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Allyl Fluorescein Ethers as Promising Fluorescent Probes for Carbon Monoxide Imaging in Living Cells Shumin Feng, Dandan Liu, Weiyong Feng, and Guoqiang Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00135 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017

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Analytical Chemistry

Allyl Fluorescein Ethers as Promising Fluorescent Probes for Carbon Monoxide Imaging in Living Cells Shumin Feng, Dandan Liu, Weiyong Feng, and Guoqiang Feng* Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, P. R. China. *Corresponding author. Tel.: +86 27 67867958; fax: +86 27 67867955; E-mail: [email protected]. ABSTRACT: Recently, the fluorescent detection of carbon monoxide (CO) in living cells has attracted great attention. However, due to the lack of effective ways to construct fluorescent CO probes, fluorescent detection of CO in living cells is still in its infancy. In this paper, we report for the first time of using allyl ether as reaction site for construction of fluorescent CO probes. By this way, two readily available allyl fluorescein ethers were prepared, which were found to be highly selective and sensitive probes for CO in the presence of PdCl2. These probes have the merits of good stability, good water-solubility, and rapid and distinct colorimetric and remarkable fluorescent turn-on signal changes. Moreover, a very low dose of these two probes can be used to detect and track CO in living cells, indicating that these two probes could be very promising biological tools for CO detection in living systems. Overall, this work provided not only two new promising fluorescent CO probes, but also a new way to devise fluorescent CO probes.

INTRODUCTION Carbon monoxide (CO) is a well-known highly toxic and lethal gas to mammals.1 Because of its colorless, tasteless, odorless and particularly, hard to sense nature, CO is often called the "silent killer". Despite of this deadly reputation, it is now evident that CO can be endogenously produced in our human body during the haem catabolism, and more importantly, it was proved to be an important cell signaling molecule with substantial therapeutic potential protecting from vascular, inflammatory, or even cancer diseases.2-4 This has attracted unprecedented attention for CO, and with the deepening research of CO in biology, it is of great scientific interest to develop selective and sensitive methods for real-time tracking of this small gas molecule in living systems.5-10 Although several traditional methods, such as gas chromatography11 electrochemical analysis12 and colorimetric detection13-16 have been developed for CO sensing, these methods are difficult for real-time tracking CO in living systems in a noninvasive manner. In contrast, fluorescence detection using fluorescent probes is more attractive due to its superiorities of convenience, high sensitivity, real-time and nondestructive detection.17-19 However, due to the lack of effective methods, the construction of fluorescent probes for CO is still at a very early stage and the reported fluorescent CO probes are very limited so far.9 In fact, it was only until very recently that a few fluorescent CO probes have been reported to be able to detect CO in living cells.20-26 Although this greatly contributed to the development of fluorescent probes for CO, these reported probes mainly used the genetic encoding protein20 and the organic palladium complexes21,22,26, which either needs extensive complex processing, or are difficult to synthesize. In addition, these probes show long response time (30-60 min), which is comparatively long. Therefore, the biological detection of CO still urgently needs probes with improved properties. To this end, de-

velopment of new methods to construct readily available fluorescent probes for CO with excellent sensing properties is desperately needed. Scheme 1. Fluorescent Detection of CO by Probes FL-CO-1, 1 and 2 Previous work: the probe has excellent sensing property, but lacks good stability PdCl2

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We recently used the cheap, highly fluorescent fluorescein and the well-known Pd0-mediated Tsuji–Trost reaction to have developed a readily available fluorescent turn-on probe (FLCO-1 in Scheme 1) for CO.27 This probe is allylcarbonate functionalized, which shows excellent sensing properties for CO and can be conveniently used for CO detection in living cells. However, due to the lack of sufficient stability of the allylcarbonate protection group, FL-CO-1 is not very stable. Although this probe can be stored well in solid state or in DMSO solution, it is found that this probe is sensitive to pH and UV light (see below), which prompted us to develop more stable CO probes. To sort out this problem, herein we prepared and investigated the second generation of fluorescein-based probes for CO, which uses allyl ether as the protection group and reaction site (probe 1 and 2 in Scheme 1). Compared to allylcarbonate, allyl

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ether should be more stable. In addition, the allyl group can also be readily removed by Pd0-mediated Tsuji–Trost reaction.28-30 Therefore, we envisaged that these allyl fluorescein ether-based probes should have great potential for CO detection based on the same mechanism.23, 27, 31 Indeed, our studies found that these allyl ethers showed satisfactory stability, and more importantly, they can be conveniently used to detect CO both in vitro and in living cells with excellent sensing properties. As far as we know, this is the first case of using allyl ether as reaction site for construction of fluorescent CO probes. In view of the easy preparation of allyl ethers and the diversity of fluorophores, this work provided a new way to devise fluorescent CO probes.

EXPERIMENTAL SECTION Materials and Chemicals. All reagents and chemicals were purchased from commercial suppliers and used without further purification. PBS buffers were prepared with distilled water that had been passed through a Millipore-Q ultrapurification system. All pH measurements were made with a Sartorius basic pH-meter PB-10. TLC analysis was performed using precoated silica plates. Melting points were determined using an X-4 apparatus and are not corrected. NMR spectra were recorded on Varian Mercury 400 or 600 instruments. High-resolution mass spectrometry (HR-MS) spectra were obtained on a Bruker microTOF-Q instrument. UV-Vis and fluorescence spectra were recorded on an Agilent Cary-100 UV-Vis spectrophotometer and an Agilent Cary Eclipse fluorescence spectrophotometer, respectively. Cell imaging was performed in an inverted ﬂuorescence microscopy with a 20× objective lens. Synthesis of Probe 1. To a solution of fluorescein (1.00 g, 3.0 mmol) in DMF (5 mL) was added K2CO3 (1.46 g, 10.5 mmol, 3.5 eq) and the mixture was stirred at room temperature for 15 min. Allyl bromide (3.63 g, 30 mmol, 10 eq) was then added. After stirring at room temperature for 30 min, the resulting mixture was heated to 70 °C for 5 h. The mixture was diluted with water (50 ml) after cooled down and extracted with ethyl acetate (50 mL × 4). The combined organic phase was washed with saturated NaHCO3 solution and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude solid product was purified via column chromatography (petroleum ether:ethyl acetate =15:1, v/v) to afford probe 1 as a white crystal (236 mg, yield 19%). Mp: 120-122℃. TLC (silica plate): Rf ~0.66 (petroleum ether:ethyl acetate 3:1, v/v). 1 H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 7.3 Hz, 1H), 7.61 (dt, J = 21.6, 7.4 Hz, 2H), 7.14 (d, J = 7.6 Hz, 1H), 6.75 (d, J = 1.9 Hz, 2H), 6.65 (d, J = 8.7 Hz, 2H), 6.60 (dd, J = 9.0, 1.9 Hz, 2H), 6.02 (ddd, J = 16.2, 10.4, 5.2 Hz, 2H), 5.41 (d, J = 17.2 Hz, 2H), 5.30 (d, J = 10.4 Hz, 2H), 4.55 (d, J = 5.1 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ 169.01, 159.80, 152.64, 151.98, 134.58, 132.14, 129.26, 128.64, 126.37, 124.53, 123.52, 117.71, 111.69, 110.92, 101.26, 82.80, 68.60. HR-MS (ESI): calcd for C26H21O5+ (M + H+): 413.1384; found 413.1383. Synthesis of Probe 2. Probe 2 (120 mg, yield 10%) was obtained as a white solid from the reaction of 2, 7-dichlorofluorescein and allyl bromide using the same procedure for probe 1. Mp: 182-184 °C. TLC (silica plate): Rf ~0.67 (petroleum ether:ethyl acetate 3:1, v/v). 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.2 Hz, 1H), 7.73–7.59 (m, 2H), 7.12 (d, J = 7.2 Hz, 1H), 6.76 (s, 2H), 6.71 (s, 2H), 6.09 – 5.99 (m, 2H), 5.47 (d, J = 17.1 Hz, 2H), 5.34 (d, J = 10.6 Hz, 2H), 4.64 (d, J = 4.9 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ168.49, 155.10, 151.64, 150.11, 135.07, 131.29, 129.87, 128.32, 125.95, 124.96, 123.42,

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118.21, 118.06, 110.96, 101.11, 81.45, 69.41. HR-MS (ESI): calcd for C26H19Cl2O5+ (M + H+): 481.0604; found 481.0605. Optical Studies. Stock solutions of probe 1 (2 mM), probe 2 (2 mM), PdCl2 (2 mM) and CORM-3 (10 mM) were separately prepared in HPLC grade DMSO and used freshly. Stock solutions (5-10 mM) of the analytes including NaF, NaCl, NaBr, NaI, NaN3, Na2SO4, NaHSO4, NaAcO, NaIO4, LiClO4, NaNO3, NaSCN, KCN, NaHCO3, NaHSO3, NaHS, cysteine (Cys), homocysteine (Hcy), glutathione (GSH), alanine (Ala), leucine (Leu), tryptophan (Trp), glycine (Gly), isoleucine (Ile), serine (Ser), phenylalanine (Phe), glutamine (Gln), glutamic acid (Glu), and lysine (Lys) were prepared in ultrapure water. ROS/RNS species such as ClO−, H2O2, NO2−, HNO, NO, ROO•, t BuOO• and •OH were prepared according our published procedure and used freshly.32-33 For a typical optical study, a 3.0 mL solution containing probe 1 (5 μM) and PdCl2 (5 μM) in PBS buffer (10 mM, pH 7.4, with 0.5% DMSO, v/v) was prepared in a quartz cuvette. The UV-Vis or fluorescent spectra were then recorded after addition of an analyte of interest at 37 °C (controlled by a temperature controller). Unless otherwise noted, the excitation wavelength for probe 1 and 2 was set at 490 and 493 nm, respectively, and slit width was set at dex = dem = 2.5 nm. Cytotoxicity of the Probes. This was assessed by the standard MTT assay. In brief, HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose medium, supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin in a 5% CO2, water saturated incubator at 37 °C. HeLa cells were then seeded in 96-well plates and cultured overnight. Various concentrations of the probe were added into the 96-well plate. After 24 h of treatment, 20 μL of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in FBS-free medium) were administered into the each well. After 4 h of incubation at 37 °C, all media were removed from wells and 200 μL of DMSO was added into each well to dissolve the intracellular formazan crystals. Absorbance of the solution was then measured at 490 nm with a microplate reader. The cell viability was expressed as the optical density ratio of the treatment to control. Values were calculated according to the formula: percentage of cell viability = OD treatment group/OD control group × 100%. Imaging of CO in Living Cells. HeLa cells were seeded in a 24-well culture plate for one night before cell imaging experiments. In a typical experiment of cell imaging, as controls, living cells were incubated with probe 1 (1 μM) and a mixture of probe 1 and PdCl2 (1 μM each) at 37 °C for 30 min, respectively, and they were imaged after washing with PBS for three times. For imaging of exogenous CO, HeLa cells were pre-treated with CORM-3 (1, 5, and 10 μM, respectively) for 30 min at 37 °C, and then were incubated with a mixture of probe 1 and PdCl2 (1 μM each) for 30 min at 37 °C. After washing the cells with PBS buffer, the cells were imaged. For imaging of heme stimulation produced CO, the procedure is almost the same to that of imaging exogenous CO, but using heme (100 μM) instead of CORM3 in the cell pre-treatment. Similar procedures were applied for cell imaging experiments of probe 2.

RESULTS AND DISCUSSION Probe Synthesis. The probe 1 and 2 were prepared directly from the commercially available fluorescein and 2, 7-dichlorofluorescein with allyl bromide in DMF under basic conditions (Scheme 2). Both probes can be isolated and purified by column


increasing of the concentration of CORM-3, both probe solutions showed progressive fluorescence increases around 520 nm until gradually close to saturation. Satisfactory linear relationships between the fluorescence changes and the CORM-3 concentrations in the range of 0-50 µM were observed for both probes (Fig. 2). The detection limits of the probe 1 and 2 systems for CO were thus determined to be about 46 nM (~1.3 ppb) and 29 nM (~0.8 ppb), respectively, based on signal-to-noise ratio S/N = 3. These results showed that both probe 1 and 2 are highly sensitive for CO. (a)

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Figure 1. Fluorescence spectra changes of the probe system (5 µM probe + 5 µM PdCl2) upon addition of CORM-3 (100 μM) in PBS buffer (10 mM, pH 7.4, with 0.5% DMSO, v/v) at 37 °C. (a) The probe 1 system upon addition of CORM-3. (b) Fluorescent intensity ratio (F/F0) changes of probe 1 at 516 nm as a function of time. (c) The probe 2 system upon addition of CORM-3. (d) Fluorescent intensity ratio (F/F0) changes of probe 2 at 527 nm as a function of time. All the spectra were recorded per min. Emission color changes under a light of 365 nm were inserted in (a) and (c), respectively. (a) 150

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The Stability of Probe 1 and 2. With probe 1 and 2 in hand, we first compared their stability with our first generation of fluorescein-based CO probe (FL-CO-1). Delightedly, like FLCO-1, almost pure PBS buffer can be used as solvent to investigate the properties of both probe 1 and 2. As shown in Fig. S1, all the fresh probe solutions in PBS buffer are non-fluorescent due to the lactone form of the fluorescein structures. However, probe FL-CO-1 showed considerable fluorescent enhancement over time when standing in PBS buffer (especially at more basic conditions), indicating it is sensitive to pH (Fig. S1a-c). Besides, FL-CO-1 is also sensitive to UV light (Fig. S1d). In contrast, both probe 1 and 2 did not show any changes under the same conditions. This result indicates that probe 1 and 2 showed the desired good stability and excellent photostability, which is much better than FL-CO-1. In addition, as expected, both probe 1 and 2 are not responsive to Pd2+ (PdCl2 was used), but showed rapid and significant fluorescence enhancement to Pd0 (Pd(PPh3)4 was used, Fig. S2). Considering that Pd2+ can be rapidly reduced to Pd0 by CO in situ,27,31 we envisaged that both 1 and 2 can be used as candidate probes for CO detection. The Optical Responses of Probe 1 and 2 for CO. The response of probe 1 and 2 for CO was then investigated in the presence of PdCl2. For safety reasons, CORM-3, a commercially available and water soluble CO-releasing molecule was used as the easy-to-handle CO source20-27,31 (it has been reported that 1 mole of CO can be liberated per mole of CORM-335). As shown in Fig. 1, upon addition of CORM-3, both the probe solutions (5 μM probe + 5 μM PdCl2) in PBS buffer (10 mM, pH 7.4, with 0.5% DMSO, v/v) showed rapid fluorescent turn-on responses, and within 20 min, the fluorescent intensities of both probe solutions were all enhanced by more than 150 times, which are remarkable. As a result, strong yellow-green fluorescence can be observed in both the probe 1 and 2 solutions (inserted in Fig. 1a and c, respectively). Meanwhile, in the UV-Vis spectra, both probe 1 and 2 solutions also showed significant responses to CORM-3, accompanied by distinct color changes from colorless to bright yellow (Fig S3). Clearly, these results indicate that both probe 1 and 2 can be used as visual and fluorescent turn-on probes for rapid detection of CO in aqueous solution under mild conditions. The Sensitivity. Different concentrations of CORM-3 were then added to the probe 1 and 2 solutions. Kinetic experiments showed that the reactions of 1 and 2 after addition of CORM-3 (10-400 μM) all performed smoothly, generating dose and time dependent fluorescence enhancement (Fig. S4). In the full fluorescent spectra changes, as shown in Fig. S5, upon progressive


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chromatography and their structures were proved by NMR and Mass analyses. Synthetic details and product characterizations can be found in the experimental section and the Supporting Information. Although the yield of probe 1 and 2 in this work is low due to formation of the undesired ether-ester product34, the synthetic procedure is direct and simple. Given all the starting materials are cheap and the isolation is easy, this one step reaction to obtain 1 and 2 is more convenient than the previously reported method,30 which used multi-step reactions. Scheme 2. The Synthesis of Probe 1 and 2


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Figure 2. (a) Fluorescent spectra changes of the probe 1 system (probe 1 + PdCl2, 5 µM each) upon addition of different concentrations of CORM-3 (0-50 µM). (b) A linear relationship of the fluorescence intensity changes of the probe 1 system at 516 nm against the concentration of CORM-3 from 0 to 50 µM (fluorescence intensity changes against 0-2 µM of CORM-3 are inserted). (c) Fluorescent spectra changes of the probe 2 system (probe 2 + PdCl2, 5 µM each) upon addition of different concentrations of CORM-3 (0-



50 µM). (d) A linear relationship of the fluorescence intensity changes of the probe 2 system at 527 nm against the concentration of CORM-3 from 0 to 50 µM (fluorescence intensity changes against 0-2 µM of CORM-3 are inserted). All data were collected in PBS buffer (10 mM, pH 7.4, with 0.5% DMSO, v/v) at 37 °C and each spectrum was obtained 20 min after mixing. The linear fitting equations are inserted in (b) and (d), respectively.

Moreover, as shown in Fig. S6, both probe systems can be used in a much lower dosage to detect CO efficiently. One can see that, even the dose of the probe system (probe + PdCl2) was reduced to 1 μM each or even lower, the probe system still showed distinct fluorescence enhancement after addition of small amount of CORM-3. This indicates that both probe systems can be used in a very low dosage to detect CO sensitively, which should be able to minimize the potential toxic effect caused by palladium ions. The Selectivity. The selectivity of the probe 1 and 2 systems was then investigated by their fluorescence responses to nearly 40 different analytes including common anions such as F−, Cl−, Br−, I−, AcO−, HCO3−, N3−, NO3−, SO42−, HSO4−, HSO3−, HS−, SCN−, CN−, ClO4−, IO4−, PO43−, and HPO42−, biothiols such as Cys, Hcy, and GSH, amino acids such as Ser, Trp, Ala, Phe, Gln, Glu, Lys, Leu, Gly, and Ile, reactive oxygen/nitrogen species (ROS/RNS) such as ClO−, H2O2, NO2−, NO, HNO, ROO•, t BuOO• and •OH. As shown in Fig. 3, only the addition of CORM-3 induced the probe 1 and 2 solutions significant fluorescence enhancements, while the other analytes showed almost no effect. Clearly, these results indicate that both probe 1 and 2 systems are highly selective for CO. Moreover, this highly selective CO detection process can be directly observed with the naked eye (Fig. S7, Supporting Information). (b) 180

the reaction mixtures of probe 1 and 2 were subjected to mass and TLC analyses (Fig. S8 for probe 1, Fig. S9 for probe 2). Mass analysis of the reaction mixture (probe 1 + PdCl2 + CORM-3) at 5 min showed a clear peak at 333.0779, which can be assigned to fluorescein (calcd 333.0757), indicating the reaction did produce fluorescein. In addition, a peak found at 373.1103 can be assigned to the mono allyl fluorescein ether, which has a calcd. mass of 373.1071 (Fig. S8a). The mono allyl fluorescein ether was then synthesized (Supporting Information) and used as reference sample for TLC analysis. The results of TLC analyses clearly indicated that the reaction underwent a mono allyl ether intermediate, and then transformed into fluorescein as the final product (Fig. S8b). Similar results can be found in the mass and TLC analyses of the reaction mixture for probe 2 (Fig. S9). Overall, all these results indicate that the sensing mechanism of the probe system for CO is most likely the process as proposed in Scheme 3. In this process, when the probe solution was treated with CO, Pd2+ was reduced to Pd0, which subsequently mediated the Tsuji–Trost reaction of the probe to remove the allyl group and simultaneous release the mono allyl fluorescein ether intermediate and the final product of the highly fluorescent fluorescein (or 2, 7-dichlorofluorescein), thus producing the observed optical changes. Another evidence to prove this sensing mechanism is that both probe 1 and 2 are not responsive for CORM-3 when Pd2+ is not present (Fig. S10). Scheme 3 The Proposed Sensing Mechanism for CO Detection with Probe 1 and 2

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Figure 3. Fluorescent spectra and fluorescent intensity responses of the probe 1 and 2 solutions for various analytes (100 µM). (a) and (b): the probe 1 solution (probe 1 + PdCl2, 5 µM each). (c) and (d): the probe 2 solution (probe 2 + PdCl2, 5 µM each). Various analytes are: 1. SO42−, 2. HSO4−, 3. HSO3−, 4. AcO−, 5. F−, 6. Cl−, 7. Br−, 8. I−, 9. ClO−, 10. IO4−, 11. ClO4−, 12. NO3−, 13. SCN−, 14. CN−, 15. N3−, 16. HCO3−, 17. ROO•, 18. NO, 19. H2O2, 20. tBuOO•, 21. HNO, 22. •OH, 23. HS−, 24. Cys, 25. Hcy, 26. GSH, 27. Ser, 28. Trp, 29. Ala, 30. Phe, 31. Gln, 32. Glu, 33. Lys, 34. Leu, 35. Gly, 36. Ile, 37. pd2+, 38. CORM-3. All spectra were monitored 20 min after mixing in PBS buffer (10 mM, pH 7.4, with 0.5% DMSO, v/v) at 37 ℃.

The Sensing mechanism. The optical changes of both probe systems for CO indicated the formation of the corresponding highly fluorescent fluorescein.27, 36-38 To confirm this,

The pH Effect. The pH effect of these two probes for sensing of CO was also tested. As shown in Fig. S11, both probe systems were stable at a wide pH range from 2-11, however, in the presence of CO, they showed significant fluorescence enhancements at pH 6-11. This indicates that both probes can be used for detection of CO over a wide pH range. Moreover, the best pH was found around a physiological relevant pH for both probes, which is highly favorable for their biological applications. Imaging of CO in Living Cells. Encouraged by the excellent properties, the potential applications of probe 1 and 2 for imaging of CO in living cells were investigated. MTT assays showed that the probe 1 system has very low cytotoxicity to living cells even it was used up to 50 μM (Fig. S12a and b), however, the probe 2 system showed a degree of cytotoxicity to living cells probably due to the presence of halogen atoms (Fig. S12c and d). Nevertheless, since both probe 1 and 2 are highly sensitive,


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we can use the probe at a very low dosage (1 μM) for fluorescent imaging of CO in living cells. Fig. 4 shows the results of using 1 μM of probe 1 for imaging of CO in living HeLa cells. One can see that, when HeLa cells were incubated with probe 1 or probe 1 with PdCl2 (1 µM each) for 30 min, no fluorescence can be observed (A1 and B1). However, when the cell culture was pre-incubated CORM-3, then incubated with the probe system (probe 1 + PdCl2, 1 µM each), a dose-dependent intracellular fluorescence was observed (C1-E1). Similar results can be observed when 1 μM of probe 2 was used (Fig. S13). These results clearly indicate that both probe 1 and 2 can be used to detect CO in living cells.

In summary, we reported for the first time of using allyl ether as reaction site to have developed two new readily available fluorescent probes for CO. These two CO probes exhibit much better stability over our recently reported allylcarbonate-based fluorescent CO probes, and more importantly, both of them showed excellent sensing properties for CO in the presence of PdCl2, giving rapid and distinct colorimetric and fluorescent turn-on signal changes. Moreover, they can be conveniently used to image CO in living cells. Overall, this work not only provided two new promising fluorescent CO probes, but also provided a new way to develop effective fluorescent probes for CO detection in living cells.

ASSOCIATED CONTENT Supporting Information Structure characterizations of probe 1 and 2 and other related compounds, and additional data. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 4. Fluorescent imaging of CO in HeLa cells by the probe 1 system (probe 1 + PdCl2, 1 µM each). Top row A-E: bright field images. Bottom row A1-E1: fluorescent images of AE, respectively, with excitation wavelength at 450-480 nm. A and A1: The cells were incubated with probe 1 (1 µM) for 30 min. B and B1: The cells were incubated with probe 1 and PdCl2 (1 µM each) for 30 min. C and C1, D and D1, and E and E1: cells were pre-incubated with 1, 5 and 10 μM of CORM-3 for 30 min, then with probe 1 and PdCl2 (1 µM each) for 30 min, respectively. Scale bar = 20 μm.

Since it was reported that heme can stimulate more heme oxygenase (HO)-derived CO in living cells,39 we also investigated the capability of probe 1 and 2 to detect the endogenous produced CO by heme treatment.27 As shown in Fig. 5, when the cells were pre-incubated with heme (100 μM) prior to incubation with a mixture of probe 1 and PdCl2 (1 µM each), the cells showed time dependent fluorescence enhancement (A1 to C1). This indicates that the process of endogenous produced CO by heme treatment can be tracked by probe 1. Similar results can be observed if probe 2 was used (Fig. S14). Therefore, these results indicated that both probe 1 and 2 have great potential to image endogenous produced CO.

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

ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (Grant Nos. 21472066 and 21672080), the Natural Science Foundation of Hubei Province (No. 2014CFA042) and the self-determined research funds of CCNU from the colleges’ basic research and operation of MOE (CCNU16A02028 and CCNU16JCCZX02).

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Figure 5. Fluorescent imaging of CO produced via heme stimulation in HeLa cells using the probe system (probe 1 + PdCl2). Top row A-C and bottom row A1-C1 are bright field images and the corresponding fluorescent images, respectively, with excitation wavelength at 450-480 nm. The cells were pre-incubated with 100 μM of heme for 1 h (A and A1), 5 h (B and B1) and 10 h (C and C1), then with the probe system (probe 1 + PdCl2, 1 µM each) for 30 min, respectively. Scale bar = 20 μm.

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CONCLUSION



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