Oxidation of phenothiazine based fluorescent probe for hypochlorite and its application to live cell imaging

Accepted Manuscript Title: Oxidation of Phenothiazine Based Fluorescent Probe for Hypochlorite and Its Application to Live Cell Imaging Authors: Mani Vedamalai, Dhaval Kedaria, Rajesh Vasita, Iti Gupta PII: DOI: Reference:

S0925-4005(18)30349-6 https://doi.org/10.1016/j.snb.2018.02.071 SNB 24166

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

4-9-2017 8-2-2018 8-2-2018

Please cite this article as: Mani Vedamalai, Dhaval Kedaria, Rajesh Vasita, Iti Gupta, Oxidation of Phenothiazine Based Fluorescent Probe for Hypochlorite and Its Application to Live Cell Imaging, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.02.071 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Oxidation of Phenothiazine Based Fluorescent Probe for Hypochlorite and Its Application to Live Cell Imaging

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Mani Vedamalaia,c, Dhaval Kedariab, Rajesh Vasitab and Iti Guptaa*

aIndian

Institute of Technology Gandhinagar, Village Palaj, Simkheda, Gandhinagar, Gujarat- 382355, India. *Corresponding author, e-mail: [email protected] bSchool of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India cDepartment of Chemistry, Bharath Institute of Higher Education and Research, Selaiyur, Chennai-600073, Tamil Nadu, India.

Highlights

Phenothiazine functionalized BODIPY has been synthesized and examined for

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Graphical abstract

hypochlorite detection.

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The probe selectively detects the hypochlorite over other ROS which was indicated by 47 nm blue shifted absorbance band and strong turn-on fluorescence emission.

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Sensing ability was almost stable throughout pH range from 3 to 10.

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Exogenous and endogenous presence of hypochlorite was successfully mapped in RAW 264.7 cells using phenothiazine functionalized BODIPY.

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Abstract New phenothiazine appended BODIPY derivative has been synthesized and characterized for the determination of hypochlorite in live cells. The fluorescent probe has exhibited 72 fold fluorescence enhancement upon addition of hypochlorite over other reactive oxygen species.

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Further, the studies proved that the probe has ability to detect the hypochlorite even at extremely low level (4.1 nM). Having less cytotoxicity and negligible auto-fluorescence, the probe was successfully used to monitor external addition and internal secretion of hypochlorite in RAW 264.7 cells.

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Keywords: BODIPY, Fluorescent probe, Hypochlorite, Myeloperoxidase, Phenothiazine.

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1. Introduction

Hypochlorite is strong Reactive Oxygen Species (ROS) which is produced by Myeloperoxidase

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(MPO) catalyzed reaction between hydrogen peroxide and chloride ion. MPO acts as antimicrobial agent in neutrophil and it would be less harmful in presence or absence of

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hydrogen peroxide.[1] In co-existence with halides or pseudo halides, it produces highly reactive

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ROS in cytoplasmic region.[2] The as-generated ROS can attack oxidisable groups such as IronSulfur cluster, proteins, sulfur containing ether groups, and fatty acids, as a result there would be

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interruption of bio-signaling and dissipation of pathological processes.[3, 4] Excessive generation of hypochlorite induces uncontrollable chain reaction in both inter and intra cellular

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region. Impairment of MPO reduces strength of immune defense system, causes chronic kidney disease and Atherosclerosis.[5, 6] Detection and monitoring of MPO together with hypochlorite will be expedient to study inflammation and genetic disorders. Apart from its role in immune

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system, MPO also plays important role in regulative functions such as elastase release, gene expression.[7, 8]. ROS produced in cell has been implicated in diseases such as cancer, aging, Hypertension, Alzheimer’s disease, Parkinson disease, diabetes mellitus, and inflammation. Importantly, ROS damages iron sulfur clusters, and enzymes. Hence, production and scavenging of ROS in intracellular region need to be balanced for successful pathological processes. There have been challenges and difficulties for the detection of highly reactive and short lived ROS 2

species selectively in in vivo. Fluorescence based sensory systems are ideal tool for in vivo and in vitro studies to probe disease diagnosis and biochemical pathology.[9] Therefore, fluorescent probes for ROS gain attentions to study kinetics of ROS production, cellular response and signaling. Among the fluorescent probes, near IR emitting probes could reveal vital information as they can penetrate deep into tissues and are less harmful. Remarkably, the fluorescence signal

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of near IR emitting probes can be easily visualized without any interference from green fluorescence emitting biomolecules; thus the problem of interference can be minimized. Boron dipyrromethene (BODIPY) derivatives have emerged as inevitable tool to elucidate biochemical reactions and biophysical interactions. Many research groups around the world have been working on BODIPY

based fluorescent probes for biological, and environmental

applications.[10] Metal homeostasis, oxidative burst, protein deposits, identification of disease

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affected cells, and enzymes have been studied successfully by BODIPY based probes.[11]

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Further, the spectral and electrochemical properties of BODIPY derivatives can be easily tuned by structural modifications of the boron-dipyrromethene core.[12-15] Recently, we have reported

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fluorescent probes based on innovative approach for detection of metal ions and tau

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aggregation.[12, 13] Generally hypochlorite detecting probes generate the readable distinct signal upon either oxidation of atoms such as sulfur, selenium and tellurium or hydrolysis of

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functional groups such as hydroxyl amine, hydrazide, di-azo bonds, and lactones.[14-20] Among

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those probes, sulfur based fluorescent probes are highly advantageous as they could expose more information on oxidation of sulfur containing biomolecules during the oxidative stress.[21]

byproduct.

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Probes which operates on hydrolysis mechanism may generate highly reactive metabolites as a

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Herein, we report a turn-on near IR emitting fluorescent probe for the hypochlorite. In this work, phenothiazine group has been attached at the C-3 position of BODIPY skeleton that ensures extended π conjugation in the molecule. Phenothiazine appended BODIPY is

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successfully applied as ROS probe to selectively determine hypochlorite both in vitro and in vivo in RAW 264.7 cells. 2. Experimental 2.1 Synthesis of HCP

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Compound 1 was prepared according to method optimized by us.[13] Compound 1 (1 mmol), Nbutylphenothiazine carboxaldehyde (1 mmol), glacial acetic acid (10 mmol) and piperidine (10 mmol) were dissolved in ethanol (10 mL). Then, the reaction mixture was irradiated in Microwave oven for 20 minutes at 100°C. Organic volatile impurities and solvents in the reaction mixture were removed using vacuum evaporator. Crude mixture was washed with water

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and extracted by using dichloromethane. Neutral Alumina packed column was used to purify the crude compound. Blue colored fractions were collected and concentrated. Title compound was obtained as blue crystals. ESI-MS: [M+H]+ C33H29BF2N3OS calculated: 592.2405 found: 592.2392; 1H NMR (500 MHz, CDCl3,  in ppm): 0.96 (t, 3H, J = 7.5 Hz), 1.47 (sex, 2H, J = 7.5 Hz), 1.80 (quint, 2H, J = 7.0 Hz), 2.69 (s, 3H), 3.87 (t, 2H, J = 7.0 Hz) 3.89 (s, 3H), 6.73 (d, 1H, J = 4.0 Hz) 6.81-6.84 (m, 2H), 6.86 (d, 2H, J = 5.5 Hz ), 6.92 (t, 1H, J = 7.0 Hz), 7.0 (d, 2H, J =

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8.5 Hz), 7.12-7.16 (m, 2H), 7.18 (d, 1H, J = 16.0 Hz), 7.35 (d, 1H, J = 2.0 Hz), 7.39 (dd, 1H, J =

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2.0, 8.5 Hz), 7.46 (d, 2H, J = 9.0 Hz), 7.57 (d, 1H, J = 16.0 Hz). 13C NMR (125 MHz, CDCl3, 

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in ppm): 161.2, 156.1, 155.1, 146.0, 144.5, 140.6, 135.9, 135.2, 134.8, 132.0, 131.0, 131.0, 130.1, 129.2, 127.5, 127.3, 127.0, 126.9, 126.2, 125.1, 124.2, 122.7, 118.9, 117.5, 116.0, 115.5,

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115.3, 113.8, 55.4, 47.4, 29.0, 20.1, 14.9, 13.8.

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2.2 Cell culture

The cell line of murine macrophage RAW 264.7 was procured from NCCS, India. The cell lines

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were grown in Dulbecco's Modified Eagle's Medium (DMEM)-high glucose, supplemented with

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10% fetal bovine serum (FBS) at 37°C and 5% CO2.

2.3 Alamar blue assay

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8 X103 cells were seeded in 96 well plate and allowed to grow for 24 hours. After that, cells were treated with 2, 20 and 40 µM of HCP in 200 µL of media and incubated for 48 hours. Following the treatment, cells were incubated with 10 % v/v of Alamar blue solution in fresh media at 37°C

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in dark for 1 hr. 200 µL of suspension was taken out after incubation in 96 well plate and absorbance was measured at 570 nm to evaluate cell viability. In order to get more accurate values, all the experiments were done in triplicate. Mean and Standard Error of Mean was calculated with GraphPad Prism software for each data set. Two tailed t-test was implemented to compare each dose with control at P = 0.05.

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2.4 Fluorescence imaging The fluorescence imaging of HCP was performed in 0.1 M phosphate-buffered saline (PBS) with NaOCl (2 µM). Briefly, cells were treated with 2 mL of HCP having final concentration of 2 µM dissolved in DMSO and incubated for 30 min at 37°C. For control experiments, cells were observed directly after incubation with HCP. To monitor the exogenous presence of

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hypochlorite, cells were washed with 0.1 M PBS (2 mL × 3) to remove remaining unbound HCP. 2 mL of RPMI 1640 media was added to the cell culture and treated with 2 µM solution of NaOCl (prepared in sterilized H2O). The cells were incubated for 30 min at 37°C, followed by washing with 0.1 M PBS (2 mL × 3). For determination of PMA induced HOCl production, Raw 264.7 cells were treated with PMA (2 µgL-1) after treatment with HCP in culture medium for 2 h. Then, the culture medium was removed and cells were washed with 0.1 M PBS (2 mL × 3).

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For enzyme impairment study, MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH, 100 µM)

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was added to HCP treated cells followed by the addition of PMA. Fluorescence imaging of cells was performed with a confocal laser scanning microscope (Zeiss, Germany), the 20 X objective

3. Results and Discussion

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lens was used and cells were visualized at 488/530 nm.

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3.1 Synthesis and photophysical properties of HCP

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Design and synthesis of phenothiazine appended BODIPY probe has been mentioned in scheme 1. The probe HCP was prepared by the reaction between compound 1 and N-butylphenothiazine aldehyde. The desired product was obtained as blue colored solid. It has exhibited strong

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absorbance band around at 634 nm and low intensity band at 395 nm. HCP was non-fluorescent due to effective charge transfer from phenothiazine to BODIPY core. Photophysical properties

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and sensing studies were done in methanol-water system (6:4, v/v, 20 mM PBS) at pH 7.4.

Insert Scheme 1 here 3.2 ROS sensing study We have examined HCP to monitor ROS as chalcogen could easily be oxidized by ROS. Sensing ability of HCP was tested against singlet oxygen, sodium hypochlorite, hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrate. Among those ROS, 5

hypochlorite selectively produced strong fluorescence emission (Fig. 1). Oxidation of sulfur atom by hypochlorite changes charge polarization in a molecule by which intra-molecular charge transfer would be disturbed, as a result emission of the probe was not quenched. Remaining other ROS did not change the fluorescence intensity. Metal ions, reactive nitrogen species, reductants and important biomolecules were also incubated with HCP to check the effect of interference.

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As shown in figure S1, metal ions did not change the emission profile of the probe. NO2-, NO3-, N3-, SCN-, 1, 2-benezene methane thiol, ascorbic acid, citric acid, glucose, sucrose, and glutathione were added to the probe to examine the selectivity. These molecules did not oxidize the phenothiazine moiety and no enhanced fluorescence was observed (Fig. S2). L-Alanine, LArginine, L-Cysteine, L- Glycine, L-Histidine, L-Phenylalanine L-Seine, L-Threonine, LTryptophan, L-Tyrosine and L-Valine were co-incubated with HCP to test if there is interference

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from amino acids. However, amino acids did not affect HCP emission (Fig. S3). Fluorometric

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titrations were carried out to establish detection range for the sensing studies. As shown in figure 2, strong fluorescence emission was observed while adding various concentration of

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hypochlorite. The fluorescence enhancement was linear up to the addition of 2 µM of

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hypochlorite. Further addition of hypochlorite did not cause significant enhanced emission as almost all HCP molecules were transformed to oxidized form. Fluorescence enhancement due to

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oxidation of the probe by hypochlorite was measured quantitatively. HCP showed weak

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emission in the given experimental condition and its fluorescent quantum yield was found to be 0.003. In the presence of one equivalent of hypochlorite, fluorescence quantum yield was calculated and found to be 0.216. Hypochlorite induced oxidation of phenothiazine has been

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mentioned in scheme 2. Oxidation pattern was very similar to already reported phenothiazine based fluorescent probes.[24, 25] To validate the origin of strong fluorescence enhancement

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upon addition of hypochlorite, the samples were analyzed by High Resolution Mass Spectrometry (HRMS). A peak at m/z 608.2349 (calcd. 608.2348) in figure S4 corresponding to

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[HCPO+H]+ confirmed the chemical reaction proposed in the scheme 2. Limit of detection for hypochlorite was calculated at signal to noise ratio 3 and found to be 4.1 nM. Insert Figure 1 here Insert Figure 2 here Insert Scheme 2 here

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3.3 UV-Visible titration Upon addition of sodium hypochlorite, the absorbance band of HCP at 634 nm was gradually blue shifted to 587 nm (Fig.3). Besides blue shift, formation of new shoulder peak at 514 nm was observed. Blue shifted absorbance band was apparent due to the oxidation of sulfur atom, which in turn reduces π-conjugation in a molecule. As shown in figure 4, deep blue color solution of

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HCP was turned to dark pink color due to annihilation of charge transfer. The change in emission intensity was measured as the function of pH to check the probe’s stability and capability for biosensing (Fig. 5). Though the emission intensity was stable throughout pH 3 to 10, the emission intensity was slightly higher in basic region. Fluorescence emission of HCP (2 µM) in the presence of various concentration of hypochlorite was measured with respect to incubation time (Fig. S5). The oxidation reaction was very fast and within 2 minutes all the

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hypochlorite ions transformed HCP to HCPO. Further increase on incubation time did not cause

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noteworthy fluorescence change.

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Insert Figure 3 here Insert Figure 4 here

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Insert Figure 5 here 3.4 Alamar blue test

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HCP probe was assessed for cytotoxicity to RAW 264.7 cells by Alamar blue assay. It contains

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fluorescent dye resazurin that serve as an oxidation-reduction indicator in cellular metabolism. Resazurin can be used quantitatively to evaluate cell viability and cytotoxicity by detecting the level of oxidation in the cell. The results showed that after 48 hours of treatment of probe, the

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cell viability was ≥ 100 % at all concentrations. The difference between all dosages with control for cell viability was non-significance at 95 % confident interval. As depicted in figure 6, this

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result indicated that there was no toxic effect of probe and it was biocompatible to the cells at provided concentrations.

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Insert Figure 6 here

3.5 Cell internalization Raw 264.7 cells were used as a model cell line. The fluorescence images showed that no fluorescence was observed for cells with only HCP (Fig. 7a). After treatment with NaOCl, strong fluorescence signal was observed in the cells. The overlaid image of bright-field and 7

fluorescence demonstrate that the fluorescence signals were confined in the cytoplasm, specifying an intracellular distribution of HOCl and reasonable HCP cell-membrane permeability (Fig. 7b). Next, HCP was used to determine PMA-induced HOCl production in Raw 264.7 cells cells. After stimulation with PMA in the presence of HCP, strong red fluorescence was observed in the cells (Fig. 7c). These results confirm that HCP could visualize

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PMA-induced endogenous HOCl production in Raw 264.7 cells. Interestingly, co-incubation of inhibitor ABAH with PMA produced negligible fluorescence (Fig. 7d). These results clearly indicated that only the occurrence of HOCl could give the significant fluorescence enhancement in cells, whereas other ROS and RNS could not produce fluorescence signals. Insert Figure 7 here

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4. Conclusion

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We have described the synthesis, characterization, selectivity and biological applications of

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phenothiazine appended BODIPY derivative. Oxidation of sulfur in phenothiazine moiety

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provides a chemoselective approach for the detection of hypochlorite. The probe has exhibited highly selective and sensitive nature towards hypochlorite. The better selective and high cell permeability of the probe may offer the possibility of mapping production and transport of ROS

Conflict of interest

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None

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in live systems.

Acknowledgement

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MV and IG are grateful to IIT Gandhinagar and SERB, Govt. of India (Grant No: EMR/2015/000779) for providing financial support.

DK and RV thank Department of

Biotechnology

Govt.

of

India)

and

GSBTM,

of

Gujarat

(Grant

No:

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(Govt.

GSBTM/MD/Projects/SSA/1431/2014-15), respectively for fellowship and fiscal support.

Appendix A. Supplementary data: Supplementary data associated with this article is provided.

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References [1] S.J. Klebanoff, Myeloperoxidase: friend and foe, J Leukoc Biol, 77(2005) 598-625. [2] J.C. van Dalen, W.M. Whitehouse, C.C. Winterbourn, J.A. Kettle, Thiocyanate and chloride as competing substrates for myeloperoxidase, Biochem J, 327(1997) 487-92.

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[3] D. Scalia, N. Lacetera, U. Bernabucci, K. Demeyere, L. Duchateau, C. Burvenich, In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability, J Dairy Sci, 89(2006) 147-54.

[4] H.J. Forman, M. Torres, Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling, Am J Respir Crit Care Med, 166(2002) S4-S8.

[5] E.A. Podrez, H.M. Abu-Soud, S.L. Hazen, Myeloperoxidase-generated oxidants and

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atherosclerosis, Free Radic Biol Med, 28(2000) 1717-25.

[6] J. Himmelfarb, Relevance of oxidative pathways in the pathophysiology of chronic kidney

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disease, Cardiol Clin, 23(2005) 319-30.

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[7] V. Papayannopoulos, K.D. Metzler, A. Hakkim, A. Zychlinsky, Neutrophil elastase and

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myeloperoxidase regulate the formation of neutrophil extracellular traps, J Cell Biol, 191(2010) 677-91.

[8] A.P. Kumar, W.F. Reynolds, Statins downregulate myeloperoxidase gene expression in

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macrophages, Biochem Biophys Res Commun, 331(2005) 442-51.

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[9] D. Roy, J. Quiles, D. Gaze, P. Collinson, J. Kaski, G. Baxter, Role of reactive oxygen species on the formation of the novel diagnostic marker ischaemia modified albumin, Heart, 92(2006)

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113-4.

[10] T. Kowada, H. Maeda, K. Kikuchi, BODIPY-based probes for the fluorescence imaging of

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biomolecules in living cells, Chem Soc Rev, 44(2015) 4953-72. [11] Y. Marfin, A. Solomonov, A. Timin, E. Rumyantsev, Recent advances of individual BODIPY and BODIPY-based functional materials in medical diagnostics and treatment, Curr

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Med Chem, (2017). [12] M. Vedamalai, D. Kedaria, R. Vasita, S. Mori, I. Gupta, Design and synthesis of BODIPYclickate based Hg2+ sensors: the effect of triazole binding mode with Hg2+ on signal transduction, Dalton Trans, 45(2016) 2700-8.

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[13] V. Mani, V.G. Krishnakumar, S. Gupta, S. Mori, I. Gupta, Synthesis and characterization of styryl-BODIPY derivatives for monitoring in vitro Tau aggregation, Sens Actuators B-Chem, 244(2017) 673-83. [14] S.-R. Liu, S.-P. Wu, Hypochlorous acid turn-on fluorescent probe based on oxidation of diphenyl selenide, Org Lett, 15(2013) 878-81.

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[15] S.T. Manjare, J. Kim, Y. Lee, D.G. Churchill, Facile meso-BODIPY annulation and selective sensing of hypochlorite in water, Org Lett, 16(2013) 520-3.

[16] S.-R. Liu, M. Vedamalai, S.-P. Wu, Hypochlorous acid turn-on boron dipyrromethene probe based on oxidation of methyl phenyl sulfide, Anal Chim Acta, 800(2013) 71-6.

[17] X. Jin, L. Hao, Y. Hu, M. She, Y. Shi, M. Obst, et al., Two novel fluorescein-based fluorescent probes for hypochlorite and its real applications in tap water and biological imaging,

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Sens Actuators B-Chem, 186(2013) 56-60.

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[18] Y.-R. Zhang, Y. Liu, X. Feng, B.-X. Zhao, Recent progress in the development of fluorescent probes for the detection of hypochlorous acid, Sens Actuators B-Chem, 240(2017)

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18-36.

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[19] L. Long, Y. Wu, L. Wang, A. Gong, F. Hu, C. Zhang, A fluorescent probe for hypochlorite

Commun, 51(2015) 10435-8.

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based on the modulation of the unique rotation of the N–N single bond in acetohydrazide, Chem

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[20] L. Long, D. Zhang, X. Li, J. Zhang, C. Zhang, L. Zhou, A fluorescence ratiometric sensor for hypochlorite based on a novel dual-fluorophore response approach, Anal Chim Acta, 775(2013) 100-5.

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[21] H. Rosen, S.J. Klebanoff, Y. Wang, N. Brot, J.W. Heinecke, X. Fu, Methionine oxidation contributes to bacterial killing by the myeloperoxidase system of neutrophils, Proc Natl Acad

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Sci, 106(2009) 18686-91.

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1. M. Vedamalai received his M.Sc., in Inorganic Chemistry from University of Madras and Ph.D. degree from National Chiao Tung University, Taiwan. His research interests focus on developing fluorescent chemosensors for metal ions and biomolecules.

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2. Dhaval Kedaria is a graduate research student at Central University of Gujarat, India. He is working as a Senior Research Fellow of the Department of Biotechnology-Govt. of India. His research work is focusing on fabrication of polymer based hydrogel scaffold to create 3D microenvironment of tumor. His research interests include: bio-mimetic materials, tissue material interactions and modulating physical properties of hydrogels. His expertise includes functionalization of polymeric biomaterials, fluorescent imaging, hydrogel fabrication and 3D cell culture.

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3. Rajesh Vasita is a faculty member at School of Life Sciences, Central University of Gujarat and leading a research group in Biomaterials & Biomimetic Laboratory. The focus of his research group includes surface engineering of nano-biomaterials and investigation of structure-property-function relationships of these materials. The applications of these materials include tissue regeneration in development and disease, stem cell fate regulation, and drug delivery. Current projects include creating in vitro niche for homing meshenchymal stem cells for bone tissue engineering and designing scaffold for 3D tumor.

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5. Iti Gupta obtained PhD in Chemistry from Indian Institute of Technology Bombay, India. She received JSPS-fellowship from Japan and did postdoctoral research at Kyushu University, Fukuoka where she worked on expanded porphyrins. Later she joined BITS-Pilani KKBirla Goa campus as faculty in Chemistry (2007-2009). Currently, she is Associate Professor at Indian Institute of Technology Gandhinagar. Her research interests lie in the design and synthesis of D-A systems based on porphyrins, corroles and boron based fluorescent dyes for bioimgaing and diagnostic applications.

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Figure 1. Emission spectra of HCP (2 µM, 6/4 methanol-water, v/v) in the presence of various ROS (2 µM) in 20 mM PBS at pH 7.4. ROS used were singlet oxygen, sodium hypochlorite, hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrate. Excitation wavelength was 590 nm.

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Figure 2. Emission spectra of HCP (2 µM, 6/4 methanol-water, v/v) in the presence of various concentration of hypochlorite in 20 mM PBS at pH 7.4. Hypochlorite concentrations were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, and 2.5 µM. Excitation wavelength was 590 nm. Inset: emission intensity (a. u.) versus hypochlorite concentrations (in µM).

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HCP HCP + 2 M NaOCl HCP + 3 M NaOCl HCP + 4 M NaOCl

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Figure 3. UV-visible spectra of HCP (4 µM, 6/4 methanol-water, v/v) in the presence of various concentration of hypochlorite in 20 mM PBS at pH 7.4. Hypochlorite concentrations were 0, 2, 3, and 4 µM.

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Figure 4. Colorimetric (top) and fluorescence (bottom) change of HCP (4 µM) in the presence of various ROS (4 µM).

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Figure 5. Emission intensity of HCP (2 µM) in the absence and presence of hypochlorite in methanol-water (6:4, v/v, 20 mM PBS) as function of different pH values. Excitation wavelength was 590 nm. Black circle denotes emission intensity of free HCP and red colored squares denotes fluorescence intensity of HCP with hypochlorite.

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Figure 6. Cell viability test using various concentrations of HCP.

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Figure 7. Fluorescence microscopic image of HCP incubated Raw 264.7 cells. Bright field image (top), fluorescence image (middle), merged image (bottom). (a) The cells incubated with HCP (2 µM), (b) Subsequent exogenous addition of NaOCl (2 µM) to the cells, (c) the cells treated with PMA for 2 hours in the presence of HCP (2 µM), (d). cell treated with HCP (2 µM), ABAH and PMA.

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OHC

Piperidine Gl. AcOH Ethanol 110°C MW

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Scheme 1. Synthesis of fluorescent probe HCP.

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Scheme 2. Oxidation of the probe by hypochlorite.

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