Catalytic Phenol Hydroxylation with Dioxygen: Extension of the Tyrosinase Mechanism Beyond the Protein Matrix

A pinnacle of bio-inorganic chemistry is the ability to leverage insights gleaned from metalloenzymes toward the design of small analogs capable of effecting catalytic reactivity outside the context of the natural system.^[1,2] Structural mimicry of active sites is an attempt to insert a synthetic catalyst into an enzymatic mechanism. Such a mechanism evolves by selection pressures for efficiency and traverses an energetic path with barriers and wells neither too high nor too deep in energy – a critical factor of catalytic turnover.^[3] An advantage of metalloenzymes over small metal complexes is the site-isolation of the metal center in the protein matrix with its attendant ability to attenuate destructive decay processes – reaction sinks. This protection provides access to thermal regimes that allows barriers and wells to be traversed. Synthetic complexes too must avoid any deleterious reactions, often necessitating deliberate incorporation of protective superstructures.^[4,5] Such limitations make reproducing enzymatic catalytic reactivity in a synthetic complex with native substrates a significant challenge, as evidenced by the dearth of good examples, despite decades of effort.

The intellectual investment of translating a catalytic mechanism from an active site to a synthetic system is justified by a comparison of Nature’s dexterity with dioxygen to perform catalytic monooxygenase-type chemistry^[6–9] and the dependence of synthetic chemists on exotic reagents for oxygen-atom insertion reactions. Tyrosinase is a ubiquitous dinuclear copper enzyme that catalyzes the hydroxylation of phenols to catechols and the subsequent oxidation of catechols to quinones by activating dioxygen in the form of a side-on bonded peroxide dicopper(II) species, crystallographically^[10] and spectroscopically identified.^[11,12] The seemingly simple, regiospecific transformation mediated by tyrosinase is not reproduced easily by synthetic methods, though its importance has been acknowledged by recent efforts to move beyond stoichiometric oxidants,^[13] as these sometimes multistep syntheses are often low to moderate yielding and frequently unselective.^[14–17] Limited examples of stoichiometric phenolate hydroxylation exist with synthetic, tyrosinase-like side-on peroxide complexes^[18–22] formed by oxygenation of copper(I) complexes. Only two complexes achieve significant catalytic phenol hydroxylation using dioxygen: a dinucleating, polydentate imine complex reported by Réglier,^[23] and a mononucleating analogue reported by Tuczek^[24] both capable of ca. 16 turnovers in the presence of triethylamine. Neither study could identify the operative oxygenated copper species nor illuminated key mechanistic hallmarks that support a tyrosinase-like mechanism. The possibility of a synthetic side-on peroxide as a catalytic oxidant is suggested by the work of Casella with a dinucleating copper-benzimidazole complex, achieving 1.2 turnovers with readily oxidized phenols.^[21,25]

Here we report a synthetic catalyst, capable of hydroxylating a wide variety of phenols using dioxygen, that proceeds through a room temperature (RT) stable analog of oxygenated tyrosinase – a side-on peroxide complex, possessing ligation and spectroscopic attributes similar to those of the enzymatic active site. Efficient stoichiometric oxidation of phenolates to catecholates at −78°C is shown, as well as catalytic oxidation of phenols to quinones at RT with triethylamine, through a reaction pathway consistent with the generally accepted enzymatic mechanism.^[11] Conditions are shown in which catalytic turnover is halted and restored at catecholate-product adduct. This investigation demonstrates that simple structural mimicry suffices to not only move the inherent enzymatic catalytic reactivity into a synthetic complex, but that bioinspiration is a viable stategy of pursuing selective transformations of substrates beyond scope of the enzyme.^[26]

Oxygenation of [Cu(I)bis(3-tert-butyl-pyrazolyl)pyridylmethane]SbF₆ in dichloromethane (CH₂Cl₂) at −78°C results in near quantitative formation (vide infra) of [Cu₂O₂(HC(3-tBuPz)₂(Py))₂](SbF₆)₂ (1), a side-on peroxide dicopper(II) complex (Figure 1A), as evidenced by the characteristic O-O stretch at 750 cm⁻¹ in its resonance Raman spectrum, which shifts by 39 cm⁻¹ upon ¹⁸O₂ substitution (Figure 1C).^[27,28] The expected mass signature and isotope pattern observed by cryo-ESI-TOF also shifts appropriately upon oxygenation with ¹⁸O₂ (Figure S3). The ligand-to-metal-charge-transfer (LMCT) features at 350 nm (20 mM⁻¹ cm⁻¹) and 550 nm (1 mM⁻¹ cm⁻¹) have a 20:1 intensity ratio,^[22,27] similar to those of oxy-tyrosinase and oxy-hemocyanin,^[29] and the feature near 412 nm (0.9 mM⁻¹ cm⁻¹) is tentatively assigned to a pyrazole/pyridine π*→d_xy charge transfer transition, based on a natural transition orbital analysis of a TD-DFT calculated spectrum (Figure 1B).^[22,30] The DFT optimized structure of 1 predicts a planar Cu₂O₂ core with a Cu-Cu separation of 3.57 Å, in line with the 3.51 Å distance determined by Cu K-edge extend x-ray absorption fine structure models (Table S2 and Figure S5).^[27,28] Taken together, these data fully support the structural homology between tyrosinase and 1.

Figure 1.

A) Preparation of the side-on peroxide species 1. B) Absorption spectra of 1; (inset) TD-DFT predicted optical spectrum of 1. C) Resonance Raman spectra of 1 in acetone with 412 nm excitation (red: ¹⁶O₂, black: ¹⁸O₂, asterisks (*): solvent peaks); (inset) isotopic shift of 750 cm⁻¹ feature.

The formation of 1 is effectively quantitative (> 95%) in a variety of solvents at −78°C, as assessed by iodometric titrations of the released peroxide after treatment with trifluoroacetic acid.^[22,31] The complex is stable for weeks in CH₂Cl₂ at −78°C, yet decays within 1 day in tetrahydrofuran or acetone. Compound 1 reacts within 60 min with a wide variety of sodium phenolate salts (5 equiv), both electron-rich and deficient, efficiently consuming the side-on peroxide oxidant and forming catechol products after an acidic workup (Table 1A). Using a 1:1 oxidant:phenolate stoichiometry, impressive catecholate yields (> 90%) are possible at −78°C, albeit reaction times of nearly 1 week are required. Expectedly, the mass of the p-methoxy-1,2-catechol product is shifted by 2 a.u. if 1 is formed with ¹⁸O₂, indicating dioxygen as the oxygen atom source.^[32]

Table 1.

A) Reaction of Phenolic Substrates and 1 under a N₂ atmosphere at −78°C; B) Catalytic Oxidation of Phenols to Quinones.

A)	R	X+	Equiva	Time	% catecholb
	OMe	Na+	5	5 min	>95
	Me	Na+	5	5 min	>95
	Cl	Na+	5	5 min	>95
	H	Na+	5	5 min	>95
	F	Na+	5	5 min	>95
	COOMe	Na+	5	60 min	>95
	CN	Na+	5	60 min	>95
	NO2	Na+	5	60 min	>90
	estronea	Na+	5	5 min	>90
	8-hydroxyquinolinea	Na+	5	5 min	>90
	N-acetyl-tyrosine-ethylester	Na+	5	5 min	>90
	OMe	HNEt3+	5	10 min	>95
	Me	HNEt3+	5	10 min	>95
	OMe	Na+	1	7 days	>90
	OMe	HNEt3+	1	10 days	>90
	estrone	Na+	1	8 days	>90
	8-hydroxyquinoline	Na+	1	8 days	>90
	N-acetyl-tyrosine-ethylester	Na+	1	5 days	>90

B)	Substrates	Time	Turn-oversa
	p-methoxyphenol	1 h	10
	N-acetyl-tyrosine-ethylester	16 h	15
	estrone	6 h	4
	8-hydroxyquinoline	16 h	8

All reactions performed in CH₂Cl₂ at 1 mM [Cu] and quenched with acid at −78°C after the given reaction time.

^aEquiv relative to 1.

^bYields based on the concentrations of 1. No quinone or biphenol products were observed.

All reactions performed in CH₂Cl₂ at 25°C under 1 atm of O₂ with 25 equiv of substrate, 50 equiv NEt₃ and [Cu] = 1.0 mM.

^aTurnover numbers based on the concentration of 1.

From kinetic data, the hydroxylation of phenolate to catecholate by 1 is best understood as a second order process: first order in [1] and first-order in [phenolate], with a pre-equilibrium binding event and a rate limiting oxidation step, presumably C-O bond formation. An intramolecular competitive kinetic isotope effect of 1.2(2) measured at −78°C with 2-D-4-tert-butylphenolate excludes rate limiting C-H bond cleavage. The observed rate constants k_obs saturate with respect to added phenolate (Figure 2), consistent with an initial phenolate-binding equilibrium K_eq, followed by an intramolecular rate-determining oxidation step k_ox from a substrate-complex adduct (Table S1),^[18,32–33] as electron-deficient phenolates clearly react more slowly. A plot of ln(k_ox) versus σ_p⁺ for a variety of phenolates gives a Hammett ρ = −0.99 (Figure 2), consistent with the trend reported for tyrosinase (ρ = −1.8 to −2.2) ^[34–35] and in line with an electrophilic aromatic substitution mechanism.

Figure 2.

Reactivity at −78°C in CH₂Cl₂. Left: Substrate-binding kinetics of the stoichiometric hydroxylation reaction with 4-fluorophenolate. Right: Hammett plot for the stoichiometric hydroxylation reaction with 1 – 20 equiv of various p-substituted phenolates.

At RT in CH₂Cl_2, 1 is quantitatively formed but irreversibly decays with a half-life of 30 min. However, this rare stability allows for the catalytic hydroxylation of phenols to quinone products at RT. Only two other synthetic side-on peroxide species are more thermally stable, but no exogenous substrate reactivity has been reported.^[36,37] With 25 equiv of p-methoxyphenol and 50 equiv of triethylamine under 1 atm of O₂,^[23,24,38] 10 equiv of quinone are formed in 1 hr or 15 equiv in 24 hr (Table 1B), as assessed by the characteristic 400 nm quinone optical feature (Figure S1). Slower catalytic reaction rates are observed with more electron-deficient phenols. At these higher temperatures, the oxidation of the catechol to quinone and its subsequent release is proposed to drive the reduction of the Cu(II) centers back to a Cu(I) state, permitting re-oxygenation to 1 (Figure 3).

Figure 3.

Proposed catalytic mechanism of phenol oxidation by 1 in the presence of triethylamine.

Three conditions appear to be necessary for catalytic turnover and support operation through a tyrosinase mechanism: ambient reaction temperatures (25°C), proton management, and excess dioxygen. Under catalytic reaction conditions at −78°C, rapid formation of a green species is observed, tentatively assigned as a Cu(II)-catecholate complex (Figure 3), as one equiv of p-methoxy-1,2-catechol is formed as the sole product upon an acidic quench at this temperature. If warmed to 25°C, this green species yields quinone rather than a catecholate product, suggesting catecholate-binding inhibition at low temperatures (Figure 3). Phenol deprotonation and binding to a copper center is essential in the catalytic oxidation process, as exclusion of triethylamine returns unreacted phenol at −78°C over hours. After phenol deprotonation, the resultant triethylammonium cation presumably provides the necessary proton for hydroxide protonation to water as catecholate reduces Cu(II) to Cu(I).

Beyond the ability to catalytically oxidize a tyrosinase substrate, N-acetyl-tyrosine methyl ester,^[39] to its dopaquinone form, the oxidative reactivity of 1 can be extended to more complicated phenols, outside the typical substrate scope of the enzyme (Table 1B). Estrone, an estrogenic hormone, is regioselectively ortho-hydroxylated to 3,4-estrone-o-catechol in >90% yield at −78°C in less than 5 min with 5 equiv of its sodium salt relative to 1 (Table 1A). The 3,4-estrone-o-quinone is formed under the catalytic conditions at 25°C with 4 turnovers.^[32] While mushroom tyrosinase itself can oxidize estrone,^[40] 8-hydroxyquinoline is not a viable substrate,^[32] possibly due to the extreme steric demands of the fused ring structure positioned ortho to the phenol oxygen (Table 1B).^[26] Compound 1 oxidizes 8-hydroxyquinoline both stoichiometrically to the catechol (7,8-dihydroxyquinoline) at −78°C, and catalytically to its quinone at 25°C (quinoline-7,8-dione; 8 turnovers; Table 1B), extending the substrate scope beyond that possible in the enzymatic system.^[2] The substrate flexibility of 1 may thus make it a potentially useful reagent for efficient stoichiometric conversion of a wide variety of phenolates to catechol or a catalyst for the multi-turnover oxidation of phenols by dioxygen to quinones, which are readily reduced back to catechols.^[17]

Oxygen-insertion reactions that use dioxygen directly are extremely limited, despite the indisputable advantages to using Earth’s ready supply of dioxygen. The difficulty lies in directing the oxidative power of dioxygen and in assuring that reactions profiles do not have insurmountable barriers or thermodynamically over-stabilized intermediates. Here, the essence of the tyrosinase enzyme mechanism, founded on the oxidizing power of dioxygen, is translated from its protein environment into a small complex through only approximate structural mimicry of the oxygenated active site, yet selective and efficient catalytic ortho-hydroxylation reactivity results. This strategy of copying pre-existing reactivity from Nature opens the door not only to challenging organic transformations, but for developing useful synthetic tools with substrate scopes beyond those of biological systems.