Orientation on the Mechanistics of Electron-Transfer on Oxidation of Chondroitin-4-Sulfate as Sustainable Sulfated Polysaccharide by Permanganate Ion in Aqueous Perchlorate Solutions

A spectrophotometric investigation of oxidation of chondroitin-4-sulfate (CS) as a sustainable and biodegradable sulfated macromolecule by oxyanion permanganate ion as multi-equivalent oxidant in acid perchlorate solutions at a constant ionic strength of 2.0 mol dm has been studied. The experimental results on the influence of the concentration of the reactants the reaction rates revealed first-order dependence in MnO4 , fractional second-order in [CS] and fractional first-order with respect to the hydrogen ion concentration. The hydrogen acid concentration dependence of the rate constants indicated that the oxidation rates were increased with increasing the acid concentration which means that the oxidation reaction is of acid-catalyzed nature. The oxidation of all other polysaccharides by this oxidant in acidic solutions indicated that the oxidation processes were proceeding throughout two distinct stages. The first stage was relatively slow, followed by a more fast reaction in the second stage. The addition of Mn and F ions to the reaction mixtures indicated that the Mn and/or Mn as the reduced forms of permanganate were the reactive species in the second fast stage. Here, neither formation of Mn and/ or Mn transient species nor presence of two stages in the oxidation reaction were detected in the oxidation process, and this result was found to be on contrary to that observed for oxidation of all other polysaccharides by this oxidant. Formation of 1:2 coordination biopolymer intermediate complexes prior to the rate-determining step was revealed, kinetically. Identification of the oxidation product revealed the formation of keto-acid as derivative precursor of CS oxidation. This product was identified by formation of 2,4-dinitrophenyhydrazone and dioxime as wells as by the bands at frequencies 3430 (OH of COOH group); 1760-1730 cm (broad) for C=O of diketone; 1639 cm of νas, OCO; 1418 cm -1 of νs OCO and 1338 cm -1 of C-O-C, respectively, in the FTIR spectra. Two-electron transfer process of inner-sphere nature in the slowest step has been suggested. A tentative reaction mechanism in terms of the kinetic parameters have been suggested and discussed.

The kinetics of oxidation of some sulfated macromolecules such as carrageenans [11,12] and chondroitin-4-sulfate [13] have been investigated in alkaline permanganate in more details and discussed previously. However, the oxidation of polysaccharides including sulfated carrageenans has been investigated in acidic permanganate [14][15][16][17][18][19], no attention was paid to the oxidation of chondroitin-4sulfate by this oxidant in acidic solutions up till now. Pseudo first-order plots of oxidation of polysaccharides in acidic solutions showed two distinct separate stages of sigmoidal inverted S-shapes curves named induction and auto-acceleration periods. The initial rates were relatively slow at the early stages, followed by an increase in the oxidation rates over longer time periods. Formation of coordination biopolymer intermediate complexes in both two stages was revealed, kinetically. Again, added salts such as Mn 2+ to the reaction mixtures indicated that both formed Mn 3+ and/or Mn 4+ as transient species of reduced permanganate play the important role in the reaction kinetics of oxidation.
Preliminary experiments on the oxidation of chodroitin-4-sulfate polysaccharide by acidic permanganate was found to be much surprising where the oxidation process indicated the absence of such two stages or the formation of Mn 3+ and Mn 4+ as transient species during the reaction course. This behavior was quite different from that observed for the oxidation of other polysaccharides by this oxidant [14][15][16][17][18][19].
In view of the above discrepancies and our interest in the kinetics of oxidation of polysaccharides by various oxidants [20][21][22][23][24][25], the present work oxidation of CS by acidic permanganate seems to be of great importance and merits an investigation with the aims of shedding some light on the nature of electrontransfer and transition states in the rate-determining step and to compare the results obtained with that reported earlier for oxidation of other polysaccharides by this oxidant [14][15][16][17][18][19]. Again, a comparison of the kinetic parameters for oxidation of CS and other polysaccharides in acidic media by this oxidant is presented and discussed. This work may enhance the elucidation of a suitable reaction mechanism of oxidation of such alcoholic macromolecules in acidic solutions. Again, a novel ketoacid-chondroitin coordination biopolymer chelating agent was synthesized as a precursor derivative of oxidation.

Materials
All materials used were of analytical grade. Doubly distilled water was redistilled from alkaline permanganate and degassed by bubbling through nitrogen, boiling and cooling under atmosphere [26].
Stock solutions of chondroitin-4-sulfate (ICN Biomedicals, Inc.) were prepared by stepwise addition of the reagent powder to doubly-distilled water whilst rapidly stirring the solution to avoid the formation of aggregates, which swell with difficulty.
A stock solution of KMnO 4 was prepared and standardized by the conventional methods as described elsewhere [26,27]. Then, the stock solution was stored in a dark bottle away from light to avoid photoreduction and was re-standardized spectrophotometrically before each run.
All other reagents were prepared by dissolving the requisite amounts of the sample in doubly distilled water.
The ionic strength was controlled by addition of NaClO 4 as a non-complexing agent. The temperature was controlled within 0.05°C.

Kinetic Measurements
Absorbancetime plots showed that the initial part (~7-10%) was too fast to be followed by the conventional spectrophotometer used. Therefore, the cited kinetic measurements were concerned with the remained part (~90-93%) of the reaction completion. Pseudo-first-order conditions in the presence of a large excess of [CS] over that of permanganate concentration was applied in all kinetic measurements. Sodium perchlorate, NaClO 4 , as an inert electrolyte was used to maintain the ionic strength constant. The measurements of absorbance changes absorbance were made in a thermostated cell compartment at the desired temperature on a Shimadzu UV-2101 double-beam spectrophotometer using cells of path length 1 cm. Some experimental runs have been carried out in nitrogen atmosphere in order to decide whether permanganate ion or dissolved oxygen is the reactive oxidizing agent for oxidation of the formed aldehyde to its corresponding acid at the final stage. The same product was obtained in both two cases. This means that the dissolved oxygen has no influence on the oxidation process.
The procedure of measurements was the same as described elsewhere [26][27][28]. The spectral changes during the oxidation reaction are shown in Fig. 1.

Polymerization Test
The possibility of formation of free-radicals during the progress of oxidation process was examined by adding 10% (v/v) acrylonitrile to the partially oxidized reaction mixture. The formation of a heavy white precipitate after the lapse of 15 min on warming indicating that the oxidation reaction of the present work was proceeding via free-radical intervention mechanism.

Stoichiometry
Determination of the reaction stoichiometry is of great importance since the kinetics of this present redox reaction is different from that reported for oxidation of other sulfated polysaccharides such as carrageenans by this oxidant and seems to be of   14 S -) n represent the chondroitin-4-sulfate and its corresponding keto-acid derivative, respectively. The products were separated by the method described elsewhere [29,30]. The keto-acid derivatives were characterized by the formation of 2,4-dinitrophenylhydrazone and dioxime derivatives on addition of 2,4-dinitrophenylhydrazine and hydroxyl amine respectively as following analysis: Again, the product was also identified by the IR spectral bands observed at frequencies of 1760-1730 cm -1 that characterize the carbonyl group of α-diketones;1639 cm -1 of ν as , OCO ; 1418 cm -1 of ν s OCO and 1338 cm -1 of C-O-C, respectively [31]. The enhancement of the absorption band of OH group at wavelength of 1700 nm in the IR spectra of the product may indicate oxidation of OH groups present in CS monomers to its corresponding keto-forms as shown in Fig. 2.

Dependence of Reaction Rate on [MnO 4 -] and [CS]
Plots of ln (Absorbance) vs. time gave good straight lines for more than two-half-lives of reaction completion indicating that the oxidation reaction is first-order in permanganate ion concentration. This dependency was confirmed not only by the pseudo first-order plots but also by the independence of the reaction rates on various initial concentration of permanganate used ranging between 2 × 10 -4 and 8 × 10 -4 mol dm -3 . A fractional-second-order in [CS] was obtained from the plots of double logarithm of the rate constants and concentration (k obs = [CS] n ). Again, when the reciprocals rate constants, 1/k obs were plotted against 1/[CS] 2 , straight lines with positive intercept on the 1/k obs axis was obtained. This behavior indicates that the present redox system exhibits the Michaelis-Menten kinetics for formation of 1:2 intermediate complexes (Fig. 3).
Again, the small shift in absorbance of permanganate ion to shorter UV-spectra at wavelength of 300 nm may confirm the formation of an intermediate complex prior to the rate-determining step. From the line weaver-Burk plot, the Michaelis-Menten constant (K m ) was calculated and found to be 5.86 × 10 2 dm 3 mol -1 where the maximum rate (γ max ) = 4.17 × 10 -3 s -1 . The large value obtained for (K m ) may confirm the fast formation of the intermediate complex prior to the rate-determining step.

Dependence of Reaction Rate on [H + ]
Kinetic measurements were performed in HClO 4 -NaClO 4 solutions with different [H + ] and constant ionic strength and temperature to clarify the influence of [H + ] on the reaction rates as well as to elucidate a suitable reaction mechanism. An increase in the acid concentration was found to accelerate the oxidation rates. This means that the oxidation reaction is acid-catalyzed (Tab. 1). Fractional first-order dependence with respect to [H + ] was observed (lnk obs -ln [H + ] plots).

Dependence of Reaction Rate on Ionic Strength
In order to shed some lights on the reactive species in the rate-determining step, kinetic runs were performed at constant [H + ] = 1.0 mol dm -3 as the NaClO 4 concentration was increased to 2.5 mol dm -3 . The value of k obs was found to increase with increasing the ionic strength as shown in Fig. 4. This result indicated the catalytic salt effect which was supported by the linearity of the Debye-Hückel relationship (ln k obs vs. I 0.5 plot). The ionic strength dependence is qualitatively as expected when considering the charges involved despite the used ionic strength was far from the applied Debye-Huckel range [32,33]. This means that the oxidation reaction may occur between a cation and a neutral molecule.

Dependence of Reaction Rate on Added Salts
The influence of Mn 2+ on the rate of reaction should be examined since it is one of the oxidation products. It is reported [34,35] that acidified permanganate ion is reduced by Mn 2+ to Mn 3+ and Mn 4+ as transient species according to the Eq. (2), Addition of either [Mn 2+ ] or [F -] ions to the reaction mixtures, was found to has no appreciable changes of the reaction rates under our experimental conditions. This negative result may indicate the absence of formation of either Mn 3+ or Mn 4+ as transient-species intermediates throughout the oxidation reaction. This result is opposite to that observed in the oxidation of other carbohydrates by this oxidant [14][15][16][17][18][19].

Discussion
Although, a considerable amount of work have been reported on the kinetics of oxidation of organic and inorganic substrates as well as the alcoholic macromolecules by permanganate ion as a multi-equivalent oxidant in acidic solutions, some unanswered questions concerning the mechanisms of oxidation in terms of the nature of electron-transfer process and the transition states in the rate-determining steps still remains not complete and poor understand [36]. A question of basic interest may be arisen here, is whether the transfer of electrons, proceeds through successive one-electron transfer process Mn VII → Mn VI → Mn V in a sequence or by simultaneous two-electron changes Mn VII → Mn V → Mn III in a single step. In addition, is whether the mechanism of electron-transfer process exhibits outer-sphere or inner-sphere nature. These difficulties may be arisen from the reactions complexity with respect to the formation of unstable intermediates through the passage of manganese ion from heptavalent to divalent state in acidic solutions. Therefore, a variety of reaction mechanisms has been suggested for oxidation of various substrates by this oxidant. Some of these reactions tend to proceed by the formation of ion pairs [26] and intermediate complexes of inner-sphere nature through free-radicals [18,27,37] or without free-radicals intervention [38]. Other reactions were proceeding through outer-sphere mechanisms in the absence of free-radical intervention [39].
In view of the above discrepancies of the kinetic results in comparison to that reported for other polysaccharides oxidized by this oxidant, the most suitable reaction mechanism which may be suggested for oxidation of CS by permanganate ion involves the protonation of both reactants to give the more reactive permanganic acid (HMnO 4 ) and alkoxnium ion (CSH + ), respectively, where K and K 1 are the protonation constants of permanganate ion and CS substrate, respectively. This suggestion was based on the observed hydrogen ion dependence of the reaction rates. Again, the dependence of the observed pseudo first-order rate constants on [CS] which obeys the Michaelis-Menten kinetics (Fig. 3) This intermediate complex (C 1 ) picks up a further protonated substrate molecule to give further intermediate complex (C 2 ) with substraction of hydronium ions prior to the rate-determining step as follows where K 2 and K 3 are the formation constants of the complexes C 1 and C 2 , respectively. Then, the formed complex (C 2 ) is slowly decomposed in the rate-determining step to give the substrate radical (C 2 . ) and Mn V as initial oxidation products.
A formed substrate radical is rapidly oxidized by picking up either further oxidant molecule or the formed Mn V in Step (7) to give rise to the final oxidation products.
The change of the rate constants with the change in the hydrogen ion and CS substrate concentrations may be expressed by the following rate-law, where [CS] T is the analytical total concentration of CS substrate.
Under our experimental conditions in the presence of a large excess of [CS] over that of the oxidant, the rate-law expression is, where k obs is the observed pseudo first-order rate constant and [OX] represents to the permanganate ion oxidant.
On comparison of Eqs. (9) and (10) and rearrangement, one concludes that, According to Eq. The small intercept observed in Fig. 3 may lead us to simplify Eqs. (11) to (12), which is considered as the appropriate rate-law expression for oxidation of CS by permanganate ion.
where k a ′ = k a KK 1 K 2 K 3 , k a ′′ = k a KK 2 K 3 and k n is the second-order rate constant. According to Eq. (12), a plot [CS] 2 /k obs against 1/ [H + ] gave straight lines with the positive intercepts (Fig. 5) from whose slopes and intercepts, the values of the apparent rate constants k a ′ and k a ′′ as were well as the protonation constant K 1 can be evaluated. These values calculated by using the least-squares method and found to be 0.67, 0.62, and 0.51 at 25, 30, and 35°C, respectively. The calculated values of protonation constants (K 1 ) were found to be in good agreement with that reported for oxidation of other polysaccharides by this oxidant elsewhere [14][15][16][17][18][19].
The negative values of entropies of activation (ΔS ≠ ) observed in Tab. 2 indicates the compactness of the intermediates rather than the reactants. Again, the positive values observed for ΔG ≠ may confirm the nonspontaneity of the formed intermediate complexes as was suggested by the proposed mechanism. The observed small activation energy value, E a ≠ (Tab. 2), may support that the high tendency of the reaction between the neutral molecule and the positive ion as was deduced from the ionic strength dependency of the rate constants. This means that the reactants do not need much energy to bring them together in order to form the transition states of the formed complexes.
Moreover, it has been reported previously [40][41][42][43][44] that entropy of activation tends to be more negative for reactions of inner-sphere nature, whereas the reactions of negative ΔS ≠ values proceed via an inner-sphere of one-or two-electron transfer mechanism. In view of the values observed for the entropy of activations (Tab. 2), the oxidation of CS by permanganate ion is more favorable to proceed by inner-sphere path of two-electron changes rather than by outer-sphere mechanism. In this context, it should be noted that there does not appear to be any experimental confirmation of outer-sphere two-electron transfer process [19,42].
Leffler and Grunwald suggested an isokinetic relationship between the enthalpies and entropies of activations (ΔH ≠ = α + βΔS ≠ ). Therefore, the activation parameters of the second-order rate constants for oxidation of some polysaccharides by permanganate ion in aqueous perchlorate solutions are summarized in Tab. 3.
However, the variation of the alcoholic substrate polysaccharide, and the nature of electron-transfer process and the transition states in the rate-determining steps, the values of activation enthalpies ΔG ≠ seems to be unaltered, i.e., with the same order of magnitude. This means these redox reactions should  have a correlation relationship. Therefore, an isokinetic plot of ΔH ≠ against ΔS ≠ values of (Leffler and Grunwald [45]) gave a straight line as shown in Fig. 6. This means that the oxidation products still remains the same (keto or keto-acid derivatives depending on the nature of alcoholic groups) despite the various routes and kinetic behavior of oxidation. The activation energies, E a ≠ , indicated that efficiency for transferring the reactants to overcome the energy barriers for formation of the corresponding intermediates complexes were decreased in the order KCAR > ICAR > CS ≥ LCAR ≥ CSA > CMC > MC, respectively. This means that the reactants needs more energy for overcome this barrier in the same order which is depending on the geometrical configuration of substrate, the positions of the OH groups, the magnitude of the charge formed on the complex and the reactive species of the reactants.
This linearity may indicate that the mechanism of electron-transfer in such oxidation reactions is similar which corresponds to the inner-sphere type. Again, the β value obtained (305K) is significant and may be  considered as a deserved comment for the reactivity of these redox reactions. In view of the above kinetic interpretations and the experimental observations, a tentative reaction mechanism for oxidation of chondroitin-4-sulfate by acidic permanganate may be suggested as illustrated by Scheme I.
The perturbation of the spectra change ( Fig. 1) may indicate that the initial fast part of oxidation reaction is not true electron-transfer process. Therefore, the initial rapid part of oxidation may be attributed to a fast formation of an intermediate between the reactants. Again, some trials have been performed in order to detect the formation of hypomanganate (V) intermediate as transient species. Unfortunately, all attempts were unsuccessful. This failure may be attributed to the lower absorptivity of formed Mn (V) under our experimental conditions of lower reactants concentrations or the fast reaction between the formed manganese (V) and substrate radical (Eq. (7)) to give rise to the final oxidation products.

Conclusions
The kinetics and mechanism of oxidation of CS as a sulfated macromolecule by permanganate ion oxidant in acid perchlorate solutions at a constant ionic strength of 2.0 mol dm -3 , has been studied spectrophotometrically. The experimental results of the pseudo first order plots indicated that the oxidation exhibits only one step pathway reaction; first-order in MnO 4 -, fractional second-order in [CS] and fractional first-order with respect to the hydrogen ion concentration. Acid-dependence of the reaction rates indicated that the oxidation reaction was of acid-catalyzed nature. Absence of either Mn 3+ and/or Mn 4+ transient species as involvement species in the oxidation process was confirmed by the added Mn 2+ to the reaction mixtures. Formation of 1:2 coordination biopolymer intermediate complexes prior to the rate-determining step was revealed, kinetically. These kinetic results observed here were found to be on contrary to that observed for the oxidation of other alcoholic polysaccharides by this oxidant which showed two distinct stages of S-sigmoidal curves for the pseudo first order plots. The initial stage was relatively slow (induction period), followed by a relatively fast stage (autoacceleration period which involving the Mn 3+ and/or Mn 4+ as transient species) with formation of 1:1 or 1:2 intermediate complexes prior to the rate-determining steps. A tentative reaction mechanism in terms of the kinetic observations and evaluated kinetic parameters have been suggested and discussed.