Selective Targeting of Dendritic Cell‐Specific Intercellular Adhesion Molecule‐3‐Grabbing Nonintegrin (DC‐SIGN) with Mannose‐Based Glycomimetics: Synthesis and Interaction Studies of Bis(benzylamide) Derivatives of a Pseudomannobioside

Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) and Langerin are C‐type lectins of dendritic cells (DCs) that share a specificity for mannose and are involved in pathogen recognition. HIV is known to use DC‐SIGN on DCs to facilitate transinfection of T‐cells. Langerin, on the contrary, contributes to virus elimination; therefore, the inhibition of this latter receptor is undesired. Glycomimetic molecules targeting DC‐SIGN have been reported as promising agents for the inhibition of viral infections and for the modulation of immune responses mediated by DC‐SIGN. We show here for the first time that glycomimetics based on a mannose anchor can be tuned to selectively inhibit DC‐SIGN over Langerin. Based on structural and binding studies of a mannobioside mimic previously described by us ( 2 ), a focused library of derivatives was designed. The optimized synthesis gave fast and efficient access to a group of bis(amides), decorated with an azide‐terminated tether allowing further conjugation. SPR inhibition tests showed improvements over the parent pseudomannobioside by a factor of 3–4. A dimeric, macrocyclic structure ( 11 ) was also serendipitously obtained, which afforded a 30‐fold gain over the starting compound ( 2 ). The same ligands were tested against Langerin and found to exhibit high selectivity towards DC‐SIGN. Structural studies using saturation transfer difference NMR spectroscopy (STD‐NMR) were performed to analyze the binding mode of one representative library member with DC‐SIGN. Despite the overlap of some signals, it was established that the new ligand interacts with the protein in the same fashion as the parent pseudodisaccharide. The two aromatic amide moieties showed relatively high saturation in the STD spectrum, which suggests that the improved potency of the bis(amides) over the parent dimethyl ester can be attributed to lipophilic interactions between the aromatic groups of the ligand and the binding site of DC‐SIGN.     

A Glycan Array‐Based Assay for the Identification and Characterization of Plant Glycosyltransferases

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.     

Regioselective 3-O-Substitution of Unprotected Thiodigalactosides: Direct Route to Galectin Inhibitors

The synthesis of tailored bioactive carbohydrates usually comprises challenging (de)protection steps, which lowers synthetic yields and increases time demands. We present here a regioselective single-step introduction of benzylic substituents at 3-hydroxy groups of β-d -galactopyranosyl-(1→1)-thio-β-d -galactopyranoside (TDG) employing dibutyltin oxide in good yields. These glycomimetics act as inhibitors of galectins—human lectins, which are biomedically attractive targets for therapeutic inhibition in, for example, cancerogenesis. The affinity of the prepared glycomimetics to galectin-1 and galectin-3 was studied in enzyme-linked immunosorbent (ELISA)-type assays and their potential to inhibit galectin binding on the cell surface was shown. We used our original in vivo biotinylated galectin constructs for easy detection by flow cytometry. The results of the biological experiments were compared with data from molecular modeling with both galectins. The present work reveals a facile and elegant synthetic route for the preparation of TDG-derived glycomimetics that exhibit differing selectivity and affinity to galectins depending on the choice of 3-O-substitution.     

Hemicarbasucrose: Turning off the Exoanomeric Effect Induces Less Flexibility

Hemicarbasucrose, a close congener of sucrose in which the endocyclic oxygen atom of the glucose moiety is replaced by a methylene group was synthesized for the first time. The conformational behaviour of hemicarbasucrose was studied by a combination of molecular mechanics and NMR spectroscopy (J and NOE data). It was shown that the carbadisaccharide populates two distinct conformational families in solution, the normal syn‐ψ conformation, which is the predominating conformation of the parent natural O‐glycoside, and the anti‐ψ conformation, which has not been detected for the O‐disaccharide. Interestingly, the hemicarbasucrose is less flexible than its natural congener.     

A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.     

α‐O‐Linked Glycopeptide Mimetics: Synthesis,Conformation Analysis,and Interactions with Viscumin,a Galactoside‐Binding Model Lectin

Efficient cycloaddition of a silylidene‐protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide‐mimetic scaffold. Its carbohydrate part was further extended by β1–3‐linked galactosylation. The pyranose rings retain their ⁴C₁ chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo‐anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation‐transfer‐difference NMR spectroscopy by using the galactoside‐specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared α‐O‐linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O‐glycans for lectin blocking and vaccination.     

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.     

Enhancing Potency and Selectivity of a DC-SIGN Glycomimetic Ligand by Fragment-Based Design: Structural Basis

Chemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a glycomimetic antagonist (compound 16 ) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/ 16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain.     

Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with K_a≈20 000 m ^?1, whereas the other one binds an O‐GlcNAcylated peptide with K_a≈70 000 m ^?1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts.     

sp2‐Iminosugar O‐, S‐, and N‐Glycosides as Conformational Mimics of α‐Linked Disaccharides; Implications for Glycosidase Inhibition

The synthesis of mimics of the α(1→6)‐ and α(1→4)‐linked disaccharides isomaltose and maltose featuring a bicyclic sp²‐iminosugar nonreducing moiety O‐, S‐, or N‐linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp²‐iminosugars determines the α‐stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem‐diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α‐glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O‐linked derivative being a more potent inhibitor than the N‐linked analogue; the S‐linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N‐pseudodisaccharide is more rigid than the O‐linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N‐ or O‐linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.     

A Synthetic Lectin for O‐Linked β‐N‐Acetylglucosamine

Changing employment : Receptor 1 binds β‐N‐acetylglucosaminyl (β‐GlcNAc) up to 100 times more strongly than it does glucose. This synthetic lectin shows affinities similar to wheat germ agglutinin (WGA), a natural lectin used to bind GlcNAc. Remarkably, 1 is more selective than WGA. It favors especially the glycoside unit in glycopeptide 2 , a model of the serine‐O‐GlcNAc posttranslational protein modification.

     

Carbohydrate‐Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona

Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so‐called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES‐NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of “stealth” and targeting behavior.     

Stereoselective Synthesis of α‐ and β‐Glycofuranosyl Amides by Traceless Ligation of Glycofuranosyl Azides

A highly stereoselective synthesis of α‐ or β‐glycofuranosyl amides based on the traceless Staudinger ligation of glycofuranosyl azides of the galacto, ribo, and arabino series with 2‐diphenylphosphanyl‐phenyl esters has been developed. Both α‐ and β‐isomers can be obtained with excellent selectivity from a common, easily available precursor. The process does not depend on the anomeric configuration of the starting azide but appears to be controlled by the C2 configuration and by the protection/deprotection state of the substrates. A mechanistic interpretation of the results, supported by ³¹P NMR experiments, is offered and merged with our previous mechanistic analysis of pyranosyl azide ligation reactions.     

Synthesis of an Orthogonally Protected Polyhydroxylated Cyclopentene from l‐Sorbose

The use of l ‐sorbose in the synthesis of functionalized cyclopentene derivatives was accomplished. These cyclopentene derivatives are related to those found in naturally occurring jatrophane frameworks and in other bioactive compounds. The formation of allyl α‐l ‐sorbopyranoside was a key synthetic step. Regioselective introduction of protecting groups was followed by the hydrolysis of the allyl glycoside to furnish a fully protected acyclic l ‐sorbose derivative. This acyclic intermediate was subsequently used to give an orthogonally protected polyhydroxylated cyclopentene, which has potential for further synthesis of bioactive compounds. The protected cyclopentene itself showed a clear cytotoxic activity when tested against a panel of human cancer cell lines (HT29, LS174T, SW620, A549, and HeLa cells).     

Metal‐Atom Impact on the Self‐Assembly of Cup‐and‐Ball Metalloporphyrin–Fullerene Conjugates

A fullerene ammonium derivative has been combined with different metalloporphyrin–crown ether receptors to generate very stable supramolecules. The combination of fullerene–porphyrin and ammonium–crown ether interactions leads to a strong chelate effect as evidenced by a high effective molarity (3.16 M ). The different parameters influencing the stability of the supramolecular ensembles, in particular the nature of the metal in the porphyrin moiety, have been rationalized with the help of theoretical calculations thus providing new insights into fullerene–porphyrin interactions.     

Supramolecular Complexation of Biological Phosphates with an Acyclic Triazolium‐Linked Anthracenyl‐1,3‐Diconjugate of Calix[4]Arene: Synthesis,Characterization, Spectroscopy,Microscopy, and Computational Studies

A triazolium‐anthracenyl calix[4]arene conjugate ( L ) was synthesized by methylating the precursor triazole derivative and then characterized. The potential of the cationic L to differentiate nucleoside triphosphates (NTPs) from their mono‐ and diphosphates was demonstrated. Due to its unique combination of arms with the calix‐platform, a fluorescence enhancement was observed for L with all the NTPs, whereas there is no report with such enhancement being exhibited in case of all the NTPs. This has been supported by the aggregation of L observed from microscopy. Selectivity of L towards NTPs over other phosphates was a result of specific weak interactions, namely, ion–ion, hydrogen bonding and π ??? π, present in the 1:2 complex of L and NTPs (based on ESI MS), which were absent in their congener‐phosphates as delineated by NMR and computational studies. Thus, L stands as a unique receptor for NTPs.     

Conformational Selection in Glycomimetics: Human Galectin‐1 Only Recognizes syn‐Ψ‐Type Conformations of β‐1,3‐Linked Lactose and Its C‐Glycosyl Derivative

The human lectin galectin‐1 (hGal‐1) translates sugar signals, that is, β‐galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound‐state conformations of Galβ‐C‐(1→3)‐Glcβ‐OMe ( 1 ) and its βGal‐(1→3)‐βGlc‐OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)‐NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event. Although free C‐glycoside access four distinct conformers in solution, hGal‐1 recognizes shape of a local minimum of compound 1 , the syn‐Φ/syn‐Ψ conformer, not the structure at global minimum. MD simulations were run to explain, in structural terms, the observed geometry of the complex.