6,7-Difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic acid, a newly designed fluorescence enhancement-type derivatizing reagent for amino compounds

Article abstract of DOI:10.1007/s10895-009-0596-2

A novel fluorescence enhancement-type derivatizing reagent for amino compounds, 6,7-difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic acid (FMQC), was developed. FMQC reacts with aliphatic primary amino compounds to afford strong fluorescent derivatives having high photo-and thermo-stabilities. The FMQC derivatives of amino compounds showed 12-159 times higher fluorescence quantum efficiencies than those of FMQC in aqueous and polar organic media. Additionally, the absorption and fluorescence emission wavelength of the derivatives are red-shifted from those of FMQC. These differences in the fluorescence properties between FMQC and the fluorescent derivative enabled the simple and highly sensitive determination of amino compounds without removing any excess unreacted FMQC by using a simple spectrofluorometer as well as HPLC.

Full text of DOI:10.1007/s10895-009-0596-2

J Fluoresc (2010) 20:615–624
DOI 10.1007/s10895-009-0596-2
ORIGINAL PAPER
6,7-Difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic
Acid, a Newly Designed Fluorescence Enhancement-Type
Derivatizing Reagent for Amino Compounds
Junzo Hirano & Kenji Hamase & Hiroyuki Miyata &
Kiyoshi Zaitsu
Received: 31 March 2009 /Accepted: 30 December 2009 /Published online: 28 January 2010
#
Springer Science+Business Media, LLC 2010
Abstract A novel fluorescence enhancement-type derivatiz-
ing reagent for amino compounds, 6,7-difluoro-1,4-dihydro-
1-methyl-4-oxo-3-quinolinecarboxylic acid (FMQC), was
developed. FMQC reacts with aliphatic primary amino
compounds to afford strong fluorescent derivatives having
high photo-and thermo-stabilities. The FMQC derivatives of
amino compounds showed 12–159 times higher fluorescence
quantum efficiencies than those of FMQC in aqueous and
polar organic media. Additionally, the absorption and fluo-
rescence emission wavelength of the derivatives are red-
shifted from those of FMQC. These differences in the
fluorescence properties between FMQC and the fluorescent
derivative enabled the simple and highly sensitive determina-
tion of amino compounds without removing any excess
unreacted FMQC by using a simple spectrofluorometer as
well as HPLC.
with the target molecules. When using a labeling type
reagent, it is necessary to separate a fluorescent derivative
of a target compound from the unreacted reagent. On the
other hand, the fluorescent derivatizing reagent, which
shows a drastic enhancement of the fluorescence intensity
after reacting with a target molecule, has a significant value.
By using the fluorescence enhancement-type derivatizing
reagent, a target compound could be sensitively determined
without separating the unreacted reagent. Therefore, in
order to establish simple and highly sensitive analytical
methods for various target compounds, such as physiolog-
ically active substances, pollutants, and drugs, significant
efforts have been devoted to designing fluorescence
enhancement-type derivatizing reagents.
In a previous study, we reported that 4-quinolone (4QO)
analogs are useful photo-and thermo-stable fluorophores with
high fluorescence quantum efficiencies (Φ) in aqueous media,
and developed various fluorescent reagents utilizing the 4QO
structure [4–8]. Among these reagents, [(6-methoxy-4-oxo-
1,4-dihydroquinolin-3-yl)methyl]amine (6MOQ-NH2) was a
useful fluorescent reagent with a high stability, and enabled
the sensitive determination of carboxylic acids [8]. However,
6MOQ-NH2 is a labeling-type reagent, so its application is
limited to separation analyses, such as HPLC. During the
course of our investigation focusing on the relationship
between the structure and fluorescence characteristics of
4QO derivatives in aqueous media, we found that the
fluorescence properties could be modified by introducing
electron withdrawing/donating groups to the 4QO moiety.
Especially, the Φ of 4QO derivatives in H2O drastically
changed, for example, Φ is 0.001 for 6-nitro-4-quinolone,
0.017 for 4QO, and 0.498 for 6-methoxy-4-quinolone. The
significant difference in the fluorescence characteristics of
the 4QO derivatives led us to develop a highly sensitive and
stable fluorescence enhancement-type derivatizing reagent
utilizing the 4QO structure.
.
.
Keywords Derivatizing reagent Fluorescence
.
.
4-Quinolone Amino compound HPLC
Introduction
Fluorescent derivatizing reagents enable the establishment
of highly sensitive analytical methods of non-fluorescent
compounds, and they are widely utilized in various research
fields including biochemistry, clinical chemistry, and
environmental chemistry. Up to now, a significant number
of fluorescent derivatizing reagents have been reported [1–
3]. Most of them are called fluorescent labeling reagents,
showing the same fluorescence characteristics after reacting
:
:
:
J. Hirano K. Hamase H. Miyata K. Zaitsu (*)
Graduate School of Pharmaceutical Sciences, Kyushu University,
3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan
e-mail: zaitsu@phar.kyushu-u.ac.jp

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J Fluoresc (2010) 20:615–624
In the present study, we focused on the development of a
(DMSO), and of HPLC grade for MeOH and MeCN. All
the other solvents were of guaranteed reagent grade. The
products were characterized by 1H NMR, FAB-MS and
fluorescence enhancement-type derivatizing reagent having
the 4QO structure which enables the highly sensitive
determination of amino compounds. Amino compounds
including amines and amino acids are present in various
matrices as physiologically active substances and environ-
mental substances, and therefore, numerous reagents for
fluorescence determination have been reported. However,
most of these reagents are the labeling-type ones, for example,
5-dimethylaminonaphthalene-1-sulfonylchloride (DNS-Cl)
[9], 2-(5′,6′-dimethoxybenzothiazolyl)-benzenesulfonyl chlo-
ride (BHBT-SOCl) [10], 9-fluorenylmethyl chloroformate
(Fmoc-Cl) [11], 3,4-dihydro-6,7-dimethoxy-4-methyl-3-
oxoquinoxaline-2-carbonyl chloride (DMEQ-COCl) [12],
fluorescein isothiocyanate (FITC) [13], N-succinimidyl-1-
naphthylcarbamate (SINC) [14], and 6-aminoquinolyl-N-
hydroxysuccinimidyl carbamate (AQC) [15]. On the other
hand, the fluorescence enhancement-type reagents, such as
o-phthalaldehyde (OPA) [16], fluorescamine [17], and
4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) [18] are still
limited. These reagents enable the rapid and sensitive
determination of amines, and have been widely utilized in
various analytical methods. However, these reagents are not
always satisfactory with respect to the stability of the
reagents and derivatives, showing decomposition by heating
and light irradiation [19, 20].
In the present paper, we report a novel option for the
stable fluorescence enhancement-type derivatizing reagent,
6,7-difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecar-
boxylic acid (FMQC), which is suitable for the simple and
sensitive determination of amino compounds. By reacting
with amino compounds, FMQC showed a drastic enhance-
ment of the fluorescence intensity and wavelength shift, and
therefore, enabled the highly sensitive determination of
amino compounds without separating from the unreacted
reagent. Furthermore, the synthesized FMQC derivative of
the amino compound was stable and showed a strong
fluorescence in water as well as organic solvents, such as
MeOH and MeCN. Herein, we now report the design and
synthesis of FMQC. The spectroscopic properties of FMQC
and the FMQC derivatives of amino compounds were also
indicated. As an application, the determination of amino
compounds was demonstrated using a simple spectrofluo-
rometer, and using a reversed-phase HPLC (RP-HPLC).
1
elemental analyses. The H NMR spectra were recorded
using a Varian Unity-400 spectrometer (400 MHz) in
dimethylsulfoxide-d6 (DMSO-d6). The mass spectra were
obtained using a JEOL JMS-SX102A mass spectrometer.
Syntheses
7-Fluoro-4-quinolone (1). Compound 1 was synthesized
as a colorless needle (yield 3.9%) in four steps from
m-fluoroaniline according to previously reported methods
1
[21–23]. Mp 254–256 °C. H NMR (500 MHz, DMSO-
d6): δ ppm 6.02 (1H, d, J=7.5 Hz, H-3), 7.15 (1H, td, J=
2.5, 8.8 Hz, H-6), 7.27 (1H, dd, J=2.4, 10.2 Hz, H-8),
7.89 (1H, d, J=7.3 Hz, H-2), 8.12 (1H, dd, J=6.4, 8.9 Hz,
H-5), 11.74 (1H, br s, H-1). FABMS m/z = 164 (M+H).
Anal. Calcd for C9H6NOF: C, 66.26; H, 3.71; N, 8.59.
Found: C, 65.99; H, 3.71; N, 8.60.
7-Fluoro-1-methyl-4-quinolone (2). A mixture of 1 (1.5 g,
9.2 mmol) and anhydrous K2CO3 (3.18 g, 23 mmol) in dry
DMF (15 mL) was heated at 50 °C for 2 h. MeI (2.86 mL,
46 mmol) was added, and the mixture was heated at 50 °C
for 12 h. The reaction mixture was diluted with H2O, and
extracted with CHCl3. The organic layer was dried over
MgSO4, filtered, and evaporated. The crude compound was
purified by column chromatography over silica gel with
CHCl3/MeOH (5:1) and with AcOEt/MeOH (4:1) to afford a
1
colorless powder (yield 59.7%). Mp 155–156 °C. H NMR
(400 MHz, DMSO-d6): δ ppm 3.76 (3H, s, Me), 6.03 (1H, d,
J=7.6 Hz, H-3), 7.24 (1H, td, J=2.3, 8.7 Hz, H-6), 7.50 (1H,
dd, J=2.5, 11.3 Hz, H-8), 7.95 (1H, d, J=7.6 Hz, H-2), 8.20
(1H, dd, J=6.7, 8.9 Hz, H-5). FABMS m/z = 178 (M+H).
Anal. Calcd for C10H8NOF•0.5H2O: C, 64.51; H, 4.87; N,
7.52. Found: C, 64.49; H, 4.91; N, 7.52.
6,7-Difluoro-1-methyl-4-quinolone (3). 6,7-Difluoro-4-
quinolone was prepared using 3,4-difluoroaniline as de-
scribed for 1. The methylation of 6,7-difluoro-4-quinolone
was carried out as described for 2 to afford a colorless
powder (yield 22.7%). Mp 183–184 °C. 1H NMR
(400 MHz, DMSO-d6): δ ppm 3.78 (3H, s, Me), 6.05
(1H, d, J=7.6 Hz, H-3), 7.83 (1H, dd, J=6.7, 12.5 Hz, H-5
or H-8), 7.98 (1H, d, J=7.6 Hz, H-2), 8.00 (1H, dd, J=9.2,
11.0 Hz, H-5 or H-8). FABMS m/z = 196 (M+H). Anal.
Calcd for C10H7NOF2•0.3H2O: C, 59.88; H, 3.82; N, 6.98.
Found: C, 60.02; H, 3.79; N, 7.02.
Experimental
General procedures
Commercially available materials were used without any
additional purification. The solvents used for the spectral
studies were of spectroscopic grade for dimethylsulfoxide
7-Fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid
(4). The colorless powder (yield 71.9%) of 4 was obtained as
the synthetic precursor of 1 according to previously reported

J Fluoresc (2010) 20:615–624
617
1
methods [21–23]. Mp 297–298 °C. H NMR (400 MHz,
DMSO-d6): δ ppm 7.48 (1H, td, J=2.4, 8.9 Hz, H-6), 7.57
(1H, dd, J=2.4, 9.8 Hz, H-8), 8.36 (1H, dd, J=6.1, 8.9 Hz,
H-5), 8.93 (1H, s, H-2), 13.36 (1H, br s, H-1), 15.11 (1H, s,
CO2H). FABMS m/z = 206 (M–H).
A colorless powder of FMQC (yield 76.7%) was
synthesized as described for 5 using 6,7-difluoro-1,4-
dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester. Mp
1
296.5–297.5 °C. H NMR (400 MHz, DMSO-d6): δ ppm
4.07 (3H, s, Me), 8.19 (1H, dd, J=6.7, 12.2 Hz, H-5 or
H-8), 8.26 (1H, dd, J=8.7, 10.5 Hz, H-5 or H-8), 9.05 (1H,
s, H-2), 14.85 (1H, s, CO2H). FABMS m/z = 238 (M–H).
Anal. Calcd for C11H7NO3F2: C, 55.24; H, 2.95; N, 5.86.
Found: C, 55.23; H, 2.96; N, 5.83.
7-Fluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarbox-
ylic acid (5). Compound 5 was synthesized in one step,
starting from 7-fluoro-1,4-dihydro-4-oxo-3-quinoline carbox-
ylic acid ethyl ester obtained in the following manner. A
mixture of m-fluoroaniline (4.44 g, 40 mmol) and diethyle-
thoxymethylenemalonate (DEEM)(8.65 g, 40 mmol) was
heated at 120 °C for 100 min under N2 gas flow. The
obtained liquid was added to diphenyl ether (200 mL), and
the mixture was refluxed for 1 h. After petroleum ether
(200 mL) was added to the reaction mixture, the resulting
precipitate was collected by filtration, and washed with
acetone to afford 7-fluoro-1,4-dihydro-4-oxo-3-quinoline car-
boxylic acid ethyl ester as a colorless powder (yield 85.0%).
A mixture of 7-fluoro-1,4-dihydro-4-oxo-3-quinoline
carboxylic acid ethyl ester (2.0 g, 8.5 mmol) and anhydrous
K2CO3 (2.94 g, 21.3 mmol) in dry DMF (40 mL) was
heated at 50 °C for 2 h. MeI (3.18 ml, 51 mmol) was then
added, and the mixture was heated at 50 °C for 20 h. The
reaction mixture was diluted with H2O, and extracted with
CHCl3. The organic layer was dried over MgSO4, filtered,
and evaporated. The residue was then dissolved in hot H2O-
MeOH and filtered. The resulting colorless powder was
collected by filtration. To a suspension of the powder in
H2O (20 mL), H2SO4 (2 mL) and AcOH (12 mL) was
added, and the mixture was refluxed for 30 min. The
resulting precipitate was collected by filtration, then washed
with H2O to afford a colorless powder (yield 71.5%). Mp
>300 °C. 1H NMR (400 MHz, DMSO-d6): δ ppm 4.06 (3H,
s, Me), 7.56 (1H, td, J=2.4, 8.7 Hz, H-6), 7.86 (1H, dd, J=
2.4, 11.0 Hz, H-8), 8.43 (1H, dd, J=6.4, 8.9 Hz, H-5), 9.04
(1H, s, H-2), 15.05 (1H, s, CO2H). FABMS m/z = 222 (M+
H). Anal. Calcd for C11H8NO3F: C, 59.73; H, 3.65; N,
6.33. Found: C, 59.72; H, 3.66; N, 6.30.
6-Fluoro-1,4-dihydro-1-methyl-7-n-propylamino-4-oxo-3-
quinolinecarboxylic acid (7). A mixture of FMQC (100 mg,
0.42 mmol) and n-propylamine (275 μL, 3.34 mmol) in
MeCN (10 mL) was heated at 65 °C for 5 h. The resulting
precipitate was collected by filtration, then washed with
MeCN and H2O to afford a colorless block (yield 60.2%). Mp
243–244 °C. 1H NMR (400 MHz, DMSO-d6, 50 °C): δ ppm
0.96 (3H, t, J=7.3 Hz, Me), 1.68 (2H, qt, J=7.0, 7.3 Hz,
CH2), 3.25–3.30 (2H, m, CH2), 4.00 (3H, s, Me), 6.72 (1H, d,
J=7.3 Hz, H-8), 6.87 (1H, br t, NH), 7.78 (1H, d, J=11.9 Hz,
H-5), 8.79 (1H, s, H-2), 15.69 (1H, s, CO2H). FABMS m/z =
277 (M–H). Anal. Calcd for C14H15N2O3F: C, 60.42; H, 5.43;
N, 10.07. Found: C, 59.78; H, 5.49; N, 9.95.
7-N,N-Diethylamino-6-fluoro-1,4-dihydro-1-methyl-4-
oxo-3-quinolinecarboxylic acid (8). A colorless block
(yield 34.4%) of 8 was synthesized as described for 7
1
using diethylamine. Mp 210–211 °C. H NMR (400 MHz,
DMSO-d6): δ ppm 1.19 (6H, t, J=7.0 Hz, Me), 3.50 (4H, q,
J=7.0 Hz, CH2), 4.02 (3H, s, Me), 6.87 (1H, d, J = 7.9 Hz,
H–8), 7.83 (1H, d, J = 14.7 Hz, H–5), 8.86 (1H, s, H–2),
15.54 (1H, s, CO2H). FABMS m/z = 291 (M–H). Anal.
Calcd for C15H17N2O3F: C, 61.63; H, 5.86; N, 9.58. Found:
C, 61.64; H, 5.86; N, 9.57.
Absorption and fluorescence studies
The absorption and corrected fluorescence emission spectra
were obtained using a JASCO V-530 UV/Vis spectrophotom-
eter and a JASCO F-6500 spectrofluorometer. The compounds
were dissolved in DMSO at 10 mM concentrations for 1–6,
and at 1 mM concentrations for 7 and 8. These solutions were
diluted with various solvents to the appropriate concentration
for the measurements. The relative fluorescence quantum
efficiencies were obtained using quinine sulfate in aqueous
0.05 M H2SO4, which has a quantum efficiency of 0.55.
6,7-Difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecar-
boxylic acid (6, FMQC). FMQC was synthesized in one
step, starting from 6,7-difluoro-1,4-dihydro-4-oxo-3-quino-
linecarboxylic acid ethyl ester obtained in the following
manner. A mixture of 3,4-difluoroaniline (5.16 g, 40 mmol)
and DEEM (8.65 g, 40 mmol) was heated at 120 °C for
100 min under N2 gas flow. The obtained product was
added to diphenyl ether (200 mL), and the mixture was
refluxed for 1 h. After petroleum ether (200 mL) was added
to the reaction mixture, the resulting precipitate was
collected by filtration, and washed with acetone to afford
6,7-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid
ethyl ester as a colorless powder (yield 71.8%).
Reactivity of 1–6 for n-propylamine
Using each organic solvent (DMSO, DMF, MeCN),
compounds 1–6 were prepared at a 10 mM concentration,
while triethylamine (Et3N) and n-propylamine were pre-
pared at a 400 mM concentration. To a siliconized tube,

618
J Fluoresc (2010) 20:615–624
Fig. 1 Structures of designed
candidates 1–6 as fluorescence
enhancement-type derivatizing
reagents for amino compounds
O
O
O
F
F
F
F
N
H
N
N
Me
Me
1
2
5
3
O
O
O
CO2H
CO2H
F
F
CO2H
F
F
N
H
N
N
Me
Me
4
6 (FMQC)
each 1–6 solution (100 μL), triethylamine (Et3N) solution
(5 μL), and n-propylamine solution (5 μL) were added. The
tube was capped and then heated at 65 °C for 30 min. An
aliquot (20 μL) of the reaction mixture was diluted with
50 mM phosphate buffer (pH 10) or aqueous 10 mM HCl
to the appropriate concentration for the absorption and
fluorescence measurements.
was diluted with aqueous 10 mM HCl (2.7 mL), the
emission intensity at 445 nm was measured with the
excitation wavelength at 350 nm.
RP-HPLC of amines with FMQC
To a siliconized tube, 5 mM FMQC in DMSO (100 μL), 5 mM
Et3N in DMSO (100 μL), mixed amines (200 nM each
of n-propylamine, n-butylamine, n-pentylamine, and
n-hexylamine) in DMSO (100 μL) were added. The tube
was capped and heated at 100 °C for 5 h. An aliquot (30 μL)
of the reaction mixture was diluted with 10 mM aqueous HCl
(970 μL), and 5 μL of the solution was subjected to the
HPLC system. The HPLC system (Shiseido NANOSPACE
Fluorimetric analysis of n-propylamine with FMQC
To a siliconized tube, 5 mM FMQC in DMSO (100 μL),
5 mM Et3N in DMSO (100 μL), n-propylamine (9, 45, 90,
450, 900 μM) in DMSO (100 μL) were added. The tube was
capped and heated at 100 °C for 5 h. The reaction mixture
Table 1 Absorbance and fluorescence properties of 1-6 in various solvents at 20 °C
Compund
Solvent
Absorption
max (nm)
Emission
max (nm)
Molar absorptivity
(× 104 M-1 cm-1)
ΦF
1
10 mM aqueous HCl
H2O
311
311
312
318
319
319
321
322
322
304
304
313
310
310
320
312
312
323
346
343
387
350
349
350
357
356
357
336
337
379
344
344
358
347
347
355
0.910
1.289
1.145
0.989
1.324
1.327
1.030
1.288
1.283
1.021
1.114
0.911
1.223
1.176
1.138
1.193
1.146
1.049
0.062
0.007
0.094
0.038
0.015
0.018
0.022
0.021
0.022
0.050
0.045
0.028
0.046
0.023
0.001
0.036
0.029
0.001
50 mM Na-phosphate buffer (pH 10)
10 mM aqueous HCl
H2O
2
3
4
5
6
50 mM Na-phosphate buffer (pH 10)
10 mM aqueous HCl
H2O
50 mM Na-phosphate buffer (pH 10)
10 mM aqueous HCl
H2O
50 mM Na-phosphate buffer (pH 10)
10 mM aqueous HCl
H2O
50 mM Na-phosphate buffer (pH 10)
10 mM aqueous HCl
H2O
50 mM Na-phosphate buffer (pH 10)
ΦF: Fluorescence quantum efficiency

J Fluoresc (2010) 20:615–624
619
1.5
10
8
1.5
1.0
10
8
1.5
1.0
10
8
Na-phosphate
Na-phosphate
buffer (pH 10)
Na-phosphate
buffer (pH 10)
4
5
6
buffer (pH 10)
1.0
6
6
6
4
4
4
0.5
0
0.5
0
0.5
0
2
0
2
0
2
0
210
300
Wavelength (nm)
400
450
210
300
400
500 550
210
300
400
500 550
Wavelength (nm)
Wavelength (nm)
1.5
1.0
10
8
1.5
1.0
10
8
1.5
10
8
4
5
6
10 mM HCl aq.
10 mM HCl aq.
10 mM HCl aq.
1.0
6
6
6
4
4
4
0.5
0
0.5
0
0.5
0
2
0
2
0
2
0
210
300
400
450
210
300
400
500 550
210
300
400
500 550
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Fig. 2 Absorption and fluorescence emission spectra (excitation at 280 nm) of the reaction mixtures of 4–6 in the presence (red line) and absence
of n-propylamine (blue line) in 50 mM phosphate buffer (pH 10) and 10 mM aqueous HCl
SI-2 series) consisted of a type 3101 pump, a 3007 injector, a
3014 column oven, and a 3013 fluorescence detector. A data
processing program, Shiseido EZChrom Elite, was used
to monitor the detector response. The analytical column
used was a CAPCELL PAK C18 MGII S3 (1.0 mm i.d.
x 150 mm, Shiseido) maintained at 40 °C. The mobile
phase was MeCN-TFA-water (35:0.05:65, v/v/v), and the
flow rate was 60 μL/min. The fluorescence detection was
carried out at 445 nm with the excitation wavelength at
350 nm.
fluoro-4-quinolone derivatives are reported to undergo
nucleophilic attack by amines, and form 7-amino-4-quino-
lone derivatives [24–27]. In our laboratory, we have also
synthesized 6-and/or 7-substituted 4QO derivatives, and
found that the derivatives having electron-withdrawing
groups, such as NO2, AcO, and Cl, show low Φ values,
while the derivatives having electron-donating groups, such
as MeO, EtO, and NMe2, show high Φ values. These
fundamental data strongly suggested that the 7-fluoro-4-
quinolone derivatives show weak fluorescence, and the 7-
amino-4-quinolone derivatives show strong fluorescence.
Therefore, to develop fluorescence enhancement-type deri-
vatizing reagents for amino compounds, we designed the
six candidate compounds based on the 7-fluoro-4-quino-
lone structure. Figure 1 summarizes the designed com-
pounds 1–6. Compound 1 is 7-fluoro-4-quinolone.
Compounds 2 and 3 have a Me group at the 1 position of
the 4QO moiety. Compounds 4–6 are designed to have a
COOH group at the 3 positions of 1–3, respectively, to
increase the reactivity for the amino compounds. Com-
Results and discussion
Design and synthesis of 7-fluoro-4-quinolone derivatives
as fluorescence enhancement-type derivatizing reagents
for amino compounds
A number of 4QO derivatives has been synthesized as
antibacterial agents. Among these derivatives, several 7-
Fig. 3 Syntheses of 7 and 8
O
F
CO2H
excess n-propylamine
n-Pr
MeCN, 60 ºC
N
H
N
O
Me
F
CO2H
7
F
N
O
Me
FMQC
F
CO2H
excess diethylamine
MeCN, 60 ºC
Et2N
N
Me
8

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J Fluoresc (2010) 20:615–624
Table 2 Absorbance and fluorescence properties of FMQC, 7, and 8 in various solvents at 20 ºC
Compound
Solvent
Absorption
max (nm)
Emission
max (nm)
Molar absorptivity
(× 104 M-1 cm-1)
ΦF
FMQC
10 mM aqueous HCl
312
312
323
314
315
349
348
324
343
338
363
362
328
354
349
347
347
355
349
350
442
439
411
432
416
365,444
468
436
439
479
1.193
1.146
1.049
1.253
1.207
1.011
1.020
1.379
1.024
1.004
0.577
0.867
1.332
1.078
1.052
0.036
0.029
0.001
0.018
0.020
0.451
0.599
0.159
0.527
0.243
0.009
0.001
0.014
0.001
0.004
H2O
50 mM Na-phosphate buffer (pH 10)
MeOH
MeCN
7
8
10 mM aqueous HCl
H2O
50 mM Na-phosphate buffer (pH 10)
MeOH
MeCN
10 mM aqueous HCl
H2O
50 mM Na-phosphate buffer (pH 10)
MeOH
MeCN
ΦF: Fluorescence quantum efficiency
pounds 1–6 were synthesized in high yield following
Gould-Jacobs reaction and literature methods [21–23].
Compounds 1–4 exhibited no change in the absorption
and fluorescence emission spectra obtained in both
alkaline and acidic solutions. On the other hand, for 5
and 6, drastic spectral changes were observed, and the
fluorescence enhancements (red line) were observed in the
visible region of 400–500 nm in both alkaline and acidic
solutions, indicating these two compounds were promising
candidates for fluorescence enhancement-type derivatizing
reagents for amino compounds. Concerning 5, due to the
poor solubility in H2O, a precipitate was observed in the
10 mM aqueous HCl solution. Furthermore, the fluores-
cence enhancement was significantly greater for 6 than 5
(Fig. 2). Therefore, compound 6 (6,7-difluoro-1,4-dihydro-1-
methyl-4-oxo-3-quinolinecarboxylic acid, FMQC) was
employed in a subsequent study.
Spectroscopic properties of synthesized analogs 1–6
and reactivities to amino compounds
The absorption and fluorescence properties of 1–6 were
evaluated in 10 mM aqueous HCl, H2O, and 50 mM
phosphate buffer (pH 10) (Table 1). All the compounds
exhibited similar absorption spectra with the maximum
wavelengths of ca. 300–320 nm, and the molar absorptiv-
ities were on the order of 104M−1cm−1. In the fluorescence
emission spectra, 1–6 showed very weak fluorescences as
expected; the maximum Φ of 0.094 was observed for 1 in
50 mM phosphate buffer (pH 10).
We next investigated the reactivities of 1–6 to amino
compounds. The mixture of each derivative and n-propyl-
amine, which was selected as an amino compound, was
heated in the presence of the basic catalyst Et3N, and then
diluted with 50 mM phosphate buffer (pH 10) or 10 mM
aqueous HCl for the spectral measurement. Figure 2
represents the absorption and fluorescence emission
spectra of the reaction mixtures of 4–6 in the presence
(red line) and absence (blue line) of n-propylamine.
Spectroscopic properties of the FMQC derivative
of n-propylamine
The derivative 7 described in Fig. 3 was obtained by
reacting FMQC and n-propylamine on a synthetic scale in
high yield, and the absorption and fluorescence properties
were evaluated. Table 2 summarizes the spectroscopic
properties of FMQC and 7 in five solvents. FMQC showed
Fig. 4 Acid-base equilibriums
of 7
O
O
O
F
CO2H
F
CO2H
F
CO2
H+
H+
n-Pr
n-Pr
n-Pr
+
N
H2
N
N
H
N
N
H
N
pKa < 2
pKa = 6.5
Me
Me
Me
Weak fluorescence
Strong fluorescence
Weak fluorescence

J Fluoresc (2010) 20:615–624
621
2
10
8
(a)
(b)
1.5
6
4
1
0.5
0
detection
excitation
2
0
210
250
300
Wavelength (nm)
350
400
300
400
Wavelength (nm)
500
550
Fig. 5 a Absorption spectra of FMQC (blue line, 30 μM, 0.3% DMSO) and 7 (red line, 30 μM, 3% DMSO) in 10 mM HCl aq.; b fluorescence
emission spectra (excitation at 350 nm) of FMQC (blue line, 3 μM, 0.03% DMSO) and 7 (red line, 3 μM, 0.3 % DMSO) in 10 mM aqueous HCl
a weak fluorescence even in organic solvents. On the other
hand, compound 7 showed a strong fluorescence in all the
solvents; the obtained Φ values were 0.159–0.599, which
were 12–159 times higher than those of FMQC. The
absorption maxima of 7 were 324–349 nm, ca. 30 nm
red-shifted in comparison with FMQC except when using
50 mM phosphate buffer (pH 10). The fluorescence
emission wavelengths of 7 were 411–442 nm, which were
60–90 nm red-shifted from those of FMQC. To understand
the detailed spectroscopic properties, the acid-base equilib-
riums of both compounds were investigated. FMQC
exhibited a blue-shift of the absorption spectrum and the
decrease of the fluorescence intensity with a lowering of the
pH, and the pKa of COOH at the 3 position was estimated
to be 5.9. These results indicate that FMQC is mainly the
weakly fluorescent molecular form at pHs lower than 5.9,
and is the non-fluorescent carboxylate ionic form over pH
5.9. Figure 4 shows the acid-base equilibrium of 7. Based
on the blue-shift of the absorption spectrum similar to that
of FMQC, the pKa of COOH at the 3 position of 7 was
determined to be 6.5. In the fluorescence emission
spectrum, the Φ decreased over pH 6, which was caused
by the deprotonation of the COOH group at the 3 position.
Furthermore, Φ also decreased at a pH lower than pH 4,
which was thought to be caused by the protonation of the
and 347 nm for the emission. Concerning the stability,
compound 7 exhibited no decomposition for more than a
month during storage in a clear glass vial at room
temperature. With respect to its stability, FMQC was
superior to OPA, fluorescamine, and NBD-F reported as
the fluorescence enhancement-type derivatizing reagents
for amino compounds.
Reaction selectivity for amino compounds
The reaction selectivity of FMQC for amino compounds was
investigated using aliphatic primary amino compounds
(n-butylamine, n-pentylamine, n-hexylamine), aliphatic sec-
ondary amino compounds (diethylamine, di-n-propylamine),
and aromatic amino compounds (aniline, p-methoxyaniline,
p-chloroaniline). The mixture of FMQC and each amino
compound was heated in the presence of Et3N, and diluted
with 10 mM aqueous HCl for the spectral measurements. For
all the aliphatic primary amino compounds, the absorbance
enhancements at 350 nm were observed similar to that of
n-propylamine, and ca. one-sixth of the absorbance enhance-
Table 3 Emission intensities of reaction mixtures for various amino
compounds derivatized with FMQC
Emission intensity, a.u.a
+
Amine
NH group at the 7 position, indicating that the pKa of NH2
is lower than ca. 2. These results suggested that 7 shows a
strong fluorescence as its molecular form around pH 4.
The differences in the spectroscopic properties between
FMQC and 7 indicated that the use of the appropriate
excitation and emission wavelengths enables the highly
sensitive determinations of amino compounds without
separating any excess unreacted reagent, FMQC. Figure 5
represents the absorption and fluorescence emission spectra
of FMQC and 7 in 10 mM aqueous HCl. When using an
aqueous acidic solvent, the weak fluorescence of FMQC is
quite negligible by employing the excitation wavelength of
350 nm and the emission wavelength of 445 nm, because
the wavelengths of FMQC are 312 nm for the absorption
n-Propylamine
n-Butylamine
n-Propylamine
n-Hexylamine
Diethylamine
Di-n-propylamine
Aniline
100
126
132
150
3.5
0.60
0.67
0.20
0.20
0.56
p-Methoxyaniline
p-Chloroaniline
Blank
Each amine (450 μM) was derivatized with FMQC and Et3N at 100 ºC
for 1 hr. a Emission intensity at 445 nm (excitation at 350 nm)

622
J Fluoresc (2010) 20:615–624
600
400
200
(b)
550
400
200
0
y = -3.872 + 0.5673x
(a)
900 µM
450 µM
90 µM
45 µM
9 µM
r = 0.9997
0
400
450
500
550
0
200
400
600
800
1000
Wavelength (nm)
[n-Propylamine] (µM)
Fig. 6 a Fluorescence emission spectra (excitation at 350 nm) of the FMQC-derivatives for various amounts of n-propylamine in 10 mM aqueous
HCl; b calibration line of derivatized n-propylamine
ment for the primary amino compound was obtained for
diethylamine. Whereas, for all other amino compounds, no
spectral change was obtained, suggesting that the derivatiza-
tion reaction hardly proceeded for these compounds. Table 3
shows the fluorescence emission intensities at 445 nm
detected with excitation at 350 nm. The reaction mixtures
for all the aliphatic primary amino compounds exhibited a
strong fluorescence similar to that of n-propylamine,
however, the intensity for diethylamine was very weak.
The reaction mixture for diethylamine showed a slight
fluorescence despite the change of the absorption spectrum. To
clarify this phenomenon, we synthesized the derivative 8 by
reacting FMQC and diethylamine as described in Fig. 3. The
absorption and fluorescence properties of 8 were evaluated,
and it was revealed that 8 exhibited a much weaker
fluorescence than 7, its Φ value was 0.009 in 10 mM aqueous
HCl (Table 2). These obtained results indicated that FMQC is
the fluorescence enhancement-type derivatizing reagent suit-
able for the analysis of aliphatic primary amino compounds.
n-hexylamine) using FMQC. These four amino compounds
were derivatized with FMQC under the optimized conditions,
and subjected to the RP-HPLC separation without removing
the excess FMQC. Figure 7 shows the chromatogram of the
derivatives for the four amino compounds, and the deriva-
tives could be well separated within 45 min using the
isocratic mobile phase of MeCN-TFA-water (35:0.05:65,
v/v/v). Concerning the sensitivity, the signal-to-noise ratio
(S/N) for each amino compound (10 fmol per injection) were
133, 140, 87, and 58, respectively, indicating that their lower
limits of detection (S/N=3) were subfmol per injection,
which are equivalent to that of the derivatives of NBD-F
(fmol-subfmol) [28] and more sensitive than those of the
derivatives for OPA (pmol) [29] and fluorescamine (pmol)
[30]. Considering the high photo-and thermo-stabilities of
the FMQC derivatives, FMQC could be a novel and useful
option for the fluorescence enhancement-type derivatizing
reagent for amino compounds.
2
Determination of aliphatic primary amino compounds
derivatized with FMQC
1
After the optimizations of the basic catalyst and reaction
conditions (temperature and time), the procedure indicated in
the Experimental section was selected. With the optimized
reaction conditions, various amounts of n-propylamine were
derivatized with FMQC, and the fluorimetric determination
was performed using the spectrofluorometer. Figure 6 shows
the fluorescence emission spectra with the excitation
wavelength at 350 nm. The fluorescence emission intensity
quantitatively increased in accordance with the amount of
n-propylamine added, and the calibration line for the
derivatized n-propylamine was linear over the range from
9 μM to 900 μM (r=0.9997). We also demonstrated the
separation analysis of four aliphatic primary amino
compounds (n-propylamine, n-butylamine, n-pentylamine,
3
4
0
15
30
45
Retention time (min)
Fig. 7 Chromatogram of the derivatives for four aliphatic primary
amino compounds (10 fmol): (1) n-propylamine, (2) n-butylamine, (3)
n-pentylamine, (4) n-hexylamine

J Fluoresc (2010) 20:615–624
623
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Conclusions
In the present study, we designed and synthesized six
possible candidates as fluorescence enhancement-type
derivatizing reagents for amino compounds. Among these
compounds, FMQC exhibited a reactivity to amino com-
pounds with a drastic fluorescence enhancement. The
FMQC derivative of n-propylamine showed not only a
high Φ, but also high photo-and thermo-stabilities. These
properties enabled a simple and highly sensitive analysis
using a spectrofluorometer as well as using an RP-HPLC,
and the expanded applications of FMQC to various
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Acknowledgements This work was partially supported by the
Research Fellowships of the Japan Society for the Promotion of
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