Near-infrared phosphorescent terpyridine osmium(ii) photosensitizer complexes for photodynamic and photooxidation therapy

View Article Online View Journal INORGANIC CHEMISTRY F R O N T I E R S Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: G. Chen, J. Zhu, A. Ouyang, N. Lu, Y. Wang, Q. Zhang and P. Zhang, Inorg. Chem. Front., 2020, DOI: 10.1039/D0QI00846J. This is an Accepted Manuscript, which has been through the Volume 6 | Number 1 | January 2019 Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the INORGANIC Information for Authors. 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In no event shall the Royal Society of Chemistry be held responsible for any errors rsc.li/frontiers-inorganic or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/frontiers-inorganic Page 1 of 8 PInleoarsgea ndioc Cnhoet madisjturys tF mroanrtgieinrss Inorganic Chemistry Frontiers ARTICLE Near-infrared phosphorescent terpyridine osmium(II) t p complexes photosensitizers for photodynamic and i r photooxidation therapy c Received 00th January 20xx, s Accepted 00th January 20xx u Chen Ge,‡ Jiayi Zhu,‡ Ai Ouyang,‡ Nong Lu, Yi Wang, Qianling Zhang, Pingyu Zhang* DOI: 10.1039/x0xx00000x n www.rsc.org/ Osmium(II) complexes usually exhibit an excellent NIR (near-infrared) emission, which conforms to the optical window in a a biological system. Here we designed three mitochondria-targeted NIR terpyridine Os(II) complexes photosensitizers, which M have great NIR phosphorescence emission and long phosphorescence lifetime. The emission spectra of the osmium(II) complexes were red-shifted with the increasing of the conjugated planes of their terpyridine ligand (Os3 > Os2 > Os1). d Furthermore, the Os(II) complexes, especially Os3 can produce singlet oxygen and oxidize NADH under both 465 nm and e 633 nm LED light irradiation. It was found that Os3 has certain phototoxicities upon red light irradiation, which have great t potential for NIR photodynamic therapy. p e c have been approved by FDA for clinical use as PDT agents.24 c Introduction However, the properties of these agents need to be improved to allow A for more widespread clinical applications. These include:1) aqueous Cancer is a disease that endangers human health, which is caused by genetic mutation and develops in a special environment.1,2 The solubility; 2) light absorption in the spectral range of the biological s environment; 3) selectivity towards cancer cells over normal cells; 4) success of platinum as anticancer agent has greatly stimulated the r researchers to research on the transition metal complexes as antitumor photostability; 5) avoidance of photodegradation to form bilirubin, e drugs.3,4 However, on account of the high toxicity and drug tolenrance, which can cause hepatitis; 6) clearance from the body; 7) localization i (in skin tissues they may cause severe skin sensitivity). In recent years, t cis-platinum makes people explore more efficient anticancer drugs. n inorganic metal-based complexes have been also brilliant According to the research, platinum derivatives or other transition o photosensitizers, some of which could even generate more 1O than metal complexes including osmium, gold, iridium and ruthenium 2 organic molecules.25-28 Metal-based tetrapyrrolic derivatives of PdII r were discovered to have the anticancer activity,5-14 which have F (WST11), LuIII (Lutex), and SnIV (Purlytin), RuII (TLD-1433) have aroused the scientists' intense research interest. been developed; these are currently in clinical trials as PDT agents.29 The osmium complexes could emit intense infrared y Importantly, the illuminated light is usually visible red. Most phosphorescence and high light stability which has the ability of anti- r photosensitizers strongly absorb light at 630 nm or longer. Compared biological background interference. There is a large stocks t with Ru(II) and Ir(III) complexes of inorganic metal-based complexes, s displacement between excitation and emission spectra and long- wavelength metal transfer absorption (MLCT) can avoid coloring osmium complexes are more potential near-infrared photosensitizers i m for their resistance biological background, near-infrared fluorescence interference.15-17 And the molecular structure have greater plasticity, with strong penetration and PDT treatment. Recently, McFarland and so that it has good photodynamic treatment effect and imaging e co-workers reported three osmium-based photosensitizers,30,31 function. It is potential as a research hot spot in metal complexes. h [Os(biq) (phen)]-(PF )(TLD1822, biq = 2,2'-biquinoline,phen=1,10- These advantages lay a solid foundation for the research of anticancer 2 62 phenanthroline),[Os(biq) (IP)](PF ) (TLD 1829, IP = imidazo[4,5- C drugs. 2 62 f][1,10]phenanthroline) and [Os(biq)(dppn)](PF ) (TLD1824, dppn Photodynamic therapy (PDT) is an attractive therapy method which 2 62 = benzo[i]dipyrido[3,2-':2', 3' -c]phenazine). These photosensitizers c has been used to treat cancer over the past few decades. PDT requires are panchromatic, can be activated from 200 to 900 nm and have i photosensitizers (PSs) which are not toxic to cells in the dark but toxic n strong resistance to photobleaching. In vitro studies showed after irradiation.18-23 Organic photosensitizers with similar a photodynamic therapy efficacy with both red and NIR light under tetrapyrrolic scaffold, Photofrin, Chlorine6, Visudyne and Foscan normoxic and hypoxic conditions, which translated into good in vivo g efficacy of TLD1829 in a subcutaneous murine colon cancer model. r In the present work, we explore three mitochondria targeted Os(II) o a.College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, P. R. China. E-mail: p.zhang6@szu.edu.cn (P.-Y. Z.) terpyridine osmium complexes with increasing conjugate plane of n b.‡These authors contributed equally. their terpyridine ligand (Os1-Os3). They have strong near-infrared I † Footnotes relating to the title and/or authors should appear here. phosphorescence emission. With the irradiation of blue light of 465 Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x nm wavelength and red light of 633 nm wavelength, osmium complexes could produce singlet oxygen, convert NADH to NAD+ This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins .MA 62:32:6 0202/7/9 no dnalgnE weN fo ytisrevinU yb dedaolnwoD .0202 rebmetpeS 70 no dehsilbuP View Article Online DOI: 10.1039/D0QI00846J PInleoarsgea ndioc Cnhoet madisjturys tF mroanrtgieinrss Page 2 of 8 ARTICLE Journal Name and exhibit highly phototoxicities against cancer cells. DFT calculations To obtain further understanding of the photophysical properties of Result and Discussion the complexes, density functional theory (DFT) calculations were performed for the Os(II)-terpyridine complexes. DFT calculations Synthesis and characterization were utilized to rationalize the fluorescence change of the osmium t complexes by employing the Gaussian 16 program package.33 The p The synthesis of three Os(II)-terpyridine complexes (Figure 1a) were HOMO and LUMO energy levels of the Os(II)-terpyridine complexes i described in the experimental section. They were characterized by the r Table 1. Photophysical properties of Os1-Os3 in PBS. ESI-MS, NMR and elemental analysis (Figures S1-S9). The c photophysical properties were further investigated. As shown in s Complex  /nm  /nm  a Lifetime  (1O)b abs em em 2 Figure 1b, Os1-Os3 exhibited an obvious metal-to-ligand charge u transfer (MLCT) absorption band between 420 and 580 nm with ()/ns n absorption at 476 nm, 481 nm and 492 nm, respectively (Table 1), as Vis/NIR 465 /633 465 /633 a well as a weak 1GS to 3MLCT absorption band in the NIR region (650- Os1 476 583/702 0.0096 4.68/177.32 0.222/0.137 775 nm). The complexes Os1-Os3 exhibited dual emissions at both M visible and NIR wavelength (Figure 1c) with the maximum Os2 481 588/709 0.0055 5.78/98.92 0.242/0.174 wavelengths around 583/702 nm, 588/709 nm and 583/719 nm, Os3 492 583/719 0.0025 2.78/92.19 0.229/0.240 d respectively. NIR emission is effective situated inside the biological a Φ em , the luminescence quantum yield in PBS. b Φ(1O 2 ), quantum yield for 1O 2 e window and out of the range of cellular autofluorescence. The NIR determined under 465 nm and 633 nm light irradiation, respectively. [Ru(bpy) 3 ]2+ was used as a standard (Φ = 0.028, Φ(1O) = 0.22 in water).32 t emission from Os1 to Os3 were slightly redshifted, due to the em 2 p increased electron-releasing strength of the substituent group. e in PBS solvent were calculated. Representative plots of the HOMO c and LUMO frontier orbitals are depicted in Figure 2, while the energy c gap between them are provided in Table S1. The calculated HOMO- A LUMO gap of Os1 was 9.64 eV and the HOMO majorly located at the osmium central atom and the tpy precursor while LUMO s distributed at the osmium central atom and the tpy ligand. It was r important to point out that the HOMO distribution of Os2 were e majorly located at the osmium central atom and the tpy-ph ligand, i while the LUMO of Os2 distributions at the osmium central atom and t n the tpy precursor. And the calculated HOMO-LUMO gap of Os2 was 5.38 eV. The LUMO of Os3 located at the osmium central atom and o the tpy precursor while HOMO of Os3 was distributed at the tpy-ph- r tpy ligand. And the calculated HOMO-LUMO gap of Os3 was 4.70 F Figure 1. (a) The chemical structures of the Os(II)-terpyridine complexes (Os1- eV. Therefore, with the increasing conjugate plane of the auxiliary Os3). (b) The UV-vis absorption and (c) the emission spectra of Os1-Os3 in the y PBS solution at 310 K. The wavelength of excitation was 488 nm. ligand, the contribution of the charge transfer transition between the r coordination and the main ligand gradually exists, resulting in the red t The luminescence decay experiment was studied and the results shift of the spectrum. s were exhibited in Figure S10. Os(II)-complexes exhibited much i longer luminescence lifetimes in the NIR region (650-775 nm) ( = m 117.39 ns for Os1;  = 98.92 ns for Os2;  = 92.19 ns for Os3), the luminescence lifetimes in the visible region (550-600 nm) were 4.682 e ns for Os1, 5.78 ns for Os2 and 2.78 ns for Os3. The luminescence h quantum yields () were determined by the reported method.32 The C value of  of complexes Os1-Os3 were 0.0096, 0.0055 and 0.0025 in water, respectively (Table 1). In addition, it is clear from the UV-vis c absorption and emission spectra before and after 24 hours that the i osmium complexes do not decompose significantly after one day in n PBS solution (Figure S11). Then the photo-stabilities of Os1-Os3 in a PBS solution were monitored by UV-vis spectroscopy and Figure 2. The calculated HOMO and LUMO energy levels of Os1-Os3. g fluorescence spectrometer, which recorded every 10 min under blue r and red LED light irradiation. The changes of temperature of Os1- o Os3 solution after irradiation were recorded as well (Figures S12- NIR phosphorescence imaging n S13). The results showed that they are both photo-stable under 465 Based on their excellent phosphorescence properties, we investigated I nm and 633 nm light irradiations and there are no any thermal effects the imaging application of the Os(II) complexes in living cells by under irradiation. using confocal laser scanning microscopy. Hep-G2 cells were treated 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 62:32:6 0202/7/9 no dnalgnE weN fo ytisrevinU yb dedaolnwoD .0202 rebmetpeS 70 no dehsilbuP View Article Online DOI: 10.1039/D0QI00846J Page 3 of 8 PInleoarsgea ndioc Cnhoet madisjturys tF mroanrtgieinrss Journal Name ARTICLE with 10 M Os1-Os3 for 2 h and then subjected to confocal imaging. molecule with a known quantum yield ([Ru(bpy)]2+, Ф(1O ) =0.22). 3 2 The excitation wavelength was 488 nm, and the emission wavelengths The singlet oxygen quantum yields (Ф (1O )) of Os1-Os3 were 0.222, 2 were collected at 650-800 nm (red channel) and 550-600 nm (green 0.242 and 0.229 (465 nm light) and 0.137, 0.174 and 0.240 (633 nm channel), respectively. As shown in Figure S14, cytoplasm of the light), respectively. living cells was clearly lit up in both red and green channels. Moreover, intracellular generation of 1O by the Os(II)-complexes 2 t Especially for the NIR emission, it was strongly coincident with the upon photo-irradiation was detected in live Hep-G2 cells. As shown p biological optical window. in Figure 6, bright green singlet oxygen sensor green (SOSG) i In order to study more precisely about their subcellular localization, fluorescence assignable to 1O 2 was observed after both 465 nm LED- r light (13 mW/cm2) or 633 nm LED-light (20 mW/cm2) irradiation but c did not appeared in the dark. And the intracellular 1O concentration s 2 were u n a M d e t p e Figure 4. Images of live Hep-G2 cells stained with Os1-Os3 (10 M) for 2 h and c Mito-Tracker Green (200 nM) for 30 min. Red Channel:  = 488 nm,  = 650- ex em c 800 nm. Mito-Tracker Green:  = 488 nm,  = 530-580 nm. ex em Figure 5. EPR signals of Os1-Os3 (a) trapped by TEMP and (b) trapped by DMPO A we stained living Hep-G2 cells with a commercial dye Mito-Tracker in the dark or upon 465 nm (13 mW/cm2, 1 h) or 633 nm (20 mW/cm2, 1 h) light irradiation. Green (MTG) after being treated with the Os(II) complexes. As shown s in Figure 4, the red phosphorescence spots of the Os(II) complexes r mainly overlapped with the green phosphorescence of the e mitochondria. All Os(II) complexes showed high Pearson's i t colocalization coefficient (PCC) with MTG (PCC Os1–MTG = 75%, n PCC = 89%, PCC = 74% ). Meanwhile, litter Os2–MTG Os3–MTG o colocalization for the Os(II) complexes and Lyso-Tracker Red (Figure r S15) or Hoechst 33258 was observed (Figure S16). F Determination of ROS y r Electron spin resonance (EPR) spin trapping is a powerful and t sensitive way to detect the formation of reactive oxygen species s Figure 6. Confocal microscopy imaging of the Hep-G2 cells colabeled with 10 M (ROSs). ROSs readily interact with the diamagnetic nitrone spin traps i Os1-Os3 for 1 h and singlet oxygen sensor green (SOSG, 5 M, 30 min) in the m to form stable spin adducts that can be monitored by the EPR spectrum. absence or presence of NaN (5 mM, 1 h) in the dark and under light irradiation for 3 The adducts produced by ROSs and 2,2,6,6-tetramethylpiperidine 5 min at (a) 465 nm and (b) 633 nm. e (TEMP) were detected in each Os(II)-complexes after irradiation with inhibited significantly by NaN (1O quencher). These results h 465 nm or 633 nm LED light, a three-line signal with 1:1:1 intensity 3 2 demonstrated that Os(II)-complexes had a great efficacy in the C appeared between 3480-3530 GM (Figure 5). However, there was no photosensitization of molecular oxygen, one of the prerequisites for signal observed in the dark. In addition, the signal of the adducts PDT agents. c produced by hydroxyl radicals (•OH−) or superoxide anion (•OOH−) i and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) did not be monitored n both in dark and after 465 or 633 nm light irradiation. The results Dark cytotoxicity and phototoxicity a suggested that Os1-Os3 could generate 1O but not •OH− or •OOH− 2 The phototoxicities of Os(II) complexes towards Hep-G2 cells were g after both 633 nm and 465 nm light irradiation. further investigated. The cells were treated with different The 1O production quantum yields were evaluated by a method, r 2 concentrations of Os(II) complexes for 4 h, medium was replaced with o which was based on the reaction of 1O with an imidazole derivative 2 fresh non-complexes medium in dark and light (465 nm, 13 mW/cm2, to form a trans-annular peroxide adduct, which was able to quench the n 1 h or 633 nm, 20 mW/cm2, 1 h) plates. All plates were incubated for absorbance of a probe molecule, p-nitrosodimethyl aniline (RNO). I another 43 h. As shown in Figure 7 and Table S2, all complexes were We survey RNO ultraviolet spectrum every five minutes. The results found to be noncytotoxic under dark conditions (IC ˃ 100 M). were showed in Figure S17 and Table 1 by comparing with a reference 50 However, Os3 exhibited highly toxic to Hep-G2 cells after both 465 This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3 Please do not adjust margins .MA 62:32:6 0202/7/9 no dnalgnE weN fo ytisrevinU yb dedaolnwoD .0202 rebmetpeS 70 no dehsilbuP View Article Online DOI: 10.1039/D0QI00846J PInleoarsgea ndioc Cnhoet madisjturys tF mroanrtgieinrss Page 4 of 8 ARTICLE Journal Name nm light and 633 nm light irradiation. IC values of Os3 towards Figure 7. Growth curves for the HepG-2 cells treated with (a) Os1 (b) Os2 or (c) 50 Os3 for 4 h in the dark or followed by irradiation with 465 nm (13 mW/cm2, 1 h) or Hep-G2 cancer cells were 1.23 M upon blue light irradiation and 633 nm light (20 mW/cm2, 1 h), and then incubated for a further 43 h. The cells 4.05 M upon red light irradiation. Os1 and Os2 showed weak toxic were transferred to fresh medium before irradiation. (d) The IC values of the 50 to Hep-G2 cells upon blue and red light irradiation. In addition, all complexes according to (a), (b) and (c). Os(II) complexes showed almost no cytotoxicity to normal liver LO2 t cells both in the dark and upon irradiation (IC ˃ 100 M) (Figure 50 Photooxidation of NADH p S18). i NADH is an important enzyme for mitochondrial redox in cells, and r the oxidation of NADH into NAD+ can destroy the entire respiratory c chain of cells and kill cancer cells.34-36 We have investigated whether s the 1O 2 generated by Os(II) complexes can oxidate NADH, and thus u provide a photo-oxidation pathway. The photocatalytic efficiency of n Os(II) complexes toward NADH (175 M) was quantified by a ultraviolet-visible spectroscopy. As shown in Figure 8, the absorbance M of NADH at 339 nm decreased gradually under the photoirradiation (both of 633 nm or 465 nm light). In contrast, the absorbance at 339 nm changes slightly under dark conditions. It suggested that the Os(II) d complexes can significantly reduce the enzyme activity of NADH e after light irradiation.The observed NADH oxidation turnover t numbers (TONs) of the osmium(II) complexes after illumination were p significantly higher than that in the darkness (Table S3). e c c A s r e i t n o r F y r t s i m Figure 8. Photocatalytic oxidation of NADH (175 M) by Os1-Os3 (10 M) in PBS, as monitored by UV-vis spectroscopy. The UV-vis spectra recorded every 5 min for e 1 h under blue (465 nm) or red (633 nm) LED light. And the turnover numbers (TONs) values were calculated by measuring the absorption difference at 339 nm. h C Conclusions Experimental Section c In summary, three Os(II) terpyridine complexes have been developed as mitochondria-targeted NIR photosensitizers. The complexes Materials and instruments i n exhibited specific staining ability of mitochondria can be used in near Terpyridine, 2,2':6',2''-Terpyridine,4',4''''-(1,4-phenylene)bis a infrared phosphorescence imaging. And the emission spectra were (NH )[OsCl ], NaClO , CDCl and DMSO-d6 were purchased from 42 6 4 3 g red-shifted with the increasing conjugate plane of the terpyridine Aladdin. Ethylene glycol was purchased from Macklin. Liver ligand. In addition, these complexes produced singlet oxygen, hepatocellular carcinoma (Hep-G2) and human normal liver (LO2) r o converted NADH to NAD+ and exhibited highly phototoxicities cell lines were purchased from ECACC. Roswell Park Memorial against cancer cells upon both 465 nm and 633 nm light irradiation. Institute medium (RPMI-1640), fetal calf serum (FCS) were n Overall, this study suggested that the Os(II) terpyridine complexes purchased from Sigma-Aldrich. Mito-Tracker Green and Hoechst I have a potential for developing NIR photodynamic therapy agents. 33258 were purchased from Beyotime Biotechnology. Lyso-Tracker Red was purchased from KeyGEN BioTECH. 4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 62:32:6 0202/7/9 no dnalgnE weN fo ytisrevinU yb dedaolnwoD .0202 rebmetpeS 70 no dehsilbuP View Article Online DOI: 10.1039/D0QI00846J Page 5 of 8 PInleoarsgea ndioc Cnhoet madisjturys tF mroanrtgieinrss Journal Name ARTICLE NMR spectra were recorded on a BrukerAV-500M spectrometer. acetonitrile. It was further purified upon recrystallization from Elemental analysis was performed by Exeter Analytical using a acetonitrile- water (2:1) mixture in acidic condition (10−4 M HClO). 4 CHN/O/S Elemental Analyser (CE440). Positive ion ESI-MS spectra 1H NMR (500 MHz, DMSO-d6) δ 9.47 (s, 2H), 9.08 (t, J = 8.4 Hz, were obtained using an Agilent 6130B single quad coupled to an 4H), 8.80 (d, J = 8.2 Hz, 2H), 8.37 (d, J = 7.4 Hz, 2H), 8.03 (t, J = 8.1 automated sample delivery system. UV-visible absorption spectra Hz, 2H), 7.92 – 7.84 (m, 4H), 7.75 (t, J = 7.8 Hz, 2H), 7.41 (d, J = 5.4 t were recorded on a Varian Cary 300UV-vis spectrophotometer. The Hz, 2H), 7.30 (d, J = 5.2 Hz, 2H), 7.18 (t, J = 8.4 Hz, 4H). 13C NMR p fluorescence spectra were recorded on a JASCO FP-6500 Fluorimeter. (126 MHz, DMSO-d6) δ 160.22, 160.02, 155.17, 155.13, 154.88, i 152.62, 152.56, 146.73, 138.31, 138.23, 135.95, 135.43, 130.86, r Synthesis of the osmium complexes 129.64, 128.51, 128.44, 125.46, 125.12, 123.22, 120.29. ESI-MS c Synthesis of (tpy)OsCl (NH )[OsCl ] (0.4396 g, 1 mmol) and (CH OH) m/z: 776.3 [M-2(ClO )+H+CH CN]+, 367.0 [M- s 3. 42 6 3 4 3 tripyridine (0.2369 g，1.01 mmol) in 30 mL of ethylene glycol 2(ClO 4 )]2+/2. Anal. Calcd. For C 36 H 26 Cl 2 N 6 O 8 Os: C, 46.41; H, 2.81; u solution were refluxed (120 oC) for 2 h. Cooling to room temperature N, 9.02; Found: C, 46.43; H, 2.79; N, 9.03. n and diluted with 80 mL mixed solution of ethanol and water (v : v = a 3:1), and then stored at -20 oC overnight. The dark precipitate was Synthesis of Os3. (tpy)OsCl 3 (42 mg, 0.08 mmol) and tpy-ph-tpy filtered and washed with water, and directly used in the following (2,2':6',2''-terpyridine,4',4''''-(1,4-phenylene)bis) (62 mg ， 0.115 M reactions without further purification. mmol)were mixed together in 10 mL ethylene glycol and heated at 180 °C with continuous stirring under argon atmosphere for 5 h. After d Synthesis of tpy-ph. 2-acetylpyridine (8.4 mL, 74 mmol), cooling, the reaction mixture was filtered and poured into a saturated e benzaldehyde (3.4 mL, 32 mmol) and NaOH (2.0 g) were mixed and aqueous solution of NaClO 4 . The precipitates were filtered and t stirred in water (25 mL) for 1h at room temperature. The resulting washed with water and dried. The crude product was then purified by p mixture solution was stirred with 30 mL water. The white precipitate column chromatography (alumina) eluting with acetonitrile. It was e was filtered, washed thoroughly with cold ethanol and dried in air. 1, further purified upon recrystallization from a cetonitrile:water (2:1) c 5-bis-(2-pyridinyl)-3-pentadione (2.0 g, 0.61 mmol) and ammonium mixture in acidic condition (10−4 M HClO 4 ).1H NMR (500 MHz, c acetate (2.5 g) were dissolved in 25 mL ethanol, the clarified solution DMSO-d6) δ 9.66 (s, 2H), 9.17 (d, J = 8.3 Hz, 2H), 9.15 – 9.07 (m, A was heated to 70 °C for 2 h. After the mixture cooled to room 4H), 8.88 – 8.80 (m, 6H), 8.62 (d, J = 8.4 Hz, 1H), 8.35 (d, J = 8.4 Hz, temperature, 20 mL water was slowly added to the above mixture 1H), 8.14 – 8.03 (m, 3H), 8.03 – 7.95 (m, 3H), 7.95 – 7.86 (m, 3H), s solution. The precipitate was collected and purified by 7.48 (d, J = 5.5 Hz, 2H), 7.38 (d, J = 5.4 Hz, 2H), 7.29 – 7.18 (m, 6H). r recrystallization with ethyl alcohol. 1H NMR (500 MHz, CDCl):  13C NMR (126 MHz, DMSO-d6) δ 160.25, 160.02, 156.33, 155.40, 3 e 8.80 (s, 2H), 8.78 (d, J = 4.8 Hz, 2H), 8.72 (d, J = 8.0 Hz, 2H), 7.98- 155.17, 154.81, 152.67, 149.91, 149.18, 145.69, 145.08, 139.70, i 7.91 (m, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.3 Hz, 2H), 7.44- 138.43, 138.29, 138.14, 137.42, 136.95, 135.63, 129.48, 128.98, t 7.39 (m, 2H). ESI-MS (CH OH) m/z: 310.4 [M+H] +. Anal. Calcd. 128.46, 128.23, 125.60, 125.22, 123.30, 121.60, 120.34, 118.76. ESI- n 3 For C 21 H 15 N 3 : C, 81.53; H, 4.89; N, 13.58; Found: C, 81.52; H, 4.87; MS (CH 3 OH) m/z: 965.1 [M-2(ClO 4 )+H]+. Anal. Calcd. For o N, 13.60. C 51 H 35 Cl 2 N 9 O 8 Os: C, 52.67; H, 3.03; N, 10.84; Found: C, 52.65; H, r 3.07; N, 10.79. F Synthesis of Os1. (tpy)OsCl (0.4000 g，0.76 mmol) and tpy (0.2510 3 y g，1.08 mmol) were mixed together in 10 mL ethylene glycol and Determination of singlet oxygen quantum yield heated at 180 °C with continuous stirring under argon atmosphere for r t 5 h. After cooling, the reaction mixture was filtered and poured into a An air-saturated acetonitrile solution, containing the tested s saturated aqueous solution of NaClO . The precipitates were filtered compounds (A = 0.1) at irradiation wavelength, p-nitrosodimethyl 4 i and washed with water and dried. The crude product was then purified aniline (RNO, 24 M), imidazole (12 mM) or an air-saturated PBS m by column chromatography (alumina) eluting with acetonitrile. It was buffer solution, containing the complex (A = 0.1 at irradiation further purified upon recrystallization from acetonitrile-water (2:1) wavelength) RNO (20 M), histidine (10 mM) were irradiated in a e mixture in acidic condition (10−4 M HClO ). 1H NMR (500 MHz, luminescence quartz cuvette at 420 nm, recording the absorbance at h 4 DMSO-d6)  8.71 (d, J = 3.9 Hz, 4H), 8.61 (d, J = 7.9 Hz, 4H), 8.43 various time intervals. Plot of A 0 -A at 440 nm is PBS or at 420 nm in C (d, J = 7.8 Hz, 4H), 8.08 (t, J = 7.8 Hz, 2H), 7.99 (t, J = 7.7 Hz, 4H), acetonitrile (where A 0 is the absorbance before irradiation) versus the 7.51-7.45 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 160.00, 154.84, irradiation time were prepared and the slope of the linear regression c 152.50, 138.29, 135.47, 128.44, 125.13, 123.23. ESI-MS (CH 3 OH) was calculated (S sample ). As a reference compound, [Ru(bpy) 3 ]2+ (Ф ref i m/z: 328.2 [M-2(ClO 4 )]2+/2. Anal. Calcd. For C 30 H 22 Cl 2 N 6 O 8 Os: C, (1O 2 ) = 0.22) was used to obtain S ref Equation (1) was applied to n 42.11; H, 2.59; N, 9.82; Found: C, 42.20; H, 2.57; N, 9.89. calculate the singlet oxygen quantum yields (Ф sample ) for every sample: a Ф sample = Ф ref * S sample / S ref * I ref / I sample (1) g Synthesis of Os2. (tpy)OsCl 3 (0.0848 g, 0.16 mmol) and tpy-ph I= I 0 * (1-10-A  ) (2) r (0.0714 g, 0.23 mmol) were mixed together in 10 mL ethylene glycol I (absorbance correction factor) was obtained from Equation (2), o and heated at 180 °C with continuous stirring under argon atmosphere where I 0 is the light intensity of the irradiation source in the irradiation n for 5 h. After cooling, the reaction mixture was filtered and poured interval and A  is the absorbance of the sample at 465 nm. I into a saturated aqueous solution of NaClO . The precipitates were 4 filtered and washed with water and dried. The crude product was then Phosphorescence quantum yield measurement purified by column chromatography (alumina) eluting with This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins .MA 62:32:6 0202/7/9 no dnalgnE weN fo ytisrevinU yb dedaolnwoD .0202 rebmetpeS 70 no dehsilbuP View Article Online DOI: 10.1039/D0QI00846J PInleoarsgea ndioc Cnhoet madisjturys tF mroanrtgieinrss Page 6 of 8 ARTICLE Journal Name The phosphorescence spectra was obtained with a FP-6500 complexes with different concentrations. For photocytotoxicity spectrophotometer (Jasco, Japan). The relative phosphorescence studies, after 4 h incubation with complexes, the culture media was quantum yields were determined with [Ru(bpy) ]2+ as a standard and replaced by fresh medium. The 96-well plates were then irradiated by 3 calculated using the following equation: 465 nm or 633 nm light (465 nm: 13 mW/cm2; 633 nm: 20 mW/cm2) Ф = Ф * (F /F) * (A/A) * (n /n)2 for 1 h. After irradiation, the cells continue incubation for in total 48 x s x s s x x s t where Ф represents quantum yield; F stands for integrated area under h. The photocytotoxicity was measured by standard MTT method. p the corrected emission spectrum; A is absorbance at the excitation The change in optical density (OD) at 490 nm was monitored using i wavelength; λ ex is the excitation wavelength; n is the refractive index microplate reader (Promega). r of the solution; and the subscripts x and s refer to the unknown and c the standard, respectively. Photooxidation of NADH s Complexes were evaluated for the catalytic oxidation of NADH to u NAD+ by UV−visible spectroscopy in the dark and under irradiation n Electron paramagnetic resonance (EPR) assay for 1 h in 1% DMSO/99% H O (pH = 7.1) at 298 K, The complexes 2 a The EPR measurements were carried out at ambient temperature on a concentration remained fixed at 10 μM with NADH concentrations of M Bruker EMX spectrometer. Irradiation was carried out with 465 nm 175 μM. The conversion of NADH to NAD+ was followed by or 633 nm LED lights. The samples were contained in a flat-cell absorption at 339 nm (ε(NADH) = 6220 cm−1 M−1) to allow evaluation (WG812) positioned in a TM110 cavity (ER4103 TM). For kinetic of kinetic data. TON is defined as the number of moles of NADH that d measurements, the EPR parameters were: sweep width 8 mT, 1024 a mole of complexes can convert within 30 min. e points, time constant 10.24 ms and conversion time 20.48 ms, giving t a sweep time of 30 s. Field modulation was applied at 100 kHz and p Conflicts of interest 0.05 mT, and the microwave attenuation was 18 dB (~3.2 mW). The e spin trap, 2,2,6,6-tetramethyl-piperidine (TEMP for trapping 1O 2 , 20 The authors declare no competing financial interest. c mM), was used to verify the formation of 1O generated by the 2 c complexes (50 M), and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO Acknowledgements A for trapping HO , 45 mM) was used to verify the formation of HO 2 2 2 2 generated by the complexes (50 M). We appreciate the financial support of the National Natural Science s Foundation of China (NSFC, 21701113), the Science and r Technology Foundation of Shenzhen e Cell culture (JCYJ20190808153209537), the Natural Science Foundation of i The cells were grown in DMEM with or without phenol red. All media SZU (2018036) and Peacock Talent Fund (827-000389). We t n were supplemented with 10% v/v of fetal calf serum (FCS), 1% v/v of appreciate the Instrumental Analysis Center of Shenzhen 2 mM glutamine and 1% v/v penicillin/streptomycin. 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Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 62:32:6 0202/7/9 no dnalgnE weN fo ytisrevinU yb dedaolnwoD .0202 rebmetpeS 70 no dehsilbuP View Article Online DOI: 10.1039/D0QI00846J