Abstract 1896 Optimization of Metal Coordination in Cell-Penetrating Metallopeptides

A variety of strategies have been utilized to increase the capacity for peptides to cross cellular membranes and enter the cytoplasm. It is well established that structured, positively charged peptides, or peptides with positively charged termini, are best able to cross the cellular membrane. Recent work in the literature has demonstrated the binding of metal ions at designed sites on a peptide backbone greatly enhances the ability of the metallopeptide to enter a cell. Here, we present work to establish the principles that enable metal-ion enhanced permeation. We have designed a series of fluorescein-labeled peptides with natural and non-natural amino acids to bind transition metal ions across one-, two-, or three-turns of an α-helix. For each set of metal binding ligands on a peptide backbone, the metal binding affinity and change in secondary structure upon metal binding is measured for the first row transition metals Ni2+, Co2+, Cu2+, and Zn2+. As it has been previously demonstrated that palladium-bound peptides readily cross mammalian cellular membranes, we have also tested the ability of Pd(en)2+ to bind and induce structure on each peptide. After incubation with HeLa cells, we measure peptide incorporation via flow cytometry and confocal microscopy, and metal ion incorporation via inductively coupled plasma mass spectrometry (ICP-MS). We have demonstrated that peptide internalization correlates with the degree of α-helical structure induced upon peptide binding. Further, we conclude that metallopeptides are crossing cellular membranes as metal-peptide conjugates. We also demonstrate the first example of an engineered peptide with two non-natural metal-binding groups, creating a 4-coordinate metal-binding site across 3-turns of an α-helix. -Physical Sciences Scholaram Program (NSF 1742124) -Culliton Family Fund for Undergraduate Research -Department of Chemistry at Bucknell University.