Aminomethylene peptide nucleic acid (am -PNA): Synthesis, regio-/stereospecific DNA binding, and differential cell uptake of (α/γ, R / S) am- PNA analogues

Article abstract of DOI:10.1021/jo300860f

Inherently chiral, cationic am-PNAs having pendant aminomethylene groups at α(R/S) or γ(S) sites on PNA backbone have been synthesized. The modified PNAs are shown to stabilize duplexes with complementary cDNA in a regio- and stereo-preferred manner with γ(S)-am PNA superior to α(R/S)-am PNAs and α(R)-am PNA better than the α(S) isomer. The enhanced stabilization of am-PNA:DNA duplexes is accompanied by a greater discrimination of mismatched bases. This seems to be a combined result of both electrostatic interactions and conformational preorganization of backbone favoring the cDNA binding. The am-PNAs are demonstrated to effectively traverse the cell membrane, localize in the nucleus of HeLa cells, and exhibit low toxicity to cells.

Full text of DOI:10.1021/jo300860f

Article
pubs.acs.org/joc
Aminomethylene Peptide Nucleic Acid (am-PNA): Synthesis, Regio-/
Stereospecific DNA Binding, And Differential Cell Uptake of (α/γ,R/
S)am-PNA Analogues
Roopa Mitra† and Krishna N. Ganesh*,†,‡
†Organic Chemistry Division, National Chemical Laboratory, Pune 411008, India
‡Indian Institute of Science Education and Research, 900, NCL Innovation Park, Dr Homi Bhabha Road, Pune 411008, India
S
* Supporting Information
ABSTRACT: Inherently chiral, cationic am-PNAs having
pendant aminomethylene groups at α(R/S) or γ(S) sites on
PNA backbone have been synthesized. The modified PNAs are
shown to stabilize duplexes with complementary cDNA in a
regio- and stereo-preferred manner with γ(S)-am PNA
superior to α(R/S)-am PNAs and α(R)-am PNA better than
the α(S) isomer. The enhanced stabilization of am-PNA:DNA
duplexes is accompanied by a greater discrimination of
mismatched bases. This seems to be a combined result of
both electrostatic interactions and conformational preorgani-
zation of backbone favoring the cDNA binding. The am-PNAs are demonstrated to effectively traverse the cell membrane,
localize in the nucleus of HeLa cells, and exhibit low toxicity to cells.
aqueous solubility, ambiguity in directional (parallel/antipar-
allel) selectivity of binding cDNA/RNA, and refractory for
taking up by mammalian cells.7 Several modified PNA
analogues have been rationally designed and synthesized in
order to overcome some of the limitations.8−10
INTRODUCTION
■
Peptide nucleic acids (PNAs) are a promising class of
oligonucleotide analogues with great potential for gene
therapeutic applications.1 They are effective DNA mimics
with a structurally homomorphous pseudopeptide backbone
composed of achiral N-(2-aminoethyl)glycine (aeg) units to
which a nucleobase (A, C, T, or G) is attached via a methylene
carbonyl linker (Figure 1a).2 The neutral backbone of PNA
As compared to many negatively charged analogues, neutral
aeg PNA hybridizes to cDNA/RNA targets with a greater
affinity and without loss of specificity.11 Incorporation of
positive charge in PNA variants through incorporation of N-(2-
aminoethyl)-D-lysine units has shown some advantages in terms
of improved solubility and efficient binding to cDNA.9,10 The
crystal structure of a PNA−DNA decamer heteroduplex,
comprising three residues of aminoethyl-D-lysine on the PNA
backbone, has indicated that the presence of chirality may
conformationally tune the PNA backbone by influencing the
helical direction and thereby improving the recognition
specificity of the cDNA, and the positive charge enhances the
solubility and bioavailability to bind the DNA targets.12,13
Recently, promising evidence was seen with cationic
guanidinium PNAs (GPNA) of appropriate backbone stereo-
chemistry that bind sequence specifically to RNA and traverse
the cell membrane readily.14 The X-ray structure of D-lysine-
based PNA revealed that the oxygen atom of the backbone
>CO group is within a 3.17 0.1 Å vicinity of the γ-carbon
of D-lysine side chain.15 This suggested that a shortening of the
aliphatic chain by removal of the three methylene carbons may
permit a direct interaction of side chain NH2 group with the
Figure 1. Chemical structures of (a) aeg PNA, (b) and (c) α-am, and
(d) γ-am PNA.
offers an advantage for hybridizing the negatively charged
cDNA and RNA with high affinity mediated by Watson−Crick
base pairing. The unnatural polyamide linkage makes it
resistant to degradation by both proteases and nucleases.3
The simple structure in combination with its impressive
properties and ease of synthesis has made PNA attractive as a
putative gene regulatory agent.4−6 However, in order to be used
as an efficacious antigene or antisense drug, it must be able to
traverse efficiently into the cells. Despite its remarkable
properties, the major challenges that aeg PNA face are its low
Received: April 29, 2012
Published: June 7, 2012
© 2012 American Chemical Society
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carbonyl oxygen of amide backbone of PNA. Quantum-
mechanical studies by Topham et al.16 supported such an
idea of the hydrogen bonding of the protonated side chain
methylene amino group with the carbonyl of the polyamide
backbone. Based on this hypothesis, we showed in a recent
communication that synthetic PNA constructs with a chiral
backbone bearing cationic aminomethylene (am) side-chain
substitution (Figure 1b−d) exhibit improved cDNA hybrid-
ization properties.17 Herein we report the detailed synthesis
and hybridization studies of mono- and disubstituted amino-
ethyl-α-(S-aminomethylene)glycine PNA [α-(S-am) PNA]
(Figure 1b) and aminoethyl- α-(R-aminomethylene)glycine
PNA [α-(R-am) PNA] (Figure 1c). We observe a stereo-
preferred enhancement in the binding of α-(R-am) PNA with
cDNA and higher cell permeability of am-PNAs accompanied
by a low toxicity for cells. The γ-position is a hot spot for
modifications as shown recently in γ-substituted L-lysine PNAs,
which had favorable DNA hybridization properties.18−21 The
presence of γ-(S)-substituent (derived from appropriate L-
amino acids) induces conformational preorganization of
randomly folded PNA into a right handed helix.22−24 In this
context, we also report the synthesis, biophysical, and cell
uptake studies of γ-(S-aminomethylene)aminoethylglycyl PNA
[γ-(S-am) PNA] (Figure 1d), which was found to be superior
to α-(R/S-am) PNA analogues.
8b were determined by 19F NMR of the corresponding Mosher
esters,28,29 and 8a/8b were used for the solid-phase synthesis of
(α)-(R/S-am) PNA oligomers P2−P7.
Similarly, the synthesis of γ-(S-am) PNA-T monomer 16
(Scheme 2) was attempted starting from compound 4a, which
was reduced to the corresponding alcohol 9 followed by
conversion to O-mesyl derivative 10. This was treated with
NaN3 to obtain the azido derivative 11 that was hydrogenated
to the amine 12. It was alkylated with ethyl bromoacetate to 13,
followed by N-acylation with chloroacetyl chloride to 14 and
subsequent reaction with thymine to obtain the ester 15. The
hydrolysis of the ester with LiOH afforded the γ-(S-am) PNA-T
monomer 16 that was used for the synthesis of γ-(S-am) PNA
oligomers P8−P10. All intermediate compounds and the final
1
monomers were characterized by H, 13C NMR and mass
spectroscopic data (see the Supporting Information).
Synthesis of am-PNA Oligomers. The modified am-
PNA-T monomers (8a, 8b, and 16) were incorporated at
predefined sites within the aeg PNA mixed-oligomer sequence
P1 on the solid phase using Boc chemistry on L-lysine-
derivatized MBHA resin using the standard protocol. The
synthesis was carried out using HOBt−HBTU−DIPEA as
coupling reagent. The deprotection of the Cbz protecting
group on the side-chain amine was achieved synchronously
during the final step of cleavage of the peptide from the resin
using TFA−TFMSA. The different mixed purine−pyrimidine
decamers that were synthesized are listed in Table 1 with the
site of incorporation of modified T-monomers denoted by tα(S)
(P2−P4), tα(R) (P5−P7), and tγ (P8−P10). The fluorescent
PNA analogues (cf P1, cf P4, cfP7, and cf P10) required for
monitoring the cell uptake were synthesized by coupling the
corresponding PNA oligomers on the solid phase at the N-
terminus with 5/6-carboxyfluorescein succinimidyl ester before
cleavage from the resin. All PNA oligomers were purified by
reverse-phase HPLC, and their final purity was checked on
analytical RP-HPLC (C-18). The integrity of all PNA
oligomers was confirmed by MALDI-TOF data (see the
Supporting Information).
RESULTS AND DISCUSSION
■
Synthesis of am-PNA Monomers. The synthesis of
designed PNA-T monomers, α-(S/R-am) (8a,b) and γ-(S-
am) (16), was performed starting with the conversion of N-
Boc-asparagine 1 to Nα-Boc-diaminopropionic acid 2 via
Curtius rearrangement,25,26 followed by orthogonal protection
of the side chain amine with Cbz (Scheme 1). The acid was
Scheme 1. Synthesis of α-am-PNA Monomers
Stoichiometry of am-PNA:DNA Hybrids. Although the
α-(R/S-am) PNAs have a chiral-center, single-stranded PNA
with even the double modification did not show any
appreciable CD signals (Figure 2). In contrast, the doubly
modified γ-(S-am) PNA (P10) exhibited negative CD signal at
273 nm suggesting a base-stacked helical preorganization
present in the γ-(S-am)-PNA oligomers. However, upon
binding with cDNA, all PNAs show strong CD signals whose
intensity can be used for monitoring the strength of binding.
The binding stoichiometry of am-PNA:DNA complexation was
established by a Job’s plot using CD data (ellipticity at 262 nm)
and confirmed by UV data (absorbance at 260 nm) to be 1:1
(Supporting Information).
PNA:DNA Hybridization Studies. To assess the impact of
grafting the aminomethylene group on the PNA backbone on
the stability of PNA:DNA duplexes, the synthesized am-PNAs
(P1−P10) were individually annealed with the cDNAs [DNA1,
antiparallel duplex, PNA (N→ C):DNA1 (3′−5′) and DNA2,
parallel duplex, PNA (N →C):DNA2 (5′−3′)], and the thermal
stability Tm of the derived PNA:DNA duplexes was obtained by
temperature-dependent UV-absorbance measurements. The
results shown in Table 2 indicate that the cationic am-PNAs
(P2−P10) formed much more stable duplexes than the
unmodified PNA P1, with the binding affinity dependent on
the position, stereochemistry, and the number of modifications.
converted into its ester 4, which became the common precursor
for the synthesis of all target monomers. In order to synthesize
α-(S/R-am) PNA-T monomers 8a and 8b, the N1-Boc group
in compound 4 was deprotected and the resultant amine was
reacted with N-Boc-aminoacetaldehyde27 to a Schiff base which
was in situ reduced with NaBH3CN and acetic acid. The
resulting N-protected S-(am) (5a) and R-(am) (5b) having a
side-chain substituent at the α-carbon were obtained in good
yields. These were acylated with chloroacetyl chloride to obtain
6a and 6b, respectively, which were used for alkylation of
thymine at N-1 to yield the corresponding esters 7a and 7b in
quantitative yields. The esters on hydrolysis gave the target
PNA-T monomers N-Boc-α-(S-am) 8a and N-Boc-α-(R-am)
8b, respectively. The optical purities of the monomers 8a and
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Scheme 2. Synthesis of γ-am-PNA Monomer
a
Table 1. PNA Oligomers Synthesized along with Their Molecular Weight
S. no.
oligomers
aeg PNA
sequence
calcd MW
obsd MW
2805.06
P1
H-TTACCTCAGT-LysNH2
H-TTACCTCAG tα(S)-LysNH2
H-TTACCtα(S)CAGT-LysNH2
2805.74
2834.78
2834.78
2863.82
2834.78
2834.78
2863.82
2834.78
2834.78
2863.82
3166.06
3224.14
3224.14
3224.14
P2
α-(S-am) PNA
α-(S-am) PNA
α-(S-am) PNA
α-(R-am) PNA
α-(R-am) PNA
α-(R-am) PNA
γ-(S-am) PNA
γ-(S-am) PNA
γ-(S-am) PNA
cf aeg PNA
2834.78
2831.17
2884.33 [M + Na+]
P3
P4
H-Ttα(S)A CCtα(S)C AGT-Lys NH2
H-TTACCTCAGtα(R)-LysNH2
H-TTACCtα(R)CAGT-LysNH2
H-Ttα(R)ACCtα(R)C AGT-LysNH2
H-TTACCTCAGtγ-LysNH2
P5
2833.01
P6
2834.20
2885.18 [M + Na+]
P7
P8
2834.21
P9
H-TTACCtγCAGT-LysNH2
2830.85
P10
cf P1
cf P4
cf P7
cf P10
H-TtγACCtγCA GT-LysNH2
H-cf-TTACCTCAGT-LysNH2
H-cf-Ttα(S)ACCtα(S)CAGT-LysNH2
H-cf-Ttα(R)ACCtα(R)CAGT-LysNH2
H-cf-TtγACCtγCAGT-LysNH2
2862.08
3185.83 [M + Na+]
3242.67 [M + Na+]
3222.29
cf α-(S-am) PNA
cf α-(R-am) PNA
cf γ-(S-am) PNA
3221.71
a
See the Supporting Information for mass spectra.
Table 2. UV−Tm (°C) Studies of α-am and γ-am PNA/DNA
a
Hybrids
entry
oligomers
PNA 1
DNA1 (ap) DNA2 (p) DNA3 (mismatch)
1
2
49.2
52.3
49.8
54.4
54.7
55.0
57.7
57.8
59.3
62.3
48.5
49.0
51.6
50.6
51.5
47.5
48.6
52.9
50.3
49.7
42.2
46.8
45.0
42.5
44.8
42.2
41.0
42.2
41.7
44.0
α-(S-am) PNA 2
α-(S-am) PNA 3
α-(S-am) PNA 4
α-(R-am) PNA 5
α-(R-am) PNA 6
α-(R-am) PNA 7
γ-(S-am) PNA 8
γ-(S-am) PNA 9
γ-(S-am) PNA 10
3
4
5
6
7
8
9
10
a
DNA1 (ap; antiparallel) = 5′ACTGAGGTAA3′; DNA2 (p; parallel)
= 5′AATGGAGTCA3′; DNA3 (with C:C base pair mismatch) =
5′ACTGCGGTAA3′.
Figure 2. Comparison of CD spectra of unmodified aeg PNA with
doubly modified α-(S-am)aeg, α-(R-am)aeg, and γ-(S-am)aeg PNA
sequences. All samples were prepared in buffer containing 10 mM
sodium phosphate and 10 mM NaCl at 2 μM strand concentration
each.
substantial enhancement of duplex stability (ΔTm = +5.5 to
+8.5 °C) with antiparallel DNA1 as compared to α-(S-am)
PNAs P2−P4 (entries 2−4) (ΔTm = +0.6 to +5.2 °C). The
stabilization is least when the modification is in the middle of
the sequence (P3). The degree of stabilization is also enhanced
with an increase in the number of modifications (P2 and P4,
2.1°/mod; P5 and P7, 2.8°/mod). Interestingly, a single γ-
modified PNA (P8, P9) had a more striking effect on the DNA-
binding properties of PNA by stabilizing the duplex with
antiparallel DNA1 (ΔTm = +8.6 to +10 °C) depending on the
The comparative differential Tm (ΔTm) calculated for the
modified PNA oligomers P2−P10 in relation to unmodified
PNA P1 and for the analogous antiparallel and parallel
PNA:DNA duplex pairs are shown in Figure 3.
Among the α-am-modifications, with reference to unmodi-
fied PNA P1, α-(R-am) PNAs P5−P7 (entries 5−7) show
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Figure 3. Comparative ΔTm values for aeg-PNA P1, α-(S-am) PNA
P2−P4, α-(R-am) PNA P5−P7, and γ-(S-am) PNA P8−P10 with
complementary antiparallel DNA1, 5′ACTGAGGTAA3′, and comple-
mentary parallel DNA2, 5′AATGGAGTCA3′. For each am-PNA, the
darker shade represents the amount of stabilization over that with
control (ΔTm = (TmPn − TmP1) for binding with DNA1 and the
lighter shade represents ΔTm = Tm(p) − Tm(ap).
Figure 4. Comparative ΔTm values of aeg PNA P1 and am-PNAs P2−
P10 with base pair C:C mismatch DNA3.
localization by fluorescent microscopic techniques. For the
cellular uptake assay, a monolayer of HeLa cancer cells was
incubated with each fluorescently labeled PNA (1 μM), and
after 24 h of incubation, the medium-containing cf am-PNAs
was aspirated for gentle fixing of cells. The distribution of
fluorescence was examined by fluorescence microscopy with
60× objective. Figure 5 shows images of untreated cells after
DAPI staining (Figure 5B) and cf am-PNA treated cells (Figure
5C) and the superimposed images (Figure 5D). It is seen that
both unmodified PNA cf P1 and the cationic PNAs cf P4, cf P7,
and cf P10 are localized in the nucleus (just like DAPI) even at
the lowest of concentrations studied (0.25 μM). The results
indicate that although all PNAs could enter the nucleus, the
true regio (α/γ) or stereo (R/S) effects of the modified cationic
am-PNAs could not be distinguished. This was done by flow
cytometry analysis.
HeLa cells were cultured in complete media and incubated
individually with fluorescently labeled PNA for 24 h. After
incubation, the cells were briefly washed with PBS buffer and
analyzed by flow cytometry. The experiments were done at two
different temperatures of 4 and 37 °C, and the results are
shown in Figure 6. The different mean fluorescence seen for
various am-PNAs shows differences in uptake properties. At 4
°C, only modest uptake efficiency is observed for unmodified
cf P1 and α-(S-am) PNA cf P4, both having similar mean
fluorescence values (Figure 6, x-axis). A significant increase in
the cell uptake was observed for α-(R-am) PNA cf P7 followed
by γ-am-PNA cf P10, which had 10−100 times higher uptake
efficiency at 4 °C. However, there was no difference in the
number of cells that uptake different PNAs, since the number
of events (y-axis) remained the same for all PNAs. The
corresponding results at a higher temperature (37 °C)
indicated that the number of cells that uptake the am-PNAs
almost doubled up, while the mean fluorescence decreased.
These data suggest that the (i) γ-am-PNA cf P10 is taken up by
the cells better than the unmodified PNA cfP1 and α-(R/S-am)
PNAs cf P7/cf P4 and (ii) the cell uptake is a function of both
regio- (γ > α) and stereochemical (R > S) properties of am-
PNAs.
position of modification, and with doubly modified PNAs, the
stabilization of the duplex increased by 5°/mod (P8 and P10).
Parallel vs Antiparallel Duplexes. In order to evaluate
the orientational selectivity of DNA binding in complementary
antiparallel and parallel directions, UV melting studies were
done using the parallel cDNA2 and the comparative data is
shown in Figure 3 (represented by lighter shade). It reveals that
duplexes of am-PNAs P2−P10 with cDNA2 are considerably
less stable than the analogous antiparallel duplexes. The
directional selectivity in binding is distinctly better in doubly
modified γ-(S-am) PNA 10 where the parallel duplex is
significantly destabilized (ΔTm = −12.6 °C) with cDNA2 as
compared to hybrid with antiparallel cDNA1 (ΔTm = +13 °C).
The stabilizing effects were always better in case of α-(R-am)
analogues compared to α-(S-am) with the exception of the α-
(S-am) PNA3 having modification in the middle of the
sequence wherein the derived parallel duplex with DNA2 was
slightly more stable than the duplex with opposite stereomer α-
(R-am) PNA 6.
Mismatched Duplexes. The higher binding of cationic
am-PNAs with anionic DNA could merely arise from favorable
electrostatic interactions between the two backbones. Hence, to
establish the sequence specificity of binding, the stability of
antiparallel duplexes of am-PNAs with cDNA3 (Table 2)
having a single C:C base-pair mismatch in the middle was
measured. The introduction of one base-pair mismatch allowed
the formation of duplexes but considerably destabilized the
complexes (Figure 4). The duplexes exhibited considerable
destabilization with γ-(S-am) PNAs P9 and P10 (ΔTm ≈ −15
to −18 °C) followed by R-(am) PNAs P5-P7 (ΔTm ≈ −10 to
−16 °C), S-(am) PNAs P2-P4 (ΔTm ≈ −5 to −12 °C), and
the unmodified PNA P1 (ΔTm ≈ −7 °C). The destabilization
from mismatch was higher than the stabilization gained due to
am-modifications, clearly suggesting that the higher binding
affinity of the cationic am-PNAs (P2−P10) with anionic DNA
was achieved without compromising the base specificity. Both
the regio- (α/γ) and stereospecificity (S/R) of aminomethylene
side chains are important determinants in defining the stability
of derived PNA:DNA duplexes.
Cell Viability Assay. In order to test the cellular toxicity of
different am-PNAs, MTT assay was carried out on HeLa cells
incubated with am-PNAs at various concentrations (0.1−5 μM)
in a humidified 5% CO2 atmosphere. The assays were run in
triplicate, and the percent viability of cells was determined using
standard protocol. The cytotoxicity profiles (Figure 7) indicate
that the viability of cells treated with am-PNAs is more than
Cellular Uptake of am-PNAs. The doubly modified
cationic am-PNAs were tagged with carboxy fluorescein
(cf P4, cf P7, and cf P10) for evaluating their ability to enter
living cells via flow cytometry and determine their subcellular
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Figure 5. Nuclear localization of am-PNAs in HeLa cells: (A) DIC image, (B) DAPI stained image, (C) fluorescent image, and (D) superimposed
image of (B) and (C).
Figure 6. FACS analysis of HeLa cells incubated with cf PNAs (1 μM, 24 h) at (A) 4 °C and (B) 37 °C.
85% even at the highest concentration of 5 μM. Although no
significant differences were seen among the different am-PNAs,
within the experimental limits, the γ-(S-am)-PNA was found to
show minimal toxicity at 5 μM. These data indicate that the am-
PNAs are favorably safe for use as potential therapeutic agents.
Discussion. On the basis of the reported observation that
the D-lysine-substituted PNA backbone stabilizes the derived
PNA:DNA hybrids and the theoretical studies of Topham et
al.16 suggesting intramolecular H-bonding of α-methylene-
amine side chain with the amide carbonyl of PNA backbone,
am-PNA-T monomers having an R/S-methyleneamine sub-
stituent at the α-C (8a/b) and S-substituent at γ-C (16) were
synthesized. The synthesis was started from D/L-aspargine by
reducing either the side-chain amide or the α-carboxyl function
to generate the desired cationic methyleneamine side chain.
Figure 7. Dosage-dependent cytotoxicity profile of PNA oligomers on
These were incorporated into am-PNA oligomers P2−P10
corresponding to mixed base PNA sequences having single or
double modifications at C-/N-terminus and in the middle for
the basis of MTT assay. The cell viability is represented as the percent
of viable HeLa cells incubated with 0, 0.1, 0.5, 1, and 2 μM
concentrations of indicated oligomers at 37 °C.
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studying their complexation with cDNA in parallel and
antiparallel manner. It was seen that (i) the cationic am-
PNAs stabilized the derived PNA:DNA hybrids compared to
the unmodified PNA, (ii) the stability increased with the
number of substitutions, and (iii) the antiparallel duplexes were
more stable than the corresponding parallel duplexes. The
degree of stability was in the order γ(S) > α(R) > α(S). The
reason for enhanced stability was not due to mere backbone
electrostatic interaction of cationic PNA with anionic DNA,
since even a single C:C mismatch in PNA:DNA hybrids
resulted in increased destabilization of hybrids from the am-
PNAs compared to that of unmodified PNA:DNA hybrid. The
cationic am-PNA designs possess shorter aliphatic side chains
compared to D-lysine-based PNA and hence may be less prone
to engage in nonspecific electrostatic interactions with
nucleotide phosphate groups of DNA molecules. The chiral
center created on PNA backbone may favorably preorganize
the PNA to generate hybridization competent conformations.
This is reflected in the CD profile of γ-(S-am) PNA in single
stranded form, which showed negative band of significant
intensity, which was absent in the CD of α-(R/S-am) PNA.
This may be a consequence of a better stacking of bases arising
from conformational preorganization of the backbone induced
by specific chiral center at C-γ. It is also reflected in the higher
Tm of γ-(S-am) PNA:DNA hybrids which was about 6.5 °C per
modification compared to 2.5−3.0 °C per modification for α-
(R/S-am) PNAs and 2 °C per modification for earlier reported
γ-guanidino-PNAs.23
It is noteworthy that the cationic am-PNAs also transverse
the cell membrane of HeLa cells and localize into the nucleus as
shown by the fluorescent-labeled am-PNAs (Figure 5) in their
superimposed images with DAPI stained cells. Quantitative
differences among the different am-PNAs were obtained by the
flow cytometry experimental data obtained at two different
temperatures. As seen from the fluorescence, the cells take the
γ-(S-am)-PNAs better than the α-am PNAs with the R-
stereomer better than the S-isomer. At a higher temperature,
the number of cells taking up the PNAs increased suggesting
that the cell uptake mechanism could be energy dependent,
while the accompanying decreased fluorescence may be a
consequence of quenching. The observed cell viability of more
than 85% in MTT assay, even at a higher concentration of 5
μM pointed to the low toxicity of am-PNAs, enabling the
possibility of safe delivery into cells.
Conclusions. We have comprehensively demonstrated that
the chiral, cationic am-PNAs containing a short amino-
methylene side chain on the aeg PNA backbone at either the
α or γ position stabilize hybrid duplexes with cDNA in a regio-
and stereospecific manner. The higher binding of cationic am-
PNAs is associated with stringent base specificity as seen from
severe destabilization in single mismatch duplexes, and the
antiparallel duplexes are more stable than parallel duplexes. The
am-PNA modifications are shown to improve the cellular
uptake and get localized in nucleus of HeLa cells to which they
are nontoxic. The studies represent a systematic analysis of
regio and stereospecific effects of PNA modifications on cDNA
binding, cell uptake and toxicity effects, with the γ(S)-
substituted am-PNA performing superior to α-substituted (R/
S)-PNA and the R-isomer better than S-isomer in the latter
case. Such data is of value in tuning the PNA modifications to
desired properties. These inherently cationic and intrinsically
chiral PNAs are thus promising for applications in therapeutics
and the γ-S-am-PNA has a good potential for construction of
suitable target sequences to take them to the next stage of
biological testing for antisense activity.
EXPERIMENTAL SECTION
■
All of the starting materials were used without purification. Other
details are given in the Supporting Information.
Nα-Boc-L-diaminopropionic Acid (2a). A slurry of Boc-L-
asparagine (5.0 g, 21.5 mmol) in ethyl acetate (24 mL), acetonitrile
(24 mL), water (12 mL), and iodosobenzene diacetate (8.32 g, 25.8
mmol) was cooled and stirred at 16 °C for 30 min. The temperature
was raised to 20 °C, and the reaction was stirred until completion (∼4
h). The reaction mixture was cooled to 0 °C and filtered under
vacuum. The filter cake was washed with ethyl acetate and dried in
vacuo at 65 °C to obtain 2a (3.88 g, 88% yield): mp 205−212 °C;
[α]25D +7.32 (c 0.527, MeOH); IR (KBr) ν (cm−1) 3528, 3507, 3349,
3033−2581, 2968, 1688, 1660, 1534, 1461, 1404, 1364, 1016 cm−1; 1H
NMR (200 MHz, MeOH-d) δH 7.81−7.77 (m, 1H), 7.25−7.17 (m,
1H), 4.17−4.11 (m, 1H), 3.25−3.22 (m, 2H), 1.54 (s, 9H) ppm; ESI-
MS (m/z) calcd 204.1110, obsd 205.2667 [M+ + H], 227.2656 [M +
Na]+.
Nα-Boc-D-diaminopropionic acid (2b). This compound was
prepared from Boc-D-asparagine (1b) using the similar procedure as
for 2a. mp 202−208 °C. [α]25D −8.88, (c 0.225, MeOH). IR (KBr) ν
(cm−1) 3528, 3507, 3349, 3033−2581, 2968, 1688, 1660, 1534, 1461,
1404, 1364, 1016 cm−1. 1H NMR (200 MHz, MeOH-d) δH 7.81−7.77
(m, 1H), 7.25−7.17 (m, 1H), 4.17−4.11 (m, 1H), 3.25−3.22 (m, 2H),
1.54 (s, 9H) ppm . ESI-MS (m/z) calcd 204.1110, obsd 205.2675 [M+
+ H], 227.2938 [M + Na]+.
Nα-Boc-Nβ-Cbz-amino-L-alanine (3a). To an ice-cold stirred
solution of 2a (330 mg, 1.6 mmol) in water (3.3 mL) was added
NaHCO3 (340 mg, 4 mmol) slowly in portions. To this mixture was
added CbzCl (330 mg, 1.9 mmol, 0.7 mL of a 50% solution in
toluene) in one stretch and the resulting mixture stirred vigorously for
8 h at ambient temperature. Toluene was removed under reduced
pressure, and the aqueous layer was washed with ether (10 mL × 2).
The ethereal layer was discarded, and the aqueous layer was acidified
to pH 2 with citric acid. The compound was extracted into ethyl
acetate, and the organic phase washings were pooled together, washed
with brine, dried over anhydrous Na2SO4, and then concentrated to
obtain 3a as a sticky oil (370 mg, 67% yield): [α]25 +8.4 (c 0.24,
D
1
MeOH); H NMR (200 MHz, CDCl3) δH 7.25 (s, 5H), 5.80−5.69
(br, 1H), 5.05 (s, 2H), 4.34 (s, 1H), 3.56 (m, 2H), 1.40 (s, 9H) ppm;
13C NMR (200 MHz, CDCl3) δC 173.3, 157.2, 155.8, 136.1, 128.3,
127.8, 80.0, 66.8, 54.1, 42.5, 28.1 ppm; ESI-MS (m/z) calcd 338.1478,
obsd 339.2566 [M + H] , 361.2721 [M + Na]+.
+
Nα-Boc-Nβ-Cbz-amino-D-alanine (3b). This compound was
prepared from 2b using a similar procedure as for 3a. It was isolated
in the form of white solid compound: mp 78.5−81.3 °C; [α]25 −7.6
D
1
(c 0.43, MeOH); H NMR (200 MHz, CDCl3) δH 7.32 (s, 5H), 6.24
(b, 1H), 5.96 (b, 1H), 5.09 (s, 2H), 4.39 (m, 1H), 3.64 (m, 2H), 1.43
(s, 9H) ppm; 13C NMR (200 MHz, CDCl3) δC 173.3, 157.2, 155.8,
136.1, 128.3, 127.8, 80.0, 66.8, 54.1, 42.5, 28.1 ppm; ESI-MS (m/z)
calcd 338.1478, obsd 339.25 [M + H]+, 361.27 [M + Na]+.
Nα-Boc-β-Cbz-amino-L-alanine Methyl Ester (4a). To a cold
solution of 3a (5.1 g, 15 mmol) in DMF (50 mL) was added activated
K2CO3 (2.3 g, 16 mmol). After the solution was stirred for 15 min in
an ice−water bath, MeI (3.8 mL, 59 mmol) was added to the white
suspension and stirring was continued at rt for 8 h at which point TLC
analysis indicated complete formation of methyl ester. The solvent was
removed from the reaction mixture, and the residue was partitioned
between ethyl acetate (15 mL) and water (15 mL). The organic phase
was washed with brine, dried with MgSO4, filtered, and concentrated.
The residue was purified on silica gel using light petroleum and ethyl
acetate to afford 4a as a pale amber oil which on subsequent cooling
solidified into a shiny white compound (3.8 g, 71.4% yield): mp 38−
43 °C; [α]25D +25.64 (c 1.092, CHCl3); 1H NMR (200 MHz, CDCl3)
δH 7.25 (s, 5H), 5.41 (b, 1H), 5.14 (b, 1H), 5.08 (s, 2H), 4.34 (b, 1H),
3.72 (s, 3H), 3.58 (t, 2H), 1.42 (s, 9H) ppm; 13C NMR (200 MHz,
CDCl3) δC 173.3, 157.2, 155.8, 136.1, 128.3, 127.8, 80.0, 66.8, 54.1,
5701
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42.5, 28.1 ppm; ESI-MS (m/z) calcd 352.1634, obsd 353.50 [M + H]+,
375.5088 [M + Na]+, 391.5032 [M + K]+.
Nα-Boc-Nβ-Cbz-amino-D-alanine Methyl Ester (4b). Com-
mL) under nitrogen conditions was heated at 65 °C with continuous
stirring for 1.5 h. The mixture was allowed to come to room
temperature and then kept in an ice bath. To this was added dropwise
6a (2.3 mg, 4.9 mmol) diluted in DMF (15 mL). The reaction mixture
was stirred for 8 h. DMF was removed under vacuum, and the residue
was partitioned between ethyl acetate and water. The organic phase
was washed with brine, dried with MgSO4, filtered, and concentrated.
The residue was purified on silica gel using light petroleum and ethyl
acetate to afford 7a as a foamy solid (2.2 g, 78% yield): mp 69−72 °C;
1H NMR (200 MHz, CDCl3) δH 9.19 (s, 1H), 7.33 (s, 5H), 6.86 (s,
pound 4b was prepared from 3b using a similar procedure as for 4a:
1
mp 42−46.4 °C; [α]25 −24.13 (c 0.663, CHCl3); H NMR (200
D
MHz, CDCl3) δH 7.25 (s, 5H), 5.47 (b, 1H), 5.20 (b, 1H), 5.07 (s,
2H), 4.35 (b, 1H), 3.72 (s, 3H), 3.57 (m, 2H), 1.42 (s, 9H) ppm; 13C
NMR (200 MHz, CDCl3) δC 173.3, 157.2, 155.8, 136.1, 128.3, 127.8,
80.0, 66.8, 54.1, 42.5, 28.1 ppm; ESI-MS (m/z) calcd 352.1634, obsd
375.0083 [M + Na]+, 390.9739 [M + K]+.
N-(2-Boc-aminoethyl)-N-(3-Cbz-amino-L-alanyl) Methyl Ester
(5a). Compound 4a (500 mg) was dissolved in DCM (1 mL), and
TFA (1 mL) was added at 0 °C. The mixture was stirred for 1 h.
Toluene (5 mL) was added, and volatiles were removed under
vacuum. After sufficient removal of volatiles, the TFA salt of the
compound (760 mg) was obtained as a crude oil. Without further
purification, this residue was dissolved in methanol (10 mL), and Boc-
aminoacetaldehyde (248 mg, 1.6 mmol) diluted in MeOH (2.5 mL)
was added with continuous stirring at 0 °C. DIPEA (0.7 mL, 4.3
mmol) was added to this stirred solution dropwise very slowly. After
the mixture was stirred for 30 min, glacial acetic acid (0.2 mL) and
NaCNBH3 (134 mg, 2.1 mmol) were added sequentially. The reaction
mixture was stirred for 3 h. All volatiles were removed under vacuum,
and the residue was dissolved in ethyl acetate and extracted with
saturated aqueous NaHCO3. The organic phase, after drying with
MgSO4, was removed under vacuum, and the residue was purified by
flash chromatography eluting with ethyl acetate−petroleum ether.
1H), 5.79 (m, 1H), 5.54 (m, 1H), 5.09−5.06 (q, 2H), 4.72−4.64
(maj) (d, 1H), 4.19−4.11 (min) (d, 1H), 3.88−3.79 (m, 2H), 3.72 (s,
3H), 3.54 (m, 2H), 3.34 (m, 1H), 3.18−3.13 (m, 2H), 1.87 (s, 3H),
1.43 (s, 9H) ppm; 13C NMR (200 MHz, CDCl3) δC 170.1, 167.3,
164.5, 156.9, 156.1, 151.4, 140.9, 136.5, 128.5, 128.2 ppm; ESI-MS
(m/z) calcd 561.2435, obsd 599.9580 [M + K]+.
N-(2-Boc-aminoethyl)-N-(thymine-1-acetyl)-N-(3-Cbz-amino-
D-alanyl) Methyl Ester (7b). This compound was prepared from 6b
1
using a similar procedure as for 7a: mp 70−79 °C; H NMR (200
MHz, CDCl3) δH 9.44 (s, 1H), 7.33 (s, 5H), 6.86 (bs, 1H), 5.88 (m,
1H), 5.57 (m, 1H), 5.15−5.00 (q, 2H), 4.72−4.64 (maj) (d, 1H),
4.19−4.12 (min) (d, 1H), 3.88 (m, 2H), 3.72 (s, 3H), 3.57 (m, 2H),
3.33 (m, 1H), 3.18 (m, 2H), 1.86 (s, 3H), 1.42 (s, 9H) ppm; 13C
NMR (200 MHz, CDCl3) δC 170.4, 167.6, 164.8, 157.2, 156.4, 151.6,
141.2, 136.8, 128.8, 128.5, 111.2, 80.2, 67.1, 61.3, 53.0, 49.7, 48.8, 39.9,
39.1, 28.7, 12.7 ppm; ESI-MS (m/z) calcd 561.2435, obsd 562.3598
[M + 1]+, 584.3475 [M + Na]+.
Compound 5a was obtained as a yellow oil (470 mg, 83.8% yield):
N-(2-Boc-aminoethyl)-N-(thymine-1-ylacetyl)-N-(3-Cbz-
amino)-L-alanine (8a). To compound 7a suspended in THF was
added a solution of 0.5 M LiOH in water, and the mixture was stirred
for 30 min. THF was removed under vacuum, and the aqueous layer
was washed with DCM. The aqueous layer was then neutralized with
activated Dowex H+ resin until the pH of the solution turned 4.0−5.0.
The resin was removed by filtration, and the filtrate was concentrated
to obtain the resulting Boc-protected monomer 8a in excellent yield
1
[α]25 +3.23 (c 0.5, CHCl3); H NMR (200 MHz, CDCl3) δH 7.34
D
(m, 5H), 5.28 (b, 1H), 5.09 (s, 2H), 4.91 (b, 1H), 3.71 (s, 3H), 3.49
(m, 1H), 3.34 (m, 2H), 3.16 (m, 2H), 2.75−2.58 (m, 2H), 1.42 (s,
9H) ppm; 13C NMR (200 MHz, CDCl3) δC 173.5, 156.5, 156.1, 136.4,
128.5, 128.5, 79.3, 66.9, 60.6, 52.3, 47.5, 42.8, 40.5, 28.4 ppm; ESI-MS
(m/z) calcd 395.2056, obsd 396.1504 [M + H]+, 418.0625 [M + Na]+.
N-(2-Boc-aminoethyl)-N-(3-Cbz-amino-D-alanyl) Methyl
Ester (5b). This compound was prepared from 4b using a similar
1
(>85%): H NMR (200 MHz, CDCl3) δH 7.39 (m, 5H), 7.28 (bs,
1
procedure as for 5a: [α]25 −3.46 (c 0.578, CHCl3); H NMR (200
D
1H), 5.13 (s, 2H), 4.73−4.65 (maj) (d, 1H), 4.52−4.44 (min) (d,
1H), 4.09−4.01 (t, 1H), 3.76−3.73 (d, 2H), 3.55−3.42 (m, 2H),
3.30−3.29 (m, 2H), 1.9 (s, 3H), 1.49 (s, 9H); ESI-MS (m/z) calcd
547.2278, obsd 585.9543 [M + K]+.
N-(2-Boc-aminoethyl)-N-(thymine-1-ylacetyl)-N-(3-Cbz-
amino)-D-alanine (8b). The hydrolysis procedure as described for
compound 8a was followed to afford 8b in good yield: 1H NMR (200
MHz, MeOD) δH 7.39 (m, 5H), 7.29 (bs, 1H), 5.13 (s, 2H), 4.73−
4.65 (maj) (d, 1H), 4.52−4.43 (min) (d, 1H), 4.09−4.01 (t, 1H),
3.78−3.74 (d, 2H), 3.36 (m, 2H), 3.30 (m, 2H), 1.9 (s, 3H), 1.49 (s,
9H) ppm; ESI-MS (m/z) calcd 547.2278, obsd 570.5063 [M + Na]+,
586.4932 [M + K]+.
Nα-Boc-Nβ-Cbz-aminoalaninol (9). To an ice-cooled solvent
mixture of dry THF (15 mL) and absolute ethanol (10 mL)
containing NaBH4 (269 mg, 7.1 mmol) in a three necked flask was
slowly added LiBr (617 mg, 7.1 mmol) for ∼30 min. The above
solution was stirred for 1.0 h, and the appearance of turbid milky
solution indicated the formation of LiBH4 in situ. To the above ice-
cooled solution was added 4a (500 mg, 1.4 mmol) from a dropping
funnel over a period of 30 min under N2 atmosphere, and the reaction
mixture was stirred overnight at rt. The pH was then adjusted to 7.0 by
addition of a saturated solution of NH4Cl. The solvent mixture was
removed under vacuum, and the residue was extracted into ethyl
acetate (10 mL × 3). The organic layer was washed with water
followed by brine solution, dried over anhydrous Na2SO4, and
concentrated to afford a white shiny solid product 9 (360 mg. 78%
yield, Rf = 0.65, ethyl acetate: petroleum ether 1:1): mp 77.2−80.2 °C;
[α]25D +12.6 (c 0.317, CHCl3); 1H NMR (200 MHz, CDCl3) δH 7.33
(s, 5H), 5.45 (b, 1H), 5.17 (b, 1H), 5.09 (s, 2H), 3.63 (m, 2H), 3.54
(m, 1H), 3.30 (m, 2H), 1.40 (s, 9H) ppm; 13C NMR (200 MHz,
CDCl3) δC 158.0, 156.0, 136.1, 128.6, 128.1, 79.8, 67.2, 61.7, 52.2,
41.1, 28.3 ppm; ESI-MS (m/z) calcd 324.1685, obsd 325.2679 [M +
H]+, 347.2411 [M + Na]+.
MHz, CDCl3) δH 7.33 (m, 5H), 5.34 (b, 1H), 5.08 (s, 2H), 4.95 (b,
1H), 3.71 (s, 3H), 3.48 (m, 1H), 3.35 (m, 2H), 3.16 (m, 2H), 2.75−
2.59 (m, 2H), 1.41 (s, 9H) ppm; 13C NMR (200 MHz, CDCl3) δC
172.7, 155.8, 155.5, 135.6, 127.7, 127.4, 78.6, 66.1, 59.8, 51.5, 46.7,
42.0, 39.6, 27.6 ppm; ESI-MS (m/z) calcd 395.2056, obsd 396.5209
[M + H]+, 418.5118 [M + Na]+.
N-(2-Boc-aminoethyl)-N-(chloroacetyl)-N-(3-Cbz-amino-L-
alanyl) Methyl Ester (6a). To a stirred solution of 5a (2.8 g, 7
mmol) in DCM (30 mL) and Et3N (3.9 mL, 28 mmol) cooled to 0 °C
was added chloroacetyl chloride (0.7 mL, 9.1 mmol). After 30 min,
DCM was removed under reduced pressure, and the residue was
extracted with DCM (2 × 20 mL) followed by drying under Na2SO4.
The solvent was evaporated under reduced pressure, and the residue
was purified with column chromatography (petroleum ether/ethyl
acetate) to afford chloro compound 6a as a yellow oil (2.546 g, 77%
yield): [α]25D −12.35 (c 0.648, CHCl3); 1H NMR (200 MHz, CDCl3)
δH 7.33 (s, 5H), 5.37 (b, 1H), 5.15−4.98 (q, 2H), 4.03 (m, 2H), 3.83
(m, 2H), 3.74 (s, 3H), 3.57 (m, 2H), 3.31 (m, 1H), 3.18 (m, 2H), 1.42
(s, 9H) ppm; 13C NMR (200 MHz, CDCl3) δC 170.2, 167.6, 156.8,
156.1, 136.3, 128.5, 128.2, 80.0, 67.0, 60.4, 52.7, 49.9, 41.2, 40.0, 38.6,
28.3 ppm; ESI-MS (m/z) calcd 471.1772, obsd 494.3328 [M + Na]+.
N-(2-Boc-aminoethyl)-N-(chloroacetyl)-N-(3-Cbz-amino-D-
alanyl) Methyl Ester (6b). This compound was prepared from 5b
using a similar procedure as for 6a: [α]25D +14.8 (c 0.135, CHCl3); 1H
NMR (200 MHz, CDCl3) δH 7.34 (s, 5H), 5.39 (b, 1H), 5.28 (b, 1H),
5.16−4.99 (q, 2H), 4.03 (m, 2H), 3.83 (m, 2H), 3.75 (s, 3H), 3.57 (m,
2H), 3.29 (m, 1H), 3.18 (m, 2H), 1.42 (s, 9H) ppm; 13C NMR (200
MHz, CDCl3) δC 170.2, 167.6, 156.7, 156.2, 136.2, 128.6, 128.2, 80.0,
67.0, 60.4, 52.7, 49.9, 41.1, 40.0, 38.6, 28.4 ppm; ESI-MS (m/z) calcd
471.1772, obsd 472.5432 [M + H]+, 494.5393 [M + Na]+.
N-(2-Boc-aminoethyl)-N-(thymine-1-acetyl)-N-(3-Cbz-amino-
L-alanyl) Methyl Ester (7a). A mixture of thymine (685 mg, 5.4
mmol) and anhydrous K2CO3 (750 mg, 5.4 mmol) in dry DMF (25
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Nα-Boc-Nβ-Cbz-aminoalanine Methyl Sulfonate (10). Com-
pound 9 (0.3 g, 0.7 mmol) was dissolved in dry DCM (5 mL) and
cooled to 0 °C. Et3N (0.3 mL, 2 mmol) was added dropwise added
and the mixture stirred for the next 10 min. To this ice-cooled solution
was added freshly distilled mesyl chloride (0.1 mL, 1 mmol) from a
dropping funnel over a period of 10 min under N2 atmosphere. The
reaction mixture was stirred for 30 min. DCM was removed under
reduced pressure, and the residue was extracted into ethyl acetate (5
mL × 2), washed with water, dried over Na2SO4, and concentrated to
yield almost pure (single spot on TLC) sticky mesylate derivative 10
(0.20 g, 90% crude yield, Rf = 0.7, ethyl acetate/petroleum ether1:1)
which was immediately used in the next step without further
purification.
mmol) and anhydrous K2CO3 (313 mg, 26 mmol) in dry DMF (10
mL) under N2 atmosphere was heated with stirring at 65 °C for 5 h.
After cooling, the solvent was removed under reduced pressure to
leave a residue, which was extracted into DCM (2 × 25 mL) and dried
over NaSO4. The solvent was evaporated, and the crude compound
was purified by column chromatography (MeOH/DCM) to afford a
pale white solid of 15 (935 mg, 79% yield, Rf = 0.8, 80% EtOAc/
petroleum ether): mp 141.9−144.1 °C; 1H NMR (400 MHz, CDCl3)
δH 10.10 (maj) and 9.70 (min) (s, 1H), 7.31 (s, 5H), 7.02 (maj) and
6.95 (min) (br s, 1H), 6.30 (maj) and 6.07 (min) (br s, 1H), 5.88
(maj) and 5.61 (min) (br s, 1H), 5.13−5.01 (m, 2H), 4.40−4.32 (m,
2H,), 4.25−4.22 (q, 2H), 4.16−4.11 (m, 1H), 3.91−3.75 (m, 2H),
3.58 (m, 2H), 3.40 (m, 2H), 1.87 (s, 3H), 1.40 (maj) and 1.33 (min)
(s, 9H), 1.24−1.20 (t, 3H) ppm; 13C NMR (400 MHz, CDCl3) δC
169.3 (maj) and 169.1 (min), 168.7 (maj) and 167.5 (min), 164.5,
157.8, 156.0 (min) and 155.8 (maj), 151.8 (min) and 151.3 (maj),
141.4 (min) and 141.0 (maj), 136.5 (min) and 136.3 (maj), 128.5,
128.1, 111.0, 79.8, 66.9, 62.4, 61.5, 50.5, 48.0, 41.5, 28.4, 14.1, 12.3
ppm; ESI-MS (m/z) calcd 575.2591, obsd 576.4506 [M + H]+,
598.4256 [M + Na]+.
γ-am-(Cbz)-(N-thymine-1-ylacetyl)-PNA Monomer (16). The
usual hydrolysis procedure as described for compound 8 was followed
to afford the monomer 16 in good yield: 1H NMR (200 MHz,
DMSO) δH 11.34 (br s, 1H), 7.36 (s, 5H), 6.93 (maj) and 6.69 (min)
(s, 1H), 5.06 (s, 2H), 4.75−4.47 (m, 2H), 4.16 (m, 1H), 4.0−3.85 (m,
2H), 3.53−3.34 (m, 2H), 3.15 (m, 2H), 1.77 (s, 3H), 1.38 (s, 9H)
ppm; ESI-MS (m/z) calcd 547.2278, obsd 548.4579 [M + H]+,
570.4857 [M + Na]+.
Circular Dichroism. CD spectra were recorded on CD
spectropolarimeter. The CD spectra of the PNA:DNA complexes
and the relevant single strands were recorded in 10 mM sodium
phosphate buffer, 10 mM NaCl (pH 7.2) as an accumulation of five
scans from 300 to 190 nm using a 1 cm cell, a resolution of 0.1 nm, a
bandwidth of 1.0 nm, sensitivity of 2 m deg, response of 2 s, and a scan
speed of 50 nm/min. The concentration of the samples was calculated
on the basis of absorbance from the molar extinction coefficients of the
corresponding nucleobases (i.e., T = 8.8 cm2/μmol; C = 7.3 cm2/
μmol; G = 11.7 cm2/μmol, and A = 15.4 cm2/μmol). The samples
were annealed by keeping the samples at 90 °C for 5 min followed by
slow cooling to room temperature. Then the samples were cooled by
keeping at 4 °C overnight. The CD spectra of all the samples were
recorded at 10 °C in the range 190−300 nm. The ellipticity value was
plotted as a function of the PNA mole fraction at 256 nm.
Job’s Plot. For the binding stoichiometry determination, 11
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 μM) in
sodium phosphate buffer (10 mM NaCl, pH 7.2). The samples were
annealed, and their CD spectra were recorded at 10 °C at the range
190−300 nm. The ellipticity value was plotted as a function of the
PNA mole fraction at 256 nm where the CD plots show an isodichroic
point at around 260−265 nm. The ellipticity values of all the mixtures
at 262 nm (for P3) and 266 nm (for P4) are plotted against mole
fraction of PNA. The ellipticity of PNA readily increases, reaches a
maximum, and then again decreases. The stoichiometry of the paired
strands was obtained from the intersecting point of this mixing curve,
in which the optical property at a given wavelength was plotted as a
function of mole fractions of each strand. The data were processed
using Microcal Origin 6.0.
Absorbance spectra at 260 nm were also recorded for the mixtures
of PNA:DNA in different proportions as mentioned above. A mixing
curve was plotted and absorbance at fixed wavelength (λmax 260 nm)
against mole fractions of PNA. The minimum of intersect corresponds
to the binding stoichiometric molar ratio.
UV−Tm measurements. The complexes were prepared in 10 mM
sodium phosphate buffer, pH 7.2 containing NaCl (10 mM).
Absorbance versus temperature profiles were obtained by monitoring
at 260 nm with a UV spectrophotometer equipped with a temperature
programmer and water circulator, scanning from 10 to 85 °C with
temperature increments of 0.5 °C per minute. The data were
(S)-Benzyl tert-Butyl (3-Azidopropane-1,2-diyl)dicarbamate
(11). To the solution of 10 (152 mg, 0.4 mmol) in DMF (2 mL) was
added NaN3 (350 mg, 5.4 mmol). The reaction mixture was stirred at
55 °C for 8 h. The solvent was removed under reduced pressure, and
the residue was extracted into ethyl acetate (5 mL × 3). The combined
organic layer was washed with brine, dried over anhydrous Na2SO4,
and then concentrated to yield 11 (104 mg, 83% yield Rf = 0.3; 40%
ethyl acetate/petroleum ether): mp 105.8−106.9 °C; [α]25 −6.17 (c
D
0.324, CHCl3); IR (CHCl3) ν (cm−1) 3440.22, 3019.66, 2957.18,
2927.72, 2856.13, 2400.76, 2106.59, 1712.59, 1514.91, 1455.80,
1368.47, 1215.60, 1163.85, 1064.64, 755.63 cm−1; 1H NMR (200
MHz, CDCl3) δH 7.33 (s, 5H), 5.20 (b, 1H), 5.09 (s, 2H), 3.78 (m,
1H), 3.45−3.42 (d, 2H), 3.35−3.29 (t, 2H), 1.42 (s, 9H) ppm; 13C
NMR (200 MHz, CDCl3) δC 157.1, 155.6, 136.2, 128.5, 128.1, 80.0,
67.0, 52.2, 50.6, 42.6, 28.2 ppm; ESI-MS (m/z) calcd 349.1750, obsd
350.1430 [M + H]+, 372.1436 [M + Na]+.
(R)-Benzyl tert-Butyl (3-Aminopropane-1,2-diyl)dicarbamate
(12). To a solution of 11 (100 mg) in MeOH (10 mL) taken in a
hydrogenation flask was added Raney nickel (5 mL). The reaction
mixture was hydrogenated in a Parr apparatus for 3.5 h at rt and H2
pressure of 35−30 psi. The catalyst was filtered off, and the solvent was
removed under reduced pressure to yield a residue of compound 12 as
a colorless oil (65 mg, 61% yield). This compound was used for the
next reaction without any further purification.
γ-am-(Cbz)-PNA Backbone Ethyl Ester (13). Compound 12
(0.05 g, 0.2 mmol) was treated with ethyl bromoacetate (15.5 μL, 0.14
mmol) in acetonitrile (1 mL) in the presence of Et3N (65 μL, 0.5
mmol), and the mixture was stirred at ambient temperature for 5 h.
The solid that separated was removed by filtration, and the filtrate was
evaporated to obtain the alkylated derivative 13 (0.05 g, 79% yield; Rf
= 0.63, 70% EtOAc/petroleum ether) as a colorless oil: 1H NMR (200
MHz, CDCl3) δH 7.33 (s, 5H), 5.50 (b, 1H), 5.26 (b, 1H), 5.08 (s,
2H), 4.21−4.11 (q, 2H), 3.70−3.60 (m, 1H), 3.37 (s, 2H), 3.32−3.19
(m, 2H), 2.80−2.57 (m, 2H), 1.41 (s, 9H), 1.25 (t, 3H) ppm; 13C
NMR (200 MHz, CDCl3) δC 172.4, 157.1, 156.1, 136.5, 128.5, 128.1,
79.7, 66.8, 60.9, 50.9, 50.3, 43.4, 28.4, 14.2 ppm; ESI-MS (m/z) calcd
409.2213, obsd 410.4282 [M + H]+.
γ-am-(Cbz)-(N-chloroacetyl)-PNA Backbone Ethyl Ester (14).
Compound 13 (500 mg, 1.2 mmol) was dissolved in DCM (5 mL)
under ice-cooled conditions, and Et3N (0.7 mL, 4.8 mmol) was added.
The reaction mixture was stirred for a few minutes, and then
chloroacetyl chloride (0.1 mL, 1.6 mmol) was added dropwise with
vigorous stirring. The reaction was complete within 0.5 h. The
compound was partitioned between the organic layer and aqueous
phase, and the product was extracted into the organic phase. It was
then purified on silica gel using column chromatography technique to
obtain 14 as colorless oil in good yield (400 mg, 67.4% yield; Rf = 0.6;
1
40% EtOAc/petroleum ether): H NMR (200 MHz, CDCl3) δH 7.32
(s, 5H), 5.89 (b, 1H), 5.47 (b, 1H), 5.15−5.01 (q, 2H), 4.27−4.23 (m,
1H), 4.20−4.14 (m, 2H), 3.96 (s, 2H), 3.81−3.61 (m, 2H), 3.45−3.34
(m, 2H), 3.15−2.96 (m, 2H), 1.40 (s, 9H), 1.28−1.25 (t, 3H) ppm;
13C NMR (200 MHz, CDCl3) δC 169.0, 168.6, 157.7, 155.8, 136.4,
128.5, 128.1, 79.7, 66.9, 62.2, 61.5, 51.1, 50.5, 50.3, 41.0, 28.3, 14.1
ppm; ESI-MS (m/z) calcd 485.1929, obsd 486.2656 [M + H]+,
508.3486 [M + Na]+.
γ-am-(Cbz)-(N-thymine-1-ylacetyl)-PNA Backbone Ethyl
Ester (15). A mixture of 14 (1 g, 2 mmol), thymine (286 mg, 26
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dx.doi.org/10.1021/jo300860f | J. Org. Chem. 2012, 77, 5696−5704

The Journal of Organic Chemistry
Article
processed using Microcal Origin 6.0 and Tm values derived from the
derivative curves.
Notes
The authors declare no competing financial interest.
Fluorescence Microscopy. Hela cells were maintained at 37 °C in a
humidified atmosphere containing 5% CO2 in D-MEM containing 2
mM L-glutamine, 10% FBS, and 4 μg/L gentamycin solution. They
were allowed to grow for the next 24 h, after which time the culture
medium containing PNA was discarded. The cells were washed twice
with PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2
mM MgCl2.6H20; pH 6.9) for 10 min at room temperature and then
gently fixed for 10 min using 3.5% paraformaldehyde and 0.05%
glutaraldehyde in PHEM buffer as the fixing agent. After fixation, the
cells were washed thoroughly three more times for 10 min with
PHEM. The coverslips were then mounted using mounting medium
that comprised of 90% glycerol, 0.5% N-propyl gallate (or propyl 3,4,5-
trihydroxybenzoate) and 20 mM Tris-Cl (pH 8.0) containing 0.0004
mg/mL of DAPI (or 4′, 6-diamidino-2-phenylindole) on objective
glass slides. The distribution of fluorescence was examined by
fluorescence microscopy on with 60× objective. Micrographs were
viewed and analyzed using AXIO-VISION software. Images were
acquired with the same camera settings for all cells with PNAs.
Flow Cytometry Analysis. All the PNA oligomers (cf PNAs; labeled
with carboxyfluorescein) were evaluated for cellular uptake using flow
cytometry. The cells were seeded into 60 mM plates at a density of 1−
2 × 106/dish in 3 mL media and grown in a humidified 5% CO2
atmosphere at 4 and 37 °C. The cells were then incubated with the
fluorescently labeled PNA oligomers at 1 μM for 24 h. Following
incubation, the cell suspension was centrifuged at 300g. The cell pellet
was washed twice with 1 × PBS (pH 7.4) at 300xg for 5 min. Cells
were washed once more with PBS and were finally resuspended in PBS
(500 μL). For each experiment, a control of cells that were not
incubated with PNA was also analyzed. The samples were run in
triplicate and each experiment was repeated twice within the same
week. Fluorescence analysis for both the live cell lines was performed
with Flow Cytometer using FL1 laser for excitation of carboxy-
fluorescein at 488 nm. Cell Quest software was used to analyze the
data. A minimum of 20000 events per sample was analyzed.
ACKNOWLEDGMENTS
R.M. acknowledges UGC India for the award of a fellowship.
K.N.G. thanks DST, New Delhi, for the J. C. Bose Fellowship.
■
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 2−16; HPLC and
MALDI-TOF of PNA P1−P10 and fluorescent PNA cf P1,
cf P4, cf P7, and cf P10; UV−Jobs plot, CD−Jobs plot, and
UV−melting profile of various modified PNA:DNA duplexes;
UV−absorbance spectra and fluorescence emission spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 91 (20) 2590 8021. Fax: 91 (20) 2589 9790. E-mail: kn.
ganesh@iiserpune.ac.in.
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dx.doi.org/10.1021/jo300860f | J. Org. Chem. 2012, 77, 5696−5704