A novel chemiluminescent probe based on 1,2-dioxetane scaffold for

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A novel chemiluminescent probe based on 1,2dioxetane scaffold for imaging cysteine in living mice Jinyu Sun, Zhian Hu, Sichun Zhang, and Xinrong Zhang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00936 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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A novel chemiluminescent probe based on 1,2-dioxetane scaffold for imaging cysteine in living mice Jinyu Sun, Zhian Hu, Sichun Zhang and Xinrong Zhang* Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China. ABSTRACT: A novel chemiluminescent probe for detection of cysteine (Cys) from other biothiols has been reported by utilizing the excellent chemiluminescent Schaap’s adamantylidene-dioxetane scaffold. After careful assessment, the probe CL-Cys could detect Cys with high sensitivity and total light photons increased with 28-fold after the probe was treated with Cys, with the detection limit of 7.5 x 10-8 M. Finally, CL-Cys was further utilized to the chemiluminescent imaging of endogenous Cys in living mouse. In general, this probe has a remarkable ability of detecting Cys, which provides a valuable method for interrogation the Cys roles in more biological and pathological processes.

KEY WORDS: Chemiluminescent probe; Cysteine; 1,2-dioxetane; Living animal; Chemiluminescent imaging; In vivo imaging.

Biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a key role in many physiological and pathological processes, such as redox state, signal transduction and metal detoxification 1-2. In living systems, Cys is biosynthesized from methionine under the function of cystathionine-β-synthase, cystathionine-γ-lyase and adenosylhomocysteinase. Next, Cys was catalyzed by dioxygenase to cysteinesulfinate, and then transformed into βsulfinylpyruvate by aspartate aminotransferase, and finally decomposed to pyruvate and SO2 3-4. The abnormal level of Cys in living systems is associated with many diseases 5, such as rheumatoid arthritis, parkinson's disease and alzheimer's disease 6-7. In contrast, a deficiency of Cys can lead to hematopoiesis, leukopenia, psoriasis, neurotoxicity, edema, and liver injury 8-9. As the cysteine is involved in many biological processes, to better illustrate the relationship of Cys with various diseases and promote diseases diagnosis and prognosis, it is very urgent to develop a highly efficient approach for distinguishing Cys form other biothiols (Hcy/GSH), which provides a more detailed understanding of its complex biological effects. Among the current analytical methods for Cys detection, optical spectroscopy technology, especially fluorescence technology has been widely used due to its fast response, low detection limit, low cost and strong potential capacity for bioimaging 10-12. However, fluorescent detection methods always suffer from intrinsic drawbacks such as photobleaching, phototoxicity and the interference of autofluorescence of samples themselves. Compared to fluorescence, bio- and chemiluminescence provides an attractive gateway for studying biological analytical species and related processes with the extremely low background signal and high signal-to-noise ratio. Importantly, the bio- and chemiluminescent probes do not require any excitation light source to produce light emission. Therefore, this technique is

widely used as noninvasive in vivo imaging 13-15. To date, a large amount of bioluminescent probes based on firefly luciferase-luciferin system have been developed for the visualization of different enzymes and analytes in vitro and in vivo 16-24. Bio- and chemiluminescent probes for selective detection of Cys have been rarely reported. Liang group have reported a caged bioluminescent probe acrylic ester luciferin, which could selectively response Cys in vitro and in vivo 25. Ohmiya group has presented a fast and cost-effective method for Cys detection based on the biosynthesis of firefly D-luciferin in which the starting materials are 2-cyano-6hydroxybenzothiazole (CHBT) and L-cysteine. The level of L-cysteine could be measured by the bioluminescence output signal in the presence of luciferase 26. However, these probes are based on the firefly luciferase-luciferin system and thus may bring a potential drawback to inexperienced operators since they need exogenous gene expression to produce luciferase which is necessary to catalytic chemiluminescence reaction in this experimental system. Luckily, Schaap’s adamantylidene-dioxetane is a graceful scaffold to construct probes for enzymes and chemical analytes because it contains a stable dioxetane moiety 27-29. Recently, Shabat’s group improve the effiencicy of this scaffold by attaching an electron withdrawing group (EWG), finally obtain tremendous increase in luminescence quantum yield in aqueous 30-33. The Michael reaction between acrylate ester and Cys can be utilized to construct Cys-selective probes. Many fluorescent sensors (all with an acrylate appendent) have been reported to detect Cys through the Michael addition and the further intramolecular cyclization 34-36. Inspired by this, herein, we presented development, characterization, and biological applications of a reaction-based chemiluminescence probe for Cys detection, termed as CL-Cys. More importantly, unlike the firefly luciferase-luciferin system, our probe does not require

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exogenous gene expression to promote the chemiluminescence output signal.

Scheme 1. Adamantane 1,2-dioxetane-based probe for selective chemiluminescent detection of Cys. The design strategy and decompose process of this probe was illustrated in Scheme 1. The phenol group of 1,2-dioxetane chemiluminescent scaffold is protected by acrylate, a Cysresponsive protecting group (Scheme 2). The original characterization spectra of the probe were exhibited in the SI (Fig. S6–S14).The luminescence of probe CL-Cys is initially in an off state. The acrylate moiety of this probe reacted with Cys via a Michael addition. The further intramolecular cyclization induces light emission that can be gained with high selectivity against other analytes species. Afterwards, CL-Cys has been applied for Cys detection in aqueous solution and live mice.

Scheme 2. Syntheses of CL-Cys. EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals reagents are commercially available and used without further purification. All analytics reagent were dissolved into Tris-HCl (50 mM, pH=7.4) buffer solution. A stock solution (10 mM) of CL-Cys was dissolved in DMSO. Ultrapure water purified by ULTRAPURE WATER system. All 1H NMR were measured on Varian INOVA 300 spectrometer and all 13C NMR spectra were measured on a Bruker DMX-600 spectrometer.

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Electrospray ionization mass spectra (ESI-MS) were recorded in LTQ mass spectrometry. Chemiluminescent spectra were acquired using an Ultra-Weak Luminescence Analyzer for Chemiluminescence and Bioluminescence (BPCL-GP-TGC). Chemiluminescent emission spectrum was recorded on Molecular Devices SpectraMax M3. Syntheses of 2. Compound 1 was prepared according to the references with modification 30. Compound 1 (1 mmol, 402 mg), K2CO3 (1.5 mmol, 200 mg) and TBSCl (1.1 mmol, 165 mg) dissolved in THF (30 mL) for 2 h. The mixture was removed under vacuum and the residual was diluted in 30 mL water, then extracted with ethyl acetate (30 mL x 3) and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by silica gel column chromatography to afford the desired product 2 in 78 % yield. 1H NMR (300 MHz, CDCl3) δ 8.02 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.37 (d, J = 8.2 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H), 3.34 (s, 4H), 2.36 – 1.46 (m, 14H), 1.36 (t, J = 7.2 Hz, 4H), 1.09 (s, 10H), 0.25 (s, 7H). 13C NMR (150 MHz, CDCl3) δ 166.7, 151.9, 150.9, 139.8, 137.9, 131.3, 127.5, 127.3, 124.9, 124.2, 118.6, 60.5, 56.9, 39.2, 38.9, 38.6, 38.5, 37.1, 32.9, 29.6, 28.4, 28.2, 25.9, 18.8, 14.3, -3.4, -3.5. Syntheses of 3. Compound 2 (1 mmol, 516 mg) and methylene blue were dissolved in DCM (20 mL), oxygen was bubbled through the solution while irradiating with yellow light for 2 h. The reaction was monitored by TLC. Then the reaction mixture was removed under vacuum and the residual was diluted in 30 mL water, extracted with ethyl acetate (30 mL x 3) and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by silica gel column chromatography to afford the desired product compound 3 in 43% yield. 1H NMR (300 MHz, CDCl3) δ 7.95 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.21 (s, 1H), 5.32 (s, 1H), 4.30 (q, J = 7.2 Hz, 2H), 3.34 (s, 3H), 2.41 – 1.61 (m, 12H), 1.37 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 166.9, 138.3, 133.7, 126.8, 124.7, 122.9, 122.2, 121.4, 111.5, 96.3, 60.6, 49.6, 46.9, 39.3, 36.5, 34.1, 33.4, 32.7, 32.1, 31.5, 27.4, 26.1, 25.7, 14.3. MS (ESI) m/z 433.14 [C23H27ClO6-H]. Syntheses of CL-Cys. Compound 3 (0.5 mmol, 215 mg) and DIPEA (0.6 mmol, 72 mg) dissolved in DCM (20 mL) for 30 minutes, acrylyl chloride (0.6 mmol, 60 mg) was added, and then stirred at room temperature 3 h. The mixture was removed under vacuum and the residual was diluted in 30 mL water, then extracted with ethyl acetate (30 mL x 3) and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by silica gel column chromatography to afford the desired product CL-Cys in 22 % yield. 1H NMR (300 MHz, CDCl3) δ 8.28 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.47 (d, J = 7.2 Hz, 1H), 5.11 (d, J = 2.4 Hz, 1H), 4.26 (q, J = 7.2 Hz, 1H), 3.35 (s, 3H), 1.95 (m, 6H), 1.33 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CD3OD) δ 166.6, 163.4, 147.1, 136.5, 135.3, 134.3, 131.2, 128.6, 126.5, 125.7, 123.7, 123.1, 111.6, 96.2, 61.2, 61.0, 52.2, 39.4, 37.4, 36.6, 34.1, 33.9, 33.7, 33.2, 32.4, 32.1, 31.9, 31.6, 27.5, 26.6, 26.2. HRMS (ESI) m/z calculates for C26H29ClO7, 488.1621, found 511.1487 [C26H29ClO7 + Na]+.

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Spectroscopic Measurements. 2970 μL of buffer solution, 10 μL of a 0.3 mM CL-Cys in DMSO, 10 μL of a different concentration Cys were added to glass dish. The time scanning module was used to obtain chemiluminescent kinetic curve. For the response ability of probe CL-Cys to Cys, 1 μM CLCys was treated with 0, 2.5, 5, 7.5, 10, 12.5 15, 25, 50, 100, 200 μM Cys for 30 min in buffer solution. The selectivity of CL-Cys was determined by detecting the time-dependence of total light emission. Bulk Chemiluminescent Imaging of CL-Cys. The imagings were performed using ProteinSimple (FlourChem, USA) and were analyzed by software AlphaView SA3.4.0. CL-Cys (0.2 mM, 1 L) in Tris-HCl buffer solution (190 μL, 50 mM, pH=7.4), 10 μL different concentrations of Cys solution (finally concentration, 0, 12.5, 25, 50, 100, 250, 500 and 1000 μM) were loaded into a 96-well plate. Imaging was acquired with 1 min exposure time after all reagents were added.

a typical chemiluminescent kinetics curve. The luminescence emission intensity reached maximum in less than 20 minutes after the addition of Cys, then slowly decayed over a course of 60 min. Under the physiological relevant conditions, the integrated emission intensity was observed in the presence of Cys with 28-fold increase compared to a blank control. However, when treated by GSH or Hcy, the system did not show any chemiluminescence intensity enhancement. Meanwhile, the chemiluminescent emission spectrum of CLCys was also measured (Fig. S1)., The luminescence of probe is initially in an off state. As we expected, the emission intensity showed a significant enhancement in presence of Cys. These data fully validated that our probe CL-Cys could react with Cys to generate chemiluminescence.

CCK assay. The cytotoxicity of CL-Cys was measured by CCK. A549 cells were cultured in 96-well plates at a density of 4000 cells/well and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. The probe CL-Cys (5 & 10 μM) was added. The cells were incubated with CL-Cys for 12 and 24 h, respectively. Then, 10 μL of the CCK solution was added and the absorbance at 450 nm was performed after incubation 3 h. General Animal Protocols. The Laboratory Animal Center, Animal Care and Use Committee of Tsinghua University approved these experimental programs under Animal Protocol Number (#17-ZSC1). Mice were anesthetized with 3 % isoflurane in 97% oxygen gas. SCID/BALB-C mice were used in all imaging experiments. In Vivo Imaging of CL-Cys. 0.1 mM CL-Cys and 5 mM Cys were pre-prepared. The images of living mice were obtained with 1 min exposure time after treating with 100 μL solutions from a mixture of 980 μL of 50 mM Tris-HCl, 10 μL of 0.1 mM CL-Cys and 10 μL of Cys. As control, a 100 μL of buffer solution was performed in the same way. For the images endogenous Cys of CL-Cys. Anesthetized mice were treated with 100 μL mixture solution from a solution of 990 μL of 50 mM Tris-HCl and 10 μL of 0.1 mM CL-Cys, and then images were acquired with 5 min exposure time. In the contrast, a mouse was given injected with NEM (50 M, 100 L), and then probe CL-Cys (1 M) was administrated. All experiments were repeated three times on different mice. RESULTS AND DISCUSSION Spectral Response of CL-Cys to Cys With the CL-Cys in hand, we firstly evaluated the photophysical properties and reactivity toward Cys at physiological condition. Cys-dependent chemiluminescence emission was measured in Tris-HCl buffer. The chemiluminescence emission spectra was recorded using an Ultra-Weak Luminescence Analyzer by treating CL-Cys (1 M) with various concentration Cys. Treatment of CL-Cys (1 M) with Cys (20 M) resulted in instantaneous and strong emission increase. As shown in Figure 1A, the system showed

Figure 1. (A) Chemiluminescence kinetic profile of probe CL-Cys (1 M) upon incubation with Cys (20 M) in aqueous. (B) Chemiluminescent total light emission of CL-Cys (1 M) with different concentrations of Cys for 30 min. We then investigated the response ability of probe CL-Cys to Cys, the chemiluminescence kinetics curve of different concentrations of Cys and probe CL-Cys was measured. When the concentration of Cys reached 50 M, chemiluminescence intensity had the largest degree of increase and tended to reach the plateau (Fig. 1B). Under the best conditions, the intensity of chemiluminescence increases with increasing Cys concentration. We noticed a dose-dependent relationship between the chemiluminescence emission enhancement and Cys, demonstrating that the probe could be used to Cys detection. A linear response was observed between 0-25 M Cys (R2=0.992) and the detection limit of Cys (3σ/k) was calculated to be 7.5 x 10-8 M (Fig. 1B inset), where σ is the standard deviation of blank measurement, and k is the slope from plotting the total light emission versus Cys concentrations.


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pH Effect and Selectivity Study To apply the probe in living system, the probe needs a suitable pH range. The responses of probe CL-Cys towards Cys under different pH conditions were measured. The total chemiluminescence light emission gradually increased from pH 6.5 to 9.0, especially in the pH range of 7.4 to 8.5 (Fig. 2A). Therefore, a pH value closer to physiological condition (pH=7.4) was chose to preform subsequent experiments. We then tested the selectivity of probe CL-Cys (1 M) for Cys (20 M) against other biologically relevant analytes by adding reduced glutathione (GSH, 100 M), L-Homocysteine (Hcy, 100 M), N-Acetyl-L-cysteine (NAC, 100 M), or L-ascorbic acid (Vc, 100 M), H2O2 (100 M), HClO (100 M), Arg (100 M). There was no significant emission increase after treating with various species. It is worth noting that, in the presence of Cys, the emission increased by 28-fold (Fig. 2B). Moreover, the selectivity of this CL-Cys probe towards Cys against natural amino acids, some other potential interfering biomolecules/metal ions and various enzymes in vivo were further evaluated (Fig. S2&S3). Luckily, experimental results demonstrated that these potential analytes did not interfere with the detection system. These response and selectivity data demonstrated that CL-Cys would be able to detect Cys at physiological relevant condition without interferences from other biologically relevant species.

Figure 2. (A) Chemiluminescent integrated signal upon incubation of CL-Cys (1 M) with Cys (20 M) for 30 min in buffer solutions with different pH values. (B) Selectivity of CL-Cys (1 M) towards Cys among other biologically relevant analytes (100 M) in aqueous.

Figure 3. Imaging of Cys using CL-Cys. (A) Images 5 min after adding CL-Cys (1 M) to 0, 10, 25, 50, 100, 250, 500, 1000 M of Cys in aqueous solution (n=3 wells). (B) Quantification of (A). Encouraged by the strong chemiluminescent signal intensity of products from the bulk experiments and high chemiluminescent response of the probe towards Cys in vitro tests, we set out to evaluate its potential for detecting Cys in vivo. Before that, we need to evaluate the stability of the probe and its cytotoxicity (Fig. S4&S5). Experimental results exhibited that the probe CL-Cys has excellent stability and almost no toxicity to living cells. Afterwards, anesthetized mice were injected with this potential luminescence system on the back, and then imaging was obtained through the IVIS system. An obvious chemiluminescent light output signal of a living SCID/BALB-C mouse was obtained with 1 min exposure time after a 100 L mixture solution of CL-Cys (1 M) and Cys (50 M) was injected (Fig. 4A, right). And a vehicle control aqueous (buffer solution) did not induce any luminescence (Fig. 4A, left). These results indicated that CLCys can image and detect Cys in vivo via chemiluminescence.

Chemiluminescent Visualizing Cys in Living Animal Since the probe has a good response capability to Cys, we further investigated whether the probe could be used in vivo imaging under physiological conditions. A 96-well black plate was filled with 0, 10, 25, 50, 100, 250, 500, 1000 M of Cys and CL-Cys (1 M) in buffer solution. The light output indicated a significant increase in total photon flux with increasing Cys concentration (Fig. 3A). Average of repeated experiments provided a good linear response in the range of 0100 M when imaged 5 min after exposure to Cys. It is suggested that the probe has good sensitivity and the detection limit was further calculated to be 3.9 x 10-7 M in this case.

Figure 4. Chemiluminscence imaging of Cys in vivo. (A) Image with 1 min exposure time after treat with 100 μL of a mixture reagent containing CL-Cys (1 M) and Cys (50 M) in aqueous. In the vehicle control, the living mouse was administrated with subcutaneous injections of 100 L of buffer solution. (B) Quantification of (A)


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Chemiluminescent Imaging Endogenous Cys in Living Animal Having confirmed luminescence emission of this system was capable of tissue penetration, we further interrogated the ability of CL-Cys to visualize endogenous Cys in vivo. An anesthetized SCID/BALB-C mouse was administered probe CL-Cys through subcutaneous injections on the back. Then the chemiluminescence images were obtained and the wholebody images of mouse were present in Figure 5. A strong chemiluminescent signal was detected with 5 min exposure time after the probe CL-Cys (1 M, 100 L) was injected. The signal could be attributed to the interaction of probe CLCys with endogenous Cys (Fig. 5A, right). In the control, no chemiluminescent singal was observed throughout the experiments (Fig. 5A, left). By contrast, the mouse was preinjected with excess NEM (a thiol block reagent) for 30 min and then treated with probe CL-Cys via subcutaneous injection. The chemiluminescence emission was apparently attenuated in the mouse pretreated with NEM, which was attributed to the specific Cys depletion by NEM (Fig. 5A, middle). These results indicated that CL-Cys provided increased signal to endogenous Cys.

Figure 5. Chemiluminscence imaging of endogenous Cys. (A) Image with 5 min exposure time after treat with 100 L of a solution containing CL-Cys (1 M) in Tris-HCl buffer (right). In the vehicle control, the living mouse was administrated with subcutaneous injections of 100 L of buffer solution (left). In the contrast, a mouse was given injected with NEM (50 M, 100 L), and then probe CL-Cys (1 M) was administrated (middle). (B) Quantification of (A).

CONCLUSION In summary, we have developed a novel chemiluminscent imaging probe CL-Cys for Cys-spectific detection in vitro and in vivo. It has been demonstrated that the probe is able to discriminate Cys from other biothiols, including Hcy and GSH. The selective and sensitive signal is induced by the Michael addition between Cys and CL-Cys and the further intramolecular cyclization to generate light output. A good linear relationship between 0-25 M of CL-Cys to Cys was obtained and a LOD of Cys was calculated to be 7.5 x 10-8 M. Moreover, we further evaluated the potential of probe CL-Cys for sensing endogenous Cys in living mice. We anticipate that CL-Cys might be utilized to interrogate the Cys function in various physiological and pathological processes.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.xxxx. 1HNMR, 13CNMR, HRMS data. (word). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT Financial supports from the Natural Science Foundation of China (No. 21390410, 21727813 and 21621003) were greatly acknowledged. REFERENCES (1) Reddie, K. G.; Carroll, K. S. Expanding the functional diversity of proteins through cysteine oxidation. Curr. Opin. Chem. Biol., 2008, 12 (6), 746-754. (2) Zhang, S.; Ong, C.-N.; Shen, H.-M. Critical roles of intracellular thiols and calcium in parthenolide-induced apoptosis in human colorectal cancer cells. Cancer Lett., 2004, 208 (2), 143-153. (3) Du, S.-x.; Jin, H.-f.; Bu, D.-f.; Zhao, X.; Geng, B.; Tang, C.s.; Du, J.-b. Endogenously generated sulfur dioxide and its vasorelaxant effect in rats. Acta Pharmacol. Sin., 2008, 29, 923-930. (4) Ubuka, T.; Ohta, J.; Yao, W. B.; Abe, T.; Teraoka, T.; Kurozumi, Y. l-Cysteine metabolism via 3-mercaptopyruvate pathway and sulfate formation in rat liver mitochondria. Amino Acids, 1992, 2 (1), 143-155. (5) Shahrokhian, S. Lead phthalocyanine as a selective carrier for preparation of a cysteine-selective electrode. Anal. Chem., 2001, 73 (24), 5972-5978. (6) Heafield, M. T.; Fearn, S.; Steventon, G. B.; Waring, R. H.; Williams, A. C.; Sturman, S. G. Plasma cysteine and sulphate levels in patients with motor neurone, Parkinson's and Alzheimer's disease. Neurosci. Lett., 1990, 110 (1), 216-220. (7) Wang, X. F.; Cynader, M. S. Pyruvate released by astrocytes protects neurons from copper-catalyzed cysteine neurotoxicity. J. Neurosci., 2001, 21 (10), 3322-3331. (8) H., F. J.; S., B. D.; Bhupinder, S.; Birgit, Z.; K., S. M.; Fran, N.; Rodica, B.; Raman, C.; J., F. R.; Aleksey, K.; M., H. S. Cystamine increases l-cysteine levels in Huntington's disease transgenic mouse brain and in a PC12 model of polyglutamine aggregation. J. Neurochem., 2004, 91 (2), 413422. (9) Janáky, R.; Varga, V.; Hermann, A.; Saransaari, P.; Oja, S. S. Mechanisms of L-cysteine neurotoxicity. Neurochem. Res., 2000, 25 (9), 1397-1405. (10) Chan, J.; Dodani, S. C.; Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem., 2012, 4, 973-984.

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(11) Chen, X.; Tian, X.; Shin, I.; Yoon, J. Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species. Chem. Soc. Rev., 2011, 40 (9), 4783-4804. (12) Li, X.; Gao, X.; Shi, W.; Ma, H. Design strategies for water-soluble small molecular chromogenic and fluorogenic probes. Chem. Rev., 2014, 114 (1), 590-659. (13) Badr, C. E.; Tannous, B. A. Bioluminescence imaging: progress and applications. Trends Biotechnol., 2011, 29 (12), 624-633. (14) Prescher, J. A.; Contag, C. H. Guided by the light: visualizing biomolecular processes in living animals with bioluminescence. Curr. Opin. Chem. Biol., 2010, 14 (1), 80-89. (15) Roda, A.; Guardigli, M. Analytical chemiluminescence and bioluminescence: latest achievements and new horizons. Anal. Bioanal. Chem., 2012, 402 (1), 69-76. (16) Cohen, A. S.; Dubikovskaya, E. A.; Rush, J. S.; Bertozzi, C. R. Real-time bioluminescence imaging of glycans on live cells. J. Am. Chem. Soc., 2010, 132 (25), 8563-8565. (17) Heffern, M. C.; Park, H. M.; Au-Yeung, H. Y.; Van de Bittner, G. C.; Ackerman, C. M.; Stahl, A.; Chang, C. J. In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease. Proc. Natl. Acad. Sci. USA., 2016, 113 (50), 14219-14224. (18) Hequan, Y.; Min-kyung, S.; Jianghong, R. A bioluminogenic substrate for in vivo imaging of β-lactamase activity. Angew. Chem. Int. Ed., 2007, 46 (37), 7031-7034. (19) Jones, L. R.; Goun, E. A.; Shinde, R.; Rothbard, J. B.; Contag, C. H.; Wender, P. A. Releasable luciferin−transporter conjugates: tools for the real-time analysis of cellular uptake and release. J. Am. Chem. Soc., 2006, 128 (20), 6526-6527. (20) Kindermann, M.; Roschitzki-Voser, H.; Caglič, D.; Repnik, U.; Miniejew, C.; Mittl, P. R. E.; Kosec, G.; Grütter, M. G.; Turk, B.; Wendt, K. U. Selective and sensitive monitoring of caspase-1 activity by a novel bioluminescent activity-based probe. Chem. Biol., 2010, 17 (9), 999-1007. (21) Sellmyer, M. A.; Bronsart, L.; Imoto, H.; Contag, C. H.; Wandless, T. J.; Prescher, J. A. Visualizing cellular interactions with a generalized proximity reporter. Proc. Natl. Acad. Sci. USA., 2013, 110 (21), 8567-8572. (22) Van de Bittner, G. C.; Bertozzi, C. R.; Chang, C. J. Strategy for dual-analyte luciferin imaging: in vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation. J. Am. Chem. Soc., 2013, 135 (5), 1783-1795. (23) Wehrman, T. S.; von Degenfeld, G.; Krutzik, P. O.; Nolan, G. P.; Blau, H. M. Luminescent imaging of βgalactosidase activity in living subjects using sequential reporter-enzyme luminescence. Nat. Methods, 2006, 3, 295301. (24) Wender, P. A.; Goun, E. A.; Jones, L. R.; Pillow, T. H.; Rothbard, J. B.; Shinde, R.; Contag, C. H. Real-time analysis of uptake and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice. Proc. Natl. Acad. Sci. USA., 2007, 104 (25), 10340-10345. (25) Zhang, M.; Wang, L.; Zhao, Y.; Wang, F.; Wu, J.; Liang, G. Using bioluminescence turn-on to detect cysteine in vitro and in vivo. Anal. Chem., 2018, 90 (8), 4951-4954. (26) Niwa, K.; Nakajima, Y.; Ohmiya, Y. Applications of luciferin biosynthesis: Bioluminescence assays for l-cysteine and luciferase. Anal. Biochem., 2010, 396 (2), 316-318.

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(27) Hananya, N.; Eldar Boock, A.; Bauer, C. R.; SatchiFainaro, R.; Shabat, D. Remarkable enhancement of chemiluminescent signal by dioxetane–fluorophore conjugates: turn-on chemiluminescence probes with color modulation for sensing and imaging. J. Am. Chem. Soc., 2016, 138 (40), 13438-13446. (28) Schaap, A. P.; Chen, T.-S.; Handley, R. S.; DeSilva, R.; Giri, B. P. Chemical and enzymatic triggering of 1,2dioxetanes. 2: fluoride-induced chemiluminescence from tertbutyldimethylsilyloxy-substituted dioxetanes. Tetrahedron Lett., 1987, 28 (11), 1155-1158. (29) Schaap, A. P.; Sandison, M. D.; Handley, R. S. Chemical and enzymatic triggering of 1,2-dioxetanes. 3: alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane. Tetrahedron Lett., 1987, 28 (11), 1159-1162. (30) Green, O.; Eilon, T.; Hananya, N.; Gutkin, S.; Bauer, C. R.; Shabat, D. Opening a gateway for chemiluminescence cell imaging: distinctive methodology for design of bright chemiluminescent dioxetane probes. ACS Cent. Sci., 2017, 3 (4), 349-358. (31) Bruemmer Kevin, J.; Green, O.; Su Timothy, A.; Shabat, D.; Chang Christopher, J. Chemiluminescent probes for activity-based sensing of formaldehyde released from folate degradation in living mice. Angew. Chem. Int. Ed., 2018, 57 (25), 7508-7512. (32) E., R.-K. M.; R., B. C.; Doron, S. Unprecedented sensitivity in a probe for monitoring cathepsin B: chemiluminescence microscopy cell-imaging of a natively expressed enzyme. Angew. Chem. Int. Ed., 2017, 56 (49), 15633-15638. (33) Gnaim, S.; Scomparin, A.; Das, S.; Blau, R.; SatchiFainaro, R.; Shabat, D. Direct real-time monitoring of prodrug activation by chemiluminescence. Angew. Chem. Int. Ed., 2018, 57 (29), 9033-9037. (34) Ali, F.; H. A, A.; Taye, N.; Gonnade, R. G.; Chattopadhyay, S.; Das, A. A fluorescent probe for specific detection of cysteine in the lipid dense region of cells. Chem. Commun., 2015, 51 (95), 16932-16935. (35) Nawimanage, R. R.; Prasai, B.; Hettiarachchi, S. U.; McCarley, R. L. Cascade reaction-based, near-infrared multiphoton fluorescent probe for the selective detection of cysteine. Anal. Chem., 2017, 89 (12), 6886-6892. (36) Niu, W.; Guo, L.; Li, Y.; Shuang, S.; Dong, C.; Wong, M. S. Highly selective two-photon fluorescent probe for ratiometric sensing and imaging cysteine in mitochondria. Anal. Chem., 2016, 88 (3), 1908-1914.


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ACS Sensors For TOC Only


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Scheme 1. Adamantane 1,2-dioxetane-based probe for selec-tive chemiluminescent detection of Cys. 163x118mm (300 x 300 DPI)


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ACS Sensors

Scheme 2. Syntheses of CL-Cys. 131x110mm (300 x 300 DPI)


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Figure 1. (A) Chemiluminescence kinetic profile of probe CL-Cys (1 M) upon incubation with Cys (20 M) in aqueous. (B) Chemiluminescent total light emission of CL-Cys (1 M) with different concentrations of Cys for 30 min.


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ACS Sensors

Figure 2. (A) Chemiluminescent integrated signal upon incu-bation of CL-Cys (1 M) with Cys (20 M) for 30 min in buffer solutions with different pH values. (B) Selectivity of CL-Cys (1 M) towards Cys among other biologically rele-vant analytes (100 M) in aqueous.


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Figure 3. Imaging of Cys using CL-Cys. (A) Images 5 min after adding CL-Cys (1 M) to 0, 10, 25, 50, 100, 250, 500, 1000 M of Cys in aqueous solution (n=3 wells). (B) Quanti-fication of (A).


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ACS Sensors

Figure 4. Chemiluminscence imaging of Cys in vivo. (A) Image with 1 min exposure time after treat with 100 μL of a mixture reagent containing CL-Cys (1 M) and Cys (50 M) in aqueous. In the vehicle control, the living mouse was ad-ministrated with subcutaneous injections of 100 L of buffer solution. (B) Quantification of (A)


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Figure 5. Chemiluminscence imaging of endogenous Cys. (A) Image with 5 min exposure time after treat with 100 L of a solution containing CL-Cys (1 M) in Tris-HCl buffer (right). In the vehicle control, the living mouse was administrated with subcutaneous injections of 100 L of buffer solution (left). In the contrast, a mouse was given injected with NEM (50 M, 100 L), and then probe CL-Cys (1 M) was administrated (middle). (B) Quantification of (A).


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A novel chemiluminescent probe based on 1,2-dioxetane scaffold for

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