Aminoacylases, resolutions

L-Amino acids Aminoacylase (resolution of optical isomers) Japan 65 days n.a. 

Scheme 8 Amino acids synthesized by L-aminoacylase resolution.

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. 

DL-Amino acid resolution processes for various amino acids had already been successfully pioneered by the Tanabe Seiyabu Co. using immobilised, oryzae aminoacylase acting on DL-N-acetyl acids. This step was first carried out as a continuous immobilized enzyme process in 1969. 

Although this enzymatic process fills only a niche in the L-lysine market, it is a successful example of a general method for amino acid resolution. It has some superior features compared to the Tanabe L-aminoacylase approach. The L-lysine can be extended to non-protein amino acids such as the use of P. putida aminopeptidase to resolve DL-homophenylalanine to produce precursors for the anti-hypertensive dmg Enalapril. A similar approach has also been used for the production of L-cysteine from DL-2-amino-A2-thiazohne-4-caiboxylate using Sarcina lucea, which is remarkable in that both isomers form L-cysteine. 

Sato, T. and Tosa, T. (1993) Optical resolution of aminoadds by aminoacylase in Industrial Application of Immobilized Biocatalyst (eds T. Tanaka, T. Tosa and T. Kobayashi) Marcel Dekker, New York, pp. 3-14. 

Scheme 42 Continuous flow optical resolution of acetyl-D,L-phenylalanine using an immobilized aminoacylase enzyme.

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... 

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. 

The enantioselective hydrolysis of racemic N-acetylated a-amino acids d,l-1 at De-gussa represents a long established large-scale process for the production of L-ami-no acids, l-2 [4]. This enzymatic resolution requires an L-aminoacylase as the biocatalyst. The starting materials for this process are readily available, since racemic N-acetyl amino acids d,l-1 can be economically synthesized by acetylation of racemic a-amino acids with acetyl chloride or acetic anhydride under alkaline conditions via the so-called Schotten-Baumann reaction [5]. The enzymatic resolution reaction of N-acetyl d,L-amino acids, d,l-1, is achieved by a stereospecific L-aminoacylase which hydrolyzes only the L-enantiomer and produces a mixture of the corresponding L-amino acid, l-2, acetate, and N-acetyl D-amino acid, d-1 (Fig. 4) [6],... 

Fig. 4 Enzymatic resolution of racemic N-acetyl amino acids using an L-aminoacylase.

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. 

In summary, a broad range of large-scale applicable biocatalytic methodologies have been developed for the production of L-amino acids in technical quantities. Among these industrially feasible routes, enzymatic resolutions play an important role. In particular, L-aminoacylases, L-amidases, L-hydantoinases in combination with L-carbamoylases, and /l-lactam hydrolases are efficient and technically suitable biocatalysts. In addition, attractive manufacturing processes for L-amino acids by means of asymmetric (bio-)catalytic routes has been realized. Successful examples are reductive amination, transamination, and addition of ammonia to rx,/fun-saturated carbonyl compounds, respectively. 

For a detailed review about aminoacylase-catalyzed resolutions, see a) A. S. Bom-marius in Enzyme Catalysis in Organic... 

L-Methionine has been prepared from fV-acetyl-D, L-me-thionine by deacylation with an aminoacylase.37 The lipase was supported on an ion-exchange resin. The reaction was driven to completion by chromatographic ion-exchange removal of the by-product acetic acid and unreacted substrate, the racemic mixture being fed in pulses to the column. The cross-linked enzyme crystals marketed by Altus Biologies are very useful in enzymatic resolutions, because of their enhanced stability to heat and organic solvents and because they can be reused many times. (For more detail on enantiospecific reactions with enzymes, see Chap. 9.)... 

The enzymatic hydrolysis of N-acylamino acids has been known for a century and was first detected in aqueous kidney preparations 3. Based on the finding that this enzymatic hydrolysis proceeds enantiospecifically 2, Greenstein and coworkers developed a general and very attractive procedure for the resolution of a vast number of racemic N-acylated amino acids to the corresponding L-amino acids catalyzed by aminoacylase (E.C. 3.5.1.14) whereas the N-acetyl-D-amino acid does not react13 (Fig. 12.3-1). 

Production of L-Methionine by Kinetic Resolution with Aminoacylase of Aspergillus oryzae (E.C. 3.5.1.14) I76 79 ... 

Hence resolution of enantiomers is necessary to obtain optically active L-amino acids. Among many resolution methods, the enzymatic method using mold aminoacylase developed by us proved to be one of the most advantageous procedures. An acyl-DL-amino acid is selectively hydrolyzed by aminoacylase to give L-amino acid and unhydrolyzed acyl-D-amino acid. 

Between 1954 and 1969 this enzymatic resolution method had been employed by Tanabe SeiycUcu Co., Ltd. for the production of several L-amino acids. In the 1960s we extensively studied the immobilization of aminoacylase for continuous optical resolution... 

When amino acids are synthesized in nature, only the L-enantiomer is formed (Section 5.20). However, when amino acids are synthesized in the laboratory, the product is usually a racemic mixture of D and l enantiomers. If only one isomer is desired, the enantiomers must be separated. They can be separated by means of an enzyme-catalyzed reaction. Because an enzyme is chiral, it will react at a different rate with each of the enantiomers (Section 5.20). For example, pig kidney aminoacylase is an enzyme that catalyzes the hydrolysis of A -acetyl-L-amino acids, but not Ai-acetyl-D-amino acids. Therefore, if the racemic amino acid is converted into a pair of Wacetylamino acids and the A -acetylated mixture is hydrolyzed with pig kidney aminoacylase, the products will be the L-amino acid and A -acetyl-D-amino acid, which are easily separated. Because the resolution (separation) of the enantiomers depends on the difference in the rates of reaction of the enzyme with the two A -acetylated compounds, this technique is known as a kinetic resolution. 

T. Tosa, T. Mori, N. Fuse and 1. Chibata, Studies on Continuous Enzyme Reactions. IV. Preparation of a DEAE-Sephadex-Aminoacylase Column and Continous Optical Resolution of Acyl-DL-Amino Acids, Biotechnol. Bioeng.,... 

Sato T, Tosa T (1993a) Optical resolution of racemic amino acids by aminoacylase. In Tanaka A, Tosa T, Kobayashi T (eds). Industrial application of immobilized biocatalysts. Marcel Dekker, New York, pp 1-14... 

Aminoacylases catalyze the hydrolysis of A-acyl amino acid derivatives, with the acyl groups preferably being acetyl, chloroacetyl, or propionyl. Alternatively, the corresponding A-carbamoyl- and A-formyl derivatives can be used [132], Enzymes of the amino acylase type have been isolated from hog kidney, and from Aspergillus or Penicillium spp. [133-135]. The versatility of this type of enzyme has been demonstrated by the resolution of racemic iV-acetyl tryptophan, -phenylalanine, and -methionine on an industrial scale using colunm reactors (Scheme 2.15) [136, 137]. 

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. 

A method that has been successfully applied to the resolution of p-amino acids is N-acylation or N-acyl hydrolysis. Enantioselective N-acylation of p-amino esters in an organic solvent has been carried out using Candida antarctica lipase A (CAL-A) lipase, while enantioselective hydrolysis of N-acyl p-amino acids in aqueous medium has been catalyzed by aminoacylase (Scheme 14.2). 

The aminoacylase-catalyzed resolution of N-acetyl amino adds yields enantio-merically pure amino adds by enantiosdective hydrolysis. This has been a "workhorse reaction" in enantioselective amino add chemistry and has been applied on industrial scales [2,54]. Moreover, because both l- and o-aminoacylases are available, both amino add enantiomers are accessible by means of this methodology. Due to its broad substrate scope (that has been demonstrated by several... 

Tosa, T., Mori, T., Fuse, N., and Chibata, L, "Studies on continuous enzyme reaction. IV. Preparation of a DEAE-sephadex-aminoacylase column and continuous optical resolution of acyl-DL-amino acids." Biotechnol. Bioeng., 9,603-615 (1967). 

Gizzerosine (Fig. 9) is a toxin obtained firom brown fish meal, which causes gizzard erosion or ulceration in chicks at a level of 2.2 ppm in the diet. This toxicity results in a serious disease named black vomit, notorious in poultry production. Resolution of ( )-a-aminoadipic acid via its A -chloroacetyl derivative ( )-ll by aminoacylase yielded (5)-12, which was converted to crystalline dihydrochloride of (5)-gizzerosine [41]. (R)-Giz-zerosine was biologically inactive. 

If pure enantiomers of a-amino acids are required, aminoacylase serves as ein excellent tool for resolution. 

The hydrogenation technology is now well established and eminently scalable, and indeed has been used by us to provide a very diverse set of amino acids. However, there are some cases where the use of L-aminoacylase is still the method of choice for inserting the chirality by resolution. These are generally where the side chain is susceptible to further transformation by the chemocatalyst, or where it might poison the catalyst, or where the enamide synthesis is difficult. Enamide hydrogenation substrates are generally synthesized... 

We recently used L-aminoacylase to provide the amino acids shown in Scheme 8. In these instances either the aldehydes were not available or they performed very poorly in the Erlenmeyer chemistry for enamide synthesis. These resolutions can be integrated very readily into an overall process, where the unwanted isomer can be racemized and recycled by way of treatment with acetic anhydride and racemization via the azalactone. 

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