Salivary hydrogen sulfide measured with a new highly sensitive self-immolative coumarin-based fluorescent probe

Article abstract of DOI:10.3390/molecules23092241

Ample evidence suggests that H₂S is an important biological mediator, produced by endogenous enzymes and microbiota. So far, several techniques including colorimetric methods, electrochemical analysis and sulfide precipitation have been developed for H₂S detection. These methods provide sensitive detection, however, they are destructive for tissues and require tedious sequences of preparation steps for the analyzed samples. Here, we report synthesis of a new fluorescent probe for H₂S detection, 4-methyl-2-oxo-2H-chromen-7-yl 5-azidopentanoate (1). The design of 1 is based on combination of two strategies for H₂S detection, i.e., reduction of an azido group to an amine in the presence of H₂S and intramolecular lactamization. Finally, we measured salivary H₂S concentration in healthy, 18–40-year-old volunteers immediately after obtaining specimens. The newly developed self-immolative coumarin-based fluorescence probe (C₁₅H₁₅N₃O₄) showed high sensitivity to H₂S detection in both sodium phosphate buffer at physiological pH and in saliva. Salivary H₂S concentration in healthy volunteers was within a range of 1.641–7.124 μM.

Full text of DOI:10.3390/molecules23092241

molecules
Article
Salivary Hydrogen Sulfide Measured with a New
Highly Sensitive Self-Immolative Coumarin-Based
Fluorescent Probe
Ewelina Zaorska 1, Marek Konop 1, Ryszard Ostaszewski 2, Dominik Koszelewski 2,
*
and Marcin Ufnal 1,
*
ID
1
Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical
Research, Medical University of Warsaw, 02-097 Warsaw, Poland; ewelina.zaorska@gmail.com (E.Z.);
konopmarek@gmail.com (M.K.)
Institute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland;
ryszard.ostaszewski@icho.edu.pl
2
*
Correspondence: medyk20@gmail.com (D.K.); mufnal@wum.edu.pl (M.U.); Tel.: +22-57-20-734 (M.U.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 July 2018; Accepted: 28 August 2018; Published: 3 September 2018
Abstract: Ample evidence suggests that H2S is an important biological mediator, produced by endogenous
enzymes and microbiota. So far, several techniques including colorimetric methods, electrochemical
analysis and sulfide precipitation have been developed for H2S detection. These methods provide
sensitive detection, however, they are destructive for tissues and require tedious sequences of
preparation steps for the analyzed samples. Here, we report synthesis of a new fluorescent probe
for H2S detection, 4-methyl-2-oxo-2H-chromen-7-yl 5-azidopentanoate (1). The design of 1 is
based on combination of two strategies for H2S detection, i.e., reduction of an azido group to
an amine in the presence of H2S and intramolecular lactamization. Finally, we measured salivary
H2S concentration in healthy, 18–40-year-old volunteers immediately after obtaining specimens.
The newly developed self-immolative coumarin-based fluorescence probe (C15H15N3O4) showed
high sensitivity to H2S detection in both sodium phosphate buffer at physiological pH and in saliva.
Salivary H2S concentration in healthy volunteers was within a range of 1.641–7.124 µM.
Keywords: fluorescent probe; hydrogen sulfide; azide; assay; Ellman’s Reagent; biological systems;
saliva; halitosis
1. Introduction
Ample evidence shows that H2S plays a role of a mediator in many biological systems.
For example, H2S has been found to contribute to the regulation of the circulatory system [
nervous system [ ], reproductive system [ ] and energy balance [ 10]. In mammalian tissues, H2S
is generated endogenously from cysteine and homocysteine. There are at least three enzymes that
are responsible for converting sulfur-containing molecules into H2S: cystathionine -synthase (CBS),
cystathionine -lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST) [11 12]. Furthermore,
1–3]
4,
5
6–
8
9,
β
γ
,
H2S is generated in large amounts by microbiota, which is present in the intestines and in the
oral cavity. On the one hand, microbiota-produced H2S seems to play a significant physiological
role in cardiovascular and gastrointestinal systems [13–19]. On the other hand, the excessive
bacterial production of H2S may cause medical complaints such as halitosis, a chronic bad breath
condition [20,21].
Fast catabolism and low stability of H2S results in difficulties in the accurate analysis of H2S
concentrations. Several methods have been traditionally employed for H2S detection, including
colorimetric and electrochemical assays [22], gas chromatography and sulfide precipitation [23,24].
Molecules 2018, 23, 2241; doi:10.3390/molecules23092241
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2241
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Most of these techniques require lengthy storing and/or complicated processing of analyzed
sample. Therefore, new methods that will be useful for rapid and selective evaluation of H2S
concentration in biological systems are highly desired. These requirements may be met by techniques
employing fluorescent probes, which do not involve sophisticated sample processing and chemical
treatment [25].
The goal of the study was to synthesize the probe that: (i) is facile to synthesize with
an easy purification procedure (ii) acts fast (within seconds, considering real-time imaging of
H2S-related biological processes), (iii) is chemically stable for long-term storage, (iv) shows a linear
concentration–signal relationship within physiologically relevant H2S concentrations (v) is stable in
aqueous solutions, especially in physiological pH of a body fluids. Finally, in order to confirm the
usability of the designed 4-methyl-2-oxo-2H-chromen-7-yl 5-azidopentanoate (1) for the detection of
H2S in biological samples, we aimed to establish salivary H2S concentration in healthy volunteers.
2. Results and Discussion
Here, we have designed and synthesized a new fluorescent probe (C13H11N3O4, compound 1)
based on a coumarin scaffold. The operation of compound
1 is based on the azide group to amine
group reduction mediated by H2S in combination with spontaneous intramolecular lactamization.
1
The developed probe was characterized by H-NMR, 13C-NMR (The Supplementary Materials)
Compound
detection. Compound
1
showed dhe esired characteristics for a highly sensitive fluorescent probe for H2S
showed a good aqueous solubility and worked an an optimal pH 7.0,
1
≈
the pH of most of mammalian body fluids. Data analysis revealed a linear relationship between
the fluorescence signal and the concentration of aqueous solutions of NaHS, a commonly used
H2S donor. Finally, the compound
1 was synthesized from commercially available reagents in a
straightforward procedure.
Currently, several methods for H2S detection are used, i.e., colorimetric and electrochemical
assays and metal-induced sulfide precipitation. Despite the many advantages of the abovementioned
methods their widespread use in biological systems is limited. This is due to the complex, multistep
mechanisms of H2S detection, a slow response time, poor water solubility and poor stability in aqueous
solutions of the reagents, and non-physiological pH of the reaction environment. Moreover, some of
those methods require complicated sample processing steps and the destruction of cells or tissues.
During the last years fluorescence-based probes have been attracting increasing interest as a method
for H2S detection. The fluorescence-based assays for H2S detection offer high selectivity, sensitivity
and biocompatibility, less invasiveness and enable real-time imaging [26–28]. Various fluorescence
methods for H2S detection have been elegantly reviewed by Guo et al. [29]. The synthesis and design
strategies of fluorescent probes are based on the use of specific chemical reactions and the use of
several characteristic properties of H2S. The most commonly used strategy for designing fluorescent
probes is the reduction of azide or nitro groups to amine groups [30–34]. Self-immolative probes
based on coumarin were designed and synthesized by Han and co-workers [35]. Based on a similar
strategy, Zhao and Song demonstrated a series of probes with para-azidobenzyl group attached to the
1,8-naphthalimide [36,37]. Other methods are based on unique dual nucleophilic reactions [25, ,39],
38
high binding affinity towards copper ions [40
double bond [43–45].
–42] and a specific addition reaction to unsaturated
The evaluation of H2S concentration, or more precisely, free sulfhydryl group concentration
may also be performed using Ellman’s reagent, i.e., 5,50-dithiobis(2-nitrobenzoic acid), often referred
to as DTNB [46–48]. However, the latter method requires alkaline conditions (pH 8.0) and the test
absorbance response is obtained no sooner than after 15 min. Therefore, despite the progress in the
field of detection methods, further development of highly sensitive and selective fluorescent probes for
H2S detection is still needed to provide valuable information on the functions of H2S in physiological
and pathological processes.

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Saliva is a promising and increasingly used biological material for clinical investigations [49,50].
H2S in saliva may originate from its endogenous synthesis in tissues and from oral microbiota activity.
The excessive concentration of H2S in the saliva is associated with halitosis [20,51,52].
In our study, using the newly synthesized probe we showed that the concentration of H2S in
saliva of healthy 20–40-year-old humans is in the range between 1.641 and 7.124 µM (Table 1).
Table 1. Salivary H2S concentration in healthy volunteers.
Samples of Saliva (n = 15)
Range
1.641–7.124
Mean
3.424
SE
±0.547
H2S concentration [µM]
Our results are comparable to previously reported ones, however slightly higher [53–55].
Generally, the analysis of H2S is a tricky procedure because of the instability of H2S, its high
volatility and rapid oxidation. This can lead to falsely elevated or decreased H2S concentrations.
The most used methods for H2S detection are colorimetric assays (mainly the methylene blue method),
high-performance liquid chromatography and gas chromatography [52]. However, there is much
doubt about the reliability of abovementioned methods. Differences between the above methods
and the fluorescence method using our probe include different duration of sample preparation and
processing, and different measurement conditions. In our study, the collected saliva samples were
tested instantly, whereas in other studies the samples were subjected to lengthy processing or storage.
For example, in studies by Kaneshiro et al. and Ritz et al. saliva was collected by holding a cotton swab
in the mouth for a few minutes [53,54]. Other methods require a chemical treatment with strong acid or
base before analysis of H2S [36]. Some of those treatments can lead to falsely elevated or decreased H2S
levels and/or cause irreversible destruction of the analyzed sample. For example, the methylene blue
method uses acidic conditions (pH = 2) in which so-called acid labile sulfides (ALS) are formed. This
contributes to falsely high H2S level readings. As pointed out by Siegel and Kanehira the methylene
blue method may be disturbed by interference with other colored substances that interfere with the
measurements, lowering the sensitivity of this method [55]. In contrast to the methylene blue method
our measurements do not require any chemical pretreatment of the sample. Moreover, compound
1
works in an aqueous medium at pH = 7.4. Finally, the methylene blue method is a single point assay
and does not monitor the H2S concentration in real time. There are also doubts about the repeatability
of this method [56]. Another disadvantage of currently used methods is their long incubation periods,
which are needed to achieve detection [57]. Ritz et al. analyzed H2S concentrations with the fluorescent
probe SF4 which required up to 45 min of incubation with a chemosensor. Considering that H2S is
a very volatile compound H2S concentrations may decrease significantly during such a long sample
processing time.
3. Materials and Methods
3.1. Materials and Instruments
Unless noted otherwise, reagents and solvents for synthesis were obtained from commercial
suppliers and employed without further purification. Commercial reagents for quantitating sulfhydryl
groups were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Buffer reagents
were purchased from Sigma Aldrich (Saint Louis, MO, USA) and were used without purification.
All spectroscopic measurements were performed in 0.1 mM sodium phosphate buffer (pH 7.4) or
0.1 M sodium phosphate buffer (pH 8.0). Silica gel P60 (SiliCycle, Québec, QC, Canada) was used
for column chromatography and SiliCycle 60 F254 silica gel (precoated sheets, 0.25 mm thick) was
used for analytical thin layer chromatography and visualized by fluorescence quenching under UV
light. UV/Vis spectra were recorded at ambient temperature using a U-1900 spectrophotometer

Molecules 2018, 23, 2241
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(Hitachi, Chiyoda, Tokyo, Japan) and quartz cuvettes. Fluorescence spectra were recorded at ambient
temperature in quartz cuvettes using a F7000 spectrofluorometer (Hitachi).
3.2. Synthesis and Sensing Mechanisms
In the design of the probe for H2S we used 7-hydroxy-4-methylcoumarin as a fluorophore
due to its good stability and desirable spectroscopic properties, such as large absorption extinction
coefficients, sharp fluorescence emissions and excitation and emission in visible region [58,59].
The fluorescence of 7-hydroxy-4-methylcoumarin can be easily controlled by modification of
hydroxyl group causing changes of physical and chemical properties and fluorescence quenching.
Our probe operates by H2S-mediated reduction of azide group, which generates a primary
amine, that can subsequently undergo spontaneous intramolecular lactamization to release
7-hydroxy-4-methyl-coumarin and piperidin-2-one (Figure 1). Our scientific concept is analogous
to studies reported by Zadlo-Dobrowolska et al. for self-immolative carbonate-based probes [60].
Moreover, in comparison to other fluorogenic assays, self-immolative probes provide a more stable
signal with higher signal to noise ratio.
N
O
H
O
H2S
O
H2N
HO
O
O
O
O
O
N3
n
O
O
O
n
1: n=4
2: n=2
intramolecular lactamization
no fluorescence
strong fluorescence
7-hydroxy-4-methylcoumarin
Figure 1. Mechanism of H2S detection for the self-immolative probe.
To confirm the proposed mechanism, the reaction solution was analyzed by high resolution mass
spectrometry (HRMS) and NMR analysis. MS and NMR spectra confirmed formation of piperidin-2-one as
a product of intramolecular lactamization. A major peak located at 122.2 corresponding to piperidin-2-one
(C5H9NO, [M + Na]+: 122.07) was observed (Figure 2, the supplementary materials).
Figure 2. HRMS confirmed formation of piperidin-2-one in the reaction of the compound 1 with NaHS.

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In order to check the validity of the proposed mechanism we synthesized 4-methyl-2-oxo-2H-
chromen-7-yl 3-azidopropanoate ( ) and compared the results of fluorometric measurements for the
2
4-methyl-2-oxo-2H-chromen-7-yl 5-azidopentanoate (
under the same reaction conditions. For compound
1
2
) with the results obtained for compound
a much lower fluorescence response was
(Figure 3A). The hydrolytic
is further enhanced by close location of an electron-acceptor azide
group in relation to the ester bond in the 7-position of 4-methylcoumarin. In addition, NMR and MS
analysis of the assay solution was performed. In contrast to compound , the 4-membered product of
due to hydrolysis of the
2
recorded, which could be due to progressive autohydrolysis of compound
decomposition of the compound
2
1
1
intramolecular lactamization was absent in the assay solution of compound
ester bond.
2
To confirm the proposed mechanism of compound
chromen-7-yl propionate ( ) was synthesized and tested in parallel under the same conditions.
The analysis of reaction solution of compound after addition of NaHS by fluorometry showed
a minimal fluorescence enhancement (Figure 3B). In this case minimum fluorescence was caused
the hydrolysis of the ester bond. The observed lower fluorescence enhancement of compound in
comparison to , could result from the absence of the azide group in the structure of compound 3
The different responses of 4-methyl-2-oxo-2H-chromen-7-yl propionate ( ) and compound
1 in sensing H2S, 4-methyl-2-oxo-2H-
3
3
3
2
.
3
1
highlighted a key role of the azide moiety for the H2S detection mechanism. NMR and MS analysis
confirmed the absence of the lactamization product in the reaction mixture.
500
(B)
450
400
350
300
250
200
150
100
50
1600
1400
1200
1000
800
600
400
200
0
(B)
(A)
(C)
(A)
0
375 395 415 435 455 475 495 515 535
1
2
3
4
5
6
7
8
9
10
Time [min]
λ [nm]
Figure 3. Time course experiment of compound
2 (0.1 mM, A) and 3 (0.1 mM, B) reacting with NaHS
in sodium phosphate buffer (pH = 7.4) at room temperature. Time points represent time range from 1
to 10 min after addition of NaHS (0.1 mM). Fluorescence spectra of compound
were recorded for 10 min after addition of NaHS (0.1 mM).
1 (C), 2 (A) and 3 (B)
To sum up, the compound 1 can be used for the determination of H2S levels. Furthermore, it is
characterized by a high stability and the lack of susceptibility to autohydrolysis. We showed that
the distance of the reaction site and thus the azide group as the electron-acceptor group from the
fluorophore reduces the susceptibility to autohydrolysis (Figure 4). The obtained results confirm
proposed mechanism of H2S detection for the compound 1. Detection of H2S was achieved by the
reduction of azide group mediated by H2S to amine group, then intramolecular lactamization with
simultaneous release of highly fluorescent 7-hydroxy-4-methylcoumarin.
Firstly, intermediates 3-azidopropanonic acid and 5-azidopentanonic acid were obtained by
reacting suitable ester 3-chloropropionate (a) or ester 5-bromopentanoate (b) with sodium azide in
H2O (Figure 5). The compound
1 and the compound 2 were synthesized from the corresponding
commercially available fluorescent 7-hydroxy-4-methylcoumarin (Figure 6).

Molecules 2018, 23, 2241
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1600
1400
1200
1000
800
600
400
200
0
1
Compound 3
Compound 2
Compound 1
Figure 4. Comparison of fluorescence responses for the compound
1, 2 and 3 (0.1 mM). Data were
acquired at room temperature after addition of NaHS (0.1 mM) in sodium phosphate buffer (pH = 7.4,
20% CH3CN) with excitation at 365 nm. Means
responses are presented.
±
SE from three measurements of the fluorescence
Figure 5. The synthesis of 3-azidopropanonic (a) acid and 5-azidopentanonic acid (b).
3.3. Synthesis of 3-Azidopropanoic Acid and 5-Azidopentanoic acid
A solution of sodium azide (4 equiv.) in 10 mL water was added dropwise into the ester
3-chloropropionate or ester 5-bromopentanoate (1 equiv.). The reaction mixture was stirred at room
temperature for 7 days. After this time the resulting reaction mixture was acidified with solution of HCl
(1 M). Then, the mixture was extracted with ethyl acetate for several times. The combined organic layers
were dried over anhydrous MgSO4 followed by filtration and concentrated under reduced pressure.
The obtained product was used for the next reaction without purification. Then, the azide-probe
(1) and compound 2 was readily synthesized by esterification of 7-hydroxy-4-methylcoumarin with
suitable azido acid in CH2Cl2, under a room temperature as shown in Figure 6.
Figure 6. The synthesis of compounds 1 (a) and 2 (b).

Molecules 2018, 23, 2241
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In turn 4-methyl-2-oxo-2H-chromen-7-yl propionate (
3) was synthesized from the corresponding
commercially available 7-hydroxy-4-methylcoumarin with butyric acid in CH2Cl2, under room
temperature as shown in Figure 7.
Figure 7. The synthesis of 4-methyl-2-oxo-2H-chromen-7-yl propionate (3).
3.4. Synthesis of 4-Methyl-2-oxo-2H-chromen-7-yl 5-Azidopentanoate (1)
5-Azidopentanoic acid (1.2 equiv.), 7-hydroxy-4-methylcoumarin (1 equiv.), and a catalytic amount
of N,N-dimethylpyridin-4-amine (DMAP) were dissolved in dry CH2Cl2 (15 mL). Then DCC (2 equiv.)
was added. The reaction mixture was stirred at room temperature for 12 h. The reaction was monitored
by thin layer chromatography (TLC). After the reaction was completed, the precipitate was filtered and
washed several times with CH2Cl2. The filtrate was concentrated under reduced pressure. The crude
product was purified by silica gel chromatography (ethyl acetate/n-hexane 3:7) to obtain the pure
1
product as white solid (80% yield). H-NMR (400 MHz, CDCl3)
δ 7.57 (d, J = 8.6 Hz, 1H), 7.13–6.93 (m,
2H), 6.22 (s, 1H), 3.33 (t, J = 6.6 Hz, 2H), 2.62 (t, J = 7.3 Hz, 2H), 2.39 (d, J = 1.1 Hz, 3H), 1.82 (dt, J = 12.0,
7.1 Hz, 1H), 1.76–1.64 (m, 1H); 13C-NMR (100 MHz, CDCl3)
δ
170.94, 160.38, 154.14, 153.01, 153.01,
151.93, 125.41, 117.99, 117.80, 114.45, 110.31, 77.43, 77.11, 76.79, 50.98, 33.65, 28.17, 21.93, 18.64 ppm.
Element. Anal. calcd. for C15H15N3O4: C 59.80, H 5.02, N 13.95; found C 59.72, H 4.94, N 13.84.
3.5. Synthesis of 4-Methyl-2-oxo-2H-chromen-7-yl 3-Azidopropanoate (2)
3-azidopropanoic acid (1.2 equiv.), 7-hydroxy-4-methylcoumarin (1 equiv.) and DMAP (a catalytic
amount) were dissolved in dry CH2Cl2 (15 mL). Then DCC (2 equiv.) was added. The reaction mixture
was stirred at room temperature for 12 h. The reaction was monitored by thin layer chromatography
(TLC). After the reaction was completed, the precipitate was filtered and washed several times with
CH2Cl2. The filtrate was concentrated under reduced pressure. The crude product was purified by
silica gel chromatography (ethyl acetate/n-hexane 3:7) to obtain the pure product as white solid (72%
1
yield). H-NMR (400 MHz, CDCl3)
δ 7.59 (d, J = 8.6 Hz, 1H), 7.15–7.02 (m, 2H), 6.24 (d, J = 0.8 Hz, 1H),
3.69 (t, J = 6.4 Hz, 2H), 2.86 (t, J = 6.4 Hz, 2H), 2.41 (d, J = 0.9 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
168.88, 160.32, 154.15, 152.69, 151.86, 125.49, 118.03, 117.89, 114.62, 110.30, 46.55, 34.17, 18.66 ppm.
δ
Element. Anal. calcd. for C13H11N3O4: C 57.14, H 4.06, N 15.38; found C 57.08, H 4.02, N 15.29.
3.6. Synthesis of 4-Methyl-2-oxo-2H-chromen-7-yl Propionate (3)
Butyric acid (1.2 equiv.), 7-hydroxy-4-methylcoumarin (1 equiv.), and DMAP (a catalytic amount)
were dissolved in dry CH2Cl2 (15 mL). Then DCC (2 equiv.) was added. The reaction mixture was
stirred at room temperature for 12 h. The reaction was monitored by thin layer chromatography (TLC).
After the reaction was completed, the precipitate was filtered and washed several times with CH2Cl2.
The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel
chromatography (ethyl acetate/n-hexane 2:8) to obtain the pure product as a white solid (92% yield).
1H-NMR (400 MHz, CDCl3)
2H), 2.40 (d, J = 0.8 Hz, 3H), 1.26 (t, J = 7.5 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
δ
7.58 (d, J = 8.6 Hz, 1H), 7.31–6.75 (m, 2H), 6.22 (s, 1H), 2.62 (q, J = 7.5 Hz,
172.19, 160.40,
δ
154.11, 153.20, 151.96, 125.37, 118.04, 117.67, 114.34, 110.30, 77.45, 77.13, 76.81, 27.69, 18.63, 8.88 ppm.
Element. Anal. calcd. for C13H12O4: C 67.23, H 5.21; found C 67.19, H 5.17.

Molecules 2018, 23, 2241
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3.7. Characterization of the Fluorescence of Compound 1
First, we examined the optical properties of the probe/compound
1
.
Compound
1
was
non-fluorescent in sodium phosphate buffer containing 20% CH3CN at physiological pH 7.4.
The sensing ability of H2S for the compound was investigated using aqueous solutions of NaHS, a H2S
donor. The solution was analyzed by fluorometry and spectra were recorded in selected time-points
after the addition of NaHS. Upon addition of 0.1 mM NaHS, the solution of the compound showed a
1
1
strong fluorescence enhancement, as expected. A strong emission peak at 445 nm was detected when
the reaction mixture was excited at 365 nm. The fluorescence intensity was dramatically increased
due to the reduction of azide group to amine by H2S, intramolecular lactamization and the release of
highly fluorescent 7-hydroxy-4-methylcoumarin. Within 10 min of reaction with NaHS (100 µM) the
compound 1 generated an over 1000-fold fluorescence enhancement (Figure 8).
1800
1600
1400
1200
1000
800
600
400
200
0
10 min
9 min
8 min
7 min
6 min
5 min
4 min
3 min
2 min
1 min
0 min (without the addition of NaHS)
375
395
415
435
455
475
495
515
535
λ [nm]
Figure 8. Fluorescence response of the self-immolative probe/compound
1
(0.1 mM) to 0.1 mM NaHS.
Data were acquired at room temperature in sodium phosphate buffer (pH = 7.4) with excitation at
365 nm. Emission was collected in the time range 1–10 min after the addition of 0.1 mM NaHS.
The spectrum at t = 0 min was acquired from a 0.1 mM solution of the compound
addition of NaHS.
1
without the
We examined the time courses of the fluorescence intensities of compound
1
in the presence
of 100 µM NaHS. The time courses of the fluorescence intensities of the compound 1 (0.1 mM) in
the presence of NaHS (0.1 mM) in sodium phosphate (pH = 7.4) buffered acetonitrile (20%, v/v) is
displayed in Figure 9. The fluorescence signal increased rapidly at the beginning and reached steady
state at around 10 min. When we extended reaction time to 60 min, the fluorescent intensity increased
insignificantly, thus we chose 10 min as a test time.

Molecules 2018, 23, 2241
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1700
1600
1500
1400
1300
1200
1100
1000
900
800
700
0
10
20
30
40
50
60
Time [min]
Figure 9. Time course experiment of the compound
1 (0.1 mM) reacting with NaHS (0.1 mM) in sodium
phosphate buffer (pH = 7.4) at room temperature. Time points represent time range from 1 to 60 min
after addition of NaHS (0.1 mM). Means
are presented.
±
SE from three measurements of the fluorescence responses
To evaluate the compound
examined the reactivity of the compound
buffered acetonitrile (20% v/v CH3CN, pH = 7.4) at room temperature. NaHS (NaHS concentration
from 20 M up to 100 M) was added to the test solution of the compound 1 (0.1 mM). As shown
in Figure 10, we observed almost the linear relationship of fluorescence intensity of the compound
1
for feasibility of quantitative determination of H2S concentration we
1
in different concentrations of NaHS in sodium phosphate
µ
µ
1
against varying concentrations of NaHS. The regression analyses was: FEx
7.927[H2S] + 666.62 with R2 = 0.985 in 10 min of incubation with NaHS.
(365/445 nm) =
/Em
1700
y = 7.927x + 666.62
R² = 0.9852
1500
1300
1100
900
700
500
20
40
60
Concentration of NaHS (μM)
80
100
Figure 10. The correlation between fluorescence intensity and NaHS concentration determined using
a fluorometer: the compound (0.1 mM) with NaHS (20–100 M) in sodium phosphate buffer
1
µ
(λ = 365 nm) at room temperature. The points represent the mean fluorescence responses at 10 min
ex
after the addition of NaHS.

Molecules 2018, 23, 2241
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3.8. Quantification of H2S Concentration Using Ellman’s Reagent (5,5-Dithiobis(2-Nitrobenzoic Acid) and the
Developed Probe
We determined H2S levels in 20 µM up to 100 µM NaHS solution using DTNB method and our
probe. DTNB assay was performed according to the protocol provided by the manufacturer (catalog
number: 22,582, Thermo Fisher Scientific, Waltham, MA, USA).
Figure 11 shows time-dependent UV-vis absorption spectra of SH-free DTNB solution (green
line-background) and its mixture with NaHS (0.1 mM, orange line). After 15 min incubation,
the effect reaction of DTNB with NaHS on absorption spectra was observed as gain of TNB2−
(2-nitro-5-thiobenzoate anion) and a loss of DTNB, respectively. We chose 15 min as a test time,
because after this time we did not observe any increase in the intensity of absorbance, 15 min is also the
incubation time which is required for the measurement procedure recommended by the manufacturer.
3
2.5
DTNB
2
1.5
1
0.5
0
background
15 min
TNB2- (412 nm)
300
325
350
375
400
425
450
475
500
525
550
575
600
λ [nm]
Figure 11. Time-dependent UV-vis absorption spectra of SH-free DTNB solution (green line-
background) and its mixture with NaHS (0.1 mM, orange line) in the reaction buffer (pH 8.0) at
room temperature. The absorbance at 412 nm was recorded 15 min after addition of NaHS.
To determine relationship between changes of absorbance and concentration of NaHS,
we recorded the absorbance in different concentrations of NaHS aqueous solution. Concentration of
NaHS from 20 µM up to 100 µM, were used. As shown in Figure 12, we observed an increase in the
absorbance along with increasing NaHS concentration.
1.6
1.4
1.2
1
y = 0.0111x + 0.2954
R² = 0.987
0.8
0.6
0.4
0.2
0
20
40
60
80
100
Concetration NaHS [μM]
Figure 12. The correlation between absorbance and NaHS concentration determined by the DTNB
assay in sodium phosphate buffer (pH 8.0) at room temperature. The absorbance at 412 nm was
recorded for 15 min after addition of various concentrations of NaHS from 20 to 100 µm. Values of
absorbance are given as means obtained from 3 measurements. Background values were subtracted
from the sample values.

Molecules 2018, 23, 2241
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Table 2 summarizes the results of H2S detection (aqueous solution of NaHS, a H2S donor) which
have been obtained by Ellman’s test and by fluorescence method using our probe. In Table 2 are shown
the results of H2S detection (aqueous solution of NaHS, a H2S donor) which have been obtained by
Ellman’s test and by fluorescence method using our probe.
Table 2. Comparison of detection methods for H2S.
Fluorescence Method Using the
Ellman’s Test (µM) a
Concentration of
Compound 1 (µM) b
NaHS [µM]
−1
−1
]
C H2S[µM]
V [µM s
]
C H2S[µM]
V [µM s
20
40
60
80
100
15.82
36.91
57.63
78.25
94.52
0.018
0.041
0.064
0.087
0.105
17.00
38.15
58.10
78.60
97.23
0.028
0.064
0.097
0.13
0.16
a
Conditions: 10 mM Ellman’s Reagent Solution in sodium phosphate buffer (pH 8.0), room temperature,
the absorbance at 412 nm was recorded 15 min after addition of NaHS. b Conditions: 0.1 mM the fluorogenic probe
in sodium phosphate buffer (pH 7.4), room temperature, the fluorescence intensity was recorded 10 min after the
addition of NaHS.
3.9. H2S Detection in Saliva
To determine whether the novel compound
in a biological sample we performed competition experiments in 15 samples of saliva. Additionally,
we plotted the calibration curve for the compound 1 in range concentrations of NaHS from 1
up to 10 M. As shown in Figure 13, we observed almost the linear relationship of fluorescence
intensity of the compound in this range concentration of NaHS. The regression analyses were:
FEx
(365/445 nm) = 60.405[H2S] + 240.7 with R2 = 0.9987. The obtained fluorescence data for
1 can be used for the determination of H2S concentrations
µM
µ
1
/Em
saliva samples were converted into H2S concentrations by means of a calibration curve. The obtained
results measured by fluorescence method with the compound
in Table 1.
1 for 15 samples of saliva are presented
1000
900
800
700
600
500
400
300
200
y = 60.405x + 240.7
R² = 0.9987
1
2
3
4
5
6
7
8
9
10
Concentration of NaHS (μM)
Figure 13. The correlation between fluorescence intensity and NaHS concentration determined
using a fluorometer: the compound (0.1 mM) with NaHS (1–10 M) in sodium phosphate buffer
1
µ
(λ = 365 nm) at room temperature. The points represent the mean fluorescence responses at 10 min
ex
after the addition of NaHS.

Molecules 2018, 23, 2241
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3.10. General Procedure for H2S Detection by the Fluorescence Method
NaHS solutions with appropriate concentrations were prepared using sodium phosphate buffer
as a solvent. For the assay, the various volumes of NaHS solution were added respectively to solution
of 1, 2 and 3 in sodium phosphate buffer containing 20% CH3CN (pH = 7.4). The total volume of
the solution being measured was 2000
while various concentration of NaHS were added (in range from 1 to 10
to 100 M). The fluorescence response was monitored over time. Emission spectra were collected
between 350 nm and 550 nm with ex = 365 nm. Time points represent time range from 1 to 10 min after
addition of NaHS. The spectrum at t = 0 min was acquired from a 0.1 mM solution of the compound
µL. The final concentration of the compound
1 was 0.1 mM,
µM and in range from 20
µ
λ
1
without NaHS. Fluorescence data and obtained linear calibration curves were used to calculate the
reaction rate of NaHS with the compound 1 and concentrations of H2S.
3.11. General Procedure for H2S Detection Using the Ellman’s Test
Procedure was carried out quantitating sulfhydryl groups according to the manual attached by
Thermo Fisher Scientific (Catalog number: 22,582). The procedure for the quantification of sulfhydryl
groups was performed according to the manual attached by Thermo Fisher Scientific (Catalog number:
22,582).
3.12. General Procedure for H2S Detection in Saliva Using Compound 1
In these experiments an appropriate sample of saliva (0.4 mL) were added to solution of the
compound
concentration of the compound 1 was 0.1 mM. The total volume of the solution being measured
was 2000 L. The fluorescence response of the compound was monitored over time with ex = 365 nm
and = 445 nm. The spectrum at t = 0 min was acquired from a solution of the compound without
1 in the in sodium phosphate buffer containing 20% CH3CN (pH = 7.4). The final
µ
1
λ
λ
em
1
saliva. Fluorescence data were converted into H2S concentrations in sample of saliva by means of a
calibration curve.
3.13. Collection of Saliva Samples
The study was performed in compliance with the ethical guidelines of the 1975 Declaration of
Helsinki and was approved by the Bioethics Committee of the Medical University of Warsaw (approval
no. KB/138/2018). Informed consent was obtained in every case. Samples were obtained from 15 adult
volunteers. Demographics and clinical data of the study subjects are listed in Table 3. Inclusion criteria
were as follows: healthy, 18–40 years-old, male and female. Exclusion criteria were as follows: chronic
general diseases, acute general diseases, current dental problems, halitosis, treatment with any drugs
or dietary supplement including probiotics during the last month before the study. Subjects brushed
teeth and did not drink and eat for 90 min before saliva collection. Samples were collected directly to
Eppendorf tubes after short exposition of subjects to the smell of lemon.
Table 3. Demographics and clinical data of the study subjects and H2S levels in the saliva samples
obtained using fluorescence method with the compound 1.
Participant Demographics and Physical Characteristics.
Healthy volunteers (n)
Age (years)
15
Mean
28
SE (standard error)
Range
18–40
1.6
Sex (m/f)
7/8
Ethnicity:
Caucasian
Other
100%
-

Molecules 2018, 23, 2241
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4. Conclusions
We synthesized a new fluorescent, self-immolative probe for H2S detection in biological fluids.
The design of compound was based on the combination of two strategies for H2S detection, i.e., reduction
of an azido group to an amine in the presence of H2S and spontaneous intramolecular lactamization.
The compound showed several characteristics that are desirable for evaluation of H2S concentration
1
1
in biological systems and human body fluids, including straightforward synthesis, stability, reactivity
in aqueous media at physiological pH and fast response time. Finally, we measured salivary H2S
concentration in healthy, 18–40-year-old volunteers immediately after obtaining specimens. Salivary H2S
concentration in healthy humans was within a range of 1.641–7.124 µM.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: Conception or design of the work: E.Z., R.O., D.K., M.U. Performing analyzes or
interpretation of data for the work: E.Z., M.K., D.K., M.U. Drafting the work: E.Z., D.K., M.U. All authors
have approved the final version of the manuscript and agreed to be accountable for all aspects of the work.
All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.
The authors declare no conflict of interest.
Funding: This work was supported by the National Science Centre, Poland grant no. UMO-2016/22/E/NZ5/00647.
Acknowledgments: The experiments were performed at the Institute of Organic Chemistry Polish Academy of
Sciences, Warsaw, Poland and the Laboratory of Experimental Physiology and Pathophysiology, Laboratory of the
Centre for Preclinical Research, Medical University of Warsaw, Poland.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
Abbreviations
ALS
Acid Labile Sulfide
CDCl3
CH2Cl2
CH3CN
CLSS
Deuterated chloroform
Dichloromethane
Acetonitrile
ChemiLuminescent Sulfide Sensors
13C-NMR Carbon-13 nuclear magnetic resonance
CuS
Copper sulfide
CTAB
DCC
DMAP
DTNB
HCl
Cetyltrimethylammonium bromide
N,N -Dicyclohexylcarbodiimide
0
N,N-Dimethylpyridin-4-amine
0
5,5 -Dithiobis(2-nitrobenzoic acid)
Hydrochloric acid
1H-NMR
HRMS
H2S
Proton nuclear magnetic resonance
High Resolution Mass Spectrometry
Hydrogen sulfide
MgSO4
mM
MS
Magnesium sulfate
Millimolar concentration
Mass Spectrometry
NaHS
R2
SE
Sodium hydrosulfide
Coefficient of determination
Standard Error
SF
Sulfur fluride
TLC
Thin layer chromatography
2-Nitro-5-thiobenzoate anion
Ultraviolet–visible spectroscopy
Excitation wavelength
Emission wavelength
TNB2−
UV-vis
λ
ex
λ
em

Molecules 2018, 23, 2241
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Sample Availability: Samples of the compounds 3-azidopropanonic acid, 5-azidopentanonic acid, 4-Methyl-2-
oxo-2H-chromen-7-yl 5-Azidopentanoate (1), 4-Methyl-2-oxo-2H-chromen-7-yl 3-Azidopropanoate (2) and
4-Methyl-2-oxo-2H-chromen-7-yl Propionate (3) are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).