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Molecular modelling studies of interactions between two heparanase binding domains, Lys158-Asp171 and Q270-V289, with seven different oligosaccharides were performed. The results of these studies enabled detailed characterization of 3D structures of intermolecular complexes between the target oligosaccharides and heparanase binding domains.
Furthermore, the effects of two factors, counter-ions and substituents, that influence the interactions between ligands and receptors, were also analyzed. The data, for the first time, characterize the heparanase binding domain-heparin oligosaccharide complexes at the molecular level and can be used to help to design the heparanase inhibitors.
Modeling of interactions between the heparanase binding domain Lys158-Asp171 and seven different pentasaccharides have been performed by both molecular mechanics and Monte Carlo conformational search. Results of these calculations permitted to identify specific sulfate groups of the pentasaccharide that interact with specific aminoacid residues of the peptide.
The title compound was synthesized through a complex way, which was based on the preparation of a protected disaccharide intermediate containing the idose structure, obtained in 30 overall steps from commercial D-glucosamine and D-glucose. A second intermediate, a protected form of glucose, was added to obtain a trisaccharide, which was trasformed in the title compound in two steps. The obtained trisaccharide is an advanced precursor toward the synthesis of pentasaccharides putative substrates and/or inhibitors of the heparanase enzyme.
A new protocol for the oxidation of the primary hydroxyl of mono or oligosaccharides has been developed. It consists in two steps, oxidation to aldehyde with IBX (a procedure never used before for carbohydrates), followed by further oxidation to carboxyl group. The above protocol has been used for an efficient synthesis of hexuronic acid (GlcA and IdoA)-containing disaccharides. Of these acids, the IdoA is particularly difficult to obtain through classical methods. The compound so obtained has the skeleton of a glycosaminoglycan repeating unit and is an intermediate useful to build glycosaminoglycan fragments in a controlled way.
The protected pentasaccharide GlcN-alfa-(1-4) Glc-beta-(1-4) GlcN-alfa-(1-4) IdoA-alfa-(1-4)GlcN-1-alfa-OMe, precursor of a small family of potential heparanase inhibitors, was synthesized by coupling of the properly protected disaccharide GlcN-alfa-(1-4)-Glc with the protected trisaccharide GlcN-alfa-(1-4) IdoA-alfa-(1-4)GlcN-1-alfa-OMe. This compound, by selective deprotection/sulfation, and final total deprotection, will lead to a small family of partially sulfated oligosaccharides of general structure GlcN-alfa-(1-4) Glc-beta-(1-4) GlcN-alfa-(1-4) IdoA-alfa-(1-4)GlcN-1-alfa-OMe, potential inhibitors of the heparanase enzime.
Molecular modeling studies on putative heparanase substrates and inhibitors were made. The results enabled both detailed description of 3D structures of these compounds for the first time and estimation of the influence of the factors (counter-ions and substituents) that affect their conformations. These data are important for understanding the intermolecular interactions of the compounds with the target protein heparanase at the molecular level and were used in modelling of these interactions. Results of molecular mechanics calculations can be divided in several groups according to the type of compounds analyzed:
- Heparin/HS oligosaccharides;
- Maltohexaose sulfates;
- K5-derived polysulfates;
- Other saccharides with various sulfation pattern.
Both quantum chemical and molecular mechanics calculations showed that counter-ions play an important role in stabilization of the 3D structure of sulfated oligosaccharides. Structures of all analyzed saccharides were affected by the coordination of Na+ ions between the -NSO3 and the -COO groups.
Especially in larger oligomers, pentasaccharides and hexasaccharides, conformations at the glycosidic linkages were influenced by charge-charge interactions, by interactions between the COO- and the (OH) groups and between the -NSO3 and the -2-OSO3 groups. The sulfate groups showed considerable effect upon 3D structures especially in 2,6-sulfated (or in 2,3,6-sulfated) compounds. Their linkage conformations were different with non-substituted compounds or with 6-sulfated derivatives. Quantum chemical calculations also enabled description of the character chemical bonds bond, charges, etc.
Biological and pharmacological interactions of heparin and structurally-related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues. Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by HPLC analysis of fragments obtained by enzymatic or chemical degradation. NMR spectroscopy is also used for structural characterization of GAGs.
However, monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not permit reliable quantitative measurements.
With proper choice of the analytical signals, the higher resolution achieved with two dimensional (2D) NMR methods could be exploited also for quantitative applications. Heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and of HS mimics obtained by chemical modification of the common biosynthetic precursor of heparin and HS E. Coli K5 polysaccharide.
Heparin was used as a model for assessing the influence of 1H-13C magnetic couplings on volumes of the corresponding signals. For major signals, the HSQC approach permitted to quantify additional structural features both in heparin and in a typical HS. The method was applied to profile the susbstitution patterns of K5 polysaccharide derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.
There is an increasing interest in oversulfated oligo and polysaccharides for medical application (i.e. oncology, virology). We can foresee a wide application of this approaches in characterization of these complex molecules.
The title compound was synthesized in 10 steps from commercial D-glucosamine through a (partially) novel chemo-enzymatic procedure. The product here described is a starting material for the preparation of pentasaccharides designed as potential substrates of heparanase enzyme.
Sulfated C-glycosides oligosaccharide-like mimics were synthesized via formation of a direct C-C bond involving the anomeric or the 6 positions having different flexibility. The apply synthetic methodology can provide a panel of sulfate C-glycoside oligosaccharides mimics that are a new heparanase inhibitors class. It is expected that they could be resistant to enzymatic cleavage in vivo conditions and have good pharmacokinetics.
The title compound was synthesized in six steps starting from commercial pentaacetylglucose. The procedure can be made on large scale (100 grams) and requires only a purification step. The obtained compound is a flexible intermediate, which can be used to prepare heparin-like oligosaccharides containing glucuronic acid or glucose moieties.
With the aim of establishing a simple, reliable method for studying the cleavability by heparanase of heparin/heparan sulfate oligosaccharides in the absence and in the presence of heparanase inhibitors, an on-line liquid chromatography/mass spectrometric (ESI-MS) method was set up. The method works in the nanomolar range concentration of substrate and does not need derivatization (radioactive nor fluorofore) of the substrate or the products.
The method is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal stardard (a heparin disaccharide). In its most practical version, cleavage of oligosaccharidic substrates by the enzyme was monitored from the area of HPLC/mass signals of the substrate. Substrates used to set up the method were two pentasaccharides (AGAIA and AGAIA) and two octasaccharides containing the AGAIA sequence in different locations along the chain. (AGAIA is the typical sequence of the active site of heparin/HS for antithrombin. When part of larger oligosaccharides, previous studies had shown that heparanase cleaves this sequence between the G (glucuronic acid) and A (3-O-sulfated glucosamine residues.).
The present study showed that AGAIA is cleaved by the enzyme even when it is not part of larger oligosaccharides. The commercial availability of AGAIA makes it an ideal substrate to determine the specific activity of heparanase preparations in substitution of the currently used complex and difficult to reproduce heparan sulfates or HS proteoglycans extracted from animal organs. The present method is also suitable for rapid screening of potential heparanase inhibitors.
The title disaccharide was synthesized in 10 overall steps from commercial D-glucosamine and the properly protected allyl beta-D-glucopyranoside. This disaccharide is an intermediate for the synthesis of the non reducing end of the family of pentasaccharides putative substrates and/or inhibitors of the heparanase enzyme.
Using O-sulfation and O-desulfation procedures exploiting the intrinsic reactivity of different hydroxyls three structural defined family of heparin/heparansulfate glycomimetics where obtained.
- MHSs and 6-carboxy MHSs with different degrees and patterns of sulfation were obtained.
- Several samples of hight and low molecular weight sulfated hyaluronates
- Extensively O-sulfated, N-sulfated (as well as N-acetylated) K5-PS derivatives, both high and low molecular weight.
Oversulfated samples were studied by NMR spectroscopy to assign the signals and determine the percent sulfation at each position of disaccharidic repeating units.
Some of this molecules could be define as new heparanase inhibitors. Moreover, the generation of this kind of libraries can contribute to elucidate the structure activity relationship of heparin/heparansulfate sequences versus heparanase and other HS-binding proteins and suggest new potential inhibitor structures.
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