Design and engineering of myoglobin-based biocatalysts for controlled radical polymerisations

This thesis was previously held under moratorium from 1st February 2022 until 1st February 2023.Atom transfer radical polymerisation (ATRP) has attracted a lot of attentions since it was invented decades ago. Nowadays, it has been developed into the most efficient approach to synthesise the well-defined polymers. However, like other metal catalysis reactions, the metal contamination in polymer products and environment should be addressed. Our group has successfully applied several metalloenzymes to catalyse ATRP polymerisation. As an alternative solution, it not only greatly reduced the relative contamination, but also offered other potential benefits, such as selectivity. In this presented research project, myoglobin (Mb) has been discovered to catalyse ATRP polymerisations. To continue to liberate the enzyme’s power and increase the degree of control during the polymerisation, an engineering strategy has been designed based on the specific features of Mb. In conventional ATRP, the metal-ligand complex is the catalyst. Different species of metal and their ligands have a significant impact on polymerisation. Inspired by this, the cofactor of Mb could be replaced with a panel of artificial cofactors. By screening these Mb variants on bioATRP, the relationship between metal ions, cofactor base and their performance in polymerisation might be found. This part of fundamental work would be useful to upgrade the current bioATRP system. As an ATRPase, the protein scaffold of Mb is not only a ‘cage’ to hold its cofactor, but also an extra element to introduce regulation and selectivity in polymerisations. During the reaction, the initiation and propagation happen in the active centre wrapped by protein scaffold. Thus, additional modifications on the key residues of the protein scaffold would be possible to create a suitable microenvironment for the initiator to attach and react with the metal centre. Ideally, this approach could improve the efficiency of initiation and propagation, leading to an enhanced degree of control of polymerisation Five Mb variants were identified with ATRPase functionality. Chlorin e6 (Ce6) was more supportive compared to native protoporphyrin as four variants were based on Ce6 cofactor, including CuCe6-Mb. As a side project, the peroxidase activity of Mb variants was also investigated, FeCe6-Mb and MnCe6-Mb were characterised to have higher catalytic efficiency on guaiacol and TCP oxidising reactions in comparison with wild-type Mb. A scoring system was developed for the screening of Mb mutants in bioATRP. As a result, four single mutants were characterised with first order reaction kinetics and improved degree of control of polymerisation. Double mutants and the final triple mutant were screened and some of the effects from single mutant were well maintained in them. As the first project exploring engineering of the biocatalyst in bioATRP, the outcomes essentially achieved the research aim. Modifications on metal centre and its surrounding environment have been shown to be effective in optimising controlled polymerisations. The lessons learned from the current study provided us inspiration to perfect the bioATRP system. It would also be beneficial for researchers working in other areas of chemical biology, such as metal-mediated catalysis and radical-mediated modifications on biomolecules.Atom transfer radical polymerisation (ATRP) has attracted a lot of attentions since it was invented decades ago. Nowadays, it has been developed into the most efficient approach to synthesise the well-defined polymers. However, like other metal catalysis reactions, the metal contamination in polymer products and environment should be addressed. Our group has successfully applied several metalloenzymes to catalyse ATRP polymerisation. As an alternative solution, it not only greatly reduced the relative contamination, but also offered other potential benefits, such as selectivity. In this presented research project, myoglobin (Mb) has been discovered to catalyse ATRP polymerisations. To continue to liberate the enzyme’s power and increase the degree of control during the polymerisation, an engineering strategy has been designed based on the specific features of Mb. In conventional ATRP, the metal-ligand complex is the catalyst. Different species of metal and their ligands have a significant impact on polymerisation. Inspired by this, the cofactor of Mb could be replaced with a panel of artificial cofactors. By screening these Mb variants on bioATRP, the relationship between metal ions, cofactor base and their performance in polymerisation might be found. This part of fundamental work would be useful to upgrade the current bioATRP system. As an ATRPase, the protein scaffold of Mb is not only a ‘cage’ to hold its cofactor, but also an extra element to introduce regulation and selectivity in polymerisations. During the reaction, the initiation and propagation happen in the active centre wrapped by protein scaffold. Thus, additional modifications on the key residues of the protein scaffold would be possible to create a suitable microenvironment for the initiator to attach and react with the metal centre. Ideally, this approach could improve the efficiency of initiation and propagation, leading to an enhanced degree of control of polymerisation Five Mb variants were identified with ATRPase functionality. Chlorin e6 (Ce6) was more supportive compared to native protoporphyrin as four variants were based on Ce6 cofactor, including CuCe6-Mb. As a side project, the peroxidase activity of Mb variants was also investigated, FeCe6-Mb and MnCe6-Mb were characterised to have higher catalytic efficiency on guaiacol and TCP oxidising reactions in comparison with wild-type Mb. A scoring system was developed for the screening of Mb mutants in bioATRP. As a result, four single mutants were characterised with first order reaction kinetics and improved degree of control of polymerisation. Double mutants and the final triple mutant were screened and some of the effects from single mutant were well maintained in them. As the first project exploring engineering of the biocatalyst in bioATRP, the outcomes essentially achieved the research aim. Modifications on metal centre and its surrounding environment have been shown to be effective in optimising controlled polymerisations. The lessons learned from the current study provided us inspiration to perfect the bioATRP system. It would also be beneficial for researchers working in other areas of chemical biology, such as metal-mediated catalysis and radical-mediated modifications on biomolecules