solution concentrated under N2 flow. Crude products were precipitated with cold diisopropyl ether. The solid was centrifuged and washed with diisopropyl ether twice, and then lyophilized. Synthesis of the N-Glc Peptides N-Glc peptide epitopes 2-22 (Tables 1 and 2) were synthesized on a manual in batch synthesizer, starting from Rink Amide resin (250 mg, 0.63 mmol/g). N-Glc peptide epitopes 2-22 were acetylated on the N-terminal function with Ac2O (10 equiv) and NMM (10 equiv) in DMF for 2 h. The peptides were cleaved and side-chains were deprotected as described in the general cleavage procedure. The deprotection of the hydroxyl functions of glucose was performed with MeONa/MeOH. The peptides were lyophilized and purified.
N-Glc MEPs 23-26 ( Figure 4) were synthesized, starting from Fmoc4-Lys2-Lys-β-Ala-Wang resin (100 mg, 0.2 mmol/g). Fmoc-L-Asn(β-GlcAc4)-OH (2.5 equiv), Fmoc-NH-(PEG)-COOH (19atoms spacer) (N1-(9-Fluorenylmethoxycarbonyl)-1,13-diamino-4,7,10-trioxatridecan-succinamic acid) (2.5 equiv), Fmoc-NH-(PEG)-COOH (9-atoms spacer) (Fmoc-8-amino-3,6-dioxaoctanoic acid) (2.5 equiv) were coupled with HATU (2.5 equiv) and NMM (5 equiv) in DMF for 1 h. N-Glc MEPs 23-26 were acetylated on the N-terminal function with Ac2O (10 equiv) and NMM (10 equiv) in DMF for 2 h. The peptides were cleaved and side-chains were deprotected as described in the general cleavage procedure. The deprotection of the hydroxyl functions of glucose was performed with MeONa/MeOH. The peptides were lyophilized and purified.
All peptides 1-26 were purified by semipreparative RP-HPLC on a Waters instrument (Separation Module 2695, detector diode array 2996) using a Phenomenex (Torrance, CA, USA) Jupiter column C18 (10 μm, 250 × 10 mm), at 4 mL/min with solvent system A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN). The purity of the peptides was analysed by analytical HPLC using a Waters ACQUITY HPLC coupled to a single quadrupole ESI-MS (Waters 3100 Mass Detector) supplied with a BEH C18 (1.7 μm 2.1 × 50 mm) column at 35 °C, at 0.6 mL/min with solvent system A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN).
The peptides were purified by semi-preparative RP-HPLC and characterized by RP-HPLC and ESI-MS, obtaining a final purity ≥ 98%. Data were acquired and processed using MassLynx software (Waters, Milford, MA, USA). The analytical data are reported in details in Tables S1-S3. (13) 3.69 a 530.21 (530.52) Ac-N(Glc)KH-NH2 (14) 3.69 a 601.32 (601.29) Ac-N(Glc)AT-NH2 (15) 3.64 a 508.74 (508.51) Ac-ERN(Glc)GS-NH2 (16) 3.85 b 765.48 (765.76) Ac-ERN(Glc)GT-NH2 (17) 3.80 b 779.12 (779.79) Ac-ERN(Glc)KS-NH2 (18) 3.97 b 695.02 (694.68) Ac-ERN(Glc) 3 