Introduction

The type 3 copper protein tyrosinase (Ty) catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA and the subsequent two-electron oxidation to l-DOPAquinone (monophenolase activity, Fig. 1). The active site of these proteins contains two copper centers coordinated by six histidines and binds dioxygen in a side-on bridging µ-η2:η2 geometry [1–3]. The oxygenation of tyrosine to l-DOPAquinone is the first step of melanin synthesis [4]. As melanin is ubiquitous, animals, plants, fungi and bacteria make use of tyrosinase [5]. The pigmentation of skin, the browning of fruits and the sclerotization of insect cuticles are all based on melanogenesis [6, 7].

Fig. 1
figure 1

Catalytic activities of tyrosinase and catechol oxidase

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The related enzyme catechol oxidase (CO) catalyzes the two-electron oxidation of catechols to ortho-quinones (diphenolase activity). Whereas Ty also exhibits diphenolase activity CO cannot mediate the ortho-hydroxylation step involved in monophenolase activity (Fig. 1). Ty and CO are commonly referred to as phenoloxidases [8]. Recently, we discovered that the main structural difference between Ty and CO is the lack of an asparagine (Asn) in the latter enzyme. Along with a glutamate, the Asn residue activates conserved water towards deprotonation of monophenols. If Asn is missing, only diphenolase activity is observed [9].

The reproduction of tyrosinase activity by inorganic model systems catalytically oxidizing or oxygenating external phenolic substrates has a long history. In 1990, Réglier et al. presented a catalytic tyrosinase model system containing the ligand bis-2,2′-[2-(pyrid-2-yl)ethyl]-iminobiphenyl (BiPh-(impy)2) (Fig. 2) [10].

Fig. 2
figure 2

Ligand designs used for tyrosinase reaction

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In 1991, Casella and coworkers developed another model system based on the ligand α, α′-bis{bis[2-(1-methylbenzimidazol-2-yl)ethyl]-amino}-m-xylene (L66) [11]. The role of the Cu2O2 complex as the hydroxylating species in the monophenolase reaction was demonstrated in 2003 [12]. Monooxygenation of external substrates was also studied in many other systems [13–16]. Detailed mechanistic insight into the monooxygenation of external substrates was achieved by the Cu(I)DBED system (DBED = 1,2-bis(di-tert-butyl)ethylene-diamine) [17]. Upon exposure to dioxygen the formation of a (µ-η2:η2)-peroxo-dicopper(II) species was observed. Subsequent addition of a phenolate led to a bis-µ-oxo intermediate, providing the first evidence for a “ternary intermediate” (Cu + O2 + substrate) in the course of the tyrosinase-like hydroxylation reaction. Further reaction led to a catecholato intermediate and the final product, a semiquinone complex [17, 18].

The first catalytic model system of tyrosinase based on a mononucleating ligand, [2-(pyrid-2-yl)ethyl]imino-tert-butane (L py 1) was reported by our group [19]. In 2013, Herres-Pawlis et al. presented catalytic copper(I) model systems supported by bis(pyrazolyl)methane ligands which exhibit room temperature stable peroxo intermediates [20]. More recently, Lumb et al. demonstrated catalytic conversions of different monophenols to a variety of organic products [21–26].

During the last years our group published a number of new copper(I) complexes functioning as model systems for tyrosinase [2, 19, 27–34]. Starting from the parent mononuclear complex with the ligand L py 1 containing a pyridyl moiety and an imine unit, we first replaced the N-heterocycle by benzimidazole and pyrazole to investigate the influence of the electronic structure of the ligand on the turnover number (TON) of the corresponding copper catalyst. To expand the scope of the oxygenation reactions mediated by these systems, symmetric ligands such as di(pyrid-2-yl)methane and different bispyrazolylmethane ligands such as (1,1′-methylenebis-1H-pyrazole (BPM), 1,1′-methylenebis(3-methyl-1H-pyrazole) (mBPM), and 1,1′-methylenebis(3,5-dimethyl-1H-pyrazole) (dmBPM) were developed. The copper(I) complex of di(pyrid-2-yl)methane revealed no tyrosinase activity, in contrast to the copper(I) complexes of Cu(I)BPM, Cu(I)mBPM and Cu(I)dmBPM.

In this paper, we present new ligands containing imidazole moieties to model the histidine residues in the native protein (Fig. 3).

Fig. 3
figure 3

Structure of bidentate imidazole-based ligands for catalytic reactions: L OL 1-3, L imz 3 and BIMZ

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The ligand N-(2,2-dimethylpropylidene)-2-(1-trityl-1H-imidazol-4-yl-)ethyl amine (L imz 1) contains an imidazole unit instead of the pyridine moiety of the parent L py 1 system. The incorporated trityl residue increases the solubility in dichloromethane. Reaction with tetrakis(acetonitrile)copper(I) hexafluorophosphate leads to the copper(I) complex [Cu(I)(L imz 1)(CH3CN)2]PF6 (CuL imz 1). Additionally, we prepared the complex [Cu(I)(BIMZ)(CH3CN)PF6 (CuBIMZ) which is supported by the ligand bis(1-methylimidazol-2-yl)methane (BIMZ) containing two imidazole groups [35].

Importantly, the imine function being present in the parent ligand L py 1 as well as its analogs, including L imz 1, is very unstable against hydrolysis. This could be one reason for the limited TON of the resulting copper catalysts. To prevent the hydrolysis of the imine group, we replace the labile imine function of the L imz 1 ligand by different oxazoline units. To gain insight into steric effects, we investigate three ligands containing different substituents adjacent to the oxazoline moiety; i.e., a phenyl group (4-((1H-imidazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole, L OL 1), a tert-butyl group (4-((1H-imidazol-4-yl)methyl)-2-(tert-butyl)-4,5-dihydrooxyzole, L OL 2), and a methyl group (4-((1H-imidazol-4-yl)methyl)-2-methyl-4,5-dihydrooxyzole, L OL 3).

First, the catechol oxidase activity with respect to the substrate to 3,5-di-tert-butylcatechol (3,5-DTBC-H2) is studied for all five copper(I) complexes (CuL OL 1-3, CuL imz 1 and CuBIMZ), and the corresponding kinetics is determined. Based on the experimental results, a mechanistic cycle is derived. Then, comprehensive measurements with different monophenols are performed to investigate the monophenolase activity of the new copper complexes. The oxygenation products are identified and characterized with the help of UV/Vis and NMR spectroscopy, mass spectrometry, and fluorescence measurements. Cryogenic oxygenations of all presented model systems are performed to identify the dioxygen species involved in the catalytic cycle. Correlations between the structures of the complexes and their reactivities are discussed.

Materials and methods

Materials

All solvents were of commercially available reagent grade. Acetonitrile, dichloromethane and toluene were dried by heating to reflux under nitrogen atmosphere with calcium hydride, acetone with calcium sulfate, methanol with magnesium and tetrahydrofuran with lithium aluminium hydride. All commercial available starting materials were ordered by Sigma Aldrich Co. and ABCR. Air-sensitive compounds were prepared using standard Schlenk techniques or a MBraun LABmaster glovebox filled with nitrogen. 2-(R)-amino-3-(1H-imidazol-4(5)-yl)propanol dihydrochloride (histidinol dihydrochloride) was prepared according to Gynther et al. [36], 2-(1-trityl-1H-imidazol-4-yl)-ethyl amine was prepared according to Garibay et al. [37] and ethyl pivalimidate hydrochloride was prepared according to Allmann et al. [38]. Chromatographic purifications were performed with silica gel 60 (0.04–0.063). Flash chromatography of 4-methoxy-5-(4-methoxyphenoxy)-cyclohexa-3,5-diene-1,2-dione and 4-(tert-butyl)-5-(3-(tert-butyl)phenoxy)-cyclohexa-3,5-diene-1,2-dione was performed using an Isolera One fabricated by Biotage.

Elemental analyses

Elemental analyses were performed using a EuroEA 3000 Elemantal Analyser, fabricated by Euro Vector Instruments and Software.

Spectroscopy

UV/Vis spectra were recorded at room temperature with an Agilent 8435 Technologies photodiode array spectrophotometer. Low temperature measurements were performed in solution with an Agilent Cary 5000 spectral photometer using a CryoVAC KONTI cryostat and a quartz cell with a path length of 1 cm. NMR spectra were recorded with a Bruker Avance 400 Pulse Fourier Transform spectrometer at 300 K with a 1H frequency of 400.13 MHz and a 13C frequency of 100.62 MHz. Fluorescence spectra were recorded in methanol with a Perkin Elmer LS55.

Mass spectrometry

Mass spectra (ESI/MS) were recorded using a Mariner ESI-TOF mass spectrometer fabricated by Applied Biosystems. MALDI-TOF mass spectra were recorded using a Bruker Biflex III spectrometer.

Single crystal and powder x-ray diffraction

Single crystal data were recorded using a STOE Imaging Plate Diffraction System (IPDS-2) with Mo-Kα radiation (λ = 0.71073 Å) and were corrected for absorption (T min/max: 0.8211/0.8927). The structure was solved with SHELXS-97 [39] and refined against F 2 using SHELXL-2014 [40]. All non-hydrogen atoms were refined anisotropic. The C–H and N–H H atoms were positioned with idealized geometry and refined isotropic with Uiso(H) = 1.2 Ueq(C,N) using a riding model. The absolute structure was determined and is in agreement with the selected setting [Flack x-parameter: −0.001 (18)]. One of the two crystallographically independent PF6 − anions is disordered and was refined using a split model and restraints.

CCDC-1454612 contains the supplementary crystallographic data for this paper. These data can be obtained free charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

X-ray powder pattern were measured using a Stoe Stadi-P powder diffractometer with Cu Kα-radiation (Ge(111) monochromator) what is equipped with a MYTHEN 1 K detector.

Detection of hydrogen peroxide in catalytic catechol oxidase reactions

Hydrogen peroxide formation during the catalytic oxidation was determined by the development of the characteristic I3 − band at 353 nm (ɛ = 26,000 L mol−1 cm−1) [41] which forms upon reaction of I− with H2O2. After 15 min, 1 h and 2 h, 5 mL of the reaction mixture were removed and an equal volume of water was added. The quinone was extracted three times with dichloromethane. The aqueous layer was set to pH 2 using H2SO4. Furthermore, 1 mL of an aqueous potassium iodide solution (10 %) and three drops of an ammonium heptamolybdate tetrahydrate solution (10 % in water) were added and UV/Vis measurements were performed directly afterwards and also every 10 min during the first 60 min of reaction time. The reaction proceeds according to the equation:

$${\text{H}}_{ 2} {\text{O}}_{ 2} + {\text{ 2 I}}^{ - } + {\text{ 2 H}}^{ + } \to 2 {\text{ H}}_{ 2} {\text{O }} + {\text{ I}}_{ 2}$$
$${\text{I}}_{{ 2({\text{aq}})}} + {\text{ I}}^{ - } \to {\text{I}}_{ 3}^{ - } .$$

A blank was used to account for I3 − which is formed upon the oxidation of I− by atmospheric oxygen.

Catalase activity measurements

Measurements of catalase activity were performed according to Monzani et al. [41]. A 0.1 M solution of hydrogen peroxide in methanol was divided in six parts. The copper(I) complexes of L OL 1-3, L imz 1 and BIMZ (500 µM) were added and stirred for 3 h. One part was used as blank and was also stirred for 3 h. Afterwards, 5 µL of each solutions were added to 2 mL of a solution containing water (acidified to pH 2 with H2SO4), potassium iodide (30 mM) and ammonium heptamolybdate tetrahydrate (100 nM). After 1 h the relative intensity of the I3 − absorption band at 353 nm as compared to the blank was measured. Catalase activity could be observed for all five complexes.

General procedure for the oxidation of 3,5-di-tert-butylcatechol to mimic catechol oxidase activity

Under inert atmosphere at ambient temperature, the respective copper(I) complexes were dissolved in 20 mL dichloromethane to afford a 500 µM solution. Then 10 eq. of 3,5-DTBC-H2 were added. Subsequent injection of dioxygen into the reaction mixture started the oxidation of the catechol to the corresponding quinone, which was observed via UV/Vis and NMR spectroscopy.

General procedure for the oxygenation of external substrates to mimic tyrosinase activity

Under an inert atmosphere at ambient temperature, the respective copper(I) complexes were dissolved in 20 mL dichloromethane to afford a 500 µM solution. Then 50 eq. of the external substrate (2,4-DTBP-H, 4-MeOP-H, 3-TBP-H, NATEE, 8-hydroxyquinoline) and 100 eq. triethylamine were added. Subsequent injection of dioxygen into the reaction mixture started the conversion of the various monophenols which were monitored via UV/Vis and NMR spectroscopy.

General procedure of quenching the oxidized and oxygenated solutions

An aliquot of the reaction mixture (2.5 mL) was diluted to a 25 µM solution with dichloromethane and quenched by addition of 6 M HCl (20 mL). The phases were separated, and the aqueous phase was extracted with dichloromethane (2 × 20 mL). The combined organic fractions were dried over MgSO4, filtered, and evaporated in vacuum. The residue was analyzed by NMR spectroscopy.

General procedure of kinetic measurements

Catechol oxidase activities were measured in dichloromethane saturated with dioxygen at ambient temperatures using time-dependent UV/Vis spectroscopy. In a first run solutions with concentrations of 3,5-DTBC-H2 in a range of 1.5 × 10−3–4 × 10−2 M were prepared. Afterwards a solution of each copper(I) complex in dichloromethane with a constant concentration of 1 × 10−3 M was added. The increasing double bands of 3,5-di-tert-butyl semiquinone (3,5-DTBSQ) at 377 nm and 394 nm were monitored during the first 2 min. In another series of experiments, the concentration of 3,5-DTBC-H2 was kept constant (2.5 × 10−2 M) and the concentration of copper(I) complexes was varied in the range of 7.6 × 10−5–1 × 10−3 M. Measurements were performed after 2 min.

Derivatization of N-acetyl-l-dopa ethyl ester with ortho-phenylenediamine

A 500 µM solution of CuL imz 1 in dichloromethane (20 mL) was treated with N-acetyl-l-tyrosine ethyl ester monohydrate (134.7 mg, 500.2 µmol) and triethylamine (0.14 mL 1.0 mmol). Dioxygen was bubbled through the solution continuously for 2 h. Then 10 mL of this mixture were removed and the solvent was evaporated in vacuum. Acetone (25 mL) and trifluoroacetic acid (20 mL; 1 % in water) were added and stirred for 15 min under reflux. Subsequent addition of ortho-phenylenediamine (13.50 mg, 123.8 µmol) afforded a yellow solution, which was stirred for 10 min under reflux. The solvent was evaporated and the residue was dissolved in methanol. Afterwards the prepared phenazine derivative was characterized by UV/Vis and fluorescence spectroscopy.

Synthesis of 4-((1H-imidazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole (LOL1)

To a solution of histidinol dihydrochloride (1.50 g, 7.00 mmol) in dry methanol (15 mL) ethyl benzimidate hydrochloride (1.13 g, 6.10 mmol, 1.3 eq.) and sodium methoxide (950 mg, 17.6 mmol, 2.5 eq.) were sequentially added and the mixture was heated at 78 °C for 2.5 h. The suspension was filtered and washed with methanol. Then, an aqueous Na2CO3 solution (20 mL, pH 10) was added and extracted three times with dichloromethane. The combined organic phases were dried over Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by flash chromatography with chloroform/methanol (9:1, R f  = 0.10–0.26) as the eluent to afford L OL 1 (929 mg, 4.09 mmol, 58 %) as a white powder. Anal. calcd. for C13H13N3O: C (68.70 %); H (5.77 %); N (18.49 %); found C (68.33 %); H (6.14 %); N (18.51 %). 1H-NMR (CD3OD, 400 MHz): δ = 7.90 (dd, J = 5.3, 3.2 Hz, 2H, Ar–H), 7.60 (d, J = 1.0 Hz, 1H, imidazole H-2), 7.56–7.49 (m, 1H, Ar–H), 7.47–7.41 (m, 2H, Ar–H), 6.88 (s, 1H, imidazole H-5), 4.60 (m, 1H, –O–CH2–CH–N–), 4.48 (dd, J = 9.4, 8.6 Hz, 1H, –O–CH 2 –CH–N–), 4.25 (dd, J = 8.5, 7.3 Hz, 1H, –O–CH 2 –CH–N–), 3.06 (dd, J = 14.6, 5.2 Hz, 1H, imidazole–CH 2 –CH–), 2.81 (dd, J = 14.7, 7.9 Hz, 1H, imidazole–CH 2 –CH–) ppm. 13C-NMR (CD3OD, 100 MHz): δ = 166.6 (C, 1C, –N=C–O–), 136.2 (CH, 1C, imidazole C-2), 134.8 (C, 1C, imidazole C-4), 133.0 (CH, 1C, phenyl C-4), 129.6 (CH, 2C, phenyl C-3, C-5), 129.3 (CH, 2C, phenyl C-2, C-6), 128.5 (C, 1C, phenyl C-1), 118.5 (CH, 1C, imidazole C-5), 73.3 (CH2, 1C, –O–CH2–CH–N–), 67.1 (CH, 1C, –O–CH2–CH–N–), 33.7 (CH2, 1C, imidazole–CH2–CH–) ppm.

Synthesis of 4-((1H-imidazol-4-yl)methyl)-2-(tert-butyl)-4,5-dihydrooxyzole (LOL2)

The synthesis was performed with ethyl pivalimidate hydrochloride (1.01 g, 6.10 mmol, 1.3 eq.) as previously described. The crude product was purified by flash chromatography with dichloromethane/methanol (9:1, R f  = 0.13–0.29) as the eluent to afford L OL 2 (667 mg, 3.22 mmol, 46 %) as a white powder. Anal. calcd. for C11H17N3O: C (63.43 %); H (8.71 %); N (20.17 %); found C (63.15 %); H (8.84 %); N (20.41 %). 1H-NMR (CD3OD, 400 MHz): δ = 7.59 (s, 1H, imidazole H-2), 6.85 (s, 1H, imidazole H-5), 4.40–4.31 (m, 1H, –O–CH2–CH–N–), 4.25 (dd, J = 9.4, 8.5 Hz, 1H, –O–CH 2 –CH–N–), 4.10 (dd, J = 8.5, 6.7 Hz, 1H, –O–CH 2 –CH–N–), 2.94 (ddd, J = 14.6, 4.5, 0.8 Hz, 1H, imidazole–CH 2 –CH–), 2.68 (dd, J = 14.7, 7.8 Hz, 1H, imidazole–CH 2 –CH–), 1.17 (s, 9H, –C(CH 3 )3) ppm. 13C-NMR (CD3OD, 100 MHz): δ = 177.2 (C, 1C, –N=C–O–), 136.1 (CH, 1C, imidazole C-2), 134.4 (C, 1C, imidazole C-4), 118.8 (CH, 1C, imidazole C-5), 73.0 (CH2, 1C, –O–CH2–CH–N–), 66.2 (CH, 1C, –O–CH2–CH–N–), 34.3 (CH2, 1C, imidazole–CH2–CH–), 33.4 (C, 1C, –C(CH3)3), 28.1 (CH3, 1C, –C(CH3)3) ppm.

Synthesis of 4-((1H-imidazol-4-yl)methyl)-2-methyl-4,5-dihydrooxyzole (LOL3)

The synthesis was prepared with ethyl acetimidate hydrochloride (754 mg, 6.10 mmol, 1.3 eq.) as previously described. The crude product was purified by flash chromatography with dichloromethane/methanol (9:1, R f  = 0.31) as the eluent to afford L OL 3 (393 mg, 2.38 mmol, 34 %) as a white powder. Anal. calcd. for C8H11N3: C (58.17 %); H (6.71 %); N (25.44 %); found C (57.85 %); H (6.92 %); N (25.65 %). 1H-NMR (CD3OD, 400 MHz): δ = 7.58 (s, 1H, imidazole H-2), 6.86 (s, 1H, imidazole H-5), 4.40–4.34 (m, 1H, –O–CH2–CH–N–), 4.30 (dd, J = 9.4, 8.5 Hz, 1H, –O–CH 2 –CH–N–), 4.05 (dd, J = 8.3, 7.0 Hz, 1H, –O–CH 2 –CH–N–), 2.94 (ddd, J = 14.7, 5.4, 0.7 Hz, 1H, imidazole–CH 2 –CH–), 2.69 (dd, J = 14.7, 7.7 Hz, 1H, imidazole–CH 2 –CH–), 2.69 (s, 3H, –CH 3 ) ppm. 13C-NMR (CD3OD, 100 MHz): δ = 168.4 (C, 1C, –N=C–O–), 136.1 (CH, 1C, imidazole C-2), 135.9 (C, 1C, imidazole C-4), 118.3 (CH, 1C, imidazole C-5), 73.4 (CH2, 1C, –O–CH2–CH–N–), 66.5 (CH, 1C, –O–CH2–CH–N–), 33.8 (CH2, 1C, imidazole–CH2–CH–), 13.6 (CH3, 1C, –CH3) ppm.

Synthesis of N-(2,2-dimethylpropylidene)-2-(1-trityl-1H-imidazol-4-yl-)ethyl amine (Limz1)

To synthesize L imz 1 2-(1-trityl-1H-imidazol-4-yl-)ethyl amine (1.00 g, 2.83 mmol), trimethylacetaldehyde (0.62 mL, 5.66 mmol, 2 eq.) and sodium sulfate (10.1 g, 70.7 mmol, 25 eq.) were dissolved in dry toluene (15 mL). The mixture was stirred at room temperature for at least 3 h. The residue was filtered off and washed twice with dry toluene (2 × 3 mL). The solvent was removed until a colorless solid precipitated. After 24 h the solid was filtered, washed with acetonitrile and dried in vacuum to afford L imz 1 (728 mg, 1.23 mmol, 61 %) as a white powder. Anal. calcd. for C29H31N3: C (82.62 %); H (7.41 %); N (9.97 %); found C (82.56 %); H (7.50 %); N (9.90 %). 1H-NMR (CDCl3, 400 MHz): δ = 7.46 (t, J = 1.2 Hz, 1H, imidazole H-2), 7.37–7.27 (m, 9H, Ar–H), 1.15–7.10 (m, 6H, Ar–H), 6.56 (s, 1H, imidazole H-5), 3.64 (t, J = 7.0 Hz, 2H, –CH2–CH 2 –N–), 2.82 (t, J = 7.0 Hz, 2H, –CH 2 –CH2–N–), 0.97 (s, 9H, –C(CH 3 )3) ppm. 13C-NMR (CDCl3, 100 MHz): δ = 172.5 (CH, 1C, –N=CH–C(CH3)3), 142.8 (C, 3C, phenyl C-1), 139.5 (C, 1C, imidazole C-4), 138.4 (CH, 1C, imidazole C-2), 129.9 (CH, 6C, phenyl C-3, C-5), 128.1 (CH, 6C, phenyl C-2, C-6), 128.0 (CH, 3C, phenyl C-4), 118.7 (CH, 1C, imidazole C-5), 75.2 (C, 1C, –N–C–(C6H6)3), 60.6 (CH2, 1C, –CH2–CH2–N=), 36.0 (C, 1C, =CH–C(CH3)3), 30.2 (CH2, 1C, –CH2–CH2–N=), 27.1 (C, 3C, =CH–C(CH3)3) ppm.

Synthesis of bis(1-methylimidazol-2-yl)methane (BIMZ)

Bis(1-methylimidazol-2-yl)methane was prepared according to White et al. [35]. Anal. calcd. for C9H12N4: C (61.34 %); H (6.86 %); N (31.79 %); found C (61.70 %); H (7.26 %); N (31.76 %). 1H-NMR (CDCl3, 400 MHz): δ = 6.89 (s, 2H, H-4), 6.76 (s, 2H, H-5), 4.21 (s, 2H, –CH 2 ), 3.64 (s, 6H, –CH 3 ) ppm. 13C-NMR (CDCl3, 100 MHz): δ = 143.6 (C, 2C, C-2), 127.3 (CH, 2C, C-4), 121.6 (CH, 2C, C-5), 33.2 (CH3, 2C, CH3), 27.0 (CH, 1C, CH2) ppm.

Synthesis of [Cu(LOL1)(CH3CN)]PF6

Under anaerobic conditions, L OL 1 (100 mg, 0.440 mmol) was dissolved in 8 mL of acetonitrile and tetrakis-(acetonitrile)copper(I) hexafluorophosphate (164 mg, 0.440 mmol), dissolved in 5 mL acetonitrile, were added dropwise to the solution. The reaction mixture turned yellow and was stirred for 30 min under nitrogen atmosphere at ambient temperature. Subsequent evaporation to dryness yielded a yellow crystalline powder (193 mg, 0.405 mmol, 92 %). Anal. calcd. for C15H16CuF6N4P: C (37.78 %); H (3.38 %); N (11.75 %); found C (37.82 %); H (3.43 %); N (11.83 %). 1H-NMR (CD3CN, 400 MHz): δ = 8.20 (d, J = 7.5 Hz, 2H, Ar–H), 7.68–7.34 (m, 4H, Ar–H, imidazole H-2), 6.93 (s, 1H, imidazole H-5), 4.80 (q, J = 9.1 Hz, 1H, –O–CH 2 –CH–N–), 4.40 (q, J = 10 Hz, 1H, –O–CH2–CH–N–), 4.14 (dd, J = 9.7 Hz, 1H, –O–CH 2 –CH–N–), 2.97 (t, J = 17.2 Hz, 1H, imidazole–CH 2 –CH–), 2.74 (dd, J = 14.8, 11.5 Hz, 1H, imidazole–CH 2 –CH–), 1.96 (s, 3H, –NCCH 3 ) ppm. 13C-NMR (CD3CN, 100 MHz): δ = 166.0 (C, 1C, –N=C–O–), 138.5 (C, 1C, imidazole C-4), 136.5 (CH, 1C, imidazole C-2), 133.2 (CH, 1C, phenyl C-4), 129.8 (CH, 2C, phenyl C-3, C-5), 129.3 (CH, 2C, phenyl C-2, C-6), 127.4 (C, 1C, phenyl C-1), 114.2 (CH, 1C, imidazole C-5), 73.8 (CH2, 1C, –O–CH2–CH–N–), 67.7 (CH, 1C, –O–CH2–CH–N–), 33.0 (CH2, 1C, imidazole–CH2–CH–) ppm.

Synthesis of [Cu(LOL2)(CH3CN)]PF6

[Cu(L OL 2)(CH3CN)]PF6 was prepared with L OL 2 (100 mg, 0.482 mmol) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (180 mg, 0.482 mmol) as described previously. The product was obtained as a white powder (192 mg, 0.420 mmol, 87 %). Anal. calcd. for C11H15CuF6N5P: C (34.18 %); H (4.41 %); N (12.26 %); found C (34.20 %); H (4.53 %); N (12.33 %). 1H-NMR (CD3CN, 400 MHz): δ = 7.67 (s, 1H, imidazole H-2), 6.94 (s, 1H, imidazole H-5), 4.55 (t, J = 9.2 Hz, 1H, –O–CH 2 –CH–N–), 4.18–4.10 (m, 1H, –O–CH2–CH–N–), 3.89 (t, J = 9.1 Hz, 1H, –O–CH 2 –CH–N–), 2.86 (d, J = 15.3 Hz, 1H, imidazole–CH 2 –CH–), 2.55 (dd, J = 23.1, 8.2 Hz, 1H, imidazole–CH 2 –CH–), 1.96 (s, 3H, –NCCH 3 ), 1.34 (s, 3H, –C(CH 3 )) ppm. 13C-NMR (CD3CN, 100 MHz): δ = 177.6 (C, 1C, –N=C–O–), 138.3 (C, 1C, imidazole C-4), 136.5 (CH, 1C, imidazole C-2), 114.6 (CH, 1C, imidazole C-5), 73.36 (CH2, 1C, –O–CH2–CH–N–), 67.1 (CH, 1C, –O–CH2–CH–N–), 34.6 (C, 1C, –C(CH3)3), 32.9 (CH2, 1C, –O–CH2–CH–N–), 28.3 (CH3, 1C, –C(CH3)3) ppm.

Synthesis of [Cu(LOL3)(CH3CN)]PF6

[Cu(L OL 3)(CH3CN)]PF6 was prepared with L OL 3 (100 mg, 0.605 mmol) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (226 mg, 0.605 mmol) as described previously. The product was obtained as a white powder (216 mg, 0.521 mmol, 86 %). Anal. calcd. for C11H15CuF6N5P: C (28.96 %); H (3.40 %); N (13.51 %); found C (28.81 %); H (3.43 %); N (13.34 %). 1H-NMR (CD3CN, 400 MHz): δ = 7.67 (s, 1H, imidazole H-2), 6.95 (s, 1H, imidazole H-5), 4.59 (t, J = 8.9 Hz, 1H, –O–CH 2 –CH–N–), 4.11–4.04 (m, 1H, –O–CH2–CH–N–), 3.97 (t, J = 8.8 Hz, 1H, –O–CH 2 –CH–N–), 2.88 (d, J = 14.9 Hz, 1H, imidazole–CH 2 –CH–), 2.52 (dd, J = 14.0, 12.1 Hz, 1H, imidazole–CH 2 –CH–), 2.09 (s, 3H, –CH 3 ), 1.96 (s, 3H, –NCCH 3 ) ppm. 13C-NMR (CD3CN, 100 MHz): δ = 169.2 (C, 1C, –N=C–O–), 138.5 (C, 1C, imidazole C-4), 136.9 (CH, 1C, imidazole C-2), 114.6 (CH, 1C, imidazole C-5), 72.2 (CH2, 1C, –O–CH2–CH–N–), 66.1 (CH, 1C, –O–CH2–CH–N–), 33.1 (CH2, 1C, imidazole–CH2–CH–), 14.5 (C, 1C, –CH3) ppm.

Synthesis of [Cu(Limz1)(CH3CN)2]PF6

[Cu(L imz 1)(CH3CN)2]PF6 was prepared with L imz 1 (100 mg, 0.237 mmol) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (88.4 mg, 0.237 mmol) as described previously. The product was obtained as a white powder (159 mg, 0.223 mmol, 94 %). Anal. calcd. for C11H15CuF6N5P: C (55.50 %); H (5.50 %); N (9.81 %); found C (55.11 %); H (5.32 %); N (9.95 %). 1H-NMR (CD3CN, 400 MHz): δ = 7.62 (s, 1H, –N=CH–C(CH 3 )), 7.51 (s, 1H, imidazole H-2), 7.42–7.35 (m, 9H, Ar–H), 7.20–7.11 (m, 6H, Ar–H), 6.79 (s, 1H, imidazole H-5), 3.64 (dd, J = 10.5, 5.2 Hz, 2H, –CH2–CH 2 –N–), 2.70 (dd, J = 15.1 Hz, 9.6 Hz, 2H, –CH 2 –CH2–N–), 1.96 (s, 6H, CH 3 CN), 1.09 (s, 9H, –C(CH 3 )3) ppm. 13C-NMR (CD3CN, 100 MHz): δ = 177.2 (CH, 1C, –N=CH–C(CH3)3), 141.6 (C, 3C, phenyl C-1), 140.2 (C, 1C, imidazole C-4), 139.4 (CH, 1C, imidazole C-2), 130.5 (CH, 6C, phenyl C-3, C-5), 129.3 (CH, 3C, phenyl C-4), 129.2 (CH, 6C, phenyl C-2, C-6), 118.3 (CH, 1C, imidazole C-5), 76.9 (C, 1C, –N–C–(C6H6)3), 62.4 (CH2, 1C, –CH2–CH2–N=), 36.6 (C, 1C, =CH–C(CH3)3), 29.6 (CH2, 1C, –CH2–CH2–N=), 27.4 (C, 3C, =CH–C(CH3)3) ppm.

Synthesis of [Cu(BIMZ)(CH3CN)]PF6

[Cu(BIMZ)(CH3CN)]PF6 was prepared with BIMZ (100 mg, 0.567 mmol) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (212 mg, 0.567 mmol) as described previously. The product was obtained as a white powder (232 mg, 0.545 mmol, 96 %). Anal. calcd. for C11H15CuF6N5P: C (31.03 %); H (3.55 %); N (16.45 %); found C (31.29 %); H (3.56 %); N (16.53 %). 1H-NMR (Acetone-d6, 400 MHz): δ = 7.41 (s, 2H, H-4), 7.13 (s, 2H, H-5), 4.79 (s, 2H, –CH 2 ), 3.88 (s, 6H, –CH 3 ), 2.08 (s, 3H, –NCCH 3 ) ppm. 13C-NMR (Acetone-d6, 100 MHz): δ = 145.1 (C, 2C, C-2), 127.9 (CH, 2C, C-4), 124.2 (CH, 2C, C-5), 117.5 (C, 1C, –NCCH3), 34.2 (CH3, 2C, CH3), 25.8 (CH, 1C, CH2) ppm.

Synthesis of [Cu(LOL1)2](PF6)2

[Cu(L OL 1)(CH3CN)]PF6 (35.7 mg, 750 µmol) was dissolved in dry dichloromethane (25 mL). Injection of dioxygen at −78 °C for 30 min and subsequent warming to room temperature leads to a blue solution. After 3 weeks, turquoise crystals were obtained. The single crystals were of high quality and suitable for X-ray structure determination. MS (ESI+, acetone): m/z (%) = 517.249 [2 L OL 1 + Cu]+. Anal. calcd. for C26H26CuF12N6O2P2: C (38.65 %); H (3.24 %); N (10.40 %); found C (39.02 %); H (3.57 %); N (10.29 %).

Results

Synthesis of the ligands and their copper(I) complexes

Based on 2-(R)-amino-3-(1H-imidazol-4(5)-yl)-propanol dihydrochloride [36] a condensation reaction with ethyl benzimidate hydrochloride, ethyl pivalimidate hydrochloride [38] or ethyl acetimidate hydrochloride in the presence of sodium methoxide leads to the formation of three new ligands L OL 1-3 (Scheme 1). Subsequent reaction with tetrakis(acetonitrile)copper(I) hexafluorophosphate generates the corresponding copper(I) complexes CuL OL 1-3.

Scheme 1
scheme 1

Synthesis of ligands L OL 1-3 and their corresponding copper(I) complexes CuL OL 1, CuL OL 2 and CuL OL 3

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The ligand L imz 1 was prepared by an imine condensation reaction of 2-(1-trityl-1H-imidazol-4-yl)-ethyl amine [37] with trimethylacetaldehyde (Scheme 2). Reaction with tetrakis(acetonitrile)copper(I) hexafluorophosphate leads to the corresponding copper(I) complex CuL imz 1. In contrast to CuL OL 1-3 with one acetonitrile as co-ligand, CuL imz 1 contains two acetonitrile molecules as co-ligands.

Scheme 2
scheme 2

Synthesis of ligand L imz 1 and its corresponding copper(I) complex CuL imz 1

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Bis(1-methylimidazol-2-yl)methane (BIMZ) was synthesized as published previously [35]. Reaction with tetrakis(acetonitrile)copper(I) hexafluorophosphate leads to CuBIMZ with one acetonitrile as co-ligand.

Catechol oxidase activity

The catechol oxidase activity of the new copper(I) complexes CuL OL 1, CuL OL 2, CuL OL 3, CuL imz 1, and CuBIMZ was investigated first. To this end, the catalytic efficiency of all complexes towards oxidation of the substrate 3,5-di-tert-butylcatechol (3,5-DTBC-H2) to 3,5-di-tert-butyl-o-benzoquinone (3,5-DTBQ) was determined. The reaction was monitored by UV/Vis spectroscopy based on the characteristic absorption band of 3,5-DTBQ at λ = 400 nm (ε = 1900 L mol−1 cm−1) [42]. 3,5-DTBC-H2 is well suited for catechol oxidase studies [43, 44] due to its stability, low redox potential [45–48] and the presence of bulky functional groups precluding further reactions. Consequently, it has been used in many model studies of CO as a substrate [49–55].

Catalytic runs were performed by anaerobically mixing 500 µM of the respective Cu(I) complexes in dichloromethane with 10 eq. of 3,5-DTBC-H2 and subsequent injection of dioxygen. After the concentration of 3,5-DTBQ reached saturation, HCl quenches were performed. A small volume of each reaction mixture was removed and diluted to 25 µM with dichloromethane. 6 M hydrochloric acid was added, and the resulting solution was extracted twice with CH2Cl2 to eliminate all existing copper species. After removing the solvent, the resulting residue was characterized by 1H-NMR and 13C-NMR spectroscopy.

Monitoring the catechol oxidase activity of the five copper(I) complexes by UV/Vis spectroscopy revealed the formation of 3,5-di-tert-butylsemiquinone (3,5-DTBSQ) during the initial phase of the reaction, as indicated by a band with two maxima at λ = 377 and 394 nm increasing in intensity with time (Fig. 4 and Fig. S1) [48, 56].

Fig. 4
figure 4

UV/Vis absorption spectra obtained upon oxidation a 500 µM solution of CuBIMZ in dichloromethane in the presence of 10 eq. 3,5-DTBC-H2 during 69 h; path length (l) l = 1 mm

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After initial formation of 3,5-DTBSQ, subsequent oxidation to 3,5-DTBQ was observed, as evident from the formation of a band with a single maximum at λ = 400 nm. Complete conversion of 3,5-DTBC-H2 to 3,5-DTBQ was also validated by NMR spectroscopy (Figs. S2 and S3).

In the next step, the formation of 3,5-DTBSQ was investigated kinetically applying Michaelis–Menten theory. To this end, different concentrations of the substrate DTBC-H2 (1.5 × 10−3–4 × 10−2 M) were prepared in dioxygen-saturated CH2Cl2 and a 1 × 10−3 M solution of each Cu(I) complex was added. In agreement with typical enzyme kinetics, the resulting curves exhibit saturation characteristics (Fig. 5).

Fig. 5
figure 5

Dependence of the reaction rates on the formation of 3,5-DTBSQ catalyzed by CuL OL 1-3, CuL imz 1 and CuBIMZ, varying the substrate concentration. Reactions were performed in dichloromethane saturated with O2

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From plots of the inverse rate constant against the inverse substrate concentration (Lineweaver–Burk, Fig. S4) the kinetic parameters listed in Table 1 were obtained.

Table 1 Kinetic parameters for the oxidation of 3,5-DTBC-H2 determined from Lineweaver–Burk plots
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The kinetic studies allow correlations between catecholase activity and structural parameters. The three oxazoline ligands differ in their substituents, the sterical demand increasing from methyl (L OL 3) over phenyl (L OL 1) to tert-butyl (L OL 2). In agreement with these considerations, the values of v max and k cat reflect that CuL OL 1 and CuL OL 3 are the most active catalysts. The copper center of CuL OL 3 is hardly shielded by the methyl residue such that 3,5-DTBC-H2 can bind without steric hindrance. The phenyl unit in CuL OL 1 is more bulky. However, due to its flexibility only a slightly higher value of K M is observed. The much bulkier tert-butyl group in CuL OL 2, in contrast, leads to significantly lower values for v max and k cat. Compared to CuL OL 1 and CuL OL 3, CuL imz 1 and CuBIMZ exhibited inferior catalytic activities. Moreover, the rate for CuBIMZ turned out to be almost independent of the catechol concentration. This indicates a particularly high binding constant [54].

To obtain further insight into the reaction mechanism, the dependence of the formation of 3,5-DTBSQ on the complex concentration (7.6 × 10−5–1 × 10−3 M) was studied, using an excess of substrate (2 × 10−2 M). Importantly, a linear dependence was found, implying that the reaction rate is first order in the copper complex (Fig. 6 and Fig. S5).

Fig. 6
figure 6

Dependence of the reaction rates for the formation of 3,5-DTBSQ catalyzed by CuL OL 1-3, CuL imz 1 and CuBIMZ on the complex concentration. The reactions were performed in dichloromethane saturated with O2

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In addition to the characteristic double band of semiquinone, formation of an intermediate with two optical absorption bands at λ = 523 and 900 nm was observed in all kinetic measurements. These absorption features are similar to those described earlier for a purple Cu(II)-semiquinone radical complex, which was evidenced during the catalytic oxygenation of 4-tert-butylphenol mediated by a Cu(DBED) complex [25]. Importantly, this complex exhibits an absorption band at λ = 545 nm and a weaker one at λ ~900 nm. Further characterization of the semiquinone complex had been achieved by mass spectrometry and single crystal structure determination.

We thus conclude that copper(II) semiquinone complexes are also formed as intermediates during the catechol oxidase reactions catalyzed by our complexes. For the CuL OL 1 system, the absorption bands of the semiquinone complex were found to be most intense. To spectroscopically monitor the buildup and the decay of [Cu(II)(L OL 1)(3,5-DTBSQ)]+ by UV/Vis spectroscopy, the complex was generated in situ. Therefore, a 500 µM solution of CuL OL 1 was added to 1 eq. 3,5-DTBC-H2 dissolved in dichloromethane saturated with O2 gas (Fig. 7).

Fig. 7
figure 7

UV/Vis absorption spectra obtained upon oxidation a 500 µM solution of CuL OL 1 and 1 eq. 3,5-DTBC-H2 in dichloromethane saturated with O2. Top optical spectra during the first 25 min; bottom optical spectra from 25 to 600 min; l = 1 mm

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During the first 25 min an increase of the bands of the copper(II) semiquinone complex is observed. Interestingly, the formation of the semiquinone complex is accompanied by the formation of free semiquinone (Fig. 7 top and Fig. S6). After 25 min, the intensities of the bands associated with the semiquinone complex decrease, and a gradual conversion of free 3,5-DTBSQ and 3,5-DBSQ complex, respectively, to 3,5-DTBQ takes place (Fig. 7 bottom).

Visually, the buildup of the copper(II) semiquinone complex is accompanied by a color change from light yellow to purple. Subsequent decay of the semiquinone complex leads to a brown solution.

To obtain further information regarding the constitution of the Cu(II) semiquinone intermediate, an independent synthesis of [Cu(II)(L OL 1)(3,5-DTBSQ)]PF6 was attempted at −60 °C. Isolation of this complex was, however, unsuccessful due to its instability at room temperature. Nevertheless, this complex could be generated in situ and detected by ESI/MS with a corresponding m/z = 510.0 (Fig. S7).

Combination of the experimental observations allows formulating a plausible mechanism for the catalytic pathway (Fig. 8).

Fig. 8
figure 8

Postulated mechanism for the conversion of 3,5-DTBC-H2 to 3,5-DTBQ by different copper(I) complexes

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Starting with the respective copper(I) complex and 3,5-DTBC-H2, injection of dioxygen leads to the corresponding copper(II) semiquinone intermediate with the rate constant k cat. The copper(II) semiquinone complex is in equilibrium with free semiquinone and a copper(II) species (equilibrium constant K E) whence formation of the semiquinone complex and free semiquinone occur almost simultaneously (see below). Afterwards, the semiquinone converts to the product 3,5-DTBQ via the semiquinone complex with a rate constant k′.

The oxidation of the Cu(I) complex and the catechol can in principle occur under generation of H2O or H2O2. Due to linear dependence of the reaction rate on the concentration of copper complex (see above) a mononuclear mechanism (and consequently, the formation of H2O2) is more likely. However, no H2O2 could be detected. This could be due to the fact that all copper(I) complexes exhibit catalase activity (Fig. S8).

The time-dependent formation and decay of [Cu(II)(L OL 1)(3,5-DTBSQ)]+ are plotted along with the time-dependent buildup of 3,5-DTBSQ in Fig. 9 (see also Fig. S9). Concentration of the semiquinone complex was determined based on its absorption band at λ = 900 nm (ε = 5890 L mol−1 cm−1), because an additional band at λ = 560 nm (related to 3,5-DTBQ) did not allow a quantitative determination of the absorbance at λ = 523 nm (ε = 10,830 L mol−1 cm−1). The concentration of 3,5-DTBSQ as inferred from the band at λ = 385 nm (ε = 1900 L mol−1 cm−1) exhibits saturation behavior, because the subsequent conversion to 3,5-DTBQ leads to an absorption band at about the same position with a comparable intensity.

Fig. 9
figure 9

Red Time-dependent formation and decay of [Cu(II)(L OL 1)(3,5-DTBSQ)]+ at λ = 900 nm. Blue Time-dependent formation of 3,5-DTBSQ at λ = 385 nm and subsequent conversion to 3,5-DTBQ

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Based on the given ε-values it can be inferred that 3,5-DTBSQ and the [Cu(II)(L OL 1)(3,5-DTBSQ)]+ complex are in equilibrium with a ratio of about 10:1. The initial slope of the buildup curve of 3,5-DTBSQ reveals a reaction rate of 3.8 × 10−5 mol L−1 min−1. Based on the Michaelis–Menten equation a reaction rate of 3.1 × 10−5 mol L−1 min−1 is calculated for the formation of 3,5-DTBSQ mediated by CuL OL 1 with the parameters given in Table 1 (Fig. S10).

Furthermore, the formation of the [Cu(II)(L OL 1)(3,5-DTBSQ)]+ complex as calculated from the initial slope (Fig. S10) results in a reaction rate of 5.3 × 10−6 mol L−1 min−1, about one tenth of that leading to 3,5-DTBSQ. This corresponds to the ratio of maximum concentrations of free 3,5-DTBSQ and [Cu(II)(L OL 1)(3,5-DTBSQ)]+, which are reached after 25 min reaction time (Fig. 9). The subsequent monomolecular decay of the copper(II) semiquinone complex proceeds with a first order rate constant of k′ = 1.8 × 10−2 min−1 (Fig. S10), which also corresponds to formation of the final product of the catechol oxidase reaction, 3,5-DTBQ.

Tyrosinase activity

Applying well-established reaction conditions [19, 20] phenols can be catalytically oxygenated to quinones by low molecular weight model systems of tyrosinase. Following Bulkowski [57] 50 eq. of phenolic substrate and 100 eq. of triethylamine were added anaerobically to a 500 µM solution of the respective Cu(I) complex (CuL OL 1-3, CuL imz 1, CuBIMZ) in dichloromethane, followed by injection of dioxygen at room temperature. Formation of the particular quinone was monitored by in situ UV/Vis spectroscopy based on characteristic absorption bands. After completion of the oxygenation, HCl quenches were performed and the reaction products were analyzed by NMR spectroscopy (see above).

All of the five reported copper(I) complexes were investigated regarding their ability to serve as model systems for tyrosinase. Importantly, only CuL imz 1 and CuBIMZ were found to both stoichiometrically and catalytically convert a range of phenols to ortho-quinones, whereas all three oxazoline-based copper(I) complexes (CuL OL 1-3) just mediate a stoichiometric conversion of 2,4-DTBP-H (Fig. S11). In the following, the two catalytically complexes and their reactivity towards five different substrates are examined in more detail.

The first employed substrate, 2,4-di-tert-butylphenol (2,4-DTBP-H), is sterically encumbered but widely used due to the stability of the resulting quinone. Oxygenation of CuL imz 1 leads to the formation of 3,5-DTBQ with an absorption band at λ = 403 nm (ε = 1830 L mol−1 cm−1) [19] (Fig. 10; Scheme 3).

Fig. 10
figure 10

UV/Vis absorption spectra obtained upon oxygenation a 500 µM solution of CuL imz 1 in dichloromethane in the presence of 50 eq. 2,4-DTBP-H and 100 eq. triethylamine; l = 1 mm. Insert TON and TOF

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Scheme 3
scheme 3

Schematic illustration of different substrates oxygenated by CuL imz 1 and CuBIMZ

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Oxygenation was complete after about 3 h, resulting in a turnover number of 16. Conversion of the same substrate by CuBIMZ leads to the appearance of an absorption band with two maxima at λ = 378 and 396 nm during the first 2 h, indicating formation of free 3,5-DTBSQ (Fig. S12) [47, 48, 56]. This feature changes to a single band at λ = 403 nm, indicating conversion to 3,5-DTBQ the formation of which is completed after 6 h with a TON of 9. The resulting NMR spectra (Fig. S13 and S14 for CuL imz 1; Fig. S15 and S16 for CuBIMZ) confirm the formation of 3,5-DTBQ as well as the C–C coupling product 3,3′,5,5′-tetra-tert-butyl-2,2′-biphenol and unreacted 2,4-DTBP-H in a ratio of 13:35:52 for CuL imz 1 and 5:42:53 for CuBIMZ.

For both the CuL imz 1 and the CuBIMZ system, a broad band at λ = 600 nm emerges at the end of the catalytic runs. To check whether this absorption band derives from a copper triethylamine complex that limits the turnover number, because it is unreactive towards substrate, an exchange of triethylamine against 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and subsequent oxygenation of 2,4-DTBP-H by CuL imz 1 was accomplished. The UV/Vis spectra show no absorption band at 600 nm after completion of the reaction (Fig. S17) but the same turnover number is achieved (TON = 16). This leads to the conclusion that triethylamine is involved in the formation of the absorption band, but only after the conversion of 2,4-DTBP-H to 3,5-DTBQ is completed. Consequently, the formation of the unreactive end product does not limit the activity of the catalytic system.

The next investigated substrate was 3-tert-butylphenol (3-TBP-H). Oxygenation of 3-TBP-H with CuL imz 1 and CuBIMZ first leads to a band at λ = 400 nm the maximum of which subsequently shifts to λ = 425 nm where it stays until saturation (Fig. 11 for CuL imz 1; Fig. S18 for CuBIMZ).

Fig. 11
figure 11

UV/Vis absorption spectra obtained upon oxygenation a 500 µM solution of CuL imz 1 in dichloromethane in the presence of 50 eq. 3-TBP-H and 100 eq. triethylamine; l = 1 mm. Insert TON and TOF

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This observation suggests the initial formation of 4-tert-butylquinone [58] and further reaction to the coupled ortho-quinone 4-(tert-butyl)-5-(3-(tert-butyl)phenoxy)cyclohexa-3,5-diene-1,2-dione [21, 22] with a TON of 20 for CuL imz 1 and a TON of 6 for CuBIMZ (ε = 898 L mol−1 cm−1 at λ = 425 nm). The corresponding NMR spectra (Figs. S19 and S20 for CuL imz 1; Figs. S21 and S22 for CuBIMZ) validate the presence of the coupled ortho-quinone besides unreacted 3-TBP-H (Scheme 3). Importantly, no C–C coupling product was detected. The formation of a broad band at λ = 600 nm was also observed for this system after completion of the oxygenation reaction.

To study the electronic influence of substituents in the applied substrate, catalytic runs were performed using 4-methoxyphenol (4-MeOP-H). Based on the pronounced mesomeric (+M) effect [59], very rapid conversion of 4-MeOP-H to the coupled quinone 4-methoxy-5-(4-methoxyphenoxy)cyclohexa-3,5-diene-1,2-dione was observed (Fig. 12).

Fig. 12
figure 12

UV/Vis absorption spectra obtained upon oxygenation a 500 µM solution of CuL imz 1 in dichloromethane in the presence of 50 eq. 4-MeOP-H and 100 eq. triethylamine; l = 1 mm. Insert TON and TOF

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In the case of CuL imz 1, oxygenation with subsequent reaction to the coupled product proceeds with a TON of 34 (ɛ = 524 L mol−1 cm−1, λ = 418 nm) after 60 min. NMR spectra (Figs. S23, S24) confirm the existence of the coupled quinone besides the educt. The course of the reaction was also followed for additional 3 h. Hydrolysis through water formed during the catalytic run yielded the para-quinone 2-hydroxy-5-methoxy-[1, 4] benzoquinone which was also detected by NMR spectroscopy (Fig. S25 and S26) [60].

Oxygenation of 4-MeOP-H with CuBIMZ also leads to rapid emergence of an absorption band at λ = 418 nm which saturates after 40 min (Fig. S27). Further oxygenation generates an additional absorption band at approximately λ = 465 nm, rising in intensity for 6 days. A first NMR spectrum taken after 40 min indicates that the coupled quinone 4-methoxy-5-(4-methoxyphenoxy)cyclohexa-3,5-diene-1,2-dione is formed, accompanied by 2-hydroxy-5-methoxy-[1, 4] benzoquinone and further by-products (Figs. S28, S29). A turnover number of six was calculated at this reaction time; due to the mixture of products, however, this result is problematic. After 5 h another HCl quench shows a variety of undefined products (Figs. S30 and S31). Based on these observations, CuBIMZ cannot be considered as a selective catalyst for the oxygenation of 4-MeOP-H.

The substrate N-acetyl-l-tyrosine ethyl ester monohydrate (NATEE) provides an analog to the native substrate tyrosine, and therefore, is of significant interest regarding model systems of tyrosinase. The conversion to the corresponding dopaquinone mediated by CuL imz 1 proceeds with a maximal TON of 25 (ɛ = 1188 L mol−1 cm−1, λ = 392 nm) [61] (Fig. 13). The rise the ortho-quinone absorption band at λ = 392 nm exhibits a sigmoidal fashion (Fig. S32). After 4 h, the intensity of the absorption band decreases again and its maximum shifts to λ = 358 nm, indicating conversion of the N-acetyl-l-dopa ethyl ester into a product the identity of which could not be established by NMR spectroscopy [27].

Fig. 13
figure 13

UV/Vis absorption spectra obtained upon oxygenation a 500 µM solution of CuL imz 1 in dichloromethane in the presence of 50 eq. NATEE and 100 eq. triethylamine; l = 1 mm. Insert decrease of the absorption band after 4 h

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For further identification of the primary oxygenation product N-acetyl-l-dopa ethyl ester dichloromethane was removed from the reaction mixture after 2 h and the residue analyzed with mass spectrometry. The peak at m/z = 268.2 [M + H] can be assigned to the corresponding catechol (Fig. S33). However, this result did not give any information regarding the regioselectivity of the hydroxylation. Derivatization to the corresponding phenazine using ortho-phenylenediamine permits the identification of ortho-quinones via fluorescence spectroscopy. Based on a fluorescence band at λ = 523 nm and an absorption band at λ= 380 nm in the UV/Vis (Fig. S34), which are in the range of known phenazine derivatives, ortho-hydroxylation could be confirmed unequivocally [62, 63]. Surprisingly, CuBIMZ exhibited no tyrosinase activity in the case of NATEE.

Figure 14 gives an overview of the conversion of the investigated monophenols to ortho-quinones. Importantly, CuL imz 1 globally exhibits a much higher catalytic activity than CuBIMZ. Moreover, as exemplified for the substrate 4-MeOP-H, it is much more selective. These findings are attributed to the higher flexibility of the ligand backbone of L imz 1 as compared to BIMZ which facilitates formation of the ternary intermediate required for hydroxylation of the substrate (see above) [2, 17–19, 64].

Fig. 14
figure 14

Quantification of the conversion of different monophenols to the corresponding ortho-quinones

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To expand the scope of potential substrates, 8-hydroxyquinoline was also examined [65]. Following the standard oxygenation protocol, both CuL imz 1 and CuBIMZ instantaneously produced an absorption band at λ = 412 nm which did not change in time any more (Fig. S35). NMR spectra taken after the HCl quenches revealed no indication for the formation of ortho-quinone as product. To obtain more information on this issue, the reaction product was isolated by flash chromatography with dichloromethane (R f = 0.31). The corresponding NMR spectra also exclude the formation of quinone (Figs. S36 and S37). Due to the high tendency of 8-hydroxyquinoline to form stable metal complexes we conclude that no hydroxylation occurred. Instead, the ligands L imz 1 and BIMZ are replaced by 8-hydroxyquinoline to form the homoleptic square planar complex [Cu(II)(8-quinolinol)2]. The identity of the product was clarified via mass spectrometry m/z = 415.09 and powder diffractometry; the measured pattern is in full agreement with the calculated diffraction pattern based on the published structure (Fig. S38) [66].

µ-Peroxo and other Cu(II) species

The µ-η2:η2-peroxo-dicopper(II) complex is the most important reactive intermediate in the mechanistic cycle of tyrosinase [1, 2]. For a new ligand system, it is necessary to determine the active copper peroxide species. In the case of tyrosinase, the copper(I) ions are oxidized to copper(II) ions and bind dioxygen as peroxide in a characteristic side-on geometry (µ-η2:η2) [12, 22, 65]. The intense absorption band between λ = 340–380 nm is due to an in-plane πσ* → \({\text{d}}_{{{\text{x}}^2}-{{\text{y}}^2}}\) charge transfer transition, and the less intense absorption feature between λ = 510–580 nm to an out-of-plane πv* → \({\text{d}}_{{{\text{x}}^2}-{{\text{y}}^2}}\) peroxo to copper(II) charge transfer transition [1, 2, 20].

To obtain spectroscopic information regarding these intermediates, 3 mM solutions of each copper(I) complex (CuL OL 1-3, CuL imz 1 and CuBIMZ) in acetone and dichloromethane were cooled to −85 °C under nitrogen atmosphere. After injection of O2 gas intense optical absorption bands between λ = 333 and 357 nm were observed in the UV/Vis spectra (Fig. S39). The position of the maxima differs only slightly. Specifically, the µ-η2:η2-peroxo-dicopper(II) complex of CuL OL 1 and CuL OL 3 exhibit LMCT bands at λ = 357 nm and λ = 335 nm in acetone which can be associated with in-plane πσ* → \({\text{d}}_{{{\text{x}}^2}-{{\text{y}}^2}}\)  charge transfer transitions. Additional absorption bands at λ = 600 nm in the case of CuL OL 1 and λ = 510 nm in the case of CuL OL 3 are due to out-of-plane πv* → \({\text{d}}_{{{\text{x}}^2}-{{\text{y}}^2}}\) peroxo to copper(II) charge transfer transitions. CuL OL 2 and CuBIMZ were measured in dichloromethane and exhibited absorption maxima at λ = 340 nm and λ = 610 nm for CuL OL 2 and λ = 333 nm as well as λ = 600 nm for CuBIMZ. The slight shift of the πσ* → \({\text{d}}_{{{\text{x}}^2}-{{\text{y}}^2}}\) charge transfer transition to higher energies was also noticed for copper(I) complexes with bidentate bis(oxazoline) ligands [67]. In contrast, no µ-η2:η2-peroxo-dicopper(II) complex could be found for CuL imz 1 in tetrahydrofuran, dichloromethane, and acetone.

In previous publications, we correlated a high stability of the peroxo intermediates with a low catalytic reactivity and vice versa. Specifically, for model systems with a high catalytic activity the peroxo intermediate turned out to be hardly detectible [30]. This also appears to be correct for the present study as CuL imz 1 is the most active tyrosinase catalyst described in this paper.

In an attempt to generate the peroxide core for CuL OL 1 in dichloromethane no characteristic absorption band was obtained after injection of O2. Instead, blue single crystals, suitable for single crystal structure determination were obtained from the solution within 3 weeks. The resulting complex [Cu(L OL 1)2](PF6)2 crystallizes in the orthorhombic space group P212121 with four formula units per unit cell and all atoms in general positions (Fig. 15).

Fig. 15
figure 15

Crystal structure of [Cu(II)(L OL 1)2](PF6)2; H atoms are omitted for clarity. Color code: C gray; N blue; O red; Cu turquoise

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Selected crystallographic parameters and details of the structure refinement as well as lists with selected bond lengths and angles are presented in Tables S1–S4. In the crystal structure, the copper(II) cations are fourfold coordinated by each two N-atoms of two L OL 1 ligands, within a strongly distorted square planar geometry. The Cu–N bond lengths are between 1.958 (4) and 1.996 (4) Å and are in the typical range for known bidentate homoleptic copper(II) complexes [68]. The angles range from 91.12 (18)° to 159.4 (2)°.

Summary and conclusion

Five new small molecule model systems (CuL OL 1-3, CuL imz 1 and CuBIMZ) have been synthesized and investigated regarding their catechol oxidase and tyrosinase activities. The results are summarized graphically in Fig. 16. Based on the mechanistic cycle in Fig. 8, all of these copper(I) complexes oxidize 3,5-DTBC-H2 to the corresponding quinone (Fig. 16). A copper(II) semiquinone species and free 3,5-DTBSQ have been identified as intermediates during these reactions. Kinetic measurements for the formation of semiquinone applying Michaelis–Menten approach revealed a sequence of activity in the order CuL OL 3 ≈ CuL OL 1 > CuBIMZ > CuL OL 2 > CuL imz 1.

Fig. 16
figure 16

Overview of the conversion of a variety of substrates and their corresponding products using the reported new copper(I) complexes

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Whereas CuL imz 1 and CuBIMZ also exhibit catalytic tyrosinase activity, the copper(I) complexes supported by the ligands L OL 1-3 only mediate stoichiometric conversions of 2,4-DTBP-H to the corresponding ortho-quinone. This indicates that release of the product is hindered in the stoichiometric systems, in contrast to the catalytic tyrosinase model. Importantly, µ-η2:η2-peroxo intermediates of CuL OL 1-3 and CuBIMZ could be detected, whereas this was not possible for the most active tyrosinase catalyst, CuL imz 1. These findings provide evidence for the hypothesis that the reaction pathway proceeds via side-on peroxo intermediates [12, 19, 69].

To further explore the catalytic activity of the first two systems several monophenols were employed as substrates. For the oxygenation of 2,4-DTBP-H to 3,5-DTBQ a TON of 16 was derived for CuL imz 1 and 9 for CuBIMZ. In contrast to mere oxygenation, the conversion of less substituted monophenols, such as 3-TBP-H and 4-MeOP-H led to ortho-hydroxylation with subsequent two-electron oxidation, followed by a coupling reaction in 5-position.

From a biomimetic perspective, the reactivity with regard to the substrate NATEE was of particular interest. Surprisingly, only CuL imz 1 showed tyrosinase activity with respect to this substrate, exhibiting a turnover number of 25 after 4 h. The combination of an imidazole and an imine unit thus appears to be more efficient as compared to the combination of two imidazole rings bridged by a methyl group. We again attribute the difference in tyrosinase activity to the higher flexibility of the CuL imz 1 system, which is necessary for the hydroxylation step. By contrast, the increased rigidity of CuBIMZ leads to a significantly lower reactivity, independent of the used monophenol. For the substrate 4-MeOP-H, the decreased reactivity of CuBIMZ resulted in a variety of products during the catalytic mechanism; i.e., a loss of specificity.

An irreversible deactivation was observed during the reaction of both copper(I) complexes with 8-hydroxyquinoline. The high tendency of 8-hydroxyquinoline to generate stable metal complexes led to a displacement of the respective ligands, resulting in the homoleptic [Cu(II)(8-quinolinol)2] complex. From the homoleptic complex [Cu(L OL 1)2](PF6)2, we presented a new crystal structure.

In summary, the results of our study demonstrate the importance of structural factors (flexibility of the ligand backbone and presence of bulky substituents) on the catalytic activity of model systems of tyrosinase. The catechol oxidase reaction is less sensitive with respect to these steric effects. Moreover, the activities of the model systems of tyrosinase are found to critically depend on the ability of the catalyst to release the oxygenated product as quinone. Further, fine-tuning of the catalytic activity can be achieved by variation of the electronic structure of the ligand, in particular, the donor/acceptor properties of the employed heterocyclic units.