Solution Studies of Copper ( II ) Complexes as a Contribution to the Study of the Active Site of Galactose Oxidase

Reportamos neste trabalho, a síntese, e a caracterização em solução dos compostos de coordenação de cobre(II) – [Cu(H 2 bbpeten)](NO 3 ) 2 , [H 2 bbpeten = N-(2-hidroxibenzil)N,N’-bis(2-piridilmetil)N’-(2-hidroxietil)etilenodiamina]; [Cu(H 3 bpeten)](NO 3 ) 2 , [H 3 bpten = N,N’-bis-(2-hidroxibenzil)N-(2-piridilmetil)-N’-(2-hidroxietil)etilenodiamina]; [Cu(Hnbbpeten)]NO 3 , [H 2 nbbpeten, N-(5-nitro2-hidroxibenzil)-N,N’-bis(2-piridilmetil)-N’-(2-hidroxietil)etilenodiamina] e [Cu(Hbnbpeten)], [H 3 bnbpeten = N,N’-bis-(5-nitro-2-hidroxibenzil)-N-(2-piridilmetil)-N’-(2-hidroxietil)etilenodiamina] com ligantes polidentados assimétricos N,O-doadores. Dois destes ligantes apresentam grupos NO 2 em posição para aos grupos fenólicos. Estudos eletroquímicos foram realizados para os ligantes livres e para os complexos de cobre(II).


Introduction
Organic synthesis of unsymmetrical multidentate ligands that are necessary to enforce the desired coordination environments of the metal ions, has played an important role in the design of the active site of metalloprotein analogues. 1,2opper in its various roles in biological systems displays different spectroscopic and chemical properties presumably because of the different ligand environments and coordination numbers. 3alactose oxidase (Goase) is a mononuclear copper metalloenzyme which catalyses the oxidation of several primary alcohols to aldehydes.The geometry around the monomeric copper(II) center in galactose oxidase extracted from the fungus Fusarium dendroides at pH 4.5 can be described as square pyramidal, comprised of one tyrosyl oxygen atom, two histidine nitrogen atoms and one acetate oxygen atom in the plane and of a tyrosine oxygen atom occupying the apical position of the pyramid. 4,5 the active and oxidized Goase form the enzyme contains coordinated Tyr-272.Unusual features of Tyr-272 are a thioether link in the ortho-position of the phenolate oxygen formed by the covalent binding to the S-atom of Cys-228.][8][9] Thus the study of variously substituted proligands belonging to the same series seemed an essential prerequisite to the understanding and, consequently, the control of redox properties of the corresponding copper complexes.
We describe here solution studies of four copper(II) complexes derived from four unsymmetrical N,O-donor polyfunctional ligands, two of which bear no substituent at the phenol moieties (L1 and L2) [10][11][12] and two which bear the electron-withdrawing -NO 2 group at one or two of the phenol moieties (L3 and L4).These four compounds have been designed with the aim of studying the effect of -NO 2 group on the physicochemical properties of the copper complexes.

Scheme 2.
The IR spectra of 1, 2, 3 and 4 are similar to those of the proligands.They differ only in: (a) appearance of one well defined band at 3420, 3388, 3055 and 3416 cm -1 , respectively, attributed to the ν(O-H) stretching of uncoordinated primary alcohol; (b) the δ(O-H) in-plane bending of the phenol of the compounds 3 and 4, at 1343 cm -1 , compared to those observed in the proligands, indicating that the phenol groups are deprotonated and coordinated; (c) row of bands at 1384 and 1268 cm -1 in the spectrum of 1, 2, and 3, attributed to ν(N-O) of the NO 3 counterion.
The molar conductivities of 1, 2 and 3, in DMF at 298 K are 154, 158 and 87 Ω -1 cm 2 mol -1 , respectively, which are consistent with 2:1, 2:1 and 1:1 electrolytes.The molar conductivity of 4 in DMF solution at 298K is insignificant which is consistent with it being a neutral coordination compound. 14

Electronic absorption and electron paramagnetic resonance spectra
The electronic spectra of 1, 2, 3 and 4, measured in DMF in the visible region are shown in Figure 2 and reveal the following transitions at λ max /nm (ε/L mol -1 cm -1 ): 426 (280) and 650 (111) for 1; 424 (405) and 633 (114) for 2; 387 (12,273), 473 (355) and 657 (111) for 3 and 390 (18,965) and 659 (117) for 4 that can be associated with metal-ligand coordination.Copper(II) ions can adopt square-planar, square-pyramidal, trigonal-bipyramidal, octahedral and tetrahedral geometries, which, except for the first, are generally distorted from the idealized structures.The d-d spectra shown by these coordination geometries are distinctive only in the case of the tetrahedral environment where the absorptions occur at much lower energies and generally show, in the distorted forms, wellseparated absorption peaks; all the other geometries show closely spaced absorption manifolds. 3The intense absorbance bands occurring at 387 and 390 nm for 3 and 4, respectively, are assigned to Cu II → O - axial and/or O - equatorial → Cu II charge-transfer transitions.][17] The visible absorption spectrum of galactose oxidase is characterized by three absorption bands having molar extinction coefficients of the order of 1000 L mol -1 cm -1 .These occur at 444, 630 and 775 nm, the last two being broad and overlapping.The bands at 630 and 775 nm are assigned to d-d transitions and suggest that the copper(II) ion in the enzyme is in a somewhat distorted five-coordinated environment. 3,16Thus, of the four copper (II) compounds described in this work, the [Cu(H 3 bpeten)](NO 3 ) 2 , (2) has chromophoric properties most similar to those found in the enzyme.
The X-band EPR spectrum of a frozen solution of 1, 2, 3 and 4, in DMF are shown in Figure 3.The Hamiltonian parameters obtained from the spectra of these compounds and for Goase are resumed in Table 2.
The EPR spectra of the four copper(II) coordination compounds described here, indicate axial symmetry.For all four complexes, g II > g ⊥ > 2, suggesting distorted tetragonal, square-pyramidal or square-planar geometry.Moreover, the g II and A II values of Cu[(H 2 bbpeten)](NO 3 ) 2 , [Cu(H 3 bpeten)](NO 3 ) 2 , [Cu(Hnbbpeten)]NO 3 and [Cu(Hbnbpeten)] are found in the regions characteristic of CuN 4 and CuN 3 O chromophores in the g II vs.A II diagram and near the value of galactose oxidase.Sagakushi and Addison 19 showed that the g II / A II ratio can be used as a convenient empirical index of tetrahedral distortion in CuN 4 units.This value ranges from ca. 105 to 135 cm for square-planar structure, and this quotient increases on the introduction of tetrahedral distortion to the chromophore.Further, tetrahedral distortion of a square-planar chromofore is observed when any of biomimetic (N, O, S) donors reduces A // and increases g // .Using that relationship and the results in Table 2, all the four compounds have a slightly tetrahedral distortion, that is, they seemed to be square-pyramidal.Probably the nitrogen atoms of L1 and L3 are in the equatorial plane of the respective compounds 1 and 3.For the compounds 2 and 4 the equatorial ligand sets of the copper(II) ion are composed of three nitrogen atoms and one oxygen atom.The increased g // values and decreased A // values in the compounds 2, 1, 3 and 4, respectively, show that the ligand field strength decreases in these compounds in the same order.The complex [Cu(H 3 bpeten)](NO 3 ) 2 , (2) has a stronger ligand field than [Cu(H 2 bbpeten)](NO 3 ) 2 , [Cu(Hnbbpeten)]NO 3 and [Cu(Hbnbpeten)] (A // = 186.7 vs. 182.6,181.6 and 178.6 x 10 -4 cm -1 and g // = 2.215 vs. 2.225, 2.231 and 2.241) consistent with the more energetic d d transition found in its electronic spectrum.

Electrochemistry
The redox properties of the four proligands and of the four copper(II) complexes were investigated by cyclic voltammetry in acetonitrile solution.
The electrochemical study of the four proligands focuses on the first one-electron oxidation of the phenolic units in view of studying the modulation of this property by the substituting groups.This may allow prediction of the ease of one-electron oxidation of the corresponding copper(II) complexes because the oxidation potential of the protonated free proligand is close to that of the corresponding copper(II) complex (protonation parallels metallation). 7L1, L2, L3 and L4 exhibit an irreversible oxidation wave at E pa = 0.79; 0.96; 0.91 and 1.20 V vs. Ag/AgCl (Table 3).These processes correspond to oxidation of the phenolic moieties leading to unstable radical cations, undoubtedly of the phenoxyl type.The E pa values of the ligands are consistent with the electronwithdrawing character of the substituents on the phenolic arms.Compared with L1, the substitution by an electronwithdrawing group such as NO 2 (L3), increases E pa by 0.12 V and compared with L2, the substitution by two electronwithdrawing group such as NO 2 (L4) increases E pa by 0.24 V.
The cyclic voltammograms of 1, 2, 3 and 4, in CH 3 CN at scan rate of 100 mV s -1 are shown in Figures 4 and 5.In the four complexes, the ligands stabilize the copper(II) with respect to copper(I) state, since the reduction of the complexes occurs at low potentials (E pc = -0.244;-0.700; -0.512 and -0.842 V for 1, 2, 3 and 4, respectively).The cyclic voltammograms of 1 and 3 have a quasi-reversible redox couple at E 1/2 = -0.221and -0.452 V vs. Ag/AgCl, (-0.626 and -0.857 V vs. Fc + /Fc 0 ) respectively, which can be ascribed   to the Cu 2+ Cu + redox couple.Compared with 1, the presence of an electron-withdrawing group such as NO 2 in the coordination environment of 3 increases E 1/2 by 0.231 V.The cyclic voltammograms of 2 and 4 show a irreversible reduction at E pc = -0.700and -0.842 V vs. Ag/AgCl, (-1.110 and -1.252 V vs. Fc + /Fc 0 ) respectively, which can be ascribed to the Cu 2+ → Cu + process.The reduction process is irreversible for the two complexes and leads to a deposit of copper(0) on the electrode surface, as judged by the observation of a sharp oxidation peak during the reverse scan [E pa = -0.242and -0.278 V vs. Ag/AgCl (-0.652 and -0.688 V vs. Fc + /Fc 0 ), respectively] with the typical features of a redissolution process. 9However, compared with 2, the substitution by two electron-withdrawing NO 2 group on the phenolic arms of the ligand in 4 also increases E pc by 0.142 V.The most easily one-electron oxidizable proligand is H 2 bbpeten -L1 that has one phenol moiety with no electrondonating or withdrawing group.The complex [Cu(H 2 bbpeten)](NO 3 ) 2 1 has a reduction potential more cathodically shifted than 2, 3 and 4.

Conclusions
Four Cu II complexes containing four polyfunctional ligands, where two of which bear no substituent at the phenol moieties (L1 and L2) and the other two bear an electron-withdrawing NO 2 group at one or two of the phenol moieties (L3 and L4), have been prepared and characterized electrochemically and spectroscopically.The four compounds have absorption spectra bands in the ranges 665-636 (broad and distorted) and 422-463 nm due to d-d transitions.The EPR spectra showed that the four copper(II) coordination compounds have axial symmetry and a distorted square-pyramidal environment like the galactose oxidase.Electrochemical measurements gave E pa values in agreement with the electron-withdrawing character of the substituents on the phenolic arms of the respective ligands.The substitution by -NO 2 stabilizes the copper(II) state by several hundred of millivolts.Electronwithdrawing substituent such as -NO 2 do not lower the oxidation potential of the phenol groups, just like the thioether linked in the ortho-position of the phenolate oxygen of the Tyr 272 of the enzyme Goase does.Further investigations of electron-donating substituents in the phenolic arms of the ligands are now in progress.
The major influence of the substitution of the phenolic moieties by electron-withdrawing substituent such as -NO 2 implies to us that it was not necessary to add base during the preparation of the copper complexes 3 and 4, to obtain coordinated deprotonated phenol groups due the greater acidity of the respective ligands.
The results are consistent with the structures proposed for the copper(II) compounds presented in Figure 6.

Figure 1 .
Figure 1.Design of the galactose oxidase active site, crystallografically characterized.

Table 1 .
16ectronic spectra data for the copper(II) compounds in DMF, and for GOase16