Caroline Clavel

Gold Manno‐Glyconanoparticles: Multivalent Systems to Block HIV‐1 gp120 Binding to the Lectin DC‐SIGN

The HIV envelope glycoprotein gp120 takes advantage of the high-mannose clusters on its surface to target the C-type lectin dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on dendritic cells. Mimicking the cluster presentation of oligomannosides on the virus surface is a strategy for designing carbohydrate-based antiviral agents. Bio-inspired by the cluster presentation of gp120, we have designed and prepared a small library of multivalent water-soluble gold glyconanoparticles (manno-GNPs) presenting truncated (oligo)mannosides of the high-mannose undecasaccharide Man(9)GlcNAc(2) and have tested them as inhibitors of DC-SIGN binding to gp120. These glyconanoparticles are ligands for DC-SIGN, which also interacts in the early steps of infection with a large number of pathogens through specific recognition of associated glycans. (Oligo)mannosides endowed with different spacers ending in thiol groups, which enable attachment of the glycoconjugates to the gold surface, have been prepared. manno-GNPs with different spacers and variable density of mannose (oligo)saccharides have been obtained and characterized. Surface plasmon resonance (SPR) experiments with selected manno-GNPs have been performed to study their inhibition potency towards DC-SIGN binding to gp120. The tested manno-GNPs completely inhibit the binding from the micro- to the nanomolar range, while the corresponding monovalent mannosides require millimolar concentrations. manno-GNPs containing the disaccharide Manalpha1-2Manalpha are the best inhibitors, showing more than 20 000-fold increased activity (100 % inhibition at 115 nM) compared to the corresponding monomeric disaccharide (100 % inhibition at 2.2 mM). Furthermore, increasing the density of dimannoside on the gold platform from 50 to 100 % does not improve the level of inhibition.

Multivalent manno-glyconanoparticles inhibit DC-SIGN-mediated HIV-1 trans-infection of human T cells.

Viral entry is a critical step in human immunodeficiency virus (HIV) infection that takes place through a series of sequential interactions between the envelope glycoprotein gp120, cellular receptor CD4, and coreceptors CCR5 or CXCR4 on T cells. Through a mechanism named trans-infection, dendritic cells (DCs) efficiently transfer the virus to T lymphocytes where viral replication occurs. Interference in HIV transmission in the DC–lymphocyte synapse is a preferential but complex target in the development of new compounds displaying anti-HIV activity. HIV–DC interaction is mediated by the glycans of gp120 and the C-type lectin DC-SIGN (dendritic cell-specific ICAM 3grabbing nonintegrin) expressed on DCs. DC-SIGN is tetrameric and specifically recognizes N-linked high-mannose oligosaccharides (Man9GlcNAc2) through multivalent and Ca -dependent protein–carbohydrate interactions. Mimicking the cluster presentation of the oligomannosides on the surface of the virus is a strategy for designing carbohydrate-based antiviral agents. Mannose multivalent systems based on proteins, peptides, liposomes, and dendrimers as scaffolds have recently been prepared for targeting DCs and tested as inhibitors of DC-SIGN binding to gp120 in ELISA and SPR experiments. Nevertheless, none of these systems has so far been tested in cell-based trans-infection models. Glyconanoparticles (GNPs) are polyvalent, biocompatible sugar-functionalized gold nanoclusters. 10] Glyconanoparticle platforms offer the unique potential for simultaneous incorporation of different ligands in variable densities on a single gold cluster. We have prepared a small library of GNPs (mannoGNPs) coated with sets of different structural motifs of the Nlinked high-mannose undecasaccharide Man9ACHTUNGTRENNUNG(GlcNAc)2 of gp120 (or with an unnatural heptasaccharide) and have observed that GNPs that display multiple copies of the disacchaACHTUNGTRENNUNGride Mana1-2Mana block DC-SIGN/gp120 binding at 120 nm in surface plasmon resonance (SPR) experiments. The mannoGNPs were designed to target DC-SIGN receptors present on DCs by mimicking the clustered carbohydrate display of gp120. In this work we present the results obtained with manno-GNPs (Scheme 1) as inhibitors of DC-SIGN-mediated HIV trans-infection of human activated peripheral blood mononuclear cells (PBMCs). We show that manno-GNPs coated with the linear tetrasaccharide Mana1-2Mana1-2Mana1-3Mana inhibit HIV trans-infection of human T cells similarly to GNPs coated with more complex branched pentaand heptaoligomannosides. Hybrid gold manno-GNPs displaying different densities of linear and branched mannose oligosaccharides were prepared (Scheme 1). The manno-conjugates 6–10 were synthesized by conjugation of ethylamino mannosides 1–5, obtained by the protocol of Wong et al. , with isothiocyanate linker 11 and subsequent removal of the acetyl group (Scheme 1 A). A shorter five-carbon atom aliphatic linker was used for the preparation of 5’-mercaptopentyl b-d-glucopyranoside (GlcC5S), and was hidden internally in order to allow correct presentation of the oligomannosides. Manno-GNPs with 100, 50, or 10 % densities of dimannoside (D-100, D-50, and D-10, respectively), trimannoside (T-50 and T-10), tetramannoside (Te-50 and Te-10), pentamannoside (P-50 and P-10), and heptamannoside (H-50) were prepared from mixtures of manno-conjugates 6–10 and gluco-conjugate GlcC5S by using a previously described methodology (Scheme 1 B). GNPs were characterized by transmission electron microscopy (TEM), H NMR, IR, UV/Visible, and elemental analysis as previously reported. The average number of (oligo)mannosides per GNP and their average molecular weights (Table 1) were calculated from elemental analysis and gold cluster size (1–2 nm as determined by TEM; see the Supporting Information). For trans-infection studies, we used Raji DC-SIGN transfected lymphoblastoid B cells, which can capture and transmit HIV with an efficiency similar to that of monocyte-derived DCs. Manno-GNPs are nontoxic to Raji DC-SIGN and to human activated PBMCs at concentrations of 100 mg mL , as determined by the CellTiter cell viability assay (Figure S9 in the Supporting Information). The activities of manno-GNPs against R5 or X4 HIV-1 were evaluated through an original DC-SIGN transfer assay in which inhibition of HIV-1 infection by GNPs was assessed by use of recombinant viruses carrying the Renilla reporter genes in their genomes. In this assay, inhibition of viral replication is proportional to Renilla-luciferase activity in cell lysates. Briefly, Raji DC-SIGN cells were preincubated with GNPs for 1 h and were then pulsed with JR-Renilla (R5) or NL4.3-Renilla (X4) recombinant viruses for 2 h. Afterwards, the cell cultures were extensively washed and co-cultured with activated PBMCs that would be infected through transfer of the virus bound to DC-SIGN in Raji cells. Viral replication was as[a] Dr. O. Mart nezvila, Dr. M. Marradi, Dr. C. Clavel, Prof. Dr. S. Penad s Laboratory of GlycoNanotechnology, CIC biomaGUNE/CIBER-BBN P8 Miram n 182, 20009 San Sebasti n (Spain) Fax: (+ 34) 943-00-53-01 E-mail : spenades@cicbiomagune.es [b] Dr. L. M. Bedoya, Dr. J. Alcam AIDS Immunopathology Unit, National Center of Microbiology Instituto de Salud Carlos III, Ctra.Majadahonda-Pozuelo Km. 2,200 Madrid (Spain) E-mail : ppalcami@isciii.es [c] Dr. C. Clavel Current address : IBMM, UMR 5247CNRS-Universit s Montpellier 2 et 1 ENSCM, Rue de l’Ecole Normale 8, 34296 Montpellier Cedex 5 (France) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900294.

A pH‐Sensitive Lasso‐Based Rotaxane Molecular Switch

The synthesis of a pH-sensitive two-station [1]rotaxane molecular switch by self-entanglement of a non-interlocked hermaphrodite molecule, containing an anilinium and triazole moieties, is reported. The anilinium was chosen as the best template for the macrocycle benzometaphenylene[25]crown-8 (BMP25C8) and allowed the self-entanglement of the molecule. The equilibrium between the hermaphrodite molecule and the pseudo[1]rotaxane was studied by (1)H NMR spectroscopy: the best conditions of self-entanglement were found in the less polar solvent CD(2)Cl(2) and at high dilution. The triazole moiety was then benzylated to afford a benzyltriazolium moiety, which then played a dual role. On one hand, it acts as a bulky gate to trap the BMP25C8, thus to avoid any self-disentanglement of the molecular architecture. On another hand, it acts as a second molecular station for the macrocycle. At acidic pH, the BMP25C8 resides around the best anilinium molecular station, displaying the lasso [1]rotaxane in a loosened conformation. The deprotonation of the anilinium molecular station triggers the shuttling of the BMP25C8 around the triazolium moiety, therefore tightening the lasso.

A Strategy Utilizing a Recyclable Macrocycle Transporter for the Efficient Synthesis of a Triazolium-Based [2]Rotaxane

A general synthesis of triazolium-containing [2]rotaxanes, which could not be accessed by other methods, is reported. It is based on a sequential strategy starting from a well-designed macrocycle transporter which contains a template for dibenzo-24-crown-8 and a N-hydroxysuccinimide (NHS) moiety. The sequence is: 1) synthesis by slippage of a [2]rotaxane building-block; 2) its elongation at its NHS end; 3) the delivery of the macrocycle to the elongated part of the axle by an induced translational motion; 4) the contraction process to yield the targeted [2]rotaxane and recycle the initial transporter.

A pH-Sensitive Peptide-Containing Lasso Molecular Switch

The synthesis of a peptide-containing lasso molecular switch by a self-entanglement strategy is described. The interlocked [1] rotaxane molecular machine consists of a benzometaphenylene[25]crown-8 (BMP25C8) macrocycle surrounding a molecular axle. This molecular axle contains a tripeptidic sequence and two molecular stations: a N-benzyltriazolium and a pH-sensitive anilinium station. The tripeptide is located between the macrocycle and the triazolium station, so that its conformation can be tailored depending on the shuttling of the macrocycle from one station to the other. At acidic pH, the macrocycle resides around the anilinium moiety, whereas it shuttles around the triazolium station after deprotonation. This molecular machinery thus forces the lasso to adopt a tightened or a loosened conformation.

Glyconanoparticle–DNA Interactions: An Atomic Force Microscopy Study

Glyconanoparticles which present carbohydrate and amino groups motifs at their surface were produced. These particles were highly stable and soluble in aqueous solutions. The presence of the carbohydrate groups also allowed the inclusion of more strongly binding groups, without affecting solubility. The binding of a model DNA, plasmid by these nanoparticles was studied by atomic force microscopy, transmission electron microscopy, and gel electrophoresis. Significant differences between the nanoparticles based on their affinities for the DNA were found, with implications for their potential use as nonviral gene delivery agents.

NMR studies on the conformation of oligomannosides and their interaction with banana lectin.

The conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with α1→2, and α1→3, and a penta and a heptasaccharide with α1→2, α1→3, and α1→6 linkages has been evaluated by molecular mechanics and dynamics simulations and NMR spectroscopical methods. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by banana lectin has also been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures.

Active Esters as Pseudostoppers for Slippage Synthesis of [2]Pseudorotaxane Building Blocks: A Straightforward Route to Multi‐Interlocked Molecular Machines

The efficient synthesis and very easy isolation of dibenzo[24]crown-8-based [2]pseudorotaxane building blocks that contain an active ester motif at the extremity of the encircled molecular axle and an ammonium moiety as a template for the dibenzo[24]crown-8 is reported. The active ester acts both as a semistopper for the [2]pseudorotaxane species and as an extensible extremity. Among the various investigated active ester moieties, those that allow for the slippage process are given particular focus because this strategy produces fewer side products. Extension of the selected N-hydroxysuccinimide ester based pseudorotaxane building block by using either a mono- or a diamino compound, both containing a triazolium moiety, is also described. These provide a pH-dependent two-station [2]rotaxane molecular machine and a palindromic [3]rotaxane molecular machine, respectively. Molecular machinery on both interlocked compounds through variation of pH was studied and characterized by means of NMR spectroscopy.

Multifunctional Glyconanoparticles : Applications in Biology and Biomedicine

The design and preparation of complex bio-functional glyconanoparticles (GNPs) and their application as polyvalent tools to study and intervene in carbohydrate mediated biological interactions are highlighted. As examples, the preparation and study of GNPs as anti-adhesion agents in inhibition of metastasis, as potential microbicides for blocking HIV-1 infection, or as anti-cancer vaccines are also discussed. In addition, magnetic glyconanoparticles for application in cellular labelling and magnetic resonance imaging (MRI) are also reviewed.

How Secondary and Tertiary Amide Moieties are Molecular Stations for Dibenzo-24-crown-8 in [2]Rotaxane Molecular Shuttles?

Interlocked molecular machines like [2]rotaxanes are intriguing aesthetic molecules. The control of the localization of the macrocycle, which surrounds a molecular axle, along the thread leads to translational isomers of very different properties. Although many moieties have been used as sites of interactions for crown ethers, the very straightforwardly obtained amide motif has more rarely been envisaged as molecular station. In this article, we report the use of secondary and tertiary amide moieties as efficient secondary molecular station in pH-sensitive molecular shuttles. Depending on the N-substitution of the amide station, and on deprotonation or deprotonation-carbamoylation, the actuation of the molecular machinery differs accordingly to very distinct interactions between the axle and the DB24C8.