In this research project, we develop synthetic inorganic copper complexes to understand the fundamental aspects of structure and function in Cu-dependent monooxygenase enzymes. These metalloenzymes contain 1 or 2 Cu ions in their active center and they couple the reduction of O2 with the oxidation of substrates via formation of transient Cun/O2 species. We are particularly interested in studying the reactivity of mononuclear Cu/O2 intermediates since they have been proposed as active oxidants in the hydroxylation of strong C-H bonds in enzymes such as particulate methane monooxygenases (pMMOs) and lytic polysaccharide monooxygenases (LPMOs). Many questions concerning the identity of the active Cu/O2 species remain unanswered, including: i) oxidation state of Cu (CuI vs. CuII vs. CuIII); ii) reduction/protonation state of O2 (O2 ?,(H)O22?, (H)O2?) and the pKa and redox potentials associated with these Cu/O2 species; iii) mechanism by which the Cu/O2 intermediates carry out C-H hydroxylations (e.g. O-O cleavage mechanism before or after C-H oxidation?; generation of high-valent Cu-oxyl species before substrate hydroxylation?). In this research proposal, we tackle this problem using two different approaches: 1) We utilize ligand scaffolds (L) that contain C-H substrates covalently attached to their structure (substrate- ligands) that permit us to generate and characterize LCu/O2 species and evaluate their reactivity towards intramolecular C-H hydroxylation. Substrate-ligand modifications will permit us to: i) evaluate the ability of the Cu/O2 species to oxidize sp3 C-H bonds and sp2 C-H bonds; ii) control the stereo-electronic properties of the Cu complexes by the use of different ligand donors (i.e. N2, N3, N4) that will lead to the generation of mononuclear and dinuclear LCu/O2 species, and analyze their reactivity towards intramolecular C-H hydroxylation including characterization of reaction intermediates, kinetics and computations; iii) utilize this approach (Cu-directed hydroxylations) to develop synthetic protocols to promote challenging organic transformations such as enantioselective C-H hydroxylations and one-pot synthesis of 1,3-oxazines. 2) We synthesize mononuclear Cu complexes bearing redox-active ligands with tunable H-bonds that stabilize Cu-hydroxo and Cu-oxyl cores. These unusual Cu complexes are able to reach multiple oxidation states via oxidation of the metal and/or ligand scaffold. These high-valent CuO(H) cores will be characterized by various spectroscopic methods and their ability to perform biorelevant intermolecular 2e? C-H hydroxylations will be examined systematically using the Bordwell equation (i.e. species with higher redox potential and higher pKa should be capable of oxidizing stronger C-H bonds), kinetic experiments and analysis of the reactions products derived from hydroxylation (e.g. organic product(s) and oxidation/protonation state of the final Cu complexes). Overall, these studies will contribute to a broader understanding of the biochemical role of Cu ions involved in O2 reduction and biologically relevant oxidations.
The studies described in this research proposal will lead to a broader understanding of the copper biochemistry associated with the oxidation of organic molecules (e.g. hydroxylation of methane). We are particularly interested in understanding the structure, spectroscopy and reactivity of the Cu/O2 species formed in Cu- dependent monooxygenase enzymes. The use of synthetic inorganic complexes that mimic some features of the active centers of these enzymes will allow us to generate metastable LCu/O2 species and evaluate their reactivity towards biologically relevant C-H hydroxylation reactions.
