Catechol chemistry unleashed: Spontaneous reactions in physiologically-relevant media

Catecholic compounds, characterized by their distinct dihydroxybenzene functional groups, are recognized for their diverse biological roles and emerging therapeutic potential. Despite significant advances in understanding their biotransformation and chemical reactivity, the potential for enzyme-independent reactions of catechols under physiologically relevant conditions remains underexplored. This study investigates the enzyme- and cell-free transformations of catechol in cell culture media and biocompatible buffers. In neutral to mildly alkaline (pH 7.0-8.0) phosphate buffer without additional additives, catechol transformed into dark-coloured, spectrally active species (“browning”) within 6 hours, in a temperature-dependent manner. The presence of amine-containing molecules – including amino acids (glycine, lysine, proline), biocompatible buffers (tris(hydroxymethyl)aminomethane, or Tris), and proteins (bovine serum albumin, or BSA) – dramatically accelerated browning rates in a pH-dependent manner, with visually detectable and spectrophotometrically quantifiable changes occurring within minutes. BSA-enhanced catechol browning also altered the protein's electrophoretic mobility, suggesting structural modifications induced by the browning reaction. These transformations may occur before cellular uptake or interaction with biological targets, potentially influencing the bioavailability and therapeutic efficacy of catecholic compounds. This work raises new questions about the structural-functional relationships of catecholic compounds and underscores the importance of accounting for their spontaneous reactivity in experimental design.