EPR and Semi-Empirical Studies as Tools to Assign the Geometric Structures of FeIII Isomer Models for Transferrins

A reação entre Fe(ClO 4 ) 3 .nH 2 O e N,N’-bis[(2-hidroxibenzil)-N,N’-bis(1-metilimidazol-2-ilmetil)]etilenodiamina (H 2 bbimen) resulta na formação de dois isômeros geométricos, A e B, do cátion complexo [Fe(bbimen)], os quais foram isolados e caracterizados por espectroscopia na região do infravermelho e UV-visível, espectrometria de massas, medidas de condutividade molar, voltametria cíclica, espectroeletroquímica e espectroscopia de ressonância paramagnética eletrônica (RPE). A geometria de um desses isômeros foi claramente demonstrada por análise cristalográfica de raios X de monocristal. O composto cristaliza no sistema monoclínico, grupo espacial P2 1 /c, a = 14,104 (3), b = 15,626 (3), c = 13,291 (3) Å, β = 98,07 (3), Z = 4, R 1 = 6,35% e wR 2 = 20,57%. As propriedades eletroquímicas do cátion [Fe(bbimen)] (-0,58 V versus NHE) são bastante similares às das transferrinas (-0,52 V versus NHE), indicando que o complexo é um bom modelo para as propriedades redox daquelas metaloenzimas. A espectroscopia de RPE foi a única técnica espectroscópica capaz de diferenciar os dois isômeros isolados (A e B). Os estudos de RPE revelaram que o isômero A trata-se de um complexo de Fe spin alto distorcido rombicamente (E/D ≅ 0,33) e apresenta um espectro caracterizado por uma linha estreita em g 1 ≅ 4,1 e outra larga em g 2 ≅ 9,0. O espectro de RPE do isômero B apresenta, além das linhas em g 1 ≅ 4,2 e em g 2 ≅ 9,0, um conjunto de linhas em g 1 ≅ 3,0, g 2 ≅ 3,6 e g 3 ≅ 5,1, que têm sido atribuídas a complexos de Fe com simetria tendendo à axial (E/ D ≅ 0,22). Estudos teóricos empregando cálculos semi-empíricos, combinados com os dados de RPE, foram essenciais para a atribuição das estruturas geométricas dos isômeros A e B.


Introduction
Transferrins (Tfs), or siderophilins, are a family of Fe-binding proteins belonging to the Fe-tyrosinate class.In vertebrates, they are found in blood (serum transferrin), eggs (ovotransferrin) and milk (lactoferrin).The main physiological role of serum transferrin is the Fe transport through the circulating blood and its release to the Fedependent cells.Another role is believed to be played by ovotransferrin and lactoferrin, which involves bacteriostatic effects. 1,2As the serum concentration of transferrin is about 35 μmol L -1 , and about 70% of it is in the apo-form (protein depleted of Fe), it is also proposed to be a blood transporter of several other metal ions, including vanadium, aluminum, gallium and cobalt. 3Of the Fe-dependent cells, tumor cells are probably the ones of most concern because of their high demand for iron and as a consequence they exhibit enhanced transferrin receptor (TfR) expression. 4uring the lifetime of serum transferrin in the circulation system, it is able to perform reverse uptake and deliver Fe about 100-200 times, and thus an understanding of the kinetics and the mechanism involved in the fundamental physiological process is of interest. 5It has been proposed that the first step is the coordination of Fe III to transferrin, which is coupled to the synergistic binding of a carbonate ion.The Fe III -Tf complex is then transported through the blood to the Fe-dependent cells, where the Fe III -Tf complex binds to a membrane transferrin receptor.This system is then internalized by the cell to an ATP-driven proton-pumping endosome (pH 5.4-6.0),where the metal is released as Fe II .This then leaves the endosome via a membrane metal ion transporter (DMT). 6n order to better understand this process, the full characterization of transferrins is of fundamental importance.The structures of human lactoferrin, 7 serum transferrin, 8,9 and duck ovotransferrin 10 have all been well studied using X-ray crystallography and are quite similar.They are bilobal proteins with each domain (C-and Nlobes) binding one Fe III ion.[9][10] In addition to the structural characterization, transferrins have also been investigated using other techniques.Human lactoferrin gives an electronic spectrum characterized by two ligand-to-metal charge transfer (LMCT) bands at 465 nm and 280 nm, both ascribed to tyrosinate →Fe III charge transfer transitions. 11ssbauer spectra of human lactoferrin and serum transferrin display isomer shifts of 0.39 mm s -1 and 0.38 mm s -1 respectively, which are typical of high-spin Fe III centers. 12Additionally, the serum transferrin spectrum shows a large quadrupole splitting reflecting the nonsymmetrical coordination field around the Fe III ion, as shown by the structural analysis. 8,11In agreement with the Mössbauer data, the EPR spectrum of serum transferrin displays a sharp signal at g ≅ 4.3 and a shoulder at g ≅ 9.0, both characteristics of high-spin Fe III ions. 12s the iron transport involves the uptake of Fe III and the release of Fe II ions, knowledge of the redox potentials of Fe-bonded transferrins is extremely important.Redox potentials of human serum transferrin have been investigated under physiological (7.4) and endosomal (5.8) pH conditions.Spectroelectrochemical measurements 13 revealed that E 1/2 = -0.52 (8) V versus NHE at pH 7.4.At pH 5.8, the redox potentials (E 1/2 ) of the three possible Fe III -Tf forms were measured: the diferric (E 1/2 = -0.526V versus NHE), the monoferric C-lobe (E 1/2 = -0.501V versus NHE) and the monoferric N-lobe (E 1/2 = -0.520V versus NHE) forms. 14All of these measured redox potentials are too low for reduction of Fe III by physiological agents.However, it has recently been demonstrated that the interaction of the Fe III -Tf complex with the membrane transferrin receptor (TfR) raises the potential by more than 200 mV, explaining the Fe III →Fe II reduction in physiological medium. 66][17][18][19][20] Very recently, we reported 16 the synthesis and full characterization of a series of Fe III complexes, [Fe(bbpen-X)] + , using hexadentate ligands (H 2 bbpen-X) containing pyridine and phenol pendant arms.In this paper, we show that the reaction of the analogous H 2 bbimen (Scheme 1) with Fe(ClO 4 ) 3  .nH 2 O affords a mixture of geometric isomers (A and B), which were only differentiated by EPR analysis.In order to determine the structure of the Fe III complexes, semiempirical studies were a helpful tool when combined with EPR spectroscopic data.

Materials
Electrochemical and spectroscopic data were collected in high purity solvents, and high purity argon was used when necessary to obtain inert atmosphere.All other chemicals and solvents were of reagent grade, purchased from commercial sources, and used without further purification.

Physical measurements
Infrared spectra were obtained on a FT-IR Perkin-Elmer 16PC spectrophotometer as KBr pellets or films.Elemental analyses were performed on a CHN Perkin-Elmer 2400 analyser.Molar conductivity was measured in CH 3 CN (10 -3 mol L -1 ) at 298K with a Digimed CD-21 equipament.Electrospray-ionization (ESI-MS) mass spectra were recorded in acetonitrile using a Micromass LCT time-of-flight mass spectrometer with electrospray and APCI, coupled to a Waters 1525 Binary HPLC pump.UV-visible absorption spectra were measured in CH 3 CN on a Perkin-Elmer Lambda 19 spectrophotometer.Cyclic voltammograms were recorded at room temperature using a PAR 273 (Princeton Applied Research) potentiostat in acetonitrile solution, under argon atmosphere, with TBAPF 6 (0.1 mol L -1 ) as supporting electrolyte.A standard three-electrode cell was used: a gold working electrode, a platinum wire auxiliary electrode and a SCE reference electrode.Ferrocene was used as internal standard (E 1/2 = 0.16 V versus SCE). 22Spectroelectrochemical experiments were carried out at room temperature in acetonitrile, under argon, with TBAPF 6 (0.1 mol L -1 ) as supporting electrolyte, using an optically transparent thinlayer cell constructed according a previously reported procedure. 23A gold minigrid and a platinum wire were used, respectively, as working and auxiliary electrodes, and a SCE was used as the reference electrode.Potentials were applied with a PAR 263 potentiostat/galvanostat and the spectra were recorded with a Perkin-Elmer Lambda 19 spectrophotometer.Spectral changes were registered after the establishment of equilibrium (120 s) and the experiment was stopped when no further changes were observed.Potentials were applied in the range of -0.84 to -1.06 V versus Fc + /Fc and the spectra recorded from 300 to 750 nm.The Fc + /Fc couple was used separately to monitor 22 the reference electrode (E 1/2 = 0.16 V versus SCE).X-band EPR spectra were recorded on a Bruker ESP 300E spectrometer at 77 K in CH 2 Cl 2 solution.

Synthesis of [Fe(bbimen)]ClO 4
Mixture of isomers (1) crystals suitable for X-ray crystallographic analyses were obtained by slow evaporation of a solution of 1 in methanol:ethanol:water (10:1:1).While attempting to find solvent systems to recrystallize the product, we were able to isolate two geometric isomers of [Fe(bbimen)]ClO 4 by fractional crystallization.About 60% of the mixture of isomers ( 1) is soluble in hot acetone (isomer A) and was recrystallized in this solvent, yielding sharp purple needles that were not suitable for X-ray analysis.The remaining amount (isomer B) was then recrystallized in methanol yielding a microcrystalline sample.
Isomer A. ESI Caution!Perchlorate salts of metal complexes with organic ligands are potentially explosive, and only small amounts should be carefully handled.

X-ray crystallography
A violet crystal was selected and isolated for crystallographic analysis with a CAD-4 diffractometer.Cell parameters were determined from 25 carefully centered reflections in the θ range 8.79-15.25 o using a standard procedure. 25Data were corrected for Lorentz and polarization effects 26 and for absorption 27 (transmission factors 0.7305 and 0.9546).The structure was solved with SIR97 28 and refined by full-matrix least-square methods using SHELXL97. 29The disorder in the perchlorate ion was modeled with two alternative positions for each oxygen atom.At C7, C8 and C2E, the H atoms were also placed using a standard disordered model.All non-H atoms were refined with anisotropic displacement parameters, except for O22.Hydrogen atoms were added at calculated positions and included in the structure factor calculations, with C-H = 0.93 Å (0.96 Å for methyl groups) and U iso (H) = 1.2U eq (C) or 1.5U eq (methyl C).Other selected crystallographic information is shown in Table 1.

Theoretical calculations
All geometry optimizations were performed using semi-empirical PM3 TM molecular orbital calculation using the Spartan 04 program. 33Theoretical vibrational frequencies were used to find the minimum geometries.1][32] The spin state of the Fe III isomers was stated as high-spin, ground state 6 S, according to the EPR data.The calculations were carried out on a 2.6 GHz Athlon PC, with 1 GB RAM and 40 Gb HD, under the Windows 2000 operational system.

Results and Discussion
Synthesis H 2 bbimen was prepared in good yield as previously described. 21It reacts with Fe(ClO 4 ) 3  .nH 2 O in methanol to give a mixture (1) of geometric isomers (A and B) of the stable cation complex [Fe(bbimen)] + .The mixture was separated by fractional crystallization in hot acetone.

X-ray structural characterization
A single crystal suitable for X-ray analysis was isolated from 1, i.e., from the mixture of isomers.The structure of the complex consists of a discrete mononuclear cation, [Fe(bbimen)] + , and a ClO 4 -counterion, in general positions.The elemental cell also possesses an ethanol molecule as crystallization solvent.Crystallographic data and main bond distances and angles are presented in Tables 1 and 2, respectively.X-ray crystallographic analysis shows that the Fe III center is in a pseudo-octahedral environment, with the two halves of the bbimen 2-ligand coordinated in a facial mode (Figure 2).This same arrangement (fac-N 2 O) is observed in other complexes with the analogous hexadentate bbpen 2-ligand: [Fe III (bbpen)] + , 16,20 [V III (bbpen)] + , 34 and [Mn III (bbpen)] + . 23he equatorial plane is geometrically defined by the O1-O2-N1-N2 atoms, from which the Fe III deviates only 0.001 Å.The equatorial plane is then composed of two nitrogen and two oxygen atoms from the ethylenediamine backbone and the phenolate groups, respectively.They are coordinated in such a way that the nitrogen atoms are cis to each other, and trans to the oxygen atoms.Completing the coordination sphere of the metal center are the two nitrogen atoms from the 1-methylimidazole group.The octahedral geometric distortion can be observed by the angles of the Fe III coordination sphere that deviate from 90 °(Table 2).The coordination of the ethylenediamine group results in a distorted five-membered ring (FeN2C5C6N1), in which the C5 and C6 atoms lie at opposite sides of the equatorial plane, with deviations of -0.358 and 0.202 Å, respectively.

Physical-chemical properties IR spectroscopy and ESI-MS.
The IR spectra of 1, A and B are quite similar and characterized by typical bands of the ligand skeletal, in addition to a band at 1094 cm -1 attributed to the Cl-O stretching of the perchlorate anion (Figure S1). 40A comparison with the IR spectrum of the free H 2 bbimen reveals a decrease in the band at 1372 cm -1 , which indicates the coordination of the phenol group in its deprotonated form.The ESI-MS (positive mode) spectra of 1, A and B were recorded from freshly prepared solutions in acetonitrile (Figure S2).In fact, the spectra of A and B are identical, with the base peak (100%) corresponding to [Fe(bbimen)] + at m/z + 514.2.The other four observed peaks agree quite well with the predicted isotopic distribution for a Fe center (predicted: 514.18 (100%); 515.18 (33.1%); 512.18 (6.4%); 516.18 (5.9%)  and 513.19 (1.8%)).The molar conductivities of 1, A and B in acetonitrile at 298 K are all around 130 Ω -1 cm 2 mol -1 , which agrees with 1:1 electrolyte solutions. 24Therefore, we conclude that the first coordination sphere of the Fe III center is maintained intact when the complex is dissolved in acetonitrile.
UV-visible spectra.The UV-Visible spectra of 1, A and B were recorded in acetonitrile and show the same behavior with bands at identical wavelengths.The spectrum of 1 (Figure 3) is characterized by transitions at λ max /nm (ε/mol L -1 cm -1 ): 236 (13500-shoulder); 278 (11300); 321 (7700) and 542 (4700).As the H 2 bbimen spectrum (Figure 3-inset) has bands at λ max /nm (ε/mol L -1 cm -1 ): 213 (29000) and 276 (6100), the two bands observed for 1 at higher energy are attributed to π → π * internal transitions of the aromatic rings.17][18][19][20]41 Comparing the UV-Visible data of 1 with those reported 16,20 for the analogous [Fe(bbpen)] + , it is observed that there is a hypsochromic shift of the p π → d π* LMCT band from 574 to 542 nm when pyridine groups in H 2 bbpen are replaced by 1-methylimidazole groups in H 2 bbimen.These results are interpreted as a consequence of the greater basicity of the 1-methylimidazole groups (pKa 1 ≅ 2.06) 42 compared to the pyridine groups (pKa 1 < 1.3). 439]41 The electronic spectrum of 1 was also recorded in the solid state (KBr pellets, diffuse reflectance) and revealed the same behavior observed in acetonitrile (λ max at 538 and 318 nm), indicating no changes in the coordination sphere of 1 when in solution, in agreement with the ESI-MS mass spectral data.
Electrochemistry and spectroelectrochemistry.The redox behavior of 1, A and B was investigated by cyclic voltammetry and, as observed using all other techniques discussed above, it is quite similar.Complexes A and B have one reversible one-electron redox couple at approximately -0.58 V versus NHE (-0.98 V versus Fc + / Fc) ascribed to the Fe III → Fe II redox process (Figures 4  and S4).This value is cathodically shifted (-0.16 V versus NHE) when compared to that of [Fe(bbpen)] + (-0.42 V versus NHE), 16 reflecting the decrease in the Lewis acidity on the Fe III center resulting from the ligand basicity increase. 17,18Regarding the electrochemical properties of transferrins, the redox potential of -0.52 V versus NHE is in close proximity to that observed for [Fe(bbimen)] + (-0.58 V versus NHE), which indicates that it is a good model for the redox potential of transferrins.
In order to investigate the spectral changes during the Fe III → Fe II redox process, spectroelectrochemical measurements were carried out under the same experimental conditions as those employed in the CV studies.A decrease in the LMCT phenolate → Fe III band at 542 nm, with a simultaneous increase in a new band at 420 nm was observed (Figure 5).This band (420 nm) is tentatively attributed to an Fe II → 1-methylimidazole MLCT transition.This kind of transition has been observed in other systems employing pyridine and pyrimidine groups as ligands, and corroborates this assignment. 16,44During the whole process an isosbestic point was clearly observed at 340 nm.The presence of isosbestic points provides strong evidence for only two species present in solution during the redox process.Figure 5 also presents the Nernst plot, which is in agreement with the cyclic voltammetric data, and provides a redox potential of -0.94 V versus Fc + /Fc for the transference of one electron in the process.

Figure 1 .
Figure 1.Graphic representation of the active site of human lactoferrin based on coordinates from the PDB file from reference 9 (1D3K).

Table 1 .
Selected crystal data and structure refinement for [Fe(bbimen)]