4.2. Peptide Synthesis

Caboxyamidated p1 (MW 2571) and p3 (MW 2492) were synthesized using Fmoc chemistry at the University of Texas Southwestern Medical Center (Dallas, TX, USA). For the TAMRA-labeled forms of the peptides, the fluorescent label was attached to the amino-end of the peptides before cleavage from the resin. The peptides were purified at William and Mary on a Waters HPLC system using a C18 X-Bridge Waters column (Milford, MA, USA) and acetonitrile/water gradient acidified with 0.1% trifluoroacetic acid, as previously described [77,78]. After removal of the organic phase, the peptides were lyophilized. Next, they were dissolved in dilute HCl and dialyzed to remove residual trifluoroacetic acid. The purification steps yielded 98% pure peptides based on HPLC chromatograms and mass spectrometry data. HPLC chromatograms and mass spectra collected at William and Mary on the purified peptides are included in the Supplementary Information (Figures S1 and S2). The purified peptides were dissolved in nanopure water. The concentrations of p1 and p3 were determined by amino acid analysis at the Texas A&M Protein Chemistry Center (College Station, TX, USA). Metallation of each peptide was achieved when an equimolar of CuSO₄ was added to the medium (see below). For the TAMRA-labeled peptides, the absorbance at 547 nm was used to quantify their concentrations. To avoid photobleaching of the fluorescent probe, the TAMRA-labeled peptides were protected from light by wrapping containers with foil.