Burley, Glenn

Feng, Yi

Vendrell Escobar, Marc

Alexander, Emma

2025-08-19T09:45:35Z

2025-08-19T09:45:35Z

2025-08-19

The copper-catalysed azide alkyne cycloaddition (CuAAC) is a widely used bio-orthogonal reaction. However, drawbacks include oxidative damage of biomolecules leading to cytotoxicity which limits the in vivo applications of the CuAAC reaction. Strategies to temper oxidative damage include the use of ligands and chelating azides, yet there has been little development on the alkyne design. Aromatic ynamines are alkyne analogues displaying an enhanced reactivity relative to conventional terminal alkynes. This enhanced reactivity provides rapid kinetics for the CuAAC reaction, enabling low copper loadings without using ligand or additives. However, the aromatic ynamine core – the benzimidazole heterocycle – has yet to be systematically investigated. This work aims to investigate how the structure of the ynamine influences the CuAAC reaction and solvent on reactivity. These findings will then be applied to construct a probe for calcium imaging, in an attempt to utilise the unique reactivity of the ynamine for addition of a fluorophore or an organelle targeting moiety. A palette of benzimidazole and imidazole ynamine substrates containing various electrondonating and electron-withdrawing groups were synthesised. First, hydrogen deuterium exchange (HDE) was utilised to probe how substituents affect the alkyne proton lability. It was found that the substituent changes on benzimidazole influenced the HDE, with EDGs increasing the rate of HDE and EWGs reducing the rate. Imidazole substituents were slower to exchange than the benzimidazole equivalents, and a larger difference was observed between substituents. Then HPLC analysis was used to investigate the influence of the modification on the CuAAC reaction. The experiments showed how reaction kinetics are strongly affected by varying the heterocycle and the substituents in the aromatic moiety, with a 5,6-dimethoxy benzimidazole displaying fast reaction kinetics in MeCN with 5,6-difluoro benzimidazole significantly slower. When these groups were substituted on imidazole ynamine scaffolds, the fluorine substituent gave faster reaction rates. Additionally, reaction rates and side product formation are highly dependent on solvents and copper-catalyst loading, with the catalyst percentage able to be lowered to 0.15 mol% for a benzimidazole ynamine containing two methoxy groups on the scaffold. The changes in reaction rates in solvents are substrate dependent, however, it was consistently observed that using HFIP/water resulted in no side product formation, but solubility issues were common. In addition, these results uncovered fundamental differences between imidazole and benzimidazole ynamines. This points to potential changes in rate determining step of the imidazole ynamine CuAAC. The use of the ynamine in bioconjugation applications was explored through the use of calcium probes. Synthesis of calcium probes containing varying functional groups was attempted, with the aim of conjugation an ynamine, to allow for modular click modification with fluorophores. Research initially began with the conjugation of fluorophore to ynamine to determine precedent, and research on the BAPTA focused on the synthesis of probes with a range of linkers. The findings in this thesis underpin the potential of aromatic ynamines for bioconjugation. Specifically, future ynamine probes should utilise benzimidazole substituents to avoid solubility issues and use a 5,6 methoxy group in MeCN to maximise reactivity, however, all factors need to be taken into consideration before deciding on a system. Additionally, the lack of dependence on the copper concentration of imidazoles highlights their potential for bioconjugation/imaging which has not been explored yet.

https://hdl.handle.net/1842/43828

https://strath-my.sharepoint.com/:f:/r/personal/emma_alexander_100_strath_ac_uk/Documents/Finalised %20Thesis/Appendix?csf=1&web=1&e=q2knFp

http://dx.doi.org/10.7488/era/6359

en

The University of Edinburgh

Bunschoten, R. P.; Peschke, F.; Taladriz-Sender, A.; Alexander, E.; Andrews, M. J.; Kennedy, A. R.; Fazakerley, N. J.; Jones, G. C. L.; Watson, A. J. B.; Burley, G. A. Mechanistic Basis of the Cu(OAc)2 Catalyzed Azide-Ynamine (3 + 2) Cycloaddition Reaction. J. Am. Chem. Soc. 2024, 146 (19), 13558–13570

copper-catalysed azide alkyne cycloaddition

CuAAC

aromatic ynamine

High-Performance Liquid-Chromatography

HPLC

reaction rates

calcium probes

Developing a click chemistry imaging platform using aromatic ynamines

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy