Practical Protein Chemistry - A. Darbre 1989

Polypeptide Fragmentation by Chemical Methods
Other Methods of Chemical Peptide Bond Cleavage
Cleavage at the Dehydroalanine Residue

Certain Serine and Cysteine derivatives (87) in Peptides readily undergo β-elimination, followed by Cleavage of the peptide bond at the α-carbon atom of dehydroalanyl peptides (88).

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In serine derivatives bearing strong electron-withdrawing groups on the hydroxyl—such as the O-tosyl [138] or O-diphenylphosphoryl groups [149]—the β-elimination process is induced in a basic medium. For instance, Chymotrypsin is converted into dehydrochymotrypsin by tosylation of the active-site serine residue using tosyl fluoride, followed by base Treatment [184, 201].

Cysteine is converted into dehydroalanine upon S-nitroarylation of the cysteine residue with dinitrofluorobenzene, followed by treatment with 0.1 M alkali [178]. S-Methylsulfonium is converted into dehydroalanine at lower pH values (8.5–9) compared to S-dinitrophenyl derivatives [179].

Upon treatment of peptides with alkali, the dehydroalanine residue (91) is formed directly from cystine (90). Cleavage of the disulfide group is initiated by the attack of a hydroxide ion on the proton at the α-carbon atom, yielding an S-thiocysteine residue (92), which is subsequently converted into cysteine and free sulfur [189].

C—S bond cleavage can occur on either side of the disulfide bridge; consequently, alkaline treatment of cystine-containing peptides yields a complex mixture of dehydroalanine-containing derivatives. Dehydroalanyl peptides are cleaved by means of Hydrolysis or oxidation [178]. In performic acid, dehydroalanine is oxidized to α,β-dihydroxyalanine, which is then hydrolyzed at pH 10 to yield a peptide with an amide group and a fragment bearing an N-terminal hydroxypyruvic acid residue. Cleavage can also be carried out by bromination in aqueous acetic acid, which forms labile α,β-dibromoalanine as an intermediate. In the presence of bromine, the peptide bonds of Tryptophan, Tyrosine, and Histidine residues are also cleaved [181].

At the same time, the method of peptide bond cleavage via The intermediate formation of dehydroalanine has significant drawbacks. The dehydroalanine residue (93) can react with nucleophiles, such as the ε-amino group of a Lysine residue (94), to form DL-α-amino-β-(ε-N-L-lysine)propionic acid (lysinoalanine) (95) [12]. In an alkaline medium in the presence of hydrogen peroxide, oxidation of the side chains of several Amino Acids may occur, leading to extensive Modification of the peptides. Alkali treatment can also be accompanied by Side Reactions, such as transpeptidation, hydrolysis of amide groups, and non-Specific Cleavage of peptide bonds.