David Bonnaffé

New accesses to l-iduronyl synthons

(PhS)3CLi adds with a total l-ido selectivity onto 3-O-benzyl-1,2-O-isopropylidene-α-d-xylo-dialdose 2, opening the way to the most efficient preparation of 1,2,4-tri-O-acetyl-3-O-benzyl-l-iduronyl synthon 8. Alternatively, in view of combinatorial syntheses, aldehyde 2 allows a good access to vinylic l-ido and d-gluco synthons which may be converted into uronic acid by a sequence involving a new aldehyde oxidation by m-CPBA in aqueous solution.

HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides

Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO(3) moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate-derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na](-) were easily observed on mass spectra. MALDI-MS characterization of challenging analytes such as heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide was successfully achieved.

Heparan Sulfate Mimicry A SYNTHETIC GLYCOCONJUGATE THAT RECOGNIZES THE HEPARIN BINDING DOMAIN OF INTERFERON-γ INHIBITS THE CYTOKINE ACTIVITY

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-γ (IFNγ), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNγ monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNγ. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Å-long spacer termed 2O10, displays high affinity for the cytokine and inhibits IFNγ-HS binding with an IC50 of 35–40 nm. Interestingly, this molecule also inhibits the binding of IFNγ to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNγ activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNγ recognition, providing a new strategy to inhibit IFNγ in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create”tailor-made“sequences based on the HS template to isolate therapeutic activities.

Repairing the Thiol‐Ene Coupling Reaction

Thiol-ene coupling (TEC) reactions emerged as one of the most useful processes for coupling different molecular units under reaction mild conditions. However, TEC reactions involving weak CH bonds (allylic and benzylic fragments) are difficult to run and often low yielding. Mechanistic studies demonstrate that hydrogen-atom transfer processes at allylic and benzylic positions are responsible for the lack of efficiency of the radical-chain process. These competing reactions cannot be prevented, but reported herein is a method to repair the chain process by running the reaction in the presence of triethylborane and catechol. Under these reaction conditions, a unique repair mechanism leads to an efficient chain reaction, which is demonstrated with a broad range of anomeric O-allyl sugar derivatives including mono-, di-, and tetrasaccharides bearing various functionalities and protecting groups.

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase

Background: Sulfatases are emerging as key adaptive tools of commensal bacteria to their host. Results: The first bacterial endo-O-sulfatase and three exo-O-sulfatases from the human commensal Bacteroides thetaiotaomicron, specific for glycosaminoglycans, have been discovered and characterized. Conclusion: Commensal bacteria possess a unique array of highly specific sulfatases to metabolize host glycans. Significance: Bacterial sulfatases are much more diverse than anticipated. Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.

STEREOSELECTIVITY CONTROL IN ANOMERIC O-ALKYLATION. APPLICATION TO THE SYNTHESIS OF C2 SYMMETRIC GLYCOCONJUGATES

Abstract Tetrabutylammonium salts strongly influence the stereoselectivity of O-anomeric alkylation and allows to shift from β to α selectivity. Allyl glucosaminide 7 prepared in this way, was used to synthesize the new type of C2 symmetric neoglycoconjugates 1a-c.

Mixture synthesis and "charge tagging" based demixing: an efficient strategy for the preparation of heparan sulfate libraries.

ReceiVed January 4, 2008 Heparan sulfate (HS), a member of the glycosaminoglycan family, is a linear sulfated polysaccharide interacting with and regulating the activity of numerous proteins. HS is composed of an alternation of uronic acids units (1f4) linked to R-(1f4)-linked 2-deoxy-2-aminoglucosyl units. It is one of the most heterogeneous biopolymers because various epimerization and sulfation patterns (sulfoforms) may occur along the chain. The uronic acid may be either D-glucuronic or L-iduronic, while O-sulfation may occur on position 2 of the uronic acid and 3 or 6 of the amino sugar. The glucosamine nitrogen may be sulfated, acetylated, or less frequently, unmodified, leading to 48 possible disaccharides. The diversity grows exponentially with the polymer length, leading to 2304 possible tetrasaccharides, 110592 hexasaccharides, and more than 5 × 10 octasaccharides. After the commercialization of Arixtra, a synthetic HS-like pentasaccharide that catalyzes the activity of antithrombin III, it is anticipated that new drugs will emerge from the identification of HS fragments able to control the activity of other therapeutic targets, such as cytokines, chemokines, FGFs, or other growth factors. The development of methodologies allowing access to this huge molecular diversity, that is, the preparation of HS fragments with defined structures, is thusat the forefrontofcurrent research inglycochemistry. The combinatorial nature of the HS polymer has struck several glycochemists and various modular synthesis schemes have been proposed, but to our knowledge, these strategies have not yet been implemented into multiparallel or mixture syntheses. In a domain close to HS fragment synthesis, we have shown that solution-phase split-pool synthesis was an ideal tool to generate all the disaccharide sulfoforms of chondroitin sulfate (another glycosaminoglycan). However, although this work, as well as studies performed in others fields of glycochemistry, have shown, in the late 1990s, that mixture synthesis could be efficiently used to prepare oligosaccharide libraries, there have been few further developments of such methodologies, mainly because oligosaccharide mixtures are not really suitable for biological tests. Recently, the use of mixture synthesis followed by final demixing has been introduced in other area of organic chemistry, and this is beneficial both from the advantages of mixture synthesis and from the delivery of pure compounds for biological tests. We thus decided to test whether such an approach could be used to speed up the preparation of HS fragment libraries. On the basis of chemistry we developed in the past decade in the synthesis of individual HS oligosaccharides, we planned to perform mixture syntheses of HS fragments by oligomerizing the suitably protected disaccharide building blocks 1–3 (Figure 1). For the demixing step, we decided to rely on the synthetic scheme being designed to generate, prior to the final hydrogenolysis step, mixtures of oligosaccharides containing different sulfate/benzyl groups ratios, which are thus “charge tagged”. As example, tetrasaccharide 11 contains eight charges/five Bn groups; tetrasaccharide 12 has seven charges/six Bn groups, and tetrasaccharide 13 has six charges/seven Bn groups. A mixture of those three compounds should thus be easily demixed by chromatography on a RP-C18 stationary phase. In this approach, charge tagging is used in a way similar to the fluorous tagging recently introduced by the group of D. P. Curran. As a poof of concept for this “mixture synthesis/demix” approach, we decided to prepare the small library of isolated tetrasaccharides 14–16 (scheme 1). Donor 4 can be obtained in high yield from disaccharide 1. On the other hand, treatment of disaccharides 1–3 with TFA gave acceptors 5–7 in nearly quantitative yields. We first wanted to perform some model experiments on small scale to check whether a glycosylation reaction involving one donor and three acceptors could be performed with enough synthetic efficiency for a mixture synthesis. It is indeed known that fragments from the repetitive sequence of heparin, may be assembled with high stereoselectivities and yields from building block similar to 1. However, when working on more heterogeneous structures than the repeating unit of heparin, glycosylation reactions become less predictable especially, but not only, when working with glucuronic acceptors. For these model experiments, the three acceptors 5–7 were mixed in an equimolar ratio and condensed with 1.3 equiv of imidate 4. The reactions were performed in CH2Cl2 at -30 °C in the presence of TMSOTf (Scheme 1). After three hours at this temperature, the reaction was quenched with NEt3, warmed to room temperature (RT), and directly purified by exclusion chromatography on Sephadex LH-20. The fractions containing the highest molecular weight compounds were pooled and analyzed by RP-C18 HPLC. * To whom correspondence should be addressed. Phone: 33 (0)1 69 15 72 33. Fax: 33 (0)1 69 15 47 15 . E-mail: david.bonnaffe@icmo.u-psud.fr. Figure 1. Set of disaccharide building blocks for the combinatorial synthesis of HS fragments. J. Comb. Chem. 2008, 10, 166–169 166

Efficient selective preparation of methyl-1,2,4-tri-O-acetyl-3-O-benzyl-β-l-idopyranuronate from methyl 3-O-benzyl-l-iduronate

Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-L-idopyranuronate 6beta/6alpha, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key synthon in heparin/heparan sulfate synthesis. The 1H and 13C NMR spectra of the furanose-pyranose mixture of 4, after dissolution and equilibration in d(4)-methanol, were fully assigned allowing to expect that 4 could crystallise in the beta-pyranose form. New acetylation conditions able to trap this form were subsequently devised, allowing the isolation of 83% of pure 6beta by simple crystallisation, along with 9% of the 6beta/6alpha mixture. This represents a major advantage over the previously published procedure, especially on multigram scales.

A synthetic heparan sulfate-mimetic peptide conjugated to a mini CD4 displays very high anti- HIV-1 activity independently of coreceptor usage.

The HIV-1 envelope gp120, which features both the virus receptor (CD4) and coreceptor (CCR5/CXCR4) binding sites, offers multiple sites for therapeutic intervention. However, the latter becomes exposed, thus vulnerable to inhibition, only transiently when the virus has already bound cellular CD4. To pierce this defense mechanism, we engineered a series of heparan sulfate mimicking tridecapeptides and showed that one of them target the gp120 coreceptor binding site with μM affinity. Covalently linked to a CD4-mimetic that binds to gp120 and renders the coreceptor binding domain available to be targeted, the conjugated tridecapeptide now displays nanomolar affinity for its target. Using solubilized coreceptors captured on top of sensorchip we show that it inhibits gp120 binding to both CCR5 and CXCR4 and in peripheral blood mononuclear cells broadly inhibits HIV-1 replication with an IC(50) of 1 nM.

First Synthesis of Heparan Sulfate Tetrasaccharides Containing both N‐Acetylated and N‐Unsubstituted Glucosamine—Search for Putative 10E4 Epitopes

Heparan sulfate (HS) is a linear and sulfated polysaccharide, composed of alternating d-glucosamine (GlcN) and d-glucuronic (GlcA) or l-iduronic (IdoA) acid residues. It is endowed with remarkable biological properties, as it is found at the cell surface and in the extracellular matrix, where it interacts and regulates the localization, half-lives, and bioactivities of numerous proteins. 2] HS chains are thus involved in various fundamental biological processes including embryonic development, metastasis, inflammation, viral cell entry, thrombosis as well as neurodegenerative pathologies such as Alzheimer’s disease and transmissible spongiform encephalopathies (TSEs). HS sequences are dynamically regulated. Diverse patterns of N-substitution and/or O-sulfation together with epimerization of GlcA to IdoA occur and are exposed at the cell surface allowing specific interactions with various proteins. As HS-derived oligosaccharides are often heterogeneous and difficult to purify, the use of chemically synthesized oligosaccharides is an effective way of determining unequivocally the structural requirements for binding by a given protein. This approach has led to the development of new therapeutics. The monoclonal antibody 10E4 is commonly used to detect HS in tissues and glycoconjugates. Immunohistochemical and neoglycolipid-based immunochemical studies have placed the epitope of 10E4 at the centre of prion infectivity and disease progression. It has been proposed that the minimum sequence recognized by the 10E4 antibody is an unusual nonsulfated HS tetrasaccharide containing an internal N-unsubstituted GlcN residue. It has been suggested that the nonreducing end residue is a GlcA, as heparin lyase III, used to partially depolymerize HS and release an antigen-positive tetrasaccharide, is known to act on the linkages between GlcNAc/GlcNS and GlcA as the “primary” site in the low sulfated domains, although it can also cleave GlcNS6S-IdoA in the highly sulfated domain as