Amino acids and peptides. Part 32. Total synthesis of eglin c. Part 2. Synthesis of a heptacontapeptide corresponding to the entire amino acid sequence of eglin c and of related peptides, and studies on the relationship between the structure and inhibitory activity against human leukocyte elastase, cathepsin G and α-chymotrypsin

Abstract

Commencing with a protected C-terminal triacontapeptide of eglin c, eglin c (31–70), eglin c (22–30) and eglin c (8–70) and finally eglin c were synthesized by a conventional solution method in order to allow us to study the relationship between their structure and the inhibitory activity against human leukocyte elastase, cathepsin G and α-chymotrypsin. Ten relatively small peptide fragments were coupled successively from the C-terminus by the azide method to minimize racemization and to avoid the need for protection of the side-chain functional groups of the amino acid residues as much as possible during the peptide synthesis. The protected peptides were treated with HF at 0 °C for 60 min in the presence of thioanisole and m-cresol to give the desired eglin c fragments and eglin c, which exhibited a symmetrical single peak on analytical HPLC. Although the inhibitory activity of eglin c (31–70) and eglin c (22–70) against the aforementioned enzymes did not increase dramatically, eglin c (8–70) exhibited inhibitory activity against the above enzymes with similar or rather lower K_i-values than that of N^α-acetyleglin c.

Mass spectrometry of the synthetic eglin c by electrospray ionization exhibited peaks at 1012 (M + 8H)⁸⁺, 1157 (M + 7H)³⁺ and 1349 (M + 6H)⁶⁺, supporting the view that the molecular weight of synthetic eglin c [C₃₇₃H₅₅₀N₉₆O₁₀₇] is 8090.9 [Calc. for (M + 8H)/8 = 1012.36, (M + 7H)/7 = 1156.84 and (M + 6H)/6 = 1349.48]. Furthermore, the synthetic eglin c exhibited the same inhibitory activity against human leukocyte elastase, cathepsin G and α-chymotrypsin (K_i= 5.1 × 10^–9, 1.5 × 10^–9, and 2.2 × 10^–9 mol dm^–3, respectively) as N^α-acetyleglin c synthesized genetically (K_i= 5.0 × 10^–9, 1.0 × 10^–9, and 2.3 × 10^–9 mol dm^–3, respectively).