Highly Specific Cys Fluorescence Probe for Living Mouse Brain

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Highly Specific Cys Fluorescence Probe for Living Mouse Brain Imaging via Evading Reaction with Other Biothiols Yandi Zhang, Xin Wang, Xiaoyi Bai, Ping Li, Di Su, Wen Zhang, Wei Zhang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01878 • Publication Date (Web): 29 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Analytical Chemistry

Highly Specific Cys Fluorescence Probe for Living Mouse Brain Imaging via Evading Reaction with Other Biothiols Yandi Zhang,‡ Xin Wang,‡ Xiaoyi Bai, Ping Li,* Di Su, Wen Zhang, Wei Zhang and Bo Tang*. College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institutes of Biomedical Sciences, Shandong Normal University, Jinan 250014, People’s Republic of China.

ABSTRACT: Depression is characterized by oxidative stress in the brain. As the crucial reductive biothiol, cysteine (Cys) directly regulate the occurrence of oxidative stress in brain. Despite its significance, the precise exploration of Cys in mouse brains remains a challenge, primarily owing to the limitations of Cys-monitoring tools, especially the interference from unavoidable reaction with other biothiols. Thus, we developed a novel two-photon fluorescence probe for Cys based on a new specific recognition site, thiobenzoate. Encountering Cys, the carbon-sulfur double bond in the probe formed a stable fivemembered ring via the selective nucleophilic addition reaction, triggering a remarkable fluorescence increase. Notably, this reaction can’t occur with other biothiols, which afford the probe unprecedented selectivity to Cys. With two-photon excitation at 754 nm, we achieved in situ visualization of the increased Cys in PC12 cells under dithiothreitol stimulation. Furthermore, we directly visualized the precipitous reduction of Cys in the brains of mice with depression phenotypes for the first time. This work opens up new vistas for Cys imaging and expands the understanding of pathogenesis of depression.

Depression is a global mental disorder with high prevalence.1-2 However, the current understanding of physiological and pathological of depression is still preliminary.1-2 Previous studies suggest that depression is characterized by oxidative stress in brains.3 Cysteine (Cys), primarily generated from the demethylation of methionine and reduction of cystine, maintains the conformations of proteins and the synthesis of glutathione (GSH) in the cell.4 In particular, Cys functions as protecting cells from oxidative damage though directly involved in the intracellular reduction process and phospholipid metabolism.5 Despite the significance of Cys in redox homeostasis, its precise variation in brains of mice with depression phenotypes remains poorly understood, mainly due to lack of tools for specific measurement of Cys in living brains. Fluorescence imaging technology is a robust approach for monitoring molecular events in living cells and in vivo because of its excellent spatial and temporal resolution.6-8 Recently, some fluorescence probes for imaging Cys have been developed based on the nucleophilicity and reducibility of its sulfydryl.9-22 However, these probes suffer with unavoidable reactions with other biothiols, especially GSH or Hcy. These up

the risk of false-positive signals and doses of probes. Therefore, to accurately and conveniently explore Cys in the living brain, lacking the probes with specificity, high sensitivity still is a great stumbling block.

Scheme 1. The structure and Luminescence Mechanism of TCS. To meet the demand, based on the specific nucleophilic addition reaction between thiobenzoate and Cys,23-25 we rationally constructed a novel two-photon fluorescent probe TCS (O-phenyl-N-4-methyl-2-oxo-2H-chromen-7ylcarbamothioate). Because the biological context of the brain is extremely complicated, we utilized two-photon (TP) imaging with deeper tissue penetration, lower background fluorescence, higher spatiotemporal resolution, and less photodamage.26-27 In particular, TCS as a molecular fluorescent probe, will easily cross the blood-brain barrier (BBB) and be excreted.28 In this compound, the coumarin 120 is selected as a fluorophore due to its extraordinary TP optical properties. The strong electron-withdrawing carbon-sulfur double bond in TCS educes the electron-donating ability of the amino and lessens the push-pull electron effect of coumarin, decreasing its fluorescence. In the presence of Cys, the carbon-sulfur double bond in the probe formed a stable five-membered ring via the selective nucleophilic addition reaction,23-25 restoring the push-pull electron effect of the coumarin and increasing its fluorescence (Scheme 1, Figure S2). Thus, we synthesized TCS and tested its performance. Our data indicated that TCS have the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for precise assay of Cys in the brain of mice with depression phenotypes.

EXPERIMENTAL SECTION Synthesis of TCS Coumarin 120 (0.176 g, 1.0 mmol) was first dissolved in Tetrahydrofuran (10 mL), followed by the addition of Phenyl chlorothionocarbonate (135 μL, 1.0 mmol). The mixture was then stirred at 28 °C under nitrogen for 12 h. After the reaction was completed, the mixture was concentrated under vacuum.

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The residue was purified by preparative thin layer chromatography on silica gel GF254 with cyclohexane/ethyl acetate (1:2) as the eluent, and the light yellow product was obtained. The probe yield is 60.40 %. NMR data: 1HNMR (400 MHz, d6-DMSO): δ 8.12 (s, 1H), 7.79 (d, 2H), 7.49 (t, 2H), 7.31 (t, 1H), 7.23 (d, 2H), 6.35 (s, 1H), 5.77 (s, 1H), 2.42 (s, 3H). 13C NMR (101 MHz, d6-DMSO): δ 160.27, 157.81, 153.40, 129.84, 127.33, 126.67, 122.88, 122.43, 119.28, 118.14, 116.98, 115.67, 114.19,113.71, 18.50. HRMS (ESI) m/z calcd. for C17H13NO3S [M+H]+ calculated 312.0688, found 312.0670. TCS-Cys was obtained by TCS reaction with excess Cys. HRMS (ESI) m/z calcd. for C14H12N2O4S [M-H]- calculated 303.0434, found 303.0409. 1H NMR (400 MHz, d6-DMSO): δ 9.37 (s, 1H), 7.56 (s, 1H), 6.74 (d, 2H), 6.45 (S, 1H), 5.90 (s, 1H), 2.43(s, 3H), 2.30(s, 1H), 1.23 (s, 2H), 13C NMR (101 MHz, d6-DMSO): δ 159.97, 157.94, 154.10, 153.07, 130.06, 127.61, 123.06, 119.58, 116.16, 115.44, 114.42, 22.82, 18.96, 14.42.

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correlation coefficient of 0.997 in the range of 0-35 μM, yielding a detection limit of 0.16 μΜ. This means that TCS can serve as a highly sensitive Cys probe. Moreover, time course studies showed that the fluorescence intensity plateaued in approximately 14 min and remained virtually unchanged for another 60 min (Figure S5, S6). Our data indicate that TCS have sensitivity, response rate and photostability suitable for assessing Cys.

Two-photon imaging experiments For cells imaging, living PC12 cells were detached, transplanted onto 12-well platelets with glass-bottom, and cultured for 24 h before imaging. Cys (100 μM), NEM (1.0 mM), DTT (1.0 mM) pre-treated cells for 30 min, then the cells incubation with the probe for 20 min. Fluorescence images of TCS were obtained with an excitation wavelength of 754 nm and the emission window was 390-550 nm. Analyses were performed using Zeiss software. For data analysis, the average fluorescence intensity per image under each experimental condition was obtained by selecting regions of interest. Each experiment was repeated at least three separate times with identical results. In vivo imaging experiment, Zeiss 880 NLO microscopy was employed with z-stack mode and a water objective (20×). To image for exogenous Cys, the mice were anesthetized by chloral hydrate firstly, then the mice labeled with 0.2 mg kg-1 Cys, After 10 min, 0.3 mg kg-1 TCS via intraperitoneal injection. After 10 min, the abdomen of the mice was worn thinner carefully before imaging. In addition, the mice were anesthetized by chloral hydrate, then the mice labeled with 0.3 mg kg-1 TCS via intraperitoneal injection. After 10 min, the skull of the mice was worn thinner carefully before imaging. RESULTS AND DISUSSION The synthesis and characterization of the probe were described in Supporting Information (Figure S1, S12-13). Firstly, we found the absorption spectra of TCS was centered at approximately 340 nm. After the addition of Cys, the absorption at 340 nm significantly increased (Figure S3). Figure S4 depicts the maximum excitation and fluorescence emission peaks of TCS located at 340 nm and 443 nm, respectively. In the presence of 100 μΜ Cys, the fluorescence intensity of TCS at 443 nm obviously enhanced (25-fold) upon excitation at one-photon (340 nm) or two-photon (754 nm) (Figure 1A). Accordingly, the fluorescence quantum yield increased from 0.16 to 0.49, and TP absorption cross-section from 7.39 to 18.01.29 These changes in fluorescence properties demonstrate that the TCS transformed into TCS-Cys with the stronger push-pull electronic effects via the reaction with Cys, conducing to the enhancement of fluorescence. Next, we investigated whether TCS could sensitively report Cys. Varying Cys concentration from 0 to 100 μΜ progressively increased the fluorescence intensity of TCS (Figure 1B). The dose-dependent responses of TCS to Cys revealed a linear regression equation F=17.25[Cys](μΜ)+32.46 with a

Figure 1. Photophysical properties and selectivity of TCS. (A) Two-photon emission spectra of TCS (50 μΜ) before (black line) and after (red line) the addition of Cys (100 μΜ). (B). Changes in the fluorescence of TCS (20 μM) after the addition of various concentrations of Cys (0-100 μΜ). Insert: Linear relationship of fluorescence intensity with Cys. (C) Fluorescence responses of TCS to different competing species (100 μΜ): 1. TCS, 2. Phe; 3. Ile, 4. Asp, 5. His, 6. Thr, 7. Tyr, 8. Glu, 9. Met, 10. Gly, 11. Arg, 12. Lys, 13. Ser, 14. Val, 15. Pro, 16. Cystine, 17. SeCys2, 18. SeC, 19. DTT, 20. Peptide: RGDPGC, 21. Peptide: CRGDPG, 22. Peptide: CRGDPC, 23. Cys. (D) HPLC analyses of 50 μM TCS, 50 μM TCS-Cys and 50 μΜ TCS solution after reaction with 100 μΜ Cys, Hcy or GSH. To test the specificity of TCS for Cys, we investigated its response to other competing species. As shown in Figure 1C and Figure S7A, other biologically relevant substances did not induce any detectable fluorescence responses. In contrast, TCS (50 μM) elicited evident increase in fluorescence in the presence of Cys (100 μM). Notably, we noted no obvious alteration in fluorescence of TCS after GSH (100 μM) or Hcy (100 μM) were added. We further examined if TCS reacted with GSH or Hcy. Exhilaratingly, although 50 μM TCS was preincubated with 100 μM GSH or 100 μM Hcy for 15 min, it still showed the same dramatic increase in fluorescence after the addition of 100 μΜ Cys (Figure S7C-D). Moreover, HPLC analyses further confirmed TCS did not react with GSH or Hcy at all (Figure 1D). Those results indicate that the probe exhibits unprecedented selectivity to Cys by evading reaction with other biothiols. We believe the five-membered ring produced by the reaction of Cys might be most stable. That’s why we choose thiobenzoate as the recognition sites. Fortunately, this is an excellent fluorescent probe that can specifically only react with Cys, which distinctly avoid interference from other biothiols that abundant in living system. These greatly improved accuracy of the proposed method. Moreover, altering pH with buffers (pH 6-8) resulted in faint fluorescence changes, suggesting a weak pH dependency (Figure S8). Collectively, these results highlight the fact that TCS is a sensitive, specific Cys probe.

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Analytical Chemistry Before the bioimaging employments, standard 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evidence that the TCS has low cytotoxicity under 100 μM (IC50 value) (Figure S9).30 To evaluate the performance of TCS in cultured cells, we explore the imaging of TCS in PC12 cells. Firstly, we observed that the cells incubated with Cys (100 μM) produced brighter fluorescence (3.5-fold) than the control cells (Figure 2 A, B), confirming the excellent response of TCS to Cys in living cells. Conversely, Hcy or GSH did not caused any detectable fluorescence responses (Figure 2C, D). Moreover, Cys-induced fluorescence responses were blocked by N-ethylmaleimide (NEM), a thiol scavenger (Figure 2F).31-32 In addition, to imitate oxidative stress, we used H2O2 to oxidize Cys, weaker fluorescence was observed in the cells co-incubated with Cys and H2O2, which is consistent with the in vitro result (Figure 2E, Figure S7B). To verify TCS can visualize the endogenous Cys, we treated cells with dithiothreitol (DTT),31-32 and found these cells evoked relatively larger fluorescence response (Figure 2G). As a control, application of NEM, completely blocked Cysinduced fluorescence enhancement (Figure 2H). Collectively, these data reflect that intracellular increased fluorescence was caused by Cys, indicating that TCS is suitable for precise monitoring Cys in living systems.

Figure 2. TP fluorescence images of PC12 cells. (A) PC12 cells treated with TCS (50 μΜ). (B) PC12 cells pretreated with Cys (100 μM). (C) PC12 cells incubated with Hcy (100 μM) for 20 min. (D) PC12 cells pretreated with GSH (100 μM) for 20 min. (E) PC12 cells co-treated with Cys (100 μM) and H2O2 (1.0 mM) for 20 min. (F) PC12 cells co-incubated with Cys (100 μM) and NEM (1.0 mM) for 20 min. (G) PC12 cells stimulated with DTT (1.0 mM) for 20 min. (H) PC12 cells co-incubated with DTT (1.0 mM) and NEM (1.0 mM) for 20 min. The fluorescence intensity in A-D (I) and E-H (J).

Owing to its satisfactory performance in cellular application, TCS should be particularly useful for in vivo imaging. Firstly, we confirmed satisficing imaging performance of TCS in the mouse abdomen (Figure S10). Further, we used TCS to explore the fluctuate of Cys in the brain of mice with depression-like behaviours. At first, we exposed mice to 28 consecutive days of chronic unpredictable mild stress (CUMS) (Figure S11).33 Then, we separated them into susceptible and resilient populations based on the behavior tests.34 As Figure 3 exhibited, with two-photon excitation at 754 nm, the brains of susceptible mice showed evident decrease (2.1-fold) in fluorescence intensity compare to those mice without CUMS. Reversely, the resilient mice evoked no detectable fluorescence changes (Figure 3). The brain images testified that TCS can easily traverse the BBB. These results indicate that the concentration of Cys in the brain of susceptible mice is significantly reduced, which may be due to the redox imbalance in mice with depression-like behaviors.3, 35 Overall, this result provides direct evidence for the negative correlation between Cys levels and degree of depression-like behaviors. CONCLUSIONS In summary, we created a novel two-photon fluorescent probe TCS for monitoring Cys, which possesses the merits of superior sensitivity and biocompatibility. The unique specific recognition site endows it with exclusive selectivity towards Cys, successfully avoiding any reaction with other biomolecules. These admirable traits enabled in situ visualization of endogenous Cys in PC12 cells stimulated by DTT. Importantly, utilizing TCS, we unprecedentedly traced Cys in mouse brains and successfully revealed the negative correlation between Cys levels and degree of depression-like behaviors. Altogether, the work should advance our fundamental understanding of the pathogenesis of depression and other Cys-mediated diseases.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional experimental data, including synthesis, characterization, photophysical properties, cytotoxicity, and experimental details.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Bo Tang: 0000-0002-8712-7025

Author Contributions Figure 3. In situ TP imaging in brains of mice. (A) TP imaging and 3D images of the brains of mice treated with TCS (0.3 mg kg-1) through intraperitoneal injection. First row: Imaging at 100 μm in mouse brain. Second row: 3D images in mouse mice. (B) The fluorescence intensity in A. The data are expressed as mean ± S.D, ***P< 0.001 compared to the control group.

‡These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21535004, 91753111, 21675105) and

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the Key Research and Development Program of Shandong Province (2018YFJH0502), National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2017ZX09301030004) and the Natural Science Foundation of Shandong Province of China (ZR2017ZC0225).

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