Dehydroalanine formation

Although dehydroalanine formation represents a minor side reaction, the related byproduct may serve to confirm further the assigned disulfide pattern since the Xaa-dehydroalanine bond is stable toward the base treatment. Progress of the oxidative folding of human epidermal growth factor was analyzed by trapping the intermediates via cyanylation followed by their disulfide assignments. 61 ... 

Ring closure of (3-hydroxy-a-amino acids with sulfuryl chloride/triethylamine 68 is accompanied by formation of (3-chloroalanine,16 1 whereas cyclization of urethane-protected serine and threonine by the Mitsunobu reaction 54 69 70 leads to oxazoline and dehydroalanine formation as side products. 47,71 Formation of dehydroalanine can be prevented by bulky carboxy protecting groups such as tert-butyl esters. 69 ... 

We shall present our findings on the chemical nature of so-called labile sulfur in adrenodoxin. (1) The determination of dehydroalanine formation gives a negative result in measuring pyruvate after mild acid hydrolysis of trichloroacetic acid precipitates of adrenodoxin (Table 12). 

The formation of dehydroalanine would also increase the likelihood of lanthionine formation which would be resistant to peptide bond cleavage. It is likely that dehydroalanine formation itself is responsible for the final peptide bond cleavage through migration of the double bond and subsequent hydrolysis. 

The generally accepted route of formation of LAL is through the formation of dehydroalanine from cysteine, cystine, serine or phosphoserine through e-elimination reaction followed by Michael addition between the dehydroalanine and the e-amino group of lysine. The formation of LAL from the oxidized derivatives of cystine has been reported by Finley et al. (13). It was suggested that oxidation of cystine to cystine monoxide may accelerate dehydroalanine formation and subsequent LAL formation. It was also observed that very little LAL was formed through the 6-elimination of cysteine. Mel let (14) proposed that the elimination reaction in serine residues was responsible for the formation of dehydroalanine in peptides. Whitaker and Feeney (15) have reviewed the alkaline decomposition of phosphoserine and glycosylated serine or threonine residues in proteins. 

Figure 1 summarizes the potential pathways involved in the formation of dehydroalanine. It appears that dehydroalanine can be formed in a variety of amino acids protein, suggesting that any or all of the routes in Figure 1 could be involved in dehydroalanine formation. Table 1 contains results of partial amino acid analysis of several alkaline treated proteins. The results support the suggestion that both serine and cystine or their derivatives can be sources of dehydroalanine and subsequently the lysinoalanine measured in the proteins. In casein there is substantial LAL formation with a measurable loss in serine. In isolated soy protein and lactalbumin it can be seen that cystine shows the most significant losses. It should be noted that a significant portion of the serine in casein is present as phosphoserine. The relatively rapid 6-elimination of phosphoserine (15) accounts for the formation of considerable quantities of dehydroalanine and subsequently the substantial levels of LAL found in casein. In addition, as mentioned above, the presence of calcium would accelerate dehydroalanine formation from the phosphoserine present in the casein. The variability of... 

The cited evidence for the B-elimination mechanism leading to dehydroalanine formation merits further comment. Nashef et al. (41) report that alkali-treatment of lysozyme ribonuclease and several other proteins resulted in loss of cystine and lysine residues and the appearance of new amino acids lysinoalanine, lanthionine, and B-aminoalanine. Alkali-treatment of the proteins induced an increase in absorbance at 241 nm, presumably from the formation of dehydroalanine residues. The dehydroalanine side chain can participate in nucleophilic addition reactions with the e-NH2 group of lysine to form lysinoalanine, with the SH groups of cysteine to form lanthionine, and with ammonia to form B-aminoalanine. 

Takemoto et al. applied the Pd-catalyzed coupling reaction to N-phlhaloyl dehydroalanine 102 and benzaldehyde (Scheme 27) [41]. Instead of hydrosilanes, M-BuiiSnH was capable of serving as a hydrogen donor to promote C - C bond formation giving 103. 

Formation of the very unstable dehydroalanine derivatives A p-dimethylaminophe-nyl- and Af-p-nitrophenyhnethylenedehydroalanine methyl ester could only be verified by -NMR. Because of Michael-type reactions with cuprates, the iV-arylmethylenedehy-droalanine methyl esters have been applied as building blocks in the synthesis of amino acids.[18]... 

The first reaction is p-elimination in cysteine, serine, phosphoserine, and threonine residues due to attack by hydroxide ion, leading to the formation of very reactive dehydroalanine (DHA). In a cystine residue, this results in rupturing of the disulfide bond and liberation of a sulfide ion and free sulfur (Figure 13.4). Nucleophilic additions of the s-amino group of the protein-bound lysine to the double bond of DHA residue causes crosslinking of the polypeptide chain. After hydrolysis, a mixture of L-lysino-L-alanine and L-lysino-D-alanine, with probably a small proportion of dl and dd isomers,... 

Wuts and co-workers recently reported that the Vilsmeier reagent is superior to thionyl chloride for the cyclodehydration of primary and secondary p-hydroxy amides to prepare oxazolines, in particular, for oxazoline 18b, which is used in Taxol synthesis (Scheme 8.10). Some other examples are shown in Table 8.5 (Fig. 8.3). As expected, inversion of configuration at the alcohol bearing carbon atom is observed. Of the examples examined, serine afforded low yields due to the formation of dehydroalanine. The reaction is conveniently carried out in pyridine at room temperature. p-Chloro amides are also formed, which can be converted to the oxazoline with DBU, generally using the same mixture without isolation. The... 

Scheme 6 Protected Partially Racemic Lanthionine Formation via Michael Addition of L-Cysteine to Dehydroalanine Derivatives133 ...

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). 

Friedman (21) studied the effect of pH on the amino acid composition of wheat gluten. At pH 10.6 and above (65 C, 3 hours) no cystine was present. LAL increased with pH above 10.6. Lysine decreased over the same range of pH s, while serine and threonine contents dropped sharply at pH 13.9. Friedman concluded that cystine is most sensitive to alkali and that LAL will form most readily if lysine residues are in proximity to the dehydroalanine formed from cystine. Thus, he explained that different steric considerations may explain the different susceptibilities of wheat gluten, casein, and lactalbumin to LAL formation. 

The coupling reaction by which the aromatic group from one residue of mono- or diiodotyrosine is joined in ether linkage with a second residue is also catalyzed readily by peroxidases. One dehydroalanine residue is formed for each molecule of hormone released.108 A possible mechanism involves formation of an electron-deficient radical, which can undergo (3 elimination to produce a dehydroalanine residue and an aromatic radical. The latter could couple with a second radical to form triiodothyronine or thyroxine. However, as depicted in Eq. 25-6, the radical coupling may occur prior to chain cleavage. While P elimination (pathway... 

Other conversions to unnatural residues occur when most proteins are exposed to high pH (80, 81,82). The high pH causes a -elimination of a cystine (see Figure 16) or O-substituted serine or threonine, with the formation of a dehydroalanine or a dehydro-a-aminobutyrate. Such products are subject to nucleophilic attack by the e-amino group of a lysine to form a cross-linkage, such as lysinoalanine, or attack by cysteine to form lanthionine. Walsh et al. (81) have taken advantage of the formation of these cross-links to produce avian ovomucoids that have nonreducible cross-links and have lost the antiprotease activity of one of their two inhibitory sites (see Figure 17). 

Scheme I. Formation of dehydroalanine in alkali-treated proteins and its common derivatives resulting m new amino acids in the protein hydrolysate (88, 89, 90-92). Data taken from (I) Friedman (89) (11) Bohak (88) (111) Horn et al. (91,) (TV) Ziegler et al. (92) and (V) Asquith and Skinner (90,).

Lysinoalanine formation is not restricted to alkaline conditions—it can also be formed by prolonged heat treatment. Any factor favoring lower pH and less drastic heat treatment will reduce the formation of lysinoalanine. Hurrell (1984) found that dried whole milk and UHT milk contained no lysinoalanine and that evaporated and sterilized milk contained 1,000 ppm. More severe treatment with alkali can decompose arginine into ornithine and urea. Ornithine can combine with dehydroalanine in a reaction similar to the one giving lysinoalanine and, in this case, omithinoalanine is formed. 

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