Enantiomeric glycosylated cationic block co-beta-peptides eradicate Staphylococcus aureus biofilms and antibiotic-tolerant persisters

The treatment of bacterial infections is hindered by the presence of biofilms and metabolically inactive persisters. Here, we report the synthesis of an enantiomeric block co-beta-peptide, poly(amido-D-glucose)-block-poly(beta-L-lysine), with high yield and purity by one-shot one-pot anionic-ring opening (co)polymerization. The co-beta-peptide is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA), including replicating, biofilm and persister bacterial cells, and also disperses biofilm biomass. It is active towards community-acquired and hospital-associated MRSA strains which are resistant to multiple drugs including vancomycin and daptomycin. Its antibacterial activity is superior to that of vancomycin in MRSA mouse and human ex vivo skin infection models, with no acute in vivo toxicity in repeated dosing in mice at above therapeutic levels. The copolymer displays bacteria-activated surfactant-like properties, resulting from contact with the bacterial envelope. Our results indicate that this class of non-toxic molecule, effective against different bacterial sub-populations, has promising potential for the treatment of S. aureus infections.


General Methods and Instrumentation
Commercially available materials purchased from Alfa Aesar or Aldrich were used as received. All reactions were carried out under argon using standard techniques, unless otherwise noted. THF was distilled over sodium benzophenone ketyl under argon.
Carbon nuclear magnetic resonance ( 13 C NMR) spectra were recorded on a Bruker AV400 (100 MHz) spectrometer. Protected polymer molecular weights were determined by gel permeation chromatography (GPC) versus polystyrene standards using DMF (1 mg mL -1 LiBr) as the eluent at a flow rate of 1.0 mL min -1 through two Styragel columns (HR5 and HR5E, 7.8 × 300 mm) in series at 40 º C with a refractive index detector. Circular dichroism (CD) spectra were obtained using a Chirascan circular dichroism spectrometer with samples dissolved in buffer in a 1 cm path-length quartz cuvette. Flash chromatography was performed using Merck silica gel 60 with distilled solvents. Analytical thin-layer chromatography (TLC) was carried out on Merck 60 F254 pre-coated silica gel plate (0.2 mm thickness). Visualization was performed using a UV lamp. Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) measurements were obtained using Applied Biosystems 4700 series. Biomarkers of in vivo studies were recorded using Blood Chemistry Analyzer Pointcare V2 (MNChip).

Monomer Synthesis Procedures
Synthesis of N-Cbz-β-lactam-L-hLys 1 (BLK p ) β 3 -hLys was prepared following a published procedure 1 . Starting from a commercially available protected form of amino acid L-lysine, β 3 -hLys was obtained in high yield via Arndt-Eistert reaction. Then β 3 -hLys was cyclized following the general procedure of Mukaiyama 2 to generate N-Boc-β-lactam-L-hLys. A solution of N-Boc-β-lactam-L-hLys (970 mg, 4 mmol) in dichloromethane (5 mL) was added to trifluoroacetic acid (3.1 mL, 40 mmol) dropwise at 0 º C, the mixture was stirred at room temperature for about 1 hour until the N-Boc-β-lactam-L-hLys was completely consumed (monitored S3 by TLC). After completion, the mixture was cooled to 0 º C, then quenched with a saturated aqueous solution of NaHCO3 (20 mL). The reaction was diluted with THF (10 mL) followed by the addition of benzyl chloroformate (0.7 mL, 5 mmol), the resulting mixture was stirred at room temperature for 8 hours and then extracted with diethyl ether (2 × 20 mL). The combined organic extracts were then dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (5:1 EtOAc:hexane) on silica gel and then recrystallized from EtOAc/hexane to give N-Cbz-β-lactam-L-hLys 1 (BLK p ) as a white solid (yield > 90%). Rf 0.25, (eluent: EtOAc:hexane = 5:1); 1 The cyclic sugar derived β-lactam monomer 2 (O-Bn-β-lactam-D-glucose or DGu p ) was prepared on multigram scales in moderate yield following reported methods 3 via the stereoselective cycloaddition of tri-O-benzyl-D-glucal and chlorosulfonyl isocyanate, followed by in situ reduction to remove the sulfonyl group.

General Polymerization Procedure
Stock solutions of the monomers 1 and 2 were prepared in a glove box by weighing 2 mmol of each monomer separately into oven-dried 10 mL volumetric flasks. The Into an oven-dried Schlenk tube equipped with a magnetic stir bar was placed a total of 2 mL (0.4 mmol) of monomer solution, adjusted for the desired proportion of each monomer in the polymerization feed (e.g., for PDGu p (10)-b-PBLK p (10), a 1:1 mixture S4 of 1 and 2, 1 mL of 1 and 1 mL of 2 stock solutions was used). To the mixture was then added 1 mL (0.02 mmol, 5 mol %) of tBuBzCl stock solution. The Schlenk tube was sealed, removed from the glove box and cooled to -30 º C under argon atmosphere. To the stirring reaction solution was then added 1 mL (0.05 mmol, 12.5 mol %) of LHMDS stock solution. The resulting mixture was stirred at −30 ºC for about 8 hours until the reaction finished (monitored by TLC), and was then quenched with methanol. After completion, the solution was transferred into a plastic 50 mL centrifuge tube, and the reaction vial was rinsed with a small amount of THF such that the total tube volume was about 5 mL. Hexane (40 mL) was then added to the tube, from which a white solid precipitated. The mixture was centrifuged, and the supernatant solution was decanted.
After two more repetitions of the precipitation/centrifugation procedure, the white pellet was dried overnight under a nitrogen stream to yield the protected product
Sodium was washed in toluene and hexane and cut into small pieces before addition.
The reaction mixture was warmed to -55 °C and maintained at this temperature for about 2 hours, after which a saturated aqueous solution of ammonium chloride (NH4Cl, 10 mL) was added to quench the reaction. Meanwhile, the deep blue color disappeared.
The solution was warmed to room temperature in a water bath to evaporate the ammonia. The resulting clear solution was filtered, washed with DI water and dialyzed with 1,000 MWCO tubing for 36 hours with 10 water changes. After lyophilization, PDGu(7)-b-PBLK(13) was obtained as an amorphous white solid (>90% yield). Other copolymers (PDGu(x)-b-PBLK(y), x+y=20) were synthesized using the same conditions. NMR integrations showed that the ratio of DGu to BLK in PDGu(x)-b-PBLK(y) differed from the stoichiometric ratio of added monomers 1 and 2 in the polymerization step (Table 1, Supplementary Figure 7).

Molecular weight determination using MALDI-TOF
The molecular weights of (a) homocationic homopolymer PBLK ( Da and 3391 Da for homocationic, homosguar and block copolymer respectively.

Membrane assays
Propidium iodide (PI) (L13152 Invitrogen) was used following the manufacturers' protocol. Log phase MRSA USA300 was grown as previously described and washed three times with PBS, and resuspended to a final concentration of 10 8 CFU mL -1 in PBS. Polymers were added to bacteria suspension to achieve desired concentrations and incubated for 30 minutes or 1.5 hours before washing and staining with PI dye for 15 minutes. Samples were washed twice, diluted to 10 7 CFU mL -1 in PBS and analyzed using Flow cytometry (BD Accuri C6 plus). Data were plotted as histogram of FL3-A channel. Bacteria without polymer treatment, dead bacteria (70°C, 30min) and nisin treated bacteria (128 µg mL -1 ) served as negative control, positive control and antibiotics control respectively. Data gating and analysis was done using FCS Express Liposomes were prepared by dissolving POPG (2-oleoyl-1-palmitoyl-sn-glycero-3phospho-rac-(1-glycerol), Avanti company) or POPC (2-oleoyl-1-palmitoyl-snglycero-3-phosphocholine, Avanti company) in chloroform at 10 mg mL -1 and vacuum drying at room temperature for 30 minutes. The lipid thin film was rehydrated with DI water, sonicated in hot water bath for 2 minutes, followed by vigorous vortexing for 2 minutes. The sonication-vortexing process was repeated 3 more times and the lipid suspension was extruded manually using a Mini-Extruder (Avanti ® ) through a 100nm membrane to obtain unilamellar liposomes.  Before the initiation of the polymerization, the beta-lactam (M1) is deprotonated by a base (LiHMDS) to obtain the beta-lactamate anion I; simultaneously, the betalactam is promoted to form an imide II. The initiation then involves the reaction of the anionic monomer I with the imide II to form a dimeric anion III (Step 1). Proton transfer exchange then occurs between the anion III and another beta-lactam (M1) to form a new imide IV and another beta-lactamate anion I respectively. Propagation is repeated nucleophilic addition of the beta-lactamate anion I to imide IV followed by proton transfer (Step 2). After M1 is consumed, M2 is initiated with the active terminal of anion III with proton transfer followed by nucleophilic addition of a new generated beta-lactamate anion V to form an anionic intermediate VI ( Step 3), which undergoes propagation with M2 and final termination (Step 4) to form the block copolymer VII (P1-P2).

Supplementary Note
Circular dichroism study shows that the homocationic PBLK(20) transitions from a random coil to a left-handed helix. In water, the block copolymer PDGu (

Simulation parameters
All Molecular Dynamics (MD) simulations were performed by Gromacs 4.6.3 package 11 . The CHARMM general force field 12 and CHARMM36 lipid force field 13 were used to characterize the beta-peptide sequence and the lipid molecules, respectively. During the simulation, a leap-frog algorithm was used to integrate Newton's equations of motion, and the time step was set to 2fs. The counter ions, sodium and chlorine were added to TIP3P water 14 model to neutralize the system and S39 reach a concentration of 0.15M physiological salt solution. All the covalent bonds in solutes and solvents were constrained by the LINCS algorithm 15 and the SETTLE algorithm 16 , respectively. The cut-off for the short range electrostatics and Lennard-Jones interactions were both set to 1.2 nm and the particle mesh Ewald (PME) 17 algorithm was used to calculate the long range electrostatic interactions. Before the production simulations, energy minimization of the system was done using the steepest descent algorithm, and then the system was equilibrated by 100 ps simulation in NVT ensemble and 100 ps simulation in NPT ensemble with position restraint of the peptide.
The reference temperature and pressure were set to 310K and 1 bar respectively, and the Nose-Hoover method 18 and the semi-isotropic Parrinello-Rahman method 19 (isotropic Parrinello-Rahman method for systems without membrane) were applied for temperature coupling and pressure coupling. All the visualization work was done by PyMol, version 1.5.0.3.
The well-tempered meta-dynamics simulations were performed with the help of the PLUMED 2.1.3 plug-in 20 . The system went through the same procedures as described above before the production sampling. We chose the number of the backbone H-bonds of the PBLK as a biased collective variable (CV) to enhance the production sampling.
This CV is defined by the following switching function: where ( ) gives the instantaneous distance between CV atom and atom , 0 = 0.25 defines the distance cutoff for H-bonds, and and are 8 and 12 respectively. The Gaussian-width for H-bonds was set as 0.5. The initial Gaussian height was set to 2 kJ mol -1 and the bias factor was set to 8. The Gaussian deposition was performed every 1 ps. However, this scaled-down factor is not compatible with the lipid model used in our S40 simulation system, which is described by the CHARMM 36 lipid force field with a scaling factor of 1. Thus we didn't use their parameters for compatibility reasons. In our simulation, the beta-peptide parameters are created based on similar structures that are available in the CHARMM general force field. The backbone dihedral angles φ, θ, and ψ of the beta-peptide, as depicted by a typical beta-peptide in Supplementary Figure   34a, are described by CG2O1-NG2S1-CG311-CG321, NG2S1-CG311-CG321-CG2O1 and CG311-CG321-CG2O1-NG2S1 in the CHARMM general force field, respectively, where the CG2O1 is the carbonyl carbon, NG2S1 is the peptide nitrogen, CG311 is the aliphatic carbon in CH group, and CG321 is the aliphatic carbon in CH2 group. These parameters, according to the annotation from the force field, are obtained from the alanine dipeptide parameters, alkane parameters, and alanine dipeptide parameters, respectively.
Consequently, the PDGu(7)-b-PBLK(13) exists as one continuous helix during the whole binding process, as indicated by the small RMSD value of its heavy atoms with respect to those of the initial configuration (Supplementary Figure 34k).

In vivo murine systemic toxicity (intravenous injection) study
Clinically significant biomarkers were recorded before, and 24 hours, 4 days and 7 days after the first injection and data are expressed as mean ± standard deviation in Table 3. For histopathology studies, mice were sacrificed at 48 hours post last injection, and histological analysis was performed on the tissues obtained from their harvested organs (liver, kidney, and spleen). The sections were examined by a pathologist and no pathological changes were detected in comparison of tissue sections of treated mice with that of the controls. These results provide evidence that the prepared formulation has negligible acute tissue toxicity.