Salerno, Luigia;
(2024)
Active methylene units for homogeneous cysteine bioconjugation.
Doctoral thesis (Ph.D), UCL (University College London).
Preview |
Text
ThesiscorrectionsFINAL-LS.pdf - Accepted Version Download (37MB) | Preview |
Abstract
Chemical modification of proteins is an ever-growing field, which finds applications in many different fields, from the investigation of biological functions to therapeutics and diagnostics. In particular, antibody-drug conjugates (ADCs) generated through bioconjugation have emerged as promising therapeutics for the treatment of cancer: a cytotoxic drug is linked to an antibody through a chemical linker, resulting in highly selective and potent drugs with reduced side effects. Current limitations include premature release of drug due to instability of the linker, as well as variable sites of conjugation and Drug-Antibody Ratios (DARs) due to non-selective modification. This results in heterogeneous ADCs with sub-optimal pharmacokinetic properties, thus reducing the efficacy of the drug. Consequently, the development of site-selective reactions for the synthesis of stable, homogeneous bioconjugates is highly sought after. Next Generation Maleimides (NGMs) have proved to be excellent reagents for site-selective cysteine and disulfide modification, delivering homogeneous conjugates with precise DAR control. This thesis describes the development and study of novel NGMs, synthesised through Michael addition-elimination reaction with active methylene compounds, and their application towards protein modification. In particular, it was found that β-ketonitriles are an exciting class of reagents for the site-selective modification of NGM-functionalised cysteines in proteins. Their reaction with NGM-rebridged Trastuzumab Fab fragment leads to the release of a cysteine residue, which can be reacted with electrophiles to achieve dual modification of Fab fragments. Furthermore, the products present a bathochromic shift in UV-Vis absorption compared to the starting materials, offering the opportunity for real-time monitoring of the reaction. The conjugates are stable to hydrolysis and extracellular thiol conditions for > 24 h, whilst unstable under intracellular thiol conditions, providing early proof of a promising stability profile for in vivo studies.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Active methylene units for homogeneous cysteine bioconjugation |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2024. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Department of Neuromuscular Diseases UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10188555 |
Archive Staff Only
 |
View Item |
