Kinetics and mechanism of the oxidation of disaccharides by CrVI

Article abstract of DOI:10.1139/v02-187

The oxidation of D-lactose, D-maltose, D-melibiose, and D-cellobiose by Cr^VI yields the corresponding aldobionic acid and Cr³⁺ as final products when an excess of reducing disaccharide over Cr^VI is used. The rate law for t

Full text of DOI:10.1139/v02-187

1
676
Kinetics and mechanism of the oxidation of
VI
disaccharides by Cr
Viviana Roldán, Juan Carlos González, Mabel Santoro, Silvia García,
Nieves Casado, Silvina Olivera, Juan Carlos Boggio, Juan Manuel Salas-Peregrin,
Sandra Signorella, and Luis F. Sala
VI
Abstract: The oxidation of D-lactose, D-maltose, D-melibiose, and D-cellobiose by Cr yields the corresponding
3
+
VI
aldobionic acid and Cr as final products when an excess of reducing disaccharide over Cr is used. The rate law for
VI
VI
VI
the Cr oxidation reaction is expressed by –d[Cr ]/dt = k [disaccharide][Cr ], where the second-order kinetic con-
stant, k , depends on [H ]. The relative reactivity of the disaccharides with Cr is expressed as follows: Mel > Lac >
Cel > Mal, at 33°C. In acid medium, intermediate Cr forms and reacts with the substrate faster than Cr . The EPR
H
+
VI
H
V
VI
V
spectra show that five- and six-coordinate oxo-Cr intermediates are formed, with the disaccharide acting as bidentate
ligand. Five-coordinate oxo-CrV species are present at any [H ], whereas six-coordinate ones are observed only at pH < 2,
+
V
where they rapidly decompose to the redox products. In the pH 3–7 range, where hexa-coordinate oxo-Cr species are
not observed, CrV complexes are stable enough to remain in solution from several days to several months.
Key words: chromium, saccharides, kinetics, EPR.
Résumé : Lorsqu’on utilise un excès de du disaccharide réducteur par rapport au Cr(VI), l’oxydation du D-lactose, du
D-maltose, du D-melibiose et du D-cellobiose par le Cr(VI) conduit à la formation de l’acide aldobionique correspondant
et de Cr3 comme produits finaux. La constante de vitesse de la réaction pour la réaction d’oxydation par le Cr(VI)
+
peut être exprimée par l’équation –d[Cr(VI)]/dt = kH [disaccharide] [Cr(VI)], dans laquelle la constante cinétique du
+
deuxième ordre, k , dépend de [H ]; à 33°C, les réactivités relatives des disaccharides vis-à-vis du Cr(VI) sont les
H
suivantes: Mel > Lac > Cel > Mal. En milieu acide, il se forme du Cr(V) comme intermédiaire qui réagit avec le
substrat plus rapidement que le Cr(VI). Les spectres de RPE montrent qu’il se forme des intermédiaires penta- et
hexacoordinés d’oxo-Cr(V) dans lesquels le disaccharide agit comme ligand bidentate. Les espèces pentacoordinées
+
oxo-Cr(V) sont présentes à toutes les [H ] alors qu’on n’observe les espèces hexacoordinées qu’à des pH < 2 et que
dans ces conditions elles se décomposent rapidement en produits d’oxydoréduction. À des pH allant de 3 à 7 où on
n’observe pas de formation des espèces hexacoordinées d’oxo-Cr(V), les complexes de Cr(V) sont suffisamment stables
pour être observés en solution pour des périodes allant de plusieurs jours à plusieurs mois.
Mots clés : chrome, disaccharides, cinétique, RPE.
[
Traduit par la Rédaction] Roldán et al. 1686
Introduction
considerable amount of interest in their chemistry and
biochemistry (8–10).
Compounds of CrVI represent a potential environmental
hazard because of their mammalian carcinogenicity and tox-
icity (1, 2), and they are known to bioaccumulate in flora
and fauna, creating ecological problems (3–5). The observa-
The major coordination sites involved in Cr binding are
hydroxo, alcoholato, carboxylato, and thiolato donor groups
(2, 11–12). Five-membered, O-donor, chelate ligands, such
as 1,2-diols and 2-hydroxy acids, are effective as non-
enzymatic reductants (at low pH values) and chelates for
high oxidation states of Cr (13–19). Carbohydrates consti-
tute 5–20% (average 10%) of soil organic matter, and they
V
IV
tion of Cr and Cr intermediates in the selective oxidation
VI
of organic substrates by Cr and their implication in the
mechanism of Cr-induced cancers (1, 6–7) has generated a
Received 7 June 2002. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 20 December 2002.
V. Roldán. Departamento de Ciencias Básicas, Facultad de Ciencias Veterinarias, UNL, R.P. Kreder 735, Esperanza, Argentina.
1
2
J.C. González, M. Santoro, S. Garcia, S. Olivera, S. Signorella, and L.F. Sala. Departamento de Química, Facultad de
Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 531, 2000 Rosario, Argentina.
N. Casado. Instituto de Desarrollo Tecnológico para la Industria Química, Güemes 3450, 3000 Santa Fe, Argentina.
J.C. Boggio. Departamento de Farmacología y Toxicología, Facultad de Ciencias Veterinarias, UNL, R.P. Kreder 735, Esperanza,
Argentina.
J.M. Salas-Peregrin. Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071
Granada, España.
1
Corresponding author (e-mail: signorel@infovia.com.ar).
Corresponding author (e-mail: inquibir@satlink.com).
2
Can. J. Chem. 80: 1676–1686 (2002)
DOI: 10.1139/V02-187
© 2002 NRC Canada

Roldán et al.
1677
constitute more than 50% of dry matter in plants (20). For
this reason, it is interesting to examine the ability of saccha-
rides and their derivatives to reduce or stabilize high oxida-
tion states of Cr, to understand their potential roles in the
biochemistry of Cr.
VI
V
We are studying the possible fate of Cr and Cr in bio-
VI
logical systems by examining reactions of Cr with
low-molecular-weight molecules (21–37). Our studies on the
VI
V
reduction of Cr and the intermediate Cr by aldoses (21–
6), deoxyaldoses (27–28), sugar acids (31, 32, 35), and
2
methyl glycosides (29–30) showed that the relative redox re-
activities of these saccharides toward chromate are based on
the relative rates of the oxidation vs. complexation pro-
cesses. In view of some preliminary results obtained on the
VI
(Sigma grade), D-cellobiose (Pfanshtiehl 99.9%), potassium
dichromate (Cicarelli c.a.), potassium chromate (Aldrich
grade), glutathione (Sigma grade), acrylonitrile (Aldrich
grade), perchloric acid 70% (Merck P.A.), phosphoric acid
milk–Cr system suggesting the formation of long lived lac-
tose–CrV species generated by direct reaction of CrVI with
lactose (5), we decided to evaluate the kinetics of the reac-
VI
tion of Cr with four disaccharides: D-lactose (Lac),
(
Anedra P.A.), and sulfuric acid (Merck P.A.) were used
D-maltose (Mal), D-cellobiose (Cel), and D-melibiose (Mel),
to determine the influence of the glycoside moiety attached
without further purification. Water was purified by
deionisation, followed by double distillation from a potas-
sium permanganate solution.
6
4
to the C or C of glucose (glc) on the chelating and (or) re-
dox reactivity of the saccharide with high oxidation states of
chromium.
In experiments performed at constant ionic strength (I =
.0 M) and in different hydrogen ion concentrations, mix-
1
tures of sodium perchlorate solutions and perchloric acid so-
lutions were used. Sodium perchlorate solutions were
prepared from sodium hydroxide and perchloric acid solu-
tions. The concentration of stock solutions of perchloric acid
was determined by titration using standard analytical meth-
ods.
The stability of the organic substrate under conditions
used in the kinetic studies was tested by paper chromatogra-
phy and HPLC at given hydrogen ion concentrations.
Caution: Cr compounds are human carcinogens, and CrV
complexes are mutagenic and potential carcinogens (39).
Contact with skin and inhalation must be avoided.
Acrylonitrile is a carcinogen and must be handled in a well-
ventilated fume hood (40).
VI
Spectrophotometric measurements
Kinetic measurements were performed by monitoring
absorbance changes using a Jasco V-530 spectrophotometer
with a fully thermostated cell compartment (±0.2°C). The
reactions were followed under pseudo-first-order conditions,
VI
using an excess of disaccharide over Cr . Reactant solutions
Lac, the most important representative of the ꢀ-
galactosides, is found in the milk of all mammals up to a
concentration of approximately 5% and in some plants in
low concentrations. Mal is a product of enzymatic hydroly-
sis of starch; Mel is a constituent of the trisaccharide
raffinose; and Cel is the basic repeating unit of cellulose and
were previously thermostated and transferred into a 1-cm
pathlength cell immediately after mixing. Experiments were
performed at 33°C unless otherwise stated.
The disappearance of Cr was followed at 350 nm until
at least 80% of the Cr was consumed. In the kinetic mea-
VI
VI
VI
VI
lichetin (38). In this work, we study the Cr oxidation of
surements, the concentration of Cr was kept constant at
–
4
these four disaccharides and provide information on the
mechanism of oxidation as well as on the structure and sta-
8.0 × 10 M and the disaccharide concentration was varied
from 0.08 to 0.36 M. Mixtures of sodium perchlorate and
perchloric acid were used to maintain a constant ionic
strength (I) of 1.0 M.
V
bility of the Cr species formed as redox intermediates.
The observed pseudo-first-order rate constants (kobs), de-
Experimental
termined from the slopes of plots of ln(A350 – A ) vs. time,
8
Materials
were averages of at least three determinations and were
D-Lactose monohydrate (Anedra, RA-ACS grade), D-malt-
ose monohydrate (Sigma grade, 99%, less than 1.0% of
maltotriose and less than 0.3% of D-glucose), D-melibiose
within ±5% of each other. The first-order dependence of the
VI
rate upon [Cr ] was verified by calculating the k values at
obs
VI
–4
–4
various [Cr ] (4.0 × 10 – 8.0 × 10 M) at constant
0
©
2002 NRC Canada

1
678
Can. J. Chem. Vol. 80, 2002
Table 1. Observed pseudo-first-order rate constants (kobs)a for different concentrations of disaccharide in 0.2 M HClO4.
05 kobs (s ) for
–1
1
Lac
Mal
Cel
Mel
b
c
b
c
b
c
b
c
[
disaccharide] (M)
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
570
3
50
570
350
570
350
570
350
0
0
0
0
0
0
0
0
0
0
0
0
1
.02
.04
.06
.08
.12
.125
.16
.20
.24
.28
.32
—
—
—
7.35(9)
—
—
—
—
7.03(7)
—
1.30(3)
2.42(5)
3.09(1)
4.35(1)
—
6.16(3)
—
—
—
13.9(5)
—
1.28(3)
2.36(7)
2.98(7)
4.42(4)
—
6.07(5)
—
—
—
—
1.35(4)
3.34(1)
4.6(1)
6.36(6)
8.10(3)
—
—
—
6.77(6)
8.5(2)
—
12.1(2)
—
—
—
—
—
—
—
—
10.2(5)
—
—
—
—
—
—
—
19.0(1)
25.0(5)
28.6(6)
—
—
—
—
—
14.0(1)
16.5(1)
19.2(1)
24.2(2)
28.6(2)
31.4(1)
8.6(1)
13.9(6)
16.0(3)
19.4(2)
22.8(1)
27.0(3)
31.6(6)
5.2(1)
12.2(3)
15.4(2)
17.0(3)
20.7(5)
—
19.8(1)
25.0(2)
28.0(9)
—
38.5(6)
—
—
—
12.1(2)
38.8(2)
—
.36
—
7.4(1)
—
0 k (M–1 s )
4
–1
H
Note: T = 33°C; I = 1 M.
a
Mean values from multiple determinations.
b
c
VI
–4
[Cr ] = 8 × 10 M.
0
VI –3
[
Cr ] = 8 × 10 M.
0
temperature, initial disaccharide concentration, acidity, and
ionic strength.
drolysis to be characterized (44, 45), the redox reaction be-
tween Cr and the disaccharide is extremely slow at the
temperature used in this work and does not reach comple-
tion, even after 1 year. The reaction of chromate with sac-
charides is completed in shorter periods only when the
reaction is allowed to proceed under reflux (44, 45).
VI
III
VI
The formation of Cr was monitored at 570 nm ([Cr ] =
.0 × 10 M) in the presence of excess disaccharide (0.08 to
o
–
3
8
0
.36 M) in 0.20 M HClO . Under these conditions, the first-
4
VI
order dependence of the rate upon [Cr ] was verified and
kobs were obtained following the Cr growth at 570 nm. Initial
III
Chromate esters were investigated by UV–vis spectro-
photometry in the 350–400 nm region in which these esters
show characteristic absorption bands. Reactions were per-
formed at pH 4.7, where the redox reaction is slow enough
to enable the observation of the ester formation. The instru-
ments were zeroed to both the reference and sample beams
reaction rates were obtained by fitting the time-dependent
data for the absorbance at 570 nm to a polynomial expres-
sion (41) and calculating the slope of the tangent at time
zero (r ). First-order rate constants were calculated from
i
these initial rates using the equation –r /(A – A ), with aver-
i
o
f
VI
age values of the initial rate and calculated estimates of
A – A ). The results at 570 nm are in total agreement with
passing through matched cuvettes, both containing Cr in
(
H2O at pH 4.7. The solution in the sample cell was replaced
o
f
–
4
VI
those obtained at 350 nm (Table 1). At the end of the reac-
tion, the two d-d bands ascribed to Cr were observed at
ꢁ
1
with the reaction solution containing 8 × 10 M Cr and
0.12–0.32 M Lac at pH = 4.7, I = 1.0 M, and T = 33°C.
Spectra obtained within 30 min after mixing revealed a dis-
tinctive absorption band at 377 nm.
III
–
1
–1
= 410 nm (ꢂ = 18 M cm ) and ꢁ
= 570 nm (ꢂ =
max
max
–
1
–1
5 M cm ). Both visible and UV spectral maxima and in-
II
tensities are in close agreement with those observed for the
The formation of Cr was examined by periodic scan-
3
+
[
Cr(H O) ] ion. These bands are attributable to the octahe-
ning of solutions containing 0.21 M Lac and 0.016–
2
6
dral A J 4T and A J T transitions in O symme-
try, and are distinctive of the free Cr aqua complex (42).
However, in experiments performed at 20°C the reaction of
4
4
4
0.09 mM Cr in 0.1–0.2 M HClO in the presence of
VI
2
g
1g
2g
2g
h
4
III
II
dioxygen. Under aerobic conditions if Cr forms it rapidly
converts to CrO , which has characteristic absorption
2
ꢃ
2
VI
VI
–3
Cr with Lac (33:1 ratio, [Cr ] = 12.7 × 10 M) in
bands at 290 and 245 nm (33, 46) and can be detected at
VI
0
2
.2 M HClO affords two d-d bands at ꢁ
= 409 nm (ꢂ =
low [Cr ]. No new bands were detected at these wave-
4
max
–1
–
1
–1
–1
VI
6.8 M cm ) and 574 nm (ꢂ = 21.9 M cm ) with higher
lengths in the reaction of Cr with Lac. The presence of
3
+
II
absorbance than expected for [Cr(OH ) ] . These two bands
agree closely with those of the acid solutions of the Cr
Cr was also examined by carrying out experiments with
2
6
III
VI
2+
II
–
Cr and Lac in the presence of [Co(NH ) Cl] (a Cr
3 5
aldobionic acid complex, which was independently prepared.
The intensity of these d-d bands slowly decreases with time
toward the two bands with the molar absorption coefficients
trapping agent) under anaerobic conditions (8, 47). A solu-
tion containing 0.3 M Lac, 4 mM Cr , and 4 mM
VI
2
+
[Co(NH ) Cl] in 0.2 M HClO , I = 1 M and T = 33°C,
3
5
4
3
+
II
corresponding to the [Cr(H O) ] ion. This behaviour has
was left to react, and the Co content was analyzed after
10-fold dilution with concentrated HCl, centrifuging, and
measuring the absorbance at 692 nm (the wavelength ex-
pected for the absorption band of CoCl4 ) in the
supernatant. No Co was detected spectrophotometrically.
These results suggest that the formation of Cr in the reac-
tion of Cr with Lac can be ruled out.
2
6
III
been observed previously, i.e., when a Cr complex forms
3
+
as the final redox product and then hydrolyses to the Cr
ion (29, 31, 43, 44). The hydrolysis of the CrIII – aldobionic
2ꢄ
II
acid complex in 0.2 M HClO precludes its separation, and
4
II
consequently, its structural characterization. At pH > 2,
where these kind of complexes are stable enough toward hy-
VI
©
2002 NRC Canada

Roldán et al.
1679
Product analysis
HPLC was employed to detect the reaction products under
the conditions used in the kinetic measurements (excess
Fig. 1. Effect of [disaccharide] on kobs at 33°C and 0.20 M
HClO4. Inset: Effect of acidity on kH, for Lac.
VI
disaccharide over Cr ). The chromatograms were obtained
on a KNK-500A chromatograph provided with a 7125
HPLC pump. The effluent was monitored with refractive in-
dex (ERC-7522, ERMA INC) and UV (115 UV Gilson) de-
+
tectors. The [H ] of the standard and the reaction mixture
samples was adjusted to 0.1 or 0.2 M by addition of HClO4,
and then the samples were filtered through a 0.2 ꢅm mem-
brane prior to injection into the chromatographic system.
Standard solutions of the disaccharides and aldobionic ac-
ids were prepared individually in 0.10 M HClO4 and
chromatographed separately to determine the retention time
of each sample. D-Maltobionic acid, D-cellobionic acid, and
D-melibionic acid, used as standards, were synthesized ac-
cording to the literature methods (48). The stability of the
disaccharides and their respective acids was controlled by
HPLC. Chromatograms registered after incubation of the
+
standard samples in 0.2 M HClO (the highest [H ] used in
4
the kinetic measurements) at 34°C over 48 h were identical
to those of freshly prepared samples.
Experimental conditions and retention times for the
disaccharides and oxidation products are given as supple-
mentary material.
according to the literature method (51). No precipitation oc-
3
V
IV
curred on mixing the Cr or Cr
complexes with
acrylonitrile under the same conditions as those used in the
VI
Cr + disaccharide reactions.
EPR measurements
The EPR spectra were obtained on a Bruker ESP 300 E
spectrometer. The microwave frequency was generated with
a Bruker 04 ER (9–10 GHz) and measured with a Racal-
Dana frequency meter. The magnetic field was measured
with a Bruker NMR-probe gaussmeter. All of the EPR ex-
periments were carried out at 20°C. Reactions were carried
Results
CrVI oxidation of disaccharides
Over the whole range of perchloric acid concentrations
used in the kinetic measurements, UV–vis studies showed
that the reaction of each of the studied disaccharides with
out by direct reaction of K Cr O with 16- to 33-times ex-
2
2
7
VI
cess disaccharide at a given pH or by addition of Na CrO
2
4
Cr resulted in an absorbance band at 350 nm and a shoul-
VI
(
8.3 mM) + glutathione (8.3 mM) to solutions of
der at 420–500 nm, characteristic of Cr in acidic medium.
disaccharide (83 mM) at pH 5.0 and 20 ± 1°C.
At 350 nm a monotonic decrease in absorbance was ob-
served and it could be described by a single exponential de-
cay. Table 1 summarizes the values of the pseudo-first-order
rate constants (kobs), calculated from the slopes of the
ln(A350 – A ) vs. time data, for various concentrations of
Lac, Mal, Cel, and Mel, in 0.20 M HClO . In every case,
plots of kobs vs. [disaccharide] show a first-order dependence
on [disaccharide] (Fig. 1), and the values of the second-order
rate constants (k ) were determined from the slopes of the
EPR spectra were simulated using the program PEST
WinSIM (49) with 100% Lorentzian lineshapes. The spectral
V
parameters for each Cr species were consistent within all
simulations, with maximum deviations between giso values
8
1
being ±0.0001 units. In the simulations, values for H a
iso
4
1
were included only where the H a value was greater than
the LW (line width) of the Cr species, since the signal is
not significantly affected where the H a value is ?LW.
iso
V
1
iso
H
straight lines (Table 1, bottom). Consequently, at constant
Polymerization test
[HClO ], the rate law is expressed as:
4
The presence of free radicals in the reactions of all of the
VI
VI
VI
disaccharides with Cr was tested by the acrylonitrile poly-
[1]
–d[Cr ]/dt = kobs [Cr ]T
merization test. In a typical experiment, acrylonitrile in
kH [disaccharide] [CrVI]
=
0
2
.2 M HClO (0.5 mL) at 33°C was added to a solution of
4
–
3
VI
VI
.66 × 10 M K Cr O and 0.04 M disaccharide in 0.2 M
where [Cr ] refers to the total Cr concentration. For Lac,
2
2
7
T
HClO (2.0 mL). After a few minutes a white precipitate ap-
kH shows first-order dependence with [HClO ] (inset of
4
4
+
peared. Control experiments (without K Cr O or reductant
Fig. 1), which may be expressed as k = k [H ], with k =
2
2
7
H
–
2
–1
present) did not show the formation of a precipitate. Possible
0.0047(1) M s .
V
IV
VI
reactions of Cr and Cr with acrylonitrile were tested with
As expected on the basis of the rate law, –d(ln[Cr ])/dt =
V
IV
+
Na[Cr O(ehba) ] (50) and [Cr O(ehba) ] (ehba = 2-ethyl-2-
kobs, where kobs = f([saccharide][H ]); k was essentially
2
2
obs
VI
hydroxybutanoic acid); the latter was generated in solution
constant with increasing [Cr ] .
0
3
Supplementary data may be purchased from the Depository of Unpublished Data, Document Delivery, CISTI, National Research Council
Canada, Ottawa, ON K1A 0S2, Canada (http://www.nrc.ca/cisti/irm/unpub_e.shtml for information on ordering electronically).
©
2002 NRC Canada

1
680
Can. J. Chem. Vol. 80, 2002
VI
Fig. 2. Time evolution of the UV–vis spectrum of a mixture of
Fig. 4. X-band EPR spectra of mixtures of (a) Cr –Lac = 1:33,
VI
–3
+
Lac (0.32 M) and Cr (8 × 10 M). [HClO ] = 0.20 M; T =
[H ] = 0.20 M, t = 6 min, modulated amplitude (mod. ampl.) =
4 G; (b) Cr –Lac = 1:19, pH = 5.0, t = 24 h, mod. ampl. = 0.4
4
VI
3
3°C, I = 1.0 M over a period of ca. 250 min.
G. I = 1.0 M, T = 20°C, frequency W 9.7 GHz.
VI
Fig. 3. UV–vis difference spectra of Cr –Lac solutions at
pH 4.7, showing the increasing band at 377 nm with increasing
VI
–4
[
Lac]: (a) 0.12, (b) 0.20, and (c) 0.32 M. [Cr ] = 8 × 10 M,
T = 33°C, I = 1 M. Spectra taken after 2 min.
constant of the reduction of CrVI by the saccharide (k ) was
6
V
found to be much lower than that of the reduction of Cr
(
k ). The fact that k >> k implies that the slow redox step
5 5 6
VI
involves the reduction of Cr .
Detection of the intermediate CrVI ester
VI
Differential UV–vis spectra of mixtures of Cr and Lac
exhibited an absorption band with ꢁ
consistent with that ascribed to Cr oxy-esters (52, 53). At
pH 4.7, the redox reactions of the studied disaccharides pro-
= 377 nm (Fig. 3)
max
VI
VI
ceed very slowly with negligible reduction of Cr within the
first hour. Thus, at this pH the ester formation step can be
distinguished clearly from the electron transfer reaction.
Spectra obtained within 2 min after mixing revealed a dis-
tinctive absorption band at 377 nm. Continued scanning for
3
0 min showed no further change in the spectra. Varying the
excess concentration of disaccharide at pH = 4.7 showed that
the absorbance at 377 nm increased with increasing concen-
tration of disaccharide (Fig. 3), probably as a result of a shift
toward the ester in the esterification equilibrium.
We followed the formation of CrIII at 570 nm for various
III
[
disaccharide] in 0.20 M HClO to determine the rate of Cr
4
VI
formation relative to that of Cr consumption. The pseudo-
first-order rate constants obtained at this wavelength (Ta-
VI
Intermediacy of CrV
ble 1) are coincident with those obtained from the Cr de-
cay at 350 nm, with the same dependence on [disaccharide].
This means that Cr forms as fast as Cr is consumed; this
is consistent with the observation of an isosbestic point at
Using EPR spectroscopy, we have detected the formation
III
VI
V
of intermediate Cr species in the reaction of the
VI
disaccharides with Cr . The EPR spectra of mixtures of Lac
VI
5
25 nm (Fig. 2). This result was independently confirmed by
and Cr in 0.2 M HClO show the formation of several in-
4
V
+
following the redox reaction by EPR spectroscopy. The rate
termediate Cr species (Fig. 4a). At this [H ], the EPR spec-
©
2002 NRC Canada

Roldán et al.
1681
Table 2. EPR spectral parameters for CrV intermediates in the
Fig. 5. Experimental and simulated X-band EPR spectra of mix-
VI
VI
VI
a
tures of (a) Cr –Mal = 1:19; (b) Cr –Cel = 1:16; (c) Cr –Mel =
reactions.
VI
1
:19. [Cr ] = 0.015 M, I = 1.0 M, pH 5.0, t = 24 h, T = 20°C,
aiso
frequency W 9.7 GHz, mod. ampl. = 0.4 G.
CrV species
giso
(10 cm ) (N)
4
–1
[
Cr(O)(cis-O,O-Lac)2]–
1.98
0.93 (2)
0.85 (2)
ND
1
.9794
1
2
+
[
[
[
Cr(O)(O ,O -lactobionato)(H O) ]
1.9743
1.9715
1.9795
2
3
+
Cr(O)(cis-O,O-Lac)(H O) ]
ND
2
3
1
2
–
Cr(O)(O ,O -Mal) ]
0.76 (2)
0.92 (2)
0.97 (2)
0.88 (2)
0.98 (2)
2
1
.9794
1
2
–
[
[
Cr(O)(O ,O -Cel) ]
1.9793
1.9802
2
–
Cr(O)(cis-O,O-Mel)2]
1
.9798
a
N: number of equivalent protons. ND: not determined.
In the pH 3–7 range and with a Lac–CrVI ratio of 19:1, the
EPR spectrum, taken 24 h after mixing, is composed (signals
were deconvoluted by fitting the spectra to Lorentzian deriv-
atives) of two triplets at giso1 = 1.9800 and giso2 = 1.9794 in
an ~1:1 proportion. Each of these triplets shows four weak
5
3
Cr (9.55% abundance, I = 3/2) hyperfine peaks at 16.5(3) ×
–
4
–1
1
0
cm spacing (Fig. 4b, Table 2).
In the pH 5–7 range, the EPR spectrum of a 19:1 Mal–
VI
Cr reaction mixture affords two triplets at giso1 = 1.9795
and giso2 = 1.9794 in a 1:1 ratio (Fig. 5a). In the same pH
VI
range, a 16:1 Cel–Cr reaction mixture yields one triplet at
giso = 1.9793 (Fig. 5b, Table 2).
Mel behaves similarly to Lac. The best fit of the EPR
VI
spectrum of a 19:1 Mel–Cr mixture in the pH 3–7 range
taken 24 h after mixing affords two triplets at giso1 = 1.9802
and giso2 = 1.9798 (Fig. 5c, Table 2). These two signals are
present in a 1:1 ratio independent of pH.
The ultimate fate of the chromium in these reactions is a
III
III
Cr species, and a typical broad Cr EPR signal, centred at
g ~ 1.98, is always observed at later times.
tra consist of one signal at giso = 1.9797, which persists at
higher pH, and two additional signals at g = 1.9743 and
At room temperature and pH 5.0, the reaction of chromate
with glutathione (1:1 ratio) in the presence of a 10-times ex-
iso
+
1
.9715. At this [H ], the EPR signals are not resolved be-
VI
V
cause of the high modulation amplitude required to observe
the Cr species. The signals at lower g are not observed at
cess disaccharide over Cr affords Cr EPR spectra identi-
cal to those obtained by direct reaction of the disaccharide
V
iso
VI
pH > 2, where the electron transfer reaction becomes ex-
with Cr .
V
tremely slow; by contrast, the Cr species with g = 1.9797
iso
remains stable in solution for a long time.
Discussion
+
V
At the [H ] used in the kinetics measurements, the Cr
EPR signal intensities grow and decay rapidly. For the re-
CrVI oxidation of disaccharides
VI
+
duction of Cr by Lac ([H ] = 0.2 M, [Lac] = 0.424 M,
For the ranges of substrate and acid concentrations used in
VI
V
[
Cr ] = 0.0127 M, T = 20°C), the Cr EPR signal grows
the kinetics measurements, the oxidation of the
0
VI
VI
and decays within 3 h, while the total conversion of Cr
disaccharides by Cr
is a complex reaction, yielding
III
3+
into Cr requires 20 h. Under these conditions, changes in
[Cr(H O) ] and the aldobionic acid as the final redox prod-
2 6
the EPR signal intensity give values for the rate constant of
ucts. We propose a mechanism that takes into account:
(a) the kinetic results; (b) the polymerization of acrylonitrile
added to the reaction mixture; (c) the detection of an inter-
VI
the disappearance of Cr (k ), which is ~50-times higher
6
than the corresponding rate constant for the disappearance of
V
VI
Cr (k ). The k values obtained from EPR data agree with
mediate Cr ester; (d) the detection of intermediate of
5
6
the kobs values calculated spectrophotometrically at 570 nm
under the same conditions. Thus, the fact that k > k means
that changes in absorbance at 350 nm reflect changes in Cr
concentration.
oxochromate(V) species by EPR; and (e) the formation of
the aldobionic acid as the only organic reaction product.
5
6
VI
+
VI
VI
At the [H ] and [Cr ] used in the kinetics studies, Cr
ꢄ
may exist as HCrO (54), and this species is proposed as the
4
VI
We have also performed the reaction of the disaccharides
reactive form of Cr , in agreement with the first-order de-
VI
+
VI
and Cr at a lower [H ] than was used in the kinetic mea-
pendence of the reaction rate on [Cr ]. The chromic oxida-
V
surements to obtain information on the structure of Cr spe-
cies formed as intermediates in the reduction of Cr to Cr .
tion of alcohols and glycols are preceded by the formation
of a chromate ester (52, 53). The observation of the
VI
III
©
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1
682
Can. J. Chem. Vol. 80, 2002
VI
5
VI
4
VI
VI
Scheme 1. Cr –Mel: R = O -glc, R = H; Cr –Lac: R = H, R = O -glc. Cr –Lac(Mal, Cel): R = glycoside, R = H; Cr –Mel: R =
1
VI
2
1
2
3
4
3
VI
H, R = glycoside. Cr –Lac: R = OH, R = H; Cr –Cel: R = H, R = OH. AldA = aldobionic acid; w = water.
4
5
6
5
6
absorbance band, characteristic of chromate oxy-esters, at
approximately 377 nm (a few minutes after mixing the
disaccharides and Cr under conditions where the redox re-
anomeric hydroxyl group is oxidized, it seems reasonable to
hypothesize that the complexes with the primary hydroxyl
group bound to Cr (II, III) should be the precursors of the
VI
VI
action is extremely slow) reveals that such an intermediate
Cr complex is rapidly formed prior to the redox steps.
slow redox steps.
VI
The slow redox step in the mechanism might take place
by either a one-electron or a two-electron transfer (56). The
slow step proposed in Scheme 1 involves the intramolecular
transfer of two electrons to yield Cr and the aldobionic
acid (eq. [2]). This is based on the mechanism reported for
aldoses, which are also selectively oxidized by Cr at the
hemiacetalic group with an initial two-electron transfer slow
step (21–28). After the slow redox step, Cr is predicted to
react with excess saccharide to yield Cr and a disaccharide
Thus, the first step of the mechanism proposed in Scheme 1
VI
is the formation of the disaccharide–Cr mono-chelate. The
VI
IV
disaccharide acts as a bidentate ligand bound to Cr via any
pair of properly disposed hydroxyl groups. While there is no
VI
VI
direct evidence for bidentate chelation of the sugar to Cr ,
VI
it is likely that this is the case, especially since Cr chelates
IV
have been observed by NMR spectroscopy in the oxidations
of another bidentate oxygen donor (18). The chelates pro-
III
VI
posed in Scheme 1 are those with the ligand bound to Cr
radical in a fast step (eq. [3]). The latter is supported by the
observed polymerization of acrylonitrile when it is added to
the reaction mixture. The rapid reaction of the disaccharide
3
>
4>
1
2
at the cis-O ,O -diolato (I) and cis-O ,O -diolato (II) moi-
eties of Lac and Mel and the cis-O ,O -diolato (II) moiety
of Mal and Cel. These are proposed on the basis that ꢆ-glc
1
2
VI
V
radical with Cr affords Cr (eq. [4]), which can further ox-
VI
III
forms the Cr –glc chelate faster than the ꢀ-D-anomer — a
idize the disaccharide to yield Cr and the aldobionic acid
fact attributed to the cis-1,2-diolato moiety present only in
the ꢆ-anomer — (21) and the observation of the marked
preference of the CrO3 ion for binding to cis- rather than
trans-diol groups of cyclic diols (21, 22, 29, 55). For Mal,
as the final oxidation product (eq. [5]).
The kH in the rate law corresponds to kK[H ] in this
+
+
mechanism. This means that the relative reactivity of the
disaccharides may be interpreted by taking into account both
VI
VI
Lac, and Cel, II is proposed to be in equilibrium with Cr –
the rate of formation of the Cr ester and its redox rate. Our
1
2
6>
VI
O ,O ,O -disaccharide chelates (III). Molecular models
experimental data show that the disaccharide–Cr chelate
formation is much faster than the redox steps. For certain
VI
show that Lac, Mal, and Cel can afford Cr –chelates with
VI
1
2
6>
the disaccharide bound to Cr at O ,O ,O and with reten-
aldoses the rate constant for the formation of the aldose–
4
VI
tion of the C chair conformation of the glycoside moiety.
Cr chelate (K) in 0.75 M HClO has been determined and
1
4
Taking into account that in the redox reaction only the
was found to be 500- to 2000-times higher than the redox
©
2002 NRC Canada

Roldán et al.
1683
constant (k). Thus, the redox rate should depend, essentially,
on the energy barrier the Cr chelate has to overcome to at-
The contribution to the EPR signals from other species, in-
volving trans-diolato moieties, should be small.
VI
tain the five-membered cyclic transition state for the intra-
molecular H transfer (22). The kinetics data show that the
relative reactivity of the disaccharides toward Cr is Mel >
Lactose–CrV
VI
VI
The reaction of Cr with a 20-times excess of Lac in the
Lac > Cel > Mal. If we compare the reactivity of these sac-
pH 3–7 range affords an EPR spectrum dominated by a sin-
gle detectable signal at giso = 1.9797 with the four weak Cr
VI
53
charides with glc, which is oxidized by Cr with a rate con-
stant of kH = 9.9 × 10–4
(
M
–1
s
–1
in 0.2 M HClO at 33°C
4
(9.55% abundance, I = 3/2) hyperfine peaks at 16.5 ×
4
–4
–1
22), we can observe that substitution of the O H group of
10 cm spacing; the product remains in solution from sev-
eral days to several months. When the modulation amplitude
is lowered to 0.4 G, the EPR signal resolves into two triplets
glc by the glycoside moiety results in the retardation of the
6
redox rate, while substitution of the primary O H accelerates
the redox reaction. The retardation of the redox reaction ob-
served for Mal, Cel, and Lac is probably related to the for-
at g
= 1.9800 and giso2 = 1.9794 (Aiso = 16.5(3) ×
iso1
–
4
–1
10 cm , ~50:50 giso1–giso2 ratio) (Fig. 4b, Table 2). The
giso and Aiso values are consistent with those calculated for
five-coordinate oxochromate(V) complexes with four alcoholato
VI
1
2
6>
mation of the Cr –O ,O ,O -disaccharide chelates (III). The
probability for Mel to act as a tridentate ligand is very low
because of the less favourable spatial orientation of the
galactoside moiety; therefore, it shows the highest reactivity
in the series.
–
4
–1
donors (gcalc = 1.9800, Acalc = 16.5 × 10 cm ) (1, 57). The
shf pattern indicates that the EPR signal is a composite of
V
two oxo-Cr -cis-diolato species with two (one from each
2
V
chelate ring) carbinolic protons coupled to the Cr electronic
spin. The two components can be attributed to linkage iso-
mers of the [Cr(O)(cis-O,O-Lac) ] (61). Even when three
V
Cr intermediates
The most common means of characterizing CrV com-
plexes in solution is EPR spectroscopy. The EPR spectral
–
2
3
>
4>
–
different
linkage
is–omers
[Cr(O)(cis-O ,O -Lac) ] ,
2
1
2
1
2
parameters, giso and A , together with the proton
[Cr(O)(cis-O ,O -Lac) ] , and [Cr(O)(cis-O ,O -Lac)(cis-
iso
2
3
>
4>
–
superhyperfine (shf) coupling, are useful in determining the
O ,O -Lac)] (IV–VI in Fig. 6) are possible, only two spe-
cies are required to simulate the EPR signal. It seems rea-
sonable to assume that the angles between the carbinolic
V
binding modes of sugars to the Cr center (1, 22, 24, 26, 27,
5
7, 58). An empirical relationship between the nature and
V
number of donor groups and the EPR spectral parameters of
Cr complexes has been established (1, 57). Five-coordinate
Cr species show higher g and lower Cr Aiso values than
protons and the Cr –ligand plane in two of the linkage iso-
mers of [Cr(O)(cis-O,O-Lac) ] are very similar, as we can-
not distinguish between them.
A more complex EPR spectral pattern is observed in a
V
–
2
V
53
iso
the corresponding six-coordinate species (57, 59, 60). Thus,
V
+
VI
the assignment of the structures of new oxo-Cr species in
0.2 M HClO solution. At this [H ] and a 33:1 ligand–Cr
4
solution was made according to the isotropic EPR parame-
ters (giso and Aiso values) and the superhyperfine (shf) pattern
of the signal (1, 59).
ratio, two additional signals at giso3 = 1.9743 and giso4
=
1.9715 appear, together with the signal at giso = 1.9797
(Fig. 4a, Table 2). The shf pattern of these two new signals
was not resolved, but the signal at giso = 1.9715 suggests the
presence of six-coordinate oxo-Cr species, possibly
[Cr(O)(cis-O,O-Lac)(H2O)3] (VII). The calculated giso
V
The EPR spectrum of a Cr -diolato species of six-
V
membered ring cis-diols yields a doublet, since only one
+
proton is in the plane of the unpaired electron density of the
V
V
V
Cr ion, while the EPR spectrum of a Cr -diolato species of
value for a six-coordinate oxo-Cr species with two
six-membered ring trans-diols (with no protons lying in the
alcoholato donors and three water molecules (gclc = 1.9724)
is in reasonable agreement with the observed giso4 value. The
positive charge of this species is also consistent with its ap-
ligand plane) yields a singlet (55). Consequently, the EPR
V
spectral multiplicity of the bis-chelate Cr -diolato species
2
V
+
formed between Cr and pyranosic cis- and trans-diols will
pearance at high [H ]. The signal at giso3 = 1.9743 may be
exhibit a triplet and a singlet, respectively, which arise from
the orientation of the ring protons of the coordinated diol
groups with respect to the basal plane of the complex (1, 55,
tentatively attributed to another positively charged six-
V
1
2
coordinate oxo-Cr
mono-chelate [Cr(O)(O ,O -lacto-
+
V
bionato)(H2O)3] (VIII, gclc = 1.9735), with Cr bound to
the 2-hydroxyacid moiety of the oxidized product and to
three water molecules. Coordination to the oxidized ligand is
5
7). Based on this, the isotropic EPR parameters giso and Aiso
were used to deduce the coordination number and nature of
V
VI
the donor groups of the Cr species formed, either for the
consistent with the fact that Cr is a stronger oxidant in
VI
more acidic medium. The six-coordinate oxo-CrV mono-
reaction of Cr with the disaccharides or for the reaction of
VI
Cr with glutathione in the presence of excess disaccharide,
chelate VII is possibly responsible for the higher rates ob-
V
according to a described empirical method (1, 57, 59, 60).
The cis-diolato moieties of the disaccharides under study
served for the Cr –disaccharide redox reactions at pH < 2.
V
At higher pH, where Cr mono-chelates are not observed,
V
V
are potential binding sites to give five-membered Cr
Cr complexes are stable enough to remain in solution from
species. The five-membered CrV chelates are the most
thermodynamically favoured, and CrO3 shows a marked
preference for binding to cis- rather than trans-diol groups
of cyclic diols (21, 22, 29, 55). Based on this, the EPR
spectral features of disaccharide–Cr mixtures were inter-
preted in terms of Cr -diolato species involving binding
several days to several months.
+
Maltose–CrV
VI
For the reaction of Cr with Mal, in the pH 5–7 range
V
and with a 19:1 ligand–metal ratio, the EPR spectra (Fig. 5a,
Table 2) consist of two triplets at giso1 = 1.9795 and giso2 =
1.9794. The giso values and the shf pattern indicate these sig-
nals may be attributed to two geometric isomers (typical
V
1
2
mainly through the cis-O ,O -diolato of Mal and Cel and the
cis-O ,O -diolato and cis-O ,O -diolato of Lac and Mel.
1
2
3> 4>
©
2002 NRC Canada

1
684
Can. J. Chem. Vol. 80, 2002
V
V
4
V
Fig. 6. Structures of Cr complexes with disaccharide ligands. R = CH OH, R = C O H , w = water. Cr –Lac: R = O -glc; Cr –
1
2
5
10
9
19
2
6
V
V
V
V
Mel : R = O -glc; Cr –Lac (Cr –Mal , Cr –Cel ): R = glycoside, R = H; Cr –Mel : R = H, R = glycoside.
2
2
2
2
2
3
4
2
3
4
ꢇgiso for geometric isomers are 1 × 10–4 – 2 × 10 ) (27, 30)
–4
EPR signal, probably for the same reasons as for the
Lac–Cr system.
V
1
2
V
of the five-coordinate oxo-Cr complex [Cr(O)(O ,O -
Mal) ] (V), with Mal bound to Cr via the 1,2-cis diolato
–
V
EPR spectra identical to those shown in Figs. 4b and 5a–
2
V
moiety.
5c are obtained when Cr is generated by the reaction of
VI
Cr with glutathione (1:1 ratio) at pH = 5.0 and stabilized
Cellobiose–CrV
by the presence of a 10-times excess of disaccharide over
VI
V
V
Cr . This result indicates that Cr species formed at this pH
effectively correspond to chelates formed with the
disaccharide and not with the oxidized product (aldobionic
acid).
Intermediate Cel–Cr species formed in the reaction of
Cr with Cel, in the pH 5–7 range and with a 14:1 ligand–
metal ratio, yield EPR spectra (Fig. 5b, Table 2) consisting
of one triplet at giso1 = 1.9793. The giso value corresponds to
that calculated for a five-coordinate oxochromate(V) com-
plex with four alcoholato donors, and the shf pattern indi-
VI
1
2
Conclusions
cates this signal may be assigned to the [Cr(O)(O ,O -
Cel) ] chelate (V), with Cel bound to Cr via the 1,2-cis
–
V
2
Disaccharides form stable high-valent chromium che-
+
VI
diolato moiety.
lates at pH > 2, while at higher [H ] their oxidation by Cr
V
and (or) Cr is favoured. The oxidation occurs selectively at
the hemiacetalyc C OH group, with first-order kinetics for
Melibiose–CrV
1
VI
VI
In the reaction of Cr with Mel at a pH 3–7 range and a
9:1 ligand–metal ratio, the EPR spectra of the intermediate
Cr species show two triplets at giso1 = 1.9802 and giso2
.9798 (~50:50 giso1–giso2 ratio). The giso values and the shf
both [Cr ] and [disaccharide]. The relative ability of the
VI
1
studied disaccharides to reduce Cr — Mel > Lac > Cel >
V
=
Mal — can be attributed to the increasing stability of the in-
VI
1
termediate disaccharide–Cr chelates formed with Mel
pattern reveal that these signals correspond to different link-
through Mal. The redox and complexation chemistry ob-
served for the disaccharide–Cr systems parallels that of
V
VI
age isomers of the five-coordinate oxo-Cr complex
–
[
Cr(O)(O,O-Mel) ] . As in the case of Lac, the three linkage
aldoses. The glycoside moiety can retard the redox rate of
2
3
>
4>
–
1
2
–
VI
isomers [Cr(O)(O ,O -Mel) ] (IV), [Cr(O)(O ,O -Mel) ]
the aglycone when additional binding to Cr by the
2
2
3
>
4>
1
2
–
(
V), and [Cr(O)(O ,O -Mel)(O ,O -Mel)] (VI) are possi-
glycoside moiety is possible. In the case of Mal, this effect
V
ble, but only two Cr species are required to simulate the
reduces k to one half of the value obtained for glc.
H
©
2002 NRC Canada

Roldán et al.
1685
At pH 3–7, redox reactions between CrV and
disaccharides are extremely slow, since once the bis-chelates
are formed they are long-lived. A different reactivity pattern
24. V. Daier, S. Signorella, M. Rizzotto, M.I. Frascaroli, C.
Palopoli, C. Brondino, J.M. Salas-Peregrin, and L.F. Sala. Can.
J. Chem. 77, 57 (1999).
+
25. M. Rizzotto, M.I. Frascaroli, S. Signorella, and L.F. Sala.
is observed at high [H ], where intramolecular electron-transfer
Polyhedron, 15, 1517 (1996).
26. L.F. Sala, S. Signorella, M. Rizzotto, M.I. Frascaroli, and F.
Gandolfo. Can. J. Chem. 70, 2046 (1992).
27. S. Signorella, M. Rizzotto, V. Daier, M.I. Frascaroli, C.
Palopoli, D. Martino, A. Boussecksou, and L.F. Sala. J. Chem.
Soc. Dalton Trans. 1607 (1996).
processes take place and mono-chelates are observed. The
V
observation
of
six-coordinate
[Cr O(O,O-disaccha-
+
+
ride)(OH ) ] at only high [H ], together with the selectivity
of the redox reaction, suggest that the six-coordinate Cr
2
3
V
mono-chelate VII could be the precursor of the
intramolecular electron-transfer reaction step in the redox re-
actions.
2
8. M. Rizzotto, S. Signorella, M.I. Frascaroli, V. Daier, and L.F.
Sala. J. Carbohydr. Chem. 14, 45 (1995).
2
9. S. Signorella, M.I. Frascaroli, S. García, M. Santoro, J.C.
González, C. Palopoli, N. Casado, and L.F. Sala. J. Chem. Soc.
Dalton Trans. 1617 (2000).
Acknowledgments
3
3
0. M. Rizzotto, A. Levina, M. Santoro, S. García, M.I. Frascaroli,
S. Signorella, L.F. Sala, and P.A. Lay. J. Chem. Soc. Dalton
Trans. 3206 (2002).
1. S. Signorella, M. Santoro, C. Palopoli, C. Brondino, J.M.
Salas-Peregrin, M. Quirós, and L.F. Sala. Polyhedron, 17,
We thank the National Research Council of Argentina
CONICET), the Third World Academy of Sciences
TWAS), the National University of Rosario (UNR),
ANTORCHAS Foundation, and the International Foundation
for Sciences (IFS) for financial support.
(
(
2
739 (1998).
2. S. Signorella, S. García, and L. F. Sala. Polyhedron, 16, 701
1997).
3. C. Palopoli, S. Signorella, and L.F. Sala. New J. Chem. 21,
43 (1997).
4. L.F. Sala, C. Palopoli, and S. Signorella. Polyhedron, 14, 1725
1995).
3
3
3
3
3
3
(
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