Simplest Monodentate Imidazole Stabilization of the oxy‐Tyrosinase Cu₂O₂ Core: Phenolate Hydroxylation through a Cu^III Intermediate

Abstract

Tyrosinases (Ty) are ubiquitous binuclear copper enzymes that oxygenate to Cu(II) 2 O 2 ( S P) cores bonded by three histidine Nτ-imidazoles per Cu center. Synthetic monodentate imidazole bonded S P species self-assemble in a near quantitative manner at −125°C, but Nπ-ligation has been required. Herein, we disclose the syntheses and reactivity of three Nτ-imidazole bonded S P species at solution temperatures of −145°C, which was achieved using a eutectic mixture of THF and 2-MeTHF. The addition of anionic phenolates affords a Cu(III) 2 O 2 (O) species, where the bonded phenolates hydroxylate to catecholates in high yields. Similar O intermediates are not observed using Nπ-bonded S P species, hinting that Nτ-imidazole ligation, conserved in all characterized Ty, has functional advantage beyond active-site flexibility. Substrate accessibility to the oxygenated Cu 2 O 2 core and stabilization of a high oxidation state of the copper centers are suggested from these minimalistic models. Graphical AbstractCorrespondence to: T. Daniel P. Stack. Tyrosinases (Ty) are ubiquitous binuclear Cu enzymes responsible for melanin biosynthesis via regioselective tyrosine aromatic hydroxylation to form catechols, and its subsequent oxidation to quinones. Spectroscopic and X-ray crystallographic analysis of its oxygenated form (oxyTy) reveals a side-on μ-η 2 :η 2 peroxide dicopper (II) ( S P) species ligated exclusively by six histidine residues through the τ-nitrogen (Nτ) of each imidazole ( Figure 1). [1] This results in a highly accessible S P Cu 2 O 2 core where the 4-substituent of the imidazoles are positioned remotely. This Nτ-ligated S P core is conserved in oxygenated hemocyanin (oxyHc) proteins [2] , an evolutionary descendant of Ty [3] responsible for reversible dioxygen (O 2 ) uptake and storage. [1b, 4] The reactivity difference between oxyTy and oxyHc is attributed to substrate access to the oxygenated Cu 2 O 2 core, [5] as partially denatured hemocyanin proteins can exhibit mono-oxygenase reactivity. [6] Alternatively, more sterically-demanding and less-flexible π-nitrogen (Nπ) imidazole ligation is observed exclusively in electron-transfer blue-copper sites, suggesting Nτ-ligation may serve a functional purpose such as providing flexibility for the O 2 capture, allowing substrate access to the oxygenated S P core, and potentially stabilizing higher oxidation states of copper in the mono-oxygenase reaction. [1c, 7] Synthetic S P analogues of oxyTy have received extensive attention over the last few decades. [4a, 8] Stoichiometric phenolate oxidation to catecholates is possible at the low reaction temperatures required to stabilize most synthetic S P species, although several catalytic systems operating at elevated temperatures are now known. [9] These complexes are generally supported by multi-nucleating alkyl amines or heterocycles such as pyridines, pyrazoles or benzimidazoles. However, synthetic S P species exclusively ligated by imidazoles, especially by imidazoles bearing an N-H group [10] , are...

Synthesis of Exclusively 4-Substituted β-Lactams through the Kinugasa Reaction Utilizing Calcium Carbide

“…Similar to the results obtained in pyridine (Table , entry 5), the yield of 2a drastically dropped in the absence of TBAF, emphasizing that TBAF is crucial in this reaction (Table , entry 8). It has been reported that copper–imidazole complexes are highly prone to produce peroxo copper complexes upon exposure to air . To probe the effect of air in our reaction, the preparation of 2a was carried out under argon-degassed conditions.…”

Copper–Oxygen Complexes Revisited: Structures, Spectroscopy, and Reactivity

“…Addition of excess L42a to the ( μ - η 2 : η 2 -peroxo)dicopper complex supported by 1,2-dimethylimidazole at −125 °C yields the ( trans -1,2-peroxo)dicopper complex 50 bound by L42a , a ligand-exchange process (“core capture”) 259 with precedent in copper–oxygen chemistry 261 that here also involves isomerization of the Cu 2 O 2 unit. In more recent work, 258 the previous failure to observe a ( μ - η 2 : η 2 -peroxo)dicopper complex using 1- or 4-methylimidazole or unsubstituted imidazole was obviated by an alternate synthesis involving reaction of the known 262 bis( μ -oxo)dicopper complex 51 of tetramethylpropylenediamine ( L10a ) with an excess of the imidazole at −145 °C, a temperature attained by using a 4:1 2-MeTHF:THF eutectic mixture as solvent. Addition of 4 equiv of sodium 15-crown-5,2- tert -butyl-4-cyano phenolate to the resulting ( μ - η 2 : η 2 -peroxo)dicopper complex 49 at −145 °C yielded a phenolate-bound bis( μ -oxo)dicopper species akin to 45 (Figure 36), but here in a more biomimetic and sterically unencumbered ligand environment comprising imidazoles coordinating via their N τ positions.…”

Fluorescent Bis(guanidine) Copper Complexes as Precursors for Hydroxylation Catalysis

“…Stack et al used simple imidazole ligands and observed a peroxide complex formed by self-assembly at −125 • C [17]. They later found a bis(µ-oxido) dicopper(III) intermediate prior to a hydroxylation reaction, which was supported by DFT (density functional theory) calculations [20].…”

“…Over recent years, many groups have developed model systems to mimic the efficient tyrosinase enzyme [11][12][13][14][15][16]. Instead of histidines, other N-donor groups are utilized, such as imidazoles [17][18][19][20], imines [4,[21][22][23][24], and pyrazoles [4,19,25,26]. Even though in protein systems only the peroxido species Inorganics 2018, 6, 114 2 of 22 have been observed by X-ray crystallography and Raman spectroscopy [5][6][7], Tolman and coworkers discovered the bis(µ-oxido) species of a dicopper complex as another stable intermediate [27].…”