In Vivo Probe of Lipid II‐Interacting Proteins

β‐Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin‐binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP–Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d ‐amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.     

An RCM‐Based Total Synthesis of the Antibiotic Disciformycin B

The total synthesis of the potent new antibiotic disciformycin B ( 2 ) is described, which shows significant activity against methicillin‐ and vancomycin‐resistant Staphylococcus aureus (MRSA/VRSA) strains. The synthetic route is based on macrocyclization of a tetraene substrate to the 12‐membered macrolactone core by ring‐closing olefin metathesis (RCM). Although macrocyclization was accompanied by concomitant cyclopentene formation by an alternative RCM pathway, conditions were established to give the macrocycle as the major product. Key steps in the construction of the RCM substrate include a highly efficient Evans syn‐aldol reaction, the asymmetric Brown allylation of angelic aldehyde, and the stereoselective Zn(BH₄)₂‐mediated 1,2‐reduction of an enone. The synthesis was completed by late‐stage dehydrative glycosylation to introduce the d ‐arabinofuranosyl moiety and final chemoselective allylic alcohol oxidation.     

An Activity‐Based Probe for Studying Crosslinking in Live Bacteria

Penicillin‐binding proteins (PBPs) catalyze the crosslinking of peptidoglycan (PG), an essential process for bacterial growth and survival, and a common antibiotic target. Yet, despite its importance, little is known about the spatiotemporal aspects of crosslinking—largely because of a lack of experimental tools for studying the reaction in live bacteria. Here we introduce such a tool: an activity‐based probe that enables visualization and relative quantitation of crosslinking in vivo. In Staphylococcus aureus, we show that fluorescent mimics of the natural substrate of PBPs (PG stem peptide) are covalently incorporated into the cell wall, installing fluorophores in place of natural crosslinks. These fluorescent stem peptide mimics (FSPMs) are selectively recognized by a single PBP in S. aureus: PBP4. Thus, we were able to use FSPM pulse‐labeling to localize PBP4 activity in live cells, showing that it is recruited to the septum in a manner dependent on wall teichoic acid.     

Rapid superparamagnetic‐beads‐based automated immunoseparation of Zn‐proteins from Staphylococcus aureus with nanogram yield

Pathogenic bacteria have become a serious socio‐economic concern. Immunomagnetic separation‐based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles‐based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn‐proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn‐proteins by superparamagnetic beads modified with chicken anti‐Zn(II) antibody. S. aureus culture was treated with ZnCl₂. Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle–surface interactions were tested and associated protein profiles were identified using SDS‐PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn‐protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS‐PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.     

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.     

Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.     

Enzyme‐catalyzed ring‐opening polymerization of 3(S)‐isopropylmorpholine‐2,5‐dione

The enzymatic ring‐opening polymerization of a 6‐membered cyclic depsipeptide, 3(S)‐isopropylmorpholine‐2,5‐dione in the bulk, was investigated by using lipases as catalysts at 100 and 130°C. Unchanged monomer was recovered in the absence of the enzyme or using an inactivated enzyme, indicating that the present polymerization proceeds through enzymatic catalysis. Poly(3‐isopropylmorpholine‐2,5‐dione) has a carboxylic acid group at one end and a hydroxy group at the other end.     

One‐Pot Protection‐Glycosylation Reactions for Synthesis of Lipid II Analogues

(2,6‐Dichloro‐4‐methoxyphenyl)(2,4‐dichlorophenyl)methyl trichloroacetimidate ( 3 ) and its polymer‐supported reagent 4 can be successfully applied to a one‐pot protection‐glycosylation reaction to form the disaccharide derivative 7 d for the synthesis of lipid II analogues. The temporary protecting group or linker at the C‐6 position and N‐Troc protecting group of 7 d can be cleaved simultaneously through a reductive condition. Overall yields of syntheses of lipid II ( 1 ) and neryl‐lipid II N^ε‐dansylthiourea are significantly improved by using the described methods.     

Asymmetric Hydroformylation of 4‐Vinyl‐1,3‐dioxolan‐2‐one

A chiral cyclic carbonate, 4‐vinyl‐1,3‐dioxolan‐2‐one was used as racemic substrate in asymmetric hydroformylation. The catalysts were formed in situ from “pre‐formed” PtCl₂(diphosphine) and tin(II) chloride. (2S,4S )‐2,4‐Bis(diphenylphosphinopentane ((S,S )‐BDPP)), (S,S )‐2,3‐O‐izopropylidine‐2,3‐dihydroxy‐1,4‐bis(diphenylphosphino)butane ((S,S )‐DIOP)), and (R )‐2,2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl ((R )‐BINAP)) were used as optically active diphosphine ligands. The platinum‐containing catalytic systems provided surprisingly high activity. The hydroformylation selectivities of up to 97% were accompanied by perfect regioselectivity towards the dioxolane‐based linear aldehyde. The enantiomeric composition of all components in the reaction mixture was determined and followed throughout the reaction. The unreacted 4‐vinyl‐1,3‐dioxolan‐2‐one was recovered in optically active form. The kinetic resolution was rationalized using the enantiomeric composition of the substrate and the products.     

Cathepsin B‐Specific Metabolic Precursor for In Vivo Tumor‐Specific Fluorescence Imaging

Recently, metabolic glycoengineering with bioorthogonal click reactions has focused on improving the tumor targeting efficiency of nanoparticles as delivery vehicles for anticancer drugs or imaging agents. It is the key technique for developing tumor‐specific metabolic precursors that can generate unnatural glycans on the tumor‐cell surface. A cathepsin B‐specific cleavable substrate (KGRR) conjugated with triacetylated N‐azidoacetyl‐d ‐mannosamine (RR‐S‐Ac₃ManNAz) was developed to enable tumor cells to generate unnatural glycans that contain azide groups. The generation of azide groups on the tumor cell surface was exogenously and specifically controlled by the amount of RR‐S‐Ac₃ManNAz that was fed to target tumor cells. Moreover, unnatural glycans on the tumor cell surface were conjugated with near infrared fluorescence (NIRF) dye‐labeled molecules by a bioorthogonal click reaction in cell cultures and in tumor‐bearing mice. Therefore, our RR‐S‐Ac₃ManNAz is promising for research in tumor‐specific imaging or drug delivery.     

Haloduracin α binds the peptidoglycan precursor lipid II with 2:1 stoichiometry

The two-peptide lantibiotic haloduracin is composed of two post-translationally modified polycyclic peptides that synergistically act on gram-positive bacteria. We show here that Halα inhibits the transglycosylation reaction catalyzed by PBP1b by binding in a 2:1 stoichiometry to its substrate lipid II. Halβ and the mutant Halα-E22Q were not able to inhibit this step in peptidoglycan biosynthesis, but Halα with its leader peptide still attached was a potent inhibitor. Combined with previous findings, the data support a model in which a 1:2:2 lipid II:Halα:Halβ complex inhibits cell wall biosynthesis and mediates pore formation, resulting in loss of membrane potential and potassium efflux.     

Synthesis of main chain polymeric benzophenone photoinitiator via thiol‐ene click chemistry and Its use in free radical polymerization

Main chain polymeric benzophenone photoinitiator (PBP) was synthesized by using “Thiol‐ene Click Chemistry” and characterized with ¹H NMR, FTIR, UV, and phosphorescence spectroscopies. PBP as a polymeric photoinitiator presented excellent absorption properties (ε₂₉₄ = 28,300 mol^?1L^?1cm^?1) compared to the molecular initiator BP (ε₂₅₂ = 16,600 mol^?1L^?1cm^?1). The triplet energy of PBP was obtained from the phosphorescence measurement in 2‐methyl tetrahydrofurane at 77 K as 298.3 kJ/mol and according to phosphorescence lifetime, the lowest triplet state of PBP has an n‐π* nature. Triplet–triplet absorption spectrum of PBP at 550 nm following laser excitation (355 nm) were recorded and triplet lifetime of PBP was found as 250 ns. The photoinitiation efficiency of PBP was determined for the polymerization of Hexanedioldiacrylate (HDDA) with PBP and BP in the presence of a coinitiator namely, N‐methyldiethanolamine (MDEA) by Photo‐DSC. The initiation efficiency of PBP for polymerization of HDDA is much higher than for the formulation consisting of BP. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Spectral Characterization and Antimicrobial Activity of Some Schiff Bases Derived from 4‐Chloro‐2‐aminophenol and Various Salicylaldehyde Derivatives

A series of N‐(5‐chloro‐2‐hydroxyphenyl)‐(3/4/5‐substituted)‐salicylaldimines ( I – XI ) were synthesized using appropriate synthetic route. Their structures were characterized by FT‐IR, UV‐Visible, ESI‐MS, ¹H and ¹³C NMR spectroscopic techniques and analytical methods. The crystal structure of N‐(5‐chloro‐2‐hydroxyphenyl)‐5‐bromosalicylaldimine ( V ) was determined by X‐ray diffraction at room temperature. Relationship between the melting points and the structures of the compounds was examined. Antimicrobial activity of the compounds was evaluated against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis. Antifungal activities were reported for Candida albicans. Schiff bases showed considerable antimicrobial activity against S. aureus, S. epidermidis and C. albicans. N‐(5‐Chloro‐2‐hydroxyphenyl)‐3‐hydroxy‐salicylaldimine ( II ) has the broadest and highest antimicrobial activity according to the others.     

Two 9‐alkylthiophenanthrenes at 193 K (alkyl = dodecyl and tetradecyl)

Two isostructural 9‐thiophenanthrene derivatives, 9‐dodec­ylthiophenanthrene, C₂₆H₃₄S, (I), and 9‐tetradecylthiophen­anthrene, C₂₈H₃₈S, (II), are reported. They crystallize in the monoclinic space group P2₁/c with four molecules in a unit cell. The S—C_phenanthrene bonds are 1.767 (2) and 1.772 (4) Å and S—C_alkyl bonds are 1.809 (2) and 1.804 (4) Å for (I) and (II), respectively. The bond angles at S are 104.04 (11) and 104.0 (2)° for (I) and (II), respectively.     

Synthesis and characterization of a novel soluble reactive ladder‐like polysilsesquioxane with side‐chain 2‐(4‐chloromethyl phenyl) ethyl groups

A new kind of soluble structure‐ordered ladder‐like polysilsesquioxane with reactive side‐chain 2‐(4‐chloromethyl phenyl) ethyl groups ( L ) was first synthesized by stepwise coupling polymerization. The monomer, 2‐(4‐chloromethyl phenyl) ethyltrichlorosilane ( M ), was synthesized successfully by hydrosilylation reaction with dicyclopentadienylplatinum(II) chloride (Cp₂PtCl₂) ­catalyst. Monomer and polymer structures were characterized by FT‐IR, ¹H‐NMR, ¹³C‐NMR, ²⁹Si‐NMR, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), vapor pressure osmometry (VPO) and X‐ray diffraction (XRD). This novel reactive ladder‐like polymer has promise potential applications as initiator for atom transfer radical polymerization, and as precursor for a variety of advanced functional polymers. Copyright © 2001 John Wiley & Sons, Ltd.     

Efficient Access to Peptidyl‐RNA Conjugates for Picomolar Inhibition of Non‐ribosomal FemXWv Aminoacyl Transferase

Peptidyl–RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA‐dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl–RNAs based on Huisgen–Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP‐MurNAc‐pentapeptide, respectively. Synthesis of 2′‐azido RNA helix starts from 2′‐azido‐2′‐deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22‐nt RNA helix mimicking the acceptor arm of Ala‐tRNA^Ala by T4 RNA ligase. For alkyne UDP‐MurNAc‐pentapeptide, meso‐cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L ‐Cys is converted to dehydroalanine with O‐(mesitylenesulfonyl)hydroxylamine. Reaction of but‐3‐yne‐1‐thiol with dehydroalanine affords the alkyne‐containing UDP‐MurNAc‐pentapeptide. The Cu^I‐catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1‐hydroxypropyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine provided the peptidyl‐RNA conjugate, which was tested as an inhibitor of non‐ribosomal FemX_Wv aminoacyl transferase. The bi‐substrate analogue was found to inhibit FemX_Wv with an IC₅₀ of (89±9) pM , as both moieties of the peptidyl–RNA conjugate contribute to high‐affinity binding.     

Transition‐Metal‐Ion‐Mediated Polymerization of Dopamine: Mussel‐Inspired Approach for the Facile Synthesis of Robust Transition‐Metal Nanoparticle–Graphene Hybrids

Inspired by the high transition‐metal‐ion content in mussel glues, and the cross‐linking and mechanical reinforcement effects of some transition‐metal ions in mussel threads, high concentrations of nickel(II), cobalt(II), and manganese(II) ions have been purposely introduced into the reaction system for dopamine polymerization. Kinetics studies were conducted for the Ni²⁺–dopamine system to investigate the polymerization mechanism. The results show that the Ni²⁺ ions could accelerate the assembly of dopamine oligomers in the polymerization process. Spectroscopic and electron microscopic studies reveal that the Ni²⁺ ions are chelated with polydopamine (PDA) units, forming homogeneous Ni²⁺–PDA complexes. This facile one‐pot approach is utilized to construct transition‐metal‐ion–PDA complex thin coatings on graphene oxide, which can be carbonized to produce robust hybrid nanosheets with well‐dispersed metallic nickel/metallic cobalt/manganese(II) oxide nanoparticles embedded in PDA‐derived thin graphitic carbon layers. The nickel–graphene hybrid prepared by using this approach shows good catalytic properties and recyclability for the reduction of p ‐ nitrophenol.     

Bacillus licheniformis peptidoglycan characterization by CZE–MS: Assessment with the benchmark RP‐HPLC‐MS method

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non‐soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N‐acetyl‐muramidase, cleaving the β_1→4 bound between N‐acetylglucosamine and N‐acetylmuramic acid. Here, we report the use of CZE–MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID–MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.     

New Methacryloxy N‐Vinyl‐2‐pyrrolidinone‐ and Lactone‐Based Macromers

New ω‐methacryloxy‐terminated N‐vinyl‐2‐pyrrolidinone oligomers were prepared by reaction of the corresponding ω‐hydroxy‐terminated N‐vinyl‐2‐pyrrolidinone oligomers with 2‐[(1‐imidazolyl)formyloxy] ethyl methacrylate (HEMA‐Im). The oligomeric precursor had been obtained by radical chain transfer polymerization making use of isopropoxyethanol as a solvent and a chain transfer agent. α,ω‐Dimethacryloxy‐terminated ε‐caprolactone and δ‐valerolactone oligomers were also prepared by reaction of their α‐hydroxy‐ω‐methacryloxy‐terminated precursors with HEMA‐Im. These had been in turn synthesized by ring‐opening polymerization of the corresponding lactones in the presence of 2‐hydroxyethyl methacrylate as the initiator and tin octanoate as the catalyst. Due to the presence of methacrylic functions at their chain ends, both VP and lactone oligomers participate in radical polymerization reactions and can be therefore classified as radical macromers. Both macromer families have several potential applications, such as use in the synthesis of mixed hydrophilic/hydrophobic hydrogels. All macromers were characterized by NMR spectroscopy and size‐exclusion chromatography (SEC). The polymerization kinetics of the lactone macromers were also analyzed by ¹H NMR spectroscopy.     

Synthesis and Antibacterial Activity of 3‐(Substituted arylmethyl)‐4‐acylaminomethyloxazolidin‐2‐ones and Derivatization to Symmetrical Twin‐Drug Type Molecules

In connection with our studies on antibacterial active compounds in the class of new oxazolidinones against Gram‐positive (Staphylococcus aureus) and Gram‐negative (Escherichia coli) strains, some molecular modifications were attempted. In this study, molecular modifications of 4‐aminomethyloxazolidin‐2‐ones ( 3a ) to the corresponding 4‐acylaminomethyloxazolidin‐2‐one derivatives ( 3c–d ) and preparations of the represented twin‐drug type molecules ( 10–14 ) were investigated. Some additional 4‐dialkylaminomethyloxazolidin‐2‐ones ( 2 ) were also synthesized. The synthesized compounds were evaluated for antibacterial activity with Gram‐positive (S. aureus) and Gram‐negative (E. coli) strains.