The synthesis, crystal, hydrogen sulfide detection and cell assement of novel chemsensors based on coumarin derivatives.

A series of chemsensors (1-4) containing fluorobenzene group based on coumarin derivatives have been developed for the selective and sensitive detection of H2S. The advantages of the synthesized fluorescent probe (compound 1) were the low detection limit (4 × 10-6 mol·L-1), good selectivity and high sensitivity which had been demonstrated through UV-vis, fluorescent titration experiments. Besides cytotoxicity test of compounds (1 and 2) was studied and the results indicated that compounds (1 and 2) showed almost no cytotoxicityat at a concentration of 150 μg·mL-1. The interacted mechanism was the thiolysis reaction of dinitrophenyl ether which had been confirmed by fluorescence and HRMS titration experiment. In addition, probe 1 can also detect HS- selectively by naked eye in pure DMSO solvent.

Introduction

In the past decade, we has seen a boost of research interest in hydrogen sulfide (n class="Gene">H2S), a colorless, flammable, toxic gas with unpleasant smell, which is recognized as a signal gasotransmitter in the body as same as nitric oxide (NO)[1-10] and carbon monoxide (CO)[11]. Endogenous concentration of H2S is related to some diseases such as Alzheimer’s disease, Down syndrome, liver cirrhosis and diabetes[2,9,12-17]. What’s more, the regulation of H2S levels is also a potential drug development strategy[18,19] and the importance of accurate detection of H2S cannot be over-emphasized. Therefore, it presents significant research related to track and quantify H2S inside living cells being crucial in order to understand the biological and pathological roles of H2S. Recently, some methods to determine H2S concentration in biological sample have been developed including the methylene blue, the monobromobimane (MBB), gas chromatography (GC), the sulphide ion selective electrodes (ISE) and fluorescent analysis[20-23]. Among these methods, fluorescent analysis has attracted great attention due to the high sensitivity and selectivity for the detection of H2S in many fields such as environment area, pharmacy area and so on[24-31].

In the present work, a series of “OFF-ON” probes based on n class="Chemical">coumarin derivatives to detect HS− (Fig. 1). The results of UV-vis titration experiments indicated that the synthesized compounds showed high binding ability for HS− among the tested anions (NaHS (HS−), (n-C4H9)4NAcO (AcO−), (n-C4H9)4NH2PO4 (H2PO4−), (n-C4H9)4NF (F−), (n-C4H9)4NCl (Cl−), (n-C4H9)4NBr (Br−), (n-C4H9)4NI (I−)) and amino acids (Glutathione (GSH), Cysteine (Cys), Homocysteine (Hcy)). Besides, four compounds designed and synthesized could exhibit the probes with strong electron-withdrawing groups located in 2,4-positions of fluorobenzene have strong binding ability for HS− detection which provides a good idea for the design of probe in future.

Figure 1

The synthesis route of the probes (1–4).

Results and Discussion

X-ray crystallography

Compound 2 was synthesized acn class="Chemical">cording to the route shown in Fig. 1. Fortunately, the crystallographic of compound 2 was obtained by the standing method. The suitable single light yellow crystal was obtained by volatilizing ethyl acetate containing compound 2 at room temperature. The details of the crystallographic determination, selected bond lengths and angles were given in Table 1 and supplementary material respectively.

Table 1

Crystal data and structure refinement for Compound 2.

Compound 2
Empirical formula	C17 H10 F3 N O5
Formula Weight	365.26
Temperature	293 K
Bond precision	C-C = 0.0031 A
Wavelength	0.71073
a (Å)	7.7988(10)
b (Å)	8.2075(11)
c (Å)	13.5474(14)
α (°)	75.073(10)
β (°)	81.40(1)
γ (°)	69.207(12)
V (Å3)	781.69(17)
Crystal system, Space group	Monoclinic, P-1
Crystal size (mm3)	0.45 × 0.32 × 0.23
θ range for data collection (°)	3.19 to 25.01
Z	2
Mu (mm−1)	0.138
F(000)	372.0
Refinement method	Full-matrix least-squares on F2
GOF on F2	1.027
R1 and wR2 indices [I > 2σ(I)]	R1 = 0.0446, wR2 = 0.1144
R1 and wR2 indices (all data)	R1 = 0.0645, wR2 = 0.1304
Largest diff. peak and hole (e Å−3)	0.258 and −0.250

The crystal of compound 2 suitable for X-ray crystal analysis was obtained and the structure was also n class="Chemical">confirmed (Fig. 2a). The fluoride atom in benzene cycle forms hydrogen bonds with hydrogen atom (H3) (supplementary material). The overall crystal structure features a chain type joining in through the hydrogen bonds (H….F) along the b axis (Fig. 2b). In the crystal packing of compound 2 (Fig. 2c), there are two stacked forms: (1) π-π stacking of one fluorobenzene ring with another; (2) π-π stacking of one coumarin ring with another, which connected into “chair” conformation along a axis.

Figure 2

(a) The ORTEP view of compound 2 and the hydrogen atoms are shown as small circles with arbitrary radii (ellipsoids at 50% probability); (b) Crystal packing of compound 2 along the b axis; (c) Crystal packing of compound 2 along the a axis.

(a) The ORTEP view of compound 2 and the n class="Chemical">hydrogen atoms are shown as small circles with arbitrary radii (ellipsoids at 50% probability); (b) Crystal packing of compound 2 along the b axis; (c) Crystal packing of compound 2 along the a axis.

UV-vis Titration

The UV-vis spectra of probes (1–4) were recorded after addition of amino acids (n class="Chemical">GSH, Cys, Hcy) and various anions (HS−, AcO−, H2PO4−, F−, Cl− Br− and I−) through UV-vis titration experiments in pure DMSO solution and aqueous solution (DMSO-H2O 4:1, v/v 0.04 mol·L−1 HEPES buffer at pH 7.38) respectively. The data of UV-vis titration experiments manifested only compounds (1, 2) displayed different binding abilities with the above anions and amino acids. The free 1 showed a main absorption at 320 nm, as the HS− increases in pure DMSO solution of probe 1, the absorbance at 320 nm was decreased gradually, along with the simultaneous emergence of a new absorption at 470 nm. In this process, two isosbestic points noted at 330 nm and 348 nm suggesting a clear chemical reaction. Based on the well-establish thiolysis reaction of dinitrophenyl ether, the new absorption at 470 nm could be attributed to coumarin derivative, which was also supported by fluorescence and HRMS titration experiment. Furthermore, the UV-vis spectra of probe 1 with HS− in aqueous solution were also performed (shown in Fig. 3b), however, comparison with DMSO solvent, the probe 1 showed a weak response of UV-vis spectra.

Figure 3

UV-vis spectra of compound 1 (4.0 × 10−5 mol·L−1) with the addition of HS−(0–8 × 10−6 mol·L−1)(a) in DMSO solution; (b) in aqueous solution (DMSO-H2O 4:1, v/v 0.04 mol·L−1 HEPES buffer at pH 7.38). Arrows indicate the direction of increasing HS− concentration; (c) Color changes observed with the addition of 20 equiv. various anions and Cys to DMSO solution of compound 1 respectively; (d) Fluorescent intensity of compound 1 upon the addition of HS−.

UV-vis spectra of compound 1 (4.0 × 10−5 mol·L−1) with the addition of n class="Chemical">HS−(0–8 × 10−6 mol·L−1)(a) in DMSO solution; (b) in aqueous solution (DMSO-H2O 4:1, v/v 0.04 mol·L−1 HEPES buffer at pH 7.38). Arrows indicate the direction of increasing HS− concentration; (c) Color changes observed with the addition of 20 equiv. various anions and Cys to DMSO solution of compound 1 respectively; (d) Fluorescent intensity of compound 1 upon the addition of HS−.

The additions of amino acids (Cys, n class="Chemical">GSH, Hcy) and other anions (AcO−, H2PO4−, F−, Br−, Cl− and I−) to pure DMSO solution of probe 1, only Cys induced similar changes in the UV-vis spectra compared with HS−, which exhibited Cys also interacted with compound 1 (supplementary material). However, the additions of the above amino acids and anions to aqueous solution induced almost no spectra changes of compound 1. The result indicated that compound 1 showed different binding abilities for HS− and Cys in pure DMSO solution and almost no binding abilities with above amino acids and anions in aqueous solution. Therefore, compound 1 could be used as a sensor to detect HS− in aqueous solution.

Subsequently, the colorimetric sensing capabilities of compound 1 were carried out with different anions (Fig. 3c). Obvious color changes from colorless to bright yellow was observed in the presence of HS−, while faint or no color changes happened in the presence of other anions (AcO−, H2PO4−, F−, Cl−, Br−, I− and Cys), which indicated that compound 1 can be used for the detection of HS− as a colorimetric sensor.

Moreover, the detection limit[32,33] of compound 1 (4.0 × 10−6 mol·L−1) was implemented through fluorescence titration (Fig. 3d). Further data analysis revealed an excellent linear relationship (r = 0.9963) between the fluorescence signal of the probe 1 at 392 nm and the n class="Chemical">concentration of HS− (0–8 × 10−6 mol·L−1). Therefore, the detection limit of compound 1 for HS− was determined to be 4.0 × 10−6 mol·L−1.

Next, the UV-vis titration experiments of compound 2 to various anions (HS−, AcO−, H2PO4−, F−, Br−, Cl− and I−) were tested. Upon the addition of increasing amounts of HS− (supplementary material) to DMSO solution of compound 2 a new absorption peak appeared gradually at 485 nm. While, the additions of other anions (AcO−, H2PO4−, F−, Br−, Cl− and I−) to compound 2, almost no spectra changes was observed.

Fluorescence response

The photo-physical responses of four compounds (1–4) in n class="Chemical">DMSO solvent were also investigated with addition of various amino acids and anions. Just as Fig. 4a showed, an emission peak of free 1 exhibited at about 385 nm. Upon the addition of increasing amounts of HS−, the fluorescence intensity increased obviously at about 392 nm. Similar fluorescence response of compound 1 was observed upon the addition of Cys (supplementary material) compared with HS−. Furthermore, the interactions of probe 1 with amino acids (GSH, Hcy) and other anions (AcO−, H2PO4−, F−, Br−, Cl− and I−) were also investigated. The additions of H2PO4−, AcO− and F− (supplementary material) induced the appearance of fluorescence emission bands centered at about 386 nm, however, nominal changes were induced in the presence of GSH, Hcy, F−, Cl−, Br−, I−.

Figure 4

Fluorescence responses (λex 331 nm, slit widths: 5 nm/5 nm) of compound 1 (4.0 × 10−5 mol.L−1) upon the additions of HS− (a) in pure DMSO solution; (b) in aqueous solution (DMSO-H2O 4:1, v/v 0.04 mol·L−1 HEPES buffer at pH 7.38). Arrows indicate the increasing concentration of HS−.

Fluorescence responses (λex 331 nm, slit widths: 5 nm/5 nm) of n class="Chemical">compound 1 (4.0 × 10−5 mol.L−1) upon the additions of HS− (a) in pure DMSO solution; (b) in aqueous solution (DMSO-H2O 4:1, v/v 0.04 mol·L−1 HEPES buffer at pH 7.38). Arrows indicate the increasing concentration of HS−.

Besides, the fluorescence spectral responses of compound 1 with amino acids (n class="Chemical">Cys, GSH, Hcy) and various anions (HS−, AcO−, H2PO4−, F−, Br−, Cl− and I−) were also examined in aqueous solution. From Fig. 4b, compound 1 showed very weak fluorescence at the absence of anions. After HS− was added, double emission peaks appeared and the fluorescent intensity increased gradually. Results demonstrated that compound 1 showed strong binding ability for HS−. However, weak spectral responses that even could be ignored were induced with addition of amino acids (Cys, GSH, Hcy) and H2PO4−, AcO−, F−, Cl−, Br−, I−.

For compound 2, the fluorescence intensity noted at 434 nm increasing rapidly by titration of n class="Chemical">HS− (supplementary material). No obvious responses of compound 2 were observed with titration of other anions (AcO−, H2PO4−, F−, Br−, Cl− and I−) and Cys. For compounds (3 and 4), Similar experiments were carried out, however, no significant spectral responses were observed with the addition of other anions (HS−, H2PO4−, AcO−, F−, Br−, Cl− and I−) into DMSO solution of two compounds (3 and 4) which indicated the weak binding abilities of compounds (3 and 4) and other anions (AcO−, H2PO4−, F−, Br−, Cl− and I−) and Cys could be ignored.

Binding constant

The job-pot curves suggested two compounds (1 and 2) interacted with amino acids and various anions as the ratio of 1:1 or 1:2. The UV-vis spectral data was used to n class="Gene">calculate the binding constants by non-linear least square method[34,35], and the binding constants were listed in the Table 2. Obviously, the binding ability of two compounds (1 and 2) with amino acids and various anions followed the order of HS− ≫ H2PO4−, Cys, AcO−, F−, Br−,Cl−, I−, Hcy and GSH. In general, both compound 1 and compound 2 showed the strongest binding ability for HS− among amino acids and anions. Besides, a theoretical basis and these binding constants were necessary for the optimization of sensor.

Table 2

Binding constants of compound (1 and 2) with various anions.

Anion	Compound 1 (DMSO)	Compound 1 (DMSO:HEPES = 4:1)	Compound 2 (DMSO)
HS−	(5.32 ± 0.08) × 107b	(1.43 ± 0.07) × 106b	(33.9 ± 0.8)a
Cys	(7.91 ± 0.64) × 103a	ND	ND
AcO−	ND	ND	ND
F−	ND	ND	ND
H2PO4−	ND	ND	ND
Cl− Br− or I−	ND	ND	ND

aThe binding ratio of host-guest is 1:1.

bThe binding ratio of host-guest is 1:2.

Binding constants of n class="Chemical">compound (1 and 2) with various anions.

The anion binding abilities of compounds (1 and 2) with two electron-withdrawing groups on the n class="Chemical">fluorobenzene ring were stronger than that of compounds (3 and 4) which had one electron-withdrawing group on the fluorobenzene ring. In addition, the anion binding ability of compound 1 was stronger than that of compound 2 due to the electron-withdrawing ability of nitro group was greater than the trifluoromethyl group[36]. The above results indicated that strong electron-withdrawing groups located in 2, 4-positions of fluorobenzene provides a easily site for intermolecular HS− attack. Besides, for HS−, the binding ability trend of compounds (1–4) followed the order of 1 > 2 ≫ 3, 4 also could be confirmed by comprehensive analysis of UV-vis, fluorescence titration.

Mechanism

The interacted mechanism was measured by fluorescence and HRMS titration experiment. The broad emission peak of compound 1 appeared at about 396 nm in the absence of n class="Chemical">HS−. The emission peak shifted to the short wavelength from 396 nm to 380 nm after HS− was added. The emission peak of the single 7-hydroxy-4-methylcoumarin appeared at about 380 nm. The same emission peak observed in the presence of HS− which suggested free 7-hydroxy-4-methylcoumarin was released after compound 1 interacted with HS− (Fig. 5a). Furthermore, the interacted mechanism of host-guest (1-HS−) was also carried by performing MS-HRMS after the addition of 2 equiv. NaHS. The probe 1 itself exhibited a dominant peak at m/z = 341.0551(M-H)− (supplementary material). However, the above peak vanished and a new peak at m/z = 199.0364 (M + Na)+ appeared which was the ion-peak of 7-hydroxy-4-methylcoumarin (Fig. 5b) after the addition of HS−. The MS-HRMS titration experiment suggested the binding ratio of host-guest (1-HS−) was 1:2. The above results indicated the possible interacted mechanism was thiolysis reaction of dinitrophenyl ether (Fig. 5c)[37-44].

Figure 5

(a) A comparison of fluorescence spectra in the presence of 3.2 equiv HS− between compound 1 and 7-hydroxy-4-methylcoumarin (4.0 × 10−5 mol·L−1); (b) ESI-HRMS spectrum of compound 2 after addition of 2 equiv of NaHS in DMSO solution. MS-HRMS (m/z): 199.0364 (M + Na)+; (c) The possible interacted mechanism of host-guest.

(a) A comparison of fluorescence spectra in the presence of 3.2 equiv n class="Chemical">HS− between compound 1 and 7-hydroxy-4-methylcoumarin (4.0 × 10−5 mol·L−1); (b) ESI-HRMS spectrum of compound 2 after addition of 2 equiv of NaHS in DMSO solution. MS-HRMS (m/z): 199.0364 (M + Na)+; (c) The possible interacted mechanism of host-guest.

Cytotoxicity Assay

For further biological application point of view, a quantitative cytotoxicity study of two compounds (1 and 2) in vitro was conducted using MTT assay. Owning to Glutathione peroxidase (GPx1) being an important selenoprotein and not found in human breast cancer cells (MCF-7) according to scientific research, therefore, the special cell lines (MCF-7 cell) were selected by us[45,46]. The MTT assay results indicated that compound 1 and 2 showed very low cototoxicity over a concentration range of 0–150 μg·mL−1 especial probe 2 (Fig. 6). Cellular viability was minimally affected (80%, cellular viability) with the compounds (1 and 2) ( <150 μg·mL−1). In agreement with the binding constants, compounds (1 and 2) showed a high binding capacity and low cytotoxicity, especial probe 1, which indicated that compounds (1 and 2) have a potential application to H2S detection in cells.

Figure 6

Cell viabilities were measured by MTT assay after 24 h in the presence of of fluorescence probe (0–150 μg·mL−1) incubation. Cell viability (expressed in%) was set as 100% growth in the absence of fluorescence probe.

Material and Methods

Most staring materials were obtained commercially, all reagents and solvents were of analytical grade and used as received without further purification unless otherwise stated. n class="Chemical">Sodium hydrolfide and all anions in the form of tetrabutylammonium salts (such as (n-C4H9)4NCl, (n-C4H9)4NBr, (n-C4H9)4NI, (n-C4H9)4NAcO and (n-C4H9)4NH2PO4) and amino acides (Cys, GSH and Hcy) were purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). Dimethyl sulfoxide (DMSO) was distilled in vacuum after being dried with CaH2. 1H NMR spectra were recorded using an Unity Plus-400-MHz spectrometer. HRMS was obtaioned with a Mariner apparatus. Absorption spectra were obtained on UV-vis spectrophotometer (Shimadzu, UV-2600, Japan). Fluorescence emmission spectra were taken on a Cary Eclipse Fluorescence Spectrophotometer (Agilent, USA). The binding constant (Ks) was obtained by non-linear least squares calculation method for data fitting.

The cells that were at logarithmic growth phase were seeded in a 96-well plate at a density of 2.0 × 104 cell peer well for 24 h, followed by treatment different concentration of compound at 37 °C for additional 24 h. Next, cells were collected and washed with PBS three times then 100 µL of culture medium and 20 µL of MTT solution were added to each well respectively, and the cells were incubated for 4 h. The absorbance were detected by microplate reader (Thermo Multiscan MK3, Thermo Fisher Scientific, MA, USA) at 490 nm wavelength measurement. Cell viability (expressed in%) was calculated considering 100% growth at the absence of fluorescence probe, and the viability of other groups were calculated by comparing the optical density reading with the control. The IC50 was defined as the compound concentrations required for 80% inhibition of cell growth.

Synthesis of the probes

The compounds (1–4) were synthesized acn class="Chemical">cording to the route shown in Fig. 1.

Synthesis of 7-hydroxy-4-methylcoumarin (1)

7-Hydroxy-4-methylcoumarin was synthesized acn class="Chemical">cording to the literature[47]. The experimental details were described in supplementary material.

Synthesis of compound 1

7-Hydroxy-4-methyl coumarin (2.16 g, 12 mmol) and n class="Chemical">2,4-dinitrofluorobenzene (1.5 g, 8 mmol), K2CO3 (3.39 g, 24 mmol) were dissolved in DMF(40 mL) solution with stirring. The above mixture were heated for 4 h at 90 °C at N2 atmosphere and then cooled to room temperature. The reaction was poured into ice-water (400 mL) and extracted with ethyl acetate (3 × 25 mL), dried with MgSO4 overnight. Filtration, purification with column chromatography (CH3COOCH2CH3:CH2Cl2 = 1:40). Stood overnight a yellow precipitate was obtained. Yield: 68%. m.p. 187.9–189.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1 H), 8.51 (dd, J = 9.2, 2.8 Hz, 1 H), 7.92 (d, J = 8.7 Hz, 1 H), 7.40 (dd, J = 15.6, 5.8 Hz, 2 H), 7.28 (dd, J = 8.7, 2.5 Hz, 1 H), 6.43 (s, 1 H), 2.47 (s, 3 H) (supplementary material). Elemental analysis: Calc. for C16H10N2O7: C, 56.15; H, 2.94; N, 8.18. Found: C, 56.21; H, 2.94; N, 8.16. MS-HRMS (m/z): 341.0551(M-H)- (supplementary material). IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1288 cm−1 (supplementary material).

Compounds (2–4) were also synthesized acn class="Chemical">cording to the above similar procedure.

Compound 2

Suitable single light yellow crystal for X-ray crystal structure analysis was obtained. Yield: 74%. m.p. 139.3–142.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (d, J = 2.8 Hz, 1 H), 8.51 (dd, J = 9.1, 2.8 Hz, 1 H), 7.91 (d, J = 8.7 Hz, 1 H), 7.39–7.30 (m, 2 H), 7.23 (dd, J = 8.7, 2.5 Hz, 1 H), 6.42 (s, 1 H), 2.46 (s, 3 H) (supplementary material). Elemental analysis: Calc. for C17H10FNO5: C, 55.90; H, 2.76; N, 3.83. Found: C, 55.83; H, 2.76; N, 3.84. MS-HRMS (m/z): 366.0589(M + H)+ (supplementary material). IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1274 cm−1 (supplementary material).

Compound 3

Yield: 75%. m.p. 140.9–143.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (dd, J = 8.2, 1.5 Hz, 1 H), 7.86–7.76 (m, 2 H), 7.51 (t, J = 7.8 Hz, 1 H), 7.39 (d, J = 8.3 Hz, 1 H), 7.09–7.01 (m, 2 H), 6.35 (s, 1 H), 2.43 (s, 3 H) (supplementary material). Elemental analysis: Calc. for C16H11NO5: C, 64.65; H, 3.73; N, 4.71. Found: C, 64.72; H, 3.72; N, 4.72. MS-HRMS (m/z): 320.0550(M + Na)+ (supplementary material). IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1273 cm−1 (supplementary material).

Compound 4

Yield: 78%. m.p. 165.5–167.3°C. 1H NMR (400 MHz, DMSO-d6) δ 8.35–8.27 (m, 2 H), 7.88 (d, J = 8.7 Hz, 1 H), 7.28 (dd, J = 10.2, 3.1 Hz, 3 H), 7.19 (dd, J = 8.7, 2.4 Hz, 1 H), 6.40 (s, 1 H), 2.46 (s, 3 H) (supplementary material). Elemental analysis: Calc. for C16H11NO5: C, 64.65; H, 3.73; N, 4.71. Found: C, 64.82; H, 3.74; N, 4.70. MS-HRMS (m/z): 320.0533 (M + Na)+ (supplementary material). IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1267 cm-1 (supplementary material).

A light yellow crystal of compound 2 with dimensions of 0.45 nm × 0.32 nm × 0.23 nm was mounted on a glass fiber. X-ray single-crystal diffraction data was n class="Chemical">collected on a Rigaku saturn CCD area detector at 293 K with Mo-Ka radiation (λ = 0.71073 Å). The structure was solved by direct methods and refined on F2 by full-matrix least squares methods with SHELXL-97[48].

Conclusion

In conclusion, we developed a series of fluorescence probes based on n class="Chemical">coumarin derivatives for the detection of H2S successively with OFF-ON” fluorescence response. The fluorescence probes, especially compound 1, exhibited remarkable response to H2S against other anions and amino acids in pure DMSO solvent. Otherwise, the probe 1 also showed strong binding ability for HS− in HEPES buffer solution. The interacted mechanism of host-guest was the thiolysis reaction of dinitrophenyl ether. In addition, compound (1 and 2) showed highly sensitivity and low cytotoxicity to MCF-7 cells and probe 1 can also detect HS− selectively by naked eye in pure DMSO solvent. The results of our efforts highlight that the probes, especially compound 1, hold a potential chemical tool for the detection of H2S.