Targeted cell imaging properties of a deep red luminescent iridium(iii) complex conjugated with a c-Myc signal peptide

Visualising a c-Myc nuclear localisation signal peptide using an organometallic complex.


Cell staining with intracellular probes
To generate stock solutions, Ir-CMYC and Ir-PYR were resuspended in DMSO (final concentration 10 mM) and stored at -20°C (it was noted that 3 freeze-thaws cycles did not appear to reduce the compound signal). Stock solutions were diluted to the stated

Monitoring cellular metabolic activity
Human fibroblasts were plated at 10,000 cells/well of a 96-well plate (Cellbind) and treated with the indicated concentrations of Ir-PYR, Ir-CMYC or DMSO. As a measure of cell viability and metabolic activity, at 18-hours post compound addition culture medium was removed and replaced with culture medium containing 10 µg/mL resazurin diluted in DPBS (10% V/V). After a 4 hour incubation at 37°C, fluorescence was determined using a Molecular Devices Spectramax Gemini EM plate reader (lex = 445 nm; lem = 585 nm). Circular dichroism titrations were carried out using an Applied Photophysics Chirascan spectrophotometer thermostated at 25 °C. First, 2500 uL of buffer was placed in a 1 cm pathlength quartz cuvette (Hellma) and a spectrum was recorded between 700 and 230 nm.

Imaging and localisation analysis
Next, between 10 and 15 uL (typically 12.5 uL) of the stock solution of the iridium complex in DMSO was added to the buffer and a spectrum was recorded. Subsequently, 5 aliquots of 20 μL, 4 aliquots of 50 μL and 2 aliquots of 100 μL (i.e. a cumulative added volume of 500 μL) of the DNA stock solution were added and a spectrum was recorded after every addition.

DNA binding -data analysis results
Spectra and titrations were plotted in OriginLab Origin 2019b. The titration data were analysed globally for each complex using an in-house written version of the multiple independent binding sites model which also explicitly takes ligand concentrations into account. 4 Exploratory data analysis showed that binding affinity and binding site size could not be determined separately from this data, as is usual for relatively weak binding. The binding site size (n) was therefore set to three base pairs for both complexes. The molar ellipticity of the free ligand  Table S1.

Synthesis of Ir-CMYC
The crude peptide was subsequently coupled to 4.9 eq. of Ir-COOH (see below) overnight using the coupling conditions described.