Fluorometric Assay for Selective Aldehyde Detection in Biocatalytic Oxidation of Diols

Abstract Alcohol oxidation is a key transformation in the manufacture of pharmaceuticals, fragrances, and other high-value specialty chemicals. Enzymes offer greener, more efficient manufacturing routes via chemoselective alcohol oxidation under mild conditions. However, the engineering of such chemoselective biocatalysts is limited by the lack of rapid, quantitative assays that can discriminate between different oxidation products. Here, we address this challenge by developing a fluorescence assay based on the Hantzsch reaction to distinguish aldehydes from ketone and carboxylic acid by-products which can arise from secondary alcohol oxidation or aldehyde over-oxidation. Our investigations into the applicable substrate scope of the assay show that its performance depends on both the intrinsic reactivity of the aldehyde in the Hantzsch reaction and the influence of aldehyde structure on non-radiative relaxation pathways. By studying the diol, 1-(3-(hydroxymethyl)phenyl)ethan-1-ol, as a case study substrate, we demonstrate how structure–fluorescence relationships can be exploited by tuning reaction time to bias the assay toward selective detection of mono-oxidized aldehyde products while minimizing interference from di-oxidized species that also contain aldehyde groups. The assay achieved a limit of detection of 0.01 mM, a limit of quantification of 0.04 mM ( R 2 = 0.9993), and yielded results consistent with orthogonal high-performance liquid chromatography analysis with an average deviation of 0.013 mM when applied to the screening of galactose oxidase variants. Our assay will facilitate the development of precision oxidation biocatalysts with enhanced selectivity that support greener manufacturing routes for specialty chemicals.