Stabilisation of copper(i) polypyridyl complexes toward aerobic oxidation by zinc(ii) in combination with acetate anions: a facile approach and its application in ascorbic acid sensing in aqueous solution

Article abstract of DOI:10.1039/C8DT03580F

A new and facile approach to stabilise copper(i) complexes in aqueous solution by the addition of zinc(ii) ions in combination with acetate ions (OAc^?) was demonstrated. This stability enhancement toward the aerobic oxidation of copper(i) species was investigated by various techniques including UV-vis spectroscopy, ¹H-NMR, FT-IR, and ESI-MS. Our experimental results together with DFT calculations led to a proposed structure of [(adpa)Cu-OAc-Zn(OAc)(H₂O)₂]^+/2+. It was also postulated that zinc(ii) with its Lewis acidity may attract electrons from the Cu centre through the bridging ligands (OAc^?), resulting in the lower reactivity of Cu(i) with O₂. In addition, this strategy was shown to be applicable to ascorbic acid detection by monitoring a change in the redox states of copper complexes using fluorescence spectroscopy. Moreover, it was demonstrated that the method was sensitive and accurate for the quantitative analysis of ascorbic acid in vitamin C tablets.

Full text of DOI:10.1039/C8DT03580F

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PAPER
Stabilisation of copper(I) polypyridyl complexes
toward aerobic oxidation by zinc(II) in combination
with acetate anions: a facile approach and its
application in ascorbic acid sensing in aqueous
solution†
Cite this: Dalton Trans., 2019, 48,
997
Pattira Suktanarak,a Vithaya Ruangpornvisuti,a Chomchai Suksai,b
a,c
Thawatchai Tuntulania and Pannee Leeladee
*
A new and facile approach to stabilise copper(I) complexes in aqueous solution by the addition of zinc(II)
ions in combination with acetate ions (OAc−) was demonstrated. This stability enhancement toward the
aerobic oxidation of copper(I) species was investigated by various techniques including UV-vis spec-
troscopy, 1H-NMR, FT-IR, and ESI-MS. Our experimental results together with DFT calculations led to a
proposed structure of [(adpa)Cu-OAc-Zn(OAc)(H2O)2]+/2+. It was also postulated that zinc(II) with its
Lewis acidity may attract electrons from the Cu centre through the bridging ligands (OAc−), resulting in
the lower reactivity of Cu(I) with O2. In addition, this strategy was shown to be applicable to ascorbic acid
detection by monitoring a change in the redox states of copper complexes using fluorescence spec-
troscopy. Moreover, it was demonstrated that the method was sensitive and accurate for the quantitative
analysis of ascorbic acid in vitamin C tablets.
Received 3rd September 2018,
Accepted 12th December 2018
DOI: 10.1039/c8dt03580f
rsc.li/dalton
Ascorbic acid (vitamin C, AsH2) is an essential antioxidant
playing a vital role in biological processes. In addition, the
Introduction
Copper is an abundant and redox-active metal. It is frequently AsH2 level positively correlates with several diseases including
employed as a redox catalyst in a wide range of both synthetic cardiovascular disease, viral infection and Alzheimer’s disease
and biological reactions.1–4 In nature, copper containing as well as anticancer therapy.13–16 On the other hand, the
enzymes serve as mediators for substrate conversion. Typically, lower level of AsH2 can lead to the development of diabetes or
Cu(II) centres in the enzymes will be reduced to Cu(I) reactive cognitive impairment.17–19 Also, AsH2 has been used as an
species for further catalytic processes. Interestingly, there are additive substance in food, beverages and pharmaceutical for-
several enzymes which can generate Cu(I) active intermediates mulations for oxidative protection. Because of its advantages
using ascorbic acid (vitamin C, AsH2) as a reducing agent in food quality control and healthcare, the development of a
such as dopamine β-hydroxylase (DβH), peptidylglycine simple method for AsH2 detection has gained considerable
α-hydroxylating monooxygenase (PHM) and ascorbate oxidase attention. Recently, a number of research studies reported
(AO).5,6 Likewise, synthetic metal complexes show high poten- sensors for AsH2 based on materials such as quantum dots,
tial in AsH2 oxidation.7–12 Hence, redox active metals including Au nanoparticles and C-dots/Fe(III).20–26 However, these
copper are potentially applicable as a sensor probe for AsH2 by materials could be suffering from reproducibility and charac-
monitoring the changes in the redox state of the metal.
terisation. On the other hand, molecular probes (e.g., metal
complexes) can be characterised in terms of molecular struc-
tures, chemical properties and mechanistic studies with
routine spectroscopic techniques. In addition, these methods
are quite repeatable.
aDepartment of Chemistry, Faculty of Science, Chulalongkorn University,
Bangkok 10330, Thailand. E-mail: pannee.l@chula.ac.th
bDepartment of Chemistry and Centre for Innovation in Chemistry,
Faculty of Science, Burapha University, Chonburi 20131, Thailand
cResearch Group on Materials for Clean Energy Production STAR, Department of
Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8dt03580f
Unfortunately, the utilisation of Cu complexes in the field
of molecular sensors via a redox reaction (Cu(II) → Cu(I)) is
usually hampered by the instability of the Cu(I) species in
aqueous solution under aerobic conditions.27,28 Therefore,
new strategies for the stabilisation of the active species (Cu(I))
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species in concomitance with the oxidation of AsH2 as
evidenced by the observed production of dehydroascorbic acid
(Fig. S1 and S2†). It should be noted that the same result was
obtained when the reaction was carried out in the presence of
O2, and the Cu(I) species seemed to be stable for at least 5 h.
In contrast, when the solvent was changed to H2O/CH3CN
(7 : 3 v/v) under ambient conditions, CuI(adpa) could not be
completely formed and gradually reoxidised by air to Cu(II),
suggesting its instability in aqueous solution. However,
performing the reactions in aqueous solution is essential for
the detection of biological substances including natural
reducing agents owing to their solubility in water.
Chart 1 Ascorbic acid (AsH2) and copper complexes used in this study.
Cu(I) stability enhancement toward aerobic oxidation by the
modulation of the secondary coordination sphere
From the heteronuclear metalloenzymes in nature, we learn
that Zn2+ not only serves as a mediator for catalytic substrate
conversion, but also plays a crucial role in the stabilisation of
enzymes and proteins.38 A synthetic model for CuZnSOD
demonstrated the vital role of Zn2+ in controlling the redox
potentials of Cu(II) ions through the imidazole (Im) bridge for
the desired catalytic reaction.36 From this inspiration, we came
up with the idea that Zn2+ in combination with bridging
ligands may help to facilitate Cu(II) reduction and stabilise our
Cu(I) species in aqueous solution under aerobic conditions.
Thus, we first proved our assumption by the addition of Zn2+
and Im in the reaction of CuII(adpa) + AsH2 in H2O/CH3CN
(7 : 3 v/v). As a result, it seemed that the reduction of Cu(II) to
Cu(I) was facilitated as the Cu(II) d–d band was dramatically
decreased when monitored by UV-vis spectroscopy. However,
the reaction started to be cloudy after 15 min which hampered
a prolonged monitoring of this reaction. Then, we also tried to
add histidine (His) instead of Im, and found that Cu(II) was
fully reduced to Cu(I). However, His was not well-dissolved in
our solvent system. It should be noted that His contains not
only the Im side chain, but also a carboxyl group which can
serve as a bridging ligand. Therefore, we sought for other
alternatives, and thought that acetate anions (OAc−) might be
a good candidate since they have been demonstrated as a
bridge between two metal centres in dinuclear complexes.39–43
To test our hypothesis, the reduction of CuII(adpa) by AsH2
(1 mol equiv.) in H2O/CH3CN (7 : 3 v/v) was performed under
aerobic conditions in the presence of 8 mol equiv. of Zn(OAc)2,
Zn(NO3)2 or Zn(phen)2. Fig. 2 indicates that the d–d transition
band of Cu(II) (λmax = 632 nm) completely disappeared upon
the addition of AsH2 only in the presence of Zn(OAc)2, and the
spectrum was stable for at least 60 min before the d–d band
started to come back, implying the regeneration of Cu(II). This
Fig. 1 Proposed acetate-bridged Zn(II)–Cu(I/II)adpa complex and the
mechanism for AsH2 detection.
are of particular importance for developing a simple, rapid
and accurate sensor for the detection of antioxidants. It has
been demonstrated that copper(I) can be stabilised by metal–π
interactions relying on the ligand attributes with conjugated π
systems or by coordinating solvents (i.e. acetonitrile).29–32 We
initially tried to stabilise Cu(I) using acetonitrile as the solvent
due to its facile procedure. Unfortunately, our Cu(I) complex,
CuI(adpa) (Chart 1) was not stable in aqueous solution despite
the presence of acetonitrile.
Herein, we report a new approach to stabilise Cu(I) polypyri-
dyl complexes toward aerobic oxidation by Zn(II) ions in combi-
nation with acetate anions (OAc−) as shown in Fig. 1. We pro-
posed that the positively-charged Zn2+ may stabilise Cu(I) by
attracting electrons through acetate bridging ligands. This sec-
ondary coordination sphere modulation was inspired by an
imidazolate-bridged Cu–Zn model for superoxide dismutase
(CuZnSOD) enzymes.33–37 Our simple and easy approach was
then applied for the accurate detection of AsH2 in vitamin C
tablets with good sensitivity. To the best of our knowledge,
this is the first example of copper complexes as fluorescent
probes via a reversible redox reaction for AsH2 detection.
Results and discussion
Reaction of the Cu(II) complex and ascorbic acid
To establish distinct spectroscopic patterns between Cu(I) and could suggest that Zn(OAc)2 can help facilitating the reduction
Cu(II) species and confirm the successful reaction of our of CuII(adpa) and stabilising the Cu(I) species as well.
copper complexes and AsH2, the reaction of CuII(adpa) and Moreover, the reversible d–d band of Cu(II) (Fig. S4†) con-
AsH2 was initially performed under a N2 atmosphere in firmed that no transmetalation occurred between Zn2+ and
CH3CN. Upon the addition of AsH2 to the solution of Cu(adpa) during the redox reaction of Cu(adpa) in the
CuII(adpa), a color change from blue to pale yellow was presence of Zn(OAc)2.
observed. Monitoring of this reaction by UV-vis spectroscopy
and 1H-NMR confirmed that CuII(adpa) was reduced to Cu(I) CuI(adpa) was highlighted by comparative reactions using
Furthermore, the importance of Zn2+ in stabilising
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It is worth mentioning that the influence of a Lewis acid on
the redox potential of the metal centre has been previously
demonstrated.44–46 Therefore, the electrochemical behaviours
of our Cu(I)/(II)(adpa) redox couple in the absence and pres-
ence of Zn(OAc)2 were studied by cyclic voltammetry. It was
found that the reduction potential of CuII(adpa) did not
change significantly after adding Zn(OAc)2, but Cu(I) seemed
to be reoxidized less than that in the absence of Zn(OAc)2. In
addition, when the supporting electrolyte was changed from
an acetic–acetate buffer to a non-coordinated electrolyte (pot-
assium hexafluorophosphate, KPF6), a similar result was
obtained. These electrochemical results might suggest that the
reoxidation of Cu(I) to Cu(II) is more difficult in the presence of
Zn(OAc)2.
To rule out the possibility that the pH change caused by
Lewis acid Zn(II) ions may be responsible for the stability of
Cu(I), a comparative study in the buffered solvent (acetic–
acetate buffer solution, pH 5.6) was carried out (Fig. S18†). In
the reaction of CuII(adpa) + AsH2 in the absence of Zn(II), Cu(I)
was not fully formed and gradually reoxidised to Cu(II) within
20 min. On the other hand, in the presence of Zn(II) under the
same conditions, the Cu(I) complex was stabilised up to
40 min.
Fig. 2 UV-vis spectra of (a) 2 mM CuII(adpa); (b) CuII(adpa) + AsH2
(1 mol equiv.); CuII(adpa) + AsH2 (1 mol equiv.) in the presence of 8 mol
equiv. (c) Zn(NO3)2, (d) Zn(phen)2, and (e) Zn(OAc)2 in H2O/CH3CN (7 : 3
v/v) at room temperature.
other redox inactive cations with OAc− i.e., Na+, Mg2+ and Ca2+
instead of Zn2+ (Fig. 3 and S7†). It was found that in the
presence of such metal ions, the Cu(II) d–d band was suddenly
decreased upon the addition of AsH2, suggesting that all metal
ions with OAc− are likely to facilitate the Cu(II) reduction by
AsH2. However, all Cu(II) spectra gradually returned after
5 min, except the one with Zn(OAc)2 of which the Cu(I) species
could be stabilised for at least 60 min. Therefore, it is conclus-
ive that both Zn2+ and OAc− are required to facilitate the
reduction of the Cu(II) complex and stabilise the Cu(I) redox
state. We also proposed that Zn2+ as a good Lewis acid may
help to stabilise Cu(I) by the delocalisation of electrons
through the OAc− bridge, analogous to an imidazolate-bridged
Cu–Zn model for CuZnSOD.36
Proposed structure of [(adpa)Cu-OAc-Zn(OAc)(H2O)2]+/2+
To shed some light on how Zn(OAc)2 helps to stabilise the CuI
species, we sought to investigate the interaction between
CuII(adpa) and Zn(OAc)2 by spectroscopic techniques, ESI-MS,
and DFT calculations which led to a proposed structure of
[(adpa)Cu-OAc-Zn(OAc)(H2O)2]+/2+. To begin with, our first
structural evidence on the proposed structure came from FT-IR
analysis. Since the carboxylate group can coordinate to the
metal ions in various ways, a number of research studies exam-
ined the vibrational frequencies of acetate salts to correlate
with their structure and employed this relationship to predict
the coordination mode of OAc− in metal complexes.41,42,47
Deacon and Phillips41 reported that the frequency separation
between the COO− antisymmetric and symmetric stretching
vibrations or the Δνa–s values for the species with different
binding modes usually fall in the following order:
ΔνaꢀsðunidentateÞ > ΔνaꢀsðionicÞ ꢁ ΔνaꢀsðbridgingÞ
> ΔνaꢀsðbidentateÞ:
It should be noted that OAc− has been reported as a brid-
ging ligand in both homo- and heterometallic complexes.39–43
The FT-IR spectrum of our precipitate obtained from the reac-
tion between CuII(adpa) and Zn(OAc)2(H2O)2 is shown in
Fig. 4e in comparison with those of Zn(OAc)2(H2O)2 (biden-
tate), CuII(adpa), Na(OAc) (ionic), and a solid mixture of
Zn(OAc)2(H2O)2 + CuII(adpa). It can be seen that our precipi-
tate from the reaction showed two new peaks at 1561 and
1385 cm−1 which could be assigned to asymmetric and sym-
metric COO− stretching, respectively. Since these new signals
did not match with the starting material Zn(OAc)2(H2O)2 and
free OAc− (ionic), it suggested that OAc− might form a new
Fig. 3 Change in the absorbance of Cu(II) for the reactions of 2 mM
CuII(adpa) with AsH2 (1 mol equiv.) in the presence of various redox
inactive cations as a function of time. Each reaction was performed in
H2O/CH3CN (7 : 3 v/v) at room temperature.
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ence of Cu(II) than that in the CuII(adpa) + Na(OAc) solution.
In addition, the ESI-MS data clearly showed that OAc− from
Zn(OAc)2(H2O)2 could bind to Cu(II) (see the ESI†). All of these
results support the finding that the formation of [(adpa)Cu-
OAc-Zn(OAc)(H2O)2]2+ is feasible.
In the case of the Cu(I) species, the reaction of CuII(adpa) +
Zn(OAc)2(H2O)2 and AsH2 gave rise to a prominent signal at
m/z 512.1356 in the ESI-MS spectrum, corresponding to
[CuI(adpa) + (OAc−) + H+]+. It might be implied that OAc−
could coordinate to the Cu(I) centre in a similar manner to
that found in the Cu(II) compound, CuII(adpa). Unfortunately,
the interaction of the Cu(I) complex and Zn(OAc)2(H2O)2 could
not be investigated by other spectroscopic techniques due to
the limitations of our system. Therefore, we turned our atten-
tion to studying this interaction and proposing the structure
using DFT calculations. As a result, a DFT-optimised structure
of [(adpa)Cu-OAc-Zn(OAc)(H2O)2]+, together with its frontier
orbitals, was obtained as shown in Fig. 5 and Fig. S13–15.†
Markedly, a significant change in the highest occupied mole-
cular orbital (HOMO) delocalisation on the copper(I) centre in
the presence and absence of Zn(OAc)2(H2O)2 was noted. In
[(adpa)Cu-OAc-Zn(OAc)(H2O)2]+, the HOMO orbital is deloca-
lized to the Cu(I) centre substantially less than that of the Cu
complex without Zn(OAc)2(H2O)2. This result is consistent with
our experimental finding that the Cu(I) complex is significantly
less reactive toward O2 in the presence of Zn(OAc)2. However,
when there is only OAc− bound to the Cu(I) centre without
Zn2+, the HOMO is delocalised around the Cu(I) ion and its
coordination site which is in agreement with our reactivity
study that CuI(adpa) + NaOAc seemed to react with O2 to
Fig. 4 Comparison of the FT-IR spectra of (a) Zn(OAc)2(H2O)2; (b) solid
mixture of Zn(OAc)2(H2O)2 + CuII(adpa); (c) Na(OAc); (d) CuII(adpa) and
(e) precipitate from the reaction of CuII(adpa) + Zn(OAc)2(H2O)2.
coordination. Notably, the shifts in these vibrational frequen-
cies are quite similar to those observed for acetate bridging in
several carboxylate-bridged copper complexes (e.g. Cu–Cu,
Cu–Ca).42,48–50 The Δνa–s value of 176 cm−1 from our sample is
quite close to that from the heterometallic carboxylate-bridged
Cu–Ca complex (180),42 but significantly different from that of
bidentate OAc− in Zn(OAc)2(H2O)2 (Δν = 114). These data
pointed that OAc− might serve as a bridging ligand between
Zn2+ and CuII(adpa). To support this hypothesis, an optimised
structure of [(adpa)Cu-OAc-Zn(OAc)(H2O)2]2+ was obtained
from the DFT calculations with the CPCM(UFF)/B3LYP/6-
311+G(d,p) basis set. It was found that the O–C–O angle of the
acetate bridge was 121.8° which is relatively close to that of the
heterometallic bidentate acetate bridge of the Cu–Ca complex
(121.5(3)°),43 Zn–Ca (122.9, 123.9 and 124.3°) and Zn–Mg
(123.9 and 124.9°).39
To gain further support for the proposed structure of
[(adpa)Cu-OAc-Zn(OAc)(H2O)2]2+ in the solution phase, we also
monitored the CuII(adpa) + Zn(OAc)2(H2O)2 reaction by UV-vis
1
spectroscopy and H-NMR. Interestingly, upon the addition of
Zn(OAc)2(H2O)2 to CuII(adpa), a slight shift in the d–d band of
1
Cu(II) and a significant change in the H-NMR signal of OAc−
were observed. The 1H-NMR signal of OAc− in CuII(adpa) +
Zn(OAc)2(H2O)2 was broadening and shifted to 2.6 ppm,
indicating the coordination of OAc− to Cu(II). For comparison,
1
when Na(OAc) was added to CuII(adpa), the H-NMR signal of
CH3COO− (OAc−) could not be observed (Fig. S8†). This
suggested the difference in the coordination of OAc− between
these two systems. As a bridging ligand between the Zn(II) and
Cu(II) complex, OAc− may have a weaker paramagnetic influ-
Fig. 5 Plots of the HOMOs of (a) CuI(adpa) and (b) CuI(adpa)/Zn
(OAc)2·(H2O)2, computed at the CPCM(UFF)/B3LYP/6-311+G(d,p) level of
theory.
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generate the Cu(II) product. We account for the stability of
CuI(adpa) in the presence of Zn(OAc)2 by the role of Zn2+ as a
good Lewis acid. Due to its electron-withdrawing properties,
Zn2+ may help to attract electrons from the Cu(I) active site via
the acetate bridge, leading to a less electron density on the Cu
centre. This rationale was consistent with the fact that
CuI(adpa) + Zn(OAc)2 exhibited less reactivity toward O2 when
compared to CuI(adpa) itself. Given the proposed structure, we
also speculate that the bridge through OAc− and Zn2+ may
help to provide a steric encumber which inhibits the oxidizing
agent (e.g. O2) to react with the Cu(I) centre.
All of these results are in agreement with our proposal that
OAc− coordinated to the copper centre and could serve as a
bridging ligand between the Cu(I)/(II) and Zn2+ ions resulting
in the stabilisation of the Cu(I) species under aerobic con-
ditions. Next, this approach was tested for its applicability to
AsH2 sensing.
Fig. 7 (a) Selectivity and (b) interference study for AsH2 detection using
our sensor; (c) fluorescence spectral change upon the addition of AsH2
to Cu(atpa) + Zn(OAc)2; and (d) detection of ascorbic acid in vitamin C
tablets. The concentration of Cu(atpa) = 10 μM, Zn(OAc)2 = 40 mol
equiv. and λex = 340 nm.
Application in ascorbic acid sensing by fluorescence
Our approach was applied to detect AsH2 by fluorescence spec-
troscopy because of its high sensitivity. As a molecular sensor,
Cu(adpa) contains a dipicolylamine Cu active site and anthra-
cene as a sensory unit. A change in the redox states of the Cu
centre would give a different fluorescence spectrum. As
expected, CuII(adpa) did not show fluorescence signals owing
to the disturbance of the paramagnetic species, Cu(II).51 The
To decrease the influence of the chelation of Cu ions by
analytes and to expand the pH range of our detection, we
decided to prepare CuII(atpa) (Chart 1) for our further studies.
As a tetradentate ligand, atpa was expected to bind Cu ions
more tightly than a tridentate ligand, adpa. Hence, copper
sequestration by glutathione will be less pronounced. Our
results confirmed this assumption as shown in Fig. 7. In
addition, our limit of detection (LOD) for AsH2 was deter-
mined to be 163 nM which is relatively low (3σ/S; σ is the stan-
dard deviation for the blank solution, n = 10, and S is the
slope of the calibration curve).
Moreover, our approach was then employed to measure the
AsH2 amount in vitamin C tablets to verify our accurate detec-
tion in real samples. The measurements by our sensor,
CuII(atpa), in comparison with HPLC were done using the
standard addition method. The result from our method was in
good agreement with that from standard HPLC and was close
to the amount specified on the vitamin C tablets. Our recovery
was in between 101 and 104, and the relative standard devi-
ation was 1.5–4.0. This demonstrated the high accuracy of our
method.
addition of AsH2 resulted in
a significant fluorescence
enhancement at 421 nm which is consistent with the conver-
sion of Cu(II) to a diamagnetic Cu(I) complex as illustrated in
Fig. 6.
Because ascorbic acid is a hexanoic sugar acid with two
acidic protons (pKa 4.04 and 11.34),21 the effects of pH and
interferences from common natural reducing agents (i.e., redu-
cing sugar, citric acid, and glutathione) were investigated next.
The results showed that pH had a large influence on our AsH2
detection in terms of both sensitivity and selectivity
(Fig. S19†). At pH 4, a satisfactory fluorescence intensity and
high selectivity for AsH2 detection were achieved. However, the
selectivity was significantly lower at a higher pH due to Cu
chelation by glutathione.52–56
Conclusions
In conclusion, we presented a new strategy to stabilise Cu(I)
complexes. Our experiments demonstrated that a combination
of Zn2+ ions and OAc− can stabilise Cu(I) species in aqueous
solution under aerobic conditions. This approach was sub-
sequently applied to detect AsH2 in vitamin C tablets with high
accuracy. Our strategy would also attract interest in other areas
such as catalysis for the control of stability and reactivity of
metal complexes for mechanistic investigation. Undoubtedly,
this approach would pave the way for designing copper com-
plexes as molecular sensors for biological applications.
Fig. 6 Change in (a) the fluorescence signal (excitation wavelength =
340 nm, slit setting on the instrument = 10 and PMT = 500); and (b) UV-
vis spectrum upon the addition of AsH2 (1 mol equiv.) to the CuII(adpa)
solution in the presence of Zn(II) in H2O/CH3CN (7 : 3 v/v) buffered with
ABS at pH 5.6.
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When exposed to air, this pale-yellow species was found to be
stable for at least 5 h. An NMR sample was prepared as
follows: to a solution of CuII(adpa) (10 mM) in CD3CN (550 μL)
Experimental
Materials and methods
Solvents of HPLC grade were purchased from Merck and used was added AsH2 (20 μL, 0.55 mol equiv. dissolved in 15% D2O
without further purification. Milli-Q was prepared using ultra- in CD3CN). A color change from blue to pale yellow was noted,
pure water systems. Deuterated NMR solvents were purchased consistent with the formation of CuI(adpa). When compared
from Cambridge Isotope Laboratories, Inc. NMR experiments to a broad 1H-NMR spectrum of CuII(adpa), the pale-yellow
were carried out using a 400 MHz Varian Mercury spectro- species exhibited a sharp spectrum, supporting an assignment
meter. All UV-vis spectra were recorded by using a Varian Cary of diamagnetic d10 CuI species. An analysis of the 1H-NMR
50 probe UV-visible spectrophotometer with a quartz cuvette spectrum also revealed the formation of dehydroascorbic acid
(path length = 10 mm). Fluorescence spectra were obtained (an oxidized form of ascorbic acid) after the reaction. When
using a Varian spectrofluorometer with Cary Eclipse, a pulsed the reaction was carried out in the presence of O2, the same
xenon lamp and a photomultiplier tube detector. Mass spectra result was obtained.
were obtained by Electrospray Ionization Mass Spectrometry
(ESI-MS) on a Bruker Daltonics microOTOF. IR measurements
Generation of CuI species in the presence of Zn(OAc)2
were carried out on a Thermo, Nicolet 6700 FT-IR in ATR Unless otherwise noted, the following experiments were con-
mode. Cyclic voltammetry was performed on an μAutolab ducted under ambient conditions. A stock solution of AsH2
Type III potentiostat under an N2 atmosphere. This system (80 mM) was prepared in H2O/CH3CN (7 : 3 v/v). In a typical
contained a three-electrode cell: glassy carbon as the working reaction, a solution of CuII(adpa) (2.0 mM) was combined with
electrode, Pt wire as the counter electrode and Ag/AgNO3 Zn(OAc)2 (8.0 mol equiv.) in H2O/CH3CN (7 : 3 v/v). To this
(0.01 M) as the reference electrode. For HPLC analysis, the solution mixture was added the AsH2 stock solution (1.0 mol
amounts of ascorbic acid in vitamin C tablets were determined equiv.). A color change of the solution from green to pale
using a Varian Prostar with a C-18 column (C18 4.6 × 250 mm, yellow was observed. The reaction was also monitored by UV-
5 μm, Phenomenex), pumped at a flow rate of 1.5 mL min−1
.
vis spectroscopy, which revealed a complete conversion of the
Vitamin C tablets were purchased from PT Bayer Indonesia, spectrum for CuII(adpa) (λmax = 632 nm) to the spectrum for a
Depok, Indonesia. All copper complexes and ligands were pre- CuI complex (no d–d transition band). The CuI species seemed
pared according to modified published procedures.57–61
Caution! Perchlorate is
to be stable for at least 60 min. When Zn(OAc)2 was changed to
a
potentially explosive salt. Zn(NO3)2 or Zn(phen)2; however, the reduction was not com-
Experiments should be carefully handled.
pleted as the d–d band of CuII was present to a significant
extent. In fact, they gave the same result as observed in the
reaction without Zn(OAc)2. This indicated that the presence of
Computation methods
The density functional theory (DFT) method, the hybrid OAc− is necessary to help facilitate the reduction of CuII(adpa)
density functional theory of Becke’s three parameter exchange and the stabilisation of CuII(adpa).
functional62 with the Lee–Yang–Parr correlation functional63
Attempts at characterising this new Cu complex in the pres-
(B3LYP), using the 6-311+G(d,p) basis set64,65 was employed. ence of Zn(OAc)2 by mass spectrometry have also been made.
All structure optimizations in aqueous solution using the Samples for the ESI-MS analysis were prepared in 7 : 3 (v/v)
B3LYP/6-311+G(d,p) method combination with the solvent H2O/CH3CN. A prominent peak at a mass-to-charge ratio (m/z)
effect of the polarizable continuum model (PCM)66–72 using of 511.1332 (calc. m/z 511.13) was observed in the reaction of
the CPCM (conductor-like PCM, water as a solvent) model73–75 CuII(adpa) + Zn(OAc)2(H2O)2 (1 : 8 mol equiv.), corresponding
with the UFF molecular cavity model,64 called the CPCM(UFF)/ to [CuII(adpa) + OAc]+. On the other hand, m/z at 512.1356
B3LYP/6-311+G(d,p) level of theory, were carried out. All calcu- (calc. m/z 512.14) which corresponded to [CuI(adpa) + (OAc−) +
lations were performed with the Gaussian09 program.76 The (H+)]+ was found when the solution mixture of CuII(adpa) + Zn
molecular graphics for relevant compounds and their frontier (OAc)2(H2O)2 was added to AsH2 (5 mol equiv.). This supported
orbitals (HOMOs and LUMOs) were plotted and visualised the possibility of OAc− being coordinated to Cu(II/I)adpa.
using the GaussView 5.0.9 program.77
In addition to ESI-MS, the coordination of Zn(OAc)2(H2O)2
on the CuII centre was also investigated by IR spectroscopy.
The solid sample for IR analysis was prepared from the reac-
Reaction of CuII(adpa) and ascorbic acid (AsH2) in CH3CN
All solvents were deoxygenated prior to use by purging N2 for tion of CuII(adpa) and Zn(OAc)2·(H2O)2 (8 mol equiv.) in 7 : 3
at least 1 h. A stock solution of AsH2 was prepared in a solvent (v/v) H2O/CH3CN. After being stirred for around 10 min, the
mixture of 5% H2O in CH3CN (44 mM). To a solution of solution mixture was precipitated under reduced pressure to
CuII(adpa) (2.0 mM) in CH3CN was added the AsH2 stock solu- remove CH3CN and obtain a dark green solid. The solid was
tion (0.55 mol equiv.). A color change of the solution from filtered for subsequent analysis by IR spectroscopy in the ATR
blue to pale yellow was observed. Monitoring of this reaction mode.
by UV-vis spectroscopy showed a complete reduction of
An NMR sample was prepared as follows: a solution of
CuII(adpa) (λmax = 591 nm corresponding to a d–d transition CuII(adpa) (10 mM) in CD3CN (550 μL) was combined with
band of d9 CuII species) to CuI(adpa) (no d–d transition band). Zn(OAc)2 (8.0 mol equiv. dissolved in D2O/CD3CN 7 : 3 v/v). A
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shift and broadening of a signal corresponding to OAc− was in H2O/CH3CN 7 : 3 v/v). To this solution mixture was then
noted, indicating the coordination of OAc− to the paramag- added a stock solution of AsH2 (50 μL, 5 mol equiv. dissolved
netic CuII centre. To this solution was added AsH2 (50 μL, in H2O/CH3CN 7 : 3 v/v). After being stirred for 2 min, the reac-
5.0 mol equiv. dissolved in D2O/CD3CN 7 : 3 v/v). The solution tion was monitored by fluorescence spectroscopy (fluorescence
became pale yellow, consistent with the conversion to parameters: excitation wavelength = 340 nm, slit setting on the
CuI(adpa). The yellow product gave a sharp spectrum, corres- instrument = 10 and PMT = 500 for Cu(adpa); PMT = 530 for
ponding to the CuI species.
Cu(atpa)).
For the selectivity study, the addition of AsH2 was changed
to glucose, lactose, sucrose, fructose, citric acid or glutathione.
Effect of pH
Acetic–acetate buffer solutions (ABS) with pH 4.0–5.6 and For the interference study, the addition of AsH2 was changed
phosphate buffer solution (PBS) with pH 6.0–8.0 were prepared to the mixture of AsH2 (5.0 mol equiv.) and a possible interfer-
in H2O/CH3CN (7 : 3 v/v). In a typical reaction, to a solution of ing substance (5.0 mol equiv. of glucose, lactose, sucrose, fruc-
CuII(adpa) (10 μM) in a buffer solution with the desired pH tose, citric acid or glutathione).
(2.00 mL) was added Zn(OAc)2 (100 μL, 40 mol equiv. dissolved
in H2O/CH3CN 7 : 3 v/v). To this solution mixture was then
added a stock solution of AsH2 (50 μL, 5 mol equiv. dissolved
Sample preparation for the determination of AsH2 in vitamin
C tablets
in H2O/CH3CN 7 : 3 v/v). It should be mentioned that a color Three tablets of vitamin C were dissolved in Milli-Q water
change of the solution could not be observed with the naked (500.00 mL). The solution was then filtered with a 0.45 μM
eye at this low concentration. After being stirred for 2 min, the Millipore filter to remove insoluble components. An amount
reaction was monitored by fluorescence spectroscopy, which of the filtrate (31 μL) was then transferred to a volumetric flask
revealed an emission band at λmax = 423 nm. (Fluorescence and diluted to 10.00 mL with ABS (pH 5.6) in H2O/CH3CN
parameters: excitation wavelength = 340 nm, slit setting on the (7 : 3 v/v). The amount of ascorbic acid in vitamin C tablets
instrument = 10 and PMT = 500).
was determined by the standard addition method.
To demonstrate the effect of pH on AsH2 detection in the
presence of a possible interference, a solution of glutathione
(GSH, 5.0 mol equiv.) or a mixture of AsH2 (5.0 mol equiv.) and Conflicts of interest
GSH (5.0 mol equiv.) was used instead of AsH2.
There are no conflicts of interest to declare.
Reaction in the presence of other divalent metal ions
A stock solution of AsH2 (80 mM) and M(OAc)n (M = Zn2+, Na+,
Ca2+ or Mg2+) was prepared in H2O/CH3CN (7 : 3 v/v). In a
typical reaction, CuII(adpa) (2 mM) in 2.00 mL of H2O/CH3CN
(7 : 3 v/v) was combined with M(OAc)n (8 mol equiv., 100 μL).
After being stirred for 2 min, the solution was monitored by
UV-vis spectroscopy. To this solution mixture was added the
AsH2 stock solution (1.0 mol equiv., 50 μL). An immediate
color change of the solution from green to pale yellow was
observed. The reaction was also monitored by UV-vis spectro-
scopy, showing a complete conversion of the spectrum for
CuII(adpa) (λmzx = 635 nm) to the spectrum for a CuI complex.
Acknowledgements
This work was supported by the Thailand Research Fund
(P. L., TRG 5880235) and Grants for the Development of New
Faculty Staff, Ratchadaphiseksomphot Endowment Fund. P. S.
is grateful for the scholarship from the Science Achievement
Scholarship of Thailand (SAST). Also, we would like to thank
assistant professor Prompong Pienpinijtham for the helpful
discussion.
Notes and references
Reaction in the presence of other bridging ligands
A solution mixture of CuII(adpa) (2 mM) and a bridging ligand
(16 mol equiv. of imidazole, or histidine) was prepared in
2.00 mL of H2O/CH3CN (7 : 3 v/v). A solution of Zn(NO3)2
(8 mol equiv., 100 μL) was then added. To this reaction
mixture was added the AsH2 stock solution (1.0 mol equiv.,
50 μL). The reaction was stirred for 2 min before being moni-
tored by UV-vis spectroscopy.
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