Aspartic acid cleavage

Joshi AB, Sawai M, Kearney WR, et al. Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon. /. Pharm. Sci. 2005 94 1912-1927. 

Hermann, K. Wysocki, V. Vorpagel, E.R. Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative Tris(2,4,6-Trimethoxyphenyl) Phosphonium implications of the aspartic acid cleavage mechanism. J. Am. Soc. Mass Spectrom. 2005,16, 1067-1080. 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

This ester was designed as a base-labile protective group. Monoprotection of aspartic acid was achieved using the DCC/DMAP protocol. Cleavage is... 

S. Inglis, Cleavage at aspartic acid, Methods Enzymol, 91, 324 (1983). 

Moser et al. (1968) (one of the co-authors was Clifford Matthews) reported a peptide synthesis using the HCN trimer aminomalonitrile, after pre-treatment in the form of a mild hydrolysis. IR spectra showed the typical nitrile bands (2,200 cm ) and imino-keto bands (1,650 cm ). Acid hydrolysis gave only glycine, while alkaline cleavage of the polymer afforded other amino acids, such as arginine, aspartic acid, threonine etc. The formation of the polymer could have occurred according to the scheme shown in Fig. 4.9. 

Fig. 6.27. Simplified representation of the reaction mechanisms by which aspartic acid residues can facilitate the hydrolytic cleavage of Asp-Xaa or Xaa-Asp peptide bonds. Pathway a begins as a nucleophilic attack of the C-flanking N-atom (i.e., the amide N-atom of the n+1 residue) at the carbonyl C-atom of the Asp side chain. Pathway b, which cleaves the Asp-Xaa bond, begins as a nucleophilic attack internal to the Asp residue. Pathway c, which cleaves the Xaa-Asp bond, is analogous to Pathway b, except that the attack is at the carbonyl C-atom of the N-flanking residue (i.e., the carbonyl of the n-1 residue).

Further insights into the influence of pH on the reactivity at aspartic acid residues are provided by a study of the model peptide Val-Tyr-Pro-Asp-Gly-Ala (Fig. 6.28,a) [93], At pH 1 and 37°, the tm value for degradation was ca. 450 h, with cleavage of the Asp-Gly bond predominating approximately fourfold over formation of the succinimidyl hexapeptide. At pH 4 and 37°, the tm value was ca. 260 h due to the rapid formation of the succinimidyl hexapeptide, which was slowly replaced by the iso-aspartyl hexapeptide. Cleavage of the Asp-Gly bond was a minor route. At pH 10 and 37°, the tm value was ca. 1700 h, and the iso-aspartyl hexapeptide was the only breakdown product seen. In Sect. 6.3.3.2, we will compare this peptide with three analogues to evaluate the influence of flanking residues. 

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. 

C-Flanking serine or cysteine residues can increase the rate of deamidation and, particularly, cleavage, in analogy with the mechanism discussed for aspartic acid [92], Increased reactivity can also result from the presence of a C-flanking histidine, which increases the nucleophilicity of the Asn side-chain amido group and, thus, favors Pathways f and perhaps d in Fig. 6.29 [124], N-Flanking lysine was also found to facilitate Pathway e (Fig. 6.29) in a pH-dependent maimer, likely by increasing the electrophilicity of the carbonyl C-atom in the Asn side chain [125]. 

Reaction of the carbonium ion with water could be reduced if overlap occurred with the carboxylate anion of aspartic acid-52 either during or after the glycoside-cleavage step. Since the carboxylate anion would be held adjacent to the carbonium ion in the active site, equilibrium should be far to the side of the acylal. Reaction of acylal with H2O would then very probably be ratedetermining in the forward direction. Evidence has been obtained that the solvent is directly involved in the hydrolysis of the cyclic acylal 2-(p-nitrophenoxy)phthalide where steric factors are similar... 

The reductive cleavage of hydroxylamine and its derivatives by electro-generated TP and V forming aminyl radicals and the hydroxide ions has been studied intensively. The aminyl radicals are preferably trapped with alkenes and aromatic compounds. Thus, the reaction of hydroxylamine with electro-generated Tp in the presence of maleic acid yields aspartic acid (Eqs. (66)—(69))... 

A small number of other biosynthetic pathways, which are used by both photosynthetic and nonphotosynthetic organisms, are indicated in Fig. 10-1. For example, pyruvate is converted readily to the amino acid t-alanine and oxaloacetate to L-aspartic acid the latter, in turn, may be utilized in the biosynthesis of pyrimidines. Other amino acids, purines, and additional compounds needed for construction of cells are formed in pathways, most of which branch from some compound shown in Fig. 10-1 or from a point on one of the pathways shown in the figure. In virtually every instance biosynthesis is dependent upon a supply of energy furnished by the cleavage to ATP. In many cases it also requires one of the hydrogen carriers in a reduced form. While Fig. 10-1 outlines in briefest form a minute fraction of the metabolic pathways known, the ones shown are of central importance. 

Diazepanones have been prepared on insoluble supports by intramolecular nucleophilic cleavage, by intramolecular Mitsunobu reaction of sulfonamides with alcohols, and by intramolecular acylations (Table 15.36). As in the case of azepines, these reactions do not always proceed smoothly, and care must be taken to prevent potential side reactions from occurring. For instance, intramolecular acylations in peptides containing aspartic acid (Entry 2, Table 15.36) will generally lead to the formation of suc-cinimides (see Table 13.20) unless A-alkylamino acids are used. 

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