Exchange Interactions in the Copper ( II )-N-Benzoylglycine ( Hippuric Acid ) Complex

O complexo Cu-N-benzoilglicina provê um sistema conveniente para se estudar interações de troca fracas entre elétrons desemparelhados que são transmitidas por caminhos químicos de interesse biológico. A molécula existe como um dímero onde cada Cu está coordenado a cinco ligantes, formando uma pirâmide de base quadrada distorcida. O único caminho químico entre íons Cu magneticamente não-equivalentes envolve 17 átomos diamagnéticos e uma fraca ligação de hidrogênio putativa. Esta interação foi estudada por Ressonância Paramagnética Eletrônica de amostras monocristalinas a 9,5 e 34,5 GHz, tendo sua magnitude sido calculada em 4(1) mK. Esse valor é discutido em termos de valores obtidos para caminhos similares em outros compostosmodelo e proteínas.


Introduction
Toluene and other volatile organic compounds (VOCs) 1 are widely used as organic solvents in pharmacological, plastic and rubber industries, as well as in various materials such as paints, adhesives and combustion materials. 2The exposure to high concentration of toluene and other VOCs causes adverse health effects in nervous system, 3 mucous and dermal irritations, 4 and chromosome aberrations. 5,6oluene is metabolized in human liver microsomes 7,8 or in rat hepatic microsomes 9,10 by the hepatic cytochrome P450mediated mixed function oxidase system to benzyl alcohol, to benzoic acid and finally to hippuric acid, by conjugation with glycine to yield hippuric acid or N-benzoylglycine, which is then excreted in urine. 11opper is a bioessential element with relevant oxidation states. 12,13Due to the complexity of metalobiomolecules and the associated problems, a very useful way of gaining information is examining the behavior of simpler model compounds.The study of ion complexes of copper-amino acids [14][15][16][17][18][19][20][21][22][23][24][25][26] (or peptides) [27][28][29][30][31] and copper-nucleoside (or related bases) 32,33 has been a topic of increasing interest in the last few years, manly due to their relevance in the development of new reagents for biotechnology and medicine. 34-benzoylglycine is structurally similar to a dipeptide except for the absence of the N-terminal group.Due to its high affinity for Cu II , the dimeric complex Cu II -N-benzoylglycine could form in vivo, especially in cases of increased concentrations of hyppuric acid, and could then be used as a means of controlling hyppuric acid concentration.
6][37][38][39][40] In many cases, the copper ions form dimers with identical symmetry around the paramagnetic ions and with a shared ligand mediating exchange interaction between those ions.In this case, there is a greater possibility of formation of antiferromagnetically (AFM) coupled dimers, where the singlet (S = 0) state is the ground state and the triplet is thermally populated, than the ferromagnetic (FM) interaction at different site symmetries in bimetallic systems, [41][42][43][44][45][46] as here reported.
Electron Paramagnetic Resonance (EPR) has been extensively used to study the electronic structure and the local properties of metal ions, 47 radicals, 48 and other systems bearing an unpaired electron spin. 49EPR enables the evaluation of exchange interactions between coupled metal ions or radicals.In this way, the EPR technique has been used to measure the magnitudes of those interactions, whose nature is related to the electronic structure of the chemical path connecting the copper ions.Their study provides information about pathways that is difficult to obtain when one is dealing with long covalent and noncovalent bonds, whose weak contributions are masked by stronger interactions.][53] In combination with crystal structure data, EPR studies of model compounds allow one to study the magneto-structural correlations needed to characterize properties of weak bonds.This information may be translated to similar chemical paths occurring in macromolecules.
To evaluate the exchange interaction parameters from EPR data, detailed single-crystal measurements are required.5][56] Since exchange narrowing, in general, is essentially a temperature-independent phenomenon, [57][58][59][60] the great advantage of these experiments is that exchange interactions having very small magnitudes can be measured at room temperature.
In the present paper we report studies on the magnetic properties of di-(µ-hippurato-O)-bis[diaqua(hippurato-O) copper(II)] studied using EPR spectroscopy in single crystal samples at two microwave frequencies, 9 and 34.5 GHz, and at room temperature.The synthesis of a copper complex with N-benzoylglycine (hippuric acid), the X-ray structure and the coordination properties of the complex were first studied by Brown at al. 61 The EPR results are analyzed using the theories of Anderson, 57 and Kubo and Tomita 59 for the exchange narrowing phenomenon.The magnitudes of the exchange interactions between neighbor copper ions are estimated and discussed in terms of bond properties.

EPR measurements
Room temperature EPR spectra of single crystals were recorded at 9.5 (X-band) and 34.5 GHz (Q-band), using a Varian E-109 spectrometer.The angular variation was achieved either by rotating the sample at X-band or rotating the magnet at Q-band.At both frequencies we used 100 kHz magnetic field modulation and Cr III MgO (g = 1.9797) as a field marker.The single crystal sample with dimensions (2.5mm × 1.0mm × 1.0mm) was fixed with vacuum grease to the xz face of a cubic sample holder made of cleaved KCl crystal, which defined the xyz laboratory coordinate system.The single crystal was then oriented so that the abc crystal system was aligned with the xyz laboratory coordinate system.The sample holder was positioned on a pedestal in the center of the rectangular (X-band) or cylindrical (Q-band) cavities.The magnetic field B = Bh was then applied in the ab, c*a and c*b crystal planes, where h = (sinθcosφ, sinθsinφ, cosθ) denotes the direction of the external field in the abc* axes system.Single crystal spectra were obtained at five degree intervals in those three orthogonal planes.A single resonance line was observed for all orientations of the magnetic field B in the plane c*a, while two resonances were observed in the ab and c*b planes, except for directions close to the crystal axes, where they collapse to a single resonance (Figure 1).

Exchange pathways
The compound Cu[(C 6 H 5 )CONHCH 2 COO] 2 ⋅4H 2 O reported by Brown at al. 61   path has extremely weak character, since it encompasses long contacts through weakly electronegative atoms.One possible explanation for the establishment of such path would be the existence of a water molecule in the crystal structure which has not been detected when the structure was first determined.

EPR results
A single resonance line was observed for all orientations of the magnetic field B in the plane c*a, while two resonances were observed in the ab and c*b planes, except for directions close to the crystal axes, where they collapse to a single resonance (Figure 1).The positions and peakto-peak line widths (∆B pp ) of the single resonance for the c*a plane, or the two resonances in some orientations of the ab and c*b planes, were obtained from least-squares fits of the field derivative of one or two Lorentzian lines to the observed signal (Figure 1).The angular variation of the line positions and line widths calculated from the EPR spectra are shown in Figures 4 and 5.
The data in Figures 4 and 5 can be analyzed in terms of a spin Hamiltonian written as 21,23,28 (1), where H Z and H ex are the contributions appearing from the Zeeman and exchange interactions, S iα is the spin operator corresponding to a copper ion in the (α = A, B, C, or D) position of the ith unit cell (S = 1/2), and µ B is the Bohr magneton.As described in the previous section, the compound Cu II -N-Benzoylglycine belongs to space group P2 1 /c with two dimer units per unit cell.The distances between copper ions in the crystallographic structures are greater than 7.5 Å, and thus dipole-dipole interactions were disregarded in equation 1 31 .The g α are the symmetry-related molecular g-tensors associated to each copper ion in the sites defined before.Thus, g A ≡ g B and g C ≡ g D , with g A , g C and g B and g D related by a C 2 rotation around the b axis.Considering only exchange interactions between nearest neighbor copper ions and the symmetry conditions relating the exchange interaction parameters J iα,iβ in equation 1, only few different parameters contribute, which are discussed below in terms of the crystal structure of the compound.α (θ,ϕ) tensors of each copper site (solid line) in three crystal planes of single crystals of Cu II -N-benzoylglycine.Open triangles give the experimental data at 9.5 and 34.5 GHz.The solid lines were obtained with the components of g 2 given in Table 1.The data in the c*a plane (c) display a single resonance line for all orientations of the magnetic field B, whereas ab (a) and c * b (b) planes display the collapse of the EPR lines near the crystal axes.
Based on the previous discussion, the Zeeman and exchange interactions can be written in this case as 55 (2) (3) where and , and the exchange paths associated with the exchange parameters J AA , J AB and J AD were described above.The sum over m in the first term is over the nearest neighbor copper ions in cell i.Since g A = g B and g C = g D in equation 2, the contributions to H ex proportional to J AA and J AB in equation 3 do not produce any effect on the spectra.
The angular variation of the g 2 -tensor (Figure 4) shows that, for some orientations of the magnetic field, two resonance lines are resolved in the EPR spectra, which in turn collapse for directions around the crystal axes.This scheme can be rationalized in terms of a competition between the residual Zeeman interaction, the difference in Zeeman energy of two magnetically non-equivalent Cu II sites, and the respective exchange interaction.At some orientations, J ex is greater than the residual Zeeman interaction and then a single line is observed.This situation allows us to use a perturbation approach and divide the contributions to the Spin Hamiltonian in equation 1 in H 0 and H 1 , where H 1 represents the weaker interactions perturbing the term H 0 .Two resonance regimes arise (split and collapsed regimes), whose differences rely on the interplay between exchange and residual Zeeman contributions to H 1 .

Split resonance regime
In the split resonances regime, where two resonance lines are observed (planes ab and c*b; see Figures 4 and  5), the Hamiltonians H 0 and H 1 may be written as Each one of the lines observed in the planes ab and c*b corresponds to a magnetically non-equivalent Cu II site (Cu(A) or Cu(B)).The molecular g α -tensor is obtained directly from the data in Figure 5 in the regions where the lines are well-resolved, without the necessity of the use of decomposition methods.A least-squares fitting of the experimental data in those regions, for all orientations in the c*a plane against the function  g 2 =  ⋅  g ⋅  g ⋅ , leads to the components of the g α tensors given in Table 1.These results are used to draw the solid lines in Figure 4.

Collapsed resonances regime
In the collapsed resonances regime (ab and c*b planes near the crystal axes), the unperturbed Hamiltonian H 0 should be written as (6)   where S = SS  iα is the total spin of the system and g = (g A + g B + g C + g D )/4 is the average g tensor.The exchange interaction H ex commutes with the total spin S, and thus H 0 gives rise to a single EPR line at the average g factor.The perturbation can be written in this case as 55 (7)   and the line broadening is 62 where G u accounts for the differences among the four molecular g factors.∆B pp (0) is a residual line width that considers the small line width observed for the magnetic field along the crystal axes (Figure 5).Due to the space symmetry of the compound, and neglecting the interaction between A and C type copper electron spins, the sum over u contains a single term 28,67 A least-squares fitting of the line width data against the function defined in equation 9 for the regions around the crystal axes in the ab and c*b planes of the compound was performed.The values of the off-diagonal components of the molecular g tensors g A and g D presented in Table 1 were used in this calculation.The results of these fittings are shown as solid lines in Figure 5.These values and equation 9 were used to obtain the exchange frequencies and the values of the exchange coupling parameters | J AD /k | = 4(1)mK (10)   The magnitude of J AD (4 mK, ca.0.0028 cm -1 ) is very weak, as expected from the exchange pathway analysis described above.Similar magnitudes of exchange coupling between copper ions have been found in several complexes.In dichlorobis(1-phenyl-3,5-dimethylpyrazole)copper(II), an exchange coupling of 0.00386 cm -1 (ca.5.5 mK) was found, mediated by a long path (bond distance 13.60 Å) constituted by the pyrazole ring and a hydrogen-halogen bond C−H•••Cl 64 .In the complex Cu(Gly) 2 ⋅H 2 O, Hoffman et al. 54 determined an exchange interaction of 0.004 cm -1 (ca.5.7 mK) between magnetically non-equivalent copper ions connected through a hydrogen bond between a coordinated water molecule and a glycine oxygen atom.More recently, Santana et al. 65 calculated the coupling magnitude between copper ions in the compound Cu II (L-arginine) 2 (SO 4 ).(H 2 O) 6 , finding a weak coupling of 9 mK assigned to a long path with 12 atoms (total bond distance 19.789 Å) and two hydrogen bonds.Furthermore, in the complex aqua(glycyl-L-tryptophanate)-copper(II) dehydrate, an exchange coupling of 3 mK was found and also assigned to a long path with 10 diamagnetic atoms, including a moderate hydrogen bond (total bond distance 16.69 Å).All these model systems are very nicely related to the biological electron transfer system in the bacterial photosynthetic reaction center protein of Rhodobacter sphaeroides, where the coupling between the two quinone acceptors at 17.3 Å is 3.8 mK. 66Following the classification proposed by Vieira et al., 31 the exchange coupling determined in the present work would be in their group III, which includes long paths with more than 10 diamagnetic atoms and magnitudes of the order of few milliKelvin.

Conclusions
In this article we studied the magnetic properties of the Cu II -N-benzoylglycine compound, whose interest is associated with its toxicology or ecotoxicology.The EPR technique, along with X-ray diffraction data, supplies information on the possible chemical path and the magnetic interactions established in model metal-ligand complexes.From the analysis of the experimental data, it is possible to calculate the coupling constants between the paramagnetic sites.These couplings usually present very weak magnitudes and are thought to model biologically-relevant interactions.One can readily see that room-temperature EPR measurements can thus offer unique possibilities of evaluating very small magnitude interactions, which would be directly detected by other experimental techniques only by means of very low temperature measurements.
In the Cu II -N-benzoylglycine complex, the copper coordination sphere in each site is a slightly distorted squarebased pyramid with values of g || > g ⊥ , which indicates that the ground-state orbital of the copper ions is essentially a d x 2 -y 2 state.The values obtained from the data at 34.5 GHz in the Table 1, g || = 2.3624(9), g O( 14)-O(1) = 2.0875(8), g W(1)-W(2) = 2.0545 (7), indicate that the electron ground-state orbital is the plane containing the copper ion and the hippurato oxygen and water ligands.The only interactions that we can quantify using the EPR technique are the inter-chain contacts that relate copper ions in the two different chains observed in the crystal structure.The evaluated magnitude |J/k| is 4(1) mK.This low value of |J/k| agrees well with the described chemical path discussed in the text.

Figure 1 .
Figure 1.EPR spectra at Q-band (34.5 GHz) of the compound Cu II -N-benzoylglycine, showing the phenomenon of collapse and line narrowing by exchange in the ab (b) and c*b (c) crystal planes.The c*a (a) plane presents only one line as a consequence of the symmetry of the spatial group P2 1 /c.

Figure 2 .
Figure 2. Schematic drawing of the copper coordination sphere with distances (in Angstroms) and angles indicated.

Figure 3 .
Figure 3. Two neighbor copper ions separated by path containing the hydrogen bond discussed in the text are displayed.The distance and the angle of the putative hydrogen bond between molecules are given in Angstroms (Å) and degrees ( o ), respectively.

Figure 4 .
Figure 4. Angular variation of the molecular g 2α (θ,ϕ) tensors of each copper site (solid line) in three crystal planes of single crystals of Cu II -N-benzoylglycine.Open triangles give the experimental data at 9.5 and 34.5 GHz.The solid lines were obtained with the components of g 2 given in Table1.The data in the c*a plane (c) display a single resonance line for all orientations of the magnetic field B, whereas ab (a) and c * b (b) planes display the collapse of the EPR lines near the crystal axes.

Figure 5 .
Figure 5. Angular variation of the peak to peak EPR line width observed at 9.5 and 34.5 GHz for a magnetic field applied in the three crystalline planes ab (a), c*b (b), and c*a (c) of the single crystal sample Cu II -N-benzoylglycine.The solid lines are obtained with equation (9) and parameters are included in Table1.

Table 1 .
Values of the components of the crystal and molecular g  2 tensor obtained by least-squares analyses of the data at each microwave frequency.(g 2 ) 1 , (g 2 ) 2 , and (g 2 ) 3 and n  1 , n  2 and n  3 are the eigenvalues and eigenvectors of the g  2 tensor in abc* coordinate system(a) (a)The values of g -O1* , W1 -W2 and g || = g Cu -O1 represent the molecular g value for each in oxygen-oxygen, water-water, and parallel directions, respectively.