1. Introduction Acknowledgements Abstract Objectives Methods Results Conclusions References The project was to synthesize and perform monoclonal antibody binding study of MUC1 mucin peptide epitopes derived from the large transmembrane protein MUC1 mucin. The synthesis of two cyclic mucin peptides formed by a triaziole ring, cyclo-azA-TSAPD-Pra-G and cyclo- azA-VTSAPD-Pra-G (azA = azidoalanine, Pra = propargylglycine), were carried out. The results of antibody binding study by the Saturation Transfer Difference NMR technique (STD-NMR) showed clear peaks corresponding to the methyl groups of alanine, threonine and valine. The proline side chain protons (P, P, P) on the cyclo-azA-TSAPD-Pra-G showed significant saturation transfer effects, indicating stronger antibody interactions at these groups. MUC1 mucin is a high molecular weight transmembrane glycoprotein that is expressed on apical epithelial origins. Its structure consists of an extracellular domain that contains a variable number tandem repeat domain of repeating peptide. Each variable number tandem repeat contains 20 amino acids, with the sequence of GVTSAPDTRPAPGSTAPPAH (see Fig. 1). The biological role of normal mucin is to lubricate, hydrate, carry out cell signaling and cell protection from pathogen invasions. Tumor cells also produce a version of mucin protein. The tumor MUC1 mucin is expressed on cancers of glandular epithelial origins, such as breast, pancreas, ovary, lung, gastrointestinal tract, prostate, urinary bladder and the endometrium. The difference between normal mucin and cancer mucin is that normal mucin contains a high number of carbohydrate chains whereas cancer mucin sparingly contains fewer number of carbohydrate chains (see Fig. 2) (1). This is a significant point, because with low glycosylation the core protein is exposed allowing antibodies to develop against the core tumor mucin protein, which then signals the immune system to kill off the infected cancer cell. However, over expression of MUC1 mucin exhibits immunosuppression by inhibiting cell lysis, thus rendering the immune system ineffective in attacking cancer cells (1). In using the native epitope of MUC1 mucin (e.g., epitope of tumor MUC1 mucin), containing GVTSAPD, there is potential to induce antibodies since this is a sequence residing at the region that the immune system recognizes to be foreign. Therefore, these synthesized peptides have potential application to be used in the area of vaccine to treat cancers (2, 3).  Design MUC1 epitope peptides that can be recognized by Monoclonal antibody.  MUC1 epitope peptide must contain proper residues, correct amino acid sequence and conformations.  Synthesis:  cyclo-azA-TSAPD-Pra-G Will cyclization be possible by Click chemistry?  cyclo-azA-VTSAPD-Pra-G Contain similar residue sequence as linear peptide.  Determine if cyclo-azA-TSAPD-Pra-G and cyclo-azA-VTSAPD-Pra-G are able to bind to the MUC1 antibody.  Tumor mucin contains reduced carbohydrate chains that induce an immunological response. This study examines which amino acid residue of the mucin peptide interact with the antibody and what are the strength of the interactions.  Synthesis of peptides via Solid-Phase Peptide Synthesis using Fmoc-chemistry and Wang resin.  Cyclization of cyclic peptides via Click Reaction.  Peptide purification and analysis via HPLC and LC-MS.  Antibody Binding Study achieved by Saturation Transfer Difference (STD) NMR. Synthesis of Triaziole Cyclic Mucin Peptides and Antibody Binding Study by NMR Bonnie Thao and Thao Yang Department of Chemistry, University of Wisconsin-Eau Claire CERCA May 1-2, 2013 a b Figure 1. Structure of MUC1 mucin protein showing the extracellular repeat domain in which the peptides of study are derived from (1). Figure 2. Difference between glycosylation of healthy (a) and cancer (b) MUC1 mucin proteins (1). Figure 6. Peptide-Antibody Binding Study by Saturation Transfer Difference NMR spectroscopy (STD NMR) for cyclo-azA-TSAPD-Pra-G peptide. Top panel is a full range spectrum (9.5 ppm - 0 ppm); bottom panel is an expanded spectrum between 5 ppm – 0 ppm. In each panel, the top spectrum is the usual 1 H NMR spectrum of a mixture of antibody plus cyclo-azATSAPD-Pra-G peptide; the bottom spectrum is the STD NMR spectrum of the same mixture showing peptide peaks that indicate specific protons that bind to the antibody. Black arrows indicate the same peaks on top and bottom panels. The data indicate clear STD peaks at - and proline side chain protons.  This research is supported by the UWEC ORSP Diversity Mentoring Grant.  We gratefully thank the NSF Wisconsin Alliance for Minority Participation (WiscAMP) for initial funding and support of the project.  We also thank the UWEC Chemistry Department in providing the support, equipment and materials necessary for the project. Asp Pro Ala Ser Thr azA 6x Figure 5. CH-NH region of the 2D TOCSY NMR spectrum for cyclo-azA-TSAPD-Pra-G in 20 mM phosphate buffer, 5 mM NaCl, pH 5, 10%, 90% at 7 °C with 3-5 mg of peptide (7). Figure 4. HPLC chromatogram of crude (a) and 1x purified (b) cyclo-azA-TSAPD-Pra-G. Crude HPLC of cyclic peptide shows one peak with retention time of 15 min and a mass of 754.24 u. Purified HPLC of cyclic peptide shows a single peak with the same mass of 754.24 u at a retention time of 15 min. Back panel shows LC-MS chromatogram of 1x purified cyclo-azA-TSAPD-Pra-G, with observed mass of [M + H] + = 754.24 u compared to a theoretical mass of 754.72 u. HPLC Method: acquisition time 30 minutes, flow rate 0.5 mL/min, and ACN as the organic buffer in an Grace Vydac C-18 column, 10 µm, 10 x 250 mm. LC-MS Method: acquisition time 20 minutes, flow rate 0.5 mL/min, and ACN as the organic buffer in an Agilent Extend C-18 column, 3.5 µm, 4.6 x 150 mm (6). 1.Singh, R., and Bandyopadhyay, D. “MUC1, A Target Molecule for Cancer Therapy,” Cancer Biology & Therapy 6:4, 481-486, (2007). 2.Möller, H., Serttas, N., Paulsen, H., Burchell, J. M., Taylor- Papadimitriou, J. and Meyer, B. “NMR-based determination of the binding epitope and conformational analysis of MUC1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3.” Eur. J. Biochem. 269, 1444-1455, (2002). 3.Curigliano, G., Spitaleri, G., Pietri, E., Rescigno, M., de Braud, F., Cardillo, A., Munzone, E., Rocca, A., Bonizzi, G., Brichard, V., Orlando, L. and Goldhirsch, A. “Breast cancer vaccines: a clinical reality or fairy tale?” Annals of Oncology 17, 750 - 762, (2006). 4.Chan, W. C. and White, P. D., (ed.) in “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” Basic Procedures, Oxford University Press, p. 41 - 74, (2000). 5.Turner, R. A., Oliver, A. G., and Lokey, R. S. “Click Chemistry as a Macrocyclization Tool in the Solid- Phase Synthesis of Small Cyclic Peptides.” Org. Lett., 9, 5011-5014, (2007). 6.Chan, W. C. and White, P. D., (ed.) in “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” RPHPLC using lipophilic chromatography probes, Oxford University Press, p. 269 - 276, (2000). 7.Her, C., and Yang, T., “Antibody Binding Study of Mucin Peptide Epitopes.” Division of Biological Chemistry, 243rd ACS meeting, San Diego, CA., March 25-29, 2012. 8.Mayer, M. and Meyer, B. “Group Epitope Mapping by Saturation Transfer Difference NMR To Identify Segment of a Ligand in Direct Contact with a Protein Receptor.” J. Am. Chem. Soc. 123, 6108 – 6117, (2001). (a) (b)