Bacterial cell wall components as probes for a label-free binding assay and the use thereof

Abstract

The present disclosure provides peptidoglycan precursors and the use thereof in the study of binding between antibiotic and peptidoglycan. Specifically, the peptidoglycan precursors disclosed herein are used in a method for the screening of peptidoglycan-targeting antibiotics and identifying the binding moiety on a peptidoglycan.

Description

BACTERIAL CELL WALL COMPONENTS AS PROBES FOR A LABEL- FREE BINDING ASSAY AND THE USE THEREOF FIELD OF INVENTION

[0001] The present disclosure is related to the bacterial peptidoglycan precursors, 5 the use thereof in the study of binding between antibiotics and peptidoglycans, a method for screening a peptidoglycan-targeting antibiotic, and a method for identifying a binding moiety on a peptidoglycan by using the peptidoglycan precursors. BACKGROUND OF THE INVENTION 10

[0002] Antibiotic-resistant bacteria, such as VRE (vancomycin-resistant Enterococcus), MRSA (methicillin-resistant Staphylococcus aureus), and Mycobacterium tuberculosis strains leading to the cases of MDR-TB and XDR-TB (multidrug-resistant and extensively drug-resistant tuberculosis), have emerged and imposed a global public health threat, raising the need for continuous development of 15 new antibiotics and new strategies to tackle the problem of antibiotic resistance. One of the strategies is to target peptidoglycan (PGN), which is one of the major components of the bacterial cell wall that plays a crucial role in the structural integrity of bacteria. Although some enzymes are responsible for the synthesis and polymerization of bacterial peptidoglycan, their pockets for substrate transfer are 20 spatially variable and shallow, which makes inhibitor discovery against them highly difficult. In addition, enzyme-based targets are prone to mutation during treatment with antibiotics, leading to impaired direct interactions between antibiotics and their respective targets. Due to the aforementioned reasons, a new strategy for antibiotic drug development is in process. 25

[0003] Lipid II, which is the minimal structural unit of polymeric lipid-linked peptidoglycan, serves as an important substrate for polymerization during transglycosylation and exists in very low copy numbers in bacteria. Therefore, Lipid II is also regarded as a potential target for antibiotics. To date, four classes of naturally occurring antibiotics, including glycopeptides, cyclic depsipeptide antibiotics, 30 lantibiotics, and defensins, have been reported to endorse the approach to targeting Lipid II. For instance, in the case of VRE, the interaction between vancomycin and modified Lipid II is dramatically reduced because the last two amino acids of the stem peptide part change from D-Ala-D-Ala to D-Ala-D-Lac. However, new drug discovery based on targeting Lipid II has progressed slowly due to the limited accessibility and 1 https: / / twtsartsai-my.sharepoint.com / personal / huaili_tsartsai_com_tw / Documents / TT-HuailiLu / English / 112378 / PCT / 112378PCT-engspec.docxisolation of the structurally complex Lipid II, and the tedious and complicated process used in the current binding assay platforms. The current platforms for performing binding assay, especially for molecule-molecule interactions, require immobilization of the complex on matrices. These immobilization assays often involve additional 5 synthetic transformations, and the orientation of modified Lipid II may weaken interactions with analytes. Current label-free assays, such as isothermal titration calorimetry (ITC), are not suitable either as they require certain amounts of materials and are time-consuming, making them unsuitable for screening purposes. Accordingly, there is a notable trend for developing a label-free and convenient assay to explore 10 new Lipid II binders based on a convenient and rapid platform. SUMMARY OF THE INVENTION

[0004] The present disclosure is based, at least in part, on the surprising discovery of the peptidoglycan precursors and their use in the peptidoglycan-based label-free 15 affinity screening (PLAS) platform.

[0005] One aspect of the present disclosure is to provide a method for evaluation of the binding potency between a target biomolecule from bacteria and a compound selected from peptides, natural products, and derivatives thereof through a label-free approach. According to the present disclosure, the target biomolecule is not a protein, 20 a transporter, or an enzyme.

[0006] In one embodiment, the bacteria include Gram-positive bacteria and Gram- negative bacteria, for example, mycobacteria.

[0007] In one embodiment, the method is to screen a candidate capable of binding to bacterial biocomponent via a label-free approach. 25

[0008] In one aspect, the present disclosure provides a method for screening a peptidoglycan-targeting antibiotic, comprising: contacting an antibiotic candidate with a peptidoglycan precursor to form a complex; subjecting the complex to target engagement mass spectrometry; and determining the binding affinity of the antibiotic candidate; 30 wherein the peptidoglycan precursor is selected from at least one of the compounds of the group consisting of253(15).

[0009] In another aspect, the present disclosure provides a method for identifying 5 a binding moiety on a peptidoglycan, comprising: contacting an antibiotic candidate with a first peptidoglycan precursor to form a first complex; subjecting the first complex to target engagement mass spectrometry to determine a first binding affinity; 10 contacting the antibiotic candidate with a second peptidoglycan precursor to form a second complex; subjecting the second complex to target engagement mass spectrometry to determine a second binding affinity; and comparing the first binding affinity and the second binding affinity to determine the 15 binding moiety of the peptidoglycan to the antibiotic candidate; wherein the peptidoglycan precursor is selected from at least one of the compounds set forth above.

[0010] In another embodiment, the binding affinity of the antibiotic candidate is compared to a standard antibiotic. 20

[0011] In another embodiment, the standard antibiotic is an antibiotic targeting the oligopeptide moiety or pyrophosphate of the peptidoglycan. Preferably, the standard antibiotic is selected from the group consisting of vancomycin, teicoplanin, and ramoplanin.

[0012] In another embodiment, the binding affinity of the antibiotic candidate is 25 compared to a negative control. Preferably, the negative control is an inhibitor of4transglycosylase (TGase) or an inhibitor of transpeptidase (TPase). More preferably, the negative control is selected from Moenomycin A (MoeA) or ampicillin.

[0013] In another embodiment, the target engagement mass spectrometry is electrospray ionization mass spectrometry (ESI-MS). 5

[0014] In another embodiment, immobilization of the peptidoglycan precursor on the matrix or labeling of the peptidoglycan precursor with a fluorophore is not required in the method according to the present disclosure.

[0015] In another embodiment, the binding moiety of the peptidoglycan is selected from at least one of: a disaccharide, a pyrophosphate, a lipid tail, and an 10 oligopeptide moiety.

[0016] In another embodiment, when the first peptidoglycan precursor is compound (1), and the second peptidoglycan precursor is selected from at least one of compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is 15 pyrophosphate.

[0017] In another embodiment, when the first peptidoglycan precursor is compound (1) and the second peptidoglycan precursor is selected from at least one of compound (4), compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety 20 is an oligopeptide moiety.

[0018] In still another embodiment of the present disclosure, the above method according to the present disclosure further comprises performing an analysis on a target engagement mass spectrometer. In a particular embodiment, the mass spectrometer is a hybrid linear trap / Fourier transform ion cyclotron resonance (FTICR) 25 mass spectrometer, a high-resolution accurate mass LC-MS equipped with a heat electrospray ionization probe, a high-performance liquid chromatography (HPLC) system with binary pump, and a Famos autosampler. In a particular embodiment, a sample is loaded on the target engagement mass spectrometer, wherein the sample was directly injected into a continuous flowing solvent with a flow rate of 40-60 μL / min in 30 the mobile phase consisting of IPA / H2O / NH4OH at pH 6-9. In a particular embodiment, the FTICR is operated in negative mode within a normal mass range from m / z 500-3000, showing resolution power is 100,000 at m / z 400, electrospray voltage is applied to 4.0 kV, and the capillary temperature is set at 275 ºC.5

[0019] One aspect of the present disclosure is to provide a group of peptidoglycan precursors and some embodiments thereof in the development of label-free target engagement strategies.

[0020] In one aspect of the present disclosure, the peptidoglycan precursor has a 5 formula of A-Sn-Po-Lq; wherein A is an oligopeptide moiety, S is a saccharide moiety, P is a phosphate group, 10 L is a lipid tail comprising up to 55 carbon atoms, n is 1 or 2, o is 1 or 2, and q is 0 or 1.

[0021] In one embodiment, A consists of 1 to 5 amino acids selected from the15 group consisting of Ala, Glu, m-DAP and Lys, and S is selected from N- acetylmuramic acid (MurNAc) or N-acetylglucosamine (GlcNAc).

[0022] In another embodiment, the peptidoglycan precursor is selected from at least one of:20

[0023] In another aspect, the peptidoglycan precursor according to the present disclosure is used for screening a peptidoglycan-targeting antibiotic. In another aspect, the peptidoglycan precursor according to the present disclosure is used for identifying6the moiety on a peptidoglycan to which an antibiotic binds. The antibiotic thus screened and identified by the method can be used for the treatment of bacterial diseases, especially bacterial infections caused by antibiotic-resistant bacteria.

[0024] In still another aspect of the present disclosure, an antibiotic can be 5 obtained from the method according to the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended 10 drawings. For the purposes of illustrating the invention, shown in the drawings are embodiments which are presently preferred.

[0026] In the drawings:

[0027] Fig. 1 schematically depicts the study design for the method according to the present disclosure. 15

[0028] Fig. 2 is the mass spectrum of the complex of Park’s nucleotide (20 μM) and vancomycin (20 μM) analyzed by the target engagement strategy assisted by target engagement mass spectrometry.

[0029] Fig. 3 is a graph depicting the lipid II-based label-free binding affinity study based on the peptidoglycan-based label-free affinity screening (PLAS) platform 20 according to the present disclosure. Fig. 3(a) shows the binding affinity between Neryl-Lipid II (Compound (1)) with some antibiotics; Fig. 3(b) shows the evaluation of the binding moiety of vancomycin by Lipid II analogues.

[0030] Fig.4 is a graph depicting drug test screening based on the PLAS platform according to the present disclosure. Fig.4(a) shows the binding affinity study between 25 Neryl-Lipid II (Compound (1)) (5 μM) with the antibiotic candidates (5 μM) collected from library; Fig. 4(b) shows the evaluation of the binding moieties of compound 29 (50 μM) by Lipid II analogues (50 μM). DETAILED DESCRIPTION 30

[0031] Bacterial cell wall biosynthesis is a major target of antibiotic action, with many antibiotics being designed to act on cell wall structures. Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharides. The gram- positive cell wall appears as a broad, dense wall that is 20-80 nm thick and consists of7numerous interconnecting layers of peptidoglycan. In contrast, the cell walls of gram- negative bacteria are more chemically complex, thinner, and less compact. In gram- negative bacteria, peptidoglycan makes up about 5-20% of the cell wall and is not the outermost layer, but is sandwiched between the plasma membrane and an outer 5 membrane. The peptidoglycan molecule's backbone is comprised of glucose derivatives N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), interconnected by peptides.

[0032] Lipid II is an essential component in bacterial cell wall synthesis and an established yet underused target for antibiotics currently in clinical use. Lipid II is an 10 amphipathic peptidoglycan named for the bactoprenol hydrocarbon chain which anchors it into the bacterial cell membrane from the initiation of biosynthesis. Structurally, Lipid II consists of a disaccharide (MurNAc-GlcNAc), a pyrophosphate, a lipid tail, and oligopeptide moieties (such as D-lactyl-L-alanyl-D-glutamyl-meso- diaminopimelyl (or L-lysyl)-D-alanyl-D-alanine). 15

[0033] Generally, the biosynthesis of peptidoglycan starts at the cytoplasmic side of the plasma membrane. The soluble precursor UDP-MurNAc-pentapeptide is linked to the membrane carrier bactoprenol-phosphate (C55P), yielding Lipid I. Next, the addition of N-acetylglucosamine (GlcNAc) to the N-acetylmuramic acid residue of Lipid I by MurG transferase leads to the formation of lipid II. Lipid II is transported 20 across the membrane by MurJ and is added to the growing glycan chain by peptidoglycan glycosyltransferase (GTase). This reaction is known as transglycosylation. Lastly, the DD-transpeptidase (TPase) crosslinks individual glycan chains.

[0034] Various classes of natural antibiotic peptides have been discovered to bind 25 Lipid II, including depsipeptides, lantibiotics, cyclic peptides and glycopeptides. Among them, vancomycin and its recently developed derivatives, daptomycin, oritavancin, and telavancin, have been approved as drugs for first line treatment.

[0035] The glycopeptide group of antibiotics, most commonly represented by the vancomycin, inhibits the synthesis of the cell wall in susceptible bacteria by blocking 30 the cross-linking of the sugar and peptide components of peptidoglycans during the synthesis of the bacterial cell wall. Without vigorous cross-linking, the cell wall becomes mechanically fragile, causing the bacteria to lose integrity when subjected to changes in environmental osmotic pressure. Vancomycin is known to bind with high affinity to the D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the pentapeptide portion8of the peptidoglycan precursor to impede the cross-linking. The D-Ala-D-Ala dipeptide forms complementary hydrogen bonds with the peptide backbone of vancomycin. The vancomycin-peptidoglycan complex is believed to physically block the action of the transpeptidase enzyme and thereby inhibit the formation of the 5 peptide cross-bridges that strengthens the peptidoglycan. This activity also leads to the accumulation of peptidoglycan precursors in the bacterial cytoplasm.

[0036] In general, a person skilled in the art is familiar with antibiotic glycopeptides and their mechanisms of inhibiting peptidoglycan biosynthesis in bacteria, and can easily select suitable glycopeptides from an antibiotic library for the 10 purpose of screening or identification of antibiotics in the present disclosure. Such glycopeptides are typically of a molecular weight of from 1000 to 3000 Da, and are capable of interaction with individual components of the Lipid II or bacterial peptidoglycan structure such as the Lys-D-Ala-D-Ala peptide, the Lys-D-Ala-D-Lactate depsipeptide, and components of the lipid GlcNAc-MurNAC-pentapeptide. 15 Nevertheless, there is no report showing that structural changes occur at the moiety of sugar, pyrophosphate, or prenol part of Lipid II. Accordingly, potential binding between an antibiotic candidate and a peptidoglycan precursor would be a novel strategy for developing new drugs against the threat of newly-occurring bacteria with drug resistance. 20

[0037] Disclosed herein is a versatile and innovative assay platform, peptidoglycan (PGN)-based label-free affinity screening (PLAS), which has successfully overcome the current limitations in antibiotic drug discovery, increasing throughput, and qualifying as a primary screening platform for more efficient discovery of new Lipid II binders. This advancement will accelerate research progress 25 in tackling serious antibiotic resistance.

[0038] The present disclosure is related to a method for evaluation binding potency between a binder and a biomolecule other than protein such as macromolecule, e.g. receptor or enzyme, to develop potential clinical diagnosis and treatment for conditions such as cancer or infectious disease. Compared to a targeting-based binder 30 method, the present disclosure utilizes target engagement mass spectrometry to achieve a label-free and rapid method for analysis of binder of microquantity.

[0039] The present disclosure provides a series of structurally complex PGN- related precursors, including Lipid II and its analogues or fragments, as key chemical probes. These probes were applied in the label-free target engagement (TE) strategy9employing the PGN-based label-free affinity screening platform (PLAS), which utilizes a target engagement MS-like analytic method.

[0040] The present disclosure also presents a novel MS-based binding assay platform applied specifically to target PGN precursors such as Lipid II. This novel 5 binding assay is convenient, rapid, and does not require the immobilization of Lipid II on a matrix, making it suitable for potential large-scale library screening to identify Lipid II binders. Furthermore, the platform according to the present disclosure along with these analogues would enable researchers to identify the key moiety on peptidoglycan contributing to its more potent binding interaction with candidate 10 compounds. Fig. 1 shows the general flow chart of the method according to the present disclosure which is capable of qualitatively and quantitatively analyzing the interaction of an antibiotic with a peptidoglycan or its precursor.

[0041] Various aspects of the disclosure are described in further detail in the following subsections: 15 I. Definition

[0042] The following description when being read with the accompanying drawings are made to clearly exhibit the above-mentioned and other technical contents, features, and effects of the present disclosure. As the contents disclosed herein should be readily understood and can be implemented by a person skilled in the art, all 20 equivalent changes or modifications which do not depart from the concept of the present disclosure should be encompassed by the appended claims.

[0043] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0044] As used in the specification and the appended claims, the singular forms 25 “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed. In this application, the use of “or” or “and” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, 30 “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0045] As used herein, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will10depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result, or embodiment, the term “about” means within one standard deviation per the practice in 5 the art, or a range of up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0046] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has”, “having”, “contains”, “containing”, “characterized by”, or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any 10 limitation explicitly indicated. For example, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0047] The transitional phrase “consisting of” excludes any elements, steps, or 15 ingredients not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. 20

[0048] Where applicants have defined an invention or a portion thereof with an open-ended term such as “comprising”, it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the term “consisting of”.

[0049] The term “peptidoglycan” used herein refers to a glycopeptide polymer 25 that is an essential component of bacterial cell walls, including both Gram-positive and Gram-negative bacteria. Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that form a mesh-like layer outside the plasma membrane of bacteria, forming the bacterial cell wall. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic 30 acid.

[0050] The peptidoglycan network is a dynamic structure that is cross-linked by both 4→3 and 3→3 transpeptide linkages. Both L,D- and D,D-transpeptidases are likely to be involved in the maintenance and remodeling of the peptidoglycan network.11

[0051] The peptidoglycan layer is substantially thicker in gram positive bacteria (20 to 80 nanometers) than in gram negative bacteria (7 to 8 nanometers), with the attachment of the S-layer. Peptidoglycan forms around 90% of the dry weight of gram positive bacteria but only 10% of gram negative strains, which have a thinner cell wall. 5 For both gram positive and gram negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan cell wall.

[0052] The terms “antibiotic(s)” or the like used herein refers to a compound or composition comprising the compound, which decreases the viability of a microorganism, or which inhibits the growth or reproduction of a microorganism, i.e., 10 by increasing the generation cycle time by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or preferably at least 10-fold, or more preferably at least 100-fold, or most preferably by eradicating bacterial growth and achieving total cell death. As used in this disclosure, an antibiotic is further meant to include bacteriostatic, antibacterial, or bactericidal agents. 15

[0053] The term “amino acid” used herein is read in its broadest sense and refers to either a natural and / or non-natural or synthetic amino acids, including both the D and L optical isomers, amino acid analogs, and peptidomimetics. In a preferred embodiment, the amino acid is a D-amino acid. In another embodiment, the amino acid is an L-amino acid. A peptide of three or more amino acids is commonly called 20 an oligopeptide if the peptide chain is short.

[0054] The amino acid according to the present disclosure includes but is not limited to alanine (Ala), β-alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), α,α-dimethyl-γ-aminobutyric acid, β,β-dimethyl-25 γ-aminobutyric acid, glutamine (Gln), glutamic acid (Glu), glycine (Gly), homo- cysteine, selenocysteine, homo-selenocysteine, histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), ε-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). 30

[0055] The peptidoglycan according to the present disclosure is also meant to include a peptide chain of three to five amino acids, wherein the amino acids are a modified or non-modified L- or D-amino acid. In some instances, the at least one modified amino acid is located at one or more of positions. In some embodiments, the amino acid moiety in peptidoglycan comprises natural amino acids, and does not12comprise non-natural amino acids. In some cases, only one amino acid of the peptide chain is a modified amino acid, and in other cases, at least two of the amino acids of the peptide chain are modified amino acids. For example, three amino acids of the peptide chain are modified, four amino acids of the peptide chain are modified, or five 5 amino acids of the peptide chain are modified.

[0056] The terms “polypeptide”, “peptide”, and “protein”, which may be used interchangeably herein according to the context, refer to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or 10 designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified peptide backbones. Further, one or more of the amino acids in a peptide or polypeptide may be modified, for example but not limited thereto, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for 15 conjugation, functionalization, or other modification, etc. The peptide according to the present disclosure may be derived from a natural biological source, synthesized, or produced by recombinant technology. It also may be generated in any manner, including by chemical synthesis.

[0057] The term “binding” or “bind” used herein refers to interaction between 20 molecules that may be detected using, for example, a hybridization assay, surface plasmon resonance, ELISA, competitive binding assays, isothermal titration calorimetry, phage display, affinity chromatography, rheology, or immunohistochemistry. The terms also refer to “binding” interactions between molecules. Binding may be “direct” or “indirect”. “Direct” binding comprises direct 25 physical contact between molecules. “Indirect” binding between molecules comprises the molecules having direct physical contact with one or more molecules simultaneously. For example, it is contemplated that peptidoglycan probes of the present disclosure directly bind and interact with antibiotics. This binding can result in the formation of a “complex” comprising the interacting molecules, which can 30 consist of the antibiotic and peptidoglycan probe referred to in the present disclosure. A “complex” is meant to include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces, and can be analyzed in a native complex form instead of being analyzed separately.13

[0058] The term “disease” used herein refers to any condition, infection, disorder, or syndrome that requires medical intervention or for which medical intervention is desirable. Such medical intervention can include treatment, diagnosis, and / or prevention. 5

[0059] Accordingly, the term “bacterial infection” used herein refers to the undesired invasion, presence, growth, or proliferation of pathogenic or opportunistic bacteria in a subject or host organism. This includes the excessive growth of bacteria that are normally present in or on the body of human or other organisms. More generally, a bacterial infection can be any situation in which the presence of a bacterial 10 population is damaging or toxic to a subject or results in allergy. Thus, a bacterial infection exists when excessive numbers of undesired bacteria are present in or on a subject's body, or when the effects of the presence of a bacterial population is damaging to the cells or other tissues of a subject. The undesired bacterial population may comprise of a single species of bacteria or may include various different bacterial 15 populations.

[0060] The terms “resistant” or “resistance” used herein both refer to an absence of decreased viability or inhibited growth or arrested proliferation in a microorganism when exposed to a therapeutically effective dose of antibiotic or antimicrobial agent that would have otherwise been effective in humans infected with non-resistant 20 bacterial strain. This would imply that an infection caused resistant microorganisms cannot be predictably and successfully treated with the antibiotic or antimicrobial agent used to treat the non-resistant strain.

[0061] Accordingly, the term “drug resistant” or “antibiotic resistant” or the like used herein refers to a loss or reduction in the effectiveness of an antibiotic (e.g., an 25 antimicrobial agent or an antimicrobial peptide) to inhibit the growth of a bacterial strain that was once susceptible or sensitive to the antibiotic before the bacterial strain gained resistance. As known by a person skilled in the art, treatment of patients with antibiotics has led to the development of resistance to almost all antibiotic classes (e.g., fluoroquinolones, tetracyclines, and β-lactams) and a bacterium that is generally 30 sensitive or susceptible to a class of antibiotic can develop resistance to the antibiotic through selection, thereby becoming a drug resistant strain. By definition, the selection may occur when a native strain of bacteria gains a mutation which confers inheritable traits of resistance to an / the antibiotic and becomes a fit and outstanding strain by passage. A “multi-drug resistant” bacterium is one that is resistant to more14than one antimicrobial drug. A skilled person can readily determine if a bacteria strain is drug resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic, e.g., according to the colony formation of bacteria. 5

[0062] The term “microorganism” or “bacteria”, as used herein interchangeably according to the context, refers to pathogenic or opportunistic bacteria, which include both Gram-positive and Gram-negative bacteria. By way of non-limiting examples, Gram-negative bacteria includes the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, 10 Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, Actinobacillus, Alcaligenes, Bordetella, Cedecea, Erwinia, Pantoea, Ralstonia, Stenotrophomonas, Xanthomonas and Legionella. By way of non-limiting examples, Gram-positive bacteria include the following genera: Enterococcus, 15 Streptococcus, Staphylococcus, Bacillus, Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria.

[0063] As used herein, the term “mass spectrometer” includes a device capable of identifying specific molecular species and measuring their accurate masses. The term is meant to include any molecular detector into which a polypeptide or peptide may be 20 characterized. A mass spectrometer can include three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application. The target engagement mass 25 spectrometry is capable of preserving the native structure of the analyte in the gas phase under mild conditions and therefore the native form of the complex, as discussed herein, of antibiotic and peptidoglycan precursor is maintained.

[0064] The term “liquid chromatography” used herein refers to a process in which a biological / chemical mixture carried by a liquid can be separated into components as 30 a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode15chromatography. In some cases, the sample containing the complex of peptidoglycan or its precursor of interest and antibiotic can be subjected to at least one of the aforementioned chromatographic methods. Analytes separated using chromatography will feature distinctive retention times, reflecting the speed at which an analyte moves 5 through the chromatographic column. Analytes may be compared using a chromatogram, which plots retention time on one axis and measured signal on another axis, where the measured signal may be produced from, for example, UV detection or fluorescence detection. II. General Embodiment 10

[0065] One aspect of the present disclosure is to provide a method for evaluation of the binding potency between a target biomolecule from bacteria and a compound selected from peptides, natural products, and derivatives thereof through a label-free approach. According to the present disclosure, the target biomolecule is not a protein, a transporter, or an enzyme. In one embodiment, the bacteria include Gram-positive 15 bacteria and Gram-negative bacteria, for example, mycobacteria. In one embodiment, the method is to screen a binder candidate capable of binding to bacterial biocomponent via a label-free approach. In one embodiment, the method is directed to the screening for a binder candidate which is potential to bind to a molecule on the bacterial cell wall (e.g., peptidoglycan or others) performing on target engagement 20 mass spectrometry (e.g., via screening hit, profiling structure-activity relationship (SAR), quantification (Kd), or relative quantification (complex percentage)) that can provide the profile of intermolecular correlation and binding potency.

[0066] In one aspect, the present disclosure provides a peptidoglycan precursor having a formula of A-Sn-Po-Lq; wherein 25 A is an oligopeptide moiety, S is a saccharide moiety, P is a phosphate group, L is a lipid tail comprising up to 55 carbon atoms, n is 1 or 2, 30 o is 1 or 2, and q is 0 or 1.

[0067] In one embodiment, A consists of 1 to 5 amino acids. In a further embodiment, the amino acid is natural and / or non-natural or synthetic amino acid. The amino acid can be L- or D-amino acid and can be modified or unmodified. More16preferably, the amino acid is selected from the group consisting of Ala, Glu, m-DAP and Lys.

[0068] In one embodiment, A comprises a D-Ala-D-Ala moiety.

[0069] In one embodiment, S is a monosaccharide. In another embodiment, S is a 5 disaccharide. In a further embodiment, S is selected from N-acetylmuramic acid (MurNAc) or N-acetylglucosamine (GlcNAc). In one embodiment, L is a lipid tail having a length of less than 50 carbon atoms, less than 45 carbon atoms, less than 40 carbon atoms, less than 35 carbon atoms, less than 30 carbon atoms, less than 25 carbon atoms, less than 20 carbon 10 atoms, less than 15 carbon atoms, less than 10 carbon atoms, or less than 5 carbon atoms.

[0071] According to the present disclosure, the peptidoglycan precursor is selected from the group consisting of: 1517Truncated Neryl-Lipid I (4).

[0072] In a preferred embodiment, the peptidoglycan precursor is Neryl-Lipid II:

[0073] In one aspect, the present disclosure provides a method for screening a 5 peptidoglycan-targeting antibiotic, comprising contacting an antibiotic candidate with a peptidoglycan precursor to form a complex; subjecting the complex to target engagement mass spectrometry; and determining the binding affinity of the antibiotic candidate; wherein the peptidoglycan precursor is selected from at least one of the compounds of 10 the group consisting ofe y - p ,185esstant p (),19y - p , , 5Neryl pyrophosphate (15).

[0074] In one aspect, the present disclosure provides a method for identifying a binding moiety on a peptidoglycan, comprising: 10 contacting an antibiotic candidate with a first peptidoglycan precursor to form a first complex; subjecting the first complex to target engagement mass spectrometry to determine a first binding affinity; contacting the antibiotic candidate with a second peptidoglycan precursor to form a 15 second complex;20subjecting the second complex to target engagement mass spectrometry to determine a second binding affinity; and comparing the first binding affinity and the second binding affinity to determine the binding moiety of the peptidoglycan to the antibiotic candidate; 5 wherein the peptidoglycan precursor is selected from the peptidoglycan precursors as set forth above.

[0075] In one aspect, the present disclosure provides a peptidoglycan for use in a method for screening a peptidoglycan-targeting antibiotic, wherein the method comprises the steps as set forth above. 10

[0076] In one aspect, the present disclosure provides a peptidoglycan for use in a method for identifying a binding moiety on a peptidoglycan, wherein the method comprises the steps as set forth above.

[0077] In one embodiment, the binding moiety of the peptidoglycan is selected from at least one of a disaccharide, a pyrophosphate, a lipid tail, and an oligopeptide 15 moiety. In another embodiment, the binding moiety of the peptidoglycan is a disaccharide. In another embodiment, the binding moiety of the peptidoglycan is a pyrophosphate. In another embodiment, the binding moiety of the peptidoglycan is a lipid tail, preferably an undecaprenol lipid tail. In another embodiment, the binding moiety of the peptidoglycan is an oligopeptide moiety. 20

[0078] In one embodiment, the antibiotic candidate binds to compound (1). In another embodiment, the antibiotic candidate binds to compound (4). In another embodiment, the antibiotic candidate binds to at least one of compound (5), compound (6), and compound (7).

[0079] In one embodiment, the antibiotic candidate binds to compound (1) but 25 does not binds to compound (4), compound (5), compound (6), and compound (7). In another embodiment, the antibiotic candidate binds to compound (1) but does not binds to compound (5), compound (6), and compound (7).

[0080] In one embodiment, when the first peptidoglycan precursor is compound (1), and the second peptidoglycan precursor is selected from at least one of compound 30 (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is pyrophosphate. In a further embodiment, the binding affinity to compound (1) being higher than the binding affinity to compound (5), compound (6), and compound (7) indicates that the binding moiety is pyrophosphate.21

[0081] In one embodiment, when the first peptidoglycan precursor is compound (1) and the second peptidoglycan precursor is selected from at least one of compound (4), compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is an 5 oligopeptide moiety. In a further embodiment, the binding affinity to compound (1) being higher than the binding affinity to compound (4), compound (5), compound (6), and compound (7) indicates that the binding moiety is an oligopeptide moiety.

[0082] In one embodiment, the method according to the present disclosure is employed to calculate the binding affinity of an antibiotic candidate to peptidoglycan. 10 The binding affinity is determined according to dissociation constant (KD) calculated by the equation:wherein P is the initial concentration of the antibiotic candidate, x is the initial 15 concentration of the peptidoglycan precursor, and [Complex] / [Noncomplex] is the ratio of the concentration of complex and noncomplex, wherein the complex is the complex formed by binding of the antibiotic candidate to the peptidoglycan precursor, and the noncomplex is the antibiotic candidate that does not bind to the peptidoglycan precursor. 20

[0083] In one embodiment, the binding affinity of the antibiotic candidate is compared to a standard antibiotic. In a further embodiment, the standard antibiotic is an antibiotic targeting an oligopeptide moiety or pyrophosphate of the peptidoglycan. In a specific embodiment, the standard antibiotic is selected from the group consisting of vancomycin, teicoplanin, and ramoplanin. 25

[0084] In one embodiment, the binding affinity of the antibiotic candidate is compared to a negative control. In a further embodiment, the negative control is an inhibitor of TGase or an inhibitor of TPase. In a still further embodiment, the negative control is selected from MoeA or ampicillin.

[0085] In one embodiment, the dissociation constant (KD) of the antibiotic 30 candidate is about 0.5 to about 5 μM. In a preferred embodiment, the dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM, about 1 to about224.5 μM, about 1.5 to about 4 μM, about 2 to about 3.5 μM, or about 2.5 to about 3 μM. In a preferred embodiment, the dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM, about 0.5 to about 4.5 μM, about 0.5 to about 4 μM, about 0.5 to about 3.5 μM, about 0.5 to about 3 μM, about 0.5 to about 2.5 μM, about 0.5 to 5 about 2 μM, about 0.5 to about 1.5 μM, about 0.5 to about 1 μM, about 1 to about 5 μM, about 1 to about 4.5 μM, about 1 to about 4 μM, about 1 to about 3.5 μM, about 1 to about 3 μM, about 1 to about 2.5 μM, about 1 to about 2 μM, about 1 to about 1.5 μM, about 1.5 to about 5 μM, about 1.5 to about 4.5 μM, about 1.5 to about 4 μM, about 1.5 to about 3.5 μM, about 1.5 to about 3 μM, about 1.5 to about 2.5 μM, about 10 1.5 to about 2 μM, about 2 to about 5 μM, about 2 to about 4.5 μM, about 2 to about 4 μM, about 2 to about 3.5 μM, about 2 to about 3 μM, about 2 to about 2.5 μM, about 2.5 to about 5 μM, about 2.5 to about 4.5 μM, about 2.5 to about 4 μM, about 2.5 to about 3.5 μM, about 2.5 to about 3 μM, about 3 to about 5 μM, about 3 to about 4.5 μM, about 3 to about 4 μM, about 3 to about 3.5 μM, about 3.5 to about 5 μM, about 15 3.5 to about 4.5 μM, about 3.5 to about 4 μM, about 4 to about 5 μM, about 4 to about 4.5 μM, or about 4.5 to about 5 μM.

[0086] Specifically, the dissociation constant (KD) of the antibiotic candidate is about 0.5 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM. 20

[0087] In one embodiment, the target engagement mass spectrometry is electrospray ionization mass spectrometer (ESI-MS), nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer, wherein said mass spectrometer is coupled to a liquid chromatography system. In one embodiment, the liquid chromatography system comprises reversed-phase liquid chromatography, ion 25 exchange chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed-mode chromatography, or a combination thereof.

[0088] In one embodiment, the method according to the present disclosure does not require immobilization of the peptidoglycan precursor on the matrix. 30

[0089] In one embodiment, the method according to the present disclosure does not require labeling of the peptidoglycan precursor with a fluorophore.

[0090] In still another embodiment of the present disclosure, the above method according to the present disclosure further comprises performing an analysis on target engagement mass spectrometer. In a particular embodiment, the mass spectrometer is23a hybrid linear trap / Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, a high-resolution accurate mass LC-MS equipped with a heat electrospray ionization probe, a high-performance liquid chromatography (HPLC) system with binary pump, and a Famos autosampler. In a particular embodiment, a 5 sample is loaded on the target engagement mass spectrometer, wherein the sample was directly injected into a continuous flowing solvent with a flow rate of 40-60 μL / min in the mobile phase consisting of IPA / H2O / NH4OH at pH 6-9. In a particular embodiment, the FTICR is operated in negative mode within a normal mass range from m / z 500-3000, showing resolution power is 100,000 at m / z 400, electrospray 10 voltage is applied to 4.0 kV, and the capillary temperature is set at 275 ºC.

[0091] In one aspect, the present disclosure provides a method of preparing a peptidoglycan precursor as disclosed herein, comprising: providing N-acetylglucosamine, obtaining compound (16) from N-acetylglucosamine through amide bond 15 formation,conjugating compound (16) with phosphate, oligopeptide, and optionally compound (15) to obtain a peptidoglycan precursor.

[0092] In one embodiment, the method of preparing a peptidoglycan precursor 20 further comprises: conjugating an additional saccharide to compound (16) to form a disaccharide moiety. In one embodiment, the oligopeptide is a dipeptide, tripeptide, or tetrapeptide.

[0093] In one aspect, the present disclosure provides an antibiotic obtained from the method as disclosed herein. For example, the present disclosure provides an 25 antibiotic obtained from the method of screening a peptidoglycan-targeting antibiotic, or the method of identifying a binding moiety on a peptidoglycan.

[0094] According to the present disclosure, the antibiotic candidate can be used in the treatment of bacterial diseases. One aspect provided herein is related to the antibiotic obtained from the method as disclosed herein for use in the treatment of 30 bacterial diseases. Also provided herein is related to a method for the treatment of24bacterial diseases comprising administering to a subject in need thereof an effective amount of the antibiotic obtained from the method as disclosed herein. Also provided herein is the use of the antibiotic obtained from the method as disclosed herein for the manufacture of a medicament for treating bacterial diseases. In one embodiment, the 5 bacterial disease is the bacterial infection caused by Gram-negative bacteria. In one embodiment, the bacterial disease is a bacterial infection caused by Gram-positive bacteria. In one embodiment, the bacterial disease is a bacterial infection caused by antibiotic-resistant bacteria. For example, the bacterial disease is caused by a microorganism which is vancomycin-resistant, methicillin-resistant, multidrug- 10 resistant, or extensively drug-resistant.

[0095] In one embodiment, the bacteria are resistant to antibiotic glycopeptides. In a specific embodiment, the bacteria are resistant to at least one antibiotic selected from vancomycin, teicoplanin, ramoplanin, decaplanin, oritavancin, telavancin, daptomycin, dalbavancin, ristocetin, and avoparcin. 15

[0096] In some embodiments, the bacterial disease is the infection caused by bacteria on or in the tissue or organ, including but not limited to: skin and soft tissue infection (including acne), connective tissue infection, bone infection, bacteremia, abscess, joint or muscle infection, wound infection, endovascular infection, CNS infection, abdominal infection, blood stream infection, urinary tract infection, pelvic 20 infection, invasive systemic infection, gastrointestinal infection, dental infection, food poisoning, pneumonia, meningitis, osteomyelitis, endocarditis, bacteremia, and sepsis.

[0097] Non-limiting examples of bacteria that may be the object of treatment with the antibiotic obtained from the method as disclosed herein include those belonging to the genus of Salmonella, Shigella, Campylobacter, Vibrio, Escherichia, 25 Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomyces, Nocardia, Enterobacter, Yersinia, Francisella, Pasteurella, Moraxella, Acinetobacter, Erysipelothrix, Moraxella, Actinobacillus, Streptobacillus, Listeria, Brucella, Bacillus, Clostridium, Treponema, Klebsiella, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Legionella, 30 Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia, and Mycoplasma.

[0098] The non-limiting examples of Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium,25Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium 5 marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, 10 Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, 15 Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, and Streptococcus sanguis.

[0099] The non-limiting examples of Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella 20 bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., 25 Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., 30 Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pestis.26

[0100] Abbreviations and acronyms used herein include the following as shown below:27EXAMPLES Example 1: Target engagement (TE) analysis of Park's nucleotide and vancomycin using mass spectrometry (MS) 5

[0101] The label-free target engagement mass spectrometry (MS) method, which allows for the analysis of protein complexes and their interactions while preserving their native structure in the gas phase under mild conditions, has been successfully demonstrated for studying protein-ligand interactions. The detail of the method is disclosed, for example, in Heck, A. J. R. Native Mass Spectrometry: A Bridge 10 Between Interactomics and Structural Biology. Nature Methods 2008, 5 (11), 927-933.

[0102] MS techniques were applied to study the interactions between the molecules, specifically for small molecule screening, provided that proper MS analysis conditions can be generated to enable sufficient engagement and measurable changes in the non-covalent Lipid II-analyte complex. 15

[0103] All chemicals were obtained from commercial suppliers and used without purification. Reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) on silica gel. Silica gel was used Merck Kiseslgel Si60 (40‒63 μm). TLC was performed on a glass plate coated to a thickness of 1 mm with Merck Kieselgel 60F254. NMR spectra were recorded on a dilute solution in CDCl3, CD3OD, 20 and D2O on Bruker AVENCE 600, 500, and AMX 400 spectrometers at ambient temperature. Chemical shifts are given in δ values, and coupling constant J is given in Hz. The splitting pattern is recorded as s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets), ddd (doublet of doublets of doublets), ddt (doublet of doublets of 25 triplets) and br (broad). Mass spectrometry analysis was performed by Mass Core Facility of Genomics Research Center, Academia Sinica, Taipei, Taiwan. Concentration refers to rotary evaporation.28

[0104] In the target engagement MS-like studies according to the present Example, the TE analysis between one of the PGN precursors, Park's nucleotide bearing the D- Ala-D-Ala moiety (20 μM), and vancomycin (20 μM) was performed as shown in Fig. 2. The result thereof, the vancomycin-Park nucleotide complex was successfully 5 detected under mild and neutral pH conditions. Example 2: Preparation of PGN precursors including Lipid II and Lipid I

[0105] To avoid insolubility caused by long-chain polyprenyl (C55) in naturally occurring Lipid II, Lipid II analogues with a shorter lipid tail were designed, which 10 were used to screen inhibitors of transglycosylation in inventor’s previous publications (see Liu, et al. Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl Moiety. Organic Letters 2010, 12 (7), 1608-1611 and Huang, et al. New Continuous Fluorometric Assay for Bacterial Transglycosylase Using Förster Resonance Energy 15 Transfer. Journal of the American Chemical Society 2013, 135 (45), 17078-17089), the content of which is incorporated herein by reference in its entirety.

[0106] Moreover, based on the different moieties in Lipid II, more analogues were designed and prepared using diverse synthetic strategy for peptidoglycan fragments or precursors, as shown below.2930

[0107] Among these analogues, 2S3P (5) was obtained by digestingpeptidoglycan, which had been purified from Bacillus subtilis, using lysozyme. Park'snucleotide (8) was isolated from Staphylococcus aureus. The method for isolating5 Park’s nucleotide is disclosed, for example, in Park, J. T. Uridine-5'-PyrophosphateDerivatives. III. Amino Acid-Containing Derivatives. Journal of Biological Chemistry 1952, 194 (2), 897-904, the content of which is incorporated herein by reference in itsentirety.

[0108] An exemplary scheme for preparing Neryl-Lipid II (1) is illustrated below.10Scheme 1. Preparation of Neryl-Lipid II (1)

[0109] Reagents and conditions: (a) i. BnOH, HCl, 70 ºC, 19 h; ii. PhCH(OMe)2,p-TSA, CH3CN, rt, 12 h; iii. (S)-Chloropropionic acid, NaH, 1,4-dixonane, 70 ºC, 5 h; 31iv. l-Ala-OTMSE, HBTU, Et3N, CH2Cl2 / THF, rt, 8 h, 24.7% over four steps. (b) i. p- TSA, CH3OH, 70 ºC, 1 h; ii. TBSCl, imidazole, CH2Cl2, rt, 8 h, 66.6% over two steps. (c) donor,rt, 2 h, 91.2%. (d) i. Zn, Ac2O, CH2Cl2, rt, 16 h; ii. AcOH / H2O / THF, rt, 16 h; iii. Ac2O, pyridine, 0 ºC, 12 h, 57.9% over three 5 steps. (e) i. H2, Pd(OH)2 / C, THF, rt, 16 h; ii. (iPr)2NP(OBn)2, 1H-tetrazole, CH2Cl2, rt, 2 h; iii.tBuO2H, −78 ºC to rt, 2 h; iv. TBAF, THF, rt, 2 h; v. H-D-Glu-L-Lys-D-Ala-D- Ala-OMe, HATU, DIPEA, CH2Cl2, 0 ºC to rt, 8 h; vi. H2, Pd(OH)2 / C, THF, rt, 4 h, 18.0% over six steps. (f) i. neryl phosphate, CDI, 1H-tetrazole, THF / DMF, rt, 24 h; ii. 2N LiOH(aq), CH3OH, 0 ºC to rt, 8 h, 34.1% over two steps. 10

[0110] The synthesis of the PGN precursors was carried out following the method disclosed in the previous publication with slight modifications. Please see Shih, et al. Effect of the Peptide Moiety of Lipid II on Bacterial Transglycosylase. Angewandte Chemie International Edition 2012, 51 (40), 10123-10126, the content of which is incorporated herein by reference in its entirety. 15

[0111] The intermediate compound 16 was prepared from GlcNAc through four steps including anomeric protection, 4,6-benzylidene protection, O-alkylation, and amide bond formation to give in 25% yields. To prepare the C4 position deprotected glycan acceptor, after benzylidene deprotection, TBSCl was used to selectively protect the C6 hydroxyl group to generate compound 17 in 67% yields. Disaccharide 18 was 20 given in 91% yields by treating with TMSOTf as a Lewis acid. For easy global deprotection, the trichloroethoxycarbonyl (Troc) group was reduced and protected with acetyl (Ac) by zinc dust and acetic anhydride treatment. To afford compound 19, the TBS group of the reductive product was deprotected under the acidic condition followed by treated with Ac2O in 58% yields. 25

[0112] The benzyl group on the anomeric position was hydrogenated under a hydrogen atmosphere by using Pd(OH)2 / C as a catalyst. The lactol was treated with 1H-tetrazole and dibenzyl N, N-diisopropyl phosphoramidite, and the phosphite intermediate was sequentially oxidized by tert-butyl hydroperoxide to generate the α- form phosphate. Selective deprotection of the O-TMSE group in phosphate product 30 was carried out through treating TBAF in THF, followed by coupling with the tetrapeptide D-Glu(OMe)-L-Lys(TFA)-D-Ala-D-Ala, then the benzyl groups on phosphate was hydrogenated under H2atmosphere by using Pd(OH)2 / C as a catalyst to give compound 20 in 18.0% yields over six steps. Compound 20 was conjugated with32activated neryl-phosphate and globally deprotected under basic conditions to give Neryl-Lipid II (1) in 34.1% yields over two steps.

[0113] An exemplary scheme for preparing Lipid I analogues is illustrated below.5 Scheme 2. Preparation of Lipid I analogues.

[0114] Reagents and conditions: (a) i. p-TSA, CH3OH, 70 ºC, 1 h; ii. Ac2O, pyridine, 0 ºC, 12 h, 71.0% over two steps. (b) i. H2, Pd(OH)2 / C, THF, rt, 16 h; ii. 10 (iPr)2NP(OBn)2, 1H-tetrazole, CH2Cl2, rt, 2 h; iii.tBuO2H, −78 ºC to rt, 2 h; iv. TBAF, THF, rt, 2 h; v. H-D-Glu-L-Lys-D-Ala-D-Ala-OMe, HATU, DIPEA, CH2Cl2, 0 ºC to rt, 8 h; vi. H2, Pd(OH)2 / C, THF, rt, 4 h, 17.8% over six steps. (c) i. TBAF, THF, rt, 2 h; H-D-Glu-L-Lys-D-Ala-D-Ala-OMe, HATU, DIPEA, CH2Cl2, 0 ºC to rt, 8 h, 72.3% over two steps. (d) i. H2, Pd(OH)2 / C, THF, rt, 16 h; ii. (iPr)2NP(OBn)2, 1H-tetrazole,15 CH2Cl2, rt, 2 h; iii.tBuO2H, −78 ºC to rt, 2 h; iv. TBAF, THF, rt, 2 h; v. H-D-Glu-L- Lys-OMe, HATU, DIPEA, CH2Cl2, 0 ºC to rt, 8 h; vi. H2, Pd(OH)2 / C, THF, rt, 4 h, 16.5% over six steps. (e) i. H2, Pd(OH)2 / C, THF, rt, 16 h; ii. (iPr)2NP(OBn)2, 1H- tetrazole, CH2Cl2, rt, 2 h; iii.tBuO2H, −78 ºC to rt, 2 h; iv. H2, Pd(OH)2 / C, THF, rt, 4 h, 30.2% over four steps. (f) i. neryl phosphate, CDI, 1H-tetrazole, THF / DMF, rt, 24 h; ii. 20 2N LiOH(aq), CH3OH, 0 ºC to rt, 8 h, 32.4% (2), 45.0% (4), and 36.8% (10) over two33steps. (g) 2N LiOH(aq), CH3OH, 0 ºC to rt, 8 h, 62.7%. (h) i. Ethyl phosphate, CDI, 1H- tetrazole, THF / DMF, rt, 24 h; ii. 2N LiOH(aq), CH3OH, 0 ºC to rt, 8 h, 24.3%. (i) H2, Pd(OH)2 / C, THF, rt, 16 h; ii. 2N LiOH(aq), CH3OH, 0 ºC to rt, 8 h, 26.7% over two steps. 5

[0115] To afford the Lipid I analogues containing different moieties, several peptide stems and phosphate lipids were used to couple with monosaccharide. Compound 21 was either phosphorylated or not phosphorylated, followed by coupling with a tetrapeptide to yield compounds 22 and 23, which were subsequently deprotected to yield MPP (11). Furthermore, compound 22 was coupled with Neryl- 10 phosphate, Ethyl-phosphate, or left uncoupled, and then subjected to global deprotection to generate Neryl-Lipid I (2), Ethyl-Lipid I (12), and MPP-P (3). After phosphorylation of compound 21, it was either coupled with a dipeptide or left uncoupled, followed by global deprotection to afford Tripeptide Lipid I (10) and Truncated Lipid I (4). In addition to the synthetic compounds, several PGN precursors, 15 such as Park's nucleotide, were also collected from natural sources to contribute to the PGN precursor-based probe pool.

[0116] The preparation of each compound used herein is illustrated in the following.

[0117] Compound 16 20

[0118] Conc. HCl (37%, 1.75 mL) was slowly added to a solution of N-acetyl- glucosamine (10.0 g, 45.2 mmol) in benzyl alcohol (35 mL) at rt, and the mixture was stirred at 70 ºC for 19 h. The solution was slowly cooled to 0 ºC and added Et2O (375 mL) followed by precipitation of gray solid. The precipitate was filtered off and 25 washed with diethyl ether. The solid was dried in vacuo to yield 1-O-benzyl-GlcNAc and used in the next reaction without further purification.

[0119] Benzaldehyde dimethyl acetal (10.2 mL, 67.8 mmol) and p-toluenesulfonic acid monohydrate (2.3 g, 13.6 mmol) were added to a solution of 1-O-benzyl-GlcNAc in anhydrous acetonitrile (100 mL) at rt. After 12 h, the reaction mixture was 30 concentrated to remove half of the solvent followed by added sat. NaHCO3(aq)to34neutralize and precipitate out solid. The precipitate was collected and washed with water (200 mL × 3) and Et2O (200 mL × 3) to provide 4, 6-benzylidene sugar as a gray solid and used in the next reaction without further purification.

[0120] Sodium hydride (60% dispersion in oil, 7.3 g, 182.4 mmol) was slowly 5 added to 4, 6-benzylidene sugar in dry 1,4-dioxane (250 mL) at 0 ºC. The reaction mixture was stirred at 70 ºC for 30 min followed by added the mixture of (S)-2- chloropropionic acid (9.8 mL, 113.0 mmol) and another sodium hydride (60% dispersion in oil, 7.3 g, 182.4 mmol) at 0 ºC. After stirred at 70 ºC for 5 h, the reaction mixture was quenched by adding ice and acidified with 1N HCl until pH 2 followed by 10 poured into ice water (150 mL) at 0 ºC. The precipitate was filtered off, washed with water (200 mL × 3), Et2O (200 mL × 2), and hexanes (200 mL × 2) and dried to yield muramyl compound as a white solid and used in the next reaction without further purification.

[0121] Et3N (12.6 mL, 90.4 mmol) was slowly added to muramyl compound in 15 CH2Cl2(200 mL) followed by adding HBTU (34.1 g, 90.4 mmol) and stirred at rt under Ar(g). After 15 min, H-Ala-OTMSE (12.8 g, 58.8 mmol) in THF (200 mL) was slowly added to the reaction by cannula. After stirring at rt for 8 h, the reaction mixture was filtered, concentrated, diluted with EtOAc, washed with 1N HCl, sat. NaHCO3(aq), and brine. The combined organic layer was dried over MgSO4, filtered, 20 and concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 25:1, silica gel) to yield compound 16 (7.2 g, 24.7 % over four steps) as a white solid.

[0122] 1H NMR (600 MHz, CDCl3) δ 0.03 (s, 9H), 0.99 (t, J = 8.6 Hz, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.41 (d,= 7.2 Hz, 3H), 1.94 (s, 3H), 3.67 (t, J = 9.1 Hz, 1H), 25 3.72 (t, J = 9.4 Hz, 1H), 3.79 (t, J = 10.3 Hz, 1H), 3.85-3.90 (m, 1H), 4.1 (q, J = 6.7 Hz, 1H), 4.19-4.26 (m, 3H), 4.29 (td, J = 9.2 Hz, 3.5 Hz, 1H), 4.41-4.47 (m, 1H), 4.49, (d, J = 11.8 Hz), 4.72 (d, J = 11.8 Hz, 1H), 4.98 (d, J = 3.6 Hz, 1H), 5.57 (s, 1H), 6.23 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 7.2 Hz, 1H), 7.31-7.39 (m, 8H), 7.45-7.48 (m, 2H)13C NMR (150 MHz, CDCl3) δ 172.9, 172.7, 137.1, 136.8, 129.1, 128.7 (× 2), 128.3 (× 2), 30 128.2 (× 2), 125.9 (× 2), 101.4, 97.5, 81.7, 78.3, 77.5, 70.2, 68.9, 63.8, 63.2, 53.1, 48.1, 29.7, 23.4, 19.5, 18.0, 17.3, −1.5 (× 2). HRMS calcd for [C47H74Cl3N3O18Si2−H]−1130.3469, found 1130.3401.

[0123] Compound 1735

[0124] p-Toluenesulfonic acid monohydrate (543.9 mg, 3.1 mmol) was added to compound 16 (10.0 g 15.6 mmol) in MeOH (60 mL) and stirred at 70 ºC. After 1 h, the reaction mixture was quenched with Et3N (434 μL, 3.12 mmol) followed by 5 concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 10:1, silica gel) to yield a diol product as a white solid (6.58 g, 76.3%).

[0125] TBSCl (2.7 g, 17.8 mmol) and imidazole (1.2 g, 17.8 mmol) was added to the diol (6.58 g, 11.9 mmol) in CH2Cl2(40 mL) at 0 ºC under Ar(g). After stirred at rt for 8 h, the reaction mixture was quenched with 1N HCl and diluted with CH2Cl2(200 10 mL), washed with 1N HCl, sat. NaHCO3(aq), and brine. The combined organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 25:1, silica gel) to yield compound 17 (6.9 g, 87.3%) as a white solid.

[0126] 1H NMR (600 MHz, CDCl3) δ 0.03 (s, 9H), 0.10 (s, 6H) 0.90 (s, 9H), 0.98- 15 1.02 (m, 2H), 1.43 (m, 6H), 1.91 (s, 3H), 3.22 (br, 1H), 3.54-3.59 (m, 1H), 3.63-3.69 (m, 2H), 3.77-3.88 (m, 2H), 4.13-4.24 (m, 4H), 4.42-4.49 (m, 2H), 4.69 (d, J = 12.0 Hz, 1H), 4.91 (d, J = 3.6 Hz, 1H), 6.14 (d, J = 8.5 Hz, 1H), 6.90 (d, J = 7.4 Hz, 1H), 7.29-7.39 (m, 5H).13C NMR (150 MHz, CDCl3) δ 173.1, 172.9, 170.4, 137.3, 128.7 (× 2), 128.3, 128.2 (× 2), 97.1, 80.3, 77.9, 72.8, 71.0, 69.9, 64.7, 63.9, 52.5, 48.2, 26.0 (× 20 3), 23.5, 19.4, 18.4, 18.0, 17.4, −1.3 (× 3), −5.29, −5.30. HRMS calcd for [C32H56N2O9Si2+H]+669.3597, found 669.3593.

[0127] Compound 1825

[0128] TMSOTf (163.0 μL, 0.90 mmol) was slowly added to a mixture of acceptor 17 (2.0 g, 2.99 mmol), donor (4.7 g, 7.47 mmol), and 4 Å molecular sieves in CH2Cl2at -78 ºC. The reaction was stirred at rt for 2 h followed by quenched with36dropped Et3N, filtered with celite, and concentrated. The residue was purified by column chromatography (Hexanes / EtOAc 1.5:1, silica gel) to yield compound 18 (3.1 g, 91.2%) as a white solid.

[0129] 1H NMR (600 MHz, CDCl3) δ 0.02 (s, 9H), 0.07-0.09 (m, 6H), 0.91 (s, 5 9H), 0.97-1.01 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.41 (d, J = 7.2 Hz, 3H), 1.94 (s, 3H), 2.02-2.03 (m, 9H), 3.46 (d, J = 9.8 Hz, 1H), 3.50 (t, J = 9.5 Hz, 1H), 3.56-3.61 (m, 2H), 3.69-3.76 (m, 2H), 3.82-3.87 (m, 1H), 3.96-4.00 (m, 1H) 4.05 (dd, J = 12.5 Hz, 2.0 Hz, 1H), 4.16-4.22 (m, 2H), 4.34 (dd, J = 12.5 Hz, 4.0 Hz, 1H), 4.46 (br, 1H), 4.48 (br, 1H), 4.55-4.59 (m, 1H), 4.65-4.74 (m, 3H), 4.74-4.79 (m, 1H), 5.08-5.12 (m, 2H), 10 5.12-5.15 (m, 1H), 5.25 (d, J = 9.5 Hz, 1H), 6.91 (d, J = 7.1 Hz, 1H), 7.09 (d, J = 6.2 Hz, 1H), 7.26-7.33 (m, 5H)13C NMR (150 MHz, CDCl3) δ 174.1, 172.8, 171.1, 170.8, 170.7, 169.5, 154.6, 137.8, 128.5 (× 2), 127.9 (× 2), 99.9, 97.0, 95.6, 76.3, 75.2, 74.6, 72.3, 72.2, 72.1, 71.5, 70.2, 68.2, 63.8, 61.8, 61.6, 57.0, 54.1, 48.2, 26.1 (× 3), 23.3, 20.8 (× 2), 20.7 (× 2), 19.0, 18.4, 18.1, 17.4, −1.4 (× 3), −4.6, −5.1. HRMS calcd for 15 [C47H74Cl3N3O18Si2−H]−1130.3469, found 1130.3401.

[0130] Compound 19

[0131] Ac2O (6 mL, 62.73 mmol) was added to a solution of compound 18 (3.4 g, 20 2.99 mmol) and zinc dust (9.8 g, 149.3 mmol) in CH2Cl2(60 mL) and then stir at rt. After 16 h, the reaction mixture was filtered with celite and concentrated. The residue was purified by column chromatography (Hexanes / EtOAc 1:2, silica gel) to yield the acylated disaccharide (2.4 g, 79.6%) as a white solid.

[0132] The acylated disaccharide (2.4 g, 2.38 mmol) was dissolved in 25 AcOH:H2O:THF=1:1:1 (75 mL) and stirred at rt under Ar(g)for 16 h. The reaction mixture was quenched with sat. NaHCO3(aq)and concentrated followed by diluted with EtOAc and washed with brine. The combined organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 10:1, silica gel) to yield the hydroxyl disaccharide (1.7 g, 83.2%) as a 30 white solid.37

[0133] Ac2O (747 μL, 7.90 mmol) was added to a solution of hydroxyl disaccharide (1.7 g, 1.98 mmol) in pyridine (20 mL), and the reaction was stirred at 0 ºC under Ar(g)for 12 h. The reaction mixture was concentrated, diluted with EtOAc, and washed with 1N HCl, sat. NaHCO3(aq)and brine. The combined organic layer was 5 dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to yield compound 19 (1.6 g, 87.4%) as a white solid.

[0134] 1H NMR (600 MHz, CDCl3) δ 0.02 (s, 9H), 0.99 (dd, J = 9.5 Hz, 7.7 Hz, 2H), 1.38 (d, J = 6.7 Hz, 3H), 1.41 (d, J = 7.2 Hz, 3H), 1.93 (s, 3H), 1.95 (s, 3H), 2.00 10 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.14 (s, 3H), 3.55-3.59 (m, 1H), 3.59-3.64 (m, 1H), 3.75-3.77 (m, 2H), 4.00 (ddd, J = 10.6 Hz, 6.9 Hz, 3.6 Hz, 1H), 4.03-4.09 (m, 2H), 4.10-4.14 (m, 1H), 4.14-4.22 (m, 2H), 4.30 (dd, J = 12.3 Hz, 3.7 Hz, 2H), 4.37-4.41 (m, 2H), 4.44-4.53 (m, 2H), 4.65 (d, J = 12.3 Hz, 1H), 5.06-5.13 (m, 3H), 6.17 (d, J = 9.5 Hz, 1H), 7.00 (d, J = 6.9 Hz, 1H), 7.21 (d, J = 7.4 Hz, 1H), 7.27-7.35 (m, 5H).13C 15 NMR (150 MHz, CDCl3) δ 173.6, 172.9, 171.4, 171.0, 170.8, 170.6 (× 2), 169.3, 137.4, 128.5 (× 2), 127.9 (× 2), 127.7 (× 2), 100.3, 96.8, 76.2, 75.5, 72.6, 71.9, 70.1, 69.4, 68.0, 63.7, 62.3, 61.5, 54.5, 53.4, 48.1, 23.2, 23.2, 21.0, 20.6 (× 2), 20.6, 18.6, 17.9, 17.3, −1.50 (× 3). HRMS calcd for [C42H63N3O18Si1−H]−924.3803, found 924.3782. 20

[0135] Compound 20

[0136] A mixture of compound 19 (767 mg, 0.83 mmol) and 10% Pd(OH)2 / C (767 mg) in THF (80 mL) was stirred at rt under a hydrogen atmosphere for 16h. The reaction mixture was filtered with celite and the filtrated was concentrated and purified 25 by column chromatography (CH2Cl2 / MeOH 15:1, silica gel) to yield the lactol (517 mg, 74.7%) as a white solid.

[0137] 1H-tetrazole (191 mg, 2.72 mmol) and dibenzyl N,N- diisopropylphosphoramidite (684 μL, 2.04 mmol) were added to a solution of the38lactol (517 mg, 0.68 mmol) in dry CH2Cl2(10 mL) at 0 ºC and stirred at rt for 2 h. The reaction was then cooled to −78 ºCand treated with tert-butyl hydroperoxide (408 μL, 2.96 mmol). The reaction was allowed to warm to rt and was stirred for 2 h. After the reaction was completed, the mixture was diluted with CH2Cl2and washed with 5 H2O and brine. The organic layer was dried over MgSO4, filtered, evaporated, and purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to obtain a phosphorylated disaccharide as a yellowish solid (479 mg, 70.6% over two steps).

[0138] A phosphorylated disaccharide (479 mg, 0.437 mmol) was dissolved in TBAF (2.6 mL, 1.0 M in THF) and stirred at rt for 2 h. The reaction mixture was 10 extracted with EtOAc and washed with 1N HCl and brine. The combined organic layer was dried over MgSO4, filtered, and concentrated to obtain colorless oil and used in the next reaction without further purification. The mixture of the residue and H-D- Glu(OMe)-L-Lys(TFA)-D-Ala-D-Ala-OMe (302 mg, 0.525 mmol) was dissolved in dry CH2Cl2(5 mL) followed by treated N, N-Diisopropylethylamine (305 μL, 1.75 15 mmol) and stirred at 0 ºC for 5 min. HATU (335 mg, 0.874 mmol) was added to the reaction mixture and stirred at rt for 8h. The reaction mixture was diluted with CH2Cl2, and washed with H2O and brine. The organic layer was dried over MgSO4, filtered, evaporated, and purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to obtain a peptidated disaccharide as a colorless oil (297 mg, 44.7% over two steps). 20

[0139] A mixture of peptidated disaccharide (297 mg, 0.20 mmol) and 10% Pd(OH)2 / C (30 mg) in THF (20 mL) was stirred at rt under a hydrogen atmosphere for 4 h. The reaction mixture was filtered with celite and the filtrated was concentrated and purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, silica gel) to yield compound 20 (200 mg, 76.4%) as a yellowish solid. 25

[0140] 1H NMR (600 MHz, CD3OD) δ 1.34-1.50 (m, 16H), 1.56-1.64 (m, 2H), 1.64-1.76 (m, 1H), 1.76-1.85 (m, 1H), 1.92 (s, 3H), 1.93-1.96 (m, 3H), 1.97-2.00 (m, 6H), 2.01-2.04 (m, 3H), 2.11 (s, 3H), 2.16-2.24 (m, 1H), 2.30-2.37 (m, 2H), 3.27-3.30 (m, 2H), 3.69-3.72 (m, 6H), 3.75-3.78 (m, 1H), 3.81-3.89 (m, 2H), 3.97-4.09 (m, 4H), 4.20 (t, J = 7.9 Hz, 1H), 4.26-4.30 (m, 1H), 4.33-4.43 (m, 4H), 4.43-4.51 (m, 2H), 30 4.54-4.59 (m, 2H), 4.71 (t, J = 9.4 Hz, 1H), 4.96 (t, J = 9.7 Hz, 1H), 5.29 (q, J = 9.2 Hz, 1H), 5.56 (br, 1H).13C NMR (150 MHz, CD3OD) δ 174.6, 174.4, 173.7, 173.4, 173.3, 173.1, 173.0, 172.3, 171.9, 171.8, 171.1, 170.9, 170.4, 169.9, 116.2 (q, JC-F= 286.3 Hz), 99.8, 77.4, 76.3, 75.5, 72.4, 71.4, 69.8, 68.7, 62.0, 61.5, 54.2, 51.5, 51.4, 51.4, 51.3, 39.1, 39.1, 30.8, 60.6, 28.1, 28.1, 27.2, 22.7, 21.5, 19.5, 19.4, 19.4, 19.2,3919.1, 17.9, 17.4, 17.2, 16.6, 16.4, 16.0, 15.9.31P NMR (200 MHz, CD3OD) δ −0.6. HRMS calcd for [C51H78F3N8O28P−H]−1337.4542, found 1337.4505.

[0141] Neryl-Lipid II (1) 5

[0142] 1,1’-Carbonyldiimidazole (242 mg, 1.493 mmol) was added to a solution of neryl phosphate (75 mg, 0.299 mmol) in THF (2 mL) at rt. After stirring for 2 h, anhydrous MeOH (61 μL, 1.493 mmol) was added to the reaction for quenching the excess 1,1’-carbonyldiimidazole. After another 1 h stirring, the reaction was 10 concentrated and re-dissolved in DMF / THF (9 mL, v / v = 2:1). 1H-tetrazole (22 mg, 0.314 mmol) and compound 5 (200 mg, 0.149 mmol) were added to the above solution and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated and purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, silica gel) to yield a pyrophosphate intermediate. 2N LiOH(aq)(1045 μL, 2.090 mmol) 15 was slowly added to a solution of the above intermediate in MeOH (15 mL) at 0 ºC for 8h, for the global deprotection. The reaction mixture was neutralized by 1N HCl(aq)(1254 μL, 1.25 mmol), concentrated, purified by column chromatography (CHCl3 / MeOH / H2O / NH4OH(aq)88:58:12.5:1, silica gel) and further purified by RP- HPLC on an Eclipse XDB-C18 (5 μm, 9.1 × 250 mm) column using gradient elution 20 with 50 mM NH4HCO3(aq) / MeOH (100:0 to 0:100) at a flow rate of 2.0 mL / min over 50 min to yield Neryl-Lipid II (1) (64.3 mg, 34.1% over two steps) as a white solid.

[0143] 1H NMR (600 MHz, D2O) δ 1.33-1.36 (m, 3H), 1.36-1.40 (m, 3H), 1.40- 1.48 (m, 8H), 1.62 (s, 3H), 1.65-1.73 (m, 5H), 1.73-1.85 (m, 5H), 1.85-1.94 (m, 1H), 1.97-2.02 (m, 3H), 2.02-2.07 (m, 3H), 2.08-2.20 (m, 5H), 2.27-2.38 (m, 2H), 2.97-3.03 25 (m, 2H), 3.38-3.44 (m, 2H), 3.55 (t, J = 8.3 Hz, 1H), 3.69-3.77 (m, 3H), 3.80 (t, J = 9.7 Hz, 1H), 3.88-3.97 (m, 4H), 4.09-4.17 (m, 2H), 4.17-4.36 (m, 5H), 4.44 (t, J = 6.7 Hz, 2H), 4.61 (d, J = 8.3 Hz, 1H), 5.17-5.21 (m, 1H), 5.41-5.47 (m, 2H).13C NMR (150 MHz, D2O) δ 179.3, 177.3, 175.7, 175.3, 174.4, 174.2, 174.2, 173.8, 173.6, 143.0,40133.9, 123.8, 120.3, 100.0, 94.2, 78.4, 78.0, 75.9, 73.8 (× 2), 72.4, 70.3, 62.9, 61.0, 59.7, 56.0, 54.2, 53.9, 53.5, 50.7, 50.0, 49.6, 39.2, 31.3 (× 2), 30.4, 27.6, 26.2, 26.0, 24.8, 22.7, 22.2, 22.1, 21.9, 18.7, 17.2, 17.0, 16.7, 16.4.31P NMR (200 MHz, D2O) δ −10.8 (d, JP-P= 21.0 Hz), −13.3 (d, JP-P= 21.0 Hz). HRMS calcd for 5 [C49H84N8O26P2−H]−1261.4899, found 1261.4804.

[0144] Compound 21

[0145] p-Toluenesulfonic acid monohydrate (543.9 mg, 3.1 mmol) was added to 10 compound 16 (10.0 g 15.6 mmol) in MeOH (60 mL) and stirred at 70 ºC. After 1 h, the reaction mixture was quenched with Et3N (434 μL, 3.12 mmol) followed by concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 10:1, silica gel) to yield a diol product as a white solid (6.58 g, 76.3%).

[0146] Ac2O (9.0 mL, 94.75 mmol) was added to a solution of the above diol 15 product (6.50 g, 11.84 mmol) in pyridine (40 mL), and the reaction was stirred at 0 ºC under Ar(g)for 12 h. The reaction mixture was concentrated, diluted with EtOAc, washed with 1N HCl, sat. NaHCO3(aq)and brine. The combined organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to yield compound 21 (7.04 g, 93.1%) 20 as a white solid.

[0147] 1H NMR (600 MHz, CDCl3) δ 0.03 (s, 9H), 0.97-1.01 (m, 2H), 1.32 (d, J = 6.7 Hz, 3H), 1.43 (d, J = 7.3 Hz, 3H), 1.89 (s, 3H), 2.07 (s, 3H), 2.11 (s, 3H), 3.66 (t, J = 9.9 Hz, 1H), 3.88-3.91 (m, 1H), 3.96 (q, J= 6.7 Hz, 1H), 4.03 (dd, J = 12.3 Hz, 2.3 Hz, 1H), 4.17-4.22 (m, 3H), 4.34-4.40 (m, 2H), 4.50 (d, J = 11.7 Hz, 1H), 4.69 (d, J = 25 11.7 Hz, 1H), 4.90 (d, J = 3.7 Hz, 1H), 5.07 (t, J = 9.7 Hz, 1H), 5.90 (d, J = 9.5 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 7.33-7.41 (m, 5H).13C NMR (150 MHz, CDCl3) δ 172.6, 172.1, 171.0, 170.2, 169.4, 136.7, 128.9 (× 2), 128.6, 128.4 (× 2), 97.2, 79.0, 78.5, 70.4, 69.6, 68.7, 63.8, 62.2, 53.1, 48.3, 23.5, 21.0, 20.9, 18.9, 17.6, 17.4, −1.37 (× 3). HRMS calcd for [C30H46N2O11Si2+H]+639.2949, found 639.2916. 3041

[0148] Compound 22

[0149] A mixture of compound 21 (7.04 g, 11.02 mmol) and 10% Pd(OH)2 / C (7.04 g) in THF (50 mL) was stirred at rt under a hydrogen atmosphere for 16h. The 5 reaction mixture was filtered with celite and the filtrated was concentrated and purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to yield the lactol (4.86 g, 79.2%) as a white solid.

[0150] 1H-tetrazole (1.02 g, 14.58 mmol) and dibenzyl N,N- diisopropylphosphoramidite (9.8 mL, 29.17 mmol) were added to a solution of the 10 lactol (2.0 g, 3.65 mmol) in dry CH2Cl2(40 mL) at 0 ºC and stirred at rt for 2 h. The reaction was then cooled to -78 ºC and treated with tert-butylhydroperoxide (2.2 mL, 15.90 mmol). The reaction was allowed to warm to rt and was stirred for 2 h. After the reaction was completed, the mixture was diluted with CH2Cl2and washed with H2O and brine. The organic layer was dried over MgSO4, filtered, evaporated, and 15 purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to obtain a phosphorylated saccharide as a yellowish solid (2.06 g, 69.8% over two steps).

[0151] A phosphorylated saccharide (2.06 g, 2.56 mmol) was dissolved in TBAF (15.36 mL, 1.0 M in THF) and stirred at rt for 2 h. The reaction mixture was extracted with EtOAc and washed with 1N HCl and brine. The combined organic layer was 20 dried over MgSO4, filtered, and concentrated to obtain colorless oil and used in the next reaction without further purification. The mixture of the residue and H-D- Glu(OMe)-L-Lys(TFA)-D-Ala-D-Ala-OMe (2.03 g, 3.57 mmol) was dissolved in dry CH2Cl2(25 mL) followed by treated N, N-Diisopropylethylamine (1.77 mL, 10.19 mmol) and stirred at 0 ºC for 5 min. HATU (1.95 mg, 5.09 mmol) was added to the 25 reaction mixture and stirred at rt for 8h. The reaction mixture was diluted with CH2Cl2, and washed with H2O and brine. The organic layer was dried over MgSO4, filtered, evaporated, and purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to obtain a peptidated saccharide as a colorless oil (1.61 g, 51.3% over two steps)42

[0152] A mixture of peptidated saccharide (1.61 g, 1.30 mmol) and 10% Pd(OH)2 / C (161.0 mg) in THF (20 mL) was stirred at rt under a hydrogen atmosphere for 4 h. The reaction mixture was filtered with celite and the filtrated was concentrated and purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, 5 silica gel) to yield compound 22 (860.0 mg, 62.8%) as a yellowish solid.

[0153] 1H NMR (600 MHz, CD3OD) δ 1.32 (d, J = 6.7 Hz), 1.35-1.40 (m, 4H), 1.40-1.44 (m, 7H), 1.59 (quin, J = 7.2 Hz, 2H), 1.67-1.75 (m, 1H), 1.76-1.84 (m, 1H), 1.87-1.97 (m, 4H), 2.05 (s, 3H), 2.10 (s, 3H), 2.18-2.26 (m, 1H), 2.29 (t, J = 7.6 Hz, 2H), 3.27-3.30 (m, 2H), 3.69 (s, 3H), 3.71 (s, 3H), 3.87 (t, J = 9.8 Hz, 1H), 4.10 (dd, J 10 = 12.1 Hz, 1.6 Hz, 1H), 4.14-4.20 (m, 2H), 4.21-4.30 (m, 4H), 4.34-4.42 (m, 3H), 5.06 (t, J = 9.8 Hz, 1H), 5.41-5.45 (m, 1H).13C NMR (150 MHz, CD3OD) δ 175.0, 175.0, 174.7, 174.5, 174.5, 173.5, 173.3, 172.6, 171.7, 159.1, 158.8, 117.6 (q, JC-F= 284.8 Hz), 95.3, 79.4, 79.3, 70.6, 69.9, 63.2, 55.6, 55.2, 55.2, 52.8, 52.7, 52.7, 50.9, 50.3, 40.4, 32.3, 32.1, 29.5, 28.1, 24.1, 23.1, 21.0, 20.7, 19.2, 17.9, 17.7, 17.3.31P NMR 15 (200 MHz, CD3OD) δ −1.9. HRMS calcd for [C39H61F3N7O21P−H]−1050.3537, found 1050.3517.

[0154] Compound 2520

[0155] A mixture of compound 21 (7.04 g, 11.02 mmol) and 10% Pd(OH)2 / C (7.04 g) in THF (50 mL) was stirred at rt under a hydrogen atmosphere for 16h. The reaction mixture was filtered with celite and the filtrated was concentrated and purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to yield the lactol (4.86 g, 79.2%) as a white solid. 25

[0156] 1H-tetrazole (511 mg, 7.29 mmol) and dibenzyl N,N- diisopropylphosphoramidite (1.83 mL, 5.47 mmol) were added to a solution of the lactol (1.0 g, 1.82 mmol) in dry CH2Cl2(10 mL) at 0 ºC and stirred at rt for 2 h. The reaction was then cooled to -78 ºC and treated with tert-butylhydroperoxide (1.1 mL, 7.95 mmol). The reaction was allowed to warm to rt and was stirred for 2 h. After the 30 reaction was completed, the mixture was diluted with CH2Cl2and washed with H2O43and brine. The organic layer was dried over MgSO4, filtered, evaporated, and purified by column chromatography (CH2Cl2 / MeOH 20:1, silica gel) to obtain a phosphorylated saccharide as a yellowish solid (876 mg, 59.4% over two steps).

[0157] A mixture of phosphorylated saccharide (876.0 mg, 1.04 mmol) and 10% 5 Pd(OH)2 / C (87.6 mg) in THF (20 mL) was stirred at rt under a hydrogen atmosphere for 4 h. The reaction mixture was filtered with celite and the filtrated was concentrated and purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, silica gel) to yield compound 25 (420.4 mg, 64.3%) as a yellowish solid.

[0158] 1H NMR (600 MHz, CD3OD) δ 0.06 (s, 9H), 1.02 (t, J = 8.5 Hz, 2H), 1.31 10 (d, J = 6.7 Hz, 3H), 1.41 (d, J = 7.4 Hz, 3H), 1.94 (s, 3H), 2.05 (s, 3H), 2.12 (s, 3H), 3.85 (t, J = 9.7 Hz, 1H), 4.10 (d, J = 12.2 Hz, 1H), 4.16-4.25 (m, 5H), 4.25-4.34 (m, 2H), 5.08 (t, J = 9.7 Hz, 1H), 5.52 (br, 1H).13C NMR (150 MHz, CD3OD) δ 175.1, 174.0, 173.6, 172.5, 171.5, 95.7, 79.1, 78.4, 70.7, 70.2, 64.7, 63.0, 55.1, 55.1, 22.9, 20.9, 20.7, 19.5, 18.1, 17.3, −1.5 (× 3).31P NMR (200 MHz, CD3OD) δ −1.4. HRMS 15 calcd for [C23H41N2O14PSi−H]−627.1992, found 627.1984.

[0159] Neryl-Lipid I (2)

[0160] 1,1’-Carbonyldiimidazole (464.2 mg, 2.863 mmol) was added to a solution 20 of neryl phosphate (143.9 mg, 0.573 mmol) in THF (4 mL) at rt. After stirring for 2 h, anhydrous MeOH (116 μL, 2.863 mmol) was added to the reaction for quenching the excess 1,1’-carbonyldiimidazole. After another 1 h stirring, the reaction was concentrated and re-dissolved in DMF / THF (15 mL, v / v = 2:1). 1H-Tetrazole (42.1 mg, 0.601 mmol) and compound 21 (301.0 mg, 0.286 mmol) were added to the above 25 solution and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated and purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, silica gel) to yield a pyrophosphate intermediate. 2N LiOH(aq)(1.43 mL, 2.863 mmol) was slowly added to a solution of the above intermediate in MeOH (15 mL) at 0 ºC for 8h, for the global deprotection. The reaction mixture was neutralized by 1N HCl(aq)44(1.72 mL, 1.718 mmol), concentrated, and purified by column chromatography (CHCl3 / MeOH / H2O / NH4OH(aq)88:58:12.5:1, silica gel) and further purified by RP- HPLC on an Eclipse XDB-C18 (5 μm, 9.1 × 250 mm) column using gradient elution with 50 mM NH4HCO3(aq) / MeOH (100:0 to 0:100) at a flow rate of 2.0 mL / min over 5 50 min to yield Neryl-Lipid I (2) (98.4 mg, 32.4% over two steps) as a white solid.

[0161] 1H NMR (600 MHz, D2O) δ 1.33-1.47 (m, 14H), 1.62 (s, 3H), 1.66-1.73 (m, 5H), 1.74-1.81 (m, 5H), 1.84-1.93 (m, 1H), 2.00 (s, 3H), 2.09-2.19 (m, 5H), 2.45- 2.34 (m, 2H), 2.96-3.02 (m, 2H), 3.59-3.66 (m, 1H), 3.76-3.90 (m, 3H), 3.93-3.97 (m, 1H), 4.10-4.34 (m, 7H), 4.41-4.50 (m, 2H), 5.17-5.21 (m, 1H), 5.12-5.46 (m, 2H).13C 10 NMR (150 MHz, D2O) δ 179.0, 177.2, 175.8, 175.7, 175.2, 174.1, 173.8, 173.6, 143.1, 133.9, 123.8, 120.3, 94.6, 79.8, 77.9, 72.9, 68.0, 62.8, 60.3, 54.2, 53.8, 53.3, 50.6, 49.6, 39.1, 31.6, 31.3, 30.4, 27.9, 26.2, 26.0, 24.8, 22.6, 22.1, 22.0, 21.9, 18.5, 17.1, 16.9, 16.8, 16.4.31P NMR (200 MHz, D2O) δ −9.4 (d, JP-P= 21.0 Hz), −11.9 (d, JP-P= 21.0 Hz). HRMS calcd for [C41H71N7O21P2−H]−1058.4105, found 1058.4089. 15

[0162] MPP-P (3)

[0163] 2N LiOH(aq)(495.0 μL, 0.991 mmol) was slowly added to a solution of compound 21 (104.2 mg, 0.099 mmol) in MeOH (10 mL) at 0 ºC for 8h, for the global 20 deprotection. The reaction mixture was neutralized by 1N HCl(aq)(595.0 μL, 0.595 mmol), concentrated, purified by column chromatography (nPrOH / H2O 7:3, silica gel), and further purified by PD MidiTrapTMG-10 to yield MPP-P (3) (52.4 mg, 62.7%) as a white solid.

[0164] 1H NMR (600 MHz, D2O) δ 1.31-1.46 (m, 14H), 1.65-1.73 (m, 2H), 1.74- 25 1.83 (m, 2H), 1.85-1.92 (m, 1H), 1.99 (s, 3H), 2.11-2.19 (m, 1H), 2.22-2.32 (m, 2H), 2.96-3.02 (m, 2H), 3.52-3.58 (m, 1H), 3.76-3.82 (m, 2H), 3.86-3.91 (m, 1H), 3.95-4.00 (m, 1H), 4.02-4.06 (m, 1H), 4.07-4.13 (m, 1H), 4.13-4.18 (m, 1H), 4.19-4.34 (m, 4H), 5.30 (dd, J = 7.4 Hz, 3.2 Hz, 1H).13C NMR (150 MHz, D2O) δ 179.9, 177.7, 175.9, 175.8, 175.3, 174.1, 174.0, 173.7, 92.9, 80.2, 77.9, 72.0, 68.5, 60.7, 54.2, 54.0, 53.9,4553.3, 51.0, 49.8, 19.6, 39.2, 30.6, 26.3, 22.1, 22.0, 21.9, 18.6, 17.4, 16.9, 16.4.31P NMR (200 MHz, D2O) δ 2.0. HRMS calcd for [C31H54N7O18P1−H]−842.3190, found 842.3245. 5

[0165] Truncated Neryl-Lipid I (4)

[0166] 1,1’-Carbonyldiimidazole (297.0 mg, 1.830 mmol) was added to a solution of neryl phosphate (98.2 mg, 0.366 mmol) in THF (2 mL) at rt. After stirring for 2 h, anhydrous MeOH (74.2 μL, 1.830 mmol) was added to the reaction for quenching the 10 excess 1,1’-carbonyldiimidazole. After another 1 h stirring, the reaction was concentrated and re-dissolved in THF (2 mL). 1H-tetrazole (26.9 mg, 0.384 mmol) and compound 24 (115.0 mg, 0.183 mmol) were added to the above solution and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated and purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, silica gel) to yield a 15 pyrophosphate intermediate. 2N LiOH(aq)(523 μL, 1.046 mmol) was slowly added to a solution of the above intermediate in MeOH (20 mL) at 0 ºC for 8h, for the global deprotection. The reaction mixture was neutralized by 1N HCl(aq)(677 μL, 0.677 mmol), concentrated, purified by column chromatography (CHCl3 / MeOH / H2O / NH4OH(aq)88:58:12.5:1, silica gel) and further purified by RP- 20 HPLC on an Eclipse XDB-C18 (5 μm, 9.1 × 250 mm) column using gradient elution with 50 mM NH4HCO3(aq) / MeOH (100:0 to 0:100) at a flow rate of 2.0 mL / min over 50 min to yield Truncated Neryl-Lipid I (4) (54.3 mg, 45.0% over two steps) as a white solid.

[0167] 1H NMR (600 MHz, D2O) δ 1.37-1.40 (m, 6H), 1.62 (s, 3H), 1.69 (s, 3H), 25 1.76 (s, 3H), 1.99 (s, 3H), 2.10-2.18 (m, 4H), 3.63 (t, J = 10.1 Hz, 1H), 3.78 (t, J = 9.2 Hz, 1H), 3.81-3.85 (m, 1H), 3.86-3.90 (m, 1H), 3.93-3.99 (m, 1H), 4.07-4.12 (m, 1H), 4.15-4.24 (m, 2H), 4.41-4.50 (m, 2H), 5.17-5.22 (m, 1H), 5.42-5.47 (m, 2H).13C NMR (150 MHz, D2O) δ 178.6, 175.0, 174.2, 143.0, 133.9, 123.8, 120.3, 84.6, 79.5, 77.8, 72.9, 68.2, 62.8, 60.3, 53.3, 49.9, 31.3, 26.0, 24.8, 22.6, 22.1, 18.3, 17.0, 16.9.31P46NMR (200 MHz, D2O) δ −9.5 (d, JP-P= 21.0 Hz), −11.9 (d, JP-P= 21.0 Hz). HRMS calcd for [C24H42N2O15P2−H]−659.1988, found 659.1969. 5

[0168] Neryl phosphate (C10-P)

[0169] Trichloroacetonitrile (780.0 μL, 7.78 mmol) was added to a solution of nerol (500 mg, 3.24 mmole), n-Bu4NH2PO4(2.2 g, 6.48 mmol) in CH2Cl2(65 mL). The reaction mixture was stirred at rt for 1 h. The reaction mixture was concentrated 10 and re-dissolved in THF (5 mL). NH4OH(aq)(525.6 μL, 7.78 mmol) was added to the solution and stirred for 30 min. The reaction mixture was filtered and the filtrate was concentrated. Anion exchange chromatography was conducted using a DE52 column. Compound elution was achieved using NH4OAc (0 mM, 30 mM, 150 mM) in CHCl3 / MeOH (2:1). Lipid-linked products were extracted away from salt with hexane. 15 The lipid extract was concentrated and further purified by column chromatography (CHCl3 / MeOH / H2O 60:25:4, silica gel) to yield undecaprenyl phosphate (423.7 mg, 55.8%) as a white solid.

[0170] 1H NMR (600 MHz, CD3OD 1.61 (s, 3H), 1.67 (s, 3H), 1.73 (s, 3H), 2.05- 2.13 (m, 4H), 4.39 (t, J = 6.5 Hz, 2H), 5.09-5.13 (m, 1H), 5.40 (t, J = 6.5 Hz, 1H).13C 20 NMR (150 MHz, CD3OD) 140.2, 132.7, 125.0, 123.7 (d, JC-P= 8.4 Hz), 62.8 (d, JC-P= 4.8 Hz), 33.1, 27.7, 25.9, 23.7, 17.8.31P NMR (200 MHz, CD3OD) δ 2.6. HRMS calcd for [C10H19O4P−H]−233.0948, found 233.0952. Example 3: Lipid II-based label-free binding affinity assay 25

[0171] The analysis was performed on a hybrid linear trap / Fourier transform ion cyclotron resonance (ICR) mass spectrometer (LTQFT Ultra, Thermo Electron, San Jose, CA). High-resolution accurate mass LC-MS equipped with a heat electrospray ionization (HESI-II) probe, a high-performance liquid chromatograph (HPLC) system with binary pump (Agilent 1100, Agilent Technologies, Palo Alto, CA), and a Famos 30 autosampler (LC Packings, San Francisco, CA). For flow injection analysis, the sample was directly injected (5μl) into continuous flowing solvent with a flow rate of 50 μL / min in the mobile phase consisted of 50% IPA / H2O / 0.01% NH4OH. The FTICR was operated in negative mode within a normal mass range from m / z 500-3000.47Showing resolution power was 100,000 at m / z 400. Electrospray voltage was applied to 4.0 kV and the capillary temperature was set at 275 ºC.

[0172] Several antibiotics were used to interact with Neryl-Lipid II through PLAS platform of the present disclosure. As shown in Fig.3(a), vancomycin and teicoplanin, 5 which target D-Ala-D-Ala, as well as ramoplanin, which targets pyrophosphate, formed higher complexes with Neryl-Lipid II. Conversely, MoeA and ampicillin, which are inhibitors of TGase and TPase, respectively, generated negligible complexes. Moreover, the assay platform of the present disclosure allows for not only qualitative analysis but also quantitative analysis. By titrating Neryl-Lipid II, the complex ratios 10 were fitted to the below equation to determine the dissociation constant (KD).wherein P is initial concentration of antibiotic candidate, x is initial concentration of the peptidoglycan precursor, and [Complex] / [Noncomplex] is the ratio of concentration of complex and noncomplex. The dissociation constant (KD) of each of 15 vancomycin, teicoplanin, and ramoplanin is shown in Table 1.

[0173] Table 1 Dissociation constant (KD) Vancomycin 1.54±0.06 μM Teicoplanin 1.20±0.03 μM Ramoplanin 2.91±0.12 μM

[0174] After that, vancomycin was used to confirm the effectiveness of the Lipid II analogues. The results indicated that compounds 4-7, which lack the D-Ala-D-Ala 20 moiety, could not form the complex as effectively as the other analogues (Fig.3(b)). Example 4: Drug test screening

[0175] A pilot library of in-house molecules was collected as a pilot library to screen for Lipid II binders using a developing assay platform. The results indicated 25 that compound 29 (peptide LL-37, belonging to cathelicidin family) demonstrated a binding affinity towards Lipid II, with a complex percentage of 67.6% in PLAS (Fig. 4(a)). Additionally, compound 29 was profiled using an advanced assay with a probe48pool. As shown in Fig. 4(b), compounds 5, 6, and 7 exhibited a low complex percentage, suggesting that pyrophosphate may play a potential role. Furthermore, it was determined that GlcNAc and the peptide stem did not contribute to the interaction with compound 29. 5

[0176] In conclusion, in the proof-of-concept screening test disclosed herein, compound 29 was identified as a new Lipid II binder. Subsequent advanced evaluations in PLAS revealed that compound 29 predominantly binds to the pyrophosphate moiety of Lipid II. Although the binding affinity of compound 29 is not as potent as that of vancomycin, it represents the first example of how to 10 efficiently screen a diverse library to identify new Lipid II binders and rapidly identify the possible binding moieties of Lipid II using our PGN-related chemical probes. This peptidoglycan-based label-free affinity screening (PLAS) platform allows for not only qualitative analysis but also quantitative analysis for the binding of peptidoglycan precursors to the analyzed antibiotics. 15

[0177] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the 20 invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0178] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., 25 publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims. ENUMERATED EXAMPLES: 30

[0179] The Enumerated Examples set forth below provide additional aspects of the present disclosure: 1. A method for evaluation of the binding potency between a target biomolecule from bacteria and a compound through a label-free approach, wherein the method comprising:49contacting the compound with the target biomolecule to form a complex; subjecting the complex to a target engagement mass spectrometry; and determining the binding affinity between the compound and the target biomolecule; wherein the target biomolecule is not a protein, a transporter, or an enzyme; and 5 the compound is selected from peptides, natural product, and the derivative thereof; and wherein the bacteria is selected from Gram-positive bacteria or Gram-negative bacteria, for example, mycobacteria. 2. A method for screening a peptidoglycan-targeting antibiotic, comprising: contacting an antibiotic candidate with a peptidoglycan precursor to form a 10 complex; subjecting the complex to target engagement mass spectrometry; and determining the binding affinity of the antibiotic candidate; wherein the peptidoglycan precursor is selected from at least one of the compounds of the group consisting of 15e y - p ,505esstant p (),51es stant pentapept e (13), 5Neryl pyrophosphate (15). 3. A method for identifying a binding moiety on a peptidoglycan, comprising: contacting an antibiotic candidate with a first peptidoglycan precursor to form a 10 first complex; subjecting the first complex to target engagement mass spectrometry to determine a first binding affinity; contacting the antibiotic candidate with a second peptidoglycan precursor to form a second complex;52subjecting the second complex to target engagement mass spectrometry to determine a second binding affinity; and comparing the first binding affinity and the second binding affinity to determine the binding moiety of the peptidoglycan to the antibiotic candidate; 5 wherein the peptidoglycan precursor is selected from the peptidoglycan precursors as set forth above. 4. The method according to any one of Enumerated Examples 2 to 3, wherein the binding moiety of the peptidoglycan is selected from at least one of a disaccharide, a pyrophosphate, a lipid tail, and an oligopeptide moiety. 10 5. The method according to any one of Enumerated Examples 2 to 4, wherein the binding moiety of the peptidoglycan is a disaccharide. 6. The method according to any one of Enumerated Examples 2 to 5, wherein the binding moiety of the peptidoglycan is a pyrophosphate. 7. The method according to any one of Enumerated Examples 2 to 6, wherein the 15 binding moiety of the peptidoglycan is a lipid tail. 8. The method according to any one of Enumerated Example 7, wherein the lipid tail is an undecaprenol. 9. The method according to any one of Enumerated Examples 2 to 8, wherein the binding moiety of the peptidoglycan is an oligopeptide moiety. 20 10. The method according to any one of Enumerated Examples 2 to 9, wherein the antibiotic candidate binds to compound (1). 11. The method according to any one of Enumerated Examples 2 to 10, wherein the antibiotic candidate binds to compound (4). 12. The method according to any one of Enumerated Examples 2 to 11, wherein the 25 antibiotic candidate binds to at least one of compound (5), compound (6), and compound (7). 13. The method according to any one of Enumerated Examples 2 to 4, wherein the antibiotic candidate binds to compound (1) but does not binds to compound (4), compound (5), compound (6), and compound (7). 30 14. The method according to any one of Enumerated Examples 2 to 4, wherein the antibiotic candidate binds to compound (1) but does not binds to compound (5), compound (6), and compound (7). 15. The method according to any one of Enumerated Examples 3 to 10 and 14, wherein when the first peptidoglycan precursor is compound (1), and the second53peptidoglycan precursor is selected from at least one of compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is pyrophosphate. 16. The method according to any one of Enumerated Examples 3 to 10 and 14, 5 wherein the binding affinity to compound (1) being higher than the binding affinity to compound (5), compound (6), and compound (7) indicates that the binding moiety is pyrophosphate. 17. The method according to any one of Enumerated Examples 3 to 10 and 13, wherein when the first peptidoglycan precursor is compound (1) and the second 10 peptidoglycan precursor is selected from at least one of compound (4), compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is an oligopeptide moiety. 18. The method according to any one of Enumerated Examples 3 to 10 and 13, 15 wherein the binding affinity to compound (1) being higher than the binding affinity to compound (4), compound (5), compound (6), and compound (7) indicates that the binding moiety is an oligopeptide moiety. 19. The method according to any one of Enumerated Examples 3 to 18, wherein the binding affinity is determined according to dissociation constant (KD) calculated 20 by the equation:wherein P is the initial concentration of the antibiotic candidate, x is the initial concentration of the peptidoglycan precursor, and [Complex] / [Noncomplex] is the 25 ratio of concentration of complex and noncomplex, wherein the complex is the complex formed by binding of the antibiotic candidate to the peptidoglycan precursor, and the noncomplex is the antibiotic candidate that does not bind to the peptidoglycan precursor. 20. The method according to any one of Enumerated Examples 2-19, wherein the 30 binding affinity of the antibiotic candidate is compared to a standard antibiotic.5421. The method according to any one of Enumerated Examples 2-20, wherein the standard antibiotic is an antibiotic targeting an oligopeptide moiety or pyrophosphate of the peptidoglycan. 22. The method according to any one of Enumerated Examples 2-21, wherein the 5 standard antibiotic is selected from the group consisting of vancomycin, teicoplanin, and ramoplanin. 23. The method according to any one of Enumerated Examples 2-22, wherein the binding affinity of the antibiotic candidate is compared to a negative control. 24. The method according to any one of Enumerated Examples 2-23, wherein the 10 negative control is an inhibitor of TGase or an inhibitor of TPase. 25. The method according to any one of Enumerated Examples 2-24, wherein the negative control is selected from MoeA or ampicillin. 26. The method according to any one of Enumerated Examples 2-25, wherein the dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM. 15 27. The method according to any one of Enumerated Examples 2-26, wherein the dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM, about 1 to about 4.5 μM, about 1.5 to about 4 μM, about 2 to about 3.5 μM, or about 2.5 to about 3 μM. 28. The method according to any one of Enumerated Examples 2-27, wherein the 20 dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM, about 0.5 to about 4.5 μM, about 0.5 to about 4 μM, about 0.5 to about 3.5 μM, about 0.5 to about 3 μM, about 0.5 to about 2.5 μM, about 0.5 to about 2 μM, about 0.5 to about 1.5 μM, about 0.5 to about 1 μM, about 1 to about 5 μM, about 1 to about 4.5 μM, about 1 to about 4 μM, about 1 to about 3.5 μM, about 1 to 25 about 3 μM, about 1 to about 2.5 μM, about 1 to about 2 μM, about 1 to about 1.5 μM, about 1.5 to about 5 μM, about 1.5 to about 4.5 μM, about 1.5 to about 4 μM, about 1.5 to about 3.5 μM, about 1.5 to about 3 μM, about 1.5 to about 2.5 μM, about 1.5 to about 2 μM, about 2 to about 5 μM, about 2 to about 4.5 μM, about 2 to about 4 μM, about 2 to about 3.5 μM, about 2 to about 3 μM, about 2 to about 30 2.5 μM, about 2.5 to about 5 μM, about 2.5 to about 4.5 μM, about 2.5 to about 4 μM, about 2.5 to about 3.5 μM, about 2.5 to about 3 μM, about 3 to about 5 μM, about 3 to about 4.5 μM, about 3 to about 4 μM, about 3 to about 3.5 μM, about 3.5 to about 5 μM, about 3.5 to about 4.5 μM, about 3.5 to about 4 μM, about 4 to about 5 μM, about 4 to about 4.5 μM, or about 4.5 to about 5 μM.5529. The method according to any one of Enumerate Examples 2-28, the dissociation constant (KD) of the antibiotic candidate is about 0.5 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM. 5 30. The method according to any one of Enumerated Examples 2-29, wherein the target engagement mass spectrometry is electrospray ionization mass spectrometer (ESI-MS), nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer. 31. The method according to any one of Enumerated Example 30, wherein said mass 10 spectrometer is coupled to a liquid chromatography system. 32. The method according to any one of Enumerated Example 31, wherein the liquid chromatography system comprises reversed-phase liquid chromatography, ion exchange chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction 15 chromatography, mixed-mode chromatography, or a combination thereof. 33. The method according to any one of Enumerated Examples 2-32, wherein the method does not require the immobilization of the peptidoglycan precursor on the matrix. 34. The method according to any one of Enumerated Examples 2-33, wherein the 20 method does not require labeling of the peptidoglycan precursor with a fluorophore. 35. The method according to Enumerated Examples 1 to 34, wherein the mass spectrometer is a hybrid linear trap / Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, a high-resolution accurate mass LC-MS equipped with a heat electrospray ionization probe, a high-performance liquid 25 chromatography (HPLC) system with binary pump, and a Famos autosampler. 36. The method according to Enumerated Example 35, wherein a sample is loaded on the target engagement mass spectrometer, wherein the sample was directly injected into a continuous flowing solvent with a flow rate of 40-60 μL / min in the mobile phase consisting of IPA / H2O / NH4OH at pH 6-9, wherein the FTICR is operated in 30 negative mode within a normal mass range from m / z 500-3000, showing resolution power is 100,000 at m / z 400, electrospray voltage is applied to 4.0 kV, and the capillary temperature is set at 275 ºC. 37. A peptidoglycan precursor having a formula of A-Sn-Po-Lq, wherein A is an oligopeptide moiety,56S is a saccharide moiety, P is a phosphate group, L is a lipid tail comprising up to 55 carbon atoms, n is 1 or 2, 5 o is 1 or 2, and q is 0 or 1. 38. The peptidoglycan precursor according to Enumerated Example 37, wherein A consists of 1 to 5 amino acids. 39. The peptidoglycan precursor according to Enumerated Example 38, wherein the 10 amino acid is a natural and / or a non-natural or a synthetic amino acid. 40. The peptidoglycan precursor according to any one of Enumerated Examples 38 to 39, wherein the amino acid is an L- or D-amino acid. 41. The peptidoglycan precursor according to any one of Enumerated Examples 38 to 40, wherein the amino acid is modified or unmodified. 15 42. The peptidoglycan precursor according to any one of Enumerated Examples 38 to 41, wherein the amino acid is selected from the group consisting of Ala, Glu, m- DAP, and Lys. 43. The peptidoglycan precursor according to Enumerated Examples 37 to 42, wherein A comprises a D-Ala-D-Ala moiety. 20 44. The peptidoglycan precursor according to any one of Enumerated Examples 37 to 43, wherein S is a monosaccharide. 45. The peptidoglycan precursor according to any one of Enumerated Examples 37 to 43, wherein S is a disaccharide. 46. The peptidoglycan precursor according to any one of Enumerated Examples 37 to25 43, wherein S is selected from N-acetylmuramic acid (MurNAc) or N- acetylglucosamine (GlcNAc). 47. The peptidoglycan precursor according to Enumerated Example 37, wherein the peptidoglycan precursor is selected from the group consisting of:57y -Lipid I (4). 5 48. The peptidoglycan precursor according to Enumerated Example 37, wherein the peptidoglycan precursor is Neryl-Lipid II:5849. A peptidoglycan for use in a method for screening a peptidoglycan-targeting antibiotic, comprising contacting an antibiotic candidate with a peptidoglycan precursor to form a 5 complex; subjecting the complex to target engagement mass spectrometry; and determining the binding affinity of the antibiotic candidate; wherein the peptidoglycan precursor is selected from the peptidoglycan precursors as set forth above. 10 50. A peptidoglycan for use in a method for identifying a binding moiety on a peptidoglycan, comprising: contacting an antibiotic candidate with a first peptidoglycan precursor to form a first complex; subjecting the first complex to target engagement mass spectrometry to determine 15 a first binding affinity; contacting the antibiotic candidate with a second peptidoglycan precursor to form a second complex; subjecting the second complex to target engagement mass spectrometry to determine a second binding affinity; and 20 comparing the first binding affinity and the second binding affinity to determine the binding moiety of the peptidoglycan to the antibiotic candidate; wherein the peptidoglycan precursor is selected from the peptidoglycan precursors as set forth above. 51. The peptidoglycan according to any one of Enumerated Examples 49 to 50, 25 wherein the binding moiety of the peptidoglycan is selected from at least one of a disaccharide, a pyrophosphate, a lipid tail, and an oligopeptide moiety. 52. The peptidoglycan according to any one of Enumerated Examples 49 to 51, wherein the binding moiety of the peptidoglycan is a disaccharide.5953. The peptidoglycan according to any one of Enumerated Examples 49 to 52, wherein the binding moiety of the peptidoglycan is a pyrophosphate. 54. The peptidoglycan according to any one of Enumerated Examples 49 to 53, wherein the binding moiety of the peptidoglycan is a lipid tail. 5 55. The method according to any one of Enumerated Example 54, wherein the lipid tail is an undecaprenol. 56. The peptidoglycan according to any one of Enumerated Examples 49 to 55, wherein the binding moiety of the peptidoglycan is an oligopeptide moiety. 57. The peptidoglycan according to any one of Enumerated Examples 49 to 56, 10 wherein the antibiotic candidate binds to compound (1). 58. The peptidoglycan according to any one of Enumerated Examples 49 to 57, wherein the antibiotic candidate binds to compound (4). 59. The peptidoglycan according to any one of Enumerated Examples 49 to 58, wherein the antibiotic candidate binds to at least one of compound (5), compound 15 (6), and compound (7). 60. The peptidoglycan according to any one of Enumerated Examples 49 to 58, wherein the antibiotic candidate binds to compound (1) but does not binds to compound (4), compound (5), compound (6), and compound (7). 61. The peptidoglycan according to any one of Enumerated Examples 49 to 51, 20 wherein the antibiotic candidate binds to compound (1) but does not binds to compound (5), compound (6), and compound (7). 62. The peptidoglycan according to any one of Enumerated Examples 49 to 57 and 61, wherein when the first peptidoglycan precursor is compound (1), and the second peptidoglycan precursor is selected from at least one of compound (5), compound 25 (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is pyrophosphate. 63. The peptidoglycan according to any one of Enumerated Examples 49 to 57 and 61, wherein the binding affinity to compound (1) being higher than the binding affinity to compound (5), compound (6), and compound (7) indicates that the binding 30 moiety is pyrophosphate. 64. The peptidoglycan according to any one of Enumerated Examples 49 to 57 and 60, wherein when the first peptidoglycan precursor is compound (1) and the second peptidoglycan precursor is selected from at least one of compound (4), compound (5), compound (6), and compound (7), the first binding affinity being higher than60the second binding affinity indicates that the binding moiety is an oligopeptide moiety. 65. The peptidoglycan according to any one of Enumerated Examples 49 to 57 and 60, wherein the binding affinity to compound (1) being higher than the binding affinity 5 to compound (4), compound (5), compound (6), and compound (7) indicates that the binding moiety is an oligopeptide moiety. 66. The peptidoglycan according to any one of Enumerated Examples 49 to 65, wherein the binding affinity is determined according to dissociation constant (KD) calculated by the equation: 10wherein P is the initial concentration of the antibiotic candidate, and x is the initial concentration of the peptidoglycan precursor, and [Complex] / [Noncomplex] is the ratio of the concentration of complex and noncomplex, wherein the complex is the 15 complex formed by binding of the antibiotic candidate to the peptidoglycan precursor, and the noncomplex is the antibiotic candidate that does not bind to the peptidoglycan precursor. 67. The peptidoglycan according to any one of Enumerated Examples 49 to 66, wherein the binding affinity of the antibiotic candidate is compared to a standard 20 antibiotic. 68. The peptidoglycan according to any one of Enumerated Examples 49 to 67, wherein the standard antibiotic is an antibiotic targeting an oligopeptide moiety or pyrophosphate of the peptidoglycan. 69. The peptidoglycan according to any one of Enumeratedd Examples 49 to 68, 25 wherein the standard antibiotic is selected from the group consisting of vancomycin, teicoplanin, and ramoplanin. 70. The peptidoglycan according to any one of Enumerated Examples 49 to 69, wherein the binding affinity of the antibiotic candidate is compared to a negative control. 30 71. The peptidoglycan according to any one of Enumerated Examples 49 to 70, wherein the negative control is an inhibitor of TGase or an inhibitor of TPase.6172. The peptidoglycan according to any one of Enumerated Examples 49 to 71, wherein the negative control is selected from MoeA or ampicillin. 73. The peptidoglycan according to any one of Enumerated Examples 49 to 72, wherein the dissociation constant (KD) of the antibiotic candidate is about 0.5 to 5 about 5 μM. 74. The peptidoglycan according to any one of Enumerated Examples 49 to 73, wherein the dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM, about 1 to about 4.5 μM, about 1.5 to about 4 μM, about 2 to about 3.5 μM, or about 2.5 to about 3 μM. 10 75. The peptidoglycan according to any one of Enumerated Examples 49 to 74, wherein the dissociation constant (KD) of the antibiotic candidate is about 0.5 to about 5 μM, about 0.5 to about 4.5 μM, about 0.5 to about 4 μM, about 0.5 to about 3.5 μM, about 0.5 to about 3 μM, about 0.5 to about 2.5 μM, about 0.5 to about 2 μM, about 0.5 to about 1.5 μM, about 0.5 to about 1 μM, about 1 to about 15 5 μM, about 1 to about 4.5 μM, about 1 to about 4 μM, about 1 to about 3.5 μM, about 1 to about 3 μM, about 1 to about 2.5 μM, about 1 to about 2 μM, about 1 to about 1.5 μM, about 1.5 to about 5 μM, about 1.5 to about 4.5 μM, about 1.5 to about 4 μM, about 1.5 to about 3.5 μM, about 1.5 to about 3 μM, about 1.5 to about 2.5 μM, about 1.5 to about 2 μM, about 2 to about 5 μM, about 2 to about 20 4.5 μM, about 2 to about 4 μM, about 2 to about 3.5 μM, about 2 to about 3 μM, about 2 to about 2.5 μM, about 2.5 to about 5 μM, about 2.5 to about 4.5 μM, about 2.5 to about 4 μM, about 2.5 to about 3.5 μM, about 2.5 to about 3 μM, about 3 to about 5 μM, about 3 to about 4.5 μM, about 3 to about 4 μM, about 3 to about 3.5 μM, about 3.5 to about 5 μM, about 3.5 to about 4.5 μM, about 3.5 to 25 about 4 μM, about 4 to about 5 μM, about 4 to about 4.5 μM, or about 4.5 to about 5 μM. 76. The peptidoglycan according to any one of Enumerated Examples 49 to 75, wherein the dissociation constant (KD) of the antibiotic candidate is about 0.5 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, 30 about 4 μM, about 4.5 μM, or about 5 μM. 77. The peptidoglycan according to any one of Enumerated Examples 49 to 76, wherein the target engagement mass spectrometry is electrospray ionization mass spectrometer (ESI-MS), nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer.6278. The peptidoglycan according to any one of Enumerated Example 77, wherein said mass spectrometer is coupled to a liquid chromatography system. 79. The peptidoglycan according to any one of Enumerated Example 78, wherein the liquid chromatography system comprises reversed-phase liquid chromatography, 5 ion exchange chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed-mode chromatography, or a combination thereof. 80. The peptidoglycan according to any one of Enumerated Examples 49 to 79, wherein the method does not require immobilization of the peptidoglycan 10 precursor on the matrix. 81. The peptidoglycan according to any one of Enumerated Examples 49 to 80, wherein the method does not require labeling of the peptidoglycan precursor with a fluorophore. 82. A method of preparing a peptidoglycan precursor as disclosed herein, comprising 15 providing N-acetylglucosamine, obtaining compound (16) from N-acetylglucosamine through amide bond formation,conjugating compound (16) with phosphate, oligopeptide, and optionally 20 compound (15) to obtain a peptidoglycan precursor. 83. The method according to Enumerated Example 82, wherein the method of preparing a peptidoglycan precursor further comprises: conjugating an additional saccharide to compound (16) to form a disaccharide moiety. 84. The method according to any one of Enumerated Examples 82 to 83, wherein the 25 oligopeptide is a dipeptide, tripeptide, or tetrapeptide. 85. An antibiotic obtained from the method according to any one of Enumerate Examples 1 to 36. 86. An antibiotic obtained from the method of screening a peptidoglycan-targeting antibiotic, or the method of identifying a binding moiety on a peptidoglycan 30 according to any one of Enumerated Examples 2 to 36.6387. A antibiotic obtained from the method according to any one of Enumerated Examples 2 to 36 for use in the treatment of bacterial diseases. 88. A method for the treatment of bacterial diseases comprising administering to a subject in need thereof an effective amount of the antibiotic obtained from the 5 method according to any one of Enumerated Examples 2 to 36. 89. Use of the antibiotic obtained from the method according to any one of Enumerated Examples 2 to 36 for the manufacture of a medicament for treating bacterial diseases. 90. The antibiotic according to any one of Enumerated Example 87, the method 10 according to Enumerated Example 88, or the use according to Enumerated Example 89, wherein the bacterial disease is the bacterial infection caused by Gram-negative bacteria. 91. The antibiotic according to any one of Enumerated Example 87, the method according to Enumerated Example 88, or the use according to Enumerated15 Example 89, wherein the bacterial disease is a bacterial infection caused by Gram- positive bacteria. 92. The antibiotic according to any one of Enumerated Example 87, the method according to Enumerated Example 88, or the use according to Enumerated Example 89, wherein the bacterial disease is a bacterial infection caused by 20 antibiotic-resistant bacteria. 93. The antibiotic according to any one of Enumerated Example 87, the method according to Enumerated Example 88, or the use according to Enumerated Example 89, wherein the bacterial disease is caused by a microorganism which is vancomycin-resistant, methicillin-resistant, multidrug-resistant, or extensively 25 drug-resistant. 94. The antibiotic according to any one of Enumerated Example 87, the method according to Enumerate Example 88, and the use according to Enumerate Example 89, the bacteria are resistant to antibiotic glycopeptides. 95. The antibiotic according to any one of Enumerated Example 87, the method 30 according to Enumerate Example 88, and the use according to Enumerate Example 89, the bacteria are resistant to at least one antibiotic selected from vancomycin, teicoplanin, ramoplanin, decaplanin, oritavancin, telavancin, daptomycin, dalbavancin, ristocetin, and avoparcin.6496. The antibiotic according to Enumerated Example 87, the method according to Enumerated Example 88, or the use according to Enumerated Example 89, wherein the bacterial disease is the infection caused by bacteria on or in the tissue or organ, including but not limited to: skin and soft tissue infection (including acne), 5 connective tissue infection, bone infection, bacteremia, abscess, joint or muscle infection, wound infection, endovascular infection, CNS infection, abdominal infection, blood stream infection, urinary tract infection, pelvic infection, invasive systemic infection, gastrointestinal infection, dental infection, food poisoning, , pneumonia, meningitis, osteomyelitis, endocarditis, bacteremia, and sepsis. 10 97. The antibiotic according to Enumerated Example 87, the method according to Enumerate Example 88, or the use according to Enumerate Example 89, wherein the bacteria to be treated with the antibiotic obtained from the method as disclosed herein include those selected from the group consisting of the genus of Salmonella, Shigella, Campylobacter, Vibrio, Escherichia, Streptococcus, Staphylococcus, 15 Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomyces, Nocardia, Enterobacter, Yersinia, Francisella, Pasteurella, Moraxella, Acinetobacter, Erysipelothrix, Moraxella, Actinobacillus, Streptobacillus, Listeria, Brucella, Bacillus, Clostridium, Treponema, Klebsiella, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Legionella, Pseudomonas, 20 Aeromonas, Rickettsia, Chlamydia, Borrelia, and Mycoplasma. 98. The antibiotic according to Enumerated Example 87, the method according to Enumerated Example 88, or the use according to Enumerated Example 89, wherein the Gram-positive bacteria is selected from the group consisting of: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium 25 perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium 30 haemophium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis,65Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, 5 Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, and Streptococcus sanguis. 10 99. The antibiotic according to Enumerated Example 87, the method according to Enumerated Example 88, or the use according to Enumerated Example 89, wherein the Gram-negative bacteria is selected from the group consisting of: Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella 15 bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., 20 Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, 25 Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pestis. 30

[0180] References 1. Shallcross, L. J.; Howard, S. J.; Fowler, T.; Davies, S. C. Tackling the threat of antimicrobial resistance: from policy to sustainable action. Philosophical Transactions of the Royal Society B: Biological Sciences 2015, 370 (1670), 20140082.662. Schleifer, K. H.; Kandler, O. Peptidoglycan Types of Bacterial Cell-walls and Their Taxonomic Implications. Bacteriological Reviews 1972, 36 (4), 407-477. 3. Santajit, S.; Indrawattana, N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. BioMed Research International 2016, 2016, 1-8. DOI: 5 10.1155 / 2016 / 2475067. 4. Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L. A Redesigned Vancomycin Engineered for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria. Journal of the American Chemical Society 2011, 133 (35), 13946-13949. 10 5. Chen, K.-T.; Kuan, Y.-C.; Fu, W.-C.; Liang, P.-H.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. Rapid Preparation of Mycobacterium N-Glycolyl Lipid I and Lipid II Derivatives: A Biocatalytic Approach. Chemistry - A European Journal 2013, 19 (3), 834-838. 6. Meng, F.-C.; Chen, K.-T.; Huang, L.-Y.; Shih, H.-W.; Chang, H.-H.; Nien, F.-Y.; 15 Liang, P.-H.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. Total Synthesis of Polyprenyl N-Glycolyl Lipid II as a Mycobacterial Transglycosylase Substrate.-5309. 7. Shih, H.-W.; Chen, K.-T.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. A New Synthetic Approach toward Bacterial Transglycosylase Substrates, Lipid II and 20 Lipid IV. Organic Letters 2011, 13 (17), 4600-4603. 8. Chen, K.-T.; Lin, C.-K.; Guo, C.-W.; Chang, Y.-F.; Hu, C.-M.; Lin, H.-H.; Lai, Y.; Cheng, T.-J. R.; Cheng, W.-C. Effect of the lipid II sugar moiety on bacterial transglycosylase: the 4-hydroxy epimer of lipid II is a TGase inhibitor. Chemical Communications 2017, 53 (4), 771-774. 25 9. Lin, C.-K.; Chen, K.-T.; Hu, C.-M.; Yun, W.-Y.; Cheng, W.-C. Synthesis of 1-C- Glycoside-Linked Lipid II Analogues Toward Bacterial Transglycosylase Inhibition. Chemistry - A European Journal 2015, 21 (20), 7511-7519. 10. Hsu, C.-H.; Schelwies, M.; Enck, S.; Huang, L.-Y.; Huang, S.-H.; Chang, Y.-F.; Cheng, T.-J. R.; Cheng, W.-C.; Wong, C.-H. Iminosugar C-Glycoside Analogues of 30 α-D-GlcNAc-1-Phosphate: Synthesis and Bacterial Transglycosylase Inhibition. The Journal of Organic Chemistry 2014, 79 (18), 8629-8637. 11. Shih, H.-W.; Chang, Y.-F.; Li, W.-J.; Meng, F.-C.; Huang, C.-Y.; Ma, C.; Cheng, T.- J. R.; Wong, C.-H.; Cheng, W.-C. Effect of the Peptide Moiety of Lipid II on Bacterial Transglycosylase. Angewandte Chemie International Edition 2012, 5167(40), 10123-10126. 12. Cheng, W.-C.; Liu, W.-J.; Hu, K.-H.; Tan, Y.-L.; Lin, Y.-T.; Chen, W.-A.; Lo, L.-C. Rapid Synthesis of a Natural Product-Inspired Uridine Containing Library. ACS Combinatorial Science 2020, 22 (11), 600-607. 5 13. Chen, K.-T.; Chen, P.-T.; Lin, C.-K.; Huang, L.-Y.; Hu, C.-M.; Chang, Y.-F.; Hsu, H.-T.; Cheng, T.-J. R.; Wu, Y.-T.; Cheng, W.-C. Structural Investigation of Park’s Nucleotide on Bacterial Translocase MraY: Discovery of Unexpected MraY Inhibitors. Scientific Reports 2016, 6 (1), 31579. 14. Hsieh, P. Y.; Meng, F. C.; Guo, C. W.; Hu, K. H.; Shih, Y. L.; Cheng, W. C. 10 Harnessing Fluorescent Moenomycin a Antibiotics for Bacterial Cell Wall Imaging Studies. ChemBioChem 2021, 22 (24), 3462-3468. 15. Heck, A. J. R. Native Mass Spectrometry: A Bridge Between Interactomics and Structural Biology. Nature Methods 2008, 5 (11), 927-933. 16. Liu, C.-Y.; Guo, C.-W.; Chang, Y.-F.; Wang, J.-T.; Shih, H.-W.; Hsu, Y.-F.; Chen, 15 C.-W.; Chen, S.-K.; Wang, Y.-C.; Cheng, T.-J. R.; et al. Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl Moiety. Organic Letters 2010, 12 (7), 1608- 1611. 17. Huang, S.-H.; Wu, W.-S.; Huang, L.-Y.; Huang, W.-F.; Fu, W.-C.; Chen, P.-T.; 20 Fang, J.-M.; Cheng, W.-C.; Cheng, T.-J. R.; Wong, C.-H. New Continuous Fluorometric Assay for Bacterial Transglycosylase Using Förster Resonance Energy Transfer. Journal of the American Chemical Society 2013, 135 (45), 17078- 17089. 18. Park, J. T. Uridine-5'-Pyrophosphate Derivatives. III. Amino Acid-Containing 25 Derivatives. Journal of Biological Chemistry 1952, 194 (2), 897-904. DOI: https: / / doi.org / 10.1016 / S0021-9258(18)55845-2.68

Claims

We claim:

1. A method for screening a peptidoglycan-targeting antibiotic, comprising: contacting an antibiotic candidate with a peptidoglycan precursor to form a complex; subjecting the complex to a target engagement mass spectrometry; and determining the binding affinity of the antibiotic candidate; wherein the peptidoglycan precursor is selected from at least one of the compounds of the group consisting of692. The method according to claim 1, wherein the binding affinity of the antibiotic candidate is compared to a standard antibiotic.

3. The method according to claim 1, wherein the standard antibiotic is an antibiotic targeting oligopeptide moiety or pyrophosphate of the peptidoglycan.

4. The method according to claim 1, wherein the standard antibiotic is selected from the group consisting of vancomycin, teicoplanin, and ramoplanin.

5. The method according to claim 1, wherein the binding affinity of the antibiotic candidate is compared to a negative control.

6. The method according to claim 1, wherein the negative control is an inhibitor of TGase or an inhibitor of TPase.

7. The method according to claim 1, wherein the negative control is selected from MoeA or ampicillin.

8. The method according to claim 1, wherein the target engagement mass spectrometry is electrospray ionization mass spectrometry (ESI-MS).

709. The method according to claim 1, wherein immobilization of the peptidoglycan precursor on the matrix or labeling of the peptidoglycan precursor with a fluorophore is not required.

10. The method according to claim 1, wherein the mass spectrometer is a hybrid linear trap / Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, a high- resolution accurate mass LC-MS equipped with a heat electrospray ionization probe, a high-performance liquid chromatography (HPLC) system with binary pump, and a Famos autosampler.

11. The method according to claim 10, wherein a sample is loaded on the target engagement mass spectrometer, wherein the sample was directly injected into a continuous flowing solvent with a flow rate of 40-60 μL / min in the mobile phase consisting of IPA / H2O / NH4OH at pH 6-9, wherein the FTICR is operated in negative mode within a normal mass range from m / z 500-3000, showing resolution power is 100,000 at m / z 400, electrospray voltage is applied to 4.0 kV, and the capillary temperature is set at 275 ºC.

12. A method for identifying the binding moiety on a peptidoglycan, comprising contacting an antibiotic candidate with a first peptidoglycan precursor to form a first complex; subjecting the first complex to a target engagement mass spectrometry to determine a first binding affinity; contacting the antibiotic candidate with a second peptidoglycan precursor to form a second complex; subjecting the second complex to a target engagement mass spectrometry to determine a second binding affinity; and comparing the first binding affinity and the second binding affinity to determine the binding moiety of the peptidoglycan to the antibiotic candidate; wherein the peptidoglycan precursor is selected from at least one of the compounds of the group consisting of717213. The method according to claim 12, wherein the binding moiety of the peptidoglycan is selected from at least one of a disaccharide, a pyrophosphate, a lipid tail, and an oligopeptide moiety.

14. The method according to claim 12, wherein when the first peptidoglycan precursor is compound (1), and the second peptidoglycan precursor is selected from at least one of compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is pyrophosphate.

15. The method according to claim 12, wherein when the first peptidoglycan precursor is compound (1) and the second peptidoglycan precursor is selected from at least one of compound (4), compound (5), compound (6), and compound (7), the first binding affinity being higher than the second binding affinity indicates that the binding moiety is a oligopeptide moiety.

16. The method according to claim 12, the target engagement spectrometry is electrospray ionization mass spectrometry (ESI-MS).

17. The method according to claim 12, wherein the immobilization of the peptidoglycan precursor on the matrix or labeling of the peptidoglycan precursor with a fluorophore is not required.

18. The method according to claim 12, wherein the mass spectrometer is a hybrid linear trap / Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and is73equipped with equipped with a heat electrospray ionization probe, a high-performance liquid chromatography (HPLC) system with binary pump, and a Famos autosampler.

19. The method according to claim 18, wherein a sample is loaded on the target engagement mass spectrometer, wherein the sample was directly injected into a continuous flowing solvent with a flow rate of 40-60 μL / min in the mobile phase consisting of IPA / H2O / NH4OH at pH 6-9, wherein the FTICR is operated in negative mode within a normal mass range from m / z 500-3000, showing resolution power is 100,000 at m / z 400, electrospray voltage is applied to 4.0 kV, and the capillary temperature is set at 275 ºC.

20. A peptidoglycan precursor having a formula of A-Sn-Po-Lq; wherein A is an oligopeptide moiety, S is a saccharide moiety, P is a phosphate group, L is a lipid tail comprising up to 55 carbon atoms, n is 1 or 2, o is 1 or 2, and q is 0 or 1.

21. The peptidoglycan precursor according to claim 20, wherein A consists of 1 to 5 amino acids selected from the group consisting of Ala, Glu, m-DAP and Lys, and S is selected from N-acetylmuramic acid (MurNAc) or N-acetylglucosamine (GlcNAc).

22. The peptidoglycan precursor according to claim 20, wherein the peptidoglycan precursor is selected from the group consisting of:7423. The peptidoglycan precursor according to claim 20, wherein the peptidoglycan precursor is75

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