Rationalisation of the stereochemical outcome of ene-reductase-mediated bioreduction of ,-difunctionalised alkenes Elisabetta Brenna a,∗ , Michele Crotti a , Francesco G. Gatti a , Alessia Manfredi a , Daniela Monti b , Fabio Parmeggiani a , Andrea Pugliese a , Davila Zampieri a a Politecnico di Milano, Dipartimento di Chimica, Materiali, Ingegneria Chimica, Via Mancinelli 7, I-20131 Milano, Italy b Istituto di Chimica del Riconoscimento Molecolare-CNR, Via M. Bianco 9, I-20131 Milano, Italy Received 24 October 2013 Received in revised form 17 December 2013 Accepted 21 December 2013 Available online 7 January 2014 1. Introduction The use of ene-reductases (ERs) for the enantioselective reduc- tion of activated alkenes is currently receiving great interest [1], because of the efficacy shown by this kind of transformation in the synthesis of chiral building blocks for organic chemistry appli- cations [2]. The effects due to the stereochemistry of the starting alkene, to the steric and electronic characteristics of the activating electron-withdrawing groups (EWGs), as well as of other sub- stituents are to be carefully investigated, in order to define the limits and potential of this kind of reaction. The information is essential for including this bioreduction into the pool of synthetic tools, into which chemists can delve to select the best strategy for the preparation of their target molecules, just as they are accus- tomed to do when they search among conventional reagents of organic chemistry. The availability of robust enzymatic procedures can facilitate the introduction of biocatalysed steps in modern pro- duction processes, bringing along all the advantages of enzymes for sustainability. Most of the ERs that have been identified in the last decades belong to the well-known family of Old Yellow Enzymes (OYEs), which are characterised by the presence of a flavin mononucleotide ∗ Corresponding author. Tel.: +39 02 23993077; fax: +39 02 23993180. E-mail address: elisabetta.brenna@polimi.it (E. Brenna). (FMNH 2 ) prosthetic group which imparts a yellow colour to puri- fied protein samples. It has been established that the C C double bond can only be reduced by these enzymes if it is made susceptible to the nucleophilic attack of a hydride (delivered by the reduced flavin mononucleotide prosthetic group) by the presence of an EWG, which is also able to establish hydrogen bonds within the binding pocket of the enzyme [3]. Investigations are to be devoted to define which EWGs can activate alkenes towards OYE-catalysed reductions by themselves and which ones are to be combined with other groups. Up to now, it has been established that ,- unsaturated aldehydes and ketones, nitroolefins and maleimides are good substrates for this kind of reactions, whereas the OYE- mediated reduction ,-unsaturated esters is only possible when another EWG is present on the double bond, e.g. a halogen atom linked to the same carbon atom bearing the ester function [4], or an ester [5] or a nitrile [6] group in  position. We have recently reported [6a] on the reduction of cyano esters (E)- and (Z)-1 (Scheme 1), precursors of  2 -amino acids for foldamer applications, by means of OYE1-3, and we have carried out a detailed analysis of the stereochemical course of the reaction by means of deuterium labelling. Contemporaneously, a paper has been published [6b] describing the reduction of the carbon–carbon double bond of a class of regioisomeric compounds, i.e. (E)- and (Z)-2 (Scheme 1), by means of isolated OYEs with the aim of pro- viding a biocatalytic route to precursors of GABA analogues, such as pregabalin.