A new fluorescent metal sensor with two binding moieties

Article abstract of DOI:10.1016/j.tetlet.2004.10.115

A new fluorescent chelator Oxa, having two metal-binding sites, was designed and synthesized in six steps. Oxa exhibited two distinctive dissociation constants for Zn²⁺ (Kd1=1.50 μM and Kd2=140 μM), with considerable fluorescence increase in aq

Full text of DOI:10.1016/j.tetlet.2004.10.115

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 9377–9381
A new fluorescent metal sensor with two binding moieties
Asuka Ohshima,a Atsuya Momotakeb and Tatsuo Araia,*
aGraduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1, Tennodai, Tsukuba-city, Ibaraki 305-8571, Japan
bResearch Facility Centre for Science and Technology, University of Tsukuba, 1-1-1, Tennodai, Tsukuba-city, Ibaraki 305-8571, Japan
Received 28 July 2004; revised 8 October 2004; accepted 21 October 2004
Available online 5 November 2004
Abstract—A new fluorescent chelator Oxa, having two metal-binding sites, was designed and synthesized in six steps. Oxa exhibited
two distinctive dissociation constants for Zn2+ (Kd1 ¼ 1:50lM and K2d ¼ 140lM), with considerable fluorescence increase in aqueous
buffer at pH7.2. Affinities of Oxa for the other biologically important ions such as Ca2+ (Kd1 ¼ 250lM and Kd2 ¼ 275mM) and Mg2+
(K1 ¼ 975lM and K2d could not be determined) in the same conditions were also obtained.
Ó d2004 Elsevier Ltd. All rights reserved.
Our approach of the development of a new fluorescent
sensor for biologically important ions is based on the
idea that the fluorescent probes, which have two
metal-binding sites with two distinctive dissociation con-
stants (K1d and K2d), may be applicable for measuring
ions in wide concentration range depending upon the
dissociation constants of each binding site. The first
target for the new chelator is Zn2+ since Zn2+ is one of
the most important transition metal ions in physiology.
Fluorescent probes for Zn2+ have been developed1–10,23
but they have only one metal-binding site.
shift in aqueous solution.16 When other metal-chelating
groups, which have much higher binding constants than
HBO, are introduced on the HBO skeleton, the molecule
would have two distinctive binding constants in different
concentration ranges. In this respect, Oxa was designed
and synthesized. Oxa has two chelating parts, amino-
phenol triacetic acid (APTRA) and HBO (Scheme 1).
We employed the APTRA structure for a new fluores-
cent sensor. The APTRA chelator was developed by
Levy et al.17–19 The three carboxylate groups, the anilino
nitrogen and the ether oxygen are available for
complexation at physiological pH.18 APTRA should
have much higher affinity for Zn2+ than that of HBO
due to the chelating effect and an electronic change in
the aromatic ring, which directly reflects its fluorescence
2-(20-Hydroxyphenyl)benzoxazole (HBO) is well-known
as a typical molecule which undergoes excited state
intramolecular proton transfer (ESIPT).11–15 HBO also
shows weak affinity for Zn2+ together with a wavelength
Scheme 1. Tentative structures of Zn2+- and 2Zn2+-bound forms of Oxa.
Keywords: Fluorescent sensors; Complexation.
*
Corresponding author. Tel.: +81 298 53 4315; fax: +81 298 53 6503; e-mail: arai@chem.tsukuba.ac.jp
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.10.115

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A. Ohshima et al. / Tetrahedron Letters 45(2004) 9377–9381
KOH in water yielded a fluorescence chelator, Oxa.
This aqueous solution, including potassium salt of
Oxa and little excess of KOH, was directly used as a
stock solution for this research. The complexation stud-
ies of Oxa were performed in aqueous buffer solution at
physiological pH (40mM HEPES, 100 mMKCl,
pH7.20). It is noted that Oxa can be dissolved in buffer
solution at pH7.2 without using organic solvents such as
DMSO.
When Oxa formed complexes with metal ions, the
absorption and the fluorescence spectra changed. The
change in the absorption spectra upon the addition of
Zn2+ is shown in Figure 1a–c. As illustrated in Scheme
1, Oxa showed two distinctive binding constants with
Zn2+ due to the formation of the corresponding two
Zn-complexes [Oxa+Zn–3K]ꢀ and [Oxa+2Zn–3K–H],
revealed by the observation of the three-stepabsorption
changes. First step( Fig. 1a and b): the absorbance maxi-
mum at 360nm of Oxa decreased together with the
blue-shift upon the addition of Zn2+ upto near the
[Oxa]:[Zn2+] = 1:1 ratio. The isosbestic point was ob-
served at 315nm (Fig. 1b), indicating a simple 1:1 com-
plexation equilibrium between Oxa and [Oxa+Zn–3K]ꢀ,
where Zn2+ precedently binds to the APTRA group.
The formation of [Oxa+2Zn–3K–H] is negligible as long
as the isosbestic point remains at 315nm. The apparent
dissociation constant (K1d Zn ¼ 1:50lM at pH7.2) for
Zn2+ was determined by iterative least-squares fitting
to a 1:1 model. Second step: when the concentration
of Zn2+ reaches that of Oxa, the absorption band does
not go through the isosbestic point at 313nm due to
the produced [Oxa+2Zn-3K-H]. Third Step: further
addition of Zn2+ leads to a new isosbestic point at
358nm (Fig. 1c), indicating that the concentration of
free Oxa can be negligible in the high Zn2+ concentra-
tion range and the simple equilibrium between [Ox-
a+Zn–3K]ꢀ and [Oxa+2Zn–3K–H] (Scheme 1) should
be operating. The plotting of the absorption bands at
400nm upon the addition of Zn2+ gave the second disso-
ciation constant (K2d Zn ¼ 140lM).
Scheme 2. Reagents and conditions: (a) i-Pr2NEt, NaI, bromoacetic
acid ethyl ester, acetonitrile, reflux, 15h; (b) POCl3, DMF, pyridine,
0°C, 22h; (c) H2, Pd–C, CH3CO2H, rt, 48h; (d) o-hydroxyaniline,
ethanol, reflux, 2h; (e) DDQ, chloroform, 70°C, 40h; (f) KOH,
ethanol, rt, 15h.
change, should occur when the amino nitrogen and
ether oxygen contribute to form the complex with metal
ions. Solubility to aqueous buffer at physiological pH
is necessary for the chelating reagents if the targets
are physiologically important metals. The APTRA
derivatives would give enough solubility to an aqueous
solution at medium pH.20
HBO derivatives have an extinction coefficient in the
region of 300–400nm, which is required because of the
excitation wavelength. Chemical and thermal stability
is also required for this study. In the course of our
research,21 we also tried to prepare fluorescent probes
having a salicylideneaniline as a fluorophore. However,
a salicylideneaniline analogue, such as 5 (Scheme 2), is
unstable under alkali conditions. Thus, a benzoxazole
structure is suitable from the viewpoint of not only a
highly fluorescent but also hydrolysis-proof chromo-
phore for this study.
The added Zn2+ also caused two-stepfluorescence
change. First step(Inset in Fig. 2): upon the addition
of Zn2+, slight decrease the fluorescence intensity with
the isoemissive point at 459nm was observed, indicating
Data for Oxa-ester: 1H NMR (CDCl3, 500MHz, Me4Si) d 11.20 (1H,
s, ArOH), 7.66 (1H, m, ArH), 7.55 (1H, m, ArH), 7.43 (1H, s, ArH),
7.35–7.32 (2H, m, ArH), 6.47 (1H, s, ArH), 4.63 (2H, s, ArOCH2CO),
4.30–4.20 (10H, m, CH2), 1.33–1.28 (9H, m, CH3); 13C NMR (CDCl3,
125MHz) d 170.7, 168.8, 162.8, 155.4, 149.0, 145.4, 142.4, 140.4,
124.8, 124.7, 118.8, 113.2, 110.4, 106.4, 102.4, 67.5, 61.2, 61.0, 53.9,
14.3, 14.2; Elemental Analysis. Anal. Calcd for C25H28N2O9: C,
59.99; H, 5.64; N, 5.60. Found: C, 59.84; H, 5.76; N, 5.54.
The synthesis of Oxa is shown in Scheme 2. Compound
122 was prepared from hydroquinone in four steps and
was trialkylated with bromoacetic acid ethyl ester, fol-
lowed by aromatic formylation to give aldehyde 3,
which was then reduced with catalytic Pd–C under H2
atmosphere to form 4. SchiffÕs base 5 was obtained by
coupling reaction between 4 and 2-hydroxyaniline in
refluxing ethanol. Oxidation of 5 with DDQ in chloro-
form gave Oxa-ester and following ester hydrolysis with
Oxa(acid form): Potassium salt of Oxa was reprecipitated by the
addition of 0.1N HCl in aqueous solution and the NMR of Oxa
without salt (acid form) was measured in DMSO-d6. 1H NMR
(DMSO-d6, 500MHz) d 10.88 (1H, s, ArOH), 7.77–7.73 (2H, m,
ArH), 7.40–7.36 (2H, m, ArH), 7.33 (1H, s, ArH), 6.15 (1H, s, ArH),
4.53 (2H, s, ArOCH2), 4.15 (4H, s, ArN(CH2)2). 13C NMR (CDCl3,
125MHz) d 173.7, 170.3, 162.8, 154.3, 148.6, 143.9, 141.3, 140.0,
125.1, 124.9, 118.4, 111.9, 110.8, 103.1, 99.0, 67.6, 59.3.

A. Ohshima et al. / Tetrahedron Letters 45(2004) 9377–9381
9379
Figure 1. Change in the absorption spectra of Oxa (3.50lM) upon the addition of ZnCl2 in aqueous buffer (40mM HEPES, 100mM KCl, pH7.20).
[Zn2+] = 0–558lM (a). Expanded spectra are shown in (b) and (c). Isosbestic points are observed at 315nm when [Zn2+] = 0–3.23lM (solid line in
(b)) and at 358nm when [Zn2+] = 42.1–558lM (solid line in (c)).
Figure 2. Change in the fluorescence spectra (kexcitation = 360nm) of
Oxa (3.50lM) upon the addition of ZnCl2 in buffer (40 mMHEPES,
100mM KCl, pH7.20) solution. [Zn2+] = 0–558lM (0–159equiv).
Inset shows the expanded data of Figure 2. The isoemissive point at
459nm was observed probably due to the equilibrium between Oxa
and [Oxa+Zn-3K]ꢀ.
1:1 complexation equilibrium between Oxa and [Oxa+
Zn–3K]ꢀ. Second step( Fig. 2): further addition of
Zn2+ leads to the fluorescence red-shift with dramatic in-
Figure 3. (a) Change in the absorption spectra of Oxa (3.50lM) upon
crease, which has also been observed in other chelators
the addition of CaCl2 in aqueous buffer (40 mMHEPES, 100mM KCl,
having a benzoxazole skeleton.16,23 When Zn2+ binds
pH7.20). [Ca2+] = 0–2.42M. Isosbestic points are observed at 314nm
when [Ca2+] = 0–903lM and at 360nm when [Ca2+] = 1.53–2.42M. (b)
Change in the fluorescence spectra (kexcitation = 360nm) of Oxa
(3.50lM) upon the addition of CaCl2 in buffer (40mM HEPES,
100mM KCl, pH7.20) solution. [Ca2+] = 0–2.42M.
to oxazole, the nonradiative decay pathway is inhibited
due to the electronical change in the lowest excited sin-
glet state.16 Therefore, this fluorescence increase also
strongly suggests the formation of [Oxa+2Zn–3K–H].
The emission maximum of Oxa and [Oxa+2Zn–3K–H]
is observed at 430nm and 451nm, respectively.
that HBO derivatives did not show any increase in fluo-
rescence intensity by addition of divalent cations, such
as Ca2+ and Mg2+, even at high metal concentration,16
indicating that HBO derivatives do not interact with
Ca2+ and Mg2+. In the case of Oxa, however, titration
with Ca2+ gave similar spectral change to that with
Zn2+ in both UV absorption and fluorescence measure-
ments (Fig. 3), suggesting that the HBO moiety as
The observed two distinctive dissociation constants for
Zn2+ prompted us to further explore the affinity deter-
mination of Oxa to the other biologically important
divalent cations. To Ca2+ and Mg2+, the ATPRA unit
in Oxa should have some affinities since the APTRA
chelators have originally been developed as probes for
17,18
Mg2+
.
On the other hand, Henary et al. reported

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A. Ohshima et al. / Tetrahedron Letters 45(2004) 9377–9381
In conclusion, a new fluorescence chelating reagent Oxa
was designed and synthesized and the complexation
studies were performed to give the two distinctive disso-
ciation constants for Zn2+ and Ca2+. To the best of our
knowledge, this is the first clear evidence of the spectro-
scopic properties of a chelating agent having two
chelating parts in one molecule with considerably
different binding constant capable of being dissolved in
water.
Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
tific Research on Priority Areas (417), a Grant-in-Aid
for Scientific Research (No. 16350005) and the 21st Cen-
tury COE Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of
the Japanese Government, by University of Tsukuba
Research Projects, by the Asahi Glass Foundation and
by JSR Corporation.
Figure 4. (a) Change in the absorption spectra of Oxa (3.50lM) upon
the addition of MgCl2 in aqueous buffer (40mM HEPES, 100mM
KCl, pH7.20). [Mg2+] = 0–22.8mM. Isosbestic points are observed at
300nm when [Mg+] = 0lM to 4mM (b) Change in the fluorescence
spectra (kexcitation = 360nm) of Oxa (3.50lM) upon the addition of
MgCl2 in buffer (40mM HEPES, 100mM KCl, pH7.20) solution.
[Mg2+] = 0–22.8mM.
References and notes
1. Fahrni, C. J.; OÕHalloran, T. V. J. Am. Chem. Soc. 1999,
121, 11448–11458.
2. Walkup, G. K. B.; Burdette, S. C.; Lippard, S. J.; Tsien,
R. Y. J. Am. Chem. Soc. 2000, 122, 5644–5645.
3. Burdette, S. C.; Walkup, G. K.; Springler, B.; Tsien, R.
Y.; Rippard, S. J. J. Am. Chem. Soc. 2001, 123, 1831–
1841.
well as the APTRA unit bound to Ca2+. The apparent
dissociation constant for Ca2+ was much higher than
that for Zn2+ (Kd1 Ca ¼ 250 lM and K2d Ca ¼ 275 mM).
Titration with Mg2+ gave different spectral change from
those with Zn2+ and Ca2+ (Fig. 4). In the absorption
spectra, the absorbance intensity at 360nm was
decreased with increasing Mg2+. Isosbestic point is
observed at 300nm when the concentration of Mg2+
is 0–4.0mM and the dissociation constant for Mg2+
was calculated to be 943lM, which is also much higher
than that with Zn2+. The second isosbestic point could
not be observed during the titration with Mg2+. Therefore,
the second dissociation constant for Mg2+ could not be
determined. In the fluorescence spectra, the intensity in-
creased even at low Mg2+ concentration where the Mg2+
probably not binds to HBO unit in Oxa but the reason
of fluorescence increase is still unclear. Isoemissive point
was not observed during the titration. The calculated
dissociation constants are shown in Table 1. From the
4. Pearce, D. A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. J.
Am. Chem. Soc. 2001, 123, 5160–5161.
5. Hirano, T.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Am.
Chem. Soc. 2002, 12, 6555–6562.
6. Maruyama, S.; Kikuchi, K.; Hirano, T.; Urano, Y.;
Nagano, T. J. Am. Chem. Soc. 2002, 12, 10650–10651.
7. Gee, K. R.; Zhou, Z. L.; Qian, W. J.; Kennedy, R. J. Am.
Chem. Soc. 2002, 124, 776–778.
8. Shults, M. D.; Pearce, D. A.; Imperiali, B. J. Am. Chem.
Soc. 2003, 125, 10591–10597.
9. Woodroofe, C. C.; Lippard, S. J. J. Am. Chem. Soc. 2003,
125, 11458–11459.
10. Reany, O.; Gunnlaugsson, T.; Parker, D. Chem. Commun.
2000, 473–474.
11. Arthen-Engeland, Th.; Bultmann, T.; Ernsting, M. P.;
Rodriguez, M. A.; Thiel, W. Chem. Phys. 1992, 163, 43.
12. Wang, H.; Zhang, H.; Abou-Zied, O. K.; Yu, C.;
Romesberg, F. E.; Glasbeek, M. Chem. Phys. Lett. 2003,
367, 599–608.
13. Abou-Zied, O. K.; Jimenez, R.; Thompson, E. H. Z.;
Millar, D. P.; Romesberg, F. E. J. Phys. Chem. A 2002,
106, 3665.
14. Ogawa, A. K.; Abou-Zied, O. K.; Tsui, V.; Jimenez, R.;
Case, D. A.; Romesberg, F. E. J. Am. Chem. Soc. 2000,
122, 9917–9920.
15. Yang, G.; Morelet-Savary, F.; Peng, Z.; Wu, S.; Fouas-
sier, J.-P. Chem. Phys. Lett. 1996, 256, 536–542.
16. Henary, M. M.; Fahrni, C. J. J. Phys. Chem. A 2002, 106,
5210–5220.
17. Levy, L. A.; Murphy, E.; Raju, B.; London, R. E.
Biochemistry 1988, 27, 4041–4048.
18. Otten, P. A.; London, R. E.; Levy, L. A. Bioconjug. Chem.
2001, 12, 76–83.
results, one can say that Oxa preferably binds to Zn2+
,
against Ca2+ and Mg2+ in this condition. Oxa can detect
Zn2+ in micro molar range and increase its fluorescence
in sub-milli molar range of Zn2+. The exploration of the
affinity of Oxa to the other cation species is ongoing.
Table 1. The apparent dissociation constants of Oxa for Zn2+, Ca2+
and Mg2+ in aqueous buffer (40mM HEPES, 100mM KCl, pH7.20)
Zn2+
Ca2+
Mg2+
Kd1
Kd2
1.50lM
140lM
250lM
275mM
943lM
—
19. Raju, B.; Murphy, E.; Levy, L. A.; Hall, R. D.; London,
R. E. Am. J. Physiol. 1989, 256, C540–C548.

A. Ohshima et al. / Tetrahedron Letters 45(2004) 9377–9381
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20. Cielen, E.; Stobiecka, A.; Tahri, A.; Hoornaert, G. J.; De
Schryver, F. C.; Gallay, J.; Vincent, M.; Boens, N.
J. Chem. Soc., Perkin Trans. 2 2002, 1197–1206.
21. Ohshima, A.; Momotake, A.; Arai, T. J. Photochem.
Photobiol. A, Chem. 2004, 162, 473–479.
22. Haviv, F.; Ratajczyk, J. D.; DeNet, R. W.; Kerdesky, F.
A.; Walters, R. L.; Schmidt, S. P.; Holms, J. H.; Young, P.
R.; Carter, G. W. J. Med. Chem. 1988, 31, 1719–1728.
23. Taki, M.; Wolford, J. L.; OÕHalloran, T. V. J. Am. Chem.
Soc. 2003, 126, 712–713.