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.     

Rim-differentiation vs. mixture of constitutional isomers: A binding study between pillar[5]arene-based glycoclusters and lectins from pathogenic bacteria

Macrocycle-based glycoclusters, on account of their promising anti-adhesive properties against bacteria, are potential therapeutic alternatives to classic antibiotics through the much less explored anti-adhesive strategy. In this study, a series of constitutionally-pure pentavalent glycoclusters was prepared by conjugating assorted azido-carbohydrates onto a penta-propargyl rim-differentiated pillar[5]arene (RD-P[5]) scaffold through Cu(I)-catalyzed azide–alkyne cycloaddition “click” reactions. Their binding towards therapeutically relevant bacterial lectins, such as LecA and LecB from Pseudomonas aeruginosa and concanavalin A (ConA), were evaluated subsequently by isothermal titration calorimetric studies. Most of these isomer-free RD-P[5] pentavalent glycoclusters, except the fucosylated ones, display good affinities to lectins. Nonetheless, the dissociation constants observed are similar to those displayed by an analogous pentavalent glycocluster consisting of four P[5] constitutional isomers, in which the RD-P[5] component merely accounts for 7% in the mixture. Our results revealed that high constitutional purity is not essential for achieving effective multivalent interactions between P[5]-based glycoclusters and lectins, presumably as a result of the conformationally labile nature of the P[5] scaffold. This information provides valuable design principles for low-cost and facile syntheses of glycosylated P[5]s for biomedical applications.     

Mechanistic Insight into Heptosyltransferase Inhibition by using Kdo Multivalent Glycoclusters

The synthesis of unprecedented multimeric Kdo glycoclusters based on fullerene and calix[4]arene central scaffolds is reported. The compounds were used to study the mechanism and scope of multivalent glycosyltransferase inhibition. Multimeric mannosides based on porphyrin and pillar[5]arenes were also generated in a controlled manner. Twelve glycoclusters and their monomeric ligands were thus assayed against heptosyltransferase WaaC, which is an important bacterial glycosyltransferase that is involved in lipopolysaccharide biosynthesis. It was first found that all the multimers interact solely with the acceptor binding site of the enzyme even when the multimeric ligands mimic the heptose donor. Second, the novel Kdo glycofullerenes displayed very potent inhibition (K_i=0.14 μm for the best inhibitor); an inhibition level rarely observed with glycosyltransferases. Although the observed “multivalent effects” (i.e., the enhancement of affinity of a ligand when presented in a multimeric fashion) were in general modest, a dramatic effect of the central scaffold on the inhibition level was evidenced: the fullerene and the porphyrin scaffolds being by far superior to the calix‐ and pillar‐arenes. We could also show, by dynamic light scattering analysis, that the best inhibitor had the propensity to form aggregates with the heptosyltransferase. This aggregative property may contribute to the global multivalent enzyme inhibition, but probably do not constitute the main origin of inhibition.     

Spatially Directional Resorcin[4]arene Cavitand Glycoconjugates for Organic Catalysis

The synthesis of novel spatially directional multivalent resorcin[4]arene cavitand glycoconjugates (RCGs) and their ability to catalyze organic reactions is reported. The β‐d ‐glucopyranoside moieties on the upper rim of the “bowl”‐shaped resorcin[4]arene cavitand core are capable of multiple hydrogen‐bond interactions resulting in a pseudo‐cavity, which has been investigated for organic transformations in aqueous media. The RCGs have been demonstrated to catalyze thiazole formation, thiocyanation, copper(I)‐catalyzed azide alkyne cycloaddition (CuAAC), and Mannich reactions; they impart stereoselectivity in the three‐component Mannich reaction. Thermodynamic values obtained from ¹H diffusion‐ordered spectroscopy (DOSY) experiments suggest that the upper saccharide cavity of the RCG and not the resorcin[4]arene cavity is the site of the complexation event.     

An Orthogonally Protected alpha,alpha-bis(aminomethyl)-beta-alanine building block for the construction of glycoconjugates on a solid support

Synthetic glycoclusters are extensively used as mimetics of naturally occurring, multivalent carbohydrate ligands in various glycobiological applications. Their preparation, however, is far from trivial, and it still is a limiting factor in the study of carbohydrate binding. We herein report the synthesis of an orthogonally protected building block, N-Alloc-N'-Boc-N' '-Fmoc-alpha,alpha-bis(aminomethyl)-beta-alanine (1), and its use in the preparation of triantennary peptide glycoclusters (21-24) on a solid support. The assembly of the clusters involves removal of the amino protections of the solid-supported branching unit 1 in the order Fmoc, Boc, and Alloc, and subsequent coupling of peracetylated O-(glycopyranosyl)-N-Fmoc-L-serine pentafluorophenyl esters (galactose, glucose, mannose, and ribose) to each amino group exposed.     

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.     

Chiral self-assembly of lactose functionalized perylene bisimides as multivalent glycoclusters

A chiral self-assembly has been constructed from a d-lactose functionalized perylene bisimide derivative, showing right-handed supramolecular stacking induced by the chiral d-lactose moieties. Benefiting from the grafting of D-lactose, the self-assembled multivalent glycoclusters exhibited specific binding with PNA lectin.     

Self-recognition, structure, stability, and guest affinity of pyrogallol[4]arene and resorcin[4]arene capsules in solution

In the present study, we used diffusion NMR to probe the structures and characteristics of the products obtained from the self-assembly of resorcin[4]arenes 1a and 1b and pyrogallol[4]arenes 2a and 2b in CDCl(3) solutions. It was found that all four molecules self-assemble into hexameric capsules. The hexameric capsules of pyrogallol[4]arenes 2a and 2b were found to be more stable than the capsules of resorcin[4]arenes 1a and 1b in polar media. We also studied the role of water molecules in the self-assembly of the different capsules and found that water molecules are part of the hexameric capsules of resorcin[4]arenes 1a and 1b but not in the capsules of pyrogallol[4]arenes 2a and 2b. It was found that the self-assembly process between the resorcin[4]arenes and pyrogallol[4]arenes proceeds with self-recognition. When mixing two macrocycles of different types in a chloroform solution, no heterohexamers are formed, only the capsule constructed from the same macrocycle is detected. However, when two resorcin[4]arenes (i.e., 1a and 1b) or two pyrogallol[4]arenes (i.e., 2a and 2b) are mixed, heterohexamers are formed over time. In addition, we found that resorcin[4]arenes and pyrogallol[4]arenes differ significantly in their guest affinity. The capsules of 1a and 1b can accommodate both the tertiary alkylamines and their respective ammonium salts, while the capsules of 2a and 2b encapsulate only the neutral tertiary alkylamines.     

Synthesis of porphyrin conjugates based on conformationally rigid and flexible resorcin[4]arene frameworks

Porphyrin conjugates were synthesized based on conformationally rigid, bowl shaped cavitand, and flexible unfirm resorcin[4]arenes. The influence of the resorcin[4]arene fragment on the porphyrin fluorescence is investigated.     

AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL) filaments induced by multivalent glycoclusters

Atomic force microscopy reveals that Pseudomonas aeruginosa LecA (PA-IL) and a tetra-galactosylated 1,3-alternate calix[4]arene-based glycocluster self-assemble according to an aggregative chelate binding mode to create monodimensional filaments. Lectin oligomers are identified along the filaments and defects in chelate binding generate branches and bifurcations. A molecular model with alternate 90° orientation of LecA tetramers is proposed to describe the organisation of lectins and glycoclusters in the filaments.     

Synthesis of Multivalent Carbohydrate‐Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo‐(1→6)‐β‐D ‐glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme‐linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub‐micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.     

Covalently assembled resorcin[4]arenes and molecular tweezers: a chiral recognition rationale by NMR

Chiral diamides and tetramidic resorcin[4]arenes deriving from (R,R)-1,2-diaminocyclohexane and (S,S)-1,2-diphenylethylendiamine, and a valine containing resorcin[4]arene have been compared by NMR in the enantiodiscrimination of mandelic acid. The relevance of cooperation between side arms and external surface of resorcin[4]arene core has been ascertained.     

Unprecedented gas-phase chiroselective logic gates

The gas-phase encounters between 2-aminobutane and proton-bound chiral resorcin[4]arene/nucleoside complexes behave in the gas phase as supramolecular "chiroselective logic gates" by releasing the nucleoside depending on the resorcin[4]arene and the 2-aminobutane configurations.     

Water-soluble supramolecular bowls formed by intra-clipping of resorcin[4]arene-based ligands with Pd(II) ions

Bowl-shaped superstructures have been constructed by intra-clipping of resorcin[4]arene derivatives with two equivalents of (en)Pd(NO3)2 in water. The binding phenomena of the aromatic carboxylates were proven to be both enthalpically and entropically favoured.     

Scandium triflate-catalyzed cyclocondensation of 1,3-dialkoxybenzenes with 1,3,5-trioxane. Formation of resorcin[4]arenes and confused resorcin[4]arenes

The cyclocondensation of 1,3-dialkoxybenzenes with 1,3,5-trioxane was catalyzed by Sc(OTf)₃ to produce resorcin[4]arene octaalkyl ethers as the major products. In addition, ‘confused’ resorcin[4]arene octaalkyl ethers bearing one alkoxy group at the intra-annular position were obtained as the minor products. The variable temperature ¹H NMR spectroscopy showed that the alkoxy group at the intra-annular position cannot pass through the macrocyclic annulus, indicating a rigid molecular framework. The structure of the ethoxy derivative of the confused resorcin[4]arene was determined by X-ray crystallography.     

A Novel and Effective Strategy for the Construction of "Tube-Like" Double Resorcin[4]arenes

The synthesis of rigid tube-shape structures from resorcin[4]arene unitscombined in the ``head-to-tail'' manner is described.Tetrabromomethylenecalix-[4]resorcinarene with all OH-groups connected viamethylene bridges was used as a starting material. Reaction withp-hydroxybenzaldehyde led to the tetraaldehyde derivative and the following condensation with resorcinol resulted in formation of a third aromatic rim. An electrochemical study comparing properties of both resorcin[4]arene and itstube-shaped analogs was performed.     

Sc(OTf)3-catalyzed cyclocondensation of 2-propylresorcinol with diethoxymethane. Formation and fragmentation of resorcin[n]arenes

The cyclocondensation of an equimolar amount of 2-propylresorcinol with diethoxymethane in the presence of Sc(OTf)₃ in acetonitrile produced a mixture of resorcin[n]arenes (n=4-7) with a kinetically controlled product distribution. However, the reaction with an excess amount of diethoxymethane produced the thermodynamically favored resorcin[4]arene as the major product. The fragmentation/recombination mechanism of the resorcin[n]arenes was discussed.     

Process Development for Separation of Conformers from Derivatives of Resorcin[4]arenes and Pyrogallol[4]arenes

Macrocyclic compounds, such as resorcin[4]arenes and pyrogallol[4]arenes, have proven to be useful building blocks in the construction of supramolecular organic frameworks (SOFs) because of their unique bowl‐like shape and ability to interact through variety of intermolecular interactions. Herein, we report the synthesis and crystal structures of two functionalized resorcin[4]arenes and pyroagllol[4]arenes, 4‐hydroxyphenylresorcin[4]arenes, and 4‐hydroxyphenylpyrogallol[4]arenes. These phenyl‐functionalized macrocycles usually have different conformers, such as cone, boat, chair, saddle, and diamond. The successful separation of predominant conformers from the crude product was carried out with solvent‐extraction technique. The shape and molecular arrangement of these conformers in the individual crystal structure was verified with single‐crystal X‐ray diffraction studies.     

Selective steroid recognition by a partially bridged resorcin[4]arene cavitand

The partially bridged resorcin[4]arene cavitand featuring a cleft-shaped recognition site formed by two anti-quinoxaline bridges and four convergent HO-groups was prepared in three steps and characterised by X-ray crystallography; cavitand was found to be a selective receptor for steroidal substrates in CDCl3, with the best binding observed for steroids with a flat A-ring and two H-bonding sites on rings A and C/D.     

Synthesis of Methylene-Bridged Resorcin[4]arene Dimers

Methylene-bridged resorcin[4]arene dimers were synthesized by the Sc(OTf)₃-catalyzed hydroxymethylation of partially acetylated resorcin[4]arenes, and their preliminary complexation properties with tetraethylammonium ion were examined in CD₃OD by ¹H NMR spectroscopy. The dimers adopted a closed capsular conformation in this solvent and bound the guest molecule into their cavity.