Loop-mediated fluorescent probes for selective discrimination of parallel and antiparallel G-Quadruplexes
Abstract
Herein we report simple pyridinium (1–3) and quinolinium (4) salts for the selective recognition of G-quadruplexes (G4s). Among them, the probe 1, interestingly, selectively discriminated parallel (c-KIT-1, c-KIT-2, c-MYC) G4s from anti-parallel/hybrid (22AG, HRAS-1, BOM-17, TBA) G4s at pH 7.2, through a switch on response in the far-red window. Significant changes in the absorption (broad 575 nm → sharp 505 nm) and emission of probe 1 at 620 nm, attributed to selective interaction with parallel G4s, resulted in complete disaggregation-induced monomer emission. Symmetrical push/pull molecular confinements across the styryl units in probe 1 enhanced the intramolecular charge transfer (ICT) by restricting the free rotation of C
C units in the presence of sterically less hindered and highly accessible G4 surface/bottom tetrads in the parallel G4s, which is relatively lower extent in antiparallel/hybrid G4s. We confirm that the disaggregation of probe 1 was very effective in the presence of parallel G4–forming ODNs, due to the presence of highly available free surface area, resulting in additional π-stacking interactions. The selective sensing capabilities of probe 1 were analyzed using UV–Vis spectroscopy, fluorescence spectroscopy, molecular dynamics (MD)–based simulation studies, and 1H NMR spectroscopy. This study should afford insights for the future design of selective compounds targeting parallel G4s.
Introduction
G-quadruplexes (G4s) can be classified as parallel, antiparallel, or hybrid types depending on the topologies arising from their dynamic conformations.1 Among them, parallel G4s are generally present in several oncogene promoter regions, including c-MYC, VEGF, and KRAS, forming intramolecular parallel G4s.2, 3 Considering their biological significance (e.g., in cell proliferation; their transcription factor regulatory activities; and their natural existence in genomes), intramolecular G4s have been investigated more extensively under specific conditions4, 5 than have been corresponding intermolecular G4s. Various fluorescent probes have been designed for the selective recognition of G4s over duplex [double-stranded (ds)] and single-stranded (ss) DNAs, with some of them functioning under in vitro conditions.6
Nevertheless, only a few papers have reported topology-oriented selectivities (discrimination between parallel and antiparallel/hybrid topologies) using in vitro models.7 Interestingly, a limited number of such probes have displayed topology-specific variations in their optical properties under physiological conditions.8 More commonly, the sensing strategies have relied on conjugating G4-stabilizing ligands to conventional fluorophores9, 10 or attaching quencher-free probes (e.g., squarylium,11, 12 thiazolium,13, 14 and pyridinium15, 16 units), accompanied by electron donating or withdrawing groups in both symmetrical and unsymmetrical arrangements, to induce effective push/pull effects. Considering the diverse topologies of G4 structures, various coumarin/anthracene, naphthalene diimide, and squaraine-based fluorescent materials have been developed recently to recognize parallel G4s in vitro.17, 18, 19
Although many fluorescent probes have been reported for the recognition of G4s, the structural similarity between parallel and antiparallel/hybrid topologies has made the development of probes targeting only parallel G4s still challenging, especially in the far-red window.
Based on the concept of conventional aggregation-induced quenching, and considering the variety of homo-supramolecular assemblies that are typically formed under physiological conditions through cooperative binding between monomer units, we designed probe 1 featuring dual functionalities (phenolic OH and NEt2 units as electron donors; a pyridinium core as an electron acceptor) to undergo a shift from an intramolecular mode of charge transfer to an intermolecular charge transfer process upon aggregation. Upon consideration of the conformations of the sugar and nucleobase units in each part of the G4 tetrad, especially those in the loop portion, which generally discriminate the parallel and antiparallel G4 topology, we designed our molecular materials (Scheme 1 probes 1–4) to have bent(crescent)/linear shapes, moderate flexibility, and various degrees of hydrophobicity. We validated the sensing capabilities of probe 1 through studies using UV–Vis spectroscopy, fluorescence spectroscopy, 1H NMR spectroscopy, and molecular dynamics (MD)–based simulations.
Section snippets
Results and discussion
Probes 1 and 2 were synthesized from lutidine and 2-methylpyridine scaffolds, respectively, by forming their respective 2,6-dimethylpyridinium and 2-methylpyridinium salts and then performing simple Knoevenagel condensations with N,N-diethylaminosalicylaldehyde in the presence of a catalytic amount piperidine. Probe 3 was synthesized through the condensation of 4-picolylamine and 1,8-naphthalic anhydride in EtOH and subsequent reaction with 9,10-dicholoromethylanthracene in MeCN. Similarly,
Synthesis of probes
Probe 1: A solution of lutidine (2.00 g, 18.7 mmol) and MeI (2.32 g, 37.3 mmol) in CH2Cl2 (50 mL) was stirred under a N2 atmosphere at room temperature for 5 h, monitoring through TLC. The creamy white solid was filtered off, washed with ether (3 × 15 mL), and dried under vacuum to give 1,2,6-trimethylpyridinium iodide (89%); 1H NMR [400 MHz, CDCl3, δ (ppm)] 2.97 (s, 6H), 4.03 (s, 3H), 7.74 (d, J = 7.90 Hz, 2H), 8.18 (t, J = 7.90 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 23.4, 42.6, 128.0, 144.2,
Conclusion
We have demonstrated that probe 1, a simple pyridinium-based salt, allows the identification of parallel G4s over antiparallel and hybrid G4 topologies as well as other non-canonical/canonical forms of DNA (i.e., ssDNA, dsDNA, Poly G, triplex, TWJ). Probe 1 recognized the tested parallel G4s with a selective switch on response in the far-red emission region, with excellent selectivity and sensitivity. We analyzed the selective sensing capabilities of probe 1 using UV–Vis spectroscopy,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (2017R1A2B4002398), funded by the Republic of Korea.
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