Stable luminescent iridium(iii) complexes with bis(N-heterocyclic carbene) ligands: photo-stability, excited state properties, visible-light-driven radical cyclization and CO2 reduction, and cellular imaging† †Electronic supplementary information (ESI) available: Additional experimental details, fig

Excited state properties, photo-catalysis and cellular imaging of photo-stable bis-NHC Ir(iii) complexes are described.

. Selected bond length and angles of 4 Hex b, 6 H b, 7b and cis-6 H b.

16-17
Visible-light-driven radical cyclization 17 Synthesis of substrates and general procedure for visible-light-induced radical cyclization. 17 Table S4. Characterization of substrates and products in photocatalysis. [18][19] Visible-light-driven CO2 Reduction 19 Procedure and measurements of CO2 reduction. 19 Table S5. Control experiments of CO2 reduction.             Values refer to onset of the anodic peak potential (E ox onset) and oxidation peak potential (Epa) in parenthesis at 25 o C for irreversible couples at a scan rate of 100 mV s -1 ; c Values refer to onset of the cathodic peak potential (E red onset) and reduction peak potential (Epc) in parenthesis for the irreversible reduction waves; d approximate zero-zero excitation energy E0,0 = 1240/λem (onset of emission band at 25 o C), e Calculations of approximate redox potentials of excited iridium(III) complexes: E(Ir IV/III* ) = E(Ir IV/III ) -E0,0, E(Ir III*/II ) = E(Ir III/II ) + E0,0 (eV). 2

Experimental Section
Photophysical measurements UV-vis absorption spectra were recorded on a Hewlett-Packard 8453 diode array spectrophotometer. Photo-excitation and steadystate emission spectra were obtained on a SPEX Fluorolog-3 Model FL3-21 spectrofluorometer. Solution samples for measurements were degassed on a high-vacuum line in a two-compartment cell that consisted of a pyrex bulb (10 mL) and a quartz cuvette (path length: 1 cm) and was sealed from the atmosphere by a Bibby Rotaflo HP6 Teflon Stopper. The solutions were rigorously degassed by at least five successive freeze/pump/thaw cycles. Excited state lifetime measurements of solution samples were performed with a Quanta Ray DCR-3 pulsed Nd:YAG laser system (pulse output 355 nm, 8 ns). The emission signals were detected by a Hamamatsu R928 photomultiplier tube and recorded on a Tektronix TDS 350 oscilloscope, and analysed by using a program for the exponential fits. PL values were measured 4 relative to that of a degassed acetonitrile solution of Ru(bpy)3(PF6)2 (bpy = 2,2-bipyridine) (ref = 0.062) as a standard reference and calculated by: s = r(Br/Bs)(ns/nr) 2 (Ds/Dr), where the subscripts s and r refer to sample and reference standard solutions, respectively, n is the refractive index of the solvents, D is the integrated intensity, and  is the luminescence quantum yield. The excitation intensity B was calculated by: B = 1 -10 -AL , where A is the absorbance at the excitation wavelength and L is the optical path length (L = 1 cm in all cases). Errors for  values ( 1 nm),  ( 10 %),  ( 10 %) were estimated. Nanosecond time-resolved absorption and emission measurements were performed using a LP920-KS Laser Flash Photolysis Spectrometer (Edinburgh Instruments Ltd, Livingston, UK). The excitation source was 355 nm output from a Nd:YAG laser.

Cyclic voltammetry
Cyclic voltammetry measurements were performed on a Princeton Applied Research Model 273A potentiostat. The glassy-carbon electrode was polished with 0.05 μm alumina on a microcloth, sonicated for 5 min in deionized water, and rinsed with acetonitrile before use. A Ag/AgNO3 (0.1 M in MeCN) electrode was used as reference electrode and a platinum wire as counter electrode. All solutions of samples were prepared in MeCN containing 0.1 mol dm -3 tetra(n-butyl)ammonium hexafluorophosphate ( n Bu4NPF6) as supporting electrolyte. The solutions were purged and maintained under Argon atmosphere. Scan rates were 100 mV s -1 and the potentials were reported with respect to the potential of Ag/AgNO3. Ferrocene was used as internal reference and recorded with the potential at the range of 0.05 -0.07 V for ferrocenium/ferrocene (Cp2Fe +/0 ) vs Ag/AgNO3.

Computational details
Density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations have been performed to understand the geometries and the electronic structures of Iridium complexes (1a, 1d) using Gaussian 09 package. 4 PBE0 5 /6-31G*(lanl2dz) 6 was used for the geometry optimization and PBE0/6-31+G*(lanl2dz) was used for TDDFT calculations. The Solvent effects have been studied using self-consistent reaction field (SCRF) method based on PCM models. 7 The choice of solvents (dichloromethne，a dielectric constant e = 8.93) was based on the solvent media for experiments.

X-ray crystal-structure determination
Crystals of 4 Me b (with PF6counter anion), 6 H b, cis-6 H b and 7b suitable for X-ray crystallography were obtained by slow diffusion of diethyl ether into DCM (6 H b), MeCN (4 Me b and 7b) and chloroform (cis-6 H b) solution of these complexes, respectively. The Xray diffraction data were collected on a Bruker X8 Proteum diffractometer except for 7b (Bruker D8 Venture diffractometer). The crystal was kept at 100 K during data collection. The diffraction images were interpreted and the diffraction intensities were integrated by using the program SAINT. Multi-scan SADABS was applied for absorption correction. By using Olex2, 5 the structure was solved with the ShelXS 6 structure solution program using direct Methods and refined with the XL 6 refinement package using Least Squares minimization. The positions of the H atoms were calculated on the basis of the riding mode with thermal paramet ers equal to 1.2 times that of the associated C atoms and these positions participated in the calculation of the final R indices. In the final stage of least-squares refinement, all non-hydrogen atoms were refined anisotropically. Crystallographic parameters are summarized in Table S1. CCDC 1428476-1428479 contain the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/data_request/cif. 7 were synthesized according to literature procedure. 1,1'-methylenebis(3-methyl-1H-imidazol-3-ium) diiodide, 1,1'-methylenebis(3-butyl-1H-imidazol-3-ium) diiodide were prepared according to a modification of the literature method. 8 Iridium trichloride (IrCl3) hydrate, lithium trifilate (LiOTf) and silver(I) oxide (Ag2O) were purchased from commercial sources and were used as received without further purification. Deionized (DI) water was used in the experiment procedure.
General procedure for preparation of iridium(III) complexes 1-9: Characterization for synthesis: 1 H NMR spectra were recorded using deuterated solvent on a Bruker Avance DPX-300, AV-400, or DRX-500 Fourier-Transform NMR spectrometer; chemical shifts are reported relative to tetramethylsilane. (Solvents: CDCl 3, CD3CN and CD3OD; chemical shifts:, ppm; J, Hz). Positive-ion electrospray Ionization (ESI) mass spectra were obtained on a Finnigan LCQ quadrupole ion trap mass spectrometer. Elementary analysis of the new complexes was performed on a Flash EA 1112 elemental analyzer at the Institute of Chemistry, the Chinese Academy of Sciences. These complexes were synthesized from refluxing of HC^N ligand with IrCl3 in aqueous solution of 2-methoxyethanol (75% in volume) for overnight (12-18 hours), the resulting precipitate was filtrated, washed by water, ethanol and diethyl ether and dried by air. The dichloro-bridged iridium complexes [(C^N)2IrCl]2 were used without further purification. A typical procedure is described as follows (example of 1): In a 50 mL two-neck round-bottomed flask was [(ppy)2IrCl]2 (150 mg, 0.14 mmol, 1 eq), 1,1'methylenebis(3-butyl-1H-imidazol-3-ium), 2I -(159 mg, 0.308 mmol, 2.2 eq), and silver(I) oxide (136 mg, 0.588 mmol, 4.2 eq) in 2-methoxyethanol (volume: 10 mL) to give a black suspension. The reaction was refluxing at 120 °C for overnight, 2methoxyethanol (volume: 10 mL) was removed under vacuum pump. 40 mL of DCM was added and filtrated through celite, the resulting filtrate was washed by Lithium triflate aqueous solution (0.1 g/mL) 2 times. Organic layer was dried by MgSO4. After organic solvent was removed, the residue was prepared to purify by chromatographic column, and eluted by DCM/MeCN (V/V = 20/3). After removed organic solvent, the yellowish green solid was in good yield of 82% complex of 1b: 210 mg, 0.231 mmol). δH

Complex 1a:
The synthesis procedure of bis-NHC carbene Ir(III) complexes with counter anions of PF6is similar to that of trifilate Ir(III) complexes. 1a (not purified by chromatographic column) was dissolved into MeOH and excess of NH4PF6 was added into above solution, resulting precipitate was filtrated, washed by water, MeOH and Et2O, and dried by air. The solid was prepared to purify by chromatographic column, and eluted by DCM/MeCN (V/V = 10/1 to 5/1

Complex 3a:
Yield: 19%, this complex is difficult to purify. After twice chromatograph purification procedures, the desired crude product was purified from growing crystals by diffusion diethylether into a solution of DCM. δH (400 MHz, CD3CN) = 8.

Synthesis of 2-(5-bromothiophen-2-yl)pyridine:
To a 100 mL round-bottomed flask was added 2-(thiophen-2-yl)pyridine (700 mg, 4.34mmol) in DCM to give a yellow solution. Bromine (694 mg, 4.34mmol) in 5 mL of DCM was added at ice bath. After addition for 5mins, red precipitate was formed, and it was left stirring for overnight. 75% was converted after HNMR identification. Another 150 mg of bromine was added again. The crude material was loaded on a 2.0mm plate.

Synthesis of 1-chloroisoquinoline:
To a flask was added isoquinolin-1-ol (3g, 20.67 mmol) and phosphoryl trichloride (10 mL), and heated for 4hrs at 80 o C. 100 mL of ice water was added into the cooled mixture to quench the reaction. The aqueous layer was back-extracted with DCM (30 mL× 3). Combined the organic layers and washed with water (20 mL×3). The organic layer was dried by MgSO4, filtrated and concentrated. The product was used without further purification (3.12g, 92%). 1

Synthesis of 1-(thiophene-2-yl)isoquinoline (Htiq):
In a 100 mL two-neck round-bottomed flask was 2-bromothiophene (1.95 g, 12 mmol) and magnesium (0.291 g, 12.00 mmol) in dried THF (20 mL) to give a colorless suspension. Iodine 10 mg was added to initialize the reaction and refluxing for 30 mins. In a 250 mL two-neck round-bottomed flask was 1-chloroisoquinoline (1.63 g, 10.00 mmol) and Ni(dppe)Cl2 (53 mg, 0.100 mmol) in dried THF (30 mL) to give a orange solution. The Grignard reagent of first solution was transferred by cannula at refluxing temperature, the resulting dark brown solution in the 250 mL flask was left stirring for 20 mins and refluxing for overnight. Monitoring of reaction shows product left without starting materials. H2O was added to end the reaction. The aqueous layer was back extracted with Et2O (50 mL×3). Combined the organic layers and washed with water (50 mL×2). The organic layer was dried by MgSO4, filtrated and concentrated. The crude product was added to a silica gel column and was eluted with DCM/Hex (V/V from 1/1 to 3/1). Collect fractions with Rf = 0.25 in DCM/Hex (V/V =1/1). Purified Htiq: 1.94g, Yield: 94%. 1

Synthesis of di(1H-imidazol-1-yl)methane:
In a 250 mL two-neck round-bottomed flask was 1H-imidazole (6.8 g, 100 mmol) in THF (200 mL) to give a colorless solution. Sodium hydride (4.39 g, 110 mmol) was added by three lots and resulting grey suspension was added dibromomethane (9.03 g, 51.9 mmol) by three lots under ice bath. The resulting grey suspension was heated at 50 o C for overnight, and grey solid was converted to yellow. The reaction was monitored by 1 H NMR. Upon reaction was completed, the solvent was removed, and the residue was dissolved by 100 mL of methanol and passed through celite and washed by 50 mL of methanol. The filtrate was reduced and prepared to a silica gel column and was eluted with DCM/MeOH (V/V = 4/1 to 1/1). Collected yellow fractions with Rf = 0.25 (DCM/MeOH: V/V = 4/1) which could be stained in I2 champer. 1 H NMR (400 MHz, CDCl3) δ = 7.91 (s, 2H), 7.37 (s, 2H), 6.89 (s, 2H), 6.20 ppm (s, 2H).

Synthesis of substrates:
Substrate A:

Substrate B:
General procedure for visible light-induced photocatalytic reductive cyclization of organohalides (Table 3 -7): Use substrate A1, photo-catalyst 4 Me b and N,N-diisopropylethylamine (DIPEA) as example: To a test tube (Pyrex, 15×125 mm) charged with an organohalide substrate (50 μmol) and 2mol% of 4 Me b complexes (1 μmol) were added MeCN (4 mL), 5eq of DIPEA (87 μL) and 2.5eq of HCOOH (10 μL). The mixture in test tube was degassed by nitrogen (bubbling for 10 min through septa by cannula). The test tube was placed in the irradiation apparatus equipped with blue LEDs (centred at 460 nm, 12 w). 12 . The resulting mixture was stirred at ambient temperature for specified time. The organic solvent was evaporated under reduced pressure. The n added 30 mL of Et2O and followed with 10 mL of saturated NaHCO3 (aq) solution. The mixture was extracted by Et2O (15 mL×2) and combined organic solvent was washed by water (15 mL×2), dried over MgSO4. After organic solvent is removed, the yield of product can be calculated by adding internal standard 5,5'-dimethyl-2,2'-bipyridine (with known weight) to the resulting crude residue. Table S4. The characterization of substrates and products in photocatalysis. 1 H NMR spectrum matches to reported literature. 17

Visible-light-driven CO2 Reduction
Photo-catalytic procedure of CO2 reduction In a 4 mL CH3CN/TEA (4:1, v/v; TEA = triethylamine) solution, [Co(TPA)Cl]Cl and [Ir] (4 Me b,OTf anion) (with specified concentration) was added into a Pyrex tube (Volume: 22 mL (16 (OD.)×150; 1.2 thickness (mm)) and purged with CO2 through a septum (purity ⩾ 99.8%) for 10 min, followed by 250 μL CH4 was injected to the tube prior to the irradiation using blue LEDs (centred at 460 nm, 12 w). 12 All reactions and LEDs were cooled by aluminium blocks by using cooling fans. Gas sample (200 μL) was drawn from the headspace of the tube and injected to GC-TCD for measurement.

Measurement of gases products
Gas chromatographic analysis was conducted using Agilent 7890A gas chromatography equipped with a thermal conductivity detector (TCD) and a HP-Plot 5Å column with Ar as the carrier gas. The oven temperature was held at 40 o C. Inlet and detector temperature were set at 80 o C and 150 o C respectively. Calibration curves were established separately based on the averaged results from three point of injections for CO (R 2 = 0.9997), H2 (R 2 = 1.0000) and CH4 (R 2 = 0.9996).  Biological studies: Anticancer properties: The cell lines were maintained in cell culture media (minimum essential medium for HeLa) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 o C humidified atomsphere with 5% CO2. Cell growth inhibitory effects of the iridium(III) complexes and cisplatin were determined by MTT cytotoxicity assay. Drug treated cells were incubated with MTT for 12h at 37°C in a humidified atmosphere of 5% CO2 and were subsequently lysed in solubilizing solution. Cells were then maintained in a dark, humidified chamber overnight. The formation of formazan was measured by using a microtitre plate reader at 580 nm. Growth inhibition by a drug was evaluated by IC50 (concentration of a drug causing 50% inhibition of cell growth). Each growth inhibition experiment was repeated at least three times and the results were expressed as means ± standard deviation (SD).

Fluorescence microscopic examination
HeLa cells (2×10 5 cells) were seeded in a one chamber slide (Nalgene; Nunc) with culture medium (2 mL per well) and incubated at 37 o C in a humidified atmosphere of 5% CO2/95% air for 24 h. A stock solution of iridium complex was prepared in DMSO and then diluted to 5μM into the cells glass coverlips (Mattek 35mm glass bottom dish) for imaging experiments. After treating with Ir complexes only or the mixture of Ir complexes with ER-TrackerTM (1μM) or Lysotracker ® (100 nM) or Mitotracker ® (50 nM) for