Design and Synthesis of a Long-Wavelength Latent Fluorogenic Substrate for Salicylate Hydroxylase: A Useful Fluorimetric Indicator for Analyte Determination by Dehydrogenase-Coupled Biosensors

Salicylate hydroxylase (SHL) catalyzes the production of catechol (plus CO₂ and H₂O) from salicylate, NADH, and O₂. Coimmobilization of SHL with a NAD(P)⁺-dependent dehydrogenase in front of a Clark-type oxygen electrode has been investigated in the development of a general type of dehydrogenase-based biosensors that can detect various biological analytes; however, currently, no fluorophores are available for these applications. We synthesized the first new long-wavelength latent fluorogenic substrate SHLF (3) for SHL. In the presence of NADH and under aerobic conditions, SHL catalyzes the decarboxylative hydroxylation of SHLF followed by a quinone−methide-type rearrangement reaction concomitant with the ejection of a fluorescence coumarin 2, which is spontaneous and irreversible at physiological temperatures in aqueous media. The fluorescence signal generated by this process is specific and, in the near red spectral region with an emission maximum at 595 nm, is suppressed by salicylic acid. The fluorescence response of SHLF is insensitive to various biological reactive oxygen species (ROS) and reductants. Furthermore, SHLF is a sensitive fluorimetric indicator for analyte determination in the SHL-coupled dehydrogenase assay in which NAD⁺ is converted to NADH. This novel fluorescence assay detected 3-hydroxybutyrate and cholesterol in the nanomolar range and is more sensitive than the current SHL−dehydrogenase amperometric sensors, making it applicable to the construction of a fiber-optic fluorescence biosensor for clinical diagnostic uses.