Development of a quinazoline-based chelating ligand for zinc ion and its application to validation of a zinc-ion-coordinated compound

Article abstract of DOI:10.1248/cpb.58.875

A novel fluorescent chelating ligand, 2,4-[bis-(2-hydroxy-3- methoxybenzylidene)]-dihydrazinoquinazoline (HBQZ), was synthesized, and the fluorescence characteristics of its complex with metal ions were investigated. Among the 36 different metal ions tested in this study, it was found that HBQZ emits intense fluorescence at 506 nm with an excitation wavelength of 414 nm in the presence of Zn²⁺. The fluorescence intensity was almost constant in the pH range 3.5-10.5. Complexes of other metal ions with HBQZ did not show fluorescence, and the detection limit of Zn²⁺ was approximately 250 nM (16 ppb). The proposed method was applied to the validation test of a bioactive compound containing Zn²⁺ in its structure - an antibacterial and antifungal reagent, zinc pyrithione (ZnPT). In order to effectively release Zn²⁺ from ZnPT, a pretreatment procedure involving heating with H₃PO₄ at 100 °C for 60 min was adopted. Under these conditions, a linear calibration curve was obtained in the ZnPT concentration range of 0.79-15.7 μM (0.25-5.0 ppm); the correlation coefficient and the relative standard deviation were 0.996 and within 3.1% (n=5), respectively.

Full text of DOI:10.1248/cpb.58.875

June 2010
Note
Chem. Pharm. Bull. 58(6) 875—878 (2010)
875
Development of a Quinazoline-Based Chelating Ligand for Zinc Ion and
Its Application to Validation of a Zinc-Ion-Coordinated Compound
Hiroshi YAMADA,a Akina SHIRAI,a Keisuke KATO,b Junko KIMURA,a Hideaki ICHIBA,a
a
,a
*
Takehiko YAJIMA, and Takeshi FUKUSHIMA
a Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University; and b Department of Organic
Chemistry, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan.
Received March 2, 2010; accepted March 18, 2010
A novel fluorescent chelating ligand, 2,4-[bis-(2-hydroxy-3-methoxybenzylidene)]-dihydrazinoquinazoline
(HBQZ), was synthesized, and the fluorescence characteristics of its complex with metal ions were investigated.
Among the 36 different metal ions tested in this study, it was found that HBQZ emits intense fluorescence at
506 nm with an excitation wavelength of 414 nm in the presence of Zn2ꢀ. The fluorescence intensity was almost
constant in the pH range 3.5—10.5. Complexes of other metal ions with HBQZ did not show fluorescence, and
the detection limit of Zn2ꢀ was approximately 250 nM (16 ppb). The proposed method was applied to the valida-
tion test of a bioactive compound containing Zn2ꢀ in its structure—an antibacterial and antifungal reagent, zinc
pyrithione (ZnPT). In order to effectively release Zn2ꢀ from ZnPT, a pretreatment procedure involving heating
with H3PO4 at 100 °C for 60 min was adopted. Under these conditions, a linear calibration curve was obtained in
the ZnPT concentration range of 0.79—15.7 mM (0.25—5.0 ppm); the correlation coefficient and the relative stan-
dard deviation were 0.996 and within 3.1% (nꢁ5), respectively.
Key words metal-ion complex; quinazoline; fluorescence; zinc ion; zinc pyrithione
Metal ions can act as a Lewis acid, and due to this prop- sequently, although 2,4-[bis-(2-hydroxy-3-methoxybenzyli-
erty, they have been artificially incorporated into drugs, pig- dene)]-dihydrazinoquinazoline (HBQZ, Fig. 1a) itself shows
ments, or dietary supplements1—5) in order to gain the intrin- very weak fluorescence, it can selectively bind with Zn2ꢀ to
sic activity. Metal-ion-coordinated compounds are generally emit fluorescence at 506 nm with an excitation wavelength of
validated using atomic absorption spectrometry (AAS), be- 414 nm. Further, we exploited the fluorometric properties of
cause metal ions can be selectively quantified by AAS. How- HBQZ to detect Zn2ꢀ for the validation of a Zn2ꢀ-containing
ever, a functionalized ligand that can chelate a metal ion compound, because several compounds such as drugs or sup-
selectively to emit fluorescence can also be used for such plements contain Zn2ꢀ in their chemical structure. In the
validation.
present study, an antibacterial and antifungal reagent, zinc
Recently, we developed a fluorescent chelating ligand, 2,4- pyrithione (ZnPT),10—17) in which a bidentate bis-(N-oxopyri-
[bis-(2,4-dihydroxybenzylidene)]-dihydrazinoquinazoline dine-2-thionato) ligand is coordinated with the zinc ion, was
(DBHQ), which can selectively chelate with Ga3ꢀ ions to used as the Zn2ꢀ-containing compound.
emit fluorescence at 470 nm with an excitation wavelength of
Experimental
405 nm.6) The basic structure of DBHQ is a quinazoline
skeleton with two Schiff base moieties that serve as chelating
Chemicals Anthranilic acid and hydrazine monohydrate were purchased
from Tokyo Kasei Co., Ltd. (Tokyo, Japan). Urea, dimethyl sulfoxide
sites.
In DBHQ, two 2,4-dihydroxyphenyl groups are attached to
(DMSO), phosphoryl chloride (POCl3), ZnPT, phosphoric acid, and quinine
sulfate were purchased from Wako Pure Chemical Industries Co., Ltd.
(Osaka, Japan). o-Vanillin was purchased from Kanto Kagaku Co., Ltd.
(Tokyo, Japan). H2O was used after purification using an Autopure WR600G
(Yamato Scientific Co., Ltd., Tokyo, Japan). The cations and anions tested in
the quinazoline moiety via Schiff bases, which are consid-
ered to play a role in chelation with Ga3ꢀ. This moiety may
be suitable for identifying the ionized atomic structure of
Ga3ꢀ. On the other hand, an o-vanillin moiety has recently
been incorporated into fluorescent chelating ligands such as
N-o-vanillidine-2-amino-p-cresol (OVAC),7) o-vanillin fur-
furalhydrazone (OVFH),8) and o-vanillin-8-aminoquinoline,9)
which can chelate Al3ꢀ, Ga3ꢀ, and Cr3ꢀ, respectively. The
benzaldehyde moiety of o-vanillin can be used to produce a
Schiff base via a coupling reaction with an amino group, and
the lone pair of electrons on the oxygen atom of the phenolic
hydroxyl group may act as a Lewis base for chelation with
metal ions. Similarly, o-vanillin has been used as a building
block for functionalized chelating ligands.
this study, which were purchased in the form of salts containing NO3ꢁ, SO42ꢁ
,
and Clꢁ, were of atomic absorption grade and purchased from Wako Pure
Chemical Industries.
Apparatus Excitation and fluorescence spectra were measured on a
spectrofluorometer (FP-6300; Jasco Co., Tokyo, Japan) with slit widths of
10 nm. All fluorescence and absorption data were collected in a temperature-
controlled room (25 °C). IR spectra were recorded on an FT/IR-4100 (Jasco)
instrument. Mass spectra were measured on a JMS-600H (JEOL, Ltd.,
In this study, we designed and synthesized a previously de-
veloped quinazoline-based fluorescent ligand as a structural
analog of DBHQ. In this analog, the 2,4-dihydroxybenzene
in DBHQ is replaced with the o-vanillin moiety, and its abil-
ity as a fluorescent chelating ligand was investigated. Con-
Fig. 1. Chemical Structures of (a) HBQZ and (b) Zinc Pyrithione (ZnPT)
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: t-fukushima@phar.toho-u.ac.jp

876
Vol. 58, No. 6
Electrospray Ionization (ESI) Mass Spectrometry Next, 200 ml of
1.0 mM Zn2ꢀ solutions were mixed with 200 ml of 1.0 mM HBQZ solution.
After the addition of 0.5 ml of 100 mM acetate buffer (pH 4.5), the solution
mixture was diluted with 50% MeOH in H2O containing 1% acetic acid to
10 ml. The samples were injected at a constant flow rate of 5 ml/min using a
syringe pump. All mass spectrometry (MS) data were acquired using an
LCQ ion trap mass spectrometer (ThermoFisher Scientific, Yokohama,
Japan) equipped with an electrospray ionization source, and the mass spec-
trometer was operated in the positive-ion mode. The experimental conditions
were as follows: spray needle voltage, 5 kV; heated capillary temperature,
200 °C; and sheath gas flow rate, 40 (arbitrary units). Mass spectra were
measured in the full ion scan mode for a mass-to-charge (m/z) ratio in the
range of 200—2000.
Fluorescence Quantum Yield and Calibration Curve According to a
previous method,19) the fluorescence quantum yield (f) of HBQZ in the
presence or absence of Zn2ꢀ was determined using quinine sulfate as a refer-
ence compound. In addition, the f value of HBQZ–Zn2ꢀ in the presence of
10 mM H3PO4 was also determined.
In brief, 1.0 ml of acetate buffer (pH 4.5) was added to 500 ml of 2.0 ppm
Zn2ꢀ and/or 500 ml of 20 mM H3PO4. Finally, 200 ml of 60 mM HBQZ was
added and treated as described above. In this case, a fluorescence integral
with an excitation wavelength of 414 nm was obtained, and the absorbance
at 414 nm was measured by using a Jasco V-650 spectrophotometer (Jasco,
Tokyo, Japan). A calibration curve was drawn by plotting the values of
FꢁF0 against Zn2ꢀ concentration (ppb). Then, 500 ml of a Zn2ꢀ solution
(50—1000 ppb) was used for the calibration curve (nꢂ5). Detection limit
(DL) was determined by the obtained calibration curve using the following
Eq. 2.
Tokyo, Japan). NMR spectra were measured by JEOL ECP1 (JEOL Ltd.,
Tokyo, Japan) (500 MHz for 1H and 13C) with tetramethylsilane as the inter-
nal standard. The refractive indices of the solvents were measured using an
Abbe refractometer (model ER-1, ERMA Inc., Tokyo, Japan).
Synthesis and Characterization of HBQZ 2,4-Dihydrazinoquinazo-
line (1) was synthesized according to the method described in our previous
paper.6) Compound 1 (13.0 mmol) was mixed with o-vanillin (1.16 g,
0.40 mol) in EtOH (350 ml), and the obtained solution was refluxed at 80 °C
for 3 h. After the solution was heated to dryness, the obtained brownish
residue was recrystallized using MeOH to give HBQZ in the form of a yel-
low powder. The product was recrystallized using MeOH to obtain the final
purified product (yield: 45.0%). FAB-MS: m/z 459 [MꢀH]ꢀ; Elemental
Anal. Calcd for C24H22O4N6: C, 62.87; H, 4.84; N, 18.33%. Found: C, 62.86;
H, 4.95; N, 18.36%. FT-IR (solid phase) (cmꢁ1): 3394, 3304 (nO–H), 1602
(nCꢂN), 1266, 1250 (nAr–O). 1H-NMR (CDCl3ꢀCF3CO2H) d (ppm): 3.87
(3H, s), 3.93 (3H, s), 6.88—7.04 (6H, m), 7.46 (1H, dt, Jꢂ2.0, 8.0 Hz),
7.75—7.80 (2H, m), 8.09 (1H, d, Jꢂ8.4 Hz), 8.43 (1H, s), 8.55 (1H, s). 13C-
NMR (CDCl3ꢀCF3CO2H) d (ppm): 56.0, 56.3, 109.5, 115.0, 115.3, 116.7,
117.0, 118.0, 121.4, 121.6, 122.2, 123.6, 123.9, 127.4, 137.0, 138.0, 145.2,
146.5, 147.5, 147.7, 150.2, 152.0, 154.1, 156.7. Melting point: 283—284 °C.
Screening Assay of Metal Ions Coordinated with HBQZ The proba-
bility of 36 different metal ions being coordinated with HBQZ to induce flu-
orescence emission was investigated. Solutions containing 100 ml of the
metal ions (1000 ppm), 100 ml of a 100 mM solution of HBQZ in DMSO, and
100 ml of a buffer solution at various pH values (3.5—10.5) were taken in
glass test tubes. Buffer solutions with pH 3.5—5.0, 5.5—7.0, and 7.5—10.5
were prepared from 100 mM CH3CO2Na–CH3CO2H, N-(2-hydroxyethyl)-
piperazine-Nꢃ-2-ethanesulfonic acid (HEPES)–NH3, and NH4Cl–NH3, re-
spectively. Each test tube was irradiated by UV light from a D2 lamp in a
dark room. The sample emitting intense fluorescence was concluded to be
the solution containing the fluorescent HBQZ complex. In this experiment,
the fluorescence emission was evaluated by visual assessment.
DLꢂ3.3s/s
(2)
where s and s are the standard deviation of 0 ppm Zn2ꢀ and slope of the cal-
ibration curve, respectively (nꢂ5).
Release of Zn2ꢀ from ZnPT ZnPT was dissolved in 10 mM HCl,
HNO3, H3PO4, and H2SO4 (5.0 ppm ZnPT), and was heated at 100 °C for
60 min. After the solution was cooled, 500 ml of the solution was sampled
and was treated in a similar manner (nꢂ5). ZnPT dissolved in 20 mM H3PO4
(5.0 ppm) was heated at 40, 60, 80, and 100 °C until 120 min, respectively.
Then, 500 ml of the solution at each time point was treated (nꢂ5). Next,
500 ml of 0.787—15.7 mM ZnPT in 20 mM H3PO4 were heated at 100 °C for
60 min and then used for obtaining the calibration curve for ZnPT (nꢂ5).
Sample Preparation In the case of measuring fluorescence of HBQZ–
Zn2ꢀ complex, sample preparation was performed as follows. In
a
polypropyrene test tube, 1.0 ml of 50 mM acetate buffer (pH 4.5) was added
to the sample solution. This was followed by adding 200 ml of a 60 mM
HBQZ in DMSO, and was left to stand for 10 min at room temperature.
Then, the solution was diluted tenfold with distilled 2-propanol, and the
spectrum of the diluted solution was measured in a 1.0ꢄ1.0 cm quartz cell.
Excitation and Emission Spectra For the measurement of spectra,
500 ml of a 30.8 mM (2 ppm Zn2ꢀ) solution of Zn(NO3)2 in H2O was treated
as described above.
Results and Discussion
Effect of Interfering Ions on Fluorescence In experiments involving
interfering ions, 400 ml of an aqueous solution of Zn(NO3)2 (7.69 mM, i.e.,
0.5 ppm Zn2ꢀ) and 100 ml of an aqueous solution of each metal ion or anion
(31.0—15.0ꢄ103 mM) were successively added to 1.0 ml of 50 mM acetate
buffer (pH 4.5), and treated in a similar manner in sample preparation. If a
cation or anion was found to affect the fluorescence, the procedure was re-
peated for an ion concentration diluted twice with H2O. This process was re-
peated until the ion concentration at which the fluorescence emission of the
HBQZ–Zn2ꢀ complex did not change was obtained. An error within ꢅ5% in
the measured fluorescence intensity was considered tolerable.
Synthesis and Properties of HBQZ HBQZ was suc-
cessfully synthesized in accordance with the procedure out-
lined in our previous paper,6) judging from the data of the el-
1
emental analysis, FAB-MS, H-NMR, 13C-NMR, and FT-IR.
Next, from among the 36 kinds of metal ions, the metal ion
that could coordinate with HBQZ to afford a fluorescent
complex was identified using different pH-buffered solu-
tions. Consequently, in the presence of Zn2ꢀ, intense fluores-
cence was observed upon irradiation from the D2 lamp. Thus,
Zn2ꢀ-chelating and fluorescent property of HBQZ was
mainly studied in this study.
Fluorescence Spectrum and Calibration Curve for
Zn2ꢀ Figure 2 shows the excitation and emission spectra of
HBQZ with Zn2ꢀ in 50 mM acetate buffer (pH 4.5). The opti-
mum excitation and emission wavelengths were 414 and
506 nm, respectively. As shown in Fig. 2, the intensity of flu-
orescence emitted by the HBQZ–Zn2ꢀ complex was approxi-
mately ten times that exhibited by HBQZ alone in the pres-
ence of 2 ppm Zn2ꢀ. The fluorescence intensity was almost
constant in the pH range 3.5—10.5 (data not shown), indicat-
ing that the HBQZ–Zn2ꢀ complex is formed over a broad pH
range, although most fluorescent ligands can chelate metal
ions in neutral to alkaline solutions. Among the organic sol-
vents used for the dilution of the HBQZ–Zn2ꢀ complex and
measurements of the fluorescence intensity, the most intense
fluorescence was observed in 2-propanol, and therefore, 2-
Binding Ratio of Zn2ꢀ to HBQZ Next, 500 ml of Zn2ꢀ solutions of dif-
ferent concentrations (1.54—76.9 mM) were mixed with 200 ml of 60 mM
HBQZ solution. This was followed by the addition of 1.0 ml of 100 mM ac-
etate buffer (pH 4.5). The fluorescence intensity of each solution was meas-
ured at 506 nm with an excitation wavelength of 414 nm. Further, 500 ml of a
15.4 mM (1.0 ppm) Zn2ꢀ solution was mixed with 200 ml of HBQZ solutions
(5.0—150 mM), and treated in a similar manner. The binding ratio was deter-
mined using the following molar ratio method. The fluorescence intensities
were plotted against the molar ratio of Zn2ꢀ with HBQZ, and the molar ratio
of HBQZ coordinated with Zn2ꢀ was determined stoichiometrically from the
plots.
The apparent binding constant of HBQZ with Zn2ꢀ was calculated using
the modified Benesi–Hildebrand Eq. 1 as reported by Roy et al.18)
DFꢂDFmaxꢀ(1/K[C])(DFmax
)
(1)
where DF and DFmax are equal to FꢁF0 and FmaxꢁF0, respectively. F0, F,
and Fmax are the fluorescence intensities of HBQZ, HBQZ with test Zn2ꢀ
concentrations (7.69, 12.3, 15.4, 30.8 mM), and HBQZ with the maximum
Zn2ꢀ concentration (76.9 mM), respectively. K and [C] are the apparent bind-
ing constant and the test concentration of Zn2ꢀ, respectively. DFmaxꢁDF was
plotted against 1/[C], and the value of K was obtained from the slope of the
line.

June 2010
877
propanol was used in further studies. A linear calibration and that of HBQZ to Zn2ꢀ, respectively. Both plots indicate
curve was obtained in the Zn2ꢀ (r2ꢂ0.994) concentration that HBQZ binds to Zn2ꢀ in a 1 : 1 molar ratio. In addition,
range 0.77—15.4 mM (50—1000 ppb), and the precision (rel- the ESI-MS spectrum of the solution of the HBQZ–Zn2ꢀ
ative standard deviation (RSD)) at 12.3 mM Zn2ꢀ was within complex showed the presence of ions with m/z of 520.1 (data
3.60% (nꢂ5). The detection limit of Zn2ꢀ was approximately not shown); these are molecular ions of the HBQZ–Zn2ꢀ
250 nM (16 ppb).
complex [(HBQZ–2HꢀZn)ꢀ]. This result confirms that the
Effect of Additive Ions on Fluorescence As shown in binding ratio of the HBQZ–Zn2ꢀ complex was 1 : 1. The
Table 1, the coexistence of most metal cation or anion with binding association constant K was determined by using the
Zn2ꢀ did not affect the fluorescence emission of the HBQZ– Benesi–Hildebrand method, and log K was approximately
Zn2ꢀ complex. However, the addition of more than 2.0 eq of 5.6. The f value of the HBQZ–Zn2ꢀ complex was 0.349,
Co2ꢀ, Cu2ꢀ, Fe3ꢀ, and Mn2ꢀ to Zn2ꢀ resulted in fluorescence while that of HBQZ alone was 0.021. These log K and f val-
quenching. It is suggested that these metal ions could also be ues were considered to be sufficient for the fluorescence de-
chelated by HBQZ, but the complex may not emit fluores- tection of Zn2ꢀ.
cence.
Fluorescence Detection of Zn2ꢀ from ZnPT Next,
Binding Ratio of HBQZ to Zn2ꢀ and Fluorescence HBQZ was applied to the validation of a bioactive compound
Quantum Yield Figures 3a and b show the fluorescence containing coordinated Zn2ꢀ in its structure. Many Zn2ꢀ-co-
intensity plotted against the molar ratio of Zn2ꢀ to HBQZ ordinated compounds are used as drugs, nutrients, or supple-
ments. For example, N-(3-aminopropionyl)-L-histidinato zinc
(Z-103) shows anti-ulcer activity,1) while bis(pyrrole-2-car-
boxylato)zinc shows insulinomimetic activity.2) Such Zn2ꢀ-
containing drugs are usually validated using AAS, because
AAS can achieve selective quantification of a metal ion. In
AAS measurement, however, a hollow cathode lamp is re-
quired for each metal ion; the flame from the lamp is supple-
mented with combustible acetylene gas for atomization of
the sample. Fluorescence detection using a functionalized
ligand, which can selectively chelate the metal ion, is pre-
ferred over AAS measurement due to its inherent simplicity
and safety. Therefore, in order to exploit the usefulness of the
validation test of a Zn2ꢀ-coordinated compound, in the pres-
Fig. 2. Excitation and Emission Spectra of HBQZ in the Presence (Solid
ent study, we attempted to validate an antibacterial and anti-
fungal reagent ZnPT, which has Zn2ꢀ ions coordinated bilat-
erally with electrons on the sulfur and oxygen atoms of 1-hy-
droxypyridine-2-thione (pyrithione) (Fig. 1b).10,11)
Line) or Absence (Dotted Line) of Zn2ꢀ
Table 1. Effect of Interfering Ions on the Detection of 7.69 mM (0.5 ppm)
Zn2ꢀ Using 60 mM HBQZ (Tolerable Error: ꢅ5%)
In our preliminary experiments, in which HBQZ was di-
rectly reacted with ZnPT in 50 mM acetate buffer at pH 4.0,
the observed fluorescence intensity of the 15—63 mM ZnPT
solution was considerably low—one-tenth of that of the solu-
tion containing 15—63 mM free Zn2ꢀ. This result suggests
that insufficient Zn2ꢀ ions were released from ZnPT when
the acetate buffer with pH 4.0 was used. Therefore, it was
necessary to release free Zn2ꢀ from the chelated Zn2ꢀ in
ZnPT. On the basis of the fact that pKa of pyrithione is 4.6,
Zn2ꢀ could be completely released from ZnPT in the region
with acidic pH. Thus, HCl, HNO3, H3PO4, and H2SO4 were
Tolerance ratio (m/m)
ꢆ500
250
100
50
Kꢀ, CH3CO2ꢁ, NO3ꢁ, SO42ꢁ, Clꢁ, Brꢁ, Iꢁ, PO43ꢁ, Naꢀ
Agꢀ, Ge4ꢀ
Mg2ꢀ
Be2ꢀ, Ba2ꢀ, Sr2ꢀ, Ca2ꢀ, Pb2ꢀ, Cd2ꢀ
Hg2ꢀ, ZrO2ꢀ
25
10
5
2
Ti4ꢀ
Sn2ꢀ, Al3ꢀ, Ni2ꢀ, In3ꢀ, Cr2O72ꢁ
Co2ꢀ, Cu2ꢀ, Fe3ꢀ, Mn2ꢀ
Tolerance ratio (m/m) was expressed as a ratio of the tested ion (mol) to Zn2ꢀ ion
(mol).
Fig. 3. Fluorescence Intensity of HBQZ–Zn2ꢀ Complex (a) at a Binding Ratio of Zn2ꢀ to HBQZ and (b) at a Binding Ratio of HBQZ to Zn2ꢀ

878
Vol. 58, No. 6
Fig. 4. (a) Effect of 10 mM Inorganic Acids on Fluorescence Intensity of HBQZ–Zn2ꢀ Complex Produced from ZnPT (MeanꢀS.D., nꢂ5); (b) Time
Course of Change in Fluorescence Intensity of HBQZ–Zn2ꢀ Complex Produced from ZnPT, Plotted as a Function of Heating Time in 20 mM H3PO4
(MeanꢅS.D., nꢂ5)
Closed circle: 100 °C, diamond: 80 °C, square: 60 °C, and triangle: 40 °C.
tested for identifying the type of inorganic acid required for an o-vanillin moiety was newly developed. It was shown that
the release of Zn2ꢀ from ZnPT. The highest fluorescence in- HBQZ fluoresced selectively with Zn2ꢀ and that HBQZ can
tensity of the HBQZ–Zn2ꢀ complex was observed in the be used for the validation of ZnPT after a pretreatment pro-
presence of H3PO4 (Fig. 4a). Therefore, H3PO4 was utilized cedure. The proposed fluorescent method using HBQZ will
for the release of free Zn2ꢀ from ZnPT. It is still not clear be useful for the validation test for a bioactive Zn2ꢀ-contain-
why H3PO4 was effective for the detection of free Zn2ꢀ in ing compound.
ZnPT. The cooperative coordination between the phosphoryl
Acknowledgment The authors thank Ms. Ziyu Song, Toho University,
for her help with manuscript preparation.
group in H3PO4 and Zn2ꢀ was thought to have enhanced the
fluorescence intensity of the HBQZ–Zn2ꢀ complex. Cer-
tainly, the f value of the HBQZ–Zn2ꢀ complex in the pres-
References
1) Nishide M., Yoshikawa Y., Yoshikawa E. U., Matsumoto K., Sakurai
ence of 10 mM H3PO4 was 0.712, whereas the f value of the
H., Kajiwara N. M., Chem. Pharm. Bull., 56, 1181—1183 (2008).
HBQZ–Zn2ꢀ complex alone was 0.349. Similar phenomena
2) Ito M., Tanaka T., Suzuki Y., Jpn. J. Pharmacol., 52, 513—521 (1990).
were observed in our previous study, in which a quinazoline-
type ligand, 2,4-[bis-(2,4-dihydroxybenzylidene)]-dihydrazi-
noquinazoline (DBHQ),6,20) was used. It was recently re-
ported that the structural analog, DBHQ, with Ga3ꢀ was also
enhanced by the addition of phosphoryl compounds or dihy-
drophosphate ions.20)
3) Ferruzzi M. G., Failla M. L., Schwartz S. J., J. Agric. Food Chem., 50,
2173—2179 (2002).
4) Chen J. H., Jiang S. J., J. Agric. Food Chem., 56, 1210—1215 (2008).
5) Nève J., Hanocq M., Peretz A., Khalil F. A., Pelen F., Biol. Trace Elem.
Res., 32, 201—212 (1992).
6) Kimura J., Yamada H., Ogura H., Yajima T., Fukushima T., Anal.
Chim. Acta, 635, 207—213 (2009).
Figure 4b shows the time course of the change in the fluo-
rescence intensity as a function of heating time. As shown in
Fig. 4b, heating at 100 °C resulted in high fluorescence inten-
sity after the onset of the reaction, indicating that the release
of free Zn2ꢀ from ZnPT was accelerated at 100 °C. There-
fore, in this study, heating was carried out at 100 °C for
60 min. Under these conditions, a linear calibration curve
was obtained for the ZnPT concentration range 0.79—15.7
mM (0.25—5.0 ppm); the correlation coefficient (r2) and RSD
were 0.996 and within 3.1% (nꢂ5), respectively. These data
indicate that the fluorescence method involving the use of
HBQZ is suitable for the validation of ZnPT. The detection
sensitivity of Zn2ꢀ by using HBQZ was not superior to that
by using AAS; however, fluorescence detection of Zn2ꢀ has
the advantage of simplicity. Therefore, HBQZ can be used
for the validation of a compound containing Zn2ꢀ, owing to
its capacity to capture Zn2ꢀ ions.
7) Kara D., Fisher A., Hill S. J., J. Environ. Monit., 9, 994—1000 (2007).
8) Tang B., Chen Z. Z., Zhang N., Zhang J., Wang Y., Talanta, 68, 575—
580 (2006).
9) Tang B., Yue T. X., Wu J. S., Dong Y. M., Ding Y., Wang H. J.,
Talanta, 64, 955—960 (2004).
10) Doose C.A., Ranke J., Stock F., Bottin-Weber U., Jastorff B., Green
Chem., 6, 259—266 (2004).
11) Bao V. W. W., Leung K. M. Y., Kwok K. W. H., Zhang A. Q., Lui G. C.
S., Mar. Pollut. Bull., 57, 616—623 (2008).
12) Kondoh Y., Takano S., J. Chromatogr., 408, 255—262 (1987).
13) Shih Y., Zen J. M., Kumar A. S., Chen P. Y., Talanta, 62, 912—917
(2004).
14) Thomas K. V., J. Chromatogr. A, 833, 105—109 (1999).
15) Doose C. A., Szaleniec M., Behrend P., Muller A., Jastorff B., J. Chro-
matogr. A, 1052, 103—110 (2004).
16) Yamaguchi Y., Kumakura A., Sugasawa S., Harino H., Yamada Y., Shi-
bata K., Senda T., Int. J. Environ. Anal. Chem., 86, 83—89 (2006).
17) Bones J., Thomas K. V., Paull B., J. Chromatogr. A, 1132, 157—164
(2006).
18) Roy P., Dhara K., Manassero M., Ratha J., Banerjee P., Inorg. Chem.,
46, 6405—6412 (2007).
19) Bag B., Bharadwaj P. K., J. Lumin., 126, 27—36 (2007).
20) Kimura J., Yamada H., Yajima T., Fukushima T., J. Lumin., 129,
1362—1365 (2009).
Conclusion
A novel quinazoline-based chelating ligand, HBQZ, with