Stopped-Flow Spectrophotometric Study of the Kinetics and Mechanism of CO2 Uptake by cis-[Cr(C2O4)(BaraNH2)(OH2)2]+ Cation and the Acid-Catalyzed Decomposition of cis-[Cr(C2O4)(BaraNH2)OCO2]− Anion in Aqueous Solution

The kinetics of CO2 uptake by the cis-[Cr(C2O4)(BaraNH2)(OH2)2]+ complex cation and the acid hydrolysis of the cis-[Cr(C2O4)(BaraNH2)OCO2]− complex anion (where BaraNH2 denotes methyl 3-amino-2,3-dideoxy-β-D-arabino-hexopyranoside) were studied using the stopped-flow technique. The reactions under study were investigated in aqueous solution in the 288–308 K temperature range. In the case of the reaction between CO2 and cis-[Cr(C2O4)(BaraNH2)(OH2)2]+ cation variable pH values (6.82–8.91) and the constant ionic strength of solution (H+, Na+, ClO4− = 1.0) were used. Carbon dioxide was generated by the reaction between sodium pyruvate and hydrogen peroxide. The acid hydrolysis of cis-[Cr(C2O4)(BaraNH2)OCO2]− was investigated for varying concentrations of H+ ions (0.01–2.7 M). The obtained results enabled the determination of the number of steps of the studied reactions. Based on the kinetic equations, rate constants were determined for each step. Finally, mechanisms for both reactions were proposed and discussed. Based on the obtained results it was concluded that the carboxylation (CO2 uptake) reactions of cis-[Cr(C2O4)(BaraNH2)(OH2)2]+ and the decarboxylation (acid hydrolysis) of the cis-[Cr(C2O4)(BaraNH2)OCO2]−are the opposite of each other.


Introduction
The general chemistry of carbonato complexes of transition metal ions has been described by Maccoll [1] as well as Harris and co-workers [2]. The interactions of carbon dioxide with transition metal ions like CO 2 reduction, insertion and activation have been actively pursued and the review literature on this subject is quite large [3][4][5][6][7][8]. A system consisting of transition metal ions and a bioactive organic ligand can represent a model of an enzyme and may be useful for elucidation of enzymatic reaction mechanisms. One of these classes of bioactive compounds are the aminosugars [9], which in their reactions with metal ions usually behave as monodeprotonated, bidentate ligands. Different structural factors, such as interatomic distances, bonding angles, etc., lead to differences in the steric interactions of the diastereoisomers as a result of bigger or smaller distances of the carbohydrate to the different molecules, which are sometimes located in the coordination sphere of the metal ions. The amino nitrogen is the anchoring site. Subsequently, a suitable hydroxyl group deprotonates and coordinates to form a strong chelate [10]. The stabilities of complexes of various derivatives with a particular binding mode (e.g., NH 2 , O − ) may vary by up to three orders of magnitude, depending on the relative positions of the coordinating atoms. Critical factors influencing the coordination equilibria, i.e., both stability and the structures of complexes, are as follows: (a) the number of the amino groups in the ligand; (b) the number of available hydroxyl functions; (c) the overall structure of the carbon chain, i.e., linear or cyclic; and (d) in the case of cyclic aminosugars, the number of dioxolane rings (e.g., 1,6-anhydro-derivatives) [11]. Cyclic amino sugars, like D-glucosamine or D-mannosamine [10], form efficient but simple monomeric (NH 2 , O − ) chelates that may differ considerably in complex stabilities from one aminosugar to another. 1,6-Anhydro derivatives using the same donor system as the parent sugars form a completely different set of species, including very unusual dimeric complexes [12]. Linear amino-alcohols also form very effective dimeric (only) species involving alkoxy-bridges, while linear diaminoalcohols also form dimeric complexes, but their binding mode is completely different from that of monoaminoderivatives. In the case of diaminoalcohols both amino groups act as anchoring sites for two metal ions. Thus, two independent {NH 2 ,O−} chelates are formed, leading to dimeric complexes in which two metal ions are bound to two N-terminals of the 1,5-or 1,6-diaminoalcohol. In all cases studied both ligand conformation and absolute configuration have a distinct impact on the stabilities of the complexes formed. Studies performed for four families of aminoalcohols have shown that they are very specific chelating agents for metal ions, able to also efficiently bind metal ions in a natural environment.
In our earlier investigations two anomers of methyl 3-amino-2,3-dideoxy-D-arabino-hexopyranoside [13,14] were used as bidentate (L-L) ligands [15] to obtain two coordination compounds of general formula cis-[Cr(C 2 O 4 )(L-L)(OH 2 ) 2 ] + which behave as NO 2 biosensors. These aminodeoxysugars coordinate with chromium(III) ion through the neighboring HO-4 and 3-NH 2 groups, which both adopt equatorial positions. Both coordinated anomers have a slightly distorted 4 C 1 chair conformation [13,14], compared to that of the free monosaccharide in aqueous solution. The use of these compounds in biosensors allowed us to develop a selective analytical method for the determination of the concentration of nitrogen dioxide released in biological materials [16,17].
Pyruvate, by reducing the oxygen to water molecules, activates the electron transport processes in the mitochondrial respiratory chain. As a result of disturbances in this process the formation of H 2 O 2 molecules occurs, which in a non-enzymatic reaction of pyruvate undergo an alternative conversion to CO 2 and acetate according to reaction (1). Recent studies [18] indicate that pyruvate, under myocardial ischemia and tissue protection conditions, acts as a scavenger of free radicals resulting from oxidative stress, which in turn generates the formation of certain amounts of the hydrogen peroxide. Presumably, pyruvate by reaction with H 2 O 2 undergoes an transformation to carbon dioxide and acetate, having under these conditions cytoprotective significance [19,20]. However, the mechanism of antioxidant action of pyruvate in this process is not completely understood, as oxygen is released in the form of free radicals.

Kinetics and Mechanisms of CO 2 Uptake by cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + Complex Cation
When carrying out the kinetic measurements, it was observed that the investigated carbon dioxide uptake reaction proceeded in two steps. At the beginning of the reaction a very sharp increase in the absorbance value occurred and then, after reaching the maximum, the absorbance decreased as the reaction progressed. In the first step an intermediate product was formed, which was then transformed into the characteristic final product of the second step. The data fitting and the global value analysis of the observable rate constants for both steps were based on the consecutive reactions model. Figure 1 shows the results of fitting of the rate data to the pseudo first-order kinetic equation for the assumed consecutive reaction model (A→B→C).  )(BaraNH 2 )(OH 2 ) 2 ] + cation. The observable rate constants, for first (k 1obs ) and second steps (k 2obs ), were obtained by fitting the rate data at different temperatures and different pH values studied to the same consecutive reaction model. The calculations have shown that at a fixed concentration of carbon dioxide and increasing pH value the observable rate constant for CO 2 uptake (k 1obs ) increased (Table 1) for all temperatures studied. Based on the determined acidity constants (K 1 , K 2 ) ( Table 2) and the observable rate constants (k 1obs ) a mathematical model for CO 2 After transformation of expression (2) the following equation can be obtained:  It turned out that the relationship between the first term (right hand) of Equation (3) and the concentration of hydrogen cation is linear for all temperatures studied, as shown in Figure 3.  Based on the relationships shown in Figure 3, the rate constants k 1 [s −1 M −1 ] and k 2 [s −1 M −1 ] for each temperature in the whole pH range between the measured and calculated pK 1 and pK 2 values were calculated. Activation enthalpies which were determined using Arrhenius' [21]  )] 0 occurs more easily than in the case of the monoanion complex ion, in which the central atom is linked to a carbonate ligand with two oxygen atoms. The ring closure step is much slower than the first step of CO 2 uptake and therefore it determines the rate of the process.  In this step the carbonate (or bicarbonate) anion is linked to the chromium(III) cation by one oxygen atom. The carbon dioxide uptake reaction occurs very fast since during this process no breakage of a metal-oxygen (from the hydroxyl group) bond occurs. A new bond between the carbon atoms of carbon dioxide and the oxygen atoms of the hydroxyl group of the complex ion was created [22][23][24][25][26], so it is seen that hydrogen bonding plays an important role. In this step three intermediate species exist in solution, whose concentrations are determined by the values of the acidity constants K 3 and K 4 . In the second step the final product, cis-[Cr(C 2 O 4 )(BaraNH 2 )CO 3 ] − anion, is formed. This step is disturbed by the hydrolysis reaction of the anionic product [27,28]. Due to this fact the acidity constants K 3 , K 4 and the rate constants k 3 and k 4 cannot be determined.

Acid Hydrolysis of the cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − Complex Anion
The decarboxylation reaction (the release of CO 2 from the cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − anion) as a reaction opposite to the reaction of the carbon dioxide uptake by the cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + ion also occurs in two steps. It has been confirmed by the shape of approximated curve of the absorption versus time dependence, which rises and falls biexponentially as in the case of the uptake reaction described in Section 2.1. In the first step (the opening of the carbonate ring preceded by the hydration process the intermediate product  Tables 4 and 5. It can be found that at a constant temperature and increasing [H + ] the rate constant k 1obs for the first step increases. On the other hand, the observable rate constants k 2obs in this same conditions does not change. The relationship between the 1/k 1obs and 1/[H + ] values is linear for all temperatures studied, as shown in Figure 4. Using the linear dependence shown in Figure 4 and Equation (5), the rate constants (k 1 [s −1 ] and k 2 [s −1 ]), whose values are summarized in Table 5, have been determined. As shown in Table 5, both rate constants increase with the increasing temperature. On the other hand, the constant K ~ 1.14 [M −1 ] (Table 5) describing the protonation equilibrium between the cis-[Cr(C 2 O 4 )(BaraNH 2 )(OCO 2 )] − complex anion and cis-[Cr(C 2 O 4 )(BaraNH 2 )(OCO 2 H)] 0 neutral complex has the same value at each temperature studied. This value suggests that some 50% of the chelate should exist in the protonated form in acid solution.

Proposed Mechanism for the Acid-Catalyzed Decomposition of cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − Complex Anion in Aqueous Solution
On the basis of the kinetic measurements performed, a mechanism of the hydrolysis reactions catalyzed by H + ions for the complex of chromium(III) (with the oxalate and the carbonate anions as well as the aminosugar as ligands) can be proposed (Scheme 2  (6) As shown in Scheme 2 the reaction studied proceeds in two steps. The coordination ion (cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − ) exists in solution in equilibrium with its neutral form cis-[Cr(C 2 O 4 )(BaraNH 2 )(OCO 2 H)] 0 described by acidity constant K a = 1/K. The first step of the reaction, in which the carbonate ring is opened, is slow. The constant K describes the protonation equilibrium of the [Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − anion which is established prior to the two-step hydrolysis. The first step of the reaction, in which the carbonate ring is opened, is slow. This step is dependent on the concentration of hydrogen cations. With the increased temperature the rate constant k 1 [s −1 ] is increasing, consequently, the reaction rate increases. A significant increase in the value of the rate constant of the chelate ring opening with increasing concentration of hydrogen ions is related to the protonation of CO 3 2− group and the subsequent breaking of M-OCO 2 H bond and the following substitution in one coordination site of the water molecule, which is present in an aqueous environment of this reaction. The second step is much faster than the first one-This is the hydration reaction. The rate constant of this step is not dependent on the concentration of hydrogen cations in the whole temperature range, however, depends on the temperature. The independence of the rate of the second step (hydration reaction) on the concentration of the hydrogen ion is due to the fact that it is a reaction in which the bicarbonate ligand is exchanged with a molecule of water. This exchange occurs much faster at higher temperatures, hence the temperature dependence for the second step can be determined.

Reagents
All the reagents required for the synthesis were purchased from Sigma (Poznań, Poland).

Synthesis of cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] +
In the first step the cis-K[Cr(C 2 O 4 ) 2 (OH 2 ) 2 ]·3H 2 O was synthesized according to the procedures described in [29]. Next, a solution (40 mL) of cis-K[Cr(C 2 O 4 ) 2 (OH 2 ) 2 ]·3H 2 O (1.96 g) in water was heated for 15 min at a temperature of 338-343 K. The pH of the solution was adjusted to ca. 9; to give a dark green colour. To this mixture was then added a stoichiometric quantity of methyl 3-amino-2,3dideoxy-β-D-arabino-hexopyranoside (5 mmol), dissolved in water (10 mL, pH ≈ 9). The resulting solution was stirred for ca. 15  OCO 2 ] − coordination ion were determined as described above and the content of the carbonate anion was determined quantitatively by acid-base titration using a standard solution of 0.112 M HCl in the presence of 1% aq. methyl orange.

Spectral Measurements
Spectral measurements were carried out in the UV-Vis region using a Perkin-Elmer Lambda 650 spectrophotometer equipped with a Peltier temperature control system. The system features high heating and cooling rates and excellent temperature accuracy, which is an essential requirement for measurements. The instrument has the scan accuracy of 1 nm and 1 nm slit width at a scanning rate of 120.00 nm min −1 .

Determination of Acidity Constants of cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + Complex Ion
Samples to spectrophotometric measurements were prepared immediately before recording the spectra. Thus, a aqueous solution of the cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + (1.5 mL, 0.01 M) was mixed with an equal volume of appropriate buffer solution Tris [tris-(hydroxymethyl)-aminomethane)]. pH measurements were made with a CX 731 pH-meter (reading accuracy of 0.01 pH unit) and a combined electrode manufactured by Hanna. The pK values for the acid dissociation of cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + were determined spectrophotometrically over 340/700 nm range. Then, the pK 1 and pK 2 values in the ground state were computed using Origin 8.5 program, based on absorbance variations at a selected wavelength and applying the non-linear least squares method according to the Equation (7)  Absorption spectra in the UV-visible region were measured using a Perkin-Elmer Lambda 650 spectrophotometer.

Determination of Acidity Constant for the cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − Complex Ion
The zero absorbance time (A obs ) for the reacting solution was determined by extrapolation of A t to t = 0 with a dead stirring time of 2 ms. The protonation constant K was obtained from relationship (8) described by Buckingham et al. [28] as a plot of (A 1 − A obs ) vs. [H + ] with the use of the Origin 8.5. program:

Kinetic Measurements for the Reaction of CO 2 Uptake by cis-[Cr(C 2 O 4 ) (BaraNH 2 )(OH 2 ) 2 ] +
The CO 2 uptake reaction [22] by cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + ion was investigated using an Applied Photophysics SX-17 MV stopped-flow spectrophotometer. Carbon dioxide uptake reactions were studied at a constant ionic strength of 1 M (NaClO 4 ) keeping [CO 2 ] >> [total Cr]) and over the pH and temperature ranges: 6.81 < pH < 8.91 and 278 K < T < 298 K, respectively. The measurements were carried out at five temperatures (278, 283, 288, 293 and 298 K) and at constant concentration of carbon dioxide (0.01 M). Carbon dioxide was generated by the reaction between sodium pyruvate and hydrogen peroxide according to the Equation (1). The solutions of the complex ion were prepared by mixing cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + (0.5 mL, 10 −3 M) with Tris buffer (2 mL, 0.2 M) and NaClO 4 (2 mL, 2 M) solutions. The reactions were monitored at wavelengths, which offer the largest absorbance difference between reactant and product. The observed pseudo-first-order rate constants were calculated by using a ''Glint'' program based on global analysis [31,32] and were reported as the mean of at least four kinetic runs.

Kinetic Measurements for the Reaction of Acid-Catalyzed Decomposition of cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] −
The decarboxylation reaction was investigated [24] using an Applied Photophysics SX-17 MV stopped-flow spectrophotometer. In order to carry out kinetic studies of the acid-

Conclusions
In this paper, the kinetics and mechanisms of CO 2 uptake by cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + complex cation and acid-catalyzed decomposition of cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − complex anion in aqueous solution have been studied. Kinetic investigation by the spectrophotometic stopped-flow technique was applied to track the progress of these processes. The uptake reaction of carbon dioxide by cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + indicates unambiguously that it occurs in two steps: Addition of the molecule of carbon dioxide to complex cation (first quick step-CO 2 uptake) is the first one, and the subsequent creation of the bidentate carbonate ion (second step-Ring closure) in the next step. The second one is about 10 times slower than the first step due to the fact that it consists of breaking of the chromium-oxygen bond and the subsequent creation of the new bond with the carbon dioxide.
Based on the obtained data it can be concluded that the carboxylation (the CO 2 uptake) of cis-[Cr(C 2 O 4 )(BaraNH 2 )(OH 2 ) 2 ] + complex cation and the decarboxylation (the acid hydrolysis) of cis-[Cr(C 2 O 4 )(BaraNH 2 )OCO 2 ] − complex anion are reactions opposite to each other, as is illustrated by the simplified equation (9) shown below: