UGT enzyme assays

Recombinant UGT71L1 and UGT78M1 was produced and purified as per Fellenberg et al., (2020). Enzyme assays were performed for 3 h at 30�C, in a 200-�L reaction volume in 50-mM sodium phosphate (pH 7.4). Reactions contained UDP-glucose (5 mM), substrates at a final concentration of 250 �M and 5 �g of purified recombinant protein. Reactions were halted with the addition of 20 �L 30% trichloroacetic acid (w/v), and 6 min of centrifuging at 16,100g. Supernatant was collected and stored at −20�C prior to analysis. Analysis and product identification of enzyme reaction products was conducted on a Waters Acquity UPLC LC/MS system. For analysis, an 8-�L sample injection was separated using a Waters Acquity Ethylene Bridged Hybrid (C18, pore size: 1.7 �m, dimensions: 2.1 � 50 mm) column, and glucoside products were identified based on mass and retention time. Product peak areas were determined using UV absorbance at 280 nm. Compounds were separated on a gradient binary consisting of ultra-pure water containing 0.1% formate (solvent A) and LC/MS grade acetonitrile with 0.1% formate (solvent B). The flow rate was 0.5 mL�min⁻¹. Separation gradient was 90.0% A, 0–0.5 min; 90.0%–10.0% A, 0.5–6.0 min; 10.0%–0.5% A, 6.0–7.0 min; 0.5%–10.0% A, 7.0–8.0 min; 10.0%–90.0% A, 8.0–8.5 min. A single quadrupole MS in positive ionization mode (ES+) continually scanned masses 50.0 to 1,000.0 m/z from 0.2 to 8.5 min. Cone voltage was set at 35 V. Sodium adducts of benzyl salicylate glucoside and salicyl benzoate glucoside were identified at 413 m/z. The sodium adduct of salicyl salicylate glucoside was identified at 429 m/z.