Threonine racemization

Crystallization Method. Such methods as mechanical separation, preferential crystallisation, and substitution crystallisation procedures are included in this category. The preferential crystallisation method is the most popular. The general procedure is to inoculate a saturated solution of the racemic mixture with a seed of the desired enantiomer. Resolutions by this method have been reported for histidine (43), glutamic acid (44), DOPA (45), threonine (46), A/-acetyl phenylalanine (47), and others. In the case of glutamic acid, the method had been used for industrial manufacture (48). 

The L-threonine (EC 4.1.2.5), D-threonine (EC 4.1.2.-) or L-allothreonine aldolases (EC 4.1.2.6 synonymous to S1IMT) can be used for resolution of racemic (allo)threonine mixtures by highly selective cleavage of the unwanted isomers42, but can also efficiently direct the anabolic pathways. The substrate spectrum includes propanal, butanal and dodecanal43. 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

Alkaline hydrolysis (with NaOH, KOH or more seldom with Ba(OH)2) is almost exclusively applied for the determination of tryptophan and phosphoamino acids. Serine, threonine, arginine, and cysteine are completely destroyed by alkaline hydrolysis, while other amino acids are racemized [190]. Since racemization also occurs during acid hydrolysis, when it is important to... 

Another direct approach to chiral polymeric stationary phases is the modification of commercially available polysiloxanes which contain reactive side groups. Thus, the diamide phase was linked to a modified XE-60 polysiloxane phase (Table 2). In one case (XE-60-L-Val-(/ or 5)-a-pea)124 another center of stereogenicity (R or S configuration) has been introduced in the amide group. An XE-60-L-Val-(S)-x-pea column was used for the enantiomer separation of racemic. V-rert-butoxycarbonyl amino acids after their methylation with diazomethane (serine and threonine as the O-trimethylsilyl derivatives) (Figure 12)124. 

Figure 12. Enantiomer separation of racemic A -rerf-butoxycarbonyl amino acids after lheir methylation with diazomethanc (serine and threonine as the C-trimethylsilyl derivatives) on XE-60-L-Val-(5)-a-pea (35 m fused silica capillary column, 100 180JC, 0.8 bar hydrogen)124. All D-enantiomers are eluted before the i.-enantiomers.

Beta-elimination reactions have been observed in a number of proteins. This reaction occurs primarily at alkaline pH conditions. Abstraction of the hydrogen atom from the alpha-carbon of a cysteine, serine, threonine, phenylalanine, or lysine residue leads to racemization or loss of part of the side chain and the formation of dehydroalanine (26). 

The amide bonds in peptides and proteins can be hydrolyzed in strong acid or base Treatment of a peptide or protein under either of these conditions yields a mixture of the constituent amino acids. Neither acid- nor base-catalyzed hydrolysis of a protein leads to ideal results because both tend to destroy some constituent ammo acids. Acid-catalyzed hydrolysis destroys tryptophan and cysteine, causes some loss of serine and threonine, and converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. Base-catalyzed hydrolysis leads to destruction of serine, threonine, cysteine, and cystine and also results in racemization of the free amino acids. Because acid-catalyzed hydrolysis is less destructive, it is often the method of choice. The hydrolysis procedure consists of dissolving the protein sample in aqueous acid, usually 6 M HC1, and heating the solution in a sealed, evacuated vial at 100°C for 12 to 24 hours. 

The best preventive measure against racemization in critical synthetic steps (e.g. fragment condensation, see p. 239) is to use glycine (which is achiral) or proline (no azlactone) as the activated carboxylic acid component. The next best choice is an aliphatic monoamino monocarboxylic acid, especially with large alkyl substituents (valine, leucine). Aromatic amino acids (phenylalanine, tyrosine, tryptophan) and those having electronegative substituents in the /7-position (serine, threonine, cysteine) are, on the other hand, most prone to racemization. Reaction conditions that inhibit azlactone formation and racemization are non-polar solvents, a minimum amount of base, and low temperature. If all precautions are taken, one still has to reckon with an average inversion of 1 % per condensation reaction. This means, for example, that a synthetic hectapeptide contains only 0.99100 x 100% = 37% of the fully correct diastereomer (see p. 233 f.). 

Asymmetric hydrogenation of racemic 2-substituted (3-keto esters to produce 2-substituted (3-hydroxy esters with two new chiral centers is a powerful method, and it is useful in the production of other pharmaceutical intermediates. The methodology can be used in the preparation of protected threonine derivatives 34, where 34d and 34e are key intermediates for the anti-Parkinsonian agent, L-Dops (35). 

In enzymatic reactions, the transfer proceeds via phosphorylation of the OH function of the serine residue however, threonine and tyrosine can be also involved. Hence, much attention has been paid to the fundamental study of the compounds shown in Scheme 2.36 The attractiveness of these models is due to the fact that X-ray structures both for enantiomeric and racemic forms are known (with exception of O-phospho-L-tyrosine). With the local geometry of phosphate groups and hydrogen bonding pattern taken from X-ray studies, it is possible to test the correctness of NMR analysis, the accuracy of measured structural constraints and the applicability of theoretical methods (ab initio, density functional... 

S Elimination and Racemization. There is some loss of the amino acids cystine, cysteine, serine, threonine, lysine and arginine during the alkaline treatment of proteins (12,22-30). Unlike arginine as shown above, loss of the other amino acids is not due to a hydrolytic reaction but rather to a g-elimination reaction (Equation 6). There is also some racemization of amino... 

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