Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides.

In order to gain more insight into the associations of mercury(II) with cysteinyl peptides, we investigated the effect of increasing cysteinyl residues on complex type formations. Three series of di-, tri-, and tetra-cysteinyl peptides, D[CGD]nCG (CP 2A, CP 3A, and CP 4A), E[CEG]nCG (CP 2B, CP 3B, and CP 4B) and E[CDG]nCG (CP 2C, CP 3C, and CP 4C), where n=1, 2, or 3, were prepared by microwave-assisted solid phase peptide synthesis. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides were characterized by electrospray orbitrap mass spectrometry utilizing complex specific mercury isotopic patterns. In equimolar mercury(II) to peptide ratio, all three series of di-, tri-, and tetra-cysteinyl peptides form predominantly the 1:1Hg(peptide) complex type, indicating that the intervening amino acid residues do not elicit preferential complex type formation. However, in non-equivalent mercury(II) to peptide ratio, the number of cysteinyl residues has a significant effect on the Hg:peptide stoichiometry in the complex formed. For example, in four times excess peptide, the 1:2Hg(peptide)2 and 1:1Hg(peptide) complexes are formed for di-cysteinyl peptides but not for the tri- and tetra-cysteinyl peptides. In contrast, the 2:1Hg2(peptide) and 1:1Hg(peptide) complexes are formed for the tri- and tetra-cysteinyl peptides. In excess mercury(II), CP 4C formed exclusively the 2:1Hg2(peptide) complex. The exact number of deprotonations observed for each complex could be derived from its signature mercury isotope pattern and monoisotopic peak mass. These multi-cysteinyl peptides present an attractive option for mercury chelation or environmental heavy metal remediation.