A coumarin embedded highly sensitive nitric oxide fluorescent sensor: Kinetic assay and bio-imaging applications

Article abstract of DOI:10.1039/d0ob00567c

Fluorescence spectroscopy is a significant bio-analytical technique for specific detection of nitric oxide (NO) and for broadcasting the in vitro and in vivo biological activities of this gasotransmitter. Herein, a benzo-coumarin embedded smart molecular probe (BCM) is employed for NO sensing through detailed fluorescence studies in purely aqueous medium. All the spectroscopic analysis and literature reports clearly validate the mechanistic insight of this sensing strategy i.e., the initial formation of 1,2,3,4-oxatriazole on treatment of the probe with NO which finally converted to its carboxylic acid derivative. This oxatriazole formation results in a drastic enhancement in fluoroscence intensity due to the photoinduced electron transfer (PET) effect. The kinetic investigation unveils the second and first-order dependency on [NO] and [BCM] respectively. The very low detection limit (16 nM), high fluorescence enhancement (123 fold) in aqueous medium and good formation constant (Kf = (4.33 ± 0.48) × 104 M-1) along with pH invariability, non-cytotoxicity, biocompatibility and cell permeability make this probe a very effective one for tracking NO intracellularly. This journal is

Full text of DOI:10.1039/d0ob00567c

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DOI: 10.1039/D0OB00567C
ARTICLE
A Coumarin Embedded Highly Sensitive Nitric Oxide Fluorescent
Sensor: Kinetic Assay and Bio-imaging Applications
Debjani Maitia, Abu Saleh Musha Islama, Mihir Sasmala, Ananya Duttaa, Atul Katarkarb, and
Received 00th January 20xx,
Accepted 00th January 20xx
Mahammad Ali *a,c
DOI: 10.1039/x0xx00000x
Abstract: Fluorescence spectroscopy is a significant bio-analytical technique for specific detection of Nitric Oxide (NO) and
to broadcast the in vitro and in vivo biological activities of this gasotransmitter. Herein, a benzo-coumarin embedded smart
molecular probe (BCM) is employed for NO sensing through detailed fluorescence studies in purely aqueous medium. All
the Spectroscopic analysis and literature reports clearly validate the mechanistic insight of this sensing strategy i.e., the
initial formation of 1,2,3,4-oxatriazole on treatment of the probe with NO which finally converted to its carboxylic acid
derivative. This oxatriazole formation consequences a drastic enhancement in fluoroscence intensity due to PET effect. The
kinetic investigation unveils the second and first-order dependency on [NO] and [BCM] respectively. The very low detection
limit (16 nM), high fluorescence enhancement (123 fold) in aqueous medium and good formation constant (Kf = (4.33 ±
0.48) × 104 M-1) along with pH invariability, non-cytotoxicity, biocompatibility and cell permeability recognise this probe as
very effective one for tracking of NO intracellularly.
Introduction
For
But among these analytical methods, fluorescence technique is
a few decades, biologically abundant reactive nitrogen species (RNS)
have exposed their pivotal roles as a cell signalling molecule in the
course of diverse physiological and pathological processes.1 Nitric
concerned as a most conducive one for endogenous NO detection
owing to it's high sensitivity, non-destructive and real-time imaging
properties, minimal invasiveness, sophisticated instrumentations
and high spatio-temporal resolution.15-19 Thereby, exploiting the
fluorescence spectroscopic technique a plenty of nitric oxide probes
has been emerged: i) organic probes ii) single-walled nano tube
(SWNT) probes iii) quantum dots (QD) probes iv) metal complex
based probes.20-27 The o-phenylene diamine (OPD) moiety, in the
class of organic probe, is one of the promising receptor for NO
leading to the formation of triazole with a significant enhancement
of fluorescence intensity.20-33 Though this strategy dragged a deep
attention of researchers, still it's applications are limited by some
serious shortcomings. As for examples, (a) the vicinal diamine may
suffer from oxidation by various oxidants present in biological system
, (b) the triazole formed in the reaction with NO may display different
fluorescence response at different pH due to the presence of
secondary –NH group, which may lead to misinterpretation of
fluorescence data.30,31,33 Considering all these limitations, our group
has disclosed a novel strategy for nitric oxide sensing by embedding
quinoline as a fluorophore moiety which is flunked by acyl hydrazide
group. Upon treatment with nitric oxide this probe primarily forms
1,2,3,4 oxatrizole causing a substantial enhancement in fluorescence
intensity34 which finally converted to carboxylic acid.34 In our present
work, we have used coumarin as a fluorophore which is planar, rigid
and pi-conjugated heterocycle. For a few decades it was pretty
recognized that active site at 3 position of coumarin with its benzo
counterpart are strongly fluoroscent.35 It is a naturally occurring
organic compound found in plants and oil, like oil of cassia, sweet
Oxide (NO),
a prime member of this class is a pervasive
gasotransmitter and associated with numerous physiological
processes in many bio-organisms. Endogenous production of NO
takes place through the conversion of L-arginine to L-citrulline which
is catalysed by eNOS, nNOS and iNOS (NO synthase enzymes).2
Physiologically, NO functions as (i) stimulator in vascular smooth
muscle relaxation, (ii) prevents infections functioning on immune
system and (iii) serves itself as neurotransmitter.3,4 The pathological
impact in malfunctioning of NO homeostasis is incorporated with
carcinogenesis, neurodegenerative injury, endothelial dysfunction,
diabetes, chronic liver inflammation etc.5,6 Nitric oxide is involved in
the modulation of gene transcription and acts as a controlling factor
in the translation of m-RNA.7,8 Considering all these unambiguous
significance of this bio- regulatory molecule, several methods like
chemi-luminescence, electrochemistry, colorimetric assay, electron-
paramagnetic resonance spectroscopy, fluorimetric techniques9-14
etc. are widely used to comprehend the NO biology.
a. Department of Chemistry Jadavpur University, Kolkata 700 032, India; Fax: 91-33-
2414-6223, E-mail: m_ali2062@yahoo.com, mali@chemistry.jdvu.ac.in
b. Department of Biochemistry, University of Lausanne, Ch. des Boveresses 155,
1066 Epalinges, Switzerland
c Vice-Chancellor, Aliah University, ll-A/27, Action Area II, Newtown, Action Area II,
Kolkata, West Bengal 700160; vc_au@aliah.ac.in
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clover, woodruff, tonka beans and lavender. Coumarin derivatives The fluorescence properties of the probe BCM and affinity towards
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DOI: 10.1039/D0OB00567C
are well recognized due to their plenty of activities in nitric oxide were analyzed by performing the fluorescence titration
pharmaceuticals, insecticides, agrochemicals, fragrances, polymer Fig.1. For this experiment, a fixed concentration (20 μM) of BCM was
sciences and also identified as an important organic fluorescent added in a cuvette containing 2.5 ml of aqueous HEPES buffer at 25
materials.36 It is also used in the treatment of burns, brucellosis, °C. Then, the NO solution was added progressively in the
rheumatic disease, etc.37 In respect to photophysical activities, concentration range from 0 to 40 μM. BCM alone exhibits very weak
coumarin and it’s derivatives exhibit high photostability, easy fluorescence in aqueous HEPES buffer whereas,
synthesis, extended spectral range and good solubility 38-40. So,
considering all the significance, we developed our pioneering
strategy by using coumarin fluorophore joined with benzo- counter
part which is synthesized by well-known Knoevenagel condensation
followed by hydrazination. Herein, the relevant studies to
comprehend the selectivity and sensitivity of BCM towards NO with
plausible mechanism has been performed in terms of concentration
and time dependent manner along with detailed cell imaging
applications and theoretical studies. In addition, very high sensitivity
to nitric oxide with 123 fold fluorescence enhancement in purely
aqueous medium under physiological pH, very low detection limit
(16nM) has been observed which increases efficacy of the probe for
NO detection.
Fig.1. (a) Fluorescence emission spectra recorded taking only fixed
20 μM BCM and also gradual addition of NO (0−40 μM) at 25 °C in 10
Results and discussion
mM HEPES buffer at pH 7.0, λex = 410 nm and λem=470 nm. (b) linear
plot of F.I. vs. [NO]. (c) The image of probe BCM (left) and NO treated
product (right) exposing in UV light.
Scheme-1 displays two steps synthetic pathway for the synthesis of
3-Oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, (hereafter
designated as BCM). The first step is a very well-known Knoevenagel
condensation between 2-hydroxy napthaldehyde and di-ethyl
malonate in the presence of piperidine and glacial acetic under
gradual addition of NO causes a steady generation of fluorescence
intensity resulting ∼123 fold enhancement at λem ∼470 nm (λex
=
refluxing condition. After 8 h, the reaction mixture is cooled to room
temperature whereupon pure product (L1) was formed which was
collected by filtration and well analyzed by 1H NMR (Fig. S1), 13C NMR
(Fig. S2).
410 nm). To determine the apparent formation constant we have
carried out fluorescence titration experiment and a plot of F.I. vs.
[NO], manifests an excellent linearity of the curve up to 25 μM after
which the plot gets saturated (maximum NO 40 μM). The linear part
was easily solved by utilizing the equation (1)41 under the condition
1>> cxn and n=1.
푎 + 푏 × 푐 × 푥푛
1 + 푐 × 푥푛
푦 = (
(1)
)
where, parameters a and b denote F.I. of the probe in the absence
and presence of excess NO respectively, c is the formation constant
designated as Kf. The slope (b × c) obtained from the curve gives, c =
Kf = (4.33 ± 0.48) × 104 M-1 (using b = F.I. in the presence of large
excess NO).
Mechanistic view of fluorescence sensing of receptor BCM towards
NO
Scheme 1. Synthetic Pathway for the Probe BCM.
To comprehend a neat and tidy insight into the mechanism which is
responsible for fluorescence enhancement, we collected the product
of the reaction between BCM and NO. For this purpose, BCM was
dissolved (1 mmol, 0.254 g) in minimum volume of dichloromethane
(DCM) and finally with 20 ml acetonitrile under aerobic condition.
Then nitric oxide gas was bubbled through the probe solution for 15
minutes. A vivid fluorescence was noticed throughout the solution.
Solvent was evaporated under pressure. The brown solid was
The second step comprises the treatment of corresponding ester
with hydrazine hydrate in equimolar ratio. The targeted fluorescent
probe was well characterized by common spectroscopic studies, i.e.,
1H NMR (Fig. S3), 13C NMR (Fig. S4), mass spectrum (Fig. S5), and FTIR
(Fig. S6). The receptor exhibits very high affinity and selectivity
towards NO in aqueous medium.
Photophysical response of BCM towards NO
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collected and further purified by recrystallization in ethanol. The pseudo-first-order conditions using fluorometric techVnieiqwuAertic(lFeigO.nl2in)e.
pure crystalline compound was filtered and characterized by ESI-MS+ In this study, we have taken a fixed concentration of BCM (5 μM) and
DOI: 10.1039/D0OB00567C
(Fig. S7), 1H NMR (Fig. S8) and IR spectrometry (Fig. S9). The ESI-MS+ variable [NO] (20−100 μM). The kinetic traces at 470 nm unveil it as
spectrum (Fig. S7) shows a peak at 304.0406 (1,2,3,4-oxatriazole + single exponential growth curves assigning the first-order
K+) suggesting the generation of 1,2,3,4-oxatriazole (BCM-NO) which dependency of reaction rate on [BCM]. The time dependent
was further assured by the abolishing of –NH (9.39 ppm) and –NH2 fluorescence study also shows the fluorescence intensities at 470 nm
1
(4.79 ppm) peaks in H NMR spectra (Fig. S8). The generation of IR increases with time and reach a plateau within ~5 min (Fig. S12). The
peak at 1600 cm-1 (–N=N) and 1666 cm-1 (–C=N) also strengthen the pseudo-first-order rate constants (kobs) obtained from the growth
formation of oxatriazole moiety (Fig. S9).
curves have been plotted against [NO], which yields a nonlinear
upward curveture.
Scheme 2. Possible Reaction Mechanism of BCM and NO.
The predicted mechanism is outlined in scheme-2, where NO first
reacts with O2 to generate N2O3 under aerobic condition which
nitrosylates –NH2 of BCM to generate –NH‒NO which ultimately
generates 1,2,3,4-oxatriazole (BCM-NO) as a stable product. The
formation of such electron deficient oxatriazole moiety originates a
large enhancement in fluorescence intensity at λem = 470 nm. It is
interesting to note that the formed oxatriazole is finally transformed
to carboxylic acid through the formation of azide after keeping it in
solution for a long time and was assured by Mass spectra (Fig. S10),
1H NMR spectrum (Fig. S11).
From the electronic point of view the main-stream mechanism for
NO sensing by OPD based probes is based upon the PET block
process.42-44 Very recently, our group reported a quinoline based NO
sensor containing acyl hydrazide moiety where PET effect34 was
operative. In the current report, we propose that the PET-ON
mechanism due to electron push from hydrazide part to the
coumarin fluorophore makes the probe BCM as weakly fluorescent.
However, upon reaction with NO a remarkable fluorescence
enhancement has been perceived which may arise due to the
generation of electron deficient 1,2,3,4-oxatriazole moiety that
restricts the above PET-ON process (scheme-3).
Fig.2. The kobs vs. [NO] plot gives an upward curvature (blue solid
circles) while a plot of kobs vs.[NO]2 gives straight line (red solid
circles). Experimental conditions are: [BCM] = 5 μM and [NO] =
(20−100) μM, temperature = 15 °C in 10 mM HEPES buffer (pH = 7.0,
NaCl =0.10 M,). All the data were collected using fluorescence
technique.
However, a plot of kobs vs. [NO]2 displayed a well fitted straight line
which delineates the second–order dependency upon [NO] (Fig. 2).
The log(kobs) vs. log[NO] plot furnishes a straight line with a slope =
(2.42 ± 0.08) with R = 0.997 which also supports the second order
kinetics with respect to [NO] (Fig. S13). We have accomplished a
second experiment to evaluate the dependency of rate upon [BCM]
taking a fixed 5 μM [NO] with variable [BCM] in the range between
20 and 100 μM. Here, a plot of logkobs vs. log[BCM] clearly
demonstrates a first-order dependence on [BCM] with slope = (1.19
± 0.05) (Fig. S14) and R =0.997.
Here, a provisional reaction sequence is formulated as follows:
푘1
2NO + O2 2NO2
(2)
(3)
(4)
(5)
(6)
NO2 + NO푘 N2O3
2
N2O3 + H2O 푘 2H + +2NO2―
3
Scheme 3. Pet Effect Responsible for NO Sensing.
N2O3 +L푘4L ― NO + NO2― + H +
Time dependent reaction of BCM with NO, a kinetic approach
To assure a clear view of the operative mechanism of reaction
between BCM and NO, we have thoroughly performed kinetic
studies at pH 7.0 (10 mM HEPES buffer, 0.10 M NaCl), 15 °C, under
fast
L ― NO + H + L′ + H2O
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the concerned NO probe (BCM) is highly applicable fVoierwmArotinclietoOrnilninge
DOI: 10.1039/D0OB00567C
The above reaction sequence clearly demonstrates that NO interacts
firstly with its oxidized product NO2 to form N2O3 that acts as a NO+
donor in the next step under aerobic condition. Then the formed
N2O3 nitrosylates the amine hydrazide to generate L-NO which
consequences the synthesis of a closed ring 1,2,3,4-oxatriazole
moiety in a fast step (Scheme 2). We have surveyed the literature to
afford the rate constants of the above-mentioned reactions as: k1 =
6.33 × 106 M−2 s−1,45 k2 = 1.1 × 109 M−1s−1,46 and k3[H2O] = 1.6 × 103
s−1.47 With the knowledge of these rate constants we can stipulate
that NO2 and N2O3 are present in very negligible amount as reactive
intermediates. By applying a steady-state approximation eqn. 7 can
be attained for the formation of L' (BCM-NO).
NO quantitatively in these cell types.
pH stability
To be an efficient fluorescent probe, it should show pH tolerance in
the physiological range of pH so that it can be easily applicable in bio-
organisms. For this purpose we have assessed the pH effect on
photophysical behavior of BCM in the absence and presence of
excess NO. Fig. 3 shows very high fluorescence intensity of the
푘1푘4[L]
d[L′]
=
[NO]2t [O2]t
(7)
[
]
dt
2(푘3 H2O ) + 푘4[L])
From the previously illustrated kinetic studies, it was found that the
reaction kinetics follows second and first order dependence with
respect to [NO] and [L] ([BCM]) respectively which lead us to take
an assumption that, k3[H2O] ≫ k4[L]. Now the eqn. 7 reduces to
eqn. 8.
Fig. 3. pH dependent NO detection by BCM at 470nm λex=410nm at
25 °C in 10mM HEPES buffer.
probe and its NO treated product in low pH range (2-3). Probably the
PET based probe BCM, due to presence of electron donor group (–
NH-NH2) is converted to its protonated form which would block the
PET process generating the fluorescence intensity at lower pH range.
In the pH range 4-8 the probe shows very weak fluorescence,
however on treatment with NO it displays a drastic enhancement in
fluorescence intensity due to oxatriazole formation. Moreover, at
alkaline pH (> 8) the fluorescence intensity of NO treated product is
inhibited likely due to rapid decomposition of N2O3 (eqn. 4), formed
in the reaction of NO2 and NO (eqn. 3) causing availability of lesser
and lesser amount of reactive species (N2O3) towards the probe BCM
(eqn. 5). Overall the pH study displays the well applicability of the
probe at biological pH for tracing nitric oxide.
푘1푘4[L]
d[L′]
=
[NO]2t [O2]t
(8)
[
]
dt
2푘3 H2O
As the kinetics was performed maintaining the pseudo-first-order
conditions, taking L (BCM) as a minor component (the dissolved [O2]t
= 2.5 mM at 25 °C) eqn. 8 reduces to eqn. 9, where
풌풐풃풔
= {퐝[퐋′]/퐝퐭}/[퐋]
푘obs = 푘′[NO]2t
(9)
taking,
푘1푘4
푘′ = 2푘 [H O][O2]t
(10)
3
2
The value of k4 is evaluated by utilizing the previously observed
풌′ =
values of k1 and k3[H2O] mentioned above along with
ퟖ.ퟔퟎ × ퟏퟎퟔ
M−2 s−1, achieved from the linear plot of kobs vs. [NO]2 at
풌
15 °C. From all of these data the value of ퟒ was estimated to be
ퟏퟕ.ퟑ × ퟏퟎퟓ
M−2 s−1 and found to be very much similar in the order to
some analogous reactions of N2O3 as follows: HSA (0.3 × 105 M−1 s−1),
N-acetyl cysteine (1.5 × 105 M−1 s−1), GSH (2.9 × 105 M−1 s−1), Cys (2.6
× 105 M−1 s−1), BSA (0.06 × 105 M−1 s −1). Interestingly, this result is also
in accord with our reported works. 34,48
Limit of detection
Fig.4. (a) Bar plot represents the fluorescence responses of BCM at
470 nm (λex = 410 nm) towards various biological reactive species in
HEPES buffer at pH 7.0, BCM = 20 μM, Xn- = 50 μM at 25 °C; (b)
corresponding spectral responses.
We have also performed the study to evaluate the detection limit of
NO by BCM which reveals to be very low ~16 nM (Fig. S15) making
the probe very sensitive towards NO in macrophase cultures,49
where nitric oxide is present in micromolar to nanomolar range. So
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Analyte selectivity experiment
BCM-NO were obtained at room temperature in aqueous medium
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(Fig. S21). The absorption band of BCM at 374 nm in pure aqueous
The selectivity of a fluorescent probe towards a particular analyte is medium corresponds to the peak at 354 nm (Fig. S20) obtained from
ensured by providing an appropriate binding site and some special
chemical properties. Herein, the reactivity assay of probe BCM
towards NO was conducted by using a broad array of putative
interfering species like reactive species, important metal ions, and
anions in 10 mM HEPES buffer, at 25oC at pH 7.0. Figs. S16 and S17,
S18, S19 clearly manifest that BCM is silent in fluorescence response
towards essential metal ions and anions. Likewise, the selectivity of
BCM towards NO was ensured by reacting BCM with relevant
biologically reactive species like H2O2, ClO-, KO2, ∙OH, NO, NO2 , NO3 ,
tempo, glyoxal, ONOO-, HNO, AA (ascorbic acid), HCHO which
revealed none of these potential analyte triggered fluorescence
enhancement, except NO (Fig. 4). To our delight, the important
cellular metabolites like AA, glyoxal, HCHO interacts hardly with the
concerning probe. So, this high selectivity is very much advantageous
for precise detection of NO in biological milieu.
the TDDFT calculation which is assigned to the S0 S1 electronic
transitions with significant oscillator strength (Table S3 and S4).
Whereas, for BCM-NO the theoretically evaluated absorption band
at 407 nm ascribed to the peak at 386 nm also associated with S0
S1 electronic transitions (Fig. S20). So, from this information it can be
speculated that the experimental results have good agreement with
the theoretical ones.
-
-
Geometry optimization with electronic structure
To get a theoretical overview on the interaction between BCM and
NO we have conducted the DFT and TDDFT calculations of probe
(BCM) and it’s NO mediated product (BCM-NO). Fig. 5 represents the
optimized geometry of both the compounds having C1 point group.
The structural compositions of these compounds have been
established using mass spectroscopy which denotes the formation of
oxatriazole from carbohydrazide in the presence of NO. All the
important geometrical parameters of the probe BCM and BCM-NO
are tabulated in Table S1 and S2. For BCM-NO product N-O bond
distance is 1.43 Å, whereas the C-O bond distances lie in the range
of 1.2-1.4 Å.
Fig.6. Frontier molecular orbitals of probe BCM and BCM-NO.
NO Detection in Live Cells
The fluorescence imaging studies were executed to explore the
detecting capability of BCM as NO sensors in living cell. The probe’s
cytotoxicity has been assessed in A375 and Raw 264.7 cells (Fig. 7).
The cell lines showed evidence of well admissibility of BCM (more
than 80% of the cell is viable upto 60 μM of BCM), indicating
Fig. 5. Optimized geometry of BCM and BCM-NO.
Fig. 7. Graphical picture of Cell viability study of BCM.
In ground state the density of electron clouds mainly prevails on
HOMO and LUMO molecular orbitals of the 3-Oxo-3H-
its effectiveness as NO sensor in live cells also. Exogenously
stimulated NO sensing practicability of BCM was measured in A375
cells. The live cells were treated with NO donor DEA-NONOate
followed by incubation with probe. BCM-NO showed intracellular
fluorescence compared to BCM probe only (Fig. 8). Likewise,
endogenously stimulated NO sensing of BCM probe has been
estimated in Raw 264.7 cells where, the cells were treated with LPS
(1.0 mg/mL) and IFN-γ (1000 U/mL) for 6h and then treatment with
probe BCM (Fig. 9). Very obviously, in the presence of NO stimulator
the cells exhibited high fluorescence compared to the non-
stimulated and PTIO (NO scavenger) treated ones.
benzo[f]chromene-2-carbaldehyde
and
3-Oxo-3H-benzo[f]-
chromene-2-carboxylic acid amide moiety respectively in the probe
BCM. In case of BCM-NO, the electron clouds mainly resides on
HOMO and LUMO molecular orbital of the Benzo[f]chromen-3-one
and 3-[1,2,3,4]Oxatriazol-5-yl-chromen-2-one moiety respectively
(Fig. S20). These compositions are important to know the behaviour
of the transition as well as the absorption spectra of both BCM and
BCM-NO. The calculated HOMO-LUMO energy gap associated with
BCM and BCM-NO is 3.94eV and 3.47eV respectively (Fig. 6). By
exploiting TDDFT method, the UV-Vis absorption spectra of BCM and
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Fig. 8: (A) Images of A375 cells incubated with 5 µM BCM for 30 min and then incubated with DEA-NONOate (2, 5 and 10 µM) for 30 min.
The blue fluorescence was observed owing to reaction of NO with BCM. Images were captured at 63X objective. (B) Quantification of
fluorescence intensity in response to intracellular NO interaction with BCM.
Fig. 9: Endogenous recognition of NO with flow cytometry assay: (A) Fluorescence images of Raw 264.7 macrophage cells incubated with
BCM in the absence of LPS and stimulated with LPS+IFN-γ for 6h followed by the addition of PTIO, a NO scavenger. Corresponding images
were captured at 63X objective. (B) The characteristic bar plots display the changes in fluorescence intensity of BCM upon interaction with
LPS+IFN-γ, PTIO and LPS+IFN-γ+PTIO, respectively. The experiments were performed thrice and related data are represented as mean ± SD.
For statistical importance the estimated results were verified with the student’s t test. P value ≤ 0.05 were statistically significant. (C) Flow
cytometry experiment of NO with BCM by taking the Flowing Software version 2.5.1. The representative dot plot of forward (FSC-H) versus
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side (SSC-H) scatter and the corresponding histogram plot displaying a positive response for gated Raw 264.7 macrophage cells towards NO.
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The overlap histogram plot indicates a positive shift in fluorescence intensity peak of LPS+IFN-γ stimulated NO taken by BCM compared to
without LPS+IFN-γ and PTIO.
Additionally, the Flow cytometric studies were performed in Raw
264.7 cells for the evaluation of NO, showing a positive shift in
fluorescence peak for LPS+IFN-γ stimulated cells in contrast to the
non-stimulated cells and PTIO (200 mM) (Fig. 9C). So finally it can
be concluded that, BCM showed an excellent nontoxic
fluorescence response towards NO for the purpose of cell imaging
exogenously and endogenously.
and Mass spectrum (Fig. S5). ESI-MS+ (m/z): 277 (BCM + Na+), 1H
NMR (in DMSO-d6) (δ, ppm): 9.68 ( 1H, S, -CH), 9.39 ( 1H, S, -NH),
8.58 ( 1H, d, -ArH), 8.31( 1H, d, -ArH), 8.09 (1H, d, -ArH), 7.79 (1H,
t, -ArH), 7.69-7.63 ( 2H, m, -ArH), 4.79 ( 2H, m, -NH2). 13C NMR (in
DMSO-d6) (δ, ppm): 160.2, 159.7, 154.0, 141.7, 135.4, 129.9,
128.9, 128.8, 126.5, 122.2, 117.5, 116.3, 112.6
Physical Instrumentations and Experimental Methods
The descriptions of instruments used in experiment, preparation
of solution, experimental methods to carry out this work are
supplied in the Supporting Information.
EXPERIMENTAL SECTION
Materials. The reagents like 2-Hydroxy-1-naphthaldehyde,
diethyl-malonate, piperidine, glacial acetic acid, hydrazine
hydrate are of reagent grade and purchased from Sigma Aldrich.
The sodium salts of anions, salts of metal ions (Pb2+, Mn2+, Co2+,
Ni2+, Zn2+, Cu2+, Al3+, Hg2+, Mg2+, Fe3+, La3+, Cr3+ and Cd2+) and
CONCLUSION
In brief, we are reporting herewith a smart fluoregenic probe
BCM on benzocoumarin platform which selectively recognises NO
under physiological conditions. From the linear F.I. vs. [NO] plot
−
−
biological reactive species (H2O2, O2−, TEMPO radical, NO3 , NO2 ,
ClO−, AA, NO+, DEA-NONOate (sodium salt) were obtained either
from Sigma-Aldrich or from other commercial suppliers and used
without further purification. Solvents like DMF, MeCN, etc.,
(Merck, India) were of reagent grade and dried using standard
methods.
obtained from fluorescence titration experiment gives Kf
=
(4.33±0.48) × 104 M-1. Here, the acid-hydrazide moiety of probe
BCM reacts with NO/O2 to form electron deficient 1,2,3,4-
oxatrizole moiety which is solely responsible for fluorescence
enhancement through PET blocking mechanism. The outstanding
sensing performance of BCM towards NO is also illustrated by its
very low detection limit (16 nM), specific response to NO with
desirable selectivity and sensitivity in the presence of other
biological reactive species which makes the probe highly potent
for NO detection in biological environment. The reaction
mechanism of BCM and NO was confirmed by kinetic studies
which reveals a 1st order dependence on BCM and 2nd order on
NO. The pH variation study in NO detection also supports BCM as
an efficient NO sensor in a wide range of pH. Beside these, the
bio-imaging implication of this probe is also appreciable owing to
its least cytotoxicity, bio compatibility for in vitro NO detection.
The application of BCM can also be extended to flow cytometric
analysis of exogeneous and endogeneous NO in living cells. So, all
the above-mentioned experiments and information certify BCM
as a very appropriate probe for the tracking of NO.
Synthesis of ligand
The ligand 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid
hydrazide (designated as BCM) was synthesized by two step easy
procedures.
Step-1: Synthesis of 3-Oxo-3H-benzo[f]chromene-2-carboxylic
acid ethyl ester
2-Hydroxy-1-naphthaldehyde (10 mmol, 1.72 g), di-ethyl
malonate (20 mmol, 3.2 g) is mixed in a round bottom flux in 20
ml ethanol under heating and stirring condition in presence of
1ml piperidine and 2 drops of glacial acetic acid. This reflux was
continued for 6 hrs. Cooling at room temperature, the precipitate
was filtered. After washing it with the mix solvent (hexane and
ethyl acetate)
a
light yellow colour solid was obtained,
characterized by H NMR (Fig. S1) and 13CNMR (Fig. S2).1H NMR
(in DMSO-d6) (δ, ppm): 9.24 (d, 1H,-CH), 8.50-8.47 (m, 1H, -ArH),
8.26-8.23 ( 1H, m, -ArH), 8.02 (1H, d, -ArH), 7.75-7.72 (1H, m, -
ArH), 7.63-7.60 (1H, m, -ArH), 7.53-7.50 (1H, m, -ArH), 4.34 (q, 2H,
-CH2), 1.37 (t, 3H,-CH3). 13CNMR (in DMSO-d6) (δ, ppm): 162.8,
155.9, 155.0, 143.7, 135.9, 129.6, 128.9, 126.3, 122.0, 116.4,
111.7, 61.2, 14.0.
1
Conflicts of interest
There are no conflict of interest to declare
ACKNOWLEDGMENTS
Step-2: Synthesis of 3-Oxo-3H-benzo[f]chromene-2-carboxylic
acid hydrazide (BCM)
Financial support from DST (Ref. No. 809(Sanc)/ST/P/ S&T/4G-
9/2104) West Bengal and CSIR (Ref. 01(2896)/17/EMR-II), New
Delhi, India, is gratefully acknowledged. D. Maiti is greatful to
DST-INSPIRE for the fellowship (SRF).
The compound obtained from step-1 was dissolved in 20 ml of
ethanol (5 mmol, 1.34 g) and hydrazine hydrate was added in
equimolar ratio (5 mmol) under refluxing condition for 6 h. A
yellow solid was formed during the reaction. The product BCM
was collected and analyzed by 1H NMR (Fig. S3), 13C NMR (Fig. S4)
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Journal Name
21. (a)C. J. Reinhardt, E. Y. Zhou, M. D. Jorgensen, G. Partipilo
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