BisPNA targeting to DNA: Effect of neutral loop on DNA duplex strand invasion by aepPNA-N7G/aepPNA-C substituted peptide nucleic acids

Article abstract of DOI:10.1002/ejoc.200500544

N7-Alkyl-substituted guanine (N7G) as a CH⁺ mimic has been introduced into aminoethylglycyl PNA (aegPNA) forming a hairpin through a neutral linker derived from bis(tetraethylene) glycol (teg-teg). These form pH-independent PNA₂:DNA

Full text of DOI:10.1002/ejoc.200500544

FULL PAPER
DOI: 10.1002/ejoc.200500544
BisPNA Targeting to DNA: Effect of Neutral Loop on DNA Duplex Strand
Invasion by aepPNA-N7G/aepPNA-C Substituted Peptide Nucleic Acids
[a]
[a]
[a]
Pravin S. Shirude, Vaijayanti A. Kumar,* and Krishna N. Ganesh*
Dedicated to Professor W. J. Stec on the occasion of his 65th birthday
Keywords: Peptide Nucleic Acid (PNA) / Hairpin bisPNA-DNA triplex / Strand invasion / aepPNA / N7G PNA
N7-Alkyl-substituted guanine (N7G) as a CH+ mimic has
been introduced into aminoethylglycyl PNA (aegPNA) form-
ing a hairpin through a neutral linker derived from bis(tetra-
ethylene) glycol (teg-teg). These form pH-independent
tive manner. Fluorescence assay was used to examine the
process of strand invasion of the target DNA duplex by the
modified hairpin bisPNAs with the neutral teg linker and
comparison of results of previous studies employing cationic
linkers suggested the triplex formation to be a two-step pro-
cess, with the preferred formation of HG bonds in the first
step.
2
PNA :DNA triplexes with complementary DNA sequences.
The introduction of chiral, cationic aminoethylprolyl (aep)
+
units with C and the CH mimic N7G in the backbone of the
hairpin bisPNAs with a neutral teg-teg linker, influenced the
recognition of complementary DNA in an orientation-selec-
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
The acyclic, neutral and achiral aminoethylglycyl (aeg)
In PNA :DNA triplexes, where the DNA strand is en-
2
PNAs hybridize to complementary oligonucleotides with gaged in simultaneous binding to two PNA strands, ambi-
high affinity and specificity through Watson–Crick base guity persists in establishing the specific correlation among
pairing and are true mimics in terms of base-pair recogni- the parallel/antiparallel PNA strands with the Hoogsteen
[
1]
tion. The unique properties of peptide nucleic acids (HG) or Watson–Crick (WC) binding modes (Figure 1)
[
2,3]
[16]
(PNAs)
as novel molecules designed to bind effectively with DNA.
In principle, PNA :DNA triplexes are pos-
2
with high sequence specificity to desired double-stranded sible with different combinations of parallel/antiparallel
DNA (dsDNA) targets through duplex invasion have at- strands that are not easily discernible.
tracted wide attention, warranting molecular characteriza-
tion of binding mechanisms. PNA can bind to complemen-
tary nucleic acids with Watson–Crick (WC) pairing in both
[4]
antiparallel (ap) and parallel (p) orientations, but with
slightly preferred antiparallel binding.[ Homopyrimidine
5]
oligomers such as PNA-C10 or PNA-(CT) invade double
5
stranded polynucleotides by forming PNA :DNA triplexes
2
in a pH-dependent reaction, owing to the necessity of pro-
tonation of N3 in cytosine in the third strand. Several
attempts have been made in the literature to imitate the
+
[6–15]
CH ---C hydrogen-bonding pattern by neutral mimics
2
Figure 1. H-bonding in PNA :DNA triplex: HG (Hoogsteen), WC
such as N7G (G glycosylated at N7), 8-oxo-adenine, pseu- (Watson–Crick).
doisocytosine (ψiC)[ and the J base to avoid the necessity
7]
of acidic pH for triplex formation by C-containing PNA
The ambiguity in PNA strand orientation for WC and
oligomers. Such systems exhibit pH-independent triplex HG binding with DNA in such mixed PNA :DNA com-
2
formation.
plexes can be partially resolved by conjugating the two
PNA strands with a linker to construct a bisPNA hairpin
that can form only two triplexes with a 1:1 (PNA:DNA)
[
a] Division of Organic Chemistry (Synthesis), National Chemical
[
13]
Laboratory,
composition
sibilities, we introduced the chiral aminoethylprolyl (aep)
(2S,4S)-aepPNA C and N7G] units into arm A, which is
linked to arm B of the bisPNA through a cationic peptide
(Figure 2). To identify each of these pos-
Pune 411008, India
E-mail: kn.ganesh@ncl.res.in, va.kumar@ncl.res.in
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
[
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P. S. Shirude, V. A. Kumar, K. N. Ganesh
FULL PAPER
linker.[8] This was hybridized to DNA sequences designed to study of hybridization preferences with complementary
specifically recognise arm A in either parallel or antiparallel DNA by UV-Tm at different pH values. The successful for-
mode. We found that bisPNAs consisting of chiral aepPNA- mation of complexes is supported by mobility retardation
[
7,8]
C or aepPNA-N7G in arm A, formed triplexes with higher in gel electrophoresis. A fluorescence assay
based on
stability when this arm was antiparallel to the DNA strand. ethidium bromide displacement was used to study the kinet-
As expected, the triplex formation was pH independent ics of the strand invasion of duplex DNA by different
with aepPNA-N7G, while pH dependent with the bisPNA bisPNAs, and this indicated that a two-step invasion pro-
aepPNA-C. The presence of lysines in the peptide linker cess occurs. The results, interestingly, suggest that the pre-
renders bisPNAs inherently cationic, which may disfavor ferred roles of arm A and arm B as WC or HG strands in
N3-protonation in aepPNA-C that is necessary for HG bisPNA:DNA complexes is directed by the nature of the X
bonding. The positive charges in the linker may also accel- substituent and the linker.
erate the PNA:DNA hybridization kinetics making analysis
of the relative contributing factors to triplex stability unam-
biguous.
Results and Discussion
Synthesis of PNA Monomers, Linker and BisPNA
Oligomers
The syntheses of protected aepPNA monomers 1-(N-
4
Boc-aminoethyl)-4(S)-(N -benzyloxycarbonyl cytosin-1-yl)-
2
4
(S)-proline methyl ester (1), and 1-(N-Boc-aminoethyl)-
(S)-(N -isobutyrylguanin-7-yl)-2(S)-proline methyl ester
2
(
2) were performed according to our previously reported
[
8]
procedures (Figure 4).
Figure 2. Triplex formation with bisPNAs carrying N7G as a neu-
tral C+ mimic.
In this report, we have replaced the charged peptide
[
8]
linker with a neutral linker (Figure 2) composed of two
tetraethylene glycol units to examine the true preferences in
hybridization of the two arms of PNA containing aepPNA-
C or aepPNA-N7G in parallel/antiparallel orientation to
the DNA strand (Figure 3). The hairpin PNAs (H-
TTTXTXT-teg-teg-TCTCTTT-β-alanine), containing the
neutral bis(tetraethylene) glycol (teg) linker along with
Figure 4. aeg-aepPNA monomeric units used in the synthesis of
bisPNAs.
The teg linker 11 suitable for on-line coupling in PNA
aepPNA-C or aepPNA-N7G in the X position, enable the synthesis was obtained according to Scheme 1. Tetraethyl-
Figure 3. Design of hairpin bisPNAs.
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BisPNA Targeting to DNA
FULL PAPER
3 2 2
Scheme 1. a) pTsCl, NaOH, THF, Water, 0 °C, 3 h. b) NaN , DMF, 60 °C, 5 h. c) Br–CH –COOEt, NaH, DMF, 16 h. d) Ra/Ni, H ,
2
40 psi, EtOAc, (Boc) O. e) aq. NaOH (1 ), MeOH.
ene glycol 6 was monotosylated[9] using p-toluenesulfonyl aegPNA-C, aegPNA-N7G, aepPNA-C or aepPNA-N7G
chloride followed by treatment with sodium azide in DMF monomers are added in X positions and the synthesis was
to obtain the azide alcohol 8. This was O-alkylated to the completed with other aegPNA monomers. The oligomers
azido ester 9 with ethyl bromoacetate using sodium hydride, were cleaved from the solid support using TFA-TFMSA[
followed by hydrogenation to yield an intermediate amino to yield PNAs with free “C” terminal carboxylic acids and
ester, which was protected in situ with t-Boc using di-tert- simultaneous deprotection of the benzyloxycarbonyl groups
10]
2
7
butyl dicarbonate to obtain 10. It was then hydrolyzed to on C. The N -isobutyryl groups of the N of guanine were
the desired acid 11 and all compounds were characterized removed by further treatment with aqueous ammonia at
by appropriate spectroscopic data.
55 °C or with an ethylenediamine/ethanol mixture for 20 h.
The hairpin bis PNA sequences (12–15) were designed to
The fully deprotected oligomers were desalted by size-
have aegPNA-C (12), aegPNA-N7G (13), aepPNA-C (14) exclusion chromatography over G25 Sephadex followed by
and aepPNA-N7G (15) present in the arm A of the hairpin purification using preparative reverse-phase HPLC on a C8
to bind DNA 16 in parallel and DNA 17 in antiparallel column. The purity of the oligomers was rechecked by ana-
orientations. Consequently, the unmodified arm B of the lytical reverse-phase HPLC on a C18 column and their
hairpin would bind DNA 16 and DNA 17 in opposite anti- identity confirmed by MALDI-TOF mass spectrometry
parallel and parallel orientations, respectively. The different (see Supporting Information; for details see the footnote on
PNA and DNA oligomers that were synthesized are listed the first page of this article).
in Table 1.
Table 1. Hairpin bisPNA and DNA oligomers.[a]
PNA Sequence composition
UV-Melting
The oligodeoxynucleotides 16 and 17, identical in se-
quence but reversed in a 5Ј-3Ј direction, were used to probe
the parallel/antiparallel binding preferences, studied by
thermal UV melting experiments at pH 5.8 and 7.4. The
hairpin bisPNA sequences 12–15 individually mixed with
the oligonucleotides 16 or 17 in equimolar concentrations
were annealed prior to melting. UV absorbances at 260 nm
for the different bisPNA:DNA complexes were recorded as
a function of temperature to generate percent hypochromic-
ity-temperature plots (Figure 5). The UV melting tempera-
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
H–TTTCTCT–teg-teg–TCTCTTT–NH–(CH
2
)
2
–COOH
H–TTT GT GT–teg-teg–TCTCTTT–NH–(CH –COOH
H–TTT c T c T–teg-teg–TCTCTTT–NH–(CH –COOH
–COOH
7
7
2 2
)
2 2
)
)
2 2
7
7
H–TTT gT gT–teg-teg–TCTCTTT–NH–(CH
5Ј-G C A A A G A G A C T-3Ј
5Ј-T C A G A G A A A C G-3Ј
5Ј-A G T C T C T T T G C-3Ј (complementary to 16)
5Ј-C G T T T C T C T G A-3Ј (complementary to 17)
7
7
[a] T/C/ G = aegPNA-T/C/N7G; C/ g = aepPNA-C/N7G, teg,
linker.
ture (T ) values at pH 5.8 and 7.4 were deduced from max-
m
The PNA oligomers were synthesized in a single as- ima in the first derivative plots and are given in Table 2.
sembly on Merrifield resin derivatized with β-alanine. Each The control bisPNA 12 having the aegPNA-C unit as X
coupling cycle involving successive deprotection, neutraliza- in arm A binds to either the parallel (p) DNA 16 or antipar-
tion and coupling steps was monitored for efficiency by allel (ap) DNA 17 (relative to arm A) at pH 7.4 with slightly
Kaiser’s test. Tetraethylene glycol linker 11 was coupled different affinities (21 and 27 °C, respectively) (Entry 1,
using the same procedure employing HBTU, HOBt and di- Table 2). At acidic pH 5.8, a higher Tm (29 and 42 °C) was
isopropylethylamine as the coupling agents. The oligomer seen with both DNA 16 and DNA 17 as expected from N3
synthesis was carried out starting from arm B of the hair- protonation of cytosine and Tm with DNA 17 was signifi-
pin, followed by the neutral linker and arm A in which cantly higher than that seen with DNA 16 [∆Tm = 8 °C].
Eur. J. Org. Chem. 2005, 5207–5215
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P. S. Shirude, V. A. Kumar, K. N. Ganesh
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Figure 5. UV-T
m
plots of the bisPNA:DNA complexes of (A) aegPNA-C 12, (B) aegPNA-N7G 13 (C) aepPNA-C 14 (D) aepPNA-N7G
15 with DNA 16 and DNA 17, at pH 5.8 (symbols: open squares, opens stars) and 7.4 (symbols: black filled squares, filled stars).
Table 2. UV-T
m
of bisPNA-DNA complexes.[a]
Entry
PNA
DNA 16 (p)
pH 7.4
DNA 17 (ap)
pH 7.4
pH 5.8
∆Tm(∆pH)
pH 5.8
∆Tm(∆pH)
1
2
3
4
12, aegPNA-C
21
32
23
38
29
29
49
30
+8
–3
+26
–8
27
44
42
41
42
41
52
44
+15
–3
+10
+3
13, aegPNA-N7G
14, aepPNA-C
15, aepPNA-N7G
[
a] UV-T
m
data (°C) for PNA:DNA complexes with DNA 16 and 17. Experiments were performed in 10 m sodium phosphate buffer,
values are accurate to ±1 °C and were obtained from peaks in the first derivative plots of percent hyperchromicity
pH 7.4 and 5.8. T
m
vs. temperature. Each experiment was repeated at least twice.
This protonation could be on the “C” located either in arm seen for binding of DNA 17 as compared to DNA 16 [∆Tm
A or arm B of bisPNA 12, with the protonated arm leading = 12 °C] (Figure 4, B). The T was almost pH-independent
m
to HG and the nonprotonated arm to WC H bonding. The with ∆T (∆pH) = –3 °C. The fact that N7G cannot bind
m
protonation of terminal, non-hydrogen bonded “C” in in the WC mode, rules out arm B containing C to act as an
DNA 16 and 17 does not affect the binding or recognition HG strand. Thus, although donor-acceptor H-bonding sites
event. Thus, in aegPNA 12, the two arms of bisPNA are of N7G recognize G in the HG binding mode in both p
distinguished in terms of binding in the WC or HG mode and ap orientations, the preferred binding mode is ap, with
at pH 5.8, but it is not possible to assign the protonated HG binding of arm A where N7G is located, leading to p
HG strand.
WC binding of arm B.
The replacement of aegPNA-C 12, at X in arm A by the
At an acidic pH, a slight destabilization was seen sug-
C+ mimic aegPNA-N7G (13) gave complexes of different gesting that N3 protonation of cytosine is not favored in
stabilities (Entry 2, Table 2) with the target DNA strands the arm B binding, in the parallel WC mode. In contrast to
16 and 17. At both pH values, a distinct preference was the achiral aegPNA units, introduction of a chiral aepPNA-
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BisPNA Targeting to DNA
FULL PAPER
C unit as X in arm A in bisPNA 14 leads to a significantly
stronger binding at pH 7.4 with DNA 17, as compared to
binding with DNA 16 [Table 2, Entry 3, ∆T = 19 °C]. This
m
suggests that chirality in aepPNA-C further influences the
binding orientation preference. At pH 5.8, the thermal sta-
bility was significantly enhanced in the case of both DNA
16 and DNA 17, resulting from the protonation of N3 of
C [∆T (∆pH) = 26 and 10 °C, respectively]. The additional
m
protonatable N on the pyrrolidine ring of aepPNA-C, with
a higher pK (6.8) than that of N3 of C (4.5), may perhaps
lead to the higher degree of stabilization seen at pH 5.8
a
Figure 6. Principle of fluorescence assay for strand invasion.
(Figure 4, C). Since aepPNA-C on arm A cannot carry a
positive charge simultaneously on both ring N and N3 of cence emission decay at 590 nm (λex = 490 nm) as a func-
C, the preferred mode of binding is arm A in ap WC and tion of time after individually adding the four PNAs 12–15
protonated aeg-C in arm B in p HG.
for over 60 hours. The emission intensities in each case
The bisPNA 15 containing the non-protonatable chiral showed an exponential decrease at different rates eventually
aepPNA-N7G unit as X in arm A exhibited a higher sta- reaching a plateau (Figure 7 and Figure 8). Interestingly,
bility of derived complexes at neutral pH 7.4 relative to the the decay profiles of polyoxyethylene bisPNAs exhibited bi-
chiral aepPNA-C 14. The bisPNA 15 also formed more phasic modes in contrast to the results from the cationic
[
8]
stable hybrids with DNA 17 than with DNA 16 at both pH peptide linker where the decay profile was clearly mon-
values, with lower dependence of pH on Tm for DNA 17, ophasic. In addition, the strand invasion also seems to go
similar to aegPNA-N7G 13 (Figure 5, D). This again sug- to completion. The present data on bisPNAs could be fitted
gested that arm A recognizes DNA 16 in parallel HG mode into an exponential decay of second-order. Table 3 shows
and 17 in antiparallel HG mode, with preferred binding in the two decay constants T and T computed from the data
1
2
the latter orientation. The higher Tm of aepPNA-N7G per- of Figure 7 and Figure 8.
haps arises from protonation of pyrrolidine ring N contrib-
uting to electrostatic stabilization. The overall results indi-
cate that hairpin bisPNAs bind to DNA 17 better than they
do to DNA 16.
Fluorescence: Strand Invasion Assay
The bisPNA oligomers 12–15 are appropriate for study-
ing the strand invasion properties by triplex formation when
targeted towards duplex DNA. Ethidium bromide upon in-
tercalation into a DNA duplex (16:18 or 17:19) shows an
increase in fluorescence intensity[ but is not known to in-
8]
[
8,11]
teract with PNA :DNA or PNA:PNA complexes.
2
Strand invasion of the duplex DNA saturated with ethid-
ium bromide by a triplex-forming PNA oligomer should
therefore cause a decrease in ethidium bromide fluorescence
due to the disruption of the DNA duplex-ethidium bromide
complexation. The loss of ethidium bromide fluorescence
as a function of time upon addition of triplex forming PNA
Figure 7. Ethidium bromide fluorescence as a function of time
studied for the DNA duplex 16:18 after the addition of PNA 12,
PNA 13, PNA 14 and PNA 15 at pH 7.4.
The data in Table 3 and Figure 7 and Figure 8 indicate
oligomers may be used to study the kinetics of duplex that step 1 is a faster process compared to step 2 in all cases.
strand invasion in this fluorescent intercalator displacement For duplex invasion leading to triplexation with DNA 16
assay (Figure 6).
(Figure 7, Table 3), step 1 is fastest with aegPNA-C 12
In our earlier studies with bisPNAs containing a cationic while slowest for aegPNA-N7G 13, with intermediate rates
[
8]
peptide linker we noticed that the strand invasion is for aepPNAs 14 and 15; step 2 is slowest in the case of
slower and incomplete in the case of aegPNA-N7G and aepPNA-C 14, with other PNAs having similar rates.
aepPNA at ambient temperature. These effects seem to arise
In the case of duplex invasion generating triplexes with
due to conformational distortions induced in the PNA DNA 17 (Figure 8, Table 3), step 1 is fastest with aepPNA-
backbone by the aep chiral monomer units and also the N7G 15 while slowest for aegPNA-N7G 13, with intermedi-
presence of a purine base in a pyrimidine rich strand.
ate rates for aegPNA-C 12 and 14. The decay kinetics fol-
The DNA duplexes 16:18 and 17:19 were separately satu- lows an order completely different from that seen according
rated with ethidium bromide and the kinetics of the strand to T . Thus the thermal stability of bisPNA:DNA duplexes
m
invasion process was examined by monitoring the fluores- seems to be disconnected from their kinetics of association/
Eur. J. Org. Chem. 2005, 5207–5215
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P. S. Shirude, V. A. Kumar, K. N. Ganesh
FULL PAPER
ation monitored by nondenaturing gel electrophoresis at
10 °C. Due to a higher binding affinity of PNA:DNA com-
plexes compared with DNA:DNA complexes, the added
PNA binds to the complementary DNA 16:17 in the DNA
triplex, releasing the DNA strand 18:19. This is clearly seen
in the gel electrophoresis in which the DNA duplex band is
converted into a bisPNA:DNA complex seen as a retarded
band and the released DNA strand is seen as a faster mov-
ing band (Figure 9, Lane 1–2, 4–5 and Lane 7–9). The re-
sults unambiguously prove that PNAs do competitively dis-
place one strand of a DNA duplex to form a complex with
the other complementary strand. The mobility difference
seen for ds- and ssDNA is highlighted in the supplementary
information.
Figure 8. Ethidium bromide fluorescence as a function of time
studied for the DNA duplex 17:19 after the addition of PNA 12,
PNA 13, PNA 14 and PNA 15 at pH 7.4.
Discussion
Entropic enhancement of strand invasion efficiency is
possible by use of PNA clamps or bisPNAs in which two
homopyrimidine PNA oligomers are covalently connected
by a flexible loop.
reduces a trimolecular reaction of 2×PNA and 1×DNA to
Table 3. Decay constants for duplex invasion by bisPNAs.[a]
[
12,13]
Such linking of two PNA oligomers
DNA
PNA
16:18
17:19
T
1
T
2
T
1
T
2
a PNA-DNA bimolecular reaction, accelerating the PNA
1
1
1
1
2
3
4
5
0.43
3.23
1.18
1.85
10.64
11.89
28.79
10.63
1.36
3.34
0.74
0.23
14.89
17.62
17.88
14.86
[
14]
invasion.
PNA strand invasion of dsDNA has been
shown to be accelerated by incorporation of positive
[15]
charges into PNA or bisPNA oligomers.
The use of
[
a] Decay constants obtained from Figures 7 and 8 where results bisPNAs wherein the Watson–Crick PNA strand is con-
were subjected to non-linear curve fitting: exponential decay 2 in
Microcal, origin 6.1 (T , T given in hours).
nected through ethylene glycol linkers to the Hoogsteen
1
2
[
10,13]
PNA strand
offers interesting possibilities of incorpo-
rating neutral mimitics into either of the PNA segments to
examine the relative orientations of the WC and HG pairing
strands. It has been found that the selectivity in PNA:DNA
complex formation is orientation-directed with the Watson–
Crick strand in the antiparallel configuration relative to
DNA and the Hoogsteen strand in the parallel configura-
tion.
dissociation. The faster rates of invasion seen in cationic
[
8]
linker bisPNA may presumably be partly due to the elec-
trostatic effects from the linker, which repels the positively
charged ethidium bromide. The triplex formation with
bisPNA was found to be a one-step process. The DNA du-
plex invasion by polyoxyethylene-linked bisPNA occurs in
two steps that can be kinetically delineated in contrast to
a single-step process observed for cationic peptide linked
bisPNA.
N7-Glycosylated guanine is a positional isomer of the
naturally occurring N9-guanine and recognises the target
G:C base pair occurring in major grooves through biden-
tate hydrogen bonding. Being neutral, it surpasses the
[
16–18]
requirement of acidic pH for triplexation.
N7G can
Gel Shift Assays: Competition Binding Experiments
mimic protonated cytosine in the pyrimidine motif forming
Electrophoretic gel shift assay was used to establish the stable triplexes at neutral pH with the recognition of C only
formation of bisPNA:DNA complexes. The electrophoretic being through the HG mode in either parallel and antipar-
[
13]
mobility of a DNA fragment in a native polyacrylamide gel allel orientations
is retarded upon complexation with PNA and hence can be nised in the WC binding mode. Thus incorporation of N7G
used to study the strand. units in PNA stabilizes the triplex only when engaged in the
The PNAs were individually treated with dsDNA and HG mode unlike C or J bases.
(Figure 10). However, G is not recog-
the complexation was monitored by nondenaturing gel elec-
UV-Tm studies suggest that Tm for the interaction of
trophoresis at 10 °C. The spots were visualized on a fluores- bisPNA-C (12 and 14) with DNA depends significantly on
cent TLC background. The formation of PNA:DNA tri- pH, while this is relatively insignificant for PNA-N7G (13
plexes is accompanied by displacement of one strand of and 15). The pH-dependent Tm clearly arises from the ne-
dsDNA and the appearance of a lower migrating band due cessity for N3C protonation, which should occur in arm A
to PNA:DNA complexes.
of bisPNA-C complexes of DNA 16 and arm B for bisPNA
Figure 9 shows the results of competition binding experi- complexes of DNA 17. In bisPNAs of N7G, the arm A
ments carried out by adding hairpin bisPNAs to the DNA always binds in the HG mode, since no pH-dependent Tm
duplex (16:18 or 17:19) followed by annealing and complex- was observed.
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BisPNA Targeting to DNA
FULL PAPER
Figure 9. 15% Polyacrylamide gel electrophoresis of PNA:DNA complexes.
Figure 10. N7G third strand binding in the parallel and antiparallel HG mode.
The pK ’s of cytosine N3 in aegPNA-C (3.6) and formation of a PNA:DNA Watson–Crick duplex (WC first
a
aepPNA-C (3.47) were not very different, though slightly mechanism).
lower than N3–C in cytidine (4.34). The pK of the pyrroli-
The second mechanism envisages the formation of an un-
a
dine ring is 6.7 and thus in the aepPNA-C, the protonation stable PNA:DNA triplex through Hoogsteen pairing (HG
2
of N3 of C is electrostatically unfavored due to the prior first mechanism), followed by dissociation of the duplex to
protonation of the pyrrolidine ring nitrogen (pK = 6.7). form a PNA :DNA triplex. The final outcome is the same
a
2
Thus, it is likely that C in arm B is protonated leading to for both mechanisms and the unstable intermediates are dif-
HG binding. The overall results suggest that N7G in the X ficult to detect. By using pH-dependent gel mobility shift
[
21]
position favors arm A binding in the HG mode while aep- analysis, it was demonstrated that at neutral pH, C-con-
PNA C in the X position favors arm A in the WC mode of taining PNAs undergo strand invasion by the HG first
binding.
mechanism (Figure 11).
In the literature, two plausible routes for the strand in-
Our present results based on the fluorescence assay give
vasion reaction have been postulated[
19–21]
According to direct and strong support for a two-step process, as seen by
one mechanism (Figure 11), the first stage consists of a fluc- the observance of biexponential decay. The initial step is a
tuating opening of the DNA double helix and a transient kinetically faster process over 0.2–3.4 h, while the second
Figure 11. Proposed Hoogsteen-first mechanism in strand invasion of homopyrimidine bisPNAs into dsDNA.[21]
Eur. J. Org. Chem. 2005, 5207–5215
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5213

P. S. Shirude, V. A. Kumar, K. N. Ganesh
FULL PAPER
step is slower over a subsequent 10.6 to 28.7 h. Compared Experimental Section
to aegPNA 12, the aepPNAs 14 and 15 show a T that is
1
2
[8]
N -Isobutyrylguanine,
(
(
1-(N-Boc-aminoethyl)-4-(R)-hydroxy-2-
three times lower, perhaps due to the presence of the rigid
pyrrolidine ring as part of the backbone, which sterically
slows down the initial binding of the PNA to DNA duplex.
The PNA 13 having aegPNA-N7G was the slowest, perhaps
due to additional energy barriers in the interconversion of ester,[ were prepared according to literature procedures. The syn-
syn-anti rotamers in attaining hybridization competent con- thesis of the aegPNA-T/C monomers was carried out according to
formations. The aep-PNAs 14 and 15 having the base di- the procedures described earlier.
S)-proline methyl ester,[ 1-(N-Boc-aminoethyl)-4-(R)-O-mesyl-2-
17]
S)-proline methyl ester,[ 1-(N-Boc-aminoethyl)-4-(S)-(N -benzyl-
8]
4
[8]
oxycarbonyl-cytosin-1-yl)-2-(S)-proline methyl ester, 1-(N-Boc-
aminoethyl)-4-(S)-(N -isobutyrylguanin-7-yl)-2-(S)-proline methyl
2
8]
rectly linked to the pyrrolidine rings lack such rotamers.
The first step in the case of aep-bisPNA-N7G was quite fast
2
-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethyl 4-Methylbenzenesul-
[9]
fonate(7): A solution of sodium hydroxide (2.74 g, 68.5 mmol) in
and the second step of base pair formation was inordinately water (15 mL) was added to a mixture of tetraethylene glycol (6)
slow for aep-bisPNA-C 14, for which, the reasons are not (87.8 g, 452 mmol) and THF (15 mL). After the mixture had co-
obvious from present experiments.
In the first step both DNA duplexes 16:18 and 17:19
should form transient HG pairing with arm A of bisPNAs
oled down to 0 °C, a solution of p-toluenesulfonyl chloride (8.3 g,
3.7 mmol) in THF (50 mL) was added while stirring for 1 h. After
stirring at 0 °C for 2 h, the reaction mixture was poured into ice
water (250 mL). The organic layer was separated, and the aqueous
2 2
layer was extracted with CH Cl (3×100 mL). The combined or-
4
12–15, while the second step involves formation of WC
pairing. For the duplex invasion of 17:19 by bisPNAs,
which also leads to more stable hybrids, it was noticed that
the aepPNAs exhibit faster kinetics for the first step. The
first HG mechanism gives rise to antiparallel binding with
ganic layers were washed twice with water (50 mL). Drying of the
organic layer and evaporation of the solvent under reduced pres-
sure afforded (13.74 g, 90% yield with reference to tosyl chloride)
1
of clear oil 7. H NMR (CDCl
3
): δ = 7.86–7.76 (d, 2 H), 7.35–
the DNA duplex, which is more favorable than the relatively 7.31 (d, 2 H), 4.17–4.12 (m, 2 H), 3.70–3.58 (m, 14 H), 2.43 (s, 3
slower HG parallel binding seen for the 16:18 DNA duplex. H) ppm.
For aeg-bisPNA, the first HG binding was faster in the par-
2
-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethanol (8): Monotosylate 7
allel mode than the antiparallel. Thus our results seem to
delineate the invasion process by parallel/antiparallel bind-
ing, with the latter being more favorable.
(
(
5 g, 14.4 mmol) in dry DMF (50 mL) was added to sodium azide
4.67 g, 71.85 mmol). The reaction mixture was heated at 60 °C
while stirring for 5 h. The solvent was removed under vacuum; the
residue was placed in water and extracted with CH Cl (3×50 mL).
2
2
The pooled organic extracts were washed with water and brine.
Drying of the organic layer and evaporation of the solvent under
1
reduced pressure afforded azide 8 (2.70 g, 87% yield). H NMR
Conclusions
1
3
(
CDCl
NMR (CDCl
IR (CHCl ): 2106 cm and others.
3
): δ = 3.69–3.46 (m, 14 H), 3.39–3.36 (t, 2 H) ppm.
C
3
): δ = 71.41, 69.43, 68.77, 60.28, 59.76, 49.47 ppm.
The N7G nucleobase and neutral teg linker has been suc-
cessfully incorporated into PNA using simple chemistry
and solid-phase peptide synthesis. It has proved to be a
–
1
3
Ethyl 14-Azido-3,6,9,12-tetraoxatetradecan-1-oate (9): A solution
of azide 8 (3.50 g, 16 mmol) in dry DMF (5 mL) was added to a
cooled solution of sodium hydride (0.42 g, 17.58 mmol) in dry
DMF (10 mL). The reaction mixture was stirred for 30 min and
then ethyl bromoacetate (1.96 mL, 17.5 mmol) was added dropwise,
at 0 °C. The reaction mixture was stirred for 16 h at room tempera-
ture. The reaction mixture was quenched with ice water and solvent
was removed under vacuum; the residue was placed in water and
extracted with ethyl acetate (3×50 mL). The pooled organic ex-
+
good C mimetic at neutral and acidic pH, with differences
in T being observed for DNA 16 and DNA 17. The overall
m
results suggest that bisPNAs bind better to DNA 17 than
DNA 16. Thus, triplex formation is pH dependent when
aeg/aepPNA-C units are used, while aeg/aepPNA-N7G
units permit triplex formation at physiological pH as well
as at acidic pH.
In addition, the introduction of aepPNA C units into tracts were washed with water and brine. Drying of the organic
the backbone of the hairpin bisPNAs that form triplexes, layer and evaporation of the solvent gave crude oil. Purification
1
using column chromatography afforded 9 (2.0 g, 44% yield).
NMR (CDCl
H
strongly influences the recognition of DNA in an orienta-
tion-selective manner. The more stable complexes were
formed when the arm A was antiparallel to the central
3
): δ = 4.26–4.12 (m, 4 H), 3.69–3.46 (m, 14 H), 3.39–
3.36 (m, 2 H), 1.29–1.21 (t, 3 H) ppm.
strand. In summary (i) the aegPNA HG strand prefers to Ethyl 14-tert-Butoxycarbonylamino-3,6,9,12-tetraoxatetradecan-1-
be ap to the DNA strand (ii) the effect is more pronounced oate (10): Compound 9 (2 g, 6.6 mmol) was placed in dry ethyl
acetate (2 mL) in a 250 mL hydrogenation flask and di-tert-butyl
dicarbonate (1.57 g, 7.2 mmol) was added to it. Raney Ni (1 mL,
when X is the chiral aepPNA unit (iii) aepPNA C has much
a higher UV-Tm at acidic pH values due to protonation of
suspension in ethanol) was separately washed thoroughly with ethyl
the pyrrolidine ring nitrogen in the WC mode of binding
acetate and was added to the hydrogenation flask. It was subjected
and (iv) the triplex formation by bisPNAs is a two-step pro-
to hydrogenation at 40-psi pressure for 1.5 h. The catalyst was fil-
cess which is discernible when the cationic linker is replaced
by a neutral teg-teg linker. The topological issues of speci-
tered and washed with methanol/ethyl acetate mixture. The filtrate
was concentrated under vacuum to give the crude protected amine.
ficity in PNA :DNA triplex formation in bisPNA are thus
2
Purification using column chromatography afforded 10 (1.5 g, 50%
1
resolved by employing the chiral protonatable unit yield). H NMR (CDCl ): δ = 5.20 (br. s, 1 H), 4.24–4.09 (m, 4 H),
3
aepPNA-C and the protonated C mimic aepPNA-N7G.
3.69–3.46 (m, 14 H), 3.27–3.24 (m, 2 H), 1.38 (s, 9 H), 1.26–1.19
5214
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 5207–5215

BisPNA Targeting to DNA
FULL PAPER
(
7
t, 3 H) ppm. 13C NMR (CDCl
0.20, 69.87, 68.33, 60.42, 40.10, 28.08, 13.85 ppm. MS: (ESI) 379
3
): δ = 170.11, 155.74, 78.69, 70.53,
Acknowledgments
+
+
(M) , 279 (M – Boc).
P.S.S. thanks CSIR, New Delhi for the award of a senior research
fellowship. V.A.K. acknowledges DST, New Delhi, for a research
grant. K.N.G. is an Honorary Professor at Jawaharlal Nehru Cen-
tre for Advanced Scientific Research, Bangalore.
1
4-tert-Butoxycarbonylamino-3,6,9,12-tetraoxatetradecan-1-oic
Acid (11): Aqueous 2  NaOH (2 mL) was added to a solution
of Ethyl 14-tert-butoxycarbonylamino-3,6,9,12-tetraoxatetradecan-
1
1
-oate 10 (0.4 g, 1.0 mmol) in methanol (2 mL). TLC analysis after
0 min indicated the absence of the starting material as a result
of hydrolysis of the methyl ester function. The excess NaOH was
neutralized by Dowex-50 H resin, which was then filtered off. The
methanol from the filtrate was removed under vacuum and concen-
[
1] M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M.
Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, P. E.
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2] D. R. Corey, Trends Biotechnol. 1997, 15, 224–229.
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7, 431–437.
+
trated it to dryness to obtain the product 11 (0.35 g, quantitative
[
[
1
yield) as a brown solid. H NMR (CDCl
3
): δ = 6.00 (br. s, 1 H),
4
.11 (s, 2 H), 3.69–3.46 (m, 14 H), 3.34–3.24 (m, 2 H), 1.39 (s, 9
13
H) ppm. C NMR (CDCl
7
3
): δ = 170.11, 155.74, 79.29, 71.18, [4] ap: PNA N-terminus towards the 3Ј-end and C-terminus
+
0.56, 70.27, 69.11, 40.35, 28.39 ppm. MS: (ESI) 352 (M) H, 252
towards the 5Ј-end of cDNA/RNA); p, PNA N-terminus
towards the 5Ј-end and C-terminus towards the 3Ј-end of
cDNA/RNA).
+
(M H – Boc).
UV-Melting: The concentration of the PNA oligomers was calcu-
lated on the basis of the absorption at 260 nm, assuming the molar
extinction coefficients of the nucleobases to be as in DNA, i.e., T,
[
[
[
5] M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Beg, J. Am.
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Chem. Soc. 2002, 124, 2148–2152.
–
1
–1
–1
–1
–1
–1
8
1
.8 L·cm ·mol ; C, 7.3 L·cm ·mol ; G, 11.7 L·cm ·mol and A,
–
1
–1
5.4 L·cm ·mol . The hairpin PNA oligomers (12–15) and the rel-
evant complementary DNA oligonucleotide (16/17) were mixed to-
gether in a 1:1 molar ratio in 0.01  sodium phosphate buffer, pH
[8] M. DЈCosta, V. A. Kumar, K. N. Ganesh, J. Org. Chem. 2003,
5.8 or 7.4 to obtain a final strand concentration of 1 µ. The sam-
68, 4439–4445.
ples were annealed by heating at 85 °C for 1–2 min, followed by
slow cooling to room temperature. They were kept at room tem-
perature for 30 min and then, refrigerated overnight. UV experi-
ments were performed with a Perkin–Elmer λ35 UV/Vis spectro-
photometer fitted with a Peltier temperature programmer. The
samples were heated at a rate of 0.2 °C per minute and the ab-
sorbance was recorded at every minute at 260 nm. The percent hy-
perchromicity at 260 nm was plotted as a function of temperature
and the melting temperature was deduced from the peak in the first
derivative plots.
[9] M. Smet, W. Dehaen, Molecules 2000, 5, 620–628.
[10] R. S. Hodges, R. B. Merrifield, Anal. Biochem. 1975, 65, 241–
272.
[11] W. C. Tse, D. L. Boger, Acc. Chem. Res. 2004, 37, 61–69.
[
12] M. Egholm, L. Christensen, K. L. Dueholm, O. Buchardt, J.
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13] M. C. Griffith, L. M. Risen, M. J. Greig, E. A. Lesnik, K. G.
Sprankle, R. H. Griffey, J. S. Kiely, S. M. Freier, J. Am. Chem.
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[
[14] H. Kuhn, V. V. Demidov, M. D. Frank-Kamenetskii, P. E. Niel-
sen, Nucleic Acids Res. 1998, 26, 582–587.
[15] A. G. Veselkov, V. V. Demidov, P. E. Nielsen, M. D. Frank-Ka-
Fluorescence Assay for Strand Invasion: Fluorescence measurements
were performed with a Perkin–Elmer model LS-50B spectrometer
attached to a Julabo water bath circulator for variable temperature.
The DNA duplex 16:18/17:19 (1 µ) in a 5 m sodium phosphate
buffer, pH 7.4 at 20 °C was saturated with ethidium bromide
menetskii, Nucleic Acids Res. 1996, 24, 2483–2488.
[
[
[
16] T. S. Rao, R. H. Durland, G. R. Revankar, J. Heterocycl.
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17] J. Hunziker, E. S. Priestley, H. Brunar, P. B. Dervan, J. Am.
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(0.5 µ) and then excited at 490 nm and the emission monitored at
18] H. Brunar, P. B. Dervan, Nucleic Acids Res. 1996, 24, 1987–
590 nm using a spectral bandwidth of 5 nm. The kinetics of the
1991.
strand invasion process was then examined by monitoring the fluo-
rescence decay at 590 nm as a function of time after individually
adding the four bisPNAs 12–15 (10 µ) for over 60 h.
[19] S. T. Crooke, B. Lebleu, Antisense Research and Applications,
CRC Press, Boca Raton, FL, 1993.
[20] V. V. Demidov, M. V. Yavnilovich, B. P. Belotserkovskii, M. D.
Frank-Kamenetskii, P. E. Nielsen, Proc. Natl. Acad. Sci. USA
Supporting Information Available (see also footnote on the first
page of this article): (1) HPLC profiles of PNA oligomers 12–15.
1
995, 92, 2637–2641.
[
21] H. Kuhn, V. V. Demidov, P. E. Nielsen, M. D. Frank-Kamenet-
skii, J. Mol. Biol. 1999, 286, 1337–1345.
Received: July 20, 2005
(2) Mass spectra of PNAs 12–15 (3) NMR spectra of compounds
7–11. (4) Mass spectra of compounds 2, 5, 10–11. (5) Gel electro-
phoresis of DNA complexes.
Published Online: October 13, 2005
Eur. J. Org. Chem. 2005, 5207–5215
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5215