Download ThaoSpr2013

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
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 cycloazA-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.
 Design MUC1 epitope peptides that can be recognized by Monoclonal
antibody.
 MUC1 epitope peptide must contain proper residues, correct amino
acid sequence and conformations.
Introduction
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).
Results
 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.
In t ensi t y
Objectives
2500000 754.24u
*
2000000
1500000
755.23u
1000000
500000
756.24u
0
750
760
776.21u
770
780
m/z
10400
Intensity
Abstract
7800
5200
2600
0
0
5
10
15
20
25
30
35
40
Time, min
Methods
 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.
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).
Conclusions




Synthesis of cyclo-azA-TSAPD-Pra-G was successful.
cyclo-azA-TSAPD-Pra-G does bind to the MUC1 antibody.
The binding occurred at - and at the side chains.
No binding to the T2 - CH3 .
 In addition:
 The syntheses of cyclo-azA-VTSAPD-Pra-G was inclusive.
a
References
Asp
Pro
Ala
Ser
Thr
azA
6x
b
Figure 1. Structure of MUC1 mucin protein showing the extracellular
repeat domain in which the peptides of study are derived from (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 1H 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.
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).
𝐂𝐮𝟐+ (1 equv.)
Na-ascorbate (3 equiv.)
DIPEA (6 equiv.)
18 h
Table 1. 1H chemical shifts in ppm corresponding to
the NMR spectrum.
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., TaylorPapadimitriou, 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).
Acknowledgements
(a)
 This research is supported by the UWEC ORSP Diversity Mentoring
Grant.
(b)
Figure 2. Difference between glycosylation of healthy
(a) and cancer (b) MUC1 mucin proteins (1).
Figure 3. a) Schematic illustration of solid phase peptide synthesis method used (4).
b) Illustration of Click chemistry reaction, yielding a triaziole ring as part of the
structure. Fmoc-propargylglycine (Fmoc-Pra) and Fmoc-azidoalanine (Fmoc-azA)
are used as part of the residues on the peptide chain and will be brought together at
the last step by the Click reaction via Cu2+ , Na-ascorbate and DIPEA to form the
triaziole ring structure, yielding a cyclic peptide (5).
 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.