Synthesis and Purification of Cyclic Peptides

AspH substrates were initially designed based on the sequence of EGFD1 of human coagulation factor X (hFX amino acids 86–124) (6 (link), 7 (link)); all were prepared with a C-terminal amide. The hFX–EGFD1_86–124 peptide (Fig. 1b, peptide 1/2) was synthesized by solid-phase peptide synthesis (SPPS) with the disulfide bridges being formed by thiol-oxidation in air-saturated buffer and purified by Peptide Synthetics (Peptide Protein Research Ltd, UK). The hFX–EGFD1_86–124-4Ser peptide (Fig. 1b, peptide 3) was synthesized by SPPS and purified by GL Biochem (Shanghai) Ltd (Shanghai, China).
The thioether-linked cyclic peptide hFX–CP_101–119 (hFX amino acids 101–119; Fig. 1b, peptide 4) was synthesized from the corresponding linear peptide (d-Ala replacing Cys101_hFX and Ser replacing Cys112_hFX) which was obtained by SPPS using the Fmoc-protection strategy (36 (link)). Microwave-assisted SPPS was performed using an automated peptide synthesizer (Liberty Blue, CEM Corporation) from the C to N termini on Rink Amide MBHA resin (AGTC Bioproducts; loading: 0.6–0.8 mmol/g) using iterative coupling (90 °C; 140 s; N,N-diisopropylcarbodiimide, Oxyma, Hünig's base, Fmoc-protected amino acids) and deprotection steps (90 °C; 90 s; 80/20% (v/v) DMF/piperidine). The N-terminal amine of the linear peptide was capped on the resin using N-chloroacetylsuccinimide. The linear peptide was cleaved from the resin and deprotected using a mixture of TFA, triisopropylsilane, 1,3-dimethoxybenzene, and water (92.5/2.5/2.5/2.5% (v/v/v/v), respectively). The solids were separated, and the linear peptide was precipitated from the solution using cold diethyl ether. The solid linear peptide was dissolved in water/acetonitrile, lyophilized, dissolved in aqueous triethylammonium acetate buffer (1 m, pH 8.5)/acetonitrile, and cyclized in a microwave reactor (Biotage Initiator, 10 min at 80 °C) (93 (link)). The crude product was filtered and directly purified using a semipreparative HPLC machine (JASCO) equipped with a reverse-phase column (Gemini 00G-4454-U0-AX; phase NX-C18). A linear gradient (0–30% (v/v) over 35 min) of acetonitrile in deoxygenated MQ-grade water (each containing 0.1% (v/v) TFA) was used as eluent. Fractions containing the cyclic peptide hFX–CP_101–119 (t_R = 34.4 min) were combined, lyophilized, and analyzed by MS.
The thioether-linked cyclic peptides hFVII–CP_121–139, hFIX–CP_108–126, hProC–CP_111–129, hC1r–CP_165–183, hC1s–CP_147–165, hFXII–CP_110–128, and hEGFL7–CP_152–170 were designed based on the amino acid sequence of the EGFDs of the corresponding human proteins and prepared as described above. The details are given in Fig. S4.