Styrylisoxazole-based fluorescent probes for the detection of hydrogen sulfide

Abstract

Styrylisoxazoles bearing a nitro group linked to bulky aromatic rings have been synthesized and examined for their absorption and emission studies in organic solvents and water. The molecules showed emission in the visible region with significant solvatochromic emission shifts influenced by the extended conjugation of aromatic rings and intramolecular charge transfer. These absorption and emission changes were used for the efficient and sensitive detection of trace concentrations of hydrogen sulfide (H₂S) through the reduction of the nitro group to the amine group in the presence of aqueous sodium sulfide. The experimental results indicated that the probes exhibit an excellent emission response with large shifts in the emission and sensitivity with a micromolar detection limit.

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Introduction

π-Conjugated small organic molecules bearing donor and acceptor substituents have been extensively investigated due to their unique photoresponsive ability and consequent promise in the fields of materials science and life sciences.^1–3 In particular, systems bearing a diarylethene scaffold have attracted attention as promising candidates because of their synthetic tunability, absorption and emission in solution and in the solid state, tunable redox properties, and photochemical interconversion, which have enabled their use for optoelectronic and biological applications.^4–8 Their properties can be modulated further by incorporating heteroaromatic rings.^9–12 Many biologically active substrates have a heterocyclic system as one of the components that play a key role in several biological processes. Isoxazole is a five-membered heterocyclic unit having oxygen and nitrogen atoms in the 1,2-positions, respectively. Molecules based on this heterocyclic ring are used as building blocks for the synthesis of many natural products^13–16 and were found to have multiple biological functions, such as to induce genes that support neuroendocrine and β-cell phenotypes or to act as cyclooxygenase inhibitors.^17,18 The nitro group attached to the heteroaromatic core on the vinyl chain can act as a good electron sink and has been proven to be an effective inducer of apoptosis.¹⁹ The nitroaromatics create a localization of the negative region employed to improve the binding affinity of any ligand to its putative target.²⁰ Furthermore, isoxazole derivatives coupled with calixarenes and pyrroles were utilized as chemosensors for the efficient detection of metal ion analytes,²¹ anion sensors²² or ‘turn-off’ sensors in micellar media.²³ Considering the unique electronic and biological properties of isoxazoles, in this work, we synthesized styrylisoxazoles incorporating a nitro group [Fig. 1]. We envisaged two uses of the incorporation of the nitro group. First, it acts as a strong electron withdrawing group helping in emission in the lower energy regions; second is its ability to detect and react with hydrogen sulfide (H₂S). Hydrogen sulfide (H₂S) is a cytotoxic gasotransmitter besides CO and NO produced mainly by two enzymes: cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) using L-cysteine (L-Cys) as the substrate.²⁴ The accumulation of H₂S in trace quantities in biological media was associated with some physiological and pathological processes such as vasodilatation, apoptosis, and various diseases. For the detection of H₂S, several techniques were employed that include colorimetric and fluorimetric assays,^25,26 redox or nucleophilic reactions,²⁷ and electrochemical assays.^28,29 However, considering their greater selectivity and environment sensitivity, absorption or fluorescence-based techniques became prevalent.^30–32 Fluorescence-based techniques take advantage of distinct changes in the absorption or emission process through specific structural changes.³³ Various strategies include H₂S-mediated reduction, nucleophilic addition, copper-mediated precipitations, and thiolysis among other methods.^34–40 However, most of these fluorescent probes are limited by emission in the narrower energy domains in the UV-visible region and longer response times. Despite the availability of various fluorescent probes, a design strategy with tunable emission maxima provides opportunities to develop different industrially or biologically relevant analyte sensing probes.⁴¹ Herein, we report a set of isoxazole-based fluorescent sensors (Fig. 1–4) containing a NO₂ group for the quantitative detection of H₂S that shows solvent sensitivity as well as enabling the reduction of the nitro group. Even though the presence of the nitro group yields lower fluorescence due to strong non-radiative transitions, the mild conditions required for the reduction allow the design of such fluorophores for H₂S detection. Furthermore, the strong electron withdrawing nature of the nitro group induces strong solvatochromism with emission in the lower energy regions. Taking advantage of this, we tested these fluorophores for H₂S detection. The synthesized fluorophores exhibited immediate selectivity to H₂S with a short response time correlated with the changes in the emission. The results are described in the following sections.

Fig. 1 Structures of the molecules investigated.

Fig. 2 Absorption of (a) 1–4 in acetonitrile and (b) (3) in solvents of different polarities.

Fig. 3 Normalized emission of styrylisoxazole derivatives 2 (λ_ex 350 nm), 3 (λ_ex 440 nm) and 4 (λ_ex 440 nm) in different solvents (a–c) and (d) normalized emission of styrenes (1–4) in acetonitrile.

Fig. 4 UV-vis absorption (a) and emission response (b): λ_ex 450 nm and (c): λ_ex 350 nm of probe 4 (10 μM) with increasing concentration (0 to 200 μM) of Na₂S in a DMSO + H₂O (9 : 1) binary mixture.

Experimental section

The reagents and chemicals utilized in this study were obtained from SD Fine, Sigma-Aldrich or Alfa Aesar. All the solvents employed for the synthesis and optical spectroscopic studies were dried thoroughly using reported procedures. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker (500 MHz) spectrophotometer using tetramethylsilane (TMS, δ = 0 ppm) as an internal standard with chloroform-D (CDCl₃) as the solvent. Mass-spectral data were obtained using an ESI-QToF (Waters-synapt G2S) high-resolution mass spectrometer. UV-vis absorption spectra were recorded using an Analytik Jena Specord 210 plus, and fluorescence emission studies were performed using a Horiba-Jobin Yvon Fluolorog-3 spectrofluorometer with a slit-width of 1 nm or 2 nm.

Synthesis of the compounds (1–4)

In a typical synthetic strategy, the corresponding aldehydes [benzaldehyde, naphthaldehyde, 9-anthraldehyde and pyrene-1-aldehyde (R-CHO)] were condensed with 3,5-dimethyl-4-nitroisoxazole (6) yielding the corresponding styryl derivatives (1–4) (Scheme 1). The details of the synthesis of the starting materials (pyrene aldehyde, 3,5-dimethylisoxazole and 3,5-dimethyl-4-nitroisoxazole) are given in the ESI†.

Scheme 1 Synthetic strategy for the preparation of the styrylisoxazole derivatives (1–4).

Synthesis of 3-methyl-4-nitrostyrylisoxazoles (1–4). To a solution of 3,5-dimethyl-4-nitroisoxazole (1.4 g, 10 mmol) in EtOH (10 mL) were added the desired aldehyde (1.0 equiv.) and piperidine (98.0 μl, 0.01 equiv.) successively at ambient temperature. The reaction mixture was heated at 65 °C for 1 h overnight. After cooling the reaction mixture to room temperature, the precipitate was collected by filtration and washed several times with Et₂O to give 3-methyl-4-nitro-5-alkenyl-isoxazoles (1–4) as colored solids.

(E)-3-Methyl-4-nitro-5-styrylisoxazole (1). ¹H NMR (500 MHz, CDCl₃-d) δ 7.81 (s, 1H), 7.72 (s, 1H), 7.69 (m, 3H), 7.49 (m, 3H), 2.91 (s, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 167.18, 156.14, 143.10, 134.45, 131.12, 129.18, 128.42, 110.98, 95.69, 11.84. HRMS (ESI-MS) m/z: calcd for [M + H]⁺, 230.0691; found, 231.0881.

(E)-3-Methyl-5-(2-(naphthalen-2-yl)vinyl)-4-nitroisoxazole (2). ¹H NMR (500 MHz, CDCl₃) δ 8.04 (s, 1H), 7.95 (m, 1H), 7.90 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 7.5 Hz, 1H), 7.83–7.75 (m, 2H), 7.57–7.52 (m, 2H), 2.61 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 167.20, 156.10, 143.09, 134.63, 133.35, 132.02, 130.68, 129.04, 128.75, 127.87, 127.78, 126.97, 123.33, 111.09, 11.76. HRMS (ESI-MS) m/z: calcd for [M + H]⁺, 280.0848; found, 281.1012.

(E)-5-(2-(Anthracen-9-yl)vinyl)-3-methyl-4-nitroisoxazole (3). ¹H NMR (500 MHz, CDCl₃) δ 8.78 (d, J = 16.5 Hz, 1H), 8.51 (s, 1H), 8.31 (d, J = 7.7 Hz, 2H), 8.05 (d, J = 7.5 Hz, 2H), 7.69 (d, J = 16.5 Hz, 1H), 7.60–7.50 (m, 4H), 2.67 (s, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 166.63, 156.28, 140.18, 131.34, 129.70, 129.45, 129.11, 128.88, 126.98, 125.56, 125.33, 124.81, 119.58, 11.85. HRMS (ESI-MS) m/z: calcd for [M]⁺, 330.1004; found, 330.1080.

(E)-3-Methyl-4-nitro-5-(2-(pyren-1-yl)vinyl)isoxazole (4). ¹H NMR (500 MHz, CDCl₃) δ 8.91 (d, J = 15.3 Hz, 1H), 8.50 (d, J = 9.0 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.26–8.19 (m, 4H), 8.15 (d, J = 9.0, 1H), 8.09–8.04 (m, 2H), 7.91 (d, J = 16.2 Hz, 1H), 2.64 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 167.30, 156.24, 139.27, 133.44, 131.33, 130.66, 130.31, 129.20, 127.94, 127.39, 126.48, 126.46, 126.23, 125.32, 125.01, 124.57, 124.09, 122.01, 112.23, 11.92. HRMS (ESI-MS) m/z: calcd for [M]⁺, 354.1004; found, 354.1067.

Results and discussion

Absorption and emission of (1–4)

Fig. 1 gives the structural details of the synthesized molecules. The phenyl group in (1), naphthalene in (2), anthracene in (3) and pyrene in (4) were connected to the isoxazole ring through a π-spacer (double bond). The nitro group present on the isoxazole unit acts as a strong electron acceptor. Through this arrangement, we expect to see the effect of extended conjugation on the absorption and emission properties of the styrenes. Table 1 gives the absorption data of styrenes (1–4) in organic solvents. In general, depending on the nature of the substituents, the absorption maxima were observed in the wavelength range of 345–446 nm [Fig. 2a]. Styrene (1) containing a simple phenyl ring absorbs at ∼355 nm in acetonitrile. The substitution of the naphthyl ring results in a +21 nm shift in absorption. Furthermore, an increase in conjugation through the introduction of other fused ring systems such as anthracene and pyrene results in a +88 nm increase in the absorption maxima. Despite the presence of an additional aromatic ring, the absorption of (4) shows only a slight effect on the absorption (λ_a) maxima in comparison with (3). With the change in polarity, moderate absorption changes (+4–10 nm) were noted for the substituted styrenes [Fig. 2b and Fig. S1†].

Table 1 Absorption and emission data of the synthesized molecules (1–4)

	Solvent	λ a (nm)	λ f (nm)	Stokes shift (cm^−1)		Solvent	λ a (nm)	λ f (nm)	Stokes shift (cm^−1)
(1)	Heptane	345	415	5354	(2)	Heptane	365	398, 427, 460	4618
	Dioxane	356	436	5154		Dioxane	371	461	5262
	THF	356	436	5154		THF	370	479	6150
	CH3CN	355	461	6477		CH3CN	376	524	7511
	MeOH	352	462	6764		MeOH	369	465, 496	6938
	DMSO	349	475	7600		DMSO	385	536	7319
	Water	363	464	5996		Water	347	513	9325
(3)	Heptane	441	490, 518	5464	(4)	Heptane	440	480, 513	3234
	Dioxane	441	552	4559		Dioxane	447	564	4640
	THF	441	572	5193		THF	446	574	4999
	CH3CN	434	631	7193		CH3CN	443	612	6233
	MeOH	438	595	6024		MeOH	442	593	5761
	DMSO	436	509, 637	7237		DMSO	453	623	6023
	Water	453	618	5893		Water	442	626	6649

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Fluorescence properties in solution

Styrene (1) bearing a phenyl ring shows emission (λ_f) at ∼461 nm in acetonitrile. Styrene (2) containing a naphthalene moiety emits at ∼524 nm, styrene (3) bearing an anthracene system emits at ∼631 nm and pyrenyl styrene (4) emits at 612 nm [Table 1]. The bathochromic shift in emission reflects the extended delocalization due to the increased aromaticity. Solvent polarity has moderate to strong influence on the emission behavior of these styrenes. In non-polar heptane, the structured emission is noted for (3) and the increase in solvent polarity results in bathochromic emission shifts (λ_f). Styrene (1) shows a bathochromic shift of +46 nm upon changing the solvent polarity from heptane to acetonitrile. Styrene (2) shows structured emission at 399, 427 and 460 nm in heptane but shows a significant emission (+64 nm) shift in acetonitrile [Fig. 3a]. Similarly, anthryl substituted (3) and pyrenyl substituted (4) show about +113 and +99 nm shifts in the emission with an increase in solvent polarity [Fig. 3b and c], respectively. The order is phenyl < naphthyl < pyrenyl < anthryl [Fig. 3d].

This strong solvent polarity dependent emission indicates the presence of an excited state with a strong intramolecular charge transfer (ICT) character and a large dipole moment relative to the ground state. In particular, the presence of the nitro group also has a strong influence on the intramolecular charge transfer.^42,43 These compounds show weak and blue-shifted emission in methanol compared to that in acetonitrile. This emission behavior is influenced by the involvement of solute (fluorophore)–solvent (methanol) dipole–dipole interactions and intermolecular hydrogen bonding interactions.

Detection of H₂S

H₂S, a third member of the gasotransmitter family along with NO and CO, plays an important role in some biological and pharmacological processes.^44,45 For this reason, it is important to detect the presence of H₂S, and we have utilized the spectroscopic properties of the synthesized styrylisoxazoles (1–4). The absorption and emission spectra of styrylisoxazoles were recorded in a DMSO–H₂O (9 : 1) mixture. Pyrenyl isoxazole (4) absorbs at ∼450 nm and emits at ∼631 nm in a DMSO–H₂O binary mixture. Upon titrating with aqueous sodium sulfide [typically the emission spectra are recorded after about 3 minutes post addition of Na₂S], the absorbance initially decreases (up to 50 μM) in intensity and later shows a hypsochromic shift from ∼60 μM. Beyond this concentration, the primary absorption band at ∼450 nm merely appears as a shoulder. The absorption at ∼350 nm shows a progressive increase with a well-defined isosbestic point at ∼407 nm [Fig. 4a]. This shift in absorption may indicate the formation of a new species, and the blue-shifted absorption is attributed to a loss in delocalization. This is a consequence of the shifts in the change of the structure from the initial electron withdrawing nitro (NO₂) group which is reduced to the electron donating amino group (NH₂). The changes in the emission spectra of (4) in the absence or presence of Na₂S (0–170 μM) are displayed in Fig. 4b. Under 450 nm excitation, compound (4) shows emission maxima at 631 nm in the absence of Na₂S. However, the addition of Na₂S results in a significant loss of emission intensity with the eventual loss of emission. Noticeably, the emission in the blue region of the spectrum (at or near 400–450 nm) seems to be increasing. To get a more accurate picture of this emission behavior, we excited compound (4) at 350 nm which is a dominant absorption band post addition of Na₂S. The emission shows two peaks: a long charge transfer band at ∼630 nm and a shorter wavelength structured emission at 380, 400 and 420 nm. These shorter wavelength emission peaks show an enhancement in intensity upon the addition of sodium sulfide, and a concomitant decrease in emission intensity at 630 nm was noted. Similar observations were reported for anthryl (3) and naphthyl (2) substituted isoxazoles. In the case of (3), the emission at ∼630 nm decreases in intensity, and new emissive peaks appear in shorter wavelength regions at ∼500 nm with a sudden surge in emission intensity at 80 μM [Fig. 5a]. The excitation at 350 nm shows emission peaks primarily due to the anthracene [Fig. S3†]. The naphthyl derivative (2) also shows a drop in the emission intensity of the charge transfer band (542 nm) with a slight shift of the wavelength to the blue region [Fig. 5b] at higher concentrations. The increase in the emission intensity at higher energy is conspicuous for (3) or (4) compared to that for (2). The changes in the UV-spectral profile upon the reduction of the nitro group to the amino group are shown in the ESI [Fig. S2†]. H₂S acts as a weak acid and may contribute to the observed emission changes. Our experiments showed that the emission is unaffected with pH variation [Fig S8†]. To check the sensitivity for other analytes such as Na₂SO₃, cysteine, homocysteine and glutathione, the isoxazole derivative (4) was titrated with different analyte concentrations. The results are shown in the ESI.† Similar to the earlier emission results, the emission band at ∼630 nm is quenched with the analytes used [Fig S9a†]. A comparative bar chart [Fig S9b†] for a given concentration of the analyte reveals greater sensitivity for H₂S. While this result is encouraging, the nature of product formation is uncertain. For instance, cysteine or homocysteine was earlier detected by a Michael addition, a cyclization or a sequential substitution mechanism.^46–48 The results obtained from the mass spectral analysis gave a molecular weight much higher than the sole amino product. The product formed cannot be conclusively confirmed based on the mass spectral data alone and requires further experimentation.

Fig. 5 Fluorescence response of probe (a) 3 [λ_ex 440 nm] and (b) 2 [λ_ex 380 nm] with increasing concentration (0 to 220 μM) of Na₂S in a DMSO + H₂O (9 : 1) binary mixture.

Sensing mechanism

The spectral changes involving the emission response are attributed to the loss of charge transfer due to the conversion of the electron withdrawing nitro group to the electron donating amine group [Fig. 6]. After the reaction with H₂S, the donor–acceptor electron system was inhibited by the donor–donor electron system. This expectedly disturbs the charge transfer yielding the loss in the emission intensity and the emission spectral shifts.⁴⁹ This reduction of the nitro group to the amino group is facilitated by the generation of the sulfide anion.^50,51 To prove the product formation, mass spectral analysis was performed and the HRMS analysis predicts the formation of the amino product (Fig. 7 and Fig S4–S6†). The product cannot be isolated, and NMR characterization could not be performed as the sample concentrations are too low. For qualitative visualization, the obtained products are subjected to the ninhydrin test and the products show a strong color [Fig. S7†]. These results pave the way for identifying quicker means of H₂S detection with relevance to industrial and biological applications.

Fig. 6 Schematic representation of the reduction of the nitro group for (1–4) to an amino group by H₂S in DMSO/H₂O mixtures.

Fig. 7 HRMS spectra of the amine compound obtained after the reduction of 4. The mass spectral data of other compounds are given in the ESI.†

Conclusions

In summary, we have demonstrated the detection of micromolar (60 μM) quantities of hydrogen sulfide through a rapid fluorescence response through the reduction of the nitro group to the amine functional group. The molecular systems bearing fused aromatic ring systems and the nitro group are sensitive to solvent polarity variations resulting in significant solvatochromic shifts and emission shifts attributed to intramolecular charge transfer. This unique charge transfer emission is quenched by the addition of H₂S resulting in a fast and significant emission response. The formation of the amine product is confirmed by mass spectral analysis.

Conflicts of interest

No conflict of interest to declare.

Acknowledgements

The authors acknowledge a financial grant from BRNS (Board of Research in Nuclear Sciences, India; 37(2)/14/05/2016) and IIT Gandhinagar for overall support. We thank the reviewers for useful suggestions.

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Footnote

† Electronic supplementary information (ESI) available: NMR spectral characterization data, and supporting absorption and emission figures. See DOI: 10.1039/c7pp00331e

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