Resolution, of amino acids

FIGURE 22.23 Enzymatic resolution/recycling of amino acid amides. 

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In many cases only the racemic mixtures of a-amino acids can be obtained through chemical synthesis. Therefore, optical resolution (42) is indispensable to get the optically active L- or D-forms in the production of expensive or uncommon amino acids. The optical resolution of amino acids can be done in two general ways physical or chemical methods which apply the stereospecific properties of amino acids, and biological or enzymatic methods which are based on the characteristic behavior of amino acids in living cells in the presence of enzymes. 

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

Fig. 5. Enzymatic resolution of amino acids by ring-opening reaction.

The first partial chiral resolution reported in CCC dates from 1982 [120]. The separation of the two enantiomers of norephedrine was partially achieved, in almost 4 days, using (/ ,/ )-di-5-nonyltartrate as a chiral selector in the organic stationary phase. In 1984, the complete resolution of d,l-isoleucine was described, with N-dodecyl-L-proline as a selector in a two-phase buffered n-butanol/water system containing a copper (II) salt, in approximately 2 days [121]. A few partial resolutions of amino acids and dmg enantiomers with proteic selectors were also published [122, 123]. 

Table 6-3. Resolution of amino acid derivatives on a MIP imprinted with L-phenylalanine anilide (L-PA).

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. 

Asano, Y. and Yamaguchi, S. (2005) Dynamic kinetic resolution of amino acid amide catalyzed by D-aminopeptidase and a-amino-e-caprolactam racemase. Journal of the American Chemical Society, 127 (21), 7696-7697. 

Maiorino RM, Gandolfi AJ, Brendel K, et al. 1982. Chromatographic resolution of amino acid adducts of aliphatic halides. Chem Biol Interact 38 175-188. 

H) Resolution of amino acid esters with subtilisin The commercial Prt Alcalase from... 

The resolution of ( ) amino acids isn t really a synthetic method, but it s certainly useful in the production of a particular amino acid from a racemic mixture. In the resolution of ( ) amino acids, an enzyme (a biological catalyst) interacts with only one enantiomer. (Why, you ask Because enzymes are stereoselective.) The enzyme leaves one enantiomer unchanged and modifies the other into a different compound, which makes it possible to separate the enantiomer from the other compound by a number of techniques. After the enantiomer has been separated, all that s left is to reverse the process induced by the enzyme. 

Chen, S.-T., Huang, W.-H. and Wang, K.-T. (1994) Resolution of amino acids in a mixture of 2-Methyl-2-propanol/water (19 1) catalysed by alcalase via in situ racemisation of one antipode mediated by pyridoxal 5-phosphate. J. Org. Chem., 59, 7580-7581. 

The enantioselective binding properties of certain chiral crown ethers have been employed in the resolution of amino acid racemates. The racemic amino ester is adsorbed onto silica gel as its ammonium salt and eluted by a chloroform solution of the chiral crown ether. An excellent separation of the two enantiomers is achieved by this method (74JA7100). 

Resolution of Racemic Amines and Amino Acids. Acvlases (EC 3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungal Aspergillus acylasc (AA) are commercially available, inexpensive, and stable. Amino alcohols can be resolved by a number of pathways, including hydrolysis, esterification, and transesterification. 

N Nimura, T. Kinoshita. o-Phthalaldehyde-A -acetyl-t.-cystcine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr 352 169-177, 1986. 

Another chiral aldehyde suitable for such transformations is the recently prepared pyridoxal derivative (121).323 Even more recent examples of chiral carbonyl compounds (122) have been used for the partial resolution of amino acids. The enantiomers of compounds (122) undergo reaction with racemic a-amino acids and copper(II) ions to give preferential formation of enantiomeric copper complexes. The ( -enantiomers of (122) preferentially form complexes containing the... 

Armstrong et al. [54] resolved the enantiomers of some amino acids and their derivatives on x-CD-based CSPs using 1% aqueous triethylammonium acetate (pH 5.1). The same authors also tested a /LCD CSP for the chiral resolution of amino acids [55]. In addition, they evaluated a y-CD phase for the enantiomeric resolution of some dansyl amino acids and other drugs. The mobile phase was 38% methanol with 1% triethylammonium acetate [58]. In another study, the same authors reported the chiral resolution of 25 pairs of amino acids in less than 30 min [63]. The enantiomers of some /i-adrcncrgic blockers were resolved on a /LCD stationary phase, with 1% aqueous triethylammonium acetate, containing methanol, as the mobile phase [9,48]. 

The chiral resolution on CD-based CSPs depends on the formation of inclusion complexes in the cavities and, therefore, the structures and sizes of analytes are very important for the chiral resolution of racemates on these phases. Amino acids often are considered to be the best class of racemic compounds to use in structural studies. In 1987, Han and Armstrong [55] studied the chiral resolution of amino acids on -CD-based CSPs. It was observed that different retention, separation, and resolution factor values were obtained for different amino acids under identical chromatographic conditions, which indicated that the structures and sizes of amino acids govern their chiral resolution. The same observations may be found in the work of Fujimura et al. [70]. 

The application of antibiotics as chiral selectors has resulted in the successful resolution of almost all types of neutral, acidic, and basic racemic molecule. These antibiotics have been used for the enantiomeric resolution of amino acids, their derivatives, peptides, alcohols, and other pharmaceuticals. The selectivities of the most commonly used antibiotic-based (vancomycin, teicoplanin, and ristocetin A) CSPs varied from one racemate to another and are given in Table 1. Vancomycin was used for the chiral resolution of amino acids, amines, amides, imides, cyclic amines, amino alcohols, hydantoins, barbiturates, oxazolidinones, acids, profens, and other pharmaceuticals. Teicoplanin was found to be excellent chiral selector for the enantiomeric resolution of amino acids, amino alcohols, imides, peptides, hydantoins, a-hydroxy and halo acids, and oxazolidinones, whereas ristocetin A is capable of chiral resolution of amino acids, imides, amino... 

As in the case of other CSPs, the chiral resolution is effected by the structure of the solute. The chiral resolution of amino acids may be considered as the best example for this study. The work of Fukushima et al. [20] (i.e., the chiral resolution of amino acids) indicated the different behavior of the chiral resolution on (i8 )-/V-3,5-dinitrobcnzoyl-l -naphthylglycine CSP. Altomare etal. [142] studied the chiral resolution of a series of 3-phenyl-4-(l-adamantyl)-5-A-phcnyl-A2-1,2,4-oxadiazolines on A,Af,-(3,5-dinitrobenzoyl)-l(7 ),2( )-diaminocyclohcxanc CSP. The effect of the influence of aromatic ring substituents on enantioselectivity was studied by traditional linear free-energy-related equations and comparative molecular field analysis methods. The authors reported that an increase in retention was favored by the re-basicity and the hydrophilicity of the solutes. In... 

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