Preparation and Evaluation of Small Peptide Binding Boronic Acid Probes for Bioconjugation

• Author / Creator

  Mokhtarihaj, Negar

• The study of target proteins in live cells is fundamentally important to understanding their molecular roles in biological processes. Applications of cell permeable small molecules in bioorthogonal reactions have been improved to selectively label proteins tag for the purpose of protein interaction analysis. Bioorthogonal reaction is defined as the chemical reactions that do not react or interfere with biological systems. Functional groups that involve in bioorthogonal reactions must selectively react with each other under physiologically conditions. Also using a small reactive molecule in bioorthogonal reactions can prevent the structural perturbation of the proteins that wants to be monitored. Molecule fluorophores can provide dynamic information on cell surface interactions due to their small size, low detection limits and environmental sensitivity. Boronic acids have been applied in the construction of receptors and chemosensors for a range of biologically important species, especially saccharides. In 2009, for the first time, Schepartz utilized a rhodamine–derived bisboronic acid sensor (RhoBo) as a reversible, selective and non-toxic sensor to selectively label a specific tetraserine peptide. In this thesis, simple monoarylboronic acids with potentially four covalent, reversible points of attachment were designed and synthesized to test their binding affinity to a small peptide tag in a sequence specific manner. Boronate ester formation between serines and the boronic acid part of the molecule was believed to potentially increase the selectivity and stability of imine formation between the lysine and ketone part of the molecule. Binding affinity of designed boronic acids towards a selection of chosen peptides was evaluated via the Alizarin Red S colorimetric assay and UV spectrophotometry and fluorescence spectroscopy. In Chapter 2 small boronic acid molecules with Michael acceptors were designed in which the boronic acid part of the molecule was employed as a way to increase site-specificity of the thiol-Michael addition reactions towards small peptide tag containing cysteine and serine residues. 3-Maleimidophenylboronic acid was found to be reactive towards small peptides containing cysteine and terminal serine residues. Reaction kinetics were performed to monitor its reactivity towards the peptide by HPLC-MS and 1H NMR spectroscopy.

• Subjects / Keywords

  • Bioorthogonal
  • Boronic acid

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3PZ51X5J

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. 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.