Synthesis and Characterization of Polypeptide Conjugate

The peptide, CH₁₂K₁₈, was synthesized by Fmoc-mediated solid-phase peptide synthesis using an automated peptide synthesizer (Symphony Quartet, Protein Technologies, Tucson, AZ). CH₁₂K₁₈ was purified by HPLC (Shimadzu Scientific Instruments, Columbia, MD) and molecular weights were confirmed by MALDI-TOF mass spectrometry (Voyager DE-STR, Applied Biosystems, Foster City, CA). A triblock co-polymer of poly(L-histidine)-b-poly(L-lysine)-b-PEG, CH₁₂K₁₈PEG_5k, was prepared as previously described [30 (link)]. Briefly, 5 kDa PEG was conjugated to CH₁₂K₁₈ via the reaction between the sulfhydryl group of the cysteine residue and maleimide group of PEG. Completion of the reaction was verified using a 4,4’-dithiodipyridine release assay to detect unreacted sulfhydryls. For fluorescence imaging studies, CH₁₂K₁₈PEG_5k polymer was fluorescently-labeled by conjugating the amine reactive probe Alexa Fluor 488 sulfodichlorophenol (5-SDP) ester (AF488) to the epsilon amines of lysine following the manufacturer’s protocol (reaction stoichiometry: 1 AF488 per CH₁₂K₁₈PEG_5k). Unreacted AF488 was removed by fractionating the reaction mixture on a Sephadex G25 column (GE Healthcare, Piscataway, NJ) pre-equilibrated with 50 mM ammonium acetate (AA).