Jean-Pierre Praly

Achieving High Affinity towards a Bacterial Lectin through Multivalent Topological Isomers of Calix[4]arene Glycoconjugates

A family of seven topologically isomeric calix[4]arene glycoconjugates was prepared through the synthesis of a series of alkyne-derivatised calix[4]arene precursors that are suitable for the attachment of sugar moieties by microwave-assisted copper(I)-catalysed azide-alkyne cycloaddition (CuAAC). The glycoconjugates thus synthesised comprised one mono-functionalised derivative, two 1,2- or 1,3-divalent regioisomers, one trivalent and three tetravalent topoisomers in the cone, partial cone or 1,3-alternate conformations. The designed glycoconjugates were evaluated as ligands for the galactose-binding lectin PA-IL from the opportunistic bacterium Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. Binding affinities were determined by isothermal titration calorimetry (ITC), and the interaction with the lectin was shown to be strongly dependant on both the valence and the topology. Whereas the trivalent conjugate displayed enhanced affinity when compared to a monosaccharide model, the tetravalent conjugates are to-date the highest-affinity ligands measured by ITC. The topologies presenting carbohydrates on both faces of calixarene are the most potent ones with dissociation constants of approximately 200 nM. Molecular modelling suggests that such a multivalent molecule can efficiently chelate two of the binding sites of the tetrameric lectin; this explains the 800-fold increase of affinity achieved by the tetravalent molecule. Surface plasmon resonance (SPR) experiments confirmed that this glycoconjugate is the strongest inhibitor for binding of PA-IL to galactosylated surfaces for potential applications as an anti-adhesive agent.

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.

Inhibition of Glycogen Phosphorylase in the Context of Type 2 Diabetes, with Focus on Recent Inhibitors Bound at the Active Site

Among the variety of approaches for pharmacological intervention in T2DM, the inhibition of GP with the aim of reducing hepatic glucose output is a validated and thoroughly investigated strategy. Both the academia and health companies participate in the search of potent inhibitors, that might be suitable for long-term treatment. As several inhibitory sites have been identified for GP, interest focuses mainly on structures that can bind at either the catalytic, the allosteric, or the new allosteric sites. Glucose-based motifs and azasugars that bind at the active site constitute the most populated class of GPis. During the last two years, significant progresses have been made, since newly proposed motifs have K(i) values in the low micromolar and even sub- micromolar range. Without ignoring previously reported structures, new series based on β-D-glucopyranosyl-pyrimidine, D-glucopyranosylidene-spiro-isoxazoline and D-glucopyranosylidene-spiro-oxathiazole motifs appear promising. A representative from this last series, with a 2-naphthyl residue was identified as the most potent GPi to date (K(i) = 0.16 µM). While no inhibition was found for sulfonium analogs, D-DAB remains the best inhibitor among five and six-membered iminosugars that showed inhibitory properties toward GP. A study of glucagon-induced glucose production in primary rat hepatocytes has suggested that amylo-1,6-glucosidase inhibitors in combination with GPis may lower glucose level in T2DM. Considering the limitations found for other potent GPis binding at other sites and the complexity of pharmacological development, the potential of glucose-based GPis is still not established firmly and more tests with cells, tissues, animals are required to better establish the risks and merits of these structures, as antidiabetic drugs. Further studies might also confirm other directions where glucose-based GPis could be useful.

Glucose-based spiro-isoxazolines: a new family of potent glycogen phosphorylase inhibitors.

A series of glucopyranosylidene-spiro-isoxazolines was prepared through regio- and stereoselective [3+2]-cycloaddition between the methylene acetylated exo-glucal and aromatic nitrile oxides. The deprotected cycloadducts were evaluated as inhibitors of muscle glycogen phosphorylase b. The carbohydrate-based family of five inhibitors displays K(i) values ranging from 0.63 to 92.5 microM. The X-ray structures of the enzyme-ligand complexes show that the inhibitors bind preferentially at the catalytic site of the enzyme retaining the less active T-state conformation. Docking calculations with GLIDE in extra-precision (XP) mode yielded excellent agreement with experiment, as judged by comparison of the predicted binding modes of the five ligands with the crystallographic conformations and the good correlation between the docking scores and the experimental free binding energies. Use of docking constraints on the well-defined positions of the glucopyranose moiety in the catalytic site and redocking of GLIDE-XP poses using electrostatic potential fit-determined ligand partial charges in quantum polarized ligand docking (QPLD) produced the best results in this regard.

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.

DNA-directed immobilisation of glycomimetics for glycoarrays application: Comparison with covalent immobilisation, and development of an on-chip IC50 measurement assay

Glycoarrays are powerful tools for the understanding of protein/carbohydrate interactions and should find applications in the diagnosis of diseases involving these interactions. Immobilisation of the carbohydrate probe is a key issue in the elaboration of high performance devices. In the present study, we have compared the fluorescent signal intensity and determined the lower detection limit of glycoconjugates immobilised at two concentrations (0.5 and 25 microM) by DNA-directed immobilisation (DDI), to glycoconjugates covalently immobilised on the solid support (borosilicate glass slide). At 0.5 microM, DDI led to a stronger fluorescence signal (by a factor of 4.5) and to a lower detection limit (20 nM) than covalent immobilisation (higher than 200 nM). We also report the development of an IC(50) measurement assay of DDI immobilised glycoconjugates. We found that the relative affinity per galactose residue of RCA 120 for glycoconjugates bearing one or three galactose residues was different by a factor of 23 when measured under IC(50) conditions or by direct fluorescence reading.

CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.

Photohalogenation of glycopyranosyl halides: An expedient route to c-1 gem dihalogenated sugars

Resume Free-radical halogenation of peracetylated α and β-glycopyranosyl halides of 4C1-D chair conformation takes place at C-1 or C-5 with an α-stereoselectivity. However the less reactive α-chloride and bromide moieties at first undergo a dehydrohalogenation reaction. β-Chlorides when treated with N-bromosuccinimide give new C-1 gem chlorobromo sugars in 65–70 % yield while new C-5 halogenated compounds are obtained predominantly with β -fluorides or when chlorination is carried out with sulfuryl chloride SO2Cl2. The peracetylated C-1 gem chlorobromo derivative of gluco configuration can be cleanly dehydrobrominated to yield a C-1 chlorinated glucal. It also reacts chemio and stereoselectively in the presence of silver fluoride to give the corresponding new C-1 gem chlorofluoro or difluoro derivatives, either in 70 % yield, depending on the stoechiometry. All these new compounds exhibit a good to excellent stability. 19F- n.m.r. of three peracetylated 1-halogeno- β -D-glucopyranosyl fluorides shows that the JF,C3 coupling constants increase with the increasing electronegativity of the geminal axial halogen while JF,H decreases.

Synthesis of lactosylated glycoclusters and inhibition studies with plant and human lectins

Under microwave activation, the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between an azido-functionalized lactoside and tetra-alkynylated core scaffolds (one porphyrin and three topological conformers of calix[4]arenes) afforded four lactosylated glycoclusters in high yields. The glycoclusters were then evaluated and compared to a monovalent probe as ligands of two lectins: ECA from legume plant Erythrina cristagalli and recombinant human galectin-1. Micromolar inhibition concentrations and IC(50) values were measured by inhibition of hemagglutination (HIA) or enzyme-linked lectin assays (ELLA), respectively for these glycoclusters for binding to ECA. A slight binding preference was identified for the porphyrin and the 1,3-alternate calixarene scaffolds. Similar inhibition studies were performed for galectin-1 by HIA and surface plasmon resonance (SPR) analyses. A strong selectivity was observed for the porphyrin and cone conformer topologies under HIA experimental conditions but these could not be confirmed using SPR analysis. This difference in the inhibitory properties based on two techniques confirmed the need for multiple complementary analyses for in-depth and accurate analysis of the inhibitory properties of multivalent glycoconjugates to multivalent lectins.

Specific recognition of lectins by oligonucleotide glycoconjugates and sorting on a DNA microarray.

Two glycoconjugates bearing different DNA tags are mixed in solution with lectins; both interact with their specific lectin and the resulting complexes are sorted, according to their DNA sequences, at the surface of micro-reactors bearing the immobilised complementary DNA sequences.