High-Throughput Virtual Screening of Small Molecule Quenchers for Near-Infrared Fluorophores

Energy transfer (EnT)-based near-infrared (NIR) fluorescent probes are preferred for biosensing due to their low phototoxicity, deep photon penetration, and high signal-to-noise ratios in vivo. A significant challenge in this domain is the scarcity of small-molecule quenchers (SMQs) that can effectively modulate NIR fluorescence. This study presents the first large-scale screening of SMQs, performing high-throughput virtual screening (HTVS) of 26,695 compact candidate structures (≤ 6 non-hydrogen atoms). We identified several promising molecular moieties, such as ─N═O, ─C═S, and ─B─N═ fragments, and experimentally validated the ─N═O motif as a representative proof-of-concept by constructing three red-to-NIR probes (P2-P4) that operate from ∼590 to 820 nm and successfully image nitric oxide in cells and in vivo. The resulting SMQ library includes candidates predicted to be compatible with NIR fluorophores emitting beyond 800 nm and provides a design blueprint that may accelerate the development of next-generation EnT-based NIR fluorescent probes.