Carbohydrates for Cell-Specific Targeting of Therapeutic Glycoproteins Studied by EPR Spectroscopy

UoM administered thesis: Phd

  • Authors:
  • Michael Hollas

Abstract

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Nitroxide radical spin labels combined with electron paramagnetic resonance (EPR) spectroscopy offer an effective way to monitor protein dynamics. In-order to understand such interactions, we have synthesized a nitroxide spin label that has two attachment points with orthogonal reactivity. This spin label was reacted with the thiol terminated 2-thioethyl-D-biotin amide and 2-thioethyl-α-D-mannose to give the biotin and mannoside spin labelled bioligands. These spin labelled ligands can be ligated to primary amines via reductive amination. This reactivity was demonstrated with three reagents: ethylenediamine and mono-FMOC ethylenediamine, to give amino-functionalized products that can be ligated to carboxylic acid terminated surfaces using standard peptide synthesis conditions, and propargylamine, which installs an alkyne group for “click” chemistry applications. Gold nanoparticles were created using a modified Frens procedure to give citrate capped nanoparticles, which were passivated for 24 hours with excess alkyl thiol. The amino-functionalised conjugates were directly ligated to these carboxy-terminated gold nanoparticles. Continuous wave (CW) EPR spectra were obtained for the spin labelled bioligands free in solution, and a dissociation constant obtained for the mannoside spin label with Con A. EPR spectra were also obtained for the for the spin labelled gold nanoparticles. Addition of Con A to the mannose functionalized nanoparticles led to a colour change and observable agglomeration of the nanoparticles over time. In contrast, on addition of avidin-peroxidase to the biotin functionalised nanoparticles, there was no observable agglomeration, and a dissociation constant was obtained for the biotinylated gold nanoparticles.

Details

Original languageEnglish
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Award date31 Dec 2017