Influence of pendant chiral Cγ-(alkylideneamino/guanidino) cationic side-chains of PNA backbone on hybridization with complementary DNA/RNA and cell permeability

Article abstract of DOI:10.1021/jo501639m

Intrinsically cationic and chiral Cγ-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyleneguanidino (egd)-PNA with two carbon spacers from the backbone. The relative stabilization (ΔTm) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (aeg)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability. (Graph Presented).

Full text of DOI:10.1021/jo501639m

Article
pubs.acs.org/joc
Influence of Pendant Chiral Cγ‑(Alkylideneamino/Guanidino) Cationic
Side-chains of PNA Backbone on Hybridization with Complementary
DNA/RNA and Cell Permeability
Deepak R. Jain,† Libi Anandi V,‡ Mayurika Lahiri,‡ and Krishna N. Ganesh*,†
†Chemical Biology Unit, Indian Institute of Science Education and Research, Dr Homi Bhabha Road, Pune 411008, Maharashtra,
India
‡Biological Sciences, Indian Institute of Science Education and Research, Dr Homi Bhabha Road, Pune 411008, Maharashtra, India
S
* Supporting Information
ABSTRACT: Intrinsically cationic and chiral Cγ-substituted
peptide nucleic acid (PNA) analogues have been synthesized
in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyl-
eneguanidino (egd)-PNA with two carbon spacers from the
backbone. The relative stabilization (ΔTm) of duplexes from
modified cationic PNAs as compared to 2-aminoethylglycyl
(aeg)-PNA is better with complementary DNA (PNA:DNA)
than with complementary RNA (PNA:RNA). Inherently,
PNA:RNA duplexes have higher stability than PNA:DNA
duplexes, and the guanidino PNAs are superior to amino
PNAs. The cationic PNAs were found to be specific toward
their complementary DNA target as seen from their
significantly lower binding with DNA having single base
mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated
ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to
penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the
nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency
to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both
cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show
improved cell permeability.
absence of negative charge and the higher flexibility in the
backbone. PNAs, being neither peptide nor nucleic acids,
possess resistance to both proteases and nucleases and
consequently have a longer life span in the cellular environ-
ment.5 The simplicity of chemical structure, ease of synthesis,
and superior DNA/RNA complementation properties have
made PNA an attractive gene regulatory agent.3,6,7 However,
the efficacy of PNA as an effective antisense/antigene agent has
been hampered by its low aqueous solubility, ambiguity in
binding orientation (parallel vs antiparallel), and poor cellular
uptake.8 To overcome these limitations many chemical
modifications of PNA have been attempted, which have
effected some improvements in PNA properties.9−11
INTRODUCTION
■
Peptide nucleic acids1,2 are effective DNA analogues in which
the chiral sugar−phosphate backbone is replaced with an
achiral pseudopeptide backbone consisting of 2-aminoethyl-
glycyl (aeg) linkage (Figure 1a). The nucleobases (A/T/G/C)
are attached through a methylene carbonyl linker to this
backbone at the glycyl amino nitrogen via tertiary amide
linkage. PNA hybridizes with complementary DNA/RNA via
established Watson−Crick base pairing with greater affinity
compared to equivalent DNA:DNA or DNA:RNA duplexes.3,4
The higher binding affinity of PNA is partially due to the
It has been shown that incorporation of D-lysine at the α-
position has significantly improved the solubility of PNA
oligomers in water and stabilized the derived PNA:DNA
duplexes.12 A recent crystallographic study of antiparallel
mixed-sequence PNA−DNA decamer heteroduplex, compris-
Figure 1. Chemical structures of (a) aeg-PNA, (b) γ-(S-eam) aeg-PNA,
and (c) γ-(S-egd) aeg-PNA.
Received: July 20, 2014
© XXXX American Chemical Society
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Scheme 1. Synthesis of γ-(S-eam) and γ-(S-egd) Modified PNA Monomers
ing a “chiral box” of three-residues of D-lysine, provides
evidence that introduction of a chiral center increases the
rigidity of the polyamide backbone.13,14 It has been
demonstrated that PNAs incorporating the cationic guanidi-
nium group in the side-chain at Cα (GPNA)15 or Cγ
(γGPNA)16 on the backbone result in enhanced binding
selectively to complementary RNA and DNA, respectively, and
in addition enhance the cellular uptake of the PNAs. The chiral
Cγ side-chain on the backbone is presumed to preorganize the
PNA oligomer into a right-handed helix, thereby favoring its
binding to complementary DNA/RNA.16−18 PNA carrying a
diethylene glycol side-chain at the Cγ site displayed better
hybridization property and water solubility.19 We have recently
shown that Cγ-substituted PNAs bearing an azido group via
methylene/butylene side-chains enhance the PNA:DNA hybrid
stability and are useful to append fluorophores by click
chemistry.20
Among PNA analogues bearing cationic functional groups
like amine or guanidine on pendant side-chains on the
backbone, systematic studies on the effect of alkyl spacers on
DNA/RNA complementation is lacking. This motivated us to
design, synthesize, and study PNAs incorporating a methyl-
amino side-chain at the Cα/γ site on the backbone, which
showed regio- and stereospecific dependence of PNA:DNA
hybrid stability and cell-penetrating ability.21,22 Continuing this
objective, we now report studies on Cγ-substituted PNAs
possessing ethyleneamino (eam) (Figure 1b) and ethyl-
eneguanidino (egd) (Figure 1c) side-chains to determine the
comparative effects of cationic groups and the intervening
carbon chain length on the hybridization of PNAs to
complementary DNA/RNA. It is demonstrated that the relative
degree of stabilization observed for the PNA:DNA duplexes of
cationic PNAs was higher compared to that of corresponding
PNA:RNA, while the inherent avidity of cationic amine/
guanidine modified PNAs was higher for complementary RNA.
The cationic PNAs were found to efficiently permeate the NIH
3T3 and MCF-7 cells, with the Cγ-ethyleneguanidino PNAs
being better than the Cγ-ethyleneamino PNAs.
RESULTS
■
Synthesis of Cγ-Ethyleneamino/Guanidino PNA
Monomers. The synthesis of target PNA monomers (11
and 13) containing thymine nucleobase was started from the
commercially available Boc-L-glutamine 1 (Scheme 1), which
was subjected to Hoffman rearrangement23 to obtain 4-amino-
2-(N-Boc-amino)butanoic acid 2. The side-chain amine was
protected with Cbz-group to get the orthogonally protected 4-
(Cbz-amino)-2-(Boc-amino)butanoic acid 3, which was
esterified using dimethyl sulfate to yield compound 4, followed
by reduction using NaBH4 to yield the primary alcohol 5. This
was converted to its mesylate derivative 6, which upon
treatment with NaN3 gave the azide 7. The reduction of
azide to primary amine by Raney/Ni followed by N-alkylation
of the resulting amine using ethylbromo acetate proceeded to
the N-alkylated compound 8. Upon treatment with chloroacetyl
chloride, 8 gave the chloro derivative 9, which was treated
subsequently with thymine to get the ester PNA monomer 10.
The hydrolysis of the ester with LiOH afforded the desired γ-
(S-eam) aeg-PNA-T monomer 11.
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a
Table 1. PNA Oligomers Synthesized with Their Molecular Weights
a
See the Supporting Information for mass spectra; red ta = ethyleneamino (eam) modified PNA-T unit and red tγ = ethyleneguanidino (egd)
modified PNA-T unit.
For the synthesis of the corresponding guanidinium analogue
γ-(S-egd) aeg-PNA-T monomer 13, the Cbz-group in
compound 10 was deprotected to get the free amine. It was
guanidinylated using N,N′-bis-di-Cbz-S-Me-isothiourea to
obtain the ester 12, which was hydrolyzed with NaOH to
afford the desired PNA monomer 13. All the intermediates and
the final compounds were characterized by spectroscopic (1H
and 13C NMR, mass spectroscopy) and analytical data
(Supporting Information).
Synthesis of PNA Oligomers. The modified PNA
monomers 11 and 13 were incorporated into aeg-PNA mixmer
sequence P1 at different sites in single or multiple copies. The
oligomers were synthesized by manual solid-phase peptide
synthesis using Boc-chemistry protocol24 on L-lysine-derivatized
MBHA resin utilizing the standard HOBt−HBTU coupling
procedure. At the end of the assembly, the Cbz protection on
the Cγ side-chain and the exocyclic amino group on A, G, and C
nucleobases were concurrently removed by triflouroacetic acid
(TFA)−trifluoromethanesulfonic acid (TFMSA),25 which also
cleaved the PNAs from the solid support. Various modified and
unmodified aeg-PNA oligomers that were synthesized are listed
in Table 1.
high-performance liquid chromatography (HPLC) and charac-
terized by matrix-assisted laser desorption ionization time-of-
flight (MALDI-TOF) mass spectrometric data (see Supporting
Information for HPLC and MALDI-TOF spectra).
Hybridization Studies. The comparative effects of cationic
substitutions (amine/guanidine) on the ethylene side-chain at
the Cγ position of PNA on the thermal stability (Tm) of the
composed duplex with complementary DNA/RNA were
evaluated by temperature-dependent UV absorbance studies.
The various modified PNAs 1−10 were annealed individually
with antiparallel complementary DNA 1 or complementary
RNA 1 to form the corresponding PNA:DNA/PNA:RNA
duplexes. The sequence specificity of the modified PNA
oligomers toward target DNA was also assessed from their
ability to form duplexes with DNA 2 having a single base
mismatch. The thermal stability results are presented in Table
2, and it is seen that the modified amino (P2−P5) and
guanidino (P6−P10) PNAs enhance the stability of the
corresponding PNA:DNA 1 duplexes significantly compared
to that of the unmodified aeg-PNA P1. The level of
enhancement is dependent on the nature (amine/guandine),
site (N-/C-terminus or center), and number of modifications,
with ΔTm in the range 2.5−14 °C. The relative comparisons of
differential thermal stabilities (ΔTm) of duplexes with amino-
and guanidino-modified PNAs compared with the control aeg-
PNA P1 are shown in Figure 2.
For cellular uptake studies, PNA oligomers were tagged with
5(6)-carboxyfluorescein at the N-terminus (cf P1−cf P10)
during the solid-phase synthesis to visualize the PNAs inside
the cells. All PNA oligomers were purified by reverse-phase
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hybridized with DNA 2 having a single base mismatch in the
middle of the sequence (Table 2). The introduction of one
base-pair mismatch destabilized the control duplex PNA
P1:DNA 2 by 6 °C. In comparison, the destabilization for
mismatch duplexes from cationic amino- and guanidino-
substituted PNA was significantly higher with ΔTm’s in the
range −10 to −18.5 °C depending on the number of
modifications. The guanidino PNAs showed more destabiliza-
tion compared to the amino PNAs, and the higher mismatch
duplex destabilization of cationic PNAs indicated their
inherently superior sequence specificity toward target DNA,
in addition to higher binding resulting from electrostatic
interactions.
Table 2. UV−Tm (°C) Values of PNA:DNA and PNA:RNA
a
Duplexes
entry
oligomers
aeg-PNA 1
DNA 1 (ap) DNA 2 (mm) RNA 1 (ap)
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
43.4
47.3
47.1
49.2
49.7
45.9
48.8
52.0
51.9
57.4
37.4
36.7
35.0
36.2
37.2
35.4
36.3
37.0
38.4
38.9
58.2
58.2
57.2
59.6
59.3
55.3
55.3
61.2
62.0
63.0
eam-t2a-PNA 2
eam-t6a-PNA 3
eam-t10a-PNA 4
eam-t2a,6a-PNA 5
egd-t2γ-PNA 6
egd-t6γ-PNA 7
egd-t10γ-PNA 8
egd-t2γ,6γ-PNA 9
egd-t2γ,6γ,10γ-PNA 10
The cationic PNAs were examined for their capacities to
hybridize complementary RNA, and the Tm’s of the
corresponding PNA:RNA duplexes are shown in Table 2.
The amino (P2−P5)- and guanidino (P6−P10)-modified
PNAs had a higher Tm for the derived PNA:RNA duplexes
compared to their analogous PNA:DNA duplexes by ∼10 0.5
°C. The degree of enhancement for cationic PNA:RNA
duplexes over unmodified aeg-PNA:RNA duplexes was ∼3−5
°C, except for those from P3, P6, and P7, which actually
showed destabilization.
a
DNA 1 (ap; antiparallel) = 5′ACTGAGGTAA 3′; DNA 2 (mm;
mismatch) = 5′ACTGCGGTAA 3′; RNA 1 (ap; antiparallel) = 5′
ACUGAGGUAA 3′. All samples were prepared in 10 mM sodium
phosphate buffer containing 10 mM NaCl and 0.1 mM EDTA at 2 μM
strand concentration each. The Tm’s are accurate up to 0.5 °C.
Circular Dichroic Studies and Intercalator Displace-
ment Assay. The structural effect of Cγ side-chain substitution
on the conformation of PNA:DNA and PNA:RNA duplexes
was probed by circular dichroism (CD) spectroscopy. Although
single-stranded PNA showed low-intensity CD signals,
PNA:DNA/PNA:RNA complexes exhibit characteristic CD
signatures because of the helicity induced by the chiral DNA/
RNA in corresponding hybrids (Supporting Information). The
PNA:DNA/RNA duplexes show two positive bands, one with
moderate intensity between 260 and 270 nm and another of
lower intensity between 218 and 225 nm accompanied by a
negative band near 240 nm. No significant changes were seen in
the CD profile of the duplexes from cationic PNAs compared
to that of unmodified PNA P1, suggesting conformational
similarity of all PNA:DNA and PNA:RNA duplexes.
The differential binding ability of the cationic PNAs toward
complementary DNA was examined by their competence to
displace the intercalated ethidium bromide (EtBr) from
DNA:DNA duplex26,27 (Supporting Information), monitored
by reduced fluorescence emission intensity at 610 nm. The loss
in fluorescence intensity is a consequence of strand invasion of
DNA:DNA complex by PNA resulting in disassociation of the
EtBr.28 The extent and rate of displacement of intercalator
bound to DNA:DNA duplex reflects the comparative abilities of
aeg-PNA, γ-(S-eam)-PNA, and γ-(S-egd)-PNA to invade
DNA:DNA duplex to form PNA:DNA duplex and is shown
in Figure 3. The aeg-PNA P1 displaced 84% of EtBr bound to
DNA duplex as compared to ∼76% with C-terminus single
amino-modified PNA P4. Interestingly, the analogous guanidi-
no-modified cationic PNA P8 was less effective with only 56%
displacement efficiency in spite of having higher duplex
stability. Further, the displacement efficiency did not noticeably
depend on the number of amino and guanidino modifications
in the PNA sequence (see Supporting Information). The EtBr
displacing ability of amino-modified cationic PNA (P4) was
found to be better than that of guanidino-modified PNA (P8)
but less efficient than that of control aeg-PNA. It may be
pointed out that the charge-neutral Cγ-methylene/butylene
azido PNAs were more competent than either the amino or
guanidino PNA analogues.20 The lower efficiency of cationic
Figure 2. Comparative ΔTm values for PNA:DNA and PNA:RNA
duplexes formed by γ-(S-eam, P2−P5) and γ-(S-egd, P6−P10) over
unmodified aeg-PNA at different positions on mixed decamer
sequence with complementary DNA 1, 5′ ACTGAGGTAA 3′, and
complementary RNA 1, 5′ ACUGAGGUAA 3′. ΔTm indicates the
difference in Tm with control aeg-PNA 1. Red bars = PNA:DNA, and
blue bars = PNA:RNA duplexes.
In the case of both amino and guanidino PNAs,
modifications at C-terminus [γ-(S-eam) P4] and [γ-(S-egd)
P8] stabilize the PNA:DNA duplexes (ΔTm = 5.8 and 8.6 °C,
respectively) better than the central (PNAs P3, ΔTm = 3.7; and
P7, ΔTm= 5.4 °C) and N-terminus (P2, ΔTm = 3.9; and P6,
ΔTm = 2.5 °C) modified PNAs. The degree of duplex
stabilization is enhanced with an increase in the number of
modifications in the sequence (P5, ΔTm = 6.3; P9, ΔTm = 8.5;
and P10, ΔTm = 14 °C), suggesting an average improvement in
the duplex stability by ∼5 °C/modification for guanidino and
∼3 °C/modification for amino PNAs. The duplexes from
guanidino-modified PNAs were always better stabilized than
those of the amino-modified PNAs.
The higher binding affinity of cationic PNAs toward the
negatively charged complementary DNA could arise from a
generic electrostatic interaction between the two strands. To
determine the sequence specificity, the modified PNAs were
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The duplex DNA 1:DNA 3 (lane 5) had retarded mobility
compared to the single-stranded DNAs (lanes 2 and 3) due to
its increased mass. Being negatively charged, single-stranded
DNAs moved faster on gel while the neutral aeg-PNA P1 (lane
4) and cationic PNA P10 (lane 8) did not move out of the well.
The reaction of PNA P1 to duplex DNA 1:DNA 3 results in
strand-invaded product duplex P1:DNA 1, liberating DNA 3
(lane 6). The P1:DNA 1 duplex is highly retarded in
comparison with DNA duplex (lane 5) due to a reduced
negative charge. The competition binding experiment was also
carried out by adding cationic PNA P10 to the DNA 1:DNA 3
duplex, and the resultant products PNA 10:DNA 1 duplex and
the free DNA 3 were monitored on gel. In the cationic PNA
10:DNA 1 duplex, the guanidino groups neutralize the negative
charges on DNA, decreasing the net negative charge more than
in P1:DNA 1 duplex, leading to retardation so much that it
hardly moves out of the well (lane 9). The formation of PNA
P10:DNA 1 duplex was also evident from the release of single-
stranded DNA 3 and the appearance of retarded PNA:DNA
complex (lane 10). These results suggest that the PNAs aeg-P1
and the cationic P10 invade the DNA 1:DNA 3 duplex to form
corresponding duplexes, displacing DNA 3.
Cell Uptake Studies. The effects of cationic PNAs on their
cell-penetrating abilities were investigated by treating NIH 3T3
and MCF-7 cell lines individually with the fluorescently labeled
PNAs aeg-PNA (cf P1), amino (cf P2 and cf P5) PNAs, and
guanidino (cf P6, cfP9, and cf P10) PNAs and visualization of
their intracellular location by confocal microscopy. The live
cells were incubated separately with the fluorescently labeled
PNAs (1 μM) for 24 h and processed for visualization by laser
scanning confocal microscope. Figure 5 shows confocal live cell
images for the guanidino-modified PNA cf P10 in NIH 3T3 and
MCF-7 cells (see Supporting Information for images for other
PNA oligomers). The images indicate that the cationic PNAs
enter the cells and localize inside the cells around the nucleus
irrespective of the site or type of modification and the cell line
used. A definite qualitative proof for the PNA uptake by cells is
seen from a superimposition of bright-field image (Figure 5A),
Hoechst 33342 stained image (Figure 5B), and the green
fluorescent image (Figure 5C) as shown in Figure 5D.
Figure 3. Comparative ethidium bromide displacement studies by
different PNAs from ds-DNA (azb-T10 PNA = Cγ-substituted
azidobutylene modified PNA).
PNAs in displacing EtBr from the DNA duplex is perhaps a
consequence of retarded invasion due to electrostatic repulsion
of their inherent positive charges from the positively charged
EtBr, unlike that with neutral aeg-PNA or azido-PNA. Because
of a lower pKa of side-chain amino groups in eam-PNAs than
that of guanidino function in egd-PNAs, they carry less positive
charge at pH 7.0 and, hence, are better than guanidine PNAs in
the displacement assay.
Electrophoretic Gel Mobility Shift Assay. The strand
invasion of DNA duplex by cationic PNAs was demonstrated
using electrophoretic mobility shift assay29,30 using PNA P10 as
a representative example. Control aeg-PNA P1 and the
guanidino PNA P10 were individually treated with comple-
mentary DNA 1, and the complex formations were monitored
by gel electrophoresis at ∼18 °C. The spots were visualized
through shadowing by illuminating the gel placed on a
fluorescent plate of silica gel F254 using UV light (Figure 4).
To quantitatively discern the cell-penetrating abilities of the
various modified PNA oligomers, fluorescence activated cell
sorter (FACS) analysis was performed as shown in Figure 6. In
the case of Cγ-amino-modified PNAs (Figure 6A), the doubly
modified amino-PNA cf P5 showed a slightly higher mean
fluorescence compared to the control PNA cf P1 and PNA with
single amino modification cf P2. The guanidino-modified PNAs
followed a similar pattern with mean fluorescence intensities
increasing with higher degree of substitution, in the order cfP6,
cf P9, and cf P10 (Figure 6B), all having higher fluorescence
compared to PNA (cfP1) and the amino PNA cf P2. The triply
modified guanidino PNA cfP10 showed the best fluorescence
among all PNAs.
Although the mean fluorescence intensities of PNAs with
different substitutions did not appear to vary too much, the
percentage of positive cell counts (a measure of the percentage
of the number of cells that have taken up the PNA inside that
quantifies the cellular uptake of different modified PNAs)
remarkably increased for the cationic amino- and guanidino-
modified PNAs compared to unmodified aeg-PNA P1 as shown
in Figure 7. For control aeg-PNA (cf P1), the percent positive
cell count was ∼18%, while it increased to ∼30% for single
amino modification (cf P2) and to ∼50% for double amino
Figure 4. Electrophoretic mobility shift assay and competition binding
experiment. Lane 1, Bromophenol blue; lane 2, DNA 1; lane 3, DNA
3; lane 4, PNA P1; lane 5, DNA 1:DNA 3; lane 6, PNA P1:DNA 1;
lane 7, DNA 1:DNA 3 + PNA P1; lane 8, PNA P10; lane 9, PNA
10:DNA 1; lane 10, DNA 1:DNA 3 + PNA 10 (DNA 1 = 5′
ACTGAGGTAA 3′; DNA 3 = 5′ TTACCTCAGT 3′).
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Figure 5. Distribution of three guanidino-modified PNA (cfP10) inside NIH 3T3 and MCF-7 cells: (A) bright-field image, (B) Hoechst 33342
stained image, (C) green fluorescent image, and (D) superimposed image of A, B, and C.
Figure 6. Quantitative estimation of cellular uptake in NIH 3T3 cells for (A) amino-modified PNAs and (B) guanidino-modified PNAs in
comparison with control aeg-PNA by FACS analysis.
PNAs at different concentrations. The quantitative data for
other PNA oligomers in both cell lines and the toxicity data are
given in Supporting Information.
DISCUSSION
■
PNAs can be rendered cationic through modifications either in
the backbone or conjugating a ligand through side-chains
located at α- or γ-positions on the backbone. Of these, the γ-
site has been shown to be versatile for modifications to
effectively modulate the properties of PNA.17,31,32 Continuing
our efforts to tune the effective carbon chain length and charge
on the cationic substitutions at Cγ of the PNA backbone, we
studied PNA oligomers composed of Cγ-substituted 2-carbon
spacer side-chain carrying multisite substitutions of amino and
guanidino functions. These were synthesized from the amide L-
glutamine by Hoffman rearrangement to yield amine followed
by guanidination to finally enable PNA monomers bearing
cationic functions (amino/guanidino) pendant on ethylene
side-chain at Cγ(S) of the PNA backbone. The cationic PNA
oligomers incorporating these monomers at defined, multiple
sites remarkably increased thermal stability of the derived
PNA:DNA duplexes by 2.5−14 °C depending on the position
and number of modified amino/guanidino units per PNA
strand; further, the guanidino PNAs were better than amino
PNAs, and both were much superior to unmodified PNA. The
order of stabilization for amino PNA was C-terminus > N-
terminus > middle, while that for guanidino PNA was C-
terminus > middle > N-terminus; no definite reasoning can be
attributed to these differences, although such positional effects
are common with other analogues. The higher binding of
Figure 7. Percentage of positive cells count for various modified PNAs
in NIH 3T3 cells by FACS analysis.
modifications (cf P5), a 3-fold increase in the cell permeation
compared to control PNA. The single guanidino modification
(cf P6) gave ∼42% positive cells, whereas for two guanidino
modifications (cf P9) the number of positive cells increased to
∼62%, which further increased to ∼70% for three guanidine-
modified PNAs (cf P10), thus amounting to a 4-fold increase in
the cell penetration ability over that of unmodified PNA cf P1,
clearly indicating an additive effect of cationic substitutions.
The cytotoxicity of modified cationic PNAs was assessed by
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, which showed >80% cell viability
(untreated cells, 100%) for both unmodified and modified
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cationic PNAs is clearly due to both structural and electrostatic
charge effects because a single base mismatch in the DNA
sequence destabilized the duplex stability more than that with
unmodified PNA. Thus, mere electrostatic interaction between
the cationic PNA and the anionic DNA is not the only reason
for stabilization of the duplex, and the cationic PNAs have
better base sequence discriminating ability than standard PNA.
The cationic PNA:RNA duplexes have significantly higher Tm
compared to the analogous PNA:DNA duplexes, which
assumes importance from an application perspective.
In electrophoretic experiments, the cationic PNAs retard the
derived DNA duplex on gel as expected from a net negative
charge reduction in the PNA:DNA duplex and demonstrated
successful displacement of isosequential DNA from DNA:DNA
duplex. However, the cationic PNA oligomers were less
efficient (slower) compared to unmodified aeg-PNA in
displacing the intercalated ethidium bromide from DNA:DNA
duplex through strand invasion due to the ensuing PNA-
intercalator cationic charge repulsion. The comparative ΔTm
°C/modification for PNA:DNA duplex collated from our
work21,22 and that of others16,32,33 for Cγ(S) substitution as a
function of spacer methylenes and cation type is depicted in
Table 3. It is seen that, for both amino and guanidino PNAs,
the duplex stability: a reduction of the carbon chain length
carrying cationic substitutions increases the PNA:DNA duplex
stability. The fluorescent analogues of the cationic PNAs are
also endowed with a greater efficiency of cell uptake with
localization in the vicinity of the nucleus. The results also
suggest that the cell-penetrating abilities of Cγ-substituted
PNAs depend on the nature of the cationic group, with
guanidinium being better than the amino group. Thus, the
cationic, chiral, and cell-permeable PNAs that have high DNA/
RNA binding affinity and sequence selectivity provide an
attractive platform for designing fresh PNA-based therapeutic
and diagnostic agents. These PNA analogues are akin to the
cationic peptides that are efficient cell-penetrating agents and
have the potential to cartel the necessary features for both
target inhibition and cell penetration.
EXPERIMENTAL SECTION
■
(S)-4-Amino-2-((tert-butoxycarbonyl)amino)butanoic Acid
(2). A slurry of Boc-L-glutamine 1 (5 g, 20.3 mmol), EtOAc (24
mL), CH3CN (24 mL), H2O (12 mL), and iodobenzene diacetate
(7.87 g, 24 mmol) was cooled and stirred at 16 °C for 30 min. The
temperature was maintained at 20 °C for 4 h, cooled to 0 °C, and
filtered. The solid residue was washed with EtOAc and dried in
vacuum to obtain compound 2 (2.65 g, 60% yield). mp = 200−201
°C; Rf = 0.2 EtOAc/MeOH (50:50); [α]25D + 13.6 (c 0.5, methanol);
Table 3. Comparative ΔTm °C/Modification in Thermal
1
IR (neat) 2976, 2900, 2832, 1740, 1692, 1645, 1563, 1546 cm−1; H
a
Stabilities of Cγ(S)-Substituted Cationic PNAs
NMR (400 MHz, CD3OD) δ 4.04−4.01 (m, 1H), 2.98−2.94 (t, J = 8
Hz, 2H), 2.12−1.87 (m, 2H), 1.39 (s, 9H); 13C NMR (100 MHz,
CD3OD) δ 176.3, 158.2, 80.9, 53.8, 38.3, 32.0, 28.8; HRMS (ESI-
TOF) m/z calcd for C9H18N2O4 [M + H]+ 219.1345, found 219.1342.
( S ) - 4 - ( ( ( B e n z y l o x y ) c a r b o n y l ) a m i n o ) - 2 - ( ( t e r t -
butoxycarbonyl)amino)butanoic acid (3). The solution of aq
NaHCO3 (2.3 g, 25 mL, 27 mmol) was added to an ice-cold solution
of compound 2 (2 g, 9.2 mmol) in acetone (25 mL) and stirred for 10
min at 0 °C. To this, benzyl chloroformate (3.75 g, 3.7 mL, 11 mmol)
in 50% toluene solution was added, and the reaction mixture was
stirred overnight at room temperature. Acetone was removed; the
aqueous layer was washed with Et2O (2 × 20 mL), acidified to pH 2−
3 with aq KHSO4 (sat) solution, and extracted with EtOAc (3 × 50
mL). The combined organic extracts were dried over anhydrous
Na2SO4, filtered, and concentrated to give compound 3 as a sticky oil
PNA-(CH2)n-X, n→
1
2
3
4
X = amino
6.5
3
5
NR
3
1.7
2.0
X = guanidino
NR
a
Values indicate the difference in Tm compared to analogous control
aeg-PNA; NR, not reported.
ΔTm °C/modification decreases with the increase in the
number of carbons in the spacer chain. The results have thrown
light on the structure activity relationship on the role of spacer
in determining the thermal stability of PNA:DNA hybrids.
The intrinsically cationic amine- and guanidine-substituted
PNAs were found to be efficient in cell permeability as seen by
live cell imaging of NIH 3T3 and MCF-7 cells. The green
fluorescence from the labeled PNAs (cationic and neutral aeg-
PNA) was intense in the cytoplasm around the nuclear
membrane. A similar effect was noticed by Sahu et al.16 in the
case of γ-substituted guanidino PNA (γGPNA) having 4
methylene spacers that localized within the vicinity of the
nucleus, suspected to be in the endoplasmic reticulum, in HeLa
cells. Thus, this feature seems to be a characteristic of cationic
PNAs and may have potential utility in their antisense
applications. Quantitation of cell permeation of PNAs by
FACS analysis as well as estimating percentage of cells uptaking
PNAs in both cell lines clearly established the superiority of
cationic amino/guanidino PNAs in cell penetration, with 3−4-
fold higher magnitude than unmodified PNAs.
(2.9 g, 90% yield). Rf = 0.67 EtOAc/MeOH (50:50); [α]25 − 9.2 (c
D
1, methanol); IR (neat) 2931, 2354, 2318, 1792, 1770, 1705, 1647,
1626 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.40−7.31 (m, 5H), 5.66−
5.64 (m, 1H), 5.48−5.43 (m, 1H), 5.14−5.04 (m, 2H), 4.35−4.32 (m,
1H), 3.50−3.06 (m, 2H), 2.07−1.76 (m, 2H), 1.43 (s, 9H); 13C NMR
(100 MHz, CDCl3) δ 175.7, 157.0, 156.0, 136.3, 128.5, 128.1, 80.3,
66.9, 51.1, 37.2, 33.3, 28.3; HRMS (ESI-TOF) m/z calcd for
C17H24N2O6 [M + Na]+ 375.1531, found 375.1531.
(S)-Methyl-4-(((benzyloxy)carbonyl)amino)-2-((tert-
butoxycarbonyl)amino)butanoate (4). To a stirred solution of
compound 3 (5.1 g, 14.5 mmol) in acetone (80 mL), K2CO3 (5 g, 36
mmol) was added followed by dimethyl sulfate (1.7 mL, 17.4 mmol).
The reaction mixture was heated to 55 °C for 4 h under reflux
condenser. Acetone was evaporated, and water (100 mL) was added to
the concentrate, which was extracted with EtOAc (3 × 60 mL). The
combined organic extracts were washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was
purified on silica gel (60−120 mesh) using petroleum ether and
EtOAc to give compound 4 as a white solid (5 g, 93% yield). mp =
67−69 °C; Rf = 0.5 petroleum ether/EtOAc (70:30); [α]25D − 18.8 (c
CONCLUSIONS
■
Cationic PNA analogues are capable of enhancing the inherent
cell permeability of PNAs. Comparative studies on cationic
Cγ(S)-ethyleneamino- and Cγ(S)-ethyleneguanidino-substituted
oligo PNA mixmers have shown their superior binding affinities
toward complementary DNA and RNA compared to neutral
aeg-PNA accompanied by high sequence specificity. The
current results fill an important gap on the features of spacer
chains bearing cationic substitutions at Cγ of PNA backbone on
1
0.5, methanol); IR (neat) 3341, 2975, 1698, 1518, 1446 cm−1; H
NMR (200 MHz, CDCl3) δ 7.39−7.33 (m, 5H), 5.61 (br, 1H), 5.38
(app d, J = 8 Hz, 1H), 5.2−5.11 (m, 2H), 4.44−4.34 (m, 1H), 3.73 (s,
3H), 3.55−3.07 (m, 2H), 2.15−1.65 (m, 2H), 1.45 (s, 9H); 13C NMR
(50 MHz, CDCl3) δ 172.9, 156.3, 155.7, 136.5, 128.3, 127.9, 80.0,
66.5, 52.3, 50.8, 37.0, 33.2, 28.1; HRMS (ESI-TOF) m/z calcd for
C18H26N2O6 [M + Na]+ 389.1688, found 389.1688.
G
dx.doi.org/10.1021/jo501639m | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(S)-Benzyl-tert-butyl (4-Hydroxybutane-1,3-diyl)-
dicarbamate (5). To a stirred solution of compound 4 (5 g, 13.6
mmol) in absolute EtOH (60 mL) was added NaBH4 (1.6 g, 41
mmol), and the reaction mixture was stirred for 6 h under N2
atmosphere at room temperature. EtOH was evaporated completely,
and water (100 mL) was added to the concentrate, which was
extracted with EtOAc (3 × 60 mL). The combined organic extracts
were washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was then purified on silica gel (60−120
mesh) using petroleum ether and EtOAc to give compound 5 as a
white solid (4.1 g, 89% yield). mp = 80−82 °C; Rf = 0.4 petroleum
3H), 1.64−1.42 (m, 2H), 1.34 (s, 9H), 1.21−1.14 (t, J = 7 Hz, 3H);
13C NMR (50 MHz, CDCl3) δ 171.9, 156.4, 136.6, 128.3, 127.9, 127.8,
79.4, 66.3, 60.8, 52.8, 50.4, 47.5, 37.4, 33.5, 29.5, 28.2, 14.0; HRMS
(ESI-TOF) m/z calcd for C21H33N3O6 [M + H]+ 424.2447, found
424.2444.
(S)-Ethyl-2-(N-(4-(((benzyloxy)carbonyl)amino)-2-((tert-
butoxycarbonyl)amino)butyl)-2-chloroacetamido)acetate (9).
To an ice-cold solution of compound 8 (3.1 g, 7.3 mmol) in dry
DCM (50 mL) and Et3N (2.96 g; 4 mL, 29.2 mmol) was added
chloroacetyl chloride (0.82 g; 0.58 mL, 7.3 mmol), and the reaction
mixture was stirred for 8 h. The reaction mixture was diluted with
DCM (20 mL) and washed with water (60 mL) and brine (60 mL).
The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was purified on silica gel (100−200 mesh)
using petroleum ether and EtOAc to give compound 9 as a colorless
sticky oil (2.63 g, 72%). Rf = 0.59 petroleum ether/EtOAc (40:60);
ether/EtOAc (50:50); [α]25 − 24.4 (c 0.5, methanol); IR (neat)
D
1
3334, 2974, 1689, 1519, 1453 cm−1; H NMR (400 MHz, CDCl3) δ
7.36−7.29 (m, 5H), 5.64 (br, 1H), 5.13−5.05 (m, 2H), 5.01 (br, 1H),
3.68−3.66 (app d, J = 8 Hz, 2H), 3.59−3.42 (m, 2H), 3.06−3.0 (m,
1H), 1.76−1.55 (m, 2H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3)
δ 155.6, 136.5, 128.4, 128.0, 79.7, 66.6, 65.2, 49.7, 37.6, 32.0, 28.3;
HRMS (ESI-TOF) m/z calcd for C17H26N2O5 [M + Na]+ 361.1739,
found 361.1743.
( S ) - 4 - ( ( ( B e n z y l o x y ) c a r b o n y l ) a m i n o ) - 2 - ( ( t e r t -
butoxycarbonyl)amino)butyl Methanesulfonate (6). To an ice-
cold solution of compound 5 (4 g, 12 mmol), Et3N (4.18 mL, 30
mmol) in dry dichloromethane (DCM) (50 mL) was added mesyl
chloride (1.21 mL, 15.6 mmol), and the reaction mixture was stirred
for 30 min at 0 °C under N2 atmosphere. To the reaction mixture
DCM (20 mL) was added, and it was washed with water (40 mL) and
brine (30 mL). The organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated to give compound 6 (4.5 g, 92% crude
yield). Rf = 0.53 petroleum ether/EtOAc (50:50). This compound was
used for the next step without further purification.
[α]25 − 10.8 (c 1, methanol); IR (neat) 3335, 2977, 2934, 1698,
D
1657, 1519, 1454, 1368 cm−1; 1H NMR (200 MHz, CDCl3) δ
7.337.26 (m, 5H), 5.61 (maj) 5.35 (min) (br, 1H), 5.15−5.0 (m, 2H),
4.27−4.12 (m, 4H), 3.99 (s, 2H), 3.88−3.65 (m, 2H), 3.52−3.38 (m,
2H), 3.25−2.95 (m, 2H), 1.70−1.64 (m, 2H), 1.41 (min) 1.40 (maj)
(s, 9H), 1.31−1.21 (m, 3H); 13C NMR (50 MHz, CDCl3) δ 169.1,
168.7, 168.3, 167.4, 156.5, 136.5, 128.4, 127.9, 79.4, 66.4, 62.0, 61.4,
52.8, 50.7, 49.8, 48.6, 46.8, 40.9, 37.3, 33.1, 32.3, 28.2, 14.0; HRMS
(ESI-TOF) m/z calcd for C23H34ClN3O7 [M + Na]+ 522.1982, found
522.1985.
(S)-Ethyl-2-(N-(4-(((benzyloxy)carbonyl)amino)-2-((tert-
butoxycarbonyl)amino)butyl)-2-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetamido)acetate (10). A solution of
compound 9 (1 g, 2 mmol) in dry DMF (20 mL) containing
K2CO3 (0.33 g, 2.4 mmol) and thymine (0.3 g, 2.4 mmol) was stirred
at room temperature for 12 h. To the reaction mixture water (40 mL)
was added and extracted with EtOAc (3 × 50 mL), and the organic
layer was washed with water (50 mL) and brine (30 mL). The
combined organic extracts were dried over anhydrous Na2SO4, filtered,
and concentrated. The residue obtained was purified on silica gel
(100−200 mesh) using petroleum ether and EtOAc to give compound
10 as a white solid (0.95 g, 81%). mp = 92−94 °C; Rf = 0.47 EtOAc;
[α]25D − 8.1 (c 1, methanol); IR (neat) 3332, 2978, 1671, 1519, 1464,
1369 cm−1; 1H NMR (400 MHz, CDCl3) δ 9.86 (maj) 9.56 (min) (br,
1H), 7.37−7.27 (m, 5H), 7.05 (min) 6.98 (maj) (s, 1H), 5.67−5.63
(maj) 5.58−5.56 (min) (comp, 1H), 5.11−5.04 (m, 2H), 4.82 (min)
4.78 (maj) (br, 1H), 4.47−4.37 (m, 1H), 4.28−4.13 (m, 3H), 3.94−
3.51 (m, 3H), 3.43−3.02 (m, 3H), 2.17 (br, 1H), 1.89 (min) 1.87
(maj) (s, 3H), 1.69−1.67 (comp, 1H), 1.41 (maj) 1.39 (min) (s, 9H),
1.31−1.23 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 169.1, 167.5,
164.4, 156.7, 151.5, 140.9, 136.5, 128.4, 128.0, 110.8, 79.6, 66.6, 62.3,
52.1, 51.5, 49.8, 48.6, 47.8, 47.2, 37.6, 32.9, 28.3, 14.0, 12.3; HRMS
(ESI-TOF) m/z calcd for C28H39N5O9 [M + Na]+ 612.2645, found
612.2646.
(S)-Benzyl-tert-butyl (4-Azidobutane-1,3-diyl)dicarbamate
(7). A solution of compound 6 (4.5 g, 11 mmol) and NaN3 (10.86
g, 165 mmol) in dry dimethylformamide (DMF) (50 mL) was heated
to 80 °C for 8 h. To the reaction mixture water (100 mL) was added,
which was extracted with EtOAc (3 × 70 mL). The ethyl acetate layer
was washed with water (50 mL) and brine (50 mL). The combined
organic extracts were dried over anhydrous Na2SO4, filtered, and
concentrated. The residue obtained was then purified on silica gel
(60−120 mesh) using petroleum ether and EtOAc to give compound
7 as a sticky yellowish oil (3.53 g, 87% yield). Rf = 0.73 petroleum
ether/EtOAc (50:50); [α]25D − 33.6 (c 1, methanol); IR (neat) 3332,
1
2974, 2931, 2098, 1691, 1517 cm−1; H NMR (400 MHz, CDCl3) δ
7.32−7.28 (m, 5H), 5.53 (br, 1H), 5.09−5.01 (m, 2H), 4.82 (br, 1H),
3.80−3.76 (m, 1H), 3.45−3.38 (m, 2H), 3.35−2.97 (m, 2H), 1.68−
1.50 (m, 2H), 1.40 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 156.4,
155.7, 136.5, 128.4, 128.0, 19.8, 66.5, 54.9, 47.6, 37.4, 32.8, 28.1;
HRMS (ESI-TOF) m/z calcd for C17H25N5O4 [M + Na]+ 386.1804,
found 386.1802.
(S)-Ethyl-2-((4-(((benzyloxy)carbonyl)amino)-2-((tert-
butoxycarbonyl)amino)butyl)amino)acetate (8). To a solution of
compound 8 (750 mg, 2.0 mmol) in absolute EtOH (15 mL) was
added Raney Ni (2 mL). The reaction mixture was hydrogenated in a
Parr apparatus for 6 h at room temperature and H2 pressure of 50−55
psi. The reaction mixture was filtered to remove catalyst, and the
solvent was removed under reduced pressure to yield a residue of free
amine (626 mg) as yellowish oil that was taken in CH3CN (20 mL).
To this, Et3N (0.8 mL, 5.5 mmol) was added and stirred at 0 °C for 10
min, and ethylbromo acetate (0.18 mL, 1.7 mmol) was added
dropwise followed by keeping the reaction mixture stirred for 12 h at
room temperature. CH3CN was evaporated, and water (50 mL) was
added to the concentrate. The aqueous layer was extracted with EtOAc
(3 × 40 mL), and the combined organic extracts were washed with aq
NaHCO3 (sat) and brine, dried, and concentrated. The residue
obtained was purified on silica gel (100−200 mesh) using petroleum
ether and EtOAc to give compound 8 as a yellowish oil (700 mg,
(S)-2-(N-(4-(((Benzyloxy)carbonyl)amino)-2-((tert-
butoxycarbonyl)amino)butyl)-2-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetamido)acetic Acid (11). To a stirred
solution of compound 10 (500 mg, 0.8 mmol) in MeOH was added aq
LiOH (10%), and the reaction mixture was stirred for 3 h at room
temperature. MeOH was removed under vacuum, and the aqueous
layer was washed with diethyl ether and neutralized with activated
Dowex H+ to pH ≈ 4−5. The resin was removed by filtration, and the
filtrate was concentrated to obtain the resulting compound 11 as a
white solid (0.42 g, 88%). mp = 241−245 °C; Rf = 0.5 EtOAc/MeOH
(50:50); [α]25 − 3.0 (c 0.5, methanol); IR (neat) 3341, 2938, 2354,
D
2317, 1728, 1678, 1537, 1474, 1441, 1369 cm−1; 1H NMR (400 MHz,
DMSO-d6) δ 11.30 (min) 11.25 (maj) (br, 1H), 7.35−7.26 (m, 5H),
7.21−7.15 (m, 1H), 6.91−6.89 (min) 6.78−6.76 (maj) (d, J = 8 Hz,
1H), 5.02−4.96 (m, 2H), 4.76−4.72 (maj) 4.59−4.55 (min) (d, J = 16
Hz, 1H), 4.42 (s, 2H), 3.97−3.74 (m, 4H), 3.47−3.21 (m, 2H), 3.12−
2.91 (m, 3H), 1.75 (min) 1.73 (maj) (s, 3H), 1.58−1.41 (m, 2H), 1.37
(maj) 1.36 (min) (s, 9H); 13C NMR (100 MHz, CDCl3) δ 170.6,
167.9, 167.0, 164.5, 156.0, 155.5, 151.1, 142.1,137.3, 108.0, 77.7, 65.1,
51.9, 51.1, 48.4, 47.7, 47.1, 46.5, 37.7, 32.1, 28.3, 12.0; HRMS (ESI-
80%). Rf = 0.48 petroleum ether/EtOAc (20:80); [α]25 − 10.4 (c 1,
D
methanol); IR (neat) 3329, 2929, 2354, 2318, 1738, 1705, 1694, 1647,
1529, 1516 cm−1; 1H NMR (200 MHz, CDCl3) δ 7.25−7.20 (m, 5H),
5.76 (br, 1H), 5.06−4.93 (m, 3H), 4.14−4.03 (q, J = 8 Hz, 2H), 3.65−
3.63 (m, 1H), 3.42−3.22 (m, 3H), 2.95−2.86 (m, 1H), 2.71−2.55 (m,
H
dx.doi.org/10.1021/jo501639m | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
TOF) m/z calcd for C26H35N5O9 [M + Na]+ 584.2332, found
584.2335.
Purification of PNA Oligomers by Reverse-Phase HPLC. For
the purification of peptides, a semipreparative BEH130 C18 (10 × 250
mm) column was used. Purification of PNA oligomers was performed
with the gradient elution method: A to 100% B in 20 min; A = 0.1%
TFA in CH3CN/H2O (5:95); B = 0.1% TFA in CH3CN/H2O (1:1)
with a flow rate of 3 mL/min. Control aeg-, eam-, and egd-modified
PNA oligomers were monitored at 254 nm wavelength, while all
fluorescent PNA oligomers were monitored at both 254 and 490 nm
wavelengths.
UV−Tm Measurements. UV−melting experiments were carried
out on a UV-spectrophotometer equipped with a Peltier heater. The
samples for Tm measurement were prepared by mixing the calculated
amounts of respective oligonucleotides in a stoichiometric ratio (1:1)
in sodium phosphate buffer (10 mM, pH 7.2) containing ethyl-
enediaminetetraacetic acid (EDTA) (0.1 mM) and NaCl (10 mM) to
a final concentration of 2 μM for each strand. The samples were
annealed by heating at 90 °C for 3 min followed by slow cooling to
room temperature for at least 6−8 h and then being refrigerated for at
least 4−5 h. The samples (500 μL) were transferred to a quartz cell
and equilibrated at the starting temperature for 2 min. The optical
density (OD) at 260 nm was recorded in steps from 20 to 85 °C with
a temperature increment of 1 °C/min. Each melting experiment was
repeated at least twice. The normalized absorbance at 260 nm was
plotted as a function of the temperature. The Tm was determined from
the first derivative of normalized absorbance with respect to
temperature and is accurate to 0.5 °C. The data were processed
using Microcal Origin 8.0/8.5. The concentrations of all oligonucleo-
tides were calculated on the basis of absorbance from the molar
extinction coefficients of the corresponding nucleobases, i.e., T = 8.8;
C = 7.3; G = 11.7; and A = 15.4 cm2/μmol.
Circular Dichroism Studies. CD spectra were recorded on a
spectropolarimeter connected with a Peltier heater. The calculated
amounts of PNA oligomers and the complementary DNA 1 were
mixed together in 1:1 stoichiometry in sodium phosphate buffer (10
mM, pH 7.2) containing EDTA (0.1 mM) and NaCl (10 mM) to
achieve a final concentration of 5 μM for each strand. The samples
were annealed by heating at 90 °C for 3 min followed by slow cooling
to room temperature for at least 6−8 h. The cooled samples were
transferred to a refrigerator for at least 4−5 h. The CD spectra of
PNA:DNA, PNA:RNA duplexes, and single-stranded PNAs were
recorded at 10 °C with an accumulation of five scans from 300 to 190
nm using a 2 mm cell, a resolution of 0.1 nm, a bandwidth of 1 nm, a
sensitivity of 2 m deg, a response of 2 s, and a scan speed of 50 nm/
min.
(S)-Ethyl-5-(((Benzyloxy)carbonyl)amino)-9-((tert-
butoxycarbonyl)amino)-11-(2-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetyl)-3-oxo-1-phenyl-2-oxa-4,6,11-tri-
azatridec-4-en-13-oate (12). To a solution of compound 10 (1 g,
1.7 mmol) in absolute EtOH (10 mL) was added 10% Pd/C (100 mg)
and a catalytic amount of glacial acetic acid (50 μL). The reaction
mixture was subjected to hydrogenation on a Parr hydrogenator at 60
psi for 12 h, after which it was filtered and concentrated to get free
amine (0.76 g, 1.7 mmol). It was taken in dry DMF (10 mL) to which
N,N′-bis-di-Cbz-S-Me isothiourea (0.62 g, 1.7 mmol) and a catalytic
amount of 4-dimethylaminopyridine (DMAP) were added under N2
atmosphere. The reaction mixture was stirred for 12 h at room
temperature; water (20 mL) was added and extracted with EtOAc (3
× 40 mL). The combined organic extracts were washed with water (40
mL) and brine (30 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue obtained was then purified on silica gel (100−200 mesh) using
petroleum ether and EtOAc to give compound 12 as a white solid
(0.93 g, 72% yield). mp = 99−102 °C; Rf = 0.48 EtOAc; [α]25D − 1.1
(c 1, methanol); IR (neat) 3332, 2977, 1675, 1638, 1573, 1507, 1451,
1427, 1376, 1323 cm−1; 1H NMR (400 MHz, CDCl3) δ 11.70 (s, 1H),
9.24 (br, 1H), 8.52−8.51 (app d, J = 4 Hz, 1H), 7.39−7.28 (m, 10H),
7.02 (min) 6.98 (maj) (s, 1H), 5.78 (d, J = 8 Hz, 1H), 5.22−5.09 (m,
4H), 4.84−4.80 (app d, J = 16 Hz, 1H), 4.45−4.13 (m, 5H), 3.96−
3.69 (m, 2H), 3.64−3.31 (m, 4H), 2.09 (br, 1H), 1.90 and 1.88 (s,
3H), 1.82−1.58 (m, 2H), 1.41 (maj) 1.38 (min) (s, 9H), 1.30 (min)
1.25 (maj) (t, J = 8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 169.1,
168.4, 167.5, 164.2, 163.5, 156.1, 153.6, 151.2, 141.1, 136.6, 134.5,
128.8, 128.4, 128.0, 110.6, 79.8, 68.2, 67.1, 62.2, 61.4, 52.2, 51.3, 50.0,
48.8, 47.7, 37.9, 31.9, 28.3, 14.0, 12.3; HRMS (ESI-TOF) m/z calcd
for C37H47N7O11 [M + H]+ 766.3412, found 766.3403.
( S ) - 5 - ( ( ( B e n z y l o x y ) c a r b o n y l ) a m i n o ) - 9 - ( ( t e r t -
butoxycarbonyl)amino)-11-(2-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetyl)-3-oxo-1-phenyl-2-oxa-4,6,11-tri-
azatridec-4-en-13-oic Acid (13). To a stirred solution of compound
12 (0.5 g, 0.65 mmol) in tetrahydrofuran (THF) was added aq NaOH
(2 N, 4 mL) at 0 °C, and the reaction mixture was stirred for 30 min at
0 °C. THF was evaporated, and the aqueous layer was washed with
EtOAc (2 × 20 mL). The aqueous layer was neutralized with 5 N HCl
at 0 °C to pH 3−4 and extracted with EtOAc (3 × 30 mL). The
combined organic extracts were dried over anhydrous Na2SO4, filtered,
and evaporated to give compound 13 as a white solid (0.37 g, 78%
yield). mp = 95−97 °C; Rf = 0.73 EtOAc/MeOH (50:50); [α]25
+
D
Ethidium Bromide Displacement Assay from ds-DNA.
Calculated amounts of complementary DNA strands (1:1) were
annealed to get DNA duplex (2 μM, 400 μL) in the sodium phosphate
buffer (pH 7.2). Aliquots (1 μL) of EtBr (250 μM) were added to the
annealed samples, and the fluorescence intensity at 610 nm was
recorded until it got saturated. The resulting EtBr−ds-DNA complex
was titrated against individual PNA oligomers in the aliquots of 0.4 μL,
and the change in fluorescence emission at 610 nm was recorded. The
percent change observed in fluorescence intensity (I/I0) upon
displacement of EtBr was plotted against the concentration of PNA
oligomers using Microcal Origin 8.5.
3.7 (c 1, methanol); IR (neat) 3339, 2923, 2852, 2354, 2317, 1728,
1671, 1632, 1525, 1423, 1363, 1325, 1254 cm−1; 1H NMR (400 MHz,
CDCl3) δ 11.74 (br, 1H), 10.10 (br, 1H), 8.53 (br, 1H), 7.37−7.28
(m, 10H), 6.99 (s, 1H), 6.62 (s, 1H), 5.76 (br, 1H), 5.30−5.10 (m,
5H), 4.70−4.01 (m, 4H), 3.80−3.29 (m, 6H), 1.85 (maj) 1.82 (min)
(s, 3H), 1.40 (min) 1.38 (maj) (s, 9H); 13C NMR (100 MHz, CDCl3)
δ 170.9, 170.5, 168.0, 167.4, 164.4, 163.1, 155.7, 155.2, 152.7, 151.0,
141.8, 136.9, 135.2, 128.6, 128.4, 128.1, 127.9, 126.7, 126.5, 115.7,
108.2, 78.2, 67.6, 66.4, 62.9, 51.1, 47.7, 46.9, 39.5, 38.0, 34.4, 31.0,
30.5, 28.2, 28.2, 12.0; HRMS (ESI-TOF) m/z calcd for C35H43N7O11
[M + H]+ 738.3099, found 738.3092.
Solid-Phase Synthesis Protocol. The modified and unmodified
PNA monomers were incorporated into 10-mer PNA sequence by
solid-phase synthesis on L-lysine-derivatized MBHA resin (50 mg)
having 0.35 mmol/g loading value. The solid-phase synthesis was
carried out in a glass sintered flask. The deprotection of N-t-Boc group
from the resin-bound lysine with 50% TFA in DCM (3 × 15 min) was
followed by washing with DCM and DMF (3 × 10 mL) to give a TFA
salt of amine, which was neutralized using 10% N,N-diisopropylethyl-
amine (DIPEA) in DCM (3 × 10 min) to liberate free amine. After
washing with DCM and DMF (3 × 10 mL), the free amine was
coupled with carboxylic acid of the incoming monomer (A/T/G/C; 3
equiv) in DMF (500 μL) using HOBt (3 equiv), HBTU (3 equiv), and
DIPEA (3 equiv). The reagents were then removed by filtration, and
the resin was washed with DMF.
Electrophoretic Mobility Shift Assay and Competition
Binding Experiment. The calculated amounts of PNA oligomers
(3 nmol) were individually mixed with DNA strands (3 nmol) in 1:1
ratio in sodium phosphate buffer (10 mM, NaCl 100 mM, pH 7.2; 10
μL). The samples were annealed by heating to 90 °C for 3 min
followed by slow cooling to RT and refrigeration at 4 °C overnight. To
this, 5 μL loading buffer (10 mM Tris-HCl, pH 8; 30% sucrose and
10% glycerol) was added, and samples were loaded on the gel.
Bromophenol blue (BPB) was used as the tracer dye and separately
loaded in an adjacent well. Gel electrophoresis was performed on 20%
nondenaturing polyacrylamide gel (acrylamide/N,N′-methylenebis-
(acrylamide), 19:1) with 1X-TBE containing 500 mM NaCl as the
tank buffer at a constant power supply of 14 W and 120 V for 30 h.
During electrophoresis, the temperature was maintained at ∼18 °C.
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The spots were visualized through UV shadowing by illuminating the
gel placed on a fluorescent silica gel plate, F254 using UV light.
Cellular Uptake Experiment using Confocal Microscopy.
MCF-7 and NIH 3T3 cells were plated in 8-well chambered cover
glass in 200 μL of Dulbecco’s modified Eagle medium (DMEM)
containing 10% fetal bovine serum (FBS) at the concentration of 8000
cells per well. The cells were grown by maintaining at 37 °C in a
humidified atmosphere containing 5% CO2 for 16 h. The required
amounts of 5(6)-carboxyfluorescein tagged PNA stock solutions were
added to the corresponding wells to achieve the desired final
concentration of 1 μM. The cells incubated with tagged PNA
oligomers were maintained at 37 °C in a humidified atmosphere
containing 5% CO2 for 24 h. After the incubation period was over, the
medium was aspirated and the cells were rinsed or washed thrice with
1 medium-volume equivalent of 1X phosphate-buffered saline (PBS).
The cells were then replenished with 400 μL of OPTIMEM medium
containing Hoechst 33342 at a dilution of 1/1000 of the supplied
stock solution (10 mg/mL) and left for 15 min at 37 °C. After
incubation for 15 min, the medium containing nuclear stain Hoechst
33342 was removed and rinsed with 1X PBS and replenished with
fresh OPTIMEM medium. The cells were then immediately visualized
using 40× objective of a laser scanning confocal microscope. Cells
incubated with 5(6)-carboxyfluorescein including unmodified aeg-PNA
were used as control. The results are indicative of two independent
biological replicates.
Quantification of Cellular Uptake by FACS Analysis. MCF-7
and NIH 3T3 cells were plated in 9 × 60 mm dishes in 2 mL of
DMEM medium at a concentration of 1 × 106 cells per dish. The cells
were maintained at 37 °C in a humidified atmosphere containing 5%
CO2 for 16−18 h. The required amounts of 5(6)-carboxyfluorescein-
tagged PNA stock solutions were added to the corresponding wells to
achieve the desired final concentration of 1 μM. The cells incubated
with tagged PNA oligomers were maintained at 37 °C in a humidified
atmosphere containing 5% CO2 for 24 h. After the incubation period
was over, the medium was aspirated and the cells were rinsed or
washed thrice with 1 medium-volume equivalent of ice-cold PBS. Cells
were collected by trypsinization using 0.5 mL of 0.05% trypsin−EDTA
for each dish. The trypsinization process was stopped by using 3 mL of
DMEM medium, and the cells were transferred to 15 mL tubes. These
tubes containing cells were centrifuged at 150g for 5 min at 4 °C; the
supernatant was aspirated, and the cells were washed thrice with ice-
cold 1X PBS. The cell suspension in PBS was then transferred to a
FACS tube, and the samples were immediately analyzed on flow
cytometer. The data obtained from FACS were processed using
CellQuest Pro software. The cell permeation of PNA oligomers has
been confirmed by histograms obtained from the experiment. The
percent positive cells for each individual PNA were plotted on
Microcal Origin 8.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR and 13C NMR spectra of intermediate compounds 2−
13; HPLC and MALDI-TOF spectra of PNA oligomers (PNA
1−10 and fluorescent PNAs); UV-melting profiles of various
PNA:DNA and PNA:RNA duplexes; CD spectra of all single-
stranded PNAs and duplexes of modified PNAs with DNA 1
and RNA 1; EtBr displacement by various PNAs; confocal
images of various fluorescently labeled PNAs and FACS
analysis in MCF-7 cells; MTT assay in NIH 3T3 cells. 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.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D. R. J. acknowledges IISER Pune for award of a research
fellowship. L. A. V. acknowledges INSPIRE-DST for award of
fellowship. K. N. G. is a recipient of the JC Bose Fellowship,
Department of Science and Technology, New Delhi. We thank
the Director of NCCS, Pune, for assistance and helpful
discussion in FACS analysis.
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