Mechanisms of Formation and Equilibria of 4a and 5 Adducts of an Isoalloxazine. Reaction of 10-(2′,6′-Dimethylphenyl)-3-methylisoalloxazine-6,8-disulfonate with Sulfite in Aqueous Media

The reaction of 10-(2′,6′-dimethylphenyl)-3-methylisoalloxazine-6,8-disulfonate (I) in aqueous sulfite-bisulfite buffers (30°, μ = 2.0) yields an equilibrium mixture of I plus 4a- and 5-sulfite adducts (4a, 5^–, and 5H of eq 1). From the pH dependence of the relative concentration of 4a and the kinetically apparent acid dissociation constant of 5H (i.e., 5H → 5^– with pK_a5) the pH and buffer independent equilibrium constant (K_e = [5H]/[4a]) has been calculated to be 4.12 × 10^–2. Therefore, at 30° in aqueous solution, the 4a adduct is thermodynamically favored over the neutral 5 adduct. From studies of the dependence of the equilibrium constants K_x = [4a]/[I] and K_y = [5^–]/[I] upon pH and total sulfite buffer concentration ([S]_T) it was determined that K_x was proportional to [HSO₃ ^–]^1.0 and K_y proportional to [SO₃ ^2–]^1.0. These dependencies of K_x and K_y upon concentrations of buffer species establish that the forward reactions from I to 4a and 5^– have in their rate expressions the terms [HSO₃ ^–] and [SO₃ ^2–], respectively, in excess over the retrograde reactions of 4a → I and S^– → I. The kinetics for approach to equilibrium in the conversion of I to products (4a, 5^–, and 5H) were studied under the pseudo-first-order conditions of [buffer] ≫ [I₀], The pseudo-first-order rate constants (k_obSd) were found to be dependent upon three terms (eq 14); the first contained the product [HSO₃ ^–][SO₃ ^2–], the second [SO₃ ^2–], and the third was independent of buffer species but dependent upon the mole fraction of a reactant of pK_app (pK_a5) assignable to dissociation of 5H → 5^–. With the knowledge of the dependence of the equilibrium ratios [4a]/[I] and [5^–]/[I] upon [HSO₃ ^–] and [SO₃ ^2–], the rate terms were assignable (Scheme I) to: general acid (by HSO₃ ^–) catalysis of nucleophilic attack of SO₃ ^2– upon I to yield 4a and by microscopic reversibility general base (by SO₃ ^2–) catalysis of conversion of 4a → I and unassisted nucleophilic attack of SO₃ ^2– upon I to yield 5^– with spontaneous conversion of 5^– → I. An alternate scheme that would satisfy both the thermodynamic and kinetic findings would be that of Scheme II. Here I is converted to 5H via general acid (by HSO₃ ^–) catalyzed attack of SO₃ ^2– and to 5^– by unassisted attack of SO₃ ^2–, 4a arising from rearrangement of 5H. Arguments are presented which favor the mechanism of Scheme I.