Vancomycin-Dependent Response in Live Drug-Resistant Bacteria by Metabolic Labeling

The surge in drug-resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug-resistant bacteria with temporal resolution. Vancomycin-resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell-wall precursors. A synthetic cell-wall analogue was developed to hijack the biosynthetic rewiring of drug-resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell-wall alteration in drug-resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.     

The significance of enterobacterial mutants for the chemical investigation of their cell-wall polysaccharides

The outer cell surface of Enterobacteriaceae, i.e. the cell wall, consist of a rigid structure (murein) on which additional proteins, lipids, and polysaccharides are deposited. In the bacterial wild types (S forms) the polysaccharide is species-specific and carries the serologically determinant groups of the respective O antigen. These specific polysaccharides often consist of a large number of monosaccharides; up to eight different monosaccharides may be involved. Bound to lipid A, the cell-wall lipopolysaccharides represent the endotoxins of the bacteria. During the past few years the structures of the enterobacterial cell-wall polysaccharides have been elucidated by chemical, immunochemical, biochemical, and genetic investigations, particularly in the Salmonella. Here the polysaccharides consists of a basal polysaccharide common to all species, to which (in the S form) are bound longer species-specific side chains, consisting of repeating oligosachharide units. Spontaneous S → R mutation leads to R forms which are deficient mutants of the wild types in regard to the biosynthesis of the complete cell-wall polysaccharide. In contrast to the multiplicity of the serological specificities of the somatic antigens of the S forms, only a few serological types were found among the R forms (R I, R II, etc.). These R polysaccharides correspond to intermediates in the biosynthesis of the wile-type polysaccharides. The S → R mutation leads to the loss (or block) of an enzymes (transferase, synthetase) participating in the synthesis of the S polysaccharides. Recently many deficient mutants have been isolated and analysed, and in this way numerous stages in the biosynthesis of the cell-wall polysaccharides, for example, of Salmonella minnesota, have been made accessible to direct analysis. According to these investigations, the cell-wall polysaccharides of Salmonella consist of a basic polyheptose phosphate core which is bound to lipid A via ketodeoxyoctonic acid. To the basic core are linked pentasaccharide units of (R II structure). All other R forms are structurally deficient mutants of R II. In the complete polysaccharides of Salmonella S forms (wild types), the repeating oligosaccharide units of the specific side, chain are bound to the terminal N-acetylglucosamine of the R II structure.     

Finding synthetically versatile and common intermediates for multiple useful products with the aid of a synthesis design system

With the aid of a synthesis design system, we searched for common intermediates in routes to sets of target molecules. Finding such common intermediates will be a great help in constructing cost-effective synthetic routes. We tried to look for new common intermediates with 24 investigational or commercially produced antidepressants as target molecules. Most of the common intermediates were usually found to lead to more than two targets.     

Control of bacteria adhesion by cell-wall engineering

UDP-MurNAc-pentapeptide derivative bacterial cell-wall precursors were synthesized as effective tools for surface display on living bacteria. Lactobacilli were incubated in the ketone-modified precursor-containing medium, and the ketone moiety was displayed on the bacterial surface through cell-wall biosynthesis. Oligomannose was coupled with the ketone moiety on the bacterial surface via a aminooxyl linker, thereby displaying this oligosaccharide on the surface of the bacteria. The increase in the adhesion of the sugar-displaying bacteria onto a concanavalin A-attached film compared to that of native bacteria was confirmed by microscopic observation and surface plasmon resonance measurement. The incorporation of the artificial cell-wall precursors was enhanced when incubated with fosfomycin, an inhibitor of cell-wall precursor biosynthesis.     

Rationally designed ligands as models for bacterial cell-wall recognition by vancomycin-group antibiotics

The ability of vancomycin-group antibiotics to bind cell-wall fragmentsin vitro proyides a model system for semi-quantitative studies of molecular recognition in aqueous solution. Based upon a partitioning of free energy contributions to binding, we have assessed the surface area-dependent free energy contribution of the hydrophobic effect, the free energy contribution of amide-amide and amide-carboxylate hydrogen bonds, and the cost of restricting σ-bond rotations in the binding of both natural cell-wall analogues and within a family of rationally designed cell-wall mimics. The results so far obtained may provide guiding principles for those interested in semi-quantitative studies of molecular recognition and rational drug design. Vancomycin-group antibiotics have been shown to dimerise, a process now proposed to play a functional role in the mechanism of action. Dimerisation has been shown to be cooperatively enhanced by natural cell-wall peptides. However, studies with ristocetin A show that binding of unnatural structural motifs (particularly those containing aromatic ring systems not found in the natural peptides) and dimerisation, are highly anti-cooperative phenomena.     

19F NMR in the measurement of binding affinities of chloroeremomycin to model bacterial cell-wall surfaces that mimic VanA and VanB resistance

Background: The emergence of bacteria that are resistant to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus, has made the study of the binding characteristics of glycopeptides to biologically relevant depsipeptides important. These depsipeptides, terminating in -d-alanyl-d-lactate, mimic the cell-wall precursors of resistant bacteria.Results: The use of ¹⁹F-labelled ligands in the study of the therapeutically important vancomycin series of antibiotics is demonstrated. The substantial simplification of spectra that occurs when such labelled ligands are employed is used in the measurement of binding affinities of depsipeptides to chloroeremomycin (CE). Large enhancements of binding affinities are found at a model bacterial cell-wall surface (constituted from depsipetides that are anchored into vesicles) relative to those measured in free solution.Conclusions: Surface-enhanced binding, previously shown for strongly dimerising glycopeptide antibiotics to normal -d-alanyl-d-alanine-terminating cell-wall precursors, is now demonstrated for CE to the surface of models of VanA- and VanB-resistant bacteria. The effect of depsipeptide chain length is shown to be critically important in producing and maximising this enhancement.     

Synthesis of isoxazoles en route to semi-aromatized polyketides: dehydrogenation of benzonitrile oxide-para-quinone acetal cycloadducts

A variety of highly functionalized polycyclic isoxazoles are prepared by a two-step protocol: (1) 1,3-dipolar cycloaddition of o,o'-disubstituted benzonitrile oxides to para-quinone mono-acetals, then (2) dehydrogenation. The cycloaddition proceeds in a regioselective manner, favouring the formation of the 4-acyl cycloadducts, which are suitable intermediates for the synthesis of semi-aromatized polycyclic targets derived from polyketide type-II biosynthesis.     

Synthesis of ortho-substituted nitroaromatics via improved Negishi coupling conditions

We describe modified Negishi coupling conditions that allow improved access to ortho-nitrophenylalanine derivatives. These useful amino acid intermediates can be further elaborated into biologically active lactams and cyclic hydroxamic acid targets.     

Role of the cell-wall structure in the retention of bacteria by microfiltration membranes

This experimental study investigates the retention of bacteria by porous membranes. The transfer of bacteria larger than the nominal pore size of microfiltration track-etched membranes has been studied for several kinds of bacterial strains. This unexpected transfer does not correlate to the hydrophobicity, neither to the surface charge of the microorganism, as suggested in previous reports. We conclude that, in our conditions, the kind of bacteria (Gram-positive or Gram-negative) is finally the most important parameter. As the distinction between those two types of bacteria is related to the cell-wall structure, we provide an experimental evidence, via the action of an antibiotic, that the cell-wall flexibility triggers the transfer of the bacteria through artificial membranes, when the pores are smaller in size than the cell.     

First synthesis of caerulomycin B. A new synthesis of caerulomycin C

Caerulomycins produced by Streptomyces caeruleus are bipyridinic molecules endowed with antibiotic properties. The first synthesis of caerulomycin B (1) as well as a new synthesis of caerulomycin C (2) are reported. Starting from 3-hydroxypyridine, the same methodology was used to prepare both compounds 1 and 2. Efficiently controlled reactions such as metalation to allow the synthesis of 2,6-diiodo-3,4-dialkoxypyridines, which are key intermediates, and further halogen-lithium exchange and cross-coupling to reach the targets molecules 1 and 2 have been developed.     

Direct organocatalytic hydroalkoxylation of α,β-unsaturated ketones

The direct addition of a variety of alcohols to in situ activated olefins was observed in the presence of mild bifunctional amine/acid catalysts. Unlike existing methods, the reactions proceed at room temperature and in the absence of transition metals. The use of simple commercially available catalysts, amines and acids makes this an attractive method for the preparation of β-alkoxy ketones, which are prevalent targets and intermediates in organic synthesis.     

Recent Advances in the Catalytic Synthesis of the Cyclopentene Core

Five-membered carbocycles are ubiquitously found in natural products, pharmaceuticals, and other classes of organic compounds. Within this category, cyclopentenes deserve special attention due to their prevalence as targets and as well as key intermediates for synthesizing more complex molecules. Herein, we offer an overview summarizing some significant recent advances in the catalytic assembly of this structural motif. A great variety of synthetic methodologies and strategies are covered, including transition metal-catalyzed or organocatalyzed processes. Both inter- and intramolecular transformations are documented. On this ground, our expertise in the application of C−H functionalization reactions oriented towards the formation of this ring and its subsequent selective functionalization is embedded.     

Expanding the organic toolbox: a guide to integrating biocatalysis in synthesis

This critical review presents an introduction to biocatalysis for synthetic chemists. Advances in biocatalysis of the past 5 years illustrate the breadth of applications for these powerful and selective catalysts in conducting key reaction steps. Asymmetric synthesis of value-added targets and other reaction types are covered, with an emphasis on pharmaceutical intermediates and bulk chemicals. Resources of interest for the non-initiated are provided, including specialized websites and service providers to facilitate identification of suitable biocatalysts, as well as references to recent volumes and reviews for more detailed biocatalytic procedures. Challenges related to the application of biocatalysts are discussed, including how 'green' a biocatalytic reaction may be, and trends in biocatalyst improvement through enzyme engineering are presented (152 references).     

An efficient approach to chiral nonracemic trans- and cis-decalin scaffolds for drimane and labdane synthesis

Optically active trans- and cis-ring junction decalinic intermediates, which represent useful precursors for the synthesis of more complex natural targets, have been conveniently prepared starting from the β-ketoester 2 obtained by standard chemistry from β-ionone and dimethyl carbonate. The chiral auxiliary (−)-menthol, easily attached to 2 through DMAP-catalyzed transesterification, allowed a clean separation of the diastereomers obtained in the key electrocyclization step, which were further elaborated to chiral intermediates already taken to drimane and labdane sesquiterpenes.     

Simple and practical routes to enantiomerically pure 5-(trialkylsilyl)-2-cyclohexenones

[reaction: see text] Enantiomerically pure chiral 5-silylated 2-cyclohexenones are easily prepared in high yield using as a key step kinetic resolution with a commercially available lipase. Fully active enzyme can be recovered very efficiently for reuse. The synthetic steps are outlined in Schemes 1 and 3. Enantiomerically pure 2-cyclohexenones such as 1 and 2 are versatile intermediates for the synthesis of a multitude of chiral targets by means of a variety of diastereoselective reactions such as those illustrated in Scheme 2.     

R-or-S-Thalidomide合成新工艺的研究

沙利度胺(thalidom ide)具有抗炎、免疫调节、治疗风湿性疾病和抗肿瘤等作用。近期对沙利度胺的研究热点集中于肿瘤治疗中抗血管生成,其作用机制是通过非白由基介导的DNA氧化损伤直接抑制肿瘤细胞的生长和存活,抑制肿瘤血管新生,抑制促瘤细胞生长因子的分泌,降低其活性;同时,它     

Single Molecule Characterization of Amyloid Oligomers

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.     

11-Step enantioselective synthesis of (-)-lomaiviticin aglycon

Lomaiviticins A and B are complex antitumor antibiotics that were isolated from a strain of Micromonospora. A confluence of several unusual structural features renders the lomaiviticins exceedingly challenging targets for chemical synthesis. We report an 11-step, enantioselective synthetic route to lomaiviticin aglycon. Our route proceeds by late-stage, stereoselective dimerization of two equivalent monomeric intermediates, a transformation that may share parallels with the natural products' biosyntheses. The route we describe is scalable and convergent, and it lays the foundation for determination of the mode of action of these natural products.     

"Common synthetic scaffolds" in the synthesis of structurally diverse natural products

Selected natural products have long been considered as desirable targets for total synthesis due to their unique biological properties and their challenging structural complexity. Laboratory synthesis of natural compounds usually relies on target-oriented synthesis, involving the production, isolation and purification of successive intermediates, requiring multiple steps to arrive to the final product. A far more economical approach using common synthetic scaffolds that can be readily transformed through biomimetic-like pathways to a range of structurally diverse natural products has been evolved in the last decade, leading synthesis to new directions. This tutorial review critically presents the hallmarks in this field.     

Genome-wide analysis of Excretory/Secretory proteins in root-knot nematode,Meloidogyne incognita provides potential targets for parasite control

The root-knot nematode, Meloidogyne incognita causes significant damage to various economically important crops. Infection is associated with secretion of effector proteins into host cytoplasm and interference with host innate immunity. To combat this infection, the identification and functional annotations of Excretory/Secretory (ES) proteins serve as a key to produce durable control measures. The identification of ES proteins through experimental methods are expensive and time consuming while bioinformatics approaches are cost-effective by prioritizing the experimental analysis of potential drug targets for parasitic diseases. In this study, we predicted and functionally annotated the 1889 ES proteins in M. incognita genome using integration of several bioinformatics tools. Of these 1889 ES proteins, 473 (25%) had orthologues in free living nematode Caenorhabditis elegans, 825(67.8%) in parasitic nematodes whereas 561 (29.7%) appeared to be novel and M. incognita specific molecules. Of the C. elegans homologues, 17 ES proteins had “loss of function phenotype” by RNA interference and could represent potential drug targets for parasite intervention and control. We could functionally annotate 429 (22.7%) ES proteins using Gene Ontology (GO) terms, 672 (35.5%) proteins to protein domains and established pathway associations for 223 (11.8%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG). The 162 (8.5%) ES proteins were also mapped to several important plant cell-wall degrading CAZyme families including chitinase, cellulase, xylanase, pectate lyase and endo-β-1,4-xylanase. Our comprehensive analysis of M. incognita secretome provides functional information for further experimental study.