Fig. 3: Engineering a [2 + 2] cyclase VEnT1.3 featuring a thioxanthone sensitizer.
From: Efficient and selective energy transfer photoenzymes powered by visible light
a, Chemical scheme showing the intramolecular [2 + 2] photocycloaddition of 4-(pent-4-en-1-yl)quinolin-2(1H)-one (1) which affords two enantiomeric products (+)-1a and (−)-1a. b, Bar chart showing the yield of 1 to rac-1a, as determined by UPLC, by small-molecule benzophenone and thioxanthone at different excitation wavelengths (5 min, 100 mol% catalyst loading). Error bars represent the s.d. of technical triplicate measurements. c, Reaction yield and enantioselectivity were improved along the evolutionary trajectory. Reaction conditions: 0.125 mol% catalyst, 400 µM 1, 70-s irradiation at 405 nm, 4 °C. All yields and selectivity data, including s.d., are given in Supplementary Table 2. d, A semipreparative-scale biotransformation of 1 catalysed by VEnT1.3 produced optically pure (+)-1a in essentially quantitative yield (>99% yield, >99% e.e. and 97% isolated yield). Reaction conditions: 0.5 mol% catalyst, 400 µM (12 mg) 1, 5-min irradiation at 405 nm, 4 °C. e, Left: crystal structure of VEnT1.3 (PDB: 9FYV). Blue sticks represent the mTX residue at position 173. Mutations introduced throughout directed evolution are highlighted with spheres at the Cα, coloured according to their order of introduction, corresponding to the variants shown in c. Right: close-up view of active site with docked (+)-1a shown as black sticks. f, VEnT1.3 progress curve to determine the total turnover number (TON) over 30 min. Reaction conditions: 0.05 mol% VEnT1.3, 400 µM 1, 405 nm. Error bars represent the s.d. of technical triplicate measurements. g, Light-intensity rate profile of VEnT1.3. Intensity was varied from 0% to 100% with the reactions irradiated 3 cm directly below the LED array (100% intensity corresponds to 10,960 µmol photons m2 s−1). Reaction conditions: 0.25 mol% VEnT1.3, 400 µM 1, 405 nm. E, enzyme. Error bars represent the s.d. of technical triplicate measurements. h, Comparison of reaction time courses at 395 nm for VEnT1.0 (white), VEnT1.3 (green) which both contain mTX at position 173, and EnT1.3 (purple), which bears BpA at position 173. Reaction conditions: 0.25 mol% catalyst, 400 µM 1. Error bars represent the s.d. of technical triplicate measurements.
