5-19: Multi-mode Spectroscopic High Throughput Screening (HTS) of Phenols and Monolignols

Phenolic structure is a common motif amongst monolignols. We developed multi-mode, high throughput screening (HTS)-compatible, UV-Vis and fluorescence assays for phenols and monolignols that are executed in optical “turn on” or “turn off” formats and operated under kinetic or endpoint modes. We used p-cresol as a model phenol and coniferyl alcohol as a prototype monolignol. We chose Trametes versicolor fungal laccase to oxidize p-cresol and coniferyl alcohol and thus expanded the spectroscopic tools for analyzing these molecules. Laccase activity toward p-cresol was monitored kinetically at pH 4.5 by absorption changes at 250, 274 or 297nm, and in endpoint mode by a bathochromic shift to 326nm. Laccase oxidation of p-cresol was also detected by product fluorescence at 425nm after excitation at 262 or 322nm. We optimized the kinetic parameters for p-cresol oxidation (pH optimum 4.5-5.1; 37^oC; Km = 2.2mM) resulting in laccase limits of detection and quantization (LOD, LOQ) of 25 and 75pg/mL, respectively. The p-cresol LOD was 8 micromolar. We similarly characterized the spectroscopic properties of coniferyl alcohol. Three isosbestic wavelengths were identified at 240, 242 and 262nm with S/B of ~50 for 500 micromolar coniferyl alcohol, establishing assay sensitivity. Coniferyl alcohol excitation spectrum (270 – 335nm) overlapped with its absorption spectrum. Fluorescence emission was between 360 – 500nm with peak at 416nm yielding 1 micromolar detection sensitivity. Unlike p-cresol, laccase oxidation of coniferyl alcohol quenched the fluorescence. In conclusion, orthogonal interrogation and ratiometric analysis capabilities of our assay enable high specificity while minimizing interferences during compound library screenings.