Supplementary information for

A fluorescent probe for investigating metabolic stability of active transplatin analogues

Jacek L. Kolanowski, Lucy J. Dawson, Linda Mitchell, Zelong Lim, Marcus E. Graziotto, Wojciech K. Filipek, Trevor W. Hambley, Elizabeth J. New*

School of Chemistry, The University of Sydney, NSW 2006, Australia

elizabeth.new@sydney.edu.au

Cytotoxicity measurements

A549 cells were cultured in Advanced Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific) supplemented with 2% foetal calf serum (Thermo Fisher Scientific) and 2.5 mM L-glutamine (Sigma-Aldrich) and maintained at 37 °C in 5% carbon dioxide. All Pt complex treatments were prepared by dissolving the solid complex in DMF to a concentration of 50 mM. The stock was diluted to 1 mM in sterile phosphate-buffered saline (PBS, Sigma-Aldrich) immediately before performing the assay.

96 well plates were seeded with 2000 cells per well and incubated for 36 h at 37 °C in 5% carbon dioxide. The cells were treated with varying concentrations of the appropriate Pt complex in triplicate, so that the final concentration of DMF in any of the wells was less than 0.2%. The cells were incubated for a further 72 h at 37 °C in 5% carbon dioxide. After this time, 10 L resazurin solution (0.2 mM in PBS) was added to each well, and the plate allowed to incubate for a further 3 h. After this, the fluorescence intensity of each well was measured at 590 nm with excitation at 570 nm, with a Perkin Elmer EnSpire Multimedia plate reader. The fluorescence intensities were normalised to control wells before analysis. The data were fitted to a non-linear ‘log(inhibitor) vs. response’ equation using GraphPad Prism 7 software and the IC50 was calculated by the software.

Scheme S1: Synthesis of RPt and RPt1’

Figure S1. Normalised integrated fluorescence intensity (exc = 540 nm, emission at 582-586 nm) of RPt1 (50 M in HEPES buffer, pH 7.4, 20 mM, 1% DMF) in the presence of 10 equivalents of metal ions (all as nitrate salts, except trans-Pt-pyridine, Fe(II) as Fe(NH4)2(SO4)2 and Cu(I) which has been delivered in 1:100 dilution from aa stock solution of [Cu(CH3CN)4]PF6 in acetonitrile). Bar graphs represent mean ± SD for n=3.

Figure S2. 1H NMR spectrum of RPt1.

Figure S3. 13C NMR spectrum of RPt1.

Figure S4. 1H NMR spectrum of RPt1’.

Figure S5. 13C NMR spectrum of RPt1’.