Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low‐Nanomolar Multivalent Ligands

Anti‐infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA‐targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low‐nanomolar (K_d=19 nm , microarray) ligand with a tyrosine‐based linker arm could be identified in a structure–activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.     

Rational Design and Synthesis of Optimized Glycoclusters for Multivalent Lectin–Carbohydrate Interactions: Influence of the Linker Arm

The design of multivalent glycoclusters requires the conjugation of biologically relevant carbohydrate epitopes functionalized with linker arms to multivalent core scaffolds. The multigram‐scale syntheses of three structurally modified triethyleneglycol analogues that incorporate amide moiety(ies) and/or a phenyl ring offer convenient access to a series of carbohydrate probes with different water solubilities and rigidities. Evaluation of flexibility and determination of preferred conformations were performed by conformational analysis. Conjugation of the azido‐functionalized carbohydrates with tetra‐propargylated core scaffolds afforded a library of 18 tetravalent glycoclusters, in high yields, by Cu^I‐catalyzed azide–alkyne cycloaddition (CuAAC). The compounds were evaluated for their ability to bind to PA‐IL (the LecA lectin from the opportunistic pathogen Pseudomonas aeruginosa). Biochemical evaluation through inhibition of hemagglutination assays (HIA), enzyme‐linked lectin assays (ELLA), surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) revealed improved and unprecedented affinities for one of the monovalent probes (K_d=5.8 μM ) and also for a number of the tetravalent compounds that provide several new nanomolar ligands for this tetrameric lectin.     

Development of Pseudomonas aeruginosa Lectin LecA Inhibitor by using Bivalent Galactosides Supported on Polyproline Peptide Scaffolds

LecA is a galactose‐binding tetrameric lectin from Pseudomonas aeruginosa involved in infection and biofilm formation. The emergent antibiotic resistance of P. aeruginosa has made LecA a promising pharmaceutical target to treat such infections. To develop LecA inhibitors, we exploit the unique helical structure of polyproline peptides to create a scaffold that controls the galactoside positions to fit their binding sites on LecA. With a modular scaffold design, both the galactoside ligands and the inter‐ligand distance can be altered conveniently. We prepared scaffolds with spacings of 9, 18, 27, and 36 Å for ligand conjugation and found that glycopeptides with galactosides ligands three helical turns (27 Å) apart best fit LecA. In addition, we tested different galactose derivatives on the selected scaffold (27 Å) to improve the binding avidity to LecA. The results validate a new multivalent scaffold design and provide useful information for LecA inhibitor development.     

A High‐Barrier Molecular Balance for Studying Face‐to‐Face Arene–Arene Interactions in the Solid State and in Solution

An atropisomeric molecular balance was developed to study face‐to‐face arene–arene interactions. The balance has a large central 1,4,5,8‐naphthalene diimide surface that forms intramolecular arene–arene interactions with two pendent arms. The balance adopts distinct syn and anti isomers with varying numbers of intramolecular interactions. Thus, the strength of the arene–arene interaction could be quantitatively measured by NMR spectroscopy from the anti/syn ratios. The size of the arene arms was easily varied, which allowed examination of the relationship between arene size and strength of the interaction. A nonlinear size dependence was observed in solution with larger arene arms having a disproportionately stronger arene–arene interaction. The intramolecular arene–arene interactions were also characterized in the solid state by X‐ray crystallography. These studies were facilitated by the kinetic stability of the syn and anti isomers at room temperature due to the high isomerization barrier (ΔG=27.0 kcal mol^?1). Thus, the anti isomer could be selectively isolated and crystallized in its folded conformation. The X‐ray structures confirmed that the anti isomers formed two strong intramolecular arene–arene interactions with face‐to‐face geometries. The solid‐state structure analysis also reveals that the rigid framework may contribute to the observed nonlinear size trend. The acetate linker is slightly too long, which selectively destabilizes the balances with smaller arene arms. The larger arene arms are able to compensate for the longer linker and form effective intramolecular arene–arene interactions.     

Synthesis of Simple Multivalent β-D-GalNAc-(1→4)-β-D-Gal Oligomers as Probes for Investigating the Interactions of P. Aeruginosa Pili with Multivalent Receptors

ABSTRACT

Five multivalent β-D-GalNAc-(1→4)-β-D-Gal oligomers were selected and synthesized as probes for investigating the adhesin-receptor interactions of P. aeruginosa pili with multivalent receptors. They were synthesized by the amide coupling reactions of 8-(N-2-aminoethyl)carboxamidooctyl 4-O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-β-D-galactopyranoside (7) with EDTA dianhydride, EDTA, Kemp's triacid and adipic acid with EDC, DIC and DCC combined with HOBt as coupling reagents and by the reaction of per-O-acetylated 7 with 1,3,5-benzenetricarbonyl trichloride followed by de-O-acetylation. These resulting multivalent compounds contain flexible C₉ spacer arms as linkers attached to either flexible hydrophilic moieties or rigid hydrophobic cores.     

Efficient synthesis and properties of soluble aliphatic oligo(spiroorthocarbonate)s from pentaerythritol derivatives

A series of soluble oligo(spiroorthocarbonate)s (OSOCs) were synthesized by polycondensation of tetraethylorthocarbonate with pentaerythritol derivatives. The pentaerythritol derivatives used herein were synthesized from pentaerythritol by attaching substituents on it to improve the solubility of themselves and the resulting OSOCs. The structures of the OSOCs were confirmed by NMR spectroscopy and MALDI‐TOF mass analysis. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019 , 57, 792–798     

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers

Divalent precision glycooligomers terminating in N‐acetylneuraminic acid (Neu5Ac) or 3′‐sialyllactose (3′‐SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co‐crystallization studies with the sialic acid‐binding major capsid protein VP1 of human Trichodysplasia spinulosa‐associated Polyomavirus. High‐resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.     

In vitro antibiofilm activity of Murraya koenigii essential oil extracted using supercritical fluid CO2 method against Pseudomonas aeruginosa PAO1

The antibiofilm activity of Murraya koenigii essential oil (EO) against Pseudomonas aeruginosa PAO1 was investigated in this study. A decrease in the production of rhamnolipid, extracellular polymeric substance and swarming motility was observed by the EO treatment (0.3% v/v). The static microtitre plate assay revealed 80% reduction in biofilm formation by P. aeruginosa PAO1 on M. koenigii EO treatment. Fluorescence microscopy and scanning electron microscopy analyses confirmed the reduction of biofilm formation in P. aeruginosa PAO1 when treated with M. koenigii EO. Gas chromatography–mass spectrometry analysis of the EO revealed the presence of well-known antibiofilm agents such as spathulenol (5.85%), cinnamaldehyde (0.37%) and linalool (0.04%). Cinnamaldehyde has not been previously reported in M. koenigii EO. The potent antibiofilm properties of M. koenigii EO may be effectively exploited in food and pharmaceutical industries as well as in controlling Pseudomonas biofilms on indwelling medical devices.     

基于杯[4]芳烃和S-联萘酚的荧光传感器的合成及其对N-Boc保护谷氨酸阴离子的对应选择性识别性能研究

合成了一种基于杯[4]芳烃和S-联萘酚单元的新型手性大环受体4,并用荧光光谱和核磁氢谱研究了该受体与阴离子的键合性质。非线性曲线拟合结果表明受体4与N-Boc保护L-和D-谷氨酸阴离子都能通过多重氢键形成1:1的络合物,而且对N-Boc保护谷氨酸阴离子对映体显示了较好的对应选择性识别性能（Kass(L) / Kass(D) = 4.65）。不同的荧光响应表明受体4可以用作N-Boc保护谷氨酸阴离子的对应选择性的荧光化学传感器。     

Preparation of molecularly imprinted polymeric fibers using a single bifunctional monomer for the solid‐phase microextraction of parabens from environmental solid samples

In this study, molecularly imprinted polymer fibers for solid‐phase microextraction have been prepared with a single bifunctional monomer, N,O‐bismethacryloyl ethanolamine using the so‐called “one monomer molecularly imprinted polymers” method, replacing the conventional combination of functional monomer and cross‐linker to form high fidelity binding sites. For comparison, imprinted fibers were prepared following the conventional approach based on ethylene glycol dimethacrylate as cross‐linker and methacrylic acid as monomer. The recognition performance of the new fibers was evaluated in the solid‐phase microextraction of parabens, and from this study it was concluded that they provided superior performance over conventionally formulated fibers. Ultimately, real‐world environmental testing on spiked solid samples was successful by the molecularly imprinted solid‐phase microextraction of samples, and the relative recoveries obtained at enrichment levels of 10 ng/g of parabens were within 78–109% for soil and 83–109% for sediments with a relative standard deviation <15% (n = 3).     

In vitro antibiofilm activity of the freshwater bryozoan Hyalinella punctata: a case study of Pseudomonas aeruginosa PAO1

The antibiofilm and possible antiquorum sensing effects against the strain Pseudomonas aeruginosa PAO1 of five crude extracts of the freshwater bryozoan Hyalinella punctata (Hancock, 1850) were evaluated in vitro for the first time. H. punctata ethyl acetate extract (HpEtAc) exhibited the highest antibiofilm activity reducing the biofilm formation of P. aeruginosa PAO1 in the range of 80.63–88.13%. While all tested extracts reduced the twitching motility of the aforementioned bacterial strain, HpEtAc showed to be the most effective. Finally, at a concentration of 0.5 MIC, the same extract mostly inhibited the production of pyocyanin by P. aeruginosa PAO1 (71.53%). In comparison both with the positive controls used (streptomycin and ampicillin, 67.13 and 69.77%, respectively), HpEtAc was found to inhibit pyocyanin in a higher extent. An extensive chemical characterisation of this particular extract may result in isolation and identification of novel lead compounds targeting P. aeruginosa, an opportunistic human pathogen.     

Biological activity of chitosan–sugar hybrids: specific interaction with lectin

The specific interactions between lectins and chitosan–sugar hybrids, the synthesized chitosan derivatives linking carbohydrate residue to the amino group of chitosan, were investigated. The specific bindings of chitosan‐L ‐fucose (Fuc) hybrid with Ulex europaeus agglutinin I (UEA I, a lectin specific to L ‐Fuc), and chitosan‐N‐acetyl‐D ‐glucosamine (D ‐GlcNAc) hybrid with Concanavalin A (Con A, a lectin specific to D ‐glucose, D ‐mannose and D ‐GlcNAc), were confirmed by a surface plasmon resonance technique. The microscopic observation of Pseudomonas aeruginosa, which was preincubated with the fluorescein isothiocyanate‐labeled chitosan‐L ‐Fuc hybrid, showed bacteria aggregation. The aggregation was thought to be resulted from the specific interaction of the L ‐Fuc residue of the hybrid with PA‐II lectin on the surface of P. aeruginosa. The chitosan‐L ‐Fuc hybrid inhibited P. aeruginosa growth more effectively in comparison with the other hybrids or unmodified chitosan. The enhancement of antimicrobial activity of chitosan‐L ‐Fuc hybrid could be attributed to the specific binding between PA‐II lectin of P. aeruginosa and L ‐Fuc residue of the L ‐Fuc hybrid. Copyright © 2000 John Wiley & Sons, Ltd.     

Facile Self‐Assembly of Metallo‐Supramolecular Ring‐in‐Ring and Spiderweb Structures Using Multivalent Terpyridine Ligands

A series of metallo‐supramolecular ring‐in‐ring structures was generated by assembling Cd^II ions and the multivalent terpyridine ligands ( L^1‐3 ) composed of one 60°‐bent and two 120°‐bent bis(terpyridine)s with varying alkyl linker lengths. The mechanistic study for the self‐assembly process excluded an entropically templated pathway and showed that the intramolecularly complexed species is the key intermediate leading to ring‐in‐ring formation. The next‐generation superstructure, a spiderweb, was produced in quantitative yield using the elongated decakis(terpyridine) ligand ( L⁵ ).     

Azetidine‐Containing Alkaloids Produced by a Quorum‐Sensing Regulated Nonribosomal Peptide Synthetase Pathway in Pseudomonas aeruginosa

Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine‐containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum‐sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S‐adenosylmethionine‐dependent enzyme to produce azetidine 2‐carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.     

Berberine Derivatives as Pseudomonas aeruginosa MexXY-OprM Inhibitors: Activity and In Silico Insights

The natural alkaloid berberine has been demonstrated to inhibit the Pseudomonas aeruginosa multidrug efflux system MexXY-OprM, which is responsible for tobramycin extrusion by binding the inner membrane transporter MexY. To find a structure with improved inhibitory activity, we compared by molecular dynamics investigations the binding affinity of berberine and three aromatic substituents towards the three polymorphic sequences of MexY found in P. aeruginosa (PAO1, PA7, and PA14). The synergy of the combinations of berberine or berberine derivatives/tobramycin against the same strains was then evaluated by checkerboard and time-kill assays. The in silico analysis evidenced different binding modes depending on both the structure of the berberine derivative and the specific MexY polymorphism. In vitro assays showed an evident MIC reduction (32-fold and 16-fold, respectively) and a 2–3 log greater killing effect after 2 h of exposure to the combinations of 13-(2-methylbenzyl)- and 13-(4-methylbenzyl)-berberine with tobramycin against the tobramycin-resistant strain PA7, a milder synergy (a 4-fold MIC reduction) against PAO1 and PA14, and no synergy against the ΔmexXY strain K1525, confirming the MexY-specific binding and the computational results. These berberine derivatives could thus be considered new hit compounds to select more effective berberine substitutions and their common path of interaction with MexY as the starting point for the rational design of novel MexXY-OprM inhibitors.     

Synthesis and Antibacterial Evaluation of Some Semicarbazides and 1,2,4‐Triazol‐5‐Ones Containing Thiophene Moieties

In this study, some 3‐(thiophen‐2‐ylmethyl)‐4‐substituted‐4,5‐dihydro‐1H‐1,2,4‐triazol‐5‐one derivatives were synthesized by the cyclization reaction of 1‐(thiophen‐2‐ylacetyl)‐4‐substituted semicarbazide derivatives in alkaline medium or in the immediate reaction of thiophen‐2‐yl‐acetic acid hydrazide with isocyanates. The structures of all new compounds were confirmed by analytical and spectroscopic methods. Selected derivatives were evaluated in vitro against several species of aerobic bacteria. Some of them showed activity against S. pyogenes, P. aeruginosa and S. aureus.     

Photocontrol of the Catalytic Activity of a β‐Cyclodextrin Bearing Azobenzene and Histidine Moieties as a Pendant Group

An azobenzene group was linked to β‐cyclodextrin via a histidine spacer ( 1 ) to produce a photoresponsive catalyst. The ester hydrolysis of p‐nitrophenyl acetate, Boc‐L ‐alanine‐p‐nitrophenyl ester and Boc‐D ‐alanine‐p‐nitrophenyl ester was examined in the presence of trans‐ 1 or cis‐ 1 . In the case of cis‐ 1 , the cyclodextrin cavity was used as the substrate binding site during imidazole‐catalyzed ester hydrolysis. This was not possible in the case of trans‐ 1 due to the inclusion of the trans‐azobenzene moiety in the cyclodextrin cavity. Consequently, the catalytic mechanism switches in an on‐off fashion on UV irradiation, associated with the conversion of the azobenzene moiety of 1 from trans to cis.     

Chiral sensing of Eu(III)‐containing achiral polymer complex from chiral amino acids coordination induction

The β‐diketonate‐based achiral polymer P‐1 could be synthesized by the polymerization of 3,7‐dibromo‐2,8‐dimethoxy‐5,5‐dioctyl‐5H‐dibenzo[b,d]silole ( M1 ) with (Z)?1,3‐bis(4‐ethynylphenyl)?3‐hydroxyprop‐en‐1‐one ( M2 ) via typical Sonogashira coupling reaction. The β‐diketonate unit in the main chain backbone of P‐1 can further coordinate with Eu(TTA)_x [TTA^? = 4,4,4‐trifluoro‐1‐(thiophen‐2‐yl)butane‐1,3‐dionate anion, X = 1, 2, 3] to afford corresponding Eu(III)‐containing polymer complexes. The resulting achiral polymer complex P‐2 (X = 2) can exhibit strong circular dichroism (CD) response toward both N‐Boc‐l and d‐ proline enantiomers. The CD signal was preliminarily attributed to coordination induction between chiral N‐Boc‐proline and the Eu(III) complex moiety. The linear regression analysis of CD sensing shows a good agreement between the magnitude of molar ellipticity and concentration of chiral N‐Boc‐l or d‐ proline, which indicates this kind Eu(III)‐containing achiral polymer complex can be used as a chiral probe for enantioselective recognition of N‐Boc‐l or d‐ proline enantiomers based on Cotton effect of CD spectra. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 3080–3086     

Role of the Quorum Sensing Mechanism in the Response of Pseudomonas aeruginosa to Lethal and Sublethal UVA Irradiation

The role of quorum sensing (QS) in the response of Pseudomonas aeruginosa to UVA radiation was investigated in the PAO1 strain and derivatives defective in the synthesis of the QS signals 3OC12-HSL (lasI strain), C4-HSL (rhlI strain) or both (lasI rhlI strain). Cell viability measurements demonstrated that the double mutant was significantly more sensitive to UVA than single mutants, which in turn showed reduced cell survival with regard to the PAO1 strain. Irradiation under nitrogen atmosphere and chemiluminescence measurements indicated the oxidative nature of the UVA-induced damage. The activity of the antioxidant enzymes catalase and superoxide dismutase was assayed in these strains before and after irradiation, and a strong correlation between catalase levels and UVA sensitivity was observed. When a sublethal UVA dose was applied to PAO1, a growth delay was observed and this mechanism depended on the rhl system. Moreover, low doses of UVA irradiation triplicated the level of C4-HSL in log PAO1 cells. It is concluded that QS is fundamental in the defense against the toxic effects of UVA in P. aeruginosa. The induction of the QS system by UVA independently of cell density could function as an adaptative strategy to withstand this hostile environmental condition.     

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.