5′-Amino-Formyl-Thieno[3,2-<i>b</i>]thiophene End-Label for On-Strand Synthesis of Far-Red Fluorescent Molecular Rotors and pH-Responsive Probes

The ability to label synthetic oligonucleotides with fluorescent probes has greatly expanded their nanotechnological applications. To continue this expansion, it is essential to develop approachable, modular, and tunable fluorescent platforms. In this study, we present the synthesis and incorporation of an amino-formyl-thieno[3,2-<i>b</i>]thiophene (AFTh₂) handle at the 5'-position of DNA oligonucleotides. The 5'-AFTh₂ end-label participates in both on-strand Knoevenagel and heterocyclization reactions, yielding far-red hemicyanines and pH-responsive probes with p<i>K</i>_a values in the biological regime. The Knoevenagel products, designated 5'-ATh₂Btz and 5'-ATh₂Ind, demonstrate excitation maxima beyond 640 nm with brightness up to ∼50,000 M^-1 cm^-1. Notably, 5'-ATh₂Btz demonstrates strong topology sensitivity, allowing it to probe transitions from duplex- to single-strand (SS)/G-quadruplex (GQ) topologies with an ∼9-fold increase in fluorescence in the absence of quenchers. In contrast, the heterocyclization product, 5'-ATh₂BIM, displays visible excitation and emission and is weakly fluorescent in basic solution. Upon lowering the pH from ∼8 to 5, this probe undergoes an unprecedented 400-fold light-up. Additionally, attaching 5'-ATh₂BIM to a polymorphic GQ allows for a shift in p<i>K</i>_a by ∼1.5 pH units simply by changing topology. The performance of the probes has been demonstrated in various contexts, including GQs, i-motifs, duplexes, and SS oligonucleotides. Their performance should facilitate the development of new DNA-based sensing platforms.