Modelling Lytic Polysaccharide Monooxygenases: From Small Molecules to Artificial Enzymes

Lytic polysaccharide monooxygenases (LPMOs) are Cu containing enzymes which break down lignocellulostic biomass via an oxidative mechanism. These enzymes are well studied, however the active species which performs this oxidation is unknown. The active site of LPMOs is known as the “histidine brace”, which coordinates a Cu ion via two histidine side chains and the primary amine of the N terminus of the protein. Small molecule models of enzyme active sites have been widely used within the literature to gain insight into enzymatic processes.
We have investigated a new ligand system which aims to faithfully model the histidine brace by the incorporation of a primary amine into the coordination sphere of a Cu ion, which has been unexplored in the literature so far. To this end, (2S)-1-(1H-imidazol-4-yl)-3-[(pyridin-2-yl)methoxy]propan-2-amine, 2.3, was synthesised and characterised along with its corresponding copper complexes. The electronic environment was probed using electron paramagnetic resonance spectroscopy which found that the electronics of this complex were similar to that of LPMOs (LsAA9) after substrate binding.
In order to evaluate the effect of hydrogen bonding on complex reactivity, an artificial enzyme methodology was used. A biotin-streptavidin system was devised in which 2.3 was modified via a Sonogashira cross coupling and subsequently biotinylated to form (1-H -imidazol-4-yl)propoxy]methyl)pyridin-4-yl)pro-2-yn-1-yl)-2-(2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl) pentamide , 3.12 and its corresponding Cu complex. Incorporation into streptavidin significantly changes the electronic environment as observed by EPR and UV/Visible spectroscopy. Furthermore, at high pH the system was able to stabilise a new species, similar to one which has been observed in LPMOs.
Incorporation of Cu(3.12) into streptavidin resulted in a decrease in H2O2 production compared to the free complex. However, point mutations within the streptavidin vestibule restored activity to the level of the free complex, indicating that the hydrogen bonding network provided by the protein affects the reactivity of the complexes.