Resolution of Racemic Amino Acids

The resolution of N-acetyl-D,L-amino acids to prepare non-natural l- and D-amino acids was the beginning of applied biocatalysis, and aminobutyric and di-amino-butyric acids as well as beta-D,L-phenylalanines can be resolved. A series of microbial acylases from Streptomyces, Alcaligenes, Comamonas and Pseudomonas species were produced for these applications. Immobilized acylase on Eupergit C or the use of membrane reactors allow the facile production of such chiral amino acids. 

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The broad specificity of PheDH has been applied also to the resolution of racemic amino acids or inversion of stereospecificity. Findrik and Vasic-Racki report the use of PheDH of Rhodococcus in conjunction with d-AAO to convert D-methionine into L-methionine (Scheme 7). 

Resolution of d- and L-Amino Acid Derivatives 5.03.5.4.1 Resolution of racemic amino acid esters... 

Problem 21.45 Explain the use of the enzyme deacylase in the resolution of racemic amino acids. -4... 

Two interesting approaches for resolution of racemic amino acids have been reported. Both are based on enantioseiective ring opening wilh in. silit racemization of the nonrcactive isomer (Fig. 1). 

The resolution of racemic amino acid mixtures via coordination to a metal ion has been a popular field of study. [Cu(L-aa)2] complexes can be used to resolve DL-Asp, dl-G1u and DL-His.58,59 (—)-[Co(EDTA)] has been used to resolve DL-His having first resolved the racemic [Co(EDTA)] ion using the L-histidinium cation.60 Schiff base complexes of both Co111 and Ni11 have also been used to resolve amino acids.61,62 A more esoteric finding is that the bacterium Enterobacter cloacae prefers to metabolize the A-( —) isomer of/ac-[Co(GlyO)3] rather than the A-(+) form,63 an observation reminiscent of that made by Bailar using tris(ethylenediamine)cobalt(III) salts. 

Figure 7 Preparation of chiral synthon for (3-3-receptor agonist (A) enzymatic resolution of racemic amino acid amide (8) by amidase from M. neoaurum ATCC 25795 (B) enzymatic resolution of racemic amino acid amide (10) by amidase from M. neoaurum ATCC 25795 (C) enzymatic asymmetric hydrolysis of diester (12) to the corresponding (>S)-monoester (13) by pig liver esterase.

Cellulose was the first sorbent for which the resolution of racemic amino acids was demonstrated [23]. From this beginning, derivatives such as microcrystalline triacetylcellulose and /3-cyclodextrin bonded to silica were developed. The most popular sorbent for the control of optical purity is a reversed-phase silica gel impregnated with a chiral selector (a proline derivative) and copper (II) ions. Separations are possible if the analytes of interest form chelate complexes with the copper ions such as D,L-Dopa and D.L-penicillamine [24], Silica gel has also been impregnated with (-) brucine for resolving enantiomeric mixtures of amino acids [25] and a number of amino alcohol adrenergic blockers were resolved with another chiral selector [26]. A worthwhile review on enantiomer separations by TLC has been published [27],... 

In order to increase the efficiency of biocatalytic transformations conducted under continuous flow conditions, Honda et al. (2006, 2007) reported an integrated microfluidic system, consisting of an immobilized enzymatic microreactor and an in-line liquid-liquid extraction device, capable of achieving the optical resolution of racemic amino acids under continuous flow whilst enabling efficient recycle of the enzyme. As Scheme 42 illustrates, the first step of the optical resolution was an enzyme-catalyzed enantioselective hydrolysis of a racemic mixture of acetyl-D,L-phenylalanine to afford L-phenylalanine 157 (99.2-99.9% ee) and unreacted acetyl-D-phenylalanine 158. Acidification of the reaction products, prior to the addition of EtOAc, enabled efficient continuous extraction of L-phenylalanine 157 into the aqueous stream, whilst acetyl-D-phenylalanine 158 remained in the organic fraction (84—92% efficiency). Employing the optimal reaction conditions of 0.5 gl min 1 for the enzymatic reaction and 2.0 gl min-1 for the liquid-liquid extraction, the authors were able to resolve 240 nmol h-1 of the racemate. 

Additives that specifically interact with an analyte component are also very useful in altering the electrophoretic mobility of that component. For example, the addition of copper(II)-L-histidine (12) or copper(II)-aspartame (54) complexes to the buffer system allows racemic mixtures of derivatized amino acids to resolve into its component enantiomers. Similarly, cyclodextrins have proven to be useful additives for improving selectivity. Cyclodextrins are non-ionic cyclic polysaccharides of glucose with a shape like a hollow truncated torus. The cavity is relatively hydrophobic while the external faces are hydrophilic, with one edge of the torus containing chiral secondary hydroxyl groups (55). These substances form inclusion complexes with guest compounds that fit well into their cavity. The use of cyclodextrins has been successfully applied to the separation of isomeric compounds (56), and to the optical resolution of racemic amino acid derivatives (57). 

Another example in which biocatalysis is combined with analysis is the system reported by Honda et al. [436]. A microreaction system, consisting of an enzyme-immobilized microreactor, for optical resolution of racemic amino acids was devel-... 

Figure 20.4 Resolution of racemic amino acids with enantioselective amino acid oxidase.

M. H. Hyun, J. S. Jin, and W. Lee, Liquid chromatographic resolution of racemic amino acids and their derivatives on a new chiral stationary phase based on crown ether, / Chromatogr. 822 (1998), 155. 

A single report on the resolution of racemic amino acids by TLC has been recently published. The use of silica gel TLC plates impregnated with QN (0.1%), in combination with appropriate mobile phase system, gave a successful enantior-esolution of methionine, alanine, threonine, valine, leucine, and isoleucine. This method combines the simplicity ofTLC technique with a good sensitivity (0.9-3.7 pg) and enantioselectivity (e.g., for methionine, the corresponding Ry values for d- and L-enantiomers were 25 and 50, respectively) [124],... 

Figure 16.7-6. Resolution of racemic amino acids (AA) catalyzed by (d)- and (L)-specific amino acid oxidases (AAO).

Optical resolution of racemic amino acids (methionine, phenylalanine, tryptophan, valine) by the action of L-specific amino acylase from Aspergillus oryzae (Tanabe Seiyaku Co., Ltd.). The theoretical productivity of 1000 liter immobilized amino acylase columns ranges from 214 kg per day for L-Ala to 715 kg per day for L-Met. 

Papain 3 ester Resolution of racemic amino acid derivatives 42... 

The enzymatic resolution of racemic amino acid derivatives in [BMIM][BF4] by papain was tested by Lou et al. Higher hydrolytic activity and enantioselectivity concerning asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester was achieved in ionic liquid/buffer-solution compared to organic solvent solutions. Nearly no hydrolytic activity was observed in pure ionic liquid [42]. 

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... 

Honda, T., Miyazaki, M., Yamaguchi, Y, Nakamura, H. and Maeda, H., Integrated microreaction system for optical resolution of racemic amino acids. Lab Chip, 1, 366, 2007. 

Earth s life forms. Careful analytical work proved that this optical activity was not the result of some Earth-based contaminant. In the past decade experiments have shown that with only the small amount of enantiomeric excess that these amino acids possess, some of them, such as the two shown below which have a fully substituted chirality center and cannot racemize, can effect a resolution of racemic amino acids through relatively simple processes such as crystallization. These events leave behind aqueous solutions of L-amino acids in high enantiomeric excess. Moreover, once these chiral L-amino acid solutions are generated, they can catalyze the enantiocontrolled synthesis of D-carbohydrates, which is what we all possess as well. As such, it is conceivable that the origin of chirality may well have come from outer space. 

Since the beginning of enzyme catalysis in microemulsions in the late 1970s, several biocatalytic transformations of various hydrophilic and hydrophobic substrates have been demonstrated. Examples include reverse hydrolytic reactions such as peptide synthesis [44], synthesis of esters through esterification and transesterification reactions [42,45-48], resolution of racemic amino acids [49], oxidation and reduction of steroids and terpenes [50,51], electron-transfer reactions, [52], production of hydrogen [53], and synthesis of phenolic and aromatic amine polymers [54]. Isolated enzymes including various hydrolytic enzymes (proteases, lipases, esterases, glucosidases), oxidoreductases, as well as multienzyme systems [52], were anployed. 

Active immobilized enzyme resolution of racemic amino-acids using ceramic-based enzymes... 

Amidases are also applied for the chiral resolution of racemic amino acid amides to allow the biocatalytic synthesis of nonnatural i.-amino acids, which are important building blocks for pharmaceuticals. An amidase (EC 3.5.1.4) from Pseudomonas putida has been developed for the kinetic resolution of a wide range of amino acid amides (Schmid et al. 2001). 

In an alternative process, the enz5miatic dynamic resolution of racemic amino acid 113 was also demonstrated by combining amino acid oxidase with chemical reduction. (R)-selective oxidation with Celite-immobilized (R)-amino acid oxidase from T. variabilis expressed in E. coli in combination with chemical imine reduction with borane-ammonia provided (S)-112 with a 75% process yield and 100% ee [141]. 

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