(523f) Mutants of Phenylalanine Dehydrogenase: New Bio-Catalysts in the Asymmetric Synthesis and Racemic Resolution of Non-Natural Aminocids

The market for optically pure substances such as uncommon amino acids and chiral alcohols was estimated at the end of 2003 to be in the range of $140 billion. Non-natural amino acids, in enantiopure form, are of considerable interest in the synthesis of alkaloids, peptides and other compounds with therapeutic application e.g. in HIV-protease inhibitors. Incorporation of non-natural amino acids into biologically active peptides and proteins can greatly improve their activity, stability, bioavailability and binding specificity.

The active site of phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus was mutated on the basis of homology modelling and four mutants (N145A, N145L, N145V, N145I) affecting the contact with the aromatic ring of the amino acid substrate were studied.

Further investigations have been carried out with the aim of employing these mutants to resolve racemic mixtures of non-natural aminoacids. In theory the enzyme reaction should convert the L-amino acid to the oxoacid, leaving pure D-amino acid. The separated oxoacid can then be enzymatically converted to the pure L-amino acid. This pathway would be extremely interesting for the chemical industry, decreasing the high cost of producing such synthons. The mutant enzymes are more active with some para-substituted aromatic aminoacids than the wild type. This interesting result raised a question as to whether the altered enzymes retain the strict enantioselectivity of the wild type enzyme.

UV spectrophotometry and chiral HPLC have allowed us to start answering the question. Monitoring the reactions by UV it was possible to establish when to stop the experiments, and injection of the crude mixture onto a chiral HPLC column (under optimised conditions) allowed us to assess the relative amounts of the two enantiomers: the enantioselectivity is completely retained.

The production of non-natural amino acids starting from the corresponding synthetic 2-oxo-acids likewise shows excellent enantioselectivity and can be carried out in good yield. The scale-up of these reactions involves a recycling system for the coenzyme (NAD+/NADH).