pH-Dependent Electronic and Spectroscopic Properties of Pyridoxine (Vitamin B 6 ) Mikael Ristila 1 , Jon M. Matxain, Åke Strid, and Leif A. Eriksson* Department of Natural Sciences and O ¨ rebro Life Science Center, O ¨ rebro UniVersity, S-701 82 O ¨ rebro, Sweden ReceiVed: May 8, 2006; In Final Form: June 20, 2006 The key electronic and spectroscopic properties of vitamin B 6 (pyridoxine) and some of its main charged and protonated/deprotonated species are explored using hybrid density functional theory (DFT) methods including polarized solvation models. It is found that the dominant species at low pH is the N 1 -protonated form and, at high pH, the O 3′ -deprotonated compound. Computed and experimental UV-spectra for these species (experimental spectra recorded at pH 1.7 and 11.1, respectively) show a very close resemblance. At pH 4.3, the protonated species dominates, but with onset of the zwitterionic oxo form which is also the dominant species at neutral pH. The computational studies furthermore show that neither a polarized continuum model of the polar aqueous solvent or explicit hydrogen bonding through additional water molecules are sufficient to describe accurately the spectrum at physiological pH. Instead, Na + and Cl - counterions were required to give a blue-shift of approximately 0.15 eV. I. Introduction Vitamin B 6 , or pyridoxine, is the precursor of the biologically active derivative pyridoxal-5′-phosphate and pyridoxamine-5′- phosphate, with functional roles in a number of different enzymes. 1 Pyridoxine itself is a cofactor of several enzymes that catalyze decarboxylations, transaminations, and racemations of amino acids. Bacteria, fungi, and plants produce their own vitamin B 6 , whereas parasitic organisms and higher animals have to acquire vitamin B 6 through nutrient intake. Lately, pyridoxine biosynthesis-deficient mutants of fungi and yeast have been shown to be sensitive to reactive oxygen species (ROS) such as singlet oxygen 2,3 and hydrogen peroxide. 4 This suggests that vitamin B 6 and its derivatives are also involved in stress tolerance in living organisms, especially in alleviating oxidative stress. In eukaryotes, stress resistance has been implied to involve pyridoxine-dependent singlet oxygen quenching, 5 whereby the pyridoxine itself would react with and quench the singlet oxygen. 3,5 The oxidative stress-protective effect of pyridoxine has also been described both in red blood cells and in lens cells in animals. Pyridoxine itself was found to be the most effective of the vitamin B 6 species, twice as effective as pyridoxal 5-phosphate, and as effective as vitamin E. 6 Knowledge about this novel mechanism of reaction between pyridoxine or its derivatives (cf. Figure 1) and singlet oxygen and other ROS is very small indeed. 5 However, since both the aldehyde (pyridoxal) and the amino (pyridoxamine) derivatives only to a small extent influence the rate of reaction, these moieties are probably not involved. Also, since the heteroaro- matic absorbance peak at 323 nm disappears during the reaction, at least one of the targets for singlet oxygen is most likely the core of the aromatic ring, leading to ring opening. The absorption peak at 323 nm, as well as the characteristic fluorescence of pyridoxine at 400 nm, 5 can be used to spec- trophotometrically or fluorometrically follow the degradation of vitamin B 6 . In a recent combined NMR and singlet oxygen phosphorescence decay analysis, the reaction between pyridox- ine and 1 O 2 was proposed to generate a bicyclo-octenone, with the oxygen molecule bridging across carbons C 2 and C 6 (cf. Figure 1), and the C 6 hydroperoxide as main products. 7 No mechanistic details, relative stabilities, or related, were however reported. The physiochemical properties of the different vitamin B 6 derivatives have been characterized in great detail, using fluorescence, 8 infrared, 9,10 mass, 11 NMR, 12,13 photoelectron, Raman, 14 and UV 15,16 spectroscopy techniques, and it has been concluded that the tautomeric equilibrium between the neutral hydroxy and zwitterionic oxo forms of the biologically active aldehyde derivatives pyridoxal and pyridoxal-5-phopshate (PLP), as well as 3-hydroxypyridine and pyridoxine, are strongly solvent dependent. The neutral form is dominant in a nonpolar medium, whereas the zwitterion is favored in aqueous solution. For the latter medium, a strong temperature dependency is furthermore noted on the tautomeric equilibrium. 13 Previous computational studies of a variety of hydroxypyridine and pyridoxine derivatives have primarily focused on equilibrium structures and tautomeric equilibria and range from early semiempirical investigations at AM1 and PM3 levels, 17-19 Hartree-Fock and perturbation theory (MP2) calculations, 20,21 and density functional theory (DFT), quadratic configuration interaction (QCISD(T)), and G3 studies. 22 In agreement with experimental observations from UV spectroscopy and 13 C NMR studies, the relative energies between the two tautomers depended strongly on solvent, albeit in most cases the hydroxyl form was found to be the most stable species. In order for the equilibrium to shift in favor of the zwitterionic form, additional * Corresponding author. E-mail: leif.eriksson@nat.oru.se. Figure 1. Vitamin B6 (pyridoxine) and its main derivatives. Atomic labeling is shown for pyridoxine. 16774 J. Phys. Chem. B 2006, 110, 16774-16780 10.1021/jp062800n CCC: $33.50 © 2006 American Chemical Society Published on Web 07/29/2006