Turn-on Quinoline Probe for Selective Sensing of Hypochlorite in Live Cells

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Olubiyo, Rebecca J. Burgess, Sebastian Y. S. Klu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6994428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hypochlorite is a significant reactive oxygen species (ROS) involved in various biological processes. Excessive levels of hypochlorite are linked to oxidative stress, which contributes to a number of diseases by altering the structure and function of biomacromolecules. Many existing probes for detecting hypochlorite have relatively high molecular weights and typically exhibit changes in emission wavelength upon interaction with the ROS. We have synthesized a quinoline-based probe, 5 , which is not fluorescent on its own, but becomes fluorescent at 523 nm upon reaction with hypochlorite through an oxidative mechanism. This probe exhibits time sensitivity as low as 10 seconds outside of cells. Probe fluorescence is visible to the naked eye, with intensity increasing with ROS concentration. Furthermore, probe fluorescence is detected in cells in a concentration-dependent manner. Altogether, we describe the generation and use of a quinoline-based probe to detect hypochlorite ROS in live cells. Fluorescence chemical sensors organic synthesis hypochlorite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Reactive oxygen species (ROS) are oxidative derivatives generated through complete or incomplete oxidation processes[ 1 ] and are recognized for their diverse roles in biological processes, functioning as antimicrobial agents within the immune system [ 2 ], redox signaling agents [ 3 ], and regulators of hematopoietic stem cell quiescence and self-renewal [ 4 , 5 ]. However, the overproduction of these species is directly associated with oxidative stress, which can lead to a variety of negative cellular consequences due to alterations in the structure and function of biomacromolecules [ 6 – 8 ]. These changes, evident by oxidative stress, can lead to disease and tissue damage [9.10]. Significant advances have been made to elucidate the roles and significance of these species. Technically, accurate quantification and analysis of ROS are crucial, as these species naturally occur at low concentrations [ 11 ]. Hypochlorite is an ROS that has been extensively studied because of its stability in live cells [ 12 ]. Techniques for sensing and quantifying hypochlorite include electrochemical methods, spectrophotometry, chromatography, electron spin resonance, and chemiluminescent probes, among others [ 13 – 15 ]. However, fluorescence analysis has become increasingly favored due to probe availability, high sensitivity, and low cytotoxicity [ 16 ]. The physical properties of activity-based probes, such as color, quantum yield, solubility, molar absorption coefficient, Stokes' shift, specificity, and selectivity, are all critical factors that influence their applicability [ 17 – 21 ]. Quinoline and its derivatives have been important motifs in organic chemistry since it was discovered in the 1800s [ 22 ]. Because quinoline and its derivatives contribute to binding through pi-stacking or hydrophobic interactions through the aromatic ring systems, they can intercalate DNA, disrupt biological redox systems, or chelate metal ions [ 23 ], which leads to their applications as antimalarials [ 24 , 25 ], antioxidants [ 26 , 27 ], and fluorescent dyes amongst others. The quinoline scaffold has been studied extensively as fluorescent probes for the detection and measurement of metals [ 28 – 31 ], hydrogen sulfide [ 32 ], Tau aggregates [ 33 ], and reactive oxidative species [ 34 ]. Due to the weak fluorescence of the compounds, studies on the quinoline scaffold for ROS have mostly employed addition of a fluorophore to the structure, focusing on positions 2, 3 and 4 of the motif [ 35 ]. There has been little research on the benzylic motif of the compound. Here, we have extensively studied the 7- position of the ring and synthesized a quinoline-phenothiazine structure for hypochlorite detection. We synthesized a turn-on probe that is not fluorescent by itself but responds selectively to hypochlorite while remaining unresponsive to other analytes. We show that this probe has high sensitivity and specificity and can detect hypochlorite in a concentration-dependent manner in live cells. 2. RESULTS AND DISCUSSION 2.1 Design and synthesis of probe 5 Synthesis of the precursor, 4 , was carried out in three simple steps from easily available starting materials (Scheme 1), and all intermediates and the target molecule were characterized using well established methods of 1 H NMR, 13 C NMR, mass spectrometry, and UV-Vis spectroscopy. Very little solvatochromic shift was seen in absorbance of 4 (Fig S1 SI). Precursor 4 also showed very little fluorescence in various solvents except in PBS. (Fig S2 SI). Thus, a sulfur ring closure reaction was carried out with 4 to make target molecule 5 (Scheme 2). Vedamalai et al. have determined the phenothiazine ring to be very susceptible to hypochlorite oxidation via the ICT process [36]. Their molecule had gone through a blue shift upon addition of the ROS . We hypothesized that the mechanism of action of hypochlorite on probe 5 is through the oxidation of the sulfur atom in 5 to turn into the corresponding sulfoxide, 6. Thus, we treated 5 with sodium hypochlorite and isolated compound 6 (Scheme 2). The 1 H NMR spectra of 5 and 6 were stacked (Fig 1). An obvious change in the chemical shifts of most of the protons down field shows that a strong electron-withdrawing group has been added onto the molecule. Further proof was obtained from the mass spectrometry and fluorescence results of both compounds (Fig S3 S.I and Figure S4 S.I.). Thus, 5 was further characterized for use of hypochlorite detection. 2.2 Response rate and selectivity of the probe Probe 5 shows no fluorescence in various solvents, polar and non-polar, (Fig S4 SI) at concentrations as high as 50 µM. Specificity of the probe towards different ions and ROS was tested using the following analytes: OH . , SO 3 2- , NO 3 - , H 2 O 2 , OH - , Fe 2+ , Zn 2+ , Sn 2+ , Ca 2+ , Na + , Cl - , Br - , F - , NO 2 - , and ClO - (Fig S5 SI). Probe 5 only showed fluorescence response toward ClO - , with no fluorescence detected towards all other analytes tested. Upon addition of hypochlorite, an intense fluorescence peak was observed at 523 nm, which clearly shows that the probe has selectivity towards ClO - . The sensitivity of probe 5 towards hypochlorite was determined by fluorescence titration of ClO - against the probe (Fig S6 SI). Hypochlorite concentrations between 20 µM and 90 µM were tested while the probe was kept at 5 µM (Fig S6a SI). Although probe 5 showed sensitivity to hypochlorite at as low as 35 µM, lower concentrations were below the detection limit of the fluorimeter. An increase in intensity of the peak at 523 nm proportional to the increase in molarity of hypochlorite was observed, with a steady increase in fluorescence intensity up to 70 µM of hypochlorite (Fig S6b SI). At higher concentrations, the florescence intensity began to decrease, although still well above that of lower hypochlorite concentrations. Furthermore, the fluorescence of the probe 5 when treated with ClO - could be visualized by the naked eye (Fig S7 SI). To determine the time sensitivity of the probe, a time-dependent fluorescence analysis was carried out on 5 . Probe and analyte were kept at 5 µM and 50 µM, respectively, and probe fluorescence was investigated at various times between 10 s and 20 mins. A change in intensity could be seen by 10s (Fig S8 SI). A linear relationship between intensity and time, up to 20 mins, was observed. 2.3 Detection of hypochlorite in live cells To determine if 5 could detect hypochlorite in live cells, mouse bone marrow cells were freshly isolated and incubated for 30 minutes with 5 at 5 µM, a concentration with limited cytotoxicity to live cells (Fig S9 S.I.). Cells were washed and then treated with various concentrations of exogenous ClO - for 30 minutes. The ability of cells to take up and react with exogenous hypochlorite was detected using flow cytometry and emission at 512/25nm. Compared to cells with no treatment (Mean Fluorescence Intensity (MFI): 27.6), cells with probe 5 exhibited little fluorescence (MFI: 37.7) when no hypochlorite was added (Fig 2a). In contrast, upon addition of 50 µM ClO - , a clear shift in the cells toward high probe 5 fluorescence (MFI: 2323) was detected. About 80% of single cells showed fluorescence greater than probe 5 only upon addition of ClO - . To determine the sensitivity of probe 5 , live bone marrow cells were treated with various concentrations of ClO - for thirty minutes. The hypochlorite concentrations used were validated to induce limited cytotoxicity during the course of the experiment (data not shown). The fluorescence intensity of cells treated with 5 increased with increasing concentration of ClO - , suggesting that probe 5 detects ClO - in live cells in a concentration-dependent manner (Fig 2b). Thus, probe 5 enters into live bone marrow cells and can detect exogenously added ClO - . 3. EXPERIMENTAL 1 H, 13 C, 19 F NMR were recorded in CDCl 3 with TMS as internal standard on a Bruker 500 MHz spectrometer at 500, 125, and 471 MHz, respectively, unless otherwise specified. UV-Vis Spectroscopy was carried out on a Carey Eclipse UV-vis Spectrophotometer with a 1 cm cuvette. Fluorescence was recorded on the Carey Eclipse Spectrometer with excitation and emission slits set at 5, and scan rate 600 nm /min with a 1 cm cuvette. Experimental C57BL6/J (Jackson Laboratories, strain 000664) mice at 7-12 weeks of age were used for live cell studies. Animals were housed in the Southern Illinois University Laboratory Animal Program facility, and animal procedures were approved by the Southern Illinois University Institutional Animal Care and Use Committee. Synthesis of 2 (2,4-bis(trifluoromethyl)quinolin-7-amine): To a flask containing 40 ml chloroform, was added m-phenylenediamine (5 g, 46 mmol) and 1,1,1,5,5,5-hexafluoropentane-2,4-dione (9.6 g, 46 mmol) and the resulting solution was refluxed at 90 0 C for 7 h. After completion, the solution was concentrated and 2 was recrystallized from ethanol as a fluffy yellow solid (12.8 g, 100 %). 1 H NMR (500 MHz, DMSO- d 6 ) δ 7.81 (dd, J = 9.3, 2.3 Hz, 1H), 7.58 (s, 1H), 7.33 (dd, J = 9.2, 2.4 Hz, 1H), 7.10 (d, J = 2.3 Hz, 1H), 6.46 (s, 2H). 13 C NMR (126 MHz, DMSO- d 6 ) δ 152.8, 150.7, 146.6 (q, J = 34.4 Hz), 134.9 (q, J = 31.7 Hz), 124.9 – 124.2 (m), 123.9, 122.6 (d, J = 33.7 Hz), 120.4 (d, J = 33.5 Hz), 115.7, 108.4, 106.8 19 F NMR (471 MHz, DMSO- d 6 ) δ -60.53 (d, J = 2.2 Hz), -66.68. HRMS (EI-TOF) m/z:[M] + Calcd for C 11 H 6 F 6 N 2 280.04352; Found280.04428. To a solution containing 2 (500 mg, 1.785 mmol) and NaOCH 3 (482.1 mg, 8.923 mmol) in 30 ml methanol, was added paraformaldehyde (71.42 mg, 2.230 mmol) in 5 ml methanol dropwise. The mixture was refluxed and monitored with TLC. NaBH 4 (67.53 mg, 1.785 mmol) was then added, and solution stirred for additional 2 h at reflux temperature. The crude solution was concentrated and washed with water twice. It was then extracted with dichloromethane and dried with anhyd sodium sulfate. The filtrate was concentrated and subject to column chromatography using 30 % dichloromethane in hexane to yield 3 as a bright yellow solid (178 mg, 52 %). 1 H NMR (500 MHz, DMSO- d 6 ) δ 7.84 (dq, J = 9.3, 2.2 Hz, 1H), 7.69 (s, 1H), 7.34 (dd, J = 9.3, 2.4 Hz, 1H), 7.07 (q, J = 4.9 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 2.81 (d, J = 4.9 Hz, 3H). 13 C NMR (126 MHz, DMSO- d 6 ) δ 152.8, 151.2, 147.7 – 145.8 (m), 134.8 (d, J = 31.7 Hz), 124.7, 124.2 (d, J = 17.2 Hz), 122.7 (d, J = 35.9 Hz), 120.6, 116.1, 108.7, 102.8. 19 F NMR (471 MHz, DMSO- d 6 ) δ -60.11 (d, J = 2.2 Hz), -66.32. HRMS(EI-TOF) m/z:[M] + Calcd for C 12 H 8 F 6 N 2 294.05917; Found 294.05995. Synthesis of 4 ( N -methyl-2,4-bis(trifluoromethyl)quinolin-7-amine- N -methyl- N -phenyl-2,4-bis(trifluoromethyl)quinolin-7-amine): Synthesis of 3 ( N -methyl-2,4-bis(trifluoromethyl)quinolin-7-amine): 20 ml toluene was degassed for 15 mins under argon. 3 ( N -methyl-2,4-bis(trifluoromethyl) quinolin-7-amine) (300 mg, 1.0196 mmol), bromobenzene (320mg, 2.04 mmol), palladium acetate (84 mg, 0.3732 mmol), BINAP (120 mg, 0.192 mmol) and NaO-t-Bu (1.568 g, 16.3136 mmol) was added, and the solution was stirred for 48 h at 110 0 C and the reaction was monitored with TLC. After completion, the solution was filtered and the filtrate concentrated. The filtrate was then subject to column chromatography to yield product as a solid (304.8 mg, 80.7 %). 1 H NMR (500 MHz, CDCl 3 ) δ 7.88 (dt, J = 9.0, 1.7 Hz, 1H), 7.69 (s, 1H), 7.47 (d, J = 2.6 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.30 (dd, J = 2.4, 1.1 Hz, 1H), 7.26 (ddt), J = 7.5, 6.4, 1.2 Hz, 3H), 3.46 (s, 3H). 19 F NMR (471 MHz CDCl 3 ) δ -61.32 (d, J = 2.0 Hz), -67.70 (d, J = 1.1 Hz). HRMS (ESI-TOF) m/z: [M+H] + Calcd for C 18 H 13 N 2 F 6 371.0983; Found 371.0984 . Synthesis of 5 (11-methyl-2,4-bis(trifluoromethyl)-11 H -pyrido[2,3- b ] phenothiazine): Sulfur (26.87 mg, 0.8379 mmol) and Iodine (6.72 mg,0.0265 mmol) were added to a solution of 4 (142.2 mg, 0.384 mmol) in 1,2-dichlorobenzene and the solution was stirred at 190 0 C for 48 h. The crude sample was purified using silica chromatography with 30% DCM in hexanes to yield 5 as a reddish-purple solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.96 (dd, J = 9.2, 2.1 Hz, 1H), 7.80 (s, 1H), 7.33 (d, J = 9.3 Hz, 1H), 7.22 – 7.13 (m, 2H), 6.97 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 3.48 (s, 3H). 19 F NMR (471 MHz, CDCl 3 ) δ -60.38, -67.48 (d, J = 2.3 Hz). HRMS (EI-TOF) m/z:[M+H] + Calcd for C 18 H 11 F 6 N 2 S 401.05471; Found: 401.0550. Flow cytometry analysis of live mouse bone marrow cells Mouse bone marrow cells were isolated by flushing the long bones (femurs and tibias) with a needle and syringe using staining media (Ca 2+ - and Mg 2+ -free Hank’s buffered salt solution (HBSS; Gibco) supplemented with 2% heat-inactivated bovine serum (Gibco)). Cells were gently triturated and filtered through 40-μm nylon mesh to obtain a single cell suspension. Cell counts were determined using a hemocytometer and trypan blue exclusion. For experiments, 2x10 6 cells were incubated with 200µl of probe 5 in 1X PBS at 37 o C for 30 minutes. Cells were washed with 1X PBS and treated with exogenous hypochlorite for 30 minutes at 37 o C. Cells were then washed in 1X PBS, resuspended in 500µl 1x PBS, and samples analyzed by flow cytometry using an Attune NxT flow cytometer and the 512/25 emission filter (VL2 channel). Flow cytometry was analyzed using FlowJo. Graphs and statistical analysis were generated with GraphPad Prism. 4. CONCLUSIONS A simple turn-on quinoline-based fluorescent probe has been successfully synthesized, characterized, and studied for its sensitivity towards hypochlorite. Probe 5 is a good sensor for detecting hypochlorite both in and out of live cells. Declarations Supplementary Information The online version contains supplementary material available at. Competing Interests The authors declare no competing interests. Author Contribution F.F.O. carried out experiments on chemistry part. Y.H. is the supervisor on chemistry part. S.Y.S.K. carried out the biology testing. R.J.B. is the supervisor for the biology part. All authors have contributed and reviewed the manuscript. Acknowledgements This work was supported by the Meyers Endowment Fund through Southern Illinois University Fundation (F.F.O. and Y.H.) and Southern Illinois University startup fund (S.Y.S.K and R.J.B). Data Availability No datasets were generated or analyzed during the current study. References Auten, R. L.; Davis, J. M. Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details. Pediatr. Res. 2009 , 66 (2), 121–127. https://doi.org/10.1203/PDR.0b013e3181a9eafb. Lam, P.-L.; Wong, R. S.-M.; Lam, K.-H.; Hung, L.-K.; Wong, M.-M.; Yung, L.-H.; Ho, Y.-W.; Wong, W.-Y.; Hau, D. K.-P.; Gambari, R.; Chui, C.-H. The Role of Reactive Oxygen Species in the Biological Activity of Antimicrobial Agents: An Updated Mini Review. Chem. Biol. 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K.; Ryu, H.; Cui, M.; Nam, G. “Turn-On” Quinoline-Based Fluorescent Probe for Selective Imaging of Tau Aggregates in Alzheimer’s Disease: Rational Design, Synthesis, and Molecular Docking. ACS Sens. 2021 , 6 (6), 2281–2289. https://doi.org/10.1021/acssensors.1c00338. Yang, X.; Wang, Y.; Shang, Z.; Zhang, Z.; Chi, H.; Zhang, Z.; Zhang, R.; Meng, Q. Quinoline-Based Fluorescent Probe for the Detection and Monitoring of Hypochlorous Acid in a Rheumatoid Arthritis Model. RSC Adv. 2021 , 11 (50), 31656–31662. https://doi.org/10.1039/D1RA06224G. Hooshmand, S. E.; Baeiszadeh, B.; Mohammadnejad, M.; Ghasemi, R.; Darvishi, F.; Khatibi, A.; Shiri, M.; Hussain, F. H. S. Novel Probe Based on Rhodamine B and Quinoline as a Naked-Eye Colorimetric Probe for Dual Detection of Nickel and Hypochlorite Ions. Sci. Rep. 2023 , 13 (1), 17038. https://doi.org/10.1038/s41598-023-44395-x. Vedamalai, M.; Kedaria, D.; Vasita, R.; Gupta, I. Oxidation of Phenothiazine Based Fluorescent Probe for Hypochlorite and Its Application to Live Cell Imaging. Sens. Actuators B Chem. 2018 , 263 , 137–142. https://doi.org/10.1016/j.snb.2018.02.071. Schemes Schemes 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Quinolineprobesupplementaryinformation.docx Scheme1.jpg Schemes 1 Scheme2.jpg Schemes 2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6994428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487528067,"identity":"c47fb649-b478-4b12-a6c6-9df630010eec","order_by":0,"name":"Fiyinfolu F. Olubiyo","email":"","orcid":"","institution":"Southern Illinois University Carbondale","correspondingAuthor":false,"prefix":"","firstName":"Fiyinfolu","middleName":"F.","lastName":"Olubiyo","suffix":""},{"id":487528068,"identity":"7b949d4f-5d1d-49f0-b976-4d9b085cca30","order_by":1,"name":"Rebecca J. Burgess","email":"","orcid":"","institution":"Southern Illinois University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"J.","lastName":"Burgess","suffix":""},{"id":487528069,"identity":"eddfdcda-28ad-4c17-af49-9a84babed576","order_by":2,"name":"Sebastian Y. S. Klu","email":"","orcid":"","institution":"Southern Illinois University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"Y. S.","lastName":"Klu","suffix":""},{"id":487528070,"identity":"702ed2e5-ed2f-4e57-baf4-374c55ecc3e8","order_by":3,"name":"Yuqing Hou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoUlEQVRIiWNgGAWjYBACAzBZwcADoiRI0HKGZC2MbRAOcVrM2XvMJN7Oq5MxOMB88DYPMVose86YSc7ddpjH4ABbsjVRWgxu5JhJ8247ANTCYyZNgpY5dUAt/N9I0dLADLKFjUgtZ44VW845dphH8jCbseUcorQcb954401NnT3f8eaHN94QowUIWCTA7mEmUjlY7QeivDAKRsEoGAUjFwAA+ZosWSwCqNQAAAAASUVORK5CYII=","orcid":"","institution":"Southern Illinois University Carbondale","correspondingAuthor":true,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2025-06-27 21:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6994428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6994428/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87315626,"identity":"7b60fce2-e0f1-4d39-9419-16179a694c3f","added_by":"auto","created_at":"2025-07-22 15:47:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38761,"visible":true,"origin":"","legend":"\u003cp\u003eStacked \u003csup\u003e1\u003c/sup\u003eH NMR of \u003cstrong\u003e5 \u003c/strong\u003e(below) and \u003cstrong\u003e6\u003c/strong\u003e (above) in CDCl\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/6ab36f9787f7acd94e21b97e.jpg"},{"id":87316561,"identity":"933e8a41-b0d7-46ce-988b-fee9cbffe507","added_by":"auto","created_at":"2025-07-22 15:55:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48911,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Histogram showing probe \u003cstrong\u003e5 \u003c/strong\u003efluorescence in mouse bone marrow cells at 512/25 nm by flow cytometry. (b) Fluorescence detection of probe \u003cstrong\u003e5\u003c/strong\u003e fluorescence in live bone marrow cells using flow cytometry after exogenous treatment with indicated concentrations of ClO\u003csup\u003e-\u003c/sup\u003e. Each dot represents a single sample from 6 independent experiments, where each experiment was performed on cells from a single mouse. p\u0026lt;**0.01 using one-way ANOVA.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/7695aa095afb6eaf6ae4c056.jpg"},{"id":87317902,"identity":"3cd78b77-099d-401b-8f7e-61defd2d362c","added_by":"auto","created_at":"2025-07-22 16:11:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13035,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimental section.\u003c/p\u003e","description":"","filename":"Unnumber1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/3d9af91d53b931f263cf7af6.jpg"},{"id":87315635,"identity":"f22a08cf-4521-4a15-9ad2-16403f8794b7","added_by":"auto","created_at":"2025-07-22 15:47:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14451,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimentalt section.\u003c/p\u003e","description":"","filename":"Unnumber2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/add77f0a61923159bf0ca5b3.jpg"},{"id":87316564,"identity":"483cda6a-973a-4230-8c66-b570ff377157","added_by":"auto","created_at":"2025-07-22 15:55:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18497,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimentalt section.\u003c/p\u003e","description":"","filename":"Unnumber3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/abcd24db023f453bc8c4ad50.jpg"},{"id":87315637,"identity":"da4c6d0e-a62b-4387-8402-44e25d10e302","added_by":"auto","created_at":"2025-07-22 15:47:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18236,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimentalt section.\u003c/p\u003e","description":"","filename":"Unnumber4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/d8814e8551aceffc7e4962d6.jpg"},{"id":90012400,"identity":"daded0bf-81df-49b0-9248-0acf697d7377","added_by":"auto","created_at":"2025-08-27 11:02:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":799873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/edeea97b-f63c-4805-af50-87d4d274ceb2.pdf"},{"id":87315634,"identity":"ae513bbb-8375-48ed-8ac6-8dca20639b40","added_by":"auto","created_at":"2025-07-22 15:47:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2109340,"visible":true,"origin":"","legend":"","description":"","filename":"Quinolineprobesupplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/1b2dd23077cb1ea9e292794d.docx"},{"id":87316560,"identity":"66cebe2b-0716-4e98-b7c6-5220785af85c","added_by":"auto","created_at":"2025-07-22 15:55:52","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":52997,"visible":true,"origin":"","legend":"\u003cp\u003eSchemes 1\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/5c0a1f92800da4b6f55eda37.jpg"},{"id":87315633,"identity":"ceceb8c8-11b8-4cbe-9640-2b6887693d0b","added_by":"auto","created_at":"2025-07-22 15:47:52","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":48509,"visible":true,"origin":"","legend":"\u003cp\u003eSchemes 2\u003c/p\u003e","description":"","filename":"Scheme2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6994428/v1/2250cf596654d0a7d64dde93.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Turn-on Quinoline Probe for Selective Sensing of Hypochlorite in Live Cells","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eReactive oxygen species (ROS) are oxidative derivatives generated through complete or incomplete oxidation processes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and are recognized for their diverse roles in biological processes, functioning as antimicrobial agents within the immune system [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], redox signaling agents [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and regulators of hematopoietic stem cell quiescence and self-renewal [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the overproduction of these species is directly associated with oxidative stress, which can lead to a variety of negative cellular consequences due to alterations in the structure and function of biomacromolecules [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These changes, evident by oxidative stress, can lead to disease and tissue damage [9.10]. Significant advances have been made to elucidate the roles and significance of these species. Technically, accurate quantification and analysis of ROS are crucial, as these species naturally occur at low concentrations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHypochlorite is an ROS that has been extensively studied because of its stability in live cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Techniques for sensing and quantifying hypochlorite include electrochemical methods, spectrophotometry, chromatography, electron spin resonance, and chemiluminescent probes, among others [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, fluorescence analysis has become increasingly favored due to probe availability, high sensitivity, and low cytotoxicity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The physical properties of activity-based probes, such as color, quantum yield, solubility, molar absorption coefficient, Stokes' shift, specificity, and selectivity, are all critical factors that influence their applicability [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Quinoline and its derivatives have been important motifs in organic chemistry since it was discovered in the 1800s [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Because quinoline and its derivatives contribute to binding through pi-stacking or hydrophobic interactions through the aromatic ring systems, they can intercalate DNA, disrupt biological redox systems, or chelate metal ions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which leads to their applications as antimalarials [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], antioxidants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and fluorescent dyes amongst others. The quinoline scaffold has been studied extensively as fluorescent probes for the detection and measurement of metals [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], hydrogen sulfide [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], Tau aggregates [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and reactive oxidative species [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Due to the weak fluorescence of the compounds, studies on the quinoline scaffold for ROS have mostly employed addition of a fluorophore to the structure, focusing on positions 2, 3 and 4 of the motif [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. There has been little research on the benzylic motif of the compound. Here, we have extensively studied the \u003cb\u003e7-\u003c/b\u003eposition of the ring and synthesized a quinoline-phenothiazine structure for hypochlorite detection. We synthesized a turn-on probe that is not fluorescent by itself but responds selectively to hypochlorite while remaining unresponsive to other analytes. We show that this probe has high sensitivity and specificity and can detect hypochlorite in a concentration-dependent manner in live cells.\u003c/p\u003e"},{"header":"2. RESULTS AND DISCUSSION","content":"\u003cp\u003e2.1 Design and synthesis of probe \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynthesis of the precursor, \u003cstrong\u003e4\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas carried out in three simple steps from easily available starting materials (Scheme 1), and all intermediates and the target molecule were characterized using well established methods of \u0026nbsp; \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, mass spectrometry, and UV-Vis spectroscopy.\u003c/p\u003e\n\u003cp\u003eVery little solvatochromic shift was seen in absorbance of \u003cstrong\u003e4\u003c/strong\u003e (Fig S1 SI). Precursor \u003cstrong\u003e4\u0026nbsp;\u003c/strong\u003ealso\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eshowed very little fluorescence in various solvents except in PBS. (Fig S2 SI). Thus, a sulfur ring closure reaction was carried out with \u003cstrong\u003e4\u0026nbsp;\u003c/strong\u003eto make target molecule \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003e(Scheme 2). Vedamalai \u003cem\u003eet al.\u003c/em\u003e have determined the phenothiazine ring to be very susceptible to hypochlorite oxidation via the ICT process [36]. Their molecule had gone through a blue shift upon addition of the ROS\u003cstrong\u003e.\u003c/strong\u003e We hypothesized that the mechanism of action of hypochlorite on probe \u003cstrong\u003e5\u003c/strong\u003e is through the oxidation of the sulfur atom in \u003cstrong\u003e5\u003c/strong\u003e to turn into the corresponding sulfoxide, \u003cstrong\u003e6.\u003c/strong\u003e Thus, we treated \u003cstrong\u003e5\u003c/strong\u003e with sodium hypochlorite and isolated compound \u003cstrong\u003e6\u0026nbsp;\u003c/strong\u003e(Scheme 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectra of \u003cstrong\u003e5\u003c/strong\u003e and \u003cstrong\u003e6\u003c/strong\u003e were stacked (Fig 1). An obvious change in the chemical shifts of most of the protons down field shows that a strong electron-withdrawing group has been added onto the molecule. Further proof was obtained from the mass spectrometry and fluorescence results of both compounds (Fig S3 S.I and Figure S4 S.I.). Thus, \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003ewas further characterized for use of hypochlorite detection.\u003c/p\u003e\n\u003cp\u003e2.2 Response rate and selectivity of the probe\u003c/p\u003e\n\u003cp\u003eProbe \u003cstrong\u003e5\u003c/strong\u003e shows no fluorescence in various solvents, polar and non-polar, (Fig S4 SI) at concentrations as high as 50 \u0026micro;M. Specificity of the probe towards different ions and ROS was tested using the following analytes: OH\u003csup\u003e.\u003c/sup\u003e, SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, OH\u003csup\u003e-\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Sn\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Cl\u003csup\u003e-\u003c/sup\u003e, Br\u003csup\u003e-\u003c/sup\u003e, F\u003csup\u003e-\u003c/sup\u003e, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, and ClO\u003csup\u003e-\u0026nbsp;\u003c/sup\u003e(Fig S5 SI). Probe \u003cstrong\u003e5\u003c/strong\u003e only showed fluorescence response toward ClO\u003csup\u003e-\u003c/sup\u003e, with no fluorescence detected towards all other analytes tested. Upon addition of hypochlorite, an intense fluorescence peak was observed at 523 nm, which clearly shows that the probe has selectivity towards ClO\u003csup\u003e-\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sensitivity of probe \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003etowards hypochlorite was determined by fluorescence titration of ClO\u003csup\u003e-\u0026nbsp;\u003c/sup\u003eagainst the probe (Fig S6 SI). Hypochlorite concentrations between 20 \u0026micro;M and 90 \u0026micro;M were tested while the probe was kept at 5 \u0026micro;M (Fig S6a SI). Although probe \u003cstrong\u003e5\u003c/strong\u003e showed sensitivity to hypochlorite at as low as 35 \u0026micro;M, lower concentrations were below the detection limit of the fluorimeter. An increase in intensity of the peak at 523 nm proportional to the increase in molarity of hypochlorite was observed, with a steady increase in fluorescence intensity up to 70 \u0026micro;M of hypochlorite (Fig S6b SI). At higher concentrations, the florescence intensity began to decrease, although still well above that of lower hypochlorite concentrations. Furthermore, the fluorescence of the probe \u003cstrong\u003e5\u003c/strong\u003e when treated with ClO\u003csup\u003e-\u003c/sup\u003e could be visualized by the naked eye (Fig S7 SI).\u003c/p\u003e\n\u003cp\u003eTo determine the time sensitivity of the probe, a time-dependent fluorescence analysis was carried out on \u003cstrong\u003e5\u003c/strong\u003e. Probe and analyte were kept at 5 \u0026micro;M and 50 \u0026micro;M, respectively, and probe fluorescence was investigated at various times between 10 s and 20 mins. A change in intensity could be seen by 10s (Fig S8 SI). A linear relationship between intensity and time, up to 20 mins, was observed.\u003c/p\u003e\n\u003cp\u003e2.3 Detection of hypochlorite in live cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine if \u003cstrong\u003e5\u003c/strong\u003e could detect hypochlorite in live cells, mouse bone marrow cells were freshly isolated and incubated for 30 minutes with \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003eat 5 \u0026micro;M, a concentration with limited cytotoxicity to live cells (Fig S9 S.I.). Cells were washed and then treated with various concentrations of exogenous ClO\u003csup\u003e-\u003c/sup\u003e for 30 minutes. The ability of cells to take up and react with exogenous hypochlorite was detected using flow cytometry and emission at 512/25nm. Compared to cells with no treatment (Mean Fluorescence Intensity (MFI): 27.6), cells with probe \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003eexhibited\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003elittle fluorescence (MFI: 37.7) when no hypochlorite was added (Fig 2a). In contrast, upon addition of 50 \u0026micro;M ClO\u003csup\u003e-\u003c/sup\u003e, a clear shift in the cells toward high probe \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003efluorescence (MFI: 2323) was detected. About 80% of single cells showed fluorescence greater than probe \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003eonly upon addition of ClO\u003csup\u003e-\u003c/sup\u003e. To determine the sensitivity of probe \u003cstrong\u003e5\u003c/strong\u003e, live bone marrow cells were treated with various concentrations of ClO\u003csup\u003e-\u0026nbsp;\u003c/sup\u003efor thirty minutes.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe hypochlorite concentrations used were validated to induce limited cytotoxicity during the course of the experiment (data not shown). The fluorescence intensity of cells treated with \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003eincreased with increasing concentration of ClO\u003csup\u003e-\u003c/sup\u003e, suggesting that probe \u003cstrong\u003e5\u003c/strong\u003e detects ClO\u003csup\u003e-\u003c/sup\u003e in live cells in a concentration-dependent manner (Fig 2b). Thus, probe \u003cstrong\u003e5\u003c/strong\u003e enters into live bone marrow cells and can detect exogenously added ClO\u003csup\u003e-\u003c/sup\u003e.\u003c/p\u003e"},{"header":"3. EXPERIMENTAL","content":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e19\u003c/sup\u003eF NMR were recorded in CDCl\u003csub\u003e3\u003c/sub\u003e with TMS as internal standard on a Bruker 500 MHz spectrometer at 500, 125, and 471 MHz, respectively, unless otherwise specified. UV-Vis Spectroscopy was carried out on a Carey Eclipse UV-vis Spectrophotometer with a 1 cm cuvette. Fluorescence was recorded on the Carey Eclipse Spectrometer with excitation and emission slits set at 5, and scan rate 600 nm /min with a 1 cm cuvette. Experimental C57BL6/J (Jackson Laboratories, strain 000664)\u0026nbsp;mice at 7-12 weeks of age were used for live cell studies. Animals were housed in the Southern Illinois University Laboratory Animal Program facility, and animal procedures were approved by the Southern Illinois University Institutional Animal Care and Use Committee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSynthesis of \u003cstrong\u003e2\u0026nbsp;\u003c/strong\u003e(2,4-bis(trifluoromethyl)quinolin-7-amine):\u003c/p\u003e\n\u003cp\u003eTo a flask containing 40 ml chloroform, was added m-phenylenediamine (5 g, 46 mmol) and 1,1,1,5,5,5-hexafluoropentane-2,4-dione (9.6 g, 46 mmol) and the resulting solution was refluxed at 90 \u003csup\u003e0\u003c/sup\u003eC for 7 h. After completion, the solution was concentrated and \u003cstrong\u003e2\u0026nbsp;\u003c/strong\u003ewas recrystallized from ethanol as a fluffy yellow solid (12.8 g, 100 %). \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 7.81 (dd, \u003cem\u003eJ\u003c/em\u003e = 9.3, 2.3 Hz, 1H), 7.58 (s, 1H), 7.33 (dd, \u003cem\u003eJ\u003c/em\u003e = 9.2, 2.4 Hz, 1H), 7.10 (d, \u003cem\u003eJ\u003c/em\u003e = 2.3 Hz, 1H), 6.46 (s, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 152.8, 150.7, 146.6 (q, \u003cem\u003eJ\u003c/em\u003e = 34.4 Hz), 134.9 (q, \u003cem\u003eJ\u003c/em\u003e = 31.7 Hz), 124.9 – 124.2 (m), 123.9, 122.6 (d, \u003cem\u003eJ\u003c/em\u003e = 33.7 Hz), 120.4 (d, \u003cem\u003eJ\u003c/em\u003e = 33.5 Hz), 115.7, 108.4, 106.8 \u003csup\u003e19\u003c/sup\u003eF NMR (471 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ -60.53 (d, \u003cem\u003eJ\u003c/em\u003e = 2.2 Hz), -66.68. HRMS (EI-TOF) m/z:[M]\u003csup\u003e+\u003c/sup\u003e Calcd for C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e 280.04352; \u0026nbsp;Found280.04428.\u003c/p\u003e\n\u003cp\u003eTo a solution containing \u003cstrong\u003e2\u003c/strong\u003e (500 mg, 1.785 mmol) and NaOCH\u003csub\u003e3\u003c/sub\u003e (482.1 mg, 8.923 mmol) in 30 ml methanol, was added paraformaldehyde (71.42 mg, 2.230 mmol) in 5 ml methanol dropwise. The mixture was refluxed and monitored with TLC. NaBH\u003csub\u003e4\u003c/sub\u003e (67.53 mg, 1.785 mmol) was then added, and solution stirred for additional 2 h at reflux temperature. The crude solution was concentrated and washed with water twice. It was then extracted with dichloromethane and dried with anhyd sodium sulfate. The filtrate was concentrated and subject to column chromatography using 30 % dichloromethane in hexane to yield \u003cstrong\u003e3\u003c/strong\u003e as a bright yellow solid (178 mg, 52 %). \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 7.84 (dq, \u003cem\u003eJ\u003c/em\u003e = 9.3, 2.2 Hz, 1H), 7.69 (s, 1H), 7.34 (dd, \u003cem\u003eJ\u003c/em\u003e = 9.3, 2.4 Hz, 1H), 7.07 (q, \u003cem\u003eJ\u003c/em\u003e = 4.9 Hz, 1H), 6.92 (d, \u003cem\u003eJ\u003c/em\u003e = 2.4 Hz, 1H), 2.81 (d, \u003cem\u003eJ\u003c/em\u003e = 4.9 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 152.8, 151.2, 147.7 – 145.8 (m), 134.8 (d, \u003cem\u003eJ\u003c/em\u003e = 31.7 Hz), 124.7, 124.2 (d, \u003cem\u003eJ\u003c/em\u003e = 17.2 Hz), 122.7 (d, \u003cem\u003eJ\u003c/em\u003e = 35.9 Hz), 120.6, 116.1, 108.7, 102.8. \u003csup\u003e19\u003c/sup\u003eF NMR (471 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ -60.11 (d, \u003cem\u003eJ\u003c/em\u003e = 2.2 Hz), -66.32. HRMS(EI-TOF) m/z:[M]\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCalcd for C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e294.05917; Found 294.05995.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSynthesis of \u003cstrong\u003e4\u003c/strong\u003e (\u003cem\u003eN\u003c/em\u003e-methyl-2,4-bis(trifluoromethyl)quinolin-7-amine-\u003cem\u003eN\u003c/em\u003e-methyl-\u003cem\u003eN\u003c/em\u003e-phenyl-2,4-bis(trifluoromethyl)quinolin-7-amine):\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSynthesis of \u003cstrong\u003e3\u003c/strong\u003e (\u003cem\u003eN\u003c/em\u003e-methyl-2,4-bis(trifluoromethyl)quinolin-7-amine):\u003c/p\u003e\n\u003cp\u003e20 ml toluene was degassed for 15 mins under argon. \u003cstrong\u003e3\u0026nbsp;\u003c/strong\u003e(\u003cem\u003eN\u003c/em\u003e-methyl-2,4-bis(trifluoromethyl) quinolin-7-amine) (300 mg, 1.0196 mmol), bromobenzene (320mg, 2.04 mmol), palladium acetate (84 mg, 0.3732 mmol), BINAP (120 mg, 0.192 mmol) and NaO-t-Bu (1.568 g, 16.3136 mmol) was added, and the solution was stirred for 48 h at 110 \u003csup\u003e0\u003c/sup\u003eC and the reaction was monitored with TLC. After completion, the solution was filtered and the filtrate concentrated. The filtrate was then subject to column chromatography to yield product as a solid (304.8 mg, 80.7 %).\u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 7.88 (dt, \u003cem\u003eJ\u003c/em\u003e = 9.0, 1.7 Hz, 1H), 7.69 (s, 1H), 7.47 (d, \u003cem\u003eJ\u003c/em\u003e = 2.6 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.30 (dd, \u003cem\u003eJ\u003c/em\u003e = 2.4, 1.1 Hz, 1H), 7.26 (ddt), \u003cem\u003eJ\u003c/em\u003e = 7.5, 6.4, 1.2 Hz, 3H), 3.46 (s, 3H). \u003csup\u003e19\u003c/sup\u003eF NMR (471 MHz CDCl\u003csub\u003e3\u003c/sub\u003e) δ -61.32 (d, \u003cem\u003eJ\u003c/em\u003e = 2.0 Hz), -67.70 (d, \u003cem\u003eJ\u003c/em\u003e = 1.1 Hz). HRMS (ESI-TOF) m/z: [M+H]\u003csup\u003e+\u003c/sup\u003e Calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003e 371.0983; Found 371.0984\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSynthesis of \u003cstrong\u003e5\u003c/strong\u003e (11-methyl-2,4-bis(trifluoromethyl)-11\u003cem\u003eH\u003c/em\u003e-pyrido[2,3-\u003cem\u003eb\u003c/em\u003e] phenothiazine):\u003c/p\u003e\n\u003cp\u003eSulfur (26.87 mg, 0.8379 mmol) and Iodine (6.72 mg,0.0265 mmol) were added to a solution of \u003cstrong\u003e4\u0026nbsp;\u003c/strong\u003e(142.2 mg, 0.384 mmol) in 1,2-dichlorobenzene and the solution was stirred at 190 \u003csup\u003e0\u003c/sup\u003eC for 48 h. The crude sample was purified using silica chromatography with 30% DCM in hexanes to yield \u003cstrong\u003e5\u003c/strong\u003e as a reddish-purple solid. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 7.96 (dd, \u003cem\u003eJ\u003c/em\u003e = 9.2, 2.1 Hz, 1H), 7.80 (s, 1H), 7.33 (d, \u003cem\u003eJ\u003c/em\u003e = 9.3 Hz, 1H), 7.22 – 7.13 (m, 2H), 6.97 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 1H), 6.81 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 3.48 (s, 3H). \u003csup\u003e19\u003c/sup\u003eF NMR (471 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ -60.38, -67.48 (d, \u003cem\u003eJ\u003c/em\u003e = 2.3 Hz). HRMS (EI-TOF) m/z:[M+H]\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCalcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eS 401.05471; Found: 401.0550.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis of live mouse bone marrow cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse bone marrow cells were isolated by flushing the long bones (femurs and tibias) with a needle and syringe using staining media (Ca\u003csup\u003e2+\u003c/sup\u003e- and Mg\u003csup\u003e2+\u003c/sup\u003e-free Hank’s buffered salt solution (HBSS; Gibco) supplemented with 2% heat-inactivated bovine serum (Gibco)). Cells were gently triturated and filtered through 40-μm nylon mesh to obtain a single cell suspension. Cell counts were determined using a hemocytometer and trypan blue exclusion. For experiments, 2x10\u003csup\u003e6\u003c/sup\u003e cells were incubated with 200µl of probe \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003ein 1X PBS at 37 \u003csup\u003eo\u003c/sup\u003eC for 30 minutes. Cells were washed with 1X PBS and treated with exogenous hypochlorite for 30 minutes at 37 \u003csup\u003eo\u003c/sup\u003eC. Cells were then washed in 1X PBS, resuspended in 500µl 1x PBS, and samples analyzed by flow cytometry using an Attune NxT flow cytometer and the 512/25 emission filter (VL2 channel). Flow cytometry was analyzed using FlowJo. Graphs and statistical analysis were generated with GraphPad Prism.\u0026nbsp;\u003c/p\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eA simple turn-on quinoline-based fluorescent probe has been successfully synthesized, characterized, and studied for its sensitivity towards hypochlorite. Probe \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003eis a good sensor for detecting hypochlorite both in and out of live cells.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eSupplementary Information\u003c/h2\u003e\n\u003cp\u003eThe online version contains supplementary material available at.\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eF.F.O. carried out experiments on chemistry part. Y.H. is the supervisor on chemistry part. S.Y.S.K. carried out the biology testing. R.J.B. is the supervisor for the biology part. All authors have contributed and reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Meyers Endowment Fund through Southern Illinois University Fundation (F.F.O. and Y.H.) and Southern Illinois University startup fund (S.Y.S.K and R.J.B).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAuten, R. L.; Davis, J. M. Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details. \u003cem\u003ePediatr. Res.\u003c/em\u003e \u003cstrong\u003e2009\u003c/strong\u003e, \u003cem\u003e66\u003c/em\u003e (2), 121\u0026ndash;127. https://doi.org/10.1203/PDR.0b013e3181a9eafb.\u003c/li\u003e\n\u003cli\u003eLam, P.-L.; Wong, R. S.-M.; Lam, K.-H.; Hung, L.-K.; Wong, M.-M.; Yung, L.-H.; Ho, Y.-W.; Wong, W.-Y.; Hau, D. 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Actuators B Chem.\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e263\u003c/em\u003e, 137\u0026ndash;142. https://doi.org/10.1016/j.snb.2018.02.071.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fluorescence, chemical sensors, organic synthesis, hypochlorite","lastPublishedDoi":"10.21203/rs.3.rs-6994428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6994428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypochlorite is a significant reactive oxygen species (ROS) involved in various biological processes. Excessive levels of hypochlorite are linked to oxidative stress, which contributes to a number of diseases by altering the structure and function of biomacromolecules. Many existing probes for detecting hypochlorite have relatively high molecular weights and typically exhibit changes in emission wavelength upon interaction with the ROS. We have synthesized a quinoline-based probe, \u003cb\u003e5\u003c/b\u003e, which is not fluorescent on its own, but becomes fluorescent at 523 nm upon reaction with hypochlorite through an oxidative mechanism. This probe exhibits time sensitivity as low as 10 seconds outside of cells. Probe fluorescence is visible to the naked eye, with intensity increasing with ROS concentration. Furthermore, probe fluorescence is detected in cells in a concentration-dependent manner. Altogether, we describe the generation and use of a quinoline-based probe to detect hypochlorite ROS in live cells.\u003c/p\u003e","manuscriptTitle":"Turn-on Quinoline Probe for Selective Sensing of Hypochlorite in Live Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 15:47:47","doi":"10.21203/rs.3.rs-6994428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6473d281-ad2f-45fe-9eb6-ade0c5b18559","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T12:54:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 15:47:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6994428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6994428","identity":"rs-6994428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}