Performance comparison of two cascade reaction models in fluorescence off-on detection of hydrogen sulfide

Article abstract of DOI:10.1039/c4ra13086c

Comparative studies on the performances of two cascade reaction based fluorescent H₂S probes are reported. These probes were also designed to address the solubility issues of existing probes. The Reso-N₃ probe favors the H₂S mediated azide-to-amine reduction followed by a cyclization to release the resorufin fluorophore. Reso-Br undergoes a bromide-to-thiol nucleophilic substitution followed by a similar cyclization releasing the same fluorophore. Reso-N₃ exhibited lower background fluorescence and better H₂S sensing behavior in water compared to Reso-Br. Reso-Br underwent hydrolysis in aqueous buffer conditions (pH = 7.4) while, Reso-N₃ was quite stable. Reso-N₃ displayed high selectivity and sensitivity towards H₂S. Live cell imaging of the species by the probe was also established. This journal is

Full text of DOI:10.1039/c4ra13086c

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Performance comparison of two cascade reaction
models in fluorescence off–on detection of
hydrogen sulfide†
Cite this: RSC Adv., 2015, 5, 1438
*
Tanmoy Saha, Dnyaneshwar Kand and Pinaki Talukdar
Comparative studies on the performances of two cascade reaction based fluorescent H2S probes are
reported. These probes were also designed to address the solubility issues of existing probes. The Reso-
N3 probe favors the H2S mediated azide-to-amine reduction followed by a cyclization to release the
resorufin fluorophore. Reso-Br undergoes a bromide-to-thiol nucleophilic substitution followed by a
similar cyclization releasing the same fluorophore. Reso-N3 exhibited lower background fluorescence
and better H2S sensing behavior in water compared to Reso-Br. Reso-Br underwent hydrolysis in
aqueous buffer conditions (pH ¼ 7.4) while, Reso-N3 was quite stable. Reso-N3 displayed high selectivity
and sensitivity towards H2S. Live cell imaging of the species by the probe was also established.
Received 24th October 2014
Accepted 24th November 2014
DOI: 10.1039/c4ra13086c
www.rsc.org/advances
cardiovascular,
reperfusion injury
and inammatory
Introduction
diseases.10,11 Overexpression of H2S producing enzymes12,13 is
related to various diseases e.g. Alzheimer's disease,14 Down's
syndrome,15 diabetes,16 and liver cirrhosis.17
Hydrogen sulde (H2S) is a colorless, toxic, inammable,
corrosive gas, and it is produced mainly from geological and
microbial activities.1,2 The species has a characteristic smell of
rotten eggs and its detectability threshold by a healthy human
nose ranges from 0.005–0.3 ppm. This gaseous species when
exposed or inhaled in excess, can cause respiratory failure, loss
of consciousness, sudden cardiac death, hepatic2 and olfactory
paralysis. Endogenously, H2S can be produced during metabo-
lism of cysteine (Cys) catalyzed by two pyridoxal 50-phosphate
dependent enzymes i.e. cystathionine-b-synthase (CBS),3 and
cystathionine-g-layse (CSE)4 and one pyridoxal 50-phosphate
independent enzyme i.e. 3-Mercaptopyruvate sulfurtransferase
(3-MST).5 3-MST is a mitochondrial and cytosolic enzyme (found
in brain tissues)6 whereas CBS and CSE are exclusively cytosolic
(found in liver and brain tissues). Although, H2S has been
viewed primarily as a noxious chemical species, recent studies
have established its signicance as the third most essential
gasotransmitter (aer NO and CO)6–8 which controls various
physiological processes in nervous, respiratory, endocrine and
immune systems.6,7 It also dilates blood vessels and lowers blood
pressure by acting as a smooth muscle reluctant and K-ATP
channel opener. Furthermore, the effects of H2S in gastrointes-
tinal insulin signalling and as a regulator of energy production
in mammalian cells under stress condition are also known.9 In
recent times, H2S releasing prodrugs are used for treatments of
Because of the increasing interest in H2S research, proper
understanding of its production, mode of action and
consumption is very essential. However, its optimal level in
biological systems is debatable, as the concentration varies in
the range over 105 orders.18 Very short half-life of the species is a
critical limitation for its accurate determination. Therefore,
reliable and accurate determination methods for H2S sensing in
biological samples have appeared as burgeoning interests to
acquire better knowledge of physiological and pathological
functions. Several uorescent probes were reported for in vitro
as well as in vivo detection of the species. These probes were
designed to accomplish rapid response, selectivity and sensi-
tivity towards H2S. These strategies include: (a) H2S mediated
reduction of azide to amine,19–29 (b) H2S trapping by nucleo-
philic addition,30–34 (c) copper sulde precipitation,35–37 and (d)
thiolysis of dinitrophenyl ether.38 Among these, the azide
reduction strategy has been extensively applied in numerous
uorescent probes. H2S (pKa ¼ 6.9) is a better nucleophile32
compared to other biothiols such as Cysteine (Cys, pKa ¼ 8.4)
and glutathione (GSH, pKa ¼ 9.7).39 Moreover, H2S also exhibits
dual nucleophilic character which discriminates it from other
biothiols.40 These features of H2S were helpful in the design of
various cascade reaction based probes. In 2011, Xian and co-
workers reported uorescence off–on probes based on cascade
reaction demonstrating the selective and sensitive detection of
H2S.33,34
Department of Chemistry, Indian Institute of Science Education and Research, Pune,
India. E-mail: ptalukdar@iiserpune.ac.in; Fax: +91 20 2590 8186; Tel: +91 20 2590
8098
The rst strategy incorporating H2S mediated azide-to-amine
reduction, in cascade reaction model was reported by Han et al.41
Based on their design, the probe 1 (Fig. 1A) upon reduction of
† Electronic supplementary information (ESI) available: Additional experimental
procedures, uorescence spectra. See DOI: 10.1039/c4ra13086c
1438 | RSC Adv., 2015, 5, 1438–1446
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the azide group facilitated an intramolecular cyclization to inuenced by the nature of uorophore. Therefore, introduc-
release a uorescent coumarin derivative (Fig. 1B, route a). tion of an identical uorophore was proposed to minimize
However, use of organic co-solvent (acetonitrile 20% v/v), long structural differences and establish a better comparison
response time (20–40 min), low sensitivity, high detection limit between two aforementioned reaction mechanisms. Herein, we
(100 mM) and blue emission of the coumarin uorophore limits report design, synthesis and H2S sensing abilities of two probes
its further biological applications. Guo and co-workers reported Reso-N3 and Reso-Br (Fig. 1A) to carry out a performance
an alternate cascade reaction based probe 2 (Fig. 1A) which comparison of two cascade reaction models. Resorun was
works via nucleophilic substitution of iodide by H2S, leading to a selected as a uorophore owing to its excellent chromogenic
cyclization and release of uorescein (Fig. 1B, route b). This and uorogenic properties.44–51 Photophysical properties in the
strategy partially addressed the limitation related to blue-shied visible region, water solubility and excellent biocompatibility
emission in the probe 1.42 Although, the probe 2 displayed a have already established the signicance of resorun in the
faster response time (5 min) and a lower detection limit (0.1 mM) development of uorescence probes with in vitro and in vivo
compared to 1, the sensitivity of the probe was not signicant applications. Quenched uorescence states for these probes
(50-fold) compared to other existing probes. The use of high was rationalized easily because, esterication of the hydroxyl
concentration of a surfactant (20 mM cetrimonium bromide group at C7-position of resorun is known to provide non-
[CTAB]) in sensing studies limits its applicability in biological uorescent species.47
systems.43
The use of dissimilar uorophores in these probes also
impedes the comparison of two cascade reaction mechanisms
Results and discussion
Addressing water solubility of Reso-N3 and Reso-Br
because; physicochemical property of the probe is largely
To ensure the water solubility and cell membrane permeation
of the designed probes, Reso-N3 and Reso-Br, respective
C log P (the logarithm of the partition coefficient between
water and 1-octanol) values were predicted using ChemBio-
Draw 14.0 program and values were also compared with those
of probes 1 and 2. The program provided C log P ¼ 4.54, 4.21,
4.93 and 6.48 for Reso-N3, Reso-Br, 1 and 2, respectively
(Fig. S1†). Considering the Lipinski's rule of ve,52,53 the Reso-N3
and Reso-Br probes were expected to be more appropriate
for sensing H2S in the water and better cell permeable over
probes 1 and 2. The bromo leaving group in Reso-Br instead of
an iodo was considered because the manipulation provides
improved C log P value compared to the corresponding iodo
derivative (Fig. S1†).
Synthesis of Reso-N3 and Reso-Br
Synthesis of Reso-N3 and Reso-Br were carried out from acids 6
and 9, respectively. Prior to the synthesis of Reso-N3, ester 5 was
converted to the acid 6 via the sequential benzylic bromination,
Fig. 1 Structures of the reported probes 1–2 and proposed probes
Reso-N3 and Reso-Br (A). Cascade reaction based strategies for H2S
sensing (B).
Scheme 1 Synthesis of Reso-N3 (A) and Reso-Br (B).
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nucleophilic substitution of bromide by azide and ester and t1/2 ¼ 2.4 min (Fig. 3B and S5B†). Sensing processes were
hydrolysis.54,55 Acid 6 was then converted to acid chloride which completed within 6 min and 10 min for Reso-N3 and Reso-Br,
was further treated with 7 to furnish Reso-N3 (54%) as red solid. respectively. These experiments conrm the faster response of the
Pthalide 8 was rst converted to acid 9 following a reported probe Reso-N3 compared to Reso-Br in water. Fluorescence
protocol.56 Compound 9 was treated further with SOCl2 followed kinetics experiments also suggest the stability and inertness of
by addition of resorun 7 to obtain Reso-Br as red solid (51%) each probe, Reso-N3 and Reso-Br towards other biothiols (Fig. 3),
(Scheme 1).
Cys (5 mM) and GSH (5 mM).
Quantitative response of Reso-N3 and Reso-Br towards H2S in
water
Comparison of photophysical properties between Reso-N3 and
Reso-Br
Responses of probes Reso-N3 (10 mM) and Reso-Br (10 mM)
towards H2S were evaluated by UV-visible spectroscopy. Na2S
(0–5 mM) was added into the solution of each probe (10 mM),
and UV-visible spectra were recorded aer 10 min of each
addition. For Reso-N3 probe, the changes of 318, 344 and
419 nm bands were less prominent but, the formation of a
572 nm band indicated the formation resorun 7 during the
sensing process (Fig. 4A and S2†). For Reso-Br, sharp decrease
in absorption bands 364 and 443 nm was observed (Fig. 4B).
The sensing of H2S by Reso-Br also resulted in the appearance
of a strong band at 572 nm that corresponds to the formation of
resorun 7 (Fig. S2†).
Aer synthesizing Reso-N3 and Reso-Br, their background
uorescence properties were compared. Based on dynamic light
scattering (DLS) studies, both probes (0–100 mM) were
conrmed to be soluble in water. Therefore, photophysical
properties and H2S sensing properties of these probes were
determined in water. UV-visible spectrum of Reso-N3 (10 mM) in
water (Fig. 2A) exhibited weaker absorption signals at 318 nm
(3 ¼ 2400 Mꢀ1 cmꢀ1), 344 nm (3 ¼ 2000 Mꢀ1 cmꢀ1) and 419 nm
(3 ¼ 1100 Mꢀ1 cmꢀ1). On the other hand, Reso-Br (10 mM)
exhibited absorption maxima at lmax ¼ 364 nm (3 ¼ 9000
Mꢀ1 cmꢀ1) and a less intense peak at 443 nm. When uores-
cence intensities were monitored at l ¼ 585 nm (lex ¼ 540 nm),
probe Reso-N3 displayed 0.17 times lower background
uorescence compared to probe Reso-Br (Fig. 2B). Therefore,
Reso-N3 was expected to exhibit better off–on response during
sensing of H2S compared to Reso-Br because, both probes are
identical uorophore releasers.
Kinetics of H2S sensing by Reso-N3 and Reso-Br in water
In the next stage, response times of probes Reso-N3 (10 mM) and
Reso-Br (10 mM) during H2S sensing were determined under
pseudo rst order reaction conditions. Emission spectra (lex
¼
540 nm) were acquired at denite time intervals for each probe
aer addition of Na2S (5 mM) and uorescence intensities at
585 nm were plotted against time. For Reso-N3, a pseudo rst
order reaction kinetics was observed with rate constant,
k ¼ 0.6 minꢀ1 and half-life t1/2 ¼ 1.2 min (Fig. 3A and S5A†). A
Fig. 3 Fluorescence kinetics of probes Reso-N3 (A) and Reso-Br
similar kinetic prole was observed for Reso-Br with k ¼ 0.28 minꢀ1 (B) towards H2S, Cys and GSH in water, recorded at 585 nm (lex
¼
540 nm).
Fig. 2 UV-visible spectra of Reso-N3 and Reso-Br (10 mM each) in
water (A); comparison of fluorescence intensity at 585 nm (lex ¼ 540 Fig. 4 UV-Visible titration spectra of Reso-N3 (A) and Reso-Br (B) with
nm) of both probes (10 mM) in water (B).
increasing concentration of Na2S in water.
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Next stage, uorometric titrations were carried out to esti- uorescence enhancements were observed in the case of any
mate quantitative responses of probes Reso-N3 (Fig. 5A) and analyte (Fig. 7 and S8†). When Na2S was added further to each of
Reso-Br (Fig. 5B) towards H2S. Sharp enhancements in uo- aforementioned solutions, the observed uorescence enhance-
rescence intensities at 585 nm (lex ¼ 540 nm) were observed ments were similar to that of only Na2S addition. This data
when increasing concentration of Na2S (0 to 5 mM) was added conrm the selectivity of each probe towards H2S. Addition of a
to each probe in the water. Reso-N3 exhibited 614-fold uores- thiol containing amino acid (either of Cys, GSH and Hcy) to
cence enhancement, which is signicantly better than 414-fold each probe did not show any signicant increment of uores-
jump determined for Reso-Br (Fig. S4†). The limit of detection cence. But, further addition of Na2S to these solutions did not
(LOD) for H2S sensing by Reso-N3 was 440 nM (Fig. S7A and exhibit the expected uorescence enhancement. These results
Table S1†). The outcome was slightly better than the LOD ¼ were surprising considering the established selectivity of two
550 nM calculated for Reso-Br (Fig. S7B and Table S2†).
cascade mechanisms towards H2S. A plausible explanation of
Encouraged by fast response time and low LOD values of the unanticipated results can be attributed to the Michael
probes Reso-N3 and Reso-Br, water sample analyses were con- addition of aliphatic thiols on resorun leading to the forma-
ducted to detect trace amounts of H2S (or S2ꢀ).57 As expected, no tion of adducts that are nonuorescent.58
H2S was detected in the collected samples of tap water. When
The reduction-cyclization and nucleophilic substitution-
these water samples were spiked with a measured quantity of cyclization mechanisms for detection of H2S by Reso-N3 and
Na2S, quantitative responses were observed for both Reso-N3 Reso-Br were further studied by 1H NMR titrations. Upon
(Fig. 6A) and Reso-Br (Fig. 6B).
stepwise addition of Na2S to the solutions of probes, decrease of
High sensitivity of probes Reso-N3 and Reso-Br during H2S peaks corresponds to Reso-N3 (Fig. S13†) and Reso-Br
detection led us to evaluate their specicity to the analyte in an (Fig. S14†) with a simultaneous appearance of new peaks cor-
isolated and competitive environment with other biorelevant responding to resorun 7 was observed in both the cases.
species. Each probe (10 mM) was treated with an analyte (NaF,
NaCl, NaBr, NaI, Na2SO3, Na2SO4, Na2S2O3, NaSCN, NaNO2,
H2S sensing properties of Reso-N3 under physiological
NaNO3, Cys, GSH and Hcy; 5 mM each), and uorescence
conditions
spectra were recorded aer 10 min of addition. No signicant
Further assessments of these two probes were carried out under
physiological conditions. Probe Reso-Br was not stable in varied
aqueous buffer (pH ¼ 7.4) conditions and resulted in the rapid
release of uorophore 7 with the time (Fig. S9 and S10†). The
instability of the Reso-Br probe can be corroborated to its
solvolysis under the weakly nucleophilic media59,60 followed by
cyclization to release resorun 7 (Scheme S1†). This outcome is
not unanticipated for a benzyl bromide system with carboxylic
Fig. 5 Fluorescence titration spectra of Reso-N3 (A) and Reso-Br (B)
with Na2S in water. In each experiment, 10 mM of probe was treated
with Na2S (0–5 mM) and fluorescence spectrum (lex ¼ 540 nm) was
recorded after 10 min of addition.
Fig. 7 Relative fluorescence intensity enhancements at 585 nm for, (A)
Reso-N3 (10 mM) and (B) Reso-Br (10 mM) towards Na2S (5 mM) in
Fig.
6 Bar diagram, representing relative fluorescence intensity water. Front row: change in intensities in the presence of various
enhancements at 585 nm (lex ¼ 540 nm) for Reso-N3 (A) and Reso-Br analytes (5 mM); rear row: Na2S was added in the presence of
(B) towards various tap water samples having different amount of Na2S. respective analyte.
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buffer (5 mM, pH ¼ 7.4), addition of Na2S results in a gradual
increase in uorescence intensity (Fig. 9B). When uorescence
intensity at 585 nm was plotted against the concentration of
added Na2S an excellent linear correlation (R2 ¼ 0.98095) was
obtained (Fig. S11†). A detection limit of 4.15 mM was calculated
based on signal to noise ratio, S/N ¼ 3 (Table S3†).
Further, the selectivity of Reso-N3 towards H2S over other
analytes was evaluated under comparable conditions. When the
probe was treated with ranges of analytes (NaF, NaCl, NaBr, NaI,
Na2SO3, Na2SO4, Na2S2O3, NaSCN, NaNO2, NaNO3, Ala, Ser, Cys,
GSH or Hcy) in phosphate buffer (5 mM, pH ¼ 7.4), no signif-
icant increment in uorescence intensity was observed
(Fig. 10A, front row). On the other hand, strong enhancement in
uorescence intensity was observed when Na2S was added to
the probe (Fig. 10A, the lemost column of the rear row).
Addition of Na2S to solutions of the probe and competing
analyte resulted in strong uorescence intensity enhancements
(Fig. 10A, rear row). The response of Na2S towards the probe in
the presence of Cys improved than that observed in pure water.
Cuvette images taken under ambient light suggests a change in
color from yellow to pink upon addition of only Na2S to the
probe Reso-N3 (Fig. 10B). Similarly, the appearance of orange
uorescent for the probe in the presence of Na2S under the
hand held UV-lamp (lex ¼ 365 nm) also corroborates to the
selectivity of probe towards H2S over other analytes (Fig. 10C).
Fig. 8 Fluorescence spectra of Reso-N3 (10 mM) upon addition of
Na2S (5 mM) in phosphate buffer (5 mM, pH ¼ 7.4) recorded with
increasing time (A) and corresponding fluorescence kinetics diagram
(B) generated by plotting intensity at l ¼ 585 nm (lex ¼ 540 nm) versus
time. Stability of probe Reso-N3 in phosphate buffer (5 mM, pH ¼ 7.4),
in absence and in presence of Cys (5 mM), GSH (5 mM) are also
presented.
ester moiety (an electron withdrawing group) at the ortho-
position of aryl ring. Reso-N3 in contrast, was stable under
aqueous buffer (pH ¼ 7.4) conditions (Fig. 8B and S9†).
The probe Reso-N3 (10 mM) upon reaction with Na2S (5 mM) in
phosphate buffer (5 mM, pH ¼ 7.4) exhibited a pseudo rst
order reaction kinetics with rate constant, k ¼ 0.13 minꢀ1 and
t1/2 ¼ 5.33 min (Fig. S6†). The sensing process completes within
18 min aer addition of H2S (Fig. 8A). The uorescence
enhancement for Reso-N3 probe was limited to only H2S and
addition of either GSH or Cys provided no signicant uores-
cence enhancement (Fig. 8B).
Live cell imaging of intracellular H2S by Reso-N3
To evaluate the ability of Reso-N3 for detecting H2S in the bio-
logical systems, live cell imaging studies were carried out using
The quantitative response of Reso-N3 towards H2S was eval-
uated in buffer (5 mM, pH ¼ 7.4) by UV-visible spectroscopy and
uorometric titrations. A gradual decrease in absorption
maxima at 318 nm with sharp increment at 572 nm peak was
observed with stepwise addition of Na2S (Fig. 9A). When uo-
rometric titration was carried out for Reso-N3 in phosphate
Fig. 10 Relative fluorescence intensity enhancements at 585 nm for
Reso-N3 (10 mM) towards Na2S (5 mM) and other analytes in phosphate
buffer (A). Front row: change in intensities in the presence of various
analytes (5 mM); rear row: Na2S was added in the presence of
Fig. 9 UV-visible (A) and fluorescence spectra (B) of Reso-N3 (10 mM) respective analyte. Cuvette images of Reso-N3 (50 mM) in phosphate
with increasing concentration of Na2S (0 to 5 mM) in phosphate buffer buffer (5 mM, pH ¼ 7.4) in presence of various analytes taken under
(5 mM, pH ¼ 7.4).
ambient light (B) and under hand held UV lamp, lex ¼ 365 nm (C).
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by H2S followed by cyclization to release the same uorophore.
The C log P guideline based on Lipinski's rule of ve was also
considered in the design, to favour water solubility and cell
membrane permeation of these molecules. Reso-N3 was
prepared from methyl 2-methylbenzoate in three steps with
overall 27% yield. Reso-Br probe was synthesized from pthalide
in two steps with overall 25% yield. Photophysical properties of
these probes in water provided a lower background uorescence
for Reso-N3 compared to Reso-Br. H2S sensing by Reso-N3 under
the pseudo rst order reaction kinetics provided rate constant,
k ¼ 0.6 minꢀ1 and t1/2 ¼ 1.2 min. Response of the probe Reso-Br
towards H2S under similar conditions was signicantly faster
with k ¼ 0.28 minꢀ1 and half life t1/2 ¼ 2.4 min. Reso-N3 dis-
played 614-fold off–on uorescence with detection limit ¼
440 nM. For Reso-Br, considerably reduced sensitivity of 414-
Fig. 11 Live cell imaging of HeLa cell: DIC (A), fluorescence (B) and
overlay (C) images of cells, incubated with Reso-N3 (10 mM) for 15 min.
(D–F) are respective DIC, fluorescence and overlay image of HeLa
cells, preincubated with Reso-N3 (10 mM) followed by incubation with
Na2S (1 mM) for 15 min.
1
fold and detection limit ¼ 580 nM were determined. H NMR
titration experiments were carried out to prove the sensing
mechanisms for each probe. Moreover under physiological
conditions (in aqueous buffer, pH ¼ 7.4) Reso-Br was found to
be unstable, possibly due to its solvolysis under the weakly
nucleophilic media. Reso-N3 on the other hand was stable
under the comparable conditions. During H2S sensing under
these conditions, the Reso-N3 probe displayed k ¼ 0.13 minꢀ1
,
t1/2 ¼ 5.33 min and sensitivity of 110-fold. Live cell imaging
studies demonstrated applicability of Reso-N3 in the detection
of intracellular H2S.
Experimental section
I. General methods
Fig. 12 Fluorescence images of HeLa cell acquired at different time
intervals (0, 5, 10, 15 and 20 min) after incubating with Na2S (A–E). Bar
diagram was plotted by the average pixel intensity of selected ROI's
versus time (F).
All reactions were conducted under a nitrogen atmosphere. All
the chemicals were purchased from commercial sources and
used as received unless stated otherwise. Solvents: petroleum
ether and ethyl acetate (EtOAc) were distilled prior to thin layer
and column chromatography. Dichloromethane (DCM) was
pre-dried over calcium hydride and then distilled under
vacuum. Column chromatography was performed on Merck
silica gel (100–200 mesh). TLC was carried out with E. Merck
silica gel 60-F254 plates.
HeLa cells. When cells were incubated with only Reso-N3
(10 mM, in PBS buffer, pH ¼ 7.4) for 15 min, very low uores-
cence was observed. The weak uorescence is attributed to the
low levels of intracellular H2S. Strong uorescence was observed
when cells, preincubated with probe, were incubated with Na2S
(1 mM, in PBS buffer, pH ¼ 7.4) for 15 min (Fig. 11).
Finally, the quantitative response of probe Reso-N3 towards
intracellular H2S was studied. Quantication was done by
recording the pixel intensity of the selected region of interest
(ROI) from images acquired at different time intervals (0, 5, 10,
15, 20 min), aer incubation with Na2S. When pixel intensity
values were plotted in a bar diagram against time, an increase in
intensity was observed up to 20 min of incubation (Fig. 12).
The gradual increment in intensity corresponds to an increase
in intracellular H2S concentration.
II. Physical measurements
The 1H and 13C spectra were recorded on 400 MHz Jeol ECS-400
(or 100 MHz for 13C) spectrometers using either residual
solvent signals as an internal reference or from internal tetra-
methylsilane on the d scale (CDCl3 dH, 7.24 ppm, dC 77.0 ppm).
The chemical shis (d) were reported in ppm and coupling
constants (J) in Hz. The following abbreviations were used: m
(multiplet), s (singlet), br s (broad singlet), d (doublet), t (triplet)
dd (doublet of doublet). High-resolution mass spectra were
obtained from MicroMass ESI-TOF MS spectrometer. FT-IR
spectra were obtained using NICOLET 6700 FT-IR spectropho-
Conclusions
In summary, we have developed two uorescent probes Reso-N3 tometer as KBr disc and reported in cmꢀ1. Melting points were
and Reso-Br for sensing of H2S under physiological conditions. measured using a VEEGO Melting point apparatus. All melting
Reso-N3 undergoes azide-to-amine reduction by H2S followed by points were measured in open glass capillary and values are
cyclization to release resorun uorophore. On the other hand, uncorrected. Absorption spectra were recorded on a Perki-
Reso-Br probe facilitates nucleophilic substitution of bromide nElmer, Lambda 45 UV-Vis spectrophotometer. Steady State
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uorescence experiments were carried out in a micro uores- the suspension of resorun 7 (100 mg, 0.425 mmol) in dry
cence cuvette (Hellma, path length 1.0 cm) on a Fluoromax dichloromethane (5 mL) and the reaction mixture was stirred at
4 instrument (Horiba Jobin Yvon). Cell images were taken in room temperature for 16 h. At completion, the reaction mixture
35 mm (diameter) dishes. The media (DMEM) and PBS buffer was dried under reduce pressure and then subjected to column
were purchased from commercial sources. Fluorescence images chromatography over silica gel (eluent: 18% EtOAc in petroleum
were taken using Olympus Inverted IX81 equipped with ether) to obtain pure Reso-N3 (76 mg, 54%) as dark red solid.
Hamamatsu Orca R2 microscope. ChemBio Draw Ultra and M.p.: 168–169 ꢁC (decomposed); IR (KBr): nmax cmꢀ1 3450, 3045,
Image J soware were used for drawing structure and for pro- 2103, 1739, 1614, 1509, 1438, 1242, 1033, 862; 1H NMR
cessing cell image respectively.
(400 MHz, CDCl3): d 8.28 (dd, J ¼ 7.8, 1.3 Hz, 1H), 7.87
(d, J ¼ 8.6 Hz, 1H), 7.70 (td, J ¼ 7.6, 1.3 Hz, 1H), 7.61 (d, J ¼ 6.9
Hz, 1H), 7.57–7.49 (m, 1H), 7.46 (d, J ¼ 9.9 Hz, 1H), 7.31–7.21
(m, 3H), 6.88 (dd, J ¼ 9.8, 2.0 Hz, 1H), 6.35 (s, 1H), 4.88 (s, 2H).
13C NMR (100 MHz, CDCl3): d 186.78, 164.64, 153.87, 149.72,
148.88, 144.87, 139.07, 135.67, 135.27, 134.54, 132.26, 131.86,
III. Synthesis of probes Reso-N3 and Reso-Br
Synthesis of methyl 2-(azidomethyl)benzoic acid
(C8H7N3O2)54,55
6
Step A. To the solution of 2-methylbenzoate 5 (1.00 g, 131.72, 130.66, 128.94, 127.34, 119.89, 110.40, 107.74, 77.80,
6.65 mmol) in dry CCl4 (10 mL) were added N-bromosuccini- 77.48, 77.16, 53.56. HRMS (ESI): calc. for C20H12N4O4Na+:
mide (1.30 g, 7.32 mmol), and benzoyl peroxide (0.032 g, 395.0756 [M + Na]+; found: 395.0757.
0.133 mmol). The reaction mixture was stirred at 80 ꢁC for 8 h.
Synthesis of 2-(bromomethyl) benzoic acid 9 (C8H7BrO2).56
Subsequently, the reaction mixture was cooled to room To a 25 mL round bottom ask was added pthalide 8 (500 mg,
temperature and placed in an ice-water bath to allow the 3.37 mmol) and dissolved in glacial acetic acid (5 mL). To the
precipitation of succinimide. The reaction mixture was diluted resulting solution was added drop wise a solution of 33% HBr-
with dichloromethane (50 mL) and ltered. The ltrate was AcOH (10 mL) and stirred at room temperature for 2 h. Subse-
ꢁ
washed with saturated NH4Cl (30 mL). The organic layer was quently, the reaction mixture was heated at 70 C for 1.5 h fol-
dried over Na2SO4 and concentrated under reduced pressure to lowed by stirring at room temperature for overnight.
afford 2-(bromomethyl) benzoate as yellow oil. The crude The reaction mixture was then poured into ice-water (100 mL)
product was used for the next reaction without further and the resulting white precipitate was ltered. The residue was
purication.
washed with ice-water and dried under reduced pressure to
Step B. To the solution of 2-(bromomethyl)benzoate in DMF obtain compound 9 (400 mg, 50%) as white solid. The crude
(10 mL) was added sodium azide (0.570 g, 8.72 mmol), and the product was used for the next reaction without further puri-
reaction mixture was stirred at 70 ꢁC for 16 h. At the completion cation. M.p. 157–158 ꢁC; 1H NMR (400 MHz, CD3OD): d 7.96
of the reaction, the solution was cooled to room temperature. (d, J ¼ 7.4 Hz, 1H), 7.55–7.45 (m, 2H), 7.44–7.34 (m, 1H), 5.02
To the reaction mixture was added water (30 mL) and the (s, 2H). 13C NMR (100 MHz, CD3OD): d 169.57, 140.66, 133.24,
product extracted with ethyl acetate (3 ꢂ 30 mL). The combined 132.60, 132.20, 129.31, 49.00, 31.75.
organic layer was washed with brine (40 mL), dried over
Synthesis of 3-oxo-3H-phenoxazin-7-yl 2-(bromomethyl)
Na2SO4, and concentrated under reduced pressure to obtain benzoate, Reso-Br (C20H12BrNO4). In a oven dried 25 mL round
methyl 2-(azidomethyl)benzoate (0.665 g) as yellow oil. The bottomed ask were added 2-(bromomethyl)benzoic acid 9
crude product was used for the next reaction without further (100 mg, 0.29 mmol) and SOCl2. The reaction mixture was
purication.
reuxed under inert atmosphere for 6 h. The reaction mixture
Step C. In a 50 mL round bottomed ask, was placed methyl was cooled to room temperature and concentrated under
2-(azidomethyl)benzoate and 20 mL of 2 N NaOH/MeOH (1 : 1) reduced pressure to obtain crude 2-(bromomethyl)benzoic acid
was added. Aer 1 h stirring at room temperature the reaction chloride as yellowish oil. The crude oil was added dropwise to
mixture was acidied with 1 N HCl (upto pH ¼ 1) and product the suspension of resorun 7 (100 mg, 0.425 mmol) in dry
was extracted with of chloroform (3 ꢂ 30 mL). The combined dichloromethane (5 mL) and the reaction mixture was stirred at
organic layer was dried over Na2SO4 and concentrated under room temperature for 16 h. At completion, the reaction mixture
reduced pressure to afford 2-(azidomethyl)benzoic acid 6 (0.67 was dried under reduce pressure and then subjected to column
1
g, 52%) as white solid. H NMR (400 MHz, CD3OD): d 8.03 (d, chromatography over silica gel (Eluent: 18% EtOAc in petro-
J ¼ 7.8 Hz, 1H), 7.56 (dd, J ¼ 11.8, 4.3 Hz, 1H), 7.52–7.38 (m, 2H), leum ether) to obtain pure Reso-Br (80 mg, 51%) as dark red
4.79 (s, 2H). 13C NMR (100 MHz, CD3OD): d 169.92, 138.47, solid. M.p.: 180–181 ꢁC (decomposed); IR (KBr): nmax cmꢀ1 3432,
1
133.59, 132.33, 131.33, 131.01, 129.27, 53.91.
3043, 1730, 1630, 1558, 1312, 1241, 1120, 1027, 870. H NMR
Synthesis of 3-oxo-3H-phenoxazin-7-yl 2-(azidomethyl) (400 MHz, CDCl3): d 8.21 (dd, J ¼ 7.8, 1.3 Hz, 1H), 7.87 (d, J ¼ 8.5
benzoate, Reso-N3 (C20H12N4O4). In a oven dried 25 mL round Hz, 1H), 7.63 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.57–7.48 (m, 2H), 7.46 (d,
bottomed ask were added 2-(azidomethyl)benzoic acid 6 J ¼ 9.7 Hz, 1H), 7.35–7.28 (m, 2H), 6.88 (dd, J ¼ 9.8, 2.0 Hz, 1H),
(100 mg, 0.29 mmol) and SOCl2. The reaction mixture was 6.36 (d, J ¼ 1.9 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 186.7,
reuxed under inert atmosphere for 6 h. The reaction mixture 164.5, 149.7, 148.7, 144.8, 140.7, 140.3, 135.6, 135.3, 134.3,
was cooled to room temperature and concentrated under 132.4, 132.2, 131.7, 129.2, 127.8, 119.9, 110.4, 107.7, 31.64.
reduced pressure to obtain crude 2-(azidomethyl)benzoic acid HRMS (ESI): calc. for C20H12BrNO4H+: 410.0028 [M + H]+; found:
chloride as yellowish oil. The crude oil was added dropwise to 410.0052.
1444 | RSC Adv., 2015, 5, 1438–1446
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23 G. Zhou, H. Wang, Y. Ma and X. Chen, Tetrahedron, 2013, 69,
867–870.
Acknowledgements
24 S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han and B. R. Cho,
Chem. Commun., 2012, 48, 8395–8397.
25 M.-Y. Wu, K. Li, J.-T. Hou, Z. Huang and X.-Q. Yu, Org.
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We acknowledge IISER Pune and DST-SERB (Grant no. SR/S1/
OC-65/2012) for nancial supports. T.S. thanks University
Grant Commission (UGC) and D.K. thanks Council for Scientic
and Industrial Research (CSIR) for research fellowships.
26 Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem. Commun.,
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27 R. Wang, F. Yu, L. Chen, H. Chen, L. Wang and W. Zhang,
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