Amino acids isomers

D-Amino acid oxidase D-Amino acids (see p. 5) are found in plants and in the cell walls of microorganisms, but are not used in the synthesis of mammalian proteins. D-Amino acids are, hew ever, present in the diet, and are efficiently metabolized by 1he liver. D-Amino acid oxidase is an FAD-dependent enzyme that catalyzes the oxidative deamination of these amino acid isomers. The resulting a-ketoacids can enter the general pathways of amino acid metabolism, and be reaminated to L-isomers, or cafe balized for energy. 

It may also be surprising how easily this racemization may occur. Friedman and Liardon (126) studied the racemization kinetics for various amino acid residues in alkali-treated soybean proteins. They report that the racemization of serine, when exposed to 0.1M NaOH at 75°C, is nearly complete after just 60 minutes. However, caution must be used when examining apparent racemization rates for protein-bound amino acids. Liardon et al. (127) have also reported that the classic acid hydrolysis, employed to liberate constituent amino acids, causes amino acids to racemize to various degrees. This will necessarily result in D-isomer determinations that are biased high. Widely applicable correction factors are not possible since the racemization behavior of free amino acids is different from that of amino acid residues in proteins (which can be further affected by sequence). Of course, this is not a problem for free amino acid isomer determinations since the acid hydrolysis is unnecessary. Liardon et al. also describe an isotopic labeling/mass spectrometric method for determining true racemization rates unbiased by the acid hydrolysis. For an extensive and excellent review of the nutritional implications of the racemization of amino acids in foods, the reader is directed to a review article written by Man and Bada (128). 

If amino acid isomers can be distinguished, the operation remains possible when differentiating isomeric fragment ions (m/z 303 Leu-Leu] and He-Ile] ), produced for example in the decomposition of polypeptides such as Leu-Leu-Leu and Ile-Ile-Ile. 

Maruyama A, Adachi N, Takatsuki T, Torii M, Sanui K, Ogata N. Enantioselective permeation of a-amino acid isomers through poly(amino acid)-derived membranes. Macromolecules 1990 23 2748-2752. 

The most widely used technique for the estimation of amino acid isomers is gas-liquid chromatography. Two basic strategies have been used to separate isomers by this technique. Both approaches appeared in the mid-1960 s, and both involve derivatiza-tion of the amino acids to suitably volatile acyl-esters (46,47). One method is similar in concept to the separation of disastereo-mers by ion exchange chromatography discussed above. In one step of the two step derivatization, the amino acids are esterified with an optically active agent. This procedure creates molecules with two centers of asymmetry which can be separated on a non-optically active liquid (stationary) phase (46). This method allows any laboratory equipped with a gas chromatograph to perform Isomer analyses. 

In 1960, a patent was granted for the separation of D,L-prollne on a lactose column (56). Davankov s laboratory was the first to report separation of amino acid Isomers on polymeric resins derlvatlzed with optically active amino acids (57). However, separation of amino acid enantiomers by these techniques has been hampered by long separation times (ca. 10 hr) and the difficulty In synthesizing supports of sufficient quality for modern HPLC (spherical particles, small size, uniform chemical modification). Separation of amino acid Isomers on a column consisting of silica bonded with L-amlno acids and complexed with copper (II) has been reported by Gubltz and Jellenz (42). Short analysis times for separation of mixtures of single D,L-amlno acids were reported (ca. 30 min), but complex mixtures have not been separated. 

With further development It Is likely that a HPLC method will be able to separate complex mixtures of amino acid Isomers. The high speed and efficiency of HPLC, coupled with the ability to run samples without prior derlvatlzatlon would be an Ideal situation. The likelihood that such a method could be scaled up for commercial preparation of pure Isomers Is also a strong Impetus for Its successful development. 

As detected by NMR, in each case a pair of isomers was formed, derived from d- and L-amino acids. Isomers (25b-30b) are soluble in dichloromethane or chloroform, whereas compounds (25a-30a) are practically insoluble in these solvents but are soluble in polar solvents such as dimethyl sulfoxide. 

Eleven dansylated amino acid isomers were separated on a -cyclodextrin column (A = 254nm) using methanol/water (0.2 M ionic strength phosphate buffer at pH 6.5) mobile phases [465]. The level of methanol needed to generate a separation varied fiom 15% to 25%. At 15% methanol, four sets of enantiomers (glutamine, serine, norvaline, norleucine) are partially resolved in <30 min. The aufliors claim that water is an essential component of the mobile phase, but the next study clearly argues against this statement. 

Crown ethers can be introduced into the ion chromatographic system via the mobile and/or the stationary phase. Sousa et al [67] used them for the first time as mobile-phase additives describing a separation of amino acid isomers separated with dinaphthal-18-crown-6. But even simple inorganic anions can be separated with this method [68]. However, the number of applicable crown ethers is limited because of their solubilities. Moreover, crown ethers are relatively expensive, so their application at higher concentrations is not justified. 

Enzymes capable of interconverting d- and L-amino acid isomers are widely distributed in microorganisms. Racemases have been reported for... 

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