2.2. Synthesis of Peptides

Standard Fmoc-based solid-phase peptide synthesis techniques were used to generate the peptides NCoH and HisCol starting from H-Rink amide resin and using six equivalents of the desired amino acid, six equivalents of HBTU, and 12 equivalents of DIEA for each coupling. The resin was washed with DMF, DCM, MeOH, again with DCM, and again with DMF. The Fmoc-protecting group was removed using 25% piperidine in DMF (v/v). Couplings were monitored using the Kaiser [28] or the chloranil tests [29]. This process was continued until the sequence (POG)₉-HH-NH-resin was synthesized. For HisCol, two additional histidine residues were then coupled to the N-terminus to form HH-(POG)₉-HH-NH-resin. Upon completion of the final coupling and subsequent washing, the peptide was capped using 5% acetic anhydride and 8.5% DIEA in DMF with agitation for one hour. For NCoH, the final acylation of the free amine occurred with NTA (4 eq.) in the presence of HBTU (4 eq.) and DIEA (8 eq.). For the His-tagged peptide fluorophore rhodamine-(Gly)₃-(His)₆, known as Rho-HIS (Figure 1C), solid-phase peptide synthesis proceeded as described above until the sequence (Gly)₃-(His)₆-NH-resin was formed. The final acylation was accomplished by loading the flask with NHS-rhodamine (1.5 eq.), HBTU (6 eq.), and DIEA (12 eq.) in DMF and agitating in the dark for 3 h. The solution was drained, and the resin was washed as before. Coupling was verified with the Kaiser test. A TFA cleavage cocktail (95% TFA, 2.5% TIPS, 2.5% H₂O, 15 mL) was added to each of the resin-bound peptides. The resin was agitated at room temperature for three hours, the solvent was removed in vacuo, and the peptide was precipitated with cold diethyl ether (50 mL × 2 × 30 min). The solution was centrifuged at high speed for 10 min, and the solvent was removed by decanting. This process was repeated once. The crude material was characterized by matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS) and analytical high-pressure liquid chromatography (HPLC). The desired peptides were purified to homogeneity by HPLC on a semi-prep Luna C18 column (Phenomenex, Torrance, CA, USA). The pure peptide fractions were collected, and the solvent was removed in vacuo to afford pure NCoH, HisCol, and Rho-HIS, which were characterized by MALDI-TOF mass spectrometry: NCoH: calculated: 2938.02 Da, observed: 2936.02 Da; HisCol: calculated: 3013.20 Da, observed: 3013.55 Da; Rho-His: calculated: 1424.49 Da, observed 1424.76 Da.