Kinetics and Mechanism of Oxidation of L-Cysteine by Bis-3-di-2-pyridylketone-2-thiophenylhydrazone- iron(III) Complex in Acidic Medium

The kinetics of oxidation of L-cysteine by 3-di-2-pyridylketone-2thiophenylhydrazone-iron(III), [Fe(DPKTH)2] 3+ complex in acidic medium was studied spectrophotometrically at 36 C temperature. The molar ratios of DPKTH to iron(III) and iron(II) individually, were found to be [2:1] [DPKTH : iron(III)/(II)]. The reaction was stroked to be first-order with respect to iron(III) and L-cysteine, second-order with respected to DPKTH ligand and reversed second-order with respected to hydrogen ion concentration. Added salts did not affect the rate and no free radical was detected when radical detector was placed in the reaction mixture. Ethanol solvent ratio was found to effect both the initial rate and the maximum absorbance (λmax) of [Fe(DPKTH)2] 2+ complex. The initial rate rose when the temperature was increased which empowered to calculate the activation parameters. A suitable reaction mechanism was proposed.


Introduction
In recent years, the study of the highest oxidation state of transition metals has become a central topic in the development of inorganic and organometallic chemistry 1,2 .Transition metals in a higher oxidation state can generally be stabilized by suitable chelation of polydentate ligands to establish the proper complexes.Such of these complexes with ligand systems containing multidentate nitrogen or/ and phosphine-donor atoms have been used successfully to promote the transformation of organic compounds [3][4][5][6][7][8] .As oxidizing reagents metal chelates complexes such as hexacyanoferrate(III), dihydroxydiperdatonickelate(IV), bisenylendiamine-cobalt(II), chloramines-B and biphenyl-diamineruthenium(II) [9][10][11][12] have been used to oxidize cysteine in a medium with an appropriate pH value.The relation between the structure of the ligands and the physicochemical properties of the corresponding metal complexes has been the subject of many investigations in order to understand and enhance the reactivity as well as find new applications of such of these complexes 13 .Reactions of sulfur-containing amino acids such as L-cysteine and L-methionine appear to play key roles in the biological chemistry of Pt(II) and Pt(IV), Fe(III) and Ru(III) anticancer agents [14][15][16] .The concentration of the cysteine-containing tripeptide glutathione is elevated in some platinum-resistant cell lines and thiols appear to be involved in the reduction of Pt(IV), Ru(III) and Fe(III) complexes to active Pt(II), Ru(II) and Fe(II) species [14][15][16] .
In this work, [Fe(DPKTH) 2 ] 3+ complex as an oxidizing agent using 3-di-2pyridylketone-2-thiophenylhydrazone (DPKTH) as nitrogen electrons bidentate-donor ligand was employed to oxidize L-cysteine amino acids under mild acidic condition.Herein, we aspired to perform a helpful kinetic study of oxidation of amino acid using Fe 3+ -DPKTH as new oxidation reagent.

Experimental
Absorbance measurements of the kinetics were performed using AUNICAM UV2_Shimadzou computerized UV-visible single-beam spectrophotometer with a 1 cm length quart cell.It has got vision software and a circulation thermostat water bath (C-85A) to the control temperature.All pH measurements were made using a calibrated HANNA E I8521 pH meter with combined glass electrode.Oxidation product (cystine) was identified as the corresponding amino acid by spot test 17 .Computerized Fourier transform infrared spectrophotometer (Shimadzou FTIR 820PC), with Hypride software was used to characterize the product.Standard buffer solutions were made available to standardize the set before any runs have been carried out.

Reagents
All the chemicals used were analytical reagents or of pure grade; distilled water and 95-100% redistilled ethanol were also used.FeCl 3 .6H 2 O was obtained from Aldrich Company. 1 x 10 -2 M was prepared in distilled water and served as stock solution; other concentrations were obtained from it by dilution.The stock solution was sealed in brown bottle to protect it from light and stored in cold place.L-cysteine was pursed from Aldrich, solution of 1 x 10 -3 M was daily prepared using deionized oxygen removed water.A stock solution of 1 x 10 -2 M of DPKTH 18,19 was prepared using ethanol as solvent.The Buffer sodium acetate and acetic acid buffer system was prepared using the manual 20 .

Synthesis and procedures
Preparation of nitrogen multi-electrons donor (DPKTH) ligand 3-di-2-pyridylketone-2thiophenylhydrazone (C 16 H 12 N 4 OS, DPKTH) was synthesized by treating of 3-di-2pyridylketone with of 2-thiophenylhydrazone in equivalent amount 18,19 .All the reactions runs were carried out in the 12 mL quartz cell of the UV-spectrophotometer directly to save the reaction time; care also was taken to exclude walls air bubbles formation.Fixed general procedure was adopted to achive the best reproducibility, taking in consideration the best sequence of order which found to be: buffer-water (EtOH) solution, DPKTH ligand, Fe 3+ followed by L-cysteine, respectively.1 mL of (5 x 10 -3 M) DPKTH was added to 7.5 mL of buffer-water solution, 0.5 mL of Fe 3+ (2 x 10 -3 M), the complex was shacked for 1 minute, which provided to be enough for Fe 3+ -DPKTH complex-formation, finally the mixture was treated with 1 mL of (2 x 10 -3 M) L-cysteine.The total reaction mixture volume was 10 mL.By this sequence of addition and by employment of suitable acidic medium care was taken to prevent any expected direct reaction between Fe 3+ and L-cysteine.These reagents concentrations were carefully calculated to be fit with the acceptable absorption range through trial and error technique.Together the appearing of green color (Fe 2+ -DPKTH) formed complex at λ max = 656 nm parallel to the disappearing of the yellowish color of (Fe 3+ -DPKTH) consumed complex at λ max = 364 nm indicated the oxidation reaction launching, directly by L-cysteine addition.

General investigations
Because of poor solubility of DPKTH in water and high solubility in ethanol, the complexation processes between Fe(III) and DPKTH and Fe(II) and DPKTH, respectively were performed individually in water-alcohol medium.The absorption spectra of the products showed λ max = 364 nm exhibited the yellowish Fe 3+ -DPKTH, while λ max = 656 nm revealed the green Fe +2 -DPKTH complexes formation.When the yellowish complex Fe 3+ -D PKTH was treated with L-cysteine in ethanol-water solvent λ max at 364 nm was granularly disappeared due to reduce of iron(III) to iron(II) which formed the green Fe 2+ -DPKTH complex with λ max = 656 nm.The study of L-cysteine oxidation by Fe 3+ -DPKTH complex has been carried out at 656nm (λ max of Fe 2+ -DPKTH), selected from the spectra scanned at suitable pH.The rate of reaction was modified to be measurable by suitable hydrogen ions concentration.
The molar ratios at their proper corresponding absorptions maxima of Fe 3+ -DPKTH, Fe 2+ -DPKTH complexes and Fe 3+ -DPKTH complex in the presence of L-cysteine individually, have been carried out using job's continuous variation method 20 to determent the stoichiometry of the Iron(III)/(II)-DPKTH complexes as well as to predict the real complexes structures formation.The result showed the existence of [1:2] [Iron(III)/(II): DPKTH] respectively as in Figure 1 and 3.

Optimum conditions
The oxidation rates of L-cysteine to cystine using Fe 3+ -DPKTH complex as an oxidizing agent were influenced by the pH of the buffer medium, reagents concentrations, ionic strength, ethanol ratio and temperature.The optimum conditions of the method were investigated as follows.

Effect of pH on the rate of the reaction
The effect of pH on the initial rate of the reaction was studied at different pH values between 2.2 and 8.0 at 36 o C temperature, the optimum pH lies between 4.5 and 5.5 shown in Figure 3.The absorption-time curves of the reaction in different H + concentration (0.18-1.6 x 10 -4 M, 3.8-4.7 pH) indicated that the reaction rate was increased by pH increasing (Figure 4).In this pH range the acid acted as inhibitor.A straight linear with slope = -1.94 was recorded (Figures 5) when log [initial rate] vs. log [H + ] was plotted.

Effect of ionic strength
It was observed that no sufficient effect on the initial rate was detected when NaClO 4 as selected salt was employed; the initial rate remained constant with NaClO 4 concentrations were increased even when more of 1000 fold was tried.The dependence of initial rate on the reagents concentration

Effect of iron(III) concentration
At constant temperature 36 o C and by fixing all the reaction conditions, initial rate increased by raising the concentration of Fe 3+ .The plot of log [initial rate] vs. log [Fe 3+ ] was found to be a straight linear with slope = 0.99 (Figure 6).

Effect of DPKTH ligand concentration
Positive effect of DPKTH concentration on the initial rate was noted and presented in Figure 7

Effect of L-cysteine concentration
Figure 8 shows an increase in the initial rate when L-cysteine concentration was increased; the plot of log [initial rate] vs. log [cysteine] revealed a straight linear with slope = 0.98 (Figure 8).

Effect of ethanol as solvent on the reaction
The effect of ethanol on the initial rate of reaction was studied, it can be seen that the initial rate was increased with increasing in EtOH %.Due to best salve of the ligand the λ max of belong to Fe 2+ -DPKTH was shifted positively from 656 nm to 667 nm when ethanol ratios were raised from 10-50% the result are summarized in Table 1.The dependence of reaction rate on temperature The dependence of the reaction rate on temperature was investigated between 36 and 64 o C, higher temperatures were not attempted for fear of decomposition of the complex.The reaction initial rate was increased linearly by the temperature increasing, which fulfilled Arrhenius equation and empowered to calculate the activation parameters (Table 2).

Effect of acrylonitrile as free radical detector
The addition of acrylonitrile to the reaction mixture at 36 and 60 o C did not alter the rate and there was absolutely no polymer or change in the viscosity was detected, showing the absence of free radicals in the reaction mechanism.

The suggested mechanism
We made every effort depending on the literature and the above observed result to suggest steps formed the mechanism of this oxidation process which illustrated in Scheme 1.
The first step is the formation of [Fe(DPKTH) 2 ] 3+ A [1:2] complex between Fe 3+ and DPKTH ligand this step was proved stoichiometrically and have been confirmed by separation, solubility and extraction tests.Fe 3+ concentration was low enough to prevent any dimmers or hydroxyl complexes formation.The second step involved the charge transfer intermediate complex formation [Fe(DPKTH) 2 SR] 2+ [1:2:1] B between Fe 3+ , DPKTH and L-cysteine, respectively this step was proved also stoichiometrically.The slowly intermediate B complex decomposition which gave the final complex product [Fe(DPKTH) 2 ] 2+ C was suggested to be the rate-determining step.Two electrons were transferred from the L-cysteine anion to form C complex during the charge-transfer intermediate B complex desiccation.This fact was provided by no free radicals effects were detected by acrylonitrile addition.The absorbance of C complex formation by function of time found to be very suitable stoichiometrically.Step four is very fast and provided to form Disulfide Bridge (RS-SR) cystine as final product formation.

Conclusion
Kinetic of oxidation of L-cysteine by [Fe(DPKTH) 2 ] 3+ complex was carried out in acidic medium.The differential rate method was found to be suitable for such of these investigations.The method is based on mixing DPKTH ligand with iron(III) to form yellowish Fe +3 -DPKTH complex with λ max = 364 nm.[Fe(DPKTH) 2 ] 3+ complex was reduced to [Fe(DPKTH) 2 ] 2+ at λ max = 656 nm in the presence of L-cysteine, this change in the color was followed spectrophotometrically in order to study the kinetic relations.The reaction found to be first-order with respect to iron(III) and L-cysteine, second-order with respected to DPKTH ligand and reversed second-order with respected to concentrations hydrogen ion.No salts effect or radical species have been detected.Ethanol solvent ratio was found to increase both the initial rate and the absorbance maxima.The activation parameters of the rate-determining step have been calculated, the mechanism was proposed to contained a pre-equilibrium of an adduct formation between L-cysteine and [Fe(DPKTH) 2 ] 3+ complex.