A Twice-As-Smart Synthetic G‑Quartet: PyroTASQ Is Both a Smart Quadruplex Ligand and a Smart Fluorescent Probe Aure ́ lien Laguerre, † Loic Stefan, † Manuel Larrouy, † David Genest, † Jana Novotna, †,‡ Marc Pirrotta, † and David Monchaud* ,† † Institute of Molecular Chemistry, University of Dijon, ICMUB CNRS UMR6302, 21078 Dijon, France ‡ Department of Analytical Chemistry, Institute of Chemical Technology, 166 28 Prague, Czech Republic * S Supporting Information ABSTRACT: Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex- promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as “twice-as-smart” quadruplex ligands. ■ INTRODUCTION G-quadruplex ligands 1 represent a new class of anticancer agents, the mechanism of which relies on the stabilization of an unusual DNA structure termed G-quadruplex DNA. 2 The quadruplex/ligand assemblies create DNA damages and trigger DNA damage signaling and repair machineries, 3 the so-called DNA damage response (DDR). 4 Most cancer cells being DDR- impaired, 5 or easily chemically impaired (synthetic lethality strategy), 3,6 they are found more sensitive to DNA damaging drugs than their healthy counterparts. G-quadruplex ligands can therefore be considered as a novel class of DNA damaging agents, whose effectiveness rests neither on DNA modifications (i.e., alkylating agents) nor on induction of DNA strand breaks (i.e., antitumor antibiotics), but on the targeting of a noncanonical DNA structures: 7 when stabilized, these higher- order DNA structures pose a serious challenge to DNA transactions, impeding the normal DNA/RNA polymerases processivity, which provides a strong DNA damage signal that leads to the recruitment of the DDR machinery. 3,6,8 Quadruplex ligands are not sequence but structure selective therapeutic agents, therefore displaying a higher, easier to control level of selectivity. Although indisputable progress has been made over the past years in the use of quadruplex ligands as therapeutic agents, 9 a novel challenge is now taking shape on how to take advantage of the wealth of knowledge gained in the therapeutic arena to design multitasking compounds capable of both interacting with and detecting quadruplex structures, in a cellular context a fortiori. Since an increasing number of DNA/RNA sequences able to fold into stable quadruplex structures are currently identified in key regions of both genome and transcriptome, 10 sensing the existence and consequences 11 of quadruplexes in cells is a chemical biology quest of particular significance and strategic relevance. Several successful small-molecule-based approaches have been recently devised, including an out- standing in cellulo fluorescent labeling (AlexaFluor) of a ligand (pyridostatin-α) already bound to quadruplexes in living cells, 3 or via the design of fluorescent probes whose spectroscopic properties are turned on upon interaction with quadruplexes, known as light-up probes. 12 The molecular and/or electronic mechanisms underlying the quadruplex-promoted fluorescence enhancement of the light-up dyes are rather ill-defined but mainly fall into two categories, originating either in restriction of intramolecular rotation (RIR), including cyanine (e.g., thiazole orange), 13 benzimidazole (e.g., BPBC), 14 and carbazole derivatives (e.g., BMVC) 15 as well as aggregation-induced emission (AIE) luminogens (e.g., TBE) 16 , or in a protection provided by the DNA matrix against water-mediated non- radiative deactivation, including routinely used nucleic acid Received: June 24, 2014 Published: August 7, 2014 Article pubs.acs.org/JACS © 2014 American Chemical Society 12406 dx.doi.org/10.1021/ja506331x | J. Am. Chem. Soc. 2014, 136, 12406-12414