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J. Phys. Chem. B 2006, 110, 16774-16780
pH-Dependent Electronic and Spectroscopic Properties of Pyridoxine (Vitamin B6) Mikael Ristila1 , 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 B6 (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 N1-protonated form and, at high pH, the O3′-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 B6, 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 B6, whereas parasitic organisms and higher animals have to acquire vitamin B6 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 oxygen2,3 and hydrogen peroxide.4 This suggests that vitamin B6 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 B6 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 heteroaromatic 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 spectrophotometrically or fluorometrically follow the degradation of vitamin B6. In a recent combined NMR and singlet oxygen phosphorescence decay analysis, the reaction between pyridox* Corresponding author. E-mail:
[email protected].
Figure 1. Vitamin B6 (pyridoxine) and its main derivatives. Atomic labeling is shown for pyridoxine.
ine and 1O2 was proposed to generate a bicyclo-octenone, with the oxygen molecule bridging across carbons C2 and C6 (cf. Figure 1), and the C6 hydroperoxide as main products.7 No mechanistic details, relative stabilities, or related, were however reported. The physiochemical properties of the different vitamin B6 derivatives have been characterized in great detail, using fluorescence,8 infrared,9,10 mass,11 NMR,12,13 photoelectron, Raman,14 and UV15,16 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 13C 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
10.1021/jp062800n CCC: $33.50 © 2006 American Chemical Society Published on Web 07/29/2006
pH-Dependent Properties of Pyridoxine stabilizing water molecules and polarized continuum solvent models were required at the B3LYP/6-311+G(d)//B3LYP/ 6-31G(d) level.22 To elucidate in more detail the protective biological function of pyridoxine against oxidative stress and to facilitate the elucidation of the chemical reaction mechanisms between pyridoxine and ROS, we herein report on a combined theoretical and experimental study of the dependency of the UV absorption spectrum of pyridoxine on solvent pH vs. protonation/deprotonation states of the molecules. In addition, various basic electronic properties such as localization of unpaired spin in the radical systems and change in atomic charges for the different protonated species are outlined. In subsequent work, the explicit reaction mechanisms between pyridoxine and a range of ROS will be addressed. II. Methodology A. Theory. Pyridoxine in its neutral ground state, its radical anion and cation, and its N- and O-protonated and OHdeprotonated forms (cf. Figure 1) was explored using the hybrid DFT functional B3LYP.23-25 Geometries were optimized in vacuo at the B3LYP/6-311+G(d,p) level, followed by frequency calculations at the same level of theory to ensure that these were stationary structures on their respective energy surfaces and to extract zero-point vibrational energy corrections (ZPE). Implicit solvent effects (aqueous solution) were modeled using the integral equation formalism of the polarized continuum model (IEF-PCM) of Tomasi and co-workers.26,27 Excited-state calculations were performed within the time-dependent (TD) DFT framework,28-30 using the same method and basis set as listed above. For the excited-state modeling, solvent effects were for computational reasons modeled using the conductorlike PCM model (C-PCM),31,32 previously employed very successfully by, e.g., Russo et al., to include solvent effects on excitation spectra.33 All calculations were performed using the Gaussian03 program.34
J. Phys. Chem. B, Vol. 110, No. 33, 2006 16775 From the calculations, we report key geometric parameters, adiabatic electron affinities and ionization potentials in solution, charge relocalization, distribution of unpaired spin in the radical systems, relative stabilities (in terms of Gibbs free energies ∆G(aq)298), and excitation energies. We will throughout use primes to denote substituents associated to a particular ring carbon (e.g., O3′ denotes the hydroxylic oxygen attached to C3, and so forth). In the case of the charged species, we include data for solvated electrons and protons in aqueous solution, as previously developed in our group.35 According to this scheme, a solvated proton carries the energy of -268.68 kcal/mol, whereas the solvated electron has an energy of -38.37 kcal/mol. The approach has, e.g., been employed successfully to the redox properties of DNA bases.36,37 B. Experiment. Spectra (200-500 nm) were obtained by measuring 200 µM solutions of pyridoxine hydrochloride (>99% purity, Fluka, Sigma-Aldrich Corp., St Louise, MO). Four different solvents were used in order to generate different protonation states: 100 mM KCl-HCl, pH 1.7; 100 mM NaPi, pH 7.0; 100 mM Na2HPO4-NaOH, pH 11.1; and milli-Q H2O (giving a final pH in the hydrochloride solution of 4.3). Several additional neutral buffers (pH 7) were also tested: Tris-HCOOH, MOPS-KOH, PIPES, using pyridoxine-HCl salt or pure pyridoxine. The different spectra recorded at pH ) 7 were identical, irrespective of the buffer used. All spectra were recorded on a Shimadzu UV1601 UV-visible spectrophotometer (Shimadzu Corp., Kyoto, Japan). III. Results and Discussion A. Geometric and Electronic Features. In Figure 2, we display the optimized structures of the neutral ground state and radical anion/cation of pyridoxine, as well as the key protonated and deprotonated species. With the exception of the neutral zwitterion (2f), the most stable rotamers in all cases involve
Figure 2. Optimized structures of (a) pyridoxine, (b) its radical anion, (c) its radical cation, (d) the N1-protonated species, (e) the O3′ deprotonated species, and (f) the neutral N1-O3′ oxo zwitterion, respectively. All data was obtained at the B3LYP/6-311+G(d,p) level in a vacuum.
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TABLE 1: Calculated Energies at the (IEFPCM)-B3LYP/6-311+G(d,p) Levela compound
E(aq)b
ZPE
∆Gcorr298
∆G(aq)298 c
∆∆G(aq)298
B6 (2a) B6•- (2b) B6•+ (2c) B6+ - (2f) B6-N1H+ (2d) B6-O3′H+ d B6-O4′H+ B6-O5′H+ B6-O3′- (2e) B6-O4′B6-O5′-
-592.090 321 -592.152 893 -591.867 145 -592.081 515 -592.546 098 n/a -592.509 915 -592.508 098 -591.618 107 -591.605 374 -591.586 313
0.186 894 0.181 676 0.184 226 0.186 037 0.200 081
0.149 153 0.142 716 0.144 511 0.147 038 0.162 301
-591.941 168 -592.010 177 -591.722 634 -591.934 477 -592.383 797
0.197 361 0.197 473 0.172 992 0.170 750 0.169 965
0.159 555 0.160 190 0.135 772 0.133 615 0.131 638
-592.350 360 -592.347 908 -591.482 335 -591.471 759 -591.454 675
0 -43.30 137.13 4.20 0 n/a 20.98 22.52 0 6.64 17.36
a Atomic labels refer to protonated/deprotonated atoms. All data is in atomic units except ∆∆G(aq)298 (in kilocalories/mole). b Gas-phase optimized structure, including IEFPCM solvation energy. c ∆G(aq)298 ) E(aq) + ∆Gcorr298, where the latter is the thermal correction to the free energy at 298 K. d Rearranges into B6-O4′H+.
TABLE 2: Experimental UV Absorptions (nm) of Pyridoxine at Different pH Valuesa pH 1.7
pH 4.3
pH 7.0
290.1 (1.714) s 230 (0.50) sh 207.5 (2.754) s
324.5 (0.360) w 291.0 (1.406) s 225 (1.100) w sh 200-220 (>2.0) s
323.5 (1.433) s 290-300 (0.40) w sh 253.9 (0.748) i 210-225 (3.175) s
pH 11.1 314.5 (1.030) s 245.0 (0.840) i-s 212.5 (2.723) s
a Values in parentheses are the corresponding absorbances. Values in italics are deduced by direct reading from the spectrum. Absorption indices s, i, w, and sh indicate strong, intermediate, weak, or shoulder.
intramolecular hydrogen bonding, in most cases, between the C3 and C4 hydroxyl groups. The OH groups at C4 and C5 are in all cases rotated out of the plane of the aromatic ring, which remains planar irrespective of charge or protonation state. Within the aromatic ring of neutral ground state pyridoxine, the bonds are all indicative of a fully conjugated structure with C-C distances in the range 1.39-1.41 Å and C-N distances of 1.331.34 Å. For the radical anion of pyridoxine (2b), the excess electron will occupy an antibonding π* orbital leading to lengthened N1-C2, C3-C4, C4-C5, and C4-O4′ bonds and shortened C2-C3 and C4-C4′ bonds (prime denoting the substituent). All changes are within 0.05 Å. Also, the hydrogen bond between O3′H - - O4′ increases, in this case by 0.1 Å. The least affected bonds are those to C6. The structural changes are also reflected in that the additional electron is primarily localized to C3 (∆q ) -0.87 e-), with a slight increase in negative charge also on N1 and the C5 substituent (approximately -0.20 e- on each), compared with the neutral system, whereas the unpaired spin in the anion is found on the opposing N1 (0.27 e) and C4 (0.43 e), with minor components also on C2 and C5. In the cation 2c, ionization occurs primarily from a bonding π orbital of the aromatic ring, resulting in elongated C2-C3, C3-C4, and C5-C6 bonds (by 0.03-0.05 Å) and in a decreased C4-C5 bond by the same amount. In addition, the C3-O3′ bond is substantially shortened (0.06 Å), as is the O3′H - - O4′ intramolecular hydrogen bond (by 0.28 Å) concomitant with an increased O3′-H bond length. The C-N1 bonds are essentially unaffected by the ionization. The increase in positive charge occurs at C4 and C4′ (∆q ) +1.2 and +0.7 e-, respectively), in part compensated for by drastically increased negative charges on C3 and C5 (by -0.8 and -0.6 e-, respectively). The unpaired electron is distributed over several centers, with maximum components (0.30-0.36 e) found on C2 and C6, and small contributions on C3 and O3′, respectively. Overall, very large charge redistributions occur in the different systems, depending on oxidation or reduction. The anion and cation behave rather differently in their response. Turning to the effects of different pH values on the compounds, different protonation states were explored as follows: To mimic the case of very low pH, position N1 (2d)
Figure 3. Experimental UV spectra (nm) of pyridoxine at pH 1.7 (solid), pH 4.3 (dashed), pH 7.0 (dot-dashed), and pH 11.1 (dotted). Each individual spectrum is renormalized.
or either of the three oxygens were protonated (see next section). However, as the latter were more than 20 kcal/mol less stable than the N1-protonated species, these will not be considered further. Protonation at N1 results in increased positive charge on most ring atoms except C5, which becomes 0.4 e- more negative. The largest buildup of positive charge is seen on N1 (∆q ) +0.5 e-). The geometric changes are within 0.02 Å throughout, except for the O3′H - - O4′ hydrogen bond that becomes 0.16 Å shorter. In the case of high pH, systems with one proton fully removed from either OH group were considered. Deprotonation of either O4′ or O5′ results in a highly localized charge to the oxygen in question (and a minor component on the neighboring carbon atom), accompanied by a reduction of the corresponding C-O bond length by approximately 0.1 Å. When the ring-bound hydroxyl group is deprotonated (2e), we also see a 0.1 Å shorter C3-O3′ bond, and a highly localized negative charge on O3′ (∆q ) -0.7 e-). In this case, C3 also attains a more negative charge, only partly balanced by an increased positive charge on C4. For this system, elongated C2-C3 and C3-C4 bonds are observed, along with a shift in intramolecular hydrogen bond to O4′H - - O5′.
pH-Dependent Properties of Pyridoxine
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TABLE 3: Main Absorptions (nm), Orbital Compositions, and Oscillator Strengths, Calculated at the CPCM/ TD-B3LYP/6-311+G(d,p) Level abs (nm)
orbital components
osc strengths
N1-protonated (2d) 278.4 242.5 222.0 208.4 205.1
H f L (0.64) H - 2 f L (0.64) H - 3 f L (0.46), H f L + 1 (0.48) H - 1 f L + 1 (0.62) H -3 f L (0.43), H - 2 f L + 1(0.29), H f L + 1 (-0.31)
0.202 0.023 0.070 0.066 0.217
264.3 253.9 226.6 211.5 203.4
neutral (2a) H f L (0.63) H - 1 f L (0.67) H - 1 f L (0.43), H f L + 1 (0.44) H - 3 f L (0.52) H - 2 f L (0.30), H - 3 f L (0.39), H - 4 f L (-0.34)
0.148 0.013 0.011 0.088 0.265
342.9 255.1 250.8 228.7 223.9 221.0 211.0
neutral zwitterion (2f) H f L (0.63) H f L + 1 (0.54), H f L + 2 (-0.32) H f L + 2 (0.60) H - 3 f L (0.51), H - 4 f L (0.36) H f L + 4 (0.61) H f L + 5 (0.68) H - 4 f L (0.52)
0.198 0.069 0.034 0.110 0.017 0.015 0.240
298.6 271.0 250.7 240.9 236.9 217.7 214.2 210.0 207.1 202.6
O3′-deprotonated (2e) H f L (0.65) H f L + 1 (0.57), H f L + 2 (0.40) H f L + 1 (-0.34), H f L + 2 (0.53) H f L + 4 (0.69) H f L + 3 (0.66) H - 2 f L (0.30), H - 3 f L (0.58) H f L + 8 (0.68) H - 4 f L (0.54), H - 1 f L (-0.35) H - 1 f L + 4 (0.49), H - 1 f L + 3 (-0.37) H - 2 f L (0.32), H f L + 10 (-0.34)
0.169 0.010 0.087 0.014 0.083 0.019 0.017 0.045 0.089 0.229
Figure 4. Computed absorption spectra (nm) of the N1-protonated (solid), neutral (dashed), zwitterionic (dot-dashed), and O3′-deprotonated (dotted) pyridoxine moieties. Each individual spectrum is renormalized.
a
The main orbital component(s) of the absorptions are given relative to the highest occupied (H) and lowest unoccupied (L) orbitals and their respective weights.
In addition to the above systems, the neutral zwitterion of pyridoxine, with N1 protonated and O3′ deprotonated, was also investigated (2f). In this case, the largest geometric effects occur around C3 (shorter C3-O3′ bond and longer C3-C bonds), and in slightly increased N1-C bonds. Large charge redistributions occur in the zwitterion, compared with the neutral ground state 2a, with the main positive centers now being N1, C4, C4′, and C6 (charge increase ∆q ) +0.50, +1.09, +0.20, and +0.44 e-, respectively), whereas the largest charge reductions are found at C3, C5, C2′, and O3′ (∆q ) -1.16, -0.24, -0.35, and -0.53 e-, respectively). B. Energetics. In Table 1, we list the energetics of the different compounds of Figure 2, along with the various isomeric protonated or deprotonated species. The adiabatic ionization free energy and electron affinity are 137.3 and 43.3 kcal/mol, respectively. Ionizing pyridoxine will thus require substantial energy input (more than 6 eV)seven when taking into account the solvation energy of a free electron in aqueous solution (-38.37 kcal/mol),35 whereas the calculations indicate that vitamin B6 readily takes up solvated electrons created by, e.g., radiolysis, with a net energy gain of 4.9 kcal/mol. We furthermore note that the zwitterionic structure 2f, with O3′ deprotonated and N1 protonated, is only 4 kcal/mol less stable than the neutral ground state form 2a, in close agreement with previous work on 3-hydroxypyridine21 and pyridoxal.22 As mentioned above, the different oxygen-protonated species are all at least 20 kcal/mol less stable than the N1-protonated form. It is thus reasonable to assume that these compounds will not be formed under normal physiological conditions, and very
Figure 5. (a) Experimental UV spectrum (nm) at pH 1.7 (solid) and computed spectrum for N1-protonated species (dashed). (b) Experimental UV spectrum at pH 11.1 (solid) and computed spectrum for O3′-deprotonated species (dashed).
rarely so even at low pH. The free energy difference between 2d and the neutral pyridoxine 2a is 277.75 kcal/mol which, based on the estimated solvation energy of H+ of 268.68 kcal/ mol,35 indicates that pyridoxine in the presence of protons readily will become (N1) protonated (exergonic by 9 kcal/mol). The remaining positions instead display endergonic protonation free energies by 12-14 kcal/mol, which again speaks against protonation at these sites. For the deprotonated species, the most stable form is obtained by removing the proton from O3′. Interestingly, the O4′ deprotonated form is only some 6.6 kcal/mol less stable and may contribute to a minor extent. Deprotonation at O5′, on the other hand, appears less likely. The summed free energy of the O3′-
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Figure 6. Optimized structures of (a) neutral pyridoxine with 2 water molecules, (b) zwitterionic pyridoxine with 2 water molecules, (c) neutral pyridoxine with Na+ and Cl- counterions, and (d) zwitterionic pyridoxine with Na+ and Cl- counterions.
deprotonated species and a solvated proton lies 19.2 kcal/mol above the free energy of pyridoxine alone. Thus, for this chemical species to deprotonate, the assistance of a base is required. C. Experimental and Computed UV Spectra. UV spectra were recorded for pyridoxine at four different pH values: 1.7, 4.3, 7.0, and 11.1. The tabulated pKa values for pyridoxine are approximately 4, 9, and 15,38 corresponding in a nonpolar solvent to deprotonation of the ring nitrogen, the ring-bound hydroxyl group, and one of the C4′/C5′ hydroxyl substituents, respectively. As the current spectra are recorded in aqueous solution, we may expect that UV spectra at pH 1.7 will correspond to the N1/O3′-protonated species, pH 4.3 will represent a mixture of the neutral and N1-protonated compounds, pH 7.0 will refer to the neutral O3′ hydroxyl and/or zwitterionic oxo species (or a mixture of neutral, N1-protonated, and O3′deprotonated compounds), and pH 11.1 mainly will involve the N1/O3′-deprotonated moiety. In Table 2, we list the recorded experimental absorptions for pyridoxine at different pH. For simplicity, we will throughout the remainder of the text use the neutral hydroxyl species as the basis for our notation, such that the species at low pH will be referred to as the N1-protonated form, and at high pH the O3′-deprotonated form. In Figure 3, we display the renormalized experimental spectra at different pH values (main peaks listed in Table 2). From the experimental data, several interesting observations can be made. Starting with the system at low pH, there are two main absorptions, at 290 and 208 nm, and a weak/intermediate shoulder at ca. 225 nm which, however, is buried in the very strong peak at short
wavelengths. Already at pH 4.3, the onset of a second set of absorptions is noted, as a weak absorption at 324 nm, broadening of the shoulder around 230 nm, and a broadening of the strong absorption band at 200-220 nm. The peak at 291 nm is reduced in intensity and broadened. At neutral pH, the absorption at 324 nm is strong and a new peak at 254 nm has appeared. Of the strong absorption at 291 nm, only a weak shoulder remains, and the short wavelength band is red-shifted by approximately 20 nm. At pH 11.1, finally, the absorption at 291 nm has vanished completely and we see three strong (or intermediate/ strong) absorptions, at 315, 245, and 213 nm. Compared with the main absorptions at pH ) 7.0, the same overall absorption pattern is observed, but with all peaks blue-shifted some 1015 nm. It is not unlikely that the different ionic and chemical environments provided by the buffers to some extent interfere with, or influence, the recorded spectra. The main absorptions for the different species, computed using TD-DFT in aqueous medium (CPCM), are given in Table 3, and the corresponding UV spectra are displayed in Figure 4. The main conformers of each protonation state are included: the N1-protonated species 2d, the two neutral systems 2a and 2f, and the O3′-deprotonated form (2e). The energetically close-lying O4′-deprotonated species was also examined but was found to have an absorption spectrum too far from the experimentally observed ones to be of interest. For all systems, the lowest-lying absorption corresponds to the HOMO f LUMO transition, followed by several smaller absorptions with varying orbital components in the 210-270 nm range and a sharp absorption band at 200-210 nm often
pH-Dependent Properties of Pyridoxine involving excitation from lower-lying orbitals to the LUMO (cf. Table 3). As seen from the plotted spectra (Figure 4), the four systems display large shifts depending on protonation state. Of particular interest is the very different spectra seen for the two neutral species (2a dashed and 2f dot-dashed, respectively), where the zwitterion has a strong absorption at 343 nm and clearly resolved absorptions at 255 and 229 nm, compared with the neutral species 2a that besides the strong absorption at short wavelengths only displays one sharp absorption (at 278 nm). D. Analysis of UV Spectra. To unveil the abundance of the different species at different pH values, we display in Figure 5a the experimental spectrum at pH 1.7 along with the N1-protonated species, and in Figure 5b we display the spectrum at pH 11.1 along with the O3′-deprotonated species. A very close correlation in overall appearance between the experimental and computed spectra is seen. We also note the well-known fact that computed spectra obtained at the TD-DFT level tend to give approximately 0.2 eV too high excitation energies (too short wavelengths). Correcting for an overestimation of 0.2 eV corresponds to a blue-shift of the computed absorptions of approximately 15 nm at 300 nm, 10 nm at 250 nm, and 7 nm at 200 nm. That is, a very close agreement between theory and experiment. The situations at pH 4.3 and 7.0 are more complex. Analysis of the spectra reveal that, at pH 4.3, the spectrum of the N1protonated species dominates (cf. solid vs dashed lines in Figure 3), with a weak onset of a second species with a small peak at approximately 325 nm. At pH 7.0 (dot-dashed line in Figure 3), on the other hand, we have a spectrum dominated by the species with an absorption at 324 nm, a shoulder at 290 nm, and a peak of intermediate strength at 234 nm. The shoulder at 290 nm could possibly be a slight reminiscence of the N1-protonated form. Comparing with the computed spectra in Figure 4, we see that none of these show an as good overlap with the experimental spectrum at pH 7.0, as were the cases at pH 1.7 and 11.1. To analyze the spectrum at pH 7.0 further, two additional models were investigated: the neutral hydroxyl and zwitterionic oxo species with additional water molecules hydrogen bonding to the N1 and O3′ ends and the neutral and zwitterionic species with Na+ and Cl- ions interacting at the same positions. The optimized structures of these four systems are displayed in Figure 6. The systems complexated with water molecules are highly similar to those found by Kiruba and Wong for pyridoxal.22 Compared with the nonsolvated structures 2a and 2f, the structural changes within the pyridoxine molecules are very small from the additional hydrogen bonding (geometric parameters altering less than 0.01 Å were not included in Figure 6) and highly localized to O3′ and its closest neighboring atoms. Energetically, the neutral hydroxyl form is still the most favorable species, with a ∆∆G(aq)298 2.8 kcal/mol more stable than the zwitterion. The results are in contrast with the findings of Kiruba and Wong,22 which in part can be related to the different PCM models employed and in part to the different (lower level) basis sets employed for the optimizations in ref 22. In the NaCl complex to the zwitterion, the structural effects are somewhat larger, albeit still very localized. The N1-H bond increases the most, from the influence of the chloride anion, by 0.1 Å. In the neutral hydroxyl system, the chloride anion moves over from the N1 site to interact directly with Na+, with very little influence on the geometric parameters of the pyridoxine. The two counterions carry net Mulliken charges of (0.7-0.9 e-, respectively, and the local charges on the
J. Phys. Chem. B, Vol. 110, No. 33, 2006 16779
Figure 7. (a) Experimental UV spectrum (nm) at pH 7.0 (solid line) together with computed spectra for neutral (dot-dashed) and zwitterionic (dashed) pyridoxine with two additional water molecules. (b) Experimental UV spectrum at pH 7.0 (solid line) together with computed spectra for neutral (dot-dashed) and zwitterionic (dashed) pyridoxine stabilized by Na+/Cl- counterions.
pyridoxine species are influenced to a relatively small extent by the presence of the counterions. The largest changes are seen for the O3′ atom in the zwitterion, that has 0.1 e- more negative charge in the NaCl complex than when complexating with the two water molecules. For the NaCl-complexated systems, the hydroxyl form is still the more stable of the two, by approximately 5.1 kcal/mol. The UV spectra of the hydroxyl and oxo forms with the additional water molecules are displayed in Figure 7a. Adding explicit water molecules has a small but essentially negligible effect on the UV spectra, and neither of the two models (“pure” molecules in PCM vs hydrogen-bonded water complexes in PCM) is capable of reproducing fully the recorded spectrum at pH 7.0. It hence appears that the PCM bulk solvation model itself captures the essential features of the polar environment on the excitations but that there are more factors that contribute to the experimental spectrum. The effects of adding counterions to the complexes are considerably larger. For the zwitterionic species, the entire spectrum is blue-shifted by approximately 0.15 eV, so that the low-energy (HOMO f LUMO) excitation overlaps perfectly with the experimental value of 324 nm. The small peak at 254 nm is also blue-shifted to about 245 nm. For the hydroxyl form 2a complexated with NaCl, the effects are much smaller, only around 0.05 eV for the blue-shift, albeit a better match is seen with the small 254 nm peak. On the basis of the UV spectra, it appears that the zwitterionic oxo form is the predominant species at neutral pH, in accordance with earlier spectroscopic studies, and that counterions do influence the exact positioning of the UV spectra. Hydrogen bonding and/or polar bulk solvation alone is not sufficient to fully describe this feature.
16780 J. Phys. Chem. B, Vol. 110, No. 33, 2006 IV. Conclusions The possible protonation states of pyridoxine (vitamin B6) have been explored, to determine its structural, electronic, and spectroscopic properties at different pH values. Large structural differences are noted for the different charged species, compared with the neutral ground state form, both concerning the aromatic ring and the length of the intramolecular hydrogen bond. Protonation and deprotonation also have implications for the structure, although the effects in these cases are more localized. The atomic charges and, for the radical species, the distribution of unpaired spin also show large variations. Energetically, protonation at N1 renders the most stable conformer among the protonated species, and deprotonation will primarily occur at the ring-bound O3′. Protonation is energetically favorable, whereas, based on the energy of -268.68 kcal/ mol for a solvated proton,35 the deprotonated species is less readily formed. The ionization free energy in aqueous solution is 137 kcal/mol (5.9 eV), whereas the radical anion is more stable than the neutral species by 43 kcal/mol (1.9 eV). Computed absorption data, obtained at the CPCM/TDB3LYP/6-311+G(d,p) level and compared with measured UV spectra, reveal a direct correlation between the N1-protonated species at low pH and the O3′-deprotonated form at high pH. At physiological pH, the zwitterionic oxo form dominates, although strong interaction with counterions was required in the theoretical treatment to obtain a sufficient blue-shift of the spectrum. Neither explicit hydrogen bonding to additional water molecules nor polarized continuum was sufficient to catch this feature. Acknowledgment. The authors acknowledge funding from the Swedish Science Research Council (VR), the Wood Ultrastructure Research Center (WURC) at SLU, and the Faculty of Medicine, Natural Science and Technology at O ¨ rebro University. Generous grants of computing time at the supercomputing facilities in Linko¨ping (NSC) and Stockholm (PDC) are also gratefully acknowledged. References and Notes (1) Schneider, G.; Ka¨ck, H.; Lindqvist, Y. Structure 2000, 8, R1. (2) Ehrenshaft, M.; Jenns, A. E.; Chung, K. R.; Daub, M. E. Mol. Cell 1998, 1, 603. (3) Osmani, A. H.; May, G. S.; Osmani, S. A. J. Biol. Chem. 1999, 274, 23565. (4) Rodriguez-Navarro, S.; Llorente, B.; Rodriguez-Manzaneque, M. T.; Ramne, A.; Uber, G.; Marchesan, D.; Dujon, B.; Herrero, E.; Sunnerhagen, P.; Perez-Ortin, J. E. Yeast 2002, 19, 1261. (5) Bilski, P.; Li, M. Y.; Ehrenshaft, M.; Daub, M. E.; Chignell, C. F. Photochem. Photobiol. 2000, 71, 129. (6) Ehrenshaft, M.; Bilski, P.; Li, M.; Chignell, C. F.; Daub, M. E. Proc. Natl. Acad. Sci. 1999, 96, 9374. (7) Ohta, B. K.; Foote, C. S. J. Am. Chem. Soc. 2002, 124, 12064.
Ristila¨ et al. (8) Bridges, J. W.; Creaven, P. J.; Davis, D. S.; Williams, R. T. Biochem. J. 1963, 88, 65. (9) Buyl, F.; Smets, J.; Maes, G.; Adamowicz, L. J. Phys. Chem. 1995, 99, 14967. (10) Person, W. B.; Del Bene, J. E.; Szajda, W.; Szczepaniak, K.; Szczesniak, M. J. Phys. Chem. 1991, 95, 2770. (11) Baldwin, M. A.; Langley, G. J. Chem. Soc., Perkin Trans. 2 1988, 347. (12) Llor, J.; Lopez-Mayorga, O.; Munoz, L. Magn. Reson. Chem. 1993, 31, 552. (13) Llor, J.; Munoz, L. J. Org. Chem. 2000, 65, 2772. (14) Mehdi, K. C. Indian J. Phys. 1984, 58B, 328. (15) Metzler, C. M.; Cahill, A.; Metzler, D. E. J. Am. Chem. Soc. 1980, 102, 6075. (16) Llor, J.; Asensio, S. B. J. Solution Chem. 1995, 24, 1293. (17) Scalan, M. J.; Hillier, I. H.; MacDowell, A. A. J. Am. Chem. Soc. 1983, 105, 3568. (18) Karelson, M. M.; Katritzky, A. R.; Szafran, M.; Zerner, M. C. J. Org. Chem. 1989, 54, 6030. (19) Fabian, W. M. F. J. Comp. Chem. 1991, 12, 17. (20) Wong, M. W.; Wiberg, K. B.; Frisch, M. J. J. Am. Chem. Soc. 1992, 114, 1645. (21) Wang, J.; Boyd, R. J. J. Phys. Chem. 1996, 100, 16141. (22) Kiruba, G. S. M.; Wong, M. W. J. Org. Chem. 2003, 68, 2874 and references therein. (23) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (24) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785. (25) Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. J. Phys. Chem. 1994, 98, 11623. (26) Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 106, 5151. (27) Tomasi, J.; Mennucci, B.; Cance`s, E. J. Mol. Struct. (THEOCHEM) 1999, 464, 211. (28) Casida, M. E.; Jamorski, C.; Casida, K. C.; Salahub, D. R. J. Chem. Phys. 1998, 108, 4439. (29) Bauernschmitt, R.; Ahlrichs, R. Chem. Phys. Lett. 1996, 256, 454. (30) Stratmann, R. E.; Scuseria, G. E.; Frisch, M. J. J. Chem. Phys. 1998, 109, 8218. (31) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995. (32) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Comp. Chem. 2003, 24, 669. (33) Petit, L.; Quartirolo, A.; Adamo, C.; Russo, N. J. Phys. Chem. B 2006, 110, 2398. (34) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Pittsburgh, PA, 2003. (35) Llano, J.; Eriksson, L. A. J. Chem. Phys. 2002, 117, 10193. (36) Llano, J.; Eriksson, L. A. Phys. Chem. Chem. Phys. 2004, 6, 2426. (37) Llano, J.; Eriksson, L. A. Phys. Chem. Chem. Phys. 2004, 6, 4707. (38) Harris, C. M.; Johnson, R. J.; Metzler, D. E. Biochim. Biophys. Acta 1976, 421, 181.
