Thesis

Development of ynamines as next-generation bio-orthogonal tagging reagents

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2022
Thesis identifier
  • T16280
Person Identifier (Local)
  • 201785044
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • The copper-catalysed alkyne azide cycloaddition (CuAAC) or “click” reaction is a powerful bio-orthogonal tool used throughout chemical biology. However, its use in vivo is hindered by an abundance of biological functionalities and small molecules such as glutathione (GSH) which bind copper. This results in the need for super-stoichiometric amounts of the copper catalyst, which in turn exacerbates copper toxicity. Several mitigation strategies such as chelating ligands and azides have been developed to reduce copper loading and increase reactivity. In contrast, little progress has been made to improve reaction efficiency and lower the copper loading by exploring novel alkyne reagents. Aromatic ynamines have previously outcompeted traditional alkynes under standard organic synthesis CuAAC reaction conditions. The enhanced chemical reactivity of ynamines promises superior reactivity in bioconjugation, making it a potential reagent for bio-orthogonal chemistry. Glutathione, the major antioxidant and nucleophile in cells, reduces the efficiency of the CuAAC reaction by sequestering copper and causing side-reactions with alkynes. The impact of GSH on the CuAAC reaction at physiologically-relevant concentrations (0.1 – 10 mM) has not been explored. This thesis aims to investigate the use of aromatic ynamines as bioorthogonal reagents, first by comparing the influence of GSH – both stability and reactivity – on CuAAC and bio-orthogonal reactions and then moving into investigating aromatic ynamine reactivity in cell lysate. Common bio-orthogonal reagents, alkynes and ynamines displayed good stability (> 80%) in the presence of GSH (10 mM) after 24 h and the reactivity of SPAAC and IEDDA reagents remained unaffected. In contrast, GSH dominated CuAAC reactivity and the Cu:GSH ratio could be used to tune the reaction from full conversion to complete inhibition over of 24 h. Uniquely, GSH could be used to accelerate the ynamine-CuAAC reaction and in combination with fluorinated solvents (HFIP or TFE) optimised to reach full conversion within 10 min. These conditions were successfully applied to the labelling of cell-penetrating peptides with an ynamine-desthiobiotin probe. The trend of superior ynamine reactivity continued using fluorescent “turn on” azide reagents in buffer and cell lysate, where 80% conversions were reached after 2 h with as little as 1 µM Cu(OAc)2 and over 80% conversion in <15 min with 30 µM copper in cell lysate. Conventional alkynes remained unreactive under these conditions. After these promising results the ynamine is poised for in vivo bioconjugation, which will be explored in the future. [See thesis for image: Scheme. Reaction of an aromatic ynamine with an azide to form a triazole]
Advisor / supervisor
  • Burley, Glenn
Resource Type
Note
  • This thesis was previously held under moratorium from 25/11/2021 until 25/11/2025.
DOI
Embargo Note
  • This thesis is restricted to Strathclyde users only until 25 November 2026.

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