Peptides deamidation

Lai, M.C., Hageman, M.J., Schowen, R.L., Borchardt, R.T., and Topp, E.M. 1999a. Chemical stability of peptides in polymers. 1. Effect of water on peptide deamidation in poly(vinyl alcohol) and poly(vinyl pyrrolidone) matrixes. J. Pharm. Sci. 88, 1073-1080. 

Lai MC, Hageman MJ, Schowen RL, Borchardt RT, and Topp EM. Chemical Stability of Peptides in Polymers. 1. Effect of water on Peptide Deamidation in Poly(vinyl alcohol) and Poly(vinyl pyrrolidone) Matrixes. J Pharm Sci 1999a 88 1073-1080. 

MC Lai, MJ Hageman, RL Schawen RT, Borchardt, BB Laird, EM Topp. Chemical stability of peptides in polymers. 2. Discriminating between solvent and plasticizing effects of water on peptide deamidation in poly (vinylpyrro-lidone), J Pharm Sci 88 1081-1089, 1999. 

Deamidation of soy and other seed meal proteins by hydrolysis of the amide bond, and minimization of the hydrolysis of peptide bonds, improves functional properties of these products. For example, treatment of soy protein with dilute (0.05 A/) HCl, with or without a cation-exchange resin (Dowex 50) as a catalyst (133), with anions such as bicarbonate, phosphate, or chloride at pH 8.0 (134), or with peptide glutaminase at pH 7.0 (135), improved solubiHty, whipabiHty, water binding, and emulsifying properties. 

T. Geiger and S. Clarke, Deamidation, isomerization and racemization at asparaginyl and aspartyl residues in peptides, J. Biol. Chem, 262, 785 (1987). 

R. Tyler-Cross and V. Schirch, Effects of amino acid sequence, buffers and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides, J. Biol. Chem, 266, 22549 (1991). 

The tripeptides in Fig. 6.17 underwent a few breakdown reactions (N-ter-minus elimination, Qm formation, peptide bond hydrolysis), some of which will be considered later in this section. Of relevance here was that, of the two amidated tripeptides, the amide at the C-terminus underwent deamidation predominantly (Fig. 6.17, Reaction a), which, perhaps, explains the somewhat lesser stability compared to the free carboxylic acid forms. While the hexapeptide (6.52, Fig. 6.17) followed a different pattern of decomposition [76b], deamidation was also a predominant hydrolytic reaction at all pH values. Thus, the procedure to extrapolate results from small model peptides to larger medicinal peptides appears to be an uncertain one, since small modifications in structure can cause large differences in reactivity. 

Diketopiperazine formation was also examined in the peptides 6.50, 6.51, and 6.52 shown in Fig. 6.17 [76a], Clear evidence for the formation of a diketopiperazine product was obtained only for Arg-Trp-Phe (6.50, R=OH). In this case, the product was cyclo(Arg-Trp) (Reaction b). The diketopiperazine formed from Phe-Trp-Arg (6.51, R=OH) was not seen directly, but the presence of Trp-Phe together with Phe-Trp afforded indirect evidence. Interestingly, diketopiperazine formation occurred during acid-catalyzed degradation but not under basic conditions, and, as explained, was restricted to the two deamidated tripeptides. 

A medicinal example is provided by klerval (Fig. 6.18). The aspartic acid residue in this tripeptide analogue is also a site of chemical instability. At pH 1, cleavage of the Asp-Xaa bond (Fig. 6.18, Reaction b) was second in importance after C-terminal deamidation (see Sect. 6.3.2.1), and cleavage of the Xaa-Asp bond (Fig. 6.18, Reaction c) was third. At pH 4, cleavage of the Asp-Xaa bond was the major reaction and was accompanied by the formation of the succinimide and the iso-aspartyl peptide cleavage of the Xaa-Asp bond was minor. At pH 7, the major products were the L-iso-Asp and D-iso-Asp peptides, together with minor amounts of the D-Asp peptide. 

The simplest degradation displayed by asparagine and glutamine is direct hydrolytic deamidation of the side-chain carboxamido group (Fig. 6.29, Pathway d). Such a reaction, however, is seen only at low pH values, and its biological significance appears negligible. Its product is the Asp peptide (6.62) whose further reactions have been presented in Fig. 6.27. 

Under acidic conditions, the uncleaved. aspartyl peptide formed by direct deamidation (Fig. 6.29, Pathway d) ... 

A similar case is that of salmon calcitonin (see Fig. 6.22 in Sect. 6.3.2.4). In acidic solution, this peptide undergoes deamidation of Gin14 and Gin20 to yield the corresponding (and active) [Glu14] calcitonin and [Glu20] calcitonin [85],... 

The influence of secondary structure on reactions of deamidation has been confirmed in a number of studies. Thus, deamidation was inversely proportional to the extent of a-helicity in model peptides [120], Similarly, a-hel-ices and /3-turns were found to stabilize asparagine residues against deamidation, whereas the effect of /3-sheets was unclear [114], The tertiary structure of proteins is also a major determinant of chemical stability, in particular against deamidation [121], on the basis of several factors such as the stabilization of elements of secondary structure and restrictions to local flexibility, as also discussed for the reactivity of aspartic acid residues (Sect. 6.3.3). Furthermore, deamidation is markedly decreased in regions of low polarity in the interior of proteins because the formation of cyclic imides (Fig. 6.29, Pathway e) is favored by deprotonation of the nucleophilic backbone N-atom, which is markedly reduced in solvents of low polarity [100][112],... 

In the case of the influence of adjacent residues, there are clear mechanistic analogies between activation of aspartic acid (Sect. 6.3.3.2) and asparagine sites. The presence of a C-flanking glycine residue consistently increases deamidation of peptides, for the reasons discussed in Sect. 6.3.3.2 [6], Replacement of glycine with a more bulky residue such as valine, leucine, or proline can decrease reactivity more than tenfold [99]. 

In neutral and alkaline solutions, the iso-Asp/Asp-peptide products were always formed in a ca. 3 1 ratio. In the Val-Xaa-Asn-Ser-Val series, the nature of the N-flanking residue had little effect on the rate of deamidation, regardless of size. However, the actual effect may have been partly masked by cleavage of the Asn-Ser bond (Sect. 6.3.2A), which proceeds at a rate ca. 10% that of deamidation. In the Val-Ser-Asn-Xaa-Val series, the nature of the C-flanking residue did have a major influence. As expected, the most unstable peptide was Val-Ser-Asn-Gly-Val (tll2 ca. 6 h at pH 7.3 and 60°). A second group of peptides had t1/2 values in the range of 25-75 h, which increased in the order Xaa=His[Pg.325]

Peptide stability in solids has been briefly presented in Sect. 63.2.5. It is important to note here that deamidation reactions can also play a major role in the degradation of peptides in solid matrixes. While deamidation in the solid state has received less attention than deamidation in solution, there is enough evidence to suggest that the mechanisms and pathways are comparable if not similar in the two types of media [8] [130],... 

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