Fig. 4: Engineering a photoenzyme SpEnT1.3 for spirocyclic β-lactam synthesis.
From: Efficient and selective energy transfer photoenzymes powered by visible light
a, Chemical scheme showing the photochemical conversion of 2 to afford four stereoisomeric spirocyclic β-lactam products ((S,S)-2a, (R,R)-2a, (R,S)-2a and (S,R)-2a), and two side products (3 and 4). b, Reaction yield, diastereoselectivity and enantioselectivity were improved along the evolutionary trajectory. These improvements coincided with suppression of the undesired debenzylation side reaction giving rise to 3 and 4. The ratio of syn-2a, anti-2a and side products is represented as a pie chart and e.e. is given for (S,S)-2a. Reaction conditions: 2 mol% catalyst, 200 µM 2, 1-min irradiation at 405 nm. All yields and selectivity data, including s.d., are given in Supplementary Table 6. c, Left: crystal structure of SpEnT1.3 (PDB: 9G65). Blue sticks represent the mTX residue at position 244. 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 b. Right: close-up view of active site with docked (S,S)-2a, shown as black sticks. d, Top: reverse-phase UPLC chromatograms comparing the reaction profiles of light-induced conversion of 2 by small-molecule thioxanthone and SpEnT1.3. Bottom: chiral normal-phase UPLC chromatograms comparing a stereoisomeric mixture of product 2a and the product profile achieved by SpEnT1.3. e, Light-intensity rate profile of SpEnT1.3. 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% SpEnT1.3, 400 µM 1, 405 nm. Error bars represent the s.d. of technical triplicate measurements. f, Kinetic transients showing the decay of the triplet state of thioxanthone (TX), SpEnT1.0 and SpEnT1.3 at 4 °C. Data shown are the average of five traces and have been normalized for initial intensity. Data were fitted to a double-exponential equation (black lines).
