Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) for targeted antibody blocking

Long peptide DCAF enables high selectivity to target harmful antibodies, providing new thoughts for antibody-induced disease intervention.


S-2
Chemical synthesis procedure

Synthesis of Fc-III mimetics:
The Fc-III peptide (1), DC(Acm)AWHLGELVWC(Acm)TAA-NHNH 2 , was synthesized by Solid Phase Peptide Synthesis (SPPS). The Acetamidomethyl (Acm) group was used to protect the Cys2 and Cys12 at Fc-III peptide from desulfurization. The 2-Cl-(Trt)-Cl resin (625 mg, 0.25 mmol) was washed (3×DMF, 3×CH 2 Cl 2 , 3×DMF), and followed by swollen with CH 2 Cl 2 /DMF (1/1, v/v) for 30 min. Then add 5% NH 2 NH 2 in DMF (v/v) to the resin for hydrazination at 30℃ for 30 min. Repeat this step for once. After another washing step, add 5% MeOH in DMF (v/v) to the resin to cap the unreacted sites. Wash the resin again, then the resin can be used to couple amino acids under microwave conditions. Each coupling cycle includes Fmoc deprotection using 20% piperidine in DMF (15 s at 75°C, 50 s at 90 °C) and amino acid coupling using 4-fold excess of 0.2 M Fmoc-protected amino acid in DMF, 0.5 M DIC in DMF, and 1.0 M Oxyma in DMF (120 s at 25°C, 240 s at 50 °C for His and Cys, 1500 s at 25°C, 120 s at 75°C for Arg, 15 s at 75°C, 110 s at 90 °C for other residues).
After the amino acids elongation, the finished peptide was cleaved from the resin with a mixture of 92.5% TFA, 2.5% water, 2.5% DODT and 2.5% TIPS. After 3 h, the resin was removed by filtration and washed with neat TFA. The combined solution was concentrated under N 2 flowing. Then, the crude peptide hydrazide was obtained by precipitating in cold Et 2 O and centrifugation for three times. Finally, the desired peptides were obtained after lyophilization.  Figure S2A).

Total chemical synthesis of DCAF molecules:
Ligation of peptide 1 and peptide 2 of DCAF1.
Peptide 1 (0.9 mg, 0.5 μmol) was dissolved in 0.2 mL acidified ligation buffer (aqueous solution of 6 M GnHCl and 0.2 M NaH 2 PO 4 , pH 3.0). The mixture was cooled in an ice-salt bath (-15 ºC) for 15 min, and 20 μl of 0.5 M NaNO 2 in acidified ligation buffer was added. The activation reaction system was kept in ice-salt bath with stirring for 15 min, after which 6.8 mg MPAA in 0.2 mL acidified ligation solution with 5.5 mg peptide 2 was added. The mixture was kept in ice-salt bath for 5 min under stirring, then the pH of the solution was adjusted to 6.8-7.0 at room temperature. After 12 h, 0.2 ml of 0.1 M TCEP in a ligation buffer (pH 7.0) was added to dilute the system and the reaction system was kept for 20 min under stirring. Finally, the ligation product was analyzed and purified by RP HPLC to furnish peptide 3 in 56% isolated yield (3.6 mg).  Figure S2D).

Desulfurization of peptide 3 of DCAF1
Peptide 3 (3.4 mg, 0.3 μmol) was dissolved in 50 μl phosphate neutral buffer (pH 7.0) containing 6.0 M Gn·HCl, 0.2 M NaH 2 PO 4 . To the above solution, 50 μl of 1 M TCEP, 10 μl t BuSH and 5 μl VA-044 solution (0.1 M in phosphate neutral buffer) was added. The final pH of the solution was adjusted to 6.9 and kept at 37 ºC with stirring about 5 hours. Finally, the desulfurization product was analyzed and purified by RP HPLC to furnish peptide 4 in 41% isolated yield (1.48 mg).  Figure S2E).

Remove of ACM in peptide 4 of DCAF1.
Peptide 4 (1.35 mg, 0.11 μmol) was dissolved in 200 μl of 32 mM AgOAc(in 50% CH 3 COOH ), and the S-4 reaction was stirred at r.t. for 4 hours. Subsequently, 1-3 μl of 1M Dithiothreitol (DTT) dissolved in 0.2 M phosphate solution containing 6 M Gn·HCl (pH 7.0) was added to convert the silver thiolates on proteins to free thiols. After stirred violently, the supernatant was purified and analyzed by RP-HPLC to furnish peptide 5 in 30% isolated yield (0.45 mg).  Figure 2C).
The ligation method is same with mentioned above. In brief, peptide 1 (1.5mg, 0.8 μmol) was dissolved in 0.2 mL acidified ligation buffer. The mixture was cooled at -15 ºC for 15min, and add 20 μl of 0.5 M NaNO 2 . After another -15 ºC for 15 min, 6.8 mg MPAA with 5 mg peptide 2 in 0.2 mL acidified ligation solution was added. The mixture was adjusted the pH to 6.8-7.0 at room temperature. After 12 h, 0.2 ml of 0.1 M TCEP was added to dilute the system and the reaction system was kept for 20 min under stirring.
Finally, the ligation product was analyzed and purified by RP HPLC to furnish peptide 3 in 39% isolated yield (2.3 mg).   Figure S4B);

Desulfurization of peptide 3 of DCAF2
The method is same with mentioned above. In brief, peptide 3 (2.3mg, 0.5 μmol) was dissolved in 50 μl phosphate neutral buffer (pH 7.0). To the above solution, 50 μl of 1 M TCEP, 10 μl t BuSH and 5 μl VA-044 solution was added. The final pH of the solution was adjusted to 6.9 and kept at 37 ºC with stirring about 5 hours. Finally, the desulfurization product was analyzed and purified by RP HPLC to furnish peptide 4 in 25% isolated yield (0.87 mg).  Figure S4C);

Remove of ACM in peptide 4 of DCAF2.
The method is same with mentioned above. In brief, peptide 4 (0.87 mg, 0.07 μmol) was dissolved in 200 μl of 32 mM AgOAc (in 50% CH 3 COOH), and the reaction was stirred at r.t. for 4 hours. Subsequently, 1-3 μl of 1M Dithiothreitol (DTT) was added. After stirred violently, the supernatant was purified and analyzed by RP-HPLC to furnish peptide 5 in 28% isolated yield (0.24 mg).  Figure S4D).
The ligation method is same with mentioned above. In brief, peptide 1 (0.8mg, 0.4 μmol) was dissolved in 0.2 mL acidified ligation buffer. The mixture was cooled at -15 ºC for 15min, and add 20 μl of 0.5 M NaNO 2 . After another -15 ºC for 15 min, 6.8 mg MPAA with 3.5 mg peptide 2 in 0.2 mL acidified ligation solution was added. The mixture was adjusted the pH to 6.8-7.0 at room temperature. After 12 h, 0.2 ml of 0.1 M TCEP was added to dilute the system and the reaction system was kept for 20 min under stirring.
Finally, the ligation product was analyzed and purified by RP HPLC to furnish peptide 3 in 31% isolated yield (1.3 mg).