Sulfation Code and Conformational Plasticity of l-Iduronic Acid Homo-Oligosaccharides Mimic the Biological Functions of Heparan Sulfate.

Recently, the activity of heparan sulfate (HS) has led to the discovery of many drug candidates that have the potential to impact both medical science and human health. However, structural diversity and synthetic challenges impede the progress of HS research. Here, we report a library of novel l-iduronic acid (IdoA)-based HS mimics that are highly tunable in conformation plasticity and sulfation patterns to produce many of the functions of native HS oligosaccharides. The NMR analysis of HS mimics confirmed that 4-O-sulfation enhances the population of the 1C4 geometry. Interestingly, the 1C4 conformer becomes exclusive upon additional 2-O-sulfation. HS mimic microarray binding studies with different growth factors showed that selectivity and avidity are greatly modulated by the oligosaccharide length, sulfation code, and IdoA conformation. Particularly, we have identified 4-O-sulfated IdoA disaccharide (I-21) as a potential ligand for vascular endothelial growth factor (VEGF165), which in a multivalent display modulated endothelial cell proliferation, migration, and angiogenesis. Overall, these results encourage the consideration of HS mimics for therapeutic applications.

Saturated tetrasaccharide profile of enoxaparin. An additional piece to the heparin biosynthesis puzzle.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Re-expression of glucuronyl C5-epimerase in the mutant MEF cells increases heparan sulfate epimerization but has no influence on the Golgi localization and enzymatic activity of 2-O-sulfotransferase.

Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure-function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.

Heparan Sulfate Mimetics Differentially Affect Homologous Chemokines and Attenuate Cancer Development.

Achieving selective inhibition of chemokine activity by structurally well-defined heparan sulfate (HS) or HS mimetic molecules can provide important insights into their roles in individual physiological and pathological cellular processes. Here, we report a novel tailor-made HS mimetic, which furnishes an exclusive iduronic acid (IdoA) scaffold with different sulfation patterns and oligosaccharide chain lengths as potential ligands to target chemokines. Notably, highly sulfated-IdoA tetrasaccharide (I-45) exhibited strong binding to CCL2 chemokine thereby blocking CCL2/CCR2-mediated in vitro cancer cell invasion and metastasis. Taken together, IdoA-based HS mimetics offer an alternative HS substrate to generate selective and efficient inhibitors for chemokines and pave the way to a wide range of new therapeutic applications in cancer biology and immunology.

Conformational Modulation of Iduronic Acid-Containing Sulfated Glycosaminoglycans by a Polynuclear Platinum Compound and Implications for Development of Antimetastatic Platinum Drugs.

1 H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1 C4 :2 S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.

Efficient selective deacetylation of complex oligosaccharides using the neutral organotin catalyst [tBu2SnOH(Cl)]2.

Tetra-tert-butyl-3-chloro-1-hydroxydistannoxane has been found to selectively cleave with high efficiency primary acetates on complex oligosaccharides containing esterified l-iduronic acid and bearing an anomeric acetate. This tin based catalyst was found much more effective than magnesium methoxide to carry out selective deacetylation.

A non-anticoagulant heparin-like snail glycosaminoglycan promotes healing of diabetic wound.

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

Degeneracy of the Antithrombin Binding Sequence in Heparin: 2-O-Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid.

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this "canonical" GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1 C4 -2 S0 equilibrium in the unbound saccharide, shift to full 2 S0 and full 1 C4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.

Elucidating the unusual reaction kinetics of D-glucuronyl C5-epimerase.

The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3'-phosphoadenosine 5'-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis-Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.

Synthesis of Heparan Sulfate- and Dermatan Sulfate-Related Oligosaccharides via Iterative Chemoselective Glycosylation Exploiting Conformationally Disarmed [2.2.2] l-Iduronic Lactone Thioglycosides.

Heparan sulfate (HS) and dermatan sulfate (DS) are l-iduronic acid containing glycosaminoglycans (GAGs) which are implicated in a number of biological processes and conditions including cancer and viral infection. Chemical synthesis of HS and DS is required to generate structurally defined oligosaccharides for a biological study. Herein, we present a new synthetic approach to HS and DS oligosaccharides using chemoselective glycosylation which relies on a disarmed [2.2.2] l-ido lactone motif. The strategy provides a general approach for iterative-reducing end chain extension, using only shelf-stable thioglycoside building blocks, exploiting a conformational switch to control reactivity, and thus requires no anomeric manipulation steps between glycosylations.

d-Idose, d-Iduronic Acid, and d-Idonic Acid from d-Glucose via Seven-Carbon Sugars.

A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.

Structure and heparanase inhibitory activity of a new glycosaminoglycan from the slug Limacus flavus.

A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N-acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA2R-(α1,4)-D-GlcNAc-(α1-, in which R was -OH (˜80%) or -OSO3- (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC50, 0.10 µM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity.

Replacement of the L-iduronic acid unit of the anticoagulant pentasaccharide idraparinux by a 6-deoxy-L-talopyranose - Synthesis and conformational analysis.

One critical part of the synthesis of heparinoid anticoagulants is the creation of the L-iduronic acid building block featured with unique conformational plasticity which is crucial for the anticoagulant activity. Herein, we studied whether a much more easily synthesizable sugar, the 6-deoxy-L-talose, built in a heparinoid oligosaccharide, could show a similar conformational plasticity, thereby can be a potential substituent of the L-idose. Three pentasaccharides related to the synthetic anticoagulant pentasaccharide idraparinux were prepared, in which the L-iduronate was replaced by a 6-deoxy-L-talopyranoside unit. The talo-configured building block was formed by C4 epimerisation of the commercially available L-rhamnose with high efficacy at both the monosaccharide and the disaccharide level. The detailed conformational analysis of these new derivatives, differing only in their methylation pattern, was performed and the conformationally relevant NMR parameters, such as proton-proton coupling constants and interproton distances were compared to the corresponding ones measured in idraparinux. The lack of anticoagulant activity of these novel heparin analogues could be explained by the biologically not favorable 1C4 chair conformation of their 6-deoxy-L-talopyranoside residues.

A combinatorial approach towards the synthesis of non-hydrolysable triazole-iduronic acid hybrid inhibitors of human α-l-iduronidase: discovery of enzyme stabilizers for the potential treatment of MPSI.

Preparation of substituent-diverse, triazole-iduronic acid hybrid molecules by click reaction of an azido iduronic acid derivative with randomly chosen alkynes is described. Library members were screened for their ability to inhibit α-l-iduronidase, and hit molecules and analogues were then investigated for their ability to stabilize rh-α-IDUA in a thermal denaturation study. This work resulted in the discovery of the first small molecules that can be used to stabilize exogenous rh-α-IDUA protein in vitro.

MS/IR, a new MS-based hyphenated method for analysis of hexuronic acid epimers in glycosaminoglycans.

We report an original MS-based hyphenated method for the elucidation of the epimerization in GAG fragments. It consists of measuring simultaneously the MS/MS spectrum and the gas phase IR spectrum to gain direct structural information. This is possible using a customized MS instrument, modified to allow injection of a tunable IR laser inside of the instrument for in situ spectroscopy of trapped ions. The proof of principle of this approach is performed in the case of a hyaluronic acid tetrasaccharide standard. In addition, we provide the reference IR fingerprint of glucuronic and Iduronic monosaccharide standards. Remarkably, we show that the gas phase IR fingerprint of reference hexuronic acid monosaccharides proves to be transposable to oligosaccharides. Therefore, the method presented here is predictive and allows structural elucidation of unknown GAG fragments, even in the absence of referenced standards.

13C-Labeled Idohexopyranosyl Rings: Effects of Methyl Glycosidation and C6 Oxidation on Ring Conformational Equilibria.

An ensemble of JHH, JCH, and JCC values was measured in aqueous solutions of methyl α- and ß-d-idohexopyranosides containing selective 13C-enrichment at various carbons. By comparing these J-couplings to those reported previously in the α- and ß-d-idohexopyranoses, methyl glycosidation was found to affect ring conformational equilibria, with the percentages of 4C1 forms based on 3JHH analysis as follows: α-d-idopyranose, ∼18%; methyl α-d-idopyranoside, ∼42%; methyl ß-d-idopyranoside, ∼74%; ß-d-idopyranose, 82%. JCH and JCC values were analyzed with assistance from theoretical values obtained from density functional theory (DFT) calculations. Linearized plots of the percentages of 4C1 against limiting JCH and JCC values in the chair forms were used to (a) determine the compatibility of the experimental JCH and JCC values with 4C1/1C4 ratios determined from JHH analysis and (b) determine the sensitivity of specific JCH and JCC values to ring conformation. Ring conformational equilibria for methyl idohexopyranosides differ significantly from those predicted from recent molecular dynamics (MD) simulations, indicating that equilibria determined by MD for ring configurations with energetically flat pseudorotational itineraries may not be quantitative. J-couplings in methyl α-l-[6-13C]idopyranosiduronic acid and methyl α-d-[6-13C]glucopyranosiduronic acid were measured as a function of solution pH. The ring conformational equilibrium is pH-dependent in the iduronic acid.

Enzyme overexpression - an exercise toward understanding regulation of heparan sulfate biosynthesis.

Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.

Uncovering the Relationship between Sulphation Patterns and Conformation of Iduronic Acid in Heparan Sulphate.

The L-iduronic acid (IdoA) residue is a critically important structural component in heparan sulphate polysaccharide for the biological functions. The pyranose ring of IdoA is present in (1)C4-chair, (2)SO-skew boat, and less frequently, in (4)C1-chair conformations. Here, we analyzed the conformation of IdoA residue in eight hexasaccharides by NMR. The data demonstrate a correlation between the conformation of IdoA and sulphations in the surrounding saccharide residues. For the 2-O-sulpho IdoA residue, a high degree of sulphation on neighboring residues drives ring dynamics towards the (2)SO-skew boat conformer. In contrast, the nonsulphated IdoA residue is pushed towards the (1)C4-chair conformer when the neighboring residues are highly sulphated. Our data suggest that the conformation of IdoA is regulated by the sulphation pattern of nearby saccharides that is genetically controlled by the heparan sulphate biosynthetic pathway.

A method for measuring disease-specific iduronic acid from the non-reducing end of glycosaminoglycan in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder arising from deficiency of iduronate-2-sulfatase (IDS), which results in progressive accumulation of glycosaminoglycans (GAGs) in multiple tissues. Accumulated GAGs are generally measured as the amount of total GAGs. However, we recently demonstrated that GAG accumulation in the brain of MPS II model mice cannot be reliably detected by conventional dye-binding assay measuring total GAGs. Here we developed a novel quantitative method for measurement of disease-specific GAGs based on the analysis of 2-sulfoiduronic acid levels derived from the non-reducing terminal end of the polysaccharides by using recombinant human IDS (rhIDS) and recombinant human iduronidase (rhIDUA). This method was evaluated on GAGs obtained from the liver and brain of MPS II mice. The GAGs were purified from tissue homogenates and then digested with rhIDS and rhIDUA to generate a desulfated iduronic acid from their non-reducing terminal end. HPLC analysis revealed that the generated iduronic acid levels were markedly increased in the liver and cerebrum of the MPS II mice, whereas the uronic acid was not detected in wild-type mice. These results indicate that this assay clearly detects the disease-specific GAGs in tissues from MPS II mice.

Synthetic Approaches to L-Iduronic Acid and L-Idose: Key Building Blocks for the Preparation of Glycosaminoglycan Oligosaccharides.

L-Iduronic acid (IdoA) is an important monosaccharide component of glycosaminoglycans (GAGs) such as heparin, heparan sulfate and dermatan sulfate. GAGs are complex, highly sulfated polysaccharides that mediate a multitude of physiological and pathological processes via their interactions with a range of diverse proteins. The main challenge in the synthesis of GAG oligosaccharides is the efficient gram-scale preparation of IdoA building blocks since neither IdoA nor L-idose is commercially available or readily accessible from natural sources. In this review, the different synthetic approaches for the preparation of IdoA and its derivatives, including L-idose, are presented and discussed. Derivatives of the latter are often used in GAG synthesis and are elaborated to IdoA via selective oxidation at C-6 after incorporation into a GAG chain. Particular focus will be given to the preparation of IdoA synthons most commonly used for GAG oligosaccharide synthesis, and on the progress made since the last systematic review in this area.