Catalytic activity of thiacalix[4]arenetetrasulfonate metal complexes on modified anion-exchangers for ascorbic acid oxidation

Article abstract of DOI:10.1248/cpb.c13-00241

The catalysis of ascorbic acid (AsA) oxidation by anion-exchangers modified with metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4] _A-500, Me=Mn³⁺, Fe³⁺, Co³⁺, Ce ⁴⁺, Cu²⁺, Zn²⁺, Ni²⁺, and H ₂) were investigated. Me-TCAS[4]_A-500 (Me=Mn³⁺, Fe³⁺, Ce⁴⁺, and Cu²⁺) all exhibited the ability to catalyze the oxidative reaction of AsA to dehydroascorbic acid. However, in the presence of high concentrations of AsA, only Cu ²⁺-TCAS[4]_A-500was capable of complete oxidation of the acid. Moreover, after six repeat uses, Cu²⁺-TCAS[4]_A-500 maintained high and relatively constant catalytic activity. Prior treatment of glucose solutions with Cu²⁺-TCAS[4]_A-500even in the presence of high AsA concentrations, enabled the satisfactory determination of glucose without interference by AsA. Cu²⁺-TCAS[4]_A-500 will therefore be applicable as an artificial substitute for ascorbate oxidase, and may be useful as a means to eliminate AsA interference during the analysis of vital compounds such as glucose and uric acid.

Full text of DOI:10.1248/cpb.c13-00241

September 2013
Regular Article
Chem. Pharm. Bull. 61(9) 927–932 (2013)
927
Catalytic Activity of Thiacalix[4]arenetetrasulfonate Metal Complexes on
Modified Anion-Exchangers for Ascorbic Acid Oxidation
Junichi Odo,* Tomomi Hirashima, Tomoko Hayashida, Asuka Miyauchi, Mami Minemoto,
Masato Iuchi, and Masahiko Inoguchi
Department of Biochemistry, Faculty of Science, Okayama University of Science; 1–1 Ridai-cho, Kita, Okayama
700–0005, Japan.
Received March 27, 2013; accepted July 4, 2013
The catalysis of ascorbic acid (AsA) oxidation by anion-exchangers modified with metal complexes of
3
ꢁ
3ꢁ
3ꢁ
4ꢁ
2ꢁ
2ꢁ
2ꢁ
thiacalix[4]arenetetrasulfonate (Me-TCAS[4]
, MeꢀMn , Fe , Co , Ce , Cu , Zn , Ni , and H )
2
3ꢁ 4ꢁ 2ꢁ
A-500
3
ꢁ
were investigated. Me-TCAS[4]A-500 (MeꢀMn , Fe , Ce , and Cu ) all exhibited the ability to catalyze the
oxidative reaction of AsA to dehydroascorbic acid. However, in the presence of high concentrations of AsA,
only Cu -TCAS[4]A-500 was capable of complete oxidation of the acid. Moreover, after six repeat uses, Cu
2ꢁ
2ꢁ
-
TCAS[4]
maintained high and relatively constant catalytic activity. Prior treatment of glucose solutions
A-500
2
ꢁ
with Cu -TCAS[4]A-500, even in the presence of high AsA concentrations, enabled the satisfactory determina-
2ꢁ
tion of glucose without interference by AsA. Cu -TCAS[4]A-500 will therefore be applicable as an artificial
substitute for ascorbate oxidase, and may be useful as a means to eliminate AsA interference during the
analysis of vital compounds such as glucose and uric acid.
Key words ascorbic acid; thiacalix[n]arene; ascorbate oxidase; Cu(II) complex; oxidative reaction; catalytic
activity
Calix[n]arenes have been extensively investigated within ingly, if Me-TCAS[4]A-500 is found to be capable of catalyzing
the field of supramolecular chemistry because of their high this same reaction, it may have applications as an artificial
selectivity with regard to the formation of host–guest com- substitute for ascorbate oxidase.
1
,2)
plexes.
In particular, a significant amount of work has
2
ascorbic acid+O
2
⎯ ⎯→ 2 dehydroascorbic acid+2H
(λmax : < 200nm)
2
O
focused on calix[n]arenes or their derivatives as receptors
for various metal ions and molecules. Thiacalix[n]arenes,
(
λmax : 265 nm at pH 5 – 9)
(1)
3)
which are formed by the replacement of the methylene units
In clinical analyses, the concentrations of important vital
of the parent calix[n]arenes by S linkages, have recently been compounds such as glucose and uric acid in body fluids are
4
–6)
19)
synthesized
and exhibit the interesting ability to form routinely determined using enzymatic methods. For ex-
very stable metal complexes without prior modification of ample, glucose in blood and urine is determined using a com-
7
–11)
their upper- and/or lower-rims.
To date, some thiacalix[n]- bination of two enzymatic processes which apply reactions 2
arene derivatives have been applied as analytical reagents and 3. However, since the presence of AsA may interfere with
6
,11,12)
for the separation of metal ions and molecules.
In ad- these reactions, samples must be treated with ascorbate oxi-
2
0)
dition, we have recently demonstrated that metal complexes dase to eliminate this potential interference.
of thiacalix[4]arenetetrasulfonate (TCAS[4]) on modified
glucose oxidase
glucose+O ⎯⎯⎯⎯⎯→gluconic acid+H O
(2)
2
2
2
anion-exchangers (Me-TCAS[4]A-500) exhibit peroxidase-like
13,14)
3+
and catalase-like catalytic activities.
Fe -TCAS[4]
peroxidase
A-500
H O +chromogen ⎯⎯⎯⎯→quinoid dye+2H O (3)
2
2
2
demonstrated especially high peroxidase-like catalytic activ-
ity, and was applicable as a practical analytical reagent not Therefore, if Me-TCAS[4]A-500 will catalyze reaction 1 in a
only for the determination of H O but also for the deter- manner similar to ascorbate oxidase, it may be used in the
2
2
mination of glucose and uric acid in serum as a substitute same manner to eliminate interference by AsA in these tests.
15–17)
for peroxidase in clinical analyses.
It was of significant
interest to observe that the Me-TCAS[4]A-500 compounds were Experimental
able to function as practical replacements for enzymes such as
Materials and Reagents A sample of sodium thiacalix[4]-
peroxidase and catalase, in spite of the fact that they are small arenetetrasulfonate (Fig. 1, TCAS[4]), prepared according
5
)
molecules with simple structures compared to the correspond- to a literature method, was received as a gift from the
ing enzymes. Cosmo Research Institute. Sodium calix[4]arenetetrasulfonate,
In this study, to further expand the scope of the catalytic sodium calix[6]arenehexasulfonate and sodium calix[8]-
abilities of Me-TCAS[4]A-500, its catalytic activity for the oxi- areneoctasulfonate (Fig. 1, CAS[n]; n=4, 6, and 8, respec-
dation of ascorbic acid (AsA) was investigated. Thus far, there tively) were purchased from the Sugai Kagaku Kogyo Co.
have been no reports concerning the catalytic activity of (Wakayama, Japan). DEAE cellulofine A-500 (a cellulose-type
metal complexes of thiacalix[n]arenes or calix[n]arenes for the anion-exchange material with diethylaminoethyl groups) pur-
oxidation of AsA. It is, however, well known that AsA is oxi- chased from the Seikagaku Kogyo Co. (Tokyo, Japan) was
dized according to reaction 1 to produce dehydroascorbic acid, washed with water several times and dried over P O under
2
5
18)
a reaction which is catalyzed by ascorbate oxidase. Accord- reduced pressure. Ascorbic acid (AsA, reduced form), Pretest
bII test strips and peroxidase (POD; EC 1.11.1.7; from horse-
4
The authors declare no conflict of interest.
radish; Type VI; activity 100units/mL) were purchased from
*
To whom correspondence should be addressed. e-mail: odo@dbc.ous.ac.jp
© 2013 The Pharmaceutical Society of Japan

9
28
Vol. 61, No. 9
Fig. 1. Structures of TCAS[4] and CAS[n], and Proposed Structure of Cu2+-TCAS[4]A-500
2+
the Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Glu- at room temperature for an additional 30min, the Cu -
2+
cose oxidase (GOD; Aspergillus niger; activity 100–250units/ TCAS[4]A-500 was filtered off. The amount of Cu ion remain-
mL) was purchased from Dojindo Laboratories (Kumamoto, ing in the 5.0mL supernatant was determined by titration with
Japan). All other reagents were of analytical grade and used a 0.1M EDTA solution using murexide as a metal indicator.
2
+
as-received without further purification.
Since AsA is readily oxidized in solution, metaphosphoric in the Cu -TCAS[4]
acid was used as an anti-oxidant when preparing AsA solu-
Based on these results, the molar ratio of Cu ion to TCAS[4]
2+
was indirectly determined.
A-500
2
+
Application of Cu -TCAS[4]
to Glucose Determina-
A-500
tions. Sample AsA solutions were prepared by diluting 0.01M tion via the GOD/POD Method The GOD/POD method
2+
AsA in a 5% metaphosphoric acid solution to the desired con- was applied to evaluate the extent to which Cu -TCAS[4]
A-500
centration with distilled water.
will reduce the interference of AsA. This technique is typi-
19)
2+
Preparation of Me-TCAS[4]A-500 Anion-exchangers mod- cally used to determine glucose in clinical samples. Cu -
3+
3+
ified with Me-TCAS[4] (Me-TCAS[4]
; Me=Mn , Fe , TCAS[4]A-500 (20mg) was added to 5.0mL of a buffered
A-500
3+
4+
2+
2+
2+
Co , Ce , Cu , Zn , Ni , and H ) were prepared accord- solution containing glucose and AsA and the mixture was
2
14,15)
ing to the method described in the literature.
In each case, incubated for 30min at room temperature, following which
2+
100 µmol of Me-TCAS[4] was loaded per 1g dry anion-ex- the Cu -TCAS[4]
was removed by filtration. The glucose
A-500
changer. No elution of Me-TCAS[4] from the modified anion- content of the supernatant was then analyzed using the GOD/
exchangers was observed under the reaction conditions in this POD process. A mixture of 1.0mL of the supernatant with
study. Only in the presence of the excess AsA, a color fading 5.0mL of GOD/POD reagent solution was incubated at room
3+
4+
of Me-TCAS[4]A-500 (Me=Fe , Ce ) was observed.
temperature for 10min, after which the glucose concentration
Evaluation of the Catalytic Activity of Me-TCAS[4]A-500 was determined by measuring the absorbance (at 505nm) of
If Me-TCAS[4]A-500 is capable of catalyzing reaction 1, AsA the quinoid dye produced through reactions 2 and 3, corrected
will be oxidized to produce dehydroascorbic acid and so the using a reagent blank solution. In reaction 3, a combination
activity of Me-TCAS[4]A-500 for the oxidation of AsA was of 4-aminoantipyrine (4-AAP) and phenol was used as the
evaluated by measuring the amount of unreacted AsA remain- chromogen, and the corresponding quinoid dye exhibited
ing in reaction solutions. The pKa1 and pKa2 of AsA are 4.17 a λmax of 505nm. The reagent solution used consisted of a
and 11.6, respectively, and AsA in aqueous solution exhibits 1:1:0.5:0.5:2 (v/v) mixture of 32units/mL GOD, 25units/
maximum UV-visible absorbance at 265nm under the condi- mL POD, 2mg/mL 4-AAP, 10mg/mL phenol and pH 7 borate
tions applied in this study (pH of 5–9), whereas dehydro- buffer solutions.
2
+
ascorbic acid absorbs only below 200nm. For this reason, the
Application of Cu -TCAS[4]
to Pretest 4bII Pre-
A-500
concentration of unreacted AsA was determined by measuring test 4bII (shown in Fig. 2) was also used to evaluate whether
2
+
the absorbance of solutions at 265nm.
Cu -TCAS[4]
is capable of eliminating AsA interference.
A-500
Me-TCAS[4]A-500 (100 µmol/g, 20mg) was added to a mix- This test is normally used to screen urine samples for glucose,
19)
2+
ture of 1.0mL of AsA solution with 5.0mL buffer solution, protein, pH and AsA in clinical analysis. Cu -TCAS[4]
A-500
and the combined mixture was incubated at room temperature (20mg) was added to a sample solution containing glucose,
for 30min. After incubation, the Me-TCAS[4]A-500 was filtered human serum albumine as the protein and AsA, the mixture
2+
off and the absorbance of the solution at 265nm was measured was incubated for 30min, and the Cu -TCAS[4]
was
A-500
against a reagent blank. The buffer solution in this study was filtered off. A Pretest 4bII test strip was dipped in the super-
.1 M KH PO /0.05M Na B O , adjusted to produce pH values natant and the subsequent color changes were examined to
0
2
4
2
4
7
between 5 and 9.
determine the presence of glucose and AsA. As a reference
Ethylenediaminetetraacetic Acid (EDTA) Titration check, a Pretest 4bII test strip was dipped in a sample solu-
2
+
2+
to Assess the Molar Ratio of Cu to TCAS[4] in Cu - tion containing no AsA and its color change also examined.
TCAS[4]A-500 The EDTA titration method was used to The color changes of both strips were compared to determine
2
+
2+
determine the molar ratio of Cu ion to TCAS[4] in Cu - whether or not the AsA interference had been eliminated by
2+
2+
TCAS[4]A-500. A 10mL volume of a 0.1M Cu ion solution treatment with Cu -TCAS[4]A-500.
was added to TCAS[4]A-500 (20mg) suspended in 10mL of Apparatus All UV-visible absorption spectra were recorded
water while stirring. After the mixture had been stirred on a Shimadzu UV-1600 PC double beam spectrophotometer

September 2013
929
Fig. 3. Effects of pH on the Activity of Me-TCAS[4]A-500 (100 µmol/g,
2
0mg) for the Oxidation of 0.50µmol AsA
The conditions are the same as those in the text. ●, Cu2+-TCAS[4]
; ○, Mn3+
-
A-500
4+
3+
Fig. 2. Color Tones (Excerpt) of Pretest 4bII for Each Test
TCAS[4]
(
; △, Ce -TCAS[4]
; □, Fe -TCAS[4]A-500; ■, Me-TCAS[4]A-500
A-500
A-500
3+
2+
2+
Me=Co , Ni , Zn , H2).
using 10mm quartz cells.
Results and Discussion
Catalytic Activity of Me-TCAS[4]A-500 The activity of
Me-TCAS[4]A-500 for the oxidation of AsA was investigated
using sample solutions containing 0.5µmol AsA, buffered
at pH values ranging from 5 to 9, at a Me-TCAS[4] to AsA
molar ratio of 1:0.25. The results are summarized in Fig. 3,
2+
3+
4+
3+
where it can be seen that only Cu -, Mn -, Ce -, and Fe -
TCAS[4]A-500 exhibited oxidation activity at each pH, whereas
the other Me-TCAS[4]
Cu -, Mn -, and Ce -TCAS[4]
did not show any activity at all.
A-500
4+
2+
3+
showed especially high
A-500
activity within the pH region tested.
In order to determine whether these Me-TCAS[4]
Fig. 4. Effects of Incubation Time on the Activity of Me-TCAS[4]A-500
100 µmol/g, 20mg) for the Oxidation of 3.0µmol AsA at pH 7.0
A-500
(
2+
3+
4+
(
Me=Cu , Mn , and Ce ) are capable of catalyzing the
The conditions are the same as those in the text. ●, Cu2+-TCAS[4]
; △, Ce4+
-
A-500
oxidation of higher concentrations of AsA, similar trials were
3+
TCAS[4]A-500; ○, Mn -TCAS[4]
.
A-500
performed using sample solutions containing 3.0µmol AsA,
at a Me-TCAS[4] to AsA molar ratio of 1:1.5 and pH 7. As associated with redox-potentials of Me-TCAS[4] on the anion-
2
+
shown in Fig. 4, only Cu -TCAS[4]
resulted in complete exchangers. Unfortunately, their redox-potentials were difficult
A-500
4+
oxidation of the AsA under these conditions, whereas Ce - to be investigated, because Me-TCAS[4] was loaded on the
3+
2+
and Mn -TCAS[4]
only oxidized about half of the AsA, anion-exchangers. In sharp contrast, Cu -TCAS[4]
was
A-500
A-500
even after prolonged incubation up to 120min. In the course very stable without color fading after incubation for a long
of the oxidation of excess AsA, Me-TCAS[4]A-500 will be time even in the presence of the excess AsA. This high stabil-
2
+
reasonable to exhibit their catalytic activity through a redox ity of Cu -TCAS[4]
will lead to hold its high catalytic
A-500
n+
(n−1)+
cycle of Me -TCAS[4]
⇋Me
-TCAS[4]A-500. If the activity. Moreover, considering that copper centers play an im-
-TCAS[4]A-500 is relatively stable and has no or weak portant role in the catalytic activity of ascorbate oxidase, it
A-500
(n−1)+
21)
Me
oxidation activity, this Me
oxidation activity of the corresponding Me-TCAS[4]
(
n−1)+
2+
-TCAS[4]A-500 would cause its is not unexpected that Cu -TCAS[4]
exhibited the highest
A-500
to catalytic activity of Me-TCAS[4]A-500 tested.
A-500
4+
3+
14)
weaken. Moreover, Me-TCAS[4]A-500 (Me=Ce and Mn )
As previously described in the literature, thiacalix[n]-
exhibited brown and yellow coloration, respectively, prior to arenes have the ability to form very stable metal complexes
the oxidation reaction. Their colors began to fade after 30min without any modification of their upper- and/or lower-rims,
of incubation in the presence of the excess AsA (3.0µmol), because the S of the epithio groups of these compounds is able
while no color fading was observed in the presence of 0.5µmol to coordinate firmly to metal ions. According to X-ray analy-
3
+
2+
AsA. This color fading may be due to the reduction to Ce - sis of Cu complexes of TCAS[4] and p-tert-butylthiacalix[4]-
2+
22–24)
2+
and Mn -TCAS[4]
by the excess AsA, respectively. How- arene (t-Bu-TCA[4]),
Cu ions were coordinated to the
A-500
ever, the faded Me-TCAS[4]A-500 remained unaltered even after phenolic oxygen and the S of the epithio groups in a 5- or a
2+
being allowed to stand for a long time in contact with air. Thus 6-coordinate environment. Moreover, plural Cu ions were
4+
3+
it appears that Ce - and Mn -TCAS[4]
were partially coordinated to one ligand in a manner such that the pheno-
A-500
decomposed by the excess AsA such that the metal ions were lic oxygen and/or the S of the epithio groups act as a bridge
2+
released from the Me-TCAS[4] on the anion-exchangers. As linking the adjacent two Cu ions, indicating the molar ratio
4
+
3+
2+
a result, Ce - and Mn -TCAS[4]
would lose the ability of Cu to ligand was 2:1 or 4:1. In contrast, according to
A-500
2+
to fully catalyze the oxidation of the excess AsA by partially X-ray analysis of Cu complex of calix[4]arenetetrasulfonate
decomposition of Me-TCAS[4] on the anion-exchangers and/or (CAS[4]) with no epithio group, no coordination of the Cu
the reduction of Me -TCAS[4]
Of course, the catalytic activity of Me-TCAS[4]A-500 should be titrations in this study showed that the molar ratio of Cu
2+
n+
(n−1)+
25)
to Me
-TCAS[4]A-500
.
ion to the phenolic oxygen was found. The results of EDTA
A-500
2+

9
30
Vol. 61, No. 9
2
+
ion to TCAS[4] in Cu -TCAS[4]
was approximately 1:1
A-500
2+
and, although it is possible for Cu ions to coordinate to
the diethylaminoethyl {–CH CH NH(C H ) } groups of the
anion-exchangers, coordination of the Cu ions in this man-
ner was not actually observed to any extent. Accordingly, it
can be concluded that the active species responsible for the
catalytic activity of Cu -TCAS[4]
Cu -TCAS[4] complex on the modified anion-exchangers,
as shown in Fig. 1. Interestingly, coordination of Cu ion in
2
2
2
5 2
2
+
2+
is the mononuclear
A-500
2+
2+
2
+
2+
Cu -TCAS[4]
was very different from those in the Cu
A-500
complexes of TCAS[4] and t-Bu-TCA[4].
As described above, the molar ratio of Cu ion to TCAS[4]
2+
Fig. 5. Effects of Repeated Use on the Activity of Cu2+-TCAS[4]
A-500
(100 µmol/g, 20mg) for the Oxidation of 3.0µmol AsA at pH 7.0
2+
in Cu -TCAS[4]
was very different from those in the
A-500
2+
Cu complexes of TCAS[4] and t-Bu-TCA[4] with a cone-like
confomation. In general, TCAS[4] and t-Bu-TCA[4] unmodi-
fied on their lower-rims are known to be conformationally
flexible in solutions and take a certain preferred conformation,
such as ‘cone’ and ‘partial cone,’ depending on the orienta-
The conditions are the same as those in the text.
2
)
2+
tion of each aryl group. Accordingly, in the Cu complexes
of TCAS[4] and t-Bu-TCA[4], this flexibility in conformation
2+
will make it possible to coordinate plural Cu ions to one
2+
ligand. However, the TCAS[4] in Cu -TCAS[4]
should
A-500
be conformationally rigid because it is tightly loaded on the
anion-exchangers through ionic-bonding between the –SO3−
groups of TCAS[4] and the diethylaminoethyl groups. So, the
flexibility in conformation of TCAS[4] in solution or on the
anion-exchangers will affect the coordination of Cu ions to
TCAS[4].
Fig. 6. Proposed Reaction Mechanism for the Activity of Cu2+-
TCAS[4]A-500
2+
2
+
Additionally, anion-exchangers modified with Me-CAS[n] Cu -TCAS[4]
not only shows high catalytic activity for
A-500
(
Me-CAS[n]A-500; n=4, 6, and 8) were investigated. Although reaction 1 but may also be used repeatedly while maintaining
each CAS[n] (n=4, 6, and 8) was loaded on the anion- that pronounced catalytic activity. 2
+
exchanger, it was not possible to prepare Me-CAS[n]
To further examine whether Cu -TCAS[4]
truly cata-
A-500
A-500
n+
4+
because none of the Me ions, with the exception of Ce , lyzes reaction 1, the dehydroascorbic acid produced in the
would coordinate to the calix[n]arene moiety of CAS[n]A-500
.
reaction solution was analyzed colorimetrically using 2,4-dini-
This is the reason why calix[n]arenes such as CAS[n] (n=4, trophenylhydrazine, a method which allows AsA and dehydro-
2
6,27)
6
, and 8) do not exhibit the ability to form stable metal com- ascorbic acid to be independently determined.
The results
plexes without modification of their upper- or lower-rims, indicated 2.94µmol dehydroascorbic acid in a reaction solu-
while TCAS[4] shows this ability to a remarkable extent. It tion which had originally contained 3.0µmol AsA, indicating
4
+
was further determined that Ce -CAS[n]
(n=4, 6, and 8) that reaction 1 was indeed catalyzed quantitatively in a man-
A-500
showed no catalytic activity for the oxidation of AsA at high ner analogous to the action of ascorbate oxidase. As shown in
2
+
concentrations (data not shown).
Fig. 6, it is reasonable to expect that Cu -TCAS[4]
exhib-
A-500
2+
2
+
+
Catalytic Activity of Cu -TCAS[4]
As an initial its its catalytic activity through the redox cycle Cu ⇋Cu ,
A-500
step, the effects of several factors on the catalytic activity because AsA is a powerful reductant. The O dissolved in
2
2
+
of Cu -TCAS[4]
were investigated, based on tests using the reaction solution will likely play a role in the subsequent
to oxidize 3.0µmol AsA in pH 7 buffered oxidation of Cu -TCAS[4]A-500 back to Cu -TCAS[4]A-500, and
solutions. The absorbance at 265nm was observed to fall to consequently will be reduced to O . In fact, a very small
A-500
2+
+
2+
Cu -TCAS[4]
A-500
−
2
almost zero at temperatures between 30 and 50°C after 30min amount of H O was determined in the reaction solutions,
2
2
−
of incubation time, indicating complete oxidation of the AsA which would be produced through the dismutation of O as
2
2
+
−
+
by Cu -TCAS[4]
over this temperature range.
follows: 2O +2H →O +H O . So far, catalytic activities
A-500
2
2+
2
2
2
2+
2+
Next, to determine whether Cu -TCAS[4]
can be of free Cu ion and Cu complexes for oxidation of AsA
A-500
2
8–30)
used repeatedly and continue to show unreduced catalytic have been investigated.
activity for reaction 1, the effects of repeated use on the ac- tion to produce dehydroascorbic acid and H O . Jiang et al.
tivity was investigated. In these trials, the same sample of demonstrated that when free Cu ion was in solution, the
was reused a number of times after being hydroxyl radical ( OH) was formed through the decomposition
separated from the reaction mixture, washed with water and of H O produced, interestingly any formation of OH was not
dried. As shown in Fig. 5, the oxidative activity of the sample found in the Cu complexes. This is because free Cu ion
All of them catalyzed the reac-
2
8)
2
2
2+
2
+
•
Cu -TCAS[4]
A-500
•
2
2
2
+
2+
was maintained after six repeated uses, although its efficiency is known to catalyze the decomposition of H O through the
2
2
decreased by approximately 20%. Moreover, unlike the Me- Harber–Weiss reaction and/or the Fenton reaction producing
4
+
3+
2+
•
31)
2+
TCAS[4]A-500 (Me=Ce and Mn ), Cu -TCAS[4]
was
OH. Considering Cu -TCAS[4] on the anion-exchangers
A-500
very stable and remained a yellow-brown in color without is very stable even in the presence of the excess AsA, no
fading even after repeated uses. It is therefore evident that formation of OH through the decomposition of H O by free
•
2
2

September 2013
931
Table 1. Effects of AsA on the Determination of Glucose Using the POD/GOD Method
AsA (mg/mL)
0
Cu2+-TCAS[4]
(mg)
Molar ratio (glucose:AsA)
Glucose obsd. (µmol)
Recovery (%)
A-500
0
—
5.55
4.05
1.73
5.37
5.39
4.91
4.50
100
a)
0.50
0.75
1.00
1.50
2.00
3.00
0
1.00:0.51
1.00:0.77
1.00:1.02
1.00:1.54
1.00:2.05
1.00:3.07
73.0
31.1
96.7
97.2
88.5
81.1
a)
0
20
20
20
20
Glucose added: 1.00mg/mL (5.55µmol/mL). a) Treated without Cu +-TCAS[4]A-500
2
.
Cu ion derived from Cu 2+-TCAS[4]A-500 would be found in
2+
this study. Moreover, Taqui Kahn and Martell suggested that
+
2+
the produced H O played a role on oxidation of Cu to Cu
2
2
2
9)
in Cu complexes. Based on these results, also in this study
the produced H O would be used for the oxidation of Cu -
TCAS[4]A-500 to Cu -TCAS[4]A-500, and consequently such an
+
2
2
2+
efficient redox cycle will further accelerate the oxidation of
2
+
AsA by Cu -TCAS[4]
.
A-500
2
+
Application of Cu -TCAS[4]
to the GOD/POD
A-500
Method The elimination of interference by AsA is very
important in clinical analyses, because AsA frequently affects
the results of methods using redox enzymes and may con-
sequently lead to erroneous diagnostic results. Accordingly,
2+
we investigated the application of Cu -TCAS[4]
to the
A-500
Fig. 7. Effects of the Activity of Cu2+-TCAS[4]
tion of Glucose Using the GOD/POD Method in the Presence of AsA
for the Determina-
A-500
elimination of AsA interference during the determination of
glucose using the GOD/POD method. In this study, 4-AAP
and phenol were applied as the chromogen in reaction (3), and
●
, without AsA; ○,1.50mg/mL AsA; □, 3.00mg/mL AsA.
Table 2. Effects of Cu2+-TCAS[4]A-500 on Pretest 4bII
Color change after being dipped
the quinoid dye thus produced had its λ
at 505nm. Table
max
2+
1
shows the effects of Cu -TCAS[4]
on the interference
A-500
2+
by AsA for the determination of glucose. Without Cu -
TCAS[4]A-500, severe interference by AsA was observed, as
shown by the observed glucose recoveries which decreased to
Inspection items
Before use
Withouta) Cu2+
With Cu2+
a)
-
-
TCAS[4]A-500
TCAS[4]A-500
73.0 and 31.1% in the presence of 0.50 and 0.75mg of AsA,
Glucose
Protein
pH
Yellow
Yellow
Green
respectively, at glucose to AsA molar ratios of 1:0.51 and
2+
Pale green
Orange
Blue-green
Orange
Blue-green
Orange
1
:0.77. Incorporating Cu -TCAS[4]
produced satisfac-
A-500
tory results without interference by AsA, even in the presence
of 1.50mg of AsA (glucose:AsA=1:1.54). Even in solutions
containing over 2.00mg of AsA, the concentration of glucose
could be satisfactorily determined without interference by
AsA simply by increasing the incubation time.
AsA
Navy blue
Orange
Navy blue
a) The amounts of glucose, protein, and AsA in sample solution were 100mg/dL,
1
0mg/dL, and 80mg/dL, respectively, and pH of sample solution was 5.0.
2+
The effects of Cu -TCAS[4]
on calibration curves for paper and are impregnated with reagents whose color changes
A-500
the determination of glucose in the presence of AsA are pre- indicate some characteristic of the specimen. For example,
sented in Fig. 7. In the presence of 1.50mg of AsA, almost no Pretest 4bII is routinely used to test urine samples for glucose,
interference by AsA is observed, even at the low 0.25mg/mL protein, pH and AsA, as shown in Fig. 2. Since the redox en-
glucose concentration (glucose to AsA molar ratio of 1:6.13). zymes GOD and POD used for the analysis of glucose will be
However, in the presence of 3.00mg of AsA, relatively se- affected by any reductant such as AsA, the amount of AsA in
vere interference was observed, especially at lower glucose a sample will normally be ascertained, so as not to produce
concentrations, such as 0.50 and 0.25mg/mL, at which the erroneous results for glucose.
2
+
molar ratios were 1:6.13 and 1:12.26, respectively. As such,
satisfactory results were not always obtained in the presence changes of Pretest 4bII strips associated with sample solutions
of 3.00mg of AsA.
containing glucose (100mg/dL), protein (10mg/dL of HSA)
Based on these results, the catalytic activity of Cu - and AsA (80mg/dL) at pH 5. Without pretreatment of the sam-
Table 2 shows the effects of Cu -TCAS[4]
on the color
A-500
2+
2+
TCAS[4]A-500 is applicable to the elimination of interference ple solution with Cu -TCAS[4]A-500, the test strip colors for
by AsA in place of ascorbate oxidase, during the determina- glucose and AsA were yellow and orange, respectively. The
tion of compounds such as glucose and uric acid using redox color for glucose remained unchanged in spite of a 100mg/
enzymes.
Application of Cu -TCAS[4]
dL glucose concentration in the sample solution, indicating
to Pretest 4bII In that glucose was not properly determined because of the in-
2
+
A-500
clinical analyses, blood or urinary analysis is usually carried terference by AsA. However, when the solution was pretreated
2+
out using several types of dipsticks, which are usually made of with Cu -TCAS[4]A-500, the color for glucose changed to

9
32
Vol. 61, No. 9
1
0) Iki N., Kabuto C., Fukushima T., Kumagai H., Takeya H., Miyanari
green, which corresponds exactly to the true concentration of
00mg/dL glucose, and thus the glucose level was determined
S., Miyashi T., Miyano S., Tetrahedron, 56, 1437–1443 (2000).
1
11) Iki N., J. Incl. Phenom. Macrocycl. Chem., 64, 1–13 (2009).
precisely, even in the presence of AsA. Moreover, the color
for AsA changed to navy blue, which suggests complete oxi-
dation of the AsA to dehydroascorbic acid, meaning that the
interference by AsA was totally eliminated by the activity of
12) Matsumiya H., Iki N., Miyano S., Hiraide M., Anal. Bioanal.
Chem., 379, 867–871 (2004).
13) Odo J., Kawahara N., Inomata Y., Inoue A., Takeya H., Miyanari S.,
Kumagai H., Anal. Sci., 16, 963–966 (2000).
Odo J., Yamaguchi H., Ohsaki H., Ohmura N., Chem. Pharm. Bull.,
52, 266–269 (2004).
2+
Cu -TCAS[4]A-500.
14)
In conclusion, among the modified anion-exchange materi-
2+
als with Me-TCAS[4] tested, only Cu -TCAS[4]
showed 15) Odo J., Matsumoto K., Shinmoto E., Hatae Y., Shiozaki A., Anal.
A-500
high catalytic activity for the oxidation of AsA to dehydro-
Sci., 20, 707–710 (2004).
6) Odo J., Inomata Y., Takeya H., Miyanari S., Kumagai H., Anal. Sci.,
7, 1425–1429 (2001).
17) Odo J., Shinmoto E., Shiozaki A., Hatae Y., Katayama S., Jiao G., J.
Health Sci., 50, 594–599 (2004).
2+
1
ascorbic acid. Since Cu -TCAS[4]
showed catalytic activ-
A-500
1
ity for reaction 1, a process normally catalyzed by ascorbate
2+
oxidase, Cu -TCAS[4]
is applicable as an artificial sub-
A-500
stitute for ascorbate oxidase, and will be useful for the elimi-
nation of AsA interference during the analysis of important
vital compounds, such as glucose and uric acid, in clinical
analyses.
18) Kroneck P. M. H., Armstrong F. A., Merkle H., Marchesini A., Adv.
Chem. Ser., 200, 223–248 (1982).
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ed. by Kanai I., Kanai M., Kanehara and Co., Ltd., Tokyo, 1998.
2
0) Martinello F., Luiz da Silva E., Clin. Chim. Acta, 373, 108–116
Acknowledgment The authors wish to thank the Cosmo
(2006).
Research Institute for kindly supplying sodium thiacalix[4]- 21) Messerschmidt A., Ladenstein R., Huber R., Bolognesi M., Avi-
arenetetrasulfonate.
gliano L., Petruzzelli R., Rossi A., Finazzi-Agro A., J. Mol. Biol.,
2
24, 179–205 (1992).
2
2
2
2) Wu M., Yuan D., Huang Y., Wei W., Gao Q., Jiang F., Hong M.,
Cryst. Growth Des., 7, 1446–1451 (2007).
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Commun., 7, 467–470 (2004).
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