Novel glycodendrimers self-assemble to nanoparticles which function as polyvalent ligands in vitro and in vivo.

Abstract

The recognition of oligosaccharides by proteins represents the basis of many biologically important events.[1] Individual protein±carbohydrate interactions are generally weak (KD1⁄4 10 3±10 4m 1).[2] To overcome this, such processes often involve polyvalent binding, which is characterized by the simultaneous contact of multiple ligands (oligosaccharides) on one biological entity to multiple receptors (proteins) on another.[3] Polyvalent carbohydrate±protein interactions occur frequently in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions rendering it difficult to control them with individual small molecules.[4] Therefore, complex macromolecules have been used as polyvalent antagonists, however, both characterization and preparation of these nonuniform entities is demanding.[4] Here we present an alternative concept for the polyvalent presentation of ligands based on the supramolecular chemistry[5] of small molecules that fulfil single-molecule entity criteria (Figure 1). Novel dendrons capped with carbohydrate ligands (glycodendrimers[6]) were found to self-assemble to noncovalent nanoparticles which function as polyvalent ligands. We demonstrate that these particles–not the individual molecules–efficiently inhibit polyvalent interactions, such as IgM binding (IgM1⁄4 immunoglobulin), to the aGal-epitope[7] (a-d-Gal-(1!3)-b-d-Gal(1!4)-d-GlcNAc), both in vitro and in vivo. As self-assembly is dynamic, optimization of size and shape of the polyvalent ligand could occur utilizing the receptor as a template. Dendrimer cores were prepared by a convergent TMoutsidein∫ approach[8] based on a single building block 1a which was obtained frommethyl 3,5-diaminobenzoate and 4-(tert-butoxycarbonylaminomethyl)benzoic acid (Scheme 1a). Selective deprotection furnished 1b and 1c (first-generation dendrimer core, two end-groups). A one-pot procedure comprising coupling of 1c (1 equiv) and 1b (0.5 equiv) followed by methyl ester cleavage gave 2c (second-generation dendrimer core, four end groups).[9] The third-generation dendrimer 3c (eight end groups) was obtained from 2c (1 equiv) and 1b (0.5 equiv).[9] Applying the same procedure repetitively gave dendrimers with up to 64 end groups (4c, fourth generation, 16 end groups; 5c, fifth generation, 32 end groups; 6c, sixth generation, 64 end groups). Dendrimers 1c±6c were deprotected (!1d±6d) and transformed into their chloroacetamide derivatives (1e±6e) to allow subsequent introduction of thiolated oligosaccharides such as aGal-SH[10] and Lac-SH (Figure 2b) furnishing water-soluble glycodendrimers 1 f±6 f and 3g which were purified by ultrafiltration. Compound 7, which is similar to 2 f but contains butylene chains instead of the disubstituted aromatic rings, was also prepared (Scheme 1a). The integrity of all compounds was established by 1H NMR spectroscopy. Accordingly, the firstto third-generation dendrimers exist as single molecules (purity > 95%). The fourthto sixth-generation dendrimers possibly contain minute quantities of smaller fragments. The 500 MHz 1H NMR spectra of compound 3 f in [D6]DMSO demonstrates the remarkable purity of these compounds (Figure 2). The first indication that our glycodendrimers were aggregating in water came from 1H NMR spectroscopy of 2 f in D2O. At ambient temperature, we observed very broad signals which sharpened at elevated temperatures. The aggregation was quantified using multiangle light scattering (MALS; Table 1). The first-generation dendrimer 1 f forms small aggregates (50 kDa) whereas 2 f forms large particles of 7000 kDa (more than 1500 individual molecules per particle). Interestingly, the particle weight obtained for 3 f±6 f drops (2200 to 200 kDa) with increasing mass of the individual molecule. The root-mean-square radii of the particles formed by 2 f±6 f showed the same trend (for 2 f, 3 f, and 4 f 49, 34, and 12 nm, respectively; for 5 f and 6 f the radii were below the detection limit of 10 nm). Core-modified second-generation glycodendrimer 7 (4 iaGal), which is of comparable size and lipophilicity as 2 f but contains butylene chains instead of the disubstituted aromatic rings, does not form aggregates. The third-generation compounds 3g (8 iLac; 1900 kDa) and 3 f ZUSCHRIFTEN