Design, Synthesis and 13 C‐ and 1 H‐N.M.R. Investigation of a Cyclic Octapeptide to Mimic the Zinc‐binding Site of Carboxypeptidase A

Abstract

Considering the three‐dimensional structure and the native Zn(II)‐binding ligands of carboxypeptidase A followed by extensive model building, a cyclic octapeptide, cyclo‐(Gly‐L‐Glu‐Gly‐Gly‐L‐His‐Gly‐L‐His‐Gly) was designed to mimic the Zn(II)‐binding site of carboxypeptidase A. The cyclic octapeptide was prepared by high dilution technique from the corresponding linear octapeptide, N‐Boc‐Gly‐γ‐OBut‐L‐Glu‐Gly‐Gly‐L‐His‐Gly‐L‐His‐Gly‐OBzlNO2 via the azide method. The linear octapeptide was obtained by coupling of the two tetrapeptide fragments: N‐Boc‐Gly‐γ‐OBut‐L‐Glu‐Gly‐Gly‐ONp and L‐His‐Gly‐L‐His‐Gly‐OBzlNO2. The cyclic peptide was purified to homogeneity by the method of countercurrent distribution. The product obtained was both ninhydrin negative and Pauli's reagent positive. Further confirmation of this material was obtained by the proper amino acid ratio of its acid hydrolysate and by the proton magnetic resonance spectrum in which the various kinds of protons of this peptide were accounted for. A detailed 13C‐ and 1H‐n.m.r. investigation was undertaken to determine the Zn(II)‐binding ligands of the cyclo‐octapeptide. The assignments for all the resonances were attempted by pH titration, by employing homonuclear decoupling experiments and by synthesis of cyclo‐octapeptide containing specifically deuterated amino acids cyclo‐(Gly‐L‐Glu‐Gly‐d2‐Gly‐d2‐L‐His‐Gly‐L‐His‐Gly). Titration results of Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)‐binding, the proton resonances were shifted downfield, the largest change being that of the histidine residues and more particularly C(2)‐H protons. The chemical shifts induced on glutamic acid residue were also observed for Glu CH2γ. In the case of 13C resonances, the maximum change in chemical shift was observed in the histidine residues and especially in the imidazole ring upon complexation. Two methylenes of the glutamic acid residue showed a large change in chemical shift upon ligating to the metal ion. The most significant observation was the deshielding effect of the Glu COO‐ group. The results demonstrate that Zn(II) binds both the imidazoles of the two histidine residues and the carboxyl side chain of the glutamic acid residue of the designed cyclic octapeptide.