A dark luminol analogue: HRP-catalyzed oxidation of 3-indazolinones to salicylic acid derivatives

Horseradish peroxidase (HRP)-catalyzed oxidation of luminol is a cornerstone chemiluminescent reaction, proceeding through nitrogen extrusion to generate an electronically excited 3-aminophthalate species. Here we report that 3-indazolinones, a structurally related class of heterocycles, undergo an analogous HRP-catalyzed oxidative pathway to yield salicylic acid derivatives, yet do so without any detectable light emission. This "dark" reactivity is observed across ten structurally diverse indazolinone substrates, including the amino-substituted analogue most closely resembling luminol itself, and proceeds efficiently under both alkaline and physiological buffer conditions. In contrast to the inefficient, self-limiting oxidation of luminol, indazolinone substrates are consumed nearly quantitatively, although salicylic acid accounts for only a fraction of the theoretical yield, indicating that competing radical-mediated pathways divert much of the reactive flux to unassigned side products. We further demonstrate that this newly uncovered chemistry can be harnessed analytically: a nitro-substituted indazolinone derivative serves as a real-time absorbance-based turn-off probe, while a methyl-acrylate-substituted derivative serves as a fluorescence-based turn-on probe, for monitoring HRP activity. These readouts track substrate consumption more directly than luminol-based chemiluminescent assays. These findings expand the known scope of peroxidase-catalyzed oxidative transformations and establish 3-indazolinones as a versatile new substrate class for both mechanistic enzymology and the design of enzyme-responsive optical probes.