Whole‐Cell‐Catalyzed Multiple Regio‐ and Stereoselective Functionalizations in Cascade Reactions Enabled by Directed Evolution

Abstract

Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E. coli whole cells for regio- and stereoselective cascade sequences that are not possible using man-made catalysts. The approach is illustrated using P450-BM3 in combination with appropriate alcohol dehydrogenases as catalysts in either two-, three-, or four-step cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone, respectively, leading to either (R,R)-, (S,S)-, or meso-cyclohexane-1,2-diol. The one-pot conversion of cyclohexane into (R)- or (S)-2-hydroxycyclohexanone in the absence of ADH is also described.

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

“…have recently reported the production of 4‐hydroxy‐α‐isophorone (HIP, 6 , Scheme ) from α‐IP to a pilot scale using the well‐known P450‐BM3 from Bacillus megaterium (CYP102A1), paving the way for a sustainable P450‐based KIP production. On the other hand, several reports deal with the application of P450s to catalyse the initial C−H activation in one‐pot multi‐enzymatic transformations: examples are the oxyfunctionalisation of linear hydrocarbons (both in vitro and in vivo),– the production of cycloalkanones or lactones from the corresponding cycloalkanes,– the synthesis the grapefruit flavour (+)‐nootkatone, the conversion of cyclohexane to cyclohexanediols, and the stereoselective benzylic amination of ethylbenzene derivatives . Therefore, considering these biocatalytic strategies, we designed a cascade system to access KIP from α‐IP through a double allylic oxidation (Scheme ), employing a P450 in the first step to afford HIP, followed by further oxidation by an alcohol dehydrogenase (ADH).…”

Whole‐Cell Cascade Biotransformations for One‐Pot Multistep Organic Synthesis

“… Cascade biotransformations of alkanes with E. coli cells. a) Oxidation of cyclooctane to cyclooctenone; b) conversion of octane to octylamine; c) conversion of cyclohexane to ( R , R )‐, ( S , S )‐, or meso ‐cyclohexane‐1,2‐diol …”

“…Recently, Reetz and co‐workers presented another excellent example of artificial whole‐cell cascade (Scheme c) . Directed evolution of P450‐BM3 gave two unique mutants, P450A82F and P450A82F/A328F, catalyzing the 3‐step oxidation of cyclohexane to ( R )‐ and ( S )‐2‐hydroxycyclohexanone, respectively.…”

Tandem Reactions Combining Biocatalysts and Chemical Catalysts for Asymmetric Synthesis

“…However, there are very few known processes that integrate bio-orthogonal reactions catalyzed by transition-metal catalysts into multi-step biocatalytic systems in cells for complex transformations. The progress of developing biocompatible transition-metal complexes that function in cells, together with strategies of engineering enzymatic multi-step catalysis in vivo [112,113], will open new windows for creating new cell factories for chemical production.…”