TY - JOUR
T1 - Design and synthesis of a long-wavelength latent fluorogenic substrate for salicylate hydroxylase
T2 - A useful fluorimetric indicator for analyte determination by dehydrogenase-coupled biosensors
AU - Huang, Sheng Tung
AU - Teng, Chun Jen
AU - Lee, Yu Hui
AU - Wu, Jui Yu
AU - Wang, Kun Li
AU - Lin, Chun Mao
PY - 2010/9/1
Y1 - 2010/9/1
N2 - 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.
AB - 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.
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U2 - 10.1021/ac101281p
DO - 10.1021/ac101281p
M3 - Article
C2 - 20695438
AN - SCOPUS:77956252905
SN - 0003-2700
VL - 82
SP - 7329
EP - 7334
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 17
ER -