Deracemization of racemic chiral tertiary amines has been achieved by combination of an enantioselective amine oxidase, obtained through directed evolution, and ammonia borane in a one-pot process.

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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 ...

The thermodynamic stability and solution conformational properties of three intramolecular triple helices based on the sequence AGAAGA-x-TCTTCT-x-TCTTCT (x is a non-nucleotide linker) comprising a DNA duplex and DNA, RNA, or 2‘-OMeDNA third strands have been compared. The most stable triple helix contains the 2‘-OMe third strand, followed by the triplex containing RNA in the third strand. Comparison of the NMR spectroscopic data for the RNA hybrid triplex with those of the all-DNA triplex shows that the duplex parts of the structure are very similar; the major difference is that the RNA strand is characterized by C3‘-endo sugars (except the two terminal residues). In the all-DNA triplex γ has a substantial fraction of the trans rotamer for both of the internal adenine residues (A3 and A4), whereas in the free duplex γ is g + for these residues. In the RNA-containing triplex, only A3 shows the presence of γ(t), and in the 2‘-OMe state, both A3 and A4 are γ(g + ). In addition, the 2‘-OMe triplex shows conformational heterogeneity. Thus, there are sugar-dependent differences in the degree of distortion in the purine strand imposed by the third strand binding. The helical parameters for the underlying duplexes are very similar in all three triplexes. However, the helical parameters for the third strands are different for the DNA versus the RNA and 2‘-OMe strands, reflecting their different sugar conformations. The lower degree of distortion of the underlying duplex in the presence of the 2‘-OMe third strand is consistent with higher thermodynamic stability of this triplex compared with the greater distortion of the duplex induced by both DNA and RNA third strands.

Directed evolution has been employed to generate new enzymes for the deracemisation of chiral amines.