ИСТИНА

Penicillin acylase from Escherichia coli and its mutants stabilized to inactivation in alkaline medium and by high substrate concentrations have been used for preparative stereoselective synthesis of (R)- and (S)-mandelyl derivatives of alpha-amino acids which then after activation of their carboxyl group by N,N’-dicyclohexylcarbodiimide were subjected to intramolecular cyclization to produce corresponding 2,5-diketomorpholines. Biocatalytic acylation in aqueous alkaline medium was very effective: for example at pH 9.5 acylation of 0.1 M D,L-phenylalanine by 0.2 M (R)-mandelamide was 99% (yield calculated per amount of L-enantiomer of amino acid, synthesis/hydrolysis ratio 35) and due to the high stereoselectivity of enzyme to nucleophile (E ≈ 1000) N-(R)-mandelyl-(S)-phenylalanine (e.e. 99.9%) was produced. Using this methodology different cyclodidepsipeptides were synthesized with good overall yield: (3S,6S)-3-benzyl-6-phenyl-morpholine-2,5-dione (94%), (3S,6R)-3-benzyl-6-phenyl-morpholine-2,5-dione (93%), (3S,6S)-3-methyl-6-phenyl-morpholine-2,5-dione (81%), (3S,6R)-3-methyl-6-phenyl-morpholine-2,5-dione (83%), (3S,6S)-3-(indole-3-yl-methyl)-6-phenyl-morpholine-2,5-dione (91%), (3S,6R)-3-(indole-3-yl-methyl)-6-phenyl-morpholine-2,5-dione (92%), (3S,6S)-3-carbamoyl-methyl-6-phenyl-morpholine-2,5-dione (82%), (6S)-6-phenyl-morpholine-2,5-dione (80%), (3S,6S)-3-(2-methylpropyl)-6-phenyl-morpholine-2,5-dione (92%). Earlier we had reported chemoenzymatic route to stereoisomerically pure diketopiperazines based on penicillin acylase-catalyzed synthesis of dipeptides of the general formula D-phenylglycyl-L-X, where X is alpha-amino acid. Stabilized mutants of penicillin acylase from Escherichia coli appear to be much more effective catalysts of preparative synthesis of both 2,5-diketomorpholines and diketopiperazines compared to the wild type enzyme.