A symbiotic supramolecular approach to the design of novel amphiphiles with antibacterial properties against MSRA

The co-formulation of supramolecular self-associating amphiphiles (SSAs) enhances solution state physicochemical properties and increases efficacy against methicillin-resistant Staphylococcus aureus.


Experimental Procedures
General Experimental: A positive pressure of nitrogen and oven dried glassware were used for all reactions. All solvents and starting materials were purchased from known chemical suppliers or available stores and used without any further purification unless specifically stipulated. The NMR spectra were obtained using a Burker AV2 400 MHz or AVNEO 400 MHz spectrometer. The data was processed using ACD Labs or Topspin software. NMR Chemical shift values are reported in parts per million (ppm) and calibrated to the centre of the residual solvent peak set (s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet). Tensiometry measurements were undertaken using the Biolin Scientific Theta Attension optical tensiometer. The data was processed using Biolin OneAttension software. A Hamilton (309) syringe was used for the measurements. The melting point for each compound was measured using Stuart SMP10 melting point apparatus. High resolution mass spectrometry was performed using a Bruker microTOF-Q mass spectrometer and spectra recorded and processed using Bruker's Compass Data Analysis software. Infrared spectra were obtained using a Shimadzu IR-Affinity 1 model Infrared spectrometer. The data are analysed in wavenumbers (cm -1 ) using IRsolution software. Fluorescence emission and excitation spectra were obtained using Agilent Technology Cary Eclipse Fluorescence Spectrophotometer and processed using Eclipse ADL (Advanced Reads) software, the results were reported in nm. DLS and Zeta Potential studies were carried out using Anton Paar Litesizer TM 500 and processed using Kalliope TM Professional. Cellular growth curve measurements obtained using Thermo Scientific Multiscan Go 1510-0318C plate reader and recorded using the SkanIt Software 4.0.
Mass Spectrometry: Approximately 1 mg of each compound or mixture of compounds was dissolved in 1 mL of methanol. This solution was further diluted 100-fold before undergoing analysis. 10 μL of each sample was then injected directly into a flow of 10mM ammonium acetate in 95% water (flow rate = 0.02 mL/min).
Fluorometry studies: All samples were prepared by serial dilution from an initial stock solution.
Tensiometry Studies: All the samples were prepared in a EtOH:H2O (1:19) solution. All samples underwent an annealing process in which the various solutions were heated to approximately 40 °C before being allowed to cool to room temperature, allowing each sample to reach a thermodynamic minimum. All samples were prepared through serial dilution of the most concentrated sample. Three surface tension measurements were obtained for each sample at a given concentration, using the pendant drop method. The average values were then used to calculate the critical micelle concentration (CMC).

DLS Studies:
All vials used for preparing the samples were clean dry. All solvents used were filtered to remove any particulates that may interfere with the results obtained. Samples of differing concentrations were obtained through serial dilution of a concentrated solution. All samples underwent an annealing process, in which they were heated to 40 ⁰C before being allowed to cool to 25 ⁰C. A series of 5 runs were recorded at 40 ⁰C to check for sample stability before a series of 10 runs were recorded at 25 ⁰C. Zeta Potential Studies: All vials used for preparing the samples were clean dry. All solvents used were filtered to remove any particulates that may interfere with the results obtained. All samples underwent an annealing process in which the various solutions were heated to approximately 40 °C before cooling to room temperature for 30 mins, allowing each sample to reach a thermodynamic minimum. The final zeta potential value given is an average of the number of experiments conducted at 25 °C. Samples of differing concentrations were obtained through serial dilution of an initial stock solution.
Single Crystal X-ray Studies: A suitable crystal of each amphiphile was selected and mounted on a Rigaku Oxford Diffraction Supernova diffractometer. Data were collected using Cu Kα radiation at 100 K or 293 K as necessary due to crystal instability at lower temperatures. Structures were solved with the ShelXT 1 or ShelXS structure solution programs via Direct Methods and refined with ShelXL 2 by Least Squares minimisation. Olex2 3 was used as an interface to all ShelX programs (CCDC 1866274-1866275).

X-ray Powder Diffraction Studies:
Bulk solid-state phase purity was examined using powder X-ray diffraction carried out on a PANalytical Empyrean diffractometer (40.0 kV, 30.0 mA) operating in θ-2θ reflection geometry and equipped with monochromated Cu Kα1, λ = 1.5406 Å, X-rays, and a X'Celerator 1D detector. The sample was held on a glass plate at room temperature with data collected in intervals of 0.017° over the course of 14hrs. The weakly diffracting nature of the sample together with the small quantity available results in a powder diffraction pattern with only modest signal to noise on top of an undulating background, which is consistent with that observed from a pattern of the glass plate. Powder diffraction patterns were calculated from the structures of Structures 1, 2 and 3 using Crystal Diffract 6.5.5 ( Figure S117). 4 The observed diffraction pattern was very similar to that of Structure 3, with all observed peaks and peak splitting being consistent with this being the dominant phase in the solid state. In contrast there were significant differences between the diffraction pattern of Structure 1 and the experimentally observed diffraction pattern suggesting this phase is not present. The moderate quality of the data and similarity of the powder diffraction pattern of Structure 1 and 2 prevents us from concluding that the sample is completely anhydrous but if present Structure 1 is likely a minority phase as several of the diffraction peaks expected from this phase are not observed.
Fluorescence and Transmission Microscopy Studies: All samples were visualized using an Olympus XI71 microscope with a PlanApo 100x OTIRFM-SP 1.49 NA lens attached to a PIFOC z-axis focus drive (Physik Instrumente, Karlsruhe, Germany), which was placed onto a ASI motorised stage (ASI, Eugene, OR). The objective lens, the environmental chamber along with the sample holder sustained the required temperature. The samples were illuminated using LED light sources (Cairn Research Ltd, Faversham, UK) using filters suitable for each sample (Chroma, Bellows Falls, VT). Metamorph software (Molecular Devices) software was used to control the settings and analyze the images, visualized using Zyla 5.5 (Andor) CMOS camera. 10 µl of each of the appropriate samples was pipetted onto the centre of an agarose pad. Coverslip was used to cover the pipetted sample and was secured in place. Each of the agarose pads was labelled. Filters used in the studies: GFP excitation 480 nm and emission 510 nm, DAPI excitation 360 and emission 460nm.
MIC50 studies 5 : Preparation of luria broth media (LB): Yeast extract (5 g), tryptone (10 g) and sodium chloride (10 g) were dissolved in milli-q H₂O (1000 mL) then divided into 400 mL bottles and autoclaved. Preparation of luria broth (LB) Agar plates: Agar (6 g) was added to LB (400 mL) and autoclaved. Once cool, the LB agar was poured into sterile petri dishes under sterile conditions and allowed to set. LB plates were stored at 4 °C until use. Preparation of bacterial plates: Sterile LB agar plates were streaked using the desired bacteria (MRSA USA300) then incubated in the 25 °C incubator overnight. Preparation of antimicrobial compounds for MIC50 studies: Stock solutions of compounds 1, 2 and 4 were prepared in a 1:19 EtOH:milli-q H₂O mixture the day of experiment. Eight concentrations of each compound/mixture were then prepared from the stock solution in the same solvent mixture. Preparation of inoculum: A starter culture was produced through the inoculation of LB media (5 mL) with ≥ 4 single colonies of the desired bacteria under sterile conditions and incubated at 37 °C overnight. The following day, a subculture was made using LB (5 mL) and the starter culture (100 µL), then incubated at 37 °C until the culture had reached an optical density of 0.4 at 600 nm. Cellular density was then adjusted using sterile milli-q H₂O to equal a 0.5 McFarland Standard (10 7 -10 8 cfu/mL), then a 1:10 dilution was carried out using sterile milli-q H₂O (900 µL) and the McFarland adjusted suspension (100 µL). A final dilution (1:100) was carried out on the 1:10 suspension (150 µL) using LB (14.85 mL) before use (10 5 cfu/mL). Preparation of 96 well Microplate: The 1:100 suspension (90 µL) was pipetted into the desired wells under sterile conditions, then solutions containing 1, 2, 4 or 1:1 mixtures of 1, 2 and 4 (90 µL) were added to the wells to equal a total volume of 180 µL. The plates were sealed using Parafilm, then incubated at 37 °C in a microplate reader for 18-25 hours. An absorbance reading was taken at 600nm every 15 minutes. Each experiment was repeated three times on two different days giving six repetitions in total. Calculation of MIC50: Growth curves were plotted using the average of the six comparative absorbance readings in Microsoft Excel. The MIC50 value was determined by plotting the average absorbance reading obtained at 900 minutes for each compound concentration in Origin. The resulting curve was [normalized] and fitted using the Boltzmann fit, and the equation from this fit was used to calculate the MIC50. Figure S1. Chemical structures of compounds 1-5. TBA = Tetrabutylammonium.

Compound 1:
A solution of 2-aminoanthraquinone (0.67 g, 3.01 mM) and triphosgene (0.445 g, 1.50 mM) in ethyl acetate (30 mL) was heated at reflux for four hours. TBA aminomethanesulfonate (1.06 g, 3.01 mM) in ethyl acetate (10 mL) was then added to the reaction mixture, which was then heated at reflux overnight. The resultant mixture was filtered and the solid isolated was dissolved in methanol (15 mL). Any remaining solid was removed by filtration and the filtrate taken to dryness. The resultant solid was re-dissolved in chloroform (20 mL) and washed with water (1 x 10 mL). The organic layer was then taken to dryness to give the final product as an orange solid (0.

Compound 2:
This compound was synthesised in line with previously published methods. 6 The proton spectrum matches previously reported data. 1                                         [a] Ion not observed. Table S3. An overview of species observed by high resolution ESI -ve mass spectrometry for mixtures containing 1, 2 and 4 in a 1:1 ratio. Ma and Mb represent the anionic component of that amphiphilic salts contained within the mixtures analysed.     As it is probable that larger aggregate structures of 4 exist within the DMSO solution that may not be visible using NMR, self-association constants were not determined using these data.                                      .

Particle size (nm)
Particle size (nm) Figure S94. The average intensity particle size distribution calculated using 10 DLS runs for compound 4 (55.56 mM) in a DMSO solution at 298 K. Figure S95. The average intensity particle size distribution calculated using 10 DLS runs for compounds 1 and 2 in a 1:1 mix (total concentration 55.56 mM) and a DMSO solution at 298 K.

Particle size (nm)
Particle size (nm) Figure S96. The average intensity particle size distribution calculated using 10 DLS runs for compounds 1 and 4 in a 1:1 mix (total concentration 55.56 mM) and a DMSO solution at 298 K.               . Disorder within the tetrabutylammonium counter cations has meant that the carbons atom from three of the eight butyl arms have been modelled isotopically, without the presence of the associated hydrogen atoms. This provides the most accurate model based on the limitations placed on these data by those single crystal samples obtained. Table S7. Hydrogen bond distances and angles observed for hydrogen bonded complex formation, calculated from single crystal X-ray structure, Figure S115.   Figure S117). Table S8. Hydrogen bond distances and angles observed for hydrogen bonded complex formation, calculated from single crystal X-ray structure, Figure S116.