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H2020

MetalCell Report Summary

Project ID: 659489
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - MetalCell (Transition-Metal Mediated Chemistry in Cells)

Reporting period: 2015-12-01 to 2017-11-30

Summary of the context and overall objectives of the project

The problem being addressed - This covers three main avenues of exploration: (i). The fabrication of active palladium catalysts conjugated to antibodies and small targeting peptides; (ii) Development of assays to allow the activity of the active catalysts to be determined both in vitro and inside cells and (iii). The application of these active palladium catalysts enable the conjugation of two drug “halves” to allow the in situ synthesis (inside cells) of active anticancer drugs.

Important for society - Bioorthogonal reactions are selective chemical processes which take place in biological systems and are mediated by “chemical reagents” without interfering with the biotic components of the system. These precise reactions, exemplified by Bertozzi and other groups using so-called click chemistries e.g. copper-free Huisgen 1,3-dipolar cycloaddition or the Staudinger ligation, etc. were developed because of the need to label, study and/or modulate biomolecules and their function within their biological environment. The development of this elite class of chemical reactions has provided the basis for a wide range of groundbreaking investigations. Bioorthogonal reactions occur among an exclusive collection of chemical entities with very specific reactivities, namely the ability to remain unaltered in the presence of endogenous biological functionalities while showing reactivity towards specific chemical “mates” in aqueous media at biocompatible pH’s and temperatures. For applications involving live cells or organisms, these chemicals materials have to be non-toxic and biochemically benign. This has largely precluded researchers the use of non-biological metals as a catalytic devices to mediate bioorthogonal reactions in living systems, mainly limiting the available “bioorthogonal chemical toolbox” to highly energetic “spring loaded” reactants (e.g. azides and ring-strain promoted alkynes for copper-free 1,3-dipolar cycloaddition; azides and triarylphosphines modified with methyl ester groups for Staudinger ligation, etc.). To move beyond these limits, the Bradley group recently developed a truly heterogeneous Pd(0)-catalysts with the ability to enter cells in culture, stay harmlessly within the cytosol and mediate efficient bioorthogonal chemistries.

The overall objectives of the project - MetalCell was to develop transition metal based bioorthogonal chemistry in cells. The rational of the project was the exploration of the low toxic transition metals, such as palladium, as a biocompatible catalysts to activate fluorophores and anticancer prodrugs in cancer cells lines. The previous studies from the Bradley research groups had established the foundation for the project and had shown that palladium nanoparticles supported within polymeric resins could act as biocompatible catalysts for the cellular applications.

The current project begun with the aim of synthesis of an active palladium catalyst based on N-heterocyclic carbenes (NHC) and screening of the catalysts at the in vitro level with plans for cellular applications. We developed a new synthetic methodology based on solid phase peptide synthesis (SPPS) and a systematic approach was followed for the optimization for the synthesis of Pd catalysts. This approach was further applied to the generation of a library of catalysts. We selected a few highly active and biocompatible Pd catalysts after performing various catalyst screening procedures.

The active catalysts were then applied in living cells to activate a protected fluorophore (caged fluorophore) and also enabled the activation of a caged anticancer drug (prodrug) in cancer cell lines. For future cell specific targeting, the synthesis method of the active N-hydroxisuccinimide ester of the catalysts was optimised. The catalysts were synthesized and characterized by various methods, stability studies were performed and in vitro level studies in cancer cells were reported.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The goal of the project MetalCell was to develop transition metal based bioorthogonal chemistry in cells. The rational of the project was the exploration of the low toxic transition metals, such as palladium, as a biocompatible catalysts to activate fluorophores and anticancer prodrugs in cancer cells lines. The previous studies from the Bradley research groups had established the foundation for the project and had shown that palladium nanoparticles supported within polymeric resins could act as biocompatible catalysts for the cellular applications.

The current project begun with the aim of synthesis of an active palladium catalyst based on N-heterocyclic carbenes (NHC) and screening of the catalysts at the in vitro level with plans for cellular applications. We developed a new synthetic methodology based on solid phase peptide synthesis (SPPS) and a systematic approach was followed for the optimization for the synthesis of Pd catalysts. This approach was further applied to the generation of a library of catalysts. We selected a few highly active and biocompatible Pd catalysts after performing various catalyst screening procedures.

The active catalysts were then applied in living cells to activate a protected fluorophore (caged fluorophore) and also enabled the activation of a caged anticancer drug (prodrug) in cancer cell lines. For future cell specific targeting, the synthesis method of the active N-hydroxisuccinimide ester of the catalysts was optimised. The catalysts were synthesized and characterized by various methods, stability studies were performed and in vitro level studies in cancer cells were reported.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

"Palladium catalysts were prepared using a N-heterocyclic carbene structure. These were generated via an original solid-phase method. This allowed their attachment to a variety of peptides and fabrication by solid-phase methods - and resulted in a streamed-lined method of generating ""active esters"" of peptide-N-heterocyclic carbenes-Pd. These could be prepared en masse and were stable to storage - thus allow conjugation to a variety of ligands.

Analysis of the library of ligands allowed an optimal peptide sequence to be identified (in terms of optimal catalytic activity) - both in vitro and also in vivo (cells based assays).

No IP has been filed to date. Two papers are in various states of preparation - one is almost ready for submission - the second is in draft form."

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