An asymmetric sp3-sp3 cross-electrophile coupling using 'ene'-reductases.

The catalytic asymmetric construction of Csp3-Csp3 bonds remains one of the foremost challenges in organic synthesis1. Metal-catalysed cross-electrophile couplings (XECs) have emerged as a powerful tool for C-C bond formation2-5. However, coupling two distinct Csp3 electrophiles with high cross-selectivity and stereoselectivity continues as an unmet challenge. Here we report a highly chemoselective and enantioselective Csp3-Csp3 XEC between alkyl halides and nitroalkanes catalysed by flavin-dependent 'ene'-reductases (EREDs). Photoexcitation of the enzyme-templated charge-transfer complex between an alkyl halide and a flavin cofactor enables the chemoselective reduction of alkyl halide over the thermodynamically favoured nitroalkane partner. The key C-C bond-forming step occurs by means of the reaction of an alkyl radical with an in situ-generated nitronate to form a nitro radical anion that collapses to form nitrite and an alkyl radical. An enzyme-controlled hydrogen atom transfer (HAT) affords high levels of enantioselectivity. This reactivity is unknown in small-molecule catalysis and highlights the potential for enzymes to use new mechanisms to address long-standing synthetic challenges.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

Enantioselective Synthesis of α-Thiocarboxylic Acids by Nitrilase Biocatalysed Dynamic Kinetic Resolution of α-Thionitriles.

The enantioselective synthesis of α-thiocarboxylic acids by biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyse the DKR of racemic α-thionitriles under mild conditions, affording the corresponding carboxylic acids with high conversions and good-to-excellent ee. The ammonia produced in situ during the biocatalytic transformation favours the racemization of the nitrile enantiomers and, in turn, the DKR without the need of any external additive base.

Photo-biocatalytic One-Pot Cascades for the Enantioselective Synthesis of 1,3-Mercaptoalkanol Volatile Sulfur Compounds.

The synthesis of enantiomerically pure 1,3-mercaptoalkanol volatile sulfur compounds through a one-pot photo-biocatalytic cascade reaction is described. Two new KRED biocatalysts with opposite enantioselectivity were discovered and proved to be efficient on a wide range of substrates. The one-pot cascade reaction combining photocatalytic thio-Michael addition with biocatalytic ketoreduction in an aqueous medium provides a green and sustainable approach to enantiomerically pure 1,3-mercaptoalkanols in high yields with excellent enantioselectivity.

Fluorogenic kinetic assay for high-throughput discovery of stereoselective ketoreductases relevant to pharmaceutical synthesis.

Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry's demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols.