Fig. 1. Preparation and characterization of VHH–MUC1 conjugate. (a) Solid phase synthesis of sortaggable MUC1(Tn) glycopeptide and generation of VHH conjugates by sortagging. Reagents and conditions: (i) coupling: HATU, DIPEA, DMF, 5 eq., 2 h (For Thr-Tn(3Ac), 2 eq., overnight); deprotection: 20% piperidine in DMF; (ii) TFA/TIPS/EDT/H2O (90/5/2.5/2.5, v/v); (iii) 5% hydrazine in H2O, 1 h, then direct HPLC; (iv) SrtA (pentamutant), 10 mM CaCl2, 50 mM Tris, pH = 7.4, 12 °C, 2–4 h, >95% conversion based on LC-MS, ∼70% recovery at 1 mg scale after Ni-NTA bead adsorption and PD-10 column purification; (b) SDS-PAGE analysis of VHH–MUC1 conjugates. The integrity of synthesized conjugates was further confirmed by LC-MS, sequence analysis by LC/MS/MS and immunoblot (two independent syntheses gave comparable results); the reason for the doublet seen on SDS-PAGE remains unexplained, as a single mass was observed and both species are immunoreactive (c) LC-MS analysis shows intact mass of conjugates.
