Solid-phase peptide synthesis

All peptides used in this study were synthesized as C-terminal amides using Rink amide MBHA resin by standard Fmoc chemistry. Before use, the resin was pre-swelled in DMF for 20 min. Before the first coupling, an Fmoc deprotection procedure was performed using 20% piperidine in dimethylformamide (DMF) for 30 min. The resin was then washed successively with DMF, DCM, and DMF. For the coupling reactions, 3 equiv. of Fmoc-AA-OH and 3 equiv. of PyBOP were dissolved in DMF/DCM (1:1). This mixture was added to the resin, followed by the addition of 6 equiv. of DIEA. Coupling reactions were carried out for 60-90 min. Coupling efficiency was evaluated using the ninhydrin test. For peptides 14 and 19, Fmoc-Lys(Biotin)-OH was used. For peptide 23, after peptide assembly on the solid phase, the Boc group on the lysine side chain amine was removed with 1 M HCl in DCM for 30-40 min, then 5(6)-carboxyfluorescein was coupled to the side-chain free amine using PyBOP as the coupling reagent. The peptides were cleaved from the resin with a cocktail containing 95% TFA, 2.5% water, and 2.5% TIS for 2 h. After precipitation with cold diethyl ether, the crude peptides were purified using HPLC. The desired peptides were obtained in powder form after lyophilization. All peptides were characterized by electrospray ionization mass spectrometry.

List of peptides prepared in the study (letters in lower case denote D-amino acids):

Peptide 1: Ac-KKLAVINHV; 1061.01 (observed), 1062.27 (calculated).

Peptide 2: GIGGIKA; 613.68 (observed), 613.74 (calculated).

Peptide 4: YKANGL; 664.26 (observed), 664.67 (calculated).

Peptide 5: GFGGIKA; 648.38 (observed), 648.52 (calculated).

Peptide 7: Ac-KKLAVINGF; 1031.34 (observed), 1031.56 (calculated).

Peptide 9: Fluorescein-GRANGI; 944.52 (observed), 944.97 (calculated).

Peptide 11: GIGGFKGG-klaklakklaklak; 2197.07 (observed), 2197.72 (calculated).

Peptide 14: HVGGRIK(Biotin)GA; 1119.89 (observed), 1118.61 (calculated).

Peptide 19: GVGGRIK(Biotin)GA; 1039.61 (observed), 1038.58 (calculated).

Peptide 23: GIGGIRK(Fluorescein); 1057.65 (observed), 1057.35 (calculated).

Peptide 25: GFLGVK(COCH₂ONH₂)ANHV; 1113.90 (observed), 1113.29 (calculated).

The numbering and illustrative structures of peptides and proteins prepared in this study are shown in Figure S1.