Cationic pentaheteroaryls as selective G-quadruplex ligands by solvent-free microwave-assisted synthesis

Article abstract of DOI:10.1002/chem.201202097

We report herein a solvent-free and microwaved-assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4-oxadiazole moieties (1-7). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4-FID) and CD spectroscopy. Among the G-quadruplexes considered, attention was focused on telomeric repeats together with the proto-oncogenic c-kit sequences and the c-myc oncogene promoter. Compound 1, and to a lesser extent 2 and 5, preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole (TOxaPy), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties. Copyright

Full text of DOI:10.1002/chem.201202097

FULL PAPER
DOI: 10.1002/chem.201202097
Cationic Pentaheteroaryls as Selective G-Quadruplex Ligands by
Solvent-Free Microwave-Assisted Synthesis
Michele Petenzi,[a] Daniela Verga,[b] Eric Largy,[b] Florian Hamon,[b] Filippo Doria,[a]
Marie-Paule Teulade-Fichou,*[b] Aurore Guꢀdin,[c] Jean-Louis Mergny,[c]
Mariella Mella,[a] and Mauro Freccero*[a]
Abstract: We report herein a solvent-
free and microwaved-assisted synthesis
of several water soluble acyclic penta-
heteroaryls containing 1,2,4-oxadiazole
moieties (1–7). Their binding interac-
tions with DNA quadruplex structures
were thoroughly investigated by FRET
melting, fluorescent intercalator dis-
placement assay (G4-FID) and CD
spectroscopy. Among the G-quadru-
plexes considered, attention was fo-
cused on telomeric repeats together
with the proto-oncogenic c-kit sequen-
ces and the c-myc oncogene promoter.
Compound 1, and to a lesser extent 2
and 5, preferentially stabilise an anti-
parallel structure of the telomeric
DNA motif, and exhibit an opposite
binding behaviour to structurally relat-
ed polyoxazole (TOxaPy), and do not
bind duplex DNA. The efficiency and
selectivity of the binding process was
remarkably controlled by the structure
of the solubilising moieties.
Keywords: 1,2,4-oxadiazoles
·
DNA recognition · G-quadruplexes ·
microwave
chemistry
·
pentaheteroaryls
Introduction
Organic compounds capable of recognising and stabilising
the G-quadruplex supramolecular structure of nucleic acids
(G4) have attracted great attention as selective probes and
anticancer agents.[1] Beside a large array of reversible li-
gands,[1b,2] selective G4 alkylating agents are beginning to
emerge.[3] Dual selectivity towards quadruplex versus duplex
DNA and different quadruplex architectures is a key aspect
for developing effective diagnostic and pharmacological ap-
plications for G4 ligands.[4] The selectivity issue is still pro-
pelling the design of new G4 binders. In spite of the large
variety of synthetic ligands created to date, the natural mac-
rocycle telomestatin (Scheme 1) still exhibits a unique neu-
tral molecular architecture, combining excellent binding, se-
lectivity and remarkable antitumor activity.[5] Its structural
features are still inspiring the design and synthesis of a large
number of both cyclic and acyclic polyheterocyclic ligands.[6]
Scheme 1. Cyclic and acyclic polyheteroaryl ligands based on oxazoles
and the prototype BOxAzaPy (R=H) together with the water soluble
analogues (R=H, aminoalkyls) embedding 1,2,4-oxadiazole moieties
(BOxAzaPy).
Several of them, including the recent TOxaPy, exhibit poly-
oxazole structures.[7] The potentials of acyclic derivatives as
G4 ligands have been highlighted by an unexpected specific-
ity of TOxaPy (Scheme 1), resulting probably from groove
interactions.[7] The binding properties were strongly depend-
ent on the length of the oligomeric ligand, as the shorter
pentaheteroaryl BOxaPy did not bind to G4 targets. In
order to systematically investigate the full potential of acy-
clic polyheterocyclic structures as G4 ligands, we began a
joint project in which some of us designed and synthesised a
family of a fairly similar scaffold containing 1,2,4-oxadiazole
moieties.
[a] M. Petenzi, Dr. F. Doria, Prof. M. Mella, Prof. M. Freccero
Dipartimento di Chimica, Universitꢀ di Pavia
V.le Taramelli 10, 27100 Pavia (Italy)
Fax : (+39)0382987668
E-mail: mauro.freccero@unipv.it
[b] Dr. D. Verga, Dr. E. Largy, Dr. F. Hamon, Dr. M.-P. Teulade-Fichou
UMR176 CNRS, Institut Curie, Centre de Recherche
Centre Universitaire, 91405 Orsay (France)
Fax : (+33)169-07-53-81
E-mail: marie-paule.teulade-fichou@curie.fr
[c] A. Guꢁdin, Dr. J.-L. Mergny
Water solubility of the resulting pentaheteroaryls was ach-
ieved by two symmetric and structurally modular amino-
alkyl or hydroxy-alkyl groups (BOxAzaPy compounds,
Scheme 1), introduced by an efficient microwave-assisted
Universitꢁ de Bordeaux, INSERM U869
IECB. 2 rue Robert Escarpit, 33600 Pessac (France)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202097.
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functionalisation of the prototype BOxAzaPy scaffold. The
replacement of the oxazoles by 1,2,4-oxadiazoles should, in
principle: 1) allow the formation of a nitrogen binding
cavity in the bent conformer, such as in the telomestatin-
like ligands, and 2) erase the CH–CH steric repulsions
within the oxazole-based oligoheteroaryls, like BOxaPy and
TOxaPy. The above structural features, including the cation-
ic appendages, could have a strong impact on the binding
mode of the new polyheterocycles toward G4. This aspect
has been thoroughly investigated in the present study. Final-
ly, in order to fully compare the oxazole and oxadiazole
series, we prepared three cationic derivatives of BOxaPy
(8–10) bearing side chains similar to those embedded in the
BOxAzaPy series.
Scheme 3. Stepwise and one-pot synthesis of BOxAzaPy-Br. a) 1 equiv
NH2OH.HCl, Na2CO3, EtOH/H2O 2:1, room temperature, 1 h; b) CDI,
DMF, 30 min, room temperature; 12 h, T=1508C; c) identical to step a;
d) cyclodehydration, see step b; e) 2.2 equiv NH2OH.HCl, Na2CO3,
EtOH/H2O 2:1, room temperature, 1 h; f) cyclodehydration, see step b.
Results and Discussion
set of basic or protic moieties at the C-2 positions on both
the terminal pyridines in the prototype BOxAzaPy scaffold.
Previous reports suggest that aromatic nucleophilic substitu-
tion (SNAr) with halo-aromatics, such as 2-fluoropurines
with amines, requires a temperature higher than 1008C, and
an extended reaction time (12–24 h). Reaction time could
be significantly shortened (15 min) and reaction yields im-
proved following a microwave-assisted protocol in polar
aprotic solvents, such as ACN (acetonitrile) or ACN/NMP
(N-methylpyrrolidinone) mixture.[9] In order to define the
best synthetic protocol for the SNAr reactions on the BOx-
AzaPy-Br (Scheme 4), we carried out a comparative evalua-
One step synthesis of the prototype BOxAzaPy: Several
methods are currently available for the synthesis of mono-
functional 1,2,4-oxadiazoles. Among these, the condensation
of amidoxime with carboxylic acids in the presence of a cou-
pling reagent is often considered particularly attractive.[8]
We chose 1,1’-carbonyldiimidazole (CDI) as a coupling re-
agent because the subsequent cyclodehydration can be
easily performed one-pot in the presence of the same CDI,
in DMF, without isolation of the resulting O-acylbenzami-
doxime (Scheme 2). In spite of the fairly low yield (25%),
the protocol was very effective as the resulting BOxAzaPy
crystallised from the crude with high purity.
Scheme 4. Synthesis of water soluble BOxAzaPy compounds (series I) by
a solvent-free and microwave-assisted aromatic nucleophilic substitution
(SNAr).
Scheme 2. Synthesis of the prototype BOxAzaPy: a) CDI, DMF, 30 min,
room temperature; b) 12 h, T=1508C (25%; yield).
Stepwise synthesis of the precursor for the water soluble
BOxAzaPy compounds: As a first attempt, a very similar
one-pot strategy was followed for the synthesis of the
bromo analogue BOxAzaPy-Br starting from N’2,N’6-dihy-
droxypyridine-2,6-bis(carboximidamide) and 6-bromopyri-
dine-2-carboxylic acid (Scheme 3).
Unfortunately, such a direct approach yielded the BOx-
AzaPy-Br as by-product in a sloppy mixture with very low
yield (<3%). A much better result was achieved with a
stepwise synthesis, by performing the O-acylation and the
cyclodehydration to the oxadiazole moieties in two sequen-
tial steps (Scheme 3, b and d).
tion of the thermal and the microwaved reactions in both
DMF and in neat amine for compound 1. Poor conversions
were observed with both DMF as solvent and neat amine
coupled with the thermal treatment. The results summarised
in Table 1 unambiguously suggest the solvent-free micro-
wave-assisted approach as the most effective. Therefore, this
synthetic method was extended to an array of primary
amines (Table 1). The resulting water soluble BOxAzaPy
compounds were purified by preparative HPLC (gradient
ACN/H2O; 0.1% CF3COOH), subsequently exchanging the
trifluoroacetate with chloride anion, to yield the BOxAzaPy
compounds as hydrochlorides 1–7 (Table 1).
Solvent-free microwave-assisted synthesis of the water solu-
ble BOxAzaPy compounds (series I): In order to improve
water solubility, we specifically desired access to a diverse
Synthesis of cationic BOxaPys (series II): In order to inves-
tigate the full potential of these polyheterocyclic structures,
and to compare their activity as G4 binders as a function of
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Table 1. Reactivity of several amines with BOxAzaPy-Br.
BOxAzaPy
Amines
Conditions[a]
Yields
[%][b]
Yields
[%][c]
mW, neat amine
mW, DMF
D, neat amine
D, DMF
21.4
27.8
ꢀ2
–
–
–
1
2
11.0
6.3
mW, neat amine
24.6
53.3
3
4
5
mW, neat amine
mW, neat amine
mW, neat amine
28.7
46.5
27.7
54.4
56.8
51.4
6
7
mW, neat amine
mW, neat amine
21.9
22.6
66.7
47.6
[a] Microwave (mW), standard conditions in 5 mL microwave vials:
amine (2 mL) in the presence of BOxAzaPy-Br (100 mg, 0.19 mmol), mi-
crowaved 8 min at T=1008C, P=462 kPa; D, standard conditions in
2 mL of amine or DMF (with 2 equiv amine), 24 h at T=1008C.
[b] Yields measured on the basis of isolated products by preparative
HPLC, followed by trifluoroacetate/chloride anion exchange. [c] Yields
measured on the basis of HPLC integration.
the nature of the five-member heterocycle, water soluble
BOxaPy derivatives were synthesised. The already described
dialdehyde precursor BOxaPy-CO (Scheme 5)[7] was trans-
Figure 1. Quantitative analysis of the FRET-melting competition experi-
ments with the telomeric sequence F21T. Stabilisation in: A) K+, or
B) Na+ is indicated for the prototype BOxAzaPy, 1–10, and the referen-
ces TOxaPy and BOxaPy in the absence (black bars) or presence of
double-stranded DNA (ds26) at 3 mm (grey bars) or 10 mm (white bars).
the heptaryl TOxaPy (Figure 1), which bound the telomeric
quadruplex much better in Na+.[7] However, the latter is
neutral and has two more oxazole units. Strikingly, all the
other compounds of the BOxAzaPy series were inactive
(DT1/2 < +1.08C), despite exhibiting the same scaffold. Thus,
the nature of the chain clearly is a prominent structural fea-
ture in the binding of the series.
Scheme 5. Synthesis of BOxaPy analogues 8–10 (series II).
The lack of both side chains in the prototype BOxAzaPy
and fully protonated ending groups (3 and 4) at the experi-
mental pH, resulted in a negligible stabilisation. This may
be the result of a lower solubility and a deficiency in electro-
static interactions with the quadruplex phosphate back-
bone.[11] This observation is fully consistent with our previ-
ous results showing that the deaza analogue BOxaPy, is
unable to stabilise the human telomeric quadruplex.[7] In ad-
dition, the importance of the terminal amino group has al-
ready been described in the case of acridine derivatives and
it was shown that larger ring substituents, like piperidines or
piperazine, deteriorate the affinity for quadruplexes.[11a,12]
This trend may explain the complete absence of binding of
6, which still represents a striking result. The best solubilis-
ing groups for the BOxAzaPy series are therefore basic pyr-
rolidine and dimethylamine, as already observed for other
scaffolds.[12b,c,13] The length of the chain was also important,
as 1 is a better stabiliser than 2, perhaps because the termi-
nal ammonium has a better target accessibility.[14]
formed into the symmetric bisamines (8–10) by a one-pot
reductive amination, in the presence of NaBH4 in absolute
ethanol (Scheme 5). Compounds 8–10 were purified by silica
gel chromatography (CH2Cl2/MeOH 9:1, NH3 0.1%; see the
Supporting Information).
FRET-melting assays: Stabilisation of the telomeric se-
quence by the BOxAzaPy and the BOxaPy series was first
investigated with FRET-melting assays (Figure 1).[10] Com-
pounds 1, 2 and 5 were found to be potent stabilisers of
F21T in potassium-rich buffer (DT1/2 = +15.5, 10.5 and
16.08C, for 1, 2 and 5, respectively). Their stabilising effect
was significantly lessened in sodium-rich buffer (+5.2, 5.5,
7.08C, respectively); this suggests a marked structure prefer-
ence for the K+ forms. This trend is not unexpected and has
been observed for most classes of quadruplex ligands, how-
ever, it is more pronounced in the present case.[7] On the
other hand, this selectivity is inverted compared to that of
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Importantly, the best ligands 1, 2 and 5, as well as 8–10,
fully retained their stabilising properties when an excess of
duplex DNA competitor was added, irrespective of the
chain length, ending group nature, and buffer (potassium- or
sodium-rich buffer). This is even more remarkable as the
quadruplex stabilising effect is absolutely not affected by
the duplex competitor; this indicates that the three ligands
are completely devoid of affinity for the double-stranded
form under these conditions. Concerning the three deriva-
tives of series II (8–10), they exhibit the same trends, but
appear clearly less efficient. As they bear the same side
chains as 1, 2 and 5 (differing only by one CH2), these data
evidence the advantage of the BOxAzaPy scaffold. The rest
of the study was thus more focused on compounds 1, 2 and
5. The latter were studied more comprehensively with a set
of quadruplex-forming sequences (DNA and RNA human
telomeric sequences 21AG and 21RNAG, modified human
telomeric sequence 21CTA, human minisatellite sequence
25Ceb, proto-oncogenes c-myc, c-kit1 and c-kit2) and a
duplex-forming sequence, dx (for sequences see the Experi-
mental Section). Similar trends were observed for the three
ligands. In more detail, 21AG, 21CTA, c-kit1 and c-kit2 were
the least stabilised structures in sodium-rich buffer, whereas
the difference with other sequences was significantly lower
or inexistent in potassium-rich conditions (Figure 2). Se-
quences 21RNAG, 25Ceb and c-myc seem to be stabilised in
a comparable way in both conditions. Moreover, the duplex
sequence (dx) was not stabilised, regardless of the ligand or
the cation; this is in accord with the competitive FRET-
melting results. Overall, these results suggest that the bind-
ing of BOxAzaPy ligands is controlled by the quadruplex
nucleic acid structure, rather than by the cation nature itself.
Moreover, the data demonstrate that duplex matrices offer
no binding sites for the new ligands in spite of their dica-
tionic charge.
Figure 2. FRET-melting stabilisation by derivatives 1, 2 and 5 (DT1/2, 8C)
of the quadruplexes 21AG, 21RNAG, 25Ceb, 21CTA, c-myc, c-kit1, c-kit2
and of the duplex dx in: A) K+-, or B) Na+-rich buffers. [DNA]=0.2 mm;
[ligands]=1 mm.
Generally, 1 appeared significantly more efficient than all
other compounds with regard to the set of quadruplexes in-
vestigated. Such a difference in behaviour is remarkable as
the three ligands have very close structures; in particular 1
differs from 2 only by one CH2 group in the side chains.
However, although more active than 2 and 5, 1 displays a
moderate ability to displace TO as compared to the planar
High-throughput fluorescent intercalator displacement (HT-
G4-FID) assays: The ligandsꢃ affinity for quadruplex and
duplex oligonucleotides were tested by HT-G4-FID.[15] Brief-
ly, the assay is based on the competitive displacement of the
fluorescent probe TO (thiazole orange), which binds various
nucleic acid structures. A variety of G-quadruplex structures
(c-myc, c-kit1, c-kit2, 21CTA, TBA and 22AG in potassium-
or sodium-rich buffers, called 22AG.K and 22AG.Na, respec-
tively, hereafter) and one control duplex matrix, ds26, were
used. The BOxAzaPy compounds 1, 2 and 5 and the
BOxaPy derivative 10 appeared to interact preferentially
with the telomeric sequence in potassium-rich buffer, where-
as a very weak interaction was observed in sodium-rich
buffer (Figure 3, for selected results) thus paralleling the
FRET-melting data. The BOxaPy compounds 9–10 exhibit-
ed a systematic lower affinity and a poor selectivity toward
different quadruplexes (22AG.K, 22AG.Na, 21CTA, c-myc,
c-kit1, c-kit2, TBA, see Figure S1 in Supporting Information
for full data). Therefore, the BOxapy series was discarded
for further experiments.
ligand Pt-ttpy {[PtACTHNUTRGNE(NUG 4’-(4-methylphenyl)-2,2’:6’,2’’-terpyridi-
ne)Cl]Cl} used as benchmark (Figure 3A and B).[16] Indeed,
the displacement curves never reach saturation (the probe
being displaced by up to 60–80% in the best cases), and the
shape of the curve is irregular, thereby reflecting a slow
and/or complex process. This type of curve may reflect
either a poor affinity for the TO sites, or an indirect compe-
tition between TO and the ligand due to different binding
sites. In addition, conformational changes of the G4 target
that in turn may modify the TO/G4 equilibrium cannot be
excluded.[7] Finally, the FID data unambiguously confirm
the absence of binding to duplex structures (Figure 3D).
The high and structurally selective stabilisation properties of
these compounds, together with their moderate TO displac-
ing properties, suggest a binding mode different from the
usual p stacking on external quartets. It is likely that com-
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Cationic Pentaheteroaryls as Selective G-Quadruplex Ligands
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Figure 3. G4-FID plots of 1 ( ), 2 ( ), 5 (&) and 10 ( ) with: A) 22AG.K, B) 22AG.Na, C) c-myc, and D) ds26. Pt-ttpy (&sstarf, A and B) is used as a
benchmark as it is a strong TO displacer for these G4 structures and has a stabilising effect in a comparable range (158C).
pound 1, due to its flexible and non-planar structure, is well-
suited to interact with loops and grooves. Interestingly, a
similar behaviour (strong G4 stabilisation, partial displace-
ment of TO, discrimination between various G4 matrices, no
duplex binding) has been observed for the structurally relat-
ed heptaryl TOxaPy,[7] for which molecular modelling stud-
ies supported the possibility of interaction in the grooves. It
might well be that these polyheteroaryl series make use of a
groove-binding mode or of mixed binding modes (loop-
quartet, groove-quartet) to achieve recognition of quadru-
plex matrices, as already reported for the ribbon-like dista-
mycin derivatives.[17] Finally, we noticed again that the FID
assay emphasises differences between ligands that are not
detected by FRET melting (compare 1 and 5) and thus the
two assays nicely complement each other to give a more
complete picture of the ligand-binding profile.
submillimolar nucleoside concentration, that is, a maximum
at 290 nm (usually observed with antiparallel strands), a
shoulder around 265 nm and a minimum at 240 nm (both
observed with parallel strands; Figure 4A).[18]
This spectrum may correspond to an antiparallel basket
arrangement,[18c] (3+1) hybrid structures[18b] or a mixture of
parallel and antiparallel structures.[18b,19] Secondary struc-
tures of the telomeric sequence, and corresponding CD
spectra, are very concentration dependent in potassium-rich
buffers: high concentrations result in (3+1) hybrid struc-
tures as observed with NMR spectroscopy,[18c,20] whereas an
antiparallel basket arrangement seems to be favoured at low
concentrations.[18c] Upon addition of 1, a strong increase of
the 290 nm band, together with the conversion of the posi-
tive shoulders to a negative band at 260 nm, was observed
(Figure 4A), suggesting a 1–22AG.K complex formation.
This non-conservative positive exciton couplet signature is
characteristic of an antiparallel structure, and its induction
by ligands like sanguinarine[21] or telomestatin[5b] has already
been observed. The presence of two neat isoelliptical points
at 285 and 245 nm supports the existence of a single equili-
brium converting the free DNA structure(s) into a bound
species.
Circular dichroism (CD) spectroscopy: In an attempt to
gain insight into the interaction of BoxAzaPy ligands with
G4-DNA, we conducted CD experiments. We first verified
that the achiral compound 1 did not display any CD signal
in its absorbance region (Figure S2 in the Supporting Infor-
mation). The spectrum of the prefolded human telomeric se-
quence, 22AG, in potassium-rich buffer was consistent with
results reported in the literature for the same sequence at
An apparent 2:1 stoichiometry can be inferred by plotting
the variation of intensity of the 290 and 260 nm bands as a
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Figure 4. CD spectra of 22AG alone and upon addition of increasing amounts of 1 (indicated by the arrows; A, B and C) or 5 (D) in potassium-rich
buffer (A, D), sodium-rich buffer (B) or in the absence of salts (C). Inset A: magnification of the 310–420 nm region, corresponding to an induced CD
of the achiral ligand 1.
function of 1 concentration (Figure S3A in the Supporting
Information). Global fitting by using nonlinear regression of
both scatter plots with the Hill equation, with shared bind-
ing and Hill constants, was successfully achieved, and yield-
value (Figure S4B in the Supporting Information). There is
no clear isoelliptic point; therefore, we can infer that the
spectrum obtained by the addition of two equivalents of 1
may arise from an intermediate structure or the coexistence
of more than two bound structures.
ed a shared apparent dissociation constant Kd =5.3
G
10À6 m (Figure S3B in the Supporting Information). Since
DNA does not absorb above 320 nm, the change in elliptici-
ty monitored in the 320–400 nm region has to be assigned to
induced CD (ICD) of the achiral ligand, as a result of 1
sensing the quadruplex chirality in the complex (Figure 4A,
insets). ICD was also observed for compound 1 with two
quadruplex-forming sequences the G4 structures of which
are not cation dependent: namely the tetramolecular quad-
ruplex [5’-TG4T-3’]4 and c-myc (data not shown). The same
experiment was carried out in sodium-rich buffer. The telo-
meric antiparallel structure is characterised by positive
bands at 245 and 290 nm and a negative band at 260 nm
(Figure 4B and Figure S4A in the Supporting Information,
dark blue line).[18b,22] Upon addition of 1, a strong increase
of the 290 nm band was recorded, together with a 3 nm hyp-
sochromic shift. The first two equivalents of ligand induced
a decrease of the intensity of the 260 nm band, but higher
ligand concentrations almost restored the initial ellipticity
Finally, the interaction between 22AG and 1 was studied
in the absence of cations (Figure 4C and Figure S5A in the
Supporting Information), as already described.[5b] Results
obtained with the oligonucleotide prepared in the absence
of monovalent cations are very similar to the ones obtained
in potassium-rich buffer, that is: 1) an increase of the
290 nm band, 2) the formation of a negative ellipticity band
at 260 nm, 3) the presence of an isoelliptic point at 285 nm,
and 4) the appearance of an ICD band. We can thus con-
clude that 1 binds to the telomeric sequence and preferen-
tially induces a single antiparallel structure, which seems
similar to the one obtained by complexation of (R)- or (S)-
telomestatin.[5b] Global fitting was also achieved successfully
and shows a comparable dissociation constant (Kd =1.1-
A
(Figure S6B in the Supporting Information). Interestingly,
the spectra obtained after complexation of 1 to the random-
ly structured (absence of metallic salt) or the prefolded telo-
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Cationic Pentaheteroaryls as Selective G-Quadruplex Ligands
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poured into water to induce the precipitation of a white solid (0.12 g;
meric sequence in sodium or potassium converged to an
analogous pattern, although they differ greatly in the free-
state (Figure S6 in the Supporting Information).
yield 25%), which was filtered and characterised as pure product; m.p.>
1
3008C (dec.); H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=8.92–8.89
(m, 2H), 8.48–8.45 (m, 4H), 8.12 (t, 3J
ACHTUNGTRENNUNG
3J
G
E
ACHTUNGTRENNUNG
In order to gain additional insight into the SAR (struc-
ture–activity relationship) of the BOxAzaPy series, we also
investigated the behaviour of 5 with the telomeric sequence
in potassium- or sodium-rich buffer (Figure 4D and Fig-
ure S6 in the Supporting Information). The trends were the
same as those already monitored for 1, in the DNA absorb-
ing region, but to a significantly lesser extent. Thus, in potas-
sium-rich conditions, the 290 nm band intensity increases
moderately and a 260 nm negative band only begins to
appear. In sodium-rich conditions, the 260 nm band grows
but the 290 nm band remains unchanged. Compared to 1, 5
seems to induce the formation of a similar antiparallel struc-
ture (Figure S6 in the Supporting Information, dashed lines
vs. solid lines) regardless of the cation nature, but is a
poorer inducer. Finally, it is worth noting that no ICD is de-
tectable in the 320–400 nm region for 5 (Figure 4D and Fig-
ure S6 in the Supporting Information). This might result
from a slightly different binding interaction both in terms of
affinity and structure, which would be consistent with the
different FID behaviour exhibited by the two compounds.
6 Hz; 3J
ACHTUNGTRENNUNG
[D6]DMSO, 258C, TMS): d=175.1, 168.3, 150.6, 146.9, 143.3, 138.3, 137.3,
126.8, 125.2, 124.6 ppm; elemental analysis calcd (%) for C19H11N7O2: C
61.79, H 3.00, N 26.55; found: C 61.64, H 3.01, N 26.48.
Stepwise synthesis of BOxAzaPy-Br: The experimental details refer to
steps a)–d) in Scheme 3.
a) 6-Cyano-N-hydroxy-pyridine-2-carboxamidine:[24] Hydroxylamine hy-
drochloride (0.53 g, 7.7 mmol) and Na2CO3 (0.41 g, 3.9 mmol) in water
(50 mL) were added dropwise to a solution containing pyridine-2,6-dicar-
bonitrile[24] (1.00 g, 7.7 mmol) dissolved into EtOH (100 mL). The reac-
tion mixture was stirred at room temperature for 1 h. The organic solvent
was removed and the white solid was filtered and washed with water.
The crude product was purified by MPLC (Isolera ONE; eluent CHCl3/
MeOH gradient) and the product (Z)-6-cyano-N-hydroxy-pyridine-2-car-
boxamidine was obtained as a white pale solid (0.52 g; yield 41%) and
used for the next step without further purification. 1H NMR (300 MHz,
[D6]DMSO, 258C, TMS): d=10.25 (s, 1H), 8.15–8.11 (m, 1H), 8.07–8.04
(m, 2H), 5.93 ppm (s, 1H); 13C NMR (75 MHz, [D6]DMSO, 258C, TMS):
d=151.8, 148.4, 138.3, 131.2, 129.3, 123.6, 117.3 ppm.
b) 6-[5-(6-Bromopyridin-2-yl)-[1,2,4]oxadiazol-3-yl]-pyridine-2-carboni-
trile: 6-Bromopyridine-2-carboxylic acid (0.33 g, 1.6 mmol) and CDI
(0.26 g, 1.6 mmol) were dissolved in DMF (8 mL) and stirred at room
temperature for 30 min. Then, (Z)-6-cyano-N’-hydroxypicolinimidamide
(0.26 g, 1.6 mmol) was added and the reaction mixture was stirred at
room temperature, overnight. CDI (0.26 g, 1.6 mmol) was further added
and the reaction mixture was refluxed for 6 h. After being cooled, the re-
action mixture was poured into water to induce the precipitation of the
white solid (0.16 g, yield 59%), which was filtered and used without puri-
fication; m.p. 270–2728C; 1H NMR (300 MHz, [D6]DMSO, 258C, TMS):
Conclusion
Using a microwave-assisted solvent-free synthetic protocol
we have created and investigated a new family of water
soluble oxadiazole-based acyclic quadruplex ligands. The
promising binding properties of three of these ligands (1, 2
and 5) together with the selectivity toward both quadruplex
versus duplex and within different G-quadruplexes have
been highlighted by FRET melting, HT-G4-FID and circular
dichroism experiments. Our data reveal the unexpected po-
tential of these acyclic derivatives. In fact, the on/off quad-
ruplex binding properties are clearly controlled by the struc-
tural features of the cationic moieties and optimised by the
oxadiazole core, as the oxazole analogues are much less effi-
cient G4 ligands. The sharp structure–activity relationships,
the conformational flexibility and the highly hydrophobic
character of the pentameric core are in favour of specific in-
teractions that may be established in the grooves of the
quadruplex, at least for ligand 1, as it exhibited ICD. How-
ever, a thorough structural investigation is needed to unam-
biguously confirm the proposed binding mode.
3
3
3
d=8.48 (d, J
(H,H)=8 Hz, 1H), 8.24 (d, 3J
1H), 7.99 ppm (d, 3J(H,H)=8 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO,
G
ACHTUNGTRENNUNG
E
G
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
258C, TMS): d=173.9, 167.5, 146.9, 143.2, 141.9, 141.1, 139.9, 133.4,
132.1, 130.9, 127.1, 124.1, 116.7 ppm.
c) 6-[5-(6-Bromopyridin-2-yl)-[1,2,4]oxadiazol-3-yl]-N-hydroxy-pyridine-
2-carboxamidine: A mixture of hydroxylamine hydrochloride (0.13 g,
1.9 mmol) and of Na2CO3 (0.1 g, 0.9 mmol) in water (25 mL) was added
dropwise to a solution containing 6-[5-(6-bromopyridin-2-yl)-[1,2,4]oxa-
diazol-3-yl]-pyridine-2-carbonitrile (0.3 g, 0.9 mmol) in EtOH (75 mL)
and the reaction mixture was stirred at room temperature, overnight. The
organic solvent was removed and the solid was filtered, washed with
water and dried. The desired product was obtained as a white pale solid
(0.25 g, yield 75%), m.p. 225–2308C; 1H NMR (300 MHz, CDCl3, 258C,
TMS): d=10.15 (s, 1H), 8.39 (d, 3J(H,H)=7 Hz, 1H), 8.19 (t, 3J
ACTHNUTRGENNUG ACHTUNGTRENNUNG(H,H)=
5 Hz, 1H), 8.08–8.05 (m, 3H), 8.01 (d, 3J
ACTHNUTRGNE(UNG H,H)=8 Hz, 1H), 5.89 ppm (s,
2H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=173.4, 168.2, 150.7,
148.8, 144.1, 143.3, 141.9, 141.3, 138.3, 132.1, 124.1, 123.7, 121.9 ppm.
d) 2,6-Bis(5-(6-bromopyridin-2-yl)-1,2,4-oxadiazol-3-yl)pyridine (BOxAz-
aPy-Br): 6-Bromopyridine-2-carboxylic acid (0.18 g, 0.9 mmol) and CDI
(0.15 g, 0.9 mmol) were dissolved in DMF (5 mL) and stirred at room
temperature for 30 min. Then, (Z)-6-(5-(6-bromopyridin-2-yl)-1,2,4-oxa-
diazol-3-yl)-N’-hydroxypicolinimidamide (0.28 g, 1.6 mmol) was added
and the reaction mixture was stirred at room temperature, overnight.
CDI (0.15 g) was further added and the reaction mixture was heated to
reflux for 6 h. After being cooled, the reaction mixture was poured into
water and the product was filtered as a white solid (0.25 g; 0.47 mmol);
m.p.>3008C (dec.); 1H NMR (300 MHz, CDCl3, 258C, TMS): d=8.62–
Experimental Section
Synthesis of 2,6-bis(5-(pyridin-2-yl)-1,2,4-oxadiazol-3-yl)pyridine (BOx-
AzaPy): Pyridine-2,6-dicarboxylic acid (0.35 g, 2.8 mmol) and 1,1’-carbon-
yldiimidazole (CDI, 0.46 g, 2.8 mmol) were dissolved in DMF (14 mL)
and stirred at room temperature for 30 min. (2Z,6Z)-N’2,N’6-Dihydroxy-
pyridine-2,6-bis(carboximidamide)[23] (0.25 g, 1.28 mmol) was added and
the reaction mixture was stirred at room temperature, overnight. CDI
(0.46 g, 2.8 mmol) was further added and the reaction mixture was
heated at 1508C for 6 h. After being cooled, the reaction mixture was
8.48 (m, 4H), 8.20 (t, 3J
ACTHUNTGRENNG(U H,H)=8 Hz, 1H), 8.02–7.88 ppm (m, 4H);
13C NMR (75 MHz, CDCl3, 258C, TMS): d=173.0, 168.4, 139.4, 138.4,
134.5, 131.9, 130.1, 126.4, 125.4, 123.4 ppm.
Solvent-free microwave-assisted synthesis of the water soluble BOxAza-
Py compounds (1–7): general procedure: BOxAzaPy-Br (0.1 g,
0.19 mmol) was dissolved in neat amine (2 mL) and the mixture was irra-
Chem. Eur. J. 2012, 00, 0 – 0
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M.-P. Teulade-Fichou, M. Freccero et al.
diated for 8 min in a closed-vessel microwave oven (power=200 W,
max =1008C, max =462 kPa; CEM single-mode microwave reactor).
ethanol (5 mL) and the desired amine (8–10; 0.709 mmol) was added
under nitrogen atmosphere. The reaction mixture was stirred at room
temperature for 24 h. NaBH4 (80.5 mg, 2.13 mmol) was added in one por-
tion. The solution was stirred for another 6 h until the composition of the
reaction mixture no longer changed. The solution was concentrated
under reduced pressure and to the residue was mixed with water
(20 mL). The aqueous solution was extracted with CHCl3 (3ꢄ50 mL).
The combined organic phase was dried over Na2SO4 and the solvent was
removed under reduced pressure. The obtained reaction mixture was pu-
rified by column chromatography CH2Cl2/MeOH 9:1 with NH3 (1%).
The product was obtained as a pale yellow oil.
T
P
Excess solvent was removed under high vacuum (<7 Pa). The crude
product, was dissolved in methanol and purified by preparative HPLC,
by using a C-18 reverse phase column (CH3CN/H2O, 0.1% TFA, as
eluent). Addition of HCl solution (1m; 0.5 mL) to each chromatographic
portion and subsequent solvent evaporation under vacuum afforded ad-
ducts 1–7, as hydrochlorides.
Compound 1: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
3
[D6]DMSO, 258C, TMS): d=10.83 (s, 2H), 8.39–8.35 (m, 3H), 7.76 (t, J-
3
(H,H)=7 Hz, 2H), 7.59 (d, 3J
E
N
2H), 3.72 (bs, 4H), 3.36 (bs, 4H), 2.98 ppm (s, 12H); 13C NMR (75 MHz,
[D6]DMSO, 258C, TMS): d=175.4, 167.0, 158.5, 146.1, 139.8, 139.6, 138.4,
125.7, 114.4, 113.5, 57.1, 42.7, 36.5 ppm; elemental analysis calcd (%) for
C27H33Cl2N11O2: C 52.77, H 5.41, Cl 11.54, N 25.07; found: C 52.67, H
5.46, Cl 11.44, N 24.96.
N1,N1’-(6,6’-(5,5’-(Pyridine-2,6-diyl)bis(oxazole-5,2-diyl))bis(pyridine-6,2-
diyl))bis(methylene)bis(N2,N2-dimethylethane-1,2-diamine)
yellow oil (32.8 mg, 23.0% yield); 1H NMR (300 MHz, CDCl3, 258C,
TMS): d=8.10 (d, 3J
(H,H)=9 Hz, 2H), 7.99 (s, 2H), 7.81–7.94 (m, 5H),
7.52 (d, 3J(H,H)=9 Hz, 2H), 4.10 (s, 4H), 2.81 (t, 3J
(H,H)=6 Hz, 4H),
2.51 (t, 3J(H,H)=6 Hz, 4H), 2.29 ppm (s, 12H); 13C (75 MHz, CDCl3,
(8):
Pale
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
Compound 2: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
AHCTUNGTRENNUNG
3
258C, TMS): d=160.9, 151.3, 147.3, 145.3, 137.9, 137.4, 127.9, 123.6,
120.8, 118.9, 59.1, 55.1, 46.9, 45.5 ppm; m/z (%): 590 (100) [M+ +Na+],
568 (26) [M+], 295 (81.6) [(M+ +Na+)/2]; HRMS (ESI-MS): m/z (%):
568.3141; calcd for C31H38N9O2: 568.3148.
[D6]DMSO, 258C, TMS): d=10.54 (s, 2H), 8.42–8.34 (m, 3H), 7.70 (t, J-
3
(H,H)=7 Hz, 2H), 7.54 (d, 3J
E
N
2H), 3.47–3.43 (m, 4H), 3.21–3.14 (m, 4H), 2.81 (s, 12H), 2.07–1.99 ppm
(m, 4H); 13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=175.5, 167.9,
158.9, 146.4, 140.2, 139.7, 137.9, 125.6, 113.4, 113.0, 54.5, 42.1, 37.8,
24.0 ppm; elemental analysis calcd (%) for C29H37Cl2N11O2: C 54.20, H
5.80, Cl 11.03, N 23.98; found: C 53.98, H 5.82, Cl 11.08, N 24.07.
N1,N1’-(6,6’-(5,5’-(Pyridine-2,6-diyl)bis(oxazole-5,2-diyl))bis(pyridine-6,2-
diyl))bis(methylene)bis(N3,N3-dimethylpropane-1,3-diamine) (9): Pale
yellow oil (30.0 mg, 21.3% yield); 1H NMR (300 MHz, CDCl3, 258C,
TMS): d=8.12 (d, 3J
7.50 (d, 3J
2.44 (t, 3J(H,H)=6 Hz, 4H), 2.28 (s, 12H), 1.80 ppm (m, 3J
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
Compound 3: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
3
A
ACHTUNGTRENNUNG
[D6]DMSO, 258C, TMS): d=11.11 (s, 2H), 8.40–8.34 (m, 3H), 7.74 (t, J-
3
(H,H)=8 Hz, 2H), 7.61 (d, 3J
N
(H,H)=8 Hz,
E
4H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=160.4, 160.7, 151.4,
147.3, 145.4, 138.0, 137.5, 127.9, 123.7, 120.9, 119.0, 56.0, 55.1, 51.5, 48.1,
45.5 ppm; m/z (%): 618 (41.5) [M+], 596 (100) [M++Na+]; HRMS (ESI-
MS): m/z (%): 596.3450; calcd for C33H42N9O2: 596.3461.
2H), 4.00–3.97 (m, 4H), 3.89–3.82 (m, 8H), 3.64 (d, 3J
ACHTUNGTRENUN(NG H,H)=12 Hz,
4H), 3.38 (d, 3J(H,H)=4 Hz, 4H), 3.20–3.16 ppm (m, 4H); 13C NMR
ACHTUNGTRENNUNG
(75 MHz, [D6]DMSO, 258C, TMS): d=175.5, 167.9, 158.4, 146.4, 140.3,
139.6, 138.1, 125.6, 113.8, 113.7, 63.1, 56.0, 55.5, 51.4, 35.1 ppm; elemental
analysis calcd (%) for C31H37Cl2N11O4: C 53.30, H 5.34, Cl 10.15, N 22.05;
found: C 53.04, H 5.30, Cl 10.26, N 22.28.
N,N’-(6,6’-(5,5’-(Pyridine-2,6-diyl)bis(oxazole-5,2-diyl))bis(pyridine-6,2-
diyl))bis(methylene)bis(2-(pyrrolidin-1-yl)ethanamine) (10): Pale yellow
oil (54.7 mg, 37.4% yield); 1H NMR (300 MHz, CDCl3, 258C, TMS): d=
Compound 4: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
3
[D6]DMSO, 258C, TMS): d=8.40–8.33 (m, 3H), 7.67 (t, 3J
ACHTUNGTRENNUNG
8.06 (d, J
G
T
(H,H)=6 Hz, 4H), 2.70 (t, 3J-
ACHTUNGTRENNUNG
2H), 7.52 (d, 3J(H,H)=7 Hz, 2H), 6.88 (d, 3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
3.56 (m, 4H), 3.47–3.43 ppm (m, 4H); 13C NMR (75 MHz, [D6]DMSO,
258C, TMS): d=175.4, 167.8, 158.9, 146.4, 140.1, 139.6, 137.8, 125.6,
113.2, 112.7, 59.9, 43.5 ppm; elemental analysis calcd (%) for
C23H21N9O4: C 56.67, H 4.34, N 25.86; found: C 56.84, H 4.32, N 25.81.
AHCTUNGTRENNUNG
(75 MHz, CDCl3, 258C, TMS): d=161.0, 160.9, 151.3, 147.2, 145.3, 137.8,
137.4, 127.9, 123.6, 120.8, 118.9, 56.0, 55.2, 54.2, 48.0, 23.4 ppm; m/z (%):
642 (44.6) [M++Na+], 620 (100.0) [M+]; HRMS (ESI-MS): m/z (%):
620.3467; calcd for C35H42N9O2: 620.3461.
Compound 5: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
3
FRET-melting experiments: FRET-melting measurements were per-
formed as described previously.[10] Experiments were carried out in 96-
well plates on a real-time PCR apparatus Mꢄ3005P Stratagene as follow:
5 min at 258C, then increase of 18C every minute until 958C. Each exper-
imental condition was tested in duplicate, in a volume of 25 mL for each
sample. Oligonucleotides (see below), equipped with FRET partners at
each extremity, were prepared at 0.2 mm, ligands were at 1 mm and com-
petitors at 3 or 10 mm final concentration. Measurements were made with
excitation at 492 nm and detection at 516 nm in a buffer of lithium caco-
dylate (10 mm, pH 7.2), NaCl (100 mm) or KCl (10 mm; completed by
90 mm LiCl) then heated at 908C for 2 min and finally put in ice. The se-
quences are:
[D6]DMSO, 258C, TMS): d=10.67 (s, 2H), 8.41–8.35 (m, 3H), 7.76 (t, J-
3
(H,H)=7 Hz, 2H), 7.62 (d, 3J
E
N
2H), 3.42–3.38 (m, 4H), 3.17–3.12 (m, 4H), 2.02–1.91 ppm (m, 8H);
13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=175.5, 167.9, 158.5,
146.4, 140.3, 139.6, 138.1, 125.7, 113.9, 113.6, 53.3, 40.3, 38.7, 22.7 ppm; el-
emental analysis calcd (%) for C31H37Cl2N11O2: C 55.86, H 5.59, Cl 10.64,
N 23.11; found: C 55.51, H 5.62, Cl 10.68, N 23.19.
Compound 6: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
[D6]DMSO, 258C, TMS): d=11.75 (s, 2H), 8.63 (s, 4H), 8.55–8.45 (m,
3H), 8.05 (t, 3J
3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(H,H)=7 Hz, 2H), 7.87 (d, 3J
ACHTUNGTRENNUNG
8H), 3.35–3.28 ppm (m, 4H); 13C NMR (75 MHz, [D6]DMSO, 258C,
TMS): d=175.1, 167.9, 157.9, 146.3, 140.5, 139.6, 139.5, 125.7, 114.8,
112.1, 52.9, 50.9, 41.6, 33.3 ppm; elemental analysis calcd (%) for
C31H41Cl4N13O2: C 48.38, H 5.37, Cl 18.43, N 23.66; found: C 48.43, H
5.40, Cl 18.55, N 23.82.
F21T: FAM-G3ACHTUNGRTNE[UNG T2AG3]3-Tamra
FmycT: FAM-T2GAG3TG3TAG3TG3TA2-Tamra
FKit1T: FAM-G3AG3CGCTG3AG2AG3-Tamra
FKit2T: FAM-G3CG3CGCGAG3AG4-Tamra
F25CebT: FAM-AG3TG3TGTA2GTGTG3TG3T-Tamra
Compound 7: pale yellow crystals; m.p.>3008C; 1H NMR (300 MHz,
[D6]DMSO, 258C, TMS): d=11.14 (s, 2H), 7.96–7.87 (m, 3H), 8.24 (s,
F21RT: FAM-G
[U2AG3]3-Tamra
3A[CTAG3]3-Tamra
4H), 7.71 (t, 3J
3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(H,H)=7 Hz, 2H), 7.58 (d, 3J
F21CTAT: FAM-G CTHUNGTRENNUNG
ACHTUNGTRENNUNG
4H);13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=175.5, 167.8, 158.7,
146.4, 140.3, 139.6, 137.9, 125.7, 113.8, 113.5, 41.2, 38.5 ppm; elemental
analysis calcd (%) for C23H25Cl2N11O2: C 49.47, H 4.51, Cl 12.70, N 27.59;
found: C 49.33, H 4.53, Cl 12.62, N 27.65.
FdxT: FAM-[TA]2GC[TA]2-hexaethyleneglycol-[TA]2GC[TA]2-Tamra
HT-G4-FID experiments: HT-G4-FID measurements were performed as
described previously.[15b] TO (thiazole orange) and cacodylic acid were
purchased from Aldrich and used without further purification. Stock sol-
utions of TO (2 mm in DMSO) and ligands (500 mm in DMSO) were used
for G4-FID assay. Oligonucleotides purified by reverse phase HPLC
Synthesis of BOxaPy derivatives by reductive amination (8–10): general
procedure: BOxaPy-CO (100 mg, 0.236 mmol) was dissolved in absolute
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Cationic Pentaheteroaryls as Selective G-Quadruplex Ligands
were purchased from Eurogentec (Belgium). Sequences: 22AG corre-
FULL PAPER
Alcaro, A. Artese, S. N. Richter, M. Freccero, Org. Biomol. Chem.
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sponds to the human telomeric repeat: [5’-AG
3ACHTUNGTRENUN(GN T2AG3)3-3’]; 21CTA to a
modified human telomeric sequence [5’-G3A
G
G3AG3CGCTG3AG2AG3-3’] and c-kit2 [5’-G3CG3(CG)2AG3AG4-3’] are
two sequences of the c-kit oncogene promoter and c-myc [5’-
T2GAG3TG3TAG3TG3TA2-3’] of the c-myc oncogene promoter; TBA is
the thrombin binding aptamer sequence [5’-GGTTGGTGTGGTTGG-
3’]; ds26 is the duplex obtained with the self-complementary sequence
[5’-CA2TCG2ATCGA2T2CGATC2GAT2G-3’]. HT-G4-FID measurements
were performed on a FLUOstar Omega microplate reader (BMG Lab-
tech) with a 96-wells black quartz microplate (Hellma). The percentage
of displacement was calculated from the fluorescence intensity (FA) by
using: percentage of displacement=100À
ACHTUGNTERN[NUNG (FA/FA0)ꢄ100], FA0 being the
fluorescence from TO bound to DNA without added ligand. The percent-
age of displacement was then plotted as a function of the concentration
of added ligand.
CD spectroscopy: CD measurements were carried out at 228C with a
JASCO J-710 spectropolarimeter equipped with a Peltier temperature
controller (Jasco PTC-348WI) interfaced to a PC, by using rectangular
quartz cells of 1 cm path (1 mL reaction volume). The scans were record-
ed from 220 to 500 nm with the following parameters: 100 mdeg sensitivi-
ty, 1 nm data pitch, 200 nmminÀ1 scanning speed, 1 s response, 1 nm band
width, 4 accumulations. Solutions containing a constant concentration
(5 mm) of G-quadruplex DNA (22-mer human telomeric sequence 22AG)
were titrated with increasing amounts of ligands (0 to 6 equiv; 10 min sta-
bilisation time) in either lithium cacodylate (10 mm) supplemented with
potassium or sodium chloride (100 mm), pH 7.2, or Tris buffer (50 mm),
pH 7.4, in the absence of metallic cation. The scan of the buffer was sub-
tracted from the average scan for each sample. Global fitting (nonlinear
regression by the Levenberg–Marquardt algorithm) of scatter plots was
performed by using the Hill equation (corrected with fixed offsets) with
shared binding and Hill constants. Significance of global fit models was
verified by Fisherꢃs and Studentꢃs tests.
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Acknowledgements
This work was supported by MIUR: PRIN 2009 (2009MFRKZ8), and by
the University of Pavia. M.-P.T.-F. gratefully acknowledge Chi-Hung
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Received: June 13, 2012
Published online: && &&, 2012
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Cationic Pentaheteroaryls as Selective G-Quadruplex Ligands
FULL PAPER
DNA Recognition
M. Petenzi, D. Verga, E. Largy,
F. Hamon, F. Doria,
M.-P. Teulade-Fichou,* A. Guꢀdin,
J.-L. Mergny, M. Mella,
M. Freccero* .................. &&&& — &&&&
Cationic Pentaheteroaryls as Selective
G-Quadruplex Ligands by Solvent-
Free Microwave-Assisted Synthesis
Oxadiazoles improve binding: Micro-
wave-assisted synthesis of water solu-
ble acyclic pentaheteroaryls containing
1,2,4-oxadiazoles (1–7; see structure)
was achieved. The binding with DNA
quadruplex structures was investigated
by FRET melting, G4-FID and CD
spectroscopy. The oxadiazole contain-
ing ligands 1, 2, and 5 were better
binders than the structurally related
oxazoles (8–10).
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
11
&
ÞÞ
These are not the final page numbers!