Acylase amino acid synthesis

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The most established method for enzymatic L-amino acid synthesis is the resolution of racemates of N-acetylamino acids by acylase I from AspergiUus oryzae fungus. The N-acetyl-L-amino acid is cleaved to yield L-amino acid whereas the N-acetyl-D-amino acid does not react. After separation of the L-amino acid through ion exchange chromatography or crystallization, the remaining N-acetyl-D-amino acid can be... 

Enzymatic hydrolysis of A/-acylamino acids by amino acylase and amino acid esters by Hpase or carboxy esterase (70) is one kind of kinetic resolution. Kinetic resolution is found in chemical synthesis such as by epoxidation of racemic allyl alcohol and asymmetric hydrogenation (71). New routes for amino acid manufacturing are anticipated. 

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. 

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. 

In fluorine-18 chemistry some enzymatic transformations of compounds already labelled with fluorine-18 have been reported the synthesis of 6-[ F] fluoro-L-DOPA from 4-[ F]catechol by jS-tyrosinase [241], the separation of racemic mixtures of p F]fluoroaromatic amino acids by L-amino acylase [242] and the preparation of the coenzyme uridine diphospho-2-deoxy-2-p F]fluoro-a-o-glucose from [ F]FDG-1-phosphate by UDP-glucose pyrophosphorylase [243]. In living nature compounds exhibiting a carbon-fluorine bond are very rare. 

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). 

McCague R, Taylor SJC. Integration of an acylase biotransforma-tion with process chemistry a one-pot synthesis of N-t-Boc-L-3-(4-thiazolyl)alanine and related amino acids. In Chirality in Industry n. Collins AN, Sheldrake GN, Crosby J, eds. 1997. John Wiley Sons, New York. pp. 184—206. 

The application of the non-urethane PhAc blocking group results in ca. 6% racemization during the construction of the phenylacetamido-protected dipeptides by chemical activation of the phenylacetamido amino acids. This disadvantage can be overcome by forming the peptide bonds enzymatically, e.g. with trypsin [26,27], chymotrypsin [26,28], or carboxypeptidase Y [26,29]. An interesting example is the biocatalyzed synthesis of leucine-enkephalin tert-butyl ester [25e], in which phenylacetamides are introduced and cleaved by means of penicillin G acylase, and the elongation of the peptide chain is carried out with papain or a-chymotrypsin. 

Several hydrolytic enzymes other than esterases have been applied for synthetic purposes. One important subject is the chemoenzymatic preparation of amino acids. An industrial method for the synthesis of unnatural d- or L-amino acids employs the enzymatic hydrolysis of hydantoins, prepared by Bucherer-Bergs condensation using either D- or L-hydantoinase (cf Section 3.2.1.4) [33]. Another efficient method of preparing natural and unnatural amino acids is the two-step synthesis which features a Pd-catalyzed amidocarbonylation (eq. (2) cf Section 2.1.2.4) to afford racemic A-acyl amino acids followed by enantioselective hydrolysis using various acylases [34]. 

A classic example of a typical enzymatic resolution on an industrial scale is the acylase-mediated production of L-methionine. This method has also been applied for the production of L-phenylalanine and L-valine. In addition to acylases, amidases, hydantoinases, and /i-lactam hydrolases represent versatile biocatalysts for the production of optically active L-amino acids. A schematic overview of the different type of enzymatic resolutions for the synthesis of L-amino acids is given in Fig. 2. 

In addition, the amino acylase process can be also applied in the production of other proteinogenic and non-proteinogenic L-amino acids such as L-valine and l-phenylalanine. It is worth noting that racemases have recently been developed by several companies which allow (in combination with the L-aminoacylases) an extension of the existing process towards a dynamic kinetic resolution reaction [10]. It should be mentioned that the same concept can be also applied for the synthesis of D-amino acids when using a D-aminoacylase as an enzyme. 

Kinetic resolution of alcohols and esters with hydrolases has opened up a new dimension for the synthesis of enantiomerically pure alcohols, esters and carboxylic acids, and in consequence the importance of resolution as a method for the attainment of enantiomerically pure compounds has been increased considerably. Hydrolase-catalyzed resolution is amenable to large-scale production l33-351, as was impressively demonstrated much earlier by the acylase-catalyzed racemate separation of N-acyl amino acids (not discussed in this chapter)ls4]. 

Because the resolution with acylase gave a theoretical maximum yield of only 50% and required separation of the desired product from the unreacted enantiomer at the end of the reaction, we next tried to prepare the amino acid by reductive amination of the corresponding ketoacid, a process with a theoretical maximum yield of 100%. A variety of ketoacids can be converted to L-amino acids by treatment with ammonia, reduced nicotinamide adenine dinucleotide (NADH), and a suitable amino acid dehydrogenase. 2-Keto-6-hydroxyhexanoic acid (in equilibrium with its cyclic hemiketal form) was prepared by chemical synthesis starting from 4-chloro-l-butanol, which was... 

Phenylalanine, synthesized from [ C]cyanide by the Bucherer modification of the Strecker synthesis, was resolved into its l- and D-iso-mers by the action of the d- and L-amino acid oxidases, respectively. The optically active amino acid was separated from phenylpyruvic acid by cation exchange chromatography (3). Similarly, DL-[ F]acyl-p-fluoro-phenylalanine has been subjected to stereospecific deacylation with the fungal enz)nne, L-amino acylase enz)nnatically generated L-[ F]p-fluoro-phenylalanine was separated from the D-acyl amino acid by column chromatography (4). 

Enzymatic methods offer in principle the possibility of a direct enantioselective synthesis of amino acids. Enzymes are often used for separation of racemic mixtures, as examplified in the case of methionine. Although racemic methionine is adequate for the animal feed sector, other applications require the enan-tiomerically pure (L)-form. For the resolution, (L)-acylases from Aspergillus sp. are often used, since they can accept a broad spectrum of substrates, are highly active, and very stable under the production conditions. [62]... 

There are stUl other opportunities in using elegant enzymatic synthesis methods, for instance, the hydrolysis ofN-acetyl-(D)-amino acids from racemic mixtures by (L)-acylase cleavage, by dynamic kinetic resolution, or by employing a race-mase and a hydantoinase. 

Needless to say that the product of synthesis is a racemate, from which the L and D enanthiomers have to be separated. Of the numerous methods of resolution a fairly general approach, enzyme catalyzed hydrolysis of acetyl-amino acids should be given special consideration. Acylase greatly enhances the rate of removal by hydrolysis of the acetyl group from L-amino acids, but leaves the (negligible) hydrolysis rate of the acetyl-D-amino acid unaffected. Hence, after completion of the hydrolysis, concentration of the neutral solution and dilution with absolute ethanol yields the L-amino acid, a dipolar ion insoluble in alcohol, while the salt of the unchanged acetyl derivative of the D-enanthiomer remains in solution. 

Biocatalytic resolution plays a major role in the industrial scale synthesis of a wide variety of optically pure amino acids. Tanabe uses an L-spe-cific aminoacylase for the manufacture of several L-amino acids, immobilized on DEAE-Sephadex. Degussa on the other hand, uses the free acylase in a membrane bioreactor. The process is highly efficient in enzyme use, and racemisation of the D-isomer is straightforward, thus providing good economics, and virtually no waste (Scheme 7.4). The process can be further refined by the use of racemase enzymes, which makes dynamic kinetic resolution feasible. 

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