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Triarylboron anchored luminescent probes: Selective detection and imaging of thiophenols in the intracellular environment SUDHAKAR PAGIDI, Neena K Kalluvettukuzhy, and Pakkirisamy Thilagar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01036 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Triarylboron anchored luminescent probes: Selective detection and imaging of thiophenols in the intracellular environment Sudhakar Pagidi, Neena K Kalluvettukuzhy and Pakkirisamy Thilagar* Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore-560012, India. Email: [email protected] KEY WORDS. boron, sensor, thiophenol, fluorescence, cell imaging ABSTRACT: The advances in boron incorporated organics has captured overwhelming interest on account of their outstanding properties and promising applications in various fields. Mostly, triarylborane compounds (TAB) are exploited as sensors of F- and CN- anions at the expense of intrinsic Lewis acidic nature of boron. New molecular probes 1 and 2 for detection of toxic thiophenol were designed by conjugating highly fluorescent borylanilines with the luminescent quencher 2,4-dinitrobenzene based sulfonamides (DNBS), wherein the electrophilicity of DNBS moiety have been modulated by fine-tuning the intrinsic Lewis acidity of boron. The interplay between PET (photoinduced electron transfer) and ICT have been employed for developing the TAB tethered turn-on fluorescent sensor for thiophenol with high selectivity for the first time. The newly developed probes showed very fast response towards thiophenol (within ~ 5 min) with limits of detection (LOD) lies in the micromolar ranges, clearly points to their potentiality. Further, compounds 1 and 2 were explored for detecting thiophenol in the intracellular environment by discriminating biothiols. DFT and TD-DFT calculations were performed to support the sensing mechanism.

1. Introduction Organic thiols play key roles in chemical, biological and environmental sciences. Aliphatic thiols such as cysteine, homocysteine and glutathione are associated with a variety of physiological processes for example, redox homeostasis. Variations in the concentration of these thiols in biological fluids have been associated with a number of diseases such as liver damage, Alzheimer’s disease and cardiovascular diseases.1-11 On the other hand aromatic thiols (thiophenols) play an important role in organic synthesis and are widely used as precursors for the preparation of agrochemicals, pharmaceuticals, and various industrial products. Despite their synthetic utility, thiophenols are highly toxic to human beings and environment. Prolonged exposure to thiophenols induces a burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea and vomiting and at the extreme, it could be fatal.12-20 Studies have shown that thiophenols possess a median lethal dose (LC50) of 0.01 to 0.4 mM for fish.21-24 Thus, selective detection and discrimination of thiophenols from biologically important aliphatic thiols is a pivotal area of research.25-42 Numerous thiol sensors have been developed by exploiting their strong nucleophilicity and affinity towards transition metal ions.28-36 Most of the probes are reaction based thiol dosimeters and are constructed by conjugating an electron deficient fluorescence quencher 2,4dinitrobenzenesulfonylchloride (DNBS) with a luminophore via sulfonamide bond. The high electron deficiency of DNBS moiety acts as an electron sink and induces photoinduced electron transfer (PET), resulting in the quenching of fluorescence from luminophore.33-42

Chart 1. Chemical structures of the TAB based thiol probes (1 and 2) and the luminophores (1a and 2a) generated upon addition of thiophenol. In recent times, boron compounds have attracted a lot of attention owing to their potential applications in light emitting diodes and displays, solar cells and energy harvesting systems, non-linear optical materials and cell imaging.43-56 Among various boron compounds, triarylborane (TAB) based compounds holds a special position, in view of their numerous applications in materials and biology.57-62 Due to their bright and tunable luminescence features, TAB compounds are widely exploited as sensors and external stimuli responsive materials.6368 However, boron based sensors have not been explored exclusively for the detection of thiols.69,70 We are interested in the design and development of strongly luminescent materials and efficient molecular probes for detecting various ions and neutral molecules.71-76 Recently, we demonstrated that borylanilines, the simplest TAB based D-A systems, are highly luminescent and a small change in the dihedral angle between donor amine unit (-NH2) and acceptor boryl moiety (-BMes2) has a dramatic effect on their optical properties.75,76 It was envisioned that the availability of reactive primary amine (-NH2) group on borylanilines can be conveniently exploited for the attachment of an electron deficient fluorescence quencher, DNBS via a sulfonamide bond. The attachment DNBS to boryl aniline would quench

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the fluorescence of borylanilines via PET process. The addition of thiols could regenerate the borylanilines by cleaving the sulfonamide bond; consequently, a turn-on fluorescence response can be realized. Depending upon the electrophilic and nucleophilic nature of respective thiol probe and thiols (thiophenols or biothiols), selective turn-on fluorescence detection would be possible. Accordingly, TAB based thiol probes 1 and 2 have been designed and synthesized and their selective turn-on luminescence discrimination of thiophenol over biothiols were investigated. Further, the newly developed probes showed a selective turn-on fluorescence response toward thiophenol in presence of biothiols in the intra-cellular environments.

2. Experimental Section 2.1 Materials and methods Chemicals required were purchased from dealers (SigmaAldrich, USA; Merck, Germany; SDFCL, India), and used as such without any further purification, unless otherwise mentioned. Standard schlenk-line techniques were used for performing all the reactions under inert atmosphere of argon. THF was refluxed over sodium-benzophenone until to get deep purple in color and then distilled under argon atmosphere. DCM is distilled from calcium hydride and stored over activated 4Å molecular sieves prior to use. Bruker Avance 400 MHz NMR spectrometer was used for recording 1H and 13C NMR spectra with operating frequency of 400 and 100 MHz respectively, and the corresponding spectra were referenced internally to the deuterated solvent signals (TMS signal at 0.00 ppm for 1H and CDCl3 signal at ~ 77.6 ppm for 13C). Chemical shift multiplicities are denoted as singlet (s), doublet (d) and doublet of doublets (dd). Micromass Q-TOF High Resolution Mass Spectrometer by electrospray ionization (ESI) method using argon (6kV, 10 mA) as the FAB gas was used for recording High resolution mass spectra (HRMS). UV-vis absorption spectra were recorded on a Perkin Elmer LAMBDA 750 UV/visible spectrophotometer. Fluorescence measurements were carried out on Horiba JOBIN YVON Fluoromax-4 spectrometer. Spectrophotometric grade solvents, microbalance (± 0.1 mg precision) and standard volumetric glass were used for preparing the solutions of compounds for spectral measurements. For solution state spectral measurements, quartz cuvettes with sealing screw caps were used. DFT calculations for all the molecules were carried out using the Gaussian G09 program employing hybrid B3LYP (hybrid Becke 3Lee-Yang-Parr) exchange-correlation functional with 6-31G (d) basis set. Ground state geometry optimizations followed by subsequent frequency test is carried out to make sure that molecule has minimum energy on the potential energy surface. TD-DFT calculations were performed on the ground state optimized structures.77-80 2.2 Synthetic procedures Borylanilines 1a and 2a are synthesized using our recently reported methodology.75,76 Compound 1: 2,4-Dinitrobenzenesulfonyl chloride (174 mg, 0.65 mmol) was added to dichloromethane (15 mL) solution of compound 1a (200 mg, 0.54 mmol) and 2,6-lutidine (76 µL, 0.65 mmol) at 0 °C under stirring. The reaction mixture was slowly warmed to room temperature and stirring was continued for another 24 hrs. The solvent was removed under vacuum and the residue was subjected to column chromatog-

raphy over silica gel with EtOAc/hexane (1:2) as eluent to obtain 1 as yellow solid. Yield: 70%. 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 8.66 (d, J = 2.24 Hz, 1H), 8.39 (dd, J1 = 8.50 Hz, J2 = 2.28 Hz, 1H), 8.06 (d, J = 8.64 Hz, 1H), 7.15 (s, 1H), 6.75 (s, 1H), 6.73 (s, 2H), 6.71 (s, 2H), 2.25 (s, 6H), 1.94 (s, 6H), 1.93 (s, 6H), 1.81 (s, 6H); 13C NMR (100 MHz, CDCl3, 25 °C): δ (ppm) 150.4, 148.9, 146.9, 143.8, 142.9, 141.2, 140.5, 140.2, 137.8, 134.5, 133.9, 129.3, 129.2, 126.7, 121.9, 121.0, 53.9, 23.3, 23.1, 21.6. Anal. Cald. for C32H34BN3O6S: C, 64.11; H, 5.72; N, 7.01. Found C, 64.08; H, 5.69; N, 7.05. ESI-MS (positive ion mode): Mcalc. = 599.2261 Da; found: 622.2595 Da [M+Na]+. Compound 2. A similar procedure described for 1 was used for the synthesis of 2. Quantities used for the reaction were; 2a (200 mg, 0.56 mmol), 2,6-lutidine (85µL, 0.73 mmol), 2,4dinitrobenzenesulfonyl chloride (194 mg, 0.73 mmol). Compound 3 was obtained as a yellow color solid. Yield: 85%. 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 8.61 (d, J = 2.0 Hz, 1H), 8.33 (dd, J1 = 8.88 Hz, J2 = 2.4 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.38-7.31 (m, 3H), 7.23 (s, 1H), 7.05 (s, 1H), 6.76 (s, 4H), 2.30 (s, 6H), 1.85 (s, 12H); 13C NMR (100 MHz, CDCl3, 25 °C): δ (ppm) 150.3, 148.7, 141.4, 141.0, 139.8, 138.4, 135.1, 134.8, 133.7, 130.5, 130.0, 128.7, 127.9, 127.1, 121.0, 23.7, 21.6. Anal. Cald. for C30H30BN3O6S: C, 63.05; H, 5.29; N, 7.35. Found C, 63.06; H, 5.25; N, 7.32. ESI-MS (positive ion mode): Mcalc. = 571.1948 Da; found: 594.3358 Da [M+Na]+.

2.3 Reactivity of probes with thiols Reactivity of compounds 1 and 2 towards different thiols were carried out by titrating 10 µM solution of compounds in 4:1 methanol-water mixture with thiols (12 µM, 4:1 methanolwater mixture). Thiophenol sensing studies were carried out by titrating 10 µM solution of compounds in 4:1 methanolwater mixture with thiophenol (12 µM, 4:1 methanol-water mixture). Then emission spectral measurements were carried out with different time intervals. Further, emission spectral measurements were carried out by gradually increasing the concentration of thiophenol and spectra were collected ~ 5 min after the addition of thiophenol. The detection limit was calculated from the fluorescence titration curve in presence of thiophenol. The detection limit was calculated using the equation, limits of detection (LOD) = 3ߪ/k; where ߪ is the standard deviation of the blank measurements and k is the slope of the curve obtained by plotting relative fluorescence intensity and analyte concentration. 2.4 Cell culture and fluorescence imaging HeLa cells were grown in Dulbecco’s modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% penicillin, and 10000 units/mL of streptomycin at 37 °C in a humidified air containing 5% CO2. The cells were incubated with 1 and 2 (10 µM in 1:200 DMSO-DMEM v/v pH = 7.4) for 30 min at 37 °C, and used directly for fluorescence imaging as a control experiment. For another control experiment, cells were treated with thiolphenol (1.25 µM, 1:1000 DMSODMEM v/v pH = 7.4) for 30 min at 37 °C and used directly for fluorescence imaging. The cells treated with thiophenol was washed with PBS three times to remove the remaining thiophenol and then co-incubated with compounds 1 and 2 (10 µM in 1:200 DMSO-DMEM v/v pH = 7.4)) for 30 min at 37 °C. Finally cells were washed with PBS to remove the remain-


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Langmuir ing compounds and images were taken under an inverted fluorescence microscope (Olympus, 1X71).

matched with the energy of the fluorescence peaks observed for respective borylanilines, which directly corroborate the above inference (Figure S11).

3. Results and discussion 3.1 Synthesis and Characterization Compounds 1 and 2 were synthesized by lutidine mediated condensation of 2,4-dinitrobenzenesulfonylchloride and the corresponding borylaniline (1a and 2a for 1 and 2, respectively, Scheme 1). Compounds 1a and 2a were prepared using our recently reported methodology.75,76 Compounds 1 and 2 were characterized by multi-nuclear NMR spectroscopy and high resolution mass (HRMS) spectrometry. The 1H resonances corresponding to –NH2 group (~ 3.61 and ~ 3.59 ppm for 1a and 2a, respectively)75.76 disappeared and a new peak corresponding to sulfonamide appeared around ~ 8.66 ppm and ~ 8.61 ppm, respectively for 1 and 2, confirming the formation of the target molecules. Compounds were stable under ambient conditions. R4

R4

R3

R3

R2

SO2Cl NO2

R1 B

2,6-lutidine

R2

R1

NH O O S

B

+ CH2Cl 2, r.t., NO2

NO2

DNBSA = NO2

1a. R1 = R2 = CH3, R3 = H, R4 = -NH2 2a. R1 = R2 = R4 = H, R3 = -NH2

1. R1 = R2 = CH3, R3 = H, R4 = -DNBSA 2. R1 = R2 = R4 = H, R3 = -DNBSA

Scheme 1. Synthesis of TAB based molecules 1 and 2.

3.2 Optical Properties – Studies on the fluorometric response of probes toward thiophenol Compounds 1 and 2 showed a broad absorption in the region 275-400 nm in MeOH solution and were weakly fluorescent (Figure S7). The efficient PET between DNBS and TAB moieties could be the reason for the non-emissive nature of these compounds. The observations are in line with the optical properties of donor-acceptor systems having DNBS moiety.3342 The above inference was further supported by the TD-DFT calculations (vide-infra). To check the applicability of 1 and 2 as luminescent sensors for compounds containing thiol (-SH) functional groups, they were allowed to react with various thiols (cysteine, homocysteine, glutathione and thiophenol) and the reaction was monitored by fluorescence spectroscopy. Compounds 1 and 2 showed a turn-on fluorescence response with a green (λem = ~ 520 nm) and cyan blue (λem = ~ 490 nm) color intense luminescence towards thiophenol and did not show any changes with other biothiols (Figure 1). Further, the selectivity of probes towards thiophenols was checked in the presence of 10 times excess of biothiols (50 µM) compared to thiophenol (5 µM). The compounds 1 and 2 did not show any response in presence of excess of biothiols and showed a fluorescence response only when thiophenol were added (Figure S10). This clearly inferred us that the developed TAB based probes are selective turn-on fluorescent sensors for thiophenol over biothiols. The insensitivity of probes towards aliphatic thiols could be due to their higher pKa values (ca. 8.5) compared to that of thiophenols (ca. 6.5). To prove that, the luminescence response of 1 and 2 towards thiophenol are due to the regeneration of respective borylanilines 1a and 2a, by cleaving the sulfonamide bond, the luminescence spectra of 1 and 2 after addition of thiophenol were compared with that of freshly prepared 1a and 2a. The energy of the fluorescence peaks of 1 and 2 after addition of thiophenol were exactly

Figure 1. Emission spectral changes of probe 1 (top) and 2 (bottom) upon addition of different thiols (concentration of thiol probes = 10 μM, concentration of different thiol analytes = 12 μM and λex = 350 nm) in methanol-water (v/v = 4:1) mixture.

To get further insight into the reaction between the compounds and thiophenol, the fluorescence of the reaction mixture was monitored at different time intervals (Figure 2). Compounds 1 and 2 display an instantaneous fluorescence response (within seconds) toward thiophenol and reach a plateau within ~ 4 and ~ 5 min, respectively (Figure S8). This could be due to the enhanced electrophilic nature of the sulfonamide bond owing the presence of highly Lewis acidic TAB substituent. Further, the fluorescence intensity of the probes increased progressively with concomitant increase in the concentrations of thiophenol (Figure 3). Upon addition of 2 µM (0.2 equivalents) of thiophenol, the fluorescence intensity of 1 and 2 showed ~ 6 and ~ 262 fold increase in their fluorescence intensity, respectively, compared to the solutions of corresponding compounds without adding thiophenol. The changes in the fluorescence spectra of 1 and 2 cease after addition of 13 µM (1.3 equivalents) and 15 µM (1.5 equivalents) of thiophenol, respectively (Figure S9). The limits of detection (LODs) of thiophenol for 1 and 2 were calculated using the fluorescence titration data and the values were found to be ~ 0.08 µM and ~ 0.15 µM, respectively (Figure S12). Very small reaction time and lower detection limits in the micromolar ranges clearly indicate the potential of newly developed TAB based compounds as luminescent probes for sensing thiophenol in practical applications such as intracellular environments and contaminated (or polluted) samples like water or soils.


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Figure 2. Time dependent emission spectra of 1 (left) and 2 (right) upon addition of thiophenol (concentration of thiol probes = 10 µM, concentration of thiophenol = 12 µM and λex = 350 nm) in methanol-water (v/v = 4:1) mixture.

Figure 4. Emission spectra of compounds 1 (top) and 2 (bottom) with different analytes (concentration of thiol probes = 10 µM, concentration of different analytes = 20 µM and λex = 350 nm) in methanol-water (v/v = 4:1) mixture.

3.3 Detection of thiophenol in the intracellular environment

Figure 3. Emission spectra of 1 (top) and 2 (bottom) upon addition of increasing concentration of thiophenol (concentration of thiol probes = 10 µM and λex = 350 nm, inset shows the photographs of compounds before and after addition of thiophenol) in methanol-water (v/v = 4:1) mixture.

Selectivity is always important for designing a good probe. To evaluate the selectivity of probes 1 and 2 towards thiophenol in the presence of other competing thiols and other nucleophiles, the PL spectra of these probes were recorded in competing environments (Figure 4). No prominent changes in the luminescence spectra of 1 and 2 were observed even in the presence of excess aliphatic thiols and other competing nucleophiles. The fluorescence intensity was enhanced only when thiophenol was added. Thus, these probes are very selective for thiophenol even in the competing media. Hence the utility of these probes for the detection of thiophenol in the intracellular environment was evaluated.

To determine the cell permeability as well as the use of 1 and 2 for the detection of thiophenol in intracellular environments, preliminary in vitro studies were carried out using HeLa cells. MTT assay experiment was performed on HeLa cells to check the cytotoxicity of probes 1 and 2 (Figure S13). The results indicated that 1 and 2 are biocompatible and showed low cytotoxicity for living cells. Further, we also checked the viability of cells in the presence of thiophenol of concentration 1.25 µM and found that cells are healthy under the experimental conditions (87 % of viability after 3 hrs of incubation). When cells were treated only with 1 and 2 (10 µM in 1: 200 DMSODMEM v/v pH = 7.4) at 37 °C for 30 min, no significant change in fluorescence intensity was observed (Figure 5A and B). The insensitivity of 1 and 2 towards intracellular aliphatic thiols could be the reason for the absence of fluorescence. Upon co-incubation of 1 and 2 for 30 min at 37 °C (10 µM, 1: 200 DMSO-DMEM v/v pH = 7.4) with the cells pre-treated with thiophenol (1.25 µM, 1: 1000 DMSO-DMEM v/v pH = 7.4 for 30 min at 37 °C), showed green and blue colour fluorescence, respectively (Figure 5E and F). This clearly indicates that 1 and 2 are cell permeable and can selectively react with thiophenol in the intracellular environment. Thus, the TAB based thiol probes 1 and 2 are useful for the selective detection of highly toxic thiophenol in the intracellular environment by discriminating biothiols.


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Figure 5. Fluorescence imaging of thiophenol in living cells: (A and B) cells treated with probes 1 and 2 (10 µM) for 30 min at 37° C; (C and D) cells treated with thiophenol (1.25 µM) for 30 min at 37° C; and (E and F) then co-incubated with probes (10 µM) 1 and 2 for 30 min at 37° C.

3.4 Rationalization of the fluorescence Off–On sensing mechanism using DFT/TDDFT calculations To gain insights into the electronic properties and to account for the “off-on” fluorescence response of 1 and 2 towards thiophenol, DFT/TD-DFT calculations have been performed using B3LYP method and 6-31G (d) basis set incorporated in Gaussian 09 software (Figure 6). In case of 1 and 2, highest occupied molecular orbital (HOMO) is delocalized on the aryl groups attached to B and lowest unoccupied molecular orbital (LUMO) is localized only on the DNBS moiety. The oscillator strength (f) obtained from TD-DFT calculations corresponding to the lowest energy transitions was found to be very small, the values are f = 0.0001 and 0.0004 for 1 and 2, respectively. This negligibly small value of oscillator strength and the noncrossing nature of the frontier molecular orbitals clearly indicate that the S1 state of 1 and 2 is a “dark state” and the transition from S1 to S0 is forbidden. As discussed in earlier sections, 1 and 2 upon treatment with thiophenol generate the respective borylanilines 1a and 2a. HOMO of 1a and 2a is delocalized mostly on aniline fragment; on the other hand, LUMO of 1a and 2a is completely delocalized over the entire molecule, indicating the charge transfer characteristics of a donor-acceptor system.14-16,20 TD-DFT calculations for the borylanilines showed that their S1 state is an emissive state, confirmed by the considerably higher f values such as 0.1730, and 0.0503, respectively for 1a and 2a. The allowed S1 to S0 transition is characterized as HOMO-LUMO transition and confirms borylanilines as fluorescent species.

Figure 6. Frontier molecular orbitals and electronic transitions of TAB based thiophenol probe 1 (top right) and 2 (bottom right) and the corresponding borylaniline 1a (top left) and 2a (bottom left) furnished upon the addition of thiophenol; illustrating the mechanism of turn-on fluorescence response towards thiophenol.

4. Summary and Conclusions The intrinsic Lewis acid characteristics of triarylborane (TAB) have been judiciously employed to modulate the electrophilicity of 2,4-dinitrobenzene based sulfonamides (DNBS) for the development of a new class of “off-on” fluorescent probes for selective detection of thiophenol over biothiols, for the first time. The probes were constructed by conjugating the TAB based luminophore borylanilinies with the luminescent quencher 2,4-dinitrobenzenesulfonyl (DNBS) moiety. The limits of detection (LOD) of 1 and 2 towards thiophenol lie in the range of fractions of micromoles; ~ 0.08 µM and ~ 0.15 µM, respectively. The preliminary in vitro cell imaging studies shows that the organoboron based thiol probes 1 and 2 can detect highly toxic thiophenol selectively in the intracellular environment by discriminating biothiols. Very small reaction time (~ 5 min), lower detection limits in the micromolar ranges and the selective thiophenol detection in the intracellular environment clearly demonstrate the potential of the newly developed TAB based compounds and would pave the way for the use of these probes in practical applications.

ASSOCIATED CONTENT Supporting Information Additional spectral characterization and spectroscopic data are given in the Supporting Information and “This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author


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* [email protected]

Funding Sources Science and Engineering Research Board (SERB), New Delhi, India.

ACKNOWLEDGMENT

19.

20.

PT and PS thanks Science and Engineering Board (SERB), New Delhi, India for financial support and KKN thanks IISc for research fellowship.

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