ISSN 00360244, Russian Journal of Physical Chemistry A, 2013, Vol. 87, No. 1, pp. 44–48. © Pleiades Publishing, Ltd., 2013. Original Russian Text © D.S. Salnikov, I.A. Dereven’kov, E.N. Artyushina, S.V. Makarov, 2013, published in Zhurnal Fizicheskoi Khimii, 2013, Vol. 87, No. 1, pp. 52–56.

PHYSICAL CHEMISTRY OF SOLUTIONS

Interaction of Cyanocobalamin with SulfurContaining Reducing Agents in Aqueous Solutions D. S. Salnikov, I. A. Dereven’kov, E. N. Artyushina, and S. V. Makarov Ivanovo State University of Chemistry and Technology, Ivanovo, 153000 Russia email: [email protected] Received December 12, 2011

Abstract—The kinetics and mechanism of cyanocobalamin reduction by sodium hydroxymethanesulfinate and dithionite in alkaline media are studied. It is established that the character of the ratedetermining step depends on the concentration of the reducing agents: when they are in excess, it is a step of elimination of cyanocobalamin, at lower concentrations of reducing agents a ratedetermining is a step of their addition to cobalamin. Keywords: reaction mechanism, reduction, kinetics, cyanocobalamin. DOI: 10.1134/S0036024413010226

INTRODUCTION

by sodium dithionite (we named this form B12rs). In this work, we investigate the reaction kinetics of two sulfurcontaining reducing agents, sodium dithionite and hydroxymethanesulfinate.

Perhaps no another vitamin has a history of discov ery and establishing its structure as interesting as that of vitamin B12 (cobalamin, Cbl). Despite numerous investigations and the impressive results obtained ear lier in the laboratories of famous universities and other scientific centers, cobalamins and their analogs still draw the attention of chemists, biochemists, and phy sicians. This is due primarily to the uncovering of many unknown properties and functions of cobal amins in the body and the prospects for their practical use. It has been shown in particular that cobalamin has the properties of the superoxide dismutase [1]. The reaction of Cbl(II) with nitric oxide was stud ied in detail [2], and a number of works have been per formed to investigate the properties of the structural cobalamin analog cobinamide as an antidote [3, 4]. Based on the results from spectroscopic investigations of the socalled baseoff (having no Codimethylben zimidazole bond) form of cobalamin, it was shown that the transition from Cbl(II) to Cbl(I) occurs via a fourcoordinated Cbl(II) complex rather than a five coordinated Cbl(I) complex [5]. Cobalamin proper ties depend substantially on the degree of central atom oxidation. It is known that a transition from Cbl(III) to Cbl(II) and further to Cbl(I) is observed in the pres ence of strong reducing agents [6]. It is assumed that Cbl(III), Cbl(II), and Cbl(I) form six, five, and fourcoordinated complexes, respectively [7]. Despite considerable progress, there are still many unsolved problems in the chemistry of cobalamins. Until recently, it was unclear if Cbl(II) forms stable six coordinated complexes. We established [8] that such a complex, containing a SO 2− anion radical, is formed upon the reduction of hydroxocobalamin HOCbl(III)

EXPERIMENTAL Cyanocobalamin CNCbl (≥98.5%), sodium dithion ite Na2S2O4 (≥86%), sodium hydroxymethanesulfinate dihydrate HOCH2SO2Na 2H2O (≥98.0%) (SigmaAld rich) were used without additional purification. Other substances used in this work were of reagent grade. Argon was used to create anaerobic conditions. All of our kinetic investigations were carried out under anaerobic conditions on a Specord M40 device equipped with a LT 100 thermostat (±0.1°С). A hermetic quartz cuvette 1 cm thick was used. The rate of cyanocobalamin reduction reactions was controlled by changing the opti cal density at a wavelength of 550 nm, which corresponds to the absorption maximum of CNCbl(III). It was shown by preliminary experiments that the addition of sodium chloride (0–0.5 mol/L) has no effect on the rate of reduction of the indicated cobal amins. All subsequent experiments were therefore per formed without the addition of NaCl. RESULTS AND DISCUSSION It is known that cobalamins are unstable in alkaline media [9–11]. There are, however, no kinetic data on cobalamin decomposition in alkaline media. In this work, the stability of CNCbl(III) was therefore investi gated over wide ranges of pH and temperatures. It was shown that CNCbl(III) is stable in the pH range of 6 to 12 and at temperatures of 25 to 45°С. A considerable 44

INTERACTION OF CYANOCOBALAMIN

45

Abs 1.50 308 1.25 1.00

362

0.75 386

0.50

550 0.25 0 300

400

500

600

800 λ, nm

700

Fig. 1. Spectral changes during the reaction of CNCbl with hydroxymethanesulfinate. [CNCbl]0 = 5 × 10–5 mol/L; [HMS]0 = 0.1 mol/L; pH 11.4; 25°C; anaerobic conditions. The spectra were recorded at 0.3 min intervals.

increase in the rate of cobalamin decomposition was observed in 0.1 M NaOH at temperatures above 35°С. The reduction of CNCbl(III) by sodium hydroxymethanesulfinate in weakly alkaline media is accompanied by the appearance of the spectral absorption maxima at 308, 385, and 452 nm (Fig. 1) characteristic of sixcoordinated B12rs [8]. Maxima at 385 and 452 nm also emerge during the reduction of CNCbl(III) by dithionite. We obtained the same results while investigating the reaction between hydroxocobalamin and dithionite [8]. A typical kinetic curve for the reduction of CNCbl to Co(II) by hydroxymethanesulfinate is presented in Fig. 2. Processing the kinetic data in semilogarithmic coordinates revealed that the order for cyanocobal amin is equal to unity. The dependence of the observed rate constant for the cyanocobalamin reduction reaction on the concentra tion of hydroxymethanesulfinate (HMS) is nonlinear at pH 11.4. However, it is linearized in the coordinates 1/kobs–1/HMS (Fig. 3), a plateau being reached in the >0.8 mol/L range of HMS concentrations. It is established that the observed rate constant for CNCbl(III) reduction at pH 11.4 (3.0 × 10–2 s–1 at 25°С) at [HMS] > 0.8 mol/L is close to the constant of dimethylbenzimidazole (DMBI) elimination from CNCbl(III) (4.2 × 10–2 s–1 at 25°C [12]). For the CNCbl(III) reaction with hydroxymethanesulfinate, activation parameters ΔH≠ = 103 ± 6 kJ/mol, ΔS≠ = +82 ± 6 J/(K mol) were found; they are virtually equal to those for the reaction of DMBI elimination from CNCbl(III): ΔH≠ = 105 ± 6 kJ/mol, ΔS≠ = +81 ± 6 J/(K mol) [12]. We may thus assume that DMBI RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

elimination is the ratedetermining step of the CNCbl(III) reduction process at [HMS] > 0.8 mol/L. It is known [13] that reduction by hydroxymethane sulfinate can proceed with the participation of both the HOCH 2SO 2− anion and the product of its decomposition via reaction (1), sulfoxylate SO 2H −(SO 22− ): –

HOCH2 SO 2 ↔ SO2H– + CH2O.

(1)

Using data on the temperature dependence of the rate constant for the reaction between HMS and A550 0.4

0.3

0.2

0

200

400

600

τ, s

Fig. 2. Changes of absorbance at 550 nm during CNCbl reduction by sodium hydroxymethanesulfinate under anaerobic conditions. [CNCbl]0 = 5 × 10–5 mol/L, [HMS]0 = 0.1 mol/L; pH 11.4; 25°C. Vol. 87

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the range of concentrations of S2O 42− > 0.8 mol/L), but it is linearized in the coordinates 1/kobs–1/[S2O 42−] (Fig. 6). The observed reaction rate constant at [S2O 42−] > 0.8 mol/L is virtually equal to the rate con stant of reduction by hydroxymethanesulfinate (k1 = 3.0 × 10–2 s–1 at 25°С). The similarity between the kinetic and activation parameters of CNCbl reactions with sodium hydroxymethanesulfinate and dithionite and the reac tion of DMBI elimination from CNCbl shows that the reduction of CNCbl by the indicated sulfurcontain ing compounds proceeds via an innersphere mecha nism, the step of DMBI elimination being the rate determining step upon large excesses of DMBI. With a rise in pH, the reaction rate of CNCbl(III) with hydroxymethanesulfinate increases, reaching a plateau in the range pH > 12 (Fig. 7). It was estab lished, however, that the reaction rate of CNCbl(III) with dithionite does not depend on the pH. We may thus assume that the dependence presented in Fig. 7 is due to the acid–base properties of the reducing agent, sodium hydroxymethanesulfinate. Based on the above data, we propose the follow ing scheme of CNCbl reduction by sodium hydroxymethanesulfinate and dithionite:

indigo carmine [14], for which the ratedetermining step is the decomposition of HMS with the formation of sulfoxylate (direct reaction (1)), it was established that the rate constant of direct reaction (1) is 1.5 × 10 ⎯6 s–1 at [HMS] = 1 mol/L, pH 8.0, and 45°C, while the rate constant for the reaction between CNCbl(III) and HMS at 25°C is 1.3 × 10–3 s–1 ([HMS] = 1 mol/L, pH 8.0). We may thus assume that hydroxymethane sulfinate itself (rather than its decomposition product, sulfoxylate) participates in the reaction with cyanoco balamin in alkaline media. This is confirmed by the product of reaction (1), formaldehyde, having no effect on the rate of the reduction of cyanocobalamin, as was found in this work. A typical kinetic curve of CNCbl reduction by dithionite is presented in Fig. 4. Processing the kinetic data in semilogarithmic coordinates showed that the order for cyanocobalamin is equal to unity ([Na2S2O4] > 10–3 mol/L, [CNCbl] = 5 × 10–5 mol/L). At concentrations of dithionite from 0 to 0.015 mol/L, the dependence of the observed rate of cyanocobalamin reduction on the reducing agent’s concentration is linear (Fig. 5). Upon a considerable excess of dithionite, as in the case of HMS, the indi cated dependence is nonlinear (a plateau is reached in

CN CoIII N k–1 k1

(2) CN HOCH2SO2H

Ka +H+

HOCH2SO2– +

CN k2

CoIII N H2O

CoII

•

•

+ HOCH2SO2 + CN–,

N

– e

N O HOCH2S O

•

k3

CoIII

k–2

•

(3)

CH2O + SO2H(SO2– )

HOCH2SO2

CN CoIII N k–1 k1

(4)

CN 2SO2–

S2O42– +

CoIII N H2O

CN k2 k–2

k5

CoIII 2−

N S2O4

– e

CoII

+ S2O4– + CN –,

N

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SO2− CoII

+ SO2–

N

N

In this scheme, it is assumed that in the case of dithionite the reducing agent is dianion S2O 42− itself rather than anion–radical SO 2− , as in the case of hydroxocobalamin reduction [8]. This is demon strated by the linear dependence of the reaction rate

on the concentration of dithionite. It should also be noted that the latter reaction (the formation of a sta ble Cbl(II) complex with SO 2− ) takes place upon reduction with both hydroxymethanesulfinate and dithionite.

1/kobs, s

A550

1200

0.4

800

(5)

CoIII

0.3

400 0.2 0 20

60

100 1/[HMS], L/mol

Fig. 3. Dependence of 1/kobs on 1/[HMS]. [CNCbl]0 = 5 × 10–5 mol/L; pH 11.4; 25°C.

kobs × 103, s−1

0

400

800

1200

τ, s

Fig. 4. Changes of absorbance at 550 nm vs. time during the CNCbl reaction with dithionite under anaerobic conditions. 2−

[CNCbl]0 = 5 × 10–5 mol/L; [S2O4 ]0 = 1.7 × 10 pH 11.4; 25°C.

−2

mol/L;

1/kobs, s 1200

4 3

800

2 400

1 0

0 0

5

10 15 [Na2S2O4] × 103, mol/L 2−

Fig. 5. Dependence of kobs on [S2O4 ] under anaerobic conditions. [CNCbl]0 = 5 × 10–5 mol/L; pH 11.4; 25°C. RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

0

100

200

400 300 1/[S2O42−], L/mol 2−

Fig. 6. Dependence of 1/kobs on 1/[S2O4 ] under anaerobic conditions. [CNCbl] = 5 × 10–5 mol/L; pH 11.4; 25°C. Vol. 87

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kobs × 103, s−1

2

1

0 6

8

10

12

14 pH

Fig. 7. Dependence of the observed rate constant for the CNCbl reaction with sodium hydroxymethanesulfinate on pH under anaerobic conditions. [CNCbl]0 = 5 × 10 ⎯ 5 mol/L; [HMS]0 = 0.02 mol/L; 25°C.

ACKNOWLEDGMENTS This work was supported by the Russian Founda tion for Basic Research, project no. 110300132a. REFERENCES 1. E. SuarezMoreira, J. Yun, C. S. Birch, J. H. Williams, A. McCaddon, and N. E. Brasch, J. Am. Chem. Soc. 131, 15078 (2009).

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