Ruthenium-centred btp glycoclusters as inhibitors for Pseudomonas aeruginosa biofilm formation

Carbohydrate-decorated clusters (glycoclusters) centred on a Ru(ii) ion were synthesised and tested for their activity against Pseudomonas aeruginosa biofilm formation. These clusters were designed by conjugating a range of carbohydrate motifs (galactose, glucose, mannose and lactose, as well as galactose with a triethylene glycol spacer) to a btp (2,6-bis(1,2,3-triazol-4-yl)pyridine) scaffold. This scaffold, which possesses a C2 symmetry, is an excellent ligand for d-metal ions, and thus the formation of the Ru(ii)-centred glycoclusters 7 and 8Gal was achieved from 5 and 6Gal; each possessing four deprotected carbohydrates. Glycocluster 8Gal, which has a flexible spacer between the btp and galactose moieties, showed significant inhibition of P. aeruginosa bacterial biofilm formation. By contrast, glycocluster 7, which lacked the flexible linker, didn't show significant antimicrobial effects and neither does the ligand 6Gal alone. These results are proposed to arise from carbohydrate–lectin interactions with LecA, which are possible for the flexible metal-centred multivalent glycocluster. Metal-centred glycoclusters present a structurally versatile class of antimicrobial agent for P. aeruginosa, of which this is, to the best of our knowledge, the first example.

As a control experiment, the ability of ligand 6Gal, (the precursor to complex 8Gal) which showed anti-biofilm activity was also assessed. The ligand did not show any statistically significant inhibition of biofilm formation by PAO1 under conditions analogous to those described in the main body of the manuscript S1 . S1 G. A. O'Toole, J. Vis. Exp. 2011, 47, 2437.

Minimum Inhibitory Concentration (MIC) Testing
Ligand 6Gal and complexes 7 and 8Gal were tested for their ability to inhibit growth of P. aeruginosa (PAO1) at various concentrations. In brief: PAO1 was seeded into wells at 10 6 CFU/ml. Serial dilutions of each of the compounds was then added to the wells and a set of control wells with no compound was also set up. The plates were incubated for 24hrs at 37 o C before absorption readings at 600nm were taken. The Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that inhibits the visible in-vitro growth of microorganisms. Each experiment was performed in triplicate. Compounds at high concentration were very coloured but turbidity could be observed in all wells (See images alongside MIC graphs below).

Minimum Bactericidal Concentration (MBC) Testing
After 24 hours incubation of PAO1 with various concentrations of ligand 6Gal or complexes 7 and 8Gal, samples from wells were applied to TSA (Tryptic Soy Agar) for culturing colonies. Since turbidity was clear in all wells, only the three highest compound concentration wells for each compound were plated out. The Minimum bactericidal concentration (MBC) is defined as the lowest concentration of antibiotic that kills 99.9% of the inoculum. It is determined by subculturing the last clear MIC tube onto growth medium and examining for bacterial growth. All plates returned TNTC (Too numerous to count) meaning none of the compounds tested had any bactericidal effect on PAO1 (Pseudomonas aeruginosa).  Complexes 7 and 8Gal are non-toxic to HeLa cells. HeLa cells were treated for 24h with a range of concentrations of the indicated compounds in a 96-well plate. After the required incubation period, alamar blue dye (20 µL) was added to each well and samples were incubated for 4h. Values represent the mean ± S.E.M. of two independent experiments performed in triplicate. Cytotoxicity testing was carried out by Dr Sandra Bright (Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland) as described below.
Cell culture: HeLa (human cervical cancer) cells were grown in Dulbecco's Modified Eagle Medium (Glutamax) supplemented with 10% fetal bovine serum and 50 µg/ml penicillin/streptomycin at 37˚C in a humidified atmosphere of 5% CO2.
Alamar blue viability assay: HeLa cells were seeded at a density of 5x10 3 cells/well in 96-well plates and treated with the indicated compounds for 24h. Alamar blue (20 µL) was then added to each well and incubated at 37°C in the dark for 4h. Plates were then read on a fluorescence plate reader (SpectraMax Gemini, Molecular Devices) with excitation and emission wavelengths of 544nm and 590nm respectively. Activity is expressed as percentage cell viability compared to vehicle treated controls. All data points (expressed as means ± S.E.M.) were analysed using GRAPHPAD Prism (Graphpad software Inc., San Diego, CA). The following ALERTS were generated. Each ALERT has the format test-name_ALERT_alert-type_alert-level. Click on the hyperlinks for more details of the test.

Alert level B
THETM01_ALERT_3_B The value of sine(theta_max)/wavelength is less than 0.575 Calculated sin(theta_max)/wavelength = 0.5562 Author Response: The reflectivity of this sample was weak, therefore copper wavelength was used in order to improve the data quality. The resolution was pushed to the limit but even though the resolution was limited.  0 Info 0 ALERT level A = Most likely a serious problem -resolve or explain 4 ALERT level B = A potentially serious problem, consider carefully 9 ALERT level C = Check. Ensure it is not caused by an omission or oversight 19 ALERT level G = General information/check it is not something unexpected 2 ALERT type 1 CIF construction/syntax error, inconsistent or missing data 10 ALERT type 2 Indicator that the structure model may be wrong or deficient 7 ALERT type 3 Indicator that the structure quality may be low 13 ALERT type 4 Improvement, methodology, query or suggestion 0 ALERT type 5 Informative message, check It is advisable to attempt to resolve as many as possible of the alerts in all categories. Often the minor alerts point to easily fixed oversights, errors and omissions in your CIF or refinement strategy, so attention to these fine details can be worthwhile. In order to resolve some of the more serious problems it may be necessary to carry out additional measurements or structure refinements. However, the purpose of your study may justify the reported deviations and the more serious of these should normally be commented upon in the discussion or experimental section of a paper or in the "special_details" fields of the CIF. checkCIF was carefully designed to identify outliers and unusual parameters, but every test has its limitations and alerts that are not important in a particular case may appear. Conversely, the absence of alerts does not guarantee there are no aspects of the results needing attention. It is up to the individual to critically assess their own results and, if necessary, seek expert advice.

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