Cyclodimerization of immunosuppressive fragment of HLA-DR molecule. Design, synthesis and ESI-MS/MS analysis

• Published in: Journal of peptide science 2016-08, Vol.22 (8), p.525-532
• Main Authors: Biernat, Monika, Cydzik, Marzena, Stefanowicz, Piotr, Kluczyk, Alicja, Artym, Jolanta, Zimecki, Michał, Szewczuk, Zbigniew
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies - drug effects; Cross-Linking Reagents - chemistry; Cyclization; cyclodimer; Dimerization; Drug Design; Erythrocytes - cytology; Erythrocytes - immunology; ESI-MSMS; Fluorobenzenes - chemistry; HLA-DR analogs; HLA-DR Antigens - chemistry; HLA-DR Antigens - immunology; Humans; Immunity, Humoral - drug effects; Immunosuppressive Agents - chemical synthesis; Immunosuppressive Agents - pharmacology; Lymphocytes - cytology; Lymphocytes - drug effects; Lymphocytes - immunology; Male; Mice; Mice, Inbred CBA; PEG linker; Peptide Fragments - chemical synthesis; Peptide Fragments - pharmacology; Peptides; Phenols - chemistry; Polyethylene Glycols - chemistry; Primary Cell Culture; Protein Structure, Secondary; Sheep; Solid-Phase Synthesis Techniques; Spectrometry, Mass, Electrospray Ionization
• ISSN: 1075-2617; 1099-1387
• DOI: 10.1002/psc.2898

The nonapeptide fragment of the HLA‐DR molecule, located in the exposed loop of the alpha‐chain (164–172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three‐dimensional structure of the HLA‐DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C‐termini by spacer of (Gly5)2‐Lys‐NH2 and the N‐termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5)2K‐resin analog was synthesized using solid‐phase peptide synthesis protocols. The cyclization was achieved by cross‐linking the N‐terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega‐bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Solid phase cyclodimerization of VPRSGEVYT sequence using polyethylene glycol for head‐to‐head and (Gly5)2‐Lys spacer for tail‐to‐tail cyclization resulted in a 132 atom macrocycle with enhanced immunosuppressive activity. The structure of cyclodimer was unambiguously confirmed by detailed ESI‐MS/MS analysis.