Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate–protein interactions in microtiter plates

Maza, Susana; Macchione, Giuseppe; Ojeda, Ricardo A.; López-Prados, Javier; Angulo, Jesús; Paz, José L. de; Nieto, Pedro M.

doi:10.1039/c2ob06607f

Cited by 29 publications

(33 citation statements)

References 56 publications

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“…Next, we studied the interactions between midkine and our collection of chemically synthesised heparin and CS oligosaccharides (Figure ) . The library comprises CS‐like di‐ and tetrasaccharides, which differ in sulfation pattern and sequence, including compounds 14 and 15 .…”

Section: Resultsmentioning

confidence: 99%

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three‐Dimensional Structure and Interaction with Midkine

Solera

Macchione

Maza

et al. 2016

Chemistry A European J

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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.

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Section: Resultsmentioning

confidence: 99%

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three‐Dimensional Structure and Interaction with Midkine

Solera

Macchione

Maza

et al. 2016

Chemistry A European J

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“…Thus, removal of the silylene group was followed by basic hydrolysis and selective N ‐acetylation to give 36 . Treatment with SO 3 · NMe 3 complex at 100 °C using microwave irradiation47 gave hepta‐ O ‐sulfated tetrasaccharide 37 . The introduction of the sulfate groups at the desired positions was confirmed by 1 H and 13 C NMR spectroscopic data, which showed typical downfield shifts for the sulfated positions (Tables 11 and 2).…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Chondroitin Sulfate Oligosaccharides Using N‐(Tetrachlorophthaloyl)‐ and N‐(Trifluoroacetyl)galactosamine Building Blocks

et al. 2014

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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.

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“…In addition, aiming to test the effect of the length of the GAG ligand on binding, we have studied the interaction of langerin with a HEP hexasaccharide containing an extra sulfate group at position 4 of the non-reducing ring (Hexa9, Scheme 1). 21 The introduction of a sulfate group at that position generates a non-natural sulfate distribution. In summary, this work reports on the surprising ligand size dependence of GAG binding to two different sites in langerin, defining at atomistic detail the boundaries at langerin trimeric interfaces involved in GAG molecular recognition, and describes the key contacts governing binding.…”

Section: For These Cells In Vivomentioning

confidence: 99%

Langerin–Heparin Interaction: Two Binding Sites for Small and Large Ligands As Revealed by a Combination of NMR Spectroscopy and Cross-Linking Mapping Experiments

et al. 2015

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Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.

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Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate–protein interactions in microtiter plates

Cited by 29 publications

References 56 publications

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three‐Dimensional Structure and Interaction with Midkine

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three‐Dimensional Structure and Interaction with Midkine

Synthesis of Chondroitin Sulfate Oligosaccharides Using N‐(Tetrachlorophthaloyl)‐ and N‐(Trifluoroacetyl)galactosamine Building Blocks

Langerin–Heparin Interaction: Two Binding Sites for Small and Large Ligands As Revealed by a Combination of NMR Spectroscopy and Cross-Linking Mapping Experiments

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