1.       Introduction

HCF is an octahedral stable complex anion[1]. Hexacyanoferrate (III) has the moderate reduction potential(0.41) and is a mild oxidizing agent[2]. It is mainly used in the oxidation of many organic compounds(oxygenated, nitrated, sulfated, unsaturated). Methionine is an essential amino acid that can not be synthesized in our body and must be supplemented through food. Its per day dietary requirement is 2.0 gm. Sulfur atom present in it, helps to neutralize free radicals, which are formed as a result of various metabolic process in our body , hence it is a powerful antioxidant and also acts as methyl group donor in biological process in the body. It plays a significant role in metabolism and precursor of other amino acids. It contributes to supply mineral sulfur and protect the cells from several diseases caused by airborne pollutants. It contributes to some other compounds like SAM (S-adenosyl methionine), which plays key role in transfer of labile active methyl group and sulfur for several biochemical reactions which are important for normal functioning of brain.[3-6]. Although its deficiency diseases are rare but may lead to liver damage, reduced growth rate, skin lethargy, edema and muscle loss etc. The inability to absorb methionine from gut may lead to various methionine malabsorption syndrome.  The multifunctional role of methionine includes its participation in the synthesis of creatine, glutathione, nucleic acids, polyamines, neurotransmitters, as well as serve as a substrate for protein synthesis. Methionine is a sulfur containing amino acid. It has three coordination sites: N, S and O among these S is the most susceptible for oxidation[7]. Extensive studies on oxidation of Methionine (Met) by several oxidants reports that, due to presence of electron rich Sulfur centre its behavior towards many other oxidants is different. Study of this biologically important amino acid is important because it reveals the mechanism of amino acid metabolism[8-10]. For variety of organic reactions HCF (Hexa Cyano Ferrate) has been an efficient oxidant, because for several substitution reactions CN ligands are resistant to substitution, hence outer sphere electron transfer mechanism is preferred, there by oxidation reactions are clean, devoid of side reactions, easy to monitor. Literature reveals that for several uncatalysed reactions order of the reaction for oxidants and reductants were reported to be one whereas for oxidation catalysed by Ru(III) has been reported as independent of HCF concentration. Hexacyanoferrate also exits as ion pair with K⁺ and Na⁺ present in the reaction mixture[11-12]. Oxidation of some α-amino acids such as phenyl alanine, Leucine,Glycine  and Valine by HCF yields keto acids and ammonia. Oxidation by Mn(III) and catalytic oxidation by HCF yields different products[13-15]. It has been also reported in various studies that oxidation of Methionine in alkaline medium by Osmium (VIII) catalysed HCF is 1,20,000 times faster than the uncatalyzed reaction[16]. Kinetics of Oxidation have received considerable attention due to their application in various biological processes in which metal ions participate.  To understand the mechanistic aspects of a particular oxidation - reduction reaction, catalyst and transition metal ions play an important role.

2.       Experimental

Solution preparation: Solution of DL-Methionine which is a colourless crystalline compound (E-Merck), Hexacyanoferrate [K₃Fe(CN)₆](BDH), alkali and Potassium chlorate were prepared by dissolving requisite amount of reagents in double distilled water.  In order to maintain required temperature solutions were kept at thermostat. HCF solution was standardized iodometrically. Aqueous solution of Sodium hydroxie and Potassium Chlorate were prepared to maintain the Hydroxide ion concentration and ionic strength respectively. All other chemicals used were of reagent grade[17-19].

Kinetic measurements: The required volume of DL-Methionine, HCF, Sodium Hydroxide and Potassium chlorate (to maintain ionic strength) were introduced into a three necked vessel. The reaction vessel was kept in a thermostat maintained at the desired temperature. The kinetics were followed by monitoring the reaction at 420 nm (lambda max of the complex formed) using a sampling technique. Pseudo first order conditions were maintained in all kinetic measurements. The pseudo first order rate constants were obtained from slopes of log (A∞ - A_t) vs time for two half lives, such plots were linear and duplicate measurements agreed to 2%.Conditions were maintained in all kinetic runs by using a large excess (tenfold) of DL- Methionine .

3.       Result Analysis

 Stoichiometry: At constant temperature (30^.c) and constant ionic strength different combinations of DL-Methionine and HCF and alkali were kept to react. After completion of reaction the amount of ferrate(II) formed (which is equal to amount of ferrate(III) consumed) was determined spectrophotometrically. It was observed that 2 moles of oxidant (HCF) was consumed by 1 mole of amino acid (DL-Methionine) as in equation below:

2[HCF]^3- + DL-Methionine             Methionine Sulfoxide  + 2[HCF]^4-  +  H ₂O

Effect of varying oxidant concentration:-

The effect of oxidant concentration was studied by varying concentration of HCF at constant concentration of substrate, alkali and temperature. It was observed that for most of the different concentration of Hexacyanoferrate, slope between log k versus time remained constant. Which was confirmed by the linearity of the plot shown in Fig 1, Table 1. Hence the order of the reaction was considered as unity with respect Hexacyanoferrate.

Table 1: Effect of variation in concentration of oxidant, amino acid and alkali on rate constant at 30^.C.                                                                                             I= 1.0 mol dm-³

 Fig 1: Effect of oxidant concentration on rate constant

Effect of varying Substrare concentration:- The concentration of Dl-Methionine was varied in the range of 0.5 x 10² mol dm^-3 to 6.0 x 10² mol dm^-3 Fig 2, Table 1. It was observed that rate of the reaction increases with increase in concentration of substrate. Order of the reaction was calculated by plotting a graph between logk versus log[ DL-methionine] , which came out less than 1, that is 0.5.

Fig 2: Effect of Substrate concentration on Rate constant

Effect of varying Alkali concentration:- The effect of  varying [OH^-]  in concentration on the reaction rate (Fig 3, Table 1) was studied by keeping concentration of all other reactants constant. It was noticed that reaction rate increases with increase in the concentration of [OH^-]. A graph was plotted between log(k) versus log[[OH^-]  and slope of the plot lead the order less than unity i.e.0.55.

 Fig 3: Effect of Alkali concentration on Rate constant

Effect of ionic strength and solvent polarity

The effect of ionic strength was studied by varying concentration of Potassium Chlorate and keeping others concentration constant. It was observed that ionic strength had no influence on reaction rate.

Polymerization study:-  During the progress of the reaction , the intervention of free radical was tested by adding Acrylonitrile, which is a  a free radical scavenger. A copious precipitate was formed by diluting the reaction mixture with methyl alcohol .Formation of precipitate indicates intervention of free radical during the oxidation of substrate. No precipitate was observed by treating acrylonitrile with substrate in alkali, HCF in alkali or alkali alone with methyl alcohol.

Effect of temperature on rate constant: Under same experimental conditions reaction was carried out at different temperatures. Rate constant increases with increase in temperature. (Fig 4, Table 2)

Table 2: Table between Temperatures vs. rate constant 

Fig 4: Effect of temperature on rate constant

Table 3: Calculation of Activation parameters

 Product Analysis: Methionine Sulfoxide was found to be oxidation product, which was characterized by the spot test analysis and by IR spectra. In spot test analysis sodium carbonate was added to reaction mixture with vigorous stirring along with drop wise addition of benzyl chloride solution to give a precipitate of N-benzoyl methionine sulfoxide. The product Methionine sulfoxide was precipitated by addition of an acetone–ethanol mixture of 1:1 volume ratio to the reaction solution previously brought to pH 4.0, whose identity was confirmed by its melting point (183^°C). It was subjected to IR analysis. Strong bands at 1070 cm⁻¹was shown along with the normal characteristic bands of the ionized carboxylic group and of an amine salt occurring between 3130 and 2500 cm⁻¹. The band at 1070 cm⁻¹ indicates that dl- Methionine is oxidized to sulfoxide without affecting any other part of the carbon chain leading to the formation of keto acid or aldehyde Fig 5.

Fig: 5: FTIR Spectra of Methionine Oxidation by Hexacyanoferrate

4.       Conclusion

 From above study it can be concluded that the reaction between DL-Methionine by alkaline HCF follows is an outer-sphere electron transfer mechanism. Complex is formed between HCF and DL-Methionine which is evidenced by the rapid fall of absorbance of HCF in presence of DL-Methionine at the initial stage of the reaction. Product of the reaction is identified as Methionine Sulfoxide .The reaction between oxidant substrate is much slower than the reaction reported in presence of catalyst like Ru, Cr, Ir or Os etc. In our present study rate of the reaction with respect to oxidant was found to be unity whereas with respect to substrate and alkali were found to be 0.5 and 0.55 respectively. Alteration of ionic strength had no effect on rate of reaction and free radicals are formed in the slow step of reaction.