Access to bifunctionalized biomolecular platforms using oxime ligation.

This paper describes an efficient oxime ligation strategy to prepare multivalent conjugates wherein peptides alone or in combination with carbohydrate or oxime groups were coupled to a cyclopeptide scaffold. To demonstrate the versatility of this approach, two classes of conjugates have been prepared. In one class, we attached two or four peptide sequences to the cyclopeptide core together with free oxime groups, while the second class contains an additional substitution with four or two monosaccharides. The well-defined structure of these conjugates was confirmed by high-resolution mass spectrometry.

Re-evaluation of binding properties of recombinant lymphocyte receptors NKR-P1A and CD69 to chemically synthesized glycans and peptides.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

Facile synthesis of nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranosides from ManNAc-oxazoline.

The synthetic procedures for a large-scale preparation of o- and p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside are described. The synthetic pathway employs the glycosylation of phenol with ManNAc oxazoline, followed by nitration of the aromatic moiety yielding a separable mixture of the o- and p-nitrophenyl derivative in a 2:3 ratio.

Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors.

This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a ß-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental ß1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental ß1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.

Dendri-RAFTs: a second generation of cyclopeptide-based glycoclusters.

Synthetic glycoclusters and their related biological applications have stimulated increasing interest over the last decade. As a prerequisite to discovering active and selective therapeuticals, the development of multivalent glycoconjugates with diverse topologies is faced with inherent synthetic and structural characterisation difficulties. Here we describe a new series of molecularly-defined glycoclusters that were synthesized in a controlled manner using a robust and versatile divergent protocol. Starting from a Regioselectively Addressable Functionalized Template (RAFT) carrier, either a polylysine dendritic framework or a second RAFT, then 16 copies of ßGal, αMan, ßLac or cancer-related Thomsen-Freidenreich (αTF) antigen were successively conjugated within the same molecule using oxime chemistry. We thus obtained a new generation of dendri-RAFTs glycoclusters with high glycosidic density and variable spatial organizations. These compounds displaying 16 endgroups were unambiguously characterized by NMR spectroscopy and mass spectrometry. Further biological assays between a model lectin from Canavalia ensiformis (ConA) and mannosylated glycoclusters revealed a higher inhibition potency than the tetravalent counterpart, in particular for the hexadecavalent polylysine skeleton. Together with the efficiency of the synthetic and characterisation processes, this preliminary biological study provided clear evidence of promising properties that make the second generation of cyclopeptide-based glycoclusters attractive for biomedical applications.

Deoxynojirimycin and its hexosaminyl derivatives bind to natural killer cell receptors rNKR-P1A and hCD69.

Deoxynojirimycin (1) and two new related 4-O-hexosaminyl-containing disaccharide mimics, beta-d-TalNAc-(1-->4)-DNJ (4) and beta-d-ManNAc-(1-->4)-DNJ (5), have been studied as agonists of natural killer (NK) cell receptors. As a positive and unexpected result, DNJ (1) displayed a remarkable activation effect towards both NKR-P1A (rat) and CD69 (human) receptors, and a quite similar activity was found for 4 and 5. The synthesis of the two disaccharide mimics is based on an approach that avoids the glycosylation step using known intermediates arising from lactose. The key stage of the synthesis involves the construction of the DNJ unit through an initial C-5 oxidation of the reducing d-glucopyranosyl unit followed by a stereoselective double-reductive aminocyclization of the 1,5-dicarbonyl disaccharide intermediates.

Synthesis of multivalent glycoconjugates containing the immunoactive LELTE peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells.

Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

Carboxylated calixarenes bind strongly to CD69 and protect CD69(+) killer cells from suicidal cell death induced by tumor cell surface ligands.

We have recently identified a new class of high affinity ligands for CD69 leukocyte membrane receptor, carboxylated calixarenes. Of the three compounds investigated here, thiacalix[4]arene had the highest affinity for CD69 in direct binding assays, and proved to be the most specific inhibitor of CD69 identified so far in receptor precipitation and cellular activation experiments. Carboxylated calixarenes also proved effective at protection of CD69(high) lymphocytes from apoptosis triggered by a multivalent ligand or antibody. Thus, carboxylated calixarenes set a new paradigm for noncarbohydrate ligands for CD69 making them attractive for protection of killer cells in combined animal tumor therapies.

Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase--a way to new glycodrugs and materials.

Human placental beta1,4-galactosyltransferase-I (EC 2.4.1.38) transfers the galactosyl moiety from UDP-Gal to various GlcNAc or Glc acceptors in vivo. Here, we describe the construction of its Y284L mutant as a His(6)propeptide-catbeta4GalT1 construct, in which the Gal-transferase activity was totally abolished in favor of its GalNAc-transferase activity. We used this mutant in the synthesis of three mono- and bivalent LacdiNAc glycomimetics with good yields. These compounds proved to be powerful ligands of two activation receptors of natural killer cells, NKR-P1 and CD69. A synthetic bivalent tethered di-LacdiNAc is the best currently known precipitation agent for both of these receptors and has promising potential for the development of immunoactive glycodrugs.

Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces.

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized beta-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human beta1,4-galactosyltransferase-1 and beta1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His(6)CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.

N-acetyl-D-glucosamine substituted calix[4]arenes as stimulators of NK cell-mediated antitumor immune response.

A series of calixarenes substituted with 2-acetamido-2-deoxy-beta-D-glucopyranose linked by a thiourea spacer was prepared and tested for binding activity to heterogeneously expressed activation receptors of the rat natural killer cells NKR-P1, and the receptor CD69 (human NK cells, macrophages). In the case of NKR-P1, the binding affinity of beta-D-GlcNAc-substituted calixarenes carrying two or four sugar units was in a good agreement with the inhibitory potencies of the linear chitooligomers (chitobiose to chitotetraose) reported previously. The influence of GlcNAc substitution of the calixarene skeleton on binding affinity for CD69 receptor was more profound and the 5,11,17,23-tetrakis[N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-thioureido]-25,26,27,28-tetrapropoxycalix[4]arene (cone) (1) proved to be the best CD69 ligand identified to date. Lower GlcNAc substitution led to dramatic decrease of the binding activity (by about 1.5 order of magnitude per one GlcNAc unit). The immunostimulating activity results with the newly synthesized GlcNAc tetramers on calixarene scaffolds exhibited stimulation of natural cytotoxicity of human PBMC in concentrations 10(-4) and 10(-8)M. These calix-sugar compounds were superior to the previously tested PAMAM-GlcNAc(8)5.