Fig. 3: Detailed studies on the epoxide ring opening reactions of 1 by isotope labeling, intermediate characterization and cofactor compatibility.
From: Flavin-enabled reductive and oxidative epoxide ring opening reactions
a NMR analysis of 2H or 18O incorporation in products and intermediates as well as potential interconversions. Deuterium incorporation from 2H2O at C-3 of 7 and 8 was inferred from 1H NMR spectroscopic analysis. The O18-labeling in 3-18O and 9-18O was supported by HRMS analysis. The assigned structures of 12 and 14 are shown, whereas the proposed structure of 13 remains speculative on account of its poor stability. b HPLC analysis of reactions under varying conditions. (i) 1 std.; (ii) 1 + FAD + NADH (in air); (iii) 1 + FAD + NADH (under 18O2); (iv) 1 + FAD + NADH (under N2); (v) 7 in 50 mM borax/NaOH buffer (pH 10) at 30 °C for 12 h; (vi) 100 μM 1 + 10 μM FAD + 2 mM NADH (30 °C for 30 min); (vii‒ix) incubation of 13 collected from (vi) with (vii) H2O; (viii) FAD; (ix) FAD + NADH; the reactions of (viii) and (ix) were performed in O2 at 30 °C for 30 min in 50 mM PBS buffer (pH 7.0); (x) 14 in H2O; (xi) 14 + FAD; (xii) 14 + NADH; (xiii) 14 + FAD + NADH. The reactions of (x-xiii) were performed at 30 °C for 30 min. The reactions of (xiv‒xvii) involved incubation of 1 and NADH with different flavin cofactors: (xiv) FAD; (xv) FMN; (xvi) riboflavin; (xvii) isoalloxazine at 30 °C for 30 min. (xviii) The reaction containing 1, F420, glucose 6-phosphate and FGD was incubated at 30 °C for 10 h. HPLC analysis was run with UV detection at 304 nm using a polar column (traces i-v & x-xviii) or a reversed phase C18 column (traces vi-ix).
