NMR spectra were recorded using a Varian Mercury-200 spectrometer (200 MHz for 1H), Varian Mercury-400 spectrometer (400 MHz for 1H, 100 MHz for 13C), Bruker Avance-400 spectrometer (400 MHz for 1H, 100 MHz for 13C), Varian Inova-500 spectrometer (500 MHz for 1H, 125 MHz for 13C) or Varian VNMRS-700 spectrometer (700 MHz for 1H, 175 MHz for 13C). Chemical shifts are quoted in ppm relative to residual proteo-solvent resonances. 1H NMR spectra assignment was assisted by 1H-1H COSY (correlation spectroscopy, through-bond interactions). 13C NMR spectra were recorded with proton decoupling and assigned with the aid of 1H-13C HSQC (heteronuclear single quantum correlation) and 1H-13C HMBC (heteronuclear multiple bond correlation) experiments.
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Low resolution electrospray mass spectra (ES MS) were recorded with a micromass LCT spectrometer or Thermo-Finnigan LTQ FT spectrometer using either acetonitrile or methanol as the carrier solvent and MALDI mass spectra were recorded using an Applied Biosystems Voyager-DE STR instrument or a Bruker Daltonics Autoflex II ToF/ToF instrument. High resolution ES mass spectra were recorded using a Micromass LCT spectrometer or a Thermo- Finnigan LTQ FT spectrometer. Electron ionisation (EI) mass spectra were recorded by the EPSRC National Mass Spectrometry Service at the University of Wales, Swansea. Gas chromatography-mass spectrometry (GCMS) was performed using a Thermo-Finnigan Trace GCMS instrument. Infrared (IR) spectra were measured using a Perkin Elmer FTIR Paragon 1000 with ATR attachment.
Elemental analysis was carried out using an Exeter Analytical E-440 elemental analyser and melting points were determined using a Gallenkamp 889339 capillary melting point apparatus.
5.1.3 Photophysical measurements
UV/Visible spectra were measured with a Biotek Instruments XS spectrometer operating with LabPower software. Samples were held in a quartz cuvette with 1 cm pathlength and measurements were performed against a reference of pure solvent in a matched cuvette. Exctinction coefficients were determined through graphical application of the Beer-Lambert law (Equation 2) using dilution techniques.254
( ) ( )
A cl
Equation 2
A(λ) is the absorbance at a specific wavelength, c is the concentration of the absorbing species (mol dm-3), l is the path length and ε is the extinction coefficient at the given wavelength (dm3mol-1cm-1).
Steady state emission and excitation spectra were measured with a Jobin Yvon FluoroMax-2 spectrofluorimeter, equipped with a red-sensitive Hamamatsu
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R928 photomultiplier tube (PMT) using DataMax operating software. To minimise inner filter effects, solutions being studied were sufficiently dilute as to have absorbance below 0.1 at the excitation wavelength. Samples in aerated solutions were contained in quartz cuvettes of 1 cm pathlength while samples run in degassed solution were contained in quartz cuvettes modified for attachment to a high-vacuum line. These samples were degassed with a minimum of three freeze-pump-thaw cycles leaving a final vapour pressure of ≤ 8×10-2
mbar at 77 K. The acquired emission spectra were corrected for dark count and the spectral response of the detector, while excitation spectra were automatically corrected for lamp output through use of a beam splitter which directs 8% of the excitation light to a reference photodiode.
The quantum yields were calculated by comparison to a reference standard of ruthenium(II) tris(2,2'-bipyridine) chloride in aerated aqueous solution which has Φst = 0.028.255 The quantum yield (Φ) of the sample is calculated relative to the
standard using Equation 3.
2 st st st st A I n I A n Equation 3
Where I is the integrated emission intensity, A is the absorbance at the wavelength of excitation and n is the refractive index of the solvent. Subscript „st‟ refers to the respective values of the standard.
Excited state lifetimes were measured using time-correlated single-photon counting (TCSPC) with an Edinburgh Instruments OB 920 fluorimeter, using a pulsed-diode laser as the excitation source with 374 nm excitation for iridium and dansyl compounds or 475 nm excitation for the [PtLn(thiolate)] complexes. The laser repetition rate was selected so that the pulse period was at least 5-10 times longer than the measured lifetime. The emission was detected at 90° to the excitation source, after passage through a monochromator using a Peltier- cooled R928 PMT. The observed photon count at a given time is proportional to the concentration of the excited state and can be expressed by a monoexponential decay of the form shown in Equation 4.
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t
CC e
Equation 4
Where C is the counts at time, t, C0 is the initial count after excitation and τ is the excited state lifetime. τ values were obtained from a least-squares fit to such a monoexponential decay.
5.1.4 Electrochemistry
Cyclic voltammetry was carried out using a µAutolab Type III potentiostat with GPES Manager software. Samples were made to ~1 mM concentration in CH2Cl2 with 0.1 M [Bu4N][PF6] as the supporting inert electrolyte. A three-
electrode assembly was employed, consisting of a glassy-carbon working electrode, and platinum wire counter and reference electrodes. Solutions were purged with solvent-saturated nitrogen gas for 5 minutes with stirring, prior to measurements being taken without stirring. The voltammograms were referenced to the ferrocence-ferrocenium couple (E1/2 = 0.42 V versus SCE).
5.1.5 DFT
Calculations were performed with Gaussian03 software using the B3LYP hybrid functional. The platinum and iridium core orbitals were modelled with a relativistic effective core potential (ECP), with the outer core and valence orbitals (5s, 5p and 5d) represented using the LANL2DZ basis set. All other elements were represented using the 6-31G* or 6-31G basis sets. The results from calculations were visualised using Gaussview 4.1 software.
5.1.6 Cellular studies
A Chinese hamster ovary (CHO) adherent cell line was maintained in exponential growth as a monolayer in appropriate F-12 (Ham) medium supplemented with 10% foetal bovine serum (FBS) and 1% penicillin and streptomycin. To prevent overcrowding and build up of toxins (both of which
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could cause uncharacteristic behaviour of the cells) the cells were sub-cultured every three-to-four days.
Sub-culturing was performed as follows: in preparation, sterile solutions of pH 7.5 phosphate buffered saline (PBS), trypsin and fresh growth medium were warmed to 37°C. The old medium was removed from the culture flask and the cells were rinsed with PBS followed by addition of 1.5 mL trypsin. The cells were then incubated for 3 min until they became detached from the flask surface. Fresh medium was then added and the suspension of cells was centrifuged at 1500 rpm for 3 min. The medium containing residual trypsin was removed by pipette and the cells were resuspended in 4 mL medium. Approx 0.5 mL of this suspension was transferred to a fresh culture flask containing 10 mL medium while the remaining cells could be transferred to microplates for assays.
5.1.6.1 Cytotoxicity measurements
A suspension containing approximately 1 × 106 cells in media (10 mL) was prepared from the 4 mL stock solution during sub-culturing. This was transferred to a 96-well microplate in 100 µL aliquots giving approximately 1 × 104 cells per well and the cells were incubated at 37 °C and 5% CO2
overnight, during which time adhesion occurred. The cells were then dosed with a range of concentrations of the test compounds (with each concentration repeated in 3 separate wells) and incubated for a further 24 h. The dosing media was then removed and the cells were rinsed with sterile PBS (pH 7.5, 100 µL), and 100 µL fresh media was added. The cells were incubated for 4 h with a 1.0 mM solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After this time, the culture medium was removed, and DMSO (150 µL) was added to each well. The plate was shaken for 2 min and the absorbance of each well at 541 nm was measured against a blank microplate containing 150 µL DMSO. Measurement of the absorbance was performed using an Analytic Jena FLASHScan 530, using WinFLASH version 1.5 software. The proportion of live cells in each well was calculated by the average absorbance for a given concentration divided by the average absorbance of wells containing un-dosed cells. The IC50 values for the compounds were
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obtained graphically from a plot of the proportion of live cells versus log(concentration).
5.1.6.2 Fluorescence microscopy
Cells were seeded into 12 well plates with each well containing a microscope coverslip and allowed to grow to around 70% confluence in an incubator. The old medium was removed and replaced by fresh medium dosed with the test compounds and incubated for 3 h. The coverslips were then removed and mounted on microscope slides. The slides were imaged immediately using Zeiss Axiovert 200M epifluorescence microscope with a digital camera, and were processed using the Zeiss Axiovision software. A × 40 oil-immersion objective lens was used to visualize the image. The filter sets used were G365 (Zeiss) for excitation in all compounds while FITCem and 12803 filters were used
for emission with the iridium-based and dansyl-based luminophores respectively.
5.1.6.3 Flow Cytometry
Cells for analysis were seeded in twelve-well plates and grown to around 80% confluence. The old medium was removed and replaced with fresh medium dosed with varying concentrations of compound and the cells were incubated for 3 h at 37 °C and 5% CO2. The cells were then treated with 200 µL trypsin
for 3 min at 37°C and the resulting suspension was centrifuged at 1500 rpm for 3 min. The dosed medium was removed and the cell plug was resuspended in 2 mL sterile PBS and stored on ice. The samples were filtered before measurement to remove cell clumps.
Flow cytometry measurement was conducted using a DakoCytomation Inc. MoFlo multi-laser flow cytometer (Fort Collins, CO, USA) operating with a 70 µM nozzle at 60 Psi. Samples were interrogated with a UV-laser operating at 50 mW. Luminescence signals were detected through interference filters with bandpasses of 530/40 or 630/30 for compounds with dansyl or iridium luminophores respectively. Fluorescence signals were collected in the logarithmic mode and the data was analysed using Summit v4.3 (Beckman Coulter) software.
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5.2 Synthesis of compounds
In many cases procedures were repeated several times. In these cases, masses and yields given are from a typical example.
5.2.1 Compounds from Chapter 2