Amine acylase

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

This amide, readily formed from an amine and the anhydride, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This depro-tection procedure works on peptides as well as on nonpeptide substrates. 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

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. 

Penicillin acylase catalyzes the hydrolysis of phenylacetamides and has been used in peptide synthesis for the cleavage of protecting groups [46—47]. In linker (40) developed by Flitsch et al. [41—42] (Scheme 10.8) the group -XR represents the alcohol or amine group of the target molecule. Hydrolysis of the phenylaceta-... 

This process has many benefits in the context of green chemistry it involves two enzymatic steps, in a one-pot procedure, in water as solvent at ambient temperature. It has one shortcoming, however-penicillin acylase generally works well only with amines containing an aromatic moiety and poor enantioselectivities are often observed with simple aliphatic amines. Hence, for the easy-on/easy-off resolution of aliphatic amines a hybrid form was developed in which a hpase [Candida antarctica hpase B (CALB)] was used for the acylation step and peniciUin acylase for the deacylahon step [22]. The structure of the acyl donor was also optimized to combine a high enanhoselectivity in the first step with facile deacylation in the second step. It was found that pyridyl-3-acetic acid esters gave optimum results (see Scheme 6.8). 

As VAO is able to perform an oxidative deamination of capsaicin-derived vanillyl amine, vanillin can be produced by the pathway described in the previous subsection. Van den Heuvel et al. [83] pointed out this biocatalytic route of synthesis in 2001 using penicillin G acylase to obtain vanillyl alcohol from natural capsaicin (Scheme 22.6). As the vanillin obtained can be labelled as natural. 

The seminal work of Klibanov in the early 1980s [46,47] made it clear that enzymes can be used in hydrophobic organic solvents, although at the price of a severely reduced reaction rate [48, 49]. Indeed, many Upases, as well as some proteases and acylases, are so stable that they maintain their activity even in anhydrous organic solvents. This forms the basis for their successful application in non-hydrolytic reactions, such as the (enantioselective) acylation of alcohols and amines, which now are major industrial applications [50]. 

Pencillin G acylase from E. coli is functionally, but not structurally, related to lipases. The enzyme would find wider use if it could be rendered tolerant of low-water media, which is the kind of problem that ionic liquids were expected to solve. It was found, however, that a covalently immobilized penicillin acylase, PGA 450, required aw = 0.8, which also was the minimum in toluene, to stay active in the ionic liquids [BMIm][BF4], [OMIm][BF4], and [BMIm][PF6] [67]. In a simple amine acylation test reaction (Figure 10.4), PGA 450 was somewhat less active in ionic liquids than in toluene. 

Resolution of Racemic Amines and Amino Acids. Acvlases (EC 3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungal Aspergillus acylasc (AA) are commercially available, inexpensive, and stable. Amino alcohols can be resolved by a number of pathways, including hydrolysis, esterification, and transesterification. 

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. 

Enzymatic treatment with Penicillin G acylase of the phenylacetamide of a RSMA leads to the specific hydrolysis of one enantiomer forming the amine in good yield and high enantiomeric purity (92%).236 Absolute configuration (R) and enantiomeric purity are determined by H1 NMR, after derivatization with (S)-a-trifluoromethylphenylacetic acid (Mosher s salt).302... 

E.C. 3.5.1.4) used to prepare amino acids, usually through resolution, and also penicillin G acylase (penicillin G amidohydrolase) (E.C. 3.5.1.11), used in the manufacture of semisynthetic penicillins.152 153 Immobilized penicillin G acylase has most recently been used to catalyze the formation of. V-a-phenylacetyl amino acids, which can then be used in peptide coupling reactions (see Section 19.2.3.2).154 Bacterial aminoacylase I (.V-acyl-i.-amino acid amidohydrolase, E.C. 3.5.1.14) has also been used to acylate chiral amines with poor to moderate enantioselectively.155... 

Recently a number of enzymatic systems have been developed at several chemical companies including Upases (synthesis of enantiotrope alcohols, R-amid, S-amin), nitrilases (R-mandehc acid), amidases (non-proteinogenic L-amino acids), aspartic acid ammonia lyase (L-aspartic add), penicilin acylase (6-Aminopenicilanic acid), acylases (semisynthetic penicillins), etc.( Koeller and Wong, 2001 and references therin). 

For example, we showed that penicillin acylase from Alcaligenes faecalis could be used for acylation of amines in an aqueous medium at high pH. After extraction of the remaining amine isomer, the pH was reduced to 7 and the amide hydrolyzed to afford the other amine enantiomer (Fig. 9.20). 

In order to broaden the scope we also examined [30] a combination of lipase-catalyzed acylation with penicillin acylase-catalyzed hydrolysis (deacylation). Good results (high enantioselectivity in the acylation and smooth deacylation) were obtained, with a broad range of both aliphatic amines and amines containing an aromatic moiety, using pyridylacetic acid ester as the acyl donor (Fig. 9.21). 

The phenylacetyl N-protecting group can be removed using penicillin G acylase under mild conditions which preserve sensitive peptide bonds, C-terminal esters and phosphate groups. A novel phosphate prodrug system 46 for amines, amino acids and peptides has been reported. 

Nondynamic resolution processes for the production of chiral amines are based on selective N-acylation by either lipases from Burkholderia plantarii (Scheme 4.5C) or Alcaligenes faecalis penicillin G acylases (Scheme 4.5D). The former reaction is optimal with ethylmethoxyacetate as acylating agent [30 a], whereas fhe acylase is most selective with the natural substrate phenylacetic acid [30b]. 

Several multi-ton industrial processes still use enzymatic resolution, often with lipases that tolerate different substrates. BASF, for example, makes a range of chiral amines by acylating racemic amines with proprietary esters. Only one enantiomer is acylated to an amide, which can be readily separated from the unreacted amine. Many fine chemicals producers also employ acylases and amidases to resolve chiral amino acids on a large scale. l-Acylases, for example, can resolve acyl d,l-amino acids by producing the I-amino acids and leaving the N-acyl-l-amino acid untouched after separation, the latter can be racemized and returned to the reaction. d-Acylase forms the alternative product. Likewise, DSM and others have an amidase process that works on the same principle d,l-amino acid amides are selectively hydrolyzed, and the remaining d-amino acid amide can be either racemized or chemically hydrolyzed. 

Alternative hydrolytic enzymes, such as amidases and acylases, have also been employed for the resolution of racemic amines. Unlike lipases, these enzymes are typically not commercially available but they can be isolated from microorganisms. 

Acylases have also been applied to the kinetic resolution of amines. Aminoacylase I from Aspergillus melleus was used for the resolution of a range of arylalkylamines and amino alcohols via acylation with methyl 2 methoxyacetate (Figure 14.13) [19]. Excellent chemoselectivity was also observed in all cases, as the amino group was preferentially acylated in the presence of a primary alcohol functionality. However, poor to moderate enantioselectivity was observed, with values <10. The best result (E = 9.3) was obtained with 1 aminoindane 10 during the conversion to ester 38. 

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... 

Returning to the resolution of amines, an enzymatic acylation of racemic amines in aqueous solution by a penicillin acylase (enzymes used in industry in the synthesis of penicillins) from Alicaligenes faecalis has recently been reported.40 The best acylating agent is the amide 161 of phenylacetic acid. There is a big advantage here. Unlike the ester formations and hydrolysis we discussed earlier, no amide exchange occurs with simple amides like 161. The amide (/ )-162 was formed in 45% yield and 98.5% ee. Once it has been separated from free (S)-2, it can be hydrolysed to free (R)-2 with the same enzyme This automatically perfects the ee. 

Amide formation and hydrolysis is of course also catalysed by enzymes and the recent report that the penicillin acylase from Alcaligenes faecalis catalyses an efficient kinetic resolution of racemic primary amines is very promising. A typical case is our old friend a-methylbenzylamine 57 (see chapter 22). One equivalent of an acyl donor 58 is needed and nearly 50% of the amide 59 can be isolated in excellent ee. The E value is 350 for this amine.20... 

The use of an expandase to convert penicillins to cephalosporins (9.36) also replaced a more expensive multistep chemical synthesis.238 Penicillin acylase has also been used to remove phenylacetyl protecting groups from amines in the synthesis of oligonucleotides.239... 

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