- Email: [email protected]
Design of BODIPY-based near-infrared fluorescent probes for H2S
Accepted Manuscript Title: Design of BODIPY-based Near-Infrared Fluorescent Probes for H2 S Authors: Qiang Fei, Mimi Li, Jian Chen, Ben Shi, Ge Xu, Chunchang Zhao, Xianfeng Gu PII: DOI: Reference:
S1010-6030(17)30902-4 http://dx.doi.org/10.1016/j.jphotochem.2017.08.043 JPC 10818
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date: Revised date: Accepted date:
28-6-2017 14-8-2017 18-8-2017
Please cite this article as: Qiang Fei, Mimi Li, Jian Chen, Ben Shi, Ge Xu, Chunchang Zhao, Xianfeng Gu, Design of BODIPY-based Near-Infrared Fluorescent Probes for H2S, Journal of Photochemistry and Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.08.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Design of BODIPY-based Near-Infrared Fluorescent Probes for H2S Qiang Fei,†,‡ Mimi Li,† Jian Chen,† Ben Shi,‼ Ge Xu,‼ Chunchang Zhao,‼ Xianfeng Gu†,* †
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203
China ‡
Food and Pharmaceutical Engineering Institute, Guiyang University, Guizhou, 550005, China
‼
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and
Molecular Engineering, East China University of Science & Technology, Shanghai 200237, China. E-Mail: [email protected]
Graphical Abstract
Highlights
A highly selective and sensitive probe for detecting H2S has been designed and synthesized.
H2S is able to trigger the probe to light-up NIR emission.
The probe is suitable for applications in living cells imaging.
ABSTRACT Fluorescent probes with near-infrared (NIR) emission are of great interest in biology imaging, owing to their minimal autofluorescence from biological samples and deep tissue penetration. Here, we reported two NIR fluorescence probes by masking the free hydroxyl group in a NIR fluorescent dye. We demonstrated that the designed probes showed highly selective and sensitive responsiveness to H2S. Owing to H2S triggered light-up NIR emission, the probes were suitable for applications in living cells imaging.
1. Introduction Fluorescence imaging with near-infrared (NIR, 650−900 nm) dyes are attractive for visualizing biological targets of interest in living systems [1-3], because NIR light endows the advantages of deep tissue penetration, decreased interference from autofluorescence background and minimum photo-damage to biological samples [4-7]. Accordingly, the development of novel fluorescent scaffolds that emit in the NIR region is critically required but still an important challenge. Fluorescent chromophores containing a characteristic hydroxyl group are among the most widely studied traditional dyes [8-10]. It is facile to modify the hydroxyl group in these derivatives for construction of chemodosimeters for molecular optical imaging of analytes. In this respect, we have devised a novel class of reaction-based probes full utilization of 6hydroxyindole-based BODIPY as a scaffold due to its tunable photophysical properties [11-15]. However, the absorption and emission wavelengths of these derivatives only fall into the visible region, which retards their practical applications in living system. We also reported the further modification of 6-hydroxyindole-based BODIPY to afford a NIR fluorescent dye (NIR-BOD) [26]. Interestingly, this NIR-BOD is superior to 6-hydroxyindole-based BODIPY with absorption and emission in the NIR region under neutral PBS conditions. Importantly, the free hydroxyl group in NIR-BOD can be readily modified for development of fluorescent sensors for in vitro and in vivo imaging applications. H2S as an endogenous gasotransmitter plays vital roles in exerting anti-inflammation, modulating blood pressure and regulating other fundamental processes [16-18]. On the other hand, abnormal levels of H2S may cause series of diseases, such as Alzheimer’s disease and Parkinson’s disease [19-20]. Therefore, it is of high scientific interest to develop molecular probes for detection of H2S. To data, many reaction-based fluorescent scaffolds have been reported [21-25]. However, the development of NIR fluorescent H2S probes is still an important challenge but highly desirable. Considering the NIR emission of NIR-BOD and the facile masking of the hydroxyl group, a new fluorescent probe can be established by grafting a special trigger onto the NIR-BOD moiety. Thus
two light-up NIR fluorescent probes (Scheme 1) for H2S were designed, in which p-azidobenzyl is incorporated to NIR-BOD through an ether or carbonate linkage. Interestingly, H2S-triggered reduction of azide generated the amino function, which was followed by 1,6-elimination to release the active fluorophore NIR-BOD, enabling the NIR tracking H2S in living systems. 2. Experimental 2.1. Material All chemical reagents and solvents for synthesis were purchased from commercial suppliers and were used without further purification. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer with chemical shifts reported in ppm at room temperature. Mass spectra were measured on a HP 1100 LC-MS spectrometer. UV-vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra were measured with a Varian Cary Eclipse Fluorescence spectrophotometer. 2.2. Synthesis of N3-BOD-E and N3-BOD-C probes
Scheme 1 Synthetic route for N3-BOD-E and N3-BOD-C. The synthetically related compounds are depicted in Scheme 1. Compound 2 was synthesized according to literature procedures [26]. NIR-BOD was obtained according to the previous work [27]. N3-BOD-E and N3-BOD-C were prepared as shown in Scheme 1. The structure was fully characterized by 1H and 13C NMR and high-resolution mass spectrometry (HRMS) (Fig. S10-S15).
2.2.1. Synthesis of 4-azidobenzyl (4-nitrophenyl) carbonate (3) To a solution of Compound 1 (693 mg, 3.35 mmol) in dichloromethane were added 4nitrophenyl chloroformate (1.87 g, 6.7 mmol), cooled to 0 °C for 30 min. Then Et3N was added dropwise, and the reaction was stirred at room temperature over night. The reaction mixture was diluted with water and extracted with dichloromethane. The organic layer was separated and washed with 1 % NaOH aqueous solution, saturated brine solution and water, respectively. Then dried over anhydrous Na2SO4, proceeded evaporation under vacuum, the residue was purified by silica gel chromatography (petroleum ether-ethyl acetate), a white solid was obtained (460 mg, 31.5 %). 1H NMR (CDCl3, 400 MHz, ppm) δ 8.29-8.27 (d, J = 8.8 Hz, 2H), 7.46-7.44 (d, J = 8.0 Hz, 2H), 7.39-7.37 (d, J = 8.8 Hz, 2H), 7.08-7.06 (d, J = 8.0 Hz, 2H), 5.26 (s, 2H). 2.2.2. Synthesis of 8-((4-azidobenzyl)oxy)-9-(benzo[d]thiazol-2-yl)-2-ethyl-5,5-difluoro-1, 3,11trimethyl-12-phenyl-5H-pyrrolo[1',2':3,4][1,3,2]diazaborinino[1,6-a]indol-4-ium-5-uide (N3-BODE) NIR-BOD (60 mg, 0.112 mmol), Compound 3 (47.3 mg, 0.223 mmol), and potassium carbonate (46.4 mg, 0.336 mmol) were dissolved in the flask with dry acetone (10 mL). The reaction mixture was continuously stirred over night at room temperature. After completion, the mixture was diluted with CH2Cl2. The organic layer was separated, washed with water (3 × 50 mL), and dried over anhydrous Na2SO4. After the solvent was evaporated, the residue was purified by silica gel chromatography (petroleum ether - ethyl acetate), a deep red solid (10.3 mg, 13.8 %) was obtained. 1H NMR (CDCl3, 400 MHz, ppm) δ 8.68 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.57-7.56 (m, 3H), 7.47-7.43 (m, 1H), 7.37-7.35 (m, 3H), 7.34-7.30 (m, 1H), 7.11 (d, J = 8.8 Hz, 2H), 5.40 (s, 2H), 2.72 (s, 3H), 2.39 (q, J = 7.6 Hz, 2H), 1.70 (s, 3H), 1.40 (s, 3H), 1.05 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz, ppm) δ 157.6, 146.8, 142.5, 141.7, 140.17, 137.7, 136.5, 134.9, 133.5, 132.3, 131.7, 131.1, 130.5, 130.4, 130.0, 129.5, 129.4, 128.5, 128.2, 126.9, 126.0, 124.4, 123.7, 122.0, 121.2, 119.2, 119.1, 96.2, 70.9, 29.7, 17.2, 14.2, 12.3, 11.5. HRMS (ESI): calcd for C38H31BF2N6OS: 669.2419; found: 669.2430 [M+H]+.
2.2.3. Synthesis of 8-((((4-azidobenzyl)oxy)carbonyl)oxy)-9-(benzo[d]thiazol-2-yl)-2- ethyl-5,5difluoro-1,3,11-trimethyl-12-phenyl-5H-pyrrolo[1',2':3,4][1,3,2]diazaborinino[1,6-a]indol-4-ium-5uide (N3-BOD-C) NIR-BOD (50 mg, 0.093 mmol), Compound 3, and DMAP (17.0 mg, 0.14 mmol) were set in a flask and dissolved in dried CH2Cl2. The reaction mixture was stirred at room temperature. After completion, the solvent was removed under reduced pressure, the obtained residue was purified by silica gel chromatography (petroleum ether-dichloromethane-ethyl acetate) to afford a brown red solid (10.5 mg, 15.8 %). 1H NMR (CDCl3, 400 MHz, ppm) δ8.37 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.67 (s, 1H), 7.59-7.57 (m, 3H), 7.47-7.44 (m, 1H), 7.38-7.34 (m, 5H), 6.936.91 (d, J = 8.4 Hz, 2H), 5.24 (s, 2H), 2.73 (s, 3H), 2.42-2.37(q, J = 7.6 Hz, 2H), 1.69 (s, 3H), 1.42 (s, 3H), 1.05 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz, ppm): δ 166.9, 164.0, 153.0, 148.6, 144.5, 143.5, 141.8, 140.3, 138.8, 137.6, 135.2, 134.6, 131.5, 130.9, 130.3, 129.8, 129.6, 129.5, 128.1, 126.1, 124.8, 123.4, 122.8, 121.2, 119.8, 119.1, 108.5, 69.9, 29.7, 17.2, 14.0, 12.4, 11.2. HRMS (ESI): calcd for C39H32BF2N6O3S+: 713.2318; found: 713.2346 [M+H]+. 3. Results and Discussion 3.1. Photophysical Properties of NIR-BOD The spectroscopic characteristics of NIR-BOD with varying pH were initially performed. NIRBOD displayed a ratiometric response to pH in aqueous-organic mixed media, as evidenced in the absorption spectra (Fig. 1a). Enhancing pH value from acidic to basic condition, a reduction in the absorption band of the phenolic form at 546 nm was noted, accompanied with a concomitant increase of a new band at 647 nm which was attributed to the phenolate form of NIR-BOD. Notably, a well-defined isosbestic point at 576 nm was observed, indicative of the pH controllable equilibrium of the phenolic and phenolate forms of NIR-BOD. In accordance with the emission spectra, NIR-BOD could also give phenol/phenolate switchable fluorescence profiles within the pH range of 8.4-10.2. Changing the pH value from 8.4 to 10.2, the spectrum showed a decrease of the intensity of fluorescence band at 583 and 740 nm with the simultaneous emergence of a new emission band at 700 nm (Fig. 1b). More
interestingly, NIR-BOD only displayed NIR fluorescence at 740 nm under physiological conditions (acetonitrile/PBS buffer, 1:1, v/v, pH 7.22-8.10), which was attributed to the occurrence of the ESIPT reaction in NIR-BODI to form the ESIPT tautomer [27]. 3.2. The optical responses of N3-BOD-E and N3-BOD-C to H2S With the probes in hand, we then evaluated the optical response to H2S under PBS condition (pH = 7.4, PBS buffer, CTAB, 1 mM). Here, CTAB was used to enhance the solubility of the probe in PBS and facilitate the response rate [28-29]. As shown in Fig. 2, N3-BOD-E exhibited an absorption peak at 552 nm. Upon treatment with 100 μM NaHS, the absorption peak at 552 nm was reduced, and a concomitant new absorption band at 670 nm emerged, which is in good agreement with the absorption of the phenolate form of NIR-BOD. In the fluorescence spectra, dramatic changes were also noted. Upon excitation at 620 nm, a remarkable NIR fluorescence turn-on at 700 nm was observed, showing 200-fold intensity enhancement. Moreover, N3-BOD-E showed good selectivity for H2S over other related analytes, including reactive oxygen species (ROS), and reactive nitrogen species (RNS). As shown in Fig. 3a, only H2S introduced the significant fluorescence turn-on at 700 nm upon excitation at 620 nm. In sharp contrast, other competing species trigger minimal fluorescence signals at the same wavelength. Importantly, the turn-on response was found to be maintained even in the presence of 1 mM Cys, Hcy and GSH, indicative of the high selectivity to H2S over related biothiols. Notably, N3BOD-C also showed good optical response to H2S under PBS condition (Fig. S2), however, the poor selectivity toward H2S limited its further applications (Fig. 3b), which maybe due to the instability of carbonate unit in this system. N3-BOD-E also displayed a dose dependent response to H2S. As shown in Fig. 4b, a good linear correlation of the fluorescence intensity at 700 nm and H2S concentrations could be obtained, from which the detection limit was determined as 46 nM (3σ / k), suggesting the high sensitivity to H2S. For gaining insight into the activation of N3-BOD-E with H2S, 1H NMR and HRMS characterization was performed (Fig. S4, Fig. S5). Both identified the generation of NIR-BOD after
the incubation of N3-BOD with H2S. Based on the previously well-known chemistry of H2Smediated reduction of azides to amines [30-31], it is rational that N3-BOD-E was firstly reduced and followed by self- elimination to release the NIR-BOD in phenolate form, which emits in the NIR region (Scheme 2).
Scheme 2. Mechanism of the conversion of N3-BOD-E to NIR-BOD based on the reduction by H2S. 3.3. Image of N3-BOD-E in living cells Finally, the potential application of N3-BOD-E for living cells imaging was investigated (Fig. 5). Cardiomyocytes incubated with N3-BOD-E (10 μM) for 30 min gave minimal red fluorescence signals. However, probe-treated cells afforded patent intracellular red fluorescence upon addition of NaHS. These imaging results demonstrated that probe N3-BOD-E could be utilized for monitoring H2S in living cells. 4. Conclusion In summary, masking the free hydroxyl group in NIR-BOD by an analyte-related trigger provided a convenient approach for development of NIR fluorescent sensors. This design strategy was demonstrated here by the synthesis of two H2S-responsive fluorescent probes for detection in NIR fluorescence turn-on model. The azide function in the probes could be reduced to the amino unite which subsequently introduced 1,6-elimination to release the active fluorophore NIR-BOD, enabling the highly selective and sensitive detection of H2S with a detection limit of 46 nM . Living cells imaging experiments demonstrated the capability of the probe for applications in biological systems. Acknowledgement
We gratefully acknowledge the financial support by the National Science Foundation of China (grant numbers 21572039, 21372083, 21672062). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at… References [1] L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He, S. Zhu, A unique approach to development of near-infrared fluorescent sensors for in vivo imaging, J. Am. Chem. Soc. 134 (2012) 1351013523. [2] L.Y. Niu, Y.Z. Chen, H.R. Zheng, L.Z. Wu, C.H. Tung, Q.Z. Yang, Design strategies of fluorescent probes for selective detection among biothiols, Chem. Soc. Rev. 44 (2015) 61436160. [3] V. Ntziachristos, J. Ripoll, L.V. Wang, R. Weissleder, Looking and listening to light: the evolution of whole-body photonic imaging, Nat. Biotechnol. 23 (2005) 313-320. [4] L. Xiong, A.J. Shuhendler, J. Rao, Self-luminescing BRET-FRET near-infrared dots for in vivo lymph-node mapping and tumour imaging, Nat. Commun. 3 (2012) 1193. [5] N. Karton-Lifshin, L. Albertazzi, M. Bendikov, P.S. Baran, D. Shabat, “Donor-two-acceptor” dye design: a distinct gateway to NIR fluorescence, J. Am. Chem. Soc. 134 (2012) 2041220420. [6] R. Weissleder, A clearer vision for in vivo imaging, Nat. Biotechnol. 19 (2001) 316-317. [7] Y.S. Guan, L.Y. Niu, Y.Z. Chen, L.Z. Wu, C.H. Tung, Q.Z. Yang, A near-infrared fluorescent sensor for selective detection of cysteine and its application in live cell imaging, RSC Adv. 4 (2014) 8360-8364. [8] A. Razgulin, N. Ma, J. Rao, Strategies for in vivo imaging of enzyme activity: an overview and recent advances, Chem. Soc. Rev. 40 (2011) 4186-4216. [9] K. Gu, Y. Xu, H. Li, Z. Guo, S. Zhu, S. Zhu, P. Shi, T.D. James, H. Tian, W.H. Zhu, Real-time tracking and in vivo visualization of β-galactosidase activity in colorectal tumor with a ratiometric NIR fluorescent probe, J. Am. Chem. Soc. 138 (2016) 5334-5340. [10] C. Zhao, X. Li, F. Wang, Target-triggered NIR emission with a large stokes shift for the detection and imaging of cysteine in living cells, Chem. Asian. J. 9 (2014) 1777-1781. [11] C. Zhao, P. Feng, J. Cao, Y. Zhang, X. Wang, Y. Yang, Y. Zhang, J. Zhang, 6-Hydroxyindolebased borondipyrromethene: synthesis and spectroscopic studies, Org. Biomol. Chem. 10 (2012) 267-272. [12] C. Zhao, P. Feng, J. Cao, X. Wang, Y. Yang, Y. Zhang, J. Zhang, Y. Zhang, Borondipyrromethene-derived Cu2+ sensing chemodosimeter for fast and selective detection, Org. Biomol. Chem. 10 (2012) 3104-3109. [13] Q. Fei, L. Zhou, F. Wang, B. Shi, C. Li, R. Wang, C. Zhao, Rational construction of probes rendering ratiometric response to the cancer-specific enzyme NQO1, Dyes Pigm. 136 (2017) 846-851. [14] F. Wang, Y. Zhu, L. Zhou, L. Pan, Z. Cui, Q. Fei, S. Luo, D. Pan, Q. Huang, R. Wang, C. Zhao, H. Tian, C. Fan, Fluorescent in situ targeting probes for rapid imaging of ovarian-cancerspecific γ-glutamyltranspeptidase, Angew. Chem. Int. Edit. 54 (2015) 7349-7353.
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Fig. 1. The spectroscopic characteristics of NIR-BOD (5μM, acetonitrile/PBS buffer, 1:1, v/v) with varying pH (7.22, 7.53, 7.82, 8.10, 8.40, 8.70, 8.99, 9.44, 9.83, 10.21). a) absorption, b) emission.
Fig. 2. Time-dependent absorption (a) and emission (b) changes of N3-BOD-E (2 μM) in the presence of NaHS (100 μM).
Fig. 3. Fluorescence responses (λex = 620 nm) of (a) N3-BOD-E (2 μM); (b) N3-BOD-C (2 μM) toward NaHS (100 uM), and other biologically relevant reactive species (500 uM), each data were acquired 2 h after addition of analytes under PBS condition (pH = 7.4, PBS buffer, CTAB, 1 mM). a, Free; b, GSH; c, Cys; d, Hcy; e, Ser; f, Gly; g, HPO42-; h, NO;i, S2O32-; j, SO42-; k, OAc-; l, N3-; m, CO32-; n, NO2-; o, F-; p, Cl-; q, Br-; r, I-; s, H2O2; t, ClO-; u, NaHS; v, Cys(1 mM) + NaHS; w, GSH(1 mM) + NaHS; x, Hcy(1 mM) + NaHS.
Fig. 4. (a) The turn-on fluorescence response of N3-BOD-E toward different concentrations of NaHS under PBS condition (pH = 7.4, PBS buffer, CTAB, 1 mM), (b) Plot of fluorescence intensity changes at 700 nm as function of NaHS concentrations.
Fig. 5. Detection of H2S in cardiomyocytes using confocal fluorescence imaging. (A) Cells incubated with N3-BOD-E for 30 min; (C) Cells treated with N3-BOD-E for 30 min followed by further treated with 100 μM NaHS for 30 min; (B) and (D) are the bright field images. Scale bar represents 20 μm.
S1010-6030(17)30902-4 http://dx.doi.org/10.1016/j.jphotochem.2017.08.043 JPC 10818
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date: Revised date: Accepted date:
28-6-2017 14-8-2017 18-8-2017
Please cite this article as: Qiang Fei, Mimi Li, Jian Chen, Ben Shi, Ge Xu, Chunchang Zhao, Xianfeng Gu, Design of BODIPY-based Near-Infrared Fluorescent Probes for H2S, Journal of Photochemistry and Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.08.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Design of BODIPY-based Near-Infrared Fluorescent Probes for H2S Qiang Fei,†,‡ Mimi Li,† Jian Chen,† Ben Shi,‼ Ge Xu,‼ Chunchang Zhao,‼ Xianfeng Gu†,* †
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203
China ‡
Food and Pharmaceutical Engineering Institute, Guiyang University, Guizhou, 550005, China
‼
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and
Molecular Engineering, East China University of Science & Technology, Shanghai 200237, China. E-Mail: [email protected]
Graphical Abstract
Highlights
A highly selective and sensitive probe for detecting H2S has been designed and synthesized.
H2S is able to trigger the probe to light-up NIR emission.
The probe is suitable for applications in living cells imaging.
ABSTRACT Fluorescent probes with near-infrared (NIR) emission are of great interest in biology imaging, owing to their minimal autofluorescence from biological samples and deep tissue penetration. Here, we reported two NIR fluorescence probes by masking the free hydroxyl group in a NIR fluorescent dye. We demonstrated that the designed probes showed highly selective and sensitive responsiveness to H2S. Owing to H2S triggered light-up NIR emission, the probes were suitable for applications in living cells imaging.
1. Introduction Fluorescence imaging with near-infrared (NIR, 650−900 nm) dyes are attractive for visualizing biological targets of interest in living systems [1-3], because NIR light endows the advantages of deep tissue penetration, decreased interference from autofluorescence background and minimum photo-damage to biological samples [4-7]. Accordingly, the development of novel fluorescent scaffolds that emit in the NIR region is critically required but still an important challenge. Fluorescent chromophores containing a characteristic hydroxyl group are among the most widely studied traditional dyes [8-10]. It is facile to modify the hydroxyl group in these derivatives for construction of chemodosimeters for molecular optical imaging of analytes. In this respect, we have devised a novel class of reaction-based probes full utilization of 6hydroxyindole-based BODIPY as a scaffold due to its tunable photophysical properties [11-15]. However, the absorption and emission wavelengths of these derivatives only fall into the visible region, which retards their practical applications in living system. We also reported the further modification of 6-hydroxyindole-based BODIPY to afford a NIR fluorescent dye (NIR-BOD) [26]. Interestingly, this NIR-BOD is superior to 6-hydroxyindole-based BODIPY with absorption and emission in the NIR region under neutral PBS conditions. Importantly, the free hydroxyl group in NIR-BOD can be readily modified for development of fluorescent sensors for in vitro and in vivo imaging applications. H2S as an endogenous gasotransmitter plays vital roles in exerting anti-inflammation, modulating blood pressure and regulating other fundamental processes [16-18]. On the other hand, abnormal levels of H2S may cause series of diseases, such as Alzheimer’s disease and Parkinson’s disease [19-20]. Therefore, it is of high scientific interest to develop molecular probes for detection of H2S. To data, many reaction-based fluorescent scaffolds have been reported [21-25]. However, the development of NIR fluorescent H2S probes is still an important challenge but highly desirable. Considering the NIR emission of NIR-BOD and the facile masking of the hydroxyl group, a new fluorescent probe can be established by grafting a special trigger onto the NIR-BOD moiety. Thus
two light-up NIR fluorescent probes (Scheme 1) for H2S were designed, in which p-azidobenzyl is incorporated to NIR-BOD through an ether or carbonate linkage. Interestingly, H2S-triggered reduction of azide generated the amino function, which was followed by 1,6-elimination to release the active fluorophore NIR-BOD, enabling the NIR tracking H2S in living systems. 2. Experimental 2.1. Material All chemical reagents and solvents for synthesis were purchased from commercial suppliers and were used without further purification. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer with chemical shifts reported in ppm at room temperature. Mass spectra were measured on a HP 1100 LC-MS spectrometer. UV-vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra were measured with a Varian Cary Eclipse Fluorescence spectrophotometer. 2.2. Synthesis of N3-BOD-E and N3-BOD-C probes
Scheme 1 Synthetic route for N3-BOD-E and N3-BOD-C. The synthetically related compounds are depicted in Scheme 1. Compound 2 was synthesized according to literature procedures [26]. NIR-BOD was obtained according to the previous work [27]. N3-BOD-E and N3-BOD-C were prepared as shown in Scheme 1. The structure was fully characterized by 1H and 13C NMR and high-resolution mass spectrometry (HRMS) (Fig. S10-S15).
2.2.1. Synthesis of 4-azidobenzyl (4-nitrophenyl) carbonate (3) To a solution of Compound 1 (693 mg, 3.35 mmol) in dichloromethane were added 4nitrophenyl chloroformate (1.87 g, 6.7 mmol), cooled to 0 °C for 30 min. Then Et3N was added dropwise, and the reaction was stirred at room temperature over night. The reaction mixture was diluted with water and extracted with dichloromethane. The organic layer was separated and washed with 1 % NaOH aqueous solution, saturated brine solution and water, respectively. Then dried over anhydrous Na2SO4, proceeded evaporation under vacuum, the residue was purified by silica gel chromatography (petroleum ether-ethyl acetate), a white solid was obtained (460 mg, 31.5 %). 1H NMR (CDCl3, 400 MHz, ppm) δ 8.29-8.27 (d, J = 8.8 Hz, 2H), 7.46-7.44 (d, J = 8.0 Hz, 2H), 7.39-7.37 (d, J = 8.8 Hz, 2H), 7.08-7.06 (d, J = 8.0 Hz, 2H), 5.26 (s, 2H). 2.2.2. Synthesis of 8-((4-azidobenzyl)oxy)-9-(benzo[d]thiazol-2-yl)-2-ethyl-5,5-difluoro-1, 3,11trimethyl-12-phenyl-5H-pyrrolo[1',2':3,4][1,3,2]diazaborinino[1,6-a]indol-4-ium-5-uide (N3-BODE) NIR-BOD (60 mg, 0.112 mmol), Compound 3 (47.3 mg, 0.223 mmol), and potassium carbonate (46.4 mg, 0.336 mmol) were dissolved in the flask with dry acetone (10 mL). The reaction mixture was continuously stirred over night at room temperature. After completion, the mixture was diluted with CH2Cl2. The organic layer was separated, washed with water (3 × 50 mL), and dried over anhydrous Na2SO4. After the solvent was evaporated, the residue was purified by silica gel chromatography (petroleum ether - ethyl acetate), a deep red solid (10.3 mg, 13.8 %) was obtained. 1H NMR (CDCl3, 400 MHz, ppm) δ 8.68 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.57-7.56 (m, 3H), 7.47-7.43 (m, 1H), 7.37-7.35 (m, 3H), 7.34-7.30 (m, 1H), 7.11 (d, J = 8.8 Hz, 2H), 5.40 (s, 2H), 2.72 (s, 3H), 2.39 (q, J = 7.6 Hz, 2H), 1.70 (s, 3H), 1.40 (s, 3H), 1.05 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz, ppm) δ 157.6, 146.8, 142.5, 141.7, 140.17, 137.7, 136.5, 134.9, 133.5, 132.3, 131.7, 131.1, 130.5, 130.4, 130.0, 129.5, 129.4, 128.5, 128.2, 126.9, 126.0, 124.4, 123.7, 122.0, 121.2, 119.2, 119.1, 96.2, 70.9, 29.7, 17.2, 14.2, 12.3, 11.5. HRMS (ESI): calcd for C38H31BF2N6OS: 669.2419; found: 669.2430 [M+H]+.
2.2.3. Synthesis of 8-((((4-azidobenzyl)oxy)carbonyl)oxy)-9-(benzo[d]thiazol-2-yl)-2- ethyl-5,5difluoro-1,3,11-trimethyl-12-phenyl-5H-pyrrolo[1',2':3,4][1,3,2]diazaborinino[1,6-a]indol-4-ium-5uide (N3-BOD-C) NIR-BOD (50 mg, 0.093 mmol), Compound 3, and DMAP (17.0 mg, 0.14 mmol) were set in a flask and dissolved in dried CH2Cl2. The reaction mixture was stirred at room temperature. After completion, the solvent was removed under reduced pressure, the obtained residue was purified by silica gel chromatography (petroleum ether-dichloromethane-ethyl acetate) to afford a brown red solid (10.5 mg, 15.8 %). 1H NMR (CDCl3, 400 MHz, ppm) δ8.37 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.67 (s, 1H), 7.59-7.57 (m, 3H), 7.47-7.44 (m, 1H), 7.38-7.34 (m, 5H), 6.936.91 (d, J = 8.4 Hz, 2H), 5.24 (s, 2H), 2.73 (s, 3H), 2.42-2.37(q, J = 7.6 Hz, 2H), 1.69 (s, 3H), 1.42 (s, 3H), 1.05 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz, ppm): δ 166.9, 164.0, 153.0, 148.6, 144.5, 143.5, 141.8, 140.3, 138.8, 137.6, 135.2, 134.6, 131.5, 130.9, 130.3, 129.8, 129.6, 129.5, 128.1, 126.1, 124.8, 123.4, 122.8, 121.2, 119.8, 119.1, 108.5, 69.9, 29.7, 17.2, 14.0, 12.4, 11.2. HRMS (ESI): calcd for C39H32BF2N6O3S+: 713.2318; found: 713.2346 [M+H]+. 3. Results and Discussion 3.1. Photophysical Properties of NIR-BOD The spectroscopic characteristics of NIR-BOD with varying pH were initially performed. NIRBOD displayed a ratiometric response to pH in aqueous-organic mixed media, as evidenced in the absorption spectra (Fig. 1a). Enhancing pH value from acidic to basic condition, a reduction in the absorption band of the phenolic form at 546 nm was noted, accompanied with a concomitant increase of a new band at 647 nm which was attributed to the phenolate form of NIR-BOD. Notably, a well-defined isosbestic point at 576 nm was observed, indicative of the pH controllable equilibrium of the phenolic and phenolate forms of NIR-BOD. In accordance with the emission spectra, NIR-BOD could also give phenol/phenolate switchable fluorescence profiles within the pH range of 8.4-10.2. Changing the pH value from 8.4 to 10.2, the spectrum showed a decrease of the intensity of fluorescence band at 583 and 740 nm with the simultaneous emergence of a new emission band at 700 nm (Fig. 1b). More
interestingly, NIR-BOD only displayed NIR fluorescence at 740 nm under physiological conditions (acetonitrile/PBS buffer, 1:1, v/v, pH 7.22-8.10), which was attributed to the occurrence of the ESIPT reaction in NIR-BODI to form the ESIPT tautomer [27]. 3.2. The optical responses of N3-BOD-E and N3-BOD-C to H2S With the probes in hand, we then evaluated the optical response to H2S under PBS condition (pH = 7.4, PBS buffer, CTAB, 1 mM). Here, CTAB was used to enhance the solubility of the probe in PBS and facilitate the response rate [28-29]. As shown in Fig. 2, N3-BOD-E exhibited an absorption peak at 552 nm. Upon treatment with 100 μM NaHS, the absorption peak at 552 nm was reduced, and a concomitant new absorption band at 670 nm emerged, which is in good agreement with the absorption of the phenolate form of NIR-BOD. In the fluorescence spectra, dramatic changes were also noted. Upon excitation at 620 nm, a remarkable NIR fluorescence turn-on at 700 nm was observed, showing 200-fold intensity enhancement. Moreover, N3-BOD-E showed good selectivity for H2S over other related analytes, including reactive oxygen species (ROS), and reactive nitrogen species (RNS). As shown in Fig. 3a, only H2S introduced the significant fluorescence turn-on at 700 nm upon excitation at 620 nm. In sharp contrast, other competing species trigger minimal fluorescence signals at the same wavelength. Importantly, the turn-on response was found to be maintained even in the presence of 1 mM Cys, Hcy and GSH, indicative of the high selectivity to H2S over related biothiols. Notably, N3BOD-C also showed good optical response to H2S under PBS condition (Fig. S2), however, the poor selectivity toward H2S limited its further applications (Fig. 3b), which maybe due to the instability of carbonate unit in this system. N3-BOD-E also displayed a dose dependent response to H2S. As shown in Fig. 4b, a good linear correlation of the fluorescence intensity at 700 nm and H2S concentrations could be obtained, from which the detection limit was determined as 46 nM (3σ / k), suggesting the high sensitivity to H2S. For gaining insight into the activation of N3-BOD-E with H2S, 1H NMR and HRMS characterization was performed (Fig. S4, Fig. S5). Both identified the generation of NIR-BOD after
the incubation of N3-BOD with H2S. Based on the previously well-known chemistry of H2Smediated reduction of azides to amines [30-31], it is rational that N3-BOD-E was firstly reduced and followed by self- elimination to release the NIR-BOD in phenolate form, which emits in the NIR region (Scheme 2).
Scheme 2. Mechanism of the conversion of N3-BOD-E to NIR-BOD based on the reduction by H2S. 3.3. Image of N3-BOD-E in living cells Finally, the potential application of N3-BOD-E for living cells imaging was investigated (Fig. 5). Cardiomyocytes incubated with N3-BOD-E (10 μM) for 30 min gave minimal red fluorescence signals. However, probe-treated cells afforded patent intracellular red fluorescence upon addition of NaHS. These imaging results demonstrated that probe N3-BOD-E could be utilized for monitoring H2S in living cells. 4. Conclusion In summary, masking the free hydroxyl group in NIR-BOD by an analyte-related trigger provided a convenient approach for development of NIR fluorescent sensors. This design strategy was demonstrated here by the synthesis of two H2S-responsive fluorescent probes for detection in NIR fluorescence turn-on model. The azide function in the probes could be reduced to the amino unite which subsequently introduced 1,6-elimination to release the active fluorophore NIR-BOD, enabling the highly selective and sensitive detection of H2S with a detection limit of 46 nM . Living cells imaging experiments demonstrated the capability of the probe for applications in biological systems. Acknowledgement
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Fig. 1. The spectroscopic characteristics of NIR-BOD (5μM, acetonitrile/PBS buffer, 1:1, v/v) with varying pH (7.22, 7.53, 7.82, 8.10, 8.40, 8.70, 8.99, 9.44, 9.83, 10.21). a) absorption, b) emission.
Fig. 2. Time-dependent absorption (a) and emission (b) changes of N3-BOD-E (2 μM) in the presence of NaHS (100 μM).
Fig. 3. Fluorescence responses (λex = 620 nm) of (a) N3-BOD-E (2 μM); (b) N3-BOD-C (2 μM) toward NaHS (100 uM), and other biologically relevant reactive species (500 uM), each data were acquired 2 h after addition of analytes under PBS condition (pH = 7.4, PBS buffer, CTAB, 1 mM). a, Free; b, GSH; c, Cys; d, Hcy; e, Ser; f, Gly; g, HPO42-; h, NO;i, S2O32-; j, SO42-; k, OAc-; l, N3-; m, CO32-; n, NO2-; o, F-; p, Cl-; q, Br-; r, I-; s, H2O2; t, ClO-; u, NaHS; v, Cys(1 mM) + NaHS; w, GSH(1 mM) + NaHS; x, Hcy(1 mM) + NaHS.
Fig. 4. (a) The turn-on fluorescence response of N3-BOD-E toward different concentrations of NaHS under PBS condition (pH = 7.4, PBS buffer, CTAB, 1 mM), (b) Plot of fluorescence intensity changes at 700 nm as function of NaHS concentrations.
Fig. 5. Detection of H2S in cardiomyocytes using confocal fluorescence imaging. (A) Cells incubated with N3-BOD-E for 30 min; (C) Cells treated with N3-BOD-E for 30 min followed by further treated with 100 μM NaHS for 30 min; (B) and (D) are the bright field images. Scale bar represents 20 μm.