D-acylase

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. 

In this way, (L)-valine, (L)-alanine, (L)-phenylalanine and (L)-tryptophan, but also rare amino acids like (L)-propargylglycine, (L)-p-fluorophenylalanine or (L)-3-(r-naphthyl)alanine are prepared. (D)-acylases are used to obtain (D)-propargylglycine, (D)-tryptophan or (D)-p-chlorophenylalanine. [62]... 

D-amino acids DL-acylamino acids D-amino acylase 206... 

In section 6.6.1, we described how enzymatic methods have come to dominate the production of the important intermediates used in the manufacture of semi-synthetic -lactams. In principle, the hydrolytic penicillin acylases may be used in the reverse direction to add acyl groups to 6-APA. For example, a two-step enzymatic process has been described for the preparation of ampiciilin (D-(-)-a-aminobenzylpenidllin structure shown in Figure 6.17). 

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

Several L-amino acids are produced on a large scale by enzymatic resolution of N-acetyl-D,L-amino adds (Figure A8.4). Acylase immobilised on DEAE-Sephadex is for example employed in a continuous process while Degussa uses the free acylase retained in a membrane reactor. In the latter process the advantage of reuse of the enzyme and homogeneous catalysis are combined. 

The Japanese firm Tanabe Inc Ltd has been operating, since 1969, the optical resolution of DL-amino acids using aminoacylase. The prindple is based on the asymmetrical hydrolysis of N-acyl-DL-amino add by amino acylase which gives the L-amino add and the unhydrolysed acyl-D-amino add. 

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. 

Fig. 18. Comparison of results from various particle systems for stirred vessel with baffles and bubble columns Activity a/ao of Acylase resin after t = 300 h, equilibrium drop diameter dg of silicon oil-water-surfactant emulsion and reference floe diameter dpv of floe system in dependency on specific power P/V H/D = 1 D = 0.15 m 0.4 m...

Penicillin acylases or amidohydrolases, which cleave the amide side chain of penicillin G, have been known for almost 50 years. " As one of the first enzymes to be developed for use at scale in the pharmaceutical industry, penicillin G acylase (PGA) has often been used as a model system for academic studies from molecular biology to biochemical engineering. Despite extensive screening, however, for decades there was no equivalent enzyme to generate 7-ACA by cleaving the polar D-a-aminoadipoyl side chain from cephalosporin C. 

Hsu et have cloned two enzymes from Deimcoccus radiodurans for overexpression in E. coli in order to carry out a dynamic kinetic resolution to obtain L-homophenylalanine, frequently required for pharmaceutical synthesis. The starting material is the racemic mixture of A acetylated homophenylalanine, and the two enzymes are an amino acid A -acylase, which specifically removes the acetyl group from the L-enantiomer, and a racemase, which interconverts the D- and L-forms of the A acyl amino acids. The resolution was carried out successfully using whole-cell biocatalysts, with the two enzymes either expressed in separate E. coli strains or coexpressed in the same cells. 

The preparation of protected derivatives of D-allo- and L-fl//o-threonine by enzymatic hydrolysis of 5(47/)-oxazolones using hog kidney acylase has also been described. This methodology has been extended to a wide variety of amino acids and, at present, constitutes a general procedure to prepare non-quaternary... 

For resolution of the racemate 12 two different procedures can be applied 124 the en-antioselective enzymatic deacylation of chloroacetyl-DL-a-aminosuberic acid at pH 7.2 with Taka-acylase or the enantioselective salt precipitation of Z-dl-Asu-OH with D-tyrosine hydrazide according to the method of Vogler et alJ25 Complete enzymatic digestion is achieved in about ten days at 37 °C, and the optically pure L-enantiomer is obtained in 80% yield but the overall efficiency is lower than that of the chemical method. Fractional crystallization affords in good yields the Z-l-Asu-OH derivative 13 which is then used directly as a suitably protected intermediate in subsequent derivatization steps (see Scheme 6). Moreover, the recovery of the D-enantiomer from the mother liquors is also easy in this case. 

ACA cephalosporin C D-amino acid oxidase + glutaryl acylase... 

Other biocatalytic methods of producing D-p-hydroxyphenylglycine have not proved competitive, for instance transaminase based processes require glutamate to be supplied. Others include the hydrolysis of N-acyl derivatives by acylase and amides by aminopeptidase (DSM), the use of L-specrfic hydantoinases and immobilised subtilisin for the resolution of D,L-2-acetamido-/>-hydroxyphenylacetic acid methyl ester (Bayer). 

Aspartame is synthesized using the L enantiomer of phenylalanine. The L enantiomer is separated from the D enantiomer, the racemic mixture, by reacting it with acetic anhydride (CH3C0)20) and sodium hydroxide. The product of this reaction is then treated with the enzyme porcine kidney acylase. An organic extraction with acid yields the L enantiomer in the aqueous layer and the D enantiomer in the organic layer. The L-phenylalanine is reacted with methanol and hydrochloric acid to esterify the COOH group on phenylalanine. The esterified L-phenyalanine is then reacted with aspartic acid, while using other chemicals to prevent unwanted side reactions, to produce aspartame. 

Another interesting example of resolution through formation of diastereo-mers is the isolation of four stereoisomers of 3-amino-2-methyl-3-trifluoro-methyl butanoic acid [55]. In this process, the chemical-enzymatic method by the combination of chemical and enzymatic reaction is a very convenient. At first, -phenylacetyl derivatives 61a and 61b were prepared in excellent isolated yields via the Schotten-Baumann procedure. After these materials were hydrolysed with penicillin acylase (EC 3.5.1.11) from Escherichia coli until attainment of 50% conversion, enzymatically unconverted -phenylacetyl derivatives 62 a and 62 b (organic layer) and amino acids 63 b and 63 d (aqueous layer) were separated. Acidic hydrolysis of unconverted materials produced other stereoisomers 63 a and 63 c in high optical pure form. 

Bartlett K, Gompertz D (1974) The specificity of glycine-N-acylase and acylglycine excretion in the organic acidaemias. Biochem Med 10 15-23... 

C. King The Use of Coenzymes in Biochemical Reactors. - C. Wandrey, E Flaschel Process Development and Economical Aspects in Enzyme Engineering Acylase-L-Methionine System. - D.J. Graves, Yun-Tai Wu The Rational Design of Affinity Chromatography Separation Processes. 

The aqueous filtrate from the acylase hydrolysis described above was acidified to pH 2.4 with 1M HC1. The crystals were isolated (2.91 g) and recrystallized from boiling EtOAc (500 mL) to give N-acetyl-4-benzoyl-D-phenylalanine(d-76) yield 2.72g (87%) mp 186.5-187°C [a]D25 -49.0 0.8 (c 1.05, EtOH). Reaction with Ac20 converted a small aliquot of 4-benzoyl-L-phenylalanine (l-3) into /V-acetyl-4-benzoyl-L-phenylalanine (l-76) mp 187-188 °C [a]D25 +50.9 0.8 (c 0.98, EtOH). 

The best-established method for the enzymatic production of L-amino acids is the separation of racemates of N-acetyl-DL-amino acids by acylase I (aminoacylase E.C. 3.5.1.14). N-Acetyl-L-amino acid is cleaved and yields L-amino acids whereas N-acetyl-D-amino acid does not react (Figure 7.10). 

After separation of the L-amino acid through ion exchange or by a crystallization step, the remaining N-acetyl-D-amino acid is recycled by thermal racemization under drastic conditions or by a racemase to achieve an overall yield of around 45% of L-amino acid (50% is the theoretical maximum) (Figure 7.11). D-Amino acids are also accessible by chemical hydrolysis of the N-acetyl-D-amino acid or directly by use of D-selective acylases. 

Lactams, i.e., penicillins and cephalosporins, represent the most important class of antibiotics. Penicillins consist of a common core, 6-aminopenicillanic acid (6-APA) and different side chains. Penicillin G (pen G), with a phenyl acetate side chain, and penicillin V (pen V), with a phenoxyacetate side chain, are fermented from the fungus Penicillium chrysogenum all the others are produced from 6-APA, which nowadays is produced mostly from pen G via penicillin G amidase (pen G amidase, pen G acylase, PGA, E.C. 3.5.1.11 Figure 7.33). Cephalosporins feature 7-aminocephalosporanic acid (7-ACA) or its deacetyl-form, 7-aminodesacetyl-cephalosporanic add (7-ADCA) as their common core cephalosporin C (Ceph C) is obtained through fermentation, and all the others are derived from 7-A(D)CA. 

D. Roche, K. Prasad, and O. Repic, Enantio-selective acylation of /3-aminoesters using penicillin G acylase in organic solvents, Tetrahedron Lett. 1999, 40, 3665-3668. 

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