A New Lysosome-Targetable Turn-On Fluorogenic Probe for Carbon

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A New Lysosome Targetable Turn-On Fluorogenic Probe for Carbon Monoxide Imaging in Living Cells Koushik Dhara, Somenath Lohar, Ayan Patra, Priya Roy, Swadhin Kumar Saha, Gobinda Chandra Sadhukhan, and Pabitra Chattopadhyay Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05331 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Analytical Chemistry

A New Lysosome Targetable Turn-On Fluorogenic Probe for Carbon Monoxide Imaging in Living Cells

Koushik Dhara,§* Somenath Lohar,¶ Ayan Patra,¶ Priya Roy,† Swadhin Kumar Saha,¶ Gobinda Chandra Sadhukhan,‡ and Pabitra Chattopadhyay¶

§

Department of Chemistry, Sambhu Nath College, Labpur, Birbhum 731303, West

Bengal, India, Email: [email protected] (KD) ¶

Department of Chemistry, The University of Burdwan, Golapbag, Burdwan 713104,

West Bengal, India †

Parasitology Laboratory, Department of Zoology, Visva-Bharati University,

Santiniketan-731235, West Bengal, India ‡

Former Director, UGC-HRDC, Jadavpur University, Kolkata 700 032, West Bengal,

India

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT A lysosome targetable fluorogenic probe, LysoFP-NO2, was designed and synthesized based on naphthalimide fluorophore that can detect selectively carbon monoxide (CO) in HEPES buffer (pH 7.4, 37 °C) through the transformation of nitro to the amino functionalized system in presence of CO. LysoFP-NO2 triggered a ‘turn-on’ fluorescence response to CO with a simultaneous increase of fluorescence intensity by more than 75times. The response is selective over a variety of relevant reactive nitrogen, oxygen and sulfur species. Also, the probe is an efficient candidate for monitoring changes in intracellular CO in living cells (MCF7) and the fluorescence signals specifically localize in lysosomes compartment.

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INTRODUCTION Carbon monoxide (CO) has long been known as a toxic pollutant due to its strong affinity to bind with hemoglobin that could lead to fatal consequences on inhalation.1,2 CO is often called the "silent killer" because of it’s tasteless, colourless, odorless and particularly hard to sense nature.3,4 Despite its deadly status, it is evident from various studies that CO can be continuously produced in our human body via endogenous degradation of heme by a family of inducible (HO-1) and constitutive (HO-2) heme oxygenase enzymes.5-8 Thus CO, a gasotransmitter molecules, was considered to be an important versatile signaling bio-molecule with significant therapeutic potential protecting from inflammatory, vascular or even cancer diseases.9,10 It has an essential controlling role in a variety of physiological and pathophysiological processes that take place within the nervous, cardiovascular and immune systems.11 CO generated in the vessel by heme oxygenase enzyme has been revealed to prevent both acute and chronic hypertension, and also vasoconstriction through the stimulation of soluble guanylate cyclase.12,13 CO gas is known to accelerate potent anti-inflammatory effects in the concentration rang 10 to 500 ppm.14 Endogenous CO inhibits human airway smooth muscle cell proliferation,15 prevents endothelial cell apoptosis16,17 and protects against hyperoxic as well as ischemic lung injury.18 Also, it seems to regulate sinusoidal tone in the hepatic circulation.19 These have fascinated unparalleled attention for deepening research of CO in biology. Many chemical and biological aspects of CO remain elusive owing to having the lack of ways for selective monitoring of this transient small molecule. Thus the development of the selective method for direct tracking of this small molecule in living systems is of great scientific concern.

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Although some traditional methods, such as electrochemical analysis20 gas chromatography21 and colorimetric detection22-25 have been established for sensing CO, these methods are not able to selectively detect CO in living systems in a noninvasive manner. In contrast, detection by fluorescence techniques is highly attractive due to having its high sensitivity, real-time detection approach in a nondestructive manner. Recent reports showed that a carbazole-coumarin fused two-photon platform,26 a genetically encoded fluorescent platform,27 and a few recently designed fluorescent chemosensors28-31 are capable of CO detection in living cells. We recently reported a fluorogenic probe for highly selective detection of CO in living cells based on Pd(0) mediated reaction.32 In fact, very recently, a few fluorescent CO probes have been reported to be able to detect CO in living cells and all these methods were designed based on transition metal species (e.g. Pd, Rh, Fe etc.) mediated reaction,27,30,33-38 but still highly selective and sensitive ‘targetable’ type ‘turn-on’ systems are greatly desired. Therefore, there is a strong need to develop methods that can track directly the production, transportation and distribution of CO in biological systems in order to elucidate its mechanistic behavior in physiological and pathological roles. To the best of our knowledge, to date, there has been no report on any targetable fluorogenic probe that can selectively detect lysosomal CO in living cells. In this research, for the first time, we have designed and developed a simple and effective

lysosome-targetable

fluorogenic

probe,

LysoFP-NO2,

with

3-nitro

naphthalimide as a fluorophore where the nitro group behaves as CO responsive moiety and the tagged morpholine fragment as the lysosome targetable entity. LysoFP-NO2 enables the selective detection of CO in the aqueous buffer of pH 7.4 through the

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transformation of the nitro group to highly fluorescent amino functionalized derivative, LysoFP-NH2. The probe is also efficient to detect lysosomal CO in living cells. It represents a unique chemical tool that features a selective ‘turn-on’ response to CO over reactive, nitrogen, oxygen and sulfur species and thus it can be applied to detect this gasotransmitter molecule in aqueous buffer medium.

EXPERIMENTAL SECTION Materials and methods. All chemicals and reagents were purchased from Sigma and used without further purification. Solvents used for spectroscopic experiments were purified and dried by standard procedures. Fluorescence spectra were performed using a HITACHI F-4600 Fluorescence Spectrophotometer attached with temperature controlling unit. Electron spray ionization (ESI) mass spectra were recorded on a Qtof Micro YA263 mass spectrometer. NMR spectra of organic compounds were obtained on a Bruker Advance DPX 400. General method of fluorescence studies. LysoFP-NO2 was dissolved in DMSO to obtain stock solutions for fluorometric analyses. Desired volume of DMSO stock was taken to dilute in 10 mM HEPES buffer (1 % DMSO) of pH 7.4 at 37 °C to reach the final concentration (10 µM) of the probe. All the fluorescence spectra of the probe were recorded at an excitation wavelength of 440 nm. HPLC analyses. Reversed-phase HPLC analysis was performed using Agilent 1200 Infinity series with C18 column. HPLC experiments were carried out with an increasing ratio of buffer B (0.1% CH3COOH in acetonitrile) to buffer A (0.1% CH3COOH in H2O). All samples were evaluated by increasing the amount of buffer B

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from 10 to 80% over 20 min. Cell culture. Human breast adenocarcinoma cells (MCF7) were cultured in DMEM medium supplemented with FBS (fetal bovine serum, 10%) and penicillinstreptomycin antibiotic solution (1%) in a humidified CO2 incubator (5 % CO2). The cells were incubated with trypsin-EDTA (ethylenediaminetetraacetic acid) solution for the detachment of cells from the culture flask and were washed with growth medium. Followed by several times of washing, the cells were seeded on 14 mm glass cover slips into a 6-well microtiter plate for the purpose of cellular imaging and permitted 24 h for complete adherence. Fluorescence imaging study. Cells grown on glass cover slips were incubated with various ingredients in DMEM (1 % DMSO) of pH 7.4 at 37 ºC for desired period. After that, cells were washed thrice with DMEM medium and again incubated with 50 and 100 µM CORM-3 for 45 min at 37 ºC. After every incubation, cells were washed with DMEM for three times. Bright field and fluorescence images of MCF7 cells were taken by a Confocal laser scanning Fluorescence Microscope Leica TCS SP8. Synthesis of LysoFP-NO2 (Scheme 1). A ethanolic (30 ml) solution of 4-(2aminoethyl)morpholine (0.156 g, 1.3 mmol) was added drop wise to a 50 ml ethanolic solution of 3-Nitro-1,8-naphthalic anhydride (0.243 g, 1 mmol). The solution mixture was then stirred for 30 min and then allowed to reflux for another 3 h. The reaction mixture was then cooled to room temeprature and then filtered. The filtrate was taken for next few weeks under slow evaporation. X-ray single quality crystals were obtained after few weeks. The crystals were used to perform all the characterization and spectroscopic analyses and other studies (calculated yield 77%). 1H NMR (400 MHz, CDCl3):δ 2.57-

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2.61 (t, 4H), 2.71-2.74 (t, 2H), 3.65-3.68 (t, 4H), 4.35-4.39 (t, 2H), 7.94-7.98 (t, 1H), 8.43-8.45 (d, 1H), 8.77-8.79 (d, 1H), 9.15 (s, 1H) and 9.31 (s, 1H).

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C (100 MHz, d6-

DMSO) δ 37.5,53.8, 55.8, 66.5, 122.7, 123.2, 124.2, 129.7, 130.1, 131.1, 132.3, 134.4, 136.7, 146.1, 162.6 and 163.2. HRMS (m/z) found 378.1003 Calculated for [M+Na+]+ 378.1066, where M formulated as C18N17N3O5, LysoFP-NO2. Synthesis of LysoFP-NH2. A 0.5 ml DMSO solution of LysoFP-NO2 (0.088 g, 0.25 mmol) is diluted with 50 ml 10 mM HPEPS buffer of pH 7.4. Then a 0.5 ml DMSO soultion of CORM-3 (0.736 g, 2.5 mmol) was added drop wise to the previous solution under stirring condition and the mixture was then heated to 37 0C for one hour. After that, the sovent was evaporated in rotary evaporator under reduced pressure. The residue was then dissolved in DCM and washed three times with water. Finally the residue in DCM layer was then purified by column chromatography with 40% DCM in hexane. (calculated yield 63%). 1H NMR (400 MHz, CDCl3):δ 2.57-2.61 (t, 4H), 2.68-2.71 (t, 2H), 3.60-3.62 (t, 4H), 4.31-4.34 (t, 2H), 7.31 (s, 1H), 7.59-7.63 (t, 1H), 7.93-7.95 (d, 1H), 8.02 (s, 1H) and 8.30-8.32 (d, 1H).

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C (100 MHz, d6-DMSO) δ 37.5, 53.7, 55.8,

66.6, 121.5, 122.1, 123.0, 128.4, 128.6, 129.1, 130.1, 133.16, 135.5, 142.1, 162.8, and 163.3.

HRMS (m/z) found 326.1460; calculated for [M+H+]+ 326.1505, where M

formulated as C18N20N3O3, LysoFP-NH2.

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Scheme 1. Synthetic route to obtain LysoFP-NO2 in ethanol medium under reflux condition for 3 h.

RESULTS AND DISCUSSION

Probe design and stability. The synthetic route for the preparation of LysoFPNO2 is shown in Scheme 1. In the preparation, the lysosome targetable morpholinemoiety was introduced as amide coordination by the reaction of 3-nitro-1,8-naphthalic anhydride and N-(2-aminoethyl)morpholine in ethanol medium under reflux condition for several hours and finally the final, compound was isolated in a satisfactory yield. Single crystals of LysoFP-NO2 for X-ray analysis were obtained from the methanolic solution of the compound under slow evaporation after few weeks. The molecular view of the title compound with atom labeling scheme is shown in Figure 1. First, we checked the stability of the probe in reaction buffer (10 mM HEPES, 1% DMSO) of pH 7.4 at 37 °C to establish the working ability of the probe in aqueous buffer medium. The incubated samples (up to 12 h) were examined with time to time by reversed-phase HPLC analysis (Figure S3) which displayed only one peak of LysoFPNO2 at 12.8 min suggesting the purity is maintained after the long time incubation in the aqueous medium. And there is no indication of any kind of hydrolyzed and/or decomposed products during the incubation. This result demonstrates the sufficient 8


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stability of the probe in the reaction buffer of interest. The probe was designed in such a way that 3-nitro-1,8-naphthalimide moiety displays very weak fluorescence because of the electron withdrawing nature of nitro group can quench the fluorescence of the fluorophore. The suppression of fluorescence behaviour may also be restored due to photoinduced electron transfer (PET) process from naphthalimide fluorophore to nitro group and the weakened intra-molecular charge transfer (ICT) process.39

Figure 1. ORTEP view of LysoFP-NO2 with atom numbering scheme.

Fluorescence detection and proposed mechanism for sensing. The probe, LysoFP-NO2 displays maximal absorption at around 440 nm (ε = 4,500 M-1cm-1) in 10 mM HEPES buffer. The fluorescence spectrum of LysoFP-NO2 exhibits very week fluorescence emission in aqueous buffer at pH 7.4 when excited at 440 nm. This nonemissive (ΦF = 0.0016) nature is due to the photoinduced electron transfer (PET) process from naphthalimide fluorophore to nitro group. After being treated with 100 µM CORM3 (CORM-3: carbon monoxide releasing molecule-3, [Ru(CO)3Cl(glycinate)]), the intensity dramatically enhanced with the progress of time and finally achieved its highest

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value with a quantum yield, ΦF = 0.1025, after incubation for 45 min at 37 °C in reaction buffer (Figure 2). Here the water soluble complex [Ru(CO)3Cl(glycinate)], (CORM-3) has been used as an easy-to-handle CO source.40 LysoFP-NO2 initiated a ‘turn-on’ fluorescence response to CO with a concomitant increase in fluorescence intensity by more than seventy five times when 100 µM CORM-3 was added to the reaction medium. We studied dose dependent responses of CO with the addition of various concentration of CORM-3 (3 µM, 5 µM, 10 µM, 20 µM, 30 µM, 50 µM and 100 µM) incubated for 45 min at 37 °C in reaction buffer. The fluorescence spectrum was measured every 5 min duration up to 45 min for each and every concentration of CORM-3 (Figure 3). The proposed mechanistic route for detection is shown in Scheme 2. The enhanced fluorescence can be achieved by the transformation of the 3-nitro moiety of the probe to a possible highly fluorescent amino functionalized 1,8-naphthalimide moiety whereas the analyte, CO can be oxidized to carbon dioxide. Here the nitro group served as the CO reaction site, which can be easily converted to the possible amino compound. The proposed mechanistic41 steps of the possible reduction of nitro group by CO were depicted in Figure S4. After the conversion of nitro to the amino functionalized moiety, the PET process disrupts and simultaneously strengthens the possible intra-molecular charge transfer (ICT) process and thus a turn-on signal can be monitored.42,43 The progress of this transformation was also monitored by using reversed-phase HPLC (Figure 4). For the transformation, the probe peak intensity at 12.8 min was decreased gradually as CO is consumed with the reaction of LysoFP-NO2 and also the concomitant rise of a new peak was observed at 8.5 min. The newly generated compound at 8.5 min is due to the formation of a new product, LysoFP-NH2. Isolation of this final product,

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LysoFP-NH2 was further confirmed by ESI-MS from the HPLC analyses (Figure S5). Further we have fully characterized the final product, LysoFP-NH2 by NMR experiment (Figure S6). These results confirm the formation of the amino-functionalized 1,8naphthalimide derivative. The LOD (limit of detection) was calculated as 0.60 µM from the calibration curve (Figure S7) using the 3σ method.44,45

Figure 2. Fluorescence spectrum of LysoFP-NO2 (10 µM) upon treatment with CORM-3 (100 µM) with the progress of time in reaction buffer [10 mM HEPES, 1% DMSO] of pH 7.4 at 37 °C (ex: 440 nm).

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Figure 3. LysoFP-NO2 (10 µM) showed a strong and selective turn-on response to CO in reaction buffer pH 7.4 at 37 °C (ex: 440 nm) to various levels of CORM-3 observed with the progress of time under the following experimental conditions: (1) only LysoFP-NO2, (2) only CORM-3, (3) 3 µM CORM-3, (4) 5 µM CORM-3, (5) 10 µM CORM-3, (6) 20 µM CORM-3, (7) 30 µM CORM-3, (8) 50 µM CORM-3 and (9) 100 µM CORM-3

Figure 4. Reversed-phase HPLC analyses of the reaction progress of LysoFP-NO2 with CO in reaction buffer of pH 7.4 at 37 °C (characteristic absorbance band at 440 nm).

Scheme 2. Possible mechanistic route of LysoFP-NO2 for CO detection through the transformation of nitro to the amino functionalized system.

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Selectivity study. The selectivity of this probe towards CO detection was tested with the variety of biologically relevant reactive species that may hamper the sensing study. The result shows that the enhanced fluorescence intensity was achieved only when it can be triggered by CO and no significant changes were obtained when various biologically significant reactive nitrogen, oxygen and sulfur species e.g. NO, NaOCl, tertbutyl hydroperoxide (tBuOOH), H2O2, superoxide (O2.−), and H2S were introduced (Figure 5). Thus selectivity study indicates that the probe should have potential applications to detect CO over a variety of reactive species.

Figure 5. Fluorescence responses of 10 µM LysoFP-NO2 to CO with the progress of time in the presence various species: (1) None, (2) CORM-3 (100 µM), (3) NO (source NOCl3), (4) NaOCl, (5) tBuOOH, (6) H2O2, (7) O2.− (source KO2) and (8) H2S (source NaHS).

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Imaging study. Based on the spectral response toward carbon monoxide in aqueous buffered medium, we further examined the effectiveness of probe LysoFP-NO2 for CO detection in live cells and also targetable localization in the lysosome. In the control experiments, cells treated with only 10 µM probe (Figure 6a) and 100 µM CORM-3 (Figure 6b) separately exhibit almost no fluorescence. For the imaging study, cells were incubated with 10 µM LysoFP-NO2 for 20 min and then with CORM-3 (50 µM and 100 µM) for another 60 min. After washing several time with PBS, cells exhibit exhibited strong fluorescence images with high contrast (Figure 6c and d). Finally, the subcellular distribution of LysoFP-NO2 in MCF7 cells was examined. The cells were coincubated with the probe LysoFP-NO2 along with the addition of 100 µM CORM-3 and Lyso-Tracker Blue (50 nM). As shown in Figure 7b the fluorescence patterns in the green channel of LysoFP-NO2 signal manifested and specifically localize in lysosomes due to sensing of CO. Moreover, the blue fluorescence signal in the blue channel due to the staining of the lysosomes by Lyso-Tracker Blue was obtained (Figure 7c). The merged image signified that the green fluorescence overlaps very well with the blue fluorescence (Figure 7d). These results demonstrate that the newly designed probe Lyso-FP-NO2 could display a brilliant lysosome-targetable property and detect CO in the lysosomes. In addition to that, 10 µM of Lyso-FP-NO2 did not display any significant cytotoxic effect (Figure S8) on HEPG2 human liver cancer cell line for at least up to 5 h of its treatment. Although significant cytotoxicity was detected for the incubation at higher doses after 5 h onward. These results implied that the fluorogenic probe, Lyso-FP-NO2, is an efficient candidate for monitoring changes in intracellular CO.

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Figure 6. Confocal laser scanning Fluorescence Microscope (Leica TCS SP8) images of MCF7 cells for CO detection using LysoFP-NO2. Control experiment with (a) 10 µM LysoFP-NO2 and (b) 100 µM CORM-3, Imaging experiment: (c) 10 µM LysoFP-NO2 + 50 µM CORM-3; (d) 10 µM LysoFP-NO2 + 100 µM CORM-3 and (e) 50 nM LysoTracker-Blue. The upper, middle and bottom row indicate the bright-field, fluorescence and merged images. Scale bar 10 µm (image: a-d), 20 µm for image: e.

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Figure 7. The images of the living MCF7 cells co-incubated with the probe 10 µM LysoFP-NO2, 100 µM CORM-3 and Lyso-Tracker Blue (50 nM). (a) Bright-field image of the MCF7 cells treated with LysoFP-NO2 (10 µM) and Lyso-Tracker Blue; (b) The fluorescence image of the green channel due to the probe sensing CO; (c) The fluorescence image of the blue channel due to the known Lyso-Tracker Blue and (d) The merged image of a, b, and c. Scale bar: 10 µm.

CONCLUSION In summary, we designed and synthesized lysosome targetable naphthalimidebased fluorogenic probe, LysoFP-NO2 and introduced a new approach for CO detection by fluorescence method in aqueous buffer medium through the transformation of nitro to amino functionalized system in presence of CO. LysoFP-NO2 triggered a ‘turn-on’ fluorescence response to CO with a simultaneous increase of fluorescence intensity by 75-times. The response is selective over a variety of relevant reactive nitrogen, oxygen and sulfur species. Also, the probe is able to monitor the changes in intracellular CO by inverted fluorescence microscopy and the fluorescence signals specifically localize in lysosomes compartment.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on

the

ACS

Publications

website

at

DOI:

10.1021/acs.analchem.xxxxxxx.

Crystallographic information for LysoFP-NO2 in CIF format (CCDC 1573524), Figure S1–S8.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACKNOWLEDGMENT K. Dhara acknowledges Department of Science and Technology (DST), Fast track research grand, New Delhi (vide project no. SB/FT/CS-142/2012) and University Grants Commission, Govt. of India (UGC, vide project no. PSW-026/14-15) for financial assistance. S. Lohar is thankful to UGC, New Delhi, India for financial support as SRF. A. Patra acknowledges SERB National Post-doctoral fellowship (vide file no. PDF/2016/000381) for financial support. The authors are highly grateful to Prof. Santi P. Sinha Babu, Dept. of Zoology, Visva Bharati University, India and DST-FIST program of Dept. of Zoology, Visva Bharati, India for carrying out confocal microscopy.

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