Flavonol dioxygenase chemistry mediated by a synthetic nickel superoxide

Nature exploits transition metal centers to enhance and tune the oxidizing power of natural oxidants such as O₂ and H₂O₂. The design and interrogation of synthetic metallocomplexes with similar reactivity to metalloproteins provides one strategy for gaining insight into the mechanistic underpinnings of oxygen-activating enzymes such as oxidases, oxygenases, and dioxygenases like Ni-quercetinase (Ni-QueD). Ni-QueD catalyzes the oxidative ring opening of the polyphenol quercetin, a natural product with antioxidant properties. Herein, we report the synthesis and characterization of Ni(13-DOB), a Ni(II) species complexed by an N4-macrocycle that has been characterized by single crystal X-ray crystallography. Ni(13-DOB) forms a Ni-superoxide intermediate (Ni(13-DOB)O₂^•-) upon treatment with H₂O₂ and Et₃N, as verified by resonance Raman spectroscopy. We demonstrate through UV/vis and LCMS that Ni(13-DOB)O₂^•- is capable of the 1-electron oxidation of flavonols, including both 3-hydroxyflavone (3-HF, the simplest flavonol) and quercetin itself. Incorporation of two O-atoms into the flavonol radical via superoxide from Ni(13-DOB)O₂^•- precedes oxidative cleavage of the flavonol scaffold in each case, consistent with quercetinase ring cleavage by Ni-QueD in Streptomyces sp. FLA. Conversion of 3-HF into 2-hydroxybenzoylbenzoic acid was accomplished with catalytic turnover of Ni(13-DOB) at ambient temperature, as confirmed by HPLC timecourses and GCMS analysis of isotopic labeling studies. The Ni(13-DOB)-mediated oxidative cleavage of quercetin to the corresponding biomimetic phenolic ester was also verified through ¹⁸O-isotopic labeling studies. Through the HPLC characterization of both on- and off-pathway products of flavonol dioxygenation by Ni(13-DOB)O₂^•-, the stringent reaction pathway control provided by enzyme active sites is highlighted.