Photoenzymatic Reduction of CC Double Bonds

Abstract

A simplified procedure for cell-free biocatalytic reductions of conjugated C=C double bonds using old yellow enzymes (OYEs) is reported. Instead of indirectly regenerating YqjM (an OYE homologue from B. subtilis) or NemA (N-ethylmale-A C H T U N G T R E N N U N G imide reductase from E. coli) via regeneration of reduced nicotinamide cofactors, we demonstrate that direct regeneration of catalytically active reduced flavins is an efficient and convenient approach. Reducing equivalents are provided from simple sacrificial electron donors such as ethylenediaminetetra-A C H T U N G T R E N N U N G acetate (EDTA), formate, or phosphite via photocatalytic oxidation. This novel photoenzymatic reaction scheme was characterized. Up to 65% rates of the NADH-driven reaction were obtained while preserving enantioselectivity. The chemoselectivity of the novel approach was exclusive. Even when using crude cell extracts as biocatalyst preparations, only C=C bond reduction was observed while ketone and aldehyde groups remained unaltered. Overall, a simple and practical approach for photobiocatalytic reductions is presented.

Ene‐reductases and their Applications

“…Illumination of YqjM with visible light in the presence of free flavin and EDTA as a sacrificial electron donor quantitatively converted 4-ketoisophorone (4a) to (R)-levodione (4b, Figure 18.6a) [57]. Similar studies were performed by Hollmann et al [58] for the photoenzymatic flavin reduction using YqjM and NEMR (N-ethylmaleimide reductase from E. coli) [58]. The alternative hydride sources used were EDTA, formate, or phosphite, and the reduction was performed under strict anaerobic conditions to avoid rapid reoxidation of the highly reactive reduced flavin by molecular oxygen.…”

“…For the conversion of 4a to 4b up to 65% of the specific activities in comparison to the NADH-driven reaction were obtained when flavin reduction occurred with light while preserving the enantioselectivity. Additionally, undesired side reactions, such as reduction of the carbonyl groups by contaminating ADHs, could be circumvented [58].…”

Photobiocatalytic chemistry of oxidoreductases using water as the electron donor

“…A more elegant approach is to deliver the reducing equivalents directly from the cosubstrate to the oxidoreductases, thereby shortcutting the complicated natural electron transport chain. Ever since the pioneering work by Vilker and coworkers 14 , a range of direct regeneration approaches have been reported [15][16][17][18][19][20][21][22][23][24][25][26][27][28] .…”

Alternative Hydride Sources for Ene‐Reductases: Current Trends

“…This NAD(P)H‐free system was successful in converting ketoisophorone to ( R )‐levodione, albeit with lower product enantiopurity. Similarly, Hollmann and co‐workers described the photoenzymatic flavin reduction of YqjM and NEMR ( N ‐ethylmalemide reductase from E. coli ) by using alternative sacrificial electron donors formate and phosphite . These methods required strict anaerobic conditions to prevent the rapid reoxidation of reduced FMN by molecular oxygen …”