Fig. 1. Developing biocatalytic strategies to access organic azides. (a) Photoexcited ene-reductases catalyzed redox-neutral radical hydrofunctionalization. (b) Biocatalytic approaches for azidation of alkenes. (c) This work: enantioselective hydroazidation of alkenes by synergistic dual photo/ene-reductase catalysis. EREDs = ene-reductases, FMNox = oxidized form of flavin mononucleotide, FMNox* = excited state FMNox, SET = single electron transfer, HAT = hydrogen-atom transfer, cyan LEDs, an emission wavelength centered in the 500-510 nm range.

Fig. 2. Development of the photobiocatalytic radical hydroazidation of alkenes. (a) Discovery of hydroazidation activity using ene-reductase-based photobiocatalytic single catalysis. (b) Photocatalyst effects on enantioselective radical hydroazidation of alkenes. (c) Evaluation of the key reaction parameters. aConditions: 1a (0.005 mmol), NaN3 (0.025 mmol), ERED (1.0 mol%), Eosin Y (5 mol%), DMSO (5 v/v %) in Tris buffer (50 mmol L-1, pH 6.5) were stirred at room temperature for 12 hours under N2 atmosphere with the irradiation of cyan LEDs (500-510 nm). The total volume of the reaction is 1 mL. bYield was determined by HPLC. cEnantiomeric ratio (e.r.) was determined by HPLC analysis on a chiral stationary phase. n.d. = not determined, YersER = ene-reductase from Yersinia bercovieri, PETNr = ene-reductase from Enterobacter cloacae, CsER = ene-reductase from Caulobacter segnis, OYE1 = ene-reductase from Saccharomyces pastorianus, GluER = ene-reductase from Gluconobacter oxydans.

Fig. 3. The scope and derivatization of the photobiocatalytic products. The standard conditions shown in Fig. 2(c), entry 1, were applied. HPLC determined yields as the average of duplicates using two different batches of enzyme. e.r. = enantiomeric ratio was determined by HPLC analysis on a chiral stationary phase. aYields were obtained using Eosin Y as the photocatalyst. bYields were obtained using Phloxine B as the photocatalyst. cIndicates isolated yield based on a 0.1 mmol scale-up reaction.

Fig. 4. Mechanistic studies. (a) UV-vis absorption spectra. YersER (0.05 mmol L-1), Eosin Y (0.0125 mmol L-1), NaN3 (25 mmol L-1). (b) Luminescence quenching experiments. NaN3 (0-25 mmol L-1) was added to the YersER solution (0.05 mmol L-1) in Tris buffer. Samples were excited at 455 nm, and emission was measured at 540 nm (c) Transformations with apoenzyme. (d) Radical trapping experiment with TEMPO. (e) Deuterium labeling experiments.

Fig. 5. Proposed catalytic mechanism for the photobiocatalytic hydroazidation. FMNox/sq = oxidation/semiquinone state of flavin mononucleotide, HAT = hydrogen-atom transfer.