Chemo-enzymatic synthesis of amino acids and derivatives

A short overview is given of new developments in the chemo-enzymatic synthesis of amino acids. Amino acid resolution procedures based on stereoselective enzymatic hydrolysis of amino acid amides have been developed. Two enzymes, an aminopeptidase from Pseudomonas putida and an amidase from Hycobacterium neoaurum have been purified to homogeneity. In addition, methods for the stereoselective synthesis of a-alkyl-substituted hydroxy acids are given. INTRODUCTION Amino acids and derivatives are becoming increasingly important as intermediates for pharmaceuticals and agro-chemicals. Well-known examples include D-phenylglycine and D-p-hydroxy-phenylglycine as building blocks for the antibiotics Ampicillin respectively Amoxycillin and L-phenylalanine for the dipeptide sweetener Aspartame. More recently, L-homophenylalanine and derivatives are used for the synthesis of ACE-inhibitors, L-valine as a precursor for the immuno-depressant Cyclosporin A and D-valine in the production of Fluvalinate, a pyrethroid insecticide. At DSM a generally applicable resolution process based on the enzymatic hydrolysis of amino acid amides with an aminopeptidase from Pseudomonas putida has been developed. The principle step of the process is depicted in scheme 1 (R2= H). Also visualized in this scheme is the resolution of a-disubstituted amino acids (R2= alkyl) with the aid of an L-amidase preparation from Hycobacterium neoaurum. Both processes have been extensively reviewed (ref.1). Recent developments include new approaches towards the synthesis of the starting materials of the enzymic resolution (a-H and a-alkylsubstituted amino acid amides), enzyme purification and the development of other aminopeptidase or amidase preparations with different substrate and/or stereoselectivities. Some of these developments will be discussed in the sequel. Moreover, some applications of the amino acid derivatives will be given. Finally, the enzymatic resolution of a-H and a-alkyl substituted hydroxy-acids will be discussed. R2 0 R2 0 I II I I1 Scheme 1 R2 0 I It I I NHZ NH2 R,-C-C-N€I2 ___) R,-C-C-OH + R,-C-C-NH2 I NHZ D,L-amino acid umide L-acid D-amide 1171 1172 H. E. SCHOEMAKER eta/. ENZYME PURIFICATION In the process for the production of a H amino acids crude enzyme preparations from Pseudomonas putida ATCC 12633 are used. From purification studies it became clear that the whole cells contained a number of aminopeptidase activities. Thus, after French pressure cell desintegration of the whole cells and ammonium sulfate fractionation a cell-free extract was obtained. This cell-free extract was subsequently submitted to Mono Q anion-exchange chromatography. Four distinct fractions with activity could be observed whilst one of these fractions showed a strictly Mn2+-dependent enzyme activity. From the fact that the different classes of proteinase activity showed different relative activities towards two representative substrates (L-valine and L-phenylglycine) the presence of different L-aminopeptidase activities in the cell-free extract of Pseudomonas putida ATCC 12633 with overlapping substrate specificity could be inferred. Noteworthy, the Mn2+-dependent enzyme still was capable to stereoselectively hydrolyze a large number of different substrates. The L-amidase enzyme in Mycobacterium neoaurum ATCC 25795 was purified to homogeneity by the following purification procedure: French pressure cell desintegration of the whole cells, ammonium sulfate fractionation of the crude extract, Mono Q anion-exchange chromatography and Mono S cation-exchange chromatography. The estimated molecular weight of the L-amidase is approximately 136 kD (gelfiltration) and 40 kD on SDS page. The enzyme shows an optimal pH of 7.5-9.5. The temperature optimum is 45OC. In table 1, K, and V,,, values for the purified L-amino amidase from Mycobacterium neoaurum ATCC 25795 are given.