Tyrosinase and catechol oxidase activity of copper(I) complexes supported by imidazole-based ligands: structure–reactivity correlations

J Biol Inorg Chem

DOI 10.1007/s00775-016-1370-y

ORIGINAL PAPER

Tyrosinase and catechol oxidase activity of copper(I) complexes

supported by imidazole‑based ligands: structure–reactivity

correlations

1

Franziska Wendt · Christian Näther · Felix Tuczek

Received: 25 February 2016 / Accepted: 8 June 2016

©

SBIC 2016

Abstract Four new imidazole-based ligands, 4-((1H-imi-

dazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole (L_O_L1),

(3-TBP-H), 4-methoxyphenol (4-MeOP-H), N-acetyl-l-ty-

rosine ethyl ester monohydrate (NATEE) and 8-hydrox-

yquinoline are employed. The oxygenation products are

identiﬁed and characterized with the help of UV/Vis and

NMR spectroscopy, mass spectrometry, and ﬂuorescence

measurements. Whereas the copper complexes with ligands

containing combinations of imidazole and imine functions

or two imidazole units (CuL_i_m_z1 and CuBIMZ) are found

to exhibit catalytic tyrosinase activity, the systems with

ligands containing oxazoline just mediate a stoichiometric

conversion. Correlations between the structures of the com-

plexes and their reactivities are discussed.

4

-((1H-imidazol-4-yl)methyl)-2-(tert-butyl)-4,5-di-

hydrooxyzole (L_O_L2), 4-((1H-imidazol-4-yl)methyl)-

-methyl-4,5-dihydrooxyzole (L_O_L3), and N-(2,2-

2

dimethylpropylidene)-2-(1-trityl-1H-imidazol-4-yl-)ethyl

amine (L_i_m_z1), have been synthesized. The correspond-

ing copper(I) complexes [Cu(I)(L_O_L1)(CH CN)]PF₆

3

(

CuL_O_L1), [Cu(I)(L_O_L2)(CH CN)]PF (CuL_O_L2), [Cu(I)

3 6

(

L_O_L3)(CH CN)]PF (CuL_O_L3), [Cu(I)(L_i_m_z1)(CH CN) ]

3

6

3

2

PF (CuL_i_m_z1) as well as the Cu(I) complex derived from

6

the known ligand bis(1-methylimidazol-2-yl)methane

(

BIMZ), [Cu(I)(BIMZ)(CH CN)]PF (CuBIMZ), are

3 6

screened as catalysts for the oxidation of 3,5-di-tert-butyl-

Keywords Type 3 copper enzymes · Tyrosinase · Catechol

catechol (3,5-DTBC-H ) to 3,5-di-tert-butylquinone (3,5-

oxidase · Kinetics · Dioxygen activation

2

DTBQ). The primary reaction product of these oxidations

is 3,5-di-tert-butylsemiquinone (3,5-DTBSQ) which slowly

converts to 3,5-DTBQ. Saturation kinetic studies reveal a

trend of catalytic activity in the order CuL_O_L3 ≈ CuL_O_L1

Introduction

>

CuBIMZ > CuL_O_L2 > CuL_i_m_z1. Additionally, the cata-

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

lytic activity of the copper(I) complexes towards the oxy-

genation of monophenols is investigated. As substrates

2

,4-di-tert-butylphenol (2,4-DTBP-H), 3-tert-butylphenol

2

µ-η :η 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 bac-

teria make use of tyrosinase [5]. The pigmentation of skin,

the browning of fruits and the sclerotization of insect cuti-

cles are all based on melanogenesis [6, 7].

Dedicated to Prof. Dr. Edward I. Solomon in honor of the ACS

Alfred Bader Award.

Electronic supplementary material The online version of this

article (doi:10.1007/s00775-016-1370-y) contains supplementary

material, which is available to authorized users.

*

Felix Tuczek

The related enzyme catechol oxidase (CO) catalyzes

the two-electron oxidation of catechols to ortho-quinones

(diphenolase activity). Whereas Ty also exhibits dipheno-

lase activity CO cannot mediate the ortho-hydroxylation

ftuczek@ac.uni-kiel.de

1

Institute of Inorganic Chemistry, Christian-Albrechts-

University, Max-Eyth-Straße 2, 24118 Kiel, Germany

1

3

J Biol Inorg Chem

Fig. 1 Catalytic activities of tyrosinase and catechol oxidase

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 resi-

due activates conserved water towards deprotonation of

monophenols. If Asn is missing, only diphenolase activity

is observed [9].

Fig. 2 Ligand designs used for tyrosinase reaction

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]-imino-

biphenyl (BiPh-(impy) ) (Fig. 2) [10].

2

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 Cu O complex as the hydroxylating

2 2

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 exter-

nal substrates was achieved by the Cu(I)DBED system

(

DBED = 1,2-bis(di-tert-butyl)ethylene-diamine) [17].

2 2

Upon exposure to dioxygen the formation of a (µ-η :η )-

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 + O + substrate) in the course of the tyrosinase-like

2

Fig. 3 Structure of bidentate imidazole-based ligands for catalytic

reactions: L_O_L1‑3, L_i_m_z3 and BIMZ

hydroxylation reaction. Further reaction led to a catecho-

lato intermediate and the ﬁnal product, a semiquinone com-

plex [17, 18].

The ﬁrst catalytic model system of tyrosinase based on

a mononucleating ligand, [2-(pyrid-2-yl)ethyl]imino-tert-

systems supported by bis(pyrazolyl)methane ligands which

exhibit room temperature stable peroxo intermediates [20].

More recently, Lumb et al. demonstrated catalytic conver-

sions of different monophenols to a variety of organic prod-

ucts [21–26].

butane (L 1) was reported by our group [19]. In 2013,

Herres-Pawlis et al. presented catalytic copper(I) model

py

1

3

J Biol Inorg Chem

During the last years our group published a number

of new copper(I) complexes functioning as model sys-

tems for tyrosinase [2, 19, 27–34]. Starting from the par-

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.

ent mononuclear complex with the ligand L 1 containing

py

a pyridyl moiety and an imine unit, we ﬁrst replaced the

N-heterocycle by benzimidazole and pyrazole to investi-

gate the inﬂuence of the electronic structure of the ligand

on the turnover number (TON) of the corresponding cop-

per catalyst. To expand the scope of the oxygenation reac-

tions 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),

Materials and methods

Materials

1

,1′-methylenebis(3-methyl-1H-pyrazole) (mBPM), and

,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 imida-

zole moieties to model the histidine residues in the native

protein (Fig. 3).

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 tech-

niques or a MBraun LABmaster glovebox ﬁlled with

The ligand N-(2,2-dimethylpropylidene)-2-(1-trityl-1H-

imidazol-4-yl-)ethyl amine (L_i_m_z1) contains an imidazole

unit instead of the pyridine moiety of the parent L 1 sys-

nitrogen.

2-(R)-amino-3-(1H-imidazol-4(5)-yl)propanol

py

tem. The incorporated trityl residue increases the solubil-

ity in dichloromethane. Reaction with tetrakis(acetonitrile)

copper(I) hexaﬂuorophosphate leads to the copper(I) com-

plex [Cu(I)(L_i_m_z1)(CH CN) ]PF (CuL_i_m_z1). Addition-

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 puriﬁca-

tions 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 per-

formed using an Isolera One fabricated by Biotage.

3

2

6

ally, we prepared the complex [Cu(I)(BIMZ)(CH CN)PF

3

6

(

CuBIMZ) which is supported by the ligand bis(1-methyl-

imidazol-2-yl)methane (BIMZ) containing two imidazole

groups [35].

Importantly, the imine function being present in the par-

ent ligand L 1 as well as its analogs, including L_i_m_z1, is

py

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_i_m_z1 ligand by different oxa-

zoline units. To gain insight into steric effects, we investi-

gate three ligands containing different substituents adjacent

to the oxazoline moiety; i.e., a phenyl group (4-((1H-imi-

dazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole, L_O_L1),

a tert-butyl group (4-((1H-imidazol-4-yl)methyl)-2-(tert-

butyl)-4,5-dihydrooxyzole, L_O_L2), and a methyl group

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 spectropho-

tometer. 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 spectrom-

(

4-((1H-imidazol-4-yl)methyl)-2-methyl-4,5-dihydrooxy-

zole, L_O_L3).

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_O_L1‑3,

CuL_i_m_z1 and CuBIMZ), and the corresponding kinetics is

determined. Based on the experimental results, a mecha-

nistic cycle is derived. Then, comprehensive measurements

with different monophenols are performed to investigate

1

eter at 300 K with a H frequency of 400.13 MHz and a

1

3

C frequency of 100.62 MHz. Fluorescence spectra were

recorded in methanol with a Perkin Elmer LS55.

1

3

J Biol Inorg Chem

−

Mass spectrometry

A blank was used to account for I which is formed

3

−

upon the oxidation of I by atmospheric oxygen.

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.

Catalase activity measurements

Measurements of catalase activity were performed accord-

ing to Monzani et al. [41]. A 0.1 M solution of hydro-

gen peroxide in methanol was divided in six parts. The

copper(I) complexes of L_O_L1‑3, L_i_m_z1 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 solu-

tions were added to 2 mL of a solution containing water

(acidiﬁed to pH 2 with H SO ), potassium iodide (30 mM)

Single crystal and powder x‑ray diffraction

Single crystal data were recorded using a STOE Imag-

ing Plate Diffraction System (IPDS-2) with Mo-K radia-

α

tion (λ = 0.71073 Å) and were corrected for absorp-

tion (T

: 0.8211/0.8927). The structure was solved

min/max

2

4

2

with SHELXS-97 [39] and reﬁned against F using

SHELXL-2014 [40]. All non-hydrogen atoms were reﬁned

anisotropic. The C–H and N–H H atoms were posi-

tioned with idealized geometry and reﬁned isotropic with

U (H) = 1.2 U (C,N) using a riding model. The abso-

and ammonium heptamolybdate tetrahydrate (100 nM).

After 1 h the relative intensity of the I₃absorption band at

353 nm as compared to the blank was measured. Catalase

activity could be observed for all ﬁve complexes.

−

iso

eq

lute structure was determined and is in agreement with

General procedure for the oxidation

of 3,5‑di‑tert‑butylcatechol to mimic catechol oxidase

activity

the selected setting [Flack x-parameter: −0.001 (18)].

−

One of the two crystallographically independent PF ani-

6

ons is disordered and was reﬁned using a split model and

restraints.

Under inert atmosphere at ambient temperature, the respec-

tive copper(I) complexes were dissolved in 20 mL dichlo-

romethane to afford a 500 µM solution. Then 10 eq. of

CCDC-1454612 contains the supplementary crystallo-

graphic 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-

3,5-DTBC-H were added. Subsequent injection of dioxy-

2

gen into the reaction mixture started the oxidation of the

catechol to the corresponding quinone, which was observed

via UV/Vis and NMR spectroscopy.

P powder diffractometer with Cu K -radiation (Ge(111)

α

monochromator) what is equipped with a MYTHEN 1 K

detector.

General procedure for the oxygenation of external

substrates to mimic tyrosinase activity

Detection of hydrogen peroxide in catalytic catechol

oxidase reactions

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. triethyl-

amine 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.

Hydrogen peroxide formation during the catalytic oxida-

tion was determined by the development of the character-

−

−1

istic I₃band at 353 nm (ɛ = 26,000 L mol cm ) [41]

−

which forms upon reaction of I with H O . After 15 min,

2

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 H SO . Furthermore, 1 mL

2

4

of an aqueous potassium iodide solution (10 %) and three

drops of an ammonium heptamolybdate tetrahydrate solu-

tion (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 pro-

ceeds according to the equation:

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

−

+

H O + 2 I + 2 H → 2 H O + I

2

(

2 × 20 mL). The combined organic fractions were dried

over MgSO , ﬁltered, and evaporated in vacuum. The resi-

4

−

I₂₍_a_q₎+ I → I .

3

due was analyzed by NMR spectroscopy.

1

3

J Biol Inorg Chem

General procedure of kinetic measurements

(CD OD, 400 MHz): δ = 7.90 (dd, J = 5.3, 3.2 Hz, 2H,

3

Ar–H), 7.60 (d, J = 1.0 Hz, 1H, imidazole H-2), 7.56–7.49

Catechol oxidase activities were measured in dichlorometh-

ane saturated with dioxygen at ambient temperatures using

time-dependent UV/Vis spectroscopy. In a ﬁrst run solu-

(m, 1H, Ar–H), 7.47–7.41 (m, 2H, Ar–H), 6.88 (s, 1H, imi-

dazole H-5), 4.60 (m, 1H, –O–CH –CH–N–), 4.48 (dd,

2

J = 9.4, 8.6 Hz, 1H, –O–CH –CH–N–), 4.25 (dd, J = 8.5,

2

tions with concentrations of 3,5-DTBC-H in a range

7.3 Hz, 1H, –O–CH –CH–N–), 3.06 (dd, J = 14.6, 5.2 Hz,

2

−3

−2

of 1.5 × 10 –4 × 10 M were prepared. Afterwards a

1H, imidazole–CH –CH–), 2.81 (dd, J = 14.7, 7.9 Hz,

2

1

3

solution of each copper(I) complex in dichloromethane

1H, imidazole–CH –CH–) ppm.

C-NMR (CD OD,

3

2

−

3

with a constant concentration of 1 × 10 M was added.

The increasing double bands of 3,5-di-tert-butyl semiqui-

none (3,5-DTBSQ) at 377 nm and 394 nm were monitored

during the ﬁrst 2 min. In another series of experiments,

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 (CH , 1C, –O–CH –

the concentration of 3,5-DTBC-H was kept constant

2

−2

(

2.5 × 10 M) and the concentration of copper(I) com-

CH–N–), 67.1 (CH, 1C, –O–CH –CH–N–), 33.7 (CH , 1C,

2 2

imidazole–CH –CH–) ppm.

2

−

5

−3

plexes was varied in the range of 7.6 × 10 –1 × 10 M.

Measurements were performed after 2 min.

Synthesis of 4‑((1H‑imidazol‑4‑yl)

Derivatization of N‑acetyl‑l‑dopa ethyl ester

with ortho‑phenylenediamine

methyl)‑2‑(tert‑butyl)‑4,5‑dihydrooxyzole (L_O_L2)

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 chroma-

A 500 µM solution of CuL_i_m_z1 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

tography with dichloromethane/methanol (9:1, R = 0.13–

f

(

0.29) as the eluent to afford L 2 (667 mg, 3.22 mmol,

OL

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. Sub-

sequent addition of ortho-phenylenediamine (13.50 mg,

46 %) as a white powder. Anal. calcd. for C H N O: C

1

17

3

(63.43 %); H (8.71 %); N (20.17 %); found C (63.15 %);

1

H (8.84 %); N (20.41 %). H-NMR (CD OD, 400 MHz):

3

δ = 7.59 (s, 1H, imidazole H-2), 6.85 (s, 1H, imidazole

H-5), 4.40–4.31 (m, 1H, –O–CH –CH–N–), 4.25 (dd,

2

1

23.8 µmol) afforded a yellow solution, which was stirred

J = 9.4, 8.5 Hz, 1H, –O–CH –CH–N–), 4.10 (dd, J = 8.5,

2

for 10 min under reﬂux. The solvent was evaporated and

the residue was dissolved in methanol. Afterwards the pre-

pared phenazine derivative was characterized by UV/Vis

and ﬂuorescence spectroscopy.

6.7 Hz, 1H, –O–CH –CH–N–), 2.94 (ddd, J = 14.6, 4.5,

2

0.8 Hz, 1H, imidazole–CH –CH–), 2.68 (dd, J = 14.7,

2

7.8 Hz, 1H, imidazole–CH –CH–), 1.17 (s, 9H, –C(CH ) )

2

3 3

1

3

ppm. C-NMR (CD OD, 100 MHz): δ = 177.2 (C, 1C,

3

–

N=C–O–), 136.1 (CH, 1C, imidazole C-2), 134.4 (C,

Synthesis of 4‑((1H‑imidazol‑4‑yl)

1C, imidazole C-4), 118.8 (CH, 1C, imidazole C-5), 73.0

methyl)‑2‑phenyl‑4,5‑dihydrooxyzole (L_O_L1)

(CH , 1C, –O–CH –CH–N–), 66.2 (CH, 1C, –O–CH –CH–

2

N–), 34.3 (CH , 1C, imidazole–CH –CH–), 33.4 (C, 1C,

2

To a solution of histidinol dihydrochloride (1.50 g,

–C(CH ) ), 28.1 (CH , 1C, –C(CH ) ) ppm.

3 3 3 3 3

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 Na CO solution (20 mL, pH 10) was added

Synthesis of 4‑((1H‑imidazol‑4‑yl)

methyl)‑2‑methyl‑4,5‑dihydrooxyzole (L_O_L3)

The synthesis was prepared with ethyl acetimidate hydro-

chloride (754 mg, 6.10 mmol, 1.3 eq.) as previously

described. The crude product was puriﬁed by ﬂash chroma-

2

3

and extracted three times with dichloromethane. The com-

bined organic phases were dried over Na SO , and the sol-

tography with dichloromethane/methanol (9:1, R = 0.31)

2

4

f

vent was removed by rotary evaporation. The crude prod-

uct was puriﬁed by ﬂash chromatography with chloroform/

as the eluent to afford L 3 (393 mg, 2.38 mmol, 34 %)

OL

as a white powder. Anal. calcd. for C H N : C (58.17 %);

8

11

3

methanol (9:1, R = 0.10–0.26) as the eluent to afford L_O_L1

H (6.71 %); N (25.44 %); found C (57.85 %); H (6.92 %);

f

1

(

929 mg, 4.09 mmol, 58 %) as a white powder. Anal. calcd.

N (25.65 %). H-NMR (CD OD, 400 MHz): δ = 7.58 (s,

3

for C H N O: C (68.70 %); H (5.77 %); N (18.49 %);

1H, imidazole H-2), 6.86 (s, 1H, imidazole H-5), 4.40–4.34

1

3

13

3

1

found C (68.33 %); H (6.14 %); N (18.51 %). H-NMR

(m, 1H, –O–CH –CH–N–), 4.30 (dd, J = 9.4, 8.5 Hz, 1H,

2

1

3

J Biol Inorg Chem

–

O–CH –CH–N–), 4.05 (dd, J = 8.3, 7.0 Hz, 1H, –O–

Synthesis of [Cu(L_O_L1)(CH CN)]PF

3 6

2

CH –CH–N–), 2.94 (ddd, J = 14.7, 5.4, 0.7 Hz, 1H, imi-

2

dazole–CH –CH–), 2.69 (dd, J = 14.7, 7.7 Hz, 1H, imi-

Under anaerobic conditions, L_O_L1 (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 yel-

low 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 C H CuF N P: C (37.78 %); H

2

1

3

dazole–CH –CH–), 2.69 (s, 3H, –CH ) ppm. C-NMR

2

3

(

CD OD, 100 MHz): δ = 168.4 (C, 1C, –N=C–O–), 136.1

3

CH, 1C, imidazole C-2), 135.9 (C, 1C, imidazole C-4),

1

18.3 (CH, 1C, imidazole C-5), 73.4 (CH , 1C, –O–CH –

2 2

CH–N–), 66.5 (CH, 1C, –O–CH –CH–N–), 33.8 (CH , 1C,

imidazole–CH –CH–), 13.6 (CH , 1C, –CH ) ppm.

2

3

Synthesis of N‑(2,2‑dimethylpropylidene)‑2‑

1

5

16

6

4

(

1‑trityl‑1H‑imidazol‑4‑yl‑)ethyl amine (L_i_m_z1)

(3.38 %); N (11.75 %); found C (37.82 %); H (3.43 %);

1

N (11.83 %). H-NMR (CD CN, 400 MHz): δ = 8.20 (d,

3

To synthesize L_i_m_z

1

2-(1-trityl-1H-imidazol-4-yl-)

J = 7.5 Hz, 2H, Ar–H), 7.68–7.34 (m, 4H, Ar–H, imida-

zole H-2), 6.93 (s, 1H, imidazole H-5), 4.80 (q, J = 9.1 Hz,

1H, –O–CH –CH–N–), 4.40 (q, J = 10 Hz, 1H, –O–CH –

ethyl amine (1.00 g, 2.83 mmol), trimethylacetaldehyde

0.62 mL, 5.66 mmol, 2 eq.) and sodium sulfate (10.1 g,

0.7 mmol, 25 eq.) were dissolved in dry toluene (15 mL).

The mixture was stirred at room temperature for at least

h. The residue was ﬁltered off and washed twice with

(

7

2

CH–N–), 4.14 (dd, J = 9.7 Hz, 1H, –O–CH –CH–N–),

2

2.97 (t, J = 17.2 Hz, 1H, imidazole–CH –CH–), 2.74 (dd,

2

3

J = 14.8, 11.5 Hz, 1H, imidazole–CH –CH–), 1.96 (s, 3H,

2

1

3

dry toluene (2 × 3 mL). The solvent was removed until

a colorless solid precipitated. After 24 h the solid was ﬁl-

tered, washed with acetonitrile and dried in vacuum to

afford L_i_m_z1 (728 mg, 1.23 mmol, 61 %) as a white pow-

der. Anal. calcd. for C H N : C (82.62 %); H (7.41 %); N

–NCCH ) ppm. C-NMR (CD CN, 100 MHz): δ = 166.0

3

(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, imi-

dazole C-5), 73.8 (CH , 1C, –O–CH –CH–N–), 67.7 (CH,

2

9

31

3

1

(

9.97 %); found C (82.56 %); H (7.50 %); N (9.90 %). H-

2

NMR (CDCl , 400 MHz): δ = 7.46 (t, J = 1.2 Hz, 1H, imi-

1C, –O–CH –CH–N–), 33.0 (CH , 1C, imidazole–CH –

2 2 2

3

dazole H-2), 7.37–7.27 (m, 9H, Ar–H), 1.15–7.10 (m, 6H,

CH–) ppm.

Synthesis of [Cu(L_O_L2)(CH CN)]PF

6

Ar–H), 6.56 (s, 1H, imidazole H-5), 3.64 (t, J = 7.0 Hz,

2

H, –CH –CH –N–), 2.82 (t, J = 7.0 Hz, 2H, –CH –

2

3

13

CH –N–), 0.97 (s, 9H, –C(CH ) ) ppm. C-NMR (CDCl ,

2

3 3

3

1

00 MHz): δ = 172.5 (CH, 1C, –N=CH–C(CH ) ), 142.8

[Cu(L_O_L2)(CH CN)]PF was prepared with L_O_L2 (100 mg,

3 6

3

(

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),

0.482 mmol) and tetrakis(acetonitrile)copper(I) hexaﬂuoro-

phosphate (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 C H CuF N P: C

1

28.1 (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–

1

15

6

5

(

C H ) ), 60.6 (CH , 1C, –CH –CH –N=), 36.0 (C, 1C,

(34.18 %); H (4.41 %); N (12.26 %); found C (34.20 %);

6

6 3

2

1

=

CH–C(CH ) ), 30.2 (CH , 1C, –CH –CH –N=), 27.1 (C,

H (4.53 %); N (12.33 %). H-NMR (CD CN, 400 MHz):

3

2

3

C, =CH–C(CH ) ) ppm.

δ = 7.67 (s, 1H, imidazole H-2), 6.94 (s, 1H, imidazole

3

H-5), 4.55 (t, J = 9.2 Hz, 1H, –O–CH –CH–N–), 4.18–

2

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

4.10 (m, 1H, –O–CH –CH–N–), 3.89 (t, J = 9.1 Hz, 1H,

2

–

O–CH –CH–N–), 2.86 (d, J = 15.3 Hz, 1H, imidazole–

2

Bis(1-methylimidazol-2-yl)methane was prepared accord-

ing to White et al. [35]. Anal. calcd. for C H N : C

(

CH –CH–), 2.55 (dd, J = 23.1, 8.2 Hz, 1H, imidazole–

2

CH –CH–), 1.96 (s, 3H, –NCCH ), 1.34 (s, 3H, –C(CH ))

9

12

4

2

3

1

3

61.34 %); H (6.86 %); N (31.79 %); found C (61.70 %);

ppm. C-NMR (CD CN, 100 MHz): δ = 177.6 (C, 1C, –

3

1

H (7.26 %); N (31.76 %). H-NMR (CDCl , 400 MHz):

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

(CH , 1C, –O–CH –CH–N–), 67.1 (CH, 1C, –O–CH –CH–

3

δ = 6.89 (s, 2H, H-4), 6.76 (s, 2H, H-5), 4.21 (s, 2H, –CH ),

2

1

3

.64 (s, 6H, –CH ) ppm. C-NMR (CDCl , 100 MHz):

3 3

2

δ = 143.6 (C, 2C, C-2), 127.3 (CH, 2C, C-4), 121.6 (CH,

N–), 34.6 (C, 1C, –C(CH ) ), 32.9 (CH , 1C, –O–CH –

3 3 2 2

CH–N–), 28.3 (CH , 1C, –C(CH ) ) ppm.

3 3 3

2

C, C-5), 33.2 (CH , 2C, CH ), 27.0 (CH, 1C, CH ) ppm.

3

2

1

3

J Biol Inorg Chem

Synthesis of [Cu(L_O_L3)(CH CN)]PF₆

3

[

Cu(L_O_L3)(CH CN)]PF was prepared with L_O_L3 (100 mg,

3 6

0

.605 mmol) and tetrakis(acetonitrile)copper(I) hexaﬂuoro-

phosphate (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 C H CuF N P: C

11 15 6 5

(

28.96 %); H (3.40 %); N (13.51 %); found C (28.81 %);

1

H (3.43 %); N (13.34 %). H-NMR (CD CN, 400 MHz):

3

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 –CH–N–), 4.11–

2

4

–

.04 (m, 1H, –O–CH –CH–N–), 3.97 (t, J = 8.8 Hz, 1H,

2

Scheme 1 Synthesis of ligands L_O_L1‑3 and their corresponding

copper(I) complexes CuL_O_L1, CuL_O_L2 and CuL_O_L

O–CH –CH–N–), 2.88 (d, J = 14.9 Hz, 1H, imidazole–

3

2

CH –CH–), 2.52 (dd, J = 14.0, 12.1 Hz, 1H, imidazole–

2

CH –CH–), 2.09 (s, 3H, –CH ), 1.96 (s, 3H, –NCCH )

2

3

1

3

ppm. C-NMR (CD CN, 100 MHz): δ = 169.2 (C, 1C, –

3

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 (CH ,

2

1

C, –O–CH –CH–N–), 66.1 (CH, 1C, –O–CH –CH–N–),

2 2

3

3.1 (CH , 1C, imidazole–CH –CH–), 14.5 (C, 1C, –CH )

2 2 3

ppm.

Synthesis of [Cu(L_i_m_z1)(CH CN) ]PF

6

3

2

[

Cu(L_i_m_z1)(CH CN) ]PF was prepared with L_i_m_z1

3 2 6

(

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 pow-

der (159 mg, 0.223 mmol, 94 %). Anal. calcd. for

C H CuF N P: C (55.50 %); H (5.50 %); N (9.81 %);

1

15

6

5

1

found C (55.11 %); H (5.32 %); N (9.95 %). H-NMR

(

CD CN, 400 MHz): δ = 7.62 (s, 1H, –N=CH–C(CH )),

3

7

3

.51 (s, 1H, imidazole H-2), 7.42–7.35 (m, 9H, Ar–H),

.20–7.11 (m, 6H, Ar–H), 6.79 (s, 1H, imidazole H-5),

.64 (dd, J = 10.5, 5.2 Hz, 2H, –CH –CH –N–), 2.70 (dd,

Scheme 2 Synthesis of ligand L_i_m_z1 and its corresponding copper(I)

complex CuL_i_m_z

1

2

J = 15.1 Hz, 9.6 Hz, 2H, –CH –CH –N–), 1.96 (s, 6H,

2

1

3

CH CN), 1.09 (s, 9H, –C(CH ) ) ppm. C-NMR (CD CN,

C H CuF N P: C (31.03 %); H (3.55 %); N (16.45 %);

11 15 6 5

3

3 3

3

1

00 MHz): δ = 177.2 (CH, 1C, –N=CH–C(CH ) ), 141.6

found C (31.29 %); H (3.56 %); N (16.53 %). H-NMR

3

(

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),

(Acetone-d , 400 MHz): δ = 7.41 (s, 2H, H-4), 7.13 (s,

6

2H, H-5), 4.79 (s, 2H, –CH ), 3.88 (s, 6H, –CH ), 2.08 (s,

2

3

1

3

1

29.3 (CH, 3C, phenyl C-4), 129.2 (CH, 6C, phenyl C-2,

3H, –NCCH ) ppm. C-NMR (Acetone-d , 100 MHz):

3

6

C-6), 118.3 (CH, 1C, imidazole C-5), 76.9 (C, 1C, –N–C–

δ = 145.1 (C, 2C, C-2), 127.9 (CH, 2C, C-4), 124.2 (CH,

2C, C-5), 117.5 (C, 1C, –NCCH ), 34.2 (CH , 2C, CH ),

(

=

C H ) ), 62.4 (CH , 1C, –CH –CH –N=), 36.6 (C, 1C,

6

6 3

2

3

CH–C(CH ) ), 29.6 (CH , 1C, –CH –CH –N=), 27.4 (C,

25.8 (CH, 1C, CH ) ppm.

3

2

3

C, =CH–C(CH ) ) ppm.

3

Synthesis of [Cu(L_O_L1) ](PF )

2

6 2

Synthesis of [Cu(BIMZ)(CH CN)]PF

3

6

[

Cu(L_O_L1)(CH CN)]PF (35.7 mg, 750 µmol) was dis-

3 6

[

(

Cu(BIMZ)(CH CN)]PF₆was prepared with BIMZ

100 mg, 0.567 mmol) and tetrakis(acetonitrile)copper(I)

solved in dry dichloromethane (25 mL). Injection of dioxy-

gen at −78 °C for 30 min and subsequent warming to room

temperature leads to a blue solution. After 3 weeks, tur-

quoise crystals were obtained. The single crystals were of

high quality and suitable for X-ray structure determination.

3

hexaﬂuorophosphate (212 mg, 0.567 mmol) as described

previously. The product was obtained as a white pow-

der (232 mg, 0.545 mmol, 96 %). Anal. calcd. for

1

3

J Biol Inorg Chem

+

MS (ESI , acetone): m/z (%) = 517.249 [2 L 1 + Cu] .

OL

Anal. calcd. for C H CuF N O P : C (38.65 %); H

2

6

26

12

6

2 2

(

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 hydrochlo-

ride [38] or ethyl acetimidate hydrochloride in the pres-

ence of sodium methoxide leads to the formation of three

new ligands L_O_L1‑3 (Scheme 1). Subsequent reaction with

tetrakis(acetonitrile)copper(I) hexaﬂuorophosphate gener-

ates the corresponding copper(I) complexes CuL_O_L1‑3.

The ligand L_i_m_z1 was prepared by an imine condensa-

tion reaction of 2-(1-trityl-1H-imidazol-4-yl)-ethyl amine

Fig. 4 UV/Vis absorption spectra obtained upon oxidation

500 µM solution of CuBIMZ in dichloromethane in the presence of

a

1

0 eq. 3,5-DTBC-H during 69 h; path length (l) l = 1 mm

2

[

37] with trimethylacetaldehyde (Scheme 2). Reaction with

tetrakis(acetonitrile)copper(I) hexaﬂuorophosphate leads to

the corresponding copper(I) complex CuL_i_m_z1. In contrast

to CuL_O_L1‑3 with one acetonitrile as co-ligand, CuL_i_m_z

1

contains two acetonitrile molecules as co-ligands.

Bis(1-methylimidazol-2-yl)methane (BIMZ) was syn-

thesized 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) com-

plexes CuL_O_L1, CuL_O_L2, CuL_O_L3, CuL_i_m_z1, 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 reac-

tion was monitored by UV/Vis spectroscopy based on the

characteristic absorption band of 3,5-DTBQ at λ = 400 nm

Fig. 5 Dependence of the reaction rates on the formation of 3,5-

^D^T^B^S^Q^c^a^t^a^l^y^z^e^d^b^y^C^u^LOL¹^‑³^,^C^u^Limz1 and CuBIMZ, varying the

substrate concentration. Reactions were performed in dichlorometh-

ane saturated with O₂

and the resulting solution was extracted twice with CH Cl

2

to eliminate all existing copper species. After removing

1

the solvent, the resulting residue was characterized by H-

−1

13

(

ε = 1900 L mol cm ) [42]. 3,5-DTBC-H is well suited

NMR and C-NMR spectroscopy.

2

for catechol oxidase studies [43, 44] due to its stability, low

redox potential [45–48] and the presence of bulky func-

tional groups precluding further reactions. Consequently, it

has been used in many model studies of CO as a substrate

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].

[

49–55].

Catalytic runs were performed by anaerobically mixing

00 µM of the respective Cu(I) complexes in dichlorometh-

5

ane with 10 eq. of 3,5-DTBC-H and subsequent injection

After initial formation of 3,5-DTBSQ, subsequent oxi-

dation to 3,5-DTBQ was observed, as evident from the for-

mation of a band with a single maximum at λ = 400 nm.

2

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,

Complete conversion of 3,5-DTBC-H to 3,5-DTBQ was

2

also validated by NMR spectroscopy (Figs. S2 and S3).

1

3

J Biol Inorg Chem

Table 1 Kinetic parameters for

the oxidation of 3,5-DTBC-H₂

determined from Lineweaver–

Burk plots

−1

k_c_a_t(min )

Complex

K_M(mol L

)

v_m_a_x(mol L min

)

−3

−4

−5

−4

−6

−4

−6

−7

−2

−3

−4

CuL_O_L

1

2

3

3.01 × 10 (± 2.50 × 10

)

2.23 × 10 (± 4.53 × 10

)

22.3 × 10 (± 4.53 × 10

)

−3

−4

−2

4.19 × 10 (± 8.60 × 10

1.11 × 10 (± 9.65 × 10

11.1 × 10 (± 9.65 × 10

−4

−2

7.43 × 10 (± 2.12 × 10

2.22 × 10 (± 1.07 × 10

22.2 × 10 (± 1.08 × 10

−3

−5

−3

CuL_i_m_z

1

1.04 × 10 (± 4.47 × 10

3.56 × 10 (± 3.35 × 10

3.56 × 10 (± 3.36 × 10

CuBIMZ 3.47 × 10⁻(± 9.01 × 10

4

1.26 × 10 (± 2.39 × 10

−4

12.6 × 10 (± 2.42 × 10

−2

In the next step, the formation of 3,5-DTBSQ was inves-

tigated kinetically applying Michaelis–Menten theory. To

this end, different concentrations of the substrate DTBC-

−

3

−2

H (1.5 × 10 –4 × 10 M) were prepared in dioxygen-

2

−

3

saturated CH Cl and a 1 × 10 M solution of each Cu(I)

2

complex was added. In agreement with typical enzyme

kinetics, the resulting curves exhibit saturation characteris-

tics (Fig. 5).

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.

The kinetic studies allow correlations between catecho-

lase activity and structural parameters. The three oxazo-

line ligands differ in their substituents, the sterical demand

ⁱⁿ^c^r^e^a^sⁱⁿ^g^f^r^o^m^m^e^t^h^y^l⁽^LOL³⁾^o^v^e^r^p^h^eⁿ^y^l⁽^LOL1) to tert-

butyl (L_O_L2). In agreement with these considerations, the

values of v_m_a_xand k_c_a_treﬂect that CuL_O_L1 and CuL_O_L3 are

the most active catalysts. The copper center of CuL_O_L3 is

hardly shielded by the methyl residue such that 3,5-DTBC-

Fig. 6 Dependence of the reaction rates for the formation of 3,5-

DTBSQ catalyzed by CuL_O_L1‑3, CuL_i_m_z1 and CuBIMZ on the com-

plex concentration. The reactions were performed in dichloromethane

^s^a^t^u^r^a^t^e^d^wⁱ^t^h^O2

H can bind without steric hindrance. The phenyl unit in

2

CuL_O_L1 is more bulky. However, due to its ﬂexibility only

a slightly higher value of K is observed. The much bulkier

M

tert-butyl group in CuL_O_L2, in contrast, leads to signiﬁ-

cantly lower values for v_m_a_xand k . Compared to CuL_O_L1

cat

and CuL_O_L3, CuL_i_m_z1 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

−

5

−3

complex concentration (7.6 × 10 –1 × 10 M) was

−

2

studied, using an excess of substrate (2 × 10 M). Impor-

tantly, a linear dependence was found, implying that the

reaction rate is ﬁrst order in the copper complex (Fig. 6 and

Fig. S5).

In addition to the characteristic double band of semiqui-

none, formation of an intermediate with two optical absorp-

tion 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

Fig. 7 UV/Vis absorption spectra obtained upon oxidation a 500 µM

solution of CuL_O_L1 and 1 eq. 3,5-DTBC-H in dichloromethane satu-

2

rated with O . Top optical spectra during the ﬁrst 25 min; bottom opti-

2

cal spectra from 25 to 600 min; l = 1 mm

1

3

J Biol Inorg Chem

radical complex, which was evidenced during the cata-

lytic oxygenation of 4-tert-butylphenol mediated by a

Cu(DBED) complex [25]. Importantly, this complex exhib-

its 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 sin-

gle crystal structure determination.

We thus conclude that copper(II) semiquinone com-

plexes are also formed as intermediates during the catechol

oxidase reactions catalyzed by our complexes. For the

CuL_O_L1 system, the absorption bands of the semiquinone

complex were found to be most intense. To spectroscopi-

cally monitor the buildup and the decay of [Cu(II)(L_O_L1)

+

(

3,5-DTBSQ)] by UV/Vis spectroscopy, the complex was

generated in situ. Therefore, a 500 µM solution of CuL_O_L

1

was added to 1 eq. 3,5-DTBC-H dissolved in dichlo-

2

romethane saturated with O gas (Fig. 7).

2

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 yel-

low to purple. Subsequent decay of the semiquinone com-

plex leads to a brown solution.

Fig. 8 Postulated mechanism for the conversion of 3,5-DTBC-H to

2

3

,5-DTBQ by different copper(I) complexes

To obtain further information regarding the constitu-

tion of the Cu(II) semiquinone intermediate, an independ-

ent synthesis of [Cu(II)(L_O_L1)(3,5-DTBSQ)]PF₆was

attempted at −60 °C. Isolation of this complex was, how-

ever, unsuccessful due to its instability at room tempera-

ture. 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 path-

way (Fig. 8).

Fig. 9 Red Time-dependent formation and decay of [Cu(II)(LOL1)

+

(

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

Starting with the respective copper(I) complex and

3

,5-DTBC-H , injection of dioxygen leads to the corre-

of copper complex (see above) a mononuclear mechanism

(and consequently, the formation of H O ) is more likely.

2

sponding copper(II) semiquinone intermediate with the

2

rate constant k . The copper(II) semiquinone complex is

However, no H O could be detected. This could be due to

cat

2 2

in equilibrium with free semiquinone and a copper(II) spe-

the fact that all copper(I) complexes exhibit catalase activ-

ity (Fig. S8).

cies (equilibrium constant K ) whence formation of the

E

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 time-dependent formation and decay of [Cu(II)

+

(L_O_L1)(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 com-

plex was determined based on its absorption band at

The oxidation of the Cu(I) complex and the catechol can

−1

in principle occur under generation of H O or H O . Due to

λ = 900 nm (ε = 5890 L mol cm ), because an addi-

tional band at λ = 560 nm (related to 3,5-DTBQ) did not

2

linear dependence of the reaction rate on the concentration

1

3

J Biol Inorg Chem

allow a quantitative determination of the absorbance at

−

1

−1

λ = 523 nm (ε = 10,830 L mol cm ). The concentration

of 3,5-DTBSQ as inferred from the band at λ = 385 nm

−

1

−1

(

ε = 1900 L mol cm ) exhibits saturation behavior,

because the subsequent conversion to 3,5-DTBQ leads to

an absorption band at about the same position with a com-

parable intensity.

Based on the given ε-values it can be inferred that

+

3

,5-DTBSQ and the [Cu(II)(L_O_L1)(3,5-DTBSQ)] com-

plex are in equilibrium with a ratio of about 10:1. The

initial slope of the buildup curve of 3,5-DTBSQ reveals

−

5

−1

a reaction rate of 3.8 × 10 mol L min . Based

on the Michaelis–Menten equation a reaction rate of

−5

−1

3

.1 × 10 mol L min is calculated for the formation

Fig. 10 UV/Vis absorption spectra obtained upon oxygenation a

500 µM solution of CuL_i_m_z1 in dichloromethane in the presence of

_a5_n0_de _Tq ._O 2 _F, 4-DTBP-H and 100 eq. triethylamine; l = 1 mm. Insert TON

of 3,5-DTBSQ mediated by CuL_O_L1 with the parameters

given in Table 1 (Fig. S10).

Furthermore, the formation of the [Cu(II)(L_O_L1)

+

(

3,5-DTBSQ)] complex as calculated from the ini-

tial slope (Fig. S10) results in a reaction rate of

−

6

−1

5

.3 × 10 mol L min , about one tenth of that lead-

ing to 3,5-DTBSQ. This corresponds to the ratio of maxi-

mum concentrations of free 3,5-DTBSQ and [Cu(II)(L_O_L1)

+

(

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

−

2

−1

rate constant of k′ = 1.8 × 10 min (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] phe-

nols 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 solu-

tion of the respective Cu(I) complex (CuL_O_L1‑3, CuL_i_m_z1,

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 inves-

tigated regarding their ability to serve as model systems for

tyrosinase. Importantly, only CuL_i_m_z1 and CuBIMZ were

found to both stoichiometrically and catalytically convert a

range of phenols to ortho-quinones, whereas all three oxa-

zoline-based copper(I) complexes (CuL_O_L1‑3) just medi-

ate 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.

Scheme 3 Schematic illustration of different substrates oxygenated

by CuL_i_m_z1 and CuBIMZ

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_i_m_z1 leads to the formation of 3,5-DTBQ with an

−

1

−1

absorption band at λ = 403 nm (ε = 1830 L mol cm )

[19] (Fig. 10; Scheme 3).

Oxygenation was complete after about 3 h, resulting

in a turnover number of 16. Conversion of the same sub-

strate by CuBIMZ leads to the appearance of an absorp-

tion band with two maxima at λ = 378 and 396 nm during

1

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J Biol Inorg Chem

Fig. 11 UV/Vis absorption spectra obtained upon oxygenation a

Fig. 12 UV/Vis absorption spectra obtained upon oxygenation a

500 µM solution of CuL_i_m_z1 in dichloromethane in the presence of

_a5_n0_de _Tq ._O 4_F -MeOP-H and 100 eq. triethylamine; l = 1 mm. Insert TON

5

00 µM solution of CuL_i_m_z1 in dichloromethane in the presence of

0 eq. 3-TBP-H and 100 eq. triethylamine; l = 1 mm. Insert TON

5

and TOF

cyclohexa-3,5-diene-1,2-dione [21, 22] with a TON of

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 forma-

tion of which is completed after 6 h with a TON of 9. The

resulting NMR spectra (Fig. S13 and S14 for CuL_i_m_z1; 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_i_m_z1 and 5:42:53 for CuBIMZ.

For both the CuL_i_m_z1 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 num-

ber, 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_i_m_z1 was accomplished. The UV/Vis spectra show

no absorption band at 600 nm after completion of the reac-

tion (Fig. S17) but the same turnover number is achieved

2

0 for CuL 1 and a TON of 6 for CuBIMZ (ε = 898

imz

−1

L mol cm at λ = 425 nm). The corresponding NMR

spectra (Figs. S19 and S20 for CuL 1; Figs. S21 and S22

imz

for CuBIMZ) validate the presence of the coupled ortho-

quinone besides unreacted 3-TBP-H (Scheme 3). Impor-

tantly, no C–C coupling product was detected. The forma-

tion 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

-MeOP-H to the coupled quinone 4-methoxy-5-(4-meth-

oxyphenoxy)cyclohexa-3,5-diene-1,2-dione was observed

4

(Fig. 12).

^Iⁿ^t^h^e^c^a^s^e^o^f^C^u^Limz1, oxygenation with subsequent

reaction to the coupled product proceeds with a TON of 34

−

1

−1

(

ɛ = 524 L mol cm , λ = 418 nm) after 60 min. NMR

spectra (Figs. S23, S24) conﬁrm the existence of the cou-

pled 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-qui-

none 2-hydroxy-5-methoxy-[1, 4] benzoquinone which was

also detected by NMR spectroscopy (Fig. S25 and S26)

(

TON = 16). This leads to the conclusion that triethyl-

amine 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 unreac-

tive end product does not limit the activity of the catalytic

system.

[60].

The next investigated substrate was 3-tert-butylphenol

Oxygenation of 4-MeOP-H with CuBIMZ also leads

(

3-TBP-H). Oxygenation of 3-TBP-H with CuL_i_m_z1 and

to rapid emergence of an absorption band at λ = 418 nm

which saturates after 40 min (Fig. S27). Further oxygena-

tion generates an additional absorption band at approxi-

mately 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

CuBIMZ ﬁrst leads to a band at λ = 400 nm the maxi-

mum of which subsequently shifts to λ = 425 nm where

it stays until saturation (Fig. 11 for CuL 1; Fig. S18 for

imz

CuBIMZ).

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)

1

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J Biol Inorg Chem

Fig. 14 Quantiﬁcation of the conversion of different monophenols to

the corresponding ortho-quinones

Fig. 13 UV/Vis absorption spectra obtained upon oxygenation a

5

00 µM solution of CuL_i_m_z1 in dichloromethane in the presence of

0 eq. NATEE and 100 eq. triethylamine; l = 1 mm. Insert decrease

of the absorption band after 4 h

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_i_m_z1 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_i_m_z1 as compared to BIMZ which facilitates formation of

the ternary intermediate required for hydroxylation of the

substrate (see above) [2, 17–19, 64].

To expand the scope of potential substrates, 8-hydrox-

yquinoline was also examined [65]. Following the stand-

ard oxygenation protocol, both CuL_i_m_z1 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 prod-

uct. To obtain more information on this issue, the reac-

tion product was isolated by ﬂash chromatography with

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 prod-

ucts, 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 can-

not be considered as a selective catalyst for the oxygena-

tion of 4-MeOP-H.

The substrate N-acetyl-l-tyrosine ethyl ester monohy-

drate (NATEE) provides an analog to the native substrate

tyrosine, and therefore, is of signiﬁcant interest regarding

model systems of tyrosinase. The conversion to the cor-

responding dopaquinone mediated by CuL_i_m_z1 proceeds

with a maximal TON of 25 (ɛ = 1188 L mol cm ,

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 absorp-

tion 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].

For further identiﬁcation of the primary oxygena-

tion product N-acetyl-l-dopa ethyl ester dichlorometh-

ane 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 hydroxyla-

tion. 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

−1

dichloromethane (R = 0.31). The corresponding NMR

f

spectra also exclude the formation of quinone (Figs. S36

and S37). Due to the high tendency of 8-hydroxyqui-

noline to form stable metal complexes we conclude that

no hydroxylation occurred. Instead, the ligands L_i_m_z

1

and BIMZ are replaced by 8-hydroxyquinoline to form

the homoleptic square planar complex [Cu(II)(8-quin-

olinol) ]. The identity of the product was clariﬁed via

2

mass spectrometry m/z = 415.09 and powder diffrac-

tometry; the measured pattern is in full agreement with

the calculated diffraction pattern based on the published

structure (Fig. S38) [66].

1

3

J Biol Inorg Chem

µ‑Peroxo and other Cu(II) species

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 coor-

dinated by each two N-atoms of two L_O_L1 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)°.

2

The µ-η :η -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 neces-

sary 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 charac-

2

teristic side-on geometry (µ-η :η ) [12, 22, 65]. The intense

absorption band between λ = 340–380 nm is due to an

in-plane π * → d

2

charge transfer transition, and the

σ

x −y

less intense absorption feature between λ = 510–580 nm to

an out-of-plane π * → d

2

peroxo to copper(II) charge

Summary and conclusion

v

x −y

transfer transition [1, 2, 20].

To obtain spectroscopic information regarding these

intermediates, 3 mM solutions of each copper(I) complex

Five new small molecule model systems (CuL_O_L1‑3,

CuL_i_m_z1 and CuBIMZ) have been synthesized and inves-

tigated 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

(

CuL_O_L1‑3, CuL_i_m_z1 and CuBIMZ) in acetone and dichlo-

romethane were cooled to −85 °C under nitrogen atmos-

phere. After injection of O gas intense optical absorption

2

bands between λ = 333 and 357 nm were observed in the

these copper(I) complexes oxidize 3,5-DTBC-H to the

2

UV/Vis spectra (Fig. S39). The position of the maxima

corresponding quinone (Fig. 16). A copper(II) semiquinone

species and free 3,5-DTBSQ have been identiﬁed as inter-

mediates during these reactions. Kinetic measurements for

the formation of semiquinone applying Michaelis–Menten

approach revealed a sequence of activity in the order

CuL_O_L3 ≈ CuL_O_L1 > CuBIMZ > CuL_O_L2 > CuL_i_m_z1.

Whereas CuL_i_m_z1 and CuBIMZ also exhibit catalytic

tyrosinase activity, the copper(I) complexes supported by

the ligands L_O_L1‑3 only mediate stoichiometric conver-

sions 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 tyrosi-

2

differs only slightly. Speciﬁcally, the µ-η :η -peroxo-

dicopper(II) complex of CuL_O_L1 and CuL_O_L3 exhibit

LMCT bands at λ = 357 nm and λ = 335 nm in acetone

which can be associated with in-plane π * → d

2

σ

x −y

charge transfer transitions. Additional absorption bands at

λ = 600 nm in the case of CuL 1 and λ = 510 nm in the

OL

case of CuL 3 are due to out-of-plane π * → d

2

per-

OL

v

x −y

oxo to copper(II) charge transfer transitions. CuL_O_L2 and

CuBIMZ were measured in dichloromethane and exhib-

ited absorption maxima at λ = 340 nm and λ = 610 nm

for CuL 2 and λ = 333 nm as well as λ = 600 nm for

OL

2

CuBIMZ. The slight shift of the π * → d

2

charge

nase model. Importantly, µ-η :η -peroxo intermediates of

CuL_O_L1‑3 and CuBIMZ could be detected, whereas this

was not possible for the most active tyrosinase catalyst,

CuL_i_m_z1. These ﬁndings provide evidence for the hypoth-

esis that the reaction pathway proceeds via side-on peroxo

intermediates [12, 19, 69].

σ

x −y

transfer transition to higher energies was also noticed for

copper(I) complexes with bidentate bis(oxazoline) ligands

2

[

67]. In contrast, no µ-η :η -peroxo-dicopper(II) complex

could be found for CuL_i_m_z1 in tetrahydrofuran, dichlo-

romethane, 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_i_m_z1 is the most active tyrosinase

catalyst described in this paper.

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_i_m_z1 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.

In an attempt to generate the peroxide core for CuL_O_L

1

in dichloromethane no characteristic absorption band was

obtained after injection of O . Instead, blue single crys-

From a biomimetic perspective, the reactivity with

regard to the substrate NATEE was of particular interest.

Surprisingly, only CuL_i_m_z1 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

2

tals, suitable for single crystal structure determination were

obtained from the solution within 3 weeks. The resulting

complex [Cu(L_O_L1) ](PF ) crystallizes in the orthorhom-

2

6 2

bic space group P2 2 2 with four formula units per unit

1

1 1

cell and all atoms in general positions (Fig. 15).

1

3

J Biol Inorg Chem

An irreversible deactivation was observed during the

reaction of both copper(I) complexes with 8-hydroxyqui-

noline. The high tendency of 8-hydroxyquinoline to gen-

erate stable metal complexes led to a displacement of the

respective ligands, resulting in the homoleptic [Cu(II)

(

[

8-quinolinol) ] complex. From the homoleptic complex

2

Cu(L_O_L1) ](PF ) , we presented a new crystal structure.

2

6 2

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 cata-

lytic 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 par-

ticular, the donor/acceptor properties of the employed het-

erocyclic units.

Fig. 15 Crystal structure of [Cu(II)(L_O_L1) ](PF ) ; H atoms are

2

6 2

omitted for clarity. Color code: C gray; N blue; O red; Cu turquoise

Acknowledgments We express our gratitude to Deutsche Forschun-

gsgemeinschaft (DFG), CAU Kiel and COST CM 1003 for support of

this research. Thanks to Miriam Schehr for the introduction to operate

with the Isolera One fabricated by Biotage, Marcel Dommaschk for

the help measuring the ﬂuorescence spectra and Michael Wendt for

performing the XRPD measurements.

Compliance with ethical standards

Conﬂict of interest Authors declare that there are no conﬂicts of

interests.

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Article Doi

Tyrosinase and catechol oxidase activity of copper(I) complexes supported by imidazole-based ligands: structure–reactivity correlations

DOI: 10.1007/s00775-016-1370-y

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Full text of DOI:10.1007/s00775-016-1370-y

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