Hydrogen sulfide sensing using an aurone-based fluorescent probe

Article abstract of DOI:10.1039/d0ra08802a

Hydrogen sulfide detection and sensing is an area of interest from both an environmental and a biological perspective. While many methods are currently available, the most sensitive and biologically applicable ones are fluorescence based. In general, these fluorescent probes are based upon large, high-molecular weight, well-characterized fluorescent scaffolds that are synthetically demanding to prepare and difficult to tune and modify. In this study, we have reported a new reduction-based, rationally designed and synthesized turn-on fluorescent probe (Z)-2-(4′-azidobenzylidene)-5-fluorobenzofuran-3(2H)-one (6g) utilizing a low molecular weight aurone fluorophore. During these studies, the modular nature of the synthesis was used to quickly overcome problems with solubility, overlap of excitation of the probe and reduced product, and rate of reaction, resulting in a final compound that is efficient and sensitive for the detection of hydrogen sulfide. The limitation of slow reaction and the reduced fluorescence in a biologically relevent medium was solved by employing cationic surfactant cetyltrimethyl ammonium bromide (CTAB). The probe features a high fluorescence enhancement, fast response (10-30 min), and good sensitivity (1 μm) and selectivity for hydrogen sulfide. This journal is

Full text of DOI:10.1039/d0ra08802a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Hydrogen sulfide sensing using an aurone-based
fluorescent probe†
Cite this: RSC Adv., 2020, 10, 45180
ab
ab
Arjun Kafle,a Shrijana Bhattarai,a Justin M. Miller
and Scott T. Handy
*
*
Hydrogen sulfide detection and sensing is an area of interest from both an environmental and a biological
perspective. While many methods are currently available, the most sensitive and biologically applicable ones
are fluorescence based. In general, these fluorescent probes are based upon large, high-molecular weight,
well-characterized fluorescent scaffolds that are synthetically demanding to prepare and difficult to tune
and modify. In this study, we have reported a new reduction-based, rationally designed and synthesized
turn-on fluorescent probe (Z)-2-(40-azidobenzylidene)-5-fluorobenzofuran-3(2H)-one (6g) utilizing
a low molecular weight aurone fluorophore. During these studies, the modular nature of the synthesis
was used to quickly overcome problems with solubility, overlap of excitation of the probe and reduced
product, and rate of reaction, resulting in a final compound that is efficient and sensitive for the
detection of hydrogen sulfide. The limitation of slow reaction and the reduced fluorescence in
a biologically relevent medium was solved by employing cationic surfactant cetyltrimethyl ammonium
bromide (CTAB). The probe features a high fluorescence enhancement, fast response (10–30 min), and
good sensitivity (1 mm) and selectivity for hydrogen sulfide.
Received 15th October 2020
Accepted 8th December 2020
DOI: 10.1039/d0ra08802a
rsc.li/rsc-advances
particularly glutathione and cysteine. A concern with existing
probes is that they are mostly based upon common bulky scaffolds
Introduction
including uorescein, xanthene, and rhodamine which have
inherent limitations in terms of cell permeability and solubility in
the cellular environment. Additionally, these molecules oen
require more lengthy synthetic sequences for their preparation
and signicant effort is required in order to modify their intrinsic
spectral range.
The detection of hydrogen sulde has been an important goal
for some time.1–5 From an environmental perspective, hydrogen
sulde is a noxious and toxic by-product of various industrial
processes, particularly the Kra pulping process.6 From a bio-
logical perspective, hydrogen sulde has increasingly been
recognized as a critical very small signaling molecule akin to
nitric oxide and carbon monoxide that can be diagnostic for
a range of pathological conditions7 including kidney disease,8
Down's syndrome,9 and cirrhosis of the liver.10 This recognition,
combined with the desire to better understand the role and
localization/transport of hydrogen sulde, has resulted in the
development of a number of probes for live cell imaging.11
Thanks to its high sensitivity, rapid response, and generally
simple sample preparation, uorescence spectroscopy has become
the most common approach for the design of new hydrogen
sulde sensors. Within these sensors, several different approaches
have been developed, particularly the reduction of nitro and azido
groups,12–22 the chelation with copper (II) complexes,23–25 nucleo-
philic thiolysis,26–40 and conjugate additions and cyclizations.41–45
For all of these approaches, one key consideration is avoiding
reaction with other common sulfur-containing compounds,
To address this situation, we envisioned using the aurone
system as an alternative uorescent scaffold. Aurones are
a small family of natural products primarily responsible for the
golden pigmentation present in plants with yellow owers such
as dahlias, snapdragons, and cosmos.46–48 Due to their unusual
exocyclic alkene, they absorb at longer wavelengths compared
to the more common avones. At the same time, this ring
system is easy to synthesize in a variety of ways, particularly the
acid or base catalyzed condensation of an aldehyde and a ben-
zofuranone.49–52 Indeed, this condensation can even be per-
formed under effectively neutral conditions, rendering it highly
functional group compatible as well as generally high yielding.
Aurones have been reported to be uorescent, with the rst
signicant study reported by Bane and co-workers.53 Since this
report on the uorescent properties of a series of amino-
substituted aurones, a few other studies have explored varia-
tions of the benzofuranone portion either experimentally or
computationally.54–56
aMolecular Bioscience Program, Middle Tennessee State University, Murfreesboro, TN
37132, USA. E-mail: justin.miller@mtsu.edu; scott.handy@mtsu.edu
bDepartment of Chemistry, Middle Tennessee State University, Murfreesboro, TN
37132, USA
Interestingly, application of aurone uorescence has been
virtually unexplored. To date, only two reports have appeared in
the literature (Fig. 1). In the rst, hydroxyaurone 1 was reported
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra08802a
45180 | RSC Adv., 2020, 10, 45180–45188
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
General procedure for the synthesis of 40-azido benzaldehyde
derivatives
as a selective sensor for mercury (II) in aqueous solution as well
as living cells.57 It exhibited high selectivity and a linear
decrease in uorescence intensity in the presence of low
micromolar concentrations of mercury (II) salts. An earlier
report noted that 4-hydroxyaurones 2 exhibited changes in their
UV and uorescence spectra in the presence of cyanide anion.58
p-Bromo-substituted compound 2b was particularly interesting
as it exhibited a dramatic increase in emission upon irradiation
at 469 nm in acetonitrile in the presence of cyanide anion as
well as a visual color change. Other anions did not induce
a similar change. As a result, aurones certainly have the
potential to be uorescent sensors.
With this in mind, we envisioned utilizing an aurone scaf-
fold to develop an azide-reduction based turn-on uorescent
probe which in the presence of H2S undergoes reduction to the
corresponding amine accompanied by a signicant shi in the
absorption and emission spectra. For this purpose, we explored
40-azidoaurone 6a as a simple model probe which ultimately on
appropriate optimization led us to the rationally designed H2S
selective probe 6g.
In a 3 dram glass vial containing a magnetic stir bar, 1.0 mmol of
uorinated aromatic aldehyde and 1.5 mmol (1.5 equivalent) of
sodium azide (NaN3) were dissolved in 2 mL of dimethylsulfoxide
(DMSO) and heated for 2–3 h at 70–90 ꢀC (70 ꢀC for multi-
uorosubstituted) in a sand bath. Aer the completion of the
reaction, it was diluted with DI water and extracted with ethyl-
acetate (ꢁ4). To remove residual DMSO the organic fraction was
further washed with DI water and brine followed by drying over
anhydrous MgSO4. The organic layer was concentrated in vacuo
and puried by ash column chromatography using 2–10% ethyl
acetate: hexane to obtain the desired 40-azido benzaldehydes.
General procedure for the synthesis of aurones (6a–6g)
All the aurones 6a–6g were synthesized based on the literature.52
In a 3-dram glass vial, benzofuranone (1 equivalent) was dis-
solved in 5 mL of glacial acetic acid containing a magnetic stir
bar. To this solution 40-azidobenzaldehyde (1.5 equivalent) and
2–3 drops (0.2 mL) of concentrated HCl were added and stirred
at room temperature for up to 3 hours. In most cases the
reaction afforded a precipitate, normally aer 30 min, indi-
cating the completion of the reaction. Aer the reaction was
complete, it was poured into ice-cold DI water. The precipitate
obtained was ltered and the residue was washed multiple
times with water and allowed to air dry. No further purication
was required. For some of the aurones, which did not precipi-
tate out efficiently, the water diluted reaction mixture was
neutralized with saturated NaHCO3 and extracted with ethyl
acetate, concentrated under reduced pressure using a Rotary
Evaporator and puried by ash column chromatography. The
characterization data for 6a–6f is provided in ESI.†
Experimental section
Materials and methods
ACS grade chemicals and solvents were used as received without
further purication. All reactions were conducted under an air
atmosphere. 1H and 13C NMR spectra were recorded on a JEOL
AS (500 or 300 MHz) NMR instrument and chemical shis were
recorded in ppm with reference to tetramethyl silane (TMS),
chloroform-d or DMSO-d6.59 The following conventions are used
for multiplicities: s, singlet; d, doublet; t, triplet; m, multiplet;
dd, doublet of doublet; dt, doublet of triplet; ddd, doublet of
doublet of doublet; br, broad. A Cary 630 FT-IR (Agilent Tech-
nologies) was used to collect IR spectra. Solid samples were
used for collecting the IR-spectra. The high-resolution mass
spectra (HRMS) were acquired on a Waters Synapt HDMS QToF
as solutions in MeOH containing 1% triuoroacetic acid.
Fluorescence spectra were collected using an F-4500 uores-
cence spectrophotometer (Hitachi, Japan) with the slit width for
excitation and emission set at 5 nm and photomultiplier voltage
set at 700v. Excitation wavelength details are provided in-text as
relevant. An HP-8452A Diode Array Spectrophotometer (Agilent
Technologies Inc.) was used to record UV-visible absorption
spectra. Reactions were monitored by Thin Layer Chromatog-
raphy (TLC). All extracts were concentrated under reduced
pressure using a Buchi Rotary Evaporator. Products were puri-
ed by ash silica gel (32-63u) column chromatography.
(Z)-2-(4-Azidobenzylidene)-5-uorobenzofuran-3(2H)-one (6g).
Reaction scale: 0.50 mmol, puried by column chromatography
(eluent ¼ 10–30% EA : hexane) yield: 50% (70 mg); yellow solid, mp
137–138 ꢀC. 1H NMR (500 MHz, CDCl3) d 7.89 (d, J ¼ 8.5 Hz, 1H),
7.45 (dd, J ¼ 6.5, 3.0 Hz, 1H), 7.38 (td, J ¼ 8.5, 3.0 Hz, 1H), 7.29 (dd, J
¼ 8.5, 3.5 Hz, 1H), 7.10 (d, J ¼ 8.5 Hz, 1H), 6.85 (s, 1H). 13C NMR
(125 MHz, CDCl3) d 183.9, 162.1, 159.0 (d, 1JC–F ¼ 244.7 Hz), 147.4,
141.9, 133.3, 129.0, 124.4 (d, 2JC–F ¼ 26.1 Hz), 122.5 (d, 3JC–F ¼ 8.0
Hz), 119.7, 114.2 (d, 3JC–F ¼ 7.8 Hz), 113.1, 110.36 (d, 2JC–F ¼ 24.3
Hz). IR (neat) 2128, 1707, 1658, 1598, 1484, 1263, 1190, 832 cmꢂ1
.
General procedure for the synthesis of amino derivatives (7a–7g)
For the synthesis of the corresponding amines (7a–7g) of the
probe candidates, one equivalent of the aurone (6a–6g) was
mixed with an excess of NaHS (4 equivalents) in a 1-dram glass
vial containing a magnetic stir bar. To the mixture, 1.5 mL of
MeCN with a few drops of DI H2O was added and stirred for 30
minutes (acetone can also be used as a solvent). Aer a few
minutes, the solution turned bright red. Once the reaction was
complete, based on the TLC, the mixture was concentrated
under vacuum and subjected to ash column chromatography
(eluted with 5% MeOH : DCM). The characterization data for
7a–7f is provided in the ESI.†
Fig. 1 Aurone-based fluorescent probes.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 45180–45188 | 45181

View Article Online
RSC Advances
Paper
cold water, the products were separated by ltration. As depic-
ted in Scheme 1, the 4-azidoaldehydes were prepared by direct
azidation of the corresponding 4-uorobenzaldehydes with
NaN3 in DMSO.60 The 40-azidoaurone derivatives 6(a–g) were
further reduced by NaHS to give the corresponding amines 7.
Based on the fact that highly water soluble H2S predomi-
nantly (>75%) exists as HSꢂ under physiological conditions,
NaHS was utilized as the source of H2S for this study.61 Although
the model probe 6a (100 mM) was found to react efficiently (ꢃ7
min) with 5 equivalent of NaHS in a 1 : 1 or higher ratio of
DMSO in phosphate buffer saline (PBS) medium, its utility was
greatly limited in highly aqueous medium (<1% DMSO) as the
probe precipitates out below 20% DMSO or MeCN in PBS.
Additionally, increasing the aqueous fraction of the solvent was
accompanied by a signicant decrease in the uorescence
intensity of the corresponding amine. Nevertheless, this
observation provided the proof of concept for the utility of an
aurone scaffold for hydrogen sulde detection.
To improve solubility with increasing ratios of water to
DMSO, we introduced a hydroxyl (–OH) group at C-6 or C-4 of
the benzofuranone moiety of the model probe 6a to give aurones
6b and 6c, respectively. As expected, this rendered them highly
soluble in largely aqueous media (as low as 5% DMSO in PBS).
Unfortunately, it also resulted in signicant spectral overlap,
limiting the selective excitation of the starting azides and/or the
corresponding amine products (Fig. 2 and S1†).
(Z)-2-(4-Aminobenzylidene)-5-uorobenzofuran-3(2H)-one
(7g). Reaction scale (0.09 mmol), Puried by column chroma-
tography (eluent ¼ 5% MeOH : DCM) yield: 85% (19.5 mg); dark
red solid, mp 178–180 ꢀC. 1H NMR (500 MHz, DMSO-D6) d 7.74
(d, J ¼ 8.5 Hz, 2H), 7.65–7.58 (m, 2H), 7.57 (dd, J ¼ 7.5, 2.5 Hz,
1H), 6.88 (s, 1H), 6.66 (d, J ¼ 8.5 Hz, 2H), 6.16 (s, 2H). 13C NMR
1
(75 MHz, DMSO-d6) d 181.6, 160.5, 158.2 (d, JC–F ¼ 240.9 Hz),
2
3
152.1, 144.3, 134.2, 123.7 (d, JC–F ¼ 26.0 Hz), 122.6 (d, JC–F
¼
3
8.1 Hz), 118.7, 116.2, 114.7 (d, JC–F ¼ 8.1 Hz), 113.9, 109.3 (d,
2JC–F ¼ 24.2 Hz). IR (neat) 3420, 3320, 3216, 1689, 1633, 1562,
1514, 1480, 1272, 1156, 817, 762 cmꢂ1. HRMS calcd for
C
15H11FNO2 256.0774, observed 256.0071.
Solution preparation method for spectroscopic measurement
Stock solutions (10, 5, and 1 mM) of the probe candidates (6a–
6g) and their respective amines were prepared in DMSO. Simi-
larly, the stock solutions of NaHS (10, 5 and 1 mM), cetyl tri-
methylammonium bromide (CTAB) (5 and 1 mM) and various
biologically relevant sulde sources L-cysteine, reduced gluta-
thione, and 2-mercaptoethanol (5 and 1 mM) were prepared in
phosphate buffered saline (PBS) (1X, pH ¼ 7.4, without Ca and
Mg). The detailed sample preparation procedures for the
experiments can be found in the ESI.† All UV-visible absorption
and emission spectra were collected in various concentrations
of DMSO (0.5, 5, 50, and 100%) and/or MeCN in PBS-buffer (1X,
pH ¼ 7.4, without Ca and Mg) in 3 mL and 1.5 mL cuvettes
respectively, in the presence and absence of 1 mM CTAB. The
detection limit was determined using different concentrations of
7g (0.1–50 mM) in different solvent systems (biological and non-
aqueous). Limits of detection were dened based on linearity
and signal-to-noise ratios for the upper and lower limits, respec-
tively. All emission intensities used to determine detection limits
were derived from triplicate experimental measurements. Rate
proles of the reduction reaction as well as the selectivity study of
the probe were performed by the incubation of 20 mM 6g with 100
mM H2S source (NaHS) in either 0.5% v/v DMSO in PBS/1 mM
CTAB (biological) or 90% v/v DMSO/10% v/v PBS (non-aqueous)
solvent systems. The reactions were excited at 465 nm and emis-
sion spectra spanning from 480 to 700 nm were collected at 2
minute intervals. All nonlinear least squares (NLLS) analyses were
performed using a single-exponential function given as:
Next, leaving the hydroxyl group intact on the benzofur-
anone moiety, the effects of substitution in the phenyl ring on
the reactivity and electronic properties of the probe molecule
were explored. As suggested by Henthorn and Pluth in their
mechanistic study of H2S-mediated reduction of aryl azides, the
introduction of uorine was reported to result in roughly a 2.2-
fold rate increase for each uorine substitution.62–65 Utilizing
this same strategy, we synthesized both mono- and di-uoro
Y ¼ A(1 ꢂ eꢂk ꢁt) + b
app
where A, kapp, t, and b represent the time course amplitude,
apparent rate constant, time, and the y-intercept, respectively.
All kinetic data were measured in triplicate to allow for statis-
tical comparisons.
Results and discussion
Probe design
All of the uorescent probe candidates 6a–6g were synthesized
as outlined in Scheme 1. The reaction involved the
Knoevenagel-type condensation of a benzofuranone 5 with a 4-
azidobenzaldehyde derivative 4 under acidic reaction condi-
Scheme 1 Aurone-based probe synthesis. (a) 1.5 equiv. NaN3, DMSO,
70–90 ꢀC, 2–5 h (b) 1.1 equiv. 4, glacial acetic acid, 3 drops conc. HCl,
rt, 3 h (c) 4 equiv. NaHS, 2 mL MeCN or acetone, few drops DI water,
tions.52 Since the reaction product generally precipitates out in 30 min.
45182 | RSC Adv., 2020, 10, 45180–45188
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 2 UV-visible spectra illustrating spectral overlap. Samples (50 mM)
were prepared in 5% DMSO in PBS (v/v) for the study.
Fig.
3
Evaluation of chromophore DMSO-sensitivity. (A and B)
substituted hydroxylated candidates 6d–6f. Despite their
outstanding aqueous solubility, all of them resulted in highly
overlapped absorption spectra with their corresponding amines
7d–7f similar to that observed with 6b and 6c. The result was
that there was still no excitation window available for the
selective excitation of reactant or product to monitor the reac-
tion (Fig. 2). This situation can be attributed to the electron
donating effect of the hydroxyl group making the carbonyl
group less electron decient which results in a reduction in the
resonance mediated electron donation from the amine.
Since, phenyl ring functionalization of aurones did not
resolve the issue of spectral overlap of the azido and amino
compounds, we decided to introduce uorine in the benzofur-
anone moiety. This addition has the potential to increase the
electrophilicity of the carbonyl carbon, which in turn will increase the
resonance meditated electron donation from amino group. This
increase in polarization should impart signicant differences in the
electronic properties of the two compounds, thereby improving their
spectral separation. The impact of uorine in the benzofuranone
moiety on the rate of azide reduction was less clear, but was expected
to be modest. As an initial test, 6g and its corresponding amine 7g
were synthesized. Interestingly, the introduction of uorine at C-5 of
benzofuranone moiety not only resolved the problem with spectral
overlap, but also afforded considerable solubility to the probe in
largely aqueous media (as low as 0.5% DMSO in PBS buffer) without
the need for any hydroxyl group. This ultimately provided us with the
desired turn-on uorescent probe 6g for the detection of H2S.
Normalized excitation and emission spectra are represented from
bottom to top of frame in order of ascending [DMSO]. All spectra are
colored blue, red, green, or black to indicate 0.5% v/v, 50% v/v, 90% v/
v, or 100% v/v DMSO, respectively. Excitation and emissions spectra
are represented as either dashed or solid lines, respectively. Peak
maxima are discussed in-text. Panel (A) and (B) represent spectral data
corresponding to reactant 6g or product 7g, respectively.
maxima of 545, 531, and 539 nm for 50%, 90%, and 100% v/v
DMSO conditions, respectively. In comparison, maximal 7g
absorption occurs at 442 nm in the presence of 0.5% DMSO,
whereas excitation maxima equal to 458, 462, and 462 nm are
observed for conditions including 50, 90, and 100% v/v DMSO,
respectively (Fig. 3B). However, maximal emissions occur at 566,
566, 563, and 561 nm for 0.5, 50, 90, and 100% v/v DMSO,
respectively, thereby indicating minimal solvent sensitivity for
product species uorescence. Comparison of the spectra in
Fig. 3 indicates that excitation of 6g/7g mixtures using wave-
lengths in the 440–460 nm range should allow for selective
observation of product emission.
Inspection of raw 7g uorescence intensities as a function of
increasing DMSO concentration suggests that an aqueous
environment may contribute to quenched product emission.
Our data indicate a 33-fold increase in uorescence intensity for
conditions including 90% versus 0.5% v/v DMSO. Thus, the use
of 6g as a uorescent sulde probe may be limited in biological
applications under these conditions. In this context, cationic
surfactant cetyltrimethylammonium bromide (CTAB) has been
utilized in the literature to enhance both the rate of the reaction
and the intensity of the uorescence.66,67 Based on this prece-
dent, we successfully employed CTAB to enhance the reactivity
of probe 6g in highly aqueous medium (0.5% DMSO in PBS).
Fig. 4 indicates that the inclusion of 1 mM CTAB in 0.5% v/v
DMSO does not impact 6g excitation properties (dashed lines in
Fig. 4A), but instead promotes a red-shi in the apparent
emission maximum from 510 nm to 550 nm in the absence
versus presence of CTAB respectively. In contrast, 7g excitation
properties in 0.5% v/v DMSO are perturbed by the inclusion of
CTAB such that maximal absorption shis from 442 nm to
462 nm in the absence versus presence of surfactant, respec-
tively (Fig. 4B). However, Fig. 4B reveals no impact of CTAB on
the wavelength of maximum emission.
Spectral characterization of 6g and 7g
To evaluate its utility as a uorescent probe, the excitation and
emission properties of probe 6g (reactant) and corresponding
amine 7g (product) were characterized under varied solvent
conditions. Fig. 3 presents excitation and emission spectra
collected with either molecule in the presence of 0.5, 50, 90, or
100% v/v DMSO and phosphate buffered saline (PBS) solution.
All 6g excitation spectra recorded in DMSO/PBS mixtures
exhibit two distinct excitation modes that are not signicantly
dependent on DMSO/PBS ratio (Fig. 3A). Excitation maxima are
observed at 346/394 nm, 362/414 nm, 346/402 nm, and 350/
406 nm for conditions of 0.5, 50, 90, or 100% v/v DMSO,
respectively (dashed lines in Fig. 3A).
Emission spectra of 6g indicate solvent sensitivity when
excited at 405 nm where maximum uorescence is observed for
0.5% DMSO/99.5% PBS conditions at 510 nm (solid lines in
Fig. 3A). This contrasts with the red-shied 6g emission
Furthermore, raw 7g uorescence intensities are observed to
increase 42-fold for 0.5% v/v DMSO conditions including CTAB
versus conditions that do not. Our data also indicate that these
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 45180–45188 | 45183

View Article Online
RSC Advances
Paper
the absence or presence of CTAB, respectively. Taken together,
we note 7g spectral properties to be largely independent of
solution pH. However, we do note an increase in raw emission
intensities for 7g when CTAB is included, where mild uo-
rophore quenching is observed at pH below 4.8. At pH $ 4.8,
emission intensities are observed to be independent of pH.
Dynamic range study
The use of any uorescent probe requires knowledge of the
dynamic range under specic working conditions. As such, we
next determined the useful working range of 7g in conditions
including either increased DMSO concentrations or 0.5% v/v
DMSO/1 mM CTAB. Knowledge of the dynamic range under
these separate conditions allows for both biological and non-
aqueous applications. Fig. 5A demonstrates that the titration
of 7g in 0.5% v/v DMSO, 99.5% v/v PBS, and 1 mM CTAB with
excitation at 465 nm yields emission spectra with maxima
observed at ꢃ565 nm. A secondary plot of maximum uores-
cence intensity versus [7g] clearly reveals non-linear behavior for
concentrations exceeding 20 mM (Fig. 5B). Our data also indi-
cate that uorescence intensities can be reliably estimated for
[7g] as low as 1 mM based on spectra signal-to-noise ratio and
deviation from linearity. Thus, the useful dynamic range for 7g
in the presence of 0.5% v/v DMSO, 99.5% PBS, and 1 mM CTAB
spans from 1 mM to 20 mM.
Fig. 4 CTAB influences product emission properties. Normalized
excitation/emission spectra are presented in the presence of 0.5% v/v
DMSO and varied buffer components. Excitation and emission spectra
are represented by dashed or solid lines, respectively. Spectra corre-
sponding to conditions including PBS buffer without or with 1 mM
CTAB are colored red or green, respectively (top panel). Spectra cor-
responding to conditions including HEPES buffer without or with 1 mM
CTAB are colored black or blue, respectively (bottom panel). Peak
maxima are discussed in-text. Panel (A) and (B) represent spectral data
corresponding to reactant 6g or product 7g respectively.
trends occur independent of buffer components included in the
aqueous fraction since the inclusion of PBS or HEPES buffers
yielded identical results. The data presented in Fig. 4A and B
collectively suggest that 7g may be utilized in aqueous solution
as a uorescent probe under conditions that include CTAB.
Fig. 5C and D reveal a similar trend when measurements are
made under conditions including 90% v/v DMSO and 10% v/v
PBS solution. However, elevated DMSO concentrations appear
to increase the useful linear concentration range such that non-
Effect of pH on spectral properties of 6g and 7g
The effect of pH on 6g and 7g spectral properties was also linearity is observed for [7g] greater than 30 mM. We note the
examined. Excitation and emission spectra were collected in observation of decreased reproducibility when 7g concentra-
PBS buffer previously pH adjusted to pH ¼ 3.0, 4.8, 7.5, 9.2, and tions are utilized that exceed 25 mM in 90% v/v DMSO/10% v/v
11 in the absence and presence of CTAB as described above. PBS as indicated by the increased standard deviation of mean
Fig. S2A and B† illustrate that 6g experiences an altered Stokes uorescence intensities presented in Fig. 5D.
shi that depends on whether CTAB is included. In the absence
of CTAB, we note excitation and emission maxima occur at 424
and 500 nm, respectively, at pH ¼ 3.0. A similar observation is
Reaction kinetics and solvent studies
made when the pH is adjusted to 4.8, where excitation and Quantication of sulde concentration in any sample using “turn-
emission maxima occur at 426 and 502 nm, respectively. At pH on” uorescent sensing requires knowledge of kinetic rates of
¼ 3.0 and 4.8, the Stokes shi is calculated as 76 nm. However, reaction under varied solution conditions. Rate proles describing
at pH 7.5 and above, we observe a blue-shi, where excitation the reduction of azide reactant to amine product were performed
maxima are observed at 400 nm when determined at pH ¼ 7.5, by the incubation of 20 mM 6g with 100 mM NaHS in either 0.5% v/v
9.2, and 11.0. Corresponding emission maxima were observed DMSO/99.5% PBS/1 mM CTAB or 90% v/v DMSO/10% v/v PBS
at 527, 528, and 528 nm for pH ¼ 7.5, 9.2, and 11.0, respectively. solvent systems at 25 ꢀC. Samples were excited at 465 nm and
The calculated Stokes shi for pH $ 7.5 is ꢃ128 nm. In emission spectra spanning from 480 to 700 nm were collected at 2
contrast, when CTAB is included, no effect of pH is observed minute intervals. Fig. 6 demonstrates a time-dependent increase in
such that maximal excitation is observed at 404 nm indepen- uorescence intensity for both conditions.
dent of pH. Emission maxima are observed at wavelengths that
Fig. 6C shows a plot of maximum uorescence intensity as
increase with increasing pH such that maxima are observed at a function of time for aqueous reaction conditions including
511 (Stokes shi ¼ 107 nm), 527 (Stokes shi ¼ 123 nm), 547 0.5% v/v DMSO/99.5% v/v PBS in the presence or absence of
(Stokes shi ¼ 143 nm), 547, and 550 (Stokes shi ¼ 146 1 mM CTAB. In the presence of 1 mM CTAB, reaction comple-
nm) nm for pH ¼ 3.0, 4.8, 7.5, 9.2, and 11.0, respectively. In tion is achieved within approximately one hour (blue spheres,
contrast 7g exhibits pH-independent excitation maxima at 440 Fig. 6C). In contrast, no reaction is observed in the absence of
and 460 nm when CTAB is absent or present, respectively. The CTAB (green spheres, Fig. 6C). By comparison, reactions per-
emission maximum for 7g is observed at 565 nm independent formed in 90% v/v DMSO/10% v/v PBS are observed to reach
of pH, yielding a Stokes shi equal to 125 or 105 depending on completion within 20 minutes (green spheres, Fig. 6D).
45184 | RSC Adv., 2020, 10, 45180–45188
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 6 Compound 6g reaction rate constant is dependent on solvent
condition. Reaction profiles describing the reduction of azide reactant
to amine product by the incubation of compound 6g with 100 mM
NaHS in the presence of either 0.5% v/v DMSO in PBS ꢄ1 mM CTAB (A)
or 90% v/v DMSO/10% v/v PBS buffer (B). Emission spectra are rep-
resented as solid lines and reveal increasing signal intensity as a func-
tion of reaction time (A and B). All data points represent average values
determined from at least three independent experiments (C and D).
Error bars indicate ꢄ standard deviation. Solid lines are the result of
nonlinear least squares (NLLS) fits with rate constants indicated.
Fig. 5 Determination of product 7g range of detection. Product 7g
was titrated separately into conditions including either 1 mM CTAB and
0.5% v/v DMSO in PBS (A and B) or 90% v/v DMSO/10% v/v PBS buffer
(C and D) with excitation at 465 nm. Emission spectra are represented
as solid lines (A and C). Signal intensity observed at 560 nm plotted as
a function of [7g] reveals linear range of detection (B and D). All data
points represent average values determined from at least three inde-
pendent experiments. Error bars indicate ꢄ standard deviation. Solid
lines are the result of Linear Least Squares (LLS) fits. For conditions with
1 mM CTAB and 0.5% v/v DMSO in PBS (B), LLS fit yields estimates of
slope and y-intercept equal to 50 ꢄ 24 (AU) and 107 ꢄ 3 AU mM,
respectively. For conditions with 90% v/v DMSO/10% v/v PBS buffer
(D), LLS fit yields estimates of slope and y-intercept equal to 68 ꢄ 24
(AU) and 60 ꢄ 1 AU mM, respectively.
480, and 500 nm with spectra collected at 2 minute intervals as
described above. Fluorescence time courses are presented in
Fig. 7A, which qualitatively demonstrate a decrease in apparent
amplitude and y-intercept with increasing excitation wave-
length. Fig. 7B predicts no signicant dependence in the
apparent rate constant describing product formation on exci-
tation wavelength. NLLS analyses of the data presented in
Fig. 7A yield estimates of the rate constant that uctuate about
Nonlinear least squares (NLLS) analysis of the data shown in
Fig. 6C estimates the apparent rate constant, amplitude, and y-
intercept as 0.078 ꢄ 0.002 minꢂ1, 842 ꢄ 8 AU, and 149 ꢄ 7 AU,
respectively. In comparison, NLLS analysis of reaction data
collected in 90% v/v DMSO/10% v/v PBS (Fig. 6D) estimates the
apparent rate constant, amplitude, and y-intercept as 0.28 ꢄ
0.02 minꢂ1, 284 ꢄ 7 AU, and 249 ꢄ 6 AU, respectively. These
data collectively indicate that reactions performed in a polar
aprotic environment occur with increased reaction rates relative
to those performed under aqueous conditions.
NLLS analyses of data presented in Fig. 6C and D yield
estimates of the y-intercept as nonzero values. Reference to
Fig. 4 illustrates that excitation at 465 nm of a mixture of 6g and
7g may yield an apparent uorescence intensity that contains
contributions from both reactant and product. Moreover, no
product could be present at time zero. Therefore, we interpret
the observation of a nonzero y-intercept to reect the contri-
bution of reactant emission to the overall observed signal.
To determine whether contaminating reactant emission
leads to errors in our estimation of reaction rate, we performed
a series of reactions using independent excitation wavelengths
wherein 20 mM 6g was incubated with 100 mM NaHS in 0.5% v/v
DMSO/99.5% PBS/1 mM CTAB. Samples were excited at 465,
a mean value equal to 0.075 ꢄ 0.006 minꢂ1
.
In addition, the apparent amplitude and y-intercept are
observed to linearly decrease as excitation wavelength is
increased, which is an observation consistent with the excita-
tion of sample at non-maximal excitation wavelengths for both
reactant and product (Fig. 7C). Given that the y-intercept
represents signal derived at time zero prior to any product
formation, we interpret the observation of a y-intercept that
decreases with increasing excitation wavelength to suggest that
longer excitation wavelengths favor decreased background
signal associated with reactant emission. Taken together, the
data presented in Fig. 7 collectively indicates that reactant
contribution to overall apparent uorescence signal does not
interfere in the estimation of reaction rate constants.
Moreover, knowledge of the dependence of time course y-
intercept on excitation wavelength allows for baseline correction to
remove reactant signal contributions, thereby allowing for
conversion of uorescence intensity values into concentration
units. By subtracting baseline emissions from subsequent time
points, the resulting emission values can be readily converted into
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 45180–45188 | 45185

View Article Online
RSC Advances
Paper
Fig. 8 All time courses can be converted into plots of [Product] versus
time. Blue spheres represent conditions including 1 mM CTAB, 0.5%
DMSO in PBS (condition A). Green squares represent conditions
including 90% DMSO and 10% PBS (condition B). The solid line
represents the results of a NLLS fit with A, kapp, and b estimated as 7.9 ꢄ
0.1, 0.08 ꢄ 0.002, and ꢂ0.03 ꢄ 0.07, respectively. The dashed line
represents the results of a NLLS fit with A, kapp, and b estimated as 4.8
ꢄ 0.1, 0.28 ꢄ 0.02, and 0.1 ꢄ 0.1, respectively. Error bars indicate ꢄ
standard deviation.
Fig. 7 Compound 6g reaction rate constant is independent of exci-
tation wavelength. (A) Kinetic time courses were collected using
excitation wavelengths equal to 465 (green spheres), 480 (red
spheres), and 500 nm (blue spheres). Reactions were performed by
incubating 20 mM 6g with 100 mM NaHS in 1 mM CTAB and 0.5% v/v
DMSO in PBS. Reaction progress was monitored by fluorescence
intensity observed at 560 nm. Solid lines are the result of nonlinear
least squares (NLLS) fits to a single-exponential function. (B) First-order
rate constants plotted as a function of excitation wavelength with
average apparent rate constant indicated. (C) estimates of amplitude
(green spheres) and y-intercept (blue spheres) describing time courses
in (A) obtained from NLLS analyses. Solid lines are the result of LLS fits
with slopes equal to ꢂ14.6 ꢄ 0.9 and ꢂ1.6 ꢄ 0.3 AU nmꢂ1, respectively.
All data points represent average values determined from at least three
independent experiments. Error bars indicate ꢄ standard deviation.
Sensing mechanism
In this study the turn-on probe 6g, in the presence of HSꢂ
undergoes an irreversible chemical transformation to amine 7g
thereby transducing signals by converting the molecular
recognition phenomenon into an intense uorescence signal.
Based on the mechanistic study by Henthorn and Pluth, this
reaction is suggested to proceed via nucleophilic attack of
hydrogen sulde anion (HSꢂ) on the terminal electrophilic
nitrogen of azido group forming an azidothiol intermediate
species which on subsequent HSꢂ intermolecular attack is
converted to the respective amine 7g.64 The amine subsequently
undergoes a spontaneous internal charge transfer (ICT) via
resonance to its corresponding highly uorescent zwitterionic
form as depicted in Fig. 10A. The presence of CTAB may further
stabilize the charged species via electrostatic interactions. This
consequently leads to the stabilization of the high quantum
yield state resulting in an enhanced emission intensity as
evident in Fig. 6A, C, and S3.† It has been suggested that CTAB
works by changing the local concentration of the reactants and
the pH value of the reaction mixture. The CTAB micelles
increase the local concentration of anions including OHꢂ and
HSꢂ around it (Fig. 10B), which increases the local pH of the
solution. This in turn decreases the [H2S]/[HSꢂ] ratio thereby
contributing an overall higher HSꢂ concentration around the
micelles.66 The effect of pH on dissociation of H2S in an aqueous
medium is summarized in Fig. 10C.61 At lower pH (ꢃ3) the
uorescence intensity of the product is expected to decrease due
to protonation of the amine which shuts down the ICT process
(Fig. S2E and S3†). This is apparent from Fig. S3† which clearly
demonstrates how the emission intensity of the product 7g is
affected by the pH although UV-absorption remains unaffected.
In this overall phenomenon, CTAB micelles serve as a scaffold
to recruit HSꢂ and probe 6g in the vicinity of each other, thereby
concentration units if a standard curve relating product concen-
tration to emission value is available. Fig. 8 highlights this ability
for time courses collected in the presence of 1 mM CTAB in 0.5%
DMSO in PBS or 90% DMSO/10% PBS. Though the estimated rate
constants are unaltered, Fig. 8 now readily allows for estimation of
hydrogen sulphide concentration in real-time.
Selectivity for sulde studies
Any use of 6g as a molecular probe will require knowledge of its
reaction specicity. To evaluate reaction specicity, a series of uo-
rescence time courses were collected using the same methodology as
described above. In place of NaHS, three potential sulde-donors were
employed: L-cysteine, reduced glutathione, and 2-mercaptoethanol.
All emission spectra reect the reaction of 100 mM sulde-donor with
20 mM 6g in 0.5% v/v DMSO, 99.5% v/v PBS, and 1 mM CTAB. As
expected, NaHS yields the observation of signicant product emission
at lEM ¼ 560 nm (Fig. 9). In contrast, the inclusion of either L-cysteine
or reduced glutathione yielded no observed product formation. Fig. 9
illustrates that a mild increase in emission was observed when 2-
mercaptoethanol was utilized as a sulde-donor. However, 2-mer-
captoethanol reactivity with 6g is much slower on the timescale
associated with NaHS:6g reaction. Taken together, we conclude that
6g chemical reactivity is selective for free sulde.
45186 | RSC Adv., 2020, 10, 45180–45188
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
the demands of various sensing applications. Based upon these
results, future optimized compounds can be designed and
prepared for a wide range of environmental as well as biological
applications. Given the ease and exibility in the synthesis of
the aurone framework, many additional sensing applications
can be envisioned, particularly applying the lessons learned in
this project. Efforts to this end are underway and will be re-
ported in due course.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors acknowledge support from the Molecular Biosci-
ences program at Middle Tennessee State University.
Fig. 9 Compound 6g displays chemical reactivity that is selective for
sulfide. Kinetic time courses were collected using an excitation
wavelength equal to 465 nm. Reactions were performed by incubating
20 mM 6g with 100 mM sulfide-donor in 1 mM CTAB and 0.5% v/v
DMSO in PBS. Sulfide-donors examined include L-cysteine (green
squares), glutathione (blue triangles), NaHS (black circles), and 2-
mercaptoethanol (red diamonds). Reaction progress was monitored
by fluorescence intensity observed at 560 nm. Solid lines are the result
of nonlinear least squares (NLLS) fits to a single-exponential function.
All data points represent average values determined from at least three
independent experiments. Error bars indicate ꢄ standard deviation.
Notes and references
1 Z. Guo, G. Chen, G. Zeng, Z. Li, A. Chen, J. Wang and L. Jiang,
Analyst, 2015, 140, 1772–1786.
2 J. Li, C. Yin and F. Huo, RSC Adv., 2015, 5, 2191–2206.
3 F. Yu, X. Han and L. Chen, Chem. Commun., 2014, 50, 12234–
12249.
4 V. S. Lin, A. R. Lippert and C. J. Chang, Methods Enzymol.,
2015, 554, 63–80.
5 B. Peng and M. Xian, Methods Enzymol., 2015, 554, 47–62.
6 S. L. M. Rubright, L. L. Pearce and J. Peterson, Nitric Oxide,
2017, 71, 1–13.
7 G. K. Kolluru, X. Shen, S. C. Bir and C. G. Kevil, Nitric Oxide,
2013, 35, 5–20.
8 A. F. Perna and D. Ingrosso, Nephrol. Dial. Transplant., 2012,
27, 486–493.
9 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi and
B. Chadefaux-Vekemans, Am. J. Med. Genet., Part A, 2003,
116, 310–311.
10 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga,
G. Rizzo, E. Distrutti, V. Shah and A. Morelli, Heptaology,
2005, 42, 539–548.
11 For a recent example, see: Y. Chen, X. Shang, C. Li, Z. Xue,
H. Chen, H. Wu and T. Wang, Sci. Rep., 2018, 8, 16159.
12 S. K. Bae, H. Heo, D. J. Choi, D. Sen, E.-H. Joe, B. R. Cho and
H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915–9923.
13 T. S. Bailey and M. D. Pluth, J. Am. Chem. Soc., 2013, 135,
16697–16704.
Fig. 10 Sensing mechanism of 6g. (A) Reaction scheme showing the
reduction of azide (–N3) to the corresponding amine. The resonance
responsible for the fluorescence is depicted in parenthesis. (B) Sche-
matic representation of CTAB micelle and its role in recruiting and
changing the local concentration of 6g and HSꢂ. (C) Reaction scheme
representing the dissociation of H2S in aqueous medium.
signicantly increasing the reaction rate (Fig. 6A and C) along 14 J. Katla and S. Kanvah, Photochem. Photobiol. Sci., 2018, 17,
with uorescent intensities.
42–50.
15 Y.-S. Zeng, R.-C. Gao, T.-W. Wu, C. Cho and K.-T. Tan,
Bioconjugate Chem., 2016, 27, 1872–1879.
16 S.-A. Choi, C. S. Park, O. S. Kwon, H.-K. Giong, J.-S. Lee,
T. H. Ha and C.-S. Lee, Sci. Rep., 2016, 6, 26203.
Conclusions
In short, an aurone-based sensor for hydrogen sulphide has
been developed that exhibits good sensitivity and selectivity. 17 Y. Guo, T. Zeng, G. Shi, Y. Cai and R. Xie, RSC Adv., 2014, 4,
Further, the development of this probe molecule has demon- 33626–33628.
strated the strength of the aurone framework in terms of the 18 K. Xiang, Y. Liu, C. Li, B. Tian, T. Tong and J. Zhang, Dyes
ease with which new compounds can be prepared to modify the Pigm., 2015, 123, 78–84.
reactivity and physical properties (solubility in this case) to t 19 Y. Jiang, Q. Wu and X. Chang, Talanta, 2014, 121, 122–126.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 45180–45188 | 45187

View Article Online
RSC Advances
Paper
20 R. Wang, F. Yu, L. Chen, H. Chen, L. Wang and W. Zhang, 43 S. K. Patra, S. K. Sheet, B. Sen, K. Aguan, D. R. Roy and
Chem. Commun., 2012, 48, 11757–11759. S. Khatua, J. Org. Chem., 2017, 82, 10234–10246.
21 H. Peng, Y. Cheng, C. Dai, A. L. King, B. L. Predmore, 44 Y. Qian, B. Yang, Y. Shen, Q. Du, L. Lin, J. Lin and H. Zhu,
D. J. Lefer and B. Wang, Angew. Chem., Int. Ed., 2011, 50,
9672–9675.
22 T. Ozdemir, F. Sozmen, S. Mamur, T. Tekinay and
E. U. Akkaya, Chem. Commun., 2014, 50, 5455–5457.
23 X. Qu, C. Li, H. Chen, J. Mack, Z. Guo and Z. Shen, Chem.
Commun., 2013, 49, 7510–7512.
Sens. Actuators, B, 2013, 182, 498–503.
45 X. Hou, X. Guo, Z. Luo, H. Zhao, B. Chen, J. Zhao and
J. Wang, Anal. Methods, 2014, 6, 3223–3226.
46 R. Haudecoeur and A. Boumendjel, Curr. Med. Chem., 2012,
19, 2861–2875.
47 C. Zwergel, F. Gaascht, S. Valente, M. Diederich, D. Bagrel
and G. Kirsch, Nat. Prod. Commun., 2012, 7, 389–394.
24 F. Hou, L. Huang, P. Xi, J. Cheng, X. Zhao, G. Xie, Y. Shi,
G. Cheng, X. Yao, D. Bai and Z. Zeng, Inorg. Chem., 2012, 48 A. V. Popova, S. P. Bondarenko and M. S. Frasinyuk, Chem.
51, 2425–2460. Heterocycl. Compd., 2019, 55, 285–299.
25 A. A. Abd-Elaal, S. M. Tawk and Y.-I. Lee, J. Mol. Liq., 2017, 49 C. Lee, E. Chew and M. Go, Eur. J. Med. Chem., 2010, 45,
247, 35–42. 2957–2971.
26 J. Lv, F. Wang, J. Qiang, X. Ren, Y. Chen, Z. Zhang, Y. Wang, 50 R. S. Varma and M. Varma, Tetrahedron Lett., 1992, 33, 5937–
W. Zhang and X. Chen, Biosens. Bioelectron., 2017, 48, 96–
100.
5940.
51 I. Hawkins and S. T. Handy, Tetrahedron, 2013, 69, 9200–
27 X. Cao, W. Lin, K. Zheng and L. He, Chem. Commun., 2012,
48, 10529–10531.
28 D. Maity, A. Raj, P. K. Samanta, D. Karthigeyan, T. K. Mundu,
9204.
52 A. T. Geissman and J. B. Harborne, J. Am. Chem. Soc., 1955,
77, 4622–4624.
S. K. Pati and T. Govindaraju, RSC Adv., 2014, 4, 11147– 53 N. Shanker, O. Dilek, M. Mukherjee, D. W. McGee and
11151. S. L. Bane, J. Fluoresc., 2011, 21, 2173–2184.
29 S. Das and P. Sahoo, Sens. Actuators, B, 2019, 291, 287–292. 54 Y. Xue, Y. Dou, L. An, Y. Zheng and Y. Liu, RSC Adv., 2016, 6,
30 Y. Chen, X. Shang, C. Li, Z. Xue, H. Chen, H. Wu and
T. Wang, Sci. Rep., 2018, 8, 1–9.
31 Y. Wang, L. Yang, X.-R. Wei, R. Sun, Y.-J. Xu and J.-F. Ge,
Anal. Methods, 2018, 10, 5291–5296.
32 G. Asaithambi and V. Periasamy, J. Photochem. Photobiol., A,
2019, 369, 97–105.
33 H. Wang, X. Wu, S. Yang, H. Tian, Y. Liu and B. Sun, Dyes
Pigm., 2019, 160, 757–764.
7002–7010.
55 C. Espinosa-Bustos, D. Coretes-Arriagada, M. A. Soto-Arriaza,
J. Robinson-Duggon, N. Pizarro, A. R. Cabrera, D. Ruentealba
and C. O. Salas, Photochem. Photobiol. Sci., 2017, 16, 1268–
1276.
56 K. Munoz-Becerra, N. Villegas-Escobar, C. Zuniga-Loyola,
D. Cortes-Arriagada and A. Toro-Labbe, Mol. Phys., 2019,
117, 1451–1458.
34 H. Wang, J. Wang, S. Yang, H. Tian, Y. Liu and B. Sun, Food 57 M. Zhang, Y.-T. Bao, W. Yang, H.-F. Xiao, Z.-X. Han, X. Wu
Chem., 2018, 257, 150–154. and L. Yang, J. Heterocycl. Chem., 2018, 55, 1130–1135.
35 J. Cui, T. Zhang, Y.-Q. Sun, D.-P. Li, J.-T. Liu and B.-X. Zhao, 58 H. Chen, Y. Sun, C. Zhou, D. Cao, Z. Liu and L. Ma,
Sens. Actuators, B, 2016, 232, 705–711. Spectrochim. Acta, Part A, 2013, 116, 389–393.
36 B. Peng, W. Chen, C. Liu, E. W. Rosser, A. Pacheco, Y. Zhao, 59 H. E. Gottlieb and V. Lotlyr, J. Org. Chem., 1997, 62, 7512–
H. C. Aguilar and M. Xian, Chem.–Eur. J., 2014, 20, 1010–
1016.
37 D. Maity, A. Raj, P. K. Samanta, D. Karthigeyan, T. K. Kundu,
7515.
60 A. Kae, S. Yossef and S. T. Handy, Tetrahedron Lett., 2020,
61, 151899.
S. K. Pati and T. Govindaraju, RSC Adv., 2014, 4, 11147– 61 Q. Li and J. R. Lancaster Jr, Nitric Oxide, 2013, 35, 21–34.
11151.
62 Z. Zhu, Y. Li, C. Wei, X. Wen and Z. Xi, Chem.–Asian J., 2016,
11, 68–71.
63 C. Wei, R. Wang, L. Wei, Z. Li, Z. Xi and L. Yi, Chem.–Asian J.,
2014, 9, 3586–3592.
64 J. Zhang, Y. Gao, X. Kang, Z. Zhu, Z. Wang, Z. Xi and L. Yi,
Org. Biomol. Chem., 2017, 15, 4212–4217.
65 H. A. Henthorn and M. D. Pluth, J. Am. Chem. Soc., 2015, 137,
15330–15336.
66 H. Tian, J. Qian, H. Bai, Q. Sun, L. Zhang and W. Zhang, Anal.
Chim. Acta, 2013, 768, 136–142.
38 J. Chao, M. Xu, Y. Zhang, F. Huo, Y. Liu, X. Wang and C. Yin,
Spectrochim. Acta, Part A, 2019, 214, 227–232.
39 H. Zhang, J. Chen, H. Xiong, Y. Zhang, W. Chen, J. Sheng and
X. Song, Org. Biomol. Chem., 2019, 17, 1436–1441.
40 J.-B. Li, Q. Wang, H.-W. Liu, L. Yuan and X.-B. Zhang, Chem.
Commun., 2019, 55, 4487–4490.
41 F. Chen, D. Han, H. Liu, S. Wang, K.-B. Li, S. Zhang and
W. Shi, Analyst, 2018, 143, 440–448.
42 X. Shang, J. Li, Y. Feng, H. Chen, W. Guo, J. Zhang, T. Wang
and X. Xu, Front. Chem., 2018, 6, 202.
67 J. Zhang and W. Guo, Chem. Commun., 2014, 50, 4214–4217.
45188 | RSC Adv., 2020, 10, 45180–45188
This journal is © The Royal Society of Chemistry 2020