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Theses and Dissertations

Light Responsive Linchpins for Accelerated Discovery of Light-Responsive Mono- and Bicyclic Peptide Ligands Open Access


Other title
Genetically Encoded Library
Cyclic peptides
Bicyclic Peptides
Phage Display
Light Responsive
Type of item
Degree grantor
University of Alberta
Author or creator
Jafari, Seyed Mohammadreza
Supervisor and department
Derda, Ratmir
Examining committee member and department
Woolley, A (Chemistry, University of Toronto)
Hall, D (Chemistry)
Campbell, R (Chemistry)
Brown, A (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
In this thesis I describe the development and application of light-responsive (LR) linchpins for efficient discovery of LR-peptides from genetically encoded peptide librar-ies. LR-linchpins have been used for dynamic control of the structure of peptides and their biochemical properties such as binding to a protein receptor. Rational design of such LR binders is not trivial and it mandates previous knowledge of the structure of the target receptor and the binding mechanism. In the first chapter of this thesis, I briefly review several linchpins that have been used to cyclize peptides. In Chapter 2 of this thesis, I describe integration of a well-known LR-linchpin 3,3'-bis(sulfonato)-4,4'-bis(chloroacetamido)azobenzene (BSBCA) in a phage displayed library of peptides for synthesis of a library of LR-macrocycles dis-played on the coat protein of the phage. Selection from this library using streptavidin as a bait resulted in identification of three cyclized LR-peptides that bind to streptavidin. We show that binding affinity decreases reversibly in response to blue light (370 nm) as measured by quantitative mass spectroscopy. In the same chapter, we also describe a method for quantification of the efficiency of cyclization of the phage library with the BSBCA linchpin. Such quantification is vital for development and screening of chemical-ly-modified phage displayed libraries, because it provides a measure for the quality of the library before selection. Appendix 1 of this thesis discusses the possible reasons of dis-crepancy observed in the reaction of phage display library and mono-clonal phage. In Ap-pendix 2, I demonstrate screening of an LR-library against anti FLAG antibody which resulted in enrichment of peptide binders against a secondary target present in the pan-ning, protein A. To extend the repertoire of LR-linchpins, in Chapter 3, I describe the synthesis of 3,3'-bis(sulfonato)-4,4'-bis(buta-2,3-dienoylamido)azobenzene (BSBDA), a new LR-linchpin that can undergo fast reaction with cysteine containing peptides – with k = 30 M-1s-1. We designed this linchpin based on BSBCA which has been used by several groups to produce cyclic LR-peptide scaffolds. We used BSBDA to cyclize a streptavidin bind-ing peptide precursor and confirmed the structure with Nuclear magnetic resonance (NMR) and mass spectroscopy (MS) analysis. We also showed that BSBDA can react with the thiols of cysteines displayed on coat protein of the phage, in the presence of re-ducing agent tris(2-carboxyethyl)phosphine (TCEP), with > 98% yield in only 20 min at pH 8. The fast reaction of linchpins with phage is essential because it reduces the expo-sure time of the phage proteins with toxic chemicals, such as TCEP. Finally, I describe the first example of a C3V symmetric LR-linchpin that can produce bicyclic LR-scaffolds from peptides consisted of unprotected natural amino ac-ids. The reaction of the linchpin with the thiols and glyoxal produced by oxidation of N-terminal serine in the peptide sequence SXnCRRRRC (where n = 1- 4) resulted in for-mation of bicyclic LR-peptides in 11-73% isolated yield. All four bicyclic peptides isom-erized to cis conformer after irradiation with 365 nm light. The switching efficiency and the thermal relaxation half-life varied for different peptide sequences. I anticipate that bicyclic LR-peptides can be used in studies where a dynamic surface and a static motif are required on the same molecule where each motif has specific biological function. One of the possible future explorations is inhibition of dimerization of EGFR by tuning the static surface for cell penetration and the dynamic surface to inhibit the coiled-coil formation of the intracellular helix of EGFR which is necessary for its function.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
M. R. Jafari, L. Deng, P. I. Kitov, S. Ng, W. L. Matochko, K. F. Tjhung, A. Zeberoff, A. Elias, J. S. Klassen, and R. Derda, “Discovery of Light-Responsive Ligands through Screening of a Light-Responsive Genetically Encoded Library”, ACS Chem Biol, 2014, 9: 443-450.

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