Tyrosinase model systems and biohybrid conjugates with copper bis(pyrazolyl)methane complexes

Liebhäuser, Patricia; Herres-Pawlis, Sonja (Thesis advisor); Okuda, Jun (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


In the last decades, small molecule transition metal complexes became useful models for analysing enzymatic mechanisms, substrate reactivities and active site structures. This doctoral thesis describes two applications of bis(pyrazolyl)methane ligands in the field of enzyme modelling. Copper complexes of these N-donor ligands are known to serve excellently as structural and functional models for the dinuclear copper enzyme tyrosinase. Furthermore, the use of bis(pyrazolyl)methane copper complexes in the field of biohybrid catalysis opens up the possibility of modelling mononuclear copper enzymes like particulate methane monooxygenase. The development of a bis(pyrazolyl)methane ligand with a substituted pyridinyl moiety (HC(3-tBuPz)2(4-CO2MePy)) established a new tyrosinase model system. The reaction with molecular oxygen displayed that the peroxide dicopper(II) complex with this substituted pyridinyl ligand exceeds the stability of the system with the former pyridinyl ligand. Simultaneously, conversion of the phenolic substrate 8-hydroxyquinoline showed an improved catalytic reactivity compared to the reported imidazolyl system with regard to yield and reaction time. The hydroxylation of sodium phenolates resulted in saturation kinetics. Moreover, an electrophilic aromatic substitution mechanism was found, which resembles that of the enzyme tyrosinase. The ligand HC(3-tBuPz)2(4-CO2MePy) was furthermore used for the synthesis and structural characterisation of copper(I) and copper(II) complexes. The stabilisation of a mononuclear superoxide copper(II) species with bis(pyrazolyl)methane ligands is rather difficult due to the mostly transient nature of superoxide complexes, which tend to form dinuclear species. This might be overcome with the help of a biohybrid conjugate. The design of maleimide-bearing ligands led to the establishment of a new application field for bis(pyrazolyl)methanes. The optimised synthesis of two maleimido-bis(pyrazolyl)methane ligands is described. Copper complexes of both ligands were analysed concerning oxygen activation and transfer reactions. This was performed with the phenolic substrates 8-hydroxyquinoline and 4-methoxyphenol. Furthermore, these maleimide ligands were conjugated into variants of the $\beta$-barrel protein nitrobindin. The formation of copper complexes inside of the protein as well as the incorporation of molecular oxygen were analysed spectroscopically. Reactivity was investigated with oxygenation, oxidation or H-atom transfer reaction substrates, but no activity could be detected yet.