); RNA 26, 50-CAUCUAGUGA-30 (p).
b Measured by CD to avoid interference from thermal transitions of single
stranded PNAs. ndZnot detected. Values in brackets are Tm for parallel
duplexes with DNA 24 and RNA 26.

T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
2325
d
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
A
B
d
c
a
b
e
f
e
f
a
c
b
20
40
60
80
20
40
60
80
Temperature (°C)
Temperature (°C)
Figure 4. UV–Tm curves of A. (a) aegPNA 13, (b) bepPNA 14, (c) bepPNA 15, (d) bepPNA 16, (e) bepPNA 17 and (f) bepPNA 18 with DNA 21.
B. (a) aegPNA 13, (b) bepPNA 14, (c) bepPNA 15, (d) bepPNA 16, (e) bepPNA 17 and (f) bepPNA 18 with RNA 22 (buffer, 10 mM sodium phosphate pH 7.0,
100 mM NaCl, 0.1 mM EDTA).
not bind to DNA (antiparallel and parallel), as there was no
transition detected in the UV melting experiment, whereas
high affinity binding was observed with RNA (antiparallel,
DTmZ26 8C). Destabilization of (DTm w7 8C) was
observed for the parallel bepPNA 20:RNA duplex indicating
the preference for antiparallel mode of binding.
intensity band at 265 nm (Fig. 6B, c). Interestingly, the CD
signature of the bepPNA 18 is more pronounced than that of
DNA 21 (Supplementary information). As expected, the
C- and N-terminus modified bepPNAs 14 and 16 with DNA
showed CD signatures similar to the control aegPNA2:DNA
triplex (Fig. 6A, a and c), whereas the CD signature of
bepPNA 15 with DNA showed broad band from
260–285 nm (Fig. 6A, b) due to weak binding interaction
as shown by gel electrophoresis assay. CD patterns of the
alternating aeg–bepPNA 17 and bepPNA 18 with DNA 21
(Fig. 6A, d and e) were found to be additive spectra of
corresponding CD signals of single stranded bepPNA and
DNA 21. Subtraction of the CD spectra of single stranded
bepPNA 17 and 18 from the CD spectra of [bepPNA 17C
DNA 21] and [bepPNA 18CDNA 21], respectively, gave
the CD spectrum corresponding to single strand DNA
21(Fig. 6B, b and d). These CD results are in complete
agreement with the results obtained by UV measurements
and gel shift assay.17 CD spectra of bepPNA–poly rA
complexes were recorded to study the structural changes
after complexation in comparison with control aegPNA
13:poly rA complex (Fig. 6C, a). The CD signatures of
singly modified bepPNAs 14, 15, and 16 with poly rA
(Fig. 6C, b–d, respectively) gave different pattern compared
to the control aegPNA 13–poly rA that was similar to
(aegPNA 13)2–DNA 21 triplex; CD profile with two
characteristic bands at 260 and 285 nm. As expected from
the UV–Tm data of aeg–bepPNA 17 and bepPNA 18, the CD
patterns were quite similar to each other as well as to that of
control aegPNA 13–poly rA (Fig. 6C, e and f and a,
respectively). The aeg–bepPNA–poly rA complexation was
accompanied by the appearance of strong positive bands at
225 and 265 nm and a low negative intensity band at 245 nm
compare to control with slight similarity. However, bepPNA
18–poly rA showed presence of single strand bepPNA 18
and strong positive band at 235 nm was also observed. Thus,
the CD spectral studies also demonstrated RNA selectivity
of bepPNAs.
1.4. CD spectroscopic studies of bepPNA:DNA
and bepPNA:RNA complexes
Achiral aegPNAs show very weak CD signatures due to the
presence of chiral linker amino acid L-lysine. However,
PNA:DNA complexes exhibit characteristic CD signatures
due to chirality induced by the DNA component. It is known
that the formation of PNA2:DNA triplexes25 accompanied
by the appearance of positive CD bands at 260 and 285 nm
that are not present in DNA (Supplementary information).
Unlike aegPNA 13, the single stranded bepPNAs (14–18),
showed distinct CD patterns depending on the number and
position of modified units present (Supplementary infor-
mation). Alternating aeg–bepPNA 17 showed a positive
lower intensity bands at 245 nm, high intensity band at
260 nm and negative intensity bands at around 225 and
275 nm region (Fig. 6B, a). The fully modified bepPNA
homo-oligomer (18) gave a CD signature with a maximum
intensity positive band at around 235 nm, and a negative
1.0
0.8
0.6
0.4
d
c
b
a
0.2
0.0
1.5. Electrophoretic gel shift assay
20
40
60
80
100
Temperature °C
An electrophoretic gel shift experiment was carried out to
prove the results obtained from UV-thermal denaturation
studies of bepPNAs with complementary DNA 21
Figure 5. Melting curves of bepPNA 20 with A: (a) DNA 23 (antiparallel),
(b) DNA 24 (parallel), (c) RNA 25 (antiparallel), and (d) RNA 26 (parallel).

2326
T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
3
3
2
c
c
2
1
a
b
a
1
0
0
–1
–2
–3
–1
–2
–3
d
b
e
A
d
B
220
240
260
280
300
320
220
240
260
280
300
320
Wavelength (nm)
Wavelength (nm)
6
4
e
f
c
2
0
b
–2
d
a
–4
C
–6
220
240
260
280
300
320
Wavelength (nm)
Figure 6. A. CD spectra of PNA2:DNA (a) bepPNA 14, (b) bepPNA 15, (c) bepPNA 16, (d) bepPNA 17 and (e) bepPNA 18 with DNA 21; B. CD spectra of
single stranded (a) bepPNA 17 and (c) bepPNA 18; subtraction spectra (b) [bepPNA 17:DNA 21–bepPNA 17] and (d) [bepPNA 18:DNA 21–bepPNA 18];
C. CD spectra of PNA2:RNA (a) aegPNA 13, (b) bepPNA 14, (c) bepPNA 15, (d) bepPNA 16, (e) bepPNA 17 and (f) bepPNA 18 with RNA 22 (buffer, 10 mM
sodium phosphate pH 7.0, 100 mM NaCl, 0.1 mM EDTA).
(Supplementary information). The various PNAs were
individually mixed with DNA 21 in buffer, and subjected
to non-denaturing gel electrophoresis26 at 10 8C. The bands
were visualized on a fluorescent TLC background. The
formation of a PNA:DNA complex was accompanied by
disappearance of the band due to single stranded DNA 21
and appearance of a lower migrating band of complex. The
terminally modified bepPNAs 14 and 16, upon mixing with
DNA migrated about the same as the control aegPNA
13:DNA complex (Fig. SI, lane 1) and much lower than that
of DNA (Fig. SI, lanes 5 and 7). The bepPNA 15 exhibited
very weak binding interaction at lower temperature, though
it was not seen during UV–Tm thermal denaturation (Fig. SI,
lane 6). Under the conditions used, the single stranded PNAs
carrying positive charge do not migrate from the well.
No complexation was observed in case of alternating
aeg–bepPNA 17 and bepPNA 18 with DNA as it can be
seen from faster moving band due to unbound single strand
DNA and hence clearly support the data obtained from
UV–Tm experiments.
properties essential for development into a therapeutic drug.
Further studies on the mixed purine–pyrimidine sequences
with bepPNA A/G/C/T units and other diastereomeric
bepPNAs are underway.
3. Experimental
3.1. General experimental and spectroscopic data
Melting points of samples were determined in open
capillary tubes using Buchi Melting point B-540 apparatus
and are uncorrected. IR spectra were recorded on an infrared
Fourier Transform spectrophotometer using KBr pellets.
Column chromatographic separations were performed using
silica gel 60–120 mesh, solvent systems gradient EtOAc/pet
ether and pure DCM to 3% MeOH/DCM. 1H and 13C
spectra were obtained using Bruker AC-200 (200 MHz) and
500 MHz NMR spectrometers. The chemical shifts are
reported in delta (d) values. The optical rotation values were
measured on Bellingham-Stanley Ltd, ADP220 polarimeter.
CD spectra were recorded on JASCO-715 Spectropolari-
meter. Mass spectra were obtained either by LCMS
techniques or by LC-TOF-MS mass spectrometry. Oligomers
were characterized by RP-HPLC, C18 column and LC-TOF-
MS mass spectrometry.
2. Summary
We have reported the design and synthesis of a novel class
of cationic pyrrolidine PNAs with extended backbone that
show improved binding affinity and selectivity towards
DNA/RNA recognition. The complementation studies with
DNA/RNA reveal that these bepPNAs bring in unprece-
dented RNA binding selectivity in triplex as well as duplex
modes. From the application perspective, this chiral,
cationic PNA analogue is shown to have very important
3.1.1. (2S,4R)-N1-(Benzyloxycarbonyl)-4-hydroxy-2-
(hydroxymethyl)-pyrrolidine (4). To an ice cooled solvent
mixture of dry THF (175 ml) and absolute ethanol (250 ml)
containing NaBH4 (3.192 g, 84.3 mmol) in a three-necked
flask, LiCl (3.58 g, 84.3 mmol) was added slowly from a

T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
2327
solid addition funnel over 30 min. The above solution was
stirred for 1.0 h and the appearance of a milky solution
indicates the formation of LiBH4 in situ. To the above ice-
cooled milky solution, (2S,4R)-N1-(benzyloxycarbonyl)-4-
hydroxyproline methyl ester (3) (9.45 g, 33.75 mmol)
dissolved in absolute ethanol (50 ml) was added from a
dropping funnel over a period of 30 min under nitrogen
atmosphere, and the reaction mixture was stirred over night
at rt. Then the pH of the reaction mixture was adjusted to 7.0
by adding saturated NH4Cl. The solvent mixture was
removed under vacuum and the residue was extracted into
ethyl acetate (25 ml!3). The organic layer was washed
with water, brine solution, dried over anhydrous Na2SO4
and concentrated to afford oily product diol 4 (7.5 g, yield
88%, RfZ0.3, ethyl acetate/petroleum ether-1:1). [a]D20
C16.8 (c 3.26, CH2Cl2); IR (neat) (n) cmK1. 3410, 3021,
1699, 1470, 1415. 1H NMR (CHCl3-d, 200 MHz); d: 1.55–
1.8 (m, 1H), 1.9–2.2 (m, 1H), 3.25–3.8 (m, 4H), 3.8–4.25
(m, 2H), 4.25–4.5 (br d, 1H), 5.12 (s, 2H), 7.4 (s, 5H). 13C
NMR (CHCl3-d, 200 MHz); d: 37.1, 55.4, 58.9, 65.5, 87.2,
89.0, 127.7, 128.3, 136.2, 159.5. Anal. Calcd (%) for
C13H17NO4: C, 62.15; H, 10.75; N, 5.57. Found C, 61.83; H,
10.91; N, 5.53; LCMS; 252.07 [MC1]C.
3.97–4.45 (m, 2H, C2H, C4H), 5.09 (s, 2H, OCH2), 7.31 (s,
5H, C6H5). 13C NMR (CHCl3-d, 200 MHz); d: 36.9, 37.5,
51.9, 53.2, 55.2, 54.8, 5, 66.6, 67.0, 68.4, 68.8, 127.2, 127.5,
128.1, 136.2, 162.5; LCMS; 277.00 [MC1]C.
3.1.4. (2S,4R)-N1-(Benzyloxycarbonyl)-4-hydroxy-2-
(aminomethyl)-pyrrolidine (7). To a solution of the azide
6 (3.5 g, 12.7 mmol) in methanol (5 ml) taken in hydrogen-
ation flask was added Raney Ni (1.5 ml). The reaction
mixture was hydrogenated in a Parr apparatus for 3.5 h at rt
and H2 of pressure 35–40 psi. The catalyst was filtered off
and then solvent was removed under reduced pressure to
yield a residue of the amine 7 as colorless oil. Yield (3.0 g,
95.0%); this compound was used for the further reaction
without any purification.
3.1.5. (2S,4R)-N1-(Benzyloxycarbonyl)-2-(tert-butyloxy-
carbonylaminomethyl)-4-hydroxy pyrrolidine (8). The
amine 7 (3.0 g, 12.0 mmol) was taken in DMSO (10 ml),
triethylamine (1.58 g, 15.6 mmol) and BocN3 (2.05 g,
14.4 mmol) were added. The reaction mixture was heated
to 50 8C for 8 h. The reaction mixture was poured into
150 ml of ice-cold water and the product extracted into ether
(20 ml!8). The combined ether layer was washed with
water, brine and then concentrated to give Boc protected
amine 8 as light yellow oil. (3.2 g, yield 76%, RfZ0.6, ethyl
acetate:petroleum ether). [a]2D0 K12.7 (c 0.7, CH2Cl2); IR
(neat) (n) cmK1. 3310, 3121, 1689, 1570. 1H NMR (CHCl3-d,
200 MHz); d: 1.41 (s, 9H, Boc), 1.6–2.3 (m, 3H, C2H,
C3H2), 3.15–3.75 (m, 4H, C5H2, CH2NH), 3.9–4.2 (m, 1H,
C4–OH), 4.3–4.5 (m, 1H, C4H), 5.11 (s, 2H, OCH2),
5.4–5.6 (br d, 1H, carbamate NH), 7.33 (s, 5H, C6H5). 13C
NMR (CHCl3-d, 200 MHz); d: 28.3, 38.04, 44.1, 54.9, 56.6,
67.0, 69.2, 79.2, 128.4, 136.4, 156.3, 159.6. Anal. Calcd (%)
for C18H26N2O5: C, 61.71; H, 7.42; N, 8.00. Found C, 61.68;
H, 7.64; N, 7.78; LCMS; 351.21 [MC1]C.
3.1.2. (2S,4R)-N1-(Benzyloxycarbonyl)-4-hydroxy-2-(p-
toluenesulfonyloxymethyl)-pyrrolidine (5). The diol 4
(7.12 g, 28.36 mmol) was dissolved in dry pyridine (200 ml)
and cooled to 0 8C. To this ice cooled solution, freshly
crystallized p-toluenesulfonyl chloride (5.95 g, 31.2 mmol)
in pyridine was added from a dropping funnel over a period
of 1.5 h under nitrogen atmosphere. The reaction mixture
was stirred for 8 h at rt. Pyridine was removed under
reduced pressure and co-evaporated with toluene (twice).
The residue was extracted into ethyl acetate (50 ml!2),
washed with water, dried over Na2SO4 and concentrated to
yield crude oily residue. The residue was purfied by column
chromatography to get monotosylate 5 as a thick oil. (8.6 g,
yield 75%, RfZ0.36, ethyl acetate/petroleum ether-1:1).
[a]D20 C27.7 (c 4.43, CH2Cl2); IR (neat) (n) cmK1. 3011,
1699,1550, 1500, 1470, 1415. 1H NMR (CHCl3-d,
200 MHz); d:1.7–2.15 (2H, C2H, C3H), 2.2–2.5 (s, 3H,
OCH3), 3.1–3.6 (m, 2H, C5H2), 3.7–4.2 (m, 3H, CH3),
4.25–4.65 (m, 2H, CH2-OTs), 4.7–5.5 (br d, 4H, C4H,
COOCH2, OH), 7.26–7.4 (s, 7H, C6H5, two CH-Ts), 7.5–7.8
(m, 2H, two CH-Ts). 13C NMR (CHCl3-d, 200 MHz); d:
21.0, 35.9, 54.5, 54.8, 66.4, 68.7, 69.8, 127.3, 128.0, 129.5,
132.3, 136.0, 144.5, 154.6. Anal. Calcd (%) for C20H23NO6S:
C, 59.25; H, 5.67; N, 3.45; S, 7.90. Found C, 58.92; H,
5.77; N, 3.37; S, 7.67; MS LCMS; 405.00 [M]C.
3.1.6. [(2S,4R)-2-(tert-Butyloxycarbonylaminomethyl)-4-
hydroxypyrrolidine (9). To a solution of the ester 8 (3.2 g,
9.5 mmol) in methanol (5 ml) in a hydrogenation flask was
added 10% Pd–C (0.32 g). The reaction mixture was
hydrogenated in a Parr apparatus for 7 h at rt and H2 at
60 psi pressure. The catalyst was filtered off and then
solvent was removed under reduced pressure to yield a
residue of the amine 9 as colorless oil. Yield (1.9 g, 95%);
this compound was used for the further reaction without any
purification.
3.1.7. Ethyl [(2S,4R)-2-(tert-butyloxycarbonylamino-
methyl)-4-hydroxypyrrolidin-1-yl]-propanoate (10). To
the cyclic amine 9 (1.9 g, 8.8 mmol) in methanol (20 ml),
was added ethyl acrylate (0.97 g, 9.68 mmol) and stirred for
3 h at rt. The reaction mixture was evaporated to dryness and
was extracted into ethyl acetate (25 ml!3). The organic layer
wasdriedoverNa2SO4 andconcentratedtogivecruderesidue,
which on column chromatography afford ester 10 as thick
colorless oil. (2.0 g, yield 72%, RfZ0.6, MeOH/CH2Cl2-1:9).
[a]2D0 K7.7 (c 3.23, CH2Cl2); IR (neat) (n) cmK1. 3410, 1730,
3.1.3. (2S,4R)-N1-(Benzyloxycarbonyl)-4-hydroxy-2-
(azidomethyl)-pyrrolidine (6). To the solution of mono-
tosylate 5 (6.0 g, 14.8 mmol) in DMF (50 ml), NaN3 (7.7 g,
118.4 mmol) was added. The reaction mixture was stirred at
55 8C for 8 h. The solvent was removed under reduced
pressure and the residue was extracted into ethyl acetate
(25 ml!3). The combined organic layer was washed with
water, brine, dried over anhydrous Na2SO4 and then
concentrated to yield azide 6 as thick oil. (3.9 g, yield
96%, RfZethyl acetate/petroleum ether-1:1). [a]2D0 C19.3
(c 2.15, CH21Cl2); IR (neat) (n) cmK1. 3016, 2360, 2106,
1697, 1419. H NMR (CHCl3-d, 200 MHz); d: 1.95–2.02
(m, 2H, C3H), 3.07–3.85 (m, 4H, 2!C5H, CH2N3, OH),
1
1697, 1410. H NMR (CHCl3-d, 200 MHz); d: 1.24 (t, 3H,
ester CH3), 1.41 (s, 9H, Boc), 1.52–1.9 (m, 2H, C3H2),
1.95–2.6 (m, 5H, –CH2–CH2–, C2H), 2.7–3.5 (m, 5H, C5H2,
CH2NH, OH), 3.7–4.2 (m, 2H, ester CH2), 4.25–4.4 (m, 1H,
C4H), 4.75–5.45 (br d, 1H, NH). 13C NMR (CHCl3-d,

2328
T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
200 MHz); d:13.9, 28.1, 33.7, 37.7, 40.7, 49.1, 60.2, 61.3,
61.6, 69.2, 78.7, 156.3, 172.4. Anal. Calcd (%) for
C15H28N2O5: C, 56.96; H, 8.86; N, 8.86. Found C, 56.65; H,
8.95; N, 8.71; LCMS; 317.00 [MC1]C.
excellent yield (O85%). In case of cytosine monomer ethyl
ester, mild base 0.5 M LiOH was used to avoid deprotection
of the exocyclic amine-protecting group.
3.3. Synthesis of PNA oligomers, incorporating
bepPNA monomers
3.1.8. Ethyl [(2S,4S)-2-(tert-butyloxycarbonylamino-
methyl)-4-(N3-benzoylthymin-1-yl)-pyrrolidin-1-yl]-
propanoate (11). To a solution of alcohol 10 (1.5 g,
4.74 mmol), N3-benzoylthymine and triphenylphosphine in
dry benzene cooled to 4 8C, was added DIAD dropwise by a
syringe under nitrogen atmosphere. The reaction mixture
was stirred for another 5 h at rt. The reaction mixture was
evaporated to dryness and the residue was purified by
column chromatography to obtain monomer ethyl ester 11
as foam. (1.8 g, yield 72%, RfZ0.76, MeOH/CH2Cl2-1:9).
[a]D20 K69.16 (c 0.50, CH2Cl2); IR (Nujol) (n) cmK1. 3019,
1730, 1710, 1697, 1550, 1415. 1H NMR (CHCl3-d,
500 MHz); dH 1.29 (t, 3H, ester CH3), 1.41 (s, 9H, Boc),
1.55–1.8 (m, 1H, C3H0), 1.96 (s, 3H, thymine-CH3),
2.15–2.3 (m, 1H, C3H), 2.35–2.7 (m, 5H, –CH2–CH2–,
C2H), 3.0–3.2 (m, 3H, C5H0, CH2NH), 3.25–3.4 (m, 1H,
C5H), 3.95–4.15 (m, 2H, ester CH2), 4.95–5.15 (m, 1H,
C4H), 5.2–5.3 (br d, 1H, NH), 7.15–7.25 (m, 2H, Ar), 7.25–
7.35 (m, 1H, Ar), 7.35–8.00 (m, 3H, thy CH, Ar). 13C NMR
(CHCl3-d, 500 MHz); dC 12.1, 13.9, 28.0, 33.2, 35.5, 39.5,
47.5, 57.3, 58.5, 60.4, 63.0, 79.0, 110.6, 128.8, 130.0, 131.6,
134.5 and 135.4, 149.6, 156 .0, 162.2, 168.9, 172.2. Anal.
Calcd (%) for C27H36N4O7: C, 61.36; H, 6.81; N, 10.60.
Found C, 61.13; H, 6.97; N, 10.43; MS LCMS; 528.01
[M]C, 428.01 [MKtBoc]C.
The modified PNA monomers were built into PNA
oligomers using standard procedure on a L-lysine deriva-
tized (4-methylbenzhydryl)amine (MBHA) resin (initial
loading 0.25 meq gK1) with HBTU/HOBt/DIEA in DMF/
DMSO as a coupling reagent.
3.4. Cleavage of the PNA oligomers from the resin
The PNA oligomers were cleaved from the resin with
TFMSA. The oligomrs were purified by RP-HPLC (C18
column) and characterized by LC-TOF-MS mass spec-
trometry. The overall yields of the raw products were
35–65%. The normal PNAs were prepared similarly as
discussed above. The oligomer attached MBHA resin
(20 mg) was stirred with thioanisole (40 ml) and 1,
2-ethanedithiol (32 ml) in an ice bath for 10 min. TFA
(240 ml) was added, and after equilibration for 10 min,
TFMSA (32 ml) was added slowly. The reaction mixture
stirred for 2.5 h at rt, filtered and concentrated under
vacuum. The product was precipitated with dry ether from
methanol and the precipitate was dissolved in water (200 ml)
and loaded over Sephadex G25 column. Fractions of 0.5 ml
were collected and the presence of oligomer was detected by
measuring the absorbance at 260 nm. Fractions containing
oligomer were freeze-dried and the purity of the fractions
was assessed by analytical RP-HPLC. If the purity is less
than 90%, oligomers were purified by preparative HPLC.
3.1.9. [(2S,4S)-2-(tert-Butyloxycarbonylaminomethyl)-4-
(thymin-1-yl)-pyrrolidin-1-yl]-propaonic acid (12). The
monomer ethyl ester 11 (1.2 g, 2.8 mmol) was dissolved in
methanol (6 ml), 2 M NaOH (6 ml) was added and the
reaction stirred for 7 h. The aqueous layer was then
neutralized with cation exchange resin (Dowex-HC). The
reaction mixture was filtered to remove the resin. The
aqueous layer was washed with ethyl acetate to remove
benzoic acid. The aqueous layer was concentrated to a
residue that on co-evaporation with dichloromethane
(10 ml!2) afforded monomer 12 as foam. (1.1 g, yield
98%), mp, 119–121 8C; [a]2D0 K78.0 8 (c 0.5, CH2Cl2); IR
(neat) (n) cmK1. 3016, 1705, 1699. 1H NMR (D2O,
500 MHz); dH 1.39 (s, 9H, Boc), 1.82 (s, 3H, thy CH3),
2.1–2.3 (m, 1H, C3H0), 2.45–2.75 (m, 2H, N–CH2–CH2–
CO), 2.8–2.9 (m, 1H, C3H), 2.95–3.15 (m, 1H, C2H), 3.4–
3.8 (m, 5H, COCH2–CH2–N, CH2NH, C5H), 3.95–4.15 (m,
1H, C5H0) 4.8 (m, 1H, C4H), 7.44 (s, 1H, thy CH). 13C
NMR (D2O, 500 MHz); dC 11.0, 27.45, 31.9, 32.4, 38.0,
50.3, 56.7, 57.5, 66.8, 81.4, 110.4, 142.6, 157.6, 158.0,
168.5, 177.7. Anal. Calcd (%) for C18H28N4O6: C, 54.54; H,
7.07; N, 14.14. Found C, 54.23; H, 7.29; N, 13.97; MS
LCMS; 397.05 [MCH]C, 297.05 [MC1KtBoc]C.
3.5. Gel filtration
The crude PNA oligomer obtained after ether precipitation
was dissolved in water (200 ml) and loaded on a gel filtration
column. This column consisted of G25 Sephadex and had a
void volume of 1 ml. The oligomer was eluted with water
and ten fractions of 1 ml volume each were collected. The
presence of the PNA oligomer was detected by measuring
the absorbance at 260 nm. The fractions containing the
oligomer were freeze-dried. The purity of the cleaved crude
PNA oligomer was determined by RP-HPLC on a C18
column. If the purity of the oligomers found to be above
96%, the oligomers were used as such for experiments
without further purification. If the purity was not satisfac-
tory, the oligomers were purified by HPLC.
3.6. HPLC (high performance liquid chromatography)
purification of PNA oligomers
Peptide purifications were performed on a Waters DELTA-
PAK-RP semi preparative C18 column attached to a
Hewlett Packard 1050 HPLC system equipped with an
auto sampler and Jasco-UV970 variable-wavelength detec-
tor. An isocratic elution method with 10% CH3CN in 0.1%
TFA/H2O was used with flow rate 1.5 ml/min and the eluent
was monitored at 260 nm. The purity of the oligomers was
further assessed by RP-C18 analytical HPLC column (25!
0.2 cm, 5 mm) with gradient elution: A to 50% B in 30 min,
3.2. Hydrolysis of the ethyl ester functions
of aegPNA monomers (general method)
The ethyl esters were hydrolyzed using 2 M aqueous NaOH
in methanol and the resulting acid was neutralized with
activated Dowex-HC until the pH of the solution was 7.0.
The resin was removed by filtration and the filtrate was
concentrated to obtain the resulting Boc-protected acids in

T. Govindaraju, V. A. Kumar / Tetrahedron 62 (2006) 2321–2330
2329
AZ0.1% TFA in H2O, BZ0.1% TFA in CH3CN/H2O 1:1
with flow rate 1 ml/min. The purities of the purified
3.11. Gel mobility shift assay
oligomers were found to be O98%.
The PNAs (13–18, Table 2) were individually mixed with
DNA 21 in 2:1 ratio (PNA strand, 0.4 mM and DNA 21,
0.2 mM) in water. The samples were lyophilized to dryness
and re-suspended in sodium phosphate buffer (10 mM,
pH 7.0, 10 ml) containing EDTA (0.1 mM). The samples
were annealed by heating to 85 8C for 5 min followed by
slow cooling to rt and refrigeration at 4 8C overnight. To
this, 10 ml of 40% sucrose in TBE buffer pH 8.0 was added
and the sample was loaded on the gel. Bromophenol blue
(BPB) was used as the tracer dye separately in an adjacent
well. Gel-electrophoresis was performed on a 15% non-
denaturing polyacrylamide gel (acrylamide/bis-acrylamide,
29:1) at constant power supply of 200 V and 10 mA, until
the BPB migrated to three-fourth of the gel length. During
electrophoresis the temperature was maintained at 10 8C.
The spots were visualized through UV shadowing by
illuminating the gel placed on a fluorescent silica gel
plate, GF254 using UV-light.
3.7. LC-TOF-MS
Purity and the integrity of all PNAs synthesized were
ascertained by HPLC/LC-TOF-MS mass spectrometry
employing electrospray ionization technique. Neat samples
were dissolved in methanol and are injected through HPLC
system. The TOF (time of flight) detector was used to
analyze the molecular ion peaks.
3.8. Binding stoichiometry
Eleven mixtures of PNA:DNA with different ratios to each
other such as 0:100, 10:90, 20:80, 30:70, 40:60, 50:50,
60:40, 70:30, 80:20, 90:10 and 100:0; all of the same total
strand concentration (2 mM) in sodium phosphate buffer
(100 mM NaCl, 0.1 mM EDTA, pH 7.0). The samples are
heated to 85 8C in water bath for 5 min, allowed to cool to rt
and then cooled further in a refrigerator overnight. CD
spectra for all the samples were recorded at 10 8C with
wavelength range from 350–190 nm with scan speed
100 nm/min, accumulation-8, response time-4 s, band
width-1 nm and sensitivity-10 mdeg. CD cell for all the
studies was of 10 mm path length. Baseline was subtracted
from all the CD spectra.
Acknowledgements
T.G. thanks CSIR, New Delhi for the award of a Research
fellowship and V.A.K. thanks Department of Science and
Technology, New Delhi for a research grant.
Supplementary data
3.9. UV–Tm measurements
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2005.12.
The concentration was calculated on the basis of absorbance
from the molar extinction coefficients of the corresponding
nucleobases (i.e., T, 8.8 cm2/mmol; C, 7.3 cm2/mmol; G,
11.7 cm2/mmol and A, 15.4 cm2/mmol). The complexes
were prepared in 10 mM sodium phosphate buffer, pH 7.0
containing NaCl (100 mM) and EDTA (0.1 mM) and were
annealed by keeping the samples at 85 8C for 5 min
followed by slow cooling to rt (annealing). Absorbance
versus temperature profiles were obtained by monitoring at
260 nm with Perkin-Elmer Lambda 35 UV–vis spectropho-
tometer scanning from 5 to 85/90 8C at a ramp rate of 0.2 8C
per min. The data were processed using Microcal Origin 5.0
and Tm values derived from the first derivative curves.
1
002. H and 13C NMR Spectra, Mass spectra of selected
compounds, HPLC profiles and LC-TOF-MS spectra of
bepPNAs and UV–Tm curves, CD curves.
References and notes
1. (a) Barrett, J. C.; Miller, P. S.; Ts’o, P. O. P. Biochemistry
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Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci.
U.S.A. 1978, 75, 285–289.
3.10. Circular dichroism
2. (a) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W.
Angew. Chem., Int. Ed. 1998, 37, 2796–2823. (b) Bennett, C. F.
In Applied Antisense Oligonucleotide Technology; Stein, C. A.,
Craig, A. M., Eds.; Wiley-Liss: New York, 1998. (c)
Braasch, D. A.; Corey, D. R. Biochemistry 2002, 41,
4503–4510. (d) Petersen, M.; Wengel, J. Trends Biotechnol.
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CD spectra were recorded on a JASCO J-715 spectro-
polarimeter. The CD spectra of the PNA:DNA complexes
and the relevant single strands were recorded in 10 mM
sodium phosphate buffer, 100 mM NaCl, 0.1 mM EDTA,
pH 7.0. The temperature of the circulating water was kept
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homothymine T8 single strands were recorded as an
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