PNA‐Encoded Synthesis (PES) of a 10 000‐Member Hetero‐Glycoconjugate Library and Microarray Analysis of Diverse Lectins

Identification of selective and synthetically tractable ligands to glycan‐binding proteins is important in glycoscience. Carbohydrate arrays have had a tremendous impact on profiling glycan‐binding proteins and as analytical tools. We report a highly miniaturized synthetic format to access nucleic‐acid‐encoded hetero‐glycoconjugate libraries with an unprecedented diversity in the combinations of glycans, linkers, and capping groups. Novel information about plant and bacterial lectin specificity was obtained by microarray profiling, and we show that a ligand identified on the array can be converted to a high‐affinity soluble ligand by straightforward chemistry.     

Lipochitin Oligosaccharides Immobilized through Oximes in Glycan Microarrays Bind LysM Proteins

Glycan microarrays have emerged as novel tools to study carbohydrate–protein interactions. Here we describe the preparation of a covalent microarray with lipochitin oligosaccharides and its use in studying proteins containing LysM domains. The glycan microarray was assembled from glycoconjugates that were synthesized by using recently developed bifunctional chemoselective aminooxy reagents without the need for transient carbohydrate protecting groups. We describe for the first time the preparation of a covalent microarray with lipochitin oligosaccharides and its use for studying proteins containing LysM domains. Lipochitin oligosaccharides (also referred to as Nod factors) were isolated from bacterial strains or chemoenzymatically synthesized. The glycan microarray also included peptidoglycan‐related compounds, as well as chitin oligosaccharides of different lengths. In total, 30 ligands were treated with the aminooxy linker molecule. The identity of the glycoconjugates was verified by mass spectrometry, and they were then immobilized on the array. The presence of the glycoconjugates on the array surface was confirmed by use of lectins and human sera (IgG binding). The functionality of our array was tested with a bacterial LysM domain‐containing protein, autolysin p60, which is known to act on the bacterial cell wall peptidoglycan. P60 showed specific binding to Nod factors and to chitin oligosaccharides. Increasing affinity was observed with increasing chitin oligomer length.     

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin‐3 Inhibitor

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent‐field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3′‐mono‐ and 2,3′‐diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin‐3 for 2′‐arylamido derivatives over ureas, thioureas, and amines and that of galectin‐7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin‐3 versus galectin‐1 and galectin‐7.     

Phosphatidyl myo-inositol mannosides mimics built on an acyclic or heterocyclic core: synthesis and anti-inflammatory properties

Phosphatidyl myo-inositol mannosides (PIMs) are constituents of the mycobacterial cell wall and possess immunomodulatory activities. Certain PIM derivatives have immunoprotective activity and are of interest as anti-inflammatory agents. In order to identify simplified analogues of PIMs that retain this interesting activity, we have prepared a series of new analogues based either on an acyclic or on a heterocyclic scaffold that replaces the inositol moiety, and evaluated these compounds for their inhibition of LPS-induced release of NO and pro-inflammatory cytokines by macrophages. It was found that the inositol moiety can be favourably replaced by an aza-cyclitol (trihydroxy-piperidine) or an oxa-cyclitol (trihydroxy-tetrahydropyran) unit, and that the configuration of the OH-carrying carbons does not play a significant role. The biological activity is reduced if the nitrogen atom is free in the aza-cyclitol unit.     

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI

Uropathogenic E. coli (UPEC) employ the mannose‐binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half‐life in vivo. In a rational design strategy, we obtained an X‐ray structure of lead mannosides and then designed mannosides with improved drug‐like properties. We show that cyclizing the carboxamide onto the biphenyl B‐ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2‐methyl‐4‐(1‐oxo‐1,2‐dihydroisoquinolin‐7‐yl)phenyl α‐d ‐mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N‐Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22 , as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria‐mediated hemagglutination and prevent biofilm formation by UPEC with single‐digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date.     

Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.     

Mercury(II) removal using polymer inclusion membranes containing Cyanex 471X

BACKGROUND: Regulatory controls to limit mercury emissions in waters have impacted on the development of membrane extraction‐based methodologies for its separation. The specific advantages (effective carrier immobilization, easy preparation, versatility, and good mechanical properties) of polymer inclusion membranes (PIMs) make them suitable for this purpose. In this work a novel procedure using PIMs for mercury separation with a commercial available extractant (Cyanex 471X) is described and evaluated through the determination of the efficiency parameters (permeability, selectivity, stability) and membrane characterization. RESULTS: Using a membrane composed of 30% cellulose triacetate (CTA), 60% 2‐nitrophenyl octyl ether (NPOE), and 10% w/w Cyanex 471X a 0.1 mmol dm^?3 Hg(II) solution prepared in 0.01 mol dm^?3 HCl was transported to a 0.05 mol dm^?3 NaCl solution at pH 12.3 with permeability values in the feed and strip phases of 0.25 and 0.15 cm min^?1, respectively. A diffusive Fickian‐type mechanism was inferred from the results. High separation factors ranging between 2 and 5900, less than 11% of competing metal ions transported, active transport of the metal ion and a successful reuse of the PIM were achieved. CONCLUSION: Optimized PIMs using Cyanex 471X represent an interesting alternative for Hg(II) removal from waters showing high efficiency factors and easy implementation. Copyright © 2009 Society of Chemical Industry     

Supported Liquid (SLM) and Polymer Inclusion (PIM) Membranes Pertraction of Copper(II) from Aqueous Nitrate Solutions by 1-Hexyl-2-Methylimidazole

The facilitated transport of Cu(II) ions from different aqueous nitrate source phases (c _Me = 0.001 M, pH = 6.0) across supported (SLMs) and polymer inclusion membranes (PIMs) doped with 1-hexyl-2-methylimidazole as ion carrier was reported. The membrane is characterized by means of atomic force microscopy (AFM). The results show that Cu²⁺ can be separated very effectively from other transition metal cations as Zn²⁺, Co²⁺, and Ni²⁺ from different equimolar mixtures of these ions. The highest initial fluxes of Cu(II) were found for PIM, while lower values were observed for SLM. However, after taking into account the morphology of the membranes (porosity, tortuosity), the values of the initial flux of Cu(II) transport across PIM is less than that across SLM. The recovery factor of Cu²⁺ ions during transport across PIM from different mixtures of cations is above 91% after 24 hrs and above 76% during transport across SLM. Also, the stability of PIM and SLM doped with 1-hexyl-2-methylimidazole was confirmed in replicate experiments.     

Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.     

Application of Polymer and Supported Membranes with 1-Decyl-4-Methylimidazole for Pertraction of Transition Metal Ions

The facilitated transport of Zn(II), Cd(II), Co(II), and Ni(II) ions from different aqueous chloride source phases (c_Me = 0.001 mol/dm³, pH = 6.0) across supported (SLMs) and polymer inclusion membranes (PIMs) doped with 1-decyl-4-methylimidazole as ion carrier was reported. The membrane is characterized by means of atomic force microscopy (AFM). The results show that Zn(II) can be separated very effectively from other transition metal cations as Cd(II), Co(II), and Ni(II) from different equimolar mixtures of such ions. The higher initial fluxes for Zn(II) were found for PIM (3.62-4.10 μmol/m².s), while the lower values were observed for SLM. However, after taking into account the morphology (porosity, tortuosity) of the membranes, the values of the initial flux of Zn(II) transport across the PIM are lower than those across the SLM. The recovery factor of Zn(II) ions during transport across PIM and SLM from different mixtures of cations is above 95% after 24 hrs. PIM containing 1-decyl-4-methylimidazole are stable for 120 hrs.     

Conformational Analysis of a High‐Mannose‐Type Oligosaccharide Displaying Glucosyl Determinant Recognised by Molecular Chaperones Using NMR‐Validated Molecular Dynamics Simulation

Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high‐mannose‐type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses ¹³C labelling and lanthanide tagging to detect conformation‐dependent paramagnetic effects by NMR spectroscopy. The NMR‐validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced‐fit mechanism.     

A Synthetic MUC1 Anticancer Vaccine Containing Mannose Ligands for Targeting Macrophages and Dendritic Cells

A MUC1 anticancer vaccine equipped with covalently linked divalent mannose ligands was found to improve the antigen uptake and presentation by targeting mannose‐receptor‐positive macrophages and dendritic cells. It induced much stronger specific IgG immune responses in mice than the non‐mannosylated reference vaccine. Mannose coupling also led to increased numbers of macrophages, dendritic cells, and CD4⁺ T cells in the local lymph organs. Comparison of di‐ and tetravalent mannose ligands revealed an increased binding of the tetravalent version, suggesting that higher valency improves binding to the mannose receptor. The mannose‐coupled vaccine and the non‐mannosylated reference vaccine induced IgG antibodies that exhibited similar binding to human breast tumor cells.     

The Tyrosine Gate of the Bacterial Lectin FimH: A Conformational Analysis by NMR Spectroscopy and X‐ray Crystallography

Urinary tract infections caused by uropathogenic E. coli are among the most prevalent infectious diseases. The mannose‐specific lectin FimH mediates the adhesion of the bacteria to the urothelium, thus enabling host cell invasion and recurrent infections. An attractive alternative to antibiotic treatment is the development of FimH antagonists that mimic the physiological ligand. A large variety of candidate drugs have been developed and characterized by means of in vitro studies and animal models. Here we present the X‐ray co‐crystal structures of FimH with members of four antagonist classes. In three of these cases no structural data had previously been available. We used NMR spectroscopy to characterize FimH–antagonist interactions further by chemical shift perturbation. The analysis allowed a clear determination of the conformation of the tyrosine gate motif that is crucial for the interaction with aglycone moieties and was not obvious from X‐ray structural data alone. Finally, ITC experiments provided insight into the thermodynamics of antagonist binding. In conjunction with the structural information from X‐ray and NMR experiments the results provide a mechanism for the often‐observed enthalpy–entropy compensation of FimH antagonists that plays a role in fine‐tuning of the interaction.     

Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One

Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and β-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.     

Solution NMR Structural Studies of Glycans

In this account, we present a summary of our progress in the study of glycan solution conformations by NMR spectroscopy. We begin by comparing the structural biology of glycans to that of proteins. We continue by defining the challenges in solution structural studies of glycans as we see them and show what we have done to help address those challenges. This includes Residual Dipolar Coupling studies and their hurdles, accurate coupling constant measurement in the presence of strong ¹H‐¹H coupling, direct detection of hydrogen bonds where the NHs, OHs or CHs are the hydrogen bond donors, and on‐cell NMR structural studies. We also suggest some future approaches to glycan structure determination.     

Discovery and development of thiazolo[3,2-a]pyrimidinone derivatives as general inhibitors of Bcl-2 family proteins

A class of compounds with a common thiazolo[3,2‐a]pyrimidinone motif has been developed as general inhibitors of Bcl‐2 family proteins. The lead compound was originally identified in a random screening of a small compound library using a fluorescence polarization‐based competitive binding assay. Its binding to the Bcl‐x_L protein was further confirmed by ¹⁵N‐HSQC NMR experiments. Structural modifications on the lead compound were guided by the outcomes of molecular modeling studies. Among the 42 compounds obtained, a number of them exhibited much improved binding affinities to Bcl‐2 family proteins as compared to the lead compound. The most potent compound, BCL‐LZH‐ 40 , inhibited the binding of BH3 peptides to Bcl‐x_L, Bcl‐2, and Mcl‐1 with inhibition constants (K_i) of 17, 534, and 200 nM , respectively.     

Carbohydrate Analogue Microarrays for Identification of Lectin‐Selective Ligands

Fifty‐five mono‐ and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin‐selective ligands. The microarray study showed that two disaccharide analogues, 28′ and 44′ , selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28′ and 44′ selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.     

Unraveling the Interaction between the LPS O‐Antigen of Burkholderia anthina and the 5D8 Monoclonal Antibody by Using a Multidisciplinary Chemical Approach,with Synthesis,NMR, and Molecular Modeling Methods

The interaction between the O‐chain from the lipopolysaccharide from Burkholderia anthina and a lipopolysaccharide‐specific monoclonal antibody (5D8) has been studied at high resolution by NMR spectroscopy. In particular, the 5D8‐bound epitope of the saccharide entity has been unraveled by a combination of saturation transfer difference (STD) and transferred NOESY (tr‐NOESY) experiments performed on the 5D8/polysaccharide complex. To dissect the fine details of the molecular recognition events, further experiments with simpler carbohydrate ligands were carried out. Thus, experiments were also performed with ad hoc synthesized trisaccharide and hexasaccharide O‐antigen repeating units. By using this multidisciplinary approach (chemical synthesis, NMR spectroscopy and molecular dynamics simulation), determination of the binding epitope and the contribution to the binding of the sugar units composing the O‐chain have been determined.     

The Molecular Basis of Inhibition of Golgi α‐Mannosidase II by Mannostatin A

Mannostatin A is a potent inhibitor of the mannose‐trimming enzyme, Golgi α‐mannosidase II (GMII), which acts late in the N‐glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation‐associated changes in cancer cell surface oligosaccharide structures. Here, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X‐ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor—an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein–ligand complexes are also important determinants of plasticity as well as selectivity of inhibitor binding.