The activity orchestration of an unprecedented cell‐free enzyme system with self‐sufficient cofactor recycling enables the stepwise transformation of aliphatic diols into ω‐hydroxy acids at the expense of molecular oxygen as electron acceptor. The efficiency of the biosynthetic route was maximized when two compatible alcohol dehydrogenases were selected as specialist biocatalysts for each one of the oxidative steps required for the oxidative lactonization of diols. The cell‐free system reached up to 100 % conversion using 100 mM of linear C5 diols and performed the desymmetrization of prochiral branched diols into the corresponding ω‐hydroxy acids with an exquisite enantioselectivity (ee>99 %). Green metrics demonstrate superior sustainability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5‐hydroxypetanoic acid. Finally, the cell‐free system was assembled into a consortium of heterogeneous biocatalysts that allowed the enzyme reutilization. This cascade illustrates the potential of systems biocatalysis to access new heterofunctional molecules such as ω‐hydroxy acids.

In vitro biosynthetic pathways that condense and reduce molecules through coenzyme A (CoASH) activation demand energy and redox power in the form of ATP and NAD(P)H, respectively. These coenzymes must be orthogonally recycled by ancillary reactions that consume chemicals, electricity, or light, impacting the atom economy and/or the energy consumption of the biosystem. In this work, we have exploited vinyl esters as dual acyl and electron donor substrates to synthesize β‐hydroxy acids through a non‐decarboxylating Claisen condensation, reduction and hydrolysis stepwise cascade, including a NADH recycling step, catalyzed by a total of 4 enzymes. Herein, the chemical energy to activate the acyl group with CoASH and the redox power for the reduction are embedded into the vinyl esters. Upon optimization, this self‐sustaining cascade reached a titer of (S)‐3‐hydroxy butyrate of 24 mM without requiring ATP and simultaneously recycling CoASH and NADH. This work illustrates the potential of in vitro biocatalysis to transform simple molecules into multi‐functional ones.

In this study, we probed the inhibition of pig heart citrate synthase (E.C. 4.1.3.7) by synthesising seven analogues either designed to mimic the proposed enolate intermediate in this enzyme reaction or developed from historical inhibitors. The most potent inhibitor was fluorovinyl thioether 9 (Ki=4.3 μm), in which a fluorine replaces the oxygen atom of the enolate. A comparison of the potency of 9 with that of its non‐fluorinated vinyl thioether analogue 10 (Ki=68.3 μm) revealed a clear “fluorine effect” favouring 9 by an order of magnitude. The dethia analogues of 9 and 10 proved to be poor inhibitors. A methyl sulfoxide analogue was a moderate inhibitor (Ki=11.1 μm), thus suggesting hydrogen bonding interactions in the enolate site. Finally, E and Z propenoate thioether isomers were explored as conformationally constrained carboxylates, but these were not inhibitors. All compounds were prepared by the synthesis of the appropriate pantetheinyl diol and then assembly of the coenzyme A structure according to a three‐enzyme biotransformation protocol. A quantum mechanical study, modelling both inhibitors 9 and 10 into the active site indicated short CF⋅⋅⋅H contacts of ≈2.0 Å, consistent with fluorine making two stabilising hydrogen bonds, and mimicking an enolate rather than an enol intermediate. Computation also indicated that binding of 9 to citrate synthase increases the basicity of a key aspartic acid carboxylate, which becomes protonated.

The reaction between configurably stable -lithiated oxiranes (and aziridines) and 3-substituted cyclobutanones allows to obtain enantiomerically enriched cyclobutanols (er > 98:2). These adducts, subjected to base-mediated Payne rearrangement lead to...

A variety of enantiomerically enriched tetrahydrofuran spirocyclic derivatives have been synthesised by a sequential enantioselective proline‐induced stereospecific resolution through aldol reaction of (E/Z)‐(phenylsulfanyl)cycloalkanecarbaldehydes followed by a ketone reduction, acid‐catalysed [1,2]‐SPh stereospecific migration, and ring‐closure reaction.