Useful hydrolytic enzymes: Proteases, lipases and nitrilases

The most recent developments in the use of hydrolytic enzymes proteases, lipases and nitrilases are summarized: Sterically demanding a,a-disubstituted carboxylic esters can be enzymatically resolved using a commercially available protease derived from Aspergillus oryzae. A significant enhancement of selectivity and activity of Candida cylindracea lipase in irreversible acyl transfer reactions may be achieved by removal of by-products or via covalent modification of the enzyme. Enzymatic hydrolysis of nitriles proceeds under mild conditions but enzyme-catalysed sidereactions such as esteror epoxide-hydrolysis may occur. INTRODUCTION About two thirds of the research reported on the biotransformation of non-natural organic compounds makes use of enzymes from the group of the hydrolases. Their main advantages, which makes them the favourite class of enzymes for organic chemists, is their lack of cofactors and their ready availability. PROTEASES-RESOLUTION OF c~,cY-DISUBSTITUTED CARBOXYLATES Numerous proteases, the most prominent of which are a-chymotrypsin, subtilisin and papain, have extensively been used to resolve not only a-amino acid derivatives of type 1 , but also many amonosubstituted carboxylic esters (type 2, ref. 1). Although the majority of the structural features (Rl, R2) of the general substrate types can be varied considerably, one restriction remains for both types of substrates: The remaining hydrogen atom on the a-carbon atom must not be replaced, since the sterically more demanding a,a-disubstituted counterparts of 1 and 2 are generally not accepted as substrates (ref. 2). A protease from Aspergillus oryzae (AOP, ref. 3) seems to be an exception to this rule by being able to accept also bulky substrate esters. From the two ester moieties of diethyl 4,5-dihydroisoxazole-3,5-dicarboxylate (3), only the 5-ester moiety was hydrolysed in a regioselective manner by AOP to give monoacid 4 (60% yield). Similarly, a &/trans-mixture of epoxy esters 5 and 6-could be separated via a regioselective hydrolysis using this protease. The reaction ceased, when the trans-isomer 6 was converted to 7, leaving the cis-counterpart 5 untouched. The potential of AOP to resolve sterically demanding esters was recently demonstrated by the enantioselective hydrolysis of a-substituted mandelic esters such as atrolactic (8) or a precursor of Mosher's acid (9, ref. 4). This concept was extended to the 5-methyl-4,5-dihydroisoxazole-5-carboxylates 10-1 2, which are precursors of ahydroxy y-amino acids (ref. 5). Depending on the size of the substituent in position 3, low to good enantioselectivities (E) were achieved. In analogy to the regioselective transformation of diester 3, the enantioselective hydrolysis of diester 12 only occurred on the 5-carboxylate by leaving the ester in position 3 untouched. Chemical transformation of the recovered unhydrolysed (R)-12 (e.e. >97%) led to optically pure (R)-citramalic acid (ref. 6). An interesting reversal of the stereochemical preference of AOP was observed with substrate 13 (ref. 7). This behaviour is not uncommon among hydrolytic enzymes and may be explained by an alternative fit of the sterically more and less hindered substrate esters (11 and 13, resp.) within the active site of the enzyme (ref. 8). The highly substituted trans-configurated heterocyclic ester 14, which represents a precursor for a rare amino acid in the antibiotic Nikkomycin (ref. 9), was resolved with excellent selectivity. The general applicability of this concept is demonstrated by replacement of the dihydroisoxazole core with a dioxolane moiety, leading to substrate 15 (ref. 10).