Hymecromone naphthoquinone ethers as probes for hydrogen sulfide detection

Article abstract of DOI:10.1016/j.dyepig.2021.109765

Hydrogen sulfide (H₂S), as one of the important gasotransmitters, plays an essential role in a many physiological and pathological processes. Commonly used methods for real-time detection and quantification of H₂S are rather complicated. This report presents a simple fluorescence and colorimetry dual-mode assay based on ethers (Nq-Cm and Nq-2Cm) derived from hymecromone (7-hydroxy-4-methylcoumarin, Cm) and naphthoquinone (Nq). In these compounds an electron-deficient naphthoquinone unit was used as a suitable reagent for nucleophilic aromatic substitution (S_NAr) reaction. Thiolysis of Nq-Cm and Nq-2Cm at physiological pH releases Cm and causes an significant fluorescence enhancement at 448 nm (with excitation at 320 nm). At the same time, the resulting naphthoquinone derivative Nq-2SH causes the solution a purple color, which allows naked-eye detection. Thus Nq-Cm and Nq-2Cm are colorimetrically selective for H₂S. The efficacy of the Nq-Cm and Nq-2Cm were demonstrated in buffer with associated submicromolar detection limit as low as 130 nM and 150 nM, respectively. Presented results suggest that both probes are excellent quantitative detection tool for H₂S.

Full text of DOI:10.1016/j.dyepig.2021.109765

Dyes and Pigments 196 (2021) 109765
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Hymecromone naphthoquinone ethers as probes for hydrogen
sulfide detection
a
a
a
a
´
Daniel Słowi n´ ski , Małgorzata Swierczy n´ ska , Aleksandra Grzelakowska , Marcin Szala ,
a
b
a,*
Jolanta Koli n´ ska , Jarosław Roma n´ ski , Radosław Podsiadły
a
Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 16, 90-537, Lodz, Poland
b
Department of Organic and Applied Chemistry, Faculty of Chemistry, University of Lodz, Tamka 12, 91-403, Lodz, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hydrogen sulfide (H S), as one of the important gasotransmitters, plays an essential role in a many physiological
2
Dual-mode detections
Coumarin-derived probe
Hydrogen sulfide
Fluorescent probe
Colorimetric probe
2
and pathological processes. Commonly used methods for real-time detection and quantification of H S are rather
complicated. This report presents a simple fluorescence and colorimetry dual-mode assay based on ethers (Nq-
Cm and Nq-2Cm) derived from hymecromone (7-hydroxy-4-methylcoumarin, Cm) and naphthoquinone (Nq). In
these compounds an electron-deficient naphthoquinone unit was used as a suitable reagent for nucleophilic
N
aromatic substitution (S Ar) reaction. Thiolysis of Nq-Cm and Nq-2Cm at physiological pH releases Cm and
causes an significant fluorescence enhancement at 448 nm (with excitation at 320 nm). At the same time, the
resulting naphthoquinone derivative Nq-2SH causes the solution a purple color, which allows naked-eye
detection. Thus Nq-Cm and Nq-2Cm are colorimetrically selective for H
Nq-2Cm were demonstrated in buffer with associated submicromolar detection limit as low as 130 nM and 150
nM, respectively. Presented results suggest that both probes are excellent quantitative detection tool for H S.
2
S. The efficacy of the Nq-Cm and
2
1
. Introduction
such as diabetes [15], liver cirrhosis [16], tumors [17], Alzheimer’s
disease [18] or Down syndrome [19]. Antiviral activity of H S has been
also demonstrated [20] and in addition, sodium thiosulfate (H S-pro-
2
Cells are the basic fundamentals of life and their metabolism is
2
regulated by many factors, such as gaseous signaling molecules or
enzyme levels [1]. Hydrogen sulfide (H S) is an endogenous gaseous
signaling compound similar to nitric oxide (NO) or carbon monoxide
CO) [2]. Hydrogen sulfide plays an essential role in a many physio-
ducing compound) has been proposed to treat patients at any stage of
the COVID-19 virus infection [21]. Thus, the last decade has brought
2
2
enormous interest in H S releasing agents [22] and its efficient scav-
(
engers [23,24]. Hence, it is significant to real time monitor H
2
S level to
logical and pathological processes [3–6]. The endogenous hydrogen
better exploration its biological role and mechanism.
sulfide is mainly produced by four enzymes: cystathionine beta-synthase
At low levels H
whereas the high concentrations of exogenous H
growth of cancer cells [27]. Thus, H S and its donors have been used in
antitumor and antimetastatic therapies [28]. Likewise, the ability of H
2
S was recognized as a tumor growth factor [25,26]
(
CBS), cystathionine gamma-lyase (CSE), 3-mercaptopyruvate sulfo-
2
S could suppress the
transferase (MST) and cysteine aminotransferase (CAT) [7,8]. Recently,
thiosulfate sulfurtransferase (TST) [9] and selenium-binding protein 1
2
2
S
(
SELENBP1) [10] are reported as H
2
S producing enzymes. It has been
S are involved in e.g.
to scavenge the reactive oxygen and nitrogen species depends on kinetic
factors i.e., rate constant and concentration of hydrogen sulfide [29].
In this context, not only the detection, but the quantitative deter-
shown that flaws in the enzymes synthesizing H
2
cancer [11] and a series of neurodegenerative diseases [4]. It supports
tissue growth by stimulating angiogenesis, regulates mitochondrial
bioenergetics, stimulates the cell cycle and has anti-apoptotic properties
2
mination of H S in the in vitro and in vivo settings is of great importance.
Methylene blue (MB) assay, ion selective electrodes (ISE), and gas
chromatography [30,31] are quite complex for real-time measurement
[
2
12,13]. Abnormal level of H S is associated with various diseases [14]
*
Corresponding author.
E-mail addresses: daniel.slowinski@dokt.p.lodz.pl (D. Słowi n´ ski), malgorzata.swierczynska@dokt.p.lodz.pl (M. Swierczynska), aleksandra.grzelakowska@p.lodz.
´
´
pl (A. Grzelakowska), marcin.szala@p.lodz.pl (M. Szala), jolanta.kolinska@p.lodz.pl (J. Koli n´ ska), jaroslaw.romanski@chemia.uni.lodz.pl (J. Roma n´ ski), radoslaw.
podsiadly@p.lodz.pl (R. Podsiadły).
https://doi.org/10.1016/j.dyepig.2021.109765
Received 10 May 2021; Received in revised form 30 August 2021; Accepted 30 August 2021
Available online 1 September 2021
0
143-7208/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Scheme 1. Synthesis of the Nq-Cm and Nq-2Cm probes.
of H
2
S release. It seems that the best method to quantify of high reactive
S, could be fluorescence based assays. Fluorescent
(Edinburgh Instruments, UK) with both excitation and emission slit
widths of 1 nm. The fluorescence quantum yields of Nq-Cm and Nq-
2Cm were determined using 7-hydroxy-4-methylcoumarin (Φ = 0.87
[53]) as reference in PB buffer (0.1 mM, pH 7.4, with 30% MeCN, v/v).
The quantum yields were calculated using the following equation (1):
species including H
2
probes are regarded as a reliable technique due to their high sensitivity,
rapid response, real-time detection ability and simple usage [32]. Up to
2
now, several fluorescent probes for H S based on azide or nitro group
reduction [33–35], copper sulfide precipitation [36,37], nucleophilic
addition [38], or thiolysis of ethers [39–41] have been reported. The
major drawback of these probes include multi-step synthesis and/or
poor stability in water solutions [42]. Conventional probes are prepared
by modifying a fluorescent core with a reactive masking moiety which is
(
)( n
)
2
Grad
GradST
x
x
ΦX = ΦST
(1)
2
n
ST
where, the subscripts ST and X denote standard and probe respectively,
is the slope of the line ob-
Φ is the fluorescence quantum yield, Grad
x
2
deprotected by H S. This masking group should release the fluorophore
tained from the plot of the area of fluorescence vs. absorbance for probe,
only by reaction with hydrogen sulfide [43]. However, only a few of
probes can be applied in real biological sample due to slow reaction rate
and/or low sensitivity.
GradST is the slope of the line obtained from the plot of the area of
fluorescence
vs.
absorbance
for
standard
(7-hydroxy-4-
methylcoumarin).
In the present work, we used an electron-deficient naphthoquinone
[
44,45] unit as a suitable reagent for nucleophilic aromatic substitution
Ar) reaction, and 4-methyl-7-hydroxycoumarin (Cm, hymecromone)
(
S
N
2.2. Synthesis of 2-chloro-3-(4-methyl-2-oxo-2H-chromen-7-yloxy)
naphthalene-1,4-dione (Nq-Cm) and 2,3-bis(2-oxo-2H-chromen-7-yloxy)
naphthalene-1,4-dione (Nq-2Cm) [47]
as a strong fluorescent dye which serve as a leaving fluorophore. This
strategy was inspiration to design and prepare hymecromone naph-
thoquinone ethers Nq-Cm and Nq-2Cm as novel probes for H
). Notably, the thiolysis of naphthoquinone ethers has not previously
been exploited as mode for fluorescent and colorimetric detection of
S. The combination of a fluorescent coumarin and a colored naph-
thoquinone derivative that are formed in the reaction of the tested ethers
with H S allows to selectively detect hydrogen sulfide and distinguish it
from other biothiols.
2
S (Scheme
1
To a solution of 7-hydroxy-4-methylcoumarin (0.50 g, 2.9 mmol) in
2 3
DMSO (20 mL), K CO (0.4 g, 2.9 mmol) was added. After 5 min of
H
2
stirring the 2,3-dichloro-1,4-napthoquinone (0.59 g, 2.6 mmol) was
added to the mixture. After stirring at room temperature for 12 h, the
resulting mixture was poured into ice water and extracted with EtOAc (2
x 30 mL). Organic layer was washed with 60 mL brine (saturated NaCl
2
solution), dried over Na
2 4
SO and concentrated in vacuo. The mixture of
2
. Experimental
.1. General
-Hydroxy-4-methylcoumarin (Cm) and 2,3-dichloro-1,4-napthoqui-
Nq-Cm and Nq-2Cm was separated by column chromatography (SiO )
2
using dichloromethane:methanol (10:1) to obtain pure solid
2
compounds.
◦
Nq-Cm: yellow solid, yield 25%, R
f
= 0.72, m.p. 214–216 C
1
7
H NMR (600 MHz) δ (ppm): 8.15 (d, J = 7.8 Hz, 1H), 8.02 (d, J =
8.4 Hz, 1H), 7.95-7.90 (m, 2H), 7.78 (d, J = 9.0 Hz, 1H), 7.41 (d, J = 2.4
none (Nq) were purchased from Merck. Dimethyl sulfoxide (DMSO,
reagent grade) used for synthesis was purchased from Chempur. For thin
layer chromatography (TLC) pre-coated aluminum-backed plates
Hz, 1H), 7.30 (dd, J
1
= 9.0 Hz, J = 2.4 Hz, 1H), 6.33 (s, 1H), 2.44 (d, J
2
1
3
= 1.2 Hz, 3H)); C NMR (151 MHz): δ (ppm): 178.5; 178.1; 160.2;
(
Merck Kieselgel 60 F254) was used. Column chromatography purifi-
159.1; 154.8; 153.6; 152.2; 135.1; 134.9; 132.1; 131.1; 127.5; 127.1;
1
+
cations were performed on Merck Silica gel 60 (70–230 mesh). H NMR
and C NMR spectra were recorded with a Bruker Avance III 600 using
as solvent DMSO‑d . The high resolution mass spectrum (HRMS) was
126.9; 116.0; 113.6; 113.0; 104.2; 18.7. HRMS [M+H] for C20
H
11ClO
5
1
3
calc. for 367.0378, found 367.0387.
◦
Nq-2Cm: bright orange solid, yield 35%, R
f
= 0.22, m.p. 219–222 C
6
1
performed using Synapt G2-Si mass spectrometer equipped with an
electrospray ionization (ESI) source and time of flight (TOF) analyzer.
The UV–Vis absorption spectra were recorded in a quartz cell of 1 cm
path length using the Jasco V-670 UV–vis/NIR spectrophotometer.
Fluorescence spectra were measured on spectrofluorometer FLS-920
H NMR (600 MHz) δ (ppm): 8.07-8.04 (m, 2H), 7.95-7.92 (m, 2H),
7.66 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 2.4 Hz, 2H), 7.22 (dd, J = 8.7 Hz,
1
1
3
J
2
= 3.0 Hz, 2H), 6.27 (d, J = 1 Hz, 2H), 2.39 (s, 6H). C NMR (151
MHz): δ (ppm): 180.2; 160.2; 159.5; 154.6; 153.5; 146.0; 134.9; 131.6;
+
127.1; 126.5; 115.8; 113.7; 112.9; 104.5; 18.6. HRMS [M+H] for
30 18 8
C H O calc. for 507.1085, found 507.1080.
2

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Fig. 1. (A) UV–Vis absorption and (B) emission spectra of Nq, Cm, Nq-Cm and Nq-2Cm recorded in a phosphate buffer (0.1 M, pH 7.4) containing acetonitrile
30%). λex = 320 nm, slites 1.0/1.0 nm.
(
2
.3. UV–vis and fluorescence measurements
where,
slope of the linear calibration plot between the fluorescence emission
intensity and the concentration of Na S.
σ
is the standard deviation of probe solution (blank), S is the
All spectroscopic measurements were performed in a phosphate
2
buffer (0.1 M, pH 7.4) containing acetonitrile (MeCN), (30%, v/v) at
room temperature. Phosphate buffer solution (0.1 M) was made by
2
.4. HPLC measurements
HPLC analyses of Cm, Nq-Cm and Nq-2Cm were performed using
mixing 0.2 M solution of Na
2
HPO
4
× 12H
2 2 4
O, 0.2 M solution of NaH PO
×
2H O and water. Compounds were dissolved in MeCN to prepare 1
2
mM stock solution. Before measurements probe stock solution was
diluted with buffer (0.1 M, pH 7.4) containing MeCN (30%) to afford the
UFLC Shimadzu equipped with UV–Vis absorption and fluorescence
detector. Analyses were done using a Kinetex C18 column (Phenomenex
final concentration of 2–80
μ
M. Stock solution of Na
2
S (1 mM) in
1
00 mm x 46 mm, 2.6
water, containing 0.1% trifluoroacetic acid (TFA). The standards and
products formed in the reaction of Nq-Cm or Nq-2Cm with H S were
μm) which was equilibrated with 10% of MeCN in
phosphate buffer (PB) was used as H
2
S source. To evaluate of H S
2
′
concentration in phosphate buffer, we used Ellman’s reagent, i.e., 5,5 -
dithiobis(2-nitrobenzoic acid) (DTNB). Detail procedure is described in
2
ꢀ
eluted by an increase of MeCN concentration from 10 to 100% over 12
Supporting Information. Various sodium salt stock solutions (1 mM; Cl ,
ꢀ
ꢀ
2ꢀ
2ꢀ
2ꢀ
2ꢀ ꢀ
min at the flow rate of 1.5 mL/min. The HPLC traces of Nq-Cm, Nq-2Cm
Br , I , CO
3
, SO
4
, S
2
O
4
, SO
3
, HSO ) were freshly prepared by
3
2
and coumarin (Cm) formed in reaction with H S were detected by
dissolving weighed portions of the corresponding salts in deionized
water. Stock solution of biothiols (1 mM): L-cysteine (L-Cys), L-gluta-
thione (L-GSH), N-acetyl-L-cysteine (NAC) were also prepared in
deionized water. Stock solution (1 mM) of chemical reducing agent:
monitoring the absorption at 330 nm.
3
. Results and discussion
.1. Synthesis and optical characterizations
Coumarins have good stability and desired spectroscopic properties
4
sodium borohydride (NaBH ) and biological reductant NADPH were
3
freshly prepared in methanol and deionized water, respectively. The
reduction of probes using sodium borohydride was performed in an
organic solvent (MeCN). After 30 min of reaction, phosphate buffer was
added and the emission spectra were measured. All measurements were
performed in a 3.5 mL quartz cuvette with 3 mL solution. The reaction
mixture was shaken uniformly at room temperature before measure-
ments. For fluorescence measurement excitation wavelength was set at
i.e. excitation and emission in UV region, large molar extinction coef-
ficient and good fluorescence quantum yield [46]. Hydroxyl group at
position C-7 in the coumarin allows for relatively easy addition of
fluorescence quenching trigger. Nq-Cm and Nq-2Cm were synthesized
from commercially available reagents by coupling of 7-hydroxy-4-me-
thylcoumarin (Cm) with 2,3-dichloro-1,4-naphthoquinone (Nq) at
3
20 nm.
The limit of the detection was calculated from equation (2):
room temperature (Scheme 1) according to slightly modified procedure
1
LOD = 3.3
σ
/S
(2)
[47]. Identity of Nq-Cm and Nq-2Cm was confirmed by means of
H
Table 1
Spectroscopic properties of Nq, Cm, Nq-Cm and Nq-2Cm in acetonitrile.
e
Compound
λ
max (nm)
ε
(λmax
)
λ
exc (nm)
λ
em (nm)
Φ
em (%)
Stokes shift (nm)
pKa
n/a
E
ꢀ
1
ꢀ 1
)
(
M
⋅cm
a
Nq
272
323
16500
12420
n/a
320
320
320
n/a
450
448
448
n/a
n/a
127
135
135
ꢀ 0.45
b
c
d
Cm
87 [53]
7.7
7.8
0.7
Nq-Cm
Nq-2Cm
279,313
279,313
24150,16000
29700, 26000
n/a
n/a
n/a
n/a
7.8
a
in a 0.1 M Tris buffer (pH 8.5) from Ref. [51].
in PBS from Ref. [52].
b
c
in PB buffer from Ref. [49].
d
e
in PBS buffer from Ref. [50].
using 7-hydroxy-4-methylcoumarin (Cm) (Φ = 0.87) as reference [53].
3

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Fig. 2. (A), (B) Time-depended absorbance spectra of Nq-Cm and Nq-2Cm (20
μ
M) recorded after bolus addition of Na
S (20 M).
2
S (80 M). (C), (D) Time depended
μ
fluorescence spectra (λex = 320 nm) of Nq-Cm and Nq-2Cm (5
μM) after bolus addition of Na
2
μ
NMR, 13C NMR and HRMS spectroscopy (see Supplementary Informa-
tion (SI), Figures S12-S19).
naphthoquinone (Nq). The absorbance and emission profiles are illus-
trated in Fig. 1. As shown in Fig. 1A, studied ethers have absorption
bands located approx. at 279 nm and 313 nm, which can be assigned to
the Nq and Cm core, respectively. Small negative reduction potentials
[48] make Nq act as good electron acceptor. The influence of Nq on
photophysical properties of Nq-Cm and Nq-2Cm is clearly visible when
we compare the fluorescence quantum yield of Cm and its ethers. The
data presented in Table 1 clearly shows that Nq-Cm and Nq-2Cm have
First, we tested the solubility of Nq-Cm and Nq-2Cm in PB buffer
solutions containing MeCN (30%, v/v). The linear dependence of the
absorbance on the concentration of the probes in the range at 2–20
μ
M
(
Fig. S1) confirms the possibility of performing all tests in such solution.
Next, we compared the spectroscopic properties of Nq-Cm and Nq-2Cm
with the 7-hydroxy-4-methylcoumarin (Cm) and 2,3-dichloro-1,4-
2
Scheme 2. The sensing mechanism of probes for H S.
4

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Fig. 3. Reaction time profiles of Nq-Cm and Nq-2Cm (5
μ
M) alone or in the presence of H
2
S (20
μM) in PB buffer (0.1 M, pH 7.4 containing 30% MeCN v/v), λex =
3
20 nm, slites 1.0/1.0 nm.
an emission band located at the same wavelength as Cm. Only the
fluorescence quantum yield of ethers is ten times lower compared to the
quantum yield of the parent coumarin. Both probes exhibit a weak
fluorescence (Φ < 0.08) in PB buffer (0.1 M, pH 7.4, with 30% MeCN,
v/v). Thus, the release of the Cm fluorophore from Nq-Cm and Nq-2Cm
will result in the activation of fluorescence.
times and may be sufficient to confirm the presence of hydrogen sulfide
or its donors in various biosystems. The resulting fluorescent spectra of
Nq-Cm and Nq-2Cm after addition of Na
cence intensity at the same wavelength as 7-hydroxy-4-methylcou-
marin. Those results suggest that H S had cleaved the ether bond in
Nq-Cm and Nq-2Cm, releasing the fluorescent coumarin (Scheme 2).
,4-Naphtoquinone unit effectively quenches the emission of fluo-
rophore via the photoinduced electron transfer (PET) effect. After
treatment with H S, the electron-donating ability of the hydroxyl group
2
S show a maximum fluores-
2
2
3
.2. UV–vis and fluorescence response of Nq-Cm and Nq-2Cm toward
2
2
H S
of Cm came into play and triggered the ICT process, and the fluorophore
induces a strong blue fluorescent signal. Moreover, the intensity of the
absorption bands located at 319 nm and 530 nm increase, which could
be assign to the formation of Cm and Nq-2SH, respectively. Addition-
ally, we confirmed that gaseous hydrogen sulfide converted the probes
To investigate our hypothesis that naphthoquinone-derived probes
are a suitable platform for creating H
S sensitive “turn-on” sensors, we
study the reactivity of Nq-Cm and Nq-2Cm in the presence of H S.
Initially, we checked if the addition of Na S (as an aqueous source of
hydrogen sulfide) will cause the release of Cm from Nq-Cm and Nq-
Cm. Fig. 2 shows the changes in the absorption and emission spectra of
the probes recorded within 20 min after mixing of tested ethers with
Na S. After addition of the analyte to the solution of Nq-Cm or Nq-2Cm
2
2
2
(
see SI, Figures S7 and S8) in the same way as Na
2
S.
To confirm the proposed sensing mechanism the reaction mixtures
2
were analyzed by high resolution mass spectrometry (HRMS). HRMS
spectra confirmed in both cases formation of 7-hydroxy-4-methylcou-
2
8 3
marin (Cm). A peak located at 175.04 corresponding to Cm (C10H O ,
we observed three alterations: (i) increase of the absorption band at 325
nm, (ii) emergence a new wide band at around 530 nm and (iii) decrease
of the absorption band at 279 nm. Both probes reacting with the
hydrogen sulfide source give a very fast fluorescent response. After the
first minutes of the reaction, the fluorescence intensity increases several
-
[
M ꢀ H] :175.16) was observed. In addition analysis confirmed the
formation of Nq-2SH (peak at 220.97; C10 ) as a second product
6 2 2
H O S
of thiolysis of both probes (see SI, Figures S17 and S19). The analysis
also showed the formation of the Nq-SH-Cm intermediate with one –SH
μ
2
Fig. 4. (A) The time-dependent fluorescence intensity at 448 nm for Nq-2Cm (2 M) in the presence of different concentrations of Na S in PB buffer (0.1 M, pH 7.4,
containing 30% MeCN, v:v), (B) The linear relationship of kobs versus Na
2
S concentrations.
5

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Fig. 5. (A), (B) Absorption spectral change of Nq-Cm and Nq-2Cm (20
μ
M) response to various concentration of Na
2
S (0–60
S (C), (D) The fluorescence change of Nq-
M) in PB buffer (0.1 M, pH 7,4, containing 30% MeCN) after 15 min of incubation,
excitation is 320 nm, slites 1.0/1.0 nm, Inset: 5 (A), (B) The plot of absorbance changes at 325 and 279 nm upon addition of Na S; (C), (D), fluorescence intensity
changes at 448 nm of Nq-Cm and Nq-2Cm as a function of Na S concentrations. Photos of the quartz cuvettes before and after the addition of Na S under UV light.
μ
M) in PB buffer (0.1 M, pH 7,4,
containing 30% MeCN, v/v) after 15 min of incubation; Photos of the quartz cuvettes before and after the addition of Na
Cm and Nq-2Cm (5 M) response to various concentration of Na
S (0–25
2
μ
2
μ
2
2
2
group (peak at 363.03, C20
H
12
O
5
S).
kinetics, as an important parameter, was only investigated for the Nq-
2Cm probe due to its slower reaction with Na S. The time-dependent
Recorded fluorescence and absorption spectra allow to determine the
time needed to release the maximum amounts of Cm and Nq-2SH from
the studied probes. Fig. 2 reveals that the maximum fluorescence
response of both tested probes occurs in approximately 15 min after the
addition of sodium sulfide. Additionally, after this time, we did not
observe any changes in the absorption spectra.
2
fluorescence at 448 nm was recorded for data analysis (Fig. 4A). The
pseudo-first order rate (kobs), was found by fitting the data with a single
2
ꢀ 1 ꢀ 1
exponential function (Fig. S4). The reaction rate k
2
(7.35 × 10 M
s )
2
was obtained by linear fitting of the kobs versus Na S concentration
(Fig. 4B). The reaction rate between probe and Na
the reaction rate of most fluorescent or colorimetric probes based on
various fluorophores [54,55], implying that Nq-2Cm can scavenge H
2
S is much faster than
The recorded time-dependent fluorescence (Fig. 3) showed that the
maximal fluorescence signals were reached within 15 min with a 32 fold
and 64 fold enhancement for Nq-Cm and Nq-2Cm, respectively. In the
absence of any analyte, no significant change of fluorescence intensity of
Nq-Cm or Nq-2Cm was observed, indicating that both probes possess a
good stability in the system. Our results indicate that sensors may be
2
S
efficiently. Because there are at least two steps in Cm release from the
probe, the rate constant should be treated as an apparent rate constant,
which is influenced by the kinetics of at least two steps of the reaction.
useful for detection of H
2
S.
3.3. Absorption and fluorescence titration
We also tried to determine the pseudo-first-order rate constant by
measuring fluorescent response of Nq-Cm after addition of Na
2
S in
In the next step, we measured the changes of ethers response towards
◦
phosphate buffer (pH 7.4) at 25 C. However, the reaction is too fast to
be measured with the aid of steady-state fluorescence spectra. Reaction
increasing Na S concentration. The absorption spectra were recorded
2
after 15 min of incubation. As depicted in Fig. 5A and B, with increasing
6

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Fig. 6. HPLC chromatogram of the standards (40
μ
M) and reaction mixtures of (A) Nq-Cm (40
μ
M) and (B) Nq-2Cm with Na
2
S (0–160
μ
M) after 15 min of in-
M) with Na S after
cubation. The traces were collected using an absorption detector set at 330 nm. HPLC-based titration of (C) Nq-Cm (40
5 min of incubation in solution. Data are means ± standard deviation of three independent experiments.
μ
M) and (D) Nq-2Cm (40
μ
2
1
Na
2
S concentration, the absorption band at 279 nm slowly decreases and
intensity after reaction of Nq-2Cm with H
fluorescence intensity during Nq-Cm titration with H
gests that two coumarin molecules are released during the reaction of
the Nq-2Cm probe with the analyte (H S).
2
S is twice as high as the
simultaneously the absorption band at 325 nm increases, along with an
appearance of new absorption band at 530 nm. The same absorption
2
S. This may sug-
band was formed when we mixed Nq with Na
2
S (see SI, Fig. S9), whereas
2
an bolus addition of Na S into the solution of Cm did not change its
2
absorption and fluorescence spectra (see SI, Fig. S11). The two-fold and
greater excess of sodium sulfide used during titration do not affect the
absorption spectrum of the Nq-Cm and Nq-2Cm ethers. Fig. 5C and D
show that intensity of fluorescence emission of probes at 448 nm also
3
.4. HPLC titrations
Our spectroscopic investigations showed that the mixing of the
probes with Na S turns on a blue fluorescence and a purple color of the
2
increase when Na
In addition, the fluorescence intensity was linearly connected with
concentrations of Na S ranging from 0 to 10 M for Nq-Cm and from 0 to
M for Nq-2Cm (insets in Fig. 5C and D). The limit of detection (LOD)
2
S was added gradually (0–5 equiv.) into the solution.
solution. Therefore, we conducted HPLC analyses to detect the formed
products and to determine the stoichiometry of the reaction between
2
μ
2
probes and Na S. HPLC chromatograms and stoichiometric analyses are
1
5
μ
shown in Fig. 6. These chromatograms show the slow disappearance of
naphthoquinone ethers (Nq-Cm and Nq-2Cm) and the formation of a
fluorescence product with the retention time of 2.9 min. Comparison of
the retention time of the authentic standard of 7-hydroxy-4-
based on fluorescent measurements was calculated to be 130 nM for Nq-
Cm and 150 nM for Nq-2Cm. These results suggest that the probes are a
2
good quantitative detection tools for H S. Additionally, the fluorescence
7

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
ꢀ
Fig. 7. The fluorescence intensity changes of probe Nq-Cm (5
μ
M) (A) and Nq-2Cm (5
μ
M) (B) upon addition of various species (25
μ
M for each). 1. Blank; 2. Cl ; 3.
ꢀ
ꢀ
2ꢀ ,
2ꢀ
2ꢀ
ꢀ
2ꢀ
Br ; 4. I ; 5. CO
3
6. SO
4
, 7. SO
3
; 8. HSO
3
; 9. S
2
O
4
; 10. L-Cys; 11. L-GSH; 12. NAC. Data are means ± standard deviation of three independent experiments.
methylcoumarin (Cm) confirms that Cm is one of the products found in
the reaction mixture. Additionally, HPLC analysis showed that Cm is not
monitor a Nq-derivative product we also recorded a HPLC trace at 530
nm (Fig. S3). At this wavelength, for both probes only one product (with
a retention time of 5.34 min) was detected, which indicates the forma-
tion of Nq-2SH as probes thiolysis product. Stoichiometric analysis
a sole product of the Na
2
S-induced Nq-Cm or Nq-2Cm conversion. The
new compound with the retention time of 5.34 min was formed. To
Fig. 8. (A), (C) HPLC chromatogram of Nq-Cm, Nq-2Cm (each 60
time was 30 min. The traces were collected using an absorption detector set at 330 nm, (B), (D) Emission of Nq-Cm and Nq-2Cm (each 5
addition of Na S, NaBH , NADPH (each 50
M) after 30 min of incubation in PB buffer (0.1 M, pH 7.4, containing 30% MeCN, v. v). Data are means ± standard
deviation of three independent experiments.
μ
M) and the reaction mixture of probes with Na
2
S, NaBH
4
, NADPH (each 600
μ
M). Incubation
μM) before and after
2
4
μ
8

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
Fig. 9. Absorption spectral changes of Nq-Cm and Nq-2Cm (each 40
μ
M) response to various sulfur species (200
μ
M) after 15 min of incubation.
shows that both probes are completely consumed by Na S. However, the
2
glutathione (L-GSH), L-cysteine (L-Cys) and N-acetyl-L-cysteine (NAC).
This result is not entirely satisfactory, however the good probe should
has a high selectivity and does not react with other compounds. On the
other hand, this result was expected, because L-Cys, L-GSH and NAC
amount of Cm released depends on the chemical structure of the probe.
Mono- and bis-hymecromone naphthoquinone ethers (Nq-Cm and Nq-
2
Cm) produce one and two equivalents of Cm, respectively (Fig. 6C and
D). Moreover, the Nq-Cm probe due to the reactivity of the chlorine
atom in the naphthoquinone ring, firstly form the Nq-SH-Cm interme-
diate followed by released of Cm in the reaction of Nq-SH-Cm with
along with H
2
S, belong to biothiols and have similar properties.
S and biothiols.
Therefore, it is difficult to design a selective probe for H
2
Fluorescence measurements with the aid of Nq-Cm and Nq-2Cm will
report total biothiols, however the usage of colorimetry and/or HPLC-
based monitoring of the Nq-derived product will help identify the ana-
lyte. It is also worth mentioning that hydrogen sulfide, due to its lower
pKa than L-cysteine, reacts faster with the probe, which causes a rapid
Na
equivalents of Na
colored compound, the differences in the stoichiometry of the changes in
2
S. As a result, the release of coumarin from Nq-Cm requires two
2
S. As the Nq-SH-Cm intermediate is nonfluorescent,
2
absorbance (1:1) and fluorescence (1:2) of Nq-Cm in response to Na S
were also observed (Fig. 5A and C).
2
fluorescence response. It may seem that probes for H S based on the
azide reduction mechanism are less sensitive to other sulfur-containing
compounds. The reactive systems used do not change the fluorescence
3
.5. Selectivity of and co-interference studies
intensity after the addition of e.g. L-Cys or H
We also examined how the probes behaved in the presence of a
chemical reductant (NaBH ) and a biological reducing agent (NADPH).
2
S [56].
Promising, described above results, allows to expand investigation
for the selectivity and sensitivity of the Nq-Cm and Nq-2Cm probes. To
study of selectivity the various analytes, including Na S, selected anions,
sulfur species and biothiols, were added to the probes solution and the
fluorescence responses were recorded. As shown in Fig. 7, Na S as ex-
4
It is well known that strong reductants can reduce carbonyl-containing
compounds such as ketones or aldehydes and quinones to alcohols and
hydroquinones [57]. Due to the potential usage of probes in biological
systems, it was necessary to test the stability of the probes in the pres-
ence of these reducing agents. Using HPLC and spectrofluorimetry we
2
2
pected induced obvious fluorescence enhancement. We also observed a
significant fluorescent signal growth in the case of biothiols: L-
Fig. 10. The fluorescence intensity of Nq-Cm and Nq-2Cm (each 5
μ
M) in the absence and presence of Na
2
S (25
μM) in different pH buffer solutions (λex = 320 nm,
slites 1.0/1.0 nm). Data are means ± standard deviation of three independent experiments.
9

D. Słowi n´ ski et al.
Dyes and Pigments 196 (2021) 109765
tested the stability of Nq-Cm and Nq-2Cm in solution with reducing
agents (Fig. 8). The addition of a 10-fold excess of reductants did not
affect the stability of the probes. HPLC chromatograms show the absence
of new product(s) formation, and the recorded fluorescence intensities
were independent of the reductants addition and were not changed
the work reported in this paper.
Acknowledgements
This work was supported by the National Center for Research and
Development (Warsaw, Poland) within the grant InterChemMed
4
during a prolonged incubation time. Thus, NaBH or NADPH had no
effect on the probes.
(
POWR.03.02.00–00-I029/16)
Additionally, we investigated the absorption changes of probes to-
ward important biothiols and sulfur species. Both probes mixed only
Appendix A. Supplementary data
2
with Na S give a new absorption band at 530 nm, that results as the
violet color of the solution (Fig. 9). This is a satisfactory result because
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109765.
the Nq-Cm and Nq-2Cm are colorimetrically selective for biothiols and
sulfur-containing anions. However, only for Na
2
S appearance of new
S from
absorption bands at 530 nm was observed, which distinguishes H
2
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