Clickable peptide nucleic acids (cPNA) with tunable affinity

Article abstract of DOI:10.1039/c0cc01081b

Peptide nucleic acids (PNAs) are functional analogues of natural oligonucleotides. Herein, we report the synthesis of PNAs bearing a triazole in lieu of the amide bond assembled using a "click" cycloaddition, their hybridization properties as well as the DNA-templated coupling of the azide and alkyne PNA fragments. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0cc01081b

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Clickable peptide nucleic acids (cPNA) with tunable affinityw
Dalila Chouikhi, Sofia Barluenga and Nicolas Winssinger*
Received 22nd April 2010, Accepted 9th June 2010
First published as an Advance Article on the web 23rd June 2010
DOI: 10.1039/c0cc01081b
Peptide nucleic acids (PNAs) are functional analogues of natural
oligonucleotides. Herein, we report the synthesis of PNAs
bearing a triazole in lieu of the amide bond assembled using a
‘‘click’’ cycloaddition, their hybridization properties as well as
the DNA-templated coupling of the azide and alkyne PNA
fragments.
Compared to natural oligonucleotides, PNAs1,2 are endowed
with higher melting temperature (Tm), greater sensitivity to
mismatches, and are more resistant to chemical and enzymatic
degradation. These features have stimulated a number of
applications ranging from biotechnology to medicine.3,4 We
have been particularly interested in PNAs as an encoding tag
for supramolecular assemblies.5,6 Herein, we report the use of
an azide–alkyne ‘‘click’’ coupling to assemble PNA fragments
and the hybridization properties of these new modified PNA.
Scheme 1 Synthesis of azido PNA 3 and alkynic PNA 6–7. (a)
The copper-catalyzed coupling of azides with alkynes7,8
has emerged as one of the most robust (bio)conjugation
technologies.9,10 Additionally, the resulting 1,4-triazole has
been shown to be a good mimic of a trans-amide bond.11–14
Furthermore, DNA bearing a triazole linkage in lieu of the
phosphate have already been reported.15,16 Based on these
precedents, we hypothesized that a triazole substitution of the
amide within the PNA backbone should be tolerated and
would provide access to PNA via click cycloadditions. To
investigate the impact of this substitution, the azido-PNA
monomers 3 (Scheme 1) were prepared from compound 1 as
previously described17 but with nucleobase-substituted acids 2
bearing Boc groups18,19 which are compatible with Mtt
deprotection conditions.20 Two alkyne bearing PNA monomers
(6 and 7, Scheme 1) with one or two carbon atoms between the
alkyne and the amide linkage to the nucleobase were prepared.
Thus reaction of propargyl chloride or tosylated homo-
propargylic alcohol with ethylene diamine21 followed by
selective Mtt protection of the primary amine afforded
compounds 4 and 5 respectively. Standard HBTU coupling
with nucleobase-substituted acids 2 afforded the desired PNA
monomers 6 and 7. This chemistry is readily scalable to obtain
gram-quantities of all four nucleic acid monomers. PNA
oligomers bearing the triazole modifications were prepared
by standard Fmoc-based PNA synthesis, using Boc-protected
nucleobases, on a PEG-based resin (NovaPEG) with a Rink
linker (Scheme 2). Oligomers were terminated with an azido
2-Aminoethylbromide (1.0 equiv.), NaN3 (1.1 equiv.), DMF, 60 1C,
3 h; then, benzyl 2-bromoacetate (0.8 equiv.), Et3N (2.0 equiv.), 0 1C,
2 h, 64%; (b) 2 (1.0 equiv.), HBTU (1.0 equiv.), Et3N (2.0 equiv.),
DMF, 23 1C, 12 h, 40–90%; (c) NaOH (4.0 equiv.), dioxane/H2O,
23 1C, 1 h, 45–75%; (d) ethylene diamine (10.0 equiv.), DBU
(1.0 equiv.), toluene, 23 1C, 4 h, 52–53%; (e) Mtt-Cl (1.3 equiv.),
Et3N (2.0 equiv.), CH2Cl2, 23 1C, 5 h, 60%.
monomer 3 yielding oligomers 8 and coupled to an alkyne
monomer 6 or 7 to obtain a t1 or t2 junction respectively
(Scheme 2). The cycloaddition (click) was carried out in a
NMP : water mixture (10 : 1) at room temperature for 12
hours with 7.5 equivalents of alkyne monomer 6 or 7
(70 mM) in the presence of copper sulfate (2.5 mM), TBTA
ligand (5 mM) and ascorbic acid (70 mM). The reactions were
found to reliably proceed to completion based on the analysis
of the cleavage product from an analytical aliquot of resin.
The Mtt was removed with hexafluoroisopropanol (HFIP)
and the standard PNA synthesis was continued with or
without reiteration of cycloaddition (click) couplings. A first
set of three 8mers containing no modification (10, Scheme 2), a
t1 linkage (11) and a t2 linkage (12) were prepared to assess the
impact of these modifications. Hybridization to DNA at 5 mM
revealed Tms of 57 1C, 55 1C and 45 1C for 10, 11 and 12
respectively. Thus, the t2 junction strongly destabilizes the
duplex while t1 has a modest impact. We next asked whether
the triazole junction affected the fidelity of hybridization. To
this end, we used a microarray bearing 625 oligonucleotides
which has been used to decode PNA-libraries.22 The 625
sequences represent all the combinatorial permutations of
four sets of codons encoding five elements of diversity each
(5 Â 5 Â 5 Â 5 = 625, see Fig. S2, ESIw) for the sequences and
their position on the array). It thus follows that for any given
spot of the microarray (i.e. sequence in the library), there will
´
´
Institut de Science et Ingenierie Supramoleculaires (ISIS—UMR
´
´
7006)Universite de Strasbourg—CNRS, 8 allee Gaspard Monge,
F67000 Strasbourg, France. E-mail: winssinger@unistra.fr;
Fax: +33 368855112
w Electronic supplementary information (ESI) available: experimental
procedures and microarray data. See DOI: 10.1039/c0cc01081b
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Scheme 2 Synthesis of PNA oligomers containing triazole linkages. (a) Alkyne monomer (7.5 equiv.), sodium ascorbate (7.5 equiv.), CuSO4 (0.25
equiv.), TBTA (0.5 equiv.), NMP/H2O 10 : 1, 23 1C, 12 h; (b) HFIP/DCE (200 mL per 10 mg of resin) 4 Â 2 min; (c) monomer (4.0 equiv.), HATU
(3.5 equiv.), 2,6-lutidine (6.0 equiv.), DIPEA (4.0 equiv.), NMP, 23 1C, 25 min; (d) Lys(Fmoc)NHBoc (4.0 equiv.), HATU (3.5 equiv.), 2,6-lutidine
(6.0 equiv.), DIPEA (4.0 equiv.), NMP, 23 1C, 25 min; (e) 20% piperidine in DMF, 5 Â 2 min; then Cy3 (5.0 equiv.), TNTU (4.5 equiv.), DIPEA
(10.0 equiv.), 20% DMSO in NMP, 40 1C, 12 h; (f) neat TFA, 23 1C, 3 h.
be 19 other spots on the array which share three of the same
codons and will have only two to three mismatches. The array
is laid out so that mismatches at the beginning (C-terminus of
the PNA) or end (N-terminus of the PNA) appear vertically or
horizontally, respectively, while mismatches on the internal
portion of the sequence appear on diagonal lines. We had
previously optimized hybridization conditions for 14-mer
PNAs and found that PNAs could be detected at or below
1 nM with reliable fidelity by hybridizing at 50 1C.22 We thus
prepared five different 14mer PNAs (13–17) with a Cy3
fluorophore18 containing different combinations of triazole
linkages to assess their impact. All probes were hybridized at
0.2, 1 and 5 nM at 50 1C on a slide containing sixteen
subarrays thus insuring homogeneous conditions for each
Fig. 1 Fidelity of hybridization for cPNA. Peptide nucleic acids
13–16 were hybridized to a microarray containing 625 different
experiment. Comparing the intensity of the perfect match vs.
mismatched hybridization at a given concentration reflects the
affinity of the PNA and its fidelity. As shown in Fig. 1,
hybridization of PNA 13 (1 nM, see Fig. S2, ESIw) for a
picture of the whole slide containing the lower and higher
concentrations) showed a strong intensity for the perfect
match (PM) and weak intensities for hybridization to se-
quences sharing three out of the four codons. Mismatched
hybridizations in the vertical line were most preponderant
reflecting the lower impact of a mismatch at the terminal
residues relatively to the internal residues. As evident from
the comparison of probes 14 and 16 containing a single
t1-linkage to unmodified PNA 13, a single t1 modification
has negligible impact on the overall hybridization equilibrium
and slightly improves specificity (comparing the ratio of the
perfect match [PM] to the most intense mismatch [MM]).
Hybridization of the PNA containing the t2 linkage leads to
a sharp decrease in intensity (8 fold) reflecting a lower affinity
concurring the Tm measurements by hypochromicity. The
PNA containing two t1 modifications separated by two pseudo-
nucleosides shows a lower intensity (three fold) without
a notable compromise in the selectivity. Whether the distance
between triazole junctions has a strong impact remains to
sequences.
be investigated. The mismatched hybridization showing the
strongest intensity corresponded to the sequence GCC GGG
À À À
CCA CCT GC (wherein the underlined residues represent the
mismatch) and no signal was detected for mismatches of
residues containing the triazole linked nucleotides.
We then turned our attention to the DNA-templated copper
catalyzed cycloaddition of alkynic PNA with azido PNA.
There is significant interest in these types of reactions for
nucleic acid sensing23–25 and to translate DNA into synthetic
polymers.26–28 While a number of copper catalyzed ‘‘click’’
cycloadditions have been reported in the presence of DNA,
our first efforts were unfruitful and led to decomposition of the
DNA.29,30 We found it essential to use a large excess of ligand
(THPTA31) relatively to copper to avoid DNA degradation.
Using 7.0 equiv. of THPTA relatively to copper sulfate (which
is reduced in situ with ascorbic acid), we observed a complete
coupling for the PNA matching the DNA template (21 + 20)
at 21 1C (Fig. 2) whereas negligible reaction was observed if
the azide component was not complementary to the DNA
template (23 + 20) when performing the reaction at or below
10 mM. A single base pair mismatch in the azide component
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This work was supported by grants from Institut Universi-
taire de France (IUF) and European Research Council (ERC)
as well as a fellowship from the Algerian Ministry of Higher
Education and Scientific Research (DC). We thank Laurence
Oswald for her technical assistance and Antoine Dieudonne-
´
Vartran for preliminary work. We thank Vincent Aucagne for
a sample of THPTA.
Notes and references
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5478 | Chem. Commun., 2010, 46, 5476–5478