Titanium(IV) and vitamin C: Aqueous complexes of a bioactive form of Ti(IV)

Article abstract of DOI:10.1021/ic301545m

Ascorbic acid is among the biorelevant ligands that render Ti(IV) stable in aqueous solution. A series of pH-dependent titanium(IV) coordination complexes of l-ascorbic acid is described. Directed by spectropotentiometric methods, important aspects of the aqueous interactions in this system are investigated, including ligand binding mode, pH-dependent metal-ligand stoichiometry, and the importance of metal ion-promoted hydrolysis and the binding of hydroxide. Stability constants are determined for all metal ion-ligand-proton complexes by a process of model optimization and nonlinear least-squares fitting of the combined spectropotentiometric titration data to the log β_MLH values in the model. A speciation diagram is generated from the set of stability constants described in the model. In the range pH 3-10, the aqueous speciation is characterized by the sequential appearance of the following complexes as a function of added base: Ti(asc)₂⁰ → Ti(asc) ₃^2- → Ti(asc)₂(OH)₂ ^2- → Ti(asc)(OH)₄^2-. These species dominate the speciation at pH < 3, pH 4-5, pH ~ 8, and pH > 10, respectively, with minimum log stability constants (β values) of 25.70, 36.91, 16.43, and -6.91. Results from electrospray mass spectrometry, metal-ligand binding experiments, and kinetics measurements support the speciation, which is characterized by bidentate chelation of the ascorbate dianion to the titanium(IV) ion via proton displacement, and a pH-dependent metal-ligand binding motif of ligand addition followed by metal ion-promoted hydrolysis, stepwise ligand dissociation, and the concomitant binding of hydroxide ion. Additionally, the kinetics of ligand exchange of titanium ascorbate with citrate are reported to understand better the possible fate of titanium ascorbate under biologically relevant conditions.

Full text of DOI:10.1021/ic301545m

Article
pubs.acs.org/IC
Titanium(IV) and Vitamin C: Aqueous Complexes of a Bioactive Form
of Ti(IV)
†
†
,‡
Katherine M. Buettner, Joseph M. Collins, and Ann M. Valentine*
†
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, Connecticut 06520-8107, United States
Department of Chemistry, Temple University, 1901 North 13th Street, Philadelphia, Pennsylvania 19122, United States
‡
*
S Supporting Information
ABSTRACT: Ascorbic acid is among the biorelevant ligands that render Ti(IV)
stable in aqueous solution. A series of pH-dependent titanium(IV) coordination
complexes of L-ascorbic acid is described. Directed by spectropotentiometric
methods, important aspects of the aqueous interactions in this system are
investigated, including ligand binding mode, pH-dependent metal−ligand
stoichiometry, and the importance of metal ion-promoted hydrolysis and the
binding of hydroxide. Stability constants are determined for all metal ion−ligand−
proton complexes by a process of model optimization and nonlinear least-squares
fitting of the combined spectropotentiometric titration data to the log βMLH values in the model. A speciation diagram is
generated from the set of stability constants described in the model. In the range pH 3−10, the aqueous speciation is
0
2‑
characterized by the sequential appearance of the following complexes as a function of added base: Ti(asc) → Ti(asc)
→
2
3
2
‑
2‑
Ti(asc) (OH)2 → Ti(asc)(OH) . These species dominate the speciation at pH < 3, pH 4−5, pH ∼ 8, and pH > 10,
2
4
respectively, with minimum log stability constants (β values) of 25.70, 36.91, 16.43, and −6.91. Results from electrospray mass
spectrometry, metal−ligand binding experiments, and kinetics measurements support the speciation, which is characterized by
bidentate chelation of the ascorbate dianion to the titanium(IV) ion via proton displacement, and a pH-dependent metal−ligand
binding motif of ligand addition followed by metal ion-promoted hydrolysis, stepwise ligand dissociation, and the concomitant
binding of hydroxide ion. Additionally, the kinetics of ligand exchange of titanium ascorbate with citrate are reported to
understand better the possible fate of titanium ascorbate under biologically relevant conditions.
INTRODUCTION
is invoked, characterized by monodentate or bidentate
coordination via the oxygen atoms on C2 or C3, with the
ligand singly or doubly deprotonated. Crystal structures are
available for metal ascorbate complexes of the soft ions
■
L-Ascorbic acid is an important molecule in both chemistry and
biology, and its complexes with metals are of particular interest
in both of these areas.
the ligand has multiple protonation and oxidation states (Figure
), because the metal−ligand complexes are in general not as
tight as one might expect for an ene-diol ligand, and because
1
−3
Their study is complicated because
2
3,4
thallium(I) and platinum(II),
coordination modes. A multinuclear mixed-valent Fe(II)Fe-
which employ various
5,6
1
(
III) complex with the related tetramethyl reductic acid ligand
2
7
was characterized by X-ray crystallography and in solution.
One study reported bidentate binding on titanium dioxide
surfaces through binding mode 6, supported by IR, EXAFS, and
the complexes have mostly defied crystallographic character-
1
ization. In most cases, coordination via one of the modes 4−6
8
XANES data.
Complexes of ascorbic acid with titanium(IV) should be
10
relatively strong, and not prone to the metal-catalyzed ligand
oxidation that renders many metal ascorbate complexes so
reactive. Accordingly, complexes of ascorbic acid with titanium,
11
first reported in 1936, have been used for detection and
1
2
quantitation of Ti(IV). Early work was reviewed by
13
Sommer, who combined spectroelectrochemistry and electro-
phoresis to address the pH-dependent speciation of titanium
ascorbate. The data supported the predominant species
Ti(Hasc)3+ (pH < 1), TiO(Hasc) (1 < pH < 1.6), TiO(Hasc)
+
2
2
‑
Figure 1. L-Ascorbic acid (H asc, 1), doubly deprotonated ascorbic
(1.6 < pH < 2.2), and Ti(asc)3 (2.5 < pH < 4.8). The latter
species, the one used for metal quantitation, was characterized
2
2
‑
9
12
acid (asc , 2) (pK of C3-OH = 4.04 and pK of C2-OH = 11.34),
a
a
dehydroascorbic acid (3), and selected metal−ligand binding modes,
including monodentate (mono)protonated (4), bidentate (mono)-
protonated (5), and bidentate deprotonated (6).
Received: July 16, 2012
Published: September 27, 2012
©
2012 American Chemical Society
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Inorganic Chemistry
Article
−
1
−1
terminal water ligands on the order of 3400 s−1 at 25 °C.19
by a broad absorption at λmax = 360 nm, ε = 6800 M cm .
The extinction coefficient was determined at a ligand/metal
ratio of nearly 2000:1, and the ligand absorption tails into this
Titanium ascorbate injected into human blood serum at pH 7.4,
for example, for an imaging application, where the ascorbate
1
3
47,48
region; it represents an upper limit. Minority species
concentration is on the order of 50 μM,
should undergo
TiO(Hasc)22 and Ti(asc) were also detected. Above pH 4.8,
‑
ligand exchange reactions with citrate, which is abundant in
2
48
the color disappeared, and hydroxo complexes were
suspected. This quantitative work agreed well with the
original qualitative titrations. Another study invoked TiO-
Hasc) (λ = 355 nm, ε = 7800 M cm ) and a second,
blood serum at 100 μM and makes quite stable titanium(IV)
1
3
49
complexes. Likewise, titanium ascorbate formulated near pH
11
5 and sprayed onto plants or fed to animals as a growth
promoter might encounter citrate or similar α-hydroxy acid
competitor ligands. It remains to be evaluated how the rates of
these exchanges, involving presumably bidentate ligands, might
compare with the benchmark value for water exchange.
In light of the demonstrated bioactivity of titanium ascorbate,
the current work revisits these complexes, to further explore
this system. This work enumerates the important complexes
and equilibria that define the aqueous speciation, particularly in
the most bioactive formulations. The ascorbate ligand binding
mode is addressed. Finally, ligand exchange reactions are
studied to understand the kinetics of such exchange under
biorelevant conditions.
+
−1
−1
(
max
anionic complex with λmax = 375−385 nm that formed at lower
14
ascorbate concentration and higher pH. Complexes formu-
−
1
−1
lated as TiO(Hasc) (λ = 364.4 nm, ε = 1202 M cm )
2
max
and TiO(OH)(Hasc) (λmax = 344.8 nm, ε = 692 M− cm )
1
−1
were prepared and characterized by elemental analysis and IR
1
15,16
and H NMR spectroscopies.
The former complex is one
predicted by Sommer to occur at low pH; the latter, a
presumed hydrolysis product prepared by methanol extraction
of the former, was not one of the species thought to
13
predominate in aqueous solution. The IR spectra were not
−1
16
reported below 1500 cm , so the presence or absence of a
TiO stretch could not be ascertained. A complex formulated
as K Ti(asc) was isolated from EtOH and characterized by
2
3
EXPERIMENTAL SECTION
■
elemental analysis, but the ligand dianion was thought to be too
17
Reagents and Chemicals. All aqueous solutions were prepared in
Nanopure-quality water (18.2 MΩ cm resistivity; Barnstead), and all
chemicals were ACS grade or better. Stock solutions of 1.0 M
potassium chloride (KCl, J.T. Baker) and 0.2000 M potassium
hydrogen phthalate (KHP, ACS primary standard, Mallinckrodt) were
prepared in volumetric flasks by weighing out an appropriate amount
of the salt, dissolving in water, and diluting to volume. Solutions of 0.1
M hydrochloric acid (HCl, 1 N volumetric solution, J.T. Baker) and
0.2 M potassium hydroxide (KOH, 1 N carbonate-free Dilut-It
Analytical Concentrate, J.T. Baker) were diluted from their respective
stock solutions volumetrically, and their concentrations were
determined accurately (4 significant figures) by titrating the base
against KHP, and the acid against the base. Phenolphthalein (1% in
basic to form in aqueous solution.
2
+
The titanyl ion, TiO (aq), is often invoked in the aqueous
coordination chemistry of Ti(IV), though the importance of
discrete, monomeric TiO units in solution has been
challenged. One study used O NMR spectroscopy to
reinvestigate the issue of the aqueous titanyl ion. The “yl”
oxygen of TiO (aq) exchanged 9 orders of magnitude faster
than the “yl” oxygen of VO (aq). This finding led to the
conclusion that, at low pH and in the absence of any stabilizing
ligand, a rapid equilibrium exists among TiO (aq), Ti-
2
+
1
8
17
1
9
2+
2
+
2
+
+
4+
(
OH)22 (aq), and Ti (aq).
The bioactivity of titanium(IV) ascorbate is well established,
but in general the complex is prepared in situ and not
characterized. In one early use of titanium ascorbate in
medicine, a solution was used to treat tumors in humans.
9
5% alcohol, Mallinckrodt) was used as an end point indicator, and the
analyses were done in triplicate. All solutions were prepared fresh, and
were stored under an inert gas (nitrogen or argon) in parafilm-
wrapped Nalgene bottles.
20
4
5
21
For the spectropotentiometric titrations, the ascorbate concen-
tration was constant and the Ti(IV) concentration was varied. An
accurately weighed amount (70.45 mg, 0.400 mmol) of L-ascorbic acid
crystals (99+ %, Sigma-Aldrich) was added to an argon-purged and
foil-wrapped 100 mL volumetric flask containing an aqueous solution
of 0.1 M KCl (aq). The titanium(IV) source for all experiments was a
Ti ascorbate has been investigated as a radiopharmaceutical.
Metal delivered as the complex by injection into rats bound to
albumin in plasma and accumulated in the spleen, liver, and
blood. 45Ti concentrated in and was used to image a glioma in
the brain of a rat. More recently, ascorbic acid has been
shown to increase the uptake of titanium in rat everted gut
Pais described the use of titanium(IV) ascorbate
as a water-soluble and relatively stable compound capable of
stimulating the growth of plants. Several reviews have covered
21
neat solution of TiCl (l) (99.9%, with SureSeal cap, Sigma-Aldrich)
4
2
2
sacs. Istvan
́
which is highly reactive with water and water vapor and was added to
the titration solutions by using a gastight glass syringe (Hamilton Co.).
Following addition of metal ion (2−15 μL, 30−170 μmol), a 50.0-mL
aliquot of this solution was transferred to the titration vessel by using a
volumetric pipet. For other applications, a 0.1000 M stock solution of
L-ascorbic acid was prepared volumetrically from the acid crystals and
stored under inert gas; additionally, this solution was stored in a foil-
wrapped bottle to prevent photo-oxidation. The Ti(IV) concentration
was determined from the remaining 50-mL titration solution by flame
atomic absorption analysis (SpectrAA-20 flame atomic absorption
spectrometer, Varian) by using the method of internal standards.
For kinetic experiments, stock solutions of 10 mM titanium
ascorbate were made by dissolving ascorbic acid crystals (0.35 g, 2.0
23
24−27
the research in this area.
The generally beneficial effects of
titanium solutions on various plants, including peppers,
2
8−30
3
1,32
33
34,35
36,37
wheat,
oats, apples,
and plums,
have been an
important focus. Titanium ascorbate was patented as a growth
promoter for plants, animals, or both. Other patents cover
38
39
40
41
42
its preparation as a solid or for aquaculture applications or
4
3−46
as a component of a complex fertilizer mixture.
The
formulation applied to organisms is not always described in
great detail, but it often comprises titanium(IV) with at least a
0-fold excess of ascorbate, with the pH adjusted to between 5
and 7. The earlier work on titanium ascorbate speciation does
not address the complex speciation under these conditions, but
instead focuses on lower pH values.
Once administered into a biological system, titanium
ascorbate complexes may undergo ligand exchange reactions.
Titanium(IV) is a labile metal ion, with exchange rates for
mmol) in 0.5 M NaCl. To this solution, neat TiCl (25 μL, 250 μmol)
2
4
was added, and allowed to mix for 1 h. The pH of the solution was
adjusted to either 7.4 or 4.5 by using NaOH. Individual samples were
diluted immediately before kinetics measurements to 125 μM by using
0
.5 M NaCl. A stock citric acid solution was made by dissolving citric
acid monohydrate (2.6 g, 12 mmol) in 0.5 M NaCl, and the pH was
adjusted to either 7.4 or 4.5 using NaOH. Further dilutions were made
from this solution to give the range of concentrations used.
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Inorganic Chemistry
Article
Spectropotentiometric Titrations. All titrations were conducted
4
9
as described previously, according to the general methods
5
0,51
described,
with minor modifications. The entire assembly was
foil-wrapped to prevent any photo-oxidation of the ligand, and kept
under positive pressure from argon to prevent oxygen contamination.
The total number of equilibrated titration points for which data were
collected was between 70 and 90. The fitting software was Specfit/
52−56
32.
Full details of the experimental procedure are given in the
Supporting Information.
Metal−Ligand Binding Stoichiometry. Ligand-to-metal stoi-
chiometry at pH 4.5 was determined independently by using an
aqueous mole ratio method, probed by UV−vis spectroscopy.
Repeated volumes of TiCl (l) were added to a solution of 4.00
4
mM H asc in 0.1 M KCl under the same conditions discussed above
2
for the titrations. The pH was readjusted to 4.5 for each successive
solution by adding acid or base, and total solution volume was
monitored. Following each equilibration, a 1.0 mL aliquot was
removed, and its absorbance was measured. After data collection, the
remaining solution was used for titanium concentration analysis. For
each point, an L/M molar ratio value was determined, and the
corresponding absorbance at 366 nm, corrected for dilution and
reported as an observed molar (Ti(IV)) absorptivity, was calculated.
Mass Spectrometry. Electrospray mass spectra were collected in
positive ion mode on a Waters/Micromass ZQ spectrometer. The
pertinent voltages were as follows: capillary, 3 kV; cone, 20 V;
extractor, 3 V; RF lens, 0.3 V. The aqueous samples were injected
directly, and molecular ions were detected in a Waters 2996
Photodiode Array Detector.
Kinetics. Citric acid solutions were prepared in 0.5 M NaCl at
concentrations ranging from 3.75 to 250 mM to ensure pseudo-first-
order kinetics. The final solutions contained 62.5 μM titanium
ascorbate and between 1.88 and 125 mM citrate.
Kinetics measurements were made by using a Cary 50 Bio UV−vis
spectrophotometer. The absorbance was measured every 6 s between
Figure 2. (A) Potentiometric titration curves, as a function of molar
equivalents of base added, for 4.00 mM L-ascorbic acid in the presence
of various concentrations of Ti(IV). (B) Ligand deprotonation (ZL)
curves, as a function of pH, for 4.00 mM L-ascorbic acid in the
presence of various concentrations of Ti(IV). In both panels, data are
as follows: no symbol, ascorbic acid alone with no titanium; open
circles (○), [Ti(IV)] = 0.297 mM (L/M = 13.5); filled circles (●),
300 and 600 nm, or every 0.6 s at 370 nm for single wavelength
measurements. Longer time scale experiments were performed over 20
h to confirm complete conversion to the citrate species. These
measurements were made every 6 s for the first 2 min, every 30 s for
the next 8 min, and then every 10 min for the remainder of the 20 h.
The pH was unbuffered except by the solution components, but was
constant to within ±0.1 pH unit over the course of the reaction.
[
1
(
Ti(IV)] = 0.530 mM (L/M = 7.5); open triangles (Δ), [Ti(IV)] =
.058 mM (L/M = 3.8); filled triangles (▲), [Ti(IV)] = 1.665 mM
L/M = 2.4). For clarity, in each case, only every fifth data point is
symbolized. Dashed lines represent equivalents of acidic ligand
protons. All titrations were conducted at 25 °C, in 0.1 M KCl, and
under an argon atmosphere.
53−56
Data were fit by using Specfit/32.
Data at pH 7.4 were best fit
to a sequential two step model. Data at pH 4.5 were best fit to a
sequential three step model. All data were collected at least in
triplicate; error bars represent the standard deviations of all trials.
the basis of both its position relative to the titration of ligand
alone and the steepness of the inflection region. For titrations
involving a diprotic acid such as L-ascorbic acid, having pKa
values that widely straddle the isopotential pH of water (pH
6.90 in the presence of 0.1 M supporting electrolyte and at 25
°C) are particularly useful, because the region of inflection is
isolated from the two ligand deprotonations. Furthermore, in
the absence of metal-ion promoted hydrolysis and subsequent
hydroxide binding by the metal ion, the inflection region will be
as steep as that seen for the titration of ligand alone.
Exactly 1 molar equivalent of base was needed to reach the
inflection point in the titration of ligand alone (Figure 1A, no
symbols). As metal ion was introduced in subsequent titrations
at constant ligand concentration, the position of the inflection
was shifted along the x-axis, reflecting the extra base needed to
consume the additional protons in solution that resulted solely
from the presence of metal ion. A comparison was made
between experimental and predicted inflection point values,
with the former determined from the graph and the latter
calculated on the assumption that the metal ion was
coordinatively saturated by anionic oxygen donors originating
from water or ascorbate. Because the calculated values
accounted for the 1 equiv of ligand proton consumed by
added base, but did not differentiate between a proton
RESULTS AND DISCUSSION
■
L-Ascorbic Acid: Protonation States and Oxidation.
The spectropotentiometry of ascorbic acid in the absence of
metal was performed, in some cases with deliberate ligand
oxidation (Figures S1−S3), so that the latter could be
recognized and avoided. In the absence of oxidation, the
measured pK values and species spectroscopic properties
a
9
agreed with those reported in the literature.
Potentiometric Data Analysis. By examining the unfitted
potentiometric data for a series of spectropotentiometric
titrations varying only in ligand-to-metal (L/M) molar ratio,
several important points relating to the aqueous interactions of
Ti(IV) with L-ascorbic acid were established. The raw
potentiometric data can be presented as plots of pH as a
function of equivalents of base added to a given amount of
ligand in the presence of varying amounts of metal ion, or as
ligand deprotonation curves showing the pH-dependent
removal of acidic ligand protons for the same data (Figure
2
). In the former case the most important observations
concerned the inflection point for the various titrations. An
inflection point in a metal ion−ligand titration is informative on
1
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Inorganic Chemistry
presumably the O(2) hydroxyl proton) of ascorbate versus
Article
+
= [H+]
− [H+]
(
[H ]
bound to L
total
free
(4)
water as the source of additional protons, the analysis was
limited by an inability to distinguish between species related to
and
[H ]
2‑
3
2‑
3
+
− [OH‐]
two limiting cases: Ti(Hasc) (OH) and Ti(asc) . This
3
= n[H L]
total
n
total
added
(5)
comparison can be described by the following equations:
In cases where the L/M ratio was greater than 3, the curves
thus generated (Figure 2B), when compared to the curve for
the ligand alone, were uniformly shifted to lower Z values as a
function of added Ti(IV), and were essentially parallel to the
curve for the ligand alone. This finding is an indication
primarily that their speciations are similar. This observation is
reinforced by comparison with the curve for the L/M 2.4
titration, which has a different overall shape from the others,
+
equiv H titrated to midpoint, experimental
mol base added to midpoint
L
=
mol ligand
(1)
(2)
+
equiv H titrated to midpoint, calculated
3
=
1 +
and which crosses into negative Z territory at pH ∼ 4. Both of
L/M molar ratio
L
these differences point to a situation where the lack of sufficient
ligand for tris-coordination of the metal ion becomes a crucial
factor in understanding the speciation of the system.
The experimental and calculated midpoint values for the three
curves (Figure 1A) describing titrations involving a minimum
of a 3-fold excess of ligand were in close agreement (exp/calc
for L/M 13.5, 7.5, 3.8 were 1.22/1.22, 1.44/1.40, 1.86/1.90,
respectively). While this finding supports the contention (see
below) that, in the region before inflection, and in the presence
of sufficient ligand for tris-coordination, the prominent metal−
To address the inability of the above plots to discriminate
conclusively between bidentate metal−ligand binding with
proton displacement and metal-promoted hydrolysis and
binding of hydroxide in mixed metal−ligand−hydroxo
complexes in the coordination sphere of Ti(IV), a further
graphical tool was employed. This tool, referred to as a Bjerrum
plot or n-plot, defines a function n as the average number of
ligands bound per metal ion. The analysis utilizes total ligand
and metal concentrations (Ltotal and Mtotal, respectively), base
concentration ([base]), initial solution volume (Vinitial), and the
conditional pK and pK values as known quantities, and can be
2
3
‑
ligand complex is the aqueous Ti(asc) species, it confirms
only that, in acidic aqueous solution, Ti(IV) complexation
involves octahedral coordination of anionic oxy-donors. The
titration at L/M ratio of 2.4, where insufficient ligand precluded
the complete tris-coordination of ascorbate by metal ion, gave
values not in agreement, with the experimental value
significantly higher than the calculated (exp/calc = 2.48/
51
w
a
expressed in terms of a diprotic acid ligand:
2.25). This finding indicates that, in the presence of insufficient
n(pH, vbase, mf
, mfHasc )
−
ascorbate for tris-coordination, coordinative saturation is
maintained, and metal-ion-promoted hydrolysis and binding
of hydroxide has occurred, with the loss of at least one bound
ligand.
The argument for metal-ion-promoted hydrolysis and
binding of hydroxide in titrations lacking sufficient ligand for
tris-coordination is supported by several further observations.
In the case of the L/M 2.4 titration, from the fact that the
inflection fell outside of the limit of titratable ligand protons
H asc
2
v
base·[base]
− 10−pH + 10pH−pK
2
Ltotal
−
w
Vinitial + vbase
Ltotal
−
2
·mfH2asc + mfHasc
−
=
Mtotal
(6)
The experimental quantities determined for each titration point
include the pH-dependent mole fractions for the protonation
states of the ligand (mfH2asc, mfHasc‑), the pH, and the volume of
titrant added (vbase). A derivation of the equation used to
generate these plots can be found in Supporting Information,
Derivation S1. The n-curves for a particular metal−ligand
system can be shown mathematically to coincide independent
of total metal and ligand concentration (and L/M ratio)
provided that the system of interest is characterized by a series
(
>2); it follows that because the sole source of protons in this
region is water, these titratable protons were necessarily
produced by hydrolytic binding of hydroxide by the metal
ion. Also, as mentioned above, for an aqueous titration in the
presence of supporting electrolyte and an acid ligand such as
ascorbate the inflection region should be both sharp and steep.
The presence of metal ion, however, alters this behavior. The
trace for the L/M 2.4 titration, for instance, and to a lesser
extent for the L/M 3.8 titration, exhibited some buffering in the
inflection region, which can be attributed to the hydrolytic
activity of the metal ion.
of ML−ML complexes. If, on the other hand, the aqueous
x
speciation is more complicated, including ternary metal−
hydroxo−ligand species and protonated metal−ligand species,
the assumptions contained in the n-plot algorithm are
invalidated, and the curves will be unique. Finally, because n
will likely have an upper limit of 3 for the Ti(IV)−ascorbate
coordination system, values exceeding this limit reflect, as noted
above, portions of the pH range for which simple ML−MLx
complexes are of minor importance.
51
̈
The Z plot, adapted from the work of Ohman, is another
L
convenient tool for visualizing the average number of protons
bound per ligand molecule as a function of pH, and, by
extension, for identifying regions where mixed ligand−hydroxo
complexes are formed, pure Ti(IV) hydroxo species having
For each experimental titration data pair (volume of base
added, pH) comprising a given titration, the n-value was
49
been shown, in previous work, to be insoluble above pH ∼ 3
2‑
generated and plotted against log[L ], which was independ-
ently derived from the speciation of the ligand alone at the
particular pH. The results are shown in Figure 3. Three of the
curves, all representing titrations with >3-fold ligand excess,
were very nearly identical, flattening at n ∼ 3 before curving
upward again above pH 7. The fourth curve, representing the
L/M 2.4 titration, was distinct from the other three, with
incomplete flattening at n ∼ 2.5. These observations are
at the millimolar concentrations used here. The function Z was
L
derived from the potentiometric data by using the following
equation:
+
H ]
Z (pH, added base) = [ [
bound to L
L
H L]
(3)
n
total
where
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Inorganic Chemistry
Article
Figure 3. Bjerrum plots, also referred to as n-plots, describing
equivalents of metal-bound asc2 as a function of log[asc ] (bottom
axis), with corresponding pH values (top axis), for titrations of 4.00
mM L-ascorbic acid in the presence of varying amounts of Ti(IV).
Symbols as follows: open circles (○), [Ti(IV)] = 0.297 mM (L/M =
‑
2‑
1
3.5); filled circles (●), [Ti(IV)] = 0.530 mM (L/M = 7.5); open
triangles (Δ), [Ti(IV)] = 1.058 mM (L/M = 3.8); filled triangles (▲),
Ti(IV)] = 1.665 mM (L/M = 2.4). For clarity, only every fifth data
[
point is symbolized.
evidence that (1) between pH 3 and 6, and in the presence of
at least a 3-fold excess of ligand, the aqueous speciation of
Ti(IV) ascorbate is characterized by the binding of a third
2‑
equivalent of asc ; (2) above pH ∼ 6 ternary metal−ligand−
hydroxo species begin to dominate, regardless of L/M ratio;
and (3) displacement of the O(2) proton leading to bidentate
2
‑
binding of asc is the characteristic aqueous binding mode of
the ligand.
Spectral Data Analysis. The UV−vis absorbance changes
associated with the titrations were examined independently. For
titrations spanning L/M 2−15, the pH-dependent trends were
similar, and reflected in several key ways the conclusions of the
potentiometric analysis.
Figure 4. Selected UV−vis absorbance spectra for the spectropo-
tentiometric titration of 4.00 mM L-ascorbic acid in the presence of
0
.297 mM Ti(IV) (L/M = 13.5). Ranges as follows: (A) pH 2.9−4.1,
(B) pH 4.1−4.8, (C) pH 4.8−5.8, (D) pH 5.8−8.8, (E) pH 8.8−10.0.
The pH-dependent UV−vis behavior for one titration (L/M
All traces were normalized against a baseline of 0.1 M KCl (25 °C).
For clarity, only six traces are shown per panel out of a total of 76
traces collected. Dashed line is at 366 nm. Dotted line is at 348 nm.
1
3.5) is shown in Figure 4. Overall, the data agree well with the
11
qualitative trends described by Ettori. From pH 3−4 (Figure
4
A), a steady increase in the absorbance at 366 nm was
Ti(asc)32 + 2H O → Ti(asc) (OH) + Hasc + H+
‐
2‐
‐
observed. This trend supports the conclusions of the
potentiometric data, specifically the n-plots, which supported
for this region the following binding event:
2
2
2
(8)
2
‐
Ti(asc) (OH) + 2H O
→
Ti(asc) 0 + Hasc‐ → Ti(asc)
2‐
+ H+
2
2
2
2
3
(7)
Ti(asc)(OH)42 + Hasc + H
‐
‐
+
(9)
The absorbance then remained unchanged from pH 4−5
Figure 4B), reflecting the stability of the putative tris-ligand
(
This qualitative description of the aqueous speciation of
Ti(IV) with L-ascorbic acid was further investigated by plotting
the observed 366 nm molar absorptivity profiles as a function of
pH for each of the L/M ratios considered (Figure S4). The
most striking observation was that, for the three titrations with
L/M > 3, a clear absorbance maximum was seen at pH 4.5. The
L/M 2.4 titration peaked slightly before pH 4.5 due to the
inability of the metal ion to coordinate fully a third ascorbate
ligand. Additionally, the apparent molar absorptivity at a given
pH increased as L/M increased. This correlation can be
explained by considering the effect of increasing L/M ratio on
any aqueous speciation. It can be seen that, as L/M increases,
complex in this region. In Figure 4C, encompassing the range
pH 5−6, the λ = 366 nm absorbance began to decrease slightly,
an indication of the onset of metal-promoted hydrolysis and
ligand loss. Finally, in the region pH 6−10 (Figure 4D,E), the
decrease was more dramatic, and was accompanied by a shift in
the LMCT absorbance to 348 nm. This result is significant
because it agrees with the λ proposed for a ternary Ti(IV)−
max
16
ascorbate−hydroxo species, and it supports the findings of the
n-plots regarding metal-promoted hydrolysis and replacement
of bound ligand by hydroxide. The binding events proposed for
this region can be summarized by the following equations:
1
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Inorganic Chemistry
Article
both of the following equilibria will be forced to the right in
response to the conditions (charges omitted for simplicity):
of L/M ratio. Looking at the values generated for L/M > 3, this
trend was linear for each particular species (Figure S9),
allowing for the reporting of stability constants with reference
to a singular L/M ratio. Because a minimum 10-fold ligand
excess is a common condition for studies of this type, and
because Figure S9 showed that asymptotic linearity began at L/
M ∼ 10, this ratio was chosen for reporting the log β values
characteristic of this system (Table 1). When applied to the
spectropotentiometric data, these results produced a speciation
that was consistent in all respects with the benchmark findings
used to direct the fitting process.
M + large excess L → ML2
M + large excess L → ML3
(10)
(11)
However, if the two equilibria are in direct competition over a
certain pH range, as they are in the speciation model being
constructed, the second equilibrium will be favored. By this
reasoning, the Ti(IV) tris-ascorbate complex will dominate the
region around pH 4.5 as L/M ratio increases by excluding the
neighboring species; this phenomenon is seen in the
experimental data as an increase in the observed absorptivity
of the species whose wavelength is being monitored. Finally,
the pH 4.5 absorbance profiles were investigated as a function
of L/M ratio (Figure S5). The results concurred with the above
discussion in that the effect of increasing L/M ratio from 1 to
The UV−vis absorbance characteristics were consistent with
those predicted from the various analyses of the spectral data,
and with the UV−vis characteristics of the species predicted by
16
Jabs et al., with binary metal−ligand and ternary metal−
ligand−hydroxo species exhibiting different λ
values and
max
extinction coefficients based on the numbers of bound ligand
12 changed the speciation, seen in the shift of the λmax from 348
and hydroxide (Table 2). Viewed graphically (Figure 5), these
to 366 nm, and in the increasing εobserved, without changing the
equilibrium model.
Table 2. Spectral Characteristics of Aqueous Complexes in
the Ti(IV) Ascorbate System Predicted for L/M = 10
pH-Dependent Speciation of Aqueous Titanium(IV)−
Ascorbate Complexes. The data were analyzed first by
model-free evolving factor analysis (EFA) (Figures S6 and S7).
Specific speciation models were proposed, on the basis of the
considerations outlined above (Figure S8) and complemented
by modeling of the data. Details of proposed models are
outlined in the Supporting Information. The optimized fit
returned the following species and stability constants (Table 1).
(
[H asc] = 4.00 mM) in 0.1 M KCl (25 °C, Argon
2
Atmosphere)
1
species
λmax (nm)
εmax (M− cm−1
)
Ti(asc) 0
370
365
348
362
243
266
299
1260
1660
1060
700
2
Ti(asc)32
‑
‑
2
Ti(asc) (OH)22
Ti(asc)(OH) 2
‑
4
H asc
9800
15 100
9700
Table 1. Minimum Stability Constants Determined for
2
−
Hasc
Aqueous Ti(IV)−Ascorbate−Hydroxo Species Using L/M =
asc2
‑
1
0 ([H asc] = 4.00 mM) in 0.1 M KCl (25 °C, Argon
2
Atmosphere)
equilibrium
⇆
Ti(asc)20
log βmin
Ti4 + 2asc
+
2‐
2‐
2
3
1
5.70
6.91
6.43
Ti4+ + 3asc ⇆ Ti(asc)3
2‐
Ti4 + 2asc + 2H O ⇆ Ti(asc) (OH) + 2H
+
2‐
2‐
+
2
2
2
2‐
Ti4 + asc + 4H O ⇆ Ti(asc)(OH) + 4H+
+
2‐
−
6.91
2
4
4
+
Because free Ti (aq) was not detected even at the lowest
pH values, these β values are best considered minimum values.
Their relative magnitudes are firm, however. An important
aspect of a series of stability constants generated for a particular
system is the confluence of these constants over a range of L/M
values. Assuming that the model is unchanged in the range of
conditions employed, the values optimized for each independ-
ent data set should produce the same speciation based on the
same log β values. This condition depends on the magnitude of
the stability constant(s), and the hydrolytic propensity of the
metal ion. For a strong complexation with a low propensity for
metal ion-promoted hydrolysis, the log β values will be virtually
identical regardless of L/M ratio. The opposite is true for values
optimized in this system, incorporating relatively weak binding
with a strong propensity toward hydrolysis (Table S1 and
Figure S9). The reasoning is a reflection of the relative
concentrations of metal ion and ligand with respect to pH,
wherein solutions with L/M < ∼50 will be differentially prone
Figure 5. (A) Speciation diagram and (B) corresponding absorbance
spectra for spectropotentiometric titration of 4.00 mM L-ascorbic acid
57
to ligand binding and hydrolysis. This effect was seen in the
log β values optimized for the Ti(IV) ascorbate system at
different L/M ratios (Table S1), for which slightly different
values were determined for each particular species as a function
in the presence of 0.297 mM Ti(IV) fitted to optimized L/M = 10 log
0
β values. Data as follows: open circles ( ), Ti(asc) (β120); filled
○
2
circles (●), Ti(asc)32 (β130); open triangles (Δ), Ti(asc) (OH)
‑
2‑
2
2
2
‑
(β12−2); filled triangles (▲), Ti(asc)(OH)4 (β ).
11−4
1
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Inorganic Chemistry
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results, applied to the L/M 13.5 titration data, produced a
speciation and absorbance paradigm that could be generalized
in form to all L/M ratios. Furthermore, the log β values
generated very similar speciation diagrams for all L/M > 3 data
sets, changing in a predictable way below this ratio for the
reason of insufficient ligand for tris-coordination (Figure S10).
These findings correspond well with the conclusions of the
present study, in that, for the pH range 2.5−10, and in the
presence of excess ligand (ascorbate), no evidence was found
for the titanyl ion. This result can be explained by considering
the lability of the “yl” oxygen, and the resulting equilibria
between the titanyl ion, the bis-hydroxo species, and the
aqueous metal ion. Specifically, as discussed below, the mos0t
acidic species described in this investigation, the Ti(asc)2
complex, was not anionic according to the potentiometric
Extrapolation of the linear portions of the data showed an
intersection at almost exactly L/M 3, with an extinction
coefficient in close agreement with that predicted from an
examination of the raw spectral data (1630 M− cm ).
Therefore, in addition to predicting the relatively weak binding
interaction between metal ion and ligand, this technique
supported a Ti(IV) tris-ascorbate species in the aqueous
speciation of the system. Additional experiments were
conducted at pH = ∼2, ∼6, and ∼9 (data not shown). The
asymptote at high L/M was not sufficiently horizontal to allow
quantitation, but qualitatively, the L/M ratio for binding in each
of these regions was clearly less than 3.
1
−1
Electrospray mass spectrometry (ESI-MS) offered an addi-
tional means of probing the formation of the tris-coordinated
species. Several processes, including ligand oxidation, degrada-
2
‑
and fitting results; a putative Ti(O)(asc)2 species was
therefore ruled out. Furthermore, the binding of a third
ascorbate ligand was not found to be accompanied by proton
tion, and protonation, and metal precipitation (as TiO ), were
2
difficult to avoid in the instrument despite the “soft” ionization
that is the hallmark of electrospray; these constraints limited
the pH range and L/M ratios under which results could be
obtained. At pH = ∼3 and L/M = ∼7.5, with low cone and
capillary voltages, however, a spectrum was detected in positive
2
‑
consumption, which would be the case if either Ti(O)(asc)
2
2
‑
(
aq) or Ti(OH) (asc) (aq) were in significant concentration
2 2
at this pH. On the contrary, the binding event in this pH region
was found to entail proton liberation from the ascorbate
monoanion. The combination of these characteristics led to the
conclusion that titanyl species are not prevalent in the pH
values surveyed.
+
ion mode depicting, based on m/z and isotope distribution, a
Ti(IV) tris-ascorbate species (Figure 7).
Metal−Ligand Stoichiometry. Several further analytical
procedures were undertaken to substantiate the existence of the
tris-coordinated titanium(IV) ascorbate species. In one experi-
ment a solution of ascorbic acid was maintained at pH 4.5 as
aliquots of TiCl (l) were added. After each addition, the pH of
4
the solution was readjusted to 4.5, and the solution was allowed
to re-equilibrate. UV−vis scans were then taken by using a
cuvette, and adjusted ligand and metal concentrations were
determined. The resulting L/M ratios were plotted against
observed molar absorbance at 366 nm, giving the plot shown in
Figure 6. The shape of the curve is indicative of a relatively
weak binding interaction, and points to the potential for
hydroxide to compete with ascorbate for metal binding.
Figure 7. Portion of an electrospray mass spectrum (positive ion
mode) for an aqueous solution of Ti(IV) ascorbate (L/M = ∼7.5, pH
=
∼3). Peaks centered on m/z = 573 are consistent with the
+
formulation H [Ti(asc) ] (C H O Ti) in terms of mass and
3
3
18 21 18
isotope distributions (dotted trace). Peak at m/z = 567 is similarly
+
consistent with the formulation K(H asc)3 (C H O K).
2
18 24 18
Rates of Ligand Exchange. The binding of Ti(IV) by
ascorbate is sufficiently strong to prevent hydrolytic precip-
itation, but weaker than binding by other common biological
1
0,49
ligands such as citrate.
Speciation models suggest that
Ti(IV) ascorbate complexes introduced to blood serum, for
58
example, would undergo ligand exchange with citrate. The
rapid exchange rates reported for Ti(IV) using water as a
−1
19
benchmark ligand (>1000 s ) suggest that this exchange
among biomolecules might be very fast. But other data, such as
the kinetics of delivery of Ti(IV) from citrate to the metal
Figure 6. Mole ratio method for ligand binding stoichiometry in the
aqueous Ti(IV) ascorbate system at pH 4.5, monitored by the
absorbance at 366 nm. Experiments were conducted at 25 °C and
59
transport protein transferrin, suggest that exchange kinetics
might be much slower, on the order of minutes or hours.
The competition for Ti(IV) binding between ascorbate and
citrate was studied under pseudo-first-order conditions of
excess citrate. Ligand exchange was probed by monitoring the
disappearance of absorbance due to the Ti(IV) ascorbate
under an argon atmosphere by adding aliquots of neat TiCl (l) to a
4
solution containing 4.00 mM L-ascorbic acid and 0.1 M KCl. The pH
4.5 ligand-alone absorbance trace was subtracted from each, and all
traces were corrected for dilution. The two solid lines intersect at L/M
−1
−1
=
3.009 and ε(366 nm) = 1632 M cm . Dashed line is at L/M = 3.
1
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Inorganic Chemistry
Article
complexes at 355 nm after citrate was introduced. Experiments
were carried out at the physiological serum pH of 7.4 and also
at pH 4.5, near the pH of formulation of the patented growth-
promoting complexes, where the tris-ascorbate species is
predicted to predominate. The dominant species predicted
match well with titanium species having two, one, and no
ascorbate ligands. At pH 4.5, a tris-ascorbate species dominates,
and the UV−vis spectra of the kinetic species match well with
titanium species having three, two, one, and no ascorbate
ligands. The rate constants at the two pH values are remarkably
similar: the first ascorbate ligand in the tris-ascorbate species
49
under these two conditions are
−1
exchanges at ∼0.2 s (t1/2 ∼ 3 s), the penultimate ascorbate at
2
‐
3‐
Ti(asc) (OH) + 3Hcit
→
−1
2
2
∼0.06−0.08 s (t ∼ 10 s), and the last ascorbate at ∼0.006
−1
1/2
Ti(cit)38 + 3Hasc (at pH 7.4)
‐
‐
s
(t ∼ 2 min). The last kinetic step does not exhibit a
(12)
1/2
concentration dependence on citrate, which is consistent with a
dissociative mechanism. At pH 4.5, for example, eq 14 is
consistent with the observed data.
Ti(asc)32 + 3H cit /H cit
‐
2‐
‐
2
3
4
‐
‐
→
Ti(Hcit) (H cit) + 3Hasc /H asc (at pH 4.5)
2
2
2
(13)
The data at pH 7.4 fit best to two sequential rate constants
Table S2). The first rate constant was dependent on citrate
(
concentration (Figure 8A) and saturable, yielding a dissociation
At pH 4.5, citrate exists as a mixture of predominantly the
2
‑
−
H cit (57%) and H cit (39%) forms, whereas ascorbate exists
2
3
‑
as Hasc (73%) and H asc (27%). These species provide
2
buffering for protonation changes during ligand exhanges. The
starting and ending species are those that predominate at pH
4
.5 under the solution conditions. The intermediate species are
highly speculative, but consistent with the data.
These data serve as a model of processes that may be
occurring in blood serum where the ligands studied in the
exchange are in relatively high concentrations. The K values
d
for the citrate-dependent steps are far higher than the
48
physiological serum citrate concentration of 0.1 mM, and
so this model predicts that in serum the conversion of titanium
ascorbate to titanium citrate complexes would occur on the
order of minutes or longer, and that the rates would depend
strongly on the citrate concentration. Physiological citrate
concentration varies in certain disease states such as epilepsy
6
0,61
Figure 8. Observed rate constants for the disappearance of the
and renal failure.
2
‑
2‑
Ti(Hasc) (OH)2 or Ti(asc)3 absorbance as a function of citrate
The time scale of this exchange is far slower than the rates
reported for monodentate ligands, the exchange of which
showed no concentration dependence on the incoming ligand,
2
concentration. Error bars represent standard deviations among at least
−
1
three trials. (A) Data at pH 7.4. (●) k : k = 0.081 ± 0.007 s , K =
1
max
d
−1
1
0 ± 4 mM (■) k = 0.0060 ± 0.009 s . (B) Data at pH 4.5. (●) k :
−1
2
1
and had forward rate constants ranging from k = 4 s to kobs
obs
−1
k
= 0.22 ± 0.02 s , K = 18 ± 5 mM (■) k : k = 0.055 ± 0.002
−1 19,62
max
d
2
max
>
100 s .
The faster rates are likely due to the difference in
−
1
−1
s , K = 5.4 ± 1.7 mM (⧫) k = 0.0056 ± 0.0008 s .
d
3
the nature of the ligands binding to the Ti center. In the current
case, ascorbate having lost one or both M−L bonds might
remain associated with the complex, rebinding rather than
exchanging for citrate.
constant of approximately 11 mM for the first detectable step of
the exchange. The second rate constant was not dependent on
citrate concentration. For experiments carried out at pH 4.5,
three steps were necessary to fit the data. The first two were
CONCLUSIONS
■
saturable, with apparent K values of 18 mM and 5.4 mM for k1
The aqueous speciation model determined for the Ti(IV)−
ascorbate complexation system under the reported conditions
gave considerable insight into several important facets of the
binding. The binding mode was suggested to feature bidentate
chelation, with the deprotonated oxy-groups at C2 and C3 as
the donors. This finding supports the stipulation that a hard
metal ion like Ti(IV) is capable of out-competing protons for
the binding of these hard donor atoms. Additionally, direct and
indirect evidence for a Ti(IV) tris-ascorbate species at pH =
∼4.5 was described. The question of metal-promoted
hydrolysis was investigated. The results indicated that, even
d
and k (Figure 8B). The third did not depend on citrate
2
concentration.
The observation that, at 7.4, two distinct rate constants were
detectable by UV−vis, whereas at pH 4.5, three steps were
detectable may be related to the titanium ascorbate speciation
at those pH values. For the former, a bis-ascorbate species
predominates, and the removal of each ascorbate may be
detectable in distinct steps. Consistent with this interpretation,
the absorbance change for each step is comparable, and the
UV−vis spectra of the kinetic species modeled by Specfit/32
1
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Inorganic Chemistry
Article
while coordinative saturation was maintained, binding of
hydroxide was indicated below physiological pH for this
metal ion. Titanium ascorbate complexes convert smoothly to
titanium citrate complexes under physiological concentrations
of the ligands in multistep processes, with exchange times on
the order of minutes. Taken together, the results reported here
delineate many thermodynamic and kinetic features of one of
the longest-known and most bioactive forms of Ti(IV).
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ASSOCIATED CONTENT
Supporting Information
(
19) Comba, P.; Merbach, A. Inorg. Chem. 1987, 26, 1315−1323.
■
*
S
(20) Arloing, F.; Morel, A.; Josserand, A. C. R. Hebd. Seances Acad.
Sci. 1937, 204, 824−825.
UV−vis spectra of ascorbic acid protonation states; potentio-
metric plots and UV−vis spectra depicting oxidation of ascorbic
acid; mathematical derivation of n-plot; graphical displays
showing effect of L/M ratio on λ(366 nm) absorbance as a
function of pH, and on pH 4.5 absorbance as a function of
wavelength; graphical representation of EFA smoothing;
comparison of EFA profiles at different L/M ratios; speciation
diagram comparison of 3- vs 4-species model; table and graph
comparing fitted stability constants as a function of L/M ratio;
comparison of speciation diagrams for different L/M ratios
using singular log β values. Full spectra kinetics and single
wavelength cuts across citrate concentrations for 4 min
exchange reactions. UV−vis absorbance of the full spectra for
overnight kinetics of exchange at pH 4.5. Specfit/32
determined species and speciation for both pH 7.4 and 4.5
kinetics data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Soil Sci. Plant Anal. 1977, 8, 407−410.
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(
1
09.
26) Pais, I.; Feher
991, 57−58, 675−680.
́
, M.; Bokori, J.; Nagy, B. Water, Air, Soil Pollut.
(27) Carvajal, M.; Alcaraz, C. F. J. Plant Nutr. 1998, 21, 655−664.
(28) Carvajal, M.; Gimenez, J. L.; Riquelme, F.; Alcaraz, C. F. Acta
Aliment. 1998, 27, 365−375.
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Carvajal, M. Acta Aliment. 2000, 29, 9−23.
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31) Lesko, K.; Stefanovits-Banyai, E.; Pais, I.; Simon-Sarkadi, L.
AUTHOR INFORMATION
Corresponding Author
E-mail: ann.valentine@temple.edu. Phone: 215-204-7836.
Notes
■
2
(
*
Novenytermeles 2001, 50, 71−81.
(32) Lesko, K.; Stefanovits-Banyai, E.; Pais, I.; Simon-Sarkadi, L. J.
Plant Nutr. 2002, 25, 2571−2581.
The authors declare no competing financial interest.
(
33) Hruby, M.; Cígler, P.; Kuzel, S. J. Plant Nutr. 2002, 25, 577−
ACKNOWLEDGMENTS
598.
■
(
(
34) Wojcik, P. J. Plant Nutr. 2002, 25, 1129−1138.
35) Wojcik, P.; Gubbuk, H.; Akgul, H.; Gunes, E.; Ucgun, K.; Kocal,
We thank the New England Division of the American Cancer
Society for a Research Scholar Award (Research Scholar Grant
RSG-06-246-01-CDD) to A.M.V., the donors of the Petroleum
Research Fund administered by the American Chemical
Society, and the Research Corporation’s Research Innovation
Award RI0961, for partial support of this research.
H.; Kucukyumuk, C. J. Plant Nutr. 2010, 33, 1914−1925.
36) Alcaraz-Lopez, C.; Botia, M.; Alcaraz, C. F.; Riquelme, F. J. Plant
Physiol. 2003, 160, 1441−1446.
37) Alcaraz-Lopez, C.; Botia, M.; Alcaraz, C. F.; Riquelme, F. J. Plant
Nutr. 2004, 27, 713−729.
38) Pais, I.; Feher, M.; Soptei, C.; Tomordi, E. Titanium Plant
(
(
(
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