Inorganic Chemistry Communications 13 (2010) 227–230
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A flexible hydroxy-bridged dicopper complex as catechol oxidase mimic Tamás Csay a, Balázs Kripli a, Michel Giorgi b, József Kaizer a,*, Gábor Speier a,* a
Department of Chemistry, University of Pannonia, 8201 Veszprém, Hungary Laboratoire de Cristallochimie et Laboratoire de Bioinorganique Structurale Université Paul, Cézanne Aix-Marseille III F.S.T. Saint-Jérôme, Service 432 Avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France b
a r t i c l e
i n f o
Article history: Received 8 September 2009 Accepted 20 November 2009 Available online 27 November 2009
a b s t r a c t A flexible hydroxy-bridged dicopper complex with isoindoline ligand has prepared and characterized and shown that it does not maintain its dimeric nature when catalyzing catechol oxidase-like reaction. Ó 2009 Elsevier B.V. All rights reserved.
Keywords: Copper Oxidation Catechol Catecholase mimics
Catechol oxidases and phenol monooxygenases contain two cooperating copper ions within their active site [1–4]. The two proximate copper ions activate the kinetically inert O2, and they both together mediate multi-electron redox reactions. One of these so-called type 3 copper centers is catechol oxidase, which catalyzes the two-electron oxidation of ortho-diphenols to the corresponding quinones [5–6]. The X-ray crystal structure of catechol oxidase from sweet potatoes showed details of the enzyme structure in different redox states, with both copper ions in N-donor (histidine) ligation [7– 8]. The two-electron substrate oxidation occurs by means of the cooperative action of the two copper ions, both undergoing redox shuttle between their +1 and +2 oxidation states. The structure of the dinuclear active site is preorganized as well many of the model systems (Scheme 1). However, a fair number of mononuclear copper systems are known which also do catalyze the enzyme-like reaction [9]. To our knowledge no catechol oxidases have been described with only one copper atom at the active site in the literature. The question arises whether in catechol oxidase the dinuclear nature of the active center is only supported by the preorganized, rather rigid protein or the protein may be folded in a way not to force the two copper ions close to each other to make cooperation possible. In order to modeling this situation we prepared a flexible l-hydroxodicopper isoindoline complex and tested its activity as well its mechanism as catechol oxidase model compared with a mononuclear copper catecholate complex (Scheme 1). * Corresponding authors. Tel.: +36 88 624 720; fax: +36 88 624 469. E-mail addresses:
[email protected] (J. Kaizer),
[email protected] (G. Speier). 1387-7003/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.inoche.2009.11.018
[Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH (1) was prepared by mixing copper(II) perchlorate with the ligand 40 MeindH (1,3bis(40 -methyl-2-pyridylimino)isoindoline) in water [10]. Its X-ray structure (Fig. 1) revealed that the two copper(II) ions are bridged by an OH-ligand. The complex contains two methanol molecules, the one coordinated to the copper(II) ion and the other a solvate. The one copper(II) ion has a bent square geometry while the other is pentacoordinated with one additional MeOH molecule coordinated to the copper(II) ion (Table 1). A s value of 0.36 was found indicating a distorted square planar pyramidal geometry [14]. The isoindoline ligands are in deprotonated form as proved by a bathochrome shift in the UV–Vis spectrum [15–17]. A mononuclear copper(II) isoindoline complex with the 3,5DTBCH2 ligands Cu(40 Meind)(3,5-DTBCH) (2) was also prepared and characterized as possible monomeric form of the catalyst in the catalytic cycle (Scheme 2). Cu(OMe)2 with 40 MeindH were dissolved in MeCN and 3,5-di-tert-butylcatechol was added to the solution to give 2 in 60.5% yield [18]. The formation of 2 in acetonitrile proceeded according to Eq. (1):
CuðOMeÞ2 þ 40 MeindH þ 3; 5-DTBCH2 ¼ Cuð40 MeindÞð3; 5-DTBCHÞ þ 2MeOH
ð1Þ
Kinetic studies on the oxidation of 3,5-DTBCH2 to 3,5-DTBQ catalyzed by 1 resulted in a Michaelis–Menten type kinetics with the parameters: VM = 3.47 107 M s1, KM = 8.44 103 M, and kcat = 6.32 103 s1 (Fig. 2), and revealed first-order dependence on the catalyst and dioxygen concentration (Table 2) [19]. Using 3,5-DTBCD2 as substrate a KIE of 3.01 was found. The answer to be obtained was whether the complex remains in its dimeric form or in a mononuclear form while catalyzing the
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T. Csay et al. / Inorganic Chemistry Communications 13 (2010) 227–230 Table 1 Summary of the crystallographic data and [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH [11]. Formula weight Crystal system Crystal description Space group Unit cell dimensions a (Å) b (Å) c (Å) a (°) b (°) c (°) Volume (Å3) Z Calculated density (g cm1) Crystal size (mm) Index ranges
Scheme 1.
Temperature (K) Radiation Absorption coefficient (mm1) F(0 0 0) Reflections collected Observed reflections [I > 2r(I)] Goodness-of-fit Final R indices R indices (all data)
structure
parameters
for
960.38 Triclinic Prism P1 12.3800(2) 12.9088(2) 13.7599(2) 80.736010 80.9590(10) 75.0983(9) 2081.81(6) 2 1.532 0.50 0.40 0.35 0 6 h 6 16 16 6 k 6 17 17 6 l 6 18 293(2) Mo Ka (k = 0.71073) 1.150 988 10,478 8031 1.145 R1 = 0.0428, wR2 = 0.1204 R1 = 0.0700, wR2 = 0.1524
Fig. 1. The molecular structure of [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH (1). Selected bond lengths (Å) and bond angles (deg): Cu(1)–N(1) 1.920(2), Cu(2)–N(6) 1.906(2), Cu(1)–N(3) 2.042(2), Cu(2)–N(8) 1.988(2), Cu(1)–N(5) 2.030(2), Cu(2)– N(10) 2.008(2), Cu(1)–O(1) 1.9696(18), Cu(2)–O(1) 1.9373(17), Cu(1)–O(3) 2.343(2), N(1)–Cu(1)–O(1) 146.62(9), N(10)–Cu(2)–N(8) 163.53(9), N(5)–Cu(1)– N(3) 171.97(9), N(6)–Cu(2)–O(1) 149.22(9), N(3)–Cu(1)–O(3) 93.18(8), O(1)–Cu(2)– N(8) 95.23(8), Cu(2)–O(1)–Cu(1) 113.83(9).
enzyme-like reaction. Under identical conditions and by the use of the same amount of the catalysts based on the Cu-content catalytic experiments were carried out in order to see eventual differences or similarities between the reaction rates and kinetics of complex 1 and 2. The result above shows that the formation rates of 3,5DTBQ followed by UV–Vis spectroscopy resulted in almost equal slopes in both cases (Table 2). The color of the reaction mixture during the catalytic reaction was reddish-brown and by EPR studies the free 3,5-di-tert-butylbenzosemiquinone (3,5-DTBSQ: g0 = 2.0043, aH = 3.48 G) could be detected. When nitroblue tetrazolium (NBT) was added to complex 2 or the reaction mixtures using 1 or 2 as catalyst its reduction to diformazan took place. This indicates clearly the concomitant formation of superoxide anion. According to these facts we assume that complex 1 is transformed in a fast reaction to complex 2 as shown in Eq. (2). That is the most persistent species during the catalytic cycle.
Fig. 2. Dependence of the reaction rates on the DTBCH2 concentrations for the oxidation reaction catalyzed by [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH. Conditions: [[Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH]0 = 5.49 105 mol dm3, under air at 50 °C in DMF.
½ð40 MeIndÞCuðl-OHÞCuð40 MeIndÞClO4 þ 23; 5-DTBCH2
Scheme 2.
¼ 2Cuð3; 5-DTBCHÞð40 MeIndÞ þ H2 O þ HClO4
ð2Þ
T. Csay et al. / Inorganic Chemistry Communications 13 (2010) 227–230 Table 2 Kinetic data for the [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH-catalyzed oxidation of DTBCH2 in DMF. 105[Cu2]a 103[DTBCH2]a 107V0 Experiment T 103[O2]a (mol dm3 s1) No. (°C) (mol dm3) (mol dm3) (mol dm3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 a b c
50 50 50 50 50 50 50 50 50 50 50 50 50 50
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 2.92 5.99 5.99
5.49 5.49 5.49 5.49 11.0b 5.49 5.49 5.49 1.83 3.66 7.32 7.32 7.32 7.32
3.75 7.49 11.20 15.40 15.40 18.70 22.50 26.00 15.40 15.40 15.40 15.40 15.40 15.40c
1.06 1.67 2.01 2.17 2.32 2.38 2.55 2.60 0.74 1.44 2.74 5.94 12.8 4.25
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are formed as initial reaction step(s) entering the catalytic cycle of the reaction. That hints to the possibility that the enzyme itself may remains in a dimeric form due to the rigid protein ligating the copper metal ions. Acknowledgements We thank the Hungarian National Research Fund (OTKA # K67871 and K75783), COST and Nagymaros Trade Ltd. for financial support. Appendix A. Supplementary material CCDC 727019 contains the supplementary crystallographic data for 1. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.inoche.2009.11.018.
In 30 ml DMF. [Cu(40 Meind)(3,5-DTBCH)]. In the presence of DTBCD2 (KIE = 3.01).
This is then followed by an intramolecular electron transfer from the ligand 3,5-DTBCH to the copper(II) ion, resulting in a complex CuI(3,5-DTBCH)(ind). This equilibrium is largely shifted to the left (K1 is rather small). The presence of the superoxide anion (NBT) suggests the concomitant formation of the [CuII(3,5DTBCH)(ind)]O2 via an intermolecular electron transfer (K2). The saturation kinetics of the substrate together with a medium large KIE may suggest that the rate-determining step is the reaction of [CuII(3,5-DTBCH)(ind)]O2 with 3,5-DTBCH2 via an 3,5-di-tertbutylbensosemiquinone (EPR, 3,5-DTBCH) and HO2. The latter undergo disproportionation to the catechol and the quinone and O2 and H2O2 (Scheme 3). The results obtained indicate that a flexible l-hydroxodicopper complex does not keep its dimeric nature during the catechol oxidase-like reaction instead of that mononuclear copper complexes
References [1] T. Klabunde, C. Eicken, J.C. Sacchettini, B. Krebs, Nat. Struct. Biol. 5 (1998) 1084. [2] W. Kaim, J. Rall, Angew. Chem., Int. Ed. Engl. 35 (1996) 43. [3] K.A. Magnus, B. Hazes, H. Ton-That, C. Bonaventura, J. Bonaventura, W.G.J. Hol, Proteins Struct. Funct. Genet. 19 (1994) 302. [4] K.A. Magnus, H. Ton-That, J.E. Carpenter, Chem. Rev. 94 (1994) 727. [5] B. Salvato, M. Santamaria, M. Beltramini, G. Alzuet, L. Casella, Biochemistry 37 (1998) 14065. [6] H. Decker, T. Schweikardt, F. Tuczek, Angew. Chem., Int. Ed. 45 (2006) 4546. [7] R. Than, A. Feldman, B. Krebs, Coord. Chem. Rev. 182 (1999) 211. [8] C. Gerdemann, C. Eicken, B. Krebs, Acc. Chem. Res. 35 (2002) 183. [9] Á. Kupán, J. Kaizer, G. Speier, M. Giorgi, M. Réglier, F. Pollreisz, J. Inorg. Biochem. 103 (2009) 389. [10] Synthesis of [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH (1): Cu(ClO4)26H2O (22.31 g (6.25 mmol) and p-MeindH (2.00 g (6.25 mmol) were dissolved in distilled water (200 cm3) and stirred for 5 min to give a yellow-greenish slurry. The pH of the mixture was adjusted to 11.5 by 0.1 M NaOH solution with darkening of the color. It was stirred for 5 h, the precipitate filtered, washed with distilled water and ether and dried in vacuum to give 1 as green powder (2.53 g (42.1%). Mp: 188–192 °C. UV–Vis (DMF) kmax (log e/
Scheme 3.
230
[11]
[12] [13] [14] [15] [16] [17]
T. Csay et al. / Inorganic Chemistry Communications 13 (2010) 227–230 dm3 mol1 cm1): 271.0 (4.457), 308.5 (4.489), 319.0 (4.455), 333.5 (4.482), 348.5 (4.346), 400.0 (4.441), 421.5 (4.560), 448.5 (4.471). IR (KBr) cm1: 3570, 3407, 2917, 1643, 1614, 1523, 1467, 1406, 1367, 1315, 1298, 1217, 1195, 1107, 941, 837, 778, 752, 709, 668, 600, 525, 464, 416 cm1. Anal. Calcd for C42H41ClCu2N10O7: C, 52.53; H, 4.30; N, 14.58. Found: C, 52.42; H, 4.21; N, 14.68. Microanalyses were done by the Microanalytical Service of the University. Single crystals of [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH suitable for an Xray diffraction study were grown from methanol by ether diffusion. The intensity data were collected with Bruker-Nonius Kappa CCD diffractometer using Mo Ka radiation (k = 0.71073 Å) at 293 K. Crystallographic data and details of the structure determination are given in Table 1, whereas selected bond lengths and angles are listed in Table 2. SHELX-97 [12] was used for structure solution and full matrix least squares refinement on F2. The structure was solved by direct and difmap methods (SIR92) [13]. Crystal structure has been deposited at the Cambridge Crystallographic Data Centre (Deposition no. CCDC 716649). G.M. Sheldrick, SHELXL97. Program for the Refinement of Crystal Structures, Univ. of Göttingen, Germany, 1997. A. Altamore, G. Cascarano, C. Giacovazzo, A. Guagliardi, M.C. Burla, G. Polidori, M.J. Camalli, J. Appl. Crystallogr. 27 (1994) 435. A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Verschoor, J. Chem. Soc., Dalton Trans. (1984) 1349. J. Kaizer, G. Baráth, G. Speier, M. Réglier, M. Giorgi, Inorg. Chem. Commun. 10 (2007) 292. J. Kaizer, J. Pap, G. Speier, M. Giorgi, M. Réglier, Transition Met. Chem. 29 (2004) 630. É. Balogh-Hergovich, J. Kaizer, G. Speier, G. Huttner, A. Jacobi, Inorg. Chem. 39 (2000) 4224.
[18] Synthesis of Cu(40 Meind)(3,5-DTBCH) (2): Cu(OMe)2 (251.2 mg, 2 mmol) and 3,5-di-tert-butylcatechol (444 mg, 2 mmol) were dissolved in MeCN under argon and the ligand 40 MeindH (1221 mg, 2 mmol) was added and stirred at room temperature for 24 h. The green precipitate was filtered washed with MeCN and dried in vacuum to give 2 (738 mg, 60.5%).UV–Vis (DMF) kmax (log e/dm3 mol1 cm1): 273.0 (4.264), 303.5 (4.304, 319.5 (4.266), 332.5 (4.244), 348.5 (4.086), 421.0 (4.316), 446.5 (4.217). IR (KBr) cm1: 3518, 3386, 3057, 2949, 2897, 2859, 2680, 1649, 1572, 1514, 1467, 1360, 1312, 1209, 1181, 1162, 1104, 1086, 1036, 967, 940, 910, 855, 812, 779, 711, 668, 646, 598, 523, 489, 459, 414 cm1. Anal. Calcd for C34H37ClCuN5O2: C, 66.81; H, 6.10; N, 11.46. Found: C, 66.52; H, 6.02; N, 11.12. [19] Kinetics of the oxidation of 3,5-di-tert-butylcatechol: In a typical experiment, [Cu2(40 MeInd)2(MeOH)(l-OH)]ClO4MeOH (5.49 105 mol dm3) and the corresponding substrate 3,5-di-tert-butylcatechol (3,5-DTBCH2) (15.40 103 mol dm3) were dissolved in 30 cm3 of DMF, under argon atmosphere in a thermostated reaction vessel with an inlet for taking samples with a syringe. The solution was then heated to the appropriate temperature (50 °C), the argon was replaced by air and the oxidation of 3,5-DTBCH2 was followed spectrophotometrically by monitoring the formation of 3,5-DTBQ at 400.5 nm (log e = 3.21) as function of time (kmax of a typical band of 3,5-DTBQ). The 3,5-DTBQ was also quantified by GC and verified by GC–MS. Dioxygen uptakes were also measured in a constant pressure gas-volumetric apparatus. The volume of absorbed dioxygen was red periodically using a gas burette. The solubility of dioxygen in DMF at 40 °C was taken as 5.4 103 mol dm3 [20]. The validity of Dalton’s law was assumed for the calculation of dioxygen concentration at different partial pressures [21]. [20] A. Kruis, Landolt-Börnstein, Band4, Teil 4, Springer, Berlin, 1976. [21] G. Ram, A.R. Sharaf, J. Ind. Chem. Soc. 45 (1968) 13.
