Molecular design of a cyclic heptapeptide to mimic the zinc-binding site of carbonic anhydrase. Synthesis and zinc-binding studies by 13C-and 1H-N.M.R. spectroscopy.

A cyclic heptapeptide [cyclo-(Gly-L-His-Gly-L-His-L-Gly-L-Gly)] was designed to mimic the Zn(II)-binding site of carbonic anhydrase. The cyclic heptapeptide was synthesized from the linear heptapeptide, Gly-L-His-Gly-L-His-Gly-L-His-Gly-OH, which in turn was obtained by coupling of the fragments, viz. BOC-Gly-L-His-Gly-N3 and L-His-GLy-L-His-Gly-OBzlNO2 followed by deblocking of amino and carboxyl protecting groups. Conversion of the linear heptapeptide to the azide by treatment with diphenylphosphoryl azide was followed by cyclization in high dilution. A homogeneous material was isolated by counter-current distribution followed by gel filtration. It was found to be ninhydrin negative. The n.m.r. spectrum of the material upon integration indicated the proper ratios of various kinds of protons to be expected of the cyclic heptapeptide. A detailed 13C- and 1H-n.m.r. investigation was undertaken to determine the Zn(II)-binding ligands of the cyclic heptapeptide. The assignments for all the resonances were attempted by spin-decoupling method, pH and solvent effects, and by comparison of resonances of similar protons and carbons of model peptides. The n.m.r. titration results of the Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)-binding, the changes in the chemical shift of the imidazole protons were relatively large, indicating that this ring is involved in the complexation. All the peptide -NH-resonances were observable and unaffected; consequently, none of these nitrogens can serve as a ligand. In the case of 13C resonances, addition of 1 equiv. of Zn(II) to the cyclic heptapeptide, te C(2), C(4), and C(5) carbon resonances of this group were dramatically affected and showed a very large change in chemical shift upon complexation. The results demonstrate that Zn(II) binds to all three imidazole residues of the designed cyclic heptapeptide.