New functional models for catechol oxidases

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Inorganica Chimica Acta 300–302 (2000) 442–452

New functional models for catechol oxidases

Pavel Gentschev, Niclas Moller, Bernt Krebs *Anorganisch-Chemisches Institut der Uni6ersitat Munster, Wilhelm-Klemm-Straße 8, D-48149 Munster, Germany

Received 23 September 1999; accepted 16 November 1999

Abstract

The new dinuclear copper(II) complexes [Cu2(L1)(m-OAc)](ClO4)2·CH3CN (1), [Cu2(L2)(CH3CN)2](ClO4)4·C2H5OH (2),[Cu2(L3)2(CH3CN)2](PF6)2 (3) and [Cu2(L4)2](PF6)2·2CH2Cl2 (4) were prepared with the ligands N,N,N %,N %-tetrakis((N-2-hydroxy-ethyl)-2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane HL1, N,N,N %,N %-tetrakis (2-methylimidazolyl)-2-hydroxy-1,3-di-aminopropane HL2, (2-pyridylmethyl)(1-hydroxypropyl)amine HL3 and (2-hydroxybenzyl)(N,N-dimethylpropyl)amine HL4. Allcomplexes were characterized by X-ray structure determination, revealing dinuclear cations and perchlorate or hexafluorophos-phate counter ions. In the complex cations the two copper(II) atoms show different coordination spheres with N4O, N3O2 or N2O2

donor sets and different bridging systems. The Cu···Cu distances vary from 2.918(2) to 4.756(2) A, . The catalytic performance ofthe oxidation of 3,5-di-tert-butylcatechol to quinone was studied using UV–Vis absorption spectra methods. Complex 3 exhibitsthe highest activity with a turnover number of 32 h−1 while the other compounds show lower rates of oxidation. A kinetictreatment on the basis of the Michaelis–Menten model was applied. © 2000 Elsevier Science S.A. All rights reserved.

Keywords: Crystal structures; Copper(II) complexes; Dinuclear complexes; Catechol oxidase; Model complexes; Catecholase activity

1. Introduction

The oxidation of organic substrates with molecularoxygen under mild conditions is of great interest forindustrial and synthetic processes both from an eco-nomical and environmental point of view [1].

Although the reaction of organic compounds withdioxygen is thermodynamically favoured it is kineticallyhindered due to the triplet ground state of O2. Thesynthesis and investigation of functional model com-plexes for metalloenzymes with oxidase or oxygenaseactivity is therefore of great promise for the develop-ment of new and efficient catalysts for oxidationreactions.

The catechol oxidases (EC 1.10.3.1) are type 3 copperenzymes containing a dinuclear copper centre [2]. Well-known representatives of these type 3 copper proteinsare hemocyanin [3,4], the dioxygen carrier forarthropods and mollusks, and tyrosinase [5]. Catecholoxidase belongs, like tyrosinase, to the polyphenol oxi-dases which oxidize phenolic compounds to the corre-

sponding quinones in the presence of oxygen. Whereastyrosinase (EC 1.14.18.1) catalyzes the hydroxylation oftyrosine to dopa (cresolase activity) and the oxidationof dopa to dopaquinone (catecholase activity) withelectron transfer to dioxygen, catechol oxidase exclu-sively catalyzes the oxidation of catechols to quinoneswithout acting on tyrosine [6]. This reaction is of greatimportance in medical diagnosis for the determinationof the hormonally active catecholamines adrenaline,noradrenaline and dopa [7].

Besides other catechol oxidases from different plantsources a catechol oxidase from sweet potatoes (Ipo-moea batatas) was purified and crystallized recently.The enzyme is a monomer with a molecular mass of 39kDa and was characterized by several spectroscopicmethods [8]. EXAFS data revealed a coordination num-ber of four for each copper atom and suggested aCu···Cu distance of 2.9 A, for the native met-form [9].The sequence of the protein was determined showingcertain homologies to the primary structures of otherdinuclear copper proteins with a single type 3 site suchas tyrosinase and hemocyanin [9]. As reported recentlythe crystal structure of the Cu(II)–Cu(II) form shows amono-hydroxo-bridged Cu(II)–Cu(II) active site with

* Corresponding author. Tel.: +49-251-833 3131; fax: +49-251-833 8366.

E-mail address: krebs@nwz.uni-muenster.de (B. Krebs)

0020-1693/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.

PII: S 0 0 2 0 -1693 (99 )00553 -8

P. Gentsche6 et al. / Inorganica Chimica Acta 300–302 (2000) 442–452 443

both copper atoms in distorted (m-OH)Cu(His)3 coordi-nation spheres.

There have been large efforts to synthesize modelcomplexes which could be seen as functional or struc-tural models for catechol oxidases or related coppercontaining enzymes [10–19]. The first systematical ex-amination of catecholase activity of copper complexeswas carried out by Nishida and co-workers [12]. Theyreported that mononuclear square-planar complexes ex-hibit only little catalytic activity while non-planarmononuclear copper(II) complexes show a high cata-lytic potential. Dinuclear complexes also catalyze theoxidation process if the Cu···Cu distance is less than 5A, . A steric match between substrate and complex isbelieved to be the determining factor: two metal centreshave to be located in close proximity to facilitate bind-ing of two hydroxyl oxygen atoms of catechol prior tothe electron transfer [12]. This theory is supported bythe observation that dinuclear copper complexes aregenerally more reactive towards the oxidation of cate-chols than corresponding mononuclear species. Al-though some general correlations of structure-reactivitypatterns have been found, the exploration of the oxida-tion chemistry of structurally well-characterized coppercomplexes is still needed to fully understand theparameters affecting their catecholase activity.

Most of the complexes synthesized so far containligands with N-donor groups like pyridine, imidazole orbenzimidazole. These polydentate ligands vary frombidentate to heptadentate systems with phenoxy oralkoxy bridging or even without endogenous bridginggroups [20]. We have previously reported that dinuclearcopper(II) complexes with pentadentate ligands showthe ability to form Cu(II)-peroxo species as well as highcatecholase activity [13,14]. Herein we report the syn-thesis, structure and characterization of dinuclear cop-per(II) complexes with new tri- and heptadentateligands as shown in Figs. 1 and 2. The design of thecomplexes is based on the natural system, the catecholoxidase, and on known structure-reactivity relation-ships. Therefore, the new ligands model the coordina-tive aspect in metalloenzymes and their function asoxidation catalysts excellently. In this report wechanged the bridging groups affording different coordi-nation moieties of the copper atoms and differentCu···Cu distances, respectively. We could observe aremarkable reactivity of these complexes towards theoxidation of 3,5-di-tert-butylcatechol attempting tomodel the activity of catechol oxidase and to obtainnew information on the mechanism of the catalyticoxidation of catechols.

Fig. 1. The heptadentate ligands HL1, HL2 and the tridentate ligands HL3, HL4.

Fig. 2. Oxidation of 3,5-di-tert-butylcatechol with dinuclear copper(II) complexes.

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2. Experimental

2.1. Materials

All chemicals were of reagent grade and used asreceived.

2.2. Physical measurements

IR absorption spectra were obtained as KBr pelletsor films on a Bruker IFS 48 spectrophotometer in therange of 4000–400 cm−1. 1H NMR were measured ona Bruker WH300 spectrometer and 13C NMR spectrawere recorded on a Bruker AC200P spectrometer. Theelectronic absorption spectra and time-dependent UV–Vis spectroscopy were recorded on a Hewlett-Packard8453 diode-array spectrophotometer using quartz cu-vettes (1 cm) and acetonitrile as solvent (c=2×10−4

mol l−1). Elemental analyses were carried out at theOrganisch-Chemisches Institut der Universitat Munsteron a Heraeus CHN-O-RAPID analyzer. Magnetic sus-ceptibilities of powdered samples were measured on aQuantum Design MPMS SQUID magnetometer overthe temperature range from 5 to 300 K. The appliedmagnetic field was about 1.5 T. The experimental sus-ceptibility data were corrected for the underlying dia-magnetism in the usual manner using Pascal’sconstants. From the magnetic susceptibility the effectivemagnetic moment meff was derived following meff[mB]=8xT.

The reaction of the complexes 1–4 with 3,5-di-tert-butylcatechol (3,5-DTBC) was monitored as follows: 1ml of a 2×10−4 mol l−1 solution of complex inacetonitrile were treated with 1 ml of a 0.01 mol l−1

solution of 3,5-DTBC in acetonitrile. Directly afteraddition of 3,5-DTBC and after 3, 6, 9, 12, 15 and 18min the UV–Vis spectra were recorded.

2.3. Ligand synthesis

2.3.1. Synthesis of N,N,N %,N %-tetrakis-((N-2-hydroxyethyl)2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane HL1

2.3.1.1. o-Nitro-N-(2-hydroxyethyl)aniline. The prepa-ration was performed by a method described by Mc-Manus and Herbst [21].

To a solution of 112.00 ml (1.86 mol) ethanolamineand 18.84 g (0.09 mol) o-nitrobromobenzene wereadded 3.09 g (0.23mol) of copper(II) chloride. Thesolution was heated at 90°C for 2 h. The hot mixturewas poured into ice and the obtained red precipitatewas dried in vacuo to afford 17.00 g (0.09 mol, 93%),m.p.: 74°C.

1H NMR (300 MHz, CDCl3): d (ppm)=1.96 (s, 1H,OH), 3.51 (q, 2H, CH2), 3.94 (t, 2H, CH2), 6.65 (m, 1H,

Ar�H), 7.44 (m, 1H, Ar�H), 8.16 (dd, 1H, Ar�H), 8.22(s, 1H, NH). IR (KBr) n(cm−1): 3468 (s), 3340 (s), 3090(b), 2959 (m), 2878 (w), 1626 (s), 1570 (s), 1512 (s), 1419(m), 1361 (m), 1310 (s), 1227 (m), 1198 (s), 1149 (s),1042 (m), 741 (m), 667 (w), 526 (s). Anal. Calc. forC8H10N2O3: C, 52.74; H, 5.53; N, 15.38. Found: C,52.50; H, 5.61; N, 14.97%.

2.3.1.2. o-Amino-N-(2-hydroxyethyl)aniline. The prepa-ration was performed by a method described by Ra-mage and Trappe [22].

A solution of 5.77 g (1.80 mol) sulfur, 147.00 g (5.40mol) sodium sulfide and 17.00 g (0.09 mol) o-Nitro-N-(2-hydroxyethyl)aniline in 135 ml water was refluxedfor 2 h. The mixture was allowed to cool to roomtemperature and the resulting white precipitate wasfiltrated and dried in vacuo to afford 7.70 g (0.05 mol,55.6%), m.p.: 106°C.

1H NMR (300 MHz, DMSO-d6): d (ppm)=3.26 (q,2H, CH2), 3.79 (t, 2H, CH2), 4.49 (s, 1H, OH), 4.57 (s,2H, NH2), 4.83 (t, 1H, NH), 6.57–6.74 (m, 4H, Ar�H).IR (KBr) n(cm−1): 3407 (s), 3342 (s), 3217 (m), 3057(b), 2949 (m), 2857 (w), 2789 (m), 1605 (s), 1516 (s),1484 (w), 1459 (m), 1349 (m), 1274 (s), 1226 (m), 1119(s), 1051 (s), 927 (m), 909 (m), 824 (w), 733 (s). Anal.Calc. for C8H12N2O: C, 63.14; H, 7.95; N, 18.41.Found: C, 62.75; H, 8.06; N, 17.91%.

2.3.1.3. N,N,N %,N %-tetrakis((N-2-hydroxyethyl)2-benz-imidazolylmethyl)-2-hydroxy-1,3-diaminopropane HL1.The ligand HL1 was prepared according to the proce-dure described by our group for related ligands [23]. Amixture of 4.00 g (26.0 mmol) o-amino-N-(2-hydroxy-ethyl)aniline and 2.80 g (6.6 mmol) 2-hydroxy-1,3-di-aminopropanetetraacetic acid was heated to 180°C for3 h until all water was evaporated. The residue wasdissolved in 150 ml ethanol, refluxed which activatedcharcoal for 1 h and the hot solution was filtered. Thebrownish precipitate was recrystallized from ethanol forat least three times to obtain HL1 as white powder.Yield: 4.00 g (5.1 mmol, 77.2%), m.p.: 134°C.

1H NMR (300 MHz, DMSO-d6): d (ppm)=2.50–2.70 (m, 4H, CH2), 3.70–4.00 (m, 8H, CH2), 4.05 (m,8H, CH2), 4.15 (m, 8H, CH2), 7.18 (m, 8H, Ar�H), 7.49(m, 8H, Ar�H). 13C NMR (75 MHz, DMSO-d6): d

(ppm)=46.42 (CH2), 51.65 (CH2), 59.62 (CH2), 59.90(CH2), 68.64 (CH), 111.07 (Ar�CH), 118.20 (Ar�CH),122.23 (Ar�CH), 122.68 (Ar�CH), 135.34 (Ar�C),140.61 (Ar�C), 52.80 (Ar�C). IR (KBr) n (cm−1): 3216(b), 2938 (m), 2839 (m), 1738 (m), 1615 (m), 1511 (m),1468 (s), 1423 (m), 1332 (m), 1289 (m), 1246 (m), 1159(w), 1075 (s), 1010 (w), 883 (w), 746 (s), 624 (w). Anal.Calc. for C43H50N10O5: C, 65.63; H, 6.40; N, 17.80.Found: C, 65.27; H, 6.10; N, 17.12%.

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2.3.2. Synthesis of N,N,N %,N %-tetrakis (2-methylimida-zolyl)-2-hydroxy-1,3-diaminopropane HL2

The ligand was prepared according to a methoddescribed by Buchanan and co-workers [24].

2.3.3. Synthesis of (2-pyridylmethyl)-(1-hydroxypropyl)amine HL3

The ligand was prepared by a reaction of 1.18 g(0.011 mol) 2-pyridylcarboxaldehyde with 1.02 g (0.01mol) 3-amino-1-propanol in 100 ml of methanol andreduction of the resulting Schiff-base with 0.30 g (0.008mol) NaBH4. The reaction mixture was acidified withHCl to pH 1 and the solvent evaporated under reducedpressure. The residue was dissolved in water, the pHwas adjusted with NaOH to pH 8 and the aqueousphase extracted five times with 50 ml CHCl3. Thecombined organic phases were dried with MgSO4 andthe solvent was evaporated. The resulting oil was dis-tilled under reduced pressure (b.p. 93°C, 0.03 mmHg)affording HL3 as yellow oil. 0.39 g (27 mmol, 27%).

1H NMR (300 MHz, CDCl3): d (ppm)=1.74 (q, 2H,(CH2)2�CH2), 2.85 (t, 2H, NH�CH2), 3.55 (s, 1H, OH),3.76 (t, 2H, O�CH2), 3.90 (s, 2H, Py�CH2), 7.16 (dt,1H, HPy), 7.28 (d, 1H, HPy), 7.64 (dt, 1H, HPy), 8.53 (d,1H, HPy). 13C NMR (75 MHz, CDCl3): d (ppm)=31.2((CH2)2�CH2), 48.2 (NH�CH2), 54.7 (Py�CH2), 62.6(O�CH2), 121.9 (CPy), 122.2 (CPy), 136.4 (CPy), 149.0(CPy), 159.0 (CPy). IR (Film) n (cm−1): 3304 (br), 3141(w), 3065 (w), 3012 (m), 2934 (br), 2849 (br), 1980 (w),1662 (m), 1593 (vs), 1570 (s), 1475 (vs), 1434 (vs), 1352(m), 1299 (m), 1224 (m), 1184 (w), 1149 (m), 1111 (m),1069 (s), 1034 (w), 1002 (m), 914 (w), 890 (w), 843 (m),756 (s), 632 (m), 613 (m), 530 (w), 491 (w). Anal. Calc.for C9H14N2O: C, 65.04; H, 8.49; N, 16.97. Found: C,65.07; H, 8.39; N, 16.92%.

2.3.4. Synthesis of (2-hydroxybenzyl)(N,N-dimethylpropyl)amine HL4

The ligand was prepared according to the proceduredescribed for HL3 using 1.34 g (0.011 mol) 2-hydroxy-benzylcarboxaldehyde and 1.02 g (0.01 mol) N,N-dimethylamino-1-propanol. The resulting oil wasdistilled under reduced pressure (b.p. 108°C, 0.03mmHg) affording HL4 as orange oil. Yield: 0.41 g (25mmol, 25%).

1H NMR (300 MHz, CDCl3): d (ppm)=1.67 (q, 2 H,(CH2)2�CH2), 2.19 (s, 6H, N(CH3)2), 2.32 (t, 2H,N�CH2), 2.70 (t, 2H, NH-CH2), 3.94 (s, 2H, Ar�CH2),6.73 (dt, 1H, Har), 6.78 (d, 1H, Har), 6.95 (d, 1H, Har),7.16 (dt, 1H, Har). 13C NMR (75 MHz, CDCl3): d

(ppm)=27.1 ((CH2)2�CH2), 45.3 (N(CH3)2)), 47.3(N�CH2), 55.3 (NH�CH2), 57.9 (Ar�CH2), 115.7 (Car),118.8 (Car), 122.6 (Car), 128.4 (Car),129.5 (Car), 157.5(Car). IR (Film)n (cm−1): 3392 (w), 3175 (w), 3050 (br),2945 (s), 2862 (m), 2819 (m), 2784 (m), 2610 (br), 1591(vs), 1474 (vs), 1412 (m), 1258 (vs), 1165 (m), 1104 (m),

1070 (m), 1038 (m), 934 (w), 880 (m), 832 (m), 816 (w),755 (vs), 693 (m), 618 (m), 571 (w), 530 (m), 512 (m),451 (m). Anal. Calc. for C12H20N2O: C, 69.19; H, 9.68;N, 13.45. Found: C, 69.37; H, 9.70; N, 13.62%.

2.4. Synthesis of the Cu(II) complexes

Caution: Perchlorate salts of metal complexes withorganic ligands are potentially explosive.

2.4.1. Synthesis of [Cu2(L1)(m-OAc)](ClO4)2·CH3CN(1)

A solution of 198 mg (0.25 mmol) of HL1 and 21 mg(0.25 mmol) sodium acetate in 5 ml ethanol was addedto a solution of 186 mg (0.5 mmol) copper(II) perchlo-rate in 5 ml acetonitrile. The resulting dark greensolution was stirred for 30 min. Crystals suitable forX-ray structure determination were grown by slow dif-fusion of diethyl ether into the solution. Yield: 130 mg(0.107 mmol, 43%); decomposition over 305°C. Anal.Calc. for Cu2C47H50N11O15Cl2: C, 46.77; H, 4.18; N,12.77. Found: C, 47.11; H, 4.23; N, 12.98%.

2.4.2. Synthesis of [Cu2(L2)(CH3CN)2](ClO4)4·C2H5OH(2)

A solution of 165.20 mg (0.25 mmol) of HL2 in 5 mlethanol was added to a solution of 186 mg (0.5 mmol)copper(II) perchlorate in 5 ml acetonitrile. The resultingdark green solution was stirred for 30 min. Crystalssuitable for X-ray structure determination were grownby slow diffusion of diethyl ether into the solution.Yield: 88 mg (0.103 mmol, 41%); decomposition over259°C. Anal. Calc. for Cu2C29H44N12O18Cl4: C, 31.17;H, 3.97; N, 15.04. Found: C, 31.31; H, 4.03; N, 15.18%.

2.4.3. Synthesis of [Cu2(L3)2(CH3CN)2](PF6)2 (3)In a Schlenk tube, 186 mg (0.5 mmol)

[Cu(CH3CN)4](PF6) were dissolved in 20 ml degassedCH2Cl2 at −78°C under argon. 83 mg (0.5 mmol) HL3in 5 ml degassed CH2Cl2 were added to this solutionand stirred for 30 min to give a light blue solution. Theatmosphere was replaced by O2, and the mixture wasallowed to warm to room temperature to afford aslightly greenish solution. After stirring overnight agreen precipitate formed, which was recrystallized fromacetonitrile. Crystals suitable for X-ray structure deter-mination were grown by slow diffusion of diethyl etherinto the acetonitrile solution. Yield: 195 mg (0.235mmol, 47%); decomposition over 152°C. Anal. Calc. forCu2C22H30N6O2P2F12: C, 31.93; H, 3.65; N, 10.16.Found: C, 32.12; H, 3.71; N, 10.34%.

2.4.4. Synthesis of [Cu2(L4)2](PF6)2·2CH2Cl2 (4)Complex 4 was prepared according to the procedure

described for 3 using 104 mg (0.5 mmol) HL4, respec-

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Table 1Details of data collection and structure refinement for complexes 1, 2, 3 and 4

1Compound 2 3 4

Cu2C29H44N12O18Cl4Formula Cu2C22H30N6O2P2F12Cu2C47H50N11O15Cl2 Cu2C26H40N4O2P2F12Cl40.10×0.10×0.40Crystal size 0.72×0.64×0.880.36×0.32×0.12 0.40×0.12×0.16213(2) 213(2)213(2) 213(2)Temperature (K)

triclinicCrystal system monoclinic monoclinic monoclinicP1( (no. 2)Space group P21 (no. 4) P21/n (no. 14) P21/n (no. 14)

13.040(3) 16.990(3)11.880(2) 13.161(3)a (A, )11.612(2)b (A, ) 11.290(2)13.280(3) 15.450(3)15.143(3) 17.100(3)18.780(4) 19.557(4)c (A, )

71.44(4)a (°) 90 90 9082.57(3)b (°) 90.49(3) 91.10(3) 96.69(3)

90 9067.03(3) 90g (°)2292.88 3279.47V (A, 3) 3949.592586.012 42 4Z

1.542Dcalc. (g cm−3) 1.619 1.676 1.6741206.97M 1117.46 827.58 999.42

1022 16641230 2016F (000)4.4552u552.052u Range (°) 4.4552u551.704.5052u552.01 4.3552u552.04−155h516 −205h520−135h514 −165h516hkl Range

−165k516 −135h513 −135h513 −185h517−235l523 −185h518 −205h520 −235h524

17886 2915820679 31287Reflections measured8399 6248Reflections used in least-squares 764294340.103 0.1050.063 0.153Rint

0.057 (0.080)R1 (wR2) 0.064 (0.115) 0.048 (0.071) 0.063 (0.122)1.028Goodness-of-fit 1.075 1.071 1.006

588 507694 546Parameters0.55, −0.37 0.63, −0.43Dr maximum, minimum (e A, −3) 0.63, −0.660.98, −0.56

tively. Crystals suitable for X-ray structure determina-tion were grown by slow diffusion of diethyl ether intoa dichloromethane solution. Yield: 254 mg (0.255mmol, 51%); decomposition over 162°C. Anal. Calc. forCu2C26H40N4O2P2F12Cl4: C, 31.25; H, 4.03; N, 5.61.Found: C, 31.51; H, 4.20; N, 5.78%.

2.5. Crystallography

The unit cell data and diffraction intensities of com-pounds 1, 2, 3, and 4 were collected on a STOE IPDSimaging plate diffraction system at 213 K usinggraphite-monochromated Mo Ka radiation (l=0.71073 A, ). The sample-to-plate distance was fixed at70 mm. All structures were solved by direct methodsusing the program system SHELXS 86. All other dataprocessing were performed with the SHELXL 97 pro-gram package and the refinement on Fo

2 was carried outby full-matrix least-squares and followed by differenceFourier syntheses [25]. An empirical absorption correc-tion was applied for compound 4. The hydrogen atomswere calculated on idealized postions using a ridingmodel with isotropic displacement parameters. All non-hydrogen atoms were refined anisotropically. In courseof the refinements, it was revealed that two perchlorateions in 2 were disordered. In 3 both hexafluorophos-

phate ions were disordered. In 4 we found one disor-dered hexafluorophosphate ion and two slightlydisordered dichloromethane solvent molecules.

Relevant crystallographic data and structural refine-ment are given in Table 1 and selected bond lengthsand angles are given in Table 2 (1), Table 3 (2), Table4 (3) and Table 5 (4).

3. Results and discussion

3.1. Crystal structure of [Cu2(L1)(m-OAc)](ClO4)2·CH3CN (1)

An ORTEP plot of the complex cation and the atomlabelling are shown in Fig. 3. Selected bond lengths andangles are given in Table 2. The unit cell of compound1 consists of two complex cations, four perchloratecounter ions and two acetonitrile solvent molecules.Both copper ions of the dinuclear complex have adistorted square pyramidal geometry sharing a corner.Each copper atom is coordinated by a N3O2 donor setprovided by a bridging acetate, an alkoxo oxygen atomof L1, a tertiary amino nitrogen and two benzimida-zolyl nitrogen atoms which are supplied by the ligandL1. The alkoxo atom O(1) bridges the corner of the two

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Table 2Selected bond lengths (A ,) and angles (°) for complex 1

1.906(3) Cu(2)�O(1) 1.905(3)Cu(1)�O(1)1.947(3)Cu(2)�O(7)Cu(1)�O(6) 1.947(3)

2.105(3) Cu(2)�N(2) 2.146(3)Cu(1)�N(1)2.012(4) Cu(2)�N(7) 2.186(4)Cu(1)�N(3)

1.958(3)2.148(4) Cu(2)�N(9)Cu(1)�N(5)Cu(1)···Cu(2) 3.456(2)

96.6(3)O(1)�Cu(1)�O(6) O(1)�Cu(2)�O(7) 95.8(1)83.4(1)O(1)�Cu(1)�N(1) O(1)�Cu(2)�N(2) 84.8(1)

141.4(2)O(1)�Cu(1)�N(3) O(1)�Cu(2)�N(7) 103.6(1)O(1)�Cu(1)�N(5) 116.1(1) O(1)�Cu(2)�N(9) 151.8(2)

95.5(1)O(7)�Cu(2)�N(9)O(6)�Cu(1)�N(3) 95.2(1)176.2(2)O(7)�Cu(2)�N(2)O(6)�Cu(1)�N(1) 171.4(2)

107.0(2)O(6)�Cu(1)�N(5) O(7)�Cu(2)�N(7) 103.2(2)80.6(1)N(1)�Cu(1)�N(5) N(2)�Cu(2)�N(7) 80.4(1)79.8(1) N(2)�Cu(2)�N(9) 82.4(1)N(1)�Cu(1)�N(3)95.2(1)N(3)�Cu(1)�N(5) N(7)�Cu(2)�N(9) 98.8(1)

Fig. 4. Ellipsoid drawing of [Cu2(L2)(CH3CN)2](ClO4)4·C2H5OH (2)with the atom numbering scheme. Thermal ellipsoids are drawn at the50% probability level.

square pyramids. The Cu(1)�O(1)�Cu(2) angle is130.0(2)° which is larger than the angle of a hydroxobridge [26,27]. The values for the Cu(1)�O(1)/Cu(2)�O(1) bond lengths are approximately 1.906 A, .The acetato ligand symmetrically bridges the two cop-per(II) atoms with identical values of bond lengthswithin the standard deviations for Cu(1)�O(6) andCu(1)�O(7) with 1.947(3) A, . The Cu�N (tertiary amine)bond lengths are Cu(1)�N(1) 2.104(3) A, andCu(2)�N(2) 2.146(3) A, . In general, compared to thetertiary amine nitrogens the nitrogen atoms of thearomatic heterocycles bond more strongly to the coppercentre because the tertiary amines have a lower abilityto act as a p-donor. With respect to the nature of the Ndonor moieties the Cu�N (benzimidazolyl amine) bondlengths exhibit the largest bond length values withsignificant differences of 0.23 A, which indicates a transeffect of the ligand as can be found in other m-acetatobridged complexes [20,28]. The complex 1 reveals aCu···Cu distance of 3.456(2) A, .

3.2. Crystal structure of[Cu2(L2)(CH3CN)2](ClO4)4·C2H5OH (2)

The ORTEP plot of the complex cation and the atomlabelling are shown in Fig. 4. Selected bond lengths andangles are given in Table 3. Compound 2 crystallizeswith two complex cations, eight perchlorate counterions and two ethanol solvent molecules. The copperatoms exhibit a N4O donor set provided by an acetoni-trile molecule, a tertiary amino nitrogen, two benzimi-dazolyl nitrogen atoms from the ligand L2 and anoxygen atom forming a distorted square pyramidalcoordination sphere [24,29]. The Cu(2) atom interactswith the alkoxo oxygen atom O(1) revealing a bonddistance of 2.427(5) A, while the Cu(1) atom exhibits alarger bond length value of 2.603(3) A, and interactswith the oxygen atom O(3) of an ethanol molecule2.463(2) A, apart from Cu(1). The Cu�O distances inthis complex are comparably large in contrast to thevalues for the Cu�O(1) bond lengths in compound 1with approximately 1.906 A, and m-acetato bridgedcomplexes with similar ligands revealing values of ap-proximately 1.946 A, . For that reason the distortion ofthe square pyramidal coordination spheres is causedpartly by the interactions of the Cu(1) atom with O(1)and O(3). The axial Cu�N (tertiary amine and acetoni-trile molecules) bond lengths are longer than the equa-torial Cu�N (benzimidazolyl amine) bond lengths withCu(1)�N(6) 2.134(6) A, and Cu(2)�N(1) 2.081(7) A, forthe tertiary amines and with Cu(1)�N(11) 2.003(8) A,and Cu(2)�N(12) 1.986(9) A, for the acetonitrilemolecules in contrast to Cu(1)�N(7) 1.911(6) A, /Cu(1)�N(9) 1.941(7) A, and Cu(2)�N(2) 1.935(7) A, /Cu(2)�N(4) 1.945(6) A, for the benzimidazolyl amines.Due to the lack of a bridging ligand a Cu···Cu separa-

Fig. 3. Ellipsoid drawing of [Cu2(L1)(m-OAc)](ClO4)2·CH3CN (1)with the atom numbering scheme. Thermal ellipsoids are drawn at the50% probability level.

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Table 3Selected bond lengths (A, ) and angles (°) for complex 2

Cu(1)�N(6) Cu(2)�O(1)2.134(6) 2.427(5)1.911(6)Cu(1)�N(7) Cu(2)�N(1) 2.081(7)

1.935(7)Cu(1)�N(9) 1.941(7) Cu(2)�N(2)2.003(8)Cu(1)�N(11) Cu(2)�N(4) 1.945(6)

Cu(1)···Cu(2) 1.986(9)Cu(2)�N(12)4.756(2)

O(1)�Cu(2)�N(1)N(6)�Cu(1)�N(7) 79.8(2)82.4(3)N(6)�Cu(1)�N(9) 81.9(3) O(1)�Cu(2)�N(2) 106.0(2)N(6)�Cu(1)�N(11) 170.6(3) 86.7(2)O(1)�Cu(2)�N(4)

162.3(3)N(7)�Cu(1)�N(9) O(1)�Cu(2)�N(12) 91.8(3)82.7(3)N(1)�Cu(2)�N(2)N(7)�Cu(1)�N(11) 99.2(3)

97.6(3)N(9)�Cu(1)�N(11) N(1)�Cu(2)�N(4) 83.8(3)170.5(3)N(1)�Cu(2)�N(12)159.5(3)N(2)�Cu(2)�N(4)95.6(3)N(2)�Cu(2)�N(12)

N(4)�Cu(2)�N(12) 100.2(3)

Fig. 5. Ellipsoid drawing of [Cu2(L3)2(CH3CN)2](PF6)2 (3) with theatom numbering scheme. Thermal ellipsoids are drawn at the 50%probability level.

tion of 4.756(2) A, can be found in the dinuclearcomplex.

3.3. Crystal structure of [Cu2(L3)2(CH3CN)2](PF6)2 (3)

The ORTEP plot of the complex cation and the atomlabelling are shown in Fig. 5. Selected bond lengths andangles are given in Table 4. The unit cell of complex 3consists of four independent cations and eight hexa-fluorophosphate counter ions. Each copper core has aN3O2 donor set forming a distorted square pyramid.The copper centres are bridged by two oxygen atoms(alkoxide oxygens of the two ligands of the dimer) inthe basal plane and two nitrogen atoms (a pyridyl andan amine nitrogen, both from the ligand L3). Thesquare pyramidal coordination sphere is completedby acetonitrile molecules in the axial position withbond lengths values of Cu(1)�N(5) 2.420(4) A, andCu(2)�N(6) 2.346(4) A, , respectively [30]. The Cu�Nbond lengths in the equatorial position show nearlyidentical values within the standard deviations between1.999(3) and 2.019(3) A, like the Cu�O bond lengthsvalues in the range of 1.932(3) and 1.939(2) A, resultingin a Cu···Cu distance of 2.918(1) A, .

3.4. Crystal structure of [Cu2(L4)2](PF6)2·2CH2Cl2(4)

The structure of the complex cation and the atomlabelling are shown in Fig. 6(a). Selected bond lengthsand angles are given in Table 5. Complex 4 crystallizeswith four independent complex cations, eight hexa-fluorophosphate counter ions and eight dichloro-methane solvent molecules. In the dinuclear complexthe two copper atoms are bridged by oxygen atoms ofthe phenolates from the ligand L3. The coordinationsphere can be best described as distorted square pyra-midal. This bridging arrangement forms a Cu2O2 corewith Cu�O bond length values in the range of 1.94(4)

Table 4Selected bond lengths (A, ) and angles (°) for complex 3

Cu(1)�O(1) 1.939(2) Cu(2)�O(1) 1.932(2)Cu(2)�O(2)1.936(2) 1.935(2)Cu(1)�O(2)Cu(2)�N(3) 2.019(3)Cu(1)�N(1) 1.999(3)

2.015(3) Cu(2)�N(4)Cu(1)�N(2) 2.013(3)Cu(2)�N(6) 2.346(4)2.420(4)Cu(1)�N(5)

2.918(2)Cu(1)···Cu(2)

Cu(1)�Cu(2)�O(1) 41.2(1)Cu(2)�Cu(1)�O(1) 41.0(1)41.1(1) Cu(1)�Cu(2)�O(2)Cu(2)�Cu(1)�O(2) 41.1(1)

Cu(1)�Cu(2)�N(3)140.5(1)Cu(2)�Cu(1)�N(1) 140.0(1)133.9(1) Cu(1)�Cu(2)�N(4) 135.3(1)Cu(2)�Cu(1)�N(2)99.4(1) Cu(1)�Cu(2)�N(6)Cu(2)�Cu(1)�N(5) 98.4(1)

O(1)�Cu(2)�O(2) 81.8(1)81.6 (1)O(1)�Cu(1)�O(2)O(1)�Cu(2)�N(3) 100.3(1)O(1)�Cu(1)�N(1) 161.4(1)

173.6(1)O(1)�Cu(2)�N(4)O(1)�Cu(1)�N(2) 92.9(1)102.5(1) O(1)�Cu(2)�N(6)O(1)�Cu(1)�N(5) 95.4(1)100.7(1) O(2)�Cu(2)�N(3)O(2)�Cu(1)�N(1) 160.5(1)

94.3(1)O(2)�Cu(2)�N(4)O(2)�Cu(1)�N(2) 171.3(1)O(2)�Cu(2)�N(6) 104.0(1)O(2)�Cu(1)�N(5) 98.5(1)

82.4(1) N(3)�Cu(2)�N(4)N(1)�Cu(1)�N(2) 81.7(1)N(3)�Cu(2)�N(6) 95.2(1)95.5(1)N(1)�Cu(1)�N(5)N(4)�Cu(2)�N(6) 90.5(2)N(2)�Cu(1)�N(5) 89.3(1)

Table 5Selected bond lengths (A, ) and angles (°) for complex 4

1.959(4) Cu(2)�O(1)Cu(1)�O(1) 1.936(4)1.948(4) Cu(2)�O(2)Cu(1)�O(2) 1.975(4)

Cu(2)�N(3) 1.998(5)2.004(5)Cu(1)�N(1)2.045(5) Cu(2)�N(4)Cu(1)�N(2) 2.045(5)

Cu(1)···Cu(2) 3.062(9)

77.0(2) O(1)�Cu(2)�O(2) 76.9(2)O(1)�Cu(1)�O(2)92.5(2) O(1)�Cu(2)�N(3)O(1)�Cu(1)�N(1) 161.9(2)

158.7(2) O(1)�Cu(2)�N(4) 100.8(2)O(1)�Cu(1)�N(2)93.0(2)O(2)�Cu(2)�N(3)O(2)�Cu(1)�N(1) 161.4(2)

101.5(2) O(2)�Cu(2)�N(4)O(2)�Cu(1)�N(2) 159.7(2)93.8(2) N(3)�Cu(2)�N(4)N(1)�Cu(1)�N(2) 93.9(2)

P. Gentsche6 et al. / Inorganica Chimica Acta 300–302 (2000) 442–452 449

Fig. 6. (a) Ellipsoid drawing of [Cu2(L4)2](PF6)2·2CH2Cl2 (4) with theatom numbering scheme. Thermal ellipsoids are drawn at the 50%probability level. (b) Ellipsoid drawing of [Cu2(L4)2]2+ cation in 4with the atom numbering scheme showing the interaction with a PF6

anion. Non-coordinating parts of the ligand L4 are omitted forclarity. Thermal ellipsoids are drawn at the 50% probability level.

of 2.693(2) A, for Cu(1)�F(11) and 2.981(2) A, forCu(2)�F(7). To our knowledge this is the first dinuclearCu(II) complex with a bidentate coordinated PF6

molecule.

3.5. Characterization

Complex 1 shows absorption bands in the region of250–270 nm in the visible spectrum. For the dinuclearcomplex 2 only one absorption band at 269 nm can beobserved. The principal absorption bands displayed inthe spectra of the dimeric complexes 1 and 2 suggest thepresence of the square-pyramidal element in the geome-try as indicated by X-ray structure determination [32].Other m-acetato bridged dinuclear Cu(II) coordinationcompounds have bands in the same region as com-pound 1 [33].

The dinuclear complex 3 shows a shoulder at 300 nmwhich can be assigned to an alkoxo Cu(II) charge-transfer transition and typically appears in the 300–340nm region. The broad band between 390 and 400 nmseems to be a superposition of different charge transfertransitions as found in other complexes of this type.Compound 4 only exhibits a shoulder at 297 nm. Theprincipal absorbance bands are similar to those foundfor related complexes previously reported [34].

The IR absorption spectra of 1 shows two character-istic bands, nasym (COO) at 1560 cm−1 and nsym (COO)at 1453 cm−1 [35]. The characteristic IR bands ofcomplexes 2, 3 and 4 occur in the fingerprint region, sothat a certain assignment of these bands is not possible.

The magnetic susceptibility for compound 1 revealsan effective magnetic moment of 2.00 per Cu(II), whichis in the range typical for magnetically diluted cop-per(II) ions [36–38]. Compound 2 reveals a similarvalue for the effective magnetic moment with 1.86 perCu(II). The complex 3 exhibits a value of 1.29 perCu(II) effective magnetic moment which is consistent todata for similar complexes [30,39]. Compound 4 revealsa value of 1.33 per Cu(II) effective magnetic momentwhich is comparable with values for complexes of thesame type [40].

The values for the magnetic moments and UV–Visabsorptions data are summarized in Table 6.

3.6. Kinetic studies

3,5-di-tert-butylcatechol (3,5-DTBC) has been em-ployed as a substrate in most of the catecholase activitystudies of model complexes so far. The oxidationproduct 3,5-di-tert-butyl-o-quinone (3,5-DTBQ) is con-siderably stable and exhibits a strong absorption at 400nm (o=1900 M−1 cm−1). Therefore, activities andreaction rates can be determined using electronic spec-troscopy by following the appearance of the absorptionmaximum of the quinone. The reactivity studies wereperformed in acetonitrile solution.

and 1.97(4) A, for the m-bridging oxygens. This is con-sistent with bond distances of 1.88–1.89 A, foranalogous complexes [31]. The Cu(1)�O(2)/Cu(2)�O(1)angles are in the range of 77.0(2) and 76.9(2)°, respec-tively. The Cu�N bond lengths show values in the rangeof 1.999(5) and 2.047(5) A, . The dinuclear complexexhibits a Cu···Cu distance of 3.062(9) A, with aninteresting bidentate binding mode of a PF6 molecule(Fig. 6(b)). Two fluor atoms of a PF6 molecule showinteractions to the copper centres completing the squarepyramidal coordination sphere with bond length values

Table 6Spectroscopic data for complexes 1, 2, 3 and 4

Complex mexp (meff=1.73) (BM) l (nm) o (M−1 cm−1)

2.00 6411, 62641 248, 27412582692 1.86

3 1.29 300 19774 1.33 297 578

P. Gentsche6 et al. / Inorganica Chimica Acta 300–302 (2000) 442–452450

Fig. 7. UV–Vis spectra of the oxidation of 3,5-DTBC by compound 3.

400 nm but shifted to lower wavelength. Additionallyan extra broad band appears between 600 and 800 nm.The broad absorption between 500 and 600 nm canalso be assigned to the quinone.

Table 7 shows the calculated activities for the fourcomplexes which were studied.

The catechol and the Cu(II) complex have to form aCu(II) catecholate intermediate before electron transferfrom catechol to the Cu(II) atoms can occur. If thecoordinating molecule is a stronger ligand than thecatechol, no oxidation will be observed. In our studies,the fifth coordination site is occupied by an acetatebridge, acetonitrile molecules and a PF6 anion, respec-tively. All these are capable of being replaced by cate-chol. The fifth ligand must be released before theassociation of oxygen and the oxidation of the catecholis possible. According to this the reaction rate dependson the lability of the fifth ligand [18].

The kinetics of the oxidation of 3,5-DTBC weredetermined by the method of initial rates by monitoringthe growth of the 400 nm band of the product 3,5-DTBQ. To determine the dependence of the rates onthe substrate concentration, solutions of the complexes1, 3 and 4 were treated with increasing amounts of3,5-DTBC. A first order dependence was observed atlow concentrations of the substrate, whereas a satura-tion kinetics was found for all three compounds 1, 3and 4 at higher concentrations. A treatment on thebasis of the Michaelis–Menten model, originally devel-oped for enzyme kinetics, was applied. In our case wecan also propose a pre-equilibrium of free complex andsubstrate on the one hand, and a complex–substrateadduct on the other hand. The irreversible conversioninto complex and quinone can be imagined as therate-determining step. Although a much more compli-cated mechanism may be involved, the results show thissimple model to be sufficient for a kinetic description.

Table 7Kinetic properties of compounds 1, 2, 3, and 4

Turnover-number (h−1)Complex

3.941no activity2

3 31.644 5.13

Prior to a detailed kinetic study, it is necessary to getan estimation of the ability of the complexes to oxidizecatechol. For this purpose 2×10−4 mol l−1 solutionsof 1–4 in acetonitrile were treated with 50 equiv. of3,5-DTBC in the presence of air. The course of thereaction was followed by UV–Vis spectroscopy overthe first 18 min. The first apparent result is a significantdifference in the reactivities of the complexes. Whereas3 shows a remarkable high catecholase activity, com-plex 1 containing a heptadentate ligand and complex 4with a tridentate ligand exhibit less activity, while 2shows no catalytic potential. The activities of com-plexes 1, 3 and 4 can be graduated in this order: theincrease of absorption at 400 nm after 18 min can beexpressed in turnovers of 1: 3.94(90.6), 3: 31.64(90.9)and 4: 5.13(90.4) h−1. Complex 2 exhibits a negligiblylow catecholase activity (turnover of 0.74 after 18 min).A detailed kinetic analysis is therefore dispensable. Fig.7 shows the absorbance versus wavelength spectra ofcompound 3 for the first 18 min of the reaction. Forma-tion of 3,5-DTBQ was monitored by the increase of theabsorbance band at 400 nm. Apart from the mainproduct we obtained a secondary product which weassume to be the semiquinone. As evidence of ourassumption the following spectrographic data is given:lmax of the main product (3,5-DTBQ) is not exactly at

P. Gentsche6 et al. / Inorganica Chimica Acta 300–302 (2000) 442–452 451

For the determination of the kinetic parameters forthe three compounds studied the substrate rate wasvaried in the range from 1×10−3 to 1×10−4 mol l−1

at a complex concentration of 1×10−4 mol l−1.

4. Conclusions

A series of dinuclear copper(II) complexes has beensynthesized. They differ by the exogenous bridging andthe N,O donor sets. The structures for all complexescould be obtained by X-ray crystallography. They rep-resent good structural models for the active sites of themet-forms of catechol oxidases. They simulate the shortCu···Cu distance of about 3 A, as well as the Cu2O2

central unit.Investigations of the crystal structures and the cata-

lytic performance of the dinuclear copper(II) complexeshave provided information which generally supportsthe argument that the rate of the catalytic oxidation ofcatechols depends on the lability of the fifth ligand andthe Cu···Cu distance of the copper cores. Correlationsare found between the coordination numbers of thecomplexes and their catalytic activities. Our activitystudies with o-diphenols such as 3,5-di-tert-butylcate-chol as substrate show that dinuclear copper(II) com-plexes with tridentate ligands such as HL3 lead to moreactive complexes than if heptadentate ligands like HL1or HL2 are used. This is consistent with former studiesrevealing a maximum of activity for dinuclear cop-per(II) model complexes with pentadentate ligands suchas 2,6-bis(morpholinyl-N-methyl)-4-methylphenol [14].Complex 1 exhibits a rather large Cu···Cu distance andan acetate bridging which is tight bounded and preventsthe molecule to react more active by blocking the fifthposition of the copper atoms. In compound 2 thecoordination spheres of both copper(II) atoms are com-pleted by weak coordinated acetonitrile molecules. Thecomplex reveals a distorted square pyramidal coordina-tion sphere for each Cu(II) ion and a large Cu···Cudistance which lead to low activity [12]. This activitycannot be described as catalytic. In contrast the com-pounds 3 and 4 with tridentate ligands show consider-ably high catalytic activity. Especially compound 3exhibits remarkable activity in catechol oxidation whichcan be assigned to a quite short Cu···Cu distance of2.918(1) A, and weak coordinated acetonitrile moleculesin the axial positions. Compound 4 also reveals acomparably short Cu···Cu distance of 3.062(9) A, . Inter-estingly, compound 4 shows lower catalytic activity.

The common characteristics of the active complexesis therefore a weak coordinated ligand at the fifthposition in combination with a Cu···Cu distance around3 A, . The dinuclear copper complexes with heptadentateligands show low catalytic activity because of the m-ac-

etate bridge in 1 and the lack of a exogenous bridgeresults in a large Cu···Cu distance of 4.756(2) A, in 2.Former studies revealed that the differences in reactiv-ity are also based on geometric factors [14]. Symmetriccomplexes in relaxed, energetically favoured conforma-tions exhibit poor catalytic activity. Strongly distortedsquare pyramidal coordination geometries are the es-sential structural units for catecholase activity. Com-plex 1 shows a stronger distortion in the coordinationgeometry than 2 because of the exogenous m-acetatebridge. In the presence of a bridging coordinationpartner with a smaller bite size like a m-hydroxo bridge,the complex would adopt from the relaxed form to astrained form. Studies with the one ligand and m-hy-droxo and m-acetate bridging systems revealed highcatalytic activities for the m-hydroxo bridged systemcompared to negligible catalytic activity for the corre-sponding m-acetate bridged complex [41].

5. Supplementary material

Further details of the crystal structure investigations,atomic coordinates, thermal parameters, complete bonddistances and angles can be obtained from the Cam-bridge Crystallographic Data Centre, CCDC nos.136738 (1), 136736 (2), 136735 (3) and 136737 (4), 12Union Road, Cambridge CB2 1EZ, UK, on quotingthe full journal citation. (fax: +44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk).

Acknowledgements

The authors would like to thank D. Mensing and A.Lindemann for X-ray data collection and M. Rauterkusfor providing material. Financial support by theDeutsche Forschungsgemeinschaft (Graduiertenkolleg‘Highly reactive multiple bond systems’ and the Sonder-forschungsbereich 424), the Ministerium fur Bildungund Forschung des Landes Nordrhein-Westfalen(Katalyseverbund NRW) and by the Fonds derChemischen Industrie is gratefully acknowledged.

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