PNAs grafted with (α/γ, R/S)-aminomethylene pendants: Regio and stereo specific effects on DNA binding and improved cell uptake

Article abstract of DOI:10.1039/c0cc03988h

PNAs grafted with cationic aminomethylene (am) pendants on the backbone at the glycyl (α) or ethylenediamine (γ) segments show regio (α/γ) and stereochemistry (R/S) dependent binding with complementary DNA. These are efficiently taken up by cells, with γ(S-am) aeg-PNA being the best in all properties.

Full text of DOI:10.1039/c0cc03988h

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PNAs grafted with (a/c, R/S)-aminomethylene pendants: Regio and
stereo specific effects on DNA binding and improved cell uptakew
Roopa Mitrab and K. N. Ganesh*ab
Received 20th September 2010, Accepted 27th October 2010
DOI: 10.1039/c0cc03988h
PNAs grafted with cationic aminomethylene (am) pendants on
the backbone at the glycyl (a) or ethylenediamine (c) segments
show regio (a/c) and stereochemistry (R/S) dependent binding
with complementary DNA. These are efficiently taken up by
cells, with c(S-am) aeg-PNA being the best in all properties.
In this context we proposed to synthesise PNAs with chiral
and cationic backbones consisting of aminomethylene (am)
substituents either at the a-C of glycine (Fig. 1b and 1c) or on
the g-C of the ethylenediamine (Fig. 1d) fragments. The
shorter side chain with one methylene reduces the non-specific
electrostatic interactions in the derived PNA–DNA duplexes
and yet still being cationic may also enhance cell uptake.
We herein report the synthesis of a-(S/R-am) aeg-PNA-T
monomers (1,2) and a g-(S-am) aeg-PNA-T monomer 3
(Scheme 1) starting from D/L-aspargine and their incorporation
into PNA oligomers P2–P4 (Table 1). It is found that g-(S-am)
aeg-PNA P4 binds to complementary DNA very strongly and
taken up in HeLa cells better than the a-(R/S-am) analogues.
PNA oligomers (P2–P4) incorporating the aminomethylene
side chains at the a or g positions as pendant groups on the
backbone were made from the appropriate monomer, 1–3,
which in turn were synthesised from L or D-asparagine (4a/b)
according to Scheme 1. The side chain a-aminomethylene
group was generated from reduction of the amide in L or
D-aspargine using iodobenzenediacetate12 to give 5. This was
transformed to the fully protected 6, which is the common
intermediate to reach both the a and g monomers 1–3 through
two different synthetic paths. Route A gave the a-(S/R)-am
monomers 1 and 2, while route B lead to g-(S)-am analogue 3
employing the reagent sequences shown in Scheme 1 (details in
ESIw). Stereochemically, L-aspargine (4a) leads to a-(S) 1 and
g-(S) 3 monomers, while D-aspargine (4b) gave the a-(R)
monomer (2).
Peptide Nucleic Acid (PNA)1 (Fig. 1a) has emerged as
a promising class of oligonucleotide mimics with potential
therapeutic and diagnostic applications. In PNA, the neutral
and structurally homomorphous pseudo-peptide backbone
(aminoethylglycine, aeg) is linked to the bases A, C, T and
G. PNA hybridizes to complementary DNA and RNA with
high affinity and selectivity is mediated by Watson–Crick base
pairing. The unnatural polyamide linkage also makes it resistant
to enzymatic degradation.1 The simplicity of its structure with
distinguishable properties, ease of synthesis and low toxicity
has made aeg-PNA attractive as a putative genetic regulatory
agent.2 But its application as an effective antisense drug is
limited by its low aqueous solubility, ambiguity in parallel or
anti parallel3 orientation of binding the target DNA or RNA
and its uptake by mammalian cells. This has lead to a wide
variety of chemical modifications.3,4
Incorporation of a positive charge in PNA variants carrying
chiral D/L-lysine units in place of glycine, has shown promising
properties in terms of improved solubility and higher binding
of single-stranded DNA.5 X-ray structure analysis of D-lysine
based PNA indicated the backbone oxygen atom of the amide
carbonyl to be in proximity of g-carbon of the side chain
(3.17 ꢀ 0.1 A).6 Based on this, Topham et al.7 predicted from
theoretical calculations that a PNA backbone composed of
3-amino-D-alanine should promote preferential formation of a
P-form helix through an intramolecular hydrogen bond
between the side chain amino function at the a-C and the
amide carbonyl of the backbone. Recent reports have indicated
that L-lysine8 and L-serine9 derived g-substituted PNA units
within unmodified PNA oligomers act as helical directors due
to conformational pre-organization. It has also been reported
that cationic guanidinium groups grafted on PNAs (GPNA)10
and the phosphate backbone of oligonucleotides11 enhance the
cell uptake of the substrates.
The monomers 1–3 were incorporated at the desired sites
(denoted by lowercase letters in Table 1) within the aeg-PNA
sequence P1 via solid phase synthesis using MBHA resin (see
ESIw) to obtain am-PNA decamers P2–P4, each having two
modified sites. The corresponding fluorescent oligomers
cfP1–cfP4 needed for cell uptake assays were synthesised by
coupling 5- and 6-carboxyfluorescein at the N-terminus
of the PNA oligomers before cleavage from the resin. All
PNA oligomers were purified by HPLC, characterized by
MALDI-TOF and used further for DNA hybridization and
cell uptake studies.
Inspite of the presence of chiral centres, a-(R/S)-am single
stranded PNAs P2 and P3 did not show appreciable CD
signals. However, the g-(S-am) aeg PNA oligomer P4
exhibited a prominent negative CD signal at 273 nm suggesting
a considerable helical pre-organization induced via base stacking
(see ESIw). The complexing ability of aeg-PNA P1 and the
chiral, cationic (a/g-am)-PNA oligomers P2–P4 with the 10-mer
complementary DNA D1 (antiparallel), D2 (parallel) and
DNA D3 (single C : C mismatch) were assessed by temperature
dependent UV-absorbance experiments. The stoichiometry of
the PNA : DNA complexes was found to be 1 : 1 by CD Jobs
a Indian Institute of Science Education and Research, 900,
NCL Innovation Park, Dr HomiBhabha Road, Pune 411008, India.
E-mail: kn.ganesh@iiserpune.ac.in; Fax: 91 (20) 2589 9790;
Tel: 91 (20) 2590 8006
b Division of Organic Chemistry, National Chemical Laboratory,
Dr Homi Bhabha Road, Pune 411008, India
w Electronic supplementary information (ESI) available: Experimental
procedures for synthesis and cell uptake experiments, MTT assay, 1H
and 13C NMR of synthetic intermediates, HPLC, MALDI-TOF
spectra of PNA oligomers, CD Jobs plot, UV-Tm data, cell uptake
results. See DOI: 10.1039/c0cc03988h
c
1198 Chem. Commun., 2011, 47, 1198–1200
This journal is The Royal Society of Chemistry 2011

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Fig. 1 Structures of PNAs (a) aeg-PNA (b) a(S-am) aeg-PNA (c)
a(R-am) aeg-PNA and (d) g(S-am) aeg-PNA.
Fig.
2
Comparative DTm values for hybrids of aeg-PNA P1,
a-(S-am)-PNA P2, a-(R-am)-PNA P3 and g-(S-am)-PNA P4 with
DNAs D1–D3. (+) and (ꢁ) signs of DTm correspond to stabilisation
and destabilisation of duplexes, respectively.
The Tm data in Table 2 indicates that the cationic am-PNAs
(P2–P4) form more stable duplexes than the unmodified
aeg-PNA P1 with the complementary DNAs D1 and D2.
Among the a-am-PNA : DNA D1 antiparallel duplexes, the
(R-am)-PNA P3 had a Tm (4.3 1C-modification) higher than
that of (S-am)-PNA P2 (2.6 1C-modification). The g-(S-am)
aeg-PNA P4 stabilised the antiparallel duplex most with
DNA D1 (6.5 1C-modification). It may be pointed out
that the Tm enhancements seen with the present am-PNA
modifications are better than B2.0 1C-modification seen with
g-guanidine-PNA.6 The results indicate that the shorter
cationic side chains enhance binding significantly probably
due to steric factors.
Scheme
1 Synthesis of a-(S/R-am) 1,2 and g-(S-am) 3 PNA
monomers.
Table 1 PNA sequences with am modifications
The orientational selectivity in binding by the chiral PNAs,
was examined using DNA D2 that can form parallel duplexes.
The unmodified PNA P1 does not show much difference in Tm
of antiparallel and parallel duplexes. The a-(R-am)-PNA P3
destabilised the parallel duplex (DTm = ꢁ9.1 1C) more than
the a-(S-am)-PNA P2 (DTm = ꢁ3.8 1C) compared to the
corresponding antiparallel duplexes. However, g-(S-am)-PNA
P4 was even better at stabilising the antiparallel duplex
(DTm = ꢁ12.6 1C) than the parallel duplex. Thus g-(S-am)
aeg-PNA P4 not only shows a higher duplex stability with
DNA, but also possess a better orientational selectivity than
the a-(R/S-am) modifications.
PNA
Sequence
aeg
P1
P2
P3
P4
cfP1
cfP2
cfP3
cfP4
H-T T A C C T C A G T-lys NH2
a-(S-am)
a-(R-am)
g-(S-am)
aeg PNA
a-(S-am)
a-(R-am)
g-(S-am)
H-T ꢁta(S) A C C ꢁta(S) C A G T-lys NH2
H-T ꢁta(R) A C C ꢁta(R) C A G T-lys NH2
H-T ꢁtc(S)A C C ꢁtc(S)C A G T-lys NH2
H-CF-T T A C C T C A G T-lys NH2
H-CF-Tta(S) A C Cta(S) C A G T-lys NH2
H-CF-T ta(R)A C C ta(R) C A G T-lys NH2
H-CF-T ꢁtc(S) A C C ꢁtc(S) C A G T-lys NH2
ta(S) = a-(S-am) aeg-PNA, ta(R) = a-(R-am) aeg-PNA, tc(S)
=
ꢁ
ꢁ
ꢁ
g-(S-am) aeg-PNA, CF = carboxyfluorescein.
The sequence specificity of binding of cationic am-PNAs
was tested with DNA D3 having one base C : C mismatching
the middle of the antiparallel PNA : DNA duplex (Fig. 2,
green bars). While the Tm of the single mismatch duplex from
unmodified aeg-PNA P1 was lower by 7 1C, for am-PNAs, the
destabilization increased in the order P2, P3 and P4. This data
indicated that the higher binding affinity of the cationic PNAs
am-PNAs P2–P4 with anionic DNA, was achieved without
sacrificing the sequence specificity. Both the site-specificity
(a/g) and the stereochemistry (R/S) of aminomethylene side
chains seem to be important determinants in defining the
stability of derived PNA : DNA duplexes.
plot (see ESI w) as expected for a duplex. The melting
temperature Tm of PNA–DNA hybrids obtained from the
UV data are shown in Table 2. The comparative differential
Tm (DTm) computed for modified P2–P4 oligomers in relation
to unmodified P1 and for analogous antiparallel, parallel and
mismatch PNA : DNA duplexes are shown in Fig. 2.
Table 2 UV-Tm (1C) studies of aeg PNA and am-PNA : DNA
duplexesa
PNA
No
DNA D1(ap)
DNA D2(p)
DNA D3(ap)
aeg PNA
a-(S-am)
a-(R-am)
g-(S-am)
P1
P2
P3
P4
49.2
54.4
57.7
62.3
48.5
50.6
48.6
49.7
42.2
42.5
41.0
44.0
The uptake of fluorophore labelled cationic PNAs
cfP1–cfP4 in HeLa cancer cells was measured with cells
cultured on adherent monolayers in D-MEM media, incubated
separately with each cf-PNA (1 mM) for 24 h. After washing
with PBS, cells were imaged after fixing (see ESIw). The control
cells not treated with PNA were stained with DAPI, a known
dye for nuclear staining (Fig. 3). It is seen from the images that
a
ap, antiparallel DNA D1 = 50ACTGAGGTAA30; p, parallel DNA
D2 = 50AATGGAGTCA30; C–C base pair mismatch DNA D3 =
50ACTGCGGTAA30. Parallel: PNA (N - C) : DNA(50 - 30);
ꢁ
antiparallel PNA (N - C) : DNA(30 - 50).
c
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stereospecificity influences the efficiency of the cell uptake. The
temperature experiments suggest this process to be energy
dependent.
In conclusion, we have presented here the design and
synthesis of regio (a/g) and stereo specifically (R/S) modified
PNAs carrying shorter cationic aminomethylene side chains
on the backbone. Biophysical studies of am-PNAs show that
the g-(S-am)-PNA P4 has the best sequence selectivity and
the best DNA duplex stabilising properties. The cationic
am-PNAs are taken up in cells better than the unmodified
PNA P1 with the g-(S-am)-PNA P4 being most efficient. It is
also significant that cell uptake depends not only the site of
modification, but also on its stereochemistry. The combination
of highly sequence specific DNA binding and cell uptake
properties along with their non-toxicity are very desirable
attributes of cationic am-PNAs for their future potential
applications. The amino function on the backbone will also
be useful for conjugation with other functional and reporter
molecules.13 Future work in terms of increasing the cationic
content, RNA complexing and cell-type specific uptake
abilities are in progress.
Fig.
3 Nuclear localisation of PNAs in cells (A) Differential
Interference Contrast (DIC) image of cells (B) Cells stained with
DAPI (C) Cells treated with cfP4 and (D) overlapping of B and C.
cf-PNA P4 is localised in the nucleus similar to DAPI
(Fig. 3C) and confirmed from the super imposed images
(Fig. 3D). cfPNAs P1–P3 also showed similar behaviour even
at the lowest of concentrations studied (0.25 mM). The results
indicated that although all PNAs could enter the nucleus,
the true effects of the cationic am-PNAs could not be
distinguished.
RM thanks UGC (New Delhi) for award of a research
fellowship. KNG is a recipient of JC Bose Fellowship from
DST (New Delhi). We thank Prof Sanjeev Galande and Dr
Mayurika Lahiri (IISER Pune) for their helpful discussions
and assistance in the biological experiments.
The live cells were analysed by flow cytometry (FACS) after
incubation with cf-PNAs P1–P4 at 4 1C and 37 1C (Fig. 4). As
seen from the fluorescence intensities, which are proportional
to relative uptake efficiency, at 37 1C aeg-PNA cfP1 and
a-(S-am)-PNA cfP2 showed only modest uptake in equal
amounts followed by a slightly higher uptake of a-(R-am)-
PNA cfP3 and a maximum uptake of g(S-am)-PNA cfP4. In
comparison, at a lower temperature of 4 1C, the fluorescence
intensities were 10 fold higher for all PNAs compared to those
at 37 1C and the cell count was lower. At a higher temperature,
the number of cells incorporating the fluorescent PNA was
higher by 2 fold, but the intensity of fluorescence was lower,
perhaps due to quenching effects. The cellular toxicity of the
am-PNAs as measured by an MTT assay at different concen-
trations (see ESIw), showed the percentage viability to be more
than 80%. These results indicate overall that not only
do the pendant aminomethylene cationic charges on the PNA
backbone improve the cell uptake but also their regio and
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1200 Chem. Commun., 2011, 47, 1198–1200
This journal is The Royal Society of Chemistry 2011