CHEMICAL BIOLOGY STRATEGIES FOR THE FORMATION AND CLEAVAGE OF CHEMICAL BONDS

Chemistry plays an essential role in biological processes within all living organisms and better understanding of how specific chemical bonds can be either formed or broken gives chemical biologists opportunities to control and regulate these processes with high precision. Therefore, with growing importance of the field of chemical biology, expansion of the toolbox of available strategies for the formation or cleavage of specific chemical bonds under biocompatible conditions is needed for future advances in diverse topics in life sciences. The presented work aims to contribute to this growing field with a versatile method for the preparation of site-specific and plasma stable DNA-protein conjugates which are suitable for cellular applications. Using the method developed, various types of ONs can be easily conjugated to a range of proteins via solvent accessible cysteine residues, including therapeutic full-length IgG antibodies. Moreover, in order to optimise and simplify the characterisation of prepared DNA-protein conjugates, an improved and straightforward LC-MS method for the analysis of bioconjugation reactions between proteins and ONs was developed. Prepared and characterised DNA-antibody conjugates were then employed as probes in cellular imaging using a super- resolution DNA-PAINT method or fluorescence microscopy. Furthermore, this thesis reports new types of metal-mediated bond-cleavage reactions triggered by an active catalytic palladium species which can be either prepared as a complex or directly generated in living cells under biocompatible conditions. The cleavage reactions described are then used for on-demand extra- or intracellular release of highly cytotoxic small molecule drugs bearing phenolic hydroxyl or amine functional groups. Essentially, the strategy presented not only allows the control of the biological activity of small molecules, but also the reactivity of palladium species involved in palladium-assisted bioorthogonal reactions. In summary, the methods and findings presented in this thesis contribute to the portfolio of chemical transformation strategies available in the interdisciplinary field of chemical biology and provide new opportunities for future therapeutic applications.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Konc, Juraj

Advisor dc:contributor.advisor

• Bernardes, Gonçalo

• bioconjugation
• bond cleavage
• bioorthogonal decaging
• chemical biology
• conjugation
• DNA modification
• LC-MS analysis
• palladium
• prodrug activation
• protein modification
• transition metal

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/846f2458-5c10-4df6-86a3-6f5d87861013/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.65546

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/318434