Colorimetric and fluorescence probe for the detection of nano-molar lysine in aqueous medium

Article abstract of DOI:10.1039/c6ob01704e

A single crystal X-ray structurally characterized BODIPY based probe, THBPY, derived from 4-hydroxy-5-isopropyl-2 methyl-isophthalaldehyde, detects nano-molar lysine in aqueous medium. In the presence of lysine, THBPY visibly changes its color and fluorescence profile due to the formation of a stable imine bond. A distinctive color change allows for facile discrimination over other amino acids in a wide range of concentrations of lysine. The detection limit for lysine is 0.001 μM by a fluorescence method and 0.01 μM by a colorimetric method. The probe shows good reversibility for multiple uses and cleanly discriminates between lysine and other amino acids. Density functional theoretical studies closely resemble experimental results.

Full text of DOI:10.1039/c6ob01704e

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DOI: 10.1039/C6OB01704E.
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Colorimetric and fluorescence probe for detection of nano molar
lysine in aqueous medium
Susanta Adhikaria*, Avijit Ghosha, Sandip Mandalb, Subhajit Guriaa, Prajna Paramita Banerjeec,
Ansuman Chatterjeec and Debasis Dasb*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Single crystal Xꢀray structurally characterized BODIPY based probe THBPY, derived from 4ꢀhydroxyꢀ5ꢀisopropylꢀ2
methylꢀisophthalaldehyde detects nano molar lysine in aqueous medium. In presence of lysine, THBPY visibly changes its
colour and fluorescence profile due to formation of a stable imine bond. Distinctive color change provides facile
discrimination over other amino acids in a wide range of concentrations of lysine. The detection limit for lysine is 0.001
µM by fluorescence method and 0.01 µM by colorimetric method. The probe shows good reversibility for multiple use and
cleanly discriminates lysine from other amino acids. Density functional theoretical studies closely resembles to the
experimental facts.
www.rsc.org/
our body to form toxic substances15 that can bind DNA forming
pyreneꢀDNA adducts to alter DNA replication with increased
risk of cancer.16
Introduction
Lysine (Lys), closely involved in Krebs–Henseleit cycle and
polyamine synthesis,1 undergoes catabolism in the liver of
mammals via saccharopine pathway to produce glutamate and
αꢀamino adipate that subsequently undergoes deamination and
oxidation.2 As human body cannot produce Lys, appropriate
amount is essential in the diet for normal metabolic functions
and weight gain.3 However, high level Lys in plasma and urine
indicates congenital metabolic disorders like cystinuria or
hyperlysinemia.4 Lys plays an important role in neurotoxicity of
αβ40 and αβ42 oligomers in Alzheimer’s disease.5 Thus,
On the other hand, remarkable photo physical properties viz.
sharp absorption and emission peaks, high fluorescence
quantum yield and stability and, nonꢀtoxic nature of BODIPY
unit insist us to include the unit for design and building a
fluorescence probe selective for Lys.17 4ꢀHydroxyꢀ5ꢀisopropylꢀ
2ꢀmethyl isophthalaldehyde (DFT)18 is synthesized from
thymol. Thymol and its derivatives being well tolerable in
mammalian systems,18, 19 its small size and lipophilic character
allows easy absorption and distribution within the sub cellular
organelles.20 In view of the above discussion, herein we report a
simple BODIPY based colorimetric and fluorescence probe,
THBPY which is highly selective for Lys at neutral conditions.
monitoring of Lysine is very demanding. Chromatography and
capillary electrophoresis,
6
two major techniques for Lys
determination suffer from tedious separation step7 for large
sample volume. Lys oxidase, widely used Lys sensing suffers
interference from ascorbic acid.8 Among several methods for
Lys determination in aqueous solution,9 fluorescence and/ or
colorimetric probe is highly desirable.10 Among them, pyrene
based colorimetric Lys sensor
11
and triphenylamine based
fluorescent probes12 offers no interference from histidine and
arginine.13 Our previous reports on pyrene based ratiometric
Lys probe,14 function through monomerꢀexcimer conversion.
However, all these pyrene and its derivatives are metabolized in
Scheme 1 Synthetic route for THBPY
Results and discussion
THBPY is synthesized from 4ꢀhydroxyꢀ5ꢀisopropylꢀ2ꢀmethyl
isophthalaldehyde (DFT)18 in three steps with 25% yield
(Scheme 1). The structures of DFT and THBPY are
1
characterized by H NMR, 13C NMR and mass spectra (Figure
Sꢀ1 to Sꢀ3 and Figure Sꢀ4 to Sꢀ6, ESI†). Moreover, the structure
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of THBPY is confirmed by single crystal Xꢀray diffraction also a modified salicylaldehyde moiety and therefore it is
analysis (Figure 1, Table Sꢀ1, ESI†).
expected to react very slowly with cysteine. Theoretical
calculation also shows that the HOMOꢀLUMO energy gap of
resulting complexes of THBPY with cysteine and
homocysteine are higher than that observed in case of Lys
(ESI†).
Fig. 1 (a) Single crystal Xꢀray structure and (b) packing diagram of
THBPY.
Photo physical studies
Figure 2a reveals that the sharp band at 503 nm of THBPY is
slightly blue shifted to 497 nm upon addition of Lys (DMSO–
HEPES buffer, 1: 9, v/v, 0.01 M, pH 7.4). However, a broad
band at 550 nm appears that increases with increasing Lys (up
to 100 equiv.).
Fig. 3 (a) Emission spectra and intensities (inset) of THBPY in
presence of various amino acids. (b) changes in emission
intensities of THBPY with increasing Lys concentration. (λex
=
371 nm). (c) Plot of emission intensity vs. Lys concentration
(linear region, up to 15 µM Lys, inset). (d) Expanded view of
3b.
UV and visible light exposed colours of THBPY with
increasing Lys concentration are shown in Figure Sꢀ7 (ESI†).
Fig. 2 Changes in the (a) UVꢀVis spectra of THBPY (10 µM,
DMSO–HEPES buffer, 1: 9, v/v, 0.01 M, pH 7.4) upon addition
of Lys (up to 100 equiv., left) and (b) colour of THBPY
solution in presence of different amino acids (100 equiv.).
Lys quenches florescence of THBPY ~110 fold 515 nm (λem
=
515 nm, λex 371 nm). Interestingly, at higher Lys
=
concentration (≥ 50 µM), a new relatively weak emission band
at 582 nm is observed (λex = 503 nm, Figure 5) associated with
a clear isoꢀemissive point at 545 nm.
Consequently, the color of the solution changes from greenish
yellow to light orange. Different colors of THBPY in presence
of various amino acids are shown in Figure 2b. The
fluorescence selectivity of THBPY for Lys is presented Figure
3a. THBPY shows strong green fluorescence (λem = 515 nm)
with high quantum yield (Φfl = 0.80, ESI†). The strong
emission intensity of free THBPY at 515 nm (λex, 371 nm) in
the same solvent quenches upon addition of Lys (0ꢀ3000 µM,
Figure 3b and 3d).
Figure 3c shows the plot of emission intensity of THBPY vs.
Lys concentration, the linear region (up to 15 µM Lys, inset) is
useful for unknown Lys determination. The disappearance of
strong green fluorescence of THBPY upon addition of Lys is
presented in Figure 4. Other naturally occurring amino acids
fail to cause any significant change of fluorescence and
absorbance of THBPY. Cysteine forms a faint red colour after
~2h. Even though cysteine forms 5ꢀmembered substituted
aliphatic ring with active aldehyde group21 but salicylaldehyde
or its derivatives with a phenolic OH function group adjacent to
the aldehyde group have not been reported so far. THBPY is
Fig. 4 UV light exposed colours of THBPY (10 µM) in
presence of different amino acids (50 µM) in DMSO–HEPES
buffer (1: 9, v/v, 0.01 M, pH 7.4).
Effect of pH
Effect of pH (pH, 2.0ꢀ10.0) on the emission properties of
THBPY in absence and presence of Lys is thoroughly
investigated (Figure Sꢀ8, ESI†) and found that THBPY is
stable at biological pH range and effective for detection of Lys
in biological samples.
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addition of Lys (50 µM), no significant change of its absorption
(λmax, 501 nm, Fig.Sꢀ23, ESI) and emission (Fig.Sꢀ24, ESI)
spectra are observed.
Fig. 5 Emission profile of THBPY (10 µM) at 582 nm (λex
=
503 nm) upon addition of Lys from 50 to 3000 µM (red, left).
Right figure shows the expanded view of the blue shed.
Recognition mechanism
To unfold the colorimetric and fluorescence recognition of Lys
Scheme 2 Synthetic route for model compounds
by THBPY
, several model compounds are synthesised,
Like THBPY, fluorescence of
is quenched in presence of Lys (50 µM, Fig.Sꢀ25, ESI†).
However, the absorption maximum of is red shifted from 456
nm to 464 nm upon addition of Lys (Fig. Sꢀ26, ESI†). While 1,
5ꢀdiaminopentane causes similar changes to as that of Lys, 6ꢀ
4 (10 µM, λex, 425 nm, λem, 470)
characterised and studied their interaction with Lys. Moreover,
interactions of THBPY with several molecules resembling Lys
have also been thoroughly studied to identify any potential
interference. Although the possibility of existence of these
molecules in biological system where Lys recognition aimed
for is rare, it is of academic interest. Our objective is to facile
and selective colorimetric recognition of Lys where other
naturally occurring amino acids do not interfere and this
recognition process may be corroborated by the fluorescence
method. In this context, butylamine instead of Lys is employed
to unveil the origin of changes of the absorption and emission
profile of THBPY. Interestingly, butylamine imparts similar
absorption profile to that of Lys (Fig. Sꢀ9, ESI†), however, the
reaction proceeds at much slower rate with generation of brown
color. On the other hand, interactions of THBPY with
molecules resembling Lys, viz. 6ꢀamino hexanoic acid and 1, 5ꢀ
diamino pentane and ethylenediamine are different (Fig. Sꢀ10,
ESI†) where ~550 nm absorption band is absent, unlike Lys.
Corresponding changes in the emission profile are presented in
Fig.Sꢀ11 (ESI†). Therefore, it may be concluded that
4
4
aminohexanoic acid remains spectator. Thus it may be
concluded that presence of OH group next to aldehyde
functionality have specific role to stabilize the adduct of
THBPY and Lys.
1H NMR titration
In order to strengthen the above mechanism, H NMR titration
1
has been performed by adding lysine to the DMSOꢀd6 solution
of THBPY. Significant spectral change has been observed
upon addition of lysine. The bare eye red colour is attributed to
the formation of a Schiff’s base between Lysine and THBPY
corroborated from 1H titration (Figure 6).
,
butylamine part of Lys is responsible to interact with THBPY
.
Further, the reversibility of imine formation between THBPY
and Lys (Figure Sꢀ12 and Sꢀ13, ESI†) is tested with H2O2 and
found positive (Scheme Sꢀ2, ESI†) as supported by the mass
spectroscopy (Figure Sꢀ14, ESI†). For deeper understanding of
the sensing mechanism, two model compounds (compound
and ) have been synthesised (Scheme 2) and tested for Lys
selectivity. Compound
is synthesised by partial reduction22,23
of isophthalaldehyde with NaBH4 in mixed THFꢀethanol media
to yield compound in good yield. Reaction of with 2, 4ꢀ
dimethyl pyrrole following the procedure of THBPY synthesis
yields . Oxidation of following procedure of Dess martin
periodinane gives the BODIPY derivative
On the other hand, modified Reimer–Tiemann formylation of
3
4
3
1
1
2
2
Fig. 6 (I) 1H NMR spectra of THBPY in DMSOꢀd6; upon
addition of (II) 0.5 equiv. Lys in D2O, (III) 1.0 equiv. Lys in
D2O, (IV) 3.0 equiv. Lys in D2O and (V) 5.0 equiv. Lys in D2O.
3
.
fluorescein gives compound
4
in good yield.24 The structures of
all intermediates and model compounds are characterized by 1H
NMR, 13C NMR and mass spectra (Figure Sꢀ15 to Sꢀ22, ESI†)
analyses.
Upon addition of Lys, the aldehyde proton (b) of THBPY at
10.26 ppm disappears with the appearance of the new imine
proton (i) in the adduct product at 8.72 ppm. Moreover,
aromatic protons of both subꢀunits are up field shifted. The
formation of imine bond is further supported from the mass
Like THBPY, the BODIPY derivative
3 possesses an aldehyde
group with emission maximum at 530 nm (λex, 425 nm). Upon
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spectrum of the THBPYꢀLys adduct (Scheme Sꢀ1, ESI†) that 10 ꢁM THBPY to MDAꢀMB 231 cells for 12h,
∼90% cells
shows m/z = 553.3160, attributed to the fragment [THBPY
lysine] (Figure Sꢀ27, ESI†).
ꢀ
remain alive. This nullifies the possibility of significant
cytotoxic influence of THBPY on MDAꢀMB 231 cells.
DFT calculations
To have deeper understanding of the sensing mechanism, timeꢀ
dependent density functional theory (TDDFT) calculations have
been performed using (B3LYP) and a 6ꢀ31G basis set with
Gaussian 09 program.25 The energy optimized structure of
THBPY is used to calculate the energetics of imine bond
formation involving Lys as both free base (neutral form) and
cation form. The energy optimized geometries with HOMOꢀ
LUMO of THBPY (Table Sꢀ2, ESI†) and its Lys adducts
(Table Sꢀ3 and Sꢀ4, ESI†) have been generated. The HOMOꢀ
LUMO energy gap of THBPYꢀLys adduct is lower than that of
free THBPY. The individual energy levels of HOMO and
LUMO of the adduct product are also lower in energy.
The energy optimized structure of THBPYꢀLys (neutral form)
shows Hꢀbonding interaction between the imine nitrogen and
adjacent OH of THBPY. The calculated OꢀH distances of free
THBPY and that of the imine adduct are approximately 1.00
and 1.053Å respectively. Thus, Hꢀbonding may be responsible
for the enhanced stability of THBPYꢀLys (neutral form)
adduct. In case of THBPYꢀLys (Lys in cationic form), Hꢀbond
exists between OH of THBPY with amine group of Lys. The
calculated OꢀH bond distance is 1.327Å, well within the
recognized Hꢀbond distance. Thus Lys in both forms can
interact with THBPY. The HOMOꢀLUMO energy gaps of
THBPY and THBPYꢀLys adduct in both forms are shown in
Fig. 7 Selected MOs of THBPY, [THBPYꢀLys (neutral)],
[
THBPYꢀLys (cationic)] (not to scale; isovalue = 0.02).
Figure 7. The energy gap between HOMOꢀLUMO in THBPY
ꢀ
Lys (neutral) is lowest. The simulated spectrum of the gas
phase structure of THBPY indicates the dominant absorption
band having an oscillator strength, f = 0.6202 a. u. is observed
at 500.8 nm. More importantly, the adduct formation between
THBPY and neutral Lys leads to decrease in the oscillator
strength from f = 0.6202 a.u. to f = 0.5512 a.u. as well as redꢀ
shift of the absorption band from 500.8 nm to 550.5 nm. The
results are qualitatively in line with the experimental spectra. It
is interesting to note that the TDꢀDFT absorption spectra of Lys
(cation) have different characteristics of the dominant band
when compared with the neutral form. Therefore, the observed
change in absorbance is attributed to the imine bond formation
between THBPY and Lys.
The HOMOꢀLUMO energy gap of resulting complexes of
THBPY with cysteine and homocysteine are higher than that
observed in case of Lys (Figure Sꢀ27, ESI†) and thus support a
very very slow reaction between them
very slow reaction between them.
In vitro cell imaging
Fig. 8 Fluorescence images of THBPY treated MDAꢀMB 231
cells: (a) bright field images of free cells; (b) fluorescence
image of cells after incubation with THBPY; (c) overlay image
of a and b; (d) fluorescence image of THBPY treated cells in
presence of Lys. The images are captured using blue filter.
THBPY is prepared in ~0.3% DMSOꢀwater (v/v).
Conclusion
In summary, we have designed and synthesized BODIPY based
excellent colorimetric and fluorescence probe for selective
detection of Lys. The new probe, THBPY shows high
sensitivity as well as visual response towards Lys. THBPY
shows reversibility in presence of peroxide. Formation of
Schiff’s base between THBPY and Lys results change of
colour from yellowꢀgreen to orange, observed by bare eye. So,
the present demonstration is a new addendum for Lys sensing
in the horizon of BODIPY based receptors.
Intracellular Lys imaging in MDAꢀMB 231 cells using THBPY
has been investigated. The cells are incubated with THBPY (10
µM) for 1h and washed thrice with PBS buffer. Lys (50 µM) is
added to the medium followed by further incubation for 30 min.
Figure 8 reveals that THBPY is cell permeable and its
fluorescence is quenched upon addition of Lys within MDAꢀ
MB 231cells when observed under fluorescence microscope.
The cytotoxicity of THBPY on MDAꢀMB 231 cells is
determined by MTT assay (Fig. Sꢀ29, ESI†). Upon exposure of
Experimental
Materials and equipment
All metal salts were used as either their nitrate or their chloride
salts. All the reagents were of analytical reagent grade and used
without further purification. H NMR spectra were recorded at
300 and 75 MHz for 13C NMR using solvent peak as internal
reference at 25°C. Thin layer chromatography (TLC) was
carried out on 0.25 mm thick preꢀcoated silica plates, and spots
were visualized under UV light. MilliꢀQ Millipore 18.2 Mꢂ
1
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cm−1 water was used whenever required. A Shimadzu Multi 128.93, 128.86, 128.79, 128.74, 128.67, 128.60, 127.59,
Spec 2450 spectrophotometer was used for recording UV−vis 127.51, 127.44, 63.77, 63.70, 63.62; QTOFꢀMS ES+ calcd. for
spectra. FTIR spectra were recorded on a Shimadzu FTIR C6H9O2 (M+H)+, 137.0602: found, 137.0602.
(model IR Prestige 21 CE) spectrophotometer. Mass spectra (3-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4, 5l4-dipyrrolo
were recorded using a QTOF XEVOꢀG2 mass spectrometer in [1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)phenyl)methanol (2)
ESI positive mode. The steady state emission and excitation 2, 4ꢀDimethylpyrrole (1g, 10.51 mmol) and 3ꢀ(hydroxymethyl)
spectra
were
recorded
with
a
Hitachi
Fꢀ4500 benzaldehyde (680 mg, 5 mmol) were dissolved in dry CH2Cl2
spectrofluorimeter. A Systronics digital pH meter (model 335) (150 mL) under argon atmosphere. Two drops of trifluoroacetic
was used for pH measurement. Fluorescence microscope acid (TFA) were added in dark, and the solution was purged
(Dewinter, Italy) was used for capturing images and processed with argon for 10 min. Then the reaction mixture was stirred for
with Bioꢀwizard 4.2 software.
Synthesis
3-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo
[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)-6-hydroxy-5-
isopropyl-2-methylbenzaldehyde (THBPY)
4h at ambient temperature at dark. Then 2, 3ꢀdichloroꢀ5, 6ꢀ
dicyanoquinone (DDQ, 1.13 g, 2 mmol) was added prior to
addition of 10 mL dry CHCl3 and the mixture was stirred for
another 4h. Then triethylamine (7 mL) was added to the
reaction mixture and stirred for 5 min. Then, boron trifluoride
2, 4ꢀDimethylpyrrole (380 mg, 4 mmol) and 3ꢀhydroxyꢀ2ꢀ etherate (7 mL) was added and the mixture was stirred for
isopropylꢀ5ꢀmethylterephthalaldehyde (412.48 mg, 2 mmol) another 40 min. The dark brown solution was partitioned with
were dissolved in dry CH2Cl2 (150 mL) under argon water (3 X 20 mL) and brine (30 mL), dried over anhydrous
atmosphere. 50 ꢁL of trifluoroacetic acid (TFA) was added in magnesium sulfate and concentrated at reduced pressure. The
dark, and the solution was purged with argon for 10 min. Then crude product was purified by silicaꢀgel flash column
the reaction mixture was stirred for 4h at ambient temperature chromatography (EtOAc–petroleum ether, 20%, v/ v) and
at dark. Then, 2, 3ꢀdichloroꢀ5, 6ꢀdicyanoquinone (DDQ, 442 recrystallized from CHCl3–hexane to give THBPY as red
mg, 2 mmol) was added prior to addition of 10 mL dry CHCl3, crystal (177 mg, yield, 10%) M. P.
>
200◦C. 1H NMR (300
and the mixture was stirred for another 4h. Then, triethylamine MHz, CDCl3), δ (ppm), 7.468ꢀ7.448 (m, 2H), 7.266ꢀ7.242 (d,
(3 mL) was added to the reaction mixture and stirred for 5 min. 1H), 7.190ꢀ7.186 (d, 1H), 5.953 (s, 2H), 4.727 (s, 2H), 2.527 (s,
Further, boron trifluoride etherate (5 mL) was added and the 6H), 1.350 (s, 6H). 13C NMR (Figure S8) (75 MHz, CDCl3), δ
mixture was stirred for another 40 min. The dark brown (ppm), 155.49, 143.02, 142.08, 141.50, 135.19, 131.37, 129.32,
solution was formed which was partitioned with water (3 X 20 127.24, 127.16, 126.39, 121.22, 64.71, 30.88, 29.23, 14.56,
mL) and brine (30 mL), dried over anhydrous magnesium 14.43; QTOFꢀMS ES+, calcd. for C40H43N4O4 (M+H)+
sulfate and concentrated at reduced pressure. The crude product 643.3278: found, 643.3276. QTOFꢀMS ES+ calcd. for
was purified by silicaꢀgel flash column chromatography C20H20BF2N2O (M+H)+, 355.1793, found, 355.1793.
(EtOAc–petroleum ether, 15%, v /v) and recrystallized from 3-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-
CHCl3–hexane to give THBPY as red crystals (150 mg, yield, c:2',1'-f][1,3,2]diazaborinin-10-yl)benzaldehyde (3)
19%) M.P.
> 2 (150 mg, 0.423 mmol) was dissolved in dry
200◦C. 1H NMR (300 MHz, CDCl3), δ (ppm), Compound
1.19ꢀ1.24 (6H, 2s), 1.44 (3H, s), 1.56 (3H, s), 2.44 (3H, s), 2.46 CH2Cl2 under argon atmosphere and stirred at 0oC for 15 min
(6H, 2s), 3.39 (1H, m), 6.01 (2H, s), 7.23 (1H, s), 10.39 (1H, s), and then DessꢀMartin periodinane (179.61 mg, 0.423 mmol)
12.58 (1H, s); 13C NMR (75 MHz, CDCl3), 195.70, 161.46, was added. The reaction mixture was stirred at r.t. for 30 min.
155.94, 142.49, 140.01, 137.32, 136.50, 133.78, 131.46, After completion of the reaction (checked by TLC), the solvent
125.74, 121.36, 118.11, 26.02, 22.34, 14.60, 14.38, 14.12. was removed under reduced pressure. Organic residue was
+
QTOF mass, m/z (M+H)+ calcd. for C24H28BF2N2O2 : extracted with EtOAc and dried over anhydrous sodium
425.2206, found, 425.2212 and m/z (M+Na)+ calcd. for sulphate. Removal of solvent gave brown oil. It was purified by
+
C24H27BF2N2NaO2 : 447.2026, found, 447.2067.
column chromatography using 40% EtOAc in petroleum ether
to give 135 mg red solid (yield, 93%) having Rf = 0.35 in 10%
3-(Hydroxymethyl)benzaldehyde (1)
To a solution of isophthalaldehyde (22.5 g, 0. 125 mol) in a EtOAc in petroleum ether. 1H NMR (300 MHz, CDCl3), δ
mixture of 95% EtOH (250 mL) and THF (350 mL), NaBH4 (ppm), 10.073 (s, 1H), 8.043ꢀ8.013 (m, 1H), 7.852ꢀ7.848 (d,
(1.2 g, 26.11 mmol) was added at –15 °С with continuous 1H), 7.731ꢀ7.680 (t, 1H), 7.608ꢀ7.578 (m, 1H), 6.004 (s, 2H),
stirring for 30 min. Then the mixture was stirred for 6h at ꢀ15 2.567 (s, 6H), 1.341 (s, 6H); 13C NMR (75 MHz, CDCl3), δ
°С and neutralized with 2M HCl to рН 5. The solvent was (ppm), 191.30, 156.16, 142.66, 139.45, 137.10, 136.14, 134.08,
removed and the residue was partitioned with EtOAc. The 131.17, 129.97, 129.52, 121.60, 29.64, 14.64, 14.58; QTOFꢀMS
organic extract was dried with anhydrous Na2SO4 and the ES+ calcd. for C20H20BF2N2O (M+H)+, 353.1636, found,
solvent was removed. The yield of crude product 2 was 15.9g 353.1636.
(95%). The crude product was purified by column 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-
1
chromatography using an EtOAcꢀhexane (1: 1, v/v) mixture. H xanthene] -4'- carbaldehyde (4)24
NMR (300 MHz, CDCl3), δ (ppm), 9.899ꢀ9.878 (d, 1H), 7.803 Fluorescein (2.5 g, 7.75 mmol) was dissolved in MeOH (3 mL)
(s, 1H), 7.737ꢀ7.699 (m, 1H), 7.593ꢀ7.568 (d, 1H), 7.485ꢀ7.417 in a 100 mL three neck round bottom flask. Next, 10g of 50%
(m, 1H), 4.700ꢀ4.683 (d, 2H). 13C NMR, (75 MHz, CDCl3) NaOH solution, 2.42 mL (30 mmol) of CHCl3 and 0.03 mL of
192.61, 192.53, 141.94, 136.11, 132.80, 132.72, 132.65, 15ꢀcrownꢀ5 were carefully added maintaining the reaction
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temperature at 55°C. The mixture was stirred at 55°C for 7h. 11 a) Y. Zhou, J. Won, J. Y. Lee, J. Yoon, Chem. Commun., 2011, 47
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DCM (15:85, v/v) gave a light yellow solid with 34.1% yield 13 (a) M. Wehner, T. Schrader, P. Finocchiaro, S. Failla, G. Consiglio,
1
(952 mg).
R
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Acknowledgement
A.G and S.G are thankful to UGC and CSIR, New Delhi for
M. Hruszkewycz, K. A. Canella, K. PeHonen, L. Kotrappa, A. Dipple,
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