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ACS Chemical Biology
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scaffolds that fall far outside the “druglike” chemical space,
high molecular weights, the induction of conformational
changes, and complicated multistep synthetic procedures
further strategies are required to rationally design G4-
interacting small optical probes as easily accessible diagnostic
tools.
To detect G4s in living cells, the probe should have both
high selectivity for specific G4 structures/sequences and good
membrane permeability. These basic characteristics, although
prerequisites, are rarely simultaneously fulfilled by the same
molecular agent. Within the class of G4-probes, coumarin dyes
are among the most studied compounds. In previous studies,
the coumarin scaffolds were usually extensively modified
through the insertion of π-extended heterocyclic aromatic
motifs in order to tune both the photophysical properties and
G4-binding properties to both DNA and RNA (Scheme
1).19,35,43−47 Even if all these molecular recognition strategies
G4s, a twisted intramolecular charge transfer (TICT) process
opened competitive radiative relaxation pathways that led to a
marked light-up fluorescence response. The resulting binding
events enabled us to discriminate parallel G4s over antiparallel
and non-G4 topologies through visible color changes
detectable by the naked eye. The structural details of the
compound’s binding interactions with parallel G4 c-MYC
promoter structures were also assessed by 1D 1H NMR
titration studies, showing stacking interactions to the terminal
G-tetrads. Moreover, we showed that DNA synthesis in cells,
studied at single-molecule level, were slowed. The slower DNA
synthesis was also confirmed by a biochemical DNA
polymerase stop assay that clearly showed the ability of this
compound to arrest DNA synthesis prior to the G4 structure.
Finally, intracellular studies indicated that this small water-
soluble optical probe decrease the viability of cancer cells, and
that it is capable of rapid cellular entry and nucleolar
localization, thus enabling detection of G4 DNA structures
in live cells.
Scheme 1. Examples of Previously Published Coumarin
Derivatives Used for G4 DNA and RNA Detection in Vitro
a
,
and in Cells.19,35,43−47
RESULTS AND DISCUSSION
■
Design of the Molecular Probes. All tested compounds
had low molecular weights (<300 Da) and obey Lipinski’s
“rule of five”, which predicts druglike properties. The
compounds were synthesized in a single step (see Scheme
S1 and Figures S1−S3) through Knoevenagel condensation of
commercially available substituted ortho-hydroxyl benzalde-
hydes (1a-c) with ethyl cyanoacetate in the presence of
ammonium acetate via in situ formation of 3-cyanocoumarin,
which on subsequent reduction led to the formation of the
desired 3-amidinocoumarin derivatives (2a-c) (Scheme 1).
Characterization of the Compounds by Solvent-
Dependent Studies. We first determined the spectroscopic
properties of the coumarin derivatives 2a−2c and performed
solvent-dependent UV/vis absorption and emission measure-
ments in order to determine how different solvent polarities
affect the optical properties of the compounds. For these
measurements, we used seven different solvents with different
polarities (water, methanol (MeOH), ethanol (EtOH),
acetonitrile (ACN), dichloromethane (DCM), chloroform
(CHCl3), ethyl acetate (EtOAc), and tetrahydrofuran
(THF)). By increasing the solvent polarity, the absorption
maximum (λmax) was increased from 409 nm to 445 nm (Δλmax
= 36 nm) and 432 to 466 nm (Δλmax = 34 nm) for 2a and 2c,
respectively (Figures 1 and S4). Emission studies in these
solvents demonstrated a shift of the λem to longer wavelengths
at increasing solvent polarities. This bathochromic shift of the
emission band was 474−486 nm (Δλem = 12 nm) and 487−
502 nm (Δλem = 15 nm) for 2a and 2c, respectively (Figures 1
and S4). The plots of solvent polarity versus absorption/
emission maximum indicated an overall positive solvatochro-
mic behavior for 2a and 2c (Figure 1), showing that the red-
shifted absorption and emission spectra of the compounds are
dependent on an increased solvent polarity.
a
The structures of the coumarin analogues reported by us in previous
studies35,47 and the structures of the three derivatives used in the
present study (2a-c). The coumarin scaffold is marked in red.
have been successfully implemented in in vitro models and, in
some cases, in fixed and live cells, the molecular sizes of some
of these newly generated probes are not ideal for membrane
permeability and might therefore hinder the detection of G4
DNA in live cells.
The most-selective G4 probes show high signal in the G4-
rich nucleoli,15,19,20,35,36 which are highly dense multifunc-
tional domains in which ribosome biogenesis occurs with a
high level of transcriptional activity involving both G4 DNA
and RNA structures.20
Inspired by our recently published results on the use of
coumarin−quinazolinone,35 coumarin−benzothiazole,47 and
quinazoline−quinazolinone15,36 compounds, we synthesized
three low-molecular-weight amidinocoumarin derivatives with
druglike characteristics that differed only by the nature of their
electron-donating substituents that determine their fluores-
cence properties (Scheme 1). By testing different G4
oligonucleotides, we found that two of these probes exhibited
topology-specific G4-binding properties, but only one of them
showed promising potential to detect G4 DNA in cells. This
molecule displayed negligible background fluorescent signal in
its unbound state. However, upon interaction with parallel
For 2b, the solvent-dependent absorption and emission
studies both indicated a negative solvatochromic effect of the
compound with λmax increasing from 417 nm to 455 nm
(Δλmax = 38 nm) and λem ranging from 451 nm to 468 nm
(Δλem = 17 nm) when the solvent polarity was decreased (see
Figure 1, as well as Figure S4 in the Supporting Information).
These findings clearly support a greater stabilization of the first
excited state of 2a and 2c, relative to the ground state, which is
associated with the increased dipolar character of the
B
ACS Chem. Biol. XXXX, XXX, XXX−XXX