Acylase-catalyzed amide hydrolysis

Scheme 19.23 One-pot synthesis of L-alanine based on metal-catalyzed hydrogenation and acylase-catalyzed amide hydrolysis.

A related group of acylases catalyzes amide formation and hydrolysis. Aspergillus is one source of such enzymes. One class of substrates is made up of di- and triesters with a-heteroatom substituents. Such substrates show selectivity for the carboxy group that is a to the heteroatom, which is the position analogous to the amide bond in peptides. 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

Penicillin G acylase (PGA, EC 3.5.1.11, penicillin G amidase) catalyzes the hydrolysis of the phenylacetyl side chain of penicillin to give 6-aminopenicillanic acid. PGA accepts only phenylacetyl and structurally similar groups (phenoxyacetyl, 4-pyridylacetyl) in the acyl moiety of the substrates, whereas a wide range of structures are tolerated in the amine part [100]. A representative selection of amide substrates, which have been hydrolyzed in a highly selective fashion, is depicted in Figure 6.36. 

Figure 6.36 Examples of amides resolved by penicillin C acylase-catalyzed hydrolysis (the fast-reacting enantiomer is shown).

Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospeciflcity of over 99%. Despite limited enzyme-substrate compatibility, specific conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using a-chymotrypsin, a peptidase isolated from bovine pancreas. 

The liberation of the (3-sulfanyl group of cysteine is achieved by means of PGA-mediated hydrolysis of phenylacetamides.P " °l For this purpose, the thiol group is masked with the phenylacetamidomethyl (Phacm) blocking function. After penicillin acylase catalyzed hydrolysis of the amide incorporated in the acylated thioacetal, the labile 5-aminomethyl compound is formed and immediately liberates the desired thiol. This method has been used in a synthesis of glutathione which is isolated as its disulfide 6 (Scheme 12). 

An enzymatic procedure for amine resolution, employing acylation by C. antarctica lipase B and deacylation by penicillin G acylase, has been demonstrated by Ismail et al. (Figure 14.14) [20]. The acylase catalyzed deacylation provides a greener process than the standard chemical deacylation as a result of the elimination of the salt waste stream typically generated by deacylating under strongly alkaline condi tions. It is also more amenable to sensitive functional groups that are not stable under basic conditions. A drawback of this approach is that the amide hydrolysis step is... 

Typical commercial enzymes reported for resolution of amino acids were tested. Whole cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids" the acylase-catalyzed resolution of Xacyl amino acids had extremely low rates toward a-dialkylated amino acids and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-ethylhexanoic amide. Finally, a large-scale screening of hydrolytic enzymes for enantioselective hydrolysis of racemic amino esters was carried out. Of all the enzymes and microorganisms screened, pig hver esterase (PLE) and Humicola langinosa lipase (Lipase CE, Amano) were the only ones found to catalyze the hydrolysis of the substrate (Scheme 9.6). 

Transition metal catalysts and biocatalysts can be combined in tandem in very effective ways as shown by the following example (Scheme 2.21). An immobilized rhodium complex-catalyzed hydrogenahon of 46 was followed by enzymatic hydrolysis of the amide and ester groups of 47 to afford alanine (S)-9 in high conversion and enanhomeric excess. Removal of the hydrogenation catalyst by filtration prior to addition of enzyme led to improved yields when porcine kidney acylase 1 was used, although the acylase from Aspergillus melleus was unaffected by residual catalyst [23]. 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

The most generally useful enzymes catalyze hydrolysis of esters and amides (esterases, lipases, peptidases, acylases) or interconvert alcohols with ketones and aldehydes (oxido-reductases). Purified enzymes can be used or the reaction can be done by incubating the reactant with an organism (e.g., a yeast) that produces an... 

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