Rapid detection of aspartic acid and glutamic acid in water by BODIPY-Based fluorescent probe: Live-cell imaging and DFT studies

Article abstract of DOI:10.1016/j.dyepig.2019.04.052

Strategically modified4-(diethylamino)-2-(pyridin-2-ylmethoxy)benzaldehyde appended BODIPY-based probe N,N-SP-BPY detects aspartic acid (Asp)and glutamic acid (Glu)by inhibition of Photoinduced electron transfer (PET)process in water. The probe shows cons

Full text of DOI:10.1016/j.dyepig.2019.04.052

Accepted Manuscript
Rapid detection of aspartic acid and glutamic acid in water by BODIPY-Based
fluorescent probe: Live-cell imaging and DFT studies
Subhajit Guria, Avijit Ghosh, Kalipada Manna, Abhishek Pal, Arghya Adhikary,
Susanta Adhikari
PII:
S0143-7208(19)30154-8
https://doi.org/10.1016/j.dyepig.2019.04.052
DYPI 7507
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 20 January 2019
Revised Date: 21 April 2019
Accepted Date: 22 April 2019
Please cite this article as: Guria S, Ghosh A, Manna K, Pal A, Adhikary A, Adhikari S, Rapid detection of
aspartic acid and glutamic acid in water by BODIPY-Based fluorescent probe: Live-cell imaging and DFT
studies, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.04.052.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Rapid Detection of Aspartic acid and Glutamic acid in Water by BODIPY-Based
fluorescent Probe: Live-Cell Imaging and DFT studies
Subhajit Guria,[a] Avijit Ghosh,[a,b] Kalipada Manna,[a] Abhishek Pal,[a] Arghya Adhikary,[b]
Susanta Adhikari,*[a]
aDepartment of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009, West Bengal,
India.
bCentre for Research in Nanoscience & Nanotechnology, (CRNN), University of Calcutta, Technology
Campus, Sector-III, Block-JD 2, Salt Lake, Kolkata 700098, West Bengal, India.
* To whom correspondence should be addressed.
E-mail address: adhikarisusanta@yahoo.com;
Graphical abstract
Highlights
1. A highly selective BODIPY probe, N,N-SP-BPY detects aspartic acid (Asp) and glutamic acid (Glu) in
water.

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2. ‘Turn On’ fluorescence can be observed by inhibition of Photoinduced Electron Transfer (PET)
process.
3. A distinct colorimetric change from pink to colorless solution can be observed to recognize Asp and
Glu.
4. N,N-SP-BPY efficiently images intracellular Asp and Glu in live cells under fluorescence microscope.
Abstract: Strategically modified4-(diethylamino)-2-(pyridin-2-ylmethoxy)benzaldehyde appended
BODIPY-based probe N,N-SP-BPY detects aspartic acid (Asp) and glutamic acid (Glu) by inhibition of
Photoinduced electron transfer (PET) process in water. The probe shows considerable and rapid
fluorescence enhancement upon addition of Asp and Glu. Additionally, a distinct colorimetric change
from pink to a colorless solution can be observed to recognize traces of Asp and Glu. The probe detects
Asp and Glu as low as 1 µM and 5 µM respectively. Furthermore, the probe can efficiently detect
intracellular Asp and Glu in live HeLa, A549, MDA-MB-468 and HEK-293T cells under fluorescence
microscope without showing any cytotoxic effect. Two model BODIPY derivatives are also synthesized
1
to establish strategic modification. H NMR titration and theoretical calculations strongly supports
experimental findings.
Keywords: BODIPY• Photoinduced electron transfer process • Fluorescent probe• DFT calculation •
Live cell imaging
1. Introduction
Aspartic acid (Asp) and glutamic acid (Glu) are dicarboxylic amino acids, and their conjugate base
aspartate and glutamate, are a major excitatory neurotransmitter in the mammalian central nervous system
(CNS). The concentration of aspartate in cerebrospinal fluid (CSF) is found to be 1.22 ± 0.21 mM, while
the normal concentration of glutamate varies from few micromolar in the extracellular fluids to 1–10mM
inside neurons of the brain [1-3].
Aspartic acid is a precursor for the synthesis of two other amino acids namely glutamic acid and
glycine [4] and is recognized for its facilitating role in the tricarboxylic acid (TCA) cycle [5]. Researchers
have also demonstrated the role of aspartic acid in hydroxyapatite [6] and osteoarthritis [7]. Excessive

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aspartic acid can be a reason for Lou Gehrig’s disease [8] and is also associated with the early-onset
dementia of the Alzheimer type [9].
Glutamic acid rich chain of bone sialoprotein, osteonectin and osteopontin are concerned in Hap
nucleation and growth [10] as indicated by earlier research. It is also responsible for the biosynthesis of γ-
aminobutyric acid (GABA) [11]. Glutamate is the prime mediator of sensory information, memory
formation, memory retrieval, emotions and motor coordination [12]. In the food industry, its monosodium
salt (MSG- monosodium glutamate) is a well-known flavour enhancer and generally found in different
foods for achieving strong, balanced and preferred product flavour. However, accumulation of glutamate
can be toxic to neurons and therefore, must be firmly regulated. The extreme intake of this flavour
enhancer may cause allergic effects (headache and stomach pain) [13,14]. Patients with acute ischemic
stroke have demonstrated high levels of glutamate in plasma and cerebrospinal fluid (CSF) [15]. In
human and in animals, epileptic tissues have shown slow metabolic processing of glutamate and down-
regulation of glutamate to glutamine cycle [16].
As these amino acids play a crucial role in many physiological pathways their biological monitoring
is of utmost importance. Chromatographic techniques based on ion exchange,[17] GC (gas
chromatography) [18], LC/MS approach [19], electrochemical [20-22] and infrared spectroscopy [23]
are some of the popular analytical procedures for detection and characterization of amino acids.
Molecules that can report the interaction of amino acids through changes in their optical properties
(emission or absorption) i.e. chemosensors, could act as good candidates for the same [24-44].Among the
copious amount of highly fluorescent dyes that are available in the literature, sets based on 4,4-difluoro-4-
bora-3a,4a-diaza-s-indacene1,2 (better known as BODIPY, Difluoroboron Dipyrromethene) shows the
highest potential because of their robustness against light and chemicals. Their optical properties can also
be easily tweaked by attachment of residues at the suitable position of the BODIPY core [45].
Herein, we report the synthesis of 4-(diethylamino)-2-(pyridin-2-ylmethoxy)benzaldehyde
appended BODIPY based probe N,N-SP-BPY which detects Aspartic acid (Asp) and glutamic
acid (Glu) by inhibition of Photoinduced electron transfer (PET) process in water. The probe N,N-
SP-BPY shows strong fluorescent enhancement and a distinct change in naked eye colour of the
solution from pink to colorless during the addition of Asp/Glu in water. The probe is also able to
efficiently image Asp or Glu in live cells. The probe is weakly fluorescent due to the effective
PET process operating from N,N-diethylamino unit of N,N-SP-BPY to BODIPY core. Inhibition
of PET process occurs in the presence of Asp/Glu, leading to fluorescence enhancement. Two
model compounds, N,N-SB-BPY, and SP-BPY were synthesized to comprehend the exact
molecular mechanism associated with the changes that were taking place in fluorescence and UV-

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Visprofile. Scheme 1 shows the synthetic procedure of the probe and model compounds. All the
intermediates and probes were characterized by 1H NMR, 13C NMR and QTOF-MS (Figure S-1 to
S-17, ESI). Theoretical calculations were performed to support the experimental observations.
Scheme-1: Synthetic route to N,N-SP-BPY, N,N-SB-BPY and SP-BPY
Figure 1: (a) Single crystal X-ray structure (CCDC 1902075), and (b) packing diagram ofN,N-SP-BPY.
2. Experimental

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2.1. Materials and Equipment:
1
Chemicals and solvents were purchased from commercial suppliers and used as received. H and 13C
NMR spectra were recorded on a Bruker Advance III HD (300 MHz) spectrometer. Chemical shifts were
reported in parts per million (ppm), and the residual solvent peak was used as an internal reference:
proton (chloroform
δ 7.26), carbon (chloroform δ 77.16) or tetramethylsilane (TMS δ 0.00) was used as a
reference. Multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). Coupling constants were reported in Hertz (Hz). High resolution mass spectra were obtained
on a Xevo G2S/Q-Tof. microTM spectrometer. For thin layer chromatography (TLC), Merck precoated
TLC plates (Merck 60 F254) were used and flash chromatography separations were performed on SRL
230-400 mesh silica gel.
2.2. In vitro cell imaging
Human cervical cancer HeLa, Breast cancer cell MDA-MB-468, Lung cancer cell A549, and normal
HEK-293T cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and 5% CO2. For in vitro imaging
studies, the cells were seeded in 12-well tissue culture plates with a seeding density of 105 cells per well.
After reaching 60%–70% confluence, the previous DMEM medium was replaced with serum-free
DMEM medium (without any amino acid), supplemented with 20 µM N,N-SP-BPY and incubated for 2h
to facilitate the probe uptake by cells. Then cells were washed three times with PBS buffer to remove any
extracellular N,N-SP-BPY. Then Asp/Glu (50 µM) was added into the medium and then further incubated
for 1 hour. After washing with PBS buffer, images of live cells were taken using an EVOS® FL Cell
Imaging System, Life Technologies, USA).
2.3. Synthesis:
4-Diethylamino-2-(pyridin-2-ylmethoxy)-benzaldehyde (N,N-SP-CHO):
4-(Diethylamino)salicylaldehyde (1 g, 5.18 mmol) and 2-(Chloromethyl)pyridine hydrochloride (0.85 g,
5.18 mmol) was taken in a 50ml round bottom flask and was dissolved in 30ml acetonitrile. After all the
reactants had been completely dissolved K2CO3 (3.5 g, 25.9 mmol) and tetrabutylammonium iodide
(0.95g, 2.5 mmol) was added and the reaction mixture was refluxed for 24 h. After completion of the
reaction as confirmed by TLC, acetonitrile was evaporated under reduced pressure. The concentrate was
extracted with ethyl acetate, dried over sodium sulphate and vacuum evaporated. The crude mass was
purified by flash chromatography using petroleum ether/ ethyl acetate (3:2 v/v) (Rf = 0.5) as eluent to give
1
N,N-SP-CHO as yellowish brown solid 42.8 % (630 mg). H NMR (300 MHz, CDCl3):
δ10.29 (S, 1H),
8.60-8.58 (dd, J=0.9 Hz, J=4.8 Hz 1H), 7.78-7.71 (m, 2H), 7.62-7.59 (d, J=7.8 Hz, 1H), 7.27-7.22(dd,

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J=3.6 Hz, J=10.3 Hz 1H), 6.31-6.27(dd, J=2.1 Hz, J=9Hz, 1H), 6.09-6.08 (d, J=2.1 Hz, 1H), 5.31 (s, 2H),
3.40-3.33 (q, J= 6.9 Hz, 4H), 1.16-1.11 (t, J=7.2 Hz, 6H); 13C NMR (75 MHz, CDCl3):
δ 186.7, 162.5,
156.8, 153.6, 148.9, 148.9, 136.9, 130.8, 122.6, 121.1, 114.1, 104.5, 93.9, 70.5, 44.7, 12.4, 12.4, 12.2
ppm; HRMS-ESI (m/z): calcd for C17H21N2O2 [M+H]+ 285.1598, found 285.1603.
4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4 dipyrrolo[1,2
yl)-N,N-diethyl-3-(pyridin-2-ylmethoxy)aniline (N,N-SP-BPY)
c:2',1'f][1,3,2]diazaborinin-10-
2,4-Dimethylpyrrole (368.2 mg, 3.87 mmol) and N,N-SP-CHO (500 mg, 1.76 mmol) were
dissolved in dry CH2Cl2 (50 mL) under an argon atmosphere. The solution was purged with argon
for 10 min followed by addition of Two drops of trifluoroacetic acid (TFA) in the dark. Then the
reaction mixture was stirred for 4 h at ambient temperature in the dark. Then 2,3-dichloro-5,6-
dicyanoquinone (DDQ, 1.13 g, 2 mmol) was added followed by addition of another 10 mL dry
CH2Cl2 and the mixture was stirred for another 4 h. Then triethylamine (5 mL) was added to the
reaction mixture and stirred for another 5 mins. Then, boron trifluorideetherate (7 mL) was added
and the mixture was stirred for another 60 min. The dark brown solution was washed with water
(3 × 20 mL) and brine (30 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by silica-gel flash column chromatography
(EtOAc–petroleum ether, (2.5:7.5 v/v)(Rf = 0.3) to give N,N-SP-BPY as red solid (283 mg, 32 %
yield). 1H NMR (300 MHz, CDCl3):
Hz 1H), 7.17-7.11 (dd, =3.9Hz, J=10Hz, 2H), 6.89-6.86 (d, J=8.4 Hz, 1H), 6.37-6.34 (dd,
Hz, =8.4 Hz 1H), 6.30-6.29 (d, =2.1 Hz, 1H), 5.95 (s, 2H), 5.15 (s, 2H), 3.37-3.30 (q,
Hz, 4H), 2.56 (s, 6H), 1.57 (s, 6H), 1.15-1.10 (t,
=6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3):
δ
8.49-8.48 (d,
J
=4.8Hz, 1H), 7.57-7.54 (dd, J=1.5 Hz,
J
J
=7.8
=2.4
J
J
J
J= 7.2
J
δ
157.5, 156.0, 154.1, 149.9, 148.4, 142.7, 140.3, 136.9, 132.5, 129.9, 122.3, 121.0, 120.4, 110.7,
105.1, 96.9, 70.6, 44.4, 36.5, 31.8, 29.6, 29.2, 24.6, 22.6, 14.5, 14.1, 13.9, 12.2 ppm; HRMS-ESI
(m/z): calcd for C29H33BFN4O [M-F]+ 483.2726, found 483.2715.Moreover, the structure of N,N-
SP-BPYwas confirmed from single crystal X-ray analysis (CCDC 1902075; Figure 1).
2-Benzyloxy-4-diethylamino-benzaldehyde (N,N-SB-CHO)
4-(Diethylamino) salicylaldehyde (1 g, 5.18 mmol) and Benzyl bromide (0.88 g, 5.18 mmol ) was
taken in a 50ml round bottom flask and was dissolved in 30ml acetonitrile. After all the reactants
had been completely dissolved K2CO3 (3.5 g, 25.9 mmol) and tetrabutylammonium iodide (0.95g,
2.5 mmol) was added and the reaction mixture was refluxed for 24 h. After completion of the
reaction as confirmed by TLC, acetonitrile was evaporated under reduced pressure. The

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concentrate was extracted with ethyl acetate, dried over sodium sulphate and vacuum evaporated.
The crude mass was purified by flash chromatography using petroleum ether/ ethyl acetate (4:1
v/v) (Rf = 0.5) as eluent to give N,N-SB-CHO as yellowish white solid 49 % (728 mg).
1H NMR (300 MHz, CDCl3):
6.28 (d, J=8.7 Hz, 1H), 6.07-6.06 (d,
1.19-1.44 (t,
=6.9 Hz, 6H);13C NMR (75 MHz, CDCl3):
δ
10.25 (S, 1H), 7.76-7.73 (d, J=9 Hz, 1H), 7.47-7.31 (m, 5H), 6.31-
=2.1 Hz, 1H), 5.18 (s, 2H), 3.42-3.35 (q, = 6.9 Hz, 4H),
186.8, 163.3, 153.8, 136.8, 130.3,
J
J
J
δ
128.7, 128.6, 128.3, 127.9, 127.1, 127.0, 126.9, 126.9, 114.4, 104.6, 94.2, 70.0, 44.8, 44.7, 12.5,
12.5 ppm; HRMS-ESI (m/z): calcd for C18H22NO2 [M+H]+ 284.1645, found 284.1645.
3-(benzyloxy)-4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-
][1,3,2]diazaborinin-10-yl)-N,N-diethylaniline (N,N-SB-BPY)
c:2',1'-
f
2,4-Dimethylpyrrole (368.2 mg, 3.87 mmol) and N,N-SB-CHO (500 mg, 1.76 mmol) were
dissolved in dry CH2Cl2 (50 mL) under an argon atmosphere. The solution was purged with argon
for 10 min followed by addition of Two drops of trifluoroacetic acid (TFA) in the dark. Then the
reaction mixture was stirred for 4 h at ambient temperature in the dark. Then 2,3-dichloro-5,6-
dicyanoquinone (DDQ, 1.13 g, 2 mmol) was added followed by addition of another 10 mL dry
CH2Cl2 and the mixture was stirred for another 4 h. Then triethylamine (5 mL) was added to the
reaction mixture and stirred for another 5 mins. Then, boron trifluorideetherate (7 mL) was added
and the mixture was stirred for another 60 min. The dark brown solution was washed with water
(3 × 20 mL) and brine (30 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by silica-gel flash column chromatography
(EtOAc–petroleum ether, (0.75:4.25 v/v) (Rf = 0.4) to give N,N-SB-BPY as red solid (204 mg,
1
22.3% yield). H NMR (300 MHz, CDCl3):
δ
7.19-7.13 (m, 5H), 6.79-6.76 (d,
=2.4Hz, J=8.5 Hz, 1H), 6.17-6.16 (d, J=2.1 Hz, 1H), 5.89 (s, 2H), 4.98 (s, 2H),
= 6.9 Hz, 4H), 2.49 (s, 6H), 1.49 (s, 6H), 1.06-1.04 (t,
=5.1 Hz, 6H);13C NMR (75
156.4, 154.1, 149.9, 142.9, 140.5, 137.6, 132.6, 129.9, 128.5, 157.6, 126.7, 120.5,
J=8.4 Hz 1H),
6.28-6.25 (dd,
J
3.27-3.20 (q,
J
J
MHz, CDCl3):
δ
111.3, 105.3, 97.8, 92.1, 70.1, 44.5, 36.2, 29.7, 28.5, 24.7, 23.8, 23.4, 17.3, 14.6, 14.2, 12.4, 7.9
ppm; HRMS-ESI (m/z): calcd for C30H35BF2N3O [M+H]+ 502.2836, found 502.2835.
.
3-(benzyloxy)-4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-
][1,3,2]diazaborinin-10-yl)-N,N-diethylaniline (N,N-SB-BPY)
c:2',1'-
f
2,4-Dimethylpyrrole (368.2 mg, 3.87 mmol) and N,N-SB-CHO (500 mg, 1.76 mmol) were
dissolved in dry CH2Cl2 (50 mL) under an argon atmosphere. The solution was purged with argon
for 10 min followed by addition of Two drops of trifluoroacetic acid (TFA) in the dark. Then the

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reaction mixture was stirred for 4 h at ambient temperature in the dark. Then 2,3-dichloro-5,6-
dicyanoquinone (DDQ, 1.13 g, 2 mmol) was added followed by addition of another 10 mL dry
CH2Cl2 and the mixture was stirred for another 4 h. Then triethylamine (5 mL) was added to the
reaction mixture and stirred for another 5 mins. Then, boron trifluorideetherate (7 mL) was added
and the mixture was stirred for another 60 min. The dark brown solution was washed with water
(3 × 20 mL) and brine (30 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by silica-gel flash column chromatography
(EtOAc–petroleum ether, (0.75:4.25 v/v) (Rf = 0.4) to give N,N-SB-BPY as red solid (204 mg,
1
22.3% yield). H NMR (300 MHz, CDCl3):
δ
7.19-7.13 (m, 5H), 6.79-6.76 (d,
=8.5 Hz, 1H), 6.17-6.16 (d, J=2.1 Hz, 1H), 5.89 (s, 2H), 4.98 (s, 2H),
= 6.9 Hz, 4H), 2.49 (s, 6H), 1.49 (s, 6H), 1.06-1.04 (t,
=5.1 Hz, 6H);13C NMR (75
156.4, 154.1, 149.9, 142.9, 140.5, 137.6, 132.6, 129.9, 128.5, 157.6, 126.7, 120.5,
J=8.4 Hz 1H),
6.28-6.25 (dd, J=2.4Hz, J
3.27-3.20 (q,
J
J
MHz, CDCl3):
δ
111.3, 105.3, 97.8, 92.2, 70.1, 44.5, 36.2, 29.7, 28.5, 24.7, 23.8, 23.4, 17.3, 14.6, 14.2, 12.4, 7.9
ppm; HRMS-ESI (m/z): calcd for C30H35BF2N3O [M+H]+ 502.2836, found 502.2835.
5,5-difluoro-1,3,7,9-tetramethyl-10-(2-(pyridin-2-ylmethoxy)phenyl)-5H-4λ4,5λ4-
dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine (SP-BPY)
2,4-Dimethylpyrrole (473 mg, 4.97mmol) and SP-CHO (530 mg, 2.48mmol) were dissolved in
dry CH2Cl2 (50 mL) under an argon atmosphere. The solution was purged with argon for 15 min
followed by addition of Two drops of trifluoroacetic acid (TFA) in the dark. Then the reaction
mixture was stirred for 4 h at ambient temperature in the dark. Then 2,3-dichloro-5,6-
dicyanoquinone (DDQ, 613 mg, 2.7mmol) was added followed by addition of another 10 mL dry
CH2Cl2 and the mixture was stirred for another 4 h. Then triethylamine (5 mL) was added to the
reaction mixture and stirred for another 5 mins. Then, boron trifluorideetherate (7 mL) was added
and the mixture was stirred for another 60 min. The dark brown solution was washed with water
(3 × 20 mL) and brine (30 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by silica-gel flash column chromatography
(EtOAc–petroleum ether, (1:4) v/v)( Rf = 0.2) to give SP-BPY as red solid (268 mg, 25 % yield).
1H NMR (300 MHz, CDCl3):
(t,
=8.1Hz, 1H), 7.26-7.11 (m, 5H), 6.02 (s, 2H), 5.26 (s, 2H), 2.63 (s, 6H), 1.48 (s, 6H);13C
NMR (75 MHz, CDCl3): 156.9, 155.2, 155.1, 148.5, 142.6, 138.8, 137.2, 131.58, 130.8, 129.6,
δ 8.56-8.55 (d, J=5.4Hz, 1H), 7.66-7.62 (t, J= 7.8Hz, 1H), 7.49-7.44
J
δ
124.4, 122.7, 122.1, 121.1, 120.9, 113.2, 70.9, 29.7, 14.6, 14.1, 13.9 ppm; HRMS-ESI (m/z):
calcd for C25H24BFN3O [M-F]+ 412.1991, found 412.2025.

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3. Results and Discussion
The recognition of anionic substrates by positively charged or electrically neutral receptor
systems has emerged as an area of active research owing to their definite roles in complex
biological systems. In a receptor-based approach, it is very important to design the receptor such a
way that appropriate positions should available for effective synergistic binding. The binding
between receptors and the amino acid guests are usually strong enough viz. H-bonding or covalent
bonding to observe photophysical changes of receptor upon binding with guests. In the present
investigation, we have chosen functionalized BODIPY as an acceptor/receptor for amino acid
binding. BODIPY dyes are often used for recognition of anionic substrates including amino acids
[46-50].Asp and Glu are carboxylic acids and therefore difficult to design receptor for these
amino acids to obtain covalent type bonding [51-53]. We have carefully chosen N,N-diethylamino
benzaldehyde as starting material for designing of these probes. Introduction of the picolyl group
at a suitable position makes perfect space for amino acids like Asp or Glu to form simultaneous
H-bonding. A theoretical calculation was performed to support the designing strategy of the
probe. In accordance with H-bonding principle, it is very important to have N donor sites for the
carboxylate group. The interaction between ‘N’ centre and Asp or Glu has been strong enough to
attain photophysical changes in the probe.
3.1. Fluorescence and absorption spectral studies
The Probe N,N-SP-BPY is weakly emissive due to an effective PET process. It showed an
emission maximum at 527nm as shown in Figure 1. Upon addition of Asp or Glu, the band at 527
nm increases rapidly upon excitation at 450 nm. This band was attributed from the blocking of the
PET process. Initially, a possible PET process was initiated from ‘N’ centre of N,N-diethylamino
moiety to BODIPY core unit. Upon addition of Asp or Glu, the ‘N’ center gets involved in weak
H-bonds with amino acid inhibiting the PET process leading to an increase in emission band at
527 nm (Figure 2). Therefore, the probe N,N-SP-BPY senses Asp or Glu as a result of inhibition
of the PET process.

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Figure 2: Changes in the emission spectra of N,N-SP-BPY(20 µM) upon gradual addition of (a) Asp (0-
600 µM) and (b) Glu (0-600 µM) in 3:97 DMSO:H2O (v/v; pH 6.5 ; λex 450 nm).
On the other hand, N,N-SP-BPY has four absorbance bands centered at 268nm, 309nm, 376nm,
and 514nm respectively. All the absorbance bands were decreased simultaneously upon addition
of Asp or Glu. Interestingly, the main absorbance band at 514nm was blue shifted to 503 nm with
the addition of increasing amount of either amino acid (Figure 3). The corresponding naked eye
color of the solution was altered from pink to colorless (Figure 4).
Figure 3: Changes in the absorbance spectra of N,N-SP-BPY (20 µM) upon gradual addition of (a) Asp
(0-600 µM) and (b) Glu (0-600 µM) in 3: 97 DMSO: H2O (v/v; pH 6.5).

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Figure 4: Naked eye and UV light exposed color change of N,N-SP-BPY(20 µM) in the
presence of different amino acids up to 600 µM .
From Job’s plot a 1:1 binding interaction was observed between probe N,N-SP-BPY and its
adduct with Asp (Figure S-18, ESI). In order to establish the selectivity of the probe for Asp and
Glu, a competitive UV-Vis and fluorescence measurement experiments were performed. Except
for Asp and Glu, amino acids like Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, Val, Glp, Sec didn't significantly alter the fluorescence profile of N,N-SP-BPY
(Figure 5). Amino acid Cys enhanced the emission maxima only by a small degree. Similarly,
only Asp and Glu was found to draw a change in absorbance profile of the probe (Figure 6). The
emission intensity of the probe N,N-SP-BPY did not change significantly in the presence of other
amino acids in the same sample (Figure S-19, ESI). A relative plot of the emission intensity of
N,N-SP-BPY in the presence of other amino acids has been incorporated in Figure S-19. A
comparison table has also been incorporated on fluorescent probes found in the literature for Asp
and Glu (Table S-1, ESI) to validate the effectiveness of the probe. Most of the reported probes in
the table lacks selectivity, and have a poor quantum yield in the biological system. The probe can
selectively detect Asp/Glu in a mixture of a sample containing each amino acid.

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Figure 5: Changes in the fluorescence profile of N,N-SP-BPY (20 µM) upon gradual addition of all other
amino acids up to 600 µM in 3:97 DMSO: H2O (v/v; pH 6.5).
Figure 6: Changes in the absorbance spectra of N,N-SP-BPY (20 µM) upon gradual addition of all other
amino acids up to 500 µM in 3: 97 DMSO: H2O (v/v; pH 6.5).
Furthermore, we had synthesized 2 model compounds N,N-SB-BPY, and SP-BPY (scheme-1) to
understand the observed PET process. In N,N-SB-BPY the picolyl group of probe N,N-SP-BPY
was replaced by simple a benzyl group and SP-BPY has no N,N-diethylamino group. Initially, we
have expected two possible PET to be active in the probe N,N-SP-BPY, one operating from N,N-
diethylamino group and another from the picolyl group to BODIPY core unit. So the fluorescence
and UV-Vis changes of these two model compounds were investigated. N,N-SB-BPY was weakly
fluorescent indicating a PET process was indeed active from N,N-diethylamino group to BODIPY

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core. The emission maximum was situated at 524nm and upon addition of either Asp/Glu the
emission maxima increases and shifted slightly to 527nm (Figure 7a).
Figure 7.Changes in the fluorescence spectra following addition of Asp (0-600 µM) in (a) N,N-SB-BPY
(20 µM) and (b) SP-BPY in 3:97 DMSO: H2O (pH v/v; pH 6.5)
On the other hand, the emission intensity of SP-BPY (524nm, λex 450 nm) decreases upon
addition of Asp or Glu (Figure 7b).
Therefore, we concluded that only one PET was operating in the probe N,N-SP-BPY. On the
other hand, PET process between a receptor and suitable electron donating moieties viz. amino
acid or anion, can quench fluorescence upon van der Waals contact and supports molecular
contact. Hence, it can be concluded that there must have van der Waals contact or H-bonding
interaction between the picolyl group of SP-BPY and the amino acid.
Hence, we concluded that the fluorescence enhancement of the probe N,N-SP-BPY arise due to
quenching of PET and the picolyl group was introduced to have a van der Waals contact or H-
bonding interaction between the ligand and amino acid.
Furthermore, UV-Vis changes of N,N-SB-BPY, and SP-BPY were carried out in the presence of
Asp. The main absorbance peak of N,N-SB-BPY was centered at 270 nm, 309 nm, 518nm
respectively (Figure 8). Upon addition of Asp or Glu, the entire absorbance band decreased
continuously. Similarly, the main characteristic absorbance band of SP-BPY at 388 nm, 501nm,
and 539 nm also decreased in the presence of either amino acid. Therefore, a strong case of H-
bonding interaction may be proposed.

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Figure 8: Changes in the Absorbance profile following addition of Asp (0-600 µM) in (a) N,N-SB-BPY
(20 µM) and (b) SP-BPY in 3: 97 DMSO: H2O (v/v; pH 6.5)
The lowest detection limit of N,N-SP-BPY as calculated from fluorescence intensity was found to
be 1 µM and 5 µM (ESI) for Asp and Glu respectively. In the plot of emission intensity vs. Asp
concentration, the linear region (up to 10 µM Asp) was useful for the determination of an
unknown concentration of Asp (Figure S-20, ESI). The quantum yield of the probe N,N-SP-BPY
and its adduct with Asp was found to be 0.031 and 0.130. The association constant of probe for
Asp was estimated to be 1.49 × 105 M−1 from fluorescence titration using modified Benesi–
Hildebrand equation [54], (Fmax− F0)/ (Fx− F0) = 1 + (1/K)(1/ [M]n), where Fmax, F0, and Fx are
fluorescence intensities of N,N-SP-BPY in the presence of Asp at saturation, free N,N-SP-BPY
,
and any intermediate Asp concentration (Figure S-21, ESI).
3.2. Influence of pH
At different pH range (pH 2.0-8.0), the emission intensity of N, N-SP-BPY in the presence and in the
absence of Asp and Glu was thoroughly investigated (Figure 9). The supporting data clearly indicated that
the difference in emission intensities of N,N-SP-BPY and its complex with Asp/Glu was significant at pH
6.5. Hence, all the experiments were carried at pH 6.5 buffer system. From figure 9, it can be seen that the
probe is non-fluorescent in the pH range5-8 due to the active PET process. However, at low pH range 2-4,
protonation at the N center of N, N-diethyl moiety occurs and subsequently, the observed PET is
inhibited, resulting in strong fluorescence. In pH range 6-6.5, Aspartic acid remains in di-anionic form,
thus participating in two simultaneous H-bonds with the probe. With an increase in pH, the H-bonding
probability decreases and therefore, no emission could be observed. Lowering the pH to 5, the anionic
form of β-COOH disappears and therefore no fluorescence can be observed.

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Figure 9: Change in emission intensity of N,N-SP-BPY and its complex with Asp and Glu at different
pH.
3.3. Mode of sensing between N,N-SP-BPY and Asp/Glu
The possible sensing mechanism of N,N-SP-BPY with Asp/Glu which lead to the change in
absorbance and fluorescence profile were shown in Figure 10. Asp and Glu form two
simultaneous H-bonds with the nitrogen of N, N-diethylamino and picolyl unit, inhibiting the PET
process. Hence strong fluorescence was observed.
Figure 10.Possible binding mechanism of Asp and Glu with N,N-SP-BPY

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In order to discriminate the PET process with competing ICT, we have measured the fluorescence
and absorbance of N,N-SP-BPY in different organic solvent systems (Figure S-22, S-23). The
most prominent distinction between PET and ICT sensors lies in the different fluorescence
response upon analyte recognition. PET probes display fluorescence enhancement or quenching
without pronounced spectral shifts so that the terms ‘off–on' and ‘on-off' fluorescent sensors are
often used. In contrast, indicators based on ICT show clear fluorescence band shifts upon analyte
binding, making ratiometric measurements possible.
Our probe N,N-SP-BPY satisfies the perpendicular arrangement of the fluorophore and the
chelator, as seen from the crystal structure of the ligand. From S-22, S-23, it could be clearly seen
that the λmax UV varies between 500-503 nm and λmax Fl from 526-535 nm. Therefore, the
sensing mechanism is based on the inhibition of PET rather than ICT.
In order to gain further insight into the mechanistic aspect of sensing of the probe N,N-SP-BPY
,
1H-NMR titrations were performed using CD3CN-d4:D2O (4:1). On adding Aspartic acid, a
downfield shift was observed in the proton signal of (b), which is present in the para position of
nitrogen atom of the pyridine ring. The value shifts from
δ 7.62 to 7.64 (Figure 11), clearly
indicating interaction between the pyridine nitrogen and the hydrogen of carboxylic acid moiety
present in the analyte (Figure 10). The proton (a) upfield shifted from 8.51 to 8.44. In the free
ligand, the protons present in the meta position (c) and (d) were merged together where as in the
complex they were separated and downfield shifted from
δ 7.22 to 7.325 (c) and from upfieldδ
7.12 to 7.09.Protons present in the N,N-diethyl benzene part of the ligand was also affected,
indicating interaction between nitrogen of the benzene ring and the analyte. Protons (e, f and g) of
N,N-diethyl aromatic moiety was upfield shifted from
6.41 to 6.39. Interaction with the analyte also affects the electron density over the BODIPY core
as indicated by slight upfield shift of the pyrrole protons (i, i’) from 6.08 to 6.07 and 5.16 to
5.14. As the electron density of the nitrogen atom of N,N-diethyl moiety changes the CH2 (k) and
δ 6.91 to 6.90, δ 6.44 to 6.33 and from δ
δ
CH3 (j) protons was also shifted from
δ 3.37 to 3.34 and from δ 1.11 to 1.05 (Figure 10).Similar
1
trend was also observed when H-NMR titration was carried out in MeOH-d4:D2O (4:1); to
support the binding mechanism between Asp/Glu acid and N,N-SP-BPY (Figure S-24, ESI and
1
Table S-3 to S-4). Thus H-NMR titration evidently supports our mechanistic proposal. All the
significant changes in 1H NMR of N,N-SP-BPY have been shown in Table S-2 to S-4, ESI.

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Figure 11.1H NMR titration of N,N-SP-BPY in CD3CN-d4:D2O (3:1); Shift of aromatic protons upon
addition of increasing amount of Asp (0, 0.5, 1, 2 equivalent respectively).
3.4. Theoretical calculations
For the purpose of understanding the sensing mechanism and to attain a better picture of the
molecular orbital framework, structural optimization of N,N-SP-BPY and its complex with
Aspartic and Glutamic acid was carried out using density functional theory (DFT) as implemented
in Gaussian 09 software [55]. Becke’s three-parameter hybrid functional with the LYP correlation
functional (B3LYP) was used with the 6-31G(d,p) basis set for geometry optimization in water
using conductor-like polarizable continuum model (C-PCM) as a solvent model.
The ground state optimized structure of N,N-SP-BPY revealed localization of HOMO on N,N
diethyl benzene moiety while the LUMO is localized on BODIPY core (Figure 12). This suggests
a strong photoinduced electron transfer (PET) process acting between BODIPY core and N,N
diethyl benzene unit. Therefore no fluorescence could be expected for N,N-SP-BPY. The HOMO

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and LUMO of N,N-SP-BPY has the energy of -5.163 eV and -2.463 eV respectively with an
energy difference of 2.7 eV (459 nm).
Addition of Asp or Glu results in the formation of hydrogen-bonded complexes in which both the
HOMO and LUMO are located in BODIPY core. Therefore, the active PET process occurring in
the ligand was inhibited, and strong green fluorescence was observed. The nitrogen atom of N,N-
diethylamino, and the picolyl unit take part in hydrogen bonding interaction with respective
amino acids. The energy of HOMO and LUMO for the complex with Asp was found to be -5.562
eV and -2.561 eV, and for Glu -5.532 eV and -2.532 eV respectively. The energy difference in
both the complex was found to be 3.0 eV (413 nm). Hence, N,N-SP-BPY could form a stable
complex with Asp and Glu (Figure 11). Furthermore, theoretical, as well as experimental studies
depict the blue shift in UV-visible spectra. All the HOMO-LUMO energy levels and optimized
structures are shown in table S-5 to S-7, ESI.
Figure 12: HOMO-LUMO energy gaps in N,N-SP-BPY Aspartic acid and Glutamic acid in Water.
3.5. Intracellular imaging study
Imaging experiments were conducted for fluorescent visualization of intracellular Asp and Glu in
live HeLa, MDA-MB-468, A549 and HEK 293T cell lines. Cells were incubated in presence of
20 µM of probe N,N-SP-BPY for 2 hours (h) at 370C and 5% CO2 in the culture medium. After

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washing with PBS (×3), to remove the remaining probe, the cells were incubated with 50 µM of
each amino acid for another 1hour. The cells were then washed with PBS (×2), and fluorescent
images of the cells were collected.
Figure 13: Representative fluorescent images of MDA-MB-468 cells exposed to (a) cells incubated with
20 µM of N,N-SP-BPY; (b) green channel images of (a); (c) Overlay of (a) and (b); (d) Images were
taken after incubation with 20 µM of N,N-SP-BPY and followed by 1hour incubation with Asp (e) green
channel image of (d); (f) Overlay of (d) and (e). The cells were visualized under a fluorescence
microscope; EVOS FL Cell Imaging System equipped with Plan Fluor, 20X, NA 0.75 objective (Life
Technologies), USA; (scale bar 200 um).
Figure 14: Imaging of Asp/Glu in A549 cells: (a) bright field image of cells after incubating with N,N-
SP-BPY (20 µM); (b) green channel image of the cells; (c) Overlay of (a) and (b); (d) bright field image
of the cells after incubation with N,N-SP-BPY (20 µM) and followed by 50 µM Asp; (e) green channel

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image of (d) ; (f) Overlay of (d) and (e); (g) bright field image of the cells after incubation with N,N-SP-
BPY (20 µM) and followed by 50 µM Glu (h) green channel image of (g); (i) Overlay of (g) and (h);
(scale bar 200 um).
No fluorescence was observed in cells (control cells; Figure 13b, 14b and 15b) that were not
previously exposed to either Asp or Glu. Cells that were incubated for 1 hr with either of the
amino acids showed a significant enhancement of fluorescence in the green channel.
Figure 15: Imaging of Asp/Glu in HeLa cells: (a) bright field image of cells after incubating with N,N-
SP-BPY (20 µM); (b) green channel image of the cells; (c) Overlay of (a) and (b); (d) bright field image
of the cells after incubation with N,N-SP-BPY (20 µM)and followed by 50 µM Asp; (e) Green channel
image of (d) ; (f) Overlay of (d) and (e); (g) bright field image of the cells after incubation with N,N-SP-
BPY (20 µM)and followed by 50 µM Glu; (h) Green channel image of (g); (i) Overlay of (g) and (h);
(scale bar 200 um).
Figure 13-15 showed that the probe N,N-SP-BPY is cell permeable and can bind to the free Asp
or Glu in the cellular system. N,N-SP-BPY is highly stable in the cellular system, and over a
range of different pH. The difference in emission intensity between the probe and its complex
with either amino acid was significant enough to carry out any cellular imaging studies.
Furthermore, we have carried out cell imaging experiment in normal HEK-293T cell line. Figure
S-26 showed that N,N-SP-BPY is highly an efficient biomarker for imaging intracellular Asp/Glu
in normal cells without any significant cytotoxic effect.

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In order to examine the efficiency of the probe in detecting Asp/Glu at different pH values, we
have performed a pH-dependent fixed cell imaging experiment with N,N-SP-BPY in presence of
Asp. The result showed that maximum background-free fluorescence intensity could be achieved
in the range of biological pH (Figure S-27).
Therefore, the probe can be used as a biomarker for the detection of free Asp/Glu in different
cancer as well as in normal cell lines. The cytotoxicity of the probe on Hela cells was determined
by MTT assay (Figure S-28). The result showed that upon treatment with 20µM of N,N-SP-BPY
for 12hrs, almost 90% of the cells remains viable, indicating the non-toxic nature of ligand.
Hence, the concentration of probe has been fixed to 20µM in all experiments. Therefore, N,N-SP-
BPY may be used as an effective probe for detection of Asp or Glu in living systems.
4. Conclusions
In summary, we have designed and synthesized BODIPY based excellent colorimetric and
fluorescence probe N,N-SP-BPY for selective detection of Asp/Glu in water. The probe detects
Asp/Glu to a concentration as low as 1 µM and 5 µM respectively. N,N-SP-BPY effectively
images intracellular Asp/Glu inA549, HeLa, MDA-MB-468 and normal HEK-293T cells.
Furthermore, the cellular imaging studies clearly suggest that probe N,N-SP-BPYhas good cell
permeability and shows better selectivity towards Asp/Glu ions. Fluorescence microscopic study
shows intracellular fluorescence emission through the formation of the N,N-SP-BPY Asp/Glu
complex.
Conflicts of interest
There are no conflicts of interest
Acknowledgements
S.G, acknowledges CSIR for fellowship. A.G and A.A acknowledges DST Nano Mission. S.A
acknowledges SERB-DST, New Delhi, India, project No. EMR/2017/002140 for funding. We
acknowledge Mr. Arghyadeep Bhattacharyya for helping us with X-ray crystallography studies.
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Highlights
1. A highly selective BODIPY probe, N,N-SP-BPY detects aspartic acid (Asp) and glutamic acid
(Glu) in water.
2. ‘Turn On’ fluorescence can be observed by inhibition of Photoinduced Electron Transfer
(PET) process.
3. A distinct colorimetric change from pink to colourless solution can be observed to recognize
Asp and Glu.
4. N,N-SP-BPY efficiently images intracellular Asp and Glu in live cells under fluorescence
microscope.