Racemization of amino acid residues

M Dzieduszycka, M Smulkowski, E Taschner. Racemization of amino acid residue penultimate to the C-terminal one during activation of N-protected peptides, in H Hanson, HD Jakubke, eds. Peptides 1972. North-Holland, Amsterdam, 1973, pp 103-107. 

Excessive heat due to roasting can cause racemization of amino acid residues (25). Most amino acids are only available in the L form. Consequently, complete racemization could be equivalent to a 50% decrease in availability for the residues affected. 

In this paper we describe seme of the factors which influence racemization of amino acid residues in food proteins and discuss toxicological and nutritional consequences of feeding alkali-treated food proteins. 

Hayase, F., Kato, H., and Fujimaki, M. (1975). Racemization of amino acid residues in proteins and poly(L-amino acids) during roasting. J. Agric. Food Chem. 23, 491-494. 

In analysis, the original compound need not be recovered after separation, and therefore primary amines such as amino acids can be derivatized and analyzed in the same manner. (S)-NEI is employed as a derivatizing reagent in an Edman-like sequencing scheme to assess the extent of racemization of amino acid residues in synthetic peptides. ... 

The enantiomeric separation of the D- from the L-stereoisomers of amino acids is an area of growing interest. It is generally recognized that heat- and alkali-treatment of proteins can result in the racemization of L-isomers of amino acid residues to the D-analogs. Almost without exception, humans cannot utilize the D-isomers of amino acids, and some are thought to be toxic (although... 

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). 

M Friedman, R Liardon. Racemization kinetics of amino acid residues in alkali-treated soybean proteins. J Agric Food Chem 33 666-672, 1985. 

This type of chemistry has not been developed further into an efficient peptide synthesis involving A-terminal addition of amino acid residues to peptide esters. The main problems are concerned with the removal of the synthesized peptide from the cobalt(III) ion without causing racemization of chiral centres or hydrolysis of peptide linkages. 

Pyridoxal is the reagent in other reactions of amino acids, all involving the inline as intermediate. The simplest is the racemization of amino acids by loss of a proton and its replacement on the other face of the enamine. The enamine, in the middle of the diagram below, can be reprotonated on either face of the prochiral inline (shown in green). Protonation on the bottom face would take us back to the natural amino acid from which the enamine was made in the first place. Protonation on the top face leads to the unnatural amino acid after hydrolysis of the inline (really transfer of pyridoxal to a lysine residue of the enzyme). 

Smith G.G., Sol B.S. (1980) Racemization of amino acids in dipeptides shows COOH > NH2 for nonsterically hindered residues. Science 207, 765-7. 

In the strategical planning that must precede the, synthesis of a larger peptide racemization is one of the most important considerations. Therefore, it seems to be appropriate to discuss the various schemes of synthesis at this point. Due to the individuality of amino acid residues and to variations in the properties of blocked intermediates it appears to be impractical to propose a general scheme (strategy) that would be applicable for any peptide. Peptide synthesis should be based on retrosynthetic analysis, starting with identification of the problems inherent in the sequence of the target compound. 

However, in certain cases we must consider the possibility that the asymmetric centers of amino acid residues may be inverted or racemized before hydrolysis of the adjacent peptide linkage has taken place. Such a mechanism is probably responsible for the racemization of phenylalanine from wool and the partial racemization of amino acids obtained from the antibiotics. It was pointed out above (page 354) that most probably peptides are more easily racemized by acid than are free amino acids. It could be suggested, therefore, that valine peptides, for example, which are particularly resistant to hydrolysis (Synge, 1945b), may be very easily racemized. But this is not necessarily the case the steric factors which hinder hydrolysis are also likely to reduce racemization in both reactions by obstructing the approach of the proton. It is likely that the rate of racemization of an asymmetric carbon atom attached to a peptide linkage will be mainly determined by inductive and mesomeric... 

Racemization of amino acids Deprotonation of the a-carbon of the amino acid leads to tautomerization of the Schiff base to yield a quinonoid ketimine. The simplest reaction that the ketimine can undergo is reprotonation at the now symmetrical a-carbon. Displacement of the substrate by the reactive lysine residue results in the racemic mixture of d- and L-amino acid. 

Since the proline residue in peptides facilitates the cyclization, 3 sublibraries each containing 324 compounds were prepared with proline in each randomized position. Resolutions of 1.05 and 2.06 were observed for the CE separation of racemic DNP-glutamic acid using peptides with proline located on the first and second random position, while the peptide mixture with proline preceding the (i-alamine residue did not exhibit any enantioselectivity. Since the c(Arg-Lys-0-Pro-0-(i-Ala) library afforded the best separation, the next deconvolution was aimed at defining the best amino acid at position 3. A rigorous deconvolution process would have required the preparation of 18 libraries with each amino acid residue at this position. 

The improvements in resolution achieved in each deconvolution step are shown in Figure 3-3. While the initial library could only afford a modest separation of DNB-glutamic acid, the library with proline in position 4 also separated DNP derivatives of alanine and aspartic acid, and further improvement in both resolution and the number of separable racemates was observed for peptides with hydrophobic amino acid residues in position 3. However, the most dramatic improvement and best selectivity were found for c(Arg-Lys-Tyr-Pro-Tyr-(3-Ala) (Scheme 3-2a) with the tyrosine residue at position 5 with a resolution factor as high as 28 observed for the separation of DNP-glutamic acid enantiomers. 

The reaction mechanism for glutamate racemase has been studied extensively. It has been proposed that the key for the racemization activity is that the two cysteine residues of the enzyme are located on both sides of the substrate bound to the active site. Thus, one cysteine residue abstracts the a-proton from the substrate, while the other detivers a proton from the opposite side of the intermediate enolate of the amino acid. In this way, the racemase catalyzes the racemization of glutamic acid via a so-called two-base mechanism (Fig. 15). 

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