Providing insight into the mechanism of action of cationic lipidated oligomers using metabolomics.

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C12-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

ROMP-based Glycopolymers with High Affinity for Mannose-Binding Lectins.

Well-defined, highly reactive poly(norbornenyl azlactone)s of controlled length (number-average degree of polymerization DPn¯ = 10 to 1,000) were made by ring-opening metathesis polymerization (ROMP) of pure exo-norbornenyl azlactone. These were converted into glycopolymers using a facile postpolymerization modification (PPM) strategy based on click aminolysis of azlactone side groups by amino-functionalized glycosides. Pegylated mannoside, heptyl-mannoside, and pegylated glucoside were used in the PPM. Binding inhibition of the resulting glycopolymers was evaluated against a lectin panel (Bc2L-A, FimH, langerin, DC-SIGN, ConA). Inhibition profiles depended on the sugars and the degrees of polymerization. Glycopolymers from pegylated-mannoside-functionalized polynorbornene, with DPn¯ = 100, showed strong binding inhibition, with subnanomolar range inhibitory concentrations (IC50s). Polymers surpassed the inhibitory potential of their monovalent analogues by four to five orders of magnitude thanks to a multivalent (synergistic) effect. Sugar-functionalized poly(norbornenyl azlactone)s are therefore promising tools to study multivalent carbohydrate-lectin interactions and for applications against lectin-promoted bacterial/viral binding to host cells.

Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases*.

Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.

An optimised Cu(0)-RDRP approach for the synthesis of lipidated oligomeric vinyl azlactone: toward a versatile antimicrobial materials screening platform.

This report details the synthesis of lipidated 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers via an optimised Cu(0)-mediated reversible-deactivation radical polymerisation approach, and the use of these oligomers as a versatile functional platform for the rapid generation of antimicrobial materials. The relative amounts of CuBr2 and Me6TREN were optimised to allow the fast and controlled polymerisation of VDM. These conditions were then used with the initiators ethyl 2-bromoisobutyrate, dodecyl 2-bromoisobutyrate, and (R)-3-((2-bromo-2-methylpropanoyl)oxy)propane-1,2-diyl didodecanoate to synthesise a library of oligo(VDM) (degree of polymerisation = 10) with ethyl, dodecyl or diglyceride end-groups. Subsequently, ring-opening of the pendant oxazolone group with various amines (i.e., 2-(2-aminoethyl)-1,3-di-Boc-guanidine, 1-(3-aminopropyl)imidazole, N-Boc-ethylenediamine, or N,N-dimethylethylenediamine) expanded the library to give 12 functional oligomers incorporating different cationic and lipid elements. The antimicrobial activities of these oligomers were assessed against a palette of bacteria and fungi: i.e. Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. The oligomers generally exhibited the greatest activity against the fungus, C. neoformans, with a minimum inhibitory concentration of 1 µg mL-1 (comparable to the clinically approved antifungal fluconazole). To assess haemocompatibility, the oligomers were assayed against erythrocytes, with the primary amine or guanidine containing C12 and 2C12 oligomers exhibiting greater lysis against the red blood cells (HC10 values between 7.1 and 43 µg mL-1) than their imidazole and tertiary amine counterparts (HC10 of >217 µg mL-1). Oligomers showed the greatest selectivity for C. neoformans, with the C12- and 2C12-tertiary amine and C12-imidazole oligomers possessing the greatest selectivity of >54-109. These results demonstrate the utility of reactive oligomers for rapidly assessing structure-property relationships for antibacterial and antifungal materials.

Azlactone-based heterobifunctional linkers with orthogonal clickable groups: efficient tools for bioconjugation with complete atom economy.

We report the efficient synthesis of a series of new azlactone-based heterofunctional linkers bearing two orthogonal clickable groups that proceed with full atom economy. These new linkers comprise an azlactone (oxazolone) group that quickly reacts with amino groups in biologically relevant medium without byproduct elimination and a (bio)orthogonal handle which further undergoes facile and selective click reactions such as thiol-ene coupling, Diels-Alder or azide-alkyne cycloadditions. As an example, the application of this methodology to lysozyme PEGylation in aqueous medium is described.

Recyclable magnetic nanocluster crosslinked with poly(ethylene oxide)-block-poly(2-vinyl-4,4-dimethylazlactone) copolymer for adsorption with antibody.

Surface modification of magnetic nanoparticle (MNP) with poly(ethylene oxide)-block-poly(2-vinyl-4,4-dimethylazlactone) (PEO-b-PVDM) diblock copolymers and its application as recyclable magnetic nano-support for adsorption with antibody were reported herein. PEO-b-PVDM copolymers were first synthesized via a reversible addition-fragmentation chain-transfer (RAFT) polymerization using poly(ethylene oxide) chain-transfer agent as a macromolecular chain transfer agent to mediate the RAFT polymerization of VDM. They were then grafted on amino-functionalized MNP by coupling with some azlactone rings of the PVDM block to form magnetic nanoclusters with tunable cluster size. The nanocluster size could be tuned by adjusting the chain length of the PVDM block. The nanoclusters were successfully used as efficient and recyclable nano-supports for adsorption with anti-rabbit IgG antibody. They retained higher than 95% adsorption of the antibody during eight adsorption-separation-desorption cycles, indicating the potential feasibility in using this novel hybrid nanocluster as recyclable support in cell separation applications.

Amine-reactive polymers synthesized by RAFT polymerization using an azlactone functional trithiocarbonate RAFT agent.

A new azlactone-derived trithiocarbonate is prepared and used as a chain-transfer agent to mediate the reversible addition-fragmentation chain transfer (RAFT) polymerization of styrene, ethyl acrylate, and N-isopropyl acrylamide. Well-defined polymers with controlled molecular weights (M(n) = 1000-7000 g mol(-1) ) and narrow molecular weight distributions (PDI = 1.05-1.10) are thus obtained that retain the azlactone functionality at the chain end. The ability of the resulting end-functionalized polymers to react quantitatively at room temperature with a stoichiometric amount of amino groups with retention of the thiocarbonylthio moiety is ascertained by using 4-fluorobenzylamine and allylamine.

Preparation of a new electro-optic polymer cross-linkable via copper-free thermal Huisgen cyclo-addition and fabrication of optical waveguides by Reactive Ion Etching.

High-quality trails of ridge waveguides were successfully fabricated using a new cross-linkable polymer (PCC01) by UV photolithography followed by Reactive-Ion Etching (RIE) process. The cross-linking reaction of PCC01 is based on the copper-free Huisgen cyclo-addition between an azide and an acetylene group. The new cross-linkable polymer (PCC01) consists of a structural modification of the previously described materials (Scarpaci et al. Polym. Chem.2011, 2, 157), because the ethynyl group is functionalized by a methyl group instead of the TMS protecting group. This feature prevents the formation of silica (SiO(2)) generated by trimethylsilyl groups and which was stopping the engraving process before completion. Herein, we describe the synthesis, the NLO characterizations, and the fabrication of a high-quality ridge waveguide with PCC01. The new cross-linkable polymer PCC01 not only solves the problems encountered with our previously described polymers, but also presents an enhancement of the electro-optic stability, because d(33) coefficients up to 30 pm/V stable at 150 °C were recorded.

Synthesis and characterization of functionalized poly(gamma-benzyl-L-glutamate) derivates and corresponding nanoparticles preparation and characterization.

For being fully efficient a targeted delivery system should associate simultaneously multiple functionalities. In this context, the association of several polymeric materials to form composite multifunctional particles can be foreseen. The present work describes the synthesis of different derivates of poly(gamma-benzyl-L-glutamate) and their use for the preparation of nanoparticles exhibiting different properties, including surface hydrophilization by PEG, fluorescence imaging by FITC and target recognition through easy attachment of desired ligands by using the avidin-biotin interaction, after the nanoparticles preparation. Four PBLG derivates were successfully obtained by ring-opening polymerization (ROP) of NCA, using various initiators corresponding to the molecules to be introduced into the copolymers. Further, nanoparticles smaller than 100 nm could be prepared using a nanoprecipitation technique and the presence of the active moieties introduced within the particles as well as their functionality has been checked. Very interestingly, it has been shown that biotin molecules could be efficiently introduced at the surface of the nanoparticles, which (for 75% of the theoretical amount) could be engaged in a complexation with avidin. It is suggested that this strategy offers the possibility to easily decorate these nanoparticles with various recognition ligands for specific targeting applications by using the well known biotin-avidin sandwich technique.

A new crosslinkable system based on thermal Huisgen reaction to enhance the stability of electro-optic polymers.

The thermal Huisgen cycloaddition reaction is a new and efficient process to crosslink electro-optic polymers, since it leads to an effective increase of the thermal alignment stability of the NLO chromophores.

Synthesis and nonlinear optical properties of a peripherally functionalized hyperbranched polymer by DR1 chromophores.

The first peripheral postfunctionalization of a hyperbranched polyimide by nonlinear optic chromophores (DR1 derivative) was achieved using two different routes. The first one consists in the esterification of the terminal carboxylic acid groups, whereas the second is based on copper-catalyzed Huisgen reaction of the terminal propargylic ester groups. The resulting polymers display good solubility in classical organic solvents and good filmability because thick films can be prepared (up to 2.7 mum). The second-order nonlinear optical properties were measured by SHG at 1064 nm and we show that these hyperbranched polymers exhibit good poling efficiency and good thermal stability since the electro-optic activity remains stable up to 130 degrees C. These results illustrate the potential of hyperbranched polymers to host second-order nonlinear optical chromophores to replace dendrimers or classical linear polymers generally used in this area.

Synthesis and ITC characterization of novel nanoparticles constituted by poly(gamma-benzyl L-glutamate)-beta-cyclodextrin.

Imparting desired technological characteristics to polymeric nanoparticles requires the development of original polymers. In the present work, the synthesis and characterization of a novel PBLG-derivative, the poly(gamma-benzyl L-glutamate)-beta-cyclodextrin (PBLG-beta-CD-50), have been carried out. Nanoparticles from either PBLG-beta-CD-50 polymer or from mixtures with PBLG have been prepared using a modified nanoprecipitation method. Spherically shaped nanoparticles with diameter in the range of 50-70 nm were obtained, as determined by dynamic laser light scattering and transmission electron microscopy. The presence of a surfactant in the suspension medium had almost no influence on these parameters and was not necessary to the shelf-stability of the suspension. Further, isothermal titration microcalorimetry (ITC) experiments have been used to show unambiguously that about 20% of the cyclodextrins remain functional within the particles. Consequently, this system may be of interest when association of large amounts of hydrophobic drugs to nanoparticles is required.

Well-defined azlactone-functionalized (co)polymers on a solid support: synthesis via supported living radical polymerization and application as nucleophile scavengers.

Wang resin has been converted to a supported initiator for transition metal-mediated living radical polymerization often called atom-transfer radical polymerization (ATRP) of 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) and styrene (S). Several "Rasta" resins with well-defined macromolecular architectures, including homopolymers PVDM, PS, statistical P(S-stat-VDM), block P(S-b-VDM), and P[S-b-(S-stat-VDM)] copolymers, have been elaborated. For the homopolymerization of VDM and S, a sacrificial initiator, benzyl 2-bromoisobutyrate (BBI), has been introduced to monitor the evolution of molar masses and polydispersity indexes (PDIs) of PS and PVDM onto the Wang resin support without cleavage. After 6 h, 86.7% conversion of VDM is reached, with the isolated PVDM chains having a molar mass of 18 000 g mol(-1) and a PDI value of 1.22. Block copolymers have been synthesized in two steps, involving the synthesis of the PS block isolated at low conversions (<15%) to maintain the bromine end-chain functionality and the subsequent synthesis of the second PVDM or P(S-stat-VDM) block. Polydispersity indexes of the cleaved (co)polymers were low (PDI = 1.11-1.44), and high azlactone loadings have been reached (loading = 6.0 mmol g(-1)). Such azlactone-functionalized Wang resins have shown high efficiency during the scavenging process of benzylamine as monitored by HPLC. Moreover, grafted statistical copolymers have shown the best behavior for removing benzylamine because of an improvement of the accessibility of azlactone rings by the dilution with styrene units.