Kinetics and Mechanistic Studies on Oxidation of Levocarnitine by Bromamine-T in HCl Medium Catalyzed by Ru(III)

Abstract

A kinetic study on RuCl₃-catalysed oxidation of levocarnitine (LC) by sodium N-bromo-p-toluenesulphonamide or bromamine-T (BAT) has been carried out in HCl medium at 303 K. The reaction rate shows a first order dependence on [BAT]₀ and fractional order with respect to both [LC]₀ and [H⁺]. Addition of the reaction product, p-toluenesulphonamide, retards the rate. The addition of RuCl₃ and chloride ions to the reaction mixture shows an increase in the rate of the reaction. The dielectric effect is positive. The variation of ionic strength of the medium has no significant effect on the rate of the reaction. The reaction fails to initiate polymerization of acrylamide. Michaelis-Menten type of kinetics has been proposed. Thermodynamic parameters have been computed from Arrhenius plot by studying the reaction at different temperatures. The reaction stoichiometry and oxidation products were identified. Based on the experimental observations a suitable mechanism was proposed and rate law deduced.

1. Introduction

Levocarnitine ((R)-(-3-carboxy-2-hydroxypropyl)trimethylammonium chloride) is a carrier molecule in the transport of long chain fatty acid across the inner mitochondrial membrane. It is a naturally occurring substance required in mammalian energy metabolism. It has been shown to facilitate long chain fatty acid entry into cellular mitochondria, thereby delivering substrate for oxidation and subsequent energy production.

Levocarnitine may also alleviate the metabolic abnormalities of patients with inborn errors that result in accumulation of toxic organic acids [1, 2].

N-haloamines are mild oxidants and generally undergo a two electron change per mole in its reactions. They act as sources of halonium cations, hypohalite species, and N-anions, which act both as bases and nucleophiles [3]. There are many reports on N-halocompounds behaving as oxidizing agents [4–7]. However, a review of literature shows that kinetic studies with Bromamine-T (BAT) are meagre [8, 9].

Literature survey shows that there is no information available on the kinetics and oxidation of levocarnitine by any oxidizing agents from the mechanistic view point. There was a need for understanding the mechanism of oxidation of this drug, so that this study may throw some light on the metabolic conversions in the biological system. The reaction of LC with BAT in HCl medium was found to be sluggish but the reaction was found to be facile in the presence of RuCl₃ catalyst. Therefore, in the present communication, we report the kinetics and mechanism of oxidation of levocarnitine by bromamime-T in HCl medium catalyzed by RuCl₃ at 303 K.

2. Experimental

Bromamine-T was prepared [10] by partial debromination of dibromamine-T (DBT). Its purity was checked by iodometry for its active bromine content. An aqueous solution of BAT was standardized iodometrically and stored in brown bottles to prevent any of its photochemical deterioration. Levocarnitine (Biocon Ltd.) was used as received. The aqueous solution of the substrate was prepared freshly each time. All the other chemicals used were of analytical grade of purity. Doubly distilled water was used for all the measurements. A constant ionic strength of the medium was maintained (𝜇=0.5 mol dm⁻³) using concentrated solution of NaClO₄.

2.1. Kinetic Procedure

The reactions were carried out under pseudo-first order conditions by keeping an excess of levocarnitine over BAT in glass-stoppered pyrex boiling tubes coated black on outside to eliminate photochemical effects. Oxidant and the requisite amounts of substrate, HCl, RuCl₃ solutions, and water (for constant total volume) taken in separate boiling tubes were thermally equilibrated at 303 K. The reaction was initiated by rapid addition of measured amount of BAT to the mixture and was shaken intermittently for uniform concentration. The progress of the reaction was monitored by iodometric estimation of unreacted BAT in a measured aliquot (5 mL) of the mixture at regular time intervals. The course of the reaction was studied up to 75 to 80% completion. The rate constants were evaluated from the plots of log [BAT] against time. The pseudo-first order rate constants (𝑘′) calculated were reproducible within ±4%.

2.2. Stoichiometric and Product Analysis

Varying ratios of levocarnitine and bromamine-T were equilibrated in presence of 10.0×10⁻³ mol dm⁻³ HCl and 6.0×10⁻⁶ mol dm⁻³ RuCl₃, for 24 h at 303 K. Determination of unreacted BAT showed 1 : 1 stoichiometry: (see Scheme 1).

Scheme 1 

The reduction product of oxidant, p-toluenesulphonamide, was extracted with ethyl acetate and detected by thin layer chromatography using petroleum ether-chloroform-1-butanol (2 : 2 : 1 v/v/v) as the solvent and iodine as the reducing agent (𝑅_𝑓 0.88). The reported 𝑅_𝑓 value is consistent with given 𝑅_𝑓 value in the literature [11]. It was also identified by its melting point 137-138°C (lit m.p. 137–140°C). The oxidation product of levocarnitine, ((R)-(-3-carboxy-2-oxopropyl)trimethylammonium chloride), was detected by spot test and confirmed by IR spectral analysis. IR spectral bands for the product were observed at 1300 cm⁻¹ (amine salt), 1390 cm⁻¹ (–CH₃), 1454 cm⁻¹ (–CH₂), 1744 cm⁻¹ (C=O), 1605 cm⁻¹ (–COO⁻) and 1014 cm⁻¹ (aliphatic C–N stretch). It was also observed that there was no further oxidation of these products under the present kinetic conditions.

3. Results and Discussion

The oxidation of LC by BAT in the presence of RuCl₃ catalyst has been kinetically investigated at different initial concentrations of the reactants in HCl medium at 303 K.

3.1. Effect of Reactant Concentrations on the Reaction Rate

Under pseudo-first order conditions, with the substrate in excess, at constant [LC]₀, [HCl], [RuCl₃], and temperature, plots of log [BAT]₀ versus time were linear indicating a first order dependence of the reaction rate on [BAT]₀. Further, these values are unaffected by a variation of [BAT]₀ (Table 1), confirming the first order dependence on [oxidant]₀. Under similar experimental conditions, an increase in [LC]₀ increased the 𝑘′ values (Table 1). A plot of log 𝑘′ versus log [LC] was linear (Figure 1; 𝑟=0.997) with a slope of 0.22, showing a fractional-order dependence of the rate on [LC]₀. The rate of the reaction increases with increase in [HCl] (Table 1) and a plot of log 𝑘′ versus log [H⁺] was linear (Figure 1; 𝑟=0.999) with a slope of 0.62 indicating a fractional-order dependence of rate on [HCl].

Table 1 

Effect of varying concentrations of oxidant, substrate, acid and Ru(III) on the reaction rate at 303 K.

Figure 1 

Plot of 5+log𝑘′ versus (a) 4+log[LC]; (b) 4+log[H+].

3.2. Effect of Catalyst on the Reaction Rate and Catalytic Activity

The reaction rate increased with increase in [Ru(III)] (Table 1). A plot of log 𝑘′ versus log [Ru(III)] was linear (Figure 2) having a slope of 0.39, indicating a fractional-order dependence on [Ru(III)]. Catalytic constant values were calculated [12] at different temperatures using the relationship between catalyzed and uncatalyzed rate constants: 𝑘₁=𝑘₀+𝐾_𝑐[Ru(III)]^𝑥, (1) where 𝑘₁ is the observed rate constant in presence of Ru(III), 𝑘₀ is the rate constant in absence of catalyst, 𝐾_𝑐 is the catalytic constant, and 𝑥 is the order of the reaction with respect to Ru(III). Plot of log 𝐾_𝑐 versus 1/𝑇 was linear and thermodynamical parameters were calculated with respect to the catalyst. The data are summarized in Table 2.

Table 2 

Catalytic constants at different temperatures.

(a)

(b)

Figure 2 

(i) Plot of 𝑘′ versus [RuCl3], (ii) Plot of 5+log𝑘′ versus 7+log[RuCl3].

3.3. Effect of Halide Ions on the Reaction Rate

At constant [H⁺], addition of NaCl resulted in positive effect on the rate of the reaction (Table 3). Plots of log 𝑘′ versus log [Cl⁻] were linear with a positive fractional slope (0.28). Addition of NaBr showed negligible effect on the rate of the reaction.

Table 3 

Effect of varying concentrations of NaCl and PTS on the reaction rate.

3.4. Effect of Added p-Toluenesulphonamide on the Rate

Addition of reduction product of the oxidant, p-toluenesulphonamide had a retardation effect on the reaction rate (Table 3). A plot of log 𝑘′ versus log [PTS] was linear with negative fractional slope (−0.45), indicating a negative fractional-order dependence on [PTS] and thus suggesting that TsNH₂ is involved in a pre-equilibrium to the rate determining step.

3.5. Effect of Ionic Strength and Dielectric Permittivity on the Rate

Variation of ionic strength of the medium using NaClO₄ (1.0×10⁻³ mol dm⁻³ to 10.0×10⁻³ mol dm⁻³) solution did not affect the rate of the reaction. The dielectric permittivity of the medium was varied by adding different proportions of methanol to the reaction mixture. The rate of the reaction increased with increase in methanol content (Table 4). The plot of log 𝑘′ versus 1/D, where D is the dielectric permittivity of the medium (D values are taken from the literature [13]), gave a straight line with a positive slope. Blank experiments with methanol indicated that oxidation of methanol by BAT was negligible under the experimental conditions employed.

Table 4 

Effect of varying dielectric constant of the medium on the reaction rate.

3.6. Effect of Temperature on the Reaction Rate

The reaction was studied at different temperatures and from the linear Arrhenius plot of log 𝑘′ versus 1/T (Figure 3; 𝑟=0.998), values of activation parameters have been computed. These data are presented in Table 5.

Table 5 

Effect of temperature on the reaction rate and activation parameters.

Figure 3 

Plot of103/T versus (a) 5 + log 𝑘′; (b) 5 + log 𝑘4.

3.7. Test for Free Radicals

Addition of acrylamide to the reaction mixtures did not initiate polymerization which indicates the absence of free radical species.

Bromamine-T (TsNBrNa, Ts=p-CH₃C₆H₄SO₂⁻) like chloramine-T behaves as a strong electrolyte [14, 15] in aqueous solutions and in acid medium forms different types of reactive species such as TsNHBr, TsNBr₂, and HOBr. If TsNBr₂ were to be the reactive species, the rate law predicts a second order dependence of rate on [BAT]₀ which is contrary to the experimental observations. The rate increases with increase in [H⁺] but is retarded by the added PTS. Further, it is well known that with aqueous haloamine solutions, TsNHBr is the likely oxidizing species in acid medium. Protonation of monohalomines at pH 2 has been reported [16, 17] and in the present case, the fractional-order dependence on [H⁺] indicates that the protonation of TsNHBr results in the formation of TsNH₂Br⁺ which is likely to be the active oxidizing species involved in the mechanism.

RuCl₃ is also known to exist in solution in various aquaforms. UV spectral studies of aqueous ruthenium(III) complexes have shown that the octahedral complex species such as [RuCl₅(H₂O)]²⁻, [RuCl₄(H₂O)₂]⁻, [RuCl₃(H₂O)₃], [RuCl₂(H₂O)₄]⁺, and [RuCl(H₂O)₅]²⁺ may not exist in aqueous solution of RuCl₃ [18, 19]. Other studies [20–22] have shown that in acid medium the following equilibrium exists for RuCl₃: [RuCl₅(H₂O)]²⁻+Cl⁻⇌RuCl₆³⁻+H₂O (2)

In the present study, positive effect with respect to Cl⁻ suggests that the following equilibrium is shifted to the right in acidic ruthenium (III) chloride solution [12, 23]. It may, therefore, be assumed that [RuCl₆]³⁻ is the reactive catalysing species in the present case. Ultraviolet spectral measurements showed that Ru(III) and BAT solutions exhibit absorption bands at 204 nm and 224 nm, respectively, in the presence of 10.0×10⁻³ mol dm⁻³ HCl and a mixture of both at 233 nm indicating the formation of complex between BAT and Ru(III) [23].

In Scheme 2, X and X^′ are the intermediate complex species whose structures are depicted in Scheme 3. A detailed mechanistic interpretation of ruthenium catalysed LC-BAT reaction in acid medium is presented in Scheme 3. An initial equilibrium involves protonation of TsNHBr forming the active oxidizing species of BAT, TsNH₂Br⁺. This step (i) supports the fractional-order dependence on [H⁺] which was observed experimentally. In the next step, TsNH₂Br⁺ coordinates to the metal centre of [RuCl₆]³⁻ to form a loosely bound metal ion complex (X). This kind of loose metal ion complex formation has been used as an intermediate in some studies involving Ru(III) catalyst [24]. The metal complex further reacts in a fast step with the substrate to form another intermediate species (X^′) and also TsNH₂. The fractional-order in [Ru(III)] and negative fractional-order in [TsNH₂] support steps (ii) and (iii), respectively, in the mechanism. Finally, X^′ in a slow step rearranges to give the product.

Scheme 2 

Scheme 3 

Based on Scheme 2 the total effective concentration of BAT is: [BAT]_𝑡=TsNH₂Br⁺+[TsNHBr]+[X]+X^′. (3) By substituting the values of [TsNHBr], [TsNH₂Br⁺], and [X] from steps (i), (ii), and (iii) of Scheme 2 in (3), we getX^′=𝐾₁𝐾₂𝐾₃[BAT]_𝑡[LC]H⁺[Ru(III)]TsNH₂1+𝐾₁H⁺+𝐾₁𝐾₂H⁺[Ru(III)]+𝐾₁𝐾₂𝐾₃[LC]H⁺(.[RuIII)] (4)From the slow and rds of Scheme 2, Rate=𝑘₄X^′. (5)

Substituting (4) in (5), we can obtain the rate law as:𝐾Rate=¹𝐾₂𝐾₃𝑘₄[BAT]_𝑡[LC]H⁺[Ru(III)]TsNH₂1+𝐾₁H⁺+𝐾₁𝐾₂H⁺[Ru(III)]+𝐾₁𝐾₂𝐾₃[LC]H⁺(.[RuIII)] (6)

The rate law (6) is in good agreement with the observed kinetic results. From the intercept of linear double reciprocal plot of 1/𝑘′ versus 1/[LC] (Figure 4), decomposition constant (𝑘₄) was calculated. The decomposition constant was found to be 4.3×10⁻⁴ s⁻¹. The reduction product of oxidant, PTS, had a retardation effect on the reaction rate indicating that TsNH₂ is involved in a pre-equilibrium to the rate determining step. The change in the ionic strength of medium does not alter the rate indicating that nonionic species are involved in the rate limiting step. The effect of varying solvent composition and dielectric constant on the rate has been described in several studies. For the limiting case of zero angle of approach between two dipoles or an ion-dipole system, Amis [25] has shown that a plot of log 𝑘′ versus 1/D is linear. The positive dielectric effect observed in the present studies clearly supports the suggested mechanism. The proposed mechanism is further supported by the moderate values of energy of activation and other activation parameters. The fairly high positive values of free energy of activation and enthalpy of activation indicate that the transition state is highly solvated, while the large negative entropy of activation suggests the formation of rigid associative transition states with less degrees of freedom.

Figure 4 

Double reciprocal plot of 1/[LC] versus 1/𝑘′.

4. Conclusions

Oxidation kinetics of LC with BAT in HCl medium catalysed by RuCl₃ has been studied at 303 K. The stoichiometry of the reaction was found to be 1 : 1. The oxidation product of LC was identified as ((R)-(-3-carboxy-2-oxopropyl)trimethylammonium chloride). [RuCl₆]³⁻ and TsNH₂Br⁺ were assumed to be the active reactive species. Activation parameters were computed. The observed results have been explained by a plausible mechanism and the related rate equation has been deduced.

Acknowledgments

The authors are grateful to the Principal and Management of Jyoti Nivas College (Autonomous), Bangalore, for providing the necessary facilities. The authors thank Professor M. A. Pasha and Professor Puttaswamy, Department of Chemistry, Bangalore University, Bangalore, for valuable suggestions.