CROSS-REFERENCE TO RELATED APPLICATION

This application refers to and claims the benefit of priority of the Singapore Patent Application No. 10201703056S filed on 13 April 2017, the content of which is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT. FIELD OF THE INVENTION

The present invention relates generally to compounds, pharmaceutical compositions, and methods for the treatment or prevention of bacterial infections and diseases resulting therefrom.

BACKGROUND OF THE INVENTION

The rise of multi-drug resistant superbugs is no longer an issue that people can afford to ignore these days. Drug-resistant infections are estimated to cause more than 2 million infections and 23,000 deaths in the United States annually, in addition to $20 billion direct loss and $35 billion additional cost. In Europe, antibiotic resistance resulted in an estimated 25,000 deaths and€1 .5 billion loss. In many developing countries, the problem was more serious. Antibiotics-resistant pathogens were responsible for 58,000 neonatal sepsis in India. In order to circumvent the problem, new safe and effective antimicrobial agents are needed. Natural antimicrobial (AMPs), also called "host defense peptides", with the common characteristics of facial amphiphilicity show high bactericidal efficiency against a broad spectrum of microbial infections. Unlike most conventional antibiotics that target bacterial/fungi cell metabolism, natural AMPs break down the cell cytoplasmic membranes and may also enhance immunity by functioning as immunomodulators. Many AMPs have also other killing mechanisms which involve intracellular targets such as apidaecin, which inhibits protein synthesis. It is generally thought that it is more difficult for bacteria to evolve resistance to the membrane-disruption mode of action, although resistance has been observed with AMPs through moderation of the LPS charge. The discovery of AMPs in the 1980s sparked extensive exploration of these natural antimicrobial agents and synthetic analogues for potential therapeutic applications. However, AMPs and their analogues are sensitive to physiological environment, display rather high toxicity to host cells and are costly to synthesize/extract. Few AMPs have thus been approved for clinical applications (and these include LL-37, Gramicidin S, Vancomycin, Nisin, Daptomycin and polymyxins).

Substantial research is also dedicated to synthetic mimics of antimicrobial peptides (SMAMPs) with the goal to overcome drawbacks of natural AMPs. The inventors have reported cationic peptidopolysaccharides of chitosan-graft-polylysine (CS-g-Κιβ) that show excellent antimicrobial activity with ultra-low hemolysis. Zhou et al synthesized copeptides of poly(KioF7.sL7.5) and poly(KioFi5) through a-amino acid N-carboxyanhydrides (NCA) ring-opening polymerization, which proved more effective against a panel of bacteria than many natural AMPs.

Compared to cr-peptides, /3-peptides more easily form different types of secondary structures that may be fine-tuned to possess facial amphiphilicity as a result of suitable arrangement of β- amino acids. Facially amphiphilic structures have been thought to be important for accentuating the antibacterial effect. Another intrinsic merit of /3-peptides stems from their stability towards enzymes in physiological environment. Advances in the synthesis of /3-amino acids and peptides makes it possible to access and study AMP mimics based on /3-peptides. Gellman et al. initially reported magainin mimic antimicrobial β-peptides (β-17) with excellent activity against a panel of pathogens. DeGrado et al. also designed a series of 3i4-helix β-oligomers with activity and selectivity comparable to those of naturally occurring AMPs. Franzyk et al. compared the influence of different backbones, i.e. a-peptides, a-peptoids, β-peptides, β- peptoids, and their hydrides, on antimicrobial activity. These studies indicated that peptidomimetics could be more active than natural a-peptides and demonstrated that incorporation of unnatural amino acid analogues not only improves the stability of antimicrobial oligomers, but also significantly influences their activity. More recently Gellman et al. synthesized a family of random nylon-3 (β-peptides) copolymers through anionic ring opening polymerization, which showed highly effective antimicrobial activity and biocompatibility. However, the MICs of these, like most other AMPs, remain higher than most commercial antibiotics. Therefore, there is still need in the art for innovations that overcome the drawbacks of existing techniques.

SUMMARY OF THE INVENTION

The present invention meets the aforementioned need in the art by providing novel compounds and compositions as well as methods of preparing and using them .

In a first aspect, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof

wherein Ar represent C1-C12 hydrocarbon, preferably aromatic hydrocarbon, more preferably Ci- C6-alkylphenyl, more preferably tert-butyl-phenyl; R represents OH or NH2, preferably OH; and m and n each independently represent an integer of at least 1 , preferably up to 20, 30, 40, 50, 55, 60, 65, or 70, more preferably up to 75, 80, 85, 90, or 95, most preferably up to 100.

In various embodiments, Ar represents 4-tert-butylphenyl, m is an integer of 1 to 20, and n is an integer of 1 to 30.

In various embodiments, Ar represents 4-tert-butylphenyl ; and m is 2 and n is 18, m is 4 and n is 16, m is 6 and n is 14, m is 8 and n is 12, m is 10 and n is 10, m is 20 and n is 30, or m is 12 and n is 8.

In preferred embodiments, Ar represents 4-tert-butylphenyl, m is 8, and n is 12.

In a second aspect, the invention relates to a pharmaceutical composition comprising the compound disclosed herein and a pharmaceutically acceptable carrier.

In various embodiments, the pharmaceutical composition comprises a further antibacterial agent.

In various embodiments, the further antibacterial agent is selected from the group consisting of Rifampicin, Chloramphenicol, Trimethoprim, Sulfamethoxazole, Carbenicillin, Ampicillin, Ceftazidime, Ciprofloxacin, Polymyxin B, Colistin, Novobiocin, Carbapenem or combinations thereof.

In a third aspect, the invention relates to a method of preparing the compound or pharmaceutical composition disclosed herein, the method comprising the steps of:

(a) preparing a compound of formula (II)

by reacting a compound of formula (III)

with a compound of formula (IV)

under suitable conditions, wherein PGi and PG2 each independently represent a protecting group; and

(b) preparing the compound of formula (I) by deprotecting the compound of formula (II) under suitable conditions.

In preferred embodiments, PGi is benzyl (Bn), and PG2 is Carboxybenzyl (Cbz).

In preferred embodiments, step (a) comprises preparing the compound of formula (II) in the presence of lithium bis(trimethylsilyl)amide (LiHMDS) and 4-tert-butylbenzoyl chloride (ArCOCI); and/or step (b) comprises deprotecting the compound of formula (II) under sodium in liquid ammonia condition.

In various embodiments, the compound of formula (III) is derived from glucose or galactose.

In a fourth aspect, the invention relates to a method of treating or preventing a bacterial infection or a disease resulting from said bacterial infection in a subject, the method comprising administering to the subject an effective amount of a compound or a pharmaceutical composition disclosed herein.

In various embodiments, the bacterium is multidrug-resistant.

In various embodiments, the method comprises administering to the subject an effective amount of a further antibacterial agent. In various embodiments, the further antibacterial agent is selected from the group consisting of Rifampicin, Chloramphenicol, Trimethoprim, Sulfamethoxazole, Carbenicillin, Ampicillin, Ceftazidime, Ciprofloxacin, Polymyxin B, Colistin, Novobiocin, Carbapenem or combinations thereof.

In various embodiments, the subject is a mammal, preferably a human.

In a fifth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as an inhibitor of cellular efflux pumps and/or bacterial membrane permeabilizer.

In a sixth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as an antibacterial agent. In a seventh aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as a medicament.

In an eighth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as a medicament for treating or preventing a bacterial infection or a disease resulting from said bacterial infection.

In a ninth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment or prevention of a bacterial infection or a disease resulting from said bacterial infection.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings. Figure 1. Kinetic investigation of polymerization. Condition: [DM(Cbz)]=0.0533 M, [AS(Bn)]=0.0355 M, activator [ArCOCI ]=0.00444 M, catalyst [LiHMDS]=0.01 1 1 M, room temperature, (a) Remaining monomer concentration and conversion evaluation with time, (b) GPC curve elution of polymerization at selected slot using DMF (1 mg/ml LiBr addition) as eluent. (c) Molecular weight (Mn) and molecular weight distribution (D) as a function of conversion. Figure 2. CD spectrum of PASm-d-PDMn in water. Sample concentration is 0.05 mg/ml and molecular simulation of 3-D conformation of PAS20, PAS8-6-PDM12, and PDM20. The data indicate that the sugar block forms a helical structure. Figure 3. Molecular simulation of helical stability in PAS6-6-PDM14, PAS7-6-PDM13 and PAS8-6-PDM12.

Figure 4. CD spectrum of PAS8-6-PDM12 over a range of pH values in PBS. Figure 5. Histological examination toxicity of main organs including liver, spleen and kidney.

Figure 6. Super resolution STED microscopy images of P. aeruginosa (PA01 ) (A), Methicillin- resistant S. aureus (BAA-40) (B), and E. coli (K12) (C) treated with [100 μ^ η\] of rhodamine- PAS8-6-PDM12 labeled, (a); rhodamine-PDM20 labeled, (b); Penetrating, loosening and lysis of membrane in PA01 (A) treated with [100 ig/m\] of rhodamine-PAS8-6-PDM12 labeled, (c). Cells were stained with membrane dye FM1 -43FX and visualized. Images were acquired after 1 h of compound treatment. Scale bars =2 μιτι. (d) Cryo-TEM images of P. aeruginosa (PA01 ) in

-1

the presence of 100 \Q ml. PAS8-6-PDM12 and PDM20, scale bar = 200nm. (e) Outer membrane permeability of PA01 treated with our copolymers and homocationic polymers was measured by NPN assay, (f) Inner membrane distribution of PA01 treated with copolymers and homocationic polymers was measured by DiSC35.

Figure 7. Drug synergy in P. aeruginosa PA01 wound infection. Female C57BL/6 mice with wounds infected with 10 ⁶ CFU P. aeruginosa PA01 . Wounds were treated at 4 hpi and 24 hpi and bacteria recovered at 28 hpi for enumeration. Each data point represents one wound sample (one mouse) and the horizontal bar represents the median. Statistical analysis using Kruskall-Wallis test with Dunn's post-test for multiple comparisons to the infection control group, ^* = P<0.05, log reduction from the infection control group are indicated. Figure 8. PAS8-0-PDM12 dependent potentiation of rifampicin in a system murine infection caused by (a) clinical multi-drug resistant P. aeruginosa PAER, (b) clinical multi-drug resistant E.coli (ECOR-1 ) and (c) clinical multi-drug resistant K. pneumoniae (KPNR-1 ). Bacterial count in the intraperitoneal fluid was determined by plating on LB agar plate. Every spot represents the bacterial count in one mice and black lines represent geometric mean of them (n=10).

Figure 9. Chequerboard broth microdilution assays showing PAS8-0-PDM12 had no influence on the potency of rifampicin two Gram-positive bacteria (a) S. aureus MRSA-BAA40 and (b) E. faecalis V583. Dark regions represent higher cell density, (c) Super resolution STED microscopy images of P. aeruginosa (PA01 ) were treated with 100 μg/ml of rhodamine-PAS8- />PDM12, scale bar 2 μm. (d) PAS8-_>PDM12 and polymyxin B showing dose-dependent increase in outer membrane permeability of P. aeruginosa (PA01 ), 1 -N-phenylnaphthylamine (NPN) used as an indicator of outer membrane permeability, (e) Hierarchical clustering analysis and a heatmap of the differentially expressed genes of P. aeruginosa (PA01 ) treated by different concentrations of rifampicin (5μg mL ¹ and 30μg mL ¹ ), PAS8-_>PDM12 (^g mL ¹ and 30μg mL ¹ ), combination of PAS8-b-PDM12 and rifampicin (PAS8-6-PDM12 15 μg mL ¹ + rifampicin 5 μg mL ¹ and PAS8-_>PDM12 30 μg mL ¹ + Rifampicin 5 μg mL ¹ ) and DMSO control, (f) Metabolomic measurement of rifampicin accumulation inside bacterial treated by rifampicin alone ^g mL ¹ ) and Combination of PAS8-_>PDM12 and rifampicin (PAS8-b-PDM12 30 μg mL ¹ + rifampicin 5 μg mL ¹ ).

Figure 10. (a). Cryo-TEM images of P. aeruginosa (PA01 ) without treatment (upper) and in the presence of 100 μg mL-1 PAS8-6-PDM12 (below), scale bar = 200nm. (b) PAS8-6-PDM12 showing dose-dependent dissipation inner membrane potential in P. aeruginosa (PA01 ). 1 mg/ml Gramicidin S was used as control. Dipropylthiadicarbocyanine iodide [DiSC3(5)] dye used to indicate the dissipated of inner membrane, (c) Dark-field/fluorescence dual-mode microscopic image of wild type P. aeruginosa (PA01), PA01 treated with 16 μg mL ¹ PAS8-b- PDM12, P. aeruginosa efflux pump off mutant PAO750 and PAO750 treated with 16 μg mL ¹ PAS8-_>PDM12, efflux pump substrate ethidium bromide (EtBr, 16 μg mL ¹ ) was used to show fluorescence in cells, scale bar=2 μm. (d) PAS8-_>PDM12 showing dose-dependent increasing the fluorescence intensity of efflux pump substrate ethidium bromide (EtBr, 16 μg mL ¹ ) in the wild type P. aeruginosa PA01 , fluorescence intensity measured by flowcytometry assay, (e) P. aeruginosa efflux pump off mutant PAO750 itself showed higher fluorescence intensity of efflux pump substrate ethidium bromide (EtBr, 16 μg mL ¹ ) than wild type P. aeruginosa PA01 and addition of different concentration of PAS8-_>PDM12 had no influence on the fluorescence intensity in PAO750. Figure 11. Chequerboard broth microdilution assays showing dose-dependent novobiocin potentiation by PAS8-_>PDM12 against (a) wild type P. aeruginosa PA01 , (b) clinical multi-drug resistant P. aeruginosa PAER, (c) clinical drug sensitive K. pneumoniae (KPNS-1 ), (d) clinical multi-drug resistant K. pneumoniae (KPNR-1 ), (e) clinical drug sensitive A.baumannii (ACBAR- 1 ), (f) clinical multi-drug resistant A.baumannii (ABR), (g) clinical drug sensitive E.coli (ECOS-1 ) and d) clinical multi-drug resistant E.coli (ECOR-1 ). Dark regions represent higher cell density. PAS8-_>PDM12 dependent potentiation of rifampicin in a system murine infection caused by (i) clinical multi-drug resistant K. pneumoniae (KPNR-1 ), (j) clinical multi-drug resistant A.baumannii (ABR) and (k) clinical multi-drug resistant E.coli (ECOR-1 ). Bacterial count in the intraperitoneal fluid was determined by plating on LB agar plate. Every spot represents the bacterial count in one mice and black lines represent geometric mean of them (n=10).

Figure 12. Gel permeation chromatography (GPC) of PASm-d-PDMn.

Figure 13. Polymer decrease swim motility of PA01 , scale bar=3.5 mm.

Figure 14. Outer membrane permeability of PAS8-6-PDM12 against other gram negative bacteria including A.baumannii ACBAS, E.coli ECOS and K. pneumonia KPNS were measured by NPN assay.

Figure 15. Chequerboard broth microdilution assays showing dose-dependent rifampicin potentiation by PAS8-6-PDM12 against clinical multi-drug resistant, aeruginosa PAER. Dark regions represent higher cell density.

Figure 16. Chequerboard broth microdilution assays showing dose-dependent rifampicin potentiation by PAS8-6-PDM12 against drug sensitive GNB (a) P. aeruginosa PA01 (b) E. coli ECOS (c) A.baumannii ACBAS (d) K. pneumoniae KPNS. Dark regions represent higher cell density.

Figure 17. Chequerboard broth microdilution assays showing PAS8-0-PDM12 had no influence on the potency of rifampicin against gram positive bacteria E. faecalis V583. Dark regions represent higher cell density. Figure 18. Chequerboard broth microdilution assays showing dose-dependent rifampicin potentiation by PAS8-6-PDM12 against P. aeruginosa pump deficient strain PAO750. Dark regions represent higher cell density.

Figure 19. In vivo synergistic effect of PAS8-0-PDM12 with rifampicin against multi drug resistant GNP P. aeruginosa PAER via a systemic infection model, a) liver, b) spleen, c) Kidney.

Figure 20. In vivo synergistic effect of PAS8-0-PDM12 with rifampicin against multi drug resistant GNP E. coli ECOR via a systemic infection model, a) liver, b) spleen, c) Kidney. Figure 21. In vivo synergistic effect of PAS8-0-PDM12 with rifampicin against multi drug resistant Gram-negative K. pneumonia KPNR (CRE) via a systemic infection model, a) liver, b) spleen, c) Kidney. Figure 22. PAS8-0-PDM12 showing dose-dependent increasing in accumulation of ethidium bromide in the GNB (a) P. aeruginosa PA01 (b) E. coli K12 and (c) A.baumanii ATCC 18883, fluorescence intensity was recorded by TECAN fluorescence spectrometer. Figure 23. Dark-field/fluorescence dual-mode microscopic image of wild type P. aeruginosa PA01 , PA01 treated with 16 xg/ml PAS8-6-PDM12 and its pump deficient strain PAO750 control, efflux pump substrate ethidium bromide was used to show fluorescence in cells, scale bar=8 μm. Figure 24. Quantification of EtBr positive bacteria in other GNB A baumanii ATCC 18883 and E. coli K12. Percentage of EtBr-positive bacteria was measured by flow cytometry.

Figure 25. Chequerboard broth microdilution assays showing activity of carbapenem antibiotics (a) etrapenem and (b) imipenem restored the effect by PAS8-6-PDM12 against its resistant GNB A.baumannii MDRAB. Dark regions represent higher cell density.

Figure 26. Chequerboard broth microdilution assays showing activity of carbapenem antibiotic restored the effect by PAS8-6-PDM12 against its resistant GNB K. pneumonia KPNR (CRE). Dark regions represent higher cell density.

Figure 27. Chequerboard broth microdilution assays showing dose-dependent novobiocin potentiation by PAS8-6-PDM12 against drug sensitive GNB (a) P. aeruginosa PA01 (b) K. pneumoniae KPNS (c) A.baumannii ACBAS (d) E.coli ECOS. Dark regions represent higher cell density.

Figure 28. Chequerboard broth microdilution assays showing dose-dependent tetracycline potentiation by PAS8-6-PDM12 against wild type (a) P. aeruginosa PA01 and (b) absence of potentiation against its efflux pump deficient strain PAO750. Figure 29. Chequerboard broth microdilution assays showing dose-dependent novobiocin potentiation by PAS8-6-PDM12 against wild type P. aeruginosa PA01 (a) and (b) its pump deficient strains PAO750, MIC of novobiocin is much lower for out membrane resistant PAO750 than both outer membrane and efflux resistant PA01 . Dark regions represent higher cell density.

Figure 30. In vivo synergistic effect of PAS8-0-PDM12 with novobiocin against multi drug resistant Gram-negative A baumanii MDRAB via a systemic infection model, a) liver, b) spleen, c) Kidney. Figure 31. In vivo synergistic effect of PAS8-0-PDM12 with novobiocin against multi drug resistant Gram-negative K. pneumonia KPNR CRE via a systemic infection model, a) liver, b) spleen, c) Kidney.

Figure 32. In vivo synergistic effect of PAS8-0-PDM12 with novobiocin against multi drug resistant Gram-negative E. coli ECOR via a systemic infection model, a) liver, b) spleen, c) Kidney. Figure 33. ¹ H NMR of PASm-6-PDMn (protected) in CDC . Figure 34. ¹ H NMR of PASm-6-PDMn in D ₂ 0.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will control. In a first aspect, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof

wherein Ar represent C1-C12 hydrocarbon, preferably aromatic hydrocarbon, more preferably Ci- C6-alkylphenyl, more preferably tert-butyl-phenyl; R represents OH or NH2, preferably OH; and m and n each independently represent an integer of at least 1 , preferably up to 20, 30, 40, 50, 55, 60, 65, or 70, more preferably up to 75, 80, 85, 90, or 95, most preferably up to 100.

By "(Cx-Cy)" (x and y being two different integers) is meant that the group contains x to y carbon atoms.

The term "pharmaceutically acceptable salt" as used herein refers to those salts that are within the scope of proper medicinal assessment, suitable for use in contact with human tissues and organs and those of lower animals, without undue toxicity, irritation, allergic response or similar and are consistent with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are technically well known. In various embodiments, Ar represents 4-tert-butylphenyl, m is an integer of 1 to 20 (namely 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and n is an integer of 1 to 30 (namely 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30). In various embodiments, Ar represents 4-tert-butylphenyl ; and m is 2 and n is 18, m is 4 and n is 16, m is 6 and n is 14, m is 8 and n is 12, m is 10 and n is 10, m is 20 and n is 30, or m is 12 and n is 8.

In preferred embodiments, Ar represents 4-tert-butylphenyl, m is 8, and n is 12.

The compound of formula (I) is a novel class of glycosylated block-like β-peptides, termed herein as PASm-d-PDMn, consisting of a helical poly-amido-saccharide (PAS) domain linked to a cationic segment PDM, which is believed to function as the killing domain. The block copolymer design is advantageous since the functions of both blocks are retained.

Without wishing to be bound to any theory, it is believed that the compound disclosed herein possesses excellent biocompatibility and good antimicrobial activity against both Gram-negative (e.g. P. aeruginosa) and Gram-positive (e.g. S. aureus) bacteria. In a second aspect, the invention relates to a pharmaceutical composition comprising the compound disclosed herein and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; sterile distilled water; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations.

The skilled person would also realize that proper formulation is dependent upon the route of administration selected for the specific application , and the proper route and mode of administering the compound of the invention to a subject should be determined on a case-by- case basis.

Accordingly, the compound of the present invention can be formulated into compositions using pharmaceutically acceptable ingredients as well as established methods of preparation (Gennaro, A.L. and Gennaro, A.R. (2000) Remington : The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). To prepare the pharmaceutical compositions, pharmaceutically inert inorganic or organic excipients can be used. To prepare e.g. pills, powders, gelatin capsules or suppositories, for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils can be used. Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols, and suitable mixtures thereof as well as vegetable oils. The formulations can be sterilized by numerous means, including filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile medium just prior to use. Without wishing to be bound to any theory, it is believed that the compound described herein can synergize with a further antibacterial agent in the antibacterial activity. By "synergistic" or "synergy" is meant that the interaction of two or more agents renders their combined effect to be greater than their individual effects. Therefore, in various embodiments, the pharmaceutical composition may comprise a further antibacterial agent.

The term "antibacterial agent" as used herein refers to any substance or combination of substances capable of: (i) inhibiting, reducing or preventing growth of bacteria; (ii) inhibiting or reducing ability of bacteria to produce infection in a subject; or (iii) inhibiting or reducing ability of bacteria to multiply or remain infective in the environment. The term "antibacterial agent" also refers to a compound capable of decreasing infectivity or virulence of bacteria. Non-limiting examples of antibacterial agents include β-lactam antibacterial agents including, e.g. ampicillin, cloxacillin, oxacillin, and piperacillin, cephalosporins and other cephems including, e.g., cefaclor, cefamandole, cefazolin, cefoperazone, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, and cephalothin; carbapenems including, e.g., imipenem and meropenem ; and glycopeptides, macrolides, quinolones, tetracyclines, and aminoglycosides.

In various embodiments, the further antibacterial agent is selected from the group consisting of Rifampicin, Chloramphenicol, Trimethoprim, Sulfamethoxazole, Carbenicillin, Ampicillin, Ceftazidime, Ciprofloxacin, Polymyxin B, Colistin, Novobiocin, Carbapenem or combinations thereof.

In a third aspect, the invention relates to a method of preparing the compound or pharmaceutical composition disclosed herein, the method comprising the steps of:

(a) preparing a compound of formula (II)

by reacting a compound of formula (III)

with a compound of formula (IV)

under suitable conditions, wherein PGi and PG2 each independently represent a protecting group; and (b) preparing the compound of formula (I) by deprotecting the compound of formula (II) under suitable conditions.

The term "protecting group" (PG) as used herein refers to a chemical group that is reacted with, and bound to, a functional group in a molecule to prevent the functional group from participating in subsequent reactions of the molecule but which group can subsequently be removed to thereby regenerate the unprotected functional group. Additional reference is made to: Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, Oxford, 1997 as evidence that "protecting group" is a term well-established in field of organic chemistry. The protecting groups that may be used in the present method include, without limitation, Benzyl, Carboxybenzyl (Cbz), tert-Butyloxycarbonyl (Boc).

In preferred embodiments, PGi is benzyl (Bn), and PG2 is Carboxybenzyl (Cbz).

In preferred embodiments, step (a) comprises preparing the compound of formula (II) in the presence of lithium bis(trimethylsilyl)amide (LiHMDS) and 4-tert-butylbenzoyl chloride (ArCOCI); and/or step (b) comprises deprotecting the compound of formula (II) under sodium in liquid ammonia condition.

Block copolymers are typically synthesized via "sequential polymerization", which involves multiple isolations and purifications, which is labor consuming and results in loss of polymer yields. By the method described herein, however, the block copolymers of the present invention are prepared via an efficient one-step reaction that takes advantage of the different monomer reactivity ratios, which significantly decreases the possibility of introducing inhibitors to the highly sensitive anionic polymerization, improving the polymerization efficacy.

In various embodiments, the compound of formula (III) is derived from glucose or galactose.

In a fourth aspect, the invention relates to a method of treating or preventing a bacterial infection or a disease resulting from said bacterial infection in a subject, the method comprising administering to the subject an effective amount of a compound or a pharmaceutical composition disclosed herein.

The subject may be any human or non-human animal beings, preferably a mammal, more preferably a human.

The term "treating" refers to having a therapeutic effect and at least partially alleviating or ameliorating an abnormal condition in the subject. The term "preventing" refers to decreasing the probability that a subject contracts or develops pathogenic infection. The term "administering" relates to a method of delivering a compound to a bacterium, cell, or tissue in a subject. Many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.

The method disclosed herein may be used for treating any bacterial infections caused by any pathogens, preferably by Gram-negative pathogens, including but not limiting to, E.coli, P. sudomonia, A. bammanni, K. pneumunia.

Diseases resulting from a bacterial infection may include, without limitation, sepsis and wound dressing.

The compound or the pharmaceutical composition of the invention may be administered via any parenteral or non-parenteral (enteral) route. Parenteral application methods include, for example, intracutaneous, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures, as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders. An overview about pulmonary drug delivery, i.e. either via inhalation of aerosols (which can also be used in intranasal administration) or intracheal instillation is given by Patton et al. Proc Amer Thoracic Soc 2004; Vol. 1 pages 338-344, for example). Non-parenteral delivery modes are, for instance, orally, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or rectally, e.g. in the form of suppositories. The compound of the invention may be administered systemically or topically in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles as desired.

A "therapeutically effective amount" is an amount of a therapeutic agent that produces a desired therapeutic effect in a subject, such as preventing or treating a target condition or alleviating symptoms associated with the condition. The precise therapeutically effective amount is an amount of the compound or the pharmaceutical composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington : The Science and Practice of Pharmacy 21 ^st Edition, U niv. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

In various embodiments, the bacterium is multidrug-resistant. The term "multidrug resistant" as used herein refers to bacteria resistant to multiple drugs of different chemical structures and/or resistant to drugs directed at different targets.

In various embodiments, the method comprises administering to the subject an effective amount of a further antibacterial agent.

As stated above, the compound disclosed herein can synergize with one or more other antibacterial agents in the treating or preventing a bacterial infection or a disease resulting therefrom. Therefore, various combinations of the compound disclosed herein and one or more any additional antibacterial agents, even if not explicitly disclosed herein, are also within the scope of the present application. Determining optimal combination of the two agents is within the knowledge of the person of average skill in the art. In various embodiments, the compound disclosed herein can be administered to a subject (e.g. a human or an animal) in need thereof in an effective amount, alone or in combination (simultaneously, sequentially or separately) with one or more other antibacterial agents. Due to the synergistic effect, a therapeutically effective dose of the compound and/or other antibacterial agents may be lower than a standard dose when are they are used alone. A therapeutically effective dose for the compound and other antibacterial agents in accordance with the co-treatment embodiments may be a dose that is a fraction or a percentage of a standard dose for that compound. In some embodiments, a therapeutically effective dose may be between about 1 % and 99% of a standard dose, between about 1 % and 90% of a standard dose, between about 1 % and 80% of a standard dose, between about 1 % and 70% of a standard dose, between about 1 % and 60% of a standard dose, between about 1 % and 50% of a standard dose, between about 1 % and 40% of a standard dose, between about 1 % and 30% of a standard dose, between about 1 % and 20% of a standard dose, between about 5% and 20% of a standard dose, between about 1 % and 10% of a standard dose or below about 10% of a standard dose for a particular agent. A therapeutically effective dose may also be between about 0.1 % and 1 % of a standard dose, between about between about 0.01 % and 1 % of a standard dose or between about 0.001 % and 1 % of a standard dose for a particular agent. In various embodiments, the further antibacterial agent is selected from the group consisting of Rifampicin, Chloramphenicol, Trimethoprim, Sulfamethoxazole, Carbenicillin, Ampicillin, Ceftazidime, Ciprofloxacin, Polymyxin B, Colistin, Novobiocin, Carbapenem or combinations thereof. In a fifth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as an inhibitor of cellular efflux pumps and/or bacterial membrane permeabilizer.

The term "cellular efflux pump" as used herein refers to a protein assembly which exports substrate molecules from the cytoplasm or periplasm of a cell, in an energy dependent fashion. Such efflux pumps are known in the art. An "efflux pump inhibitor" is a compound which specifically interferes with the ability of an efflux pump to export its normal substrate or other compounds such as antimicrobial agents. Efflux pump inhibitors are useful, for example, for treating microbial infections by reducing the export of a co-administered antimicrobial agent. Thus, a microbe which can efflux an antimicrobial agent is resistant to the inhibiting effect of the antimicrobial agent and becomes sensitive to the inhibiting effect of the same antimicrobial agent in the presence of an efflux pump inhibitor. An efflux pump inhibitor is thus distinguished from generally toxic compounds, general metabolic poisons, energy uncouplers, or other such compounds which have many direct inhibitory effects in a cell. It is recognized that an efflux pump inhibitor may have additional indirect effects.

The term "bacterial membrane permeabilizer" as used herein refers to any compound capable of reducing the integrity of or disrupting the cytoplasmic membrane of a bacterium.

In a sixth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as an antibacterial agent.

In a seventh aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as a medicament.

In an eighth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein as a medicament for treating or preventing a bacterial infection or a disease resulting from said bacterial infection.

In a ninth aspect, the invention relates to use of the compound or the pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment or prevention of a bacterial infection or a disease resulting from said bacterial infection.

The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.

EXAMPLES

Example 1 : Synthesis and Chemical Characterization of the Polymers General methods and Instrumentation

All commercially available chemical compounds were purchased from Aldrich, Alfa-Aesar, Tokyo Chemical Industry (TCI), Cambridge Isotope Laboratories or Aik Moh Paints & Chemicals. Anhydrous toluene was freshly distilled from sodium under argon. Anhydrous tetrahydrofuran (THF) was freshly distilled from sodium and benzophenone was added as indicator and oxygen scavenger under argon. Anhydrous dichloromethane (DCM) was distilled from calcium hydride under argon. Analytical thin-layer chromatography (TLC) was performed on Merck 60 F254 pre-coated silica gel plate. Visualization was performed under UV lamp, potassium permanganate stain or iodine stain. Column chromatography was carried out using Merck silica gel 60. High-purity water with a resistivity of > 15 ΜΩ cm was obtained from a Merck Millipore Integral 3 water purification system. Proton and carbon nuclear magnetic resonance ( ¹ H NMR and ¹³ C NMR) spectroscopy were recorded on a Bruker Avance DPX-300 spectrometer at 300MHz for ¹ H and 75 MHz for ¹³ C. ¹ H chemical shift was recorded relative to CDC (7.26 ppm), or HOD (4.79 ppm). ¹³ C chemical shift was recorded relative to CDCI ₃ (77.0 ppm). Monomer conversion was calculated by Agilent 6890N gas chromatography (GC) equipped with a HP-5 column using anhydrous dodecane as internal standards. The molecular weight (M _n and M _w ) and molecular weight distribution (M M _n ) of protected polymers were measured by gel permeation chromatography (GPC) on a Shimadzu liquid chromatography system equipped with a Shimadzu refractive index detector (RID-10A) and two Polargel columns operating at 40 °C using DMF (with 1 mg/ml LiBr) as the mobile phase at a flowrate of 1 ml/min. β-lactam monomer preparation

Glucose derived β-lactams were synthesized according to the reported procedure (Dane, E. L. & Grinstaff, M. W. Journal of the American Chemical Society 134, 16255-16264 (2012)). In order to deprotect all the protecting group in one step, β-lactam DM(Cbz) protected by carboxybenzyl (Cbz) instead of ferf-butyloxycarbonyl (Boc) group were synthesized by a modification of published methods (Mowery, B. P., Lindner, A. H., Weisblum, B., Stahl, S. S. & Gellman, S. H. Journal of the American Chemical Society 131 , 9735-9745 (2009)). 2.58 g (10 mmol, 1 eqv.) (±)-trans-3-phthalimidylmethyl- 4,4-dimethyl azetidin-2-one (Phth-DM) and 20 ml methanol were added into a 50 ml round-bottom flask. 1 .87 ml (30mmol, 3 eqv.) hydrazine hydrate were injected into the mixture. The resulting solution was stirred at room temperature overnight under argon protection. The slurry precipitate was filtered off through celite and washed with copious methanol. The collected solvent was removed by rotary evaporation. The collected intermediate was dissolved into a 60 ml mixture of 1 :1 THF: saturated sodium bicarbonate, to which 2.9 ml (15mmol, 1 .5 eqv.) benzyl chloroformate was adding. The reaction mixture was refluxed for 5 hours. The concentrated residue was extracted with ethyl acetate and the organic phase was washed with 1 M HCI, 1 M NaOH and saturated NaCI solution . The combined organic solution was dried by anhydrous MgSCU and then purified by chromatography using ethyl acetate and hexane as eluent (1 :1). β-lactam DM(Cbz) was first obtained as a sticky transparent liquid and was solidified by sticking. The obtained β-lactam DM(Cbz) was further purified by recrystallization from dichloromethane and hexane. General procedure of one-shot polymerization

The whole polymerization process was carried out in a glovebox. 0.4 mmol monomer solution, 0.02 mmol solution of co-initiator 4-ferf-butylbenzyl chloride and 0.02 mmol solution of catalyst bis(trimethylsilyl)amide (LiHMDS) was prepared in THF separately. Taking PAS8-6-PDM12 as example: into an oven-dried 10 ml reaction flask, 600 μΙ monomer DM(Cbz) and 400 μΙ monomer AS(Bn) solution together with 1 ml co-initiator solution were added. The mixed solution was stirred for 3 mins at room temperature in a glovebox. After that, 2.5 ml catalyst (2.5 eqv relative to the co-initiator) was added to initiate the reaction. The polymerization reaction was stirred at room temperature for 1 hour. Then the reaction flask was taken out from the glovebox and quenched with a few drops of methanol. The polymer solution was dropped into 50 ml centrifuge tubes, filled with 40 ml hexane to precipitate out. The protected polymers were collected by centrifugation and decantation of supernatant. The collected polymers were re- dissolved in 3 ml dichloromethane, and the precipitation/centrifugation was repeated for another 2 cycles. Finally, the polymers were dried under argon and then further dried in an oil pump. The protected polymers were analysis by NMR and GPC with DMF containing 1 mg/ml LiBr as mobile phase.

General procedure of one-pot global deprotection

140 mg protected polymers and 54 mg potassium tert-butoxide (KOt-Bu) were dissolved in 5.0 ml of anhydrous THF. The polymer solution was added dropwise to a rapidly stirred solution of sodium (120 mg) in liquid ammonia (-15 ml) at -78 ^S C under nitrogen. Sodium was washed in toluene and hexane and cut into small pieces before addition. The reaction was stirred at -78 °C for 1 h, after which saturated ammonium chloride was added drop by drop until the blue color disappeared. The remaining ammonia was vapored at room temperature. The resulting solution was filtered, washed with Dl water and dialyzed with 1 000 Da cutoff dialysis bag for 2 days with frequent change of water. Finally, the white powder samples were obtained by freezing dry. Kinetic investigation of the polymerization

Kinetic investigation of the polymer PAS8-6-PDM12 was carried by gas chromatography (GC) that was used to determine the unreacted monomers at specific time, and by gel permeation chromatography (GPC) that was applied for calculating the molecular weight of the polymers at the same time. The anionic ring opening polymerization was carried out in the same way as description above. Into a 10 ml reaction flask, 600 μΙ 0.4 mmol DM(Cbz) and 400 μΙ 0.4 mmol AS(Bn) monomer solution together with 1 ml co-initiator solution were added. In order to quantify the monomer residues, 10 μΙ inner standard anhydrous dodecane was added. A drop of the mixture was then transferred to a vial before addition of LiHMDS to initiate the reaction. After the polymerization was triggered, 100 μΙ reaction mixture was taken out and put into a vial filled with 100 μΙ methanol at designed time points, which quenched the polymerization immediately. After collecting the samples at all the time plot, the vials were subjected to GC to calculate the ratio of peak areas of the left β-lactams to inner standards, which was conversed to the ratio of concentration based on the premade calibration curve. The same vials were further analyzed by GPC to get the molecular weight evaluation of the polymerization.

The most common method for preparing poly-p-peptides is by anionic ring opening polymerization (AROP) of corresponding β-lactam monomers(Cheng and Deming, 2001 ; Eisenbach and Lenz, 1 976; Hashimoto, 2000). Herein, we prepared a racemic cationic β-lactam monomer and a chiral sugar derived β-lactam monomer on multigram scales in moderate yield, namely benzyl ((3,3-dimethyl-4-oxoazetidin-2-yl)methyl)carbamate (DM(Cbz)) and (1 S,3R,4S,5R,6R)-4,5-bis(benzyloxy)-3-((benzyloxy)methyl)-2-ox a-8-azabicyclo[4.2.0]octan-7- one (AS(Bn)), by the reported methods via [2+2] cycloaddition reaction between N- Chlorosulfonylisocyanate (CSI) and the corresponding alkene derivative, followed by in situ reduction to remove the sulfonyl group in the β-lactam rings(Dane and Grinstaff, 2012; Mowery et al., 2009). In order to achieve one-step global deprotection, we protected the amine in the side chain of β-lactam DM(Cbz) with Carboxybenzyl (Cbz), which will be removed together with benzyl (Bn) under sodium in liquid ammonia condition. Moving forwards, Scheme 1 shows preparation of PASm-d-PDMn copolymers. The general approach involves anionic ring opening polymerization the two β-lactams initiated with lithium bis(trimethylsilyl)amide (LiHMDS) in the presence of activator 4-tert-butylbenzoyl chloride (ArCOCI). The activator generates the corresponding imide co-initiator in situ and controls the molecular weight [e.g. 5% ArCOCI was added to reach the degree of polymerization in theory (DPtheo) = 20]. Note that even though the two monomer precursors are added concurrently one obtains a block-like structure. After termination with methanol, the anionic copolymerization yields an intermediate product, which is treated with sodium in liquid ammonia to remove carboxybenzyl (Cbz) and benzoyl (Bz) protecting groups ultimately providing the desired water soluble PASm-d-PDMn product.

Scheme 1. One shot AROP polymerization and one pot global deprotection to prepare glycosylated cationic beta peptides Gas chromatography was used to examine the relative rate of consumption of the 2 precursors upon addition of LiHMDS/ArCOCI to a premixed solution of DM(Cbz) (0.053 M) and AS(Bn) (0.035M). The results of this study are shown in a (Figure 1 ). These concentration profiles reveal that AS(Bn) is consumed almost completely within 30 seconds, while less than 5% of DM(Cbz) disappears over this period. Indeed, it took over 30 minutes for DM(Cbz) to reach the same degree of conversion relative to what is obtained with AS(Bn) in 30 seconds. Gel permeation chromatography (GPC) was used to monitor the growth of molecular weight over time. As shown in b (Figure 1 ), the absence of AS(Bn), with the concomitant the emergence of polymer is observed after 1 minute of reaction, one observes the absence of AS(Bn), the emergence of polymer and unreacted DM(Cbz). With increasing reaction time, the molecular weight of the product increases with concurrent disappearance of DM(Cbz). A plot of molecular weight (Mn) vs. conversion shows a linear relationship and the dispersity (D) of the products remains within the 1 .2 to 1 .3 range (c) (Figure 1). These observations indicate a growth of polymer that involves fast consumption of AS(Bn), followed by a slower incorporation of DM(Cbz) without significant termination or chain transfer events. Different from the traditional block copolymers that are synthesized mostly based on the sequential addition of monomers, which increases the possibility of terminating "living" chains, these fortuitous conditions allow us to prepare block-copolymer structures by the simultaneous addition of both reactants, a feature that simplifies considerably the synthetic entry into the target products. Unlike the helical structure of conventional peptides, stabilized by hydrogen bonding between the backbone(Choi et al., 2010; Johnson and Gellman, 2013), the formation of a left-hand 3i4 helix structure of PAS was investigated via both circular dichroism (CD), molecular modeling and NMR by Grinstaff et al(Chin et al., 201 6; Dane and Grinstaff, 2012; Stidham et al., 2014). They suggested the helical structure detected in the PAS polymers is derived from the conformationally restricted pyranose ring and the inter-residue hydrogen bonding played a minor role. Although we found that the hydrogen bond between sugar ring C3 (i) and carbonyl groups (i+3) on peptide backbone co-exists with the helix structure during our simulation of 20- mer PAS (Figure 2), it is not the cause of the helix but the results of the helical structure. This hypothesis is supported by simulation of copolymers with short PAS lengths (see below). As the cationic lactam monomer DM(Cbz) was racemic, the homocationic polymers PDM20 existed as random coil structure (Figure 2). Therefore, the copolymers PASm-d-PDMn were expected to exhibit helix-random coil structure. Indeed, the copolymers PAS-0-PDMn exhibited a circular dichroism spectrum similar to the PAS and the signal intensity increased with increased PAS ratio (Figure 2). However, we found that PAS6-6-PDM14, PAS7-6-PDM13 deformed quickly and PAS8-6-PDM12 could retain the helical-random coil structure same as the start (Figure 3). This indicated that PAS8 was needed to maintain a stable helix. The secondary structure of the PAS8-6-PDM12 did not significantly change over a range of pH value in phosphate buffer (Figure 4).

Example 2: Biological Characterization of the Polymers Minimum Inhibitory Concentration (MIC)

Microbial Strains, culture medium and inoculums preparation: The test microorganism strains selected to evaluate the antibacterial activity of glycosylated cationic polymers were both gram- negative and gram-positive species obtained from ATCC. The following ATCC strains were used in this study: Escherichia coli (ATCC #8739), Pseudomonas aeruginosa PA01 (gift from Scotts Rice at Nanyang Technology University), Staphylococcus aureus (ATCC #29213) and MRSA (ATCC #BAA 40). All the microbial strains were streaked on a fresh appropriate agar plate (LB agar) from the glycerol stock and incubated overnight at 37 °C. Inocula were prepared by transferring single colony from each strain to a sterile Mullet Hilton Broth (MHB) (Difco®, Becton, Dickinson and Company) and incubated at 37°C with vigorous shaking (225 rpm) to obtained culture in the logarithmic growth phase. The culture was then washed and diluted to and the optical density (OD). The final bacterial density in the microtiter plates was in the range of 10 ⁵ CFU/ml, density that was confirmed by plating on agar plates.

Determination of Minimum Inhibitory Concentration (MIC): The MIC values of the glycosylated cationic polymers against the test microorganisms were determined by broth microdilution method using 96-well microtiter plates as recommended by the CLSI guideline with slight modification. The polymers stock solutions were prepared in deionized water (Dl water). The MHB broth (50 μΙ) was added into 96-well plate and used to dilute the polymers. 50 μΙ of bacterial suspension was then added into each well and incubated at 37 °C for 1 8 hrs. Bacterial growth was determined by measuring the optical density at a wavelength of 600nm (Οϋβοο) on a TECAN, infinite F200, or by visual examination of the turbidity. MIC values were defined as the lowest concentration that leads to a reduction in bacterial growth by more than 90%, or by the absence of a defined bacterial pellet. Synergistic study

Microbial Strains, culture medium and inoculums preparation: The test microorganism strains selected to evaluate the synergistic effect was Pseudomonas aeruginosa PA01 reference strain was obtained from Scotts Rice at Nanyang Technology University. The isogenic multiple Mex pump mutant, Pseudomonas aeruginosa PAO750 (A(mexAB-oprM), A(mexCD-oprJ), A(mexEF- oprN), A(mexJK), A(mexXY), AopmH, ApscC) was kindly contributed by Ayush Kumar at University of Manitoba. Clinical isolates P. aeruginosa PAD1 and PAD25 were gift from Singapore Centre for Environmental Life Sciences Engineering (SCELSE). Clinical isolates, PAES (PAN-sensitive P. aeruginosa), PAER (multi-drug resistant P. aeruginosa), ACBAS (PAN- sensitive A.baumannii), MDRAB (multi-drug resistant A.baumannii), KPNS (PAN-sensitive K.pneumoniae), KPNR (CRE) (carbapenem resistant K.pneumoniae), ECOS (PAN-sensitive E.coli) and ECOR (multi-drug resistant E.coli) were in-kind contribution by Sanjay Ryan Menon, Rao Pooja and Sarah Maria Bergin from Tan Tock Seng Hospital. Clinical MDR K.pneumonia BAK085 was isolated in National University Hospital (NUH) and extensively drug-resistant (XDR) A. baumanii X26 was isolated in Tan Tock Seng Hospital. Gram positive strains Staphylococcus aureus (ATCC #BAA 40) and E. faecalis (ATCC #700802) were purchased from ATCC. Overnight culture of bacteria cells was prepared in Mueller Hinton Broth (Sigma- Aldrich) medium by inoculating bacteria colony from streaked plate with shaking at 37°C. Pre- culture was prepared by diluting overnight culture to Ο βοο: 0.01 (approximately 1 0 ⁶ CFU/mL) in fresh MHB media.

Checkboard assay: The checkerboard method was used to assess the synergistic activity between antibiotic and glycosylated cationic polymer against bacteria (White, R. L, Burgess, D. S., Manduru, M. & Bosso, J. A. Antimicrobial agents and chemotherapy 40, 1 914-1918 (1996)). Briefly, serial twofold dilutions of each antibiotic and glycosylated cationic polymer were prepared separately. After dilution, a total of 25 μΙ of antibiotic in MHB (along y-axis) and 25μΙ of polymer in MHB (along x-axis) was dispensed into each well of a 96-well plate. 50 μΙ of a bacterial inoculum of -1 x10 ⁶ per ml in MHB was dispensed into each well that contained antibiotic-polymer mixture in various ratios. The bacteria and antibiotic-polymer mixture was mixed thoroughly with a thermomixer at a speed of 1 ,000 rpm for 30s. The plate was incubated at 37°C for 18 h under aerobic conditions. The bacterial growth was examined by measuring the ODeoo.

Time-kill Assay

Time kill was conducted by incubating bacteria with polymer, antibiotics and their combination, and viability determination by CFU count on agar plates at various time points. Briefly, 50 μΙ 2 X concentration of polymer, antibiotics and their combination were added in 96 well plate. 50 μΙ of P. aeruginosa PA01 at a density of -1 x10 ⁶ CFU/ml in MHB medium was added before incubation. Bacteria in MHB suspension without addition of polymer and antibiotic were used as positive control. The plate was incubated aerobically under shaking at 37 °C. At t = 0 h, 1 h, 3 h, 7 h, 12 h and 18 h, bacteria viability was determined by CFU plating on LB agar plates. In vitro cytotoxicity measurement

Mammalian Cell Biocompatibility test

Cell lines: The fibroblasts 3T3 cell line obtained from ATCC (#CRL-1658™) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (glutamine (2 raM), penicillin (100 units/ml), and streptomycin (100 μg/ml). The cells were maintained at 37°C in a humidified incubator with 5% CO2 until a monolayer with greater than 80% confluence, was obtained.

Cytotoxicity assay: Synthetic glycosylated cationic polymers were tested for in vitro cytotoxicity, using 3T3 fibroblast cell lines by colorimetric assay the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT). Briefly, the cultured cell lines were harvested from the flask by trypsinization and pooled in a 50 ml vials. Then, the cells were plated at a density of 2 x10 ⁴ cells/ml per well (200μΙ) into 96-well tissue culture plates. The plates were incubated at 37°C in a humidified with 5% CO2 incubator for 24h without agitation. The polymer stock solution was prepared in PBS at a concentration of 1 0 mg/ml and diluted to desired concentrations in DMEM complete medium. Polymer in DMEM solution was added into 96-well plate (cell seeded plate) and control (without compounds) was added as a comparison. The plates were incubated at 37°C in a humidified with 5% CO2 incubator for 24h without agitation. After 24hr, the culture medium containing the compounds was discarded and the cells were rinsed with PBS (phosphate buffer saline). MTT (200 μΙ of 5mg/ml) was added into each well and the plates were incubated in a CO2 incubator for an additional 4h. The MTT was aspirated and dimethyl sulfoxide (DMSO) was added into each well and the plates were kept in the shaker (at 150rpm) until the intracellular formazan crystals were completely solubilized. The absorbance of each well was measured at 570 nm using microplate reader spectrophotometer (BIO-RAD, Benchmark Plus). All the assays were performed in triplicate. The percentage of the cell viability was calculated by the formula:

% Cell viability^ (Average abs of treated cells)/ (Average abs of controls)x100%

Hemolytic activity test

Fresh human blood (5 ml) was collected from a healthy donor (age 28, male). Erythrocytes were separated by centrifugation at 1 ,000g for 10 mins, washed three times with Tris buffer (1 0 mM Tris, 150 mM NaCI, pH 7.2) and diluted to a final concentration of 5% vol/vol. 50 μΙ of polymer solution at different concentrations were mixed with 50 μΙ of erythrocytes stock, followed by addition to a 96-well microplate and incubated for 1 h at 37 °C with shaking at 1 50 rpm. The microplate was centrifuged at 1000g for 10 mins. 80 μΙ aliquots of the supernatant were then transferred to a new 96-well microplate and diluted with another 80 μΙ of Tris buffer. Hemolytic activity was determined at 540 nm with a 96-well plate spectrophotometer (Benchmark Plus, BIO-RAD). Triton X-100 (0.1 % in Tris buffer), which is able to lyse RBCs completely, was used as positive control, while Tris buffer was used as negative control. The hemolysis percentage (H) was calculated from the following equation:

Hemolysis = [(Op - Ob)/(Ot -Ob)] x 100%

where 0 is the absorbance for the polymers, Ob is the absorbance for the negative control (Tris buffer), and Ot is the absorbance for the positive control of Triton X-100.

In vivo systemic toxicity test

All animal experiments were carried out in accordance with the Code of Practice for the Insitutional Care and Use of Animals for Scientific Purposes approved by the Ethics Committee of Union Hospital, Huazhong University of Science and Technology.

Female BALB/c mice (6-8 weeks, 1 8-22 g) were selected randomly and divided into untreated control group and treatment group (n=6 in each group). The untreated control group was injected with 200 μΙ PBS and the treatment group was injected with polymers at dosage of 10 mg/kg. Injection was given intravenously through the tail vein. The liver and kidney function as well as balance of electrolytes were determined overtime using celecare M (MNCHIP, Tianjin). Histological Examination: The mice were sacrificed 7 days after polymer injection and histological examination of main organs including liver, spleen and kidney was performed. Briefly, tissues were fixed with 4% paraformaldehyde, routinely processed, and embedded in paraffin and 5 μηι in thickness. These sections were stained with hematoxylin-eosin (H&E) for microscopic examination.

Stimulated emission depletion microscopy (STED)

To prepare samples for super resolution STED microscopy, bacterial cells were pelleted by centrifugation at 3,000 X g for 10 min, suspended in culture media at a concentration of 10 ⁸ CFU/ml and incubated for 1 h in the dark with PAS8-6-PDM12. Membrane stain FM1 -43FX (5 μς/ιηΙ; Life Technologies) was added to the samples before washing three times with PBS and resuspending in a fixative solution of 2% paraformaldehyde in PBS. Cells were fixed for 2 h at room temperature, washed three times in PBS and applied to a sterile glass bottom collagen coated dish (MatTek Corporation). STED super resolution microscopy was performed on a Leica TCS SP8 STED-3X (Leica Microsystems, Wetzlar, Germany) at SingHealth Advanced Biomaging Core. Further image processing requiring deconvolution, the Huygens Professional software (Scientific Volume Imaging, Hilversum, Netherlands) was used. Cryo-TEM microscopy

Mid-log phase bacterial cells were prepared by centrifugation at 3,000 X g for 10 min, suspended in culture media at a concentration of 10 ⁸ CFU/ml and incubated for 1 h with PAS8- 6-PDM12. Bacteria suspension was centrifuged and resuspended in PBS to prepare sample for cryo-TEM image at IMB-IMCB Joint Electron Microscopy Suite, A ^* STAR, Singapore.

Outer membrane permeability assay (NPN assay)

1 -N-phenylnaphthylamine (NPN) assay was conducted on gram negative bacteria to determine the ability of the polymers to permeabilize outer membranes2. Bacteria overnight cultures were prepared by inoculating few colonies in Mueller Hinton Broth (MHB broth) at 37°C overnight. 50μΙ_ of overnight bacteria suspension was inoculated in 5ml_ fresh MHB medium (1 :100 dilution) and incubated for 3 hrs to reach log phase. Bacterial cells were then collected by centrifugation at 3,000 X g for 10 min, washed twice with 5 mM HEPES buffer and resuspended at a concentration of 107 CFU/ml in 5 mM HEPES buffer. 50 μΙ_ of 40 μΜ NPN solution was added to 100 μΙ_ of bacteria suspension in 96 well plate (black, corning costar). 50 μΙ_ polymer in HEPES buffer solution (at desired concentration) was added, and fluorescent signal was immediately captured (TECAN, infinite F200) at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The following negative controls were also recorded: 100 μΙ_ of bacteria + 100 μΙ_ of HEPES buffer; 50 μΙ_ of 40 μΜ NPN + 150 μΙ_ of HEPES buffer; 100 μΙ_ of bacteria + 50 μΙ_ of HEPES buffer + 50 μΙ_ of 40 μΜ NPN.

Rifampicin accumulation in P. aeruginosa

Sample preparation for endometabolome extraction: A pre-culture of PA01 was prepared by diluting overnight culture to an Οϋβοο of 0.01 in fresh MHB, incubated with shaking at 37 °C, 200 rpm to log phase (OD6oo= 0.5). The bacteria culture was harvested by centrifugation at 4,000 rpm, 10 mins, washed once with fresh MHB. Rifampicin with or without PAS8-0-PDM12 was added to a bacteria culture and incubated with shaking at 37 °C, for 30 min at 200 rpm. 1 ml aliquot was quickly spun down at 1 0,000 rpm for 3 mins, supernatant was discarded, and the pellet was washed with same volume of ice cold sterile phosphate buffer saline (PBS). The pellet was collected by centrifugation at 1 0,000 rpm for 3 mins, and immediately snap froze in liquid nitrogen. Samples were stored at -80 °C until extraction of metabolites.

Extraction of endometabolome with sonication: The frozen pellets were thawed on ice followed by addition of 1 ml of of 80 % (v/v) methanol in water -20 °C, and vortexed for 20 s. Samples were sonicated in ice-water bath for 30 min at 50-60 Hz, 100% power. Samples were centrifuged at 14,000 x g for 10 mins at 4 °C. 900 μΙ_ of the supernatant was collected for each sample, diluted with 3 mL of ultrapure water in falcon tube and stored at -80 °C before further processing. The frozen samples were lyophilized at -105 °C for 24 hours, reconstituted in 200 μΙ_ of 50% (v/v) aqueous methanol, and sent for targeted LC-MS studies. DMSO-treated sample was used as a reference control to normalize the accumulation of rifampicin in the bacteria.

RNA sequencing

Sample preparation for RNA Isolation: A pre-culture of PA01 was prepared by diluting overnight culture to an OD600 of 0.01 in fresh MHB, incubated with shaking at 37 °C, 200 rpm to log phase (OD6oo= 0.5). The bacteria culture was harvested by centrifugation at 4,000 rpm, 10 mins, washed once with fresh MHB and aliquoted to ink bottles with polymers and antibiotics added accordingly. Incubated with shaking for 30 min, RNAIater was added (2 volume) to the bacteria culture and incubated for 5 min at room temperature and centrifuged as recommended by the manufacturer's protocol. Poured off the supernatant and stored at -80 °C until processing.

RNA Extraction: Total RNA was extracted using Qiagen RNeasy mini kit, followed the procedure as stated in the manufacturer's protocols. Quantified the total RNA using NanoDrop 2000 (Nanodrop Technologies) and proceeded with DNase treatment (Ambion Turbo DNase kit). Agilent screen tape for RNA was used to assess the integrity of RNA and Qubit assay was performed to further measured the quality and quantity of the RNA samples.

RNA sequencing: DNase-treated RNA samples were subjected to rRNA removal using Ribo- Zero (Bacteria) kit according to manufacturer's protocol. After DNase treatment, transferred the RNA to the washed magnetic beads and immediately mixed thoroughly by pipetting and avoid clumps. Vortex mixed at high speed and incubated at room temperature for 5 mins. Placed the samples in heat block, incubated at 50 °C for 5 mins. Tubes were removed and placed immediately on magnetic rack to remove rRNA-depleted RNA contained in the supernatant. Depleted RNA were purified by using Agencourt RNACIean up XP kit to remove any foreign residuals, followed by qualitative and quantitative measurements using RNA High Sensitivity Screen Tape and Qubit HS assay kit respectively. NEBNext RNA Strand Synthesis Module was used to generate first strand of cDNA followed by second strand cDNA synthesis using NEBNext Ultra Directional RNA Second Strand Synthesis Module as recommended by the manufacturer's protocols. Double-stranded cDNAs were purified with Agencourt AMPure XP magnetic beads and stored at -20 °C before sending for library preparation and sequencing. Data of differentially expressed genes for each treatment group were presented as 2 ^ACt and represented as a heatmap.

Membrane depolarization assay (D1SC35 assay)

Bacteria in mid-log phase of growth were centrifuged and washed twice with 5 mM HEPES buffer (pH 7.8) containing 20 mM glucose and 0.1 M KCI and diluted to a final concentration of 10 ⁷ CFU/ml. The bacteria cells were treated with 0.2 mM EDTA (pH 8.0) to permeabilize the outer membrane. In a cuvette, diSC35 solution was added to 2ml_ bacteria suspension to a final concentration of 100 nM. The diSC35 dye was allowed to gradually quench at room temperature. The polymer was added into the solution with stirring to a desired concentration. Changes in fluorescence due to the disruption of the membrane potential gradient across the cytoplasmic membrane were recorded using spectrometer (Perkin Elmer LS-55 luminescence spectrometer) at an excitation wavelength of 622 nm and an emission wavelength of 670 nm.

Efflux pump inhibition assay

Fluorometric Assay

The assessment of the accumulation of ethidium bromide (EtBr) using the fluorometric method was followed the procedure reported with minor modification (Paixao, L. et al. Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. Journal of biological engineering 3, 1 8 (2009)). Bacteria in mid-log phase of growth were centrifuged and washed twice with sterilized PBS. 100 μΙ of bacteria at a density of 10 ⁸ CFU/ml was suspended in 96 well plate (black, corning costar). 50 μΙ_ EtBr solution and 50 μΙ_ polymer solution were added to 100μΙ_ of bacteria suspension at desired concentration. Glucose was added to a final concentration of 0.4%. The fluorescence was recoded using a TECAN fluorescence spectrometer at 37 °C under the excitation of 530 nm and emission of 585 nm.

Dark-field/fluorescence dual-mode microscope imaging

Bacteria in mid-log phase of growth were adjusted to a density of 10 ⁸ CFU in 1 .5 ml micro tubes. The bacterial were loaded with 16 μg/ml EtBr in PBS without glucose to let the EtBr reach maximum accumulation. The compounds in PBS containing glucose was added at the desired concentration for 5 mins. The samples were washed with PBS and imaged with dark- field/fluorescence dual-mode microscope (Olympus 1X71 ).

Flow cytometry

Using a similar procedure as described above, intracellular ethidium bromide accumulation was followed by measuring the fluorescence on a flow cytometer BD Accuri™ C6 Plus. EtBr excited at 488 nm and the fluorescence through FL-2 channel. Data was collected for at least 10,0000 events per sample.

Swimming motility assay

The motility plates were prepared as described before (Yang, X. et al. Journal of medicinal chemistry 60, 3913-3932 (2017)). Bacteria in mid-log phase of growth were centrifuged and washed twice with sterilized PBS. 10 μΙ of 10 ⁸ CFU/ml bacterial solution was spotted onto the motility plate and incubated for 20 h before taking a picture. In vivo efficacy study in systemic infection models

Eight-week-old female C57B/6 mice (weight -20 g) (Invivos, Singapore) were used. All mice were housed on a 12-hour light-dark cycle at room temperature for one week prior to the experiment. Before infection, mice were divided from a housing cage to different treated cages with 5 mice per cage. No mouse was excluded from the analyses. Sample dosage was determined based on the results of a preliminary infection trial (n=3). Inoculation was performed by intraperitoneal injection of 300 μΙ of 10 ⁶ CFU/ml multi-drug resistant P. aeruginosa PAER, K. pneumonia KPNR, E. coli ECOR and A. baumanii MDRAB, with 5% mucin (Sigma-Aldrich). Infections were allowed to establish for 1 h, and treatment was administered by intraperitoneal injection of 200 μΙ sterilized PBS (untreated control), PAS8-6-PDM12, antibiotics and combination group. Mice were euthanized by CO2 asphyxiation followed by cervical dislocation at 1 1 hours post-treatment. Peritoneal washes were done by injecting 3.0 ml of sterilized PBS in the intraperitoneal cavity followed by a massage of the abdomen. Subsequently, intraperitoneal fluid was extracted from the abdomen for CFU count determination. Liver, kidney and spleen of every mice were also collected in 800-900 μΙ ice-cold PBS. Organs were homogenized using a high-throughput tissue homogenizer (Omni). The bacteria present in the organ and abdomen were determined by CFU plating of serially diluted samples on selective agar plates. All studies and protocols were approved by the Nanyang Technological University Institutional Care and Use Committee (NTU IACUC). Protocol No. ARF-SBS/NIE-A0363.

Biological activity and biological compatibility of polymers PASm-0-PDMn are shown in Table 1. Biological activity was gauged by measuring the minimum inhibitiory concentration (MIC) against a panel of four bacteria including both Gram-positive (S. aureus and its clinical resistant strains MRSA) and Gram-negative (E.coli and P. aeruginosa) bacteria. The pure cationic polymers (PDM20) had relatively good activity in terms of MICs (16-32 μg mL ¹ ) against P. aeruginosa and S. aureus. However, the biological compatibility was another vital consideration for clinical application. The biocompatibility was measured by the concentration causing 50% death of fibroblast (3T3) cells (IC50) using a standard MTT test and 50% lysis of human red blood cells (HC50). The biocompatibility of PDM20 was poor. With the introduction of PAS block, the biocompatibility was gradually increased with increased PAS ratio. For PAS8-6-PDM12, the IC50 was greater than 500 μg mL ¹ and HC50 was around 5000 μg mL ¹ . Besides, PAS8-6- PDM12 had relatively good antimicrobial activity in terms of MICs (64 μg mL ¹ ) against both Gram-positive bacteria S. aureus, as well as Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria, P. aeruginosa.

Consistent with the former reported, increasing the polymer molecular weight (PAS20-/ PDM30) had minor effect on the antimicrobial activity, e.g. MIC for S. aureus was decreased from 64 μg mL ^-1 to 32 μg ml_ ^-1 , and significant lysis of erythrocytes. It is well established that hemolytic activity is remarkably influenced by hydrophobicity. Increasing the molecular weight would increase the total hydrophobicity due to more hydrophobic residues were connected. To prove this hypothesis, a reversed block PDM12-/ PAS8 copolymer with the initiator tert-butyl benzoyl (hydrophobic group) connecting to cationic block (containing hydrophobic two methyl groups) was synthesized by traditional sequential addition profiles. It was expected that this reversed block PDM12-/ PAS8 had increased toxicity towards erythrocytes due to the increased total hydrophobicity of cationic block. Indeed, the hemolysis results supported this. Recent study on nylon-3 polymers indicated random copolymers had better killing activity than block copolymers (Liu, R. et al. Journal of the American Chemical Society 136, 4410-4418 (2014)). Sampson et al. also pointed out that≥ 8-10 A spacing between cationic groups along the backbone was needed to achieve good antimicrobial activity (Song, A., Walker, S. G., Parker, K. A. & Sampson, N. S. ACS chemical biology 6, 590-599 (201 1 )). Therefore, two multi- block (random) copolymers (PAS2-b-PDM3)4 and (PAS-b-PDM1 .5)e were synthesized by partial addition of premixed solution at certain ratio and the antimicrobial activity was investigated. Unexpectedly, both random copolymers had minor decrease of antimicrobial activity relative to the block architecture, while they were significantly toxic to human red blood cells. These observations were consistent with the recent studies on polymethacrylate family. Results showed that block structure had diminished toxicity to erythrocytes and minor influence on antibacterial potencies relative to random architecture. Therefore, there are no fixed laws between the antimicrobial activity and block vs random copolymers even though there are in the same family, e.g. both were nylon-3 polymers.

Carbohydrates and glycopolymers/peptides play an essential role in biological commutations, like modulating protein interaction, recognition and signaling. To investigate the function of glucose-derived PAS, galactose derived lactam monomers were prepared and polymerized to construct galactose functionalized copolymers (PAS _ga i8-£>-PDM12) for comparison. Both PASgai8-£>-PDM12 and PAS _g ic8-£>-PDM12 had comparable activity. These results indicated that there are no specific receptors for the PAS block and it may serve as shielding block to reduce the toxicity, like a PEG. However, confocal image (see below) indicated that the PAS block may have additional function besides the shielding effect.

Considering both biocompatibility and antimicrobial activity, PAS8-0-PDM12 had the optimum balance and will be the focus for the following discussion. In order to indicate the potential clinical application of our polymers, toxicity of PAS8-£>-PDM12was also measured by intravenous injection in a mouse model. Polymers were injected intravenously at a concentration of 10mg/kg body weight, and the mice condition was continuously monitored for a period of 7 days. Clinically significant biomarkers were recorded before the injection, 24 h after the injection and 7 days after the injection respectively. After 7 days post-injection of polymer, all the mice survived and were active and visual inspection of the mice did not suggest any illness or lassitude. According to the data of biochemistry panel assay, the levels of function parameters of the liver and kidney and the concentration of potassium and sodium ions were unchanged 24 h after intravenous injection (Table 2). The data indicates that the polymers do not cause significant acute damage to liver and kidney function, nor do they interfere with the electrolyte balance in the blood. Importantly, these parameters remain unchanged, even at 7 days' post-injection. To further determine whether or not the polymers cause tissue damage, inflammation, or lesions, histological assessment of vital organs was conducted. As shown in Figure 5, no apparent histopathological abnormalities or lesions were observed in liver, kidney, and spleen of polymers-treated animal. These findings collectively demonstrate that the polymers did not induce significant toxicity to the mice during the period of testing.

Super resolution STED confocal microscopy experiments were conducted to analyze the cellular distribution of the polymers. In general, PA 01 , MRSA BAA-40 and E. coli K12 were incubated in the presence of the rhodamine-labeled polymers (in red) and the plasma membrane of bacteria was stained by FM1 -43FX (in green). The yellow/orange color indicated the colocalization of polymers with bacterial plasma membrane. In the case of PA01 , the copolymer (Rho-8 PAS8-0-PDM12) was localized on the membrane as well as internalized into the bacteria cytosol (Figure 6a (A)). However, when the same bacterial strain was treated with the homocationic version of the compound (Rho-PDM20), much less internalization was observed and the compound mainly remained localized on the membrane (Figure 6b (A)). In the case of MRSA BAA-40, same effects were observed for Rho- PAS8-6-PDM12 and Rho- PDM20 as with PA 01 . The copolymer colocalized with the plasma membrane and was also internalized into the cytosol (Figure 6a (B)) whereas the homocationic polymer was mainly localized on the membrane with a weaker internalization (Figure 6b (B). Finally, E. coli K12 was incubated with Rho-PAS8-£>-PDM12 and Rho-PDM20. The rhodamine-labeled copolymer was observed to be uniformly localized all along the membrane, and it also showed some diffuse and weak intracellular red fluorescence (Figure 6a (C)). These results suggest some internalization of the compound, although much less if compared to our observations with PA01 and MRSA BAA-40, this weaker internalization correlated to the poorer potency against E. coli of copolymers. This observation is similar to a recent report done by Hossain and Wade (Li, W. et al. Chemistry & biology 22, 1250-1258 (2015)). They found that monomer (Chex-Arg20) localized uniformly around the E. coli membrane and its dimer (A3-APO) and tetramer (A3-APO disulfide-linked dimer) localized in the cytosol of bacteria. They attributed this observation to different membrane activity, non-lytic membrane of monomer vs membrane disruption of dimer and tetramer. Cryo-TEM was also used to visualize cytoplasmic membrane of PA01 before and after treatment with both copolymers and homocationic polymers. Before treatment, both the inner (IMs) and outer membranes (OMs) were intact. After treated with PAS8-0-PDM12, a clear morphology changed was observed with remarkably wrinkling both IMs and OMs as well as the OMs fragmentation (Figure 6d). In the case of copolymers, the cell membranes and cell wall were disrupted into isolated fragment and lysis of cells happened (Figure 6d). These observations were consistent with STED confocal images (Figure 6c). These results indicated the primary mechanism of our polymers were membrane disrupting agents like most of AMPs. To demonstrated this, we performed 1 -N-phenylnaphthylamine (NPN) and 3,3'- Dipropylthiadicarbocyanine iodide [DiSC3(5)] dye uptake fluorescence assays to measure IMs and OMs permeabilization. PAS8-£>-PDM12strongly increased outer membrane permeability below their MIC value (Figure 6e) as well as depolarized the cytoplasmic membrane (Figure 6f). These observations indicated the difference of internalization was not attributed to the different membrane activity. Several mechanisms have been proposed on disrupting the bacterial membrane, e.g. "carpet" model, "barrel-stave" model and "toroidal" pore model (Brogden, K. A. Nature Reviews Microbiology 3, 238-250 (2005)). The mechanism is possible to change as a function of the nature of the membrane and/ or the sequence of peptides. Although the detailed mechanism study is beyond the scope of this manuscript, we hypothesized that PAS8-6-PDM12 kill the bacteria via "wormhole" or "toroid pore" mechanism while PDM20 destroy the bacteria membrane via a detergent like "carpet" model based on the observation above. Initially, the homocationic polymers interact with the negative charged cell membrane by non-specific electrostatic interaction and accumulate on the bacteria membrane, wrinkling the membrane and lowering the membrane barrier. At the threshold concentration, the membrane was disrupted, causing death (Figure 6b and Figure 6d). Whereas, the initial binding of copolymer with bacteria are electrostatic interaction and/or hydrogen bonding with peptidoglycans within the cell wall. Due to the lack of long hydrophobic block to insert into the phospholipid bilayer, barrel-stave model is improbable. Our helix PAS block orientates and twists the membrane and finally our polymers translocate into cytosols of bacteria, resulting in osmotic lysis of the bacteria (Figure 6c and Figure 6d). As our polymers killed bacteria by membrane lysis, the evolution of resistance is expected to be low.

Since the killing mechanism against Gram-negative bacteria by PAS8-0-PDM12 to some extent involves membrane permeabilization, we explored its potential to improve the antibacterial effect of traditional antibiotics. Although lots of study on the synergistic-like enhancement in antibacterial killing activity when co-administrated with antibiotics and membrane-disturbing antimicrobial polymers, almost all the works were carried out based on a-peptides. Therefore, the physiological stability, toxicity and cost will always be the concern. To the best of our knowledge, this is the first report on synergistic study on β-peptide.

When various classes of antibiotics were tested in combination with our copolymers in checkerboard assay against P. aeruginosa PA01 , synergy was observed for most antibiotic- polymer combinations, as the FIC was indicated by calculated fractional inhibitory concentration (FIC) index of less than 0.5 (Table 3). For example, at 1/4 MIC (16 μg mL ¹ ) polymer addition, MIC* (MIC of antibiotics in combination) decreased to the sensitive range (< 8 μg mL ¹ ) and the F value (MIC of antibiotics/MIC ^* ) ranged from 8 to 32 for a wide range of antibiotics. To demonstrate that these synergistic effects were not strain specific, we also carried out the same checkerboard assay against a clinical relevant strain PAN-sensitive (PAES), clinical isolate P. aeruginosa D25 and clinical multi-drug resistant strain PAER. We found that all the strains except PAES were resistant to all the five antibiotics (Ciprofloxacin, Rifampicin, Sulfamethoxazole, Trimethoprim, Chloramphenicol) (Table 4), as the MICs of antibiotics alone were higher than against wild type PA01 . For example, MIC of Rifampicin was >64 μg mL ^-1 against PAD25 and 16 μg mL ^-1 against PA01 . However, when co-administrated with our polymers at 16 μg mL ¹ , MIC* of rifampicin was significantly decreased to < 2 μg mL ¹ , with F value ranging from 16 to 256. In addition, imipenem and ceftriaxone are the clinically used antibiotics for treatment of P. aeruginosa infection and when co-administrated with our polymers at 16 μg mL ¹ , both MICs were decreased greater than 4 times except for PAER. We found there is almost no cure for PAER except for colistin (the last resort drug against multidrug- resistant Gram-negative bacteria). However, the resistance strains were developed very fast towards colistin and there are still some debates on nephrotoxicity, neurotoxicity and neuromuscular blockade for clinical usage. Interestingly, we found that our polymers decreased the amount of colistin needed to 0.5 μg mL ^-1 . Due to much less colistin used when combined with our polymers, both the resistance and toxicity are expected to be lowered. In order to further demonstrate that the synergistic effect is not limited to just P. aeruginosa, we also did checkerboard assay with another three Gram-negative bacteria, E.coli, A. baumanmii and K. pneumonia. Similar to P. aeruginosa, MIC ^* of rifampicin was potentiated to sensitive range (< 4μg mL ¹ ) when administrated with our polymers at 16 μg mL ¹ , although MICs of our polymers against these strains are significantly higher (≥ 256 μg mL ¹ ) than against P. aeruginosa (Table 5). To demonstrate the potential for clinical application, co-administration of our polymers with three clinically used antibiotics against Klebsiella pneumoniae (BAK085) and Acinetobacter baumannii (X26) were performed. We found that both bacteria are significantly resistant to amikacin (MIC=320 μg mL ¹ ), ceftriaxone (MIC=5120 μg mL ¹ ) and ciprofloxacin (MIC=512 μg mL ¹ ) (Table 6). Importantly, when 16 μg mL ¹ of our polymers was added, ≥ 10 times decreased of MIC was observed except for ciprofloxacin (F=4).

Pseudomonas aeruginosa PA01 is frequently associated with wound infections. To evaluate the efficacy of polymer and rifampicin synergy in vivo, we treated P. aeruginosa PA01 infected wounds at 4 hours post infection (hpi), and determined the recoverable colony forming units (CFU) at 28 hpi. We observed significantly less P. aeruginosa PA01 CFUs in the wounds treated with a combination of rifampicin and polymer compared to either the rifampicin alone or polymer alone treated wounds (Figure 7). The mixed treatment yielded a log reduction in CFU greater than the combined log reductions of either treatment alone, achieving 99.5% eradiation. To further confirm the in vivo synergistic improvement of drug combination, we studied the activity of PAS8-0-PDM12 in combination with rifampicin in a systemic murine infection model. Initially, a clinical multi-drug resistant P. aeruginosa PAER was used to infect the mice. Result showed that neither PAS8-0-PDM12 alone nor rifampicin alone reduced the burden of bacteria in mice. However, the combination of PAS8-0-PDM12 and rifampicin eradiated around 99.9% bacteria compared to the infection control (Figure 8a). Moreover, the synergistic killing effect was further repeated in the major organs of the infected mice (Figure 19). To broaden the application of our combination, we infected the mice with another two clinical multi-drugs resistant Gram-negative pathogens E. coli (ECOR-1 ) and K. pneumoniae (KPNR-1 ). We observed more than 7 log reductions of bacteria burden for ECOR-1 and 4 log reductions of bacteria burden for KPNR-1 while their individual treatment group didn't show significant reduction of bacteria counts (Figure 8b and 8c). This synergistic activity was also confirmed in the organ of the infected mice (Figure 20 and 21 ). These results indicate that the PAS8-6- PDM12 synergizes with rifampicin in a systemic infection model and recapitulates in vitro data. Example 3: Meachism investigation

Rifampicin, a large hydrophobic molecule is typically blocked by outer membrane of many Gram-negative bacteria(Jammal et al., 2015; Yee et al., 1996). As no synergistic effect was observed when combination was used to treat Gram-positive bacteria (Figure 9a and 9b), S. aureus MRSA-BAA40 and E. faecalis V583, which don't have the outer membrane barriers. Moreover, MICs of Rifampicin alone are much lower against Gram-positive bacteria compared to Gram-negative bacteria. Thus, we hypothesized the observed synergistic effect was attributed to that the outer membrane barrier of Gram-negative bacteria was disturbed by our polymers, which increased the penetration of antibiotics to their target inside bacteria. The red rhodamine-labelled compound (Rho-PAS8-£>-PDM12) was seen on the peripheral of the bacterial and in the cytosol; superimposition of the red Rho-PAS8-£>-PDM12 and green membrane images results in the bacteria with orange periphery and red interior (Figure 9c), indicating that the PAS8-0-PDM12 was localized on the membrane as well as internalized into the bacteria cytosol. We further performed 1 -N-phenylnaphthylamine (NPN) dye uptake fluorescence assays to evaluate the outer membrane permeability of bacteria treated with different dosage of PAS8-0-PDM12. Increased fluorescence intensity indicated the permeability of outer membrane was increased. Interestingly, PAS8-0-PDM12 promoted the outer membrane permeability at the concentration as low as 1 μg mL ¹ (Figure 9d). The hypothesis is further confirmed by transcriptomics studies (Figure 9e) and rifampicin accumulation assays (Figure 9f). The heatmap of the genes that were differently expressed among P. aeruginosa cells after different treatments and the transcriptomes were further projected by principal component analysis (PCA). Both heatmap and PCA projection showed that treatment by 30 μg mL ¹ PAS8- 0-PDM12 in combination with 5 μg mL ¹ rifampicin has similar impacts on the P. aeruginosa global transcriptome to treatment by 30 μg mL ¹ rifampicin alone (Figure 9e). A total of 155 genes were induced and 157 of genes were reduced by both treatments. For example, both treatments induced the expression of polyamine transport protein encoding gene potD, flagellum synthesis genes (e.g. flgE, flgF) and reduced the expression of phrS and Psl polysaccharide synthesis genes (e.g. psIA), which was confirmed by qRT-PCR analysis. Treatment by 30 μg mL ^-1 PAS8-0-PDM12 alone strongly induced the expression of the anr operon and phoPQ two component systems in P. aeruginosa, which suggests that our polymers target the P. aeruginosa cells in similar manner to colistin. Expression of the pmr-gfp reporter fusion confirmed the RNA-seq analysis. The significantly enhanced concentration of rifampicin within the bacteria was supported by metabolomic study (Figure 9f). Thus, the global transcriptomic analysis and metabolomic study strongly support that our PAS8-6-PDM12 impair the P. aeruginosa cell membrane and increase the penetration of rifampicin into the cells, good synergistic effect was observed. The increase in membrane permeability after treatment with PAS8-6-PDM12 was also observed in other Gram-negative pathogens (Figure 14). This accounted for the broad spectrum synergistic effect of PAS8-6-PDM12 against multiple Gram- negative pathogens.

As our PAS8-0-PDM12 also enhanced the activity of trimethoprim, which is well documented to be intrinsically resistant by P. aeruginosa through activating efflux pumps, we propose that our polymer can also effectively deactivate the efflux pump, possibly due to interfering the efflux pump system on the cytoplasmic membrane. To support this, we tested the synergy of trimethoprim against PAO750, a mutant strain with all the efflux pumps switched off. The resistance against trimethoprim disappeared in PAO750 compared with PA01 , indicating that the intrinsic resistance of trimethoprim is mainly due to efflux pump. Moreover, the synergy of trimethoprim against PAO750 was not retained, further confirming that the synergistic mechanism is due to inactivation of efflux pumps (Table 7). Addition of our polymer didn't show synergy effect in PAO750 where all the efflux pumps were already inactivated but achieved significant synergy in wild type PA01 where all the efflux pumps were active. To accentuate that our polymers served as an efflux deactivator, two more antibiotics, moxifloxacin and tetracycline which were resistant by MexAB-OprM, MexCD-OprJ, MexJK-OprM and MexXY-OprM in P. aeruginosa , were chosen to test the synergistic effect. Same as trimethoprim, both antibiotics showed synergistic effect against PA01 while the synergistic effect disappeared when co- administrated with PAS8-6-PDM12 against PAO750 (Table 7). Cryo-TEM was also used to visualize cytoplasmic membrane of PA01 before and after treatment with PAS8-0-PDM12. Before treatment, both the inner (IMs) and outer membranes (OMs) were intact (Figure 10a upper). After treated with our polymers, we observed ripple of bacterial membrane (arrow), and there are certain areas of the membrane which are completely broken (circle) (Figure 10a below). The lysis of cytoplasmic membrane supported that our p PAS8-6-PDM12 breakdown the efflux pump which was imbedded in the cytoplasm. To further explore the influence of PAS8-0-PDM12 on the bacteria cytoplasmic membrane, we used 3,3'- Dipropylthiadicarbocyanine iodide [DiSC3(5)] as indicator to evaluate IMs dissipating activity. Interestingly, PAS8-0-PDM12 depolarized the cytoplasmic membrane over a wide range of concentration (Figure 10b). To directly prove that PAS8-6-PDM12 deactivated efflux pump system in bacteria, ethidium bromide (EtBr) was used as an indicator of efflux pump activity, which is a well-known efflux pump substrate. A dark-field/fluorescence dual-mode microscope was used to visualize EtBr inside bacteria. Almost no red EtBr fluorescence was observed in the wild type PA01 without addition of PAS8-6-PDM12 while almost all the PA01 cells were filled with EtBr when treated with PAS8-6-PDM12 (Figure 10c, first 2 row). Whereas, both the PAO750 without PAS8-6-PDM12 treatment and PAO750 treated with PAS8-6-PDM12 occupied all the cells (Figure 10c, last 2 row). We further measured the fluorescence intensity of cells with and without PAS8-6-PDM12. PA01 treated with PAS8-6-PDM12 had comparable fluorescence intensity as PAO750 (Figure022a), which indicated that PAS8-6-PDM12 deactivated the efflux pump of PA01 and made it resemble its efflux pump off mutant PAO750. A concentration dependent increase in fluorescence in PA01 was detected by flow cytometry assay (Figure 10d) while its efflux pump mutant PAO750 retained constant fluorescence intensity regardless of the concentration of PAS8-0-PDM12 used (Figure 10e). The combination of microscopic observation, fluorescence intensity measurement and flow cytometry assays strongly support that PAS8-6-PDM12 deactivated the efflux pump of PA01 and make it become PAO750, all the efflux pump off. Moreover, we observed that flagellum-dependent swimming motility of PA01 was inhibited in the presence of our PAS8-6-PDM12 at sub MICs concentration (Figure 13). As the flagellum-dependent bacterial motility requires an intact portion motive force (PMF), which also controls the function of RND-based efflux pump system in P. aeruginosa. We further found that PAS8-6-PDM12 affected the electrical component (ΔΨ) of the PMF, as the observed antagonism of PAS8-0-PDM12 with aminoglycoside antibiotics (gentamicin, streptomycin and neomycin) (Table 8), which needs the electrical component (ΔΨ) of the transmembrane electrochemical gradient for cellular uptake. The effect supported that PAS8-6- PDM12 deactivated the efflux pump system of P. aeruginosa by dissipating the PMF. To demonstrate the deactivating activity of efflux pumps was not limited to P. aeruginosa, we measure the fluorescence intensity of EtBr in another two Gram negative strains E.coli and A. baumannii. Dosage dependent increment of fluorescence intensity indicated that PAS8-6- PDM12 had a broad-spectrum efflux pump inhibition activity (Figure 22b,c).

Collectively, we supposed that the observed synergistic effect of our polymers with various antibiotics is via dual mechanisms, outer membrane disrupting and/or efflux pump deactivating. The rest data could also be well explained using our hypothesis of dual mechanisms. Both the decreased outer membrane permeability and the presence of efflux pump account for the intrinsic resistant to chloramphenicol As PAS8-0-PDM12 functioned as increasing outer membrane permeability and deactivating efflux pumps, synergistic enhanced the potency of chloramphenicol was observed. Similar to trimethoprim, the intrinsic resistance to sulfamethoxazole was also attributed to multidrug efflux system in P. aeruginosa. Due to the efflux pump deactivation, good synergistic was also observed for sulfamethoxazole when combined with our polymers, MIC ^* was decreased to 4 μg ml ¹ , with F value is 32 (Table 3). The intrinsic resistant of P. aeruginosa against /3-lactam antibiotics (carbenicillin, ampicillin, and ceftazidime) is due to both reduced outer membrane permeability and efflux system. It is not surprising that a strong synergy was observed for many /3-lactam antibiotics (Table 3). The intrinsic resistance of P. aeruginosa against fluoroquinolone antibiotics (ciprofloxacin) is mainly structural changes in target enzymes and active efflux pump. The fluoroquinolones are universal substrates for four common efflux pumps; hence they are actively extruded from the interior of bacteria cells. Synergy was observed for ciprofloxacin, though the antibiotic itself could kill PA01 at 0.125 ml ¹ (Table 3). This observation is consistent with the hypothesis that PAS8- 6-PDM12 can deactivate the efflux pump system. Novobiocin was an antibiotic resistant by both the outer membrane barriers and efflux pump excretion. The effect was also observed by the relatively high MICs value against PAO750 with only membrane barrier and even higher MICs against PA01 with both resistant mechanism (Table 7). Moreover, better potentiating effect was observed against PA01 than PAO750 (Table 7). We further extended the spectrum of synergistic effect against other clinical Gram-negative pathogens including both drug sensitive and resistant E. coli, A. baumannii and K. pneumoniae (Figure 11c-5h). Most importantly, the excellent potentiating effect was reproduced in the in vivo system infection model (Figure 11 i,11j,11 k, 30-32) same as Rifampicin.

In conclusion, a novel glycosylated cationic β-peptide was designed and synthesized by AROP of two corresponding β-lactams. Kinetics study done by a combination of GC and GPC indicated the obtained β-peptides had a block-like structure, even though the two monomer precursors are added concurrently. These fortuitous conditions allow one to prepare block- copolymer like structures by the simultaneous addition of both reactants, a feature that simplifies considerably the synthetic entry into the target, as well as decreasing the possibility of inhibitors to the highly sensitive anionic polymerization, improving the polymerization yield. Block copolymers are interesting since they could retain the functions of both blocks. Structure and activity (SAR) studies also highlighted the block-like structure had better hemo-compatibility compared to the random ones. In addition, we showed that 8PAS-0-12DM had relatively good antimicrobial activity against both Gram-negative P. aeruginosa and Gram-positive S. aureus, with MICs of 64 μg/mL and in vivo biocompatibility test using a murine intravenous toxicity model at dosage of 10 mg/kg indicated that 8PAS-b-12DM had excellent biocompatibility, with 100% animal survival after 7 days and no significant difference in all the clinically important biomarkers compared to the control groups (P>0.05). The killing mechanism was proposed to be membrane lysis and was supported by STED confocal images, Cryo-TEM and inner (DiSC35) and outer membrane (NPN) assay. Combination treatment by using more than one antibiotics is applied clinically for curing bacterial infection, e.g. ceftolozane/tazobactam is a marketed combination of β-lactam antibiotics and β-lactamase inhibitors for the treatment of complicated intra-abdominal infections and complicated urinary tract infections. Herein, we showed PAS8-0-PDM12 had good synergistic effect with a wide range of antibiotics against P. aeruginosa. For example, PAS8-6- PDM12 potentiated Rifampicin potency against wild type PA01 and its clinical relative strains PAD1 , PAD25, PAES, PAER up to a range of 1 6 to 256 times. Besides, we found the synergistic effect with Rifampicin is a broad spectrum, MIC ^* was decreased to sensitive range (<4 μg/mL) against clinical relative A. baumanmii, K. pneumonia, and E.coli. Importantly, the synergistic effect was also demonstrated for Pseudomonas in a murine wound infection model and in vivo system infection caused by P. pseudomonas ,A. baumanmii, K. pneumonia, and E.coli. Moreover, the mechanism of synergy was supposed to be via either outer membrane disruption or efflux pumps inhibition or both mechanisms.

One of today's serious threats are from Gram-negative bacteria such as multi-drug resistant Pseudomonas aeruginosa. There are very few drug leads in the horizon against Gram-negative bacteria though some potential drugs using antibiotics that bind the peptidoglycan precursor lipid II look very promising. One interesting approach is the use of AMPs to potentiate the activity of antibiotics, but there is limited success against Pseudomonas aeruginosa. Our glucose functionalized cationic block β-peptides themselves killed the Pseudomonas aeruginosa well without detectable toxicity as well as serving as antibiotic adjuvant to convert those non-activity antibiotics to high efficacy against clinically relevant Gram-negative bacteria. Besides our sugar decorated cationic block β-peptides were synthesized in one pot polymerization. All these factors make it a promising agent for real world application.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds disclosed herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. The invention illustratively disclosed herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

Example 4: Summary Advantages and improvements:

Hitherto, SMAMPS have been designed to mimic the amphiphilic properties of AMPs by finely choosing and balancing the cationic and hydrophobic amino acids, causing typically the parallel shift of the activity and toxicity towards erythrocytes with increasing hydrophobicity. We attempted to design novel AMPs based on sugar since the bacterial cell envelope components (such as the peptidoglycan, teichoic acid and Lipid A) are rich in them, and fine tuning of the interaction between sugar-based peptide and bacteria cell envelop may lead to selective bacteria killing. Carbohydrates and glycopolymers/peptides play an essential role in biological commutations, like modulating protein interaction, recognition and signaling. Compared to individual, unassembled saccharide monomers, carbohydrates and glycopolymers commonly showed improved bioactivity due to their multivalency. Further, sugar-based molecules are known to possess strong hydrogen bonding and may lead to interesting secondary structures that can attenuate antimicrobial effects.

Although AMPs had relatively good activity, their activity in terms of MICs is still higher than most antibiotics. Therefore, people are more interested in synergistic-like enhancement in antibacterial killing activity when co-administrated with antibiotics and AMPs. There are some promising studies, but almost all the works were carried out based on the a-peptides. Therefore, the physiological stability, toxicity and cost will always be the concern. To the best of our knowledge, this is the first report on synergistic study on β-peptide. Novelty of this invention:

We herewith report a new class of glycosylated block-like β-peptides, i.e. PASm-0-PDMn as shown in Scheme 1 , which consists of a helical poly-amido-saccharide (PAS) domain, linked to a cationic segment PDM anticipated to function as the killing domain. Block copolymers are interesting since they could retain the functions of both blocks. Block copolymers are typically synthesized via "sequential polymerization", which involves multiple isolations and purification of first block, which is labor consuming and results in loss of polymer yields. Our block copolymers were prepared via an efficient one step reaction that takes advantage of the different monomer reactivity ratios, which significantly decreases the possibility of introducing inhibitors to the highly sensitive anionic polymerization, improving the polymerization efficacy. Structure and activity (SAR) studies also highlighted the block-like structure had better hemo-compatibility compared to the random ones. The mechanism of killing was suggested to be membrane lysis by a combination of direct (Cryo-TEM and STED) and indirect (NPN and DiSC35 assay) techniques. Therefore, the resistance to this kind of membrane disrupting agents was expected to be low. Indeed, the frenquency of resistant to the PAS8-6-PDM12 was less than 1 .9X10 ^{Λ 9} . Synergistic studies using PAS8-6-PDM12 showed its function to potentiate other antibiotics against many clinically relevant Gram-negative bacteria, which was attributed to either outer membrane disruption or efflux pumps inhibition or both function. The content of all documents and patent documents cited herein is incorporated by reference in their entirety.