ABSTRACT
Ene-reductases allow regio- and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene-reductase FOYE-1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE-1 to produce valuable compounds. A maximum activity of (319.7±3.2)â U mg-1 with NADPH or of (206.7±3.4)â U mg-1 with 1-benzyl-1,4-dihydronicotinamide (BNAH) for the reduction of N-methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE-1 showed excellent performance in most systems with up to 20â vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200â mL biotransformation of 750â mg (R)-carvone afforded 495â mg of (2R,5R)-dihydrocarvone (>95 % ee), demonstrating the simplicity of handling and application of FOYE-1.
Subject(s)
Biocatalysis , Cyclohexane Monoterpenes/chemistry , Flavoproteins/metabolism , Oxidoreductases/metabolism , Solvents/chemistry , Kinetics , NADP/metabolism , Oxidation-Reduction , Substrate SpecificityABSTRACT
The combination of enzymes with traditional chemical catalysts unifies the high selectivity of the former with the versatility of the latter. A major challenge of this approach is the difference in the optimal reaction conditions for each catalyst type. In this work, we combined a cofactor-free decarboxylase with a ruthenium metathesis catalyst to produce high-value antioxidants from bio-based precursors. As suitable ruthenium catalysts did not show satisfactory activity under aqueous conditions, the reaction required the use of an organic solvent, which in turn significantly reduced enzyme activity. Upon encapsulation of the decarboxylase in a cryogel, the decarboxylation could be conducted in an organic solvent, and the recovery of the enzyme after the reaction was facilitated. After an intermediate drying step, the subsequent metathesis in pure organic solvent proved to be straightforward. The synthetic utility of the cascade was demonstrated by the synthesis of the antioxidant 4,4'-dihydroxystilbene in an overall yield of 90 %.
ABSTRACT
A recombinant enoate reductase was expressed in cyanobacteria and used for the light-catalyzed, enantioselective reduction of C=C bonds. The coupling of oxidoreductases to natural photosynthesis allows asymmetric syntheses fueled by the oxidation of water. Bypassing the addition of sacrificial cosubstrates as electron donors significantly improves the atom efficiency and avoids the formation of undesired side products. Crucial factors for product formation are the availability of NADPH and the amount of active enzyme in the cells. The efficiency of the reaction is comparable to typical whole-cell biotransformations in E.â coli. Under optimized conditions, a solution of 100â mg prochiral 2-methylmaleimide was reduced to optically pure 2-methylsuccinimide (99 %â ee, 80 % yield of isolated product). High product yields and excellent optical purities demonstrate the synthetic usefulness of light-catalyzed whole-cell biotransformations using recombinant cyanobacteria.
Subject(s)
Biocatalysis , Cyanobacteria/metabolism , Oxidoreductases/metabolism , Water/metabolism , Biotransformation , Oxidation-Reduction , Oxidoreductases/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Water/chemistryABSTRACT
Peroxygenases catalyze a broad range of (stereo)selective oxyfunctionalization reactions. However, to access their full catalytic potential, peroxygenases need a balanced provision of hydrogen peroxide to achieve high catalytic activity while minimizing oxidative inactivation. Herein, we report an enzymatic cascade process that employs methanol as a sacrificial electron donor for the reductive activation of molecular oxygen. Full oxidation of methanol is achieved, generating three equivalents of hydrogen peroxide that can be used completely for the stereoselective hydroxylation of ethylbenzene as a model reaction. Overall we propose and demonstrate an atom-efficient and easily applicable alternative to established hydrogen peroxide generation methods, which enables the efficient use of peroxygenases for oxyfunctionalization reactions.