Read-Out with HPLC

The mixture was quenched 1:1 with acetonitrile, and centrifuged at >14,000g for 10 min. The supernatant was analyzed by HPLC. HPLC was performed on an Agilent 1260 infinity II, equipped with a C-18 column (Mackerey-Nagel, 5 μm, ref 760.100.40) using acetonitrile (HPLC grade, Agilent) and ddH₂O (0.1% (v/v) formic acid (FA)). The program was as follows: 0 min −100% ddH₂O (0.1% (v/v) FA) 0% acetonitrile. Three min −40% ddH₂O (0.1% (v/v) FA) 60% acetonitrile. Five min −0% ddH₂O (0.1% (v/v) FA) 100% acetonitrile. Six min −0% ddH₂O (0.1% (v/v) FA) 100% acetonitrile. Seven min −100% ddH₂O (0.1% (v/v) FA) 0% acetonitrile

All samples were analyzed at 277 nm, representing the isosbestic point between indole and tryptophan, allowing for the estimation of yield by comparing the area of the substrate peak to the areas of both the substrate and product peak combined.²⁷