Development of an endoplasmic reticulum-targeting fluorescent probe for the imaging of superoxide anion in living cells

doi:10.21203/rs.3.rs-1675054/v1

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Development of an endoplasmic reticulum-targeting fluorescent probe for the imaging of superoxide anion in living cells

https://doi.org/10.21203/rs.3.rs-1675054/v1

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Superoxide anion (O₂^•−) is an important reactive oxygen species (ROS), and plays critical roles in biological systems. ER stress has close relation with many metabolic diseases, and could lead to the abnormal production of ROS including O₂^•−. Herein, we present an ER-targeting probe (ER-Tf) for the detection of O₂^•− in living cells. The probe ER-Tf used triflate as the response site for O₂^•−, and employed p-methylbenzenesulfonamide as ER-targeting moiety. In response to O₂^•−, the triflate of the probe ER-Tf converted to hydroxyl group, providing strong blue emission under the excitation of ultraviolet light. The probe ER-Tf exhibited high sensitivity and selectivity to O₂^•−. Bioimaging experiments showed that the probe ER-Tf can be applied to detect O₂^•− at ER, and also demonstrated that rotenone could increase the generation of O₂^•− in living cells, while the O₂^•− level at ER showed no remarkable change during ferroptosis.

Fluorescent probe

Endoplasmic reticulum

Superoxide anion

Fluorescent imaging

Superoxide anion(O₂^•−) is a significant reactive oxygen species (ROS), and plays many critical roles in biological system [1–6]. Generally, O₂^•− is produced endogenously during cellular respiration, and O₂^•− levels are tightly controlled by scavenging enzymes in living organisms and closely related to homeostasis [7]. O₂^•− usually serves as a precursor to generate the other ROS including H₂O₂, hydroxyl radical (·OH), hypochlorous acid (HClO),and singlet oxygen (¹O₂) [8]. At the same time, O₂^•− can also be used as a signal molecule for the redox reaction in biological systems [9, 10]. However, when excess O₂^•− is produced, autophagy or apoptotic signaling pathways may be activated, leading to cell death and ultimately to various diseases including atherosclerosis and neurodegenerative diseases [11]. Meanwhile, endoplasmic reticulum (ER) is widely found in various mammal cells, and plays significant roles in the synthesis processes of proteins, lipids, sugars, and also works in maintaining blood glucose levels [12–16]. Many studies have indicated that ER stress is bound up with obesity, insulin resistance and other metabolic diseases, which may lead to abnormal production of reactive oxygen species (ROS), including O₂^•− [17,18]. Therefore, development of sensitive and selective method to detect O₂^•− at ER is highly significant for intensively studying the pathological role of O₂^•− in living systems.

Traditionally, the test methods for the detection of O₂^•− chiefly include MS, high performance liquid chromatograph (HPLC) and electron paramagnetic resonance (EPR) [19–22]. Nevertheless, the complex instruments of these methods can damage biological samples and are unsuitable for the real-time detection of O₂^•− under the normal physiological conditions. In contrast, fluorescence imaging as an emerging detection technology, has attracted widely attention due to its advantages of high sensitivity, low detection limit, real-time and non-destructive [23–25]. Currently, many fluorescent probes have been developed to detect cellular O₂^•− [26–29]. These probes utilized benzothiazoline, catechol, triflate, diphenyl phosphate as the selective recognition sites for O₂^•−, and studied the endogenous generation of O₂^•− in many organelles including mitochondrial and lysosome. Nevertheless, the fluorescent probes for detecting O₂^•− at ER are still very uncommon [30]. Therefore, it is still urgent to construct ER-targeting fluorescence probes for detecting O₂^•− .

In this work, we have developed a turn-on fluorescent probe (ER-Tf) to detect cellular O₂^•− at ER (Scheme 1). The probe ER-Tf utilized triflate group as a recognition site for O₂^•−, and utilized p-methylbenzenesulfonamide as an ER-targeting part. ER-Tf itself displayed nearly no fluorescence under excitation of ultraviolet light. When ER-Tf responded to O₂^•−, the triflate moiety converted to hydroxyl group, accompanying by the generation of strong blue emission. The probe ER-Tf showed the high sensitivity and selectivity to O₂^•−. In addition, ER-Tf can be used as an ER-targeting probe in bioimaging experiments for detecting cellular O₂^•− at ER.

2.1. Synthesis of Compound ER-Cou

The mixture of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid(206mg,1mmol), N-(2-aminoethyl)-4-methylbenzenesulfonamide (214 mg, 1 mmol), HOBt (67.5 mg, 0.5 mmol), EDC (382 mg, 2 mmol), 150 µL diisopropylethylamine (DIEA) in 5 mL DMF was stirred vigorously in a N₂ atmosphere for 5 hours. Then, 10 mL water was slowly added to the solution, and then the solution was extracted it with CH₂Cl₂, washed with H₂O and dried it with sodium sulfate, concentrated in vacuum. The crude product was then purified by the chromatographic column method (CH₂Cl₂: MeOH = 20:1) to provide the compound ER-Cou (261 mg, 65%) as a brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.72 (m, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.71 (t, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.90 (dd, 1H), 6.82 (m, 1H), 3.35 (m, 2H), 2.92 (q, 2H), 2.24 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.14, 162.18, 161.23, 156.77, 148.52, 142.96, 137.92, 132.50, 129.99, 127.00, 114.82, 113.85, 111.54, 102.26, 42.39, 21.26. HRMS (ESI): m/z cald. for C₁₉H₁₈N₂O₆S, [M + H]⁺ 403.0958, found 403.0966.

2.2 Synthesis of the probe ER-Tf

The mixture of compound ER-Cou (201 mg, 0.5 mmol), N-phenyl-bis(trifluoromethanesulfonimide) (PhN(SO₂CF₃)₂, 357mg, 1mmol), K₂CO₃ (138mg, 1mmol) in 10 mL THF was stirred for 6 hours. Then, the mixture was concentrated in vacuum and purified by a column chromatography (CH₂Cl₂:MeOH = 20:1), and provided the probe ER-Tf as a white solid (141 mg, 53%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.85 (s, 1H), 8.72 (t, 1H), 8.21 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.73 (t, 1H), 7.67 (m, 3H), 7.33 (d, J = 8.0 Hz, 2H), 3.35 (m, 2H), 2.95 (q, 2H), 2.24 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.32, 159.88, 154.79, 151.95, 146.74, 142.99, 137.94, 132.87, 130.02, 127.02, 120.20, 119.38, 118.93, 110.76, 42.22, 21.23. HRMS (ESI): m/z cald. for C₂₀H₁₇F₃N₂O₈S₂, [M + H]⁺ 535.0451, found 535.0391.

2.3 Cell culture and fluorescence imaging

HeLa cells were obtained from the College of Life Science, Nankai University (Tianjin, China). Hela cells were used to study the properties of ER-Tf, the cells were cultured in a medium containing 10% heat-inactivated fetal bovine serum and 1% antibiotics in air at 37℃ (5% CO2). Before fluorescence imaging, the HeLa cells were inoculated into a glass cover and cultured for 24 hours to achieve the suitable density. The images were obtained by a Nikon A1R confocal microscope.

Fluorescence of imaging exogenous O₂^•−: 5 µM ER-Tf was used to stain Hela cells for 20 min, followed by washing cells with PBS. Then the HeLa cells were incubated with 50 µM O₂^•− sulution for 20 min, and subsequently washed with PBS for three times and imaged.

Fluorescence of imaging endogenous O₂^•−: Firstly, the HeLa cells were incubated with 10 µM or 20 µM rotenone for 30 min, or treated with 10 µM erastin for 2h, and then washed with PBS for three times. Whereafter, 5 µM ER-Tf was used to stain HeLa cells for another 20 min, and subsequently the cells were washed with PBS for three times and imaged.

3.1 Synthesis of ER-targeting probe (ER-Tf) for O₂^•−

In order to meet requirements for the fluorescence imaging of cellular O₂^•− at ER, the designed fluorescent probe should structurally contain three units including fluorophore, response site of O₂^•− and ER-targeting group. Accordingly, we constructed the ER-targeting fluorescent probe (ER-Tf) for detecting cellular O₂^•− at ER. In the chemical structure of ER-Tf (scheme 1), coumarin group was used as the fluorophore, p-methylsulfonamide acted as an ER-targeting part, and triflate group was selected as a reaction site for the reorganization of O₂^•− [30].The synthetic route is revealed in scheme 1, and the chemical structures were characterized by ¹H NMR, ¹³C NMR and HRMS.

3.2. Optical properties of the probe ER-Tf

Firstly, we investigated the absorption spectra of the probe ER-Tf treated with or without O₂^•−. The probe ER-Tf showed no significant absorption peak in the visible region in PBS (20 mM, 5% MeOH) (Fig. 1A). However, after 20 µM O₂^•− was added to 5µM ER-Tf solution, an absorption peak could be clearly found at 405 nm, indicating that the probe ER-Tf reacted with O₂^•−. Under 405 nm excitation, the probe ER-Tf showed nearly no fluorescence (Fig. 1B). Upon the introduction of O₂^•−, the fluorescence peak at 462 nm appeared and increased continuously along with the gradual change of O₂^•− concentration from 0 µM to 90 µM (Fig. 1C). Meanwhile, the intensity of the fluorescence at 462 nm displayed the satisfactory linear relationship (R² = 0.9909) with O₂^•− concentration in the range 5–60 µM, and the detection limit was calculated to 65 nM (Fig. 1D). In addition, after ER-Tf reacted with O₂^•−, the fluorescence intensity reached the maximum at 462 nm within 7 min (Figure S1). Hence, ER-Tf could be used as a sensitive fluorescent probe to detect O₂^•−.

Whereafter, we assessed the selective property of the probe ER-Tf to O₂^•−. As shown in Fig. 2, the fluorescence spectra of ER-Tf showed negligible change in the following the addition of different biologically related species including the other ROS (H₂O₂, ClO⁻, etc), reactive nitrogen species, Cys, GSH and a battery of ions. By contrast, when the probe ER-Tf was added to 90 µM O₂^•−, a strong fluorescent peak at 462 nm can be clearly obtained, manifesting that ER-Tf had a desirable selectivity to O₂^•−. Furthermore, ER-Tf exhibited excellent fluorescence response to O₂^•− at pH 4.0–9.0. Considering that the normal pH at ER is about 7.2 (Figure S2), ER-Tf can potentially be used as the selective fluorescent probe for the detection of cellular O₂^•− at ER.

The response mechanism to O₂^•− of ER-Tf was then explored by HRMS test. As shown in the HRMS data of the reaction product between ER-Tf with O₂^•−(Figure S3), besides the peak at 535.0391 corresponding to ER-Tf (Cald. for [M + H]⁺, 535.0451), an obvious peak at 403.0966 and 425.0785, corresponding the compound ER-Cou (Cald. for [M + H]⁺, 403.0958; Cald. for [M + Na]⁺, 425.0778), indicating that the emissive compound ER-Cou was generated after the reaction of ER-Tf and O₂^•− (Figure S3). We proposed that O₂^•− firstly attacked S atom of triflate group, and then peroxytriflate free radical was removed to provide the emissive compound ER-Cou by protonation (Scheme 2).

Then, we tried to study the ability of ER-Tf to detect O₂^•− at ER. The MTT assay showed that ER-Tf showed very low cytotoxity below the concentration of 15 µM (Figure S4). When the HeLa cells were only treated with 5 µM ER-Tf for 20 min, almost no fluorescence was found under the excitation at 405 nm. Nevertheless, after the cells were firstly incubated with 30 µM or 60 µM O₂^•−, the bright fluorescence can be found in blue channel (Fig. 3A). It indicated that ER-Tf can visualize O₂^•− at ER. Besides, the fluorescence intensity of the cells treated with 60 µM O₂^•− was more intense than that of the cells incubated with 30 µM O₂^•−, suggesting that the probe ER-Tf could evaluate the concentration of cellular O₂^•− (Fig. 3B). Hence, ER-Tf could act as a sensitive fluorescent probe to detect O₂^•− in living cells.

Subsequently, in order to explore the ER-targeting ability of the probe ER-Tf, we selected the commercial ER-specific dye (ER-tracker) as a reference to compare the positioning ability of the probe ER-Tf. The specific operation of the experiment was as follows: Firstly, the cells were treated with 5 µM ER-Tf and incubated at a constant temperature for 20 min, and then washed off the medium and incubated with 50 µM O₂^•−, finally treated with 1 µM ER-tracker. Then we observe the positioning of the probe ER-Tf under a confocal microscope, the blue channel signal generated by the reaction of ER-Tf with O₂^•− can better overlap with the red channel signal generated by the ER-tracker, and the Pearson correlation coefficient is as high as 0.89 (Fig. 4).Therefore, the co-localization experiment showed that ER-Tf possessed ER-targeting property.

Then, we assessed the ability of ER-Tf to image endogenous O₂^•− in living cells. Rotenone, as a mitochondrial electron transport chain complex I inhibitor, which can lead to apoptosis and increase the production of ROS [31]. When HeLa cells were coated with 5µM ER-Tf for 20 min, almost no fluorescence was observed in the blue channel (Fig. 5). While HeLa cells were firstly treated with 10 µM or 20µM rotenone for 30 min and then stained with 5 µM ER-Tf for 20 min, the bright fluorescence could be found in blue channel. It indicates that HeLa cells produce O₂^•− when stimulated by rotenone. In addition, we evaluate the changes of endogenously generated cellular O₂^•− during ferroptosis. Erastin is a famous iron ion activator, which can inhibit the uptake of cystine by cystine/glutamate reverse transporter (system XC), and finally lead to ferroptosis. Erastin was employed as an activator to investigate the subtle changes of O₂^•− in living cells during ferroptosis [32]. After treatment of HeLa cells with 10 µM erastin for 2 h and then 5µM ER-Tf for 20 min, the fluorescence intensity of blue channel showed nearly no change (Fig. 5). It suggests that the O₂^•− level at ER showed no remarkable change during ferroptosis. Hence, ER-Tf can be used for the fluorescence imaging of endogenously generated O₂^•− at ER.

We have developed an ER-targeting probe (ER-Tf) for the detection of cellular O₂^•−. ER-Tf used triflate group as a recognition site for O₂^•−, employed p-methylbenzenesulfonamide as an ER-targeting unit. After the response of ER-Tf to O₂^•−, triflate moiety of ER-Tf converted to hydroxyl group, providing strong blue emission under the excitation. The probe ER-Tf probes exhibited high sensitivity and selectivity to O₂^•−. The bio-imaging experiments showed the probe ER-Tf can be used as an ER-targeting probe to detect cellular O₂^•− at ER. Rotenone could increase the generation of O₂^•− in living cells, while the O₂^•− level at ER showed no remarkable change during ferroptosis.

Authors' Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by H. Wei, Y. Wang, Q. Chen, Y. Sun and B. Dong. The first draft of the manuscript was written by H. Wei and B. Dong. All authors read and approved the final manuscript."

Conflicts of interest

The authors declare they have no competing interests

Funding

This work was supported by NSFC (51602127) and the Natural Science Foundation of Shandong Province (ZR2021MB022).

Ethics Declaration statement

Not applicable.

Consent to Participate

Not applicable.

Consent for publication

Not applicable.

Data Availability

All data generated or analysed during this study are included in this published article (and its supplementary information files).

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Scheme 1 and 2 are available in the Supplementary Files section

No competing interests reported.

JFSIDong.docx
Onlinefloatimage1.png
Scheme 1. Synthesis route to the probe ER-Tf.
Onlinefloatimage4.png
Scheme 2. response mechanism of the probe ER-Tf to O₂^•−.

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You are reading this latest preprint version

Development of an endoplasmic reticulum-targeting fluorescent probe for the imaging of superoxide anion in living cells

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimentation

2.1. Synthesis of Compound ER-Cou

2.2 Synthesis of the probe ER-Tf

2.3 Cell culture and fluorescence imaging

3. Results And Discussion

3.1 Synthesis of ER-targeting probe (ER-Tf) for O₂^•−

3.2. Optical properties of the probe ER-Tf

4. Conclusion

Declarations

References

Scheme

Competing Interests

Supplementary Files

Status:

Version 1

Development of an endoplasmic reticulum-targeting fluorescent probe for the imaging of superoxide anion in living cells

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimentation

2.1. Synthesis of Compound ER-Cou

2.2 Synthesis of the probe ER-Tf

2.3 Cell culture and fluorescence imaging

3. Results And Discussion

3.1 Synthesis of ER-targeting probe (ER-Tf) for O2•−

3.2. Optical properties of the probe ER-Tf

4. Conclusion

Declarations

References

Scheme

Competing Interests

Supplementary Files

Status:

Version 1

3.1 Synthesis of ER-targeting probe (ER-Tf) for O₂^•−