A coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor for detection of hypochlorite ions in aqueous medium

A coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor for detection of hypochlorite ions in aqueous medium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor for detection of hypochlorite ions in aqueous medium Aruna Aruna, Kanchan Kanchan, Arunava Agarwala, Rahul Shrivastava This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7954085/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2026 Read the published version in Journal of Fluorescence → Version 1 posted 15 You are reading this latest preprint version Abstract A coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin chemosensor (CTH) was successfully designed and synthesized for detection of hypochlorite ion, an important reactive oxygen species (ROS) in aqueous medium. The chemosensor (CTH) exhibits significant selectivity for perchlorate ion over other ROSs with fluorescence “turn-on” response within few seconds. Moreover, emission titration experiments results revealed that reported sensor detect perchlorate ion with 16 nM detection limit. Further, mass spectrometry analysis indicated that oxidizable 2-methyl-sulphur arms of sensor CTH is responsible for detection of perchlorate ion. Coumarin perchlorate ion chemosensor detection drinking water treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The development of sensitive and selective recognition for biologically important analytical species is a central goal in fluorescence and optical chemosensing. Biocompatibility in terms of solubility and non-toxicity, low detection limit (sensitivity) and selectivity continue to be keen objectives in this research area. In view of this, determination and quantification of reactive oxygen species are exceptionally valuable in biological research since concentrations of these species such as perchlorate ion, singlet oxygen hydroxyl ( ∙ OH), superoxide (O 2− ) and lipid hydroperoxide (ROOH) play crucial roles both in disease and health [ 1 – 4 ]. Hypochlorous acid (HOCl)/hypochlorite anion is a biologically valuable reactive oxygen species (ROS) in living organisms, generated enzymatically by reaction of hydrogen peroxide and chloride ion under catalysis of heme-containing enzyme myeloperoxidase (MPO) localized in leukocytes, including macrophages, monocytes and neutrophils [ 5 – 6 ]. Mounting evidence supports that hypochlorite ion performs pivotal functions in immune defense against inflammation and microorganisms [ 7 ]. Additionally, hypochlorite ions are employed in various types of industries, for example used as bleaching agent in textile industry, pulp/paper mill, utilized to remove slime and algae in tubes and piping, used in deodorant, mild disinfectant treatment of potable water and cleaning agents in treatment of industrial wastewater. Importantly, maintaining hypochlorite ions level within physiological range is utmost necessary for several cellular functions. Nevertheless, an irregular concentration of hypochlorite ions could lead to oxidative stress and damage tissues, causing numerous diseases including nephropathy, inflammatory disease, atherosclerosis, cardiovascular disease, kidney disease, neuronal degeneration etc [ 8 – 12 ]. Considering these hazardous health impacts, it is pivotal to design selective chemosensor for measure the concentration of HOCl/ClO − with high sensitivity in biological samples and aqueous medium [ 13 ]. The fluorescent molecular sensor molecules enjoy native advantages over other types of chemosensors because of their high-level sensitivity, specificity, fast response times and excellent visualization, offering attractive methods for determination of anions/cations/substrates [ 14 – 15 ]. So far, number of small fluorescent chemosensors have been developed for detection of HOCl/OCl − ions. These fluorescent chemosensors are usually derived from fluorescent fluorophores like rhodamine, BODIPY, dicyanomethylene-4H-pyran, triphenylamine, cyanine and coumarins [ 16 – 17 ]. However, it was noticed that some of these sensors are associated with limitations like pH dependency, low sensitivity and poor water-selectivity. Therefore, it is necessary to design and synthesize attractive and innovative fluorescent chemosensors that can overrule these limitations in HOCl/OCl − detection [ 18 ]. After careful investigation of various fluorophores, coumarin molecule appears to be ideal framework for fabrication of “turn-on” fluorescent chemosensor due to its specific suitable properties and inherent advantages such as easy synthesis and functionalization, excitation and emission signals in visible region, convenient source, low price, low toxicity, excellent biocompatibility and extraordinary fluorescence performance [ 19 ]. Considering these advantages, we envisaged to utilize coumarin derivative for construction of potential molecular chemosensor for detection of reactive oxygen species (ROS) specially hypochlorite ion. In this research article, we synthesized coumarin-based chemosensor 2-methylthio-4-hydroxypyrimidine[3,4-b] coumarin ( CTH ) bearing 2-methyl-sulphur arms which may respond to hypochlorite anion for sensitive detection. Our detection strategy depends upon the substitution of oxidizable 2-methyl thioether functional group on suitable position of pyrimidine-coumarin derivative and powerful oxidizing ability of hypochlorite ion [ 20 – 21 ]. The 2-methyl-S arms allow for hypochlorite oxidation and result in the formation of oxidized product of 2-methyl-S arms which results in drastic enhancement in fluorescence response [ 22 ]. To the best of our knowledge, no report of fluorescent molecular sensor using 2-methyl thioether flanked pyrimidine-coumarin moiety was reported that could sense hypochlorite ion in aqueous media. Experimental and materials 2.1. Materials and measurements Most of chemicals and solvents were obtained from Sigma-Aldrich and Spectrochem pvt. India and used without further purification. All carbon and proton NMR spectra of synthesized organic compounds and intermediates were recorded in deuterated solvent using Advance II-400 MHz instrument. The UV-visible and fluorescence measurements were obtained by using Pharmaspec UV–1800 (Shimadzu) and Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon, NJ, USA) respectively. The mass spectrum of synthesized compounds was obtained by using Water-Q-Tof micromass mass spectrometer. Result and discussion Synthesis of coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor (CTH): The fluorescent chemosensor 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin ( CTH ) was synthesized from three step procedure. In the first step, equimolar mixture of diethyl malonate ( 1 ) and salicylaldehyde ( 2 ) was refluxed in presence of piperidine for 4.0 hours which afforded corresponding 3-ethoxycarbonylcoumarin ( 3 ) in 93% yield. In the next step, synthesized 3-ethoxycarbonylcoumarin ( 3 ) was reacted with thiourea in methanol in presence of catalytic amount of anhydrous potassium carbonate under refluxing conditions and produced potassium salt of 2-thioxo-4-hydroxypyrimidine[3,4-b]coumarin ( 4 ) in 88% yield. Further, mixture of potassium salt of 2-thioxo-4-hydroxypyrimidine[3,4-b]coumarin ( 4 ), methyl iodide and anhydrous sodium acetate was refluxed in methanol for 10.0 hours afforded targeted 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin ( CTH ) in 90% yield as depicted in scheme 1 . The structure of 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin ( CTH ) was characterized by proton, carbon NMR and mass spectrometry analysis. Absorbance and fluorescence studies of synthesized coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) for sensing of perchlorate ion The ultraviolet absorption measurement of 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) was investigated in PBS buffered solution (DMSO 5% v/v) at pH 7.4 on Pharmaspec UV–1800 (Shimadzu) spectrophotometer. The analysis of ultraviolet absorption spectrum of synthesized chemosensor CTH displayed absorption band at 322 nm with shoulder peak of 348 nm as shown in Fig. 1 . The critical analysis of interaction study with perchlorate ions with CTH revealed that ultraviolet absorption band at 322 nm was red shifted to 302 nm with development of new band 354 nm. It was found that interaction of sensor CTH with concentration gradients of perchlorate ion, absorption bands at 322 nm exhibited decreasing trend whereas peak at 354 nm gradually strengthened. This shift of absorption peak was reflected in color change of chemosensor CTH solution from pale yellow to colourless which was easily noticeable under visible light. The obtained results clearly suggest that sensor CTH is capable to detect hypochlorite ion in PBS buffered solution (5% v/v DMSO) at pH 7.4 through both colorimetric and absorption spectroscopy. To evaluate designed coumarin derived molecular chemosensor CTH for selective sensing for hypochlorite ions, fluorescence spectra of CTH with other reactive oxygen species (such as NaOCl, H 2 O 2 , KO 2 , C 6 H 5 CO 3 H, ∙OH, t -Bu-OOH) were recorded at pH 7.4 in PBS buffered solution (5% v/v DMSO). As depicted in Fig. 2 a., synthesized chemosensor CTH (3.0×10 − 5 M in PBS buffer) displayed fluorescence response at 358 nm when excited at 280 nm. Interestingly, it was found that interaction of CTH with one equivalent amount of sodium hypochlorite, new emission peak at 459 evolved along with peak at 358 nm with change in colour of CTH solution from yellow to colourless. However, none of other reactive oxygen species like H 2 O 2 , KO 2 , C 6 H 5 CO 3 H, ∙OH, t -Bu-OOH produced such type of change in fluorescence responses of CTH in process of detecting of hypochlorite ion (Fig. 2 b). Further, to get more insight about interaction of hypochlorite with synthesized sensor CTH, fluorescence titration studies was performed by sequential addition of hypochlorite ion from 1.1×10 − 4 M stock solution of hypochlorite ion solution into 3.0×10 − 5 M solution of CTH solution at pH 7.4. It was clearly noticed from titration experiment that emission intensity of sensor CTH at 459 nm (λ ex = 280 nm) was enhanced progressively upon increasing hypochlorite ion concentrations as depicted in Fig. 3 . Moreover, linear relationship was recorded between intensity at 459 nm of CTH and hypochlorite ion concentration. A linear relationship was obtained with linear equation of y = 6 10 x (R 2 = 0.9956). The 16 nM limit of detection was determined from linear relationship results using equation: LOD = 3σ/S; where S represents slope of equation and σ is relative standard deviation of 10 measurements of sensor CTH solution. These results clearly revealed that this reported sensor CTH can be effectively employed in quantitative measurement of perchlorate ion with an outstanding selectivity as well as sensitivity. Effect of pH and response time towards detection of perchlorate ion: The effect of pH on detection process of synthesized sensor CTH for hypochlorite ion was thoroughly examined via fluorescence measurements. The observed results demonstrate that synthesized sensor CTH is not pH sensitive and worked over wide range of pH. As depicted in Fig. 4 , emission intensity of sensor CTH at 459 nm remained stable value over pH range from 4 to 10 when pH was adjusted using 1.0 M HCl or 1.0 M NaOH in CTH solution. This result of studies strongly suggests that synthesized sensor CTH can efficiently perform under physiological pH conditions and thus has exceptional potential for detection of perchlorate ion in biological applications. To monitor respond time of synthesized sensor CTH for detection of hypochlorite ion, change in intensity of emission peak with time upon addition of perchlorate ion was recorded at pH 7.4. The assessment of time-dependent fluorescence intensity showed that synthesized sensor CTH responded presence of hypochlorite in very short time. As depicted in Fig. 5 , interaction of perchlorate ion with 30 µM solution of CTH, emission intensity at 459 nm was dramatically enhanced and approached maximum stable value in about few seconds. To ensure completion of reaction between CTH and perchlorate ion, fluorescence spectra were collected for 15 minutes in 1.0 minute time interval. It was found intensity of peak at 459 was immediately enhance to its maximum limit and after that no significant enhancement in fluorescence intensity at 459 nm was recorded even after one minutes. The observed quick response time exhibits that CTH can monitor perchlorate ion with real-time detection capability which is important aspect for day-to-day life applications. : Detection mechanism of sensor CTH towards perchlorate ion : To investigate sensing mechanism of synthesized sensor CTH towards hypochlorite ion, mass spectrometry analysis was carried out in PBS buffer solution containing 5% DMSO at pH 7.4. The mass spectra of sensor CTH, before and after interaction of NaOCl was recorded in similar solvent system. The mass spectrometry analysis exhibited that after interaction with hypochlorite ion, peak of sensor CTH at m/z = 261.04 disappeared whereas peak at m/z = 276.89 appeared which is similar to exact mass of sulfoxide derivative of sensor CTH. The information received from mass spectrometry clearly indicated that hypochlorite oxidized thioethers group of chemosensor CTH to sulfoxide group at much faster rates within the range of second as depicted in Fig. 6 . Application of synthesized sensor CTH for perchlorate ion detection: To monitor the sensing application of synthesized sensor CTH in real samples, hypochlorite ion (ClO-) detection was monitored in various spiked tap water samples using standard addition method. The recovery studies of sensor CTH were performed by spiking perchlorate ion to drinking water with perchlorate ion concentrations below the determined detection limit. The detection performance analysis was carried out by spiking 25.0 nM, 35.0 nM and 45.0 nM of perchlorate ion and percentage recovery was determined as depicted in Table 1 . The obtained results revealed that recoveries of hypochlorite ion in spiked tap water samples were in the range of 95.8% − 98.08% indicating capability of CTH for accurate determination of perchlorate ion in contaminated water sample Table 1 Percentage recovery of perchlorate ion in spiked water samples Sample Spiked NaOCl (nM) Percentage recovery (%) Sample-A1 25 nM 95.8% Sample-A2 35 nM 97.2% Sample-A3 45 nM 98.0% Conclusion In conclusion, molecular chemosensor 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) was designed and developed for detection of reactive oxygen species. The absorption and emission studies exhibited that developed chemosensor (CTH) can sensitively detect perchlorate ion in PBS buffered solution (DMSO 5% v/v) at pH 7.4. The emission spectrum exhibited significant enhancement in fluorescence response at 459 nm when excited at 280 nm. The detection limit was also calculated using emission titration data and was found to be 16 nM which is much lower than recommended limit. Moreover, response time was also evaluated and results revealed sensor CTH detect perchlorate ion within few second whereas pH studies strongly suggests that sensor can efficiently perform under pH range of 4 to 10. Furthermore, information received from mass spectrometry clearly indicated that sensor CTH detect hypochlorite ion through oxidization of thioethers group of chemosensor CTH to sulfoxide group at much faster rates. Declarations Funding Declaration There was no funding available. Author Contribution Aruna and Kanchan wrote the main manuscript text and A. A and R.S. prepared figures and supervised. All authors reviewed the manuscript. Acknowledgement Aruna and R. S are obliged to Manipal University Jaipur for facilitating resources infrastructures. We also would like to thankful to Central Analytical Facilities of Manipal University Jaipur for supporting instruments for Uv-visible, FT-IR and fluorescence spectroscopy References de Almeida AJPO, de Oliveira JCPL, da Silva Pontes LV, de Souza Junior JF, Goncalves TAF, Dantas SH, de Feitosa A, Silva MS, de Medeiros AO (2022) IA, Oxid Med. 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Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2026 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 28 Nov, 2025 Reviews received at journal 26 Nov, 2025 Reviews received at journal 20 Nov, 2025 Reviews received at journal 19 Nov, 2025 Reviews received at journal 15 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviews received at journal 07 Nov, 2025 Reviewers agreed at journal 02 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers invited by journal 31 Oct, 2025 Submission checks completed at journal 31 Oct, 2025 Editor assigned by journal 31 Oct, 2025 First submitted to journal 27 Oct, 2025 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. 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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-7954085","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":543591554,"identity":"56f4635d-5bc1-4645-bfe1-a240c4ebf8e2","order_by":0,"name":"Aruna Aruna","email":"","orcid":"","institution":"Manipal University Jaipur","correspondingAuthor":false,"prefix":"","firstName":"Aruna","middleName":"","lastName":"Aruna","suffix":""},{"id":543591555,"identity":"4041c0b6-7e14-4695-bff3-a3100e397a90","order_by":1,"name":"Kanchan Kanchan","email":"","orcid":"","institution":"Manipal University 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08:23:47","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55771,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/75619e048a81660f3aac8f64.html"},{"id":95732831,"identity":"5c971608-d864-4a60-853c-d32355350445","added_by":"auto","created_at":"2025-11-12 12:06:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26395,"visible":true,"origin":"","legend":"\u003cp\u003eUV-visible of coumarin based 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) for sensing of perchlorate ion\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/85099b030049a5e8f26703e5.png"},{"id":95801783,"identity":"325b8c15-616c-43f0-b6fd-ffab9d589312","added_by":"auto","created_at":"2025-11-13 08:26:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48961,"visible":true,"origin":"","legend":"\u003cp\u003eThe emission spectrum of CTH (3.0×10\u003csup\u003e-5\u003c/sup\u003e M) with sodium hypochlorite in PBS buffer solution containing 5.0% DMSO at pH 7.4. \u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/11d75a0f118516119dc462c5.png"},{"id":95732835,"identity":"5da47712-da3a-4aa8-8122-106c1c8a3d75","added_by":"auto","created_at":"2025-11-12 12:06:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66349,"visible":true,"origin":"","legend":"\u003cp\u003eThe emission spectrum of sensor CTH (3.0×10\u003csup\u003e-5\u003c/sup\u003e M) with variable concentration of sodium hypochlorite in PBS buffer solution containing 5% DMSO at pH 7.4. (λex=280 nm; λ em= 459 nm)\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/4091bb5b8ff79cfe125969d8.png"},{"id":95732842,"identity":"fe250030-c56f-4caa-a4d9-32c498262324","added_by":"auto","created_at":"2025-11-12 12:06:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18961,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of pH on intensity of sensor CTH on interaction of perchlorate ion at room temperature.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/42681cf2790e8762d8206da8.png"},{"id":95732847,"identity":"ba14565a-3b3d-4f39-9c7f-407d9108a806","added_by":"auto","created_at":"2025-11-12 12:06:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11027,"visible":true,"origin":"","legend":"\u003cp\u003eThe change of fluorescence intensity at 459 nm of probe (30 μM) and probe treated with equimolar amount of perchlorate ion with response time.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/2d2c43782da5666702733312.png"},{"id":95732839,"identity":"8e88ce77-9f02-40f1-b1b0-68018311c82c","added_by":"auto","created_at":"2025-11-12 12:06:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18715,"visible":true,"origin":"","legend":"\u003cp\u003eSensing mechanism of NaoCl sensing by CTH and mass spectra of CTH after treatment with NaOCl\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/b712ce019265b3533f732f63.png"},{"id":103766857,"identity":"15beacf3-fdae-4966-b620-30513af679a8","added_by":"auto","created_at":"2026-03-02 16:16:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":903289,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/a5dd8eab-aa86-4134-ba88-f658ad4da3b5.pdf"},{"id":95732836,"identity":"0d657de1-274b-434b-a082-ff6196753750","added_by":"auto","created_at":"2025-11-12 12:06:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":340196,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/5b77e72db6a3ad7c4c299ca6.docx"},{"id":95732833,"identity":"7c3f4cfc-8647-4172-8311-2219308aafb4","added_by":"auto","created_at":"2025-11-12 12:06:46","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e. Synthesis of coumarin based 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor (CTH).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7954085/v1/4161b649db2f574a7fc15827.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"A coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor for detection of hypochlorite ions in aqueous medium","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe development of sensitive and selective recognition for biologically important analytical species is a central goal in fluorescence and optical chemosensing. Biocompatibility in terms of solubility and non-toxicity, low detection limit (sensitivity) and selectivity continue to be keen objectives in this research area. In view of this, determination and quantification of reactive oxygen species are exceptionally valuable in biological research since concentrations of these species such as perchlorate ion, singlet oxygen hydroxyl (\u003csup\u003e∙\u003c/sup\u003eOH), superoxide (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e) and lipid hydroperoxide (ROOH) play crucial roles both in disease and health [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Hypochlorous acid (HOCl)/hypochlorite anion is a biologically valuable reactive oxygen species (ROS) in living organisms, generated enzymatically by reaction of hydrogen peroxide and chloride ion under catalysis of heme-containing enzyme myeloperoxidase (MPO) localized in leukocytes, including macrophages, monocytes and neutrophils [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mounting evidence supports that hypochlorite ion performs pivotal functions in immune defense against inflammation and microorganisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, hypochlorite ions are employed in various types of industries, for example used as bleaching agent in textile industry, pulp/paper mill, utilized to remove slime and algae in tubes and piping, used in deodorant, mild disinfectant treatment of potable water and cleaning agents in treatment of industrial wastewater. Importantly, maintaining hypochlorite ions level within physiological range is utmost necessary for several cellular functions. Nevertheless, an irregular concentration of hypochlorite ions could lead to oxidative stress and damage tissues, causing numerous diseases including nephropathy, inflammatory disease, atherosclerosis, cardiovascular disease, kidney disease, neuronal degeneration \u003cem\u003eetc\u003c/em\u003e [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Considering these hazardous health impacts, it is pivotal to design selective chemosensor for measure the concentration of HOCl/ClO\u003csup\u003e\u0026minus;\u003c/sup\u003e with high sensitivity in biological samples and aqueous medium [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The fluorescent molecular sensor molecules enjoy native advantages over other types of chemosensors because of their high-level sensitivity, specificity, fast response times and excellent visualization, offering attractive methods for determination of anions/cations/substrates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. So far, number of small fluorescent chemosensors have been developed for detection of HOCl/OCl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions. These fluorescent chemosensors are usually derived from fluorescent fluorophores like rhodamine, BODIPY, dicyanomethylene-4H-pyran, triphenylamine, cyanine and coumarins [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, it was noticed that some of these sensors are associated with limitations like pH dependency, low sensitivity and poor water-selectivity. Therefore, it is necessary to design and synthesize attractive and innovative fluorescent chemosensors that can overrule these limitations in HOCl/OCl\u003csup\u003e\u0026minus;\u003c/sup\u003e detection [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After careful investigation of various fluorophores, coumarin molecule appears to be ideal framework for fabrication of \u0026ldquo;turn-on\u0026rdquo; fluorescent chemosensor due to its specific suitable properties and inherent advantages such as easy synthesis and functionalization, excitation and emission signals in visible region, convenient source, low price, low toxicity, excellent biocompatibility and extraordinary fluorescence performance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Considering these advantages, we envisaged to utilize coumarin derivative for construction of potential molecular chemosensor for detection of reactive oxygen species (ROS) specially hypochlorite ion. In this research article, we synthesized coumarin-based chemosensor 2-methylthio-4-hydroxypyrimidine[3,4-b] coumarin (\u003cb\u003eCTH\u003c/b\u003e) bearing 2-methyl-sulphur arms which may respond to hypochlorite anion for sensitive detection. Our detection strategy depends upon the substitution of oxidizable 2-methyl thioether functional group on suitable position of pyrimidine-coumarin derivative and powerful oxidizing ability of hypochlorite ion [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The 2-methyl-S arms allow for hypochlorite oxidation and result in the formation of oxidized product of 2-methyl-S arms which results in drastic enhancement in fluorescence response [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To the best of our knowledge, no report of fluorescent molecular sensor using 2-methyl thioether flanked pyrimidine-coumarin moiety was reported that could sense hypochlorite ion in aqueous media.\u003c/p\u003e"},{"header":"Experimental and materials","content":"\u003cli\u003e\u003cp\u003e2.1. \u003cb\u003eMaterials and measurements\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eMost of chemicals and solvents were obtained from Sigma-Aldrich and Spectrochem pvt. India and used without further purification. All carbon and proton NMR spectra of synthesized organic compounds and intermediates were recorded in deuterated solvent using Advance II-400 MHz instrument. The UV-visible and fluorescence measurements were obtained by using Pharmaspec UV\u0026ndash;1800 (Shimadzu) and Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon, NJ, USA) respectively. The mass spectrum of synthesized compounds was obtained by using Water-Q-Tof micromass mass spectrometer.\u003c/p\u003e"},{"header":"Result and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor (CTH):\u003c/h2\u003e\u003cp\u003eThe fluorescent chemosensor 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (\u003cb\u003eCTH\u003c/b\u003e) was synthesized from three step procedure. In the first step, equimolar mixture of diethyl malonate (\u003cb\u003e1\u003c/b\u003e) and salicylaldehyde (\u003cb\u003e2\u003c/b\u003e) was refluxed in presence of piperidine for 4.0 hours which afforded corresponding 3-ethoxycarbonylcoumarin (\u003cb\u003e3\u003c/b\u003e) in 93% yield. In the next step, synthesized 3-ethoxycarbonylcoumarin (\u003cb\u003e3\u003c/b\u003e) was reacted with thiourea in methanol in presence of catalytic amount of anhydrous potassium carbonate under refluxing conditions and produced potassium salt of 2-thioxo-4-hydroxypyrimidine[3,4-b]coumarin (\u003cb\u003e4\u003c/b\u003e) in 88% yield. Further, mixture of potassium salt of 2-thioxo-4-hydroxypyrimidine[3,4-b]coumarin (\u003cb\u003e4\u003c/b\u003e), methyl iodide and anhydrous sodium acetate was refluxed in methanol for 10.0 hours afforded targeted 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (\u003cb\u003eCTH\u003c/b\u003e) in 90% yield as depicted in scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The structure of 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (\u003cb\u003eCTH\u003c/b\u003e) was characterized by proton, carbon NMR and mass spectrometry analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAbsorbance and fluorescence studies of synthesized coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) for sensing of perchlorate ion\u003c/h3\u003e\n\u003cp\u003eThe ultraviolet absorption measurement of 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) was investigated in PBS buffered solution (DMSO 5% v/v) at pH 7.4 on Pharmaspec UV\u0026ndash;1800 (Shimadzu) spectrophotometer. The analysis of ultraviolet absorption spectrum of synthesized chemosensor CTH displayed absorption band at 322 nm with shoulder peak of 348 nm as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The critical analysis of interaction study with perchlorate ions with CTH revealed that ultraviolet absorption band at 322 nm was red shifted to 302 nm with development of new band 354 nm. It was found that interaction of sensor CTH with concentration gradients of perchlorate ion, absorption bands at 322 nm exhibited decreasing trend whereas peak at 354 nm gradually strengthened. This shift of absorption peak was reflected in color change of chemosensor CTH solution from pale yellow to colourless which was easily noticeable under visible light. The obtained results clearly suggest that sensor CTH is capable to detect hypochlorite ion in PBS buffered solution (5% v/v DMSO) at pH 7.4 through both colorimetric and absorption spectroscopy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate designed coumarin derived molecular chemosensor CTH for selective sensing for hypochlorite ions, fluorescence spectra of CTH with other reactive oxygen species (such as NaOCl, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, KO\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003eH, ∙OH, \u003cem\u003et\u003c/em\u003e-Bu-OOH) were recorded at pH 7.4 in PBS buffered solution (5% v/v DMSO). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea., synthesized chemosensor CTH (3.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M in PBS buffer) displayed fluorescence response at 358 nm when excited at 280 nm. Interestingly, it was found that interaction of CTH with one equivalent amount of sodium hypochlorite, new emission peak at 459 evolved along with peak at 358 nm with change in colour of CTH solution from yellow to colourless. However, none of other reactive oxygen species like H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, KO\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003eH, ∙OH, \u003cem\u003et\u003c/em\u003e-Bu-OOH produced such type of change in fluorescence responses of CTH in process of detecting of hypochlorite ion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther, to get more insight about interaction of hypochlorite with synthesized sensor CTH, fluorescence titration studies was performed by sequential addition of hypochlorite ion from 1.1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M stock solution of hypochlorite ion solution into 3.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M solution of CTH solution at pH 7.4. It was clearly noticed from titration experiment that emission intensity of sensor CTH at 459 nm (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;280 nm) was enhanced progressively upon increasing hypochlorite ion concentrations as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Moreover, linear relationship was recorded between intensity at 459 nm of CTH and hypochlorite ion concentration. A linear relationship was obtained with linear equation of y\u0026thinsp;=\u0026thinsp;6\u003csup\u003e10\u003c/sup\u003ex (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9956). The 16 nM limit of detection was determined from linear relationship results using equation: LOD\u0026thinsp;=\u0026thinsp;3σ/S; where S represents slope of equation and σ is relative standard deviation of 10 measurements of sensor CTH solution. These results clearly revealed that this reported sensor CTH can be effectively employed in quantitative measurement of perchlorate ion with an outstanding selectivity as well as sensitivity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffect of pH and response time towards detection of perchlorate ion:\u003c/h3\u003e\n\u003cp\u003eThe effect of pH on detection process of synthesized sensor CTH for hypochlorite ion was thoroughly examined \u003cem\u003evia\u003c/em\u003e fluorescence measurements. The observed results demonstrate that synthesized sensor CTH is not pH sensitive and worked over wide range of pH. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, emission intensity of sensor CTH at 459 nm remained stable value over pH range from 4 to 10 when pH was adjusted using 1.0 M HCl or 1.0 M NaOH in CTH solution. This result of studies strongly suggests that synthesized sensor CTH can efficiently perform under physiological pH conditions and thus has exceptional potential for detection of perchlorate ion in biological applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo monitor respond time of synthesized sensor CTH for detection of hypochlorite ion, change in intensity of emission peak with time upon addition of perchlorate ion was recorded at pH 7.4. The assessment of time-dependent fluorescence intensity showed that synthesized sensor CTH responded presence of hypochlorite in very short time. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, interaction of perchlorate ion with 30 \u0026micro;M solution of CTH, emission intensity at 459 nm was dramatically enhanced and approached maximum stable value in about few seconds. To ensure completion of reaction between CTH and perchlorate ion, fluorescence spectra were collected for 15 minutes in 1.0 minute time interval. It was found intensity of peak at 459 was immediately enhance to its maximum limit and after that no significant enhancement in fluorescence intensity at 459 nm was recorded even after one minutes. The observed quick response time exhibits that CTH can monitor perchlorate ion with real-time detection capability which is important aspect for day-to-day life applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e:\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eDetection mechanism of sensor CTH towards perchlorate ion\u003c/b\u003e:\u003c/div\u003e\u003cp\u003eTo investigate sensing mechanism of synthesized sensor CTH towards hypochlorite ion, mass spectrometry analysis was carried out in PBS buffer solution containing 5% DMSO at pH 7.4. The mass spectra of sensor CTH, before and after interaction of NaOCl was recorded in similar solvent system. The mass spectrometry analysis exhibited that after interaction with hypochlorite ion, peak of sensor CTH at m/z\u0026thinsp;=\u0026thinsp;261.04 disappeared whereas peak at m/z\u0026thinsp;=\u0026thinsp;276.89 appeared which is similar to exact mass of sulfoxide derivative of sensor CTH. The information received from mass spectrometry clearly indicated that hypochlorite oxidized thioethers group of chemosensor CTH to sulfoxide group at much faster rates within the range of second as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eApplication of synthesized sensor CTH for perchlorate ion detection:\u003c/h3\u003e\n\u003cp\u003eTo monitor the sensing application of synthesized sensor CTH in real samples, hypochlorite ion (ClO-) detection was monitored in various spiked tap water samples using standard addition method. The recovery studies of sensor CTH were performed by spiking perchlorate ion to drinking water with perchlorate ion concentrations below the determined detection limit. The detection performance analysis was carried out by spiking 25.0 nM, 35.0 nM and 45.0 nM of perchlorate ion and percentage recovery was determined as depicted in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The obtained results revealed that recoveries of hypochlorite ion in spiked tap water samples were in the range of 95.8% \u0026minus;\u0026thinsp;98.08% indicating capability of CTH for accurate determination of perchlorate ion in contaminated water sample\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePercentage recovery of perchlorate ion in spiked water samples\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpiked NaOCl (nM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePercentage recovery (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample-A1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25 nM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e95.8%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample-A2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35 nM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e97.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample-A3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45 nM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e98.0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, molecular chemosensor 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin (CTH) was designed and developed for detection of reactive oxygen species. The absorption and emission studies exhibited that developed chemosensor (CTH) can sensitively detect perchlorate ion in PBS buffered solution (DMSO 5% v/v) at pH 7.4. The emission spectrum exhibited significant enhancement in fluorescence response at 459 nm when excited at 280 nm. The detection limit was also calculated using emission titration data and was found to be 16 nM which is much lower than recommended limit. Moreover, response time was also evaluated and results revealed sensor CTH detect perchlorate ion within few second whereas pH studies strongly suggests that sensor can efficiently perform under pH range of 4 to 10. Furthermore, information received from mass spectrometry clearly indicated that sensor CTH detect hypochlorite ion through oxidization of thioethers group of chemosensor CTH to sulfoxide group at much faster rates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eThere was no funding available.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAruna and Kanchan wrote the main manuscript text and A. A and R.S. prepared figures and supervised. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAruna and R. S are obliged to Manipal University Jaipur for facilitating resources infrastructures. We also would like to thankful to Central Analytical Facilities of Manipal University Jaipur for supporting instruments for Uv-visible, FT-IR and fluorescence spectroscopy\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Almeida AJPO, de Oliveira JCPL, da Silva Pontes LV, de Souza Junior JF, Goncalves TAF, Dantas SH, de Feitosa A, Silva MS, de Medeiros AO (2022) IA, Oxid Med. Cell Longev 1225578\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDymkowska D (2016) Postepy Biochem, 62:116\u0026thinsp;\u0026ndash;\u0026thinsp;126\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKruk J, Aboul-Enein HY, Kladna A, Bowser JE (2019) Free Radic Res. 53: 497\u0026thinsp;\u0026ndash;\u0026thinsp;521\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRay PD, Huang BW, Tsuji Y (2012) Cell Signal 24:981\u0026ndash;990\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePanizzi P, Nahrendorf M, Wildgruber M, Waterman P, Figueiredo JL, Aikawa E et al (2009) J Am Chem Soc 131:15739\u0026ndash;15744\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMalle E, Buch T, Grone HJ (2003) Kidney Int 64:956\u0026ndash;967\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu QL, Lee KA, Lee S, Lee KM, Lee WJ, Yoon JA (2013) J Am Chem Soc 135:9944\u0026ndash;9449\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePattison DI, Davies MJ (2006) Biochemistry 45:8152\u0026ndash;8162\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHazen SL, Heinecke JW (1997) J Clin Invest 99:2075\u0026ndash;2081\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHazell LJ, Arnold L, Flowers D, Waeg G, Malle E, Stocker R (1996) J Clin Invest 97:1535\u0026ndash;1544\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng L, Nukuna B, Brennan ML, Sun M, Goormastic M, Settle M et al (2004) J Clin Invest 114:529\u0026ndash;541\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRani B, Agarwala A, Behera D, Verma VP, Singh AP, Shrivastava R (2021) Dyes Pigm 194:109596\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen XQ, Tian XZ, Shin I, Yoon J (2011) Chem Soc Rev 40:4783\u0026ndash;4804\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAruna, Verma VP, Singh AP, Shrivastava R (2024) J Mol Struct 1295:136549\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSwami S, Agarwala A, Verma VP, Shrivastava R (2017) ChemistrySelect 2:11474\u0026ndash;11481\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng GH, Fan JL, Sun W, Sui K, Jin X, Wang JY et al (2013) Analyst 138:6091\u0026ndash;6096\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEmrullahoglu M, Ucuncu M, Karakus E (2013) Chem Commun 49:7836\u0026ndash;7838\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu BC, Xu YH, Liu WQ, Shao CX, Wu HF, Jiang HL et al (2014) Sens Actuators B 191:473\u0026ndash;478\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z (2017) Chem Rev 117:7762\u0026ndash;7810\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown HC, Mandal AK (1980) J Org Chem 45:916\u0026ndash;917\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdelfattah AG, Bansal S, Quaye JA, Kondengadan SM, Gadda G, Wang B (2025) Org. Lett. 27:3071\u0026thinsp;\u0026ndash;\u0026thinsp;3076\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh AP, Murale DP, Ha Y, Liew H, Lee KM, Segev A, Suh Y-H, Churchill DG (2013) Dalton Trans 42:3285\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Coumarin, perchlorate ion, chemosensor, detection, drinking water treatment","lastPublishedDoi":"10.21203/rs.3.rs-7954085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7954085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin chemosensor (CTH) was successfully designed and synthesized for detection of hypochlorite ion, an important reactive oxygen species (ROS) in aqueous medium. The chemosensor (CTH) exhibits significant selectivity for perchlorate ion over other ROSs with fluorescence \u0026ldquo;turn-on\u0026rdquo; response within few seconds. Moreover, emission titration experiments results revealed that reported sensor detect perchlorate ion with 16 nM detection limit. Further, mass spectrometry analysis indicated that oxidizable 2-methyl-sulphur arms of sensor CTH is responsible for detection of perchlorate ion.\u003c/p\u003e","manuscriptTitle":"A coumarin derived 2-methylthio-4-hydroxypyrimidine[3,4-b]coumarin fluorescent chemosensor for detection of hypochlorite ions in aqueous medium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 12:06:42","doi":"10.21203/rs.3.rs-7954085/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-28T19:04:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T06:02:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-20T13:55:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-19T09:45:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T03:53:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109932810042326898418217239753970694182","date":"2025-11-14T03:34:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172676040056143769444029332489177626214","date":"2025-11-12T07:47:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223857857034946172623221021238657796581","date":"2025-11-12T00:37:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-08T04:39:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69023416398863434046257323556634297399","date":"2025-11-03T03:35:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30018168941127671690159014324143541652","date":"2025-10-31T14:27:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-31T14:13:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-31T12:36:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-31T12:36:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-10-27T06:37:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2f076547-ad5c-42d0-959a-f46a1ad7194c","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:16:45+00:00","versionOfRecord":{"articleIdentity":"rs-7954085","link":"https://doi.org/10.1007/s10895-026-04737-0","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2026-03-01 15:57:17","publishedOnDateReadable":"March 1st, 2026"},"versionCreatedAt":"2025-11-12 12:06:42","video":"","vorDoi":"10.1007/s10895-026-04737-0","vorDoiUrl":"https://doi.org/10.1007/s10895-026-04737-0","workflowStages":[]},"version":"v1","identity":"rs-7954085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7954085","identity":"rs-7954085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}