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Extending artificial metalloenzymes for the uncaging of drugs on cells

Boris, Lozhkin. Extending artificial metalloenzymes for the uncaging of drugs on cells. 2022, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: https://edoc.unibas.ch/90879/

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Abstract

Bioorthogonal transformations are of particular interest in the context of cancer therapy. Acting like conventional batch catalysts, metal cofactors allow new-to-nature reactions inside living cells or in tumor tissue.
In this work, we present a Ru-deallylation cofactor associated with a low-molecular, highly specific binder of hCAIX (a known marker of various cancer cell lines). The ability of HeLa cells to express hCAIX exclusively under hypoxic conditions made it possible to use this cell line as a single model for healthy and diseased cells. The specificity of binding of the anchor to the tumor marker was demonstrated by FACS and super-resolution microscopy. A library of new prodrugs capable of Ru-catalyzed uncaging was also synthesized. Due to the limited applicability of conventional alloc-protection for highly potent cytotoxins, a new protecting group that allows caging of two drug molecules has been developed. The obtained results open-up prospects for further study of this catalytic system for targeted delivery of the Ru-cofactor to the cell surface, discriminating diseased cells against healthy ones.
In addition, as part of the work on designing the substrate for in vivo synthesis of bioactive molecules, we developed an approach to monosubstituted benzenes under RCM conditions. Unfortunately, this method was inapplicable for the synthesis of the tamoxifen precursor. However, this approach complements and completes the arsenal of methods for the synthesis of aromatic compounds under Ru-catalysis. A precursor of pacritinib has also been synthesized. It has been shown that this bioactive molecule can be obtained by RCM in various solvents, including water. Due to the controversial clinical status of the drug in 2019, it was not then tested on cancer cells. Even so, in early 2022 it was approved by the FDA, and therefore is a potential subject for further in vivo studies.
New approaches to caging phenols and amines relying on a Ru-cleavable protecting group have also been developed. In particular, SN-38 caged by naphthalene diolefin precursor was synthesized. A dendritic approach to caging of multitude of molecules was also demonstrated for the first time in a bioorthogonal manner. Finally, we developed a substrate for live-cell imaging of RCM using MINFLUX technology. The synthesis of the target substrate has been brought to the final stage. In the near future, all synthesized building blocks will be combined, and the resulting substrate will be tested in living cells in the presence of water-soluble ruthenium complexes.
With the aim of developping monomeric ArMsAs that can readily be evolved on a cell surface, we are collaborating with Prof. David Baker. Having designed a suitable cofactor bearing a sulfamide moiety, we have synthesized an optically active NHC as well as ruthenium and gold complexes for bioorthogonal reactions. These cofactors display a micromolar affinity to engineered de novo scaffolds. The next stage of this study includes optimization of the structure of binding sites, after which all cofactors will be tested on a wide range of substrates to evaluate regio-, atropo-, enantio- and other types of selectivity. Thereafter, each of these reactions will be individually optimized by directed evolution to achieve the best-in-class activities and selectivities.
Advisors:Ward, Thomas R. R. and Sparr, Christof and Unciti Broceta, Asier
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Bioanorganische Chemie (Ward)
05 Faculty of Science > Departement Chemie > Chemie > Organische Chemie (Sparr)
UniBasel Contributors:Sparr, Christof
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14894
Thesis status:Complete
Number of Pages:VIII, 312
Language:English
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss148943
edoc DOI:
Last Modified:01 Jan 2024 02:30
Deposited On:15 Dec 2022 13:12

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