INTRODUCTION:

Amino acids act not only as the building blocks in protein syntheses but they also play a significant role in metabolism and have been oxidized by a variety of oxidizing agents¹. The study of the oxidation of amino acids is of interest because of their biological significance and selectivity towards the oxidant to yield the different products^2–4.

Oxidation by permanganate ion is applied extensively in organic synthesis. Among the six oxidation states of manganese (+2 to+7), permanganate, Mn (VII) is the most potent oxidation state of Mn in acidic as well as in alkaline medium. The mechanism by which the multivalent oxidant oxidizes a substrate depends not only on the nature of the substrate but also on the medium^5-8.

The kinetic investigations of the oxidation of biologically important amino acids by variety of oxidant has been carried out under different experimental conditions⁹. In many cases amino acids undergo oxidative decarboxylation. But other studies with amino acids report the oxidation product as the corresponding aldehydes^10-11.

In order to explore the mechanism of oxidation by permanganate ion in a strongly alkaline medium and to check there activity of amino acids towards permanganate, L-Isoleucine has been selected as a substrate

The kinetics of fast reactions between ruthinate (VII), (RuO₄^-) and manganate (VI) (MnO₄^2-) has been studied ¹². The reaction is presumed to proceed via an outer sphere mechanism. The rapid exchange between MnO₄^2- and MnO₄^- has been studied in detail by variety of techniques¹³. In the present work the role of oxidant, L-Isoleucine and the catalyst have been investigated.

EXPERIMENTAL:

MATERIALS:

Stock solutions of L-Isoleucine (S.R.L. Chemicals) were prepared by dissolving the appropriate amount of samples in double distilled water. The solution of KMnO₄(B.D.H) was prepared and standardized with standard solution of H₂C₂O₄¹⁴. The solutions of Ruthenium (III) chloride (S.D. fine chemicals) and other reagents (AnalaR grade) were prepared by dissolving requisite amounts of samples in doubled distilled water. NaOH and NaClO₄ were used to provide the required alkalinity and to maintain ionic strength respectively. NaOH was standardized by conventional methods.

Kinetic Measurements:

All kinetic measurements were performed under pseudo first order conditions where [L-Isoleucine]_T used is at least 10- fold excess over [Permanganate]_T at a constant ionic strength of 0.5 mol. dm^-3. The reaction was initiated by mixing previously thermo stated solutions of MnO₄^- and L-Isolucine which also contain required quantity of NaOH NaClO₄ to maintain required alkalinity and ionic strength respectively. The temperature was uniformly maintained at ‘t’± 0.1^oC (where t = 25,30,35). The course of reaction was followed by monitoring the decrease in absorbance of MnO₄^-at 525nm in a 1 cm quartz cell of CECIL-7200 UV-Vis spectrophotometer. The first order rate constants, k_obs were evaluated from the slope of ln(At-A∞) Vs t plots, where At and A∞are absorbance of the reaction mixture at time t and at equilibrium respectively. The first order plots in most of the cases were liner up to 90% of the reaction and k_obs were reproducible within ±3 %.The correlation coefficient of plots used to determine k_obs were found to be 0.99 in most of the cases.

RESULTS:

Stochiometry and Reaction product:

The reaction mixture containing an excess of permanganate over L-Isoleucine and 0.05mol.dm^-3 sodium hydroxide at a constant ionic strength of 0.5mol dm^-3 was allowed to react for 2 hrs at 35±1^oCunder inertatmosphere. After completion of the reaction the remaining MnO₄^-was analyzed spectrophotometrecally. Results showed that two moles ofMnO₄^-were consumed by one mole of L-Isoleucine. So it is concluded that the stochiometry of the reaction under kinetic study is

R-CH(NH₂)COOH+2MnO₄^- +2OH^- →R-

CHO+2MnO₄^2-+NH₃+CO₂+H₂O--------------------------(1)

Where

R= C₂H₅-CH(CH₃)

The reaction products were identified as aldehydes ^15-16 by spot tests (2,4 dinitro phynile hydrazine) and NH₃by Nesslers reagent and manganate by its visible spectrum. The product aldehyde was quantitatively estimated to about 80%, which is evidenced by its 2,4-DNP derivative¹⁷. The nature of the aldehyde was conformed by its IR spectrum¹⁸. 2933 cm^-1 due to the C-H stretching of –CHO, 3422.43(s) cm^-1 and 1630(w) cm^-1 band may be due to H₂O in trace amount in KBr. Carbonyl stretching at 1759.55 cm^-1 indicates the presence of –CHO group in the product.(fig: 5). The same type of aldehyde as above was obtained when the product analysis was carried out under pseudo first order conditions. It was also observed that the aldehyde does not undergo further oxidation under the present kinetic conditions, as (L-Isolusimn) > (MnO₄^-).

The colour of the solution changed from violet to blue and then to green, excluding the accumulation of hypomanganate. The violet color originates from the pink of permanganate. The change of KMnO₄solution from violet Mn(VII) ion to dark green Mn(VI) has been observed. The spectral scans during the reaction are shown in Fig.1.

Reaction orders:

The reaction order were determined from the slopes of log k_obs verses log concentration plots by varying the concentration of reductant, catalyst alkali, while keeping others parameter constant.

The oxidant [potassium permanganate] was varied in the range 2x 10^-4 to 10x 10^-4 mol.dm^-3 as shown in table -1. The plots of log[At-A∞ ] verses time, for different initial concentrations of MnO₄^-were found to be liner and the fairly constant k_obs values indicate that the order with respect to [MnO₄^-]was unity.

The effect of [alkali] on the reaction rate was studied at constant [L-Isoleucine], [Ruthenium (III)] and [potassium permanganate] and ata constant ionic strength of 0.5 mol.dm^-3 at a desired temperature . The rate constants increased with increase in alkali concentration in a linear way (fig-2). Hence, the order with respect to [alkali] was found to be unity.

The substrate, L-Isoleucine concentration was varied in the range 1 x 10^-3 to 5 x 10^-3mol.dm^-3 at 35^oC keeping all other parameters fixed (Table:1).The rate constant k_obsalso increased with the increase in concentration of L-Isoleucine at different [OH^-] (Table:2). (fig.3 at 25^o C).

The ruthenium (III) concentration was varied in the range 1x 10^-7 to 5 x 10^-7 mol.dm^-3. The rate constants increased with increase in ruthenium (III) concentration (Table-1) when the concentration of other reactants were constant. This indicates the unit order dependence [Ruthenium (III)]_T (Fig.-4) under the condition used.

Table-1 a: Effect of variation of [MnO₄^-], [L-Isoleucine], [Ru(III)] and [OH^-] on ruthenium(III) catalysed oxidation of Isoleucine by KMnO₄ in aqueous alkaline medium at 35^oC and I=0.5 mol.dm^-3.

10⁴[MnO₄^-]

(mol.dm^-3)

10³[Isoleucine]

(mol.dm^-3)

[OH^-]

(mol.dm^-3)

10⁷ [Ru(III)]

(mol.dm^-3)

10³k_obs/s^-1

2.0

4.0

6.0

8.0

10.0

2.0

2.0
2.0

1.0

2.0

3.0

4.0

5.0

2.0

2.0
2.0

2.0

2.0
2.0

0.05

0.03

0.05

0.07

0.09

1.00

0.05

1.0

2.0

3.0

4.0

5.0

0.96

1.06

0.96

0.99

0.91

1.28

1.30

1.41

1.55

1.63

0.75

0.85

0.45

1.73

2.31

0.8

1.2

2.06

2.9

3.5

Table-1b : Effect of variation of [MnO₄^-], [L-Isoleucine], and [OH^-] on Chromium(III) catalysed oxidation of Isoleucine by KMnO₄ in aqueous alkaline medium at 35^oC and I=0.5 mol.dm^-3.

10⁴[MnO₄^-]

(mol.dm^-3)

10³[Isoleucine]

(mol.dm^-3)

[OH^-]

(mol.dm^-3)

10⁷ [Cr(III)]

(mol.dm^-3)

10³k_obs/s^-1

2.0

4.0

6.0

8.0

10.0

2.0

2.0
2.0

1.0

2.0

3.0

4.0

5.0

2.0

2.0
2.0

2.0

2.0
2.0

0.05

0.03

0.05

0.07

0.09

1.00

0.05

1.0

2.0

3.0

4.0

5.0

0.99

1.04

0.90

0.98

0.91

1.18

1.30

1.49

1.55

1.59

0.76

0.84

0.45

1.73

2.31

0.85

1.24

2.16

2.92

3.51

Table-2 :Effect of variation of [OH^-] at different concentration of [L-Isoleucine], at 25,30,35^oC,[KMnO₄]=2x10^-4mol.dm^-3, [Ru(III)] = 1x10^-7mol.dm^-3and I=0.5 mol.dm^-3.

[OH^- ]

(mol.dm^-3)

10³[Isoleucine]

(mol.dm^-3)

10³k_obs/ s^-1

25, 30, 35^oC

0.03

0.05

0.07

0.09

2.0

3.0

4.0

5.0

2.0

3.0

4.0

5.0

2.0

3.0

4.0

5.0

2.0

3.0

4.0

5.0

0.60 1.17 1.22

0.79 1.32 1.39

0.98 1.46 1.89

1.01 1.53 2.02

0.9 1.45 1.51

1.21 1.81 1.97

1.62 1.89 1.99

1.69 2.15 2.35

1.30 1.67 1.97

1.79 2.10 2.52

2.21 2.54 2.64

2.48 2.70 2.85

1.90 1.96 2.36

2.36 2.45 2.84

2.90 2.91 2.97

3.04 3.17 3.27

Table:3a Value of k,K₂ and activation parameters at various temperature(Ruthenium catalysed)

Amino acid

Temp(^oC)

k x 10²

(dm³.mol^-1s^-1)

K₂ x10^-5(dm³.mol^-1)

ΔH^≠

(kJ/mol)

ΔS^≠

(JK^-1/mol)

L-Isoleucine

1.8

2.42

3.27

2.08

3.05

3.26

48.38±9.36

-134.2±23.2

Table:3b Value of k,K₂ and activation parameters at various temperature(Chromium catalysed)

Amino acid

Temp(^oC)

k x 10²

(dm³.mol^-1s^-1)

K₂ x10^-5(dm³.mol^-1)

ΔH^≠

(kJ/mol)

ΔS^≠

(JK^-1/mol)

L-Isoleucine

1.88

2.42

3.27

2.18

3.05

3.26

49.38±9.36

-131.2±26.2

DISCUSSION:

Permanganate ion is a powerful oxidant in an aqueous alkaline medium. Under the prevailing experimental conditions at pH≥12,the reduction product of Mn (IV) is stable and further reduction of Mn (VI) might be stopped. Diode Arrey rapid scan spectrophotometric (DRRAS) studies have shown that at pH ≥12,the product of manganese (VII) is manganese (VI) and no further reduction was observed as reported ^{19, 20}. However, on prolonged standing green manganese (VI) is reduced to manganese (IV) under our experimental conditions.

It is known that in aqueous solutions, amino acid exists in Zwitterionic²¹ form, whereas in aqueous alkaline medium it exists as anionic form.

Under the conditions [OH^-]>>[Ru (III)], ruthenium (III) is mostly present²² as the hydroxylated species, [Ru(H₂O)₅OH]²⁺. Increase in rate with increase in [OH^-] indicates the presence of the hydroxylated species of ruthenium (III) as areactive species which is shown by the following equllibrium in accordance with the earlier work^23-25.

[Ru(H₂O)₆]³⁺ +OH^- ↔ [Ru(H₂O)₅OH]²⁺ +H₂O

The result suggests the formation of a complex between the amino acid and the hydroxylated ruthenium species. Such complex formation between substrate and catalyst has also been observed in earlier work. The reaction showed fractional order dependence in [amino acid]. The formation of the complex was also proved kinetically by the nonzero intercept of the plot of . The existence of two isobestic points in the UV-Vis spectrum of permanganate in alkaline medium indicates the presence of two equilibrium steps before the slow step of the mechanism . The reaction between the substrate and oxidant would provide a radical intermediate. This type of radical intermediate has also been observed in earlier work on the alkaline permanganate oxidation of amino acids. Basing on the experimental results a mechanism as in scheme-1 may be delineated. The probable structure of complex C is:

Based on the mechanism as described in Scheme-1, rate law for the reaction can be written as :

According to equation (2), the plots of [Ru(III)] /k_obsversus 1/[L-amino acid] (r >0.9988) and [Ru(III)]/kobs versus 1/[OH-] (r>0.9913, s<0.046) were linear.

When [Ru(III)]/ k_obs was plotted against 1 / [L-Isoleucine]_T then intercept is equal to and the slope is equal to _. Intercept at different [OH^-]_Twere determined when intercepts were plotted against 1/ [OH^-]_T, the new slope =1/ kK₁ and new intercept =1 /k , from the value of new intercept and slope k and K₁ were calculated. When slopes of different [OH^-] were plotted against 1 / [OH^-]_T, the next new slope =1/ kK₂K₁ and next new intercept = 1/ kK₂ , from the next new slope the value of K₂ was calculated. The value of k (Electron transfer rate constant ) and K₂(Equilibrium constant) and Activation Enthalpy and Entropy are presented in Table : 3.

Repetitive spectral scan of Ru(III) catalysed reaction of L-Isoleucine with alkaline KMnO₄.(1)-[KMnO₄] ₌2 x 10^-4 mol.dm^-3, [L-Isoleucine]_T=2 x 10^-3 mol.dm^-3,[OH^-] = 5 x 10^-2 mol.dm^-3, [Ru (III)]T=1 x 10^-7 mol.dm^-3, I=0.5mol.dm^-3, temp=35^oC at (1) 0 hour, (2) 5 minutes, (3) 10 minutes, (4) 15 minutes,(5) 20 minutes, (6) 30 minutes.

Figure : 2

Linear plots of k_obs vs [OH-]_T at different [L-Isoleucine]_T. (A) [L-Isoleucine]_T= 2 x 10^-3 mol dm^-3,(B)3 x 10^-3mol dm^-3,(C)4 x 10^-3mol dm^-3,(D)5 x 10^-3mol dm^-3at 25^oC.

Figure : 3

Plots of k_obs vs [L-Isoleucine]_T at different [OH ^-]_T. (A) [OH ^-]_T=5 x 10^-2mol dm^-3,(B)7 x 10^-2mol dm^-3,(C)9 x 10^-2mol dm^-3,(D)10 x 10^-2mol dm^-3 at 25^oC.

Figure:4

Linear plot of k_obs vs [Ru(III)]_T at 35^oC. Where [L-Isoleucine] =2.0 x10^-3, [KMnO₄]=2.0 x10^-4 moldm^-3,[OH] =0.05 moldm^-3, and ionic strength is 0.5moldm^-3.

Figure:5

I.R. spectral scan of reaction product of L-Isoleucine.

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