Resolution of DL-amino acids

The requirements for homochirally pure a-amino acids have not ruled out any of these general synthetic methods (which all give racemic products), since resolution of DL-a-amino acids and their derivatives is a simple, albeit time-consuming, solution to this need. Classical methods for resolution include physical separation of the DL-amino acids themselves (by chromatography on a chiral phase e.g. resolution of DL-tryptophan over cellulose, see Section 4.15), fractional crystallisation of certain racemates or supersaturated solutions (through seeding with crystals of one enan- 

POSITIVE c.d. centred near 390 nm NEGATIVE c.d. centred near 390 nm 

Remove thiobenzoyl . group by successive treatment with Mel and H2S 

Enzymic resolution is also generally useful. At first sight it is of restricted applicability, since most of the classical methods are based on the selectivity of a proteinase for catalysing the hydrolysis of the l enantiomer of an A-acyl derivative of a DL-amino acid (Equation (6.7)) or of a DL-amino acid ester. The normal substrates for these enzymes are derivatives of particular coded amino acids. 

However, the range of types of amino acids that can be resolved in this way is much greater than just the natural substrates (i.e. peptides made up of the twenty coded amino acids), because methods to relax the specificity of the enzymes have been found, in some cases by using organic solvents for the reactions. Penicillin acylase from Escherichia coli and an aminoacylase from Streptovercillium olivoreti- 

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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. 

It is also noted that Chibata and his colleagues [14] of Tanabe Pharmaceutical Co. started to use an immobilized enzyme for the optical resolution of DL-amino acids in 1969. The process included a fungal acylase immobilized on DEAE-Sephadex to hydrolyze N-acyl-L-amino acids selectively. This was the first industrial use of immobilized enzymes leading to the present concept of bioreactors. 

Bhushan, R., Reddy, G. P, and Joshi, S. (1994b). TLC resolution of DL amino acids on impregnated silica gel plates. J. Planar Chromatogr.—Mod. TLC 7 126-128. 

Different chiral selectors have been used for TLC resolution of variety of racemates of amino acids into enantiomers. Bhushan and Martens [20] in a review summarized various chiral selectors used as impregnating reagents, along with the mobile phases used for resolution of DL-amino acids (Table 12.2). 

Bruckner, H. and Strecker, B., Use of chiral monohalo-5-triazine reagents for the liquid chromatographic resolution of DL-amino acids, J. Chromatogr., 627, 97, 1992. 

Yam ADA, S., M. Yamamoto, and I. Chibata Optical Resolution of DL-Amino Acids by Preferential Crystallization Procedure. J. Org. Chem. 38, 4408-4412 (1973). Yamada, H., H. Yoshida, H. Nakazawa, and H. Kumagai Microbiological Synthesis of Tryptophan and its Related Amino Acids. 1st Inter. Symp. on Tryptophan Metabolism, Acta Vitam. et Enzymologica (Milan) (1975), in press. 

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. 

Crystals of a-glycine float on the surface of the solution, with one of the two flat (010) or (010) faces exposed to solution. If one enantiomer of a chiral hydrophobic amino acid (leucine or phenylalanine, for example) is now added to the solution, the crystal will become oriented so that the face that occludes amino acids of opposite chitrality is exposed to solution. D-amino acids are only incorporated in crystals with the 010 face exposed to solution, while L-amino acids only interact with the exposed (010) face. Therefore, if a floating crystal with the (010) face exposed to solution is presented with a solution of DL amino acids, the crystal will bind D-amino acids and will enrich the solution with respect to L-amino acids. If, for example, the underside of a film of a resolved amino acid with a long hydrophobic side chain [such as (5)-a-aminooctanoic acid] is exposed to a solution of a racemic mixture of amino acids, some optical resolution will occur by the principles just described (see Reference 64). 

Amino acid acylases have wide distribution in nature. They are found in animal tissues like kidney [1], molds, bacteria, yeast, and its activity is traced in plants including mushrooms. They serve as effective tools in resolution of L-amino acids from DL-amino acid racemic mixtures. L-amino acids are naturally occurring, physiologically active amino acids which are the building blocks of all the proteins. 

The polymer-aided resolution of racemic mixtures of simple compounds has relied mainly on chromatographic methods (Buss and Ver-meulen, 1968 Rogozhin and Davankov, 1%8 Losse and Kuntze, 1970 Boyle, 1971). The polymers have been used either in their traditional roles to separate diastereoisomers formed by chemical derivatization with a chiral group or to provide an asymmetric surface upon which the enantiomers may be directly separated. A great deal of the work in this area has been aimed at the separation of DL-amino acids. The succeeding sections will illustrate the techniques and supports that have been used to separate these and similar compounds. 

Retention (hRfi, hRf2) rid Resolution (a, Rs) Data of DL-Amino Acids on 20 X 20 cm Plates of Silica Gel G (Merck) Coated with Copper (II) Complex of L-Proline [11] and (1 R,3R,5/ )-2-Azabicyclo[3.3.0]Octan-3-Carboxylic Acid [12] as Chiral Selectors... 

Cyclodextrins have been used mainly as mobile phase additives for chiral resolutions of a number of racemates, including amino acids and their derivatives, but Alak and Armstrong [25] used fi-CD bounded to silica gel for resolution of dansyl derivatives of DL-amino acid enantiomers, by forming areversible inclusion complex of different stability. Dansyl DL-amino acids were better separated on fi-CDs layers than nonderivatized amino acids, because they have additionally two or more carbohydrogen rings. 

Bhushan, R. and Arora, M., Resolution of enantiomers of DL-amino acids on silica gel plates impregnated with optically pure (—)-quinine, Biomed. Chromatogr., 15,... 

This report presents various methods developed primarily at our laboratory for chromatographic resolution of racemates of several pharmaceuticals (e.g., -blockers, NSAIDS, anta-acids, DL-amino acids, Bupropion, Baclofen, Etodolac, Carnitine, Mexiletine). Recently, we developed methods for establishing molecular dissymmetry and determining absolute configuration of diastereomers (and thus the enantiomers) of (/< .S )-Baclofcn, (/d.SJ-Bctaxolol with complimentary application of TLC, HPLC, H NMR, LCMS this ensured the success of diastereomeric synthesis and the reliability of enantioseparation. 

AM Rizzi, S Cladrowa-Runge, H Jonsson, S Osla. Enantiomeric resolution of derivatized DL-amino acids by high-performance liquid chromatography using /3-cyclodextrin chiral stationary phase a comparison between derivatization labels. J Chromatogr A 710 287-295, 1995. 

C Tao, TB Huang. Resolution of DL-a-amino acids on a L-hydroxyproline chiral phase by ligand-exchange chromatography. Chin Chem Lett 6 383-384, 1995. 

The CSPs prepared by the molecular imprint technique have also been used for chiral resolution by CEC [98-100]. Lin et al. [91] synthesized L-aromatic amino acid-imprinted polymers using azobisnitriles with either photoinitiators or thermal initiators at temperatures ranging from 4°C to 60° C. Methacrylic acid (MAA) was used as the functional monomer and ethylene glycol dimethacrylate (EDMA) was used as the cross-linker. The resulting polymers were ground and sieved to a particle size less than 10 pm, filled into the capillary columns, and used for enantiomeric separations of some amino acids at different temperatures. The relationships of separation factor and column temperatures were demonstrated to be linear between the logarithm of the separation factors and the inverse of the absolute temperature (Fig. 24). The authors also compared the obtained chiral resolution with the chiral resolution achieved by HPLC and reported the best resolution on CEC. The chromatograms of the chiral resolution of dl-... 

Wong, C. H. and Wang K. T. (1978) Mutual resolution of (+/-)ephedrine and Z-DL-amino acid induced by seeding chiral salt, Tetrahedron Lett. 40, 3813-3816. 

Several iV-alkoxycarbonyl derivatives of 2-amino-2-deoxy-D-glucose have been prepared in this way by reacting D-fructose with the appropriate amino acid.84 The rearrangement is catalyzed by ammonium chloride, and the epimer with the d-gluco configuration is formed preferentially. Such derivatives, when formed from optically active amino acids, are diastereo-isomers, separable by chromatography this observation has been used for the resolution of DL-alanine, since the rearrangement can be reversed, and the amino acid can be recovered by alkaline treatment. 

Experiment 5.221 RESOLUTION OF dl-ALANINE Benzoyl DL-alanine. Dissolve lOOg (1.1 mol) of DL-alanine (Expt 5.180) in 400 ml of water containing 44.5 g (1.1 mol) of sodium hydroxide and cool the solution in an ice bath. Add 175g (1.2 mol) of benzoyl chloride and a solution of 49 g (1.2 mol) of sodium hydroxide in 200 ml of water to the stirred, cooled, amino acid solution, alternately and in portions during 2 hours continue to stir for a further 2-hour period. Boil the reaction mixture with 10 g of decolourising charcoal, filter, cool the clear yellow filtrate to 0 °C and acidify carefully to Congo red with concentrated hydrochloric acid. Triturate a portion of the oil which separates with water to induce crystallisation and then seed the bulk of the acidified solution with crystals and leave in an ice bath to complete the crystallisation process. Filter off the product, wash the filter cake with 500 ml of ice-cold water and recrystallise from about 3.5 litres of boiling water. The yield of benzoyl-DL-alanine, m.p. 162-164 °C, is 194.5 g (90%). 

Considering that sodium glutamate, like other amino acids, is contained in soy sauce, which is a traditional Japanese food, it is not surprising that Japan should have become interested very early in this type of fermentation. Firms like Ajinomoto and Kyowa Hakko dominate the world market for amino acids and particularly for glutamic acid and l-lysine. It is also through enzymes that the resolution of dl-methionine into its optical isomers is achieved since its laboratory synthesis yields the racemic form. 

Resolution of DL-alanine (1) is accomplished by heating the N-acetyl derivative (2) in weakly alkaline solution with acylase, a proteinoid preparation from porcine kidney containing an enzyme that promotes rapid hydrolysis of N-acyl derivatives of natural L-amino acids but acts only immeasurably slowly on the unnatural o-isomers. N-Acetyl-DL-alanine (2) can thus be converted into a mixture of l-( + )-alanine (3) and N-acetyl-o-alanine (4). The mixture is easily separable into the components, because the free amino acid (3) is insoluble in ethanol and the N-acetyl derivative (4) is readily soluble in this solvent. Note that, in contrast to the weakly levorotatory o-( - )-alanine (a 14.4°), its acetyl derivative is strongly dextrorotatory. 

Figure 12.3-6. L-amino acids prepared in bulk quantities by acylase I resolution of N-acetyl-DL-amino acids.

The fact that Co ions increase the activity of amino acid acylase in resolution of L-methionine from acetyl DL-methionine, shall be applied advantageously in the practical process of enzymatic resolution of DL-methionine. Usually in practical resolution, as cost of enzyme is high, it is desirable to employ a higher substrate concentration and less amount of enzyme which leads to production of L-methionine vith less cost. From the above results it can be concluded that by using Co ions we can reduce the amount of enzyme to a greater extent in enzymatic resolution of L-methionine by using amino acid acylase isolated from Sanzyme. 

Studies are in progress to investigate the effect of metal ions on enzymatic resolution of other DL-amino acids like DL-phenylalanine, DL-tryptophane and DL-alanine by using amino acylase isolated from Sanzyme. 

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. 

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