A molecular design towards sulfonyl aza-BODIPY based NIR fluorescent and colorimetric probe for selective cysteine detection

Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are essential biothiols for cellular growth, metabolism, and maintenance of a biological system. Thus, the detection of biothiols is highly important for early diagnosis and evaluation of disease progression. In this article, a series of sulfonyl aza-BODIPYs was synthesized, characterized, and examined by 1H-NMR, 13C-NMR, crystallization, photophysical properties and DFT calculation. Among these structures, a fluorescent probe, BDP-1, exhibited selective detection of Cys among various biothiols via nucleophilic aromatic substitution and typical size of Cys molecules. BDP-1 showed color change and near-infrared (NIR) fluorescence enhancement after reaction with Cys to generate BDP-OH, confirmed by HRMS. The red shift of absorption wavelength showed a similar tendency resulting in time-dependent density functional theory (TD-DFT). Furthermore, the calculated detection limit of BDP-1 toward Cys was 5.23 μM. This probe facilitates the colorimetric and fluorescent detection of Cys over other biothiols.


Introduction
Intracellular thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play an important role in a wide range of physiological and pathological processes in living organisms. 1,2 Among various biothiols, the range of free intracellular Cys concentrations varies between 30 and 200 mM. 3,4 Abnormal levels of Cys are related to several diseases such as hair depigmentation, lethargy, liver damage, muscle and fat loss, and skin lesions. 3,4 Therefore, it is critical to report changes in Cys concentrations via real-time monitoring.
Due to high selectivity, sensitivity, simplicity and fast response, uorescent probes represent powerful tools to monitor biologically relevant species in vitro or in vivo. Accordingly, uorescent probes for Cys, Hcy and GSH have been utilized in molecular recognition or thiol-specic reaction strategies. [5][6][7][8][9] However, because of the structural similarity of Cys and Hcy, the selective discrimination between the two species is a challenging task. On the other hand, many sulfonyl groupsbased probes have been reported with a lack of study compared their substitution ability with biothiols. 10,11 Aza-boron-dipyrromethene (aza-BODIPY) and its derivatives are a new class of near-infrared (NIR) organic photosensitizers with excellent stability, signicant red-shied absorption and high uorescence quantum efficiency. 12,13 The substitution of methine-bridged carbon atom with nitrogen leads to the formation of so-called aza-BODIPY with a bathochromic shi in absorption of about 90 nm with respect to the analogous derivative, which has NIR excitation. 14 Upon reaction with target molecules, the electron-decient groups are converted to electron-rich groups, resulting in a different level of absorbance/excitation energy compared with aza-BODIPY. Therefore, aza-BODIPY serves as an ideal base to induce uorescence emission in NIR. In addition, they have been widely investigated in photovoltaics, imaging, and photodynamic therapy (PDT). [15][16][17][18][19] In the current study, we synthesized and studied photophysical properties of three sulfonyl aza-BODIPY conjugates (BDP-1-BDP-3). Further, lowest energy structure of aza-BODIPY derivatives were obtained that based on single crystal structure and density functional theory (DFT) calculations. Among them, a near infrared (>700 nm) probe BDP-1 bearing 2,4-dinitrobenzenesulfonyl (DNBS) moieties with stronger nucleophilic aromatic substitution ability compared to toluene-or (dimethylamino)naphthalene-1-sulfonyl groups, which facilitate simultaneous colorimetric and uorometric detection of Cys over BDP-2 and BDP-3. Compared with the reported NIR uorescent probes for the recognition of Cys, the present probe exhibits high stability, excellent sensitivity, and signicant selectivity.

Results and discussion
BDP-OH was synthesized as described in the literature (Scheme S1 †). 20,21 The synthetic route of aza-BODIPY derivatives is depicted in Fig. 1a. The reaction of BDP-OH and several sulfonyl chloride compounds in the presence of TEA afforded BDP-1, BDP-2 and BDP-3 as dark-blue solids with an approximately 75% yield following extraction with H 2 O and drying over Na 2 SO 4 . The obtained structure was characterized using 1 H NMR, 13 C NMR, ESI HRMS and single crystal structure analysis (ESI †). Interestingly, the crystal structure of BDP-OH was obtained in dimer form, which showed the hydro interaction of -OH groups between two molecules ( Fig. 2a and S17a †). Specically, the crystal data of BDP-3 ( Fig. 2d and S17d †) exhibited alternate arrangement between two molecules via pp stacking of aromatic rings and intermolecular electrostatic interactions of B and F atoms.
As shown in Fig. 3, BDP-OH shows a broad absorption band from 600 nm to 750 nm. The central peak located at 697.8 nm is assigned from the S 0 / S 1 electron transition and corresponds to green color in THF solution (Fig. 1b). Furthermore, a shoulder absorption around 600 to 680 nm originated in the S 0 / S 3 electron transition of BDP-OH (Fig. S27 †). BDP-1, BDP-2, and BDP-3 exhibit approximately 40 nm blue-shi absorption compared to BDP-OH, which is attributed to the introduction of sulfonyl chloride groups (electron withdrawing groups, EWG) in the hydroxyl positions of BDP-OH. The sulfonyl-aza-BODIPY conjugates display a broad absorption band around 550 to 700 nm, and a central peak around 657 nm corresponding to their dark-green color in THF solution attributed to the S 0 / S 9 , S 0 / S 3 and S 0 / S 1 electron transition of BDP-1, BDP-2, and BDP-3, respectively (Fig. S25 †). In addition, a shoulder absorption around 560-640 nm originated subsequently from the S 0 / 2-singlet excited states of sulfonyl-aza-BODIPY ( Fig. S25 †). The molar absorbance coefficients of all aza-BODIPY derivatives are relatively high (7.4-8.5 Â 10 5 L mol À1 cm À1 ) in THF, with a slight decrease in other solvents such as MC, DMSO, and ACN (Table S3 †). BDP-OH exhibits the emission peak at 731 nm with   a uorescence quantum yield (F F ) of 2.58% due to strong ICT effect, which originated electron transfer from two -OH groups to aza-BODIPY core. [22][23][24] The BDP-1-BDP-3 exhibits an emission peak at about 685 nm with a F F value of 0.46-3.30% (in THF) owing to emission inhibition from strong sulfonyl acceptors (Table 1). These uorescence quantum yields were signicantly lower than that of aza-BODIPY core (F F ¼ 34%). 25,26 In addition, their emission is decreased in DMSO and ACN due to uorescence quenching in aggregate state.
The HOMO of all aza-BODIPY derivatives is probably localized in the BODIPY core and the phenyl ring at 3-/5-position. However, the LUMO of BDP-1 is concentrated in 2,4-dinitrobenzenesulfonyl (DNBS) subunits, whereas the LUMO of BDP-OH, BDP-2 and BDP-3 is mostly located in the BODIPY core (Fig. 4). Furthermore, the HOMO-LUMO energy gap E g of BDP-1 is lower than that of BDP-2 and BDP-3. Therefore, these specic phenomena are induced by DNBS moiety, which is known as an electron sink 27 and a stronger EWG compared to 5-(dimethylamino)naphthalene-1-sulfonyl and toluene sulfonyl. Natural transition-orbital (NTO) analysis was performed to visualize the nature of different molecular excited states (Fig. S26 †). 28 Although, the contribution to the central peak and shoulder absorption band by different orbitals from HOMOÀ4 to LUMO+2 varies (Table S5 †), NTOs indicate the uniformity of electronic transition in aza-BODIPY framework, which corresponds to similar UV/vis absorption spectra and naked-eye color of BDP-1-BDP-3.
The recognition of several thiols by BDP-1, BDP-2, and BDP-3 was investigated via colorimetric changes, and changes in UV absorption and uorescence emission in pH 7.4 PBS buffer/THF (5/5) (Fig. 5 and S20-S22 †). We investigated the colorimetric responses of BDP-1, BDP-2 and BDP-3 (10 mM) with H 2 S, 2-mercaptoethanol, thioglycolic acid, glutathione, cysteine (Cys), and homocysteine (Hcy), Na 2 S 2 O 7 , KSCN, cysteamine at different concentrations (50-1000 mM). Only Cys induced dark green to green color transition toward BDP-1 at 50 mM during 3 min, whereas no changes were detected with other reagents at higher concentrations or longer incubated time (Fig. 5d), demonstrated that Cys efficiently reacted with BDP-1. Furthermore, the UV-vis and uorescence spectra of BDP-1 in the high presence of Hcy or GSH (1 mM) was slightly change, indicated that Hcy and GSH reacted with BDP-1. The color of BDP-2 and BDP-3 solution was not altered by the addition of all reagents at concentrations as high as 1 mM during more than 30 min. The reaction of biothiol with sulfonyl groups based on nucleophilic aromatic substitution mechanism and the size of thiol structures (Fig. 6). The electron withdrawing ability of 2,4-dinitrobenzene is signicantly stronger than that of toluene or (dimethylamino)naphthalene, so only BDP-1 can react to Cys,   Hcy or GSH, whereas BDP-2 and BDP-3 cannot do that. On the other hand, the free -SH group plays an important role in the reaction (Fig. 6) of biothiol with BDP-1. It has known that Hcy can cyclize to give homocysteine thiolactone, a ve-membered heterocycle, 29 so Hcy barely react with BDP-1 compared to Cys even though these are small molecules. Thus, only UV-vis and uorescence emission spectra of BDP-1 were slightly changed ( Fig. S20 †), but the color of BDP-1 was unchanged in the highconcentration of Hcy (1 mM). [30][31][32] The color change of BDP-1 in the presence of Cys was conrmed by UV/vis absorption and uorescence emission spectra ( Fig. S20 and S23 †). Upon Cys addition, the peak of BDP-1 centered on the absorption band shied from 655 nm to 700 nm together with uorescence turn-on about 730 nm (l ex ¼ 700 nm), which similar to the absorption and emission spectra of BDP-OH. On the other hand, the uorescence emission spectra titrations of BDP-1 at 675 nm excitation in the presence of cysteine were investigated. In which, the uorescence emission at 685 nm gradually decreased, which was originated from the disappearance of BDP-1. It indicates that BDP-1 reacts with Cys to form BDP-OH and DNB-Cys (Fig. 5a). UV/vis absorption and uorescence emission spectra of BDP-2 and BDP-3 were mostly unchanged under the high concentrations of GSH, Cys and Hcy, corresponding to their color. These results indicate the sensitivity and selectivity of BDP-1 toward Cys. In addition, ESI HRMS peaks m/z ¼ 528.1707 [M À H] À , calc. for C 32 H 21 BF 2 N 3 O 2 (BDP-OH) and m/z ¼ 286.0145 [M + H] À , calc. for C 9 H 9 N 3 O 6 S (DNB-Cys) (Fig. S16 †) conrm the reaction of BDP-1 and Cys. Analysis of changes in UV/vis absorption spectra of BDP-1 (10 mM) in PBS buffer (pH 7.4)/THF (5/5) suggests that the original 655 nm absorption band of BDP-1 decreased together with a simultaneous increase at 700 nm upon binding with Cys (Fig. 5b). As shown in Fig. 5c, the emission intensity of BDP-1 was gradually enhanced by increasing Cys levels from 0 to 80 mM. The limit of detection (LOD) of BDP-1 for Cys was calculated at 5.23 mM (Fig. 5f). In addition, the colorimetric response value (CR) of BDP-1 (10 mM) was observed at 56.99% in the presence of 50 mM Cys (Fig. S23b †).

Synthesis
Synthesis of BDP-1-BDP-3: 11,33 sulfonyl chloride compound (1.2 mmol) was diluted in dichloromethane (10 mL) at 0 C. The mixture was slowly added a solution of BDP-OH (0.4 mmol), TEA (0.2 mL) in dichloromethane (20 mL) and then was stirred at room temperature overnight. The solution was extracted with H 2 O, dried over Na 2 SO 4 , and concentrated to give a black-blue solid as product (yield about 75%).

Computational data
The DFT calculations of the aza-BODIPY derivatives were performed using the Gaussian 09 program package. These geometric structures were optimized without imaginary frequencies using the B3LYP hybrid function together with the 6-31+G(d) basic sets for C, H, B, O, and F atoms; 6-31+G(2d,p) basic sets for S and N atoms. 34 Several important bond lengths and angles of these optimized structures are highly similar to those of crystal structures obtained.
Optical excitation energies were calculated with various functional parameters using time-dependent DFT (TD-DFT) comprising the hybrid, gradient-corrected, popular local, and functional methods. The 6-31G(d) basic sets were used for C, H, B, and O, F while 6-31G(2d,p) basic sets were used for S and N in THF solvent in the polarizable continuum model (PCM) using the integral equation formalism variant (IEFPCM). 35 Among various functionals, LSDA functional showed that the calculated excitation energies were closer to the experimental data with mean absolute deviation (MAD) as low as 0.04 (Table S4 †). This nding will facilitate current and future TD-DFT calculations about (aza-)BODIPY and/or sulfonyl chloride structures. introducing 2,4-dinitro-1-sulfonyl chloride to BDP-OH. Upon treatment with Cys, the BDP-1 displayed a selective colorimetric shi from dark-green to green, as well as red-shi enhancement of uorescence. The sulfonyl group of BDP-1 efficiently reacted with Cys, resulting in the formation of BDP-OH and DNB-Cys. BDP-1 showed a high selectivity for Cys among other biothiols due to nucleophilic aromatic substitution mechanism and small size of Cys. The HOMO-LUMO energy gap was also calculated via TD-DFT calculation and the value matched UV/vis and uorescence emission spectra precisely. The calculated detection limit of BDP-1 was 5.23 mM and the colorimetric response (CR) value was 56.99%. Finally, the reaction between BDP-1 and Cys was conrmed via mass spectroscopy.

Author contributions
Thanh Chung Pham conceived and designed the probes and calculated the DFT in the Gaussian 09 package, Yeonghwan Choi performed and collected the UV-vis spectra and uorescence spectra, Chaeeon Bae performed and collected the UV-vis spectra and uorescence spectra, Cong So Tran synthesized aza-BODIPY derivatives, Dongwon Kim obtained single crystal data, Ok-Sang Jung interpreted nal crystal structures, Yong-Cheol Kang performed the uorescence spectra and calculated quantum yield, SungYong Seo studied 1 H NMR, 13 C NMR, HRMS, Hyun Sung Kim performed computational study, Hwayoung Yun designed the scheme to synthesize probes, Xin Zhou designed aza-BODIPY derivatives for NIR probes, Songyi Lee designed BDP-OH, BDP-1, BDP-2, BDP-3 and wrote the paper.

Conflicts of interest
There are no conicts to declare.