Styrylisoxazole-based fluorescent probes for the detection of hydrogen sulfide

Article abstract of DOI:10.1039/c7pp00331e

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.

Full text of DOI:10.1039/c7pp00331e

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Styrylisoxazole based Fluorescent Probes for the Detection of Hydrogen Sulfide
Jagadish Katla and Sriram Kanvah*
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Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar
382355
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*eꢀmail:kanvah@gatech.edu
Abstract
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Styrylisoxazoles bearing 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 efficient and sensitive detection of trace
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concentrations of hydrogen sulfide (H S) through reduction of the nitro group to the amine in the
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presence of aqueous sodium sulfide. The experimental results indicated that the probes exhibited
excellent emission response with large shifts in the emission and sensitivity with micromolar
detection limit.
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Key Words: H S detection, Nitro to Amine Reduction, styryl isoxazoles, solvatochromism
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Introduction
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πꢀconjugated small organic molecules bearing donor and acceptor substituents have been
extensively investigated due to their unique photoresponsive ability and consequent promise in
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the fields of materials science and life sciences . In particular, systems bearing diarylethene
scaffold attracted attention as promising candidates because of synthetic tunability, absorption
and emission in the solution and the solid state, tunable redox properties, photochemical
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interconversion and enabled their use for optoelectronic and biological applications . The
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properties can be modulated further by incorporating heteroaromatic rings . 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 fiveꢀmembered heterocyclic unit having oxygen and
nitrogen atoms in the 1, 2ꢀpositions respectively. Molecules based on this heterocyclic ring are
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used as a building block for the synthesis of many natural products
and were ascribed
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multiple biological functions, to induce genes that support neuroendocrine and βꢀcell phenotypes
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or act as cyclooxygenase inhibitors
. 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
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apoptosis . The nitroaromatics create a localization of negative region employed to improve the
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binding affinity of any ligand to its putative target . Furthermore, isoxazole derivatives coupled
with calixarenes, pyrroles were utilized as chemosensors for the efficient detection of metal ion
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analytes , anion sensing or ‘turnꢀoff’ sensors in micellar media . Considering the potential
for unique electronic and biological properties of isoxazoles, in this work, we synthesized styryl
isoxazoles incorporating a nitro group [Figꢀ1]. We envisaged two uses for incorporation of the
nitro group. First, this is acts as a strong electron withdrawing group helping in emission in the
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lower energy regions; second is its ability to detect and react with hydrogen sulfide (H S).
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Hydrogen sulfide (H S), is a cytotoxic gasotransmitter besides CO and NO produced mainly by
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two enzymes: cystathionine–β–synthase (CBS) and cystathionineꢀγ–lyase (CSE) using Lꢀ
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cysteine (LꢀCys) as the substrate . Accumulation of H S in trace quantities in biological media
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was associated with some physiological and pathological processes such as vasodilatation,
apoptosis, and various diseases. For the detection of H S, several techniques were employed that
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include colorimetric and fluorimetric assays
, redox or nucleophilic reactions , and
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electrochemical assays
sensitivity, the absorption or fluorescenceꢀbased techniques became prevalent
fluorescenceꢀbased techniques take advantage of distinct changes in the absorption or the
. However, considering greater selectivity and environment
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. The
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emission process through specific structural changes . Various strategies include H Sꢀmediated
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reduction, nucleophilic addition, copperꢀmediated precipitations, thiolysis among other
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methods
. However, most of these fluorescent probes are limited by emission in the narrower
energy domains in the UVꢀVisible region and longer response times. Despite the availability of
various fluorescence probes, a design strategy with tunable emission maxima provides
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opportunities to develop different industrially or biologically relevant analyte sensing probes .
Herein, we report a set of isoxazole based fluorescent sensors (Figꢀ1; 1ꢀ4) containing NO group
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for quantitative detection of H S that shows solvent sensitivity as well as enable reduction of the
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nitro group. Even though the presence of nitro group yields lower fluorescence due to strong
nonꢀradiative transitions, the mild conditions required for the reduction allows the design of such
fluorophores for H S detection. Further, the strong electron withdrawing nature of the nitro
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group induces strong solvatochromism with emission in the lower energy regions. Taking
advantage of this, we tested these fluorophores for H S detection. The synthesized fluorophores
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exhibited immediate selectivity to H S with short response time correlated by the changes in the
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emission. The results are described in the following sections.
Experimental section
The reagents and chemicals utilized in this study were obtained from SD Fine, SigmaꢀAldrich or
Alfa Aesar. All the solvents employed for the synthesis and optical spectroscopic studies were
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dried thoroughly using reported procedures. H NMR and C NMR spectra were recorded on
Bruker (500 MHz) spectrophotometer using tetramethylsilane (TMS, δ = 0 ppm) as an internal
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standard with chloroformꢀD (CDCl ) as the solvent. Massꢀspectral data were obtained using ESIꢀ
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QToF (Watersꢀ synaptic G2S) highꢀresolution mass spectrometer. UVꢀVis absorption spectra
were recorded using Analytik Jena Specord 210 plus, and fluorescence emission studies were
performed using HoribaꢀJobin Yvon Fluolorogꢀ3 spectrofluorometer with a slitꢀwidth of 1 nm or
2 nm.
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Fig.1 Structures of molecules investigated.
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Synthesis of the compounds (1-4)
In a typical synthetic strategy, the corresponding aldehyde [benzaldehyde, naphthaldehyde, 9ꢀ
anthraldehyde and pyreneꢀ1ꢀaldehyde (RꢀCHO)] were condensed with 3,5ꢀdimethylꢀ4ꢀ
nitroisoxazole (6) yielding the corresponding styryl derivatives (1ꢀ4). Details of the synthesis of
the starting materials (pyrene aldehyde, 3,5ꢀdimethylisoxazole, 3,5ꢀdimethylꢀ4ꢀnitroisoxazole)
were given in electronic supporting information.
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Scheme 1. Synthetic strategy for the preparation of the styryl isoxazole derivatives (1ꢀ4)
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Synthesis of 3ꢀmethylꢀ4ꢀNitrostyrylisoxazoles (1-4): To a solution of 3,5ꢀdimethylꢀ4ꢀ
nitroisoxazole (6, 1.4g, 10 mmol) in EtOH (10 mL) was added the desired aldehyde (1.0 equiv)
and piperidine (98.0 ꢁl, 0.01 equiv) successively at the ambient temperature. The reaction
mixture was heated at 65ºC for 1 h overnight. After cooling the reaction mixture to room
temperature, the precipitate was collected by filtration and washed several times with Et O to
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give 3ꢀmethylꢀ4ꢀnitroꢀ5ꢀalkenylꢀisoxazoles (1-4) as colored solids.
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(E)ꢀ3ꢀmethylꢀ4ꢀnitroꢀ5ꢀstyrylisoxazole (1): H NMR (500 MHz, CDCl3ꢀd) δ 7.81 (s, 1H), 7.72
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(s, 1H) 7.69 (m, 3H), 7.49 (m, 3H), 2.91 (s, 3H). C NMR (125 MHz, CDCl ) δ 167.18, 156.14,
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143.10, 134.45, 131.12, 129.18, 128.42, 110.98, 95.69, 11.84. HRMS (ESIꢀMS) m/z: calcd for
+
[M+H] , 230.0691; found, 231.0881.
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(E)ꢀ3ꢀmethylꢀ5ꢀ(2ꢀ(naphthalenꢀ2ꢀyl)vinyl)ꢀ4ꢀnitroisoxazole (2): H NMR (500 MHz, CDCl ) δ
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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,
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2H), 7.57 – 7.52 (m, 2H), 2.61 (s, 3H); C NMR (125 MHz, CDCl ) δ 167.20, 156.10, 143.09,
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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 (ESIꢀMS) m/z: calcd for [M+H] , 280.0848; found, 281.1012.
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(E)ꢀ5ꢀ(2ꢀ(anthracenꢀ9ꢀyl)vinyl)ꢀ3ꢀmethylꢀ4ꢀnitroisoxazole (3): H NMR (500 MHz, CDCl ) δ
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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
1
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(d, J = 16.5 Hz, 1H), 7.60 – 7.50 (m, 4H), 2.67 (s, 3H). C NMR (125 MHz, CDCl ) δ 166.63,
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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 (ESIꢀMS) m/z: calcd for [M] ,330.1004; found, 330.1080.
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(E)ꢀ3ꢀmethylꢀ4ꢀnitroꢀ5ꢀ(2ꢀ(pyrenꢀ1ꢀyl)vinyl)isoxazole (4): H NMR (500 MHz, CDCl ) δ 8.91
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(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
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(d, J = 9.0, 1H), 8.09 – 8.04 (m, 2H), 7.91 (d, J = 16.2 Hz, 1H), 2.64 (s, 3H); C NMR (125
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MHz, CDCl ) δ 167.30, 156.24, 139.27, 133.44, 131.33, 130.66, 130.31, 129.20, 127.94,
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127.39, 126.48, 126.46, 126.23, 125.32, 125.01, 124.57, 124.09, 122.01, 112.23, 11.92. HRMS
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(ESIꢀMS) m/z: calcd for [M] ,354.1004; found, 354.1067.
Results and Discussion
Absorption and emission of (1-4)
Figureꢀ1 gives the structural details of the synthesized molecules. The phenyl group in (1),
naphthalene (2), anthracene (3) and pyrene (4) group was connected to the isoxazole ring
through a πꢀspacer (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. Tableꢀ1 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 345ꢀ446 nm [Fig. 2a]. Styrene (1)
containing a simple phenyl ring, absorbs at ~355nm in acetonitrile. Substitution of the naphthyl
ring results in +21 nm shift in absorption. Further, increase in conjugation through the
introduction of other fused rings systems such as anthracene and pyrene results in +88 nm
increase in the absorption maxima. Despite the presence of the additional aromatic ring,
absorption of (4) shows only a slight effect on the absorption (λ ) maxima in comparison to (3).
a
With the change in polarity, moderate absorption changes (+4ꢀ10 nm) were noted for the
substituted styrenes [Fig 2b and Fig. S1].
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Fig. 2 Absorption of a) (1ꢀ4) in acetonitrile; b) (3) in solvents of different polarity.
Tableꢀ1: Absorption and emission data of the synthesized molecules (1ꢀ4)
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Solvent
λa
λf
Stokes
Shift (cm )
5354
5154
5154
6477
6764
7600
5996
Solvent λa
(nm) (nm)
Heptane 365 398, 427, 460
Dioxane 371
THF 370
(2) CH CN 376
λf
Stokes
Shift (cm )
ꢀ1
ꢀ1
(
nm) (nm)
Heptane 345
Dioxane 356
1) THF
415
436
436
461
462
475
464
4618
5262
6150
7511
6938
7319
9325
461
479
524
(
356
355
352
349
363
CH CN
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3
MeOH
DMSO
Water
MeOH
DMSO
Water
369
385
347
465, 496
536
513
Heptane 441
Dioxane 441
490, 518 5464
Heptane 440
Dioxane 447
480, 513
564
574
612
593
3234
4640
4999
6233
5761
6023
6649
552
4559
5193
7193
6024
7237
5893
THF
441
434
438
436
453
572
THF
446
(
3) CH CN
631
(4) CH CN 443
3
3
MeOH
DMSO
Water
595
MeOH
DMSO
Water
442
453
442
509,637
618
623
626
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Fluorescence properties in solution
Styrene (1) bearing a phenyl ring shows emission (λ ) at ~461nm in acetonitrile. Styrene (2)
f
containing a naphthalene moiety emits at ~ 524 nm, Styrene (3) bearing an anthracene system
emits at ~631nm and pyrenyl styrene (4) emits at 612 nm [Tableꢀ1]. The bathochromic shift in
emission reflects the extended delocalization due to increased aromaticity. Solvent polarity has
moderate to strong influence on the emission behavior of these styrenes. In nonꢀpolar heptane,
the structured emission is noted for (3) and the increase in solvent polarity results in
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bathochromic emission shifts (λ ) shifts. Styrene (1) shows a bathochromic shift of + 46nm upon
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changing the solvent polarity from heptane to acetonitrile. Styrene (2) shows structured emission
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at 399, 427 and 460 nm in heptane but shows significant emission (+64 nm) shifts in acetonitrile
[Fig.3a]. Similarly, anthryl substituted (3) and pyrenyl substituted (4), shows about +113 and
+99 nm shifts in the emission with an increase in solvent polarity [Fig.3b and 3c] respectively.
The order being phenyl < naphthyl <pyrenyl <anthryl [Fig 3d].
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Fig. 3 Normalized Emission of styryl isoxazole derivatives 2 (λ 350 nm), 3 (λ 440 nm) and 4
ex
ex
(λex 440 nm) in different solvents (aꢀc) and (d) normalized emission of styrenes (1ꢀ4) in
acetonitrile.
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This strong solvent polarity dependent emission indicates the presence of an excited state with
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
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intramolecular charge transfer
. These compounds show weak and blueꢀshifted emission in
methanol as compared to solvent acetonitrile. This emission behavior is influenced by the
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involvement of solute (fluorophore)ꢀsolvent (methanol) dipoleꢀdipole interactions and
intermolecular hydrogen bonding interactions.
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Detection of H2S
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H S, a third member of gasotransmitter family along with NO and CO, plays an important role in
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some biological and pharmacological processes
. For this reason, it is important to detect the
presence of H S, and we have utilized spectroscopic properties of the synthesized
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styrylisoxazoles (1ꢀ4). The absorption and emission spectra of styryl isoxazoles were recorded
in DMSOꢀH O (9:1) mixture. Pyrenyl isoxazole (4) absorbs at ~450 nm and emits at ~631 nm in
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DMSOꢀH O binary mixture. Upon titrating with aqueous sodium sulfide [typically the emission
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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 hyposchromic 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 wellꢀdefined isosbestic point at ~407
nm [Fig 4a]. This shift in absorption may indicate the formation of a new species, and the blueꢀ
shifted absorption is attributed to a loss in delocalization. This is a consequence of shifts in the
change of the structure from initial electron withdrawing nitro (NO ) which is reduced to an
2
electron donating amino group (NH ). The changes in the emission spectra of (4) in the absence
2
or presence of Na S (0–170 ꢁM) are displayed in Fig. 4b. At 450 nm excitation, compound
2
(4) shows emission maxima at 631 nm in the absence of Na S. However, the addition of Na S
2
2
results in significant loss of emission intensity with the eventual loss of emission. Noticeably
emission at the blue region of the spectrum (at or near 400ꢀ450 nm) seems increasing. To get
more accurate picture of this emission behavior, we excited the compound (4) at 350 nm which
is a dominant absorption band post addition of Na S. The emission shows two peaks a long
2
charge transfer band at ~630 nm and a shorter wavelength structured emission at 380, 400 and
420 nm. These shorter wavelength emission peaks show enhancement in intensity upon 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 isoxazole. In the
case of (3), the emission at ~630 nm decreases in intensity, and new emissive peaks appear at
shorter wavelength regions ~500 nm with a sudden surge in emission intensity at 80µM [Fig 5a].
Excitation at 350 nm shows emission peaks primarily due to the anthracene [Fig. S3]. Naphthyl
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derivative (2) also shows a drop in emission intensity of the charge transfer band (542 nm) with
slight shift of 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) than for (2). The changes to
UVꢀspectral profile upon reduction of nitro to the amino group are given in the supporting
information [Fig. S2]. H S acts as a weak acid and may contribute to the observed emission
2
changes. Our experiments showed that the emission is unaffected with pH variation [Fig S8]. To
check the sensitivity for other analytes such as Na SO , cysteine, homocysteine and glutathione,
2
3
the isoxazole derivative (4) was titrated with different analyte concentration. The results are
shown in the supporting information. 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
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given concentration of the analyte reveals greater sensitivity for H S. While this result is
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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
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mechanism
. The results obtained from mass spectral analysis gave a molecular weight much
higher than sole amino product. The product formed cannot be conclusively confirmed based on
Mass spectral data alone and require further experimentation.
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Fig. 4 UVꢀVis absorption (a) and emission response (b): λ 450 nm and (c): λ 350 nm of probe
ex ex
4 (10 µM) with increasing concentration (0 to up to 200 µM) of Na S in DMSO + H O (9:1)
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binary mixture.
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Fig.5 Fluorescence response of probe a) 3 [λ 440 nm] and b) 2 [λ 380 nm] with increasing
ex ex
concentration (0 to 220 µM) of Na S in DMSO + H O (9:1) binary mixture.
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Sensing mechanism
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The spectral changes involving the emission response are attributed to the loss of charge transfer
due to the conversion of electron withdrawing nitro group to an electron donating amine group
[Fig. 6]. After the reaction with H S, the donorꢀacceptor electron system was inhibited by the
2
donorꢀdonor electron system. This expectedly disturbs the charge transfer yielding the loss in the
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emission intensity and the emission spectral shifts . This reduction of the nitro group to the
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0, 51
amino group is facilitated by the generation of sulfide anion
. To prove the product formation,
the mass spectral analysis was performed and the HRMS analysis predicts the formation of the
amino product (Fig. 7 and Fig S4ꢀS6). The product cannot be isolated, and NMR characterization
could not performed as the sample concentrations are too low. For qualitative visualization, the
obtained products are subjected to Ninhydrin test and the products show strong color [Fig. S7].
These results pave the way for identifying quicker means of H S detection with relevance to
2
industrial and biological applications.
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NH2
NO2
N
H S
N
2
O
O
O N
H N
2
2
DMSO/H O (9:1)
2
Ar
Ar
1
Ar=
1
2
Fig 6. Schematic representation for the reduction of the nitro group for (1ꢀ4) into an amino
3
4
group by H S in DMSO/H O mixtures.
2
2
5
6
7
8
9
0
Fig 7. HRMS spectra of the amine compound obtained after reduction of (4). The mass spectral
data of other compounds are given in the supporting information
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Conclusions
In summary, we have demonstrated detection of micromolar (60 µM) quantities of hydrogen
sulfide through a rapid fluorescence response through reduction of nitro to 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 emission
shifts attributed to intramolecular charge transfer. This unique charge transfer emission is
quenched by the addition of H S resulting in a fast and significant emission response. The
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formation of the amine product is confirmed by mass spectral analysis.
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Acknowledgements
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.
Supporting information
NMR spectral characterization data, supporting absorption and emission figures are included.
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54x190mm (96 x 96 DPI)