Asymmetric Bioreduction of Activated Alkenes Using Cloned 12‐Oxophytodienoate Reductase Isoenzymes OPR‐1 and OPR‐3 from Lycopersicon esculentum (Tomato): A Striking Change of Stereoselectivity

Abstract

The asymmetric reduction of C=C bonds goes in hand with the creation of up to two chiral centers and is thus one of the most widely employed strategies for the synthesis of chiral compounds. Whereas cis hydrogenation using homogeneous catalysts based on (transition) metals has been developed to a high standard, [1] stereocomplementary trans reduction is still at the stage of development. [2,3] The biocatalytic equivalent of this reaction is catalyzed by enoate reductases [EC 1.3.1.x], [4,5] commonly denoted as the "old yellow enzyme" family.[6] These common enzymes act through transfer of a hydride ion, derived from a flavin cofactor (FMNH 2 ), onto the b-carbon atom of an a,bunsaturated carbonyl compound, while a proton, derived from the solvent, adds from the opposite side onto the acarbon atom. As a consequence of this mechanism, the hydrogenation occurs in a trans-specific fashion. The catalytic cycle is completed by the reduction of FMN at the expense of NAD(P)H, which is regenerated by an additional redox reaction (Scheme 1). Although the mode of action of these enzymes has been elucidated in great detail, their application in preparative-scale biotransformations has been hampered for several reasons: although the best-studied enzymes from this group, which were isolated from strictly anaerobic bacteria such as Proteus and Clostridium spp., [7] were shown to be highly stereoselective, the sensitivity of these proteins towards traces of molecular oxygen prevented their practical application. As a consequence, whole-cell biotransformations using aerobic microorganisms, most prominently bakers yeast, [8] dominated the field. Although the stereoselectivities achieved were often excellent, the chemoselectivity of wholecell bioreductions with respect to the reduction of C = C against C=O bonds was notoriously plagued by the competing reduction of the carbonyl group that was catalyzed by alcohol dehydrogenases. [9,10] Since enoate reductases and alcohol dehydrogenases depend on the same nicotinamide cofactor, redox decoupling of both enzyme activities is not possible.Isolated and/or cloned enoate reductases are required in sufficient amounts, together with a suitable recycling system for the nicotinamide cofactor, to make this biotransformation practically feasible. Only recently, the first successful attempts in the chemo-and stereoselective reduction of conjugated enones were reported by using cloned old yellow enzymes from Saccharomyces carlsbergensis [11] and bakers yeast. [12] Since enoate reductases have been shown to possess a much broader substrate spectrum than their name would suggest, [13] we initiated a search for a candidate that possessed a broad substrate specificity but showed high stereoselectivity towards a wide range of C = C bonds bearing an electronwithdrawing activating group. In this context, we came across 12-oxophytodienoate reductase (OPR) which occurs in several isoforms in plants. [14] Whereas isoenzyme OPR3 is responsible for the reduction of (9S,13S)-12-oxophytodienoate to the corre...

Activated α,β‐Unsaturated Aldehydes as Substrate of Dihydroxyacetone Phosphate (DHAP)‐Dependent Aldolases in the Context of a Multienzyme System

“…The enzyme was inactive for more sterically hindered substrates such as 3-phenylcyclohexene-1-one. Similar trends have also been seen with other OYEs, [2][3][4] which is consistent with the structural similarity of the enzymes within the family. However, recent studies show that directed evolution can be used to extend the substrate specificity of OYEs towards more sterically hindered substrates.…”

“…This unexpected change of enantioselectivity for formation of 2-methylcyclopentanone 17b has also been reported for other OYEs, although no explanation for this observation was given. [2][3][4] In light of recent work by Stewart et al [5] it is possible that substrate 17a may bind in a 'flipped' orientation, while maintaining an optimal Cβ-FMN N5 distance and angle (105°), [21] similar to that seen for OYE1 mutant W116I. [5] PETNR proved to be highly efficient and highly stereoselective in the reduction of 2-methylmaleimide derivatives 1a-b giving the respective (2R)-products in quantitative yields and high enantioselectivities (>99% ee, Table 2 and Scheme 1c).…”

“…[4,22] The interpretation of the stereochemical outcome of the reduction of citral 11a [the citral 11a used in this study was a mixture of geranial (E)-11a and neral (Z)-11a in approximately 65:35 ratio (by GC)] and nitroalkenes 18a and 19a is more complex, because these substrates isomerise between their E/Z forms under the reaction conditions. [23,24] Fryszkowska et al Page 4 Adv Synth Catal.…”

Identification of enzymes responsible for the reduction of geraniol to citronellol

“…Successful attempts in the chemo-and stereoselective reduction of activated alkenes were reported by using cloned yeast OYE1 from Saccharomyces carlsbergensis, [5][6][7] , OYE2 and OYE3 from S. cerevisiae, [6][7][8] CmOYE from Candida macedoniensis; 9 and bacterial OYE homologues YqjM from Bacillus subtilis, NCR from Zymomonas mobilis; 6,10 and plant OYE homologues LeOPR1 and LeOPR3 from Lycopersicon esculentum (tomato). 10,11 The plant 12-oxophytodienoate reductase (OPR) is involved in the reduction of (9S,13S)-12-oxophytodienoate to the corresponding cyclopentanoic acid OPC-8:0 for jasmonate biosynthesis. 12 By using the cloned OPR1 and OPR3 from tomato, a striking change of stereoselectivity and a remarkably broad substrate spectrum were achieved for asymmetric reduction.…”

“…12 By using the cloned OPR1 and OPR3 from tomato, a striking change of stereoselectivity and a remarkably broad substrate spectrum were achieved for asymmetric reduction. 10,11 Although the above OYE enzymes have been biochemically well characterized, they have never been investigated for the capacity of reducing the allylic alcohol double bond in geraniol, because alcohols are not typically favored as activating groups for OYEs. 3 In our effort to broaden the applicability of plant OPR, we came across its catalytic activity of converting geraniol to citronellol as a sole product (Scheme).…”