Quinolinecarboxylic acid based fluorescent molecules: ratiometric response to Zn2+

Article abstract of DOI:10.1016/j.tet.2009.03.037

Novel fluorescent compounds based on quinolinecarboxylic acid were developed. These compounds were easily prepared in two or three steps from commercially available compounds. Compound 2 showed a ratiometric response to zinc(II) salt in aprotic solvent.

Full text of DOI:10.1016/j.tet.2009.03.037

Tetrahedron 65 (2009) 4235–4238
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Quinolinecarboxylic acid based fluorescent molecules: ratiometric
response to Zn2þ
*
Hisanaka Ito , Mai Matsuoka, Yohei Ueda, Madoka Takuma, Yoshihisa Kudo, Kazuo Iguchi
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel fluorescent compounds based on quinolinecarboxylic acid were developed. These compounds
were easily prepared in two or three steps from commercially available compounds. Compound 2
showed a ratiometric response to zinc(II) salt in aprotic solvent.
Received 10 November 2008
Received in revised form 10 March 2009
Accepted 11 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Available online 25 March 2009
1. Introduction
Based on the PRODAN structure, compounds 1 and 2 were newly
designed (Fig. 1). Compound 1 is a 6-dimethylamino-derivative of
Development of novel fluorescent molecules as chemosensors
for the detection of chemically, biologically, and environmentally
important functional molecules has received much attention in
recent years.1 Although a large number of fluorescent chemo-
sensors have been prepared based on some basic fluorophores,
development of novel poly-functionalized fluorophores is still an
important theme for creation of chemosensors. PRODAN is one of
the most fundamental fluorophores having a relatively large molar
extinction coefficient and a high fluorescence quantum yield
(Fig. 1).2 This molecule is sensitive to environmental changes, and
with an increase in dielectric constant of the solvent, the maxima of
emission spectra of the molecule shifts to longer wavelength
regions.
kynurenic acid ethyl ester. Kynurenic acid and related quinoline-
carboxylic acid derivatives have been well known not only as im-
portant compounds for neurobiology3 but also as receptors for
metal ions.4 Kynurenic acid derivatives can be prepared through
short steps including Conrad–Limpach cyclization5 and can contain
extendable functional groups such as hydroxyl and ethoxycarbonyl
groups. Moreover, the sp2 nitrogen atom serves as a Lewis base on
the fluorophore, and together with the ethoxycarbonyl group,
could be expected to behave as a coordination site for an external
acidic functional group.
Zinc ion is the second most abundant heavy metal ion and is
known to play important roles in cellular events including struc-
tural co-factors, regulators, and catalytic centers of enzymes, DNA
binding, and neuronal signal transmission.6 In relation to these
biological roles, many ratiometric fluorescent probes for zinc ion
have been already developed.7 In this paper, we report the de-
velopment of novel fluorescent compounds and their ratiometric
response to zinc(II) salt.
OH
5
8
4
N
3
2
6
7
O
N
1
O
OH
PRODAN
N
kynurenic acid
2. Results and discussions
2.1. Synthesis
OR
N
Compounds 1 and 2 were prepared in a similar manner as
the preparation of kynurenic acid derivatives8 in two or three
steps sequence from commercially available compounds and
results are shown in Scheme 1. Condensation of N,N-dimethyl-
p-phenylenediamine with oxalacetic acid diethyl ester gave in-
termediate 3 and subsequent cyclization under reflux conditions
in diphenyl ether gave fluorescent compound 1 in 76% yield.
Compound 2 was obtained by benzyl etherification of 1 in 67%
yield.
O
1: R = H
2: R = Bn
OEt
Figure 1. Structures of PRODAN, kynurenic acid and fluorescent compounds 1 and 2.
* Corresponding author. Tel.: þ81 42 676 5473; fax: þ81 42 676 7282.
E-mail address: itohisa@ls.toyaku.ac.jp (H. Ito).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.037

4236
H. Ito et al. / Tetrahedron 65 (2009) 4235–4238
O
OEt
OEt
N
N
O
toluene
reflux
+
OEt
N
OEt
HO
NH2
H
O
O
3
OH
OBn
N
N
diphenyl ether
reflux
NaH, BnBr
DMF
O
O
N
N
OEt
OEt
2 steps 76%
67%
1
2
Scheme 1. Synthesis of compounds 1 and 2 through Conrad–Limpach cyclization.
2.2. Fluorescent characteristics
The fluorescent characteristics of compounds 1 and 2 are shown
in Table 1. Excitation and emission spectra for both compounds
were strongly influenced by solvent polarity. Compound 1 having
a free phenolic hydroxyl group showed maxima of emission spectra
at 501 nm (toluene) and 552 nm (MeOH). Compound 2, in which
the hydroxyl group is protected by a benzyl group, showed maxima
of emission spectra at 422 nm (toluene) and 484 nm (MeOH). These
results indicated that in the case of compound 1, the contribution of
tautomeric isomer 4 significantly affects both excitation and
emission spectra (Scheme 2).
Figure 2. Changes in excitation (a) and emission (b) spectra of 2 in toluene with
various amounts of Zn2þ ions ([2]¼0.29
m
M, [Zn2þ]¼0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60,
Table 1
and 0.90 mM) in methanol/toluene.
Fluorescent properties of 1 and 2
Compound
Solvent
3
(Mꢀ1 cmꢀ1
)
Q
Abs (nm)
Em (nm)
decreased, and a new band centered at 503 nm arose, corre-
sponding to a lmax (emission) red-shift of 77 nm and an isosbestic
point at 472 nm (Fig. 2b).
The ratiometric fluorescent response of compound 2 in the
presence of other metal salts is shown in Figure 3. As mentioned
above, compound 2 showed a quenching response to copper(II)
salt,9 and the ratiometric response of 2 is shown only with the
zinc(II) salt.
To elucidate the stoichiometry of complex formation of 2 with
zinc(II) salt, Job’s plot analysis was examined and the result in-
dicated that the 1:2 complex of 2 and zinc(II) salt was formed in
toluene under the presented conditions (Fig. 4).
A single crystal of the complex of compound 2 with zinc bro-
mide was obtained by recrystallization in toluene. The X-ray crystal
structure demonstrated that the zinc(II) salt adopts a tetrahedral
1
1
2
2
Toluene
MeOH
Toluene
MeOH
8.06ꢁ103
4.94ꢁ103
15.42ꢁ103
17.17ꢁ103
0.35
0.06
0.77
0.38
426
434
372
387
501
552
422
484
OH
O
N
N
N
O
OEt
N
OEt
Scheme 2. Tautomerization of 1.
H
4
O
1
2.3. Recognition of metal salts
As mentioned above, the present fluorescent molecules have
a nitrogen atom on the quinoline ring and a carbonyl group in the
ester moiety, and both functional groups could serve as co-
ordination sites for a metal ion. Therefore, the behavior of com-
pounds 1 and 2 in the presence of metal salt was examined.
Addition of a solution of copper(II) salt to a solution of compound 1
or 2 in toluene, resulted in a quenching of the fluorescence. How-
ever, no significant effects during the addition of a solution of
zinc(II) salt to a solution of compound 1 in toluene were observed.
This result also supported the presence of tautomer 4, which does
not have a quinoline ring. On the other hand, zinc(II) salt gave
a ratiometric response in both excitation and emission spectra with
compound 2 in toluene (Fig. 2). Thus, upon addition of a solution of
zinc bromide in toluene, the absorption intensities of 2 at 305 and
455 nm gradually increased, accompanied by a decrease in ab-
sorption intensities at 343 and 373 nm (Fig. 2a). In the emission
spectra, the characteristic strong emission band at 426 nm
Figure 3. Ratiometric fluorescent response of 2 with various metal salts in toluene
(ex 314 nm).

H. Ito et al. / Tetrahedron 65 (2009) 4235–4238
4237
commercial suppliers and used without further purification. 1H
and 13C NMR spectra were measured on a Bruker AV-300 and
chemical shifts are given in parts per million using CH3OH
(3.34 ppm) in CD3OD for 1H NMR and CD3OD (49.0 ppm) for 13C
NMR as internal standards, respectively. IR spectra were taken
with a Perkin–Elmer PARAGON 1000 FT-IR and only noteworthy
absorptions are listed. Mass spectra were measured on a Micro-
mass LCT. Fluorescence spectroscopic studies were performed
with a RF-5300PC (Shimadzu Corp.). Quantum yield was mea-
sured on an absolute PL quantum yield measurement system
(C9920-02, Hamamatsu Photonics K.K.). All solvents for mea-
surement of photochemical properties were of analytic grade. The
solutions of 1 and 2 in toluene were prepared before use. The
solutions of metal salts were prepared from ZnBr2, MnCl2$4H2O,
FeCl2$4H2O, CoCl2, NiCl2$6H2O, CuCl2, CdCl2$5/2H2O, NaCl, KCl,
MgCl2$6H2O, CaCl2 in methanol (10 mM). The solution was di-
Figure 4. Job’s plot for the complexation equilibrium of 2 with ZnBr2 in toluene (ex
314 nm, em 505 nm).
luted with toluene (100
mM for NaCl, KCl, MgCl2, CaCl2$H2O, and
1
m
M for other metal salts) before use.
4.2. Synthesis
4.2.1. 6-Dimethylamino-4-hydroxy-2-quinolinecarboxylic acid
ethyl ester (1)
A solution of oxalacetic acid diethyl ester (1.6 g, 8.8 mmol) and
N,N-dimethyl-p-phenylenediamine (1.2 g, 8.8 mmol) in toluene
(20 mL) was stirred at reflux for 18 h. The mixture was concen-
trated under vacuum. The resulting residue was dissolved in
diphenyl ether (10 mL) and the solution was stirred at reflux for
10 min. The mixture was concentrated under vacuum and the
residue was purified by silica gel column chromatography (hexane/
isopropanol, 3:1, followed by AcOEt only) to give compound 1
(1.74 g, 6.7 mmol) as yellow crystals in 76% yield. Mp 220–222 ꢂC. IR
(KBr)
CD3OD)
n
cmꢀ1: 3066, 1727, 1508, 1382, 1272. 1H NMR (300 MHz,
; 1.47 (3H, t, J¼7.1 Hz), 3.11 (6H, s), 4.52 (2H, q, J¼7.1 Hz),
d
6.93 (1H, s), 7.37 (1H, d, J¼2.8 Hz), 7.48 (1H, dd, J¼2.8, 9.3 Hz), 7.82
(1H, d, J¼9.3 Hz). 13C NMR (75 MHz, CD3OD)
d; 14.4 (CH3), 40.6
(CH3ꢁ2), 63.9 (CH2), 103.7 (CH), 108.7 (CH), 121.5 (CH), 122.3 (CH),
128.4 (C), 133.5 (C), 137.7 (C), 149.6 (C), 163.3 (C), 179.8 (C). HRESIMS
calcd for C14H17N2O3: 261.1239 (MþH)þ, found: 261.1248. Anal.
Calcd for C14H16N2O3: C, 64.60; H, 6.20; N, 10.76. Found: C, 64.64; H,
6.35; N, 10.37.
Figure 5. ORTEP view of the complex of 2 with ZnBr2.
geometry (Fig. 5). Although the formation of 1:2 complex of 2 and
zinc(II) salt was indicated by the result of Job’s plot analysis, the 1:1
complex was obtained as a single crystal. The nitrogen atom on the
quinoline ring and carbonyl group of the ester moiety coordinate
the zinc(II) salt to form a 1:1 complex.
4.2.2. 4-Benzyloxy-6-dimethylamino-2-quinolinecarboxylic acid
ethyl ester (2)
A solution of 1 (2.0 g, 7.69 mmol) in DMF (8 mL) was added to
a suspension of NaH (60%, 369 mg, 9.23 mmol) in DMF (8 mL) at
ambient temperature. After being stirred for 30 min at the same
temperature, benzyl bromide (1.71 g, 1.19 mL, 10 mmol) was
added to the mixture at ambient temperature and the resulting
mixture was stirred at the same temperature for 2.5 h. Water was
added to the mixture and the resulting mixture was extracted
with AcOEt three times. The combined organic layer was washed
with brine, dried over MgSO4, and concentrated under vacuum.
The residue was purified by silica gel column chromatography
(hexane/AcOEt, 1:1) to give compound 2 (1.8 g, 5.14 mmol) as
3. Conclusion
Novel fluorescent molecules having a coordination site for metal
ions were developed. Compound 2 acted as a chemosensor for
zinc(II) salt and showed a ratiometric response in both excitation
and emission spectra in aprotic solvent. Compounds 1 and 2 con-
tain an ester moiety that can be easily modified to other functional
groups, enabling the introduction of the present fluorescent com-
pounds into biologically functionalized molecules. Moreover, the
present compounds are analogs of kynurenic acid, which is an
important molecule for neurobiology. Development of fluorescent
probes by the introduction of compounds 1 and 2 to biologically
active functional molecules and an application for the visualization
of kynurenic acid-dynamics in neurobiology is now underway.
yellow crystals in 67% yield. Mp 130–134 ꢂC. IR (KBr)
n
cmꢀ1
; 1.48
:
1702, 1618, 1345, 1241, 1091. 1H NMR (300 MHz, CD3OD)
d
(3H, t, J¼7.1 Hz), 3.12 (6H, s), 4.48 (2H, q, J¼7.1 Hz), 5.42 (2H, s),
7.16 (1H, d, J¼2.8 Hz), 7.35–7.54 (4H, m), 7.56–7.62 (3H, m), 8.00
(1H, d, J¼9.4 Hz). 13C NMR (75 MHz, CD3OD)
d; 14.6 (CH3), 40.5
4. Experimental
4.1. General
(CH3ꢁ2), 62.8 (CH2), 71.5 (CH2), 99.2 (CH), 102.2 (CH), 121.0 (CH),
125.2 (C), 128.7 (CH), 129.3 (CH), 129.8 (CH), 130.8 (CH), 137.5 (C),
142.7 (C), 145.0 (C), 150.9 (C), 161.6 (C), 166.7 (C). HRESIMS calcd
for C21H23N2O3: 351.1709 (MþH)þ, found: 351.1712. Anal. Calcd for
C21H22N2O3: C, 71.98; H, 6.33; N, 7.99. Found: C, 71.92; H, 6.21; N,
7.84.
All reactions were carried out under an argon atmosphere.
Unless otherwise noted, materials were obtained from

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H. Ito et al. / Tetrahedron 65 (2009) 4235–4238
4.3. Job’s plot
References and notes
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A series of solutions containing 2 and ZnBr2 were prepared
such that the sum of the total metal and total fluorophore
concentration remained constant at 0.29 mM in toluene. The
molar fraction of the fluorophore was varied between 0.1 and
1.0.
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Single crystals of compound 2–ZnBr2 complex were grown
from a toluene solution at ambient temperature. Crystallo-
graphic data for the structural analysis of 2–ZnBr2 have been
deposited with the Cambridge Crystallographic Data Centre,
CCDC 707602.
Acknowledgements
This work was supported by Dynamic Biology Project of
New Energy and Industrial Technology Development Organi-
zation. We thank Dr. Kengo Suzuki (Hamamatsu Photonics
K.K.) for measurement of the quantum yields of the present
compounds.