Based on the AspH-TPR-Ox:hFX crystal structure, stable cyclic peptides comprising the core ring residues (aa 101–110) of the hFX EGF139mer-substrate with a thioether replacing the Cys3–4 disulfide were synthesized. The D-stereochemistry of the N-terminal amino acid (aa 101) aligns the peptide side chain with the main chain of the original substrate.
Commercial Fmoc-protected amino acids (AGTC Bioproducts; Alfa Aesar; CSBio; Iris Biotech; Novabiochem; Sigma-Aldrich; TCI) were used as received. SPPS was performed using a CSBio CS336X automated peptide synthesizer following standard Fmoc-strategy: linear peptides were synthesized from the C- to N-termini on a 0.1 mmol scale using a Rink amide linker using N,N-diisopropylcarbodiimide/1-hydroxybenzotriazole for coupling.
To obtain cyclic peptides, the N-terminal Fmoc-protecting group was cleaved on the resin, and the resin suspended in DMF (4 mL) containing N-chloroacetylsuccinimide73 (link) (150 mg). The mixture was gently shaken for 3 h, filtered, and the dried resin treated with a solution of trifluoroacetic acid, triisopropylsilane, and water (4 mL; 95/2.5/2.5). After 3 h, the mixture was filtered and the solution diluted with 45 mL cold diethyl ether. The suspension was centrifuged (4000 rpm, 10 min, 4 °C), decanted, and then taken up in 1.5 mL aqueous triethylammonium acetate buffer (1 M, pH 8.5, pH readjusted with trimethylamine) and heated for 10 min at 100 °C in a microwave reactor (Biotage Initiator). Directly afterwards, the crude cyclic peptides were purified by semi-preparative HPLC (DionexTM UltiMate® 3000, Thermo Scientific) using a reverse phase column (Grace Vydac® 218TP101522) and a gradient of acetonitrile in milliQ water (each containing 0.1%v/v trifluoroacetic acid) specified in Supplementary Fig.14 .
Commercial Fmoc-protected amino acids (AGTC Bioproducts; Alfa Aesar; CSBio; Iris Biotech; Novabiochem; Sigma-Aldrich; TCI) were used as received. SPPS was performed using a CSBio CS336X automated peptide synthesizer following standard Fmoc-strategy: linear peptides were synthesized from the C- to N-termini on a 0.1 mmol scale using a Rink amide linker using N,N-diisopropylcarbodiimide/1-hydroxybenzotriazole for coupling.
To obtain cyclic peptides, the N-terminal Fmoc-protecting group was cleaved on the resin, and the resin suspended in DMF (4 mL) containing N-chloroacetylsuccinimide73 (link) (150 mg). The mixture was gently shaken for 3 h, filtered, and the dried resin treated with a solution of trifluoroacetic acid, triisopropylsilane, and water (4 mL; 95/2.5/2.5). After 3 h, the mixture was filtered and the solution diluted with 45 mL cold diethyl ether. The suspension was centrifuged (4000 rpm, 10 min, 4 °C), decanted, and then taken up in 1.5 mL aqueous triethylammonium acetate buffer (1 M, pH 8.5, pH readjusted with trimethylamine) and heated for 10 min at 100 °C in a microwave reactor (Biotage Initiator). Directly afterwards, the crude cyclic peptides were purified by semi-preparative HPLC (DionexTM UltiMate® 3000, Thermo Scientific) using a reverse phase column (Grace Vydac® 218TP101522) and a gradient of acetonitrile in milliQ water (each containing 0.1%v/v trifluoroacetic acid) specified in Supplementary Fig.
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