Near-infrared phosphorescent iridium ( III ) complex for imaging of cysteine and homocysteine in living cells and in vivo

Near-infrared phosphorescent iridium(III) complex for imaging of cysteine and homocysteine in living cells and in vivo Yongquan Wua, Renmiao Wua, Huifang Lia, Hong Zenga, Yuanyan Lia, Qiuhong Wangb, Mei Shi*b and Xiaolin Fan*a a School of Chemistry and Chemical Engineering & Key Laboratory of Organo-pharmaceutical Chemistry of Jiangxi Province, Gannan Normal University, Ganzhou, 341000, P. R. China b Department of Chemistry, Fudan University, Shanghai 200433, P. R. China Fax: +86-797-8393536, +86-21-55664621. E-mail: fanxl2013@gnnu.cn, shimei@fudan.edu.cn

collected with an AB SCIEX mass spectrometer.The UV-visible spectra were recorded on a Shimadzu UV-2007 spectrometer.Steady-state emission experiments at room temperature were measured on an Edinburgh Instruments spectrometer FS-5.
The luminescence quantum yield in air-equilibrated solution were measured with reference to tris-(2,2'-bipyridyl)-ruthenium (II) chloride hexahydrate as a standard (Φ=0.063 in DMF).Lifetime studies were performed with an Edinburgh FL 920 photo-counting system with a hydrogen filled lamp as the excitation source.

Synthesis details
The synthesis routine of ligand 1 and NIR-Ir were shown at Scheme S1.
The raw product was purified by silica gel chromatograph using CH 2 Cl 2 /PE as eluent

Measurement of photophysical properties
UV-visible spectra were recorded on Shimadzu UV-2007 spectrometer and their emission spectra were recorded on Edinburgh FS-5 spectrometer in ethanol solution at room temperature.Lifetime studies were performed with an Edinburgh FL 920 photocounting system with a hydrogen filled lamp as the excitation source.The data were analyzed by iterative convolution of the luminescence decay profile with the instrument response function using a software package provided by Edinburgh Instruments.
Quantum yields were calculated according to the literature at room temperature. 1e samples solution was diluted by ethanol, and the aerated DMF solution of tris (2,2'-bipyridyl) ruthenium (II) chloride hexahydrate (0.063 in DMF) was utilized as the reference.The quantum yields of the complexes were calculated according to eq.
where, 'N' represents the solution's refractive index, 'I' represents the integrated fluorescence intensity, 'A' represents the integrated absorbance intensity, and the subscripts 'μ' and 's' refer to the reference samples and the samples, respectively.

The amino acids titration of NIR-Ir
Spectrophotometric determination was carried out in DMSO-HEPES (pH 7.4, 4:1 v/v) at room temperature.Different concentrations of various amino acids were titrated into a solution of NIR-Ir (10 μM) in DMSO-HEPES, respectively.Before UV-vis absorption and photoluminescence spectra of the samples were measured, the solutions were kept at 37 0 C for 2 hours.For luminescence measurements, excitation was provided at 495 nm, and emission was collected from 600 to 800 nm.

Computational details
These iridium complexes were optimized with density functional theory (DFT) 1 using the Becke's three-parameter hybrid exchange functional combined with the Lee-Yang-Parr correlation functional (B3LYP) 2 , a functional that has been widely employed in previous studies of iridium complexes. 3The "double-ζ" quality basis set LANL2DZ and corresponding effective core potentials 4 were used for iridium atom, while the 6-31G(p,d) 5 basis set was used on nonmetal atoms in the gradient optimizations.All optimized configurations were confirmed to be minima on the potential energy surfaces by performing vibrational frequency calculations at the same level.In addition, a conductor-like polarizable continuum model (CPCM) 6 using ethanol (ε = 24.852)as the solvent was considered for optimization calculations of the involved geometries.Calculations were performed with Gaussian 09 (Revision D.01) 7 .
To shed more light on the nature of the excited states of these Ir(III) compounds, vertical transition energies were calculated on the basis of the optimized S 0 and T 1 structures via time-dependent DFT (TDDFT). 89Natural transition orbital (NTO) 10 analyses were performed further to examine the nature of the excited states.

Cytotoxicity assay
The HeLa cell lines were provided by the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences).The HeLa cells were grown in DEME (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (Fetal Bovine Serum) at 37 0 C and 5% CO 2 .
In vitro cytotoxicity was measured by performing methyl thiazolyl tetrazolium (MTT) assays on the HeLa cells.Cells were seeded into a 96-well cell culture plate at 5×10 3 /well, and were cultured at 37 0 C and 5 % CO 2 for 24 h.Different concentrations of NIR-Ir (0, 5, 10, 15, 20, and 25 μmol/L, diluted in DEME) were then added to the wells.The cells were subsequently incubated for 24 h at 37 0 C under 5% CO 2 .Thereafter, MTT (5 mg/mL) was added to each well and the plate was incubated for an additional 5 h at 37 0 C under 5 % CO 2 .The optical density OD570 value (Abs.) of each well, with background subtraction at 690 nm, was measured by means of a microplate reader (KHB ST360, China).The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of Abs.value of treatment group/mean of Abs.value of control) × 100%.

Confocal luminescence imaging
Confocal luminescence imaging of cells was performed with an OLYMPUS FV1000 laser scanning microscope, and a 40 oil-immersion objective lens was used.
For fluorescence imaging, MCF-7 cells were incubated on glass bottom dishes for 12 h.Excitation of the MCF-7 cells at 532 nm was carried out with a laser, and emission was collected at 700±50 nm using a PMT detector.Prior to imaging, the medium was removed.Cell imaging was carried out after washing cells with PBS (pH=7, 10 mM) three times.

In vivo imaging
Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee.In vivo luminescence imaging was performed with a modified luminescence in vivo imaging system (IVScpoe 7550, Shanghai CLINX Science Instruments Ltd., China).In this system, two external 0 − 5 W adjustable CW

Figure S5 .Figure S6 .
Figure S5.TD-NTO analysis for the dominant pair of the hole-particle wave function pairs of natural transition orbital for the S 1 state based on the optimized geometries in the ground state.The corresponding square of the singular value is denoted on the bottom.

Table S3 .
Vertical absorption and emission energies (in eV), dominated orbital excitations obtained from TD-DFT calculations.The absorption energies are based on the S 0 state equilibrium geometry.

Table S4
Comparison of Cys or Hcy imaging with Ir (III) complex probe