Structure and activity of NADPH-dependent reductase Q1EQE0 from Streptomyces kanamyceticus, which catalyses the R-selective reduction of an imine substrate.

NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99% ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossman-fold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known ß-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme.

Straightforward preparation of biologically active 1-aryl- and 1-heteroarylpropan-2-amines in enantioenriched form.

Because of the importance of developing stereoselective syntheses for single enantiomers, a selected panel of racemic biologically active 1-aryl- and 1-heteroarylpropan-2-amines has been prepared, followed by a study of their behavior in enzymatic kinetic resolution (KR) processes. For this purpose, lipase B from Candida antarctica (CAL-B) proved to be an ideal biocatalyst allowing the preparation of the corresponding enantioenriched (R)-amides and (S)-amines by aminolysis reactions. Likewise, dynamic kinetic resolutions (DKR) have been successfully achieved combining the use of CAL-B and Shvo's catalyst. This research constitutes the first example of a lipase-catalyzed DKR process of ß-substituted isopropylamines.

Chemoenzymatic synthesis of rivastigmine based on lipase-catalyzed processes.

A straightforward chemoenzymatic synthesis of enantiomerically pure rivastigmine has been efficiently carried out under mild reaction conditions, with Candida antarctica lipase B responsible for the stereoselective acetylation of the corresponding (R)-alcohol or amine. An exhaustive enzymatic study has been developed exploring the possibilities of carry out enzyme recycling, scaling up the enzymatic process and development of a dynamic kinetic resolution procedure for the production of adequate enantiomerically pure precursors of rivastigmine. Total chemoenzymatic synthesis of this pharmaceutical has been performed in good overall yield from commercially available 3-methoxyacetophenone.

Efficient access to enantiomerically pure cyclic alpha-amino esters through a lipase-catalyzed kinetic resolution.

A series of alpha-amino acid derivatives containing the 2,3-dihydroindole or octahydroindole core have been chemoenzymatically synthesized in good overall yields and high enantiomeric purity under mild reaction conditions using lipases for the introduction of chirality. Candida antarctica lipase type A has shown excellent activity and high enantiodiscrimination ability towards the two cyclic amino esters used as substrates. The selectivity of the process proved to be greatly dependent on the alkoxycarbonylating agent. Thus, the enzymatic kinetic resolution of methyl indoline-2-carboxylate has been successfully achieved using 3-methoxyphenyl allyl carbonate, whereas (2R,3aR,7aR)-benzyl octahydroindole-2-carboxylate required the less reactive diallyl carbonate.