Regioselective Cleavage of Myoglobin with Copper(II) Compounds at Neutral pH

Selective hydrolytic cleavage of myoglobin was studied with CuCl2, Cu(ClO4)2, Cu(AC)2, and binuclear Cu(II) complexes of 3,6,9,16,19,22-hexaaza-6,19-bis (2-hydroxyethyl)-tricyclo-[22,2,2,211,14]-triaconta-1,11,13,24,27,29-hexaene (1) and 3,6,9,16,19,22-hexaaza-tricyclo-[22,2,2,211,14]-triaconta-1,11,13,24,27,29-hexaene (2). The sites of cleavage were precisely determined by LC-ESIMS and further confirmed by an MS/MS method through fragmentation from both the N-terminal and C-terminal. The peptide bonds of Gln91-Ser92 and Ala94-Thr95 were remarkably cleaved by Cu(II) anchored to the side chain of the His93 residue. The data presented in this study show that Cu(II)-mediated cleavage of myoglobin is able to proceed at neutral pH, more selectively than Pd(II)-mediated cleavage, and buffer solution of phosphate and NH4HCO3 accelerates the cleavage reaction.