Juniper c18 column

The labeling reaction was carried out by dissolving the cyclic peptide in DMF and adding one equivalent of fluorescein maleimide, 2% v.v N-methylmorpholine and DMF until the peptide concentration reached 200μM. Additionally, one equivalent of TCEP with respect to the peptide was added to ensure that the thiol was reduced and readily available for labeling. The reaction was left to sit at room temperature in the dark and complete conversion was achieved in 1 hour. The reaction mixture was purified by RP-HPLC (Waters Prep LC, Juniper C18 column) to afford the labeled product. All peptides were characterized with LC-MS analysis to confirm their identities and purities. The peptide concentrations were calibrated by measuring UV absorption at 495 nm (ε_fluorescein, ₄₉₀=80000 M⁻¹ cm⁻¹, ε_peptide, ₄₉₀=80000 M⁻¹ cm⁻¹).^{18 (link)}

The NCL reaction was carried by modifying a reported procedure.¹⁵ The linear peptide was dissolved in a mixture of DMF and ligation buffer (0.2 M sodium phosphate, 3 M guanidinium chloride, 20 mM 4-mercaptophenylacetic acid, pH 8) to a concentration of 2–5 mM. An equal amount of TCEP was added with respect to peptide concentration. The reaction was allowed to sit at room temperature with complete conversion in 4 hours. The reaction was acidified with water (0.1% TFA) and purified by RP-HPLC (Waters Prep LC, Juniper C18 column) to afford the cyclic peptide in 60–70% yield. All peptides were characterized with LC-MS analysis to confirm their identities and purities. The peptide concentrations were calibrated by measuring UV absorption at 275 nm (ε_Bip, ₂₇₅=11200 M⁻¹ cm⁻¹, ε_Trp, ₂₇₅=5378 M⁻¹ cm⁻¹, ε_peptide, ₂₇₅=16578 M⁻¹ cm⁻¹).^{16 (link),17}

The cLac peptides were synthesized through Fmoc/tBu chemistry using preloaded Dawson Dbz resin (3-(Fmoc-amino)-4-aminobenzoyl AM resin, Novabiochem), which consists of a 3,4-diaminobenzoic acid attached via the carboxyl group to the resin. The syntheses were carried out on a 0.05 mmole scale using five equivalents of the Fmoc-protected amino acids and HBTU for the coupling reaction. Additionally, a main-chain Boc-protected cysteine residue was installed at the terminal position of the peptide. A mixture of 50 mg 4-nitrophenylchloroformate in 1 mL of dichloromethane was added at the end of the synthesis and allowed to shake for 2 hours to afford the C-terminal imidazolidinone Nbz moiety, which will later facilitate the thioester formation in the subsequent native chemical ligation (NCL) reaction. The peptides were cleaved off the resin and globally deprotected using Reagent K (80% TFA, 5% H₂O, 2.5% EDT, 5% Thioanisole and 7.5% Phenol). The crude products were purified by RP-HPLC (Waters Prep LC, Juniper C18 Column). All peptides were characterized with LC-MS to confirm their identities and purities.