pH-Dependent Electronic and Spectroscopic Properties of Pyridoxine (Vitamin B

6
)
Mikael Ristila1, Jon M. Matxain, ke 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
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/depro-
tonation 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 OH-
deprotonated 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 cal-
culations 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
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., O
3
denotes the hydroxylic oxygen attached to C
3
,
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 M 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 NaP
i
,
pH 7.0; 100 mM Na
2
HPO
4
-NaOH, pH 11.1; and milli-Q H
2
O
(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 spectrophotom-
eter (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.
pH-Dependent Properties of Pyridoxine J. Phys. Chem. B, Vol. 110, No. 33, 2006 16775
intramolecular hydrogen bonding, in most cases, between the
C
3
and C
4
hydroxyl groups. The OH groups at C
4
and C
5
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 and C-N distances of 1.33-
1.34 . For the radical anion of pyridoxine (2b), the excess
electron will occupy an antibonding * orbital leading to
lengthened N
1
-C
2
, C
3
-C
4
, C
4
-C
5
, and C
4
-O
4
bonds and
shortened C
2
-C
3
and C
4
-C
4
bonds (prime denoting the
substituent). All changes are within 0.05 . Also, the hydrogen
bond between O
3
H - - O
4
increases, in this case by 0.1 . The
least affected bonds are those to C
6
. The structural changes are
also reflected in that the additional electron is primarily localized
to C
3
(q ) -0.87 e
-
), with a slight increase in negative charge
also on N
1
and the C
5
substituent (approximately -0.20 e
-
on
each), compared with the neutral system, whereas the unpaired
spin in the anion is found on the opposing N
1
(0.27 e) and C
4
(0.43 e), with minor components also on C
2
and C
5
.
In the cation 2c, ionization occurs primarily from a bonding
orbital of the aromatic ring, resulting in elongated C
2
-C
3
,
C
3
-C
4
, and C
5
-C
6
bonds (by 0.03-0.05 ) and in a decreased
C
4
-C
5
bond by the same amount. In addition, the C
3
-O
3
bond
is substantially shortened (0.06 ), as is the O
3
H - - O
4
intramolecular hydrogen bond (by 0.28 ) concomitant with
an increased O
3
-H bond length. The C-N
1
bonds are es-
sentially unaffected by the ionization. The increase in positive
charge occurs at C
4
and C
4
(q ) +1.2 and +0.7 e
-
,
respectively), in part compensated for by drastically increased
negative charges on C
3
and C
5
(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
C
2
and C
6
, and small contributions on C
3
and O
3
, 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 N
1
(2d)
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 N
1
-protonated species, these will not be considered
further. Protonation at N
1
results in increased positive charge
on most ring atoms except C
5
, which becomes 0.4 e
-
more
negative. The largest buildup of positive charge is seen on N
1
(q ) +0.5 e
-
). The geometric changes are within 0.02
throughout, except for the O
3
H - - O
4
hydrogen bond that
becomes 0.16 shorter.
In the case of high pH, systems with one proton fully removed
from either OH group were considered. Deprotonation of either
O
4
or O
5
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 . When the ring-bound
hydroxyl group is deprotonated (2e), we also see a 0.1 shorter
C
3
-O
3
bond, and a highly localized negative charge on O
3
(q ) -0.7 e
-
). In this case, C
3
also attains a more negative
charge, only partly balanced by an increased positive charge
on C
4
. For this system, elongated C
2
-C
3
and C
3
-C
4
bonds are
observed, along with a shift in intramolecular hydrogen bond
to O
4
H - - O
5
.
TABLE 1: Calculated Energies at the (IEFPCM)-B3LYP/6-311+G(d,p) Level
a
compound E(aq)
b
ZPE Gcorr
298
G(aq)
298 c
G(aq)
298
B6 (2a) -592.090 321 0.186 894 0.149 153 -591.941 168 0
B6
-
(2b) -592.152 893 0.181 676 0.142 716 -592.010 177 -43.30
B6
+
(2c) -591.867 145 0.184 226 0.144 511 -591.722 634 137.13
B6
+ -
(2f) -592.081 515 0.186 037 0.147 038 -591.934 477 4.20
B6-N1H
+
(2d) -592.546 098 0.200 081 0.162 301 -592.383 797 0
B6-O3H
+ d
n/a n/a
B6-O4H
+
-592.509 915 0.197 361 0.159 555 -592.350 360 20.98
B6-O5H
+
-592.508 098 0.197 473 0.160 190 -592.347 908 22.52
B6-O3
-
(2e) -591.618 107 0.172 992 0.135 772 -591.482 335 0
B6-O4
-
-591.605 374 0.170 750 0.133 615 -591.471 759 6.64
B6-O5
-
-591.586 313 0.169 965 0.131 638 -591.454 675 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) + Gcorr
298
, where the latter is the thermal correction to the free energy at 298 K.
d
Rearranges into B6-O4H
+
.
TABLE 2: Experimental UV Absorptions (nm) of Pyridoxine at Different pH Values
a
pH 1.7 pH 4.3 pH 7.0 pH 11.1
324.5 (0.360) w 323.5 (1.433) s 314.5 (1.030) s
290.1 (1.714) s 291.0 (1.406) s 290-300 (0.40) w sh
230 (0.50) sh 225 (1.100) w sh 253.9 (0.748) i 245.0 (0.840) i-s
207.5 (2.754) s 200-220 (>2.0) s 210-225 (3.175) 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.
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.
16776 J. Phys. Chem. B, Vol. 110, No. 33, 2006 Ristila et al.
In addition to the above systems, the neutral zwitterion of
pyridoxine, with N
1
protonated and O
3
deprotonated, was also
investigated (2f). In this case, the largest geometric effects occur
around C
3
(shorter C
3
-O
3
bond and longer C
3
-C bonds), and
in slightly increased N
1
-C bonds. Large charge redistributions
occur in the zwitterion, compared with the neutral ground state
2a, with the main positive centers now being N
1
, C
4
, C
4
, and
C
6
(charge increase q ) +0.50, +1.09, +0.20, and +0.44
e
-
, respectively), whereas the largest charge reductions are found
at C
3
, C
5
, C
2
, and O
3
(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 B
6
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 O
3
deprotonated and N
1
protonated, is only 4 kcal/mol less stable
than the neutral ground state form 2a, in close agreement with
previous work on 3-hydroxypyridine
21
and pyridoxal.
22
As
mentioned above, the different oxygen-protonated species are
all at least 20 kcal/mol less stable than the N
1
-protonated form.
It is thus reasonable to assume that these compounds will not
be formed under normal physiological conditions, and very
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 (N
1
) 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 O
3
. Interestingly, the O
4
depro-
tonated form is only some 6.6 kcal/mol less stable and may
contribute to a minor extent. Deprotonation at O
5
, on the other
hand, appears less likely. The summed free energy of the O
3
-
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 H fL (0.64) 0.202
242.5 H - 2 fL (0.64) 0.023
222.0 H - 3 fL (0.46), H fL + 1 (0.48) 0.070
208.4 H - 1 fL + 1 (0.62) 0.066
205.1 H -3 fL (0.43), H - 2 fL + 1(0.29),
H fL + 1 (-0.31)
0.217
neutral (2a)
264.3 H fL (0.63) 0.148
253.9 H - 1 fL (0.67) 0.013
226.6 H - 1 fL (0.43), H fL + 1 (0.44) 0.011
211.5 H - 3 fL (0.52) 0.088
203.4 H - 2 fL (0.30), H - 3 fL (0.39),
H - 4 fL (-0.34)
0.265
neutral zwitterion (2f)
342.9 H fL (0.63) 0.198
255.1 H fL + 1 (0.54), H fL + 2 (-0.32) 0.069
250.8 H fL + 2 (0.60) 0.034
228.7 H - 3 fL (0.51), H - 4 fL (0.36) 0.110
223.9 H fL + 4 (0.61) 0.017
221.0 H fL + 5 (0.68) 0.015
211.0 H - 4 fL (0.52) 0.240
O
3-deprotonated (2e)
298.6 H fL (0.65) 0.169
271.0 H fL + 1 (0.57), H fL + 2 (0.40) 0.010
250.7 H fL + 1 (-0.34), H fL + 2 (0.53) 0.087
240.9 H fL + 4 (0.69) 0.014
236.9 H fL + 3 (0.66) 0.083
217.7 H - 2 fL (0.30), H - 3 fL (0.58) 0.019
214.2 H fL + 8 (0.68) 0.017
210.0 H - 4 fL (0.54), H - 1 fL (-0.35) 0.045
207.1 H - 1 fL + 4 (0.49), H - 1 fL + 3 (-0.37) 0.089
202.6 H - 2 fL (0.32), H fL + 10 (-0.34) 0.229
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.
Figure 4. Computed absorption spectra (nm) of the N1-protonated
(solid), neutral (dashed), zwitterionic (dot-dashed), and O3-depro-
tonated (dotted) pyridoxine moieties. Each individual spectrum is
renormalized.
Figure 5. (a) Experimental UV spectrum (nm) at pH 1.7 (solid) and
computed spectrum for N1-protonated species (dashed). (b) Experi-
mental UV spectrum at pH 11.1 (solid) and computed spectrum for
O
3-deprotonated species (dashed).
pH-Dependent Properties of Pyridoxine J. Phys. Chem. B, Vol. 110, No. 33, 2006 16777
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 pK
a
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 C
4
/C
5
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 N
1
/O
3
-protonated species, pH 4.3 will
represent a mixture of the neutral and N
1
-protonated compounds,
pH 7.0 will refer to the neutral O
3
hydroxyl and/or zwitterionic
oxo species (or a mixture of neutral, N
1
-protonated, and O
3
-
deprotonated compounds), and pH 11.1 mainly will involve the
N
1
/O
3
-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 N
1
-protonated
form, and at high pH the O
3
-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 10-
15 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 N
1
-protonated species
2d, the two neutral systems 2a and 2f, and the O
3
-deprotonated
form (2e). The energetically close-lying O
4
-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
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.
16778 J. Phys. Chem. B, Vol. 110, No. 33, 2006 Ristila et al.
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
N
1
-protonated species, and in Figure 5b we display the spectrum
at pH 11.1 along with the O
3
-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 N
1
-
protonated 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
N
1
-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 N
1
and O
3
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 param-
eters altering less than 0.01 were not included in Figure 6)
and highly localized to O
3
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 N
1
-H bond
increases the most, from the influence of the chloride anion,
by 0.1 . In the neutral hydroxyl system, the chloride anion
moves over from the N
1
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
pyridoxine species are influenced to a relatively small extent
by the presence of the counterions. The largest changes are seen
for the O
3
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 ap-
proximately 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.
Figure 7. (a) Experimental UV spectrum (nm) at pH 7.0 (solid line)
together with computed spectra for neutral (dot-dashed) and zwitter-
ionic (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.
pH-Dependent Properties of Pyridoxine J. Phys. Chem. B, Vol. 110, No. 33, 2006 16779
IV. Conclusions
The possible protonation states of pyridoxine (vitamin B
6
)
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 N
1
renders the most stable
conformer among the protonated species, and deprotonation will
primarily occur at the ring-bound O
3
. Protonation is energeti-
cally 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/TD-
B3LYP/6-311+G(d,p) level and compared with measured UV
spectra, reveal a direct correlation between the N
1
-protonated
species at low pH and the O
3
-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 Ultra-
structure 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 Linkoping (NSC) and Stockholm (PDC) are also
gratefully acknowledged.
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