Steric Effects on the Stereochemistry of Old Yellow Enzyme‐Mediated Reductions of Unsaturated Diesters: Flipping of the Substrate within the Enzyme Active Site Induced by Structural Modifications

Abstract

The ene-reductase-mediated reduction of the carbon-carbon double bond of some alkyl 2substituted butenedioates was investigated. The stereochemical outcome of the reaction was found to be influenced by steric effects. Ethyl and butyl citraconates were converted into the corresponding alkyl (R)-2-methylsuccinates with excellent enantioselectivity, whereas ethyl and butyl mesaconates were completely unreactive. Methyl 2-substituted fumarates were reduced to enantiomerically enriched methyl (S)-2-substituted succinates, whereas the (Z)-stereoisomers were left unreacted by enereductases. Labelling experiments were performed to investigate the mechanism of these bioreductions and explain their stereochemical outcome.

“A Study in Yellow”: Investigations in the Stereoselectivity of Ene‐Reductases

“…In particular, in the case of a generic substrate with two equal or similar EWGs, the situation is rather complex, since eight possible options have to be considered, as shown for example with the case of diethyl 2-methylmaleate (also known as diethyl citraconate) in Figure 2. [9] Crystallographic structures and/or docking studies are powerful tools to elucidate the binding, but in most cases they offer a rather "static" view of the active site, which only partially takes into account the dynamic and flexible structure of the protein, often oversimplifying the interactions on the basis of steric hindrance. This is obviously not true if very detailed computational simulations are carried out with high level of chemical accuracy, but these calculations are highly time-and resource-consuming and can be performed only by experienced computational scientists.…”

Investigation of the stereochemical course of ene reductase-catalysed reactions by deuterium labelling

“…In a further development of our research, we investigated the OYE-mediated reduction of 2substituted methyl fumarates and maleates (E)-and (Z)-2 with alkyl chains of increasing length at C 2 , alkyl mesaconates and citraconates (E)-and (Z)-3 showing ester moieties longer than CH 3 [13], and cyanoesters (E)-and (Z)-4 characterised by substituents of increasing steric hindrance at C 2 [14], in order to evaluate the effects due to the stereochemistry of the starting alkene and the nature of substituents. High values of enantioselectivity and conver-sions were obtained with compounds (E)-2, and (Z)-3, affording, respectively, the (S)-, and (R)-enantiomers of the corresponding reduced products.…”

“…The structural characteristics of the EWGs which are needed for the double bond reduction are still to be clearly defined, in order to establish the synthetic potential of this procedure and to include it into the enzyme tool box available to synthetic chemists. With this aim, we have recently investigated the OYE-mediated reduction of difunctionalised substrates reported in Figure 2, in order to establish whether the presence of a second EWG besides the carboxylic moiety could promote the C=C bioreduction [13,14,[18][19][20]. The comprehension of the different stereochemical outcome of the (E) and (Z) stereoisomers of the same compound required the identification of the EWG involved in the formation of the hydrogen bonds in the enzyme active site.…”

Ene‐reductases and their Applications

“…However, the addition of a second activating group can increase the C═C bond polarization and facilitate the hydrogenation. Several studies have been performed on α,β-unsaturated dicarboxylic acids and esters [41,42] and α-halo-substituted unsaturated acids and esters [19,40,[43][44][45] for determining the reaction rate and/or stereochemical outcome of the asymmetric hydrogenation using ERs. For example, an additional halogenated substituent in the α-position was shown to be beneficial for ER activity, whereas β-alkyl or β-aryl substituents were detrimental for the reactivity of nonhalogenated substrates [40,43].…”