Oxyma

Peptides were obtained by manual or semiautomatic (Biotage MultiSynTech) SPPS on a 0.3–1 mmol scale. This method allowed us to reduce the amount of solvent for each synthesis, as well as the excess of coupling reagents compared to a fully automatic SPPS (automated synthesis requires dead volumes, namely at least one equivalent of all Fmoc-amino acids and coupling reagents more than manual synthesis. Several steps of needle cleaning are also needed, with consequent waste of solvent). We used either the rink-amide resin or the 2-chlorotrityl resin preloaded with the 2-aminoalcohol L-leucinol, both commercially available. The protocol makes use of the standard fluorenylmethyloxycarbonyl (Fmoc-) protecting group, removed by treatment with 20% piperidine in DMF (2 mL for 0.75 g of resin). The ε-NH₂ groups on the side-chain of Lys residues were protected by tert-butyloxycarbonyl (Boc) groups, which can be removed in acidic conditions, such as a 3M HCl solution in methanol (or trifluoroacetic acid, TFA).
The activation of the carboxyl group of the incoming amino acid was achieved using Oxyma pure and DIC [35 (link),51 (link)] (Fmoc-AA/Oxyma/DIC 1:1:1) using 1.5 (for Gly and Ile) or 2.5 equivalents with respect to the loading of the resin, instead of the standard 4 equivalents. The standard SPPS procedure outline includes double coupling for each and every step involving Aib residues. The poor reactivity of Aib due to the steric hindrance of the gem dimethyl groups, however, mostly hampers the nucleophilic attack by Aib αNH₂ onto the incoming, activated Fmoc-Leu-OH/nOctanoic acid. Thus, only that step really needs to be repeated with fresh reagents. Therefore, we performed double couplings only to link Fmoc-Leu-OH (or nOct) onto Aib. We repeated the synthesis of all analogs several times and unequivocally established that Fmoc-Aib-OH can be successfully inserted by a single coupling with 2.5 equivalents. All washings and coupling steps were performed in dimethylsulfoxide/ethyl acetate 1:9 [40 (link)]. Peptides were released from the resin by acidic treatment [52 (link)]. The procedure must be repeated at least three times to obtain complete peptide release, and then additional treatment with HCl 3M in methanol is needed to remove the tertButyloxycarbonyl (Boc) side-chain protection (either on Lys or Api). Release from Rink-amide resin was achieved by treatment with a mixture of 95% TFA, 2.5% water, and 2.5% triisopropylsilane (2 mL for 0.75 g of resin). The collected solution was concentrated to dryness and the obtained solid (or oily) precipitate was washed twice with diethyl ether. Conversely, peptides were released from 2-chlorotrityl resin by repeated treatments with a solution of 1,1,1,3,3,3-hexafluoroisopropanol 30% in dichloromethane (3 × 2 mL for 0.75 g of resin) lasting from one hour to overnight. Such mild acidic conditions do not cleave side-chain protections. Thus, Boc protecting groups were removed in solution by treatment with 3M hydrochloric acid in methanol (2 mL for 0.75 g of resin).
Peptides were obtained with very good crude purities (85–97%) and easily purified to 95–99% purity by medium-pressure liquid chromatography (Isolera Prime, Biotage, Uppsala (Sweden)). To purify one gram of crude peptide from 80% to >95% we used 500 mL of water milliQ and 150mL of acetonitrile. We used HCl instead of TFA (1 mL of HCl 37% per 1L of eluant). After peptide elution, we washed the column with ethanol and water (300 mL in total). Peptide characterization (reported in the Supplementary Materials) was obtained through high-resolution mass spectrometry (HRMS) analysis (electrospray ionization time-of-flight, ESI-TOF), analytical HPLC (used also to determine the degree of purity), and nuclear magnetic resonance (¹H NMR). Yields obtained (after purification): always > 70%. Full characterizations for all peptides are reported in the Supplementary Materials (p. S7–S42).

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.

Fmoc‐protected amino acids for peptide synthesis were purchased from EMD Millipore. Fmoc‐azidolysine was purchased from Combi‐Blocks Inc. Dichloromethane (DCM) was purchased from Millipore Sigma. Dimethylformamide (DMF) and diethyl ether were purchased from Oakwood Chemical Inc. Piperidine was purchased from Alfa Aesar. Oxyma and diisopropyl carbodiimide (DIC) were purchased from ChemImpex. TFA was purchased from Oakwood, and Rink Amide resin was purchased from Novabiochem. Dibenzocyclooctyne‐Sulfo‐N‐hydroxysuccinimide (DBCO‐sulfo‐NHS) linker was purchased from Click Chemistry Tools. All oligonucleotides were purchased from Integrated DNA Technologies.

Fmoc-protected amino acids, N,N’-diisopropylcarbodiimide, Oxyma Pure and trifluoroacetic acid were purchased from Iris Biotech (Marktredwitz, Germany). Cl-MPA ProTide Resin was supplied by CEM (Matthews, NC, USA). Dess-Martin Periodinane was supplied by Fluorochem (Hadfield, UK). Reagents for electrophoresis were purchased from Biorad (Hercules, CA, USA). The cell culture reagents, if not otherwise stated, were purchased from Merck (Darmstadt, Germany). All general purpose solvents (dimethylformamide, dichloromethane, dimethyl sulfoxide) as well as HEPES and Tris were supplied by Fisher Scientific (Waltman, MA, USA). Acetonitrile (Supelco) was purchased from Merck (Darmstadt, Germany).