Peptide Synthesis and Purification

PG-1 was kindly provided by Prof. R. Lehrer (University of California, Los Angeles, CA, USA); it was synthesized by SynPep Corporation (Dublin, CA, USA) with assessed purity of 99%. ChBac3.4 and RFR-ChBac3.4(1–14) were produced as previously described [47 (link)] according to a standard solid-phase peptide synthesis protocol utilizing Fmoc/tBu protecting groups scheme on a Symphony X peptide synthesizer (Protein Technologies, Tucson, AZ, USA). Trifluoroacetic acid (TFA) cleavage cocktail (TFA/triisopropylsilane/water/ethandithiol = 94/1/2.5/2.5) was used for the final deprotection and cleavage of the assembled linear peptides from the 2-chlorotrityl chloride resin. Samples were purified via semi-preparative RP-HPLC using a Waters SymmetryPrep C18 column, 9 × 300 mm, 100Å, 7µm, and then verified for a purity of no less than 96% by analytical RP-HPLC using Luna C18 column, 4.6 × 250 mm, 100 Å, 5 µm on a Gilson chromatograph. The molecular weight of the peptides was checked to be as expected by MALDI-TOF mass spectrometry with alpha-cyano-4-hydroxycinnamic acid as a matrix.