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At the Molecular Level through Photophysical Studies: Structural Implications on the Reactivity of Dual-Site Sensitive Positional Isomers Toward a Gasotransmitter (H2S) M. Venkateswarulu,† Pankaj Gaur,† Sougata Sinha,† Avijit Pramanik,‡ and Subrata Ghosh*,† †

School of Basic Sciences, Indian Institute of Technology Mandi, Mandi 175001, Himachal Pradesh, India Department of Chemistry and Biochemistry, Jackson State University, 1325 J. R. Lynch Street, Jackson, Mississippi 39217, United States

‡

J. Phys. Chem. C 2015.119:19367-19375. Downloaded from pubs.acs.org by RICE UNIV on 09/03/15. For personal use only.

S Supporting Information *

ABSTRACT: A combined experimental and theoretical approach has enabled us to understand at the molecular level the importance of positional and electronic effects of chemical functionality present in molecular system that acts as an optical signaling agent. The present study demonstrates the structural implications of isomeric dual-site reactive (nitro and sulfonte ester groups) molecular probes (P1, P2, and P3) on optical signaling of hydrogen sulfide (H2S), a known emerging mediator in human physiological activities and diseases. The reactivity of these probes toward H2S was established using fluorescence signaling studies. The reductive interaction of H2S with nitro functionality of P2 resulted in the formation of orange fluorescent amine derivative P2′, while the nucleophilic S−O bond cleavage of sulfonate ester group of P3 produced sulfonothionic acid derivative P3′ as a green emissive fluorescent species. Crystal structure determination and structure−reactivity relationship studies demonstrated positional as well as electronic effects of nitro functionality on the reactivity of these probes. While the electronic effect is responsible for increasing the reactivity of sulfonate functionality, the accessibility of the reactive site by H2S is dictated by the steric factor. Although both −M (mesomeric) and −I (inductive) effects of nitro functionality are supposed to be prominent in P1 and P3, crystal structure analysis revealed a steric crowding on P1created by nitro group as well as out-of-plane arrangement of nitro group, which in turn makes P1 much less reactive than P3. In the case of P2, the probe is free from steric effect, but the weak −I effect and the absence of −M effect made sulfonate functionality nonreactive toward H2S. At the same time, slow reductive interaction of nitro group of P2 yielded orange emissive fluorescent species P2′.

1. INTRODUCTION The understanding of structural dynamics is of enormous curiosity as the precise orientation, position, and electronic effects of a substrate group are the major and fundamental concepts for evaluation and better understanding of chemistry of organic compounds. These factors play a fascinating role in reactivity determination of molecular probes toward the wellknown gaseous signaling compound like nitric oxide (NO), carbon monoxide (CO), and H2S. Out of which, H2S is a noxious gas that is not only fabulous for its unpleasant smell and toxicity but also plays an important role in a wide range of physiological activities.1−13 The recent studies revealed that overexpression of H2S-producing enzyme and continual exposure to the industrial produced H2S could be the cause of various diseases ranging from Alzheimer’s disease14 and Down’s Syndrome15 to diabetes,16 pulmonary hypertension, and liver cirrhosis.17 Hence, for the better understanding of its physiopathological importance, the development of new efficient techniques for its monitoring is a dire need. In this context, several techniques for H2S detection, such as colorimetric,18−20 metalinduced sulfide precipitations,21 and electrochemical22−24 and chromatographic assays25,26 have been developed successfully © 2015 American Chemical Society

in the past few decades. As a suitable and efficient alternative of these methods, fluorescence-emission-based optical signaling of H2S has been proven to be highly selective, sensitive, and more convenient. Recently, several groups reported the development and application of reaction-based probe for H2S detection,27−31 which inspired the researchers to design new molecular probes that can use H2S either as a nucleophile32−38 or as a reductant to reduce azide or nitro groups on masked fluorophores.39−43 While the reported probes involve some interesting chemical process as previously mentioned for H2S signaling, to our knowledge, H2S recognition through sulfonate ester bond cleavage has not been realized until date. Most importantly, although all three gasotransmitters are relatively small in size, how the signaling process gets affected by the steric as well electronic factors present in the positional isomeric probes has not yet been investigated and reported. Therefore, it is highly crucial to address these fundamental aspects through structure− reactivity relationship studies. Such studies will also reveal that Received: June 8, 2015 Revised: July 20, 2015 Published: July 21, 2015 19367

DOI: 10.1021/acs.jpcc.5b05459 J. Phys. Chem. C 2015, 119, 19367−19375

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The Journal of Physical Chemistry C

Br−, NO3−, CN−, HSO4−, PO43−, and AcO− as tetrabutylammonium salt), cysteine, homocysteine, glutathione, and H2S (as NaHS) in H2O buffered with HEPES (1 mM, pH 7.2) were used as standard solutions to record the UV−vis and fluorescence spectra. FT-IR spectra in KBr were recorded on a PerkinElmer Spectrum 2 spectrophotometer. 1H and 13C NMR spectra in CDCl3 were recorded on Jeol-ECX-500 MHz spectrometer using tetramethylsilane as an internal standard. Absorption spectra were recorded with SHIMADZU UV-2450 spectrophotometer. The fluorescence spectra were recorded with Cary Eclipse spectrophotometer with slit widths of 5 and 10 nm for excitation and emission, respectively. LC−MS−ESI spectra were recorded on a Bruker impact-HD spectrometer. X-ray diffraction analysis patterns were measured on X-ray diffractometer system, which is of Agilent Technologies using Cu Kα, λ radiation (λ = 1.5406 Å). 2.2. Single-Crystal X-ray Diffraction Studies. Single crystals of P1/P2/P3 suitable for X-ray diffraction were grown in ethanol by its slow evaporation. Diffraction studies were performed on Agilent Technologies X-ray diffractometer system by irradiating the crystals using Cu K α radiation (λ = 1.5406 Å) at 298(2) K, which revealed that P1 crystallizes in orthorhombic P212121, P2 in monoclinic P121/c1, and P3 in orthorhombic Pbca space groups. Data were collected by standard “CrysalisPro” Software (online version), and reduction was undertaken with CrysalisPro Software (offline version). The molecular structure was solved by direct methods OLEX2 and was refined using full-matrix least-squares (F2) on SHELXL-97. The positions of all non-hydrogen atoms were located and were refined anisotropically. After that, hydrogen atoms were obtained from the residual density map and refined with isotropic thermal parameters. All parameters of crystal structures are given in Table S1 (Supporting Information). 2.3. UV−vis and Fluorescence Titrations. UV−vis and fluorescence titrations were conducted using 10 μM solutions of probes (P1/P2/P3) in THF/H2O solution (1:1 v/v) buffered with HEPES (1 mM, pH 7.2). All measurements were performed using 348 nm as an excitation wavelength by keeping excitation and emission slit widths as 5/10, respectively. UV−vis and fluorescence spectra were recorded using a 3 mL quartz cuvette (path length 1 cm) by the addition of freshly prepared solutions of anions (1 mM) (Cl−, F−, Br−, HSO4−, CN−, NO3−, PO43−, ClO4−, and CH3COO− as tetrabutylammonium salts) and biothiols (cysteine, homocysteine, glutathione) (1 mM). NaHS solution was used as a source of H2S (10 mM). UV−vis and fluorescence titrations were conducted using all stock solutions in high concentration to avoid dilution error. 2.4. DFT Calculations. The geometry of all of the probes P1/P2/P3 and their derivatives were optimized at DFT with B3LYP/6-31G(d,p) basis set44,45 with no symmetry constraint using the Gaussian 09 suite of programs.46 Frequency calculation at the same level with the same basis set was performed to ensure that the geometries correspond to real minima. Gauss view software along with Chemcraft software was used for visualization purpose.47


the positional and electronic effects of functional groups cannot be overlooked while designing molecular probes for H2S signaling. Herein, we present a combined experimental and theoretical approach to understand and explain at the molecular level the importance of electronic and steric factors for a molecular optical material to be used for signaling H2S. To execute our plan, we synthesized nitro-substituted dansyl-based three isomeric molecular probes P1, P2, and P3 (Figure 1), and their

Figure 1. Chemical structures of P1, P2, and P3.

reactivity toward H2S through photophysical studies was carefully investigated. We have explored, for the first time, the reactivity of functional sulfonates toward H2S to develop new probes, which ultimately renders turn-on fluorescence upon interaction with H2S. The probes were designed such that they all contain two reactive sites for H2S viz. nitro and sulfonate ester functionalities. A substantial change in their photophysical behavior while interacting with H2S made us curious to investigate the positional/electronic effect of substituents in isomeric molecular materials. An understanding through crystal structure determination revealed that the reactivity of these probes is governed by a delicate balance between the electronic and steric factors. The photophysical behavior of these newly developed probes was investigated by studying the changes in UV−vis and fluorescence spectra and correlating these changes with their chemical structures. Interestingly, while the probe P3 (bearing nitro functionality at para position) underwent fast sulfonate bond cleavage with strong fluorescence emission upon interaction with H2S (mainly due to the presence of strong electronic effects, both −M and −I, and the absence of steric crowding, which make the sulfonate bond highly reactive), P2-containing nitro group at the m position produced a weak fluorescence signal due to the formation of an amine derivative through nitro reduction by H2S (because of the presence of weak electronic effect, only −I, and the absence of steric crowding). The third positional isomer P1 bearing nitro group in the ortho position was found to be unreactive toward H2S. Crystal structure analysis helped us to conclude that the steric factor dominates over electronic factor in P1, which, in turn, makes it silent toward H2S. TD-DFT (time-dependent density functional theory) calculation results supported this conclusion. The chemical structures of these probes were fully characterized by single-crystal X-ray analysis, NMR spectroscopy, and mass spectrometry.

3. RESULTS AND DISCUSSION 3.1. Synthesis. The reaction between dansyl chloride and nitro phenols in dichloromethane in the presence of triethylamine gave the desired compounds P1/P2/P3 in good yield (Scheme S1). The final products were fully characterized using various spectroscopic techniques such as FT-IR, NMR, ESI-MS, and X-ray single-crystal analysis.

2. EXPERIMENTAL SECTION 2.1. General Information. All chemicals were purchased from Merck, S.D. Fine, and Sigma-Aldrich and were used without further purification. THF (AR grade) was used for the spectral studies. Freshly prepared solutions of anions (Cl−, F−, 19368


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Figure 2. UV−vis spectra of probe P1/P2/P3 (10 μM) in THF/H2O (1:1, v/v) buffered with HEPES (1 mM, pH 7.2) upon the addition of NaHS (0.83 mM). Inset: UV−vis spectra of P1/P2/P3 (10 μM) in THF/H2O (1:1, v/v) buffered with HEPES (1 mM, pH 7.2).

when P2 (10 μM) was exposed with H2S (Figure S6). The origin of the fluorescence enhancement was because of the formation of a weakly fluorescent amine derivative P2′ through H2S-mediated reduction of nitro functionality (electron-withdrawing group in P2) to the amine functionality (electron-donating group in P2′) (Scheme 1). To support our conclusion, the product P2′ was isolated from the reaction mixture and fully characterized using various spectroscopic techniques (Figures S7 and S8 and detailed characterization data have been provided in the Supporting Information). In contrast with P2, we noticed an impressive fluorescence enhancement (change in fluorescence quantum yield (Φ) = 0.0038 to 0.089) with 9 nm blue shift in emission maxima (from 492 to 483 nm) when P3 was exposed to H2S (Figure 3a and Figure S9). While no spectral changes were observed in the presence of other relevant biothiols and anions, the fluorescence enhancement upon interaction with H2S strongly suggested high affinity and excellent selectivity of P2/P3 toward H2S over other relevant competitors (Figure 3b and Figures S10 and S11). The electrophilic nature of other two gassotransmitters (NO and CO) possibly makes them the unreactive toward P3 (Figure 3b). The interesting observation on H2S-directed strong fluorescence enhancement of P3 was a 9 nm blue shift in emission maxima, which was entirely different from the results obtained from the interaction between H2S and P2, where we observed 80 nm red shift in emission maxima (Figures S6 and S9). These experimental results indicated that the mode of interaction of P2 with H2S is different from the P3−H2S interaction, and hence the origins of fluorescence enhancement in both of the cases were different. As previously mentioned, we proved that the nitro group of P2 reacts with H2S and gets converted to amine functionality through a slow reduction process, which ultimately led to fluorescence enhancement. While looking at product composition in the reaction mixture of P3 and H2S, we observed that P3 undergoes sulfonate ester bond cleavage (S−O bond) through nucleophilic attack of H2S (thiolysis) at the electron-deficient S center and thus produces a strong fluorescent species sulfonothionic acid derivative P3′ (Scheme 1 and Scheme S2). All of the products were successfully isolated in pure form from the reaction mixture and were characterized using common spectroscopic techniques (Figures S12−S14 and detailed characterization data have been provided in the Supporting Information). Therefore, the origin of H2S directed fluorescence enhancement followed two different mechanisms for P2 and P3 (Scheme 1). These chemical reactions also eliminated the possibility of d-PET mechanism in the final

3.2. Photophysical Studies. We investigated first the photophysical behavior of probes P1/P2/P3 (10 μM) by UV− vis absorption studies in 50% aqueous tetrahydrofuran solution buffered with HEPES (1 mM, pH 7.2). The inset of Figure 2 represents the absorption profiles of P1/P2/P3, which indicate similar absorption profile of all of these three isomeric probes. Probe P1 showed strong absorption bands centered at 253 (ε ≈ 8.433 M−1 cm−1) and 352 nm (ε ≈ 1566 M−1 cm−1), P2 exhibited a sharp absorption peak at 256 nm (ε ≈ 10 500 M−1 cm−1) and a band at 349 nm (ε ≈ 1800 M−1 cm−1), and P3 showed absorption bands at 261 nm (ε ≈ 15 500 M−1 cm−1) and at 348 nm (ε ≈ 2600 M−1 cm−1). All these three probes were found to be weakly fluorescent (Figure S1 and Table S2 for detail spectral parameters) mainly because of photoinduced electron transfer (PET) from excited fluorophore (dansyl unit) to the strong electron-withdrawing benzene moiety (nitro phenol unit), which is also known as donor-excited PET or d-PET.48,49 After the initial photophysical studies, the probes were exposed to NaHS (0.83 mM) under the same condition. The addition of NaHS had almost no effect on the absorption profile of P1 (Figure 2a). Interestingly, significant changes were noticed in the absorption spectra of P2 and P3 in the presence of NaHS (Figure 2b,c and Figure S2). While the absorption peak of P3 at 261 nm almost disappeared with the appearance of a new strong peak at 404 nm with increasing concentration of NaHS, the generation of a broad absorption band with two absorption peaks at 301 and 370 nm was observed in the case of P2 (Figure 2b). From the absorption studies it was clearly evident that all three of these probes viz. P1/P2/P3 behave differently in the presence of NaHS, which encouraged us to explore the intramolecular structural factors that are responsible for controlling NaHS-triggered chemical modification. In contrast with NaHS, other biothiols such as cysteine, homocysteine, and glutathione had no effect on the absorption profiles of P2/ P3 (Figures S3 and S4). Moreover, the absorption spectra of P2/P3 in the presence of various anions such as HSO4−, PO43−, NO3−, CN−, F−, Cl−, and CH3COO− remained unaffected (Figures S3 and S4). The changes observed during UV−vis studies encouraged us to study the emission properties of P1/P2/P3 in the presence and absence of H2S. All of these three probes were found to be weakly fluorescent. Whereas the emission profile of P1 remained unaffected in the presence of H2S (Figure S5), only a few folds increase in emission intensity (change in fluorescence quantum yield (Φ) = 0.0024 to 0.014) along with an 80 nm red shift in emission maxima (from 473 to 553 nm) was observed 19369


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Scheme 1. Mechanism of H2S-Mediated Structural Change Followed by Fluorescence Enhancement

Figure 3. (a) Emission titration spectra of P3 (10 μM) in THF/H2O (1:1, v/v) buffered with HEPES (1 mM, pH 7.2) obtained upon gradual addition of NaHS (0 to 0.83 mM) (excitation at 348 nm). Solution was incubated for 15 min at 35 °C. (b) Emission spectra of probe P3 (10 μM) in THF/H2O (1:1, v/v) buffered with HEPES(1 mM, pH 7.2) upon the addition of NaHS, Br−, Cl−, F−,CN−, OAc−, HSO4−, ClO4−,NO3−, PO43−, CO, NO, Cys, Glu, and Homocys (0.83 mM) and excitation at 348 nm. Solution was incubated for 15 min at 35 °C.

compounds and thus resulted in impressive fluorescence signaling of H2S. The probe P3 can detect as low as 26 nM H2S, which strongly recommends the trace amount detection of H2S (Figure S15). The time-dependent fluorescence study was carried out to monitor the thiolysis reaction of probe P3 with time (Figure S16). This study revealed the immediate onset of thiolysis reaction after the addition of NaHS, and it took 15 min to complete the S−O bond cleavage process. To investigate the effect of pH on H2S-mediated thiolysis of P3, we conducted pH titration within a wide pH range of 3.0−9.0, which covers physiological pH. Interestingly, pH had no influential role in S−O bond cleavage and thus the probe P3 was stable enough in various pH (Figure S17), which implies

the working efficiency of probe P3 at different pH values. Because H2S is a weak acid, depending on the pH of the solution, it mainly exists either as H2S (pH 5 or below) or as HS− (pH 9 or above) or as a combination of H2S and HS− (pH ∼5.5−8.5).13 From the pH experiments, it is clear that P3 exhibits fluorescence enhancement in a wider pH range (pH 3−9). These results clearly indicate that both of the nucleophilic species, H2S and HS−, are reactive toward P3. Therefore, at physiological pH (at which both the species exist, H2S and HS−), the concertation refers to the total sulfide concentration that includes H2S and HS−. The findings of pH experiments revealed that although negative inductive effect (−I) operates in all three probes (P1/P2/P3) and negative 19370


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Figure 4. ORTEP diagram of isomeric probes P1 (a), P2 (c), and P3 (e) showing atomic crystallographic numbering scheme. Thermal ellipsoids are scaled to the 50% probability level. (b) Perspective 2D-packing view of P1 along c axis. (d) Packing capped stick model view of P2 along c axis. (f) Zigzag chain structure of P3 when viewed along the b axis.

mesomeric effects (−M) are supposed to be active in P2/P3, these three positional isomers behave differently while interacting with H2S; whereas P3 was found to be highly reactive toward H2S, P2 reacted slowly, and most interestingly P1 remained silent. It is known that NaHS in the presence of water dissociates into NaOH, H2S, and HS−. To ensure that H2S/HS− were responsible for these entire chemical changes but not the NaOH (as we used aqueous solution of NaHS as H2S source), we treated the probes with pure H2S gas generated at Kipp’s apparatus (to avoid the presence of NaOH) and observed similar spectral changes (Figure S18). All of these observations suggest that the reactivity of gasotransmitters inside biological body is highly dependent on the chemical/structural environment of the reacting site of molecular/macromolecular species. Such observations might also help the researchers while designing drug candidates for H2S-mediated diseases. We then became curious to understand at the molecular level the reasons for such different chemical and optical changes for different positional isomers when they interact with H2S, although all three of the probes contain dual reactive sites (nitro and sulfonate centers). The high reactivity of p-nitro-substituted probe P3 was attributed to the presence of sterically relaxed

and electronically activated sulfonyl ester as compared with P1/P2.50 3.3. Single-Crystal Analysis. To understand at the molecular level, we investigated the structural parameters obtained from single crystals analysis of P1/P2/P3. The molecular structures of the o-, m-, and p-nitro-substituted isomeric probes [C18H16N2O5S] along with the numbering scheme and 3D architectures are shown in Figure 4; whereas, o- and p-nitrosubstituted molecular probes were crystallized in the highly symmetric orthorhombic, P212121, Pbca space groups, the metanitro derivative was crystallized in monoclinic, P121/c1 space group. All of these probes contain the −NMe2 group, which shows positive mesomeric effect (+M), whereas the −NO2 group shows negative mesomeric effect (−M). These are responsible for photoinduced electron transfer (PET) process. Because of extended conjugation of the −NO2 group with the C−O bond connected to ‘S’ of sulfonate ester functionality via aromatic ring, the negative mesomeric effect (−M) is more prominent in the p isomer. The −M effect makes the O atom of C−O bond highly electron-deficient, which, in turn, enhances the reactivity/ electrophilicity of the S center of sulfonate functionality (Scheme 2 and Figure 5). On the contrary, only the −I effect of nitro group is prominent in P2. Because it is well known that the −M effect is 19371


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The Journal of Physical Chemistry C Scheme 2. Contributing Structures of P3a


a

3.58 Å for m isomer (Figure S20b), and C20−H···O12 3.17 Å, C19−H···O12 3.12 Å, and C22−H···O25 3.25 Å for p isomer (Figure S20c) play an important role in the formation of 3D architecture. 3.4. Mass Spectroscopy Studies. To further validate our conclusion on sulfonate ester bond cleavage, we conducted the mass titration experiment by taking probe P3 (1 μM) in THF/ H2O (1:1). After the addition of 30 μM NaHS to the probe solution, the reaction mixture was stirred at 30 °C for 20 min. The solution was concentrated under reduced pressure to yield the crude product. The product was dissolved in acetonitrile and analyzed by HRMS. The characteristic peak of P3 was not observed in the mass spectra of reaction mixture, but new peaks were observed at m/z 266.0303(C12H12NO2S2) and 138.0199 (C6H4NO3) which correspond to [P3′+H]+ and [P3″]+ respectively. These results strongly supported the sulfonyl ester bond cleavage of P3 upon the addition of H2S (Schemes S1 and S2 and Figure S21). Similarly, we performed a mass titration experiment for P2 under the same conditions. The mass spectra showed the complete disappearance of the molecular ion peak of P2 and the appearance of new peaks at m/z 343.1110 [C18H19N2O3S]+ and 365.0930 [C18H18N2NaO3S]+, which correspond to [P2′] + and [P2′+Na]+, respectively. These results indicated that instead of sulfonate ester cleavage the nitro group of P2 underwent reduction to produce the amine derivative P2′ (Scheme S1 and Figure S22). 3.5. Theoretical Studies. Theoretical analyses have been carried out to understand the probable structure of P1/P2/P3 along with its photophysical properties in the gas phase. Ground-state geometry optimizations and frontier molecular orbital calculations have been carried out at the B3LYP/ 6-31G(d,p) level44,45 of density functional theory using Gaussian 09 suite of programs.46 Frequency calculation at the same level with the same basis set was performed to ensure that the geometries correspond to real minima. For geometry optimization, initial structures of P1/P2/P3 have been chosen as predicted by X-ray analysis. Optimized structures of P1/P2/P3 are shown in Figure 7. It clearly indicates that all three probes hold their original structural pattern, as predicted by X-ray analysis. While the bond distances between the two oxygen atoms of the sulfonyl ester group and between the two oxygen atoms of the nitro group converge toward the molecule at the S−O−C group across the twist motif with N−O···O−S distances of 3.64 and 3.68 Å, 6.07 and 6.60 Å, and 6.78 and 8.63 Å, respectively, the S−O−C bond angles are 118.97, 119.76, and 120.31° for probe P1/P2/P3, respectively. These bond distances and bond angle values are very close to the same in solid state as predicted by X-ray analytical studies.

Extended conjugation increases electrophilicity of S-center (P3−B).

many folds stronger than the −I effect, theoretically the sulfonate group of p-nitro derivative should be highly sensitive toward the nucleophilic attack of H2S as compared with P2, which is supported by the experimental data, but the most interesting fact is the silence of P1 while interacting with H2S. Similar to P3, the −M and −I effects of the nitro group are expected to be prominent in P1. Single crystallographic results revealed that the nitro group is slightly out of the plane with respect to the plane of aromatic ring and C−O bond (Figures 5 and 6). This lack of planarity inhibits the −M effect of nitro group as effective overlapping of p orbitals (necessary for extended conjugation) gets affected; therefore, the S center does not gain any additional reactivity due to the presence of the nitro group at the o position. In addition, the nitro group induces steric crowding in the o-isomer P1 because of the close proximity of nitro and sulfonate functionalities. This effect in fact does not allow H2S to approach either of the functionalities for any chemical process. The bond distances of two oxygen atoms of the nitro group and two oxygen atoms of the sulfonyl ester group converge toward the molecule at the S−O−C group across the twist motif with N−O···O−S distances of 3.455 and 3.639 Å (Figure S19a for P1), 6.68 and 6.24 Å (Figure S19b for P2), and 6.9 and 8.55 Å (Figure S19c for P3) respectively. The interplanar angles between nitrobenzene groups and dansyl moieties of P1, P2, and P3 are 59.49, 72.15, and 84.55° (Figure 6) and the S−O−C bond angles are 118.03, 119.57, and 122.46°, respectively (Table S3). The higher interplanar angle and larger S−O−C bond angles of the p isomer also further support the absence of steric crowding in P3 as compared with P1. In addition, other supramolecular interactions, such as C−H···O, C−H···π, C16−H···O14 3.50 Å, C10−H···O14 3.25 Å, and C20−H···O14 3.41 Å for o isomer (Figure S20a), C−H··· O,C16−H···O14 3.47 Å, C8−H···O13 3.22 Å, and C25−H···π

Figure 5. (a) Out-of-plane view of aryl ether with nitro group in o-isomer (P1). (b,c) Perspective in plane views of aryl ethers with nitro groups in m-(P2) and p-(P3) isomers. 19372


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Figure 6. Interplanar angle between nitrobenzene groups and dansyl moieties of probes P1 (a), P2 (b), and P3 (c), respectively.

Figure 7. Ground-state optimized structure of P1, P2, and P3.

Probe P3 is more stable than probe P2 (1.08 kcal/mol), while it is much more stable in comparison with probe P1 (8.26 kcal/mol). Calculated dipole moments are 10.37, 9.79, and 6.71 D for P1/ P2/P3, respectively. The frontier molecular orbitals are depicted in Figures S23−S25. In all cases HOMOs are mainly formed by the N,N-dimethylaminonaphthyl ring and the LUMOs are situated mainly on the nitrobenzene ring. The HOMO−LUMO energy gaps in P1/P2/P3 are 3.66, 3.48, and 3.46 eV, respectively. Because of the strong electronwithdrawing effect induced by nitro group, the nitro-substituted rings experience lower LUMO energy level compared with unsubstituted rings and possess lowest HOMO−LUMO energy gap.51 Thus, the lower LUMO energy level allows intramolecular electron transfer from the singlet excited state fluorophore to the electron deficient benzene ring, which is commonly termed as d-PET mechanism and the main cause behind the weakly fluorescent nature of these probes.52 The well-separated charge distribution between HOMO and LUMO in probe P3 upon excitation indicates considerable charge transfer from the donor unit (N,N-dimethylaminonaphthyl group) to the acceptor unit (nitro benzene). The addition of H2S induced the thiolysis reaction in the case of probe P3 and generates a fluorescent thiol adduct (P3′). With the help of DFT calculation we also propose the optimized geometry of the thiol adduct P3′ (Figure 8). The S−S and S−H bond lengths in P3′ are 2.16 and 1.35 Å, respectively. HOMO is mainly situated on the N,N-dimethylaminonaphthyl, while the LUMOs are placed on the N,N-dimethylaminonaphthyl and −SO3SH group. Figure 9 indicates that H2S-induced thiolysis reaction brings HOMO and LUMO in the same plane, which may be the key behind the strong fluorescence nature of P3′ along with the removal of d-PET process. On the contrary, the presence of H2S converted the nitro group to the amine group in the case of P2. The optimized structure and frontier molecular orbital of the amine derivative are shown in Figures S26 and S27.

Figure 8. Ground-state optimized structure of P3′.

Figure 9. Frontier molecular orbitals of P3′ (isocontour at 0.03 au). 19373


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The Journal of Physical Chemistry C HOMO is shifted to the amine-containing benzene ring, while LUMO is located on naphthyl skeleton. TD-DFT calculation has been carried out to correlate the theoretical absorption spectra of P1, P2, P3, P3′, and P2′ with the experimental observation. Theoretical analysis reveals the absorption band at 364 and 255 nm for probe P1, which falls within the experimental major absorption band domain (254 and 354 nm) (Table S4 and Figure S28). Similarly the calculated absorption values are found to be the part of experimental absorption band domain for probe P2/P3. These values are 364, 254 for P2 and 366, 255 nm for P3 (Table S4 and Figures S29 and S30). Two more dominant vertical electronic transitions are observed at 269 and 274 nm during TD-DFT calculation of probe P3. Theoretical analysis is also in close proximity to the experimental absorption value in the case of the thiol adduct as well. UV−vis spectral analysis predicted the absorption peak at 409 and 305 nm, which is very close to the theoretical predicted value 388 and 293 nm (Table S4 and Figure S31). From the TDDFT studies it is quite evident that HOMO−LUMO transition is possible only in the case of P3′, which may be the origin of the fluorescence (Table S4). TD-DFT studies support the experimental absorption peak of the amine derivative P2′ at 372 nm (theoretical value: 357 nm) (Table S4 and Figure S32) and also show the possible HOMO−LUMO transition, which is absent in the case of its nitro derivative.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was received from the Department of Science and Technology, India (Grant No. SERB/F/2408/2012-13). M.V. thanks CSIR, India for research fellowship and P.G. is grateful to DST for his fellowship. We thankfully acknowledge the Director, IIT Mandi for research facilities. The support of Advanced Materials Research Center (AMRC), IIT Mandi, for sophisticated instrument facility is thankfully acknowledged.

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REFERENCES

(1) Szabo, C. Hydrogen Sulphide and its Therapeutic Potential. Nat. Rev. Drug Discovery 2007, 6, 917−935. (2) Abe, K.; Kimura, H. The Possible Role of Hydrogen Sulfide as an Endogenous Neuromodulator. J. Neurosci. 1996, 16, 1066−1071. (3) Eto, K.; Asada, T.; Arima, K.; Makifuchi, T.; Kimura, H. Brain Hydrogen Sulfide is Severely Decreased in Alzheimer’s Disease. Biochem. Biophys. Res. Commun. 2002, 293, 1485−1488. (4) Fiorucci, S.; Antonelli, E.; Mencarelli, A.; Orlandi, S.; Renga, B.; Rizzo, G.; Distrutti, E.; Shah, V.; Morelli, A. The Third Gas: H2S Regulates Perfusion Pressure in Both the Isolated and Perfused Normal Rat Liver and in Cirrhosis. Hepatology 2005, 42, 539−548. (5) Li, L.; Bhatia, M.; Zhu, Y. Z.; Zhu, Y. C.; Ramnath, R. D.; Wang, Z. J.; Anuar, F. B.; Whiteman, M.; Salto-Tellez, M.; Moore, P. K. Hydrogen Sulfide is a Novel Mediator of Lipopolysaccharide-Induced Inflammation in the Mouse. FASEB J. 2005, 19, 1196−1198. (6) Miller, T. W.; Isenberg, J. S.; Roberts, D. D. Molecular Regulation of Tumor Angiogenesis and Perfusion via Redox Signaling. Chem. Rev. 2009, 109, 3099−3124. (7) Nicholson, C. K.; Calvert, J. W. Hydrogen Sulfide and Ischemia Reperfusion Injury. Pharmacol. Res. 2010, 62, 289−297. (8) Papapetropoulos, A.; Pyriochou, A.; Altaany, Z.; Yang, G.; Marazioti, A.; Zhou, Z.; Jeschke, M. G.; Branski, L. K.; Herndon, D. N.; Wang, R.; Szabo, C. Hydrogen Sulfide is an Endogenous Stimulator of Angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 21972−21977. (9) Peng, Y. J.; Nanduri, J.; Raghuraman, G.; Souvannakitti, D.; Gadalla, M. M.; Kumar, G. K.; Snyder, S. H.; Prabhakar, N. R. H2S Mediates O2 Sensing in the Carotid Body. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 10719−10724. (10) Yang, G.; Wu, L.; Jiang, B.; Yang, W.; Qi, J.; Cao, K.; Meng, K.; Mustafa, A. K.; Mu, W.; Zhang, S.; Snyder, S. H.; Wang, R. H2S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine γ-Lyase. Science 2008, 322, 587−590. (11) Yang, W.; Yang, G.; Jia, X.; Wu, L.; Wang, R. Activation of KATP Channels by H2S in Rat Insulin-Secreting Cells and the Underlying Mechanisms. J. Physiol. 2005, 569, 519−531. (12) Dufton, N.; Natividad, J.; Verdu, E. F.; Wallace, J. L. Hydrogen Sulfide and Resolution of Acute Inflammation: A Comparative Study Utilizing a Novel Fluorescent Probe. Sci. Rep. 2012, 2, 1−9. (13) Hughes, M. N.; Centelles, M. N.; Moore, K. P. Making and Working With Hydrogen Sulphide the Chemistry and Generation of Hydrogen Sulfide in Vitro and its Measurement in Vivo: A Review. Free Radical Biol. Med. 2009, 47, 1346−1353. (14) Eto, K.; Asada, T.; Arima, K.; Makifuchi, T.; Kimura, H. Retraction of ″Brain Hydrogen Sulfide is Severely Decreased in

4. CONCLUSIONS In conclusion, to understand the structural implications on the reactivity of small molecules, particularly positional isomers toward a well-known gasotransmitter H2S, we have developed three new dansyl-based molecular probes bearing dual reactive sites. The aim of brining in two competitive reactive sites in single molecular species was to study, for the first time, whether the gasotransmitter reacts simultaneously with both the reactive functionalities simultaneously or chemoselectively with one of them in the case of a choice between the two. Combined theoretical and experimental studies at the molecular level revealed that the extent of reactivity of individual site is determined by the delicate balance between electronic and steric effects. Interestingly, with the help of crystal structure analysis and photophysical studies we could prove how the steric factor can govern the reactivity of a particular reaction center even for a small size nucleophile like H2S. Furthermore, UV−vis absorption and fluorescence spectroscopic studies have shown that on incubation with H2S the probe P3 renders the fluorescence signal through nucleophilic thiolysis of sulfonate ester bond, whereas the signaling of H2S by P2 is mainly due to reduction of nitro functionality and thus generates fluorescent amine derivative P2′. Because the reduction process is slower than the bond cleavage pathway, P3 has been found to be a much more efficient H2S signaling device than P2. To our knowledge, this is the first report on the exploration of sulfonate ester cleavage strategy for H2S signaling. The presence of a strong steric effect as well as a weak electronic effect due to out-of-plane arrangement of nitro functionality made the probe P1 silent toward H2S. At the end, the structural analysis robustly recommended that the intramolecular factors present in the positional isomers may have adverse effects on many biochemical processes.

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Crystallographic data in CIF or other electronic format, the selection of DFT functionals and transition characters of excited states, and characterization data and additional spectroscopy data (PDF).

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The Journal of Physical Chemistry C Alzheimer’s Disease. Biochem. Biophys. Res. Commun. 2002, 293, 1485−1488. (15) Kamoun, P.; Belardinelli, M.-C.; Chabli, A.; Lallouchi, K.; Chadefaux-Vekemans, B. Endogenous Hydrogen Sulfide Overproduction in Down Syndrome. Am. J. Med. Genet. Part A 2003, 116A, 310− 311. (16) Yang, W.; Yang, G.; Jia, X.; Wu, L.; Wang, R. Activation of KATP Channels by H2S in Rat Insulin-Secreting Cells and the Underlying Mechanisms. J. Physiol. 2005, 569, 519−531. (17) Fiorucci, S.; Antonelli, E.; Mencarelli, A.; Orlandi, S.; Renga, B.; Rizzo, G.; Distrutti, E.; Shah, V.; Morelli, A. The Third Gas: H2S Regulates Perfusion Pressure in Both the Isolated and Perfused Normal Rat Liver and in Cirrhosis. Hepatology 2005, 42, 539−548. (18) Lei, W.; Dasgupta, P. K. Determination of Sulfide and Mercaptans in Caustic Scrubbing Liquor. Anal. Chim. Acta 1989, 226, 165−170. (19) Jiménez, D.; Martinez-Manez, R.; Sancenon, F.; Ros-Lis, J. V.; Benito, A.; Soto, J. A New Chromo-Chemodosimeter Selective for Sulfide Anion. J. Am. Chem. Soc. 2003, 125, 9000−9001. (20) Hughes, M. N.; Centelles, M. N.; Moore, K. P. Making and Working With Hydrogen Sulfide the Chemistry and Generation of Hydrogen Sulfide in Vitro and its Measurement in Vivo: A Review. Free Radical Biol. Med. 2009, 47, 1346−1353. (21) Ishigami, M.; Hiraki, K.; Umemura, K.; Ogasawara, Y.; Ishii, K.; Kimura, H. A Source of Hydrogen Sulfide and a Mechanism of its Release in the Brain. Antioxid. Redox Signaling 2009, 11, 205−214. (22) Richardson, C. J.; Magee, E. A. M.; Cummings, J. H. A New Method for the Determination of Sulphide in Gastrointestinal Contents and Whole Blood by Microdistillation and Ion Chromatography. Clin. Chim. Acta 2000, 293, 115−125. (23) Searcy, D. G.; Peterson, M. A. Hydrogen Sulfide Consumption Measured at Low Steady State Concentrations Using a Sulfidostat. Anal. Biochem. 2004, 324, 269−275. (24) Doeller, J. E.; Isbell, T. S.; Benavides, G.; Koenitzer, J.; Patel, H.; Patel, R. P.; Lancaster, J. R.; Darley-Usmar, V. M.; Kraus, D. W. Polarographic Measurement of Hydrogen Sulfide Production and Consumption by Mammalian Tissues. Anal. Biochem. 2005, 341, 40− 51. (25) Radford-Knoery, J.; Cutter, G. A. Determination of Carbonyl Sulfide and Hydrogen Sulfide Species in Natural Waters Using Specialized Collection Procedures and Gas Chromatography with Flame Photometric Detection. Anal. Chem. 1993, 65, 976−982. (26) Bérubé, P. R.; Parkinson, P. D.; Hall, E. R. Measurement of Reduced Sulphur Compounds Contained in Aqueous Matrices by Direct Injection into a Gas Chromatograph With a Flame Photometric Detector. J. Chromatogr. A 1999, 830, 485−489. (27) Lippert, A. R.; New, E. J.; Chang, C. J. Reaction-Based Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in Living Cells. J. Am. Chem. Soc. 2011, 133, 10078−10080. (28) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton, A. R.; Xian, M. Capture and Visualization of Hydrogen Sulfide by a Fluorescent Probe. Angew. Chem. Int. Ed. 2011, 123, 10511−10513. (29) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.; Wang, B. A Fluorescent Probe for Fast and Quantitative Detection of Hydrogen Sulfide in Blood. Angew. Chem. Int. Ed. 2011, 123, 9846−9849. (30) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S. Y.; Zhu, H. L.; Banerjee, R.; Zhao, J.; He, C. Selective Fluorescent Probes for LiveCell Monitoring of Sulphide. Nat. Commun. 2011, 2, 495−501. (31) Sasakura, T.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura, Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T. Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide. J. Am. Chem. Soc. 2011, 133, 18003−18005. (32) Chen, S.; Chen, Z. J.; Ren, W.; Ai, H. W. Reaction-Based Genetically Encoded Fluorescent Hydrogen Sulfide Sensors. J. Am. Chem. Soc. 2012, 134, 9589−9592.

(33) Lippert, A. R.; New, E. J.; Chang, C. J. Reaction-Based Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in Living Cells. J. Am. Chem. Soc. 2011, 133, 10078−10080. (34) Montoya, L. A.; Pluth, M. D. Selective Turn-On Fluorescent Probes for Imaging Hydrogen Sulfide in Living Cells. Chem. Commun. 2012, 48, 4767−4769. (35) Xuan, W. M.; Sheng, C. Q.; Cao, Y. T.; He, W. H.; Wang, W. Fluorescent Probes for the Detection of Hydrogen Sulfide in Biological Systems. Angew. Chem., Int. Ed. 2012, 51, 2282−2284. (36) Yu, F. B. A.; Li, P.; Song, P.; Wang, B. S.; Zhao, J. Z.; Han, K. L. An ICT-Based Strategy to a Colorimetric and Ratiometric Fluorescence Probe for Hydrogen Sulfide in Living Cells. Chem. Commun. 2012, 48, 2852−2854. (37) Lin, V. S.; Lippert, A. R.; Chang, C. J. Cell-Trappable Fluorescent Probes for Endogenous Hydrogen Sulfide Signaling and Imaging H2O2-dependent H2S production. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7131−7135. (38) Liu, C. R.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton, A. R.; Xian, M. Capture and Visualization of Hydrogen Sulfide by a Fluorescent Probe. Angew. Chem., Int. Ed. 2011, 50, 10327−10329. (39) Liu, C. R.; Peng, B.; Li, S.; Park, C. M.; Whorton, A. R.; Xian, M. Reaction Based Fluorescent Probes for Hydrogen Sulfide. Org. Lett. 2012, 14, 2184−2187. (40) Wintner, E. A.; Deckwerth, T. L.; Langston, W.; Bengtsson, A.; Leviten, D.; Hill, P.; Insko, M. A.; Dumpit, R.; VandenEkart, E.; Toombs, C. F.; Szabo, C. A Monobromobimane-Based Assay to Measure the Pharmacokinetic Profile of Reactive Sulphide Species in Blood. Br. J. Pharmacol. 2010, 160, 941−957. (41) Shen, X. G.; Pattillo, C. B.; Pardue, S.; Bir, S. C.; Wang, R.; Kevil, C. G. Measurement of Plasma Hydrogen Sulfide in Vivo and in Vitro. Free Radical Biol. Med. 2011, 50, 1021−1031. (42) Qian, Y.; Zhang, L.; Ding, S.; Deng, X.; He, C.; Zheng, X. E.; Zhu, H.-L.; Zhao, J. A Fluorescent Probe for Rapid Detection of Hydrogen Sulfide in Blood Plasma and Brain Tissues in Mice. Chem. Sci. 2012, 3, 2920−2923. (43) Chen, Y. C.; Zhu, C. C.; Yang, Z. H.; Chen, J. J.; He, Y. F.; Jiao, Y.; He, W. J.; Qiu, L.; Cen, J. J.; Guo, Z. J. A Ratiometric Fluorescent Probe for Rapid Detection of Hydrogen Sulfide in Mitochondria. Angew. Chem., Int. Ed. 2013, 52, 1688−1691. (44) Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. (45) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron Density. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785−789. (46) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2009. (47) Dennington, R.; Keith, T.; Millam, J. GaussView, version 5; Semichem Inc.: Shawnee Mission, KS, 2009. (48) Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. Mechanism-Based Molecular Design of Highly Selective Fluorescence Probes for Nitrative Stress. J. Am. Chem. Soc. 2006, 128, 10640−10641. (49) Nagano, T. Development of Fluorescent Probes for Bioimaging Applications. Proc. Jpn. Acad., Ser. B 2010, 86, 837−847. (50) Kiyose, K.; Aizawa, S.; Sasaki, E.; Kojima, H.; Hanaoka, K.; Terai, T.; Urano, Y.; Nagano, T. Molecular Design Strategies for NearInfrared Ratiometric Fluorescent Probes Based on the Unique Spectral Properties of Aminocyanines. Chem. - Eur. J. 2009, 15, 9191−9200. (51) Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. Mechanism-Based Molecular Design of ighly Selective Fluorescence Probes for Nitrative Stress. J. Am. Chem. Soc. 2006, 128, 10640−0641. (52) Ueno, t.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima, H.; Kikuchi, K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. Rational Principles for Modulating Fluorescence Properties of Fluorescein. J. Am. Chem. Soc. 2004, 126, 14079−14085.

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