Formation or Cleavage of Rings via Sulfide-Mediated Reduction Offers Background-Free Detection of Sulfide

Article abstract of DOI:10.1021/acs.joc.9b01946

A set of three highly selective probes for sulfide detection has been developed. Two novel mechanistic strategies for the detection, including (a) transformation of a pro-fluorophore into an active fluorophore and (b) destruction of a fused ring to activate a fluorophore, have been explored. The structural features of the probes including azido groups ("active" and "latent") and leaving groups (with or without being attached to the fluorophore) have been investigated. During the course of the mechanistic studies, the single-crystal structures of all the probes and the products were obtained. One of the probes proved to be superior in terms of its ability to detect sulfide in pure water via an in situ formation of a fluorophore from a nonfluorescent precursor. These cheap and easy-to-prepare probes offer practical applications of sulfide recognition in environmental water samples and in the ovaries of fruit flies. A detection and quantification method using one of these probes and analysis with a smartphone enabled nonspecialists to detect sulfide reliably.

Full text of DOI:10.1021/acs.joc.9b01946

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Article
Formation or cleavage of rings via sulfide-mediated
reduction offers background-free detection of sulfide
Sanjib Ghosh, Biswajit Roy, and Subhajit Bandyopadhyay
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01946 • Publication Date (Web): 28 Aug 2019
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Sanjib Ghosha, Biswajit Roya, Subhajit Bandyopadhyay*a
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aDepartment of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur,
Nadia 741246, India
ABSTRACT: A set of three highly selective probes for sulfide detection has been developed. Two novel mechanistic strat-
egies for the detection, including (a) transformation of a pro-fluorophore into an active fluorophore, and (b) destruction
of a fused ring to activate a fluorophore, have been explored. The structural features of the probes including azido groups
(‘active’ and ‘latent’) and leaving groups (with or without attached to the fluorophore) have been investigated. During the
course of the mechanistic studies, the single crystal structures of all the probes and the products were obtained. One of
the probes proved to be superior in terms of its ability to detect sulfide in pure water via an in situ formation of a fluoro-
phore from a non-fluorescent precursor. These cheap and easy-to-prepare probes offer practical applications of sulfide
recognition in environmental water samples and in the ovaries of fruit-flies. A detection and quantification method using
one of these probes and analysis with a smartphone enabled non-specialists to detect sulfide reliably.
toxicity with HCN and CO, has resulted in mass destruc-
tion through accidental leakage.9–11 It has also been used
INTRODUCTION
as a chemical weapon.12 Alarmingly, it has become a
Hydrogen sulfide has a significant physiological and
popular tool for committing chemical suicide as well as in
pathological role in biological sysems.1–4 Nevertheless, H2S
is a hazardous sewer gas which is largely produced by
industrial processes and organic wastes that pollutes the
water-bodies. Its toxic effects depend on its concentration
and the exposure time. Exposure to sulfide concentrations
in the range of 10-500 ppm, may result in rhinitis and
acute respiratory paralysis.5 However, under a long-term
exposure or higher concentration only a few breaths are
required to cause coma, brain damage, and death.6–7 As
the ‘knock down gas’, it has caused many such mishaps of
occupational toxic exposure where mining labourers have
been knocked unconscious and killed.8 H2S, similar in
the attempt of orchestrating terror plots.13–15 While it in-
fluences the proliferation of cancer cell,16 altered levels of
H2S have been linked to Alzheimer’s disease,17 Down’s
syndrome,18 diabetes19 and liver cirrhosis.20
H2S is a silent killer. Though sulfide has a characteristic
malodor, it is not a reliable indicator. While exposure to
high level (>150 ppm) of sulfide numbs the smelling sen-
sation instantly, the minimum perceptible odor of H2S is
reported to be 0.13 ppm.21 The underlying toxicological
mechanism is yet to be explored in detail and sulfide poi-
Scheme 1. Schematic representation of the strategies adopted in this worka,b
b
aTransformation of a pro-fluorophore into another fluorescent species via cyclization and destruction of the ring fused
with the fluorophore where LG and EWG refer to ‘Leaving Group’ and ‘Electron-withdrawing Group’ respectively; (inset)
structures of probes 1, 2 and 3 are shown in circles.
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soning lacks specific therapy.22 Therefore, a fast and pre-
cise method for the detection of sulfide is of utmost im-
portance.
This work explores the detection of sulfides via two novel
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strategies. The first one deals with a sulfide-mediated
reduction of an azide and a subsequent annulation, form-
ing a new fused-ring system. The second strategy exploits
a non-fluorescent tetrazine ring system that can be for-
mally considered as a “latent (or camouflaged) azide”.
Reduction of the latent azide destroys the fused ring, en-
suing a push-pull mechanism.
The importance and applications of fluorimetric method
of sulfide detection has been reiterated in the literature.23–
29 The principal detection strategies include metal precipi-
tation, nucleophilic attack, and reduction of azide (or
nitro) groups. A popular strategy is the sulfide-induced
reduction of an azido group, directly30–35 (or indirectly,
through a spacer36–40) attached to a fluorophore, into an
amine which turns on the fluorescence generally through
a charge transfer (Scheme S1). In 2011, Chang has been
the pioneer to introduce the azide-based sulfide probes.41
Since then, more than 100 papers have been published
underlining the dominance of this detection strategy (see
Supporting Information for references). However, over-
exploitation of this method with mere change in the
fluorophore led to the loss of its novelty and academic
interest. Therefore, this vastly used method warrants im-
provisation and added features to broaden the scopes and
improve on the detection.
We envisaged that each of the pro-fluorophores can un-
dergo transformation from an open chain aryl derivative
(or a fused ring system) to an N-heterocyclic fluorescent
species with better delocalization (and perhaps greater
structural rigidity) and consequently a red-shifted emis-
sion maximum (Scheme 1). The open chain could be
flanked by a leaving or a non-leaving group. Probes 1 and
2 were thought to be an ideal manifestation of these ideas
respectively (Scheme 1a). On the other hand, the azide
group might also remain ‘latent’ as a tetrazole ring, fused
with a fluorophore that may undergo the removal of the
ring to revive the actual emission of the fluorophore.
Probe 3 could be proved to be a perfect candidate, befit-
ting this scheme (Scheme 1b).
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Figure 1. Fluorescence spectral changes of (a) probe 1, (b) probe 2 and (c) probe 3 (100 μM each) upon addition of various
analytes (0.6 mM each); (inset) visual changes of the respective probes before and after addition of sulfide under ambient
light (above) and UV light (below). Fluorescence enhancement of (d) probe 1 (e) probe 2 and (f) probe 3 (100 μM each) along
with the titration profile of each probe (shown in inset) upon addition of sulfide. Time-dependent studies of (g) probe 1, (h)
probe 2 and (i) probe 3 (100 μM each) in the presence of sulfide (400 μM); (inset) kinetic profiles for the sulfide-induced re-
action undergone by the respective probes.
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ly), at em 445, 440 and 432 nm respectively, linearly vary-
RESULTS AND DISCUSSION
1
ing with the concentration of sulfide from 0 to 4 equiv.
(Figure 1df, Supporting Information). The limits of de-
tection (LOD) were found to be 2.07, 0.67 and 1.73 M for
probes 1, 2 and 3, respectively (Figure S2, Supporting In-
formation).43
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Experiments were performed in milli-Q water (for probe
1) and water-acetonitrile mixture (for probes 2 and 3) at
pH 7.4 at 25 °C with 100 μM probe in each case unless
otherwise indicated. The ‘addition of sulfide’ is simply to
be understood as ‘addition of Na2S’ unless otherwise indi-
cated.42 The preliminary spectroscopic analyses caused
the emission bands of the probes 1, 2 and 3 to shift from
430 to 445 nm (λex = 375 nm), 425 to 440 nm (λex = 335
nm), and 414 to 432 nm (λex = 366 nm), respectively, with
a dramatic emission enhancement in the presence of Na2S
(6 equiv.) in each case (Figure 1ac). In contrast, the fluo-
rescence profile seemed to remain practically unaffected
in the presence of other anions and small molecules sug-
gesting the specific nature of the probes toward sulfide.
The time-dependent emission studies of the sufide-
induced reaction of probes 1, 2 and 3 showed that the pro-
gress of the reaction reached a plateau after 180, 300 and
60 min, respectively, and followed a pseudo-first order
kinetics with rate constants of 0.017, 0.0091, and 0.033
min-1 respectively (Figure 1g-i). Visibly, the changes in
fluorescence intensity could be observed within 30
minutes after the addition of the sulfides to the probes as
the emission intensity reached 43%, 25% and 70% of the
respective saturation point with the probes 1, 2 and 3,
respectively. Of course, the time to reach the saturation
fluorescence intensity took longer. Competitive experi-
ments with various biologically and environmentally rele-
vant analytes including oxidants (e.g. H2O2, perborate,
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Probe 1 displayed discrete absorption bands at 250, 286
and 335 nm. Addition of sulfide (0-6 equiv.) to
probe 1 exhibited a decrease in absorbance at λabs 286 nm
with a small blue shift for all the bands. While
probe 2 showed a gradual decrease in the absorbance at
280 and 330 nm, probe 3 exhibited increments at 300 and
330 nm. All these changes are in conformity with the pre-
dicted spectra of the desired species (Figure S1). The
probe 1 ( = 0.004), 2 ( = 0.0003), and 3 ( = 0.002)
(with respect to quinine sulfate) brought about small red
shifts (~15 nm for each of the probes) in the emission
maximum along with significant enhancement i.e. 70-fold
for probe 1, 10-fold for probe 2 and 70-fold for
probe 3 (with  being 0.015, 0.006 and 0.006 respective-
2-
aerial oxygen,) reductants (e.g. ascorbate, S2O32-, S2O4 ,
S2O5 ), and nucleophiles (e.g. HSO3-, CN-, SCN-), dis-
2-
played no significant disruption of the sulfide-induced
fluorescence response (Figure S3). The pH variation stud-
ies on the fluorescence response of the probes showed
steady responses over a broad pH range (3.09.0) in the
presence of sulfide, adding to the compatibility of the
probe under different conditions (Figure S4). The reaction
mixtures of each of 1, 2 and 3 with sulfide were monitored
Figure 2. Comparative FT-IR (partial) spectra of (a) probe 1, (b) probe 2 and (c) probe 3 in the presence and the absence of
1
sulfide. Comparative H NMR (partial) spectra of (d) probe 1 in CDCl3 (inset: the peak for CONH proton), (e) probe 2 in
CDCl3 (inset: comparative 13C NMR (partial) spectra before and after addition sulfide) and (f) probe 3 in DMSO-d6/CD3CN
(2:1; v/v) in the presence and the absence of sulfide.
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by TLC.
stretching frequency of probe 1 suggested a chemical
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transformation of the group in the presence of sulfide. For
probe 1, while one of the two ester carbonyls (CO2Et)
remained intact, the other one vanished, generating new
merged peaks, presumably corresponding to the C=O
stretching and the amide NH bending, respectively (Fig-
ure 2a, S5A). Probe 2 also showed the disappearance of
azido stretching with a shift in the C=O stretching (Fig-
ure 2b, S5B). Probe 3 displayed a concomitant appearance
of an –NH stretching (with a characteristic shoulder
band) and disappearance of the –C=N stretching (of te-
trazole) (Figure 2c, S5C). The ESI-MS of probes 1, 2 and 3
after the addition of Na2S played a crucial role in unravel-
ling the mechanisms of detection by displaying the ex-
pected peaks for the intermediates, 1a′ , 2a’ and 3a’ for
probes 1, 2 and 3, respectively (Scheme 2, Figure S68).
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To obtain further insights into the mechanism of sulfide
1
detection, time-dependent H NMR titration of probe 1
was also performed (Figure S9). The titration showed that
the peaks at  4.34 and 1.38 corresponding to the Hb and
Ha gradually disappeared. Apart from other minor shifts,
Hc protons went through a shift from 4.32 to 3.58. The
probe 1 displayed a new broad signal appeared at  8.27
for the Hf which gradually broadened further and eventu-
ally vanished perhaps due to the proton exchange with
the solvent. The investigation into the mechanistic detec-
tion through 1H NMR studies continued for all the probes
in order to establish the structures of the products upon
addition of the sulfide and to study the mechanism in
further details. (Figure 2df). As can be clearly seen, upon
Figure 3. Crystal structures of the probes and their cor-
responding products obtained upon addition of sulfide.
All the structures are associated with thermal ellipsoid
plot with 50% probability; All the structures are with
50% Hydrogens are omitted for clarity.
A newly generated polar, fluorescent component was seen
to exist on the TLC plate for each of the probes upon
treatment with sulfide. FTIR spectra after the addition of
sulfide displayed new bands with distinct changes. (Fig-
ure 2ac, S5). The disappearance of the azido (N3)
1
reacting with sulfide, all the H peaks of the probes 1
(CDCl3, 10 mM) and 2 (CDCl3, 10 mM) underwent down-
1
field shifts. However, the H resonances underwent up-
Scheme 2. Proposed reaction mechanismsa,b
Reaction mechanisms are shown for aprobes 1 and 2, and bprobe 3 with sulfide (inset: crystal structures of the probes and the
respective products). The probable intermediates are detected by mass spectrometry in support of the mechanism.
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field shifts for the probe 3 (DMSO-d6/CD3CN (2:1; v/v), 10 respectively (Scheme 2a). Taking cue from the previous
reports,44-45 probe 3 is envisaged to follow the mechanism
proposed in Scheme 2b because of the presence of the
electrophilic tetrazole moiety fused to the quinoline sys-
tem which underwent ring destruction (through nucleo-
philic attack by sulfide) and generated the (reduced)
NH2 group. While all the three probes supposedly generate
N2 during the reduction of the azido groups, the nucleo-
philic attacks and the subsequent departure of EtOH and
H2O from the probes 1 and 2, respectively, led to their
corresponding products 1a and 2a. Interestingly, in the
process of sulfide-induced reduction, the ‘latent’ azido
group (of probe 3) can be considered as a ‘caged-azide’
that undergoes a ring-opening reaction unlike the ‘free’
azido group (of probes 1 and 2) having a linear geometry.
For probes 1 and 2, the ring formation reaction takes place
after the reduction of the azide group, whereas for probe
3, the ring destruction occurs due the reduction of the
latent azide group.
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mM). The addition of sulfide to probe 1 induced the gen-
eration of the least shielded broad signal appeared for the
CONH proton along with a concomitant disappearance
of the peaks corresponding to one of the COOEt groups
(Figure 2d). While probe 2 displayed a downfield shift of
the proton resonances along with the disappearance of
one of the 13C resonances of the two carbonyl carbons
(Figure 2e), probe 3 showed an upfield shift with genera-
tion of a broad peak for –NH2 (Figure 2f), providing us
with a clear picture of the associated chemical changes.
All these NMR spectral studies suggested clues as to the
chemical transformation involved with probes in the
presence of sulfide.
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In the meantime, with constant efforts, the probes and
the products of all these transformations were isolated
from the reaction mixture with column chromatography
(Experimental Section) and crystallized. The crystal struc-
tures unambiguously revealed that compounds 1a, 2a and
3a contained a carboxylate, an acetyl and a cyano group,
each appended to an aromatic quinoline (or quinolone)
system (Figure 3). Moreover, it was found that sulfide
caused compounds 2a and 3a to undergo ring annulation
and ring opening respectively to possess transformed
groups, i.e. a CH3 and an NH2 groups, respectively. All
these chemical structures were in conformity with the
FTIR, ESI-MS, and NMR analyses (Figure 2, Scheme 2,
Supporting Information).
The structures of all the three pairs of probes and the
products were optimized (Figure S10) and the
HOMO−LUMO energy differences were found to be
roughly similar. (Figure S1113). Expectedly, the daughter
products, 1a, 2a and 3a, were found to be associated with
increasing ICT characteristics, as evident from their elec-
tronic distribution. The calculated absorption peaks were
also found to be in good agreement with the experimen-
tally observed absorption bands (Figure S14).
In a turn-on fluorescence response, a lumophore, which
is generated in situ by the interaction between the probe
and the analyte, supposedly offers no background for in
vitro as well as in vivo studies, and adds a special
significance to practical applications. It is important to
note that the precursor 1 does not possess a potent
fluorophore whereas upon its transformation to 1a in the
The spectroscopic and X-ray evidences provided us with
the mechanistic insights into the sulfide-induced for-
mation of 1a, 2a and 3a from their respective precursors
(Scheme 2). The presence of the ester and the acetyl
groups in the close vicinity of the primary amino group
generated by the in situ reduction of the azido group
might be the main driving forces for the probes 1 and 2,
Figure 4. Fluorescence detection of sulfide with probe 1 (a) in water samples of varying contamination (showing emission
response of the probe before (blue bar) and after (red bar) addition of sulfide), (b) in solution (with varying probe- and sul-
fide-concentration) using paper-strips, (c) with visual changes of solution under 366 nm light with a varying sulfide concen-
tration, (d) with RGB values using calibration curve (with five blind sulfide concentrations, namely U1, U2, U3, U4 and U5,
marked in red triangles) and (e) in the ovary of Drosophila in the presence and the absence of sulfide (0.1 mM).
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presence of sulfide with the additional ring fusion, a calibration plot with high accuracy (Table S1, Supporting
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fluorescent species is generated. Since the manifestation
of the fluorescence occurs only in the presence of sulfide,
the chances of false positives with these probes are
insignificant. Also the solubility of probe 1 is highest
among the three probes of thisreport. In view of these
special features, for practical applications for the
detection of sulfides, probe 1 was chosen as a potential
candidate based on its fluorescence response in aqueous
medium.
Information). This enabled the detection of sulfide of en-
vironmental samples even by a non-technical person. The
method might be proved to be cost-effective51 and com-
pact for in-field sensing.
Encouraged by the successful in vitro application of the
probe, efforts were made to detect sulfide in egg cham-
bers of Drosophila melanogaster, a standard model for
bio-imaging as a part of a live organism (Supporting In-
formation). Adult fruit flies were fattened using dry yeast
for 20-24 hours. The ovary was dissected out of adults
into 10% fetal bovine serum containing Schneider’s Dro-
sophila medium. Dissected ovaries were separated by us-
ing micropipette. Two PBS washes were given before in-
cubation with PBS along with probe 1 for 15 min. Egg
chambers were further washed with PBS, followed by ad-
dition of sulfide and incubation for 30 min. Two PBS
washes were given to the samples. Next, they were im-
aged after mounting in 60% glycerol. The imaging was
done with two concentrations of the probe (1 and 0.1 mM)
at biological pH upon addition of 4 equiv. (w.r.t. the
probe concentration) of sulfide. The imaging was done
with two concentrations of the probe 1 i.e. 0.1 mM (Figure
4e) and 1 mM (Figure S15) at biological pH upon addition
of sulfide (0.4 and 4 mM respectively). In the presence of
sulfide, the fluorescence images of the chambers loaded
with probe 1 displayed a bright intracellular luminescence
accumulated apparently in the perinuclear regions in con-
trast to the controls.
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Industrial effluents often pollute the aquatic environment
with its sulfide content. Our probe was also applied for
the detection of sulfide in water samples of varying de-
grees of contamination (Figure 4a) only to find that the
efficiency of sulfide detection was comparable in Milli-Q,
distilled, tap and tank water samples. An instrument-free
dip-stick method for the qualitative detection of sulfide
was also performed with this probe. Pieces of filter paper
(Whatman 1, 5.5 cm × 1 cm) coated with a solution of
probe 1 (10, 1 and 0.1 mM) were air-dried. The dried strips
show a negligible emission under the 366 nm UV light.
The paper-strips were consequently dipped into the solu-
tions containing sulfide (of either same or half concentra-
tion of that of the probe), and kept them in air for 30 min.
The visual fluorescence response of the probe-coated pa-
per strips changed from dark to baby blue under 366 nm
UV light in the presence of sulfide (Figure 4b).
With the easy availability and accessibility of
smartphones, a new trend of detecting analytes with
smartphones has been an emerging area, sometimes pop-
The incubation times of 15 min for only probe 1 and 30
min subsequently for sulfide elicited the sufficient fluo-
rometric changes inside the egg chamber. The overall
incubation time for probe 1 was deliberately kept small
compared to the saturation time (180 min), so as to min-
imize the effect on the Drosophila.

ularly termed as “smartphone chemistry”.46 49 Since the
visual changes of the solution of the probe 1 under the UV
light showed distinct differences in the emission intensi-
ty, depending on the concentration gradient of sulfide
(Figure 4c), we thus explored the possibility of quantifica-
tion of sulfide in tap water samples using a smartphone
Android-based app developed by our group.50 The app,
initially designed for the color-blind students, can read
out the RGB values of an object. Each of the solutions was
added to a vial and imaged using a smartphone camera
under 366 nm UV light. The smartphone app was then
used to decipher the RGB values of the images before and
after adding sulfide, and each of the relative ΔR values
was obtained by subtracting the average R values of the
blank sample (probe 1 only) from those obtained after the
addition of sulfide and dividing the resultant by the ‘blank
value’. The data were always acquired in relative terms to
remove the dependence of the absolute RGB values of
probe 1 as the background (blank) value. The concentra-
tions of five blind samples data (containing unknown
amounts of sulfide) were estimated and confirmed by the
person (not involved in the experiment) who prepared
the samples. The relative values of the red channel (ex-
tracted from the RGB dataset) were measured and corre-
lated with the known sulfide concentration. From the
standard plot (Figure 4d), the blind concentrations of the
sulfide samples were successfully determined from the
CONCLUSIONS
In conclusion, this work introduces a pair of unconven-
tional detection strategies, namely fusion and fission of
annulated rings based on sulfide-induced azide-
reduction. We have developed a set of three selective or-
ganic probes (appended with ‘active’ or ‘latent’ azido
groups) each of which can detect sulfide, following a de
novo detection mechanism. Two types of detection strat-
egies have been employed in our work: conversion of a
pro-fluorophore into another emissive species via an in
situ transformation, and destruction of the ring fused
with the fluorophore in order to restore the original emis-
sion signature. The mechanisms proposed in this work
are established based on well-characterized evidences
including crystal structures. One of these easy-to-prepare
probes offers practical applications of the detection of
sulfide with environmental water as well as in dip-stick
analysis. This probe enabled us also for the determination
of the blind concentration with RGB analysis using a
smartphone and bioimaging in the ovary of fruit-flies.
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These novel detection mechanisms may open up a new
with NaN3 in HMPA, following the literature procedure.52
The other compounds were prepared according to the
procedures described below.
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avenue for further development of this field.
EXPERIMENTAL SECTION
Diethyl 2-(2-azidobenzylidene)malonate (1). Piperidine
(0.150 mL, 1.52 mmol) was added to the solution of dieth-
Materials and Instruments. All materials and reagents
were commercially available and no further purification
was performed unless otherwise noted. Pure solvents
were used in dried condition. A dry nitrogen atmosphere
was maintained during the reactions using flame-dried
glassware, unless otherwise indicated. The structures of
the compounds were determined by NMR spectroscopy,
mass spectrometry, XRD analysis and a plethora of other
o
ylmalonate (0.261 g, 1.63 mmol) in EtOH at o C. After
stirring for 10 min, was added dropwise the ethanolic so-
lution of 2-azidobenzaldehyde (0.200 g, 1.36 mmol). After
stirring for 4 h at room temperature, the reaction mixture
was evaporated to dryness, extracted with DCM (30 mL),
and washed with brine (50 mL×2). The organic phase was
dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue on chromatography with
hexane/ethyl acetate (3:1, v/v) yielded compound 153
(0.295 g, 75%) as a yellow solid, and recrystallized from
9
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12
13
14
15
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17
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23
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45
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48
49
50
51
52
53
54
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57
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60
1
spectroscopic techniques. The H NMR spectra were rec-
orded on a 400 MHz JEOL or a 500 MHz Bruker spec-
trometer instruments. Similarly, 13C NMR experiments
were performed with 100 MHz Jeol and 125 MHz Bruker
instruments. Chemical shifts reported in this work are
values with respect to either an internal reference (TMS)
or the solvent peak. The spectroscopy-grade solvents used
for the spectroscopic experiments were devoid of any flu-
orescent impurity. Milli-Q water was used for the spectro-
scopic experiments. The solutions of anions were pre-
pared from TBAF, TBACl, TBABr, TBAI, TBACN, NaClO4,
Na2S, NaN3, Na2SO4, NaNO2, NaNO3, NaSCN, Na2CO3,
NaHCO3, Na-ascorbate, Na-benzoate, NaBO3, Na2HPO4,
NaH2PO4, Na3PO4, NaOAc, NaHSO3, Na2S2O3, Na2S2O4,
Na2S2O5 and K2S5 (a polysulfide) in water (TBA = tetrabu-
tylammonium), while the solutions of the neutral mole-
cules were prepared from H2O2, melamine, cysteine and
glutathione. IR spectroscopic data were obtained with a
Spectrum Two PerkinElmer FT-IR Spectrometer. UV–vis
spectra were recorded with a Cary 60 UV–vis spectropho-
tometer. Fluorescence measurements were carried out
with a Horiba Jobin Yvon fluorometer (Fluoromax-4, Xe-
150 W, 250–900 nm) and JASCO FP-8300 fluorometer.
Optical studies were performed water/DMSO (99:1, v/v),
water/MeCN (1:2, v/v) and water/MeCN (2:3, v/v) for
probe 1, 2 and 3, respectively and all the media were buff-
1
DCM/EtOAc mixture. H NMR (400 MHz, CDCl3): 1.21 (t,
J= 6.88 Hz, 3H, CH3), 1.33 (t, J= 6.88 Hz, 3H, CH3), 4.26 (q,
J= 6.88 Hz, 2H, CH2), 4.31 (q, J= 6.88 Hz, 2H, CH2), 7.09
(m, 1H, ArH), 7.19 (m, 1H, ArH), 7.41 (m, 2H, ArH), 7.93 (s,
13
1H, CH). C{1H} NMR (100 MHz, CDCl3): 13.7, 14.0, 61.5,
61.6, 118.4, 124.6, 124.9, 127.7, 129.1, 131.4, 137.3, 139.4, 163.8,
166.1. FT-IR (KBr, cm-1): 1697, 1738, 2136. λabs in water (nm)
250, 286, 335. HRMS (m/z): Calcd. for C14H15N3O4Na+ or
[M+Na]+ 312.0965, found 312.0965.
3-(2-azidobenzylidene)pentane-2,4-dione (2). Piperidine
(0.150 mL, 1.52 mmol) was added to the solution of acety-
o
lacetone (0.136 g, 1.63 mmol) in EtOH at 0 C. After stir-
ring for 10 min, was added dropwise the ethanolic solu-
tion of 2-azidobenzaldehyde (0.200 g, 1.36 mmol). After
stirring for 3 h at room temperature, the reaction mixture
was evaporated to dryness, extracted with EtOAc (30 mL),
and was washed with distilled water (50 mL×2), followed
by brine (50 mL×2). The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pres-
sure. The residue on chromatography with hexane/DCM
(4:1, v/v) yielded compound 254 (0.193 g, 62%) as a light-
1
yellow powder, and recrystallized from DCM/hexane. H
o
ered with TRIS (1 mM, at pH 7.4, 25 C), unless otherwise
NMR (400 MHz, CDCl3): 2.14 (s, 3H, CH3), 2.36 (s, 3H,
CH3), 7.03 (t, J= 6.88 Hz, 1H, ArH), 7.14 (d, J= 8.36 Hz, 1H,
ArH), 7.22 (d, J= 6.88, 1H, ArH), 7.37 (t, J= 7.64 Hz, 1H,
ArH), 7.62 (s, 1H, ArH). 13C{1H} NMR (100 MHz, CDCl3):
26.4, 31.6, 118.5, 124.5, 124.3, 129.9, 131.7, 134.7, 139.2, 143.4,
196.5, 204.7. FT-IR (KBr, cm-1): 1650, 1730, 2136. λabs (nm)
in water/MeCN (1:2, v/v) 256, 280, 330. HRMS (m/z):
Calcd. for C12H11N3O2Na+ or [M+Na]+ 252.0743, found
252.0735.
indicated. Fluorescence imaging experiments were carried
out using an Olympus IX 51 inverted microscope with UV
excitation and an Axio Observer with Apotome module.
HRMS data were obtained from Acquity ultra-
performance Bruker MaXis Impact liquid chromatog-
raphy instrument by positive mode electrospray ioniza-
tion (Q-TOF). pH data were recorded with a Sartorius
Basic Meter PB-11 calibrated at pH 4, 7, and 10. Reactions
were monitored by TLC with Merck plates (TLC Silica Gel
60 F254). Silica gel (100–200 mesh, Merck) was used for
column chromatographic purification. Yields refer to the
chromatographically and spectroscopically pure com-
pounds.
Tetrazolo[1,5-a]quinoline-4-carbonitrile (3). A ethanolic
solution (5 mL/mmol) of 2-azidobenzaldehyde (0.200 g,
1.36 mmol) was added dropwise to a mixture of malono-
nitrile (0.108 g, 1.63 mmol) and piperidine (0.150 mL, 1.52
o
mmol) in EtOH at 0 C. After stirring for 3 h at room
Synthesis.
temperature, the reaction mixture was evaporated to dry-
ness, extracted with DCM (50 mL), and was washed with
distilled water (50 mL×2), followed by brine (50 mL×2).
The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue on
Experimental Procedures and Spectroscopic charac-
terization of 1, 2, 3, 1a, 2a and 3a. 2-Azidobenzaldehyde
was prepared from 2-nitrobenzaldehyde upon treatment
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chromatography with hexane/DCM (1:6, v/v) yielded rotary evaporator. The crude solid product was washed
compound 355 (0.181 g, 68%) as yellow powder, and recrys-
tallized from EtOH-DCM.1H NMR (500 MHz, DMSO-
d6/CD3CN (4:1; v/v)): 7.92 (t, J= 7.65, 1H, ArH), 8.16 (t, J=
7.95, 1H, ArH), 8.31 (d, J= 7.95 Hz, 1H, ArH), 8.67 (d, J= 8.5
Hz, 1H, ArH), 9.16 (s, 1H, ArH). 13C{1H} NMR (125 MHz,
DMSO-d6/CD3CN (4:1; v/v)): 97.2, 113.9, 116.4, 122.6, 128.9,
130.8, 131.4, 134.8, 143.3, 145.7. FT-IR (KBr, cm-1): 1654, 2233,
3066 λabs (nm) in water/MeCN (2:3, v/v) 290, 298, 330.
HRMS (m/z): Calcd. for C10H5N5Na+ or [M+Na]+ 218.0437,
found 218.0435.
with dichloromethane (30 mL) and washed with distilled
water (50 mL×2), and brine (50 mL×2). The organic phase
was dried over anhydrous Na2SO4 and concentrated un-
der reduced pressure. The residue on chromatography
with hexnane/dichloromethane (1:9, v/v) yielded com-
1
2
3
4
5
6
7
8
1
pound 3a (0.603 g, 58%) as a solid yellow powder. H
NMR (400 MHz, DMSO-d6/D2O (2:1; v/v)): 2.82 (s, 3H,
CH3), 6.69 (br, 2H, NH2), 7.28 (t, J= 6.88 Hz, 1H, ArH),
7.50 (d, J= 8.4 Hz, 1H, ArH), 7.66 (t, J= 7.64 Hz, 1H, ArH),
9
1
7.74 (d, J= 7.64 Hz, 1H, ArH), 8.61 (s, 1H, ArH). H NMR
10
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(400 MHz, CDCl3): 5.36 (br, 2H, NH2), 7.33 (m, 1H, ArH),
7.66 (m, 3H, ArH), 8.30 (s, 1H, ArH). 13C{1H} NMR (100
MHz, CDCl3): 95.1, 116.2, 121.8, 124.1, 126.4, 128.1, 133.2,
144.2, 149.1, 154.8. FT-IR (KBr, cm-1): 1654, 2227, 3162, 3321,
3396. λabs (nm) in water/MeCN (2:3, v/v) 290, 298, 330.
HRMS (m/z): Calcd. for C10H8N3+ or [M+H]+ 170.0713,
found 170.0712.
Ethyl 2-oxo-1, 2-dihydroquinoline-3-carboxylate (1a). Sodi-
um sulfide (1.35 g, 17.30 mmol) in water (3 mL) was added
dropwise to a solution of compound 1 (1 g, 3.46 mmol) in
o
methanol (50 mL) at 25 C. After stirring overnight, the
reaction was neutralized with 15 mL 0.1 (N) AcOH, ex-
tracted with ethyl acetate (25 mL) and washed with dis-
tilled water (50 mL×2), and brine (50 mL×2). The organic
phase was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue on chromatography
with hexane/ethyl acetate (1:4, v/v) yielded compound 1a
1
(0.338 g, 60%) as a brown solid. H NMR (400 MHz,
CDCl3): 1H NMR (400 MHz, CDCl3): 1.43 (t, J= 7.64 Hz, 3H,
CH2CH3), 4.43 (q, J= 7.64 Hz, 2H, CH2CH3), 7.23 (t, J= 7.64
Hz, 1H, ArH), 7.51-7.63 (m, 3H, ArH), 8.54 (s, 1H, ArH),
12.64 (bs, 1H, NH). 13C{1H} NMR (125 MHz, CDCl3): 14.2,
61.3, 116.3, 118.5, 122.0, 123.0, 129.1, 133.0, 140.0, 145.6, 161.3,
164.3. FT-IR (KBr, cm-1): 1595, 1647, 1737. λabs in water (nm)
280, 330. HRMS (m/z): Calcd. for C12H11NO3Na+ or
[M+Na]+ 240.0637, found 240.0623.
Spectroscopic and spectrometric characterization of the
compounds, fluorescence detection, single crystal X-ray dif-
fraction data, and computational analysis.
Crystal data of compound 1 (CCDC 1879384)
Crystal data of compound 1a (CCDC 1879400)
Crystal data of compound 2 (CCDC 1912153)
Crystal data of compound 2a (CCDC 1912155)
Crystal data of compound 3 (CCDC 1912156)
Crystal data of compound 3a (CCDC 1912158)
1-(2-methylquinolin-3-yl)ethan-1-one (2a). Sodium sulfide
(2.55 g, 32.72 mmol) in water (4 mL) was added to a solu-
tion of compound 2 (1.5 g, 6.54 mmol) in acetonitrile-
dichloromethane (50 mL, 4:1, v/v) at 25 oC. After stirring 4
hr, the reaction was neutralized with 20 mL 0.05 (N)
AcOH, extracted with dichloromethane (30 mL) and
washed with distilled water (50 mL×2), and brine (50
mL×2). The organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The
*E-mail: sb1@iiserkol.ac.in.
Subhajit Bandyopadhyay: 0000-0001-7668-0274
residue
on
chromatography
with
hex-
†If an author’s address is different than the one given in the
affiliation line, this information may be included here.
ane/dichloromethane (1:4, v/v) yielded compound 2a
1
(0.667 g, 55%) as a solid yellow powder. H NMR (400
MHz, CDCl3): 2.60 (s, 3H, CH3), 2.82 (s, 3H, CH3), 7.44 (t,
J= 7.64 Hz, 1H, ArH), 7.67 (t, J= 8.4 Hz, 1H, ArH), 7.73 (d,
J= 7.64 Hz, 1H, ArH), 7.95 (d, J= 9.16 Hz, 1H, ArH), 8.35 (s,
1H, ArH). 13C{1H} NMR (100 MHz, CDCl3): 25.2, 28.9, 125.3,
126.4, 128.0, 128.1, 130.7, 131.5, 138.1, 147.7, 157.3, 199.5. FT-IR
(KBr, cm-1): 1680. λabs (nm) in water/MeCN (1:3, v/v) 280.
HRMS (m/z): Calcd. for C12H12NO+ or [M+H]+ 186.0913,
found 186.0914.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
The authors declare no competing financial interest.
This work is funded by a Ministry of Human Resources grant
for
Swachhata
(Clean
India)
Action
Plan
2-aminoquinoline-3-carbonitrile (3a). Compound 3 (1.20 g,
6.14 mmol) was dissolved in dichloromethane (5 mL) and
mixed with 50 mL of methanol. Then solution of sodium
sulfide (2.40 g, 30.74 mmol) in water (4 mL) was mixed
(ICSR/SAP/2018/SN-1) to S.B. B.R. and S.G. are supported by
INSPIRE and CSIR fellowship respectively. The help of Mr.
Sayanur Rahaman, Mr. Sudipta Halder and Dr. Sudipta Bar
for the biological and imaging experiments is greatly appre-
ciated. Mr. Supriya Sasmal is gratefully acknowledged for
o
dropwise to the previous mixture at 25 C. After stirring 6
hr, the solvent of the reaction mixture was removed in
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The Journal of Organic Chemistry
istry and Neurobiology. Neurochem. Int. 2008
52, 155–165.
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crystallography and Ms. Tiasha Saha Roy for her help with
data analysis.
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