Supramolecular Kandinsky circles with high antibacterial activity

Nested concentric structures widely exist in nature and designed systems with circles, polygons, polyhedra, and spheres sharing the same center or axis. It still remains challenging to construct discrete nested architecture at (supra)molecular level. Herein, three generations (G2−G4) of giant nested supramolecules, or Kandinsky circles, have been designed and assembled with molecular weight 17,964, 27,713 and 38,352 Da, respectively. In the ligand preparation, consecutive condensation between precursors with primary amines and pyrylium salts is applied to modularize the synthesis. These discrete nested supramolecules are prone to assemble into tubular nanostructures through hierarchical self-assembly. Furthermore, nested supramolecules display high antimicrobial activity against Gram-positive pathogen methicillin-resistant Staphylococcus aureus (MRSA), and negligible toxicity to eukaryotic cells, while the corresponding ligands do not show potent antimicrobial activity.


TWIM-MS.
The TWIM-MS data were collected under the following conditions: ESI capillary voltage, 3 kV; sample cone voltage, 30 V; extraction cone voltage, 3.5 V; source temperature 100 º C; desolvation temperature, 100 º C; cone gas flow, 10 L/h; desolvation gas flow, 700 L/h (N2); source gas control, 0 mL/min; trap gas control, 2 mL/min; helium cell gas control, 100 mL/min; ion mobility (IM) cell gas control, 30 mL/min; sample flow rate, 5 μL/min; IM traveling wave height, 25 V; and IM traveling wave velocity, 1000 m/s. Q was set in rf-only mode to transmit all ions produced by ESI into the triwave region for the acquisition of TWIM-MS data.

Molecular modeling. Energy minimization of the macrocycles was conducted with Materials
Studio version 4.2, using the Anneal and Geometry Optimization tasks in the Forcite module (Accelrys Software, Inc.). All counterions were omitted. An initially energy-minimized structure was subjected to 100 annealing cycles with initial and mid-cycle temperatures of 300 and 1000 K, respectively, twenty heating ramps per cycle, one thousand dynamic steps per ramp, and one femtosecond per dynamic step. A constant volume/constant energy (NVE) ensemble was used and the geometry was optimized after each cycle. Geometry optimization used a universal force field with atom-based summation and cubic spline truncation for both the electrostatic and Van der Waals parameters. 100 energy-minimized structures were selected for the calculation of theoretical collision cross-sections using MOBCAL programs.
TEM. The sample mixtures were drop-casted on to a carbon-coated Cu grid (400 mesh, purchased from SPI supplies), and the extra solution was absorbed by filter paper to avoid further aggregation. The TEM images were taken with a FEI Morgagni transmission electron microscope.
AFM. AFM imaging was performed on a Digital Instrument Nanoscope Dimension 3000 system. The sample was prepared by using DMF solution (ca. 1 × 10 −4 mol/L), dropped on freshly cleaved mica surface, rinsed with three drops of fresh acetonitrile, and then dried in air. Silicon cantilevers tip with spring constant of around 0.1 N/m was used for the experiments.

STM.
The sample was dissolved in DMF at a concentration of 5.0 mg/mL. Solution (5 µL) was dropped on HOPG surface. After 30 seconds, surface was washed slightly with water for three times and totally dried in R.T. in air. The STM images were taken with a Pico Plus SPM system with a PicoScan 3000 Controller. The obtained STM images were processed by WSxM software. 3 3D deconvolution fluorescence microscopy. A 100 µL aliquot of cultures, of mid-logarithmic phase Escherichia coli cells grown in Luria-Bertani medium and Staphylococcus aureus cells grown in tryptic soy broth medium, were incubated with synthetic compounds G2 and G3 at indicated concentrations for 5 min at room temperature. When indicated, FM4-64 (0.01 mg/mL final concentration) was added to visualize cell membrane immediately prior to imaging. 5 µL was spotted on a glass bottom culture dish (MatTek) and covered with a 1% agarose pad made with distilled water. Cells were viewed at room temperature with a DeltaVision Elite microscope system (Applied Precision/GE Healthcare) equipped with a Photometrics CoolSnap HQ2 camera and an environmental chamber. Fluorescence of FM4-64 and the synthetic compounds G2 and G3 were captured with TRITC and DAPI filters respectively. Fluorescence of supramolecule G4 was captured using TRITC filter in the absence of FM4-64. Seventeen planes were acquired every 200 nm, and the data were deconvolved using manufacturer-provided SoftWorx software.
Minimum inhibitory concentrations (MICs) against MRSA. 4 The antimicrobial activity of the compounds are tested against Methicillin-resistant S. aureus (MRSA, ATCC 33591). Briefly, a single colony of MRSA bacterium was inoculated in 3 mL TSB medium and allowed to grow overnight at 37 °C. The bacteria culture was then diluted at 1:100 and the bacteria were able to re-grow to mid-logarithmic phase in 6-8 h. Next, 50 µL compounds in 2-fold serially diluted solution with the concentrations of 0.1-25 µg/mL were added the 96-well plate containing 50 µL of bacteria suspension (1 × 10 6 CFU/mL) in each well. Following that, the plate was incubated at 37 °C for 16 h, and the absorption of those wells at 600 nm wavelength was read on a Biotek Synergy H1 microtiter plate reader. The MICs were determined as the lowest concentrations that completely inhibit the growth of MRSA in 24 h. The experiment were repeated at least three times with duplicates each time.
After cooling down to room temperature, NaHCO3 was used to neutralize the mixture. (1. 4 mg, 4.6μmol) in MeOH (4.5 mL) was added, and then the mixture was stood at 50 º C for 3 h.
After cooling to room temperature, 60 mg of NH4PF6 was added and brown precipitate was observed.

Experimental and theoretical collision cross sections
Supplementary  rvdW is the van der Waals radii of the solvent, acetonitrile, which is far less than rh of G2-G4. As a result, c ≈ 6. "fs" is the frictional coefficient, and it is derived from a and b for the oblate spheroid model (b = b > a) with the formula:  Values in the red boxes (b and a in meter) are the only ones iteratively changed  "kb" is the Boltzmann constant, "T" is the experimental temperature (298 K), and "η" is the viscosity of CD3CN (0.000367 Pa s)  "a" and "b" are the semiminor and semimajor axis of the oblate spheroid. "p" is the aspect ratio (p = b/a)  "r(cal)" is the radius of a sphere with an equivalent volume as the spheroid generated by a and b; since spheroid volume (V) has the relationship with a and b: V =  "rh(ob)/r(cal)" is the ratio of the hydrodynamic radius generated from the oblate model and the equivalent radius generated by the volume of the spheroid; this ratio was used to guide the adjustment of a and b until the volume generated by them matches the volume generated by rh(ob) (rh(ob)/r(cal) = 1)