N^ε-Cyclosuccinyllysine:  Synthesis and Formation from Hemisuccinylated Polylysine and Its Hapten Conjugates with Sulfamethoxazole during Acid Hydrolysis

Abstract

The novel amino acid N epsilon-cyclosuccinyllysine (4) forms as a side reaction during acid hydrolysis of N epsilon-hemisuccinylated succinylsulfamethoxazole-polylysine conjugates. The presence of 4 in hydrolysates can be obscured in amino acid analysis. Identification of 4 was based on chromatographic and electrophoretic comparisons with authentic N epsilon-cyclosuccinyllysine, which was synthesized in high yield by acid-catalyzed ring closure, under anhydrous conditions, of N epsilon-hemisuccinyl-L-lysine. The latter was prepared by an improved route, starting from N-alpha-(tert-butyloxycarbonyl)-L-lysine. The amount of 4 formed is influenced by the extent of succinylation and the conditions of hydrolysis and has reached 30 mol % of lysine. Both the hemisuccinyllysyl and the haptenized succinyllysyl residues participate in cyclization. This was confirmed by the synthesis and hydrolytic behavior of hemisuccinylated polylysine (n = 16) and N4,N epsilon-succinyl(sulfamethoxazole)(lysine). Formation of 4 can result in error in the estimation of the degree of ligand substitution based on lysine content in synthetic hemisuccinylated skin test antigens and peptide immunogens, especially when the degree of epitope substitution is low. 4 appears to be sufficiently stable for trial as an amino acid surrogate.