Phage Display as a Combinatorial Chemistry Platform for Discovery of Chemical Structure-Activity Relationships

• Author / Creator

  Triana Guzman, Vivian

• Optimization of chemical reactions, discovery of optimal substrates and determination of substrate scope involve exhaustive screening of conditions as well as measurements of rates and conversions for all series of structurally similar substrates. These systematic screenings used in reaction development and reaction discovery are usually performed on a group of substrates that belong to a region of the chemical space that has been targeted using chemical intuition and empiricism. The number of tests to be performed is often high and unbiased approaches that avoid contribution of the scientist’s rationalization and insight, demand for an even higher number of experiments. One-well-one-experiment approaches are the best at replicating real reaction conditions but require many work hours or expensive robotics and automation. Several technologies have been developed for one-well-multiple-experiments screenings with the aim of reducing time and cost of screening, however, sophisticated technologies for analysis of mixtures are necessary and real one-substrate conditions are not simulated.
  This thesis presents a new methodology for Structure-Activity Relationship discovery that uses phage display as a combinatorial chemistry platform for one-well-multiple-experiments screenings of substrates. As a first step, conditions for Wittig reaction between an ester stabilized ylide and libraries of glyoxaldehydes displayed on the pIII protein of M13 phage were optimized. The Wittig reaction installed an N-terminal ester of maleic/fumaric acid on phage libraries that underwent both Michael addition and Diels-Alder cycloaddition with cyclopentadiene. After demonstrating its suitability to be applied on phage, Structure-Activity Relationship of the Wittig reaction between ester stabilized ylide and glyoxaldehydes in water was performed using focused libraries that can be entirely visualized using Illumina sequencing. Enrichment and depletion of specific residues in several positions of the sequences allowed us to find slow, medium and fast reacting peptides in the Wittig reaction covering a range of 50-fold difference in rate. Sequences lacking hydrogen-bond donors in the two positions adjacent to the aldehyde (Ald-Pro-Pro-X-X) showed up to 16-fold decrease in rate compared to average reactivity, while sequences with two Trp residues in those two positions (Ald-Trp-Trp-X-X) showed up to 5-fold increase in reactivity. These observations demonstrated the importance of hydrogen-bond stabilization of the oxaphosphetane intermediate. Preliminary studies on using phage display as a combinatorial chemistry platform for discovery of cyclization-prone sequences were performed. First, we installed the α,β-unsaturated ester via the Wittig reaction and then selected for sequences able to undergo intramolecular attack by nucleophiles present in the peptide sequence. We were able to find three sequences prone to cyclization and we isolated two of the formed adducts, demonstrating the feasibility of using phage display as a tool for discovery of optimal substrates in a reaction.

• Subjects / Keywords

  • Wittig Reaction
  • Phage display
  • Reaction development
  • Structure Activity Relationships

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3BN9XK0Q

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.