Directed Evolution of an Amine Oxidase Possessing both Broad Substrate Specificity and High Enantioselectivity

Abstract

Enantiomerically pure chiral amines are of increasing value in organic synthesis, especially as resolving agents, [1] chiral auxiliaries/chiral bases, [2] and catalysts for asymmetric synthesis. [3] In addition, chiral amines often possess pronounced biological activity in their own right and hence are in demand as intermediates for agrochemicals and pharmaceuticals. [4] Current methods for the preparation of enantiomerically pure chiral amines are largely based upon the resolution of racemates, either by recrystallization of diastereomeric salts [5] or by enzyme-catalyzed kinetic resolution of racemic substrates using lipases and acylases.[6] To develop more efficient methods, attention is turning towards asymmetric approaches or their equivalent, for example, the asymmetric hydrogenation of imines [7] or the conversion of ketones into amines by using transaminases.[8] Attempts to develop dynamic kinetic resolutions, which employ enzymes in combination with transition-metal catalysts, have unfortunately been hampered by the harsh conditions required to racemize amines.[9]Recently we reported a novel catalytic method for the preparation of optically active chiral amines by deracemization of the corresponding racemic mixture (Figure 1).[10] The deracemization approach relies upon coupling an enantioselective amine oxidase with a nonselective reducing agent to effect stereoinversion of the S to R enantiomer via the intermediate achiral imine.The S enantiomer selective amine oxidase used for the deracemization of (R/S)-a-methylbenzyl amine was identified from a library of variants of the wild-type enzyme, from Aspergillus niger, by using a high-throughput colorimetric screen to guide selection.[10] The library of variants was generated by randomly mutating the plasmid harboring the amine oxidase gene by using the E. coli XL1-Red mutator strain. Using (S)-a-methylbenzylamine as the target substrate we were able to identify a variant (Asn336Ser) that possessed significantly improved catalytic activity (47 fold) and enantioselectivity (sixfold) towards this particular substrate compared to the wild type enzyme. To explore the opportunities for using this variant amine oxidase to deracemize other racemic chiral amines we decided to undertake a more detailed study of its substrate specificity. Herein we show that the Asn336Ser variant possesses broad substrate specificity and high enantioselectivity towards a wide range of chiral amines.Prior to carrying out further studies with the Asn336Ser amine oxidase, an additional mutation was introduced into the sequence (Met348Lys) that resulted in a variant enzyme (hereafter referred to as Asn336Ser) with higher specific activity and expression levels although its substrate specificity appeared unchanged (data not shown). Incorporation of an N-terminal histidine tag into the amine oxidase allowed facile purification of both the wild-type and Asn336Ser variant in one step, by a nickel-affinity column, to yield protein of > 90 % purity as evidenced by gel electrophoresis (Figure ...

Directed evolution of d -sialic acid aldolase to l -3-deoxy- manno -2-octulosonic acid ( l -KDO) aldolase

“…Hence, we tried to coax the enzyme to accept L-sialic acid by expanding the scope of its substrate specificity with an analogue, L-KDO, which is easier to synthesize (11). Although enzyme adaptation for substrate analogues is unpredictable (19), it has been demonstrated that activities of some substrate analogues do increase throughout the course of directed evolution (10,14,20). The wild-type sialic acid aldolase exhibits a low level of activity toward D-and L-KDO (5,21).…”

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

“…Finally, we believe that enzymecatalyzed enantioselective partial oxidations will become more important in the future, especially when synthetic catalysts fail completely (65). Preliminary studies in our laboratories concerning the directed evolution of a cyclohexanone monooxygenase as a biocatalyst in the Baeyer-Villiger reaction of 4-hydroxycyclohexanone show that the enantioselectivity of this synthetically interesting transformation can be increased substantially from ee ϭ 9% to ee ϭ 90%.…”

Recent trends in whole cell and isolated enzymes in enantioselective synthesis

“…Examples of each of these are described in Figure 10. [224][225][226] Improved enantioselectivity, catalytic activity at increased substrate concentration Example C: 227 Inversion of enantioselectivity Example D: 228 …”