Kinetics and Mechanism of Oxidation of L-proline by Trivalent Copper: A free radical intervention and decarboxylation

The kinetics of oxidation of L-proline by diperiodatocuprate(III) (DPC) in aqueous alkaline medium at a constant ionic strength of 0.10 mol dm was studied spectrophotometrically. The reaction between DPC and L-proline in alkaline medium exhibits 2:1 stoichiometry (DPC: L-Proline). The reaction is of first order in [DPC], less than unit order in [L-proline] and [alkali]. Periodate has no effect on the rate of reaction. The reaction rate increases with increase in ionic strength and decrease in solvent polarity of the medium. Effect of added products and ionic strength of the reaction medium have been investigated. The main products were identified by spot test and I.R spectra. A mechanism involving the DPC as the reactive species of the oxidant and a complex formation with L-proline has been proposed. The reaction constants involved in the different steps of mechanism are calculated. The activation parameters with respect to slow step of the mechanism are computed and discussed and thermodynamic quantities are also calculated.


I Introduction ntroduction
The periodate and tellurate complexes of copper in its trivalent state have been extensively used in the analysis of several organic compounds 1 .The kinetics of self-decomposition of these complexes were studied in some detail 2 .Diperiodatocuprate (III) (DPC) is a versatile one-electron oxidant for various organic compounds in alkaline medium and its use as an analytical reagent is now well recognized and also used in estimation of amino acids 3 .Movius 4 reported the reactivity of some alcohols with DPC.Copper (III) is shown to be an intermediate in the Cu(II) catalysed oxidation of amino acids by peroxdisulphate 5 .The use of diperiodatocuprate (III) as an oxidant in alkaline medium is new and restricted to a few cases due to the fact of its limited solubility and stability in aqueous medium 6 .Moreover, when the coppe (III) periodate complex is an oxidant, since multiple equilibria between the different copper(III) species are involved, it needs to be known which of species is the active oxidant.
Amino acids have been oxidised by a variety of oxidising agents 7 .The oxidation of amino acids is of interest as the oxidation products differ for different oxidants 8,9 .The study of amino acids becomes important because of their biological significance and selectivity towards the oxidant to yield different products.L-proline is a non-essential amino acid and is an important constituent of collagen.As per recent report 10 , L-proline is considered to be the world's smallest natural enzyme and it is used in catalysing the aldol condensation of the acetone to various aldehydes with high stereo-specificity that has the pace.Literature survey reveals that there are no reports on the oxidative mechanism of Lproline by diperiodatocuprate(III) (DPC) oxidant.The present study deals with the title reaction to investigate the redox chemistry of copper(III) in such media and to arrive at a plausible mechanism of the reaction on the basis of kinetic and spectral results.
The kinetic measurements were performed on a Peltier Accessory (temperature control) attached Varian CARY 50 Bio uv -vis spectrophotometer and IR studies were performed by Nicolet Impact -410 FTIR.

Materials: Chemicals
All chemicals used were of reagent grade.Double distilled water was used throughout the work.Stock solution of L-proline (sd-fine chem.) was prepared by dissolving the appropriate amount of recrystallised sample in doubly distilled water.The purity of the sample was checked by TLC.The copper(III) periodate complex was prepared by standard procedure 11 .The purity of the complex was checked by its UV/Visible spectrum, which showed a broad absorption band at 415 nm.The aqueous solution of copper(III) was standardized by back titration 12 method.Dissolving the known amount of copper sulphate (BDH) in distilled water made the Cu(II) solution.Periodate solution was prepared by weighing out the required amount of sample in hot water and it was kept for 24 hours.Its concentration was ascertained iodometrically 13 at neutral pH by phosphate buffer.KOH and KNO 3 (BDH, AR) were employed to maintain the required alkalinity and ionic strength respectively in reaction solutions.

Kinetic measurements
The oxidation of L-proline by DPC was followed under pseudo-first order conditions where L-proline was excess over [DPC] at 25 ± 0.1ºC, unless otherwise stated.The reaction was initiated by mixing the required quantities of previously thermostatted solution of L-proline and [DPC], which also contained definite quantities of KOH, KNO 3 and IO 4 -to maintain the required alkalinity, ionic strength and periodate.Here the total concentration of hydroxide ion was calculated considering the KOH in DPC as well as the KOH additionally added.Similarly, the total metaperiodate concentration was calculated by considering metaperiodate present in solution of DPC and additionally added.The course of reaction was followed by measuring the absorbance of unreacted DPC in the reaction mixture in a 1cm quartz cell located in the thermostatted compartment of a Peltier Accessory (temperature control) attached Varian CARY 50 Bio uv-vis spectrophotometer at its maximum absorption wavelength of 415 nm as a function of time.Earlier it was mixture at this wavelength.The obedience of Beer's verified that there is negligible interference from other species present in the reaction law by DPC at 415 nm was verified earlier and the molar absorbance coefficient, 'å' was found to be 6213 ± 310 dm 3 mol -1 cm -1 at this wavelength (F Figure 1 igure 1).The first order rate constants, k obs, were obtained from the plots of log [Absorbance] vs time.The plots were linear up to about 75% completion of the reaction and the rate constants were reproducible within ±5 %.Since periodate is present in excess in DPC, the possibility of oxidation of L-proline by periodate in alkaline medium at 25°C was tested.The progress of the reaction was followed iodometrically.However, it was found that there was no significant reaction under the experimental conditions employed compared to the DPC oxidation of L-proline.
The effect of dissolved oxygen on the rate of reaction was studied by preparing the reaction mixture and following the reaction in an atmosphere of nitrogen.No significant difference between the results was observed.In view of the ubiquitous contamination of basic solutions by carbonate, the effect of carbonate on the reaction was also studied.Added carbonate had no effect on reaction rate.However, fresh solutions were used during the experiments.
In view of the modest concentration of alkali used in the reaction medium, attention was also given to the effect of the surface of the reaction vessel on the kinetics.The use of polythene or acrylic ware and quartz or polyacrylate cells gave the same results, indicating that the surface does not have any significant effect on the rate.
Regression analysis of experimental data to obtain the regression coefficient r and standard deviation S of points from the regression line was performed using a Pentium-IV personal computer.

Results Results
Stoichiometry and product analysis Different sets of reaction mixtures containing excess DPC than L-proline with constant OH -and KNO 3 were kept for 6 hrs in closed vessel under nitrogen atmosphere.The remaining concentration of DPC was estimated by spectrophotometrically at 415 nm.The results indicated 2:1 stoichiometry as given in Eq(1).

NH-CH
The main reaction products were identified as the aminobutaraldehyde by spot test 14 for amine and aldehyde groups.The product, aminobutaraldehyde was also confirmed by IR spectroscopy 15 which showed bands at 3444 cm -1 for NH stretching, 1773 cm -1 for aldehydic >CO stretching and 2956 cm -1 , for aldehydic -CH stretching respectively.The only organic product obtained in the oxidation is aminobutaraldehyde,which is further confirmed by single spot in TLC.However,the other product in alkaline medium is copper(II), identified by spot test 16 and uv-visible spectra.Test for the corresponding acid was negative.It was further observed that the aldehyde does not undergo further oxidation under prevailing kinetic conditions.

Reaction orders
The order with respect to [L-proline], [alkali] and [periodate] were found by log k obs vs log concentration plots and the obtained orders were also confirmed by differential method by the plot log(-dc/dt) vs log concentration using the equation log(±dc/dt) = logk + n log c; these orders were obtained by varying the concentration of L-proline, periodate and alkali in turn while keeping others constant.
The diperiodatocuprate(III) concentration was varied in the range, 2.0 x10 -5 to 2.0 x 10 -4 mol dm -3 and linearity of plots of log[Abs] vs time (r>0.9994,S<0.026) up to 75% completion of the reaction (Figure2) indicate the order in [diperiodatocuprate(III)] as unity.This result was also confirmed by varying the [diperiodatocuprate(III)] which did not show any change in pseudo-first order rate constants (k obs ) (Table 1) (r>0.9994,S<0.026).The substrate, L-proline was varied in the range of 5.0 x 10 -4 to 5.0 x 10 -3 -mol dm -3 at 25ºC keeping all other reactants concentrations constant.The k obs values increased with increase in concentration of L-proline indicating an apparent less than unit order dependence on [L-proline] (Table 1).The effect of [alkali] on the rate of reaction was studied at constant concentrations of L-Proline, DPC and ionic strength at 0.10 mol dm -3 .The rate constants increased with increase in [alkali] and the order was found to be less than unity (Table 1 Table 1).

Effect of relative permittivity and ionic strength
The effect of relative permitivity(∈ T ) was studied by varying the t-butanol-water content in the reaction mixture with all other conditions being maintained constant.Attempts to measure the relative permittivities were not successful.However, they were computed from the values of pure liquids 17 .The solvent did not react with the oxidant under the experimental conditions.The rate constant, k obs increased with decreasing dielectric constant of the medium.The plot of log k obs versus.1/ºT was linear with a positive slope (Figure .3).The effect of ionic strength was studied by varying the KNO 3 concentration in the reaction medium.The ionic strength was varied from 0.05 to 0.25 mol dm -3 at constant concentrations of diperiodatocuprate(III), L-proline and alkali.It was found that the rate constant increased with increasing concentration of KNO 3 ; the plot of log k obs versus.I 1/2 was linear with a positive slope (Figure 3) (r>0.9978,S<0.018).

Effect of initially added products
The externally added products, Cu(II) in the form of copper sulphate and another product aminobutaraldehyde did not have any significant effect on the rate of the reaction.

Test for Free Radicals
To test the intervention of free radicals, the reaction mixture was mixed with acrylonitrile monomer and kept for 24 hours under nitrogen atmosphere.On dilution with methanol, white precipitate of polymer was formed, indicating the presence of intervention of free radicals in the reaction.The blank experiment of either DPC or L-proline in which acrylonitrile alone did not induce polymerization under the same condition as those induce with reaction mixture.Initially added acrylonitrile decreases the rate indicating the free radical intervention, which is the case in earlier work 18 .

Effect of periodate
The effect of [IO 4 -] was observed by varying the concentration from 1.0x10 -5 to1.0 x10 -4 mol dm -3 keeping all other reactants concentrations constant.It was found that the added periodate has no effect on the rate of reaction.

Effect of temperature
The rate of reaction was measured at different temperatures under varying L-proline concentration.The rate of reaction increased with the increase of temperature.The rate constants, k of slow step of Scheme 1 were obtained from intercepts of the plots of 1/k obs vs 1/ [L-proline] (r>0.9978,S<0.0162) at different temperatures and used to calculate the activation parameters.The values of k (s -1 ) are given in table 2. The activation parameters corresponding to these constants were evaluated from the plot of log k vs 1/T (r>0.9989,S<0.0135) and are tabulated in table 2. G # (kJ mol -1 ) 55±4 (D) Thermodynamic quantities

Discussion Discussion
The water soluble Cu(III) periodate complex is reported 19 to be [Cu(HIO 6 ) 2 (OH) 2 ] 7-.However, in an aqueous alkaline medium and at a high pH range as employed in the study, periodate is unlikely to exist as HIO 6 4-(as present in the complex) as is evident from its involvement in the multiple equilibria 20 (2)-(4) depending on the pH of the solution.
Periodic acid (H 5 IO 6 ) exists in acid medium and also as H 4 IO - 6 at pH 7. Thus, under alkaline conditions, the main species are expected to be H 3 IO 6 2and H 2 IO 6 3-.At higher concentrations, periodate also tends to dimerise.Hence, at the pH employed in this study, the soluble copper(III) periodate complex exists as diperiodatocuprate(III), [Cu(H 3 IO 6 ) 2 (OH) 2 ] 3-in aqueous alkaline medium, a conclusion also supported by earlier work 4 .
Periodic acid (H 5 IO 6 ) exists in acid medium and also as H 4 IO - 6 at pH 7. Thus, under alkaline conditions, the main species are expected to be H 3 IO 6 2and H 2 IO 6 3-.At higher concentrations, periodate also tends to dimerise.Hence, at the pH employed in this study, the soluble copper(III) periodate complex exists as diperiodatocuprate(III), [Cu(H 3 IO 6 ) 2 (OH) 2 ] 3-in aqueous alkaline medium, a conclusion also supported by earlier work 4 .
The reaction between the L-proline and diperiodatocuprate(III) complex in alkaline medium has the stoichiometry 1:2 with a first order dependence on the [DPC] and a less than unit order dependence on the [alkali] and [substrate].In most of the reports 4 on DPC oxidation, periodate had retarding effect and order in the [OH -] was found to be less than unity and monoperiodatocuprate(III) is considered to be the active species.However, in the present kinetic study, different observations have been obtained i.e., periodate has totally no effect on the rate of the reaction.Accordingly, the DPC is considered to be the active species.No effect of added product such as copper (II) was observed.
It is known that L-proline exists in the form of Zwitter ion 21 in aqueous medium.In highly acidic medium, it exists in the protonated form, whereas in highly basic medium, it is in the fully deprotonated form 21 .The observed fractional order in [OH -] indicate that first alkali combines with Cu 3- to form an alkali-DPC species [Cu(OH) 2 (H 3 IO 6 )(H 2 IO 6 )] 4-in a pre-equilibrium step 22 , which is also supported by the Michaelis-Menten plot (Figure 4)which is linear with a positive intercept.L-proline in the deprotonated form reacts with alkali[Cu(OH) 2 (H 3 IO 6 )(H 2 IO 6 )] 4-to form a complex C.This complex C decomposes in a slow step to give a free radical derived from decarboxylated L-proline.This radical in turn reacts with another molecule of DPC species in a fast step to yield the products (Scheme 1 Scheme 1).

NH-CH
Spectral evidence of such a complex was obtained from the uv-vis spectra of, DPC, alkali and a mixture of L-proline,alkali and DPC.A hypochromic shift, ë max of ca.about 6 nm 416 to 410 nm is observed together with hyperchromicity at 410 nm.Such complex formation between the oxidant and substrate has been observed earlier 23 .Further, the evidence for complex formation is also proved by kinetic studies (i.e., from the Michaelis-Menten plot) by the non-zero intercept (Figure 4) (r>0.9989,S<0.0135) of the plot of 1/k obs vs 1/[L-proline].The mechanism is also supported by moderate values of activation parameters (Table 2 Table 2).A high negative value of ÄS # suggests that the intermediate complex is more ordered than the reactants.The observed modest enthalpy of activation and a relatively high rate constant of the slow step indicated that the oxidation presumably occurs via inner-sphere mechanism.This conclusion is supported by earlier observation 24 .Spectral evidence of such a complex was obtained from the uv-vis spectra of, DPC, alkali and a mixture of L-proline,alkali and DPC.A hypochromic shift, ë max of ca.about 6 nm 416 to 410 nm is observed together with hyperchromicity at 410 nm.Such complex formation between the oxidant and substrate has been observed earlier 23 .Further, the evidence for complex formation is also proved by kinetic studies (i.e., from the Michaelis-Menten plot) by the nonzero intercept (Figure 4) (r>0.9989,S<0.0135) of the plot of 1/k obs vs 1/[L-proline].The mechanism is also supported by moderate values of activation parameters (Table 2 Table 2).A high negative value of ÄS # suggests that the intermediate complex is more ordered than the reactants.The observed modest enthalpy of activation and a relatively high rate constant of the slow step indicated that the oxidation presumably occurs via inner-sphere mechanism.This conclusion is supported by earlier observation 24 .The probable structure of the complex (C) is given below Since Scheme 1 is in accordance with the generally well accepted principle of non-complementary oxidations taking place in sequences of one-electron steps, the reaction would involve a radical intermediate.A free radical scavenging experiment revealed such a possibility (vide infra).This type of radical intermediate has also been observed in earlier work 25 on alkaline-DPC oxidations of amino acids.Scheme 1 leads to the rate law given in Eq. ( 5).  2) approximate to unity in view of the low concentration of DPC used (K 1 =2.64andK 2 =8588).Therefore, Eq.( 5) becomes Eq (6) can be rearranged to the following form (7), which is suitable for the verification of the rate law: According to Eq (7), other conditions being constant, the plots of 1/k obs vs 1/[L-proline] (r>0.9734,s<0.0138), 1/[OH -] (r>0.875, s<0.0217) should be linear (Figure 4), From the slopes and intercepts, the values of K 4, K 5 and k could be derived as 2.64±1.0dm 3 mol -1 , 8.58±0.3x 10 3 dm 3 mol -1 , and 2.66±0.10x 10 - 2 s -1 respectively.Using these constants, the rate constants were calculated over different experimental conditions and there is a reasonable agreement between the calculated and experimental values Table 1 Table 1, which fortifies the proposed mechanism.1) The thermodynamic quantities for the first equilibrium steps in Scheme 1 can be evaluated as follows: The hydroxyl ion concentration and L-proline concentration as in Table 1 were varied at four different temperatures and the K 4 and K 5 were determined at and are given in Table (2B).A Vant Hoff's plot was made for the variation of K 4 with temperature (i.e, log K 4 vs 1/T) (r > 0.9781, s < 0.0108) and the corresponding thermodynamic quantities are given in Table (2D).A comparison of the latter values with those obtained for the slow step of the reaction shows that these values mainly refer to the rate limiting step, supporting the fact that the reaction before the rate determining step is fairly rapid and involves only high activation energy 26 .The effect of increasing ionic strength on the rate explains qualitatively the reaction between two negatively charged ions, as seen in Scheme1.However, increasing the content of t-butyl alcohol in the reaction medium leads to the increase in the reaction rate, contrary to the expected slower reaction between like ions in the media of lower relative permitivity.Perhaps the effect is countered substantially by the formation of active reactive species to a greater extent in low relative permitivity media leading to the net increase in rate 27 .The activation parameters for the oxidation of some amino acids by Diperiodatocuprate(III) are summarized in Table 3.The entropy of the activation for the title reaction falls within the observed range.Variation in the rate within a reaction series may be caused by change in the enthalpy and / or entropy of activation.Changes in the rate are caused by changes in both ∆H # and ∆S #, but these quantities vary extensively in a parallel fashion.A plot of ∆H # versus ∆S # is linear according to equation, β is called the isokinetic temperature; ∆H # = β∆S # + constant We have calculated the isokinetic temperature as 247.80K by plotting∆H # versus ∆S # (Figure 5) (r ≥ 0.979 & s < 0.0051).The value of β (247.80K) is lower than experimental temperature range (298-313K).This indicates that the entropy of activation 28 governs the rate.The linearity and the slope of the plot obtained may confirm that the kinetics of these reactions follow similar mechanism, as previously suggested.

Conclusion Conclusion
Among various species of DPC in alkaline medium, [(Cu(OH) 2 (H 3 IO 6 )(H 2 IO 6 )] 4-is considered as active species for the title reaction.It becomes apparent that in carrying out this reaction, the role of pH in the reaction medium is crucial.The overall mechanistic sequence described here is consistent with product studies, mechanistic studies and kinetic studies.

Table 2 .
Table2.Thermodynamic activation parameters for the oxidation of diperiodatocuprate(III) by L-proline in alkaline medium with respect to the slow step of Scheme 1

Table 3 Table 3 .
Activation parameters for some amino acids (for isokinetic temperature)