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A fluorescent ESIPT probe for imaging COreleasing melecule-3 (CORM-3) in living systems Weiyong Feng, Shumin Feng, and Guoqiang Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01908 • Publication Date (Web): 10 Jun 2019 Downloaded from http://pubs.acs.org on June 10, 2019
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A Fluorescent ESIPT Probe for Imaging CO-Releasing Melecule-3 (CORM-3) in Living Systems Weiyong Feng, Shumin Feng, and Guoqiang Feng* Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, Chemical Biology Center, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, P. R. China.
ABSTRACT: CO-releasing molecule-3 (CORM-3) has been widely used in recently as a convenient and safe CO donor to release exogenous CO in living cells and to study the effects of CO on cellular systems. Accordingly, development of effective methods for detecting and tracking CORM-3 in living systems is of great significance. In this work, a readily available fluorescent probe for detection of CORM-3 was reported for the first time. This probe is based on an excited-state intramolecular proton transfer (ESIPT) dye phthalimide and uses the reducing ability of CORM-3 to convert a nitro group to an amino group, and more importantly, it can be used for rapid, highly selective and sensitive detection of CORM-3 with a distinct turn-on green fluorescence change in aqueous solution, living cells and animals, thus provided a useful tool for studying CORM-3 in living systems.
INTRODUCTION Carbon monoxide (CO) has been recognized as a critical endogenous signaling molecule with essential regulatory roles in a variety of physiological and pathophysiological processes.1 Moreover, CO also shows great potential as a drug molecule for cytoprotection, anti-inflammation, improvement of organ transplantation survival, inhibition of bacterial growth, and even anti-cancer.2-3 However, due to the inconveniency of direct using CO gas for therapeutic applications, the development and use of CO-releasing molecules (CORMs) have emerged as a new attractive research field in the past decade.4-9 CORMS are chemical agents to release and deliver CO into the cell so that they are expected to elicit various biological activities of CO and to replace the direct using CO gas in the treatment of diseases. Among various CORMs, a water soluble tricarbonylchloro(glycinato)ruthenium(II) complex, CORM-3 (Scheme 1) has received considerable attention in recent years.10-17 CORM-3 was firstly reported by Motterlini et al in 2003, which is stable in water at acidic pH but in physiological buffers rapidly liberates CO in solution.10 More importantly, growing evidence have shown that CORM-3 holds great therapeutic potential in transplantation and treatment of myocardial infarction and rheumatoid arthritis.5 Moreover, CORM-3 has also been used in recently as a convenient and safe CO donor to release exogenous CO in living cells and to study the effects of CO on cellular systems.18-21 Accordingly, effective methods for detecting and tracking CORM-3 in living systems would be highly valuable. Owing to the advantages of simplicity, high sensitivity and non-invasiveness, the detection based on fluorescent probes has become one of the most attractive detection method nowadays.22-23 In recent decades, great advances have been achieved in the development of fluorescent probes for various analytes, however, no fluorescent probes have been reported so far to detect CORM-3 specifically. Herein, we report for the first time a simple fluorescent probe (CORM3-green in Scheme 1) that can be used for effective detection of CORM-3 in both solution and living systems. This
probe can be readily prepared and is non fluorescent; however, upon addition of CORM-3, its nitro group can be reduced rapidly to the amino group, generating a highly fluorescent phthalimide (PTI in Scheme 1), which emits bright green fluorescence due to the excited-state intramolecular protontransfer (ESIPT) process.24-25 Notably, this provides a convenient, highly selective and sensitive detection method for CORM-3. Moreover, bioimaging of CORM-3 in living cells and animals with this probe was also conveniently and successfully achieved. Hence, this probe would be a useful tool for studying CORM-3 in living systems. Scheme 1. The structure of CORM-3 and a fluorescent probe (CORM3-green) for detection of CORM-3. ESIPT OFF
ESIPT ON
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EXPERIMENTAL SECTION Instruments and Materials. Chemicals and reagents used in this work were obtained from commercial suppliers (such as Sigma-Aldrich, Adams, and J&K chemicals etc.) and used as received. Zebrafishes (4 days old) were purchased from Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, Hubei province). Kunming mice (weight 20 ± 2 g) were supplied from Wuhan Center for Disease Control and Prevention. All animal experiments were proceeded with the permission of Wuhan Animal Ethics Committee. A Varian 600 NMR spectrometer (America) was used to collect the NMR spectra. A DSQ II GC/MS spectrometer (Thermo Scientific, America) and a micro TOF-Q instrument (Bruker, Germany) were used to collect the mass spectra. UVvis absorption spectra were recorded on a Cary-100
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spectrophotometer (Agilent, America). The fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Agilent, America). Cell and zebrafish imaging were collected on a TCS SP8 Laser Scanning Confocal Microscopy (Leica, Germany) with 40× and 10× objective lens, respectively. Mice imaging was collected on an In Vivo-Xtreme imaging system (Bruker, Germany). Synthesis of CORM3-green. A mixture of 3nitrophthalicanhydride (1.55 g, 80.8 mmol) and glycine (1.86 g, 3 equiv.) in 8 mL of acetic acid was stirred and heated to 120 °C for 6 h. After cooling to room temperature, the solvent was removed in vacuum. The residual solid was then purified by recrystallization using methanol to afford probe CORM3green as a white powder (1.88 g, yield 93%). Mp: 214-215 °C. TLC (silica plate): Rf ~0.7 (dichloromethane/methanol = 5:1, v/v). 1H NMR (600 MHz, DMSO-d6): δ 8.32 (dd, J = 7.3 and 3.3 Hz, 1H), 8.22 (d, J = 6.8 Hz, 1H), 8.09 (t, J = 7.0 Hz, 1H), 4.34 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 168.66, 165.52, 162.90, 144.98, 136.97, 133.42, 129.04, 127.71, 123.09, 31.15. EI-MS: m/z found 205.02 (M+-CO2H), 178.03 (M+-C2H2NO2). HR-MS calcd. for C10H6N2NaO6+ (M + Na+) 273.0118, found 273.0136. Synthesis of PTI. A mixture of CORM3-green (250 mg, 1 mmol) and Pd/C (50 mg) in 5 mL of methanol was put into a high-pressure autoclave. The autoclave was then sealed and filled with H2 until the pressure reached 1 MPa. Then the reaction mixture was heated to 60 °C for 24 h. After cooling to room temperature, the reaction mixture was filtered and the organic solvent was removed. The solid residue was purified by recrystallization in methanol to afford PTI as a yellow-green powder (210 mg, yield 96%). Mp: 217-218 °C. TLC (silica plate): Rf ~0.36 (dichloromethane/methanol = 5:1, v/v). 1H NMR (600 MHz, DMSO-d6): δ 7.44 (s, 1H), 6.99 (s, 2H), 6.48 (s, 2H), 4.20 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.38, 168.81, 167.62, 146.75, 135.45, 132.36, 121.73, 111.06, 108.93, 38.65. EI-MS: m/z found 220.06 (M+), 175.07 (M+-CO2H). HR-MS calcd.for C10H8N2NaO4+ (M + Na+) 243.0376, found 243.0380. Spectroscopic Studies. Unless otherwise stated, all the spectroscopic studies were investigated with 10 μM of probe CORM3-green at 37 °C and in 10 mM PBS buffer (pH 7.4, with 0.5% DMSO, v/v) solution. HPLC grade DMSO was used to prepare the 1 mM and 10 mM stock solutions of CORM3-green and CORM-3, respectively. According to the literature,26 reactive oxygen species/reactive nitrogen species (ROS/RNS) were prepared and used freshly in the test. The stock solutions of other analytes were prepared in ultrapure water. For fluorescent spectral measurements in solution, 420 nm was set as the excitation wavelength with the slit width dex = 5 nm, dem = 10 nm. Imaging of CORM-3 in Living Cells. Cell imaging of CORM-3 with probe CORM3-green was investigated in living HeLa cells. Living cells were cultured at 37 °C in a 5% CO2, water saturated incubator. Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin-streptomycin antibiotics were used as culture medium. The cytotoxicity of different concentrations of CORM3-green (0, 10, 20, 30, 40 and 50 μM) was first assessed by the standard MTT method before the cell imaging test. For imaging of CORM-3, HeLa cells were incubated with different concentrations of CORM-3 (0, 5, 10 and 25 μM) at 37 °C for
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30 min at first. The cells were further incubated with 10 μM CORM3-green for another 30 min at 37 °C after washing with PBS buffer for three times. Confocal imaging was then conducted with λex = 405 nm and emission was collected at a wavelength range of 450-550 nm after washing the testing cells with PBS buffer. Imaging of CORM-3 in Living Zebrafish. For imaging CORM-3 in living zebrafish, the wild type live zebrafishes (4 days old) were treated with an increasing concentrations (0, 20, and 50 μM) of CORM-3 for 30 min. After washing three times with PBS buffer, the zebrafishes were incubated with CORM3green (10 μM) for another 30 min. Finally, the zebrafishes were washed with PBS buffer, transferred into a cuvette, and then immobilized with 1% agar. Imaging test was conducted on a confocal scanning microscope with 405 nm of excitation wavelength and 450-550 nm of emission collection wavelength. Imaging of CORM-3 in Living Mice. Before imaging experiments, the living mice were divided into three groups. Then the mice were anesthetized with 200 μL of pelltobarbitalum natricum solution (1%, dissolved in saline) and their belly fur was carefully wiped with an alcohol sponge. The first group was injected nothing and imaged directly as the blank control. An intraperitoneal (i.p.) injection of 50 μL of probe CORM3-green (50 μM, in DMSO) was given to the second group, and the mouse with the probe was imaged after 20 min as the control of probe background. For the third group, the mouse was intraperitoneally injected with 50 μL of CORM3-green (50 μM, in DMSO) at first and then was injected 100 µL of CORM-3 (50 μM, in saline). After 5 and 20 min of incubation, the mouse was imaged, respectively. For the fluorescent imaging, excitation was set at 405 nm and emission was collected around 500 nm.
RESULTS AND DISCUSSION Probe Synthesis. CORM3-green and PTI can be readily prepared according to Scheme 2 by the reported method.25 Notably, the starting materials are cheap and no column chromatography is needed for the purification of the products (see the Experimental Section and the Supporting Information). Scheme 2. The synthesis of CORM3-green and PTI. NO2
O
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O
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AcOH, reflux O
O
NH2
O
OH
OH N O CORM3-green
O
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O OH
N O PTI
Fluorescent Detection of CORM-3. The basic optical spectra of CORM3-green and PTI can be found in Figure S1. One can see that, the probe CORM3-green solution (10 µM) displays almost no absorption above 390 nm and shows no fluorescence (Ф < 0.001, using quinine sulfate as reference). In contrast, the PTI solution (10 µM) displays an absorption peak at 390 nm and shows strong fluorescence at 503 nm (Ф = 0.318, using quinine sulfate as reference) with a large Stokes shift of 113 nm, exhibiting a typical fluorescence characteristic of ESIPT dyes.24,25 The response of CORM3-green for CORM-3 was investigated in 10 mM PBS buffer at pH 7.4 with 0.5% of DMSO (v/v) as co-solvent. As shown in Figure 1, upon addition of CORM-3 (100 µM), the absorption of the CORM3-green (10 µM) solution increased rapidly above 390 nm (Figure 1a. Note: the obtained absorption spectra includes the absorption of
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Figure 1. (a) UV-vis and (b) fluorescence spectral changes of CORM3-green (10 µM) at 37 ℃ after addition of CORM-3 (100 µM). Inset: emission color changes (under a light of 365 nm).
The resulting fluorescence spectrum of the CORM3green solution after addition of CORM-3 suggested that the formation of PTI is the reason for the fluorescence change (Scheme 1). To confirm this, mass spectrometry data of the reaction mixture of CORM3-green and CORM-3 was collected and analyzed. As shown in Figure S3, the reaction generated a peak with a mass of 243.0380, which can be assigned to PTI (exact mass calcd. for M + Na+, 243.0376), indicating PTI was formed. The conventional method of thin-layer chromatography (TLC) was also used to analyze the mixture of reaction, which also confirmed the formation of PTI (Figure S4). These results indicate that CORM-3 has the ability to reduce a nitro group to an amino group. The Selectivity of the CORM3-green. Various analytes were then tested to investigate the selectivity of CORM3green. As shown in Figure 2 and Figure S5, upon addition of common anions such as Cl−, HSO4−, HSO3−, S2O82−, SO32−, HCO3−, SCN−, Br−, NO3−, F−, S2O32−, I−, PO43−, CO32−, ClO4−, S2O72−, NO2−, N3−, C2O42−, S2− and HS− (all with sodium salts), metal ions such as Na+, K+, Ca2+, Cd2+, Mg2+ , Hg2+, Fe2+, Fe3+, Cu2+, Ba2+, Zn2+, Pd2+, Al3+, Mn2+, amino acids such as Gln, Thr, Asp, Tyr, Ser, His, Pyr, Arg, Val, Ala, Glu, Met, Leu, Phe, Lys, Glu, Ile, Trp, Gly, biothiols such as GSH, Hcy, Cys, and N-acetyl-cysteine (NAC), and a series of ROS/RNS such as ROO•, tBuOO•, ClO−, NO, HNO, ONOO−, and H2O2, the CORM3-green solution showed only negligible fluorescent changes. However, upon addition of CORM-3 to the probe CORM3-green solution, significant enhancement of fluorescence around 503 nm was observed, which is clearly different. The result indicates that CORM3-green is highly selective for CORM-3. Although the reduction of nitro group has been used to develop H2S probes,27 one can see that, probe CORM3-green shows high selectivity for CORM-3 over H2S (S2− and HS−). Moreover, competition experiments showed that the presence of other common analytes did not interfere the fluorescent detection of CORM-3 with CORM3-green (Figure S6), indicating
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Figure 2. (a) Fluorescent spectral and (b) fluorescent intensity (at 503 nm) responses of CORM3-green (10 µM) upon addition of various analytes. Analytes 1-70: 1. Gln, 2. Thr, 3. Asp, 4. Tyr, 5. Ser, 6. His, 7. Pyr, 8. Arg, 9. Val, 10. Ala, 11. L-Glu, 12. Met, 13. Leu, 14. Phe, 15. Lys, 16. D-Glu, 17. Ile, 18. Gly, 19. NAcetylglycine, 20. Trp, 21. Cys, 22. NAC, 23. Hcy, 24. GSH, 25. SCN−, 26. HSO3−, 27. S2O82−, 28. SO32−, 29. HCO3−, 30. HSO4−, 31. Br−, 32. NO3−, 33. F−, 34. S2O32−, 35. I−, 36. C3H3O3−, 37. PO43−, 38. CO32−, 39. ClO4−, 40. S2O72−, 41. VO4−, 42. NO2−, 43. Cl−, 44. N3−, 45. C2O42−, 46. S2−, 47. HS−, 48. Cd2+, 49. Mg2+, 50. Hg2+, 51. Fe2+, 52. Fe3+, 53. Cu2+, 54. Na+, 55. K+, 56. Ca2+, 57. Ba2+, 58. Zn2+, 59. Pd2+, 60. Al3+, 61. Mn2+, 62. H2O2, 63. ROO•, 64. tBuOO•, 65. NO, 66. HNO, 67. ONOO−, 68. ClO−, 69. CORM-3, and 70. None. Each spectrum was obtained after 30 min of incubation of probe and analyte. The concentration of each analyte is 100 M except that of GSH is 1 mM. λex = 420 nm, slit width: dex = 5 nm; dem = 10 nm.
The Sensitivity of the CORM3-green. The sensitivity of CORM3-green was investigated by means of addition of increasing concentrations of CORM-3 to the probe solution. As depicted in Figure 3, upon addition of increasing concentrations of CORM-3, the fluorescence of the CORM3-green solution at 503 nm showed a progressive increase until reached saturation. A satisfactory linear relationship between the fluorescence intensity changes at 503 nm and the CORM-3 concentrations in the range of 0-14 µM was observed. The detection limit of CORM3-green for CORM-3 was calculated to be about 16 nM according to the 3σ method (S/N = 3). This indicates that CORM3-green has high sensitivity for CORM-3. (b)
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CORM-3 and its product after release of CO). More apparently, the solution showed a marked fluorescence enhancement around 503 nm, and started to show strong green fluorescence which can be clearly observed under a 365 nm light (inset in Figure 1b). Kinetic studies showed that the fluorescent signal of CORM3-green itself was very stable, but changed rapidly after addition of CORM-3 with a vast fluorescence enhancement within 5 min both at 37 and 25 ℃ (Figure S2). These results clearly indicate that CORM3-green can be used for rapid detection of CORM-3 in almost pure aqueous solution with prominent fluorescent turn-on changes.
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Figure 3. (a) Fluorescence spectral changes of CORM3-green (10 µM) after incubation with increasing concentrations of CORM-3 (0 to 1500 µM). Each spectrum was obtained 30 min after incubation. Inset: the fluorescence saturation curve at 503 nm. (b) The linear relationship of the emission intensity at 503 nm against the concentration of CORM-3 from 0 to 14 µM. Inset: Intensity changes against the concentration of CORM-3 at 0, 0.02, 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, and 1.00 μM.
Fluorescent detection of CORM-3 with probe CORM3green was also investigated at different pHs. As shown in
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Analytical Chemistry Figure S7, CORM3-green showed significant fluorescent turnon responses for CORM-3 over a wide pH range (6-11), and the best pH for CORM-3 detection was found at ~7.4. This indicates that CORM3-green could be employed for fluorescent turn-on detection of CORM-3 around the physiological pH. This result is highly favorable for the application of CORM3-green to detect CORM-3 in biological systems. Cell Imaging of CORM-3. In view of the excellent performance of CORM3-green for detecting CORM-3 in solution, we next explored the potential of this new probe for imaging of CORM-3 in living cells. The MTT assay with standard cell viability protocols was firstly used to investigate the cytotoxicity of CORM3-green. As displayed in Figure S8, incubation of the cells with different concentrations of CORM3-green (0-50 μM) for 24 h, the cells showed high viability (>90%), suggesting CORM3-green has low cytotoxicity to living cells. Based on this, the potential of CORM3-green for imaging of CORM-3 in living cells was investigated on a Laser Scanning Confocal Microscopy. As shown in Figure 4, when HeLa cells were incubated with CORM3-green (10 µM), there showed almost no fluorescence in the green channel. However, when the living HeLa cells were incubated with CORM-3 with different concentrations (5, 10, and 25 μM, respectively), and then probed with 10 µM CORM3-green, the cells showed green fluorescence and the fluorescence intensity was dose dependent to the concentration of CORM-3 added. These results clearly indicate that CORM3-green can be applied to image CORM-3 in living cells. A
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Figure 4. Fluorescent imaging of CORM-3 in HeLa cells with CORM3-green (10 μM). A-D: Images of bright field. A1-D1 were fluorescent images with λex = 405 nm and emissions collected at 450-550 nm. A2-D2: merged images of bright field and fluorescent field. A, A1 and A2: the cells were incubated with CORM3-green (10 μM) for 30 min. Columns B-B2, C-C2, D-D2: cells were firstly treated with different concentrations of CORM-3 (5, 10, and 25 μM, respectively) for 30 min, then incubated with CORM3green (10 μM) for another 30 min, respectively. Scale bar = 10 um. Imaging of CORM-3 in Living Zebrafish. We further used zebrafish as animal models to investigate the potential of CORM3-green for imaging CORM-3 in living animals. As shown in Figure 5, probe CORM3-green showed almost no fluorescence background in living zebrafish.
However, when the zebrafish were treated with 20 and 50 μM of CORM-3, and then probed with CORM3-green, respectively, dose dependent green fluorescence was clearly observed, indicating CORM3-green has great potential for in vivo imaging applications. A
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Figure 5. Fluorescent imaging of CORM-3 in wild zebrafish with probe CORM3-green (10 μM). A-D: images of bright field. A1D1: fluorescent images with λex = 405 nm and emissions at 450550 nm. Bottom row A2-D2: the merged images of bright field and fluorescent field. A, A1 and A2: The zebrafish blank. B, B1 and B2: The zebrafish was incubated with CORM3-green (10 μM) for 30 min. Columns C-C2 and D-D2: The zebrafish were treated with 20 and 50 μM of CORM-3 for 30 min, then incubated with CORM3-green for another 30 min, respectively. Scale bar = 250 um.
Imaging of CORM-3 in Living Mice. Based on above results, imaging of CORM-3 with CORM3-green in living mice was further investigated. As shown in Figure 6, the mouse blank and the mouse injected only with CORM3green (50 μM, in 50 μL DMSO) showed almost no fluorescence in the peritoneal cavity (the injection area). However, when the mouse was given an injection of CORM3-green (50 μM, in 50 μL DMSO) and followed with CORM-3 (50 μM, in 100 μL saline), the mouse showed significant fluorescence enhancement around the injection area and the fluorescence intensified with incubation time. Apparently, these results further prove that CORM3-green has potential for imaging of CORM-3 in vivo.
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Figure 6. Fluorescent imaging CORM-3 in living mice with CORM3-green. (A) The blank mouse. (B) The mouse was only given an intraperitoneal injection of CORM3-green (50 μM, in 50
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Analytical Chemistry μL DMSO) and imaged after 20 min. (C-D) The mouse was given an intraperitoneal injection of CORM3-green (50 μM, in 50 μL DMSO), followed with injection of 50 μM CORM-3 (100 μL in saline) and then imaged respectively after 5 and 20 min of incubation. The red cycle shows the injection and the fluorescence collection area. (E) A plot of fluorescent intensity of images A-D. Excitation was set at 405 nm and emission was collected at 450550 nm
CONCLUSION In summary, we have developed a fluorescent ESIPT probe for imaging of CORM-3 in living systems. This probe is watersoluble and can be easily prepared from cheap reagents. More importantly, this probe provided a fast responsive, highly selective and sensitive fluorescent turn-on detection method for CORM-3. Moreover, this probe exhibits great potential for imaging of CORM-3 in living cells, zebrafishes and animals.
ASSOCIATED CONTENT Supporting Information Structure characterizations for CORM3-green and PTI, data for the mechanism of sensing, and additional data of UV-vis and fluorescence studies. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *Phone: +86 27 67862163; E-mail:
[email protected].
ACKNOWLEDGMENT We gratefully thank the National Natural Science Foundation of China (21672080 and 21472066) and Fundamental Research Funds for the Central Universities (CCNU19TS007) for financial support.
REFERENCES (1) (2) (3) (4) (5)
(6) (7)
(8) (9)
Verma, A.; Hirsch, D.-J.; Glatt, C.-E.; Ronnett, G.-V.; Snyder, S.-H. Carbon monoxide: a putative neural messenger, Science 1993, 259, 381–384. Wu, L.; Wang, R. Carbon monoxide: endogenous production, physiological functions, and pharmacological applications, Pharmacol Rev. 2005, 57, 585–630. Motterlini, R.; Otterbein, L.-E. The therapeutic potential of carbon monoxide, Nat. Rev. Drug Discov. 2010, 9, 728–743. Schatzschneider, U. Novel lead structures and activation mechanisms for CO-releasing molecules (CORMs), Br. J. Pharmacol. 2015, 172, 1638–1650. Ling, K.; Men, F.; Wang, W.; Zhou, Y.; Zhang, H.; Ye, D.-W. Carbon monoxide and its controlled release: therapeutic application, detection, and development of carbon monoxide releasing molecules (CORMs), J. Med. Chem. 2018, 61, 2611– 2635. Romão, C.-C.; Blättler, W.-A.; Seixas, J.-D.; Bernardes, G.-J. Developing drug molecules for therapy with carbon monoxide, Chem. Soc. Rev. 2012, 41, 3571–3583. Chakraborty, I.; Carrington, S.-J.; Mascharak, P.-K. Design strategies to improve the sensitivity of photoactive metal carbonyl complexes (photoCORMs) to visible light and their potential as CO-donors to biological targets, Acc. Chem. Res. 2014, 47, 2603–2611. Heinemann, S.-H.; Hoshi, T.; Westerhausen, M.; Schiller, A. Carbon monoxide - physiology, detection and controlled release, Chem. Commun. 2014, 50, 3644–3660. Ji, X.; Wang, B. Strategies toward organic carbon monoxide prodrugs, Acc. Chem. Res. 2018, 51, 1377–1385.
(10) Clark, J.-E.; Naughton, P.; Shurey, S.; Green, C.-J.; Johnson, T.R.; Mann, B.-E.; Foresti, R.; Motterlini, R. Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule, Circ. Res. 2003, 93, 2–8. (11) Foresti, R.; Hammad, J.; Clark, J. E.; Johnson, T. R.; Mann, B. E.; Friebe, A.; Green, C. J.; Motterlini, R. Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule, Br. J. Pharmaco. 2004, 142, 453–460. (12) Tinajero-Trejo, M.; Denby, K.-J.; Sedelnikova, S.-E.; Hassoubah, S.-A.; Mann, B.-E.; Poole, R.-K. The carbon monoxide-releasing molecule, CORM-3 (Ru(CO)3Cl(Glycinate)), targets respiration and oxidases in campylobacter jejuni, generating hydrogen peroxide, J. Biol. Chem. 2014, 289, 29471–29482. (13) Song, H.; Bergstrasser, C.; Rafat, N.; Höger, S.; Schmidt, M.; Endres, N.; Goebeler, M.; Hillebrands, J.-L.; Brigelius-Flohé, R.; Banning, A.; Beck, G.; Loesel, R.; Yard, B.-A. The carbon monoxide releasing molecule (CORM-3) inhibits expression of vascular cell adhesion molecule-1 and E-selectin independently of haem oxygenase-1 expression, Br. J. Pharmacol. 2009, 157, 769–780. (14) Song, H.; Hoeger, S.; Hillebrands, J.-L.; Mandel, I.; Loesel, R.; Beck, G.; Schilling, L.; Schnuelle, P.; Yard, B. CORMs protect endothelial cells during cold preservation, resulting in inhibition of intimal hyperplasia after aorta transplantation in rats, Transplant International. 2010, 23, 1144–1153. (15) Vadori, M.; Seveso, M.; Besenzon, F.; Bosio, E.; Tognato, E.; Fante, F.; Boldrin, M.; Gavasso, S.; Ravarotto, L.; Mann, B.-E.; Simioni, P.; Ancona, E.; Motterlini, R.; Cozzi, E. In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM3, in the xenogeneic pig-to-primate context, Xenotransplantation. 2010, 16, 99–114. (16) Santos-Silva, T.; Mukhopadhyay, A.; Seixas, J.-D.; Bernardes, G.-J.; Romão, C.-C.; Romão, M.-J. CORM-3 reactivity toward proteins: the crystal structure of a Ru(II) dicarbonyl-lysozyme complex, J. Am. Chem. Soc. 2011, 133, 1192–1195. (17) Long, R.; Salouage, I.; Berdeaux, A.; Motterlini, R.; Morin, D. CORM-3, a water soluble CO-releasing molecule, uncouples mitochondrial respiration via interaction with the phosphate carrier, Biochim. Biophys. Acta. 2014, 1837, 201–209. (18) Michel, B. W.; Lippert, A. R.; Chang, C. A reaction-based fluorescent probe for selective imaging of carbon monoxide in living cells using a palladium-mediated carbonylation, J. Am. Chem. Soc. 2012, 134, 15668–15671. (19) Feng, W.; Liu, D.; Feng, S.; Feng, G. A readily available fluorescent probe for carbon monoxide imaging in living cells, Anal. Chem. 2016, 88, 10648–10653. (20) Feng, S.; Liu, D.; Feng, W.; Feng, G. Allyl fluorescein ethers as promising fluorescent probes for carbon monoxide imaging in living cells, Anal. Chem. 2017, 89, 3754–3760. (21) Feng, W.; Feng, G. A readily available colorimetric and nearinfrared fluorescent turn-on probe for detection of carbon monoxide in living cells and animals, Sens. Actuators B. 2018, 255, 2314–2320. (22) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging, Chem. Rev. 2010, 110, 2620-2640. (23) Wu, D.; Sedgwick, A.-C.; Gunnlaugsson, T.-E.; Akkaya, U.; Yoon, J.; James, T.-D. Fluorescent chemosensors: the past, present and future, Chem. Soc. Rev. 2017, 46, 7105–7123. (24) Yang, L.; Liu, X.; Gao, L.; Qi, F.; Tian, H.; Song, X. A selective and sensitive phthalimide-based fluorescent probe for hydrogen sulfide with a large stokes shift, RSC. Adv. 2015, 5, 98154– 98159. (25) Feng, W.; Bai, L.; Jia, S.; Feng, G. A novel phthalimide-rhodolbased ESIPT-FRET system for rapid colorimetric and ratiometric fluorescent detection of palladium, Sens. Actuators B. 2017, 260, 554–568. (26) Yu, D.; Huang, F.; Ding, S.; Feng, G. Near-infrared fluorescent probe for detection of thiophenols in water samples and living cells, Anal. Chem. 2014, 86, 8835–8841.
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Analytical Chemistry (27) Yu, F.; Han, X.; Chen, L. Fluorescent probes for hydrogen sulfide detection and bioimaging, Chem. Commun. 2014, 50, 12234–12249.
For TOC only CO H2 N Ru O OC Cl
OC
ESIPT OFF
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NO2
O
H
CORM-3 OH
N O
ESIPT ON O
O
N
H O OH
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In living cells In living zebrafish in living mice
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