Optical ( DRUV-VIS ) and Magnetic ( EPR ) Behavior of Synthetic Melanins

The properties of melanins prepared from L-dopa oxidation by both chemical and electrochemical methods are reported, searching for a material with more intense optical absorption in the visible region. The characterization of the samples have been done by cyclic voltammetry (CV), cronoamperometry (CA), Fourier transformed infrared spectroscopy (FTIR), electron paramagnetic resonance spectroscopy (EPR) and diffuse reflectance ultraviolet-visible spectroscopy (DRUV-VIS) techniques. The electrochemical method enhanced the organic free radical (spin) concentration and the absorption intensity in the visible region of the spectrum. The DRUV-VIS technique along with mathematical tools, such as the Kubelka-Munk remission function, were good options for the characterization of the final products.


Introduction
Melanins constitute a widespread class of pigment in nature, commonly formed by oxidative polymerization of cathecolic species derived from tyrosine 1 .In soil, fungal and actinomycete melanins are thinking to be an important precursor for the humic substance formation 2,3 .In humans, melanins are present in the skin and in certain brain structures like the substantia nigra and red nucleus, and in the inner ear 4 .The physico-chemical characteristics of melanins turn them quite complex to be studied by standard methods.The biological functions of melanins are now more noticeably and understandable.It is believed that in many pigment cells, melanin may act as a powerful antioxidant via, for example, for heavy metal sequestration 5 .Natural melanins contain a wide variety of bound metals in vivo, and are unusual among the biopolymers because of the highly polyionic, cross-linked, and heterogeneous polymers, whose bonding patterns likely vary in subtle ways.This amorphous dark pigment, highly hygroscopic is obtained by oxidation of molecules that contain catechol function such dopamine, L-dopa 6 .Previously, melanins were considered stable compounds due to its small changes during both acid and basic hydrolysis and low solubility 7 .Nowadays melanins are faced as heterogeneous polymers containing a mix of 5,6-di-hydroxiindol moieties, with different oxidation states and attached randomically at pirrol moieties by C-C bonding 8 .Many studies have been done about the melanins polymerization mechanism to understand its biosynthesis 9 .Some chromatographic, electrochemical and spectro-electrochemical studies [10][11][12] , besides capillary electrophoresis 13 and photoelectron spectroscopy (XPS) studies 14 have been employed to determine the melanin formation mechanism.The L-dopa (3,  4-dihydroxiphenilalanine) was used as a model compound because its structure is similar to monomers found into natural melanin structure 15 allowing the biomimetic studies about it 8,16 .
In this study two synthetic melanins were obtained from both chemical and electrochemical oxidation of L-dopa, in order to observe differences in light absorption by the prepared materials on the visible region.DRUV-VIS technique along with the Kubelka-Munk remission function was used to distinguish between the absorption properties of the two synthesized solid melanins.The electron paramagnetic resonance (EPR) study allowed determination of organic free radical (OFR) concentration (spin.g - ) of the two complex organic materials 17,18 .The OFR was also identified by the EPR spectroelectrochemistry technique 19 .L-dopa in 10 mL of aqueous phosphate/NaOH 0.1 mol.L -1 (pH = 6.8) solution and stirred at room temperature.After a few minutes, a black material (SM1) was formed as a suspension, which was filtered, dried at 45-50 °C and analyzed.To obtain the SM2 sample, 50 mg of L-dopa were dissolved in 10 mL of phosphate/NaOH 0.1 mol.L -1 aqueous solution (pH = 6.8) into an electrochemical cell that was fluxed and kept under argon atmosphere.The electrochemical oxidation process was performed by application of controlled electric potential at + 620 mV for 5 minutes.The formed suspension was isolated by the same procedure of the SM1 Sample.

Electrochemical measurements
The electrochemical synthesis of the SM2 sample was carried out using a Potenciostat-Galvanostat system (Microquimica ® , model MQPG-01) connected to a threeelectrode electrochemical cell.The working and counter electrodes were Pt wire and Pt spiral, respectively.All potentials were measured against an Ag/AgCl electrode.The buffered aqueous electrolytic solution was prepared using potassium phosphate and sodium hydroxide, both in 0.1 mol.L -1 concentration (pH = 6.8).Before the experiments, the electrolytic solution was kept at ~25 °C, and under an argon atmosphere.Voltammetric profiles were obtained at initial potential of -0.6 V and final potential of 1.0 V, at a sweep rate of 100 mV.s -1 .

EPR spectroelectrochemistry
EPR spectroelectrochemistry was performed in a BRUKER ® ESP 300E spectrometer using the X-band (~9.5 GHz) with modulation frequency of 100 kHz at room temperature (~25 °C) in a quartz flat cell under inert atmosphere of argon.EPR spectra were taken using the first derivative mode for the 500 mT range.The black product formed was dried at 45-50 °C and its spectroscopic characterization was performed.Fourier transforms infrared spectroscopy (FTIR).
The FTIR spectra were registered in a Bomem Hartmann & Braun series MB100 spectrophotometer from pellets prepared and pressed from mixtures with approximately 1 mg of the sample and 99 mg of KBr spectroscopic grade.For each spectrum, 128 scans were accomplished with resolution of 2 cm -1 in the spectrum range from 4000 to 400 cm -1 .

Diffuse reflectance ultraviolet-visible spectroscopy (DRUV-VIS)
SM1 and SM2 melanin solid state samples were analyzed by the DRUV-VIS technique.The analyses of solid samples were performed using a Shimadzu UV-2401PC spectrophotometer, equipped with a 240-52454-01 integration sphere accessory.The solid material was ground in a pan grinder to 200 mesh size.Then, this material was analyzed after being compressed carefully into the spectrometer cell.The DRUV-VIS spectra were converted to the Kubelka-Munk remission function defined by, f(KM) = (1 -R) 2 /2R = k/s, where R is the reflectance, k is the absorption coefficient, and s is the scattering coefficient 9,11 .

Results and Discussion
Initially, cyclic voltammetry (Figure 1) was used to study the electrochemical behavior of L-dopa under the electrical applied potential.Figure 1 shows a first redox pair in +217 and +386 mV and one shoulder in +628 mV and one reduction signal in -69 mV versus Ag/AgCl.This difference between oxidation and reduction peaks (386 -217 mV) of 169 mV suggests that redox system is irreversible 12 .Some authors 8 described that anodic peaks presented on the cyclic voltammogram cannot be assigned totally to L-dopa oxidation.During the oxidation there are the formation of dopaquinones that may be partially reduced to L-dopa again and standing in acidic pH.It suggests a nucleophilic attack to the deprotonated amino group resulting in an indolic product 20 .It was visually observed an orange color on the electrode surface indicating a complete oxidation of L-dopa 21 .
Regarding to EPR spectroelectrochemistry, the spectra in the sweep width of 500 mT (Figure 2) presents only an intense line with the g-factor (g = 2.0036) typical of organic free radical (OFR).The spectra in the sweep width of 5 mT, carried out at room temperature (not shown), reveals that  spin density (spin.g - ) of SM2 is higher than that of SM1 (Table 1).The g factor values are similar, SM1, g = 2.0035 and SM2, g = 2.0036.Both samples present the OFR near to the oxygen atoms of the chemical structure, which suggests the involvement of these atoms in the radical formation.The line widths also are different, with SM1, ∆H pp = 0.41 mT and SM2 = 0.35 mT.
Figure 3 shows the Kubelka-Munk function for the spectral responses, by DRUV-VIS spectroscopy, obtained at wavelengths from 250 to 800 nm.The spectral curves showed some similar characteristics, with common absorptions at 260 and 310 nm.The absorption at 340 nm in the SM1 sample appears as a shoulder of the band at 310 nm in the SM2 sample.The spectrum of the SM1 sample presents a continuous decrease of the absorption intensities from 340 nm to the end, with small maxima at 360, 420 and 500 nm.The SM2 sample presents several small peaks at the same region of the spectrum and a broad absorption band with the maximum at 530 nm and extended from about 450 to 600 nm.The absorption bands in the range of 250 to 340 nm correspond to transition of electrons from π bonding to π* antibonding orbitals.These absorptions are indicative of the polymerization degree and the presence of polyaromatic and/or conjugated unsaturated aliphatic structures.The SM2 sample with the broad absorption band at 530 nm probably presents a more conjugated chemical structure.
Figure 4 presents the infrared spectra in the range of 4000 to 400 cm -1 .The SM1 sample spectrum presents absorption bands between 3600 and 3100 cm -1 , typical of the -O-H and -N-H stretching groups undergoing intermolecular hydrogen bonding.The absorption band at 1725 cm -1 is attributed to the C=O stretching of carboxylic groups.This band is more evident for the SM1 than for the SM2 sample suggesting an incomplete melanin formation by the oxidation chemical method.For the SM1 sample, the strong band at 1645 cm -1 is attributed to the asymmetrical stretching of the carboxylate groups, while the symmetrical vibration mode is observed as a weak band at 1400 cm -1 The SM2 sample presents only a broad band at 1645 cm -1 that could be attributed to the bending vibration of water molecules, in addition to the broad band above around 3100 cm -1 , which is also attributed to the stretching of water O-H groups with a diversity of hydrogen bonding.Absorption at 1090 cm -1 could be assigned to the C-O-C asymmetrical stretching vibration of ethers that are characterized by a strong band in this region.Otherwise, SM2 sample presented a broad band (not showed) around 2350 cm -1 , suggesting an overtone shifted from the 1090 cm -1 peak 22,23 .These results indicate that the oxidative electrochemical process to produce melanins is more effective and easily controlled.
Figure 5 shows as the L-dopa concentration directly affects the current peak magnitudes.Voltammetric profiles (from -0.6 V up to + 1.0 V versus Ag/AgCl at 100 mV.s -1 )    2012; 15 (2) are shown, corresponding to the L-dopa oxidation to melanin on Pt electrode using L-dopa concentrations of 5, 10, 30 and 50 mg per 100 mL of the electrolyte.As can be seen, the I versus E voltammograms present a typical profile of the oxidation and reduction processes of this organic molecule, e.g., a reduction peak around -0.18 V and an oxidation peak around 0.27 V (versus Ag/AgCl).When the L-dopa concentration is varied, it is possible to observe a reasonable increase of the anodic peak with the increase of L-dopa concentration in phosphate buffer solution, e.g., from 0.061 to 0.33 mA.It means that the anodic charges involved in this oxidation process increases and consequently, the thickness of the oxidized product also increases.These charges could be obtained by integration of the corresponding I versus E curves.

Conclusion
Results obtained have shown that spectroscopic and electrochemical techniques used in the melanin characterization were effective to assess qualitatively the two samples.The EPR spectroelectrochemistry clearly presented only one signal of OFR due to the melanin oxidation and the o-benzosemiquinone formation along the process.These measures are confirmed also to the FTIR spectra.These results suggest that the electrochemical pathway is more effective than chemical oxidation, allowing better control of the process.

Figure 2 .
Figure 2. EPR spectroelectrochemistry in magnetic field sweep width of 500 mT during the electrochemical oxidation of L-dopa by cronoamperometry in potential controlled at +620 mV versus ENH and electrolyte solution of phosphate buffer 0.1 mol.L -1 at pH = 6.8 under argon inert atmosphere.