Chapter THE KINETICS OF THE LIGAND SUBSTITUTION REACTIONS OF IODOCOBALAMIN. k 1. Co OH 2 k -1. k a k -a

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1 Chapter CHAPTER 6 THE KINETICS OF THE LIGAND SUBSTITUTION REACTIONS OF IODOCOBALAMIN 6.1 Introduction As discussed previously (Chapters 1 and 4), there has been much contention over whether the mechanism of ligand substitution in aquacobalamin proceeds through a dissociative or a dissociative interchange pathway. Reenstra and Jencks 1 reported that a D mechanism is unlikely in the hexacoordinate cobalamins because the pentacoordinate counterparts are too unstable in water. Thus in Scheme 6.1, species A would be too short-lived to actually occur. If the D mechanism is to be preferred over the I d mechanism then the addition of water must be slow in comparison with k a but fast compared with the addition of the ligand in the k 2 process. This is highly unlikely because of the unreactivity of water with most metals. Marques and co-workers have proved that the substitution of the coordinated axial water ligand by various incoming ligands in k 1 Co OH 2 Co OH 2 k -1 K OS -L L k a k -a -L L L L L Co k 1 k -1 k 2 Co OH 2 OH 2 OH 2 Scheme 6.1 The reaction of aquacobalamin with an incoming ligand, L, showing both a dissociative mechanism (upper path) and a dissociative interchange mechanism (lower path). 1

2 Chapter aquacobalamin is via an I d mechanism. 2-4 If a bulkier ligand such as I replaces the axial water ligand, then will the mechanism remain I d or will the bulky ligand prevent the incoming ligand from forming an outer-sphere complex with the metal centre? An investigation was undertaken to determine which mechanism is operational. In order to determine whether the mechanism is I d or D, at least two ligands are required that show saturating behaviour. An I d mechanism applies if the k sat values differ for these ligands because this indicates that there is nucleophilic participation in the transition state by the incoming ligand. 6.2 Results and Discussion In this study, the exogenous ligands used to displace the coordinated iodide axial ligand in iodocobalamin were S 2 O 2 3, N 3 and imidazole. Sufficient NaI was added to solutions of aquacobalamin in order to ensure that complex formation was 97% (log K for the coordination of I by B 12a = ). UV-vis specrophotometry was then used to check that the iodocobalamin complex had indeed formed. The kinetics was studied as a function of temperature and the ionic strength was maintained at 2.2 M. Figure 6.1 shows the formation of iodocobalamin when NaI was added to aquacobalamin along with the spectral shift that is seen on binding imidazole to iodocobalamin. It is not always easy to observe saturation (as mentioned in Chapters 4 and 5) because it might occur at concentrations that are experimentally unattainable. In order to maintain the ionic strength at 2.2 M, the thiosulfate concentrations were limited to <0.35 M and hence saturation was not seen for this ligand, which most likely shows saturating behaviour above these concentrations. However, saturation was seen for both N 3 and imidazole. Imidazole was problematic in that k obs became independent of the ligand concentration above 0.6 mol dm 3 and could not be fitted to any kinetic model (Figure 6.2). Self-association of imidazole may be occurring above this concentration, thereby resulting in a constant activity with increasing concentration. Therefore, the study had to be confined to concentrations <0.6 mol dm 3 as there was no way of correcting for

3 Chapter this inactivity. If for example, dimerisation of imidazole is occurring then the kinetically available concentration of imidazole would be less than the actual concentration in solution and hence its activity would be decreased. Aquacobalamin Absorbance Iodocobalamin Replacement I of the iodide Ligand by Imidazole Wavelength /nm Figure 6.1 The spectral changes showing aquacobalamin (ph = 8.519; 25 ºC); followed by addition of iodide to form iodocobalamin (97% I binding); and finally, replacement of the axial iodide ligand with imidazole (0.011 g).

4 Chapter Substitution Kinetics of Iodocobalamin with the Imidazole and Azide Ligands Despite observing saturation for N 3 and imidazole, this is incomplete saturation, as can be seen in Figures 6.2 and 6.3. Higher ligand concentrations cannot be used because of solubility limits and as a result of maintaining a constant ionic strength of 2.2 M. Therefore, reciprocal plots were used and Equation 6.1 was inverted to give the linear Equation 6.2. k cor kk [ L] [ ] 4 = 1+ L K (6.1) 1 1 = k k K [ L] cor (6.2) k k cor /s [Imidazole] /mol dm Figure 6.2 Plot of the corrected rate constant as a function of ligand concentration at 25.0 C for the replacement of iodide in iodocobalamin by imidazole.

5 Chapter [N 3 ] /mol dm Figure 6.3 Plot of the corrected rate constant as a function of ligand concentration at 24.9 C for the replacement of iodide in iodocobalamin by azide. The problem with reciprocal plots is that the low ligand concentration values can lead to significant skewing of the intercept. However, omission of any particular points may lead to bias and so points were not removed if the straight-line fit was relatively good, i.e. with a small standard deviation. k sat can then be obtained from the intercept of a plot of 1/k cor against 1/[L] (Figure 6.4). K (or K OS ), for the formation of the outer-sphere complex of an I d mechanism is obtained from the ratio of the intercept and the slope. The activation parameters H and S for the two ligands were then determined from the slope and intercept, respectively, of a plot of ln(k 4 h/k B T) against 1/T, where h and k B are the Planck and Boltzmann constants, respectively (an example of the plot can be seen for the thiosulfate ligand in Figure 6.6). The kinetic results from this study are listed in Table 6.1. By comparing the plots of imidazole and azide (Figures 6.2 and 6.3) it is clear that the saturation constants are very different; thus the incoming ligand must be participating in the transition state. The different values of H and S are a

6 Chapter further indication that an I d mechanism is in operation because these values would have been identical, within experimental error, for a D mechanism. Hence, the reaction is not proceeding via a D mechanism / k cor /s /[N 3 ] /dm mol Figure 6.4 Plot of 1/k cor against 1/[N 3 ] at 24.9 C extrapolates to 1/k sat, the reciprocal of the saturating rate constant, and the ratio of the intercept and slope gives the equilibrium constant, K OS.

7 Table 6.1 Rate constants, k 4, and outer-sphere formation constants, K OS, for the replacement of iodide in iodocobalamin with imidazole and azide, and the second order rate constant for the reaction with thiosulfate. a Ligand T/ºC k 4 /s 1 k II /dm 3 mol 1 s 1 H /kj mol 1 S /J K 1 mol 1 K OS /dm 3 mol (4) 0.80(9) Imidazole (9) 1.1(3) (9) 0.9(1) (5) 57(9) 32(25) 1.8(9) (1) 0.99(8) (3) 0.86(2) N (7) 0.8(1) (8) 73(3) 33(11) 0.88(5) (7) (2) 2 S 2 O (1) (4) 64(1) b 16(5) b d 77 c 18 c a The number in parenthesis is the standard error on the last significant figure. b Values for k II. c Estimated values for k 4. d Calculated from the values of H K and S K.

8 Chapter Substitution Kinetics of Iodocobalamin with the Thiosulfate Ligand From Figure 6.5 it can be seen that no saturation occurs for thiosulfate and hence only the second-order rate constant, k II, was obtained for the substitution of I by S 2 O k cor /s [S2O 3 ] /mol dm Figure 6.5 Plot of the corrected rate constant as a function of ligand concentration at 25.0 C for the replacement of iodide in iodocobalamin by thiosulfate. The activation parameters H and S for the ligands were determined from a plot of ln(k 4 h/k B T) against 1/T (Figure 6.6). These results are also reported in Table 6.1 along with the results for imidazole and azide.

9 Chapter ln ( khkt II / B ) / T /K Figure 6.6 Temperature dependence of k II for the reaction of iodocobalamin with the thiosulfate ligand Determining the Outer-sphere Constant, K OS, for the Thiosulfate Ligand The straight-line part of the graph, i.e. the limiting slope, before saturation is observed is equal to k 4 K OS. For the thiosulfate ligand, saturation is not observed; therefore k II = k 4 K OS. Hence if K OS is known, k 4 can be determined. The values of K OS for Equations 6.3 and 6.4 (see Scheme 6.2) can be approximately determined from previous results obtained by Marques et al. 3 (see Table 6.2). These are the K OS values that they obtained from their saturation kinetics studies. The natural logarithm of these values was plotted against the inverse of the corresponding temperature, shown in Figures 6.7 and 6.8 for the thiosulfate and iodide ligands, respectively.

10 Co Co N N Co Co N N Co Co N Scheme 6.2 N

11 Table 6.2 Equilibrium constants 3 for the coordination of the ligands thiosulfate, iodide and azide by aquacobalamin, along with the thermodynamic parameters for the formation of an outer-sphere complex between the incoming ligand and aquacobalamin calculated from these equilibrium constants. Ligand Replacing Water S 2 O I N 3 Ligand Replacing Iodide S 2 O 3 2 a Results from Ref 3. T /ºC K OS a /dm 3 mol 1 H K /kj mol 1 S K /J K 1 mol 1 T /ºC K b OS /dm 3 mol T /ºC K /dm 3 mol 1 H K c /kj mol 1 S K c /J K -1 mol 1 T /ºC K b OS /dm 3 mol 1 b Calculated from the values of H K and S K for the specific temperatures of 5.0, 15.0, 25.0 and 35.0 ºC. c Determined from a plot of ln K against 1/T

12 Chapter ln K OS / T /K Figure 6.7 Plot of ln K OS against 1/T for the ligand thiosulfate in order to obtain an estimate of the values of K OS for Equation ln K OS / T /K Figure 6.8 Plot of ln K OS against 1/T for the ligand iodide in order to obtain an estimate of the values of K OS for Equation 6.4.

13 Chapter From the plots, H K was determined (from the slope) as 12 and 25 kj mol 1 for thiosulfate and iodide, respectively, and S K as 36 and 70 J K 1 mol 1 (from the intercept) for thiosulfate and iodide, respectively. Hence, a set of K OS values could be determined for a number of temperatures (see Table 6.2) and were determined to be for Equation 6.3 (thiosulfate) and for Equation 6.4 (iodide) at 25 C. From the equations in Scheme 6.2, it can be seen that K OS (5) = K OS (3) /K OS (4), hence K OS (5) = at 25 C, for Equation 6.5. Using the temperature dependence and calculating K (5) OS at various temperatures, H and S can then be determined from a plot of ln K against 1/T as 13 kj mol (5) K OS and 34 J K 1 mol 1, respectively. = H resulting in 1 64 ( 13 ) = 77 kj mol ( 3 8 J K for the interchange between S H k 4 H k II (5) K OS (5) K OS 1 1 and similarly S k = ( 16 ) 4 ) = 1 mol, 4 2O 3 2 and I. These values of the activation parameters differ from both those of azide and imidazole and hence it can be concluded that the mechanism is indeed that of an I d and not a D mechanism. 1 and S for the reaction of aquacobalamin with the ligands S2O 2 3 and N 3 H k 4 k4 can be determined in the same manner as for the reactions with iodocobalamin above using the data in Table 6.2. The results are reported in Table 6.3. For both ligands, the entropy value is higher in the reaction for aquacobalamin than it is for iodocobalamin. The decrease in entropy for iodocobalamin is likely to be the result of the loss of degrees of freedom of the incoming ligand due to the bulkier axial iodide group.

14 Chapter Table 6.3 Calculated values of and for the reactions of both H k 4 aquacobalamin and iodocobalamin with the ligands S2O 2 3 and N 3. S k 4 Ligand H /kj mol 1 S /J K 1 mol 1 k4 k4 Aquacobalamin S 2 O 3 2 N Iodocobalamin S 2 O 3 2 N A direct comparison of the kinetic rates of iodocobalamin with aquacobalamin can only be made with the ligand S 2 O 2 3. In both these cases the second-order rate constant, k II, was obtained with no saturation being observed. This comparison can be seen in Table 6.4. Table 6.4 A comparison between the k II values and resulting activation parameters of the reaction of both aquacobalamin and iodocobalamin with thiosulfate, respectively. Compound T / o C k II /s 1 H /kj mol 1 S /J K 1 mol 1 Aquacobalamin kii 4.112(1) 12.00(1) 42.75(3) (1) 82(4) 63(12) kii Iodocobalamin (7) 2.7(2) 4.5(1) 6.8(4) 62(2) 20(6)

15 Chapter The second-order rate constants obtained at 25.0 ºC show that the rate of substitution is approximately six times faster for aquacobalamin than it is for iodocobalamin. This is attributed to the stronger bond formed by aquacobalamin with iodide than with water (log K for the coordination of I by B 12a = ). 6.3 Conclusion The values of H and S were determined for imidazole and azide as 57 and 73 kj mol 1 and 32 and 33 J K 1 mol 1, respectively. These two ligands showed saturation for the plot of the rate constant against the concentration and so the activation parameters could easily be determined. However, thiosulfate did not display this saturation with similar plots and hence only the second-order rate constant, k II, could be obtained for the substitution of I by S 2 O 2 3 ( H = 64 kj mol 1 ; S = 16 J K 1 mol 1 ). Since k II = k 4 K OS, the corresponding saturation activation parameters could be calculated, using data previously determined by Marques et al. 3, as H k 4 = 77 kj mol 1 and = 18 J K 1 mol 1. All of these activation parameters are clear evidence that a D mechanism is not operational. These would be identical, within experimental error, if the reactions were proceeding via a D mechanism. The rate of ligand exchange for thiosulfate was found to be six times faster for water exchange than for iodide exchange, which is to be expected since the iodide ligand binds more strongly to the cobalt(iii) ion than does water. Thus it can be concluded that even when a bulky ligand such as iodide is attached to the cobalt(iii) ion, substitution with various ligands proceeds through an interchange mechanism with nucleophilic participation of the incoming ligand in the transition state. This is a surprising result given that the β face of the corrin is so crowded. S k 4

16 Chapter REFERENCES FOR CHAPTER 6 1. W. W. Reenstra and W. P. Jencks, Journal of the American Chemical Society, 1979, 101, H. M. Marques, J. C. Bradley and L. A. Campbell, Journal of the Chemical Society, Dalton Transactions, 1992, H. M. Marques, O. Q. Munro, B. M. Cumming and C. de Nysschen, Journal of the Chemical Society, Dalton Transactions, 1994, H. M. Marques and L. Knapton, Journal of the Chemical Society, Dalton Transactions, 1997, J. M. Pratt, The Inorganic Chemistry of Vitamin B 12, Academic Press, London, 1972, Chapter 8.

The Effect of Solvent on the Ligand Substitution Reactions of Aquacobalamin (Vitamin B 12a

RESERCH RTICLE L. Knapton and H.M. Marques, 43 The Effect of Solvent on the Ligand Substitution Reactions of quacobalamin (Vitamin B 12a ) Leanne Knapton and Helder M. Marques* Molecular Sciences Institute,