Light‐Activated Rhenium Complexes with Dual Mode of Action against Bacteria

Abstract New antibiotics and innovative approaches to kill drug‐resistant bacteria are urgently needed. Metal complexes offer access to alternative modes of action but have only sparingly been investigated in antibacterial drug discovery. We have developed a light‐activated rhenium complex with activity against drug‐resistant S. aureus and E. coli. The activity profile against mutant strains combined with assessments of cellular uptake and synergy suggest two distinct modes of action.

MeOH to remove excess NaCl and evaporated again after filtration. The crude of 2.1 was then purified by preparative HPLC (method A).
Scheme S3. Synthetic route to L3 and complex 3.
L3 was prepared using an analogous procedure as for L1 and then used without further purification for the preparation of 3 according to the general procedure for rhenium complexes. [1] ICP-MS measurements.
ICP-MS experimental work was performed at the Environmental Geochemistry Laboratory of the School of Earth and Environmental Sciences, The University of Queensland.

Analysis
Analysis by ICP-MS (Agilent 7900) is done in collision mode through the He collision cell. Instrument parameters are as follows: RF power: 1550 W, Carrier gas: 1.08 L/min, Nebuliser pump: 0.10 rps He Flow: 5 mL/min, 115 In was used as an internal standard to monitor instrumental drift during the experiment. The ICP-MS is a highly sensitive instrument and easily damaged. Its working range is around 10 ppb, and concentrations above 30 ppb should be avoided.

Experimental Procedure
Aqueous samples are arranged in auto-sampler tubes in a numbered rack in the auto-sampler. Calibration standards are prepared to enclose the range of expected concentrations and also loaded into the auto-sampler. A separate set of standards are made up from different primary standards to that used for preparing the calibration standards, to serve as controls for the calibration and the instrument performance. These are used to test the accuracy of the analytical process, designated as the standards as unknowns. Instrument blanks (that is, solvent blanks consisting of 2% ultrapure nitric acid) are analysed at regular intervals during the experiment to determine the detection limits for the experiment. At least 7 blanks are required for a good detection limit determination. The internal standard is automatically added to each sample during analysis to correct for internal drift. A monitoring sample, similar to the samples being analysed, is also analysed at regular intervals during the experiment, to monitor any unexpected variations in instrument performance (external drift) and used to correct for this. Duplicate analysis are taken of every 10th sample at the end of the experimental run, to determine precision of analysis.

Data Reduction
Data reduction is completed after exporting the raw data to Excel and completing drift corrections, calibration and subsequent calculations in Excel. The data is processed as follows: 1.
Export the raw data from the instrument to a csv or Excel file.

2.
Pre-treat the data. Pre-treatment may include transformation, blank subtraction and internal and external drift correction. The degree of pre-treatment is dependent on the software associated with the analytical instrument.

3.
Construct calibration curves for the analysed data, using the known concentrations of the standards and the instrument response to each analyte.

4.
Determine whether the calibrations are linear or non-linear. Accurate calculations can only be done on linear calibrations. If necessary, remove the most concentrated standards from the calibration line until a linear result is achieved.

5.
Determine whether the analyte content for all samples fall within calibration range. If there are analytes beyond calibration range, the analysis will have to be repeated, using either a more concentrated standard or a more diluted sample. For ICP-MS, a more diluted sample is generally required.

6.
Once suitable calibration figures have been established, test the legitimacy of the calibration by calculating the concentrations of the calibration standards from the intensity data and the newly-constructed calibration curves. The concentration of the standards should be within 90-110% of the actual, known concentrations. For values in the ppb range, concentrations within 80%-120% of the known value, are acceptable. If not, the calibration curves will need to be refined until acceptable values are obtained. This step is known as recovery.

7.
Calculate the standards as unknowns. Their values should be within 5-10% of their expected value.

8.
Calculate the detection limits by finding the standard deviations of the intensity values for the instrument blanks and multiplying the value with 3. Then calculate the associated concentration (the detection limit) by using this value and the calibration equation for each analyte.

9.
Calculate the precision or reproducibility of the analysis by calculating the average and standard deviation on each set of duplicates. Multiply the standard deviation with 100 and divide by the average. This figure is the percentage relative standard deviation. For trace elements, they should not exceed 5%. Figure S1. Cellular uptake and distribution of rhenium in S. aureus after 60 min (incubated with 50 µM of 1). Percentage of rhenium in different fractions given as percentage of total detected rhenium.
Serially diluted compounds were added to plate containing cells and incubated for ~20 hours at 37 °C, 5% CO2.

Haemolysis Assays
Human whole blood (Australian Red Cross) was washed three times with 3 volumes of 0.9% NaCl and resuspended in a concentration of 0.5 x 10 8 cells/mL, determined by manual cell count in a Neubauer  14.9 ± 0.1 139.5 ± 0.1