Porphyrin-templated synthetic G-quartet (PorphySQ): A second prototype of G-quartet-based G-quadruplex ligand

Article abstract of DOI:10.1039/c2ob25601k

Template-assembled synthetic G-quartet (TASQ) has been reported recently as a G-quadruplex ligand interacting with DNA according to an unprecedented, nature-inspired 'like likes like' approach, based on the association between two G-quartets, one being native (quadruplex) and the other one artificial (ligand). Herein, a novel TASQ-based ligand is designed, synthesized and its quadruplex-recognition properties are evaluated in vitro: PorphySQ (for porphyrin-templated synthetic G-quartet) displays enhanced quadruplex recognition properties as compared to the very first reported prototype (DOTASQ, for DOTA-templated synthetic G-quartet), since the porphyrin template insures a more stable intramolecular G-quartet fold due to self-stabilizing interactions that may take place intramolecularly between the porphyrin ring and the formed G-quartet. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25601k

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PAPER
Porphyrin-templated synthetic G-quartet (PorphySQ): a second prototype of
G-quartet-based G-quadruplex ligand†
Hai-Jun Xu, Loic Stefan, Romain Haudecoeur, Sophie Vuong, Philippe Richard, Franck Denat,
Jean-Michel Barbe, Claude P. Gros* and David Monchaud*
Received 22nd March 2012, Accepted 17th May 2012
DOI: 10.1039/c2ob25601k
Template-assembled synthetic G-quartet (TASQ) has been reported recently as a G-quadruplex ligand
interacting with DNA according to an unprecedented, nature-inspired ‘like likes like’ approach, based on
the association between two G-quartets, one being native (quadruplex) and the other one artificial
(ligand). Herein, a novel TASQ-based ligand is designed, synthesized and its quadruplex-recognition
properties are evaluated in vitro: PorphySQ (for porphyrin-templated synthetic G-quartet) displays
enhanced quadruplex recognition properties as compared to the very first reported prototype (DOTASQ,
for DOTA-templated synthetic G-quartet), since the porphyrin template insures a more stable
intramolecular G-quartet fold due to self-stabilizing interactions that may take place intramolecularly
between the porphyrin ring and the formed G-quartet.
Introduction
Guanine is a very versatile nucleobase due to a highly malleable
H-bond creating capability, via both its Watson–Crick and
Hoogsteen edges (Fig. 1).1 Only the former is involved in the
formation of the guanine–cytosine base pair (Fig. 1) while both
the former and latter participate in the creation of alternative
assemblies, including base triplets (the basic building blocks of
triplex-DNA),2 quartets (notably G-quartets, a planar array of
four guanines (Fig. 1), the basic building blocks of quadruplex-
DNA, vide infra), and supramolecular higher-order structures
like linear ribbons (Fig. 1) or helical foldamers (for a virtually
unlimited number of guanines).3
The spontaneous self-assembly property of guanines makes
them occupy a special place in the nucleic acids community
since it enables the oligonucleotides to which the guanines
Fig. 1 Schematic representation of the guanine base H-bond donor/
acceptor capability; schematic representation of a GC base pair, a
belong to adopt peculiar secondary structures: among them,
G-quadruplex-DNA has recently attracted outstanding attention.4
G-quartet and a G-ribbon.
G-rich oligonucleotides are indeed able to form G-quartets
(intra- or inter-molecularly); in the case of contiguous guanine
stretches, the resulting juxtaposed G-quartets, self-stabilized by
both π-stacking interactions and physiologically relevant cation
trapping, form the columnar frame of a four-stranded DNA
structure termed as G-quadruplex-DNA.5 Putative quadruplex-
forming DNA sequences are widespreadly dispersed in the
genome,6 but are particularly enriched in key regions such as
telomeres7 and promoters of oncogenes;8 G-quadruplex-RNA is
also increasingly studied, notably because the 5′-untranslated
regions (5′-UTR) of mRNA are enriched in quadruplex-forming
RNA sequences.9 Consequently, it has been attempted to control
the corresponding physiological events (i.e. the chromosomal
stability (telomeric quadruplexes) and the regulation of gene
expression at both the transcriptional (quadruplex-DNA in
promoter) and translational (5′-UTR) levels) by small-molecules
Institut de Chimie Moléculaire, CNRS UMR6302, Université de
Bourgogne, Dijon, France. E-mail: claude.gros@u-bourgogne.fr,
david.monchaud@u-bourgogne.fr; Fax: (+33) 380 396 117;
Tel: (+33) 380 396 112 (CPG); (+33) 380 399 043 (DM)
†Electronic supplementary information (ESI) available: chemical syn-
thesis and characterizations (including 1H-NMR and HRMS-ESI
spectra) of PorphySQ. See DOI: 10.1039/c2ob25601k
5212 | Org. Biomol. Chem., 2012, 10, 5212–5218
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displaying high affinity and selectivity for quadruplex (G-quad-
ruplex ligands). The intensive quest of G-quadruplex ligands
has provided several promising candidates,10 among which telo-
mestatin,11 BRACO-19,12 RHPS4,13 Phen-DC14 and Pyrido-
statin15 can be cited; most of them have been designed and
studied as quadruplex-DNA interacting compounds,10,16 but
examples of quadruplex-RNA ligands are currently emerging.9,17
Importantly, in the vast majority of cases, these ligands are fine-
tuned from the known pool of duplex-ligands, according to
established guidelines (viz. extended aromatic core with water-
solubilising cationic nitrogen moieties).16
We recently reported on an original design of a G-quartet-
based quadruplex ligand named DOTASQ (for DOTA-templated
synthetic G-quartet, Fig. 2),18 thereby expanding the family of
TASQ (for template-assembled synthetic G-quartet)19 using
DOTA (for 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetra-
acetic acid) as an alternative template to the previously used
calixarene20 and macrocyclopeptide.21 The logic behind using a
synthetic G-quartet is to mimic the naturally occurring self-
assembly of contiguous G-quartets that is at the origin of the
quadruplex stability. This approach is based on the association
between two G-quartets, one being natural (quadruplex) and the
other synthetic (TASQ), according to a ‘like likes like’ process.
It was however limitedly successful since DOTASQ itself was
not a good ligand per se (vide infra), but its corresponding
terbium complex displayed satisfying quadruplex-interacting
properties (fair affinity, exquisite selectivity). This originates in
the stability of the intramolecular G-quartet fold: NMR studies
have indeed highlighted that the equilibrium between the closed
(i.e. the G-quartet is formed) and open (i.e. the G-quartet is not
formed) conformations of DOTASQ as a free base is shifted in
solution toward the open form (in an ∼2 : 8 ratio); the insertion
of a metal inside the DOTA cavity of the DOTASQ forces
the four guanines to be on the same side of the template,
thereby favouring the G-quartet formation and so, improving the
G-quadruplex recognition ability. We thus believe that one way
to improve the proficiency of G-quartet-based G-quadruplex
ligands is to design TASQ with a higher-stability G-quartet:
herein we report on a TASQ in which the template not only pro-
motes but also – and above all – stabilizes the G-quartet once
formed (this intramolecular π-stacking stabilization is symbo-
lized by the red arrow in Fig. 2). To this end, we used a
porphyrin template since i- it offers the ad hoc C4-symmetry
required for the introduction of four guanine arms, and ii- it is a
widely studied and efficient G-quartet interacting scaffold
(TMPyP4 is one of the most emblematic examples).22 We thus
herein report on the design of a porphyrin-templated synthetic
G-quartet (PorphySQ, Fig. 2) via semi-empirical calculations, its
synthesis and the evaluation of its quadruplex-recognition
properties (via a comparative analysis of FRET-melting results of
both DOTASQ and PorphySQ).
Results and discussion
Design and molecular modelling
The structure of PorphySQ was first studied in silico; a particular
attention was paid to the length of the connecting arms between
the template (in red, Fig. 2) and the guanine (in black, Fig. 2).
With DOTASQ, a 12-atom link was found long enough to
enable the G-quartet fold. The porphyrin ring being broader than
the DOTA cycle (16- vs. 12-membered ring respectively), we
envisioned that a shorter arm (9- vs. 12-atom link respectively)
may be used for constructing the PorphySQ skeleton, in order to
limit the hydrophobicity imparted by long CH2 stretches. The
ability of the thus designed PorphySQ to adopt the closed con-
formation was in silico investigated. To this end, a semi-empiri-
cal method, using the RM1 Hamiltonian,23 was employed to
perform energy minimization studies: as shown in Fig. 3, a
nearly flat G-quartet is formed, at 6.5 Å above the porphyrin
plane. The general pattern obtained for the G-quartet is similar
Fig. 2 Chemical structures of DOTASQ (upper panel) and PorphySQ
(lower panel) and schematic representation of its open (left) and closed
(right) conformation.
Fig. 3 Top- (upper panel) and side-view (lower panel) of the energy-
minimized (RM1) structure of PorphySQ.
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to that of the previously minimized one via a B3LYP DFT
study.24 Given that calculations are performed in vacuo and in a
cation-free environment, the H-bond network is different to what
is usually determined for G-quadruplex-DNA (schematically rep-
resented in Fig. 1): indeed, with both RM1 (this study) and
B3LYP calculations, the guanine interactions take place via
H-bonds created between one carbonyl group and 2 N–H hydro-
gen atoms (a strong one with the NH2 group (O⋯H, 1.74 Å) and
a weak one with the amidic proton (O⋯H, 2.42 Å), in red Fig. 3).
The difference between this H-bond pattern and the one displayed
in Fig. 1 originates in the lack of templating cations (K+, Na+),
whose presence provokes a small rotation of the guanines in order
to firmly interact with the carbonyl groups. Thus, these in silico
investigations support the PorphySQ design since its structure
enables the expected intramolecular G-quartet formation.
Prot.PorphySQ is obtained in 42% chemical yield (over 2 steps).
The final deprotection, in mild acidic conditions, leads to Por-
phySQ 6 in high chemical yield (64%) and purity (see ESI†).
Biophysical evaluations
The quadruplex-recognition properties of PorphySQ were sub-
sequently evaluated: to this end, we employed the FRET-melting
assay since it provides reliable and rapid information concerning
the ability of a candidate to stabilize a fluorescently labelled
quadruplex-forming sequence.25 This stabilization is estimated
via the temperature-induced modification of the FRET (fluore-
scence resonance energy transfer) phenomenon that takes place
between two FRET partners (herein FAM (F) and TAMRA (T)),
present at both extremities of the studied oligonucleotides. The
stabilizing property (i.e. the apparent affinity) of a candidate is
expressed as an increase in melting temperature (ΔT1/2, in °C)
imparted by the presence of the ligand. Herein, two different
doubly-labelled sequences were used, namely F21T (a 21-nt
sequence that mimics the human telomere, FAM-G3[T2AG3]3-
TAMRA)7 and F-myc-T (a 22-nt sequence found in the promoter
region of c-myc oncogene, FAM-GAG3TG4AG3TG4A2G-
TAMRA).8 As depicted in Fig. 5A and 5B, in each instance Por-
phySQ (brown line) displays significantly better stabilizing prop-
erties than DOTASQ (red line), with ΔT1/2 = 6.5 vs. 1.7 °C with
F21T, and 8.6 vs. 3.0 °C with F-myc-T. The better results of Por-
phySQ may originate in its more stable G-quartet fold, due to
the self-stabilizing porphyrin ring. To further demonstrate this,
and also to be fully confident that the good stabilizing capabili-
ties of PorphySQ do not solely originate in the presence of a
porphyrin ring that might interact autonomously with quadruplex,
we carried out FRET-melting experiments with F21T in the pres-
ence of two other compounds: the ‘naked’ porphyrin 2 (by
virtue of its stability, solubility and neutrality) and a porphyrin
structurally close to PorphySQ but unable to adopt a closed con-
formation, i.e. Prot.PorphySQ (5), in which all guanines are
Chemical synthesis
The chemical synthesis of the thus designed PorphySQ was sub-
sequently realized through the pathway described in Fig. 4 (see
ESI†). Briefly, the procedure starts with the commercially avail-
able 7-bromoheptanenitrile that is converted into 7-bromohepta-
nal 1 with DIBAL-H (84% chemical yield). Its quadruple
condensation on pyrrole, performed in presence of catalytic
amounts of BF3·Et2O provides, after oxidation by DDQ, the tetra-
(6-bromohexyl)porphyrin 2, in a chemical yield that is decent
for a porphyrin synthesis (15.5%). This compound is sub-
sequently converted into tetra(6-azidohexyl)porphyrin 3, upon
treatment with NaN3 (94% chemical yield), and the terminal
azido groups are reduced by LiAlH4 to provide the tetra-
(6-aminohexyl)porphyrin 4. This latter porphyrin was found
quite tricky to handle, it was consequently decided to make it
react, without further purification, with 2-(2-amino-(6-benzyloxy)-
9H-purin-9-yl)acetic acid,18 under classical peptidic coupling
conditions (EDCI, HOBt); the tetra-(10-(6-(2-(2-amino-6-benzyl-
oxy-9H-purin-9-yl)acetamido)hexyl))porphyrin 5 hereafter named
Fig. 4 Chemical synthesis of PorphySQ.
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O-protected and thus incapable of forming a G-quartet. As
depicted in Fig. 5A, the two compounds (grey lines for 2,
orange line for 5) impart only a very weak stabilization
(ΔT1/2 = 0.8 and 1.7 °C respectively), thereby dispelling doubts
about the origins of the performances of PorphySQ. To gain
further insight into its quadruplex-recognition, two competitive
FRET-melting experiments were performed: i- the first one with
ds26, an unlabelled self-complementary 26-nt duplex-DNA
(CA2TCG2ATCGA2T2CGATC2GAT2G):25 as depicted in Fig. 5C
(purple and magenta lines), the F21T stabilization imparted by
PorphySQ is limitedly altered by this high-level competition
(ΔT1/2 = 6.5, 6.6 and 5.8 °C with 0, 15 and 50 equiv. of ds26
respectively), implying an elevated selectivity factor (S > 0.90):
these results confirm the interest in TASQ as G-quadruplex
ligands since their shape makes them unable to interact with
duplex-DNA;18 ii- the second one with [TG5T]4, an unlabelled
tetramolecular G-quadruplex with two highly accessible G-quar-
tets: as depicted in Fig. 5C (blue and light blue lines), the
PorphySQ F21T stabilization is greatly altered by this competition
(ΔT1/2 = 6.5, 3.5 and 2.3 °C with 0, 15 and 50 equiv. of [TG5T]4
respectively), indicating that PorphySQ is sensitive to the pres-
ence of additional G-quartets and thus that its quadruplex-recog-
nition is certainly mainly driven by G-quartet interaction.18
Conclusions
We herein report on the design and the synthesis of a
novel example of water-soluble template-assembled synthetic
G-quartet (TASQ) that aims at being implemented as an innova-
tive G-quadruplex ligand. Indeed, we have recently reported on
the very first example of a TASQ-based G-quadruplex ligand
named DOTASQ (for DOTA-templated synthetic G-quartet),
whose interaction with G-quadruplex-DNA was fully original
since it hinges on the nature-inspired self-recognition of two
G-quartets, one being native (quadruplex) and the other synthetic
(TASQ) –a binding mode that we termed ‘like likes like’
approach. Herein, a second example of a G-quartet-based
G-quadruplex ligand is reported; in this novel generation, the
non-planar DOTA template of DOTASQ is substituted for a
porphyrin template: the resulting compound, named PorphySQ
(for porphyrin-templated synthetic G-quartet) is interesting in
that its intramolecular G-quartet fold is not only promoted but
also – and above all – stabilized by the C4-symmetrical
porphyrin template, the porphyrin skeleton being known to be
among the most efficient G-quartet binding motifs. The prelimi-
nary assessment of the G-quadruplex recognition properties of
PorphySQ, via an array of FRET-melting experiments, demon-
strates that this compound displays interesting and promising
capabilities, which are also admittedly modest as compared to
the best ligands reported so far (including the above-mentioned
telomestatin,11 BRACO-19,12 Phen-DC14 and Pyridostatin,15
Fig. 5 FRET-melting experiments with 0.2 μM of F21T (A, C) or
F-myc-T (B) in 10 mM lithium cacodylate buffer pH 7.2 plus 10 mM
KCl–90 mM LiCl (A, C) or 1 mM KCl–99 mM LiCl (B), without
(black curve) or with 5 μM ligand, in absence (PorphySQ (brown line),
DOTASQ (red line) porphyrin 2 (grey line) and Prot.PorphySQ (orange
line)) or presence of competitors (ds26, 3 or 10 μM (purple and
magenta lines) or [TG5T]4, 3 or 10 μM (blue and light blue lines).
Lower panel: schematic representation of the oligonucleotides used in
this study.
which all display very high stabilizing properties (i.e. ΔT1/2
>
20 °C at 1 μM dose) in similar experimental conditions).10,16
These results remain however very interesting since they keep on
supporting the hypothesis according to which artificial G-quar-
tets are valuable scaffolds for devising promising G-quadruplex
ligands. From now on, we will invest all our efforts into devising
novel PorphySQ derivatives with enhanced affinity, while
keeping the exquisite selectivity of the parent compounds.
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using CH2Cl2 as eluent and recrystallized from CHCl3–CH3OH.
Experimental
Yield: 15.5% (112 mg, 0.465 mmol). 1H NMR (CDCl3)
3
Molecular modelling
δ (ppm): 9.48 (s, 8H, H-pyrro), 4.97 (t, 8H, JHH = 6.0 Hz,
3
α-CH2–), 3.45 (t, 8H, JHH = 6.0 Hz, Br–CH2–), 2.55 (m, 8H,
RM1 calculations. The optimization was carried out without
constraint using the semi-empirical RM1 Hamiltonian23
implemented within the MOPAC2009 program (MOPAC2009,
James J. P. Stewart, Stewart Computational Chemistry, Colorado
Springs, Co, USA) with the aid of the GABEDIT graphical user
interface.26 The starting geometry was constructed using the
HYPERCHEM program (HyperChem8.0.3, Hypercube, Inc.,
1115 NW 4th Street, Gainesville, Florida 32601, USA). The
minimum energy point was checked by a frequency calculation.
β-CH2–), 1.97 (m, 8H, γ-CH2–), 1.82 (m, 8H, δ-CH2), 1.68
(m, 8H, ε-CH2–), −2.64 (s, 2H, N–H). 13C NMR (CDCl3)
δ (ppm): 144.8, 128.6 (broad s), 118.1, 38.2, 35.3, 34.0,
32.9, 29.6, 28.2. MS (MALDI-TOF) m/z = 957.96 [M]+˙,
958.14 calcd for C44H58Br4N5. UV-Vis (CH2Cl2): λmax (nm)
(ε × 10−3 L mol−1 cm−1) = 416.9 (440.7), 519.0 (14.9), 554.0
(9.8), 600.1 (4.3), 658.1 (7.3).
Tetra(6-azidohexyl)porphyrin (3). In 20 mL of THF were dis-
solved 100 mg (0.104 mmol) of tetra(6-bromohexyl)porphyrin
2, then 2 mL of H2O containing 56 mg of NaN3 were added to
the solution. The mixture was heated to 66 °C overnight. The
solvent was removed under reduced pressure. The residue was
dissolved in 100 mL CHCl3. The organic solution was washed
with H2O (3 × 10 mL) and dried over MgSO4. The organic
solvent was then removed under reduced pressure. The product
was purified by column chromatography on silica gel with
CH2Cl2–heptane (8 : 2) as eluent then recrystallized from
Synthetic chemistry
Instrumentation. 1H and 13C NMR spectra were recorded
with a Bruker DRX-300 AVANCE transform spectrometer at the
“Pôle Chimie Moléculaire (Welience, UB-Filiale)”. UV/Vis
spectra were recorded with a Varian Cary 1 spectrophotometer.
Mass spectra were obtained with a Bruker Daltonics Ultraflex II
spectrometer in the MALDI-TOF reflectron mode using dithranol
as a matrix or by ESI† on a LTQ Orbitrap XL Thermo spectro-
meter. Accurate mass measurements (HRMS) were carried out
under the same conditions as before. The measurements were
made at the “Pôle Chimie Moléculaire (Welience, UB-Filiale)”.
CHCl3–CH3OH. Yield: 94% (80 mg, 0.098 mmol). 1H NMR
3
(CDCl3) δ (ppm): 9.49 (s, 8H, H-pyrro), 4.93 (t, 8H, JHH
=
3
9.0 Hz, α-CH2–), 3.30 (t, 8H, JHH = 6.0 Hz, N3–CH2–),
2.53 (m, 8H, –CH2–), 1.81 (m, 8H, –CH2–), 1.65 (m, 16H,
–CH2–), −2.65 (s, 2H, N–H). 13C NMR (CDCl3) δ (ppm):
144.7, 128.3 (broad s), 118.1, 51.5, 38.3, 35.3, 29.9, 28.9,
26.8. MS (MALDI-TOF) m/z = 810.50 [M]+˙, 810.50 calcd for
Chemicals and reagents. Unless otherwise noted, all chemi-
cals and solvents were of analytical reagent grade and used as
received. Absolute dichloromethane, chloroform and methanol
were obtained from Carlo Erba. Silica gel (Merck; 70–120 μm)
was used for column chromatography. Analytical thin-layer
chromatography was performed with Merck 60 F254 silica gel
(precoated sheets, 0.2 mm thick). Reactions were monitored by
thin-layer chromatography, UV/Vis spectroscopy and MALDI/
TOF mass spectrometry.
C44H58N16. UV-Vis (CH2Cl2): λmax (nm) (ε × 10−3 L mol−1 cm−1
)
= 416.0 (408.6), 519.0 (14.9), 554.0 (9.8), 601.0 (4.0), 658.9
(7.2).
Tetra(6-aminohexyl)porphyrin (4). Under argon, 70 mg
(0.086 mmol) of tetra(6-azidohexyl)porphyrin 3 was dissolved
in 5 mL of dry THF and cooled to 0 °C. LiAlH4 (30 mg,
0.79 mmol) in 3 mL of dry THF was added slowly to the sol-
ution. The reaction mixture was heated to room temperature and
stirred for 6 h and 0.5 mL of ethanol was added to quench the
reaction. Then 50 mL of water was added. The porphyrin was
extracted with CHCl3 (3 × 50 mL). The collected organic
solution was washed with H2O, brine and dried over MgSO4.
The solution was concentrated to about 3 mL and used directly
7-Bromoheptanal
(1). 7-Bromoheptanenitrile
(2.0
g,
10.52 mmol) was dissolved in 15 mL of dry CH2Cl2 under argon.
The solution was cooled to −78 °C and 40 mL of diisobutyl-
aluminium hydride (1.0 M in CH2Cl2) was added dropwise. The
mixture was stirred for 1 h at −78 °C, then warmed to room
temperature for 3 h. The reaction was quenched with methanol
(20 mL) and 10 mL HCl (10%) was added dropwise at −78 °C.
The product was extracted with ether (3 × 100 mL) and washed
with distilled water then brine. The solvent was removed by
evaporation under reduced pressure to lead to compound 1
in the next step without any further purification. 1H NMR
3
(CDCl3) δ (ppm): 9.39 (s, 8H, H-pyrro), 4.87 (t, 8H, JHH
=
3
8.1 Hz, α-CH2–), 2.64 (t, 8H, JHH = 6.6 Hz, NH2–CH2–),
2.45 (m, 8H, –CH2–), 1.74 (m, 8H, –CH2–), 1.46 (m, 16H,
–CH2–), −2.70 (s, 2H, N–H). MS (MALDI-TOF) m/z =
706.36 [M]+˙, 706.54 calcd for C44H66N8.
1
in 84% yield (1.7 g, 8.80 mmol). H NMR (CDCl3) δ (ppm):
9.79 (s, 1H), 3.42 (t, 2H, 3JHH = 6.0 Hz), 2.46 (m, 2H), 1.88 (m,
2H), 1.67 (m, 2H), 1.40 (m, 4H).
Prot.PorphySQ (5). DMF (7 mL) was added to the above
solution of tetra(6-aminohexyl)porphyrin 4. Then 2-(2-amino-
6-(benzyloxy)-9H-purin-9-yl)acetic acid (110 mg, 0.367 mmol),
N-hydroxybenzotriazole (HOBt) (56 mg, 0.367 mmol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (70.5 mg,
0.367 mmol) were added. The resulting mixture was stirred at
20 °C for 4 d. After the solvent was removed under reduced
pressure, the residue was washed with water, a saturated aqueous
solution of NaHCO3, CH2Cl2, ethyl acetate and methanol repea-
tedly to give compound 5 as a solid. Yield: 42% (65 mg,
Tetra(6-bromohexyl)porphyrin (2). Under nitrogen, 3 mmol
of 7-bromoheptanal 1 (0.58 g) and 3 mmol of pyrrole (0.20 g)
were dissolved in 200 mL of chloroform. The solution was
purged by nitrogen for 2 h. Still under nitrogen, 75 μL of
BF3·Et2O were added to the solution at 20 °C. The mixture was
stirred overnight. DDQ (3 mmol) was added and the mixture
was again stirred for 2 h, then two drops of triethylamine were
added. The solvent was removed under reduced pressure. The
product was purified by column chromatography on silica gel
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1
0.036 mmol). H NMR (CDCl3) δ (ppm): 9.67 (s, 8H), 8.14
Stratagene Mx3005P real-time PCR machine, using a total reac-
tion volume of 100 μL, with 0.2 μM of tagged oligonucleotide
in a buffer containing 10 mM lithium cacodylate pH 7.2 plus
10 mM KCl–90 mM LiCl (for F21T) or 1 mM KCl–99 mM
LiCl (for F-myc-T). After a first equilibration step at 25 °C
during 30 s, a stepwise increase of 1 °C every 30 s for 65 cycles
to reach 90 °C was performed and measurements were made
after each cycle with excitation at 492 nm and detection at
516 nm. The melting of the G-quadruplex was monitored alone
or in the presence of various concentrations of compounds and/
or of competitor, either the self-complementary duplex-forming
sequence ds26 (5′CA2TCG2ATCGA2T2CGATC2GAT2G3′) or the
tetramolecular-quadruplex-forming TG5T sequence (vide infra).
Final analysis of the data was carried out using Excel and Origi-
nPro.8. Emission of FAM was normalized between 0 and 1, and
T1/2 was defined as the temperature for which the normalized
emission is 0.5. ΔT1/2 values are mean of 2 to 4 experiments.
3
(t, 4H, JHH = 6.0 Hz), 7.77 (s, 4H), 7.46–7.31 (m, 20H),
6.38 (s, 8H), 5.45 (s, 8H), 4.96 (m, 8H), 4.67 (s, 8H), 3.12 (d,
8H, JHH = 3.0 Hz), 2.41 (m, 8H), 1.77 (m, 8H), 1.50 (m, 16H),
−2.89 (s, 2H). MS (MALDI-TOF) m/z = 1832.04 [M]+˙,
1832.12 calcd for C100H110N28O8. HR-MS (ESI) m/z
=
1853.8960 [M + Na]+, 1853.8953 calcd for C100H110N28NaO8.
UV-Vis (DMF): λmax (nm) (ε × 10−3 L mol−1 cm−1) = 416.0
(242.0), 519.0 (9.9), 552.0 (6.9), 600.9 (2.9), 658.9 (5.6).
PorphySQ (6). Protected porphyrin 5 (35.0 mg, 0.019 mmol)
was taken up in a solution of MeOH saturated by HCl (20 mL).
The mixture was stirred at 20 °C for 1 h. The final product was
precipitated by addition of diethyl ether (80 mL), and washed
repeatedly with ether (20 mL). Porphyrin 6 was obtained as a
1
solid. Yield: 63% (18 mg, 0.012 mmol). H NMR (DMSO) δ
(ppm): 11.01 (s, 4H), 9.67 (s, 8H), 8.29 (s, 8H), 6.75 (s, 8H),
4.97 (s, 16H), 4.73 (s, 8H), 3.05 (s, 8H), 1.80 (s, 8H), 1.49 (s,
16H), −2.89 (s, 2H). 13C NMR (DMSO) δ (ppm): 164.7, 152.2,
153.8, 150.1, 138.2, 128.9 (broad s), 108.5, 45.7, 34.6, 29.4,
28.9, 26.4. HR-MS (ESI) m/z = 1493.7011 [M + Na]+,
1493.7076 calcd for C72H86N28NaO8. UV-Vis (DMSO):
λmax (nm) (ε × 10−3 L mol−1 cm−1) = 418.0 (238.0), 519.0
(10.0), 554.0 (6.4), 600.0 (2.5), 659.0 (4.0).
Competitive FRET-melting experiments. Experiments were
carried out with 15 and 50 equiv. of both ds26 and [TG5T]4,
expressed in motif concentrations: ds26 being a self-complemen-
tary duplex-DNA, the experiments were thus carried out with
30 and 100 equiv. expressed in strand concentration, which cor-
responds to 6 and 20 μM actual ds26 strand concentration;
[TG5T]4 being a tetramolecular-quadruplex-forming sequence,
the experiments were thus carried out with 60 and 200 equiv.
expressed in strand concentration, which corresponds to 12 and
40 μM actual TG5T strand concentration. Of note, ds26 and
[TG5T]4 have been selected since their melting temperature
(Tm, evaluated by UV-melting experiments) are >20 °C above
that of F21T (Tm = 79 and >80 °C for ds26 and [TG5T]4 respec-
tively in 10 mM lithium cacodylate pH 7.2 plus 10 mM
KCl/90 mM LiCl).
FRET-melting assay
Oligonucleotides. Labelled (F21T and F-myc-T) and
unlabelled oligonucleotides (ds26 and TG5T) were purchased
from Eurogentec (Belgium) in OligoGold purity grade at
∼200 nmol scale (purified by RP-HPLC). Oligonucleotides were
prepared by mixing 40 μL of mother solution (500 μM in strands
(except for TG5T at 2 mM in strands) in deionized water
(18.2 MΩ cm resistivity)), 152 μL of a 100 mM KCl–900 mM
LiCl solution, 152 μL of a lithium cacodylate solution (100 mM,
pH 7.2) and 456 μL of water. The final concentration of the pre-
pared aliquots was theoretically 25 μM for quadruplex-DNA and
50 μM for ds26 in a Caco.K buffer solution (comprised of
10 mM lithium cacodylate buffer (pH 7.2) + 10 mM KCl–
90 mM LiCl) and diluted aliquots (∼2.5 μM) were obtained by
addition of Caco.K buffer. The actual concentrations of aliquots
were evaluated via UV-Vis spectra analysis at 260 nm and
90 °C, using the molar extinction coefficient value provided by
the manufacturer. The higher-order structures of the aliquots
were obtained by heating the solutions at 90 °C for 5 min,
cooling in ice for 6 h to favour the intramolecular folding, and
then were stored at least overnight at 4 °C (except for both
[TG5T]4 and ds26, obtained by heating the solution at 90 °C for
5 min, cooling at 65 °C for 120 min, 50 °C for 90 min, 35 °C
for 60 min, 20 °C for 60 min and finally stored at 4 °C). Prior to
use, typical diluted solutions were F21T: 2.75 μM, F-myc-T:
3.39 μM, ds26: 51 μM and [TG5T]4: 245 μM.
Acknowledgements
The authors thank F. Cuenot for help with funding, CheMatech®
company for the assistance in DOTASQ synthesis, the CNRS,
Université de Bourgogne and Conseil Régional de Bourgogne
(3MIM project (FD, CPG, DM)) and the Agence Nationale de la
Recherche (ANR-10-JCJC-0709 (DM)).
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a high-throughput screen in a 96-well format, with F21T
(FAM-5′G3T2AG3T2AG3T2AG33′-TAMRA, with FAM (F):
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