Phenylalanine racemization

D-Phenylalanine (D-a-amino acid) From I-Phenylalanine racemization Sweet... 

While several laboratories have shown that severe racemiza-tion of proteins can occur during treatment with sodium hydroxide (6,18,22-24,61,62) the effects of other alkalis used in food processing are documented less well. Jenkins, et al. (70) have observed substantial differences in the degree of racemization caused by lime or caustic soda treatment of zein. Lime causes only 50% to 90% of the racemization observed for several amino acyl residues compared to when caustic soda is used. Because a substantial amount of calcium ion remained bound to the protein (approx. 10,000 ppm) compared to l/20th that amount of sodium ion for the caustic soda-treated zein, it is possible that divalent calcium may stabilize the protein making it less susceptible to racemization. Tovar (14) observed increases of 40% to 50% in serine and phenylalanine racemization and a decrease of 30% aspartate racemization for caustic soda-treated fish protein concentrate compared to lime-treated protein (see Table II). These studies indicate that different alkalis have different effects on racemization of proteins specifically, lime may cause less racemization than caustic soda at a similar pH. 

Eff ts of pH. Figure 14 shows the the pH dependence of the racemi-zation for the three most rapidly racemizing amino acids aspartic acid, glutamic acid, and phenylalanine. Racemization rates in the... 

Enzymatic hydrolysis is also used for the preparation of L-amino acids. Racemic D- and L-amino acids and their acyl-derivatives obtained chemically can be resolved enzymatically to yield their natural L-forms. Aminoacylases such as that from Pispergillus OTj e specifically hydrolyze L-enantiomers of acyl-DL-amino acids. The resulting L-amino acid can be separated readily from the unchanged acyl-D form which is racemized and subjected to further hydrolysis. Several L-amino acids, eg, methionine [63-68-3], phenylalanine [63-91-2], tryptophan [73-22-3], and valine [72-18-4] have been manufactured by this process in Japan and production costs have been reduced by 40% through the appHcation of immobilized cell technology (75). Cyclohexane chloride, which is a by-product in nylon manufacture, is chemically converted to DL-amino-S-caprolactam [105-60-2] (23) which is resolved and/or racemized to (24)... 

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). 

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

For the separation of amino acids, the applicability of this principle has been explored. For the separation of racemic phenylalanine, an amphiphilic amino acid derivative, 1-5-cholesteryl glutamate (14) has been used as a chiral co-surfactant in micelles of the nonionic surfactant Serdox NNP 10. Copper(II) ions are added for the formation of ternary complexes between phenylalanine and the amino acid cosurfactant. The basis for the separation is the difference in stability between the ternary complexes formed with d- or 1-phenylalanine, respectively. The basic principle of this process is shown in Fig. 5-17 [72]. 

Heteroarylphenylalanines could be smoothly obtained via microwave-promoted Suzuki reaction of heteroaryl halides with 2-amino-3-[4-(dihy-droxyboryl)phenyl]propanoic acid (Scheme 28) [46]. Interestingly, the free amino acid could be used without any protection of the amine and carboxylic acid fimctionahty. When 4-(dihydroxyboryl)-L-phenylalanine was used as organometallic partner no racemization was observed. The carboxylate anion and free amino group seem to shield the a-C - H from deprotonation and thus hmit racemization. 

Another approach for the synthesis of enantiopure amino acids or amino alcohols is the enantioselective enzyme-catalyzed hydrolysis of hydantoins. As discussed above, hydantoins are very easily racemized in weak alkaline solutions via keto enol tautomerism. Sugai et al. have reported the DKR of the hydantoin prepared from DL-phenylalanine. DKR took place smoothly by the use of D-hydantoinase at a pH of 9 employing a borate buffer (Figure 4.17) [42]. 

The production process for (S)-phenylalanine as an intermediate in aspartame perpetuates the principle of reracemization of the nondesired enantiomer (Figure 4.5) in a hollow fiber/ liquid membrane reactor. Asymmetric hydrolysis of the racemic phenylalanine isopropylester at pH 7.5 leads to enantiopure phenylalanine applying subtilisin Carlsberg. The unconverted enantiomer is continuously extracted via a supported liquid membrane [31] that is immobilized in a microporous membrane into an aqueous solution of pH 3.5. The desired hydrolysis product is charged at high pH and cannot, therefore, be extracted into the acidic solution [32]. 

Many compounds are less soluble as racemates than as their pure enantiomers. It thus appears probable that evaporation of an amino acid solution with a low ee should cause selective precipitation of the racemate crystals, which in turn should lead to an increase of the ee. Extremely simple manipulations, carried out in the chemistry department of Columbia University, led to a drastic increase in enantiomeric excess of phenylalanine 500 mg phenylalanine (with a 1 % ee of the L-component) was dissolved in water, and the resulting solution slowly evaporated until about 400 mg had crystallised out. The remaining solution contained a few mg of phenylalanine with 40% ee of the L-component (i.e., a 70 30 ratio of l to d). If 500 mg of such a solution (40% ee in water) is allowed to evaporate and is separated from the racemate, the result is about 100 mg, with 90% ee of the L-enantiomer (Breslow and Levine, 2006). 

Cyclization of racemic acylimidrazone 192, prepared from the corresponding acylhydrazone of phenylalanine, gave the protected amino acid 193 in an overall yield of 57% (Equation 60) <1999JME4331>. 

We have used a series of biocatalysts produced by site-directed mutations at the active site of L-phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus, which expand the substrate specificity range beyond that of the wild-type enzyme, to catalyse oxidoreduc-tions involving various non-natural L-amino acids. These may be produced by enantiose-lective enzyme-catalysed reductive amination of the corresponding 2-oxoacid. Since the reaction is reversible, these biocatalysts may also be used to effect a kinetic resolution of a D,L racemic mixture. ... 

Racemic mixtures of underivatized amino acids N-alkyl- and N-aryl-substitued derivates of amino acids (phenylalanine and proline) on graphitic carbon 0.001 M Cu(acetate)2 aqueous solution 229, 230... 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

ATP-dependent racemization, PHENYLALANINE RACEMASE ATP-dependent serine proteinase, PROTEASE La ATP depletion,... 

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