Flavin-enabled reductive and oxidative epoxide ring opening reactions

Abstract

Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.

Fig. 1. Representative epoxide ring-opening reactions.

a Nonenzymatic epoxide ring-opening reactions involving addition of a nucleophile (Nuc) under basic or acidic conditions. b Enzymatic epoxide ring-opening reactions by epoxide hydrolases (EHs) and oxidoreductases (ORs). c Epoxide ring-opening reactions of fluostatin C (1), either enzymatically catalyzed by the hydrolase Alp1U, or nonenzymatically mediated by FAD and NADH as reported in this study.

Fig. 2. Epoxide ring opening reactions involving FST C (1).

a HPLC analysis of reactions involving FST C (1). (i) 1 std.; (ii) 1 + Alp1U; (iii) 1 + FlsH; (iv) 1 + FlsH + Fre; (v) 1 + FlsH + NADH; (vi) 1 + FlsH + Fre + NADH; (vii) 1 + Fre + NADH; (viii) 1 + FAD + NADH; (ix) 1 + Fre; (x) 1 + NADH; (xi) 1 + FAD; (xii) 1 + FAD + NADH. HPLC was performed using a reversed phase C18 column (i–xi) or in a polar column (xii). Reactions were run for 30 min at 30 °C in 50 mM PBS buffer (pH 7.0) containing 5 μM enzyme(s) (Alp1U, FlsH or Fre), 100 μM FAD and 10 mM NADH. b The X-ray crystal structures of 7 and 10. c Experimental ECD spectrum of 9 (black line) compared with the B3LYP/TZVP PCM/MeCN // ωB97X/TZVP PCM/MeCN spectrum of (1R,2S,3R)-9 (red line). The bars represent rotational strength values for the lowest energy solution conformers.

Fig. 3. Detailed studies on the epoxide ring opening reactions of 1 by isotope labeling, intermediate characterization and cofactor compatibility.

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).

Fig. 4. Expanded substrate and product profile for the FAD/NADH-mediated epoxide ring opening reactions and comparison with the flavoenzyme RslO5-catalyzed reactions.

FST derivatives 15‒20 could undergo epoxide-ring opening reactions upon treatment with FAD/NADH to produce multiple products related to 3, 7, 8, and 9. FST Q (21) was unreactive with FAD/NADH. The epoxides 28–35 displayed no activities with FAD/NADH. Treatment of auxarthrol H (36) or menadione 2,3-epoxide (37) with FAD/NADH yielded multiple products. The epoxides 42–46 were found to be unreactive with FAD/NADH. The oxidoreductase RslO5 catalyzed a reductive epoxide ring opening reaction on 43 to produce 44 during rishirilide biosynthesis and chalcone α,β-epoxide (46) was found to be a substrate of RlsO5 to yield the single product 47.

Fig. 5. Proposed mechanisms for flavin-enabled epoxide ring opening reactions.

The reaction can proceed via reductive and oxidative pathways leading to multiple products of different redox states depending on the presence of NADH and O₂. The key transformations in the reductive reactions (yellow background) are proposed to involve a flavin N5‒C4′ adduct (49) to produce the enol intermediate 13, which can undergo tautomerization to yield 7 (major) or 8 (minor), and oxidation to produce 3 (major) or 9 (minor). 7 can be further autooxidized to 12. Whereas the oxidative ring opening is likely to involve a flavin C4a‒O‒O‒C3′ adduct (50) to give the peroxylated intermediate 14 (gray background), which can undergo reduction to 3 and 9 by NADH, and further rearrangements leading to 10 and 11.