Effects of Modifying Thioflavin T at the N(3)-Position on Its G4 Binding and Fluorescence Emission

We previously synthesized thioflavin T (ThT) with a hydroxyethyl group introduced at the N(3)-position (ThT-HE), which binds predominantly to the parallel G-quadruplex (G4) structure found in c-Myc and emits strong fluorescence. In this study, to investigate the effects of introduced substituents on G4 binding and fluorescence emission, a ThT derivative in which the hydroxyl group of ThT-HE was replaced with an amino group (ThT-AE) was synthesized for the first time. Furthermore, three other N(3)-modified ThT derivatives (ThT-OE2, ThT-SP, and ThT-OE11) having different substituent structures were synthesized by the N-acylation of the terminal amino group of ThT-AE, and their G4-binding and emission properties were investigated. The results showed that, although ThT-AE shows binding selectivity depending on the type of G4, its emission intensity is significantly decreased as compared to that of ThT-HE. However, ThT-OE11, which features an 11-unit oxyethylene chain attached to the terminal amino group of ThT-AE, regained about one-half of the emission intensity of ThT-HE while retaining selectivity for G4s. Accordingly, ThT-OE11 may be used as a key intermediate for synthesizing the conjugates of G4 binders and probes.