Abstract:The biological activity of midkine, a cytokine implicated in neurogenesis and tumorigenesis, is regulated by its binding to glycosaminoglycans (GAGs) such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, we have studied here the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine using different techniques. First, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarization competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacts with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies suggesting an interaction of the tetrasaccharide with midkine in a folded structure. Overall, our study gives valuable information on the interaction between midkine and well-defined, chemically synthesized CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.

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The synthesis of well-defined oligosaccharides is crucial for the establishment of structure-activity relationships for specific sequences of heparin, contributing to the understanding of the biological role of this polysaccharide. It is highly convenient that the synthetic oligosaccharides contain an orthogonal functional group that allows selective conjugation of the probes and expands their use as chemical tools in glycobiology. We present here the synthesis of a series of amine-functionalized heparin oligosaccharides using an n+2 modular approach. The conditions of the glycosylation reactions were carefully optimized to produce efficiently the desired synthetic intermediates with an N-benzyloxycarbonyl-protected aminoethyl spacer at the reducing end. The use of microwave heating greatly facilitates O- and N-sulfation steps, avoiding experimental problems associated with these reactions. The synthesized oligosaccharides were immobilized in 384-well microtiter plates and successfully probed with a heparin-binding protein, the basic fibroblast growth factor FGF-2. The use of hexadecyltrimethylammonium bromide minimized the amount of sugar required for attachment to the solid support. Using this approach we quantified heparin-protein interactions, and surface dissociation constants for the synthetic heparin derivatives were determined.

We have explored synthetic routes for the preparation of chondroitin sulfate (CS) oligosaccharides based on the use of N‐tetrachlorophthaloyl‐ (N‐TCP) and N‐trifluoroacetyl‐substituted (N‐TFA) galactosamine building blocks. Using N‐TCP units, we carried out the total synthesis of two CS disaccharides, demonstrating the compatibility of TCP protection with the final deprotection/sulfation steps. However, an attempted 2 + 2 coupling of N‐TCP‐containing disaccharides for the synthesis of CS tetrasaccharides failed. In contrast, a synthetic route using N‐TFA galactosamine units efficiently led to biologically relevant CS‐like oligosaccharides. The N‐TFA groups could easily be removed at the end of the synthesis, and microwave irradiation greatly facilitated the sulfation reactions. The utility of this approach is illustrated with the total synthesis of two CS‐like tetrasaccharides with different sulfate distribution patterns. Finally, we used a fluorescence polarization assay to estimate the relative abilities of the synthesized compounds to inhibit the interaction between FGF‐2 and heparin.

The synthesis of hyaluronic acid oligomers (tri- and tetrasaccharide) is described. We have followed a pre-glycosylation oxidation strategy. Glucuronic acid units were directly employed in coupling reactions with suitably protected glucosamine derivatives. In order to simplify the purification of synthetic intermediates, a fluorous-assisted strategy has been also explored. Using this approach, a hyaluronic acid trisaccharide was prepared.

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected D-glucosamine (GlcN) and D-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.