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T. Pradhan et al. / Dyes and Pigments 121 (2015) 1e6
mechanisms, fluorescence enhancement is usually preferred in
order to observe a high signal output.
spectrometers and TMS as an internal reference. Thermo Scientific
LTQ Orbitrap Mass Spectrometer was used to record the ESI-MS
spectra. The blood serum samples were provided from Dankook
University Hospital. The working solutions of compound 8 were
prepared from their stock solutions (1.0 mM) in DMSO and all the
spectra were recorded under physiological conditions (PBS buffer,
In the present work, we have designed, synthesized, and evalu-
ated the BODIPY (boron-dipyrromethene)-based fluorogenic probe 8
(Fig. 1a, Scheme 1) for ‘turn-on’ detection of UA in serum. Probe 8 is
highly selective for UA over ascorbic acid and other analytes present
in the serum. The design of probe 8 is based on complementary H-
bonding interactions between UA and the probe. Hydrogen bonding
plays an essential role in the construction and maintaining of the
three dimensional structure of many bio-logical molecules. Earlier
synthetic receptors which selectively bind and recognize the barbi-
turate family of drugs through several hydrogen bonding [27]. Like
barbiturate derivatives, uric acid also has several hydrogen bonding
sites that make it a desirable guest for forming complementary H-
bonds with suitable host. On the other hand, BODIPY derivatives
having high quantum efficiency, long wavelength emission and
excellent photostability, are good fluorogenic probes for specific
analytes [28]. Therefore, introduction of a selective chelating unit
into the BODIPY unit can make it suitable fluorogenic receptor for
uric acid. The present manuscript utilizes the H-bonding interactions
between probe 8 and UA to achieve high detection selectivity and
specificity. So far, ‘turn-on’ fluorogenic detection of UA using a simple
and effective method that can detect and discriminate UA from other
analytes has not been reported. We believe that probe 8 will be
valuable in clinical and pharmaceutical analysis of UA.
pH 7.2, 37 ꢀC). The 6
mM of compound 8 was used to measure ab-
sorption and fluorescence spectra using S-3100 (Scinco) spectro-
photometer and RFPC-5301 spectrofluorometer (Shimadzu) at
excitation wavelength of 475 nm with slit widths of 3 and 3 nm
(excitation and emission, respectively).
2.2. Synthesis
2.2.1. Synthesis of 3,5-bis(methoxycarbonyl)benzoic acid (2)
Compound 2 was synthesized as per the reported procedure
[29], yield: 55%.
1H NMR (CDCl3, 300 MHz):
d C
4.01 (brs, 6H), 8.95 (brs, 3H). 13
NMR (CDCl3, 100 MHz): 53.3, 53.4, 131.4, 132.7, 133.7, 134.0, 134.3,
165.3, 166.2, 166.3 ppm. ESI-MS: Calculated for C11H10O6 (Mþ)
240.21; found 240.95.
2.2.2. Synthesis of dimethyl 5-(hydroxymethyl)isophthalate (3)
This compound was synthesized followed by reported proce-
dure [29], yield: 45%.
1H NMR (CDCl3, 300 MHz):
d 3.94 (s, 6H), 4.81 (brs, 2H), 8.23
(brs, 2H), 8.58 (s, 1H). 13C NMR (CDCl3, 100 MHz): 52.7, 64.4, 130.0,
131.0, 132.2, 142.2, 166.4 ppm. ESI-MS: Calculated for C11H12O5
224.06; found 223.00 (Mꢁ1).
2. Experimental section
2.1. General synthetic materials and sample preparation
2.2.3. Synthesis of dimethyl 5-formylisophthalate (4)
The chemicals used for the reactions were purchased from
Aldrich, Acros Organics, Alfa-Aesar, Carbosynth, and TCI, and used
as received. Analytical TLC was performed using Merck 60 F254
silica gel. For column chromatography, silica gel 60 (Merck,
0.063e0.2 mm) was used. 1H and 13C NMR spectra were measured
using CDCl3, CD3OD and DMSO on Varian 300 and 400 MHz
A mixture of dimethyl 5-(hydroxymethyl)isophthalate (500 mg,
2.23 mmol), and 0.07 mL of HBr (48%) in DMSO (15 mL) was heated
at 100 ꢀC for 5 h in an oil bath. The reaction was monitored with TLC
and after completion, the reaction mixture was diluted with ether
and washed with brine. The solvent was evaporated under reduced
pressure and reaction mixture was purified by column chroma-
tography (Ether:Hexane 2:8e4:6) to afford compound 4 (80 mg,
16% yield) as a white solid. 1H NMR (CDCl3, 300 MHz):
d 3.92 (s, 6H),
8.61 (s, 2H), 8.80 (s, 2H), 10.05 (s, 1H). 13C NMR (CDCl3, 100 MHz):
52.9, 131.9, 134.4, 135.8, 137.0, 165.3, 190.5 ppm. HRMS: Calculated
for C11H10O5 222.0528; found 223.0608 (Mþ1).
2.2.4. Synthesis of (Z)-dimethyl 5-((3,5-dimethyl-1H-pyrrol-2-
yl)(3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)isophthalate (5)
To a mixture of compound 4 (230 mg, 1.04 mmol) and 2,4-
dimethylpyrrole (197 mg, 2.07 mmol) in 40 mL of CH2Cl2 solvent,
a catalytic amount of TFA was added. The reaction mixture was
stirred overnight at room temperature and monitored by TLC till
consumption of the aldehyde. A solution of DDQ (307 mg,
1.35 mmol) was added and stirred for another 1 h. After comple-
tion, the reaction was quenched by adding water, CH2Cl2 layer was
separated, evaporated under reduced pressure and purified by
column chromatography on silica gel (EtOAc:Hexane 1:1e9:1) to
give compound 5 (270 mg, 67% yield) as a blackish red sticky solid.
1H NMR (CDCl3, 300 MHz):
5.89 (s, 2H), 8.21 (s, 2H), 8.76 (s, 1H). HRMS: Calculated for
23H24N2O4 (Mþ1) 392.1736; found 393.1816.
d 1.23 (s, 6H), 2.35 (s, 6H), 3.94 (s, 6H),
C
2.2.5. Synthesis of 10-(3,5-bis(methoxycarbonyl)phenyl)-5,5-
difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:10,20-f][1,3,2]
diazaborinin-4-ium-5-uide (6)
Fig. 1. Structure of (a) probe 8, (b) uric acid. Optimized structure of (c) probe 8 and (d)
8euric acid, calculated using B3LYP/6-31G(d) level of theory. The red, blue, grey and
white in molecular representation indicate oxygen, nitrogen, carbon and hydrogen
atoms, respectively. Black dotted lines (aen) in the 8euric acid complex indicate H-
bonds. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Compound
5 (150 mg, 0.68 mmol) and DIPEA (1.8 mL,
10.2 mmol) were dissolved in 20 mL dichloromethane solvent and
stirred at room temperature for 10 min. BF3$OEt2 (1.45 mL,