Heparin Oligosaccharides Research Articles

Microarrays of Synthetic Heparin Oligosaccharides

We present the first preparation of microarrays containing synthetic heparin oligosaccharides in order to elucidate the heparin-protein interactions involved in a variety of biological processes. For this purpose, we have developed a novel linker strategy that is compatible with the protecting-group manipulations required for the synthesis of the highly sulfated oligosaccharides and can also be extended to an automated solid phase approach. Strategic placement of the orthogonally protected amine linker was key to the success of the array construction. These heparin chips allow for the high-throughput screening of oligosaccharides by using approximately picomoles of protein. The potential of the new method was demonstrated by probing the carbohydrate affinity of two heparin-binding growth factors, FGF-1 and FGF-2, that are implicated in the development and differentiation of several tumors.

Automated Synthesis of Oligosaccharides as a Basis for Drug Discovery

AbstractFor Abstract see ChemInform Abstract in Full Text.

Microarrays of heparin oligosaccharides obtained by nitrous acid depolymerization of isolated heparin

Heparin oligosaccharides derived by nitrous acid depolymerization of heparin have been immobilized on amine-coated glass slides. The formation of a Schiff base creates heparin chips that are a suitable platform for the high-throughput analysis of carbohydrate-protein interactions.

Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

Heparin and heparin-derived components are widely applied anticoagulant drugs used for amongst other applications medical treatment of deep vein thrombosis and pulmonary embolism. Depolymerisation of native heparin results in complex mixtures of sulfated linear oligosaccharides that are usually not well characterised. In order to further characterise such mixtures, two on-line ion-pairing reverse-phased chromatography electrospray ionisation (ESI) mass spectrometry methods have been developed. One of the systems allows the determination of more than 200 components in a medium molecular weight heparin preparation, whereas the other system can be used to separate isomeric heparin oligosaccharides after previous separation according to size. This latter system allows semi-preparative isolation of isomeric heparin oligosaccharides. The experimental setup includes on-line cation exchange in order to prevent the ion-pairing reagent from entering the mass spectrometer.

Enzymatic Redesigning of Biologically Active Heparan Sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

Cooperative Dimerization of Fibroblast Growth Factor 1 (FGF1) upon a Single Heparin Saccharide May Drive the Formation of 2:2:1 FGF1·FGFR2c·Heparin Ternary Complexes

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.

Structural specificity in a FGF7‐affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb

Variations in sulfation of heparan sulfate (HS) affect interaction with FGF, FGFR, and FGF-HS-FGFR signaling complexes. Whether structurally distinct HS motifs are at play is unclear. Here we used stabilized recombinant FGF7 as a bioaffinity matrix to purify size-defined heparin oligosaccharides. We show that only 0.2%-4% of 6 to 14 unit oligosaccharides, respectively, have high affinity for FGF7 based on resistance to salt above 0.6M NaCl. The high affinity fractions exhibit highest specific activity for interaction with FGFR2IIIb and formation of complexes of FGF7-HS-FGFR2IIIb. The majority fractions with moderate (0.30-0.6M NaCl), low (0.14-0.30M NaCl) or no affinity at 0.14M NaCl for FGF7 supported no complex formation. The high affinity octasaccharide mixture exhibited predominantly 7- and 8-sulfated components (7,8-S-OctaF7) and formed FGF7-HS-FGFR2IIIb complexes with highest specific activity. Deduced disaccharide analysis indicated that 7,8-S-OctaF7 comprised of DeltaHexA2SGlcN6S in a 2:1 ratio to a trisulfated and a variable unsulfated or monosulfated disaccharide. The inactive octasaccharides with moderate affinity for FGF7 were much more heterogenous and highly sulfated with major components containing 11 or 12 sulfates comprised of predominantly trisulfated disaccharides. This suggests that a rare undersulfated motif in which sulfate groups are specifically distributed has highest affinity for FGF7. The same motif also exhibits structural requirements for high affinity binding to dimers of FGFR2IIIb prior to binding FGF7 to form FGF7-HS-FGFR2IIIb complexes. In contrast, the majority of more highly sulfated HS motifs likely play FGFR-independent roles in stability and control of access of FGF7 to FGFR2IIIb in the tissue matrix.

Short De Novo Synthesis of Fully Functionalized Uronic Acid Monosaccharides

Short and Sweet: A convergent route leads to orthogonally protected D-glucuronic and L-iduronic acid thioglycoside building blocks, commonly used in heparin oligosaccharide assembly (see scheme). Rapid access to substantial quantities of these key intermediates feeds the growing need for monosaccharide building blocks for use in the assembly of bioactive oligosaccharides. PG=protecting group.

Heparin Octasaccharides Inhibit Angiogenesis In vivo

In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing > or =16 saccharide residues were investigated. Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

Chemokine-Glycosaminoglycan Binding: SPECIFICITY FOR CCR2 LIGAND BINDING TO HIGHLY SULFATED OLIGOSACCHARIDES USING FTICR MASS SPECTROMETRY

Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2 (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that, in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.

Automated synthesis of oligosaccharides as a basis for drug discovery

Carbohydrates present both potential and problems - their biological relevance has been recognized, but problems in procuring sugars rendered them a difficult class of compounds to handle in drug discovery efforts. The development of the first automated solid-phase oligosaccharide synthesizer and other methods to assemble defined oligosaccharides rapidly has fundamentally altered this situation. This review describes how quick access to oligosaccharides has not only contributed to biological, biochemical and biophysical investigations, but also to drug discovery. Particular focus will be placed on the development of carbohydrate-based vaccines, defined heparin oligosaccharides and aminoglycosides that have recently begun to affect drug discovery.

Separation and Analysis of Nanomole Quantities of Heparin Oligosaccharides Using On-Line Capillary Isotachophoresis Coupled with NMR Detection

Glycosaminoglycans (GAGs) are important in a number of biological processes and are structurally altered in many pathological conditions. The complete determination of GAG primary structures has been hampered by the lack of sensitive and specific analytical techniques. Nuclear magnetic resonance spectroscopy (NMR) is a powerful tool for GAG structure elucidation despite its relatively poor limits of detection. Solenoidal microcoils have greatly enhanced the mass limits of detection of NMR, enabling the on-line coupling of microseparation and concentration techniques such as capillary isotachophoresis (cITP), which can separate and concentrate analytes by 2-3 orders of magnitude. We have successfully used cITP coupled with on-line NMR detection to separate and concentrate nanomole quantities of heparin oligosaccharides. This sensitive on-line measurement approach has the potential to provide new insights into the relationships between biological function and GAG microstructures.

Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry

A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.

Heparin Sequencing Using Enzymatic Digestion and ESI-MSn with HOST: A Heparin/HS Oligosaccharide Sequencing Tool

Mass spectrometry, and specifically sequential stages of mass spectrometry (MSn), is an established tool for the analysis of carbohydrates, proteins, and more recently glycosaminoglycans. As this trend continues, the development of algorithms for the rapid and automatic interpretation of mass spectra to identify glycan structure is also expected to grow as an active field of research. The methodology described herein utilizes a combination of enzymatic digestion, ESI-MS, and MSn for the sequencing of small heparin oligosaccharides. The heparin oligosaccharide sequencing tool (HOST) is a basic software application that was developed to aid in the integration and analysis of the data generated from these experiments, facilitating the process involved in arriving at sequence information. The sequences of several heparin oligosaccharides were determined using this method to illustrate proof of principle. Tandem MS is a very rapid and efficient tool for oligosaccharide analysis when limited amounts of material are available. Having a means, such as HOST, for automating the interpretation of the MSn data generated from glycosaminoglycans, provides a practical methodology for the future analysis of heparin/HS oligosaccharides of unknown structure.

Heparin structures in FGF‐2–dependent morphological transformation of astrocytes

Fibroblast growth factor-2 (FGF-2) participates in the morphological transformation of astrocytes (stellation) during the formation of glial scars in injured brains. In the current study, we used quantitative morphometric analysis to investigate the structural requirements for heparin's enhancement of FGF-2-induced stellation of cultured cortical astrocytes. Native heparin significantly promoted FGF-2-dependent astrocytic stellation, whereas heparin hexasaccharide inhibited FGF-2-dependent stellation. Furthermore, 2-O-, 6-O-, and N-desulfated heparins were unable to promote FGF-2-dependent stellation. The stellation induced by FGF-2 or by a combination of FGF-2 and native heparin was inhibited by SU5402, an FGF receptor inhibitor. These results demonstrate that the length and sulfated position of heparin are important for its enhancement of FGF-2-dependent astrocyte stellation. In addition, our findings show that heparin oligosaccharides are useful for regulating the FGF-2-dependent astrocytic transformation.

Carbohydrates as the Next Frontier in Pharmaceutical Research

Synthetic carbohydrates and glycoconjugates are used to study their roles in biological important processes such as inflammation, cell-cell recognition, immunological response, metastasis, and fertilization. The development of an automated oligosaccharide synthesizer greatly accelerates the assembly of complex, naturally occurring carbohydrates as well as chemically modified oligosaccharide structures and promises to have major impact on the field of glycobiology. Tools such as microarrays, surface plasmon resonance spectroscopy, and fluorescent carbohydrate conjugates to map interactions of carbohydrates in biological systems are presented. Case studies of the successful application of carbohydrates as active agents are discussed, for example, fully synthetic oligosaccharide vaccines to combat tropical diseases (e.g., malaria), bacterial infections (e.g., tuberculosis), viral infections such as HIV, and cancer. Aminoglycosides serve as examples of drugs acting through carbohydrate-nucleic-acid interactions, while heparin works by carbohydrate-protein interactions. A general, modular strategy for the complete stereoselective synthesis of defined heparin oligosaccharides is presented. A carbohydrate-functionalized fluorescent polymer has been shown to detect miniscule amounts of bacteria faster than commonly used methods.

An exploratory study on the synthesis of heparin-like oligosaccharides by polycondensation

The synthesis of heparin oligosaccharide fragments by autocondensation of suitably protected D- glucosamine α1→4 L-iduronic acid disaccharide constructs containing both glycosyl- donor and glycosyl- acceptor functionalities has been explored. Trichloroacetimidate and n-pentenyl groups have been investigated for anomeric activation. The generation of building blocks equipped with a trichloroacetimidate function is seriously hampered by the presence of the free- OH glycosyl- acceptor group on the same molecule, which favors an intramolecular transesterification reaction. Using an n-pentenyl leaving group, the glycosylation promoter competes as a nucleophile with the OH glycosyl acceptor in the autocondensation process, giving rise to fast termination of the chain reaction, a low yield, and low degree of polymerization. It is concluded that, in this area, polycondensation can hardly compete with elaborate stepwise approaches as a result of the intrinsic low reactivity—both as a glycosyl donor and as glycosyl acceptor—of the D-glucosamine α1→4 L-iduronic acid building blocks.

Molecular weight determines the frequency of delayed type hypersensitivity reactions to heparin and synthetic oligosaccharides

Eczematous lesions, resulting from type IV sensitizations are well-known and relatively frequent cutaneous adverse effects of s.c. heparin therapy. If anticoagulation is further required intravenous heparin, heparinoids or lepirudin may be used as a substitute. However, these alternatives are not optimal in terms of practicability and/or safety-profiles. As molecular weight of different heparin preparations has repetitively been implied to determine the frequency of sensitization, we hypothesized, that due to its low molecular weight the pentasaccharide fondaparinux may provide a practicable and safe anticoagulant therapy in patients with delayed type hypersensitivity reactions (DTH) to heparin and other oligosaccharides. To test this concept, patients referred for diagnosis of cutaneous reactions after s.c. anticoagulant treatment underwent a series of in vivo skin allergy- and challenge-tests with unfractionated heparin, a series of low molecular weight heparins (nadroparin, dalteparin, tinzaparin, enoxaparin and certoparin), the heparinoid danaparoid and the synthetic pentasaccharide fondaparinux. In total, data from twelve patients was evaluated. In accordance with previously published data, we report a high crossreactivity among heparins and heparinoids. In contrast--and in support of our initial hypothesis--sensitization towards the synthetic pentasaccharide fondaparinux was rarely observed. Plotting the cumulative incidence against the determined molecular weight of the individual anticoagulant preparations, shows that molecular weight generally is a key determinant of sensitization towards heparins and other oligosaccharides (r2 = 0.842, p = 0.009). Hence, fondaparinux may be used as a therapeutic alternative in patients with cutaneous DTH relations towards heparin and other polysaccharides.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

Effect of 1,6-Anhydro Bicyclic Ring Structure on the Pharmacokinetic and Pharmacodynamic Behavior of Low Molecular Weight Heparin.

Introduction: Heparin cleavage under alkaline conditions results in low molecular weight heparin (LMWH) chains, a significant proportion of which contain 1,6-anhydromannosamine and/or 1,6-anhydroglucosamine at the reducing end. Despite the widespread use of the LMWHs for the prophylaxis and treatment of thrombosis, it remains unclear whether such structural modifications impact the pharmacologic activity of the drug. This study examined the in vitro anticoagulant and in vivo pharmacokinetic/pharmacodynamic (PK/PD) behavior of LMWHs containing varying levels of 1,6-anhydrosugar content.Materials and Methods: By altering the temperature and pH of the depolymerization reaction, LMWHs containing 0, 5, 10, 20 and 40% 1,6-anhydrosugar were produced. These compounds were supplemented to normal human plasma and normal primate plasma and assayed for anticoagulant (APTT and Heptest) and antiprotease (anti-IIa and anti-Xa) activity. The effect of 1,6-anhydrosugar on the PK/PD profile of LMWHs was assessed by administering the 40% 1,6-anhydro LMWH or enoxaparin (~20% 1,6-anhydrosugar) intravenously to groups of non-human primates (n=4–6) at a dose of 1 mg/kg. Blood samples were collected at baseline and at various time points up to 24 hours post-administration for determination of Heptest clotting times, anti-IIa and anti-Xa activity. The biologic activities were converted to equivalent LMWH concentrations using calibration curves prepared in normal primate plasma.Results: The molecular weight profiles of these LMWHs were comparable. No effect on anticoagulant or antiprotease activity was observed when the 1,6-anhydro content varied between 0 and 10%. When the 1,6-anhydro content was increased to 20 and 40%, a content-dependent reduction in anticoagulant activity was observed such that the prolongation of the APTT and Heptest by the 40% 1,6-anhydro LMWH was 58 and 23% less, respectively, than that produced by the LMWH lacking the 1,6-anhydro group when tested in the linear range of the concentration-response curve. This effect appears to be related primarily to an interference with antithrombin activity. Inhibition of thrombin activity in an amidolytic assay was 35% lower with the 40%-anhydro LMWH compared to the 0% anhydro compound (10 mg/ml), whereas anti-Xa activity was only 7% lower. Assay dependent variations were observed in the PK/PD profiles of the 40% anhydro LMWH and enoxaparin. As expected, the half-life of antithrombin activity was considerably shorter than that of the anti-Xa activity. The pharmacokinetic behavior of the 40% 1,6-anhydro LMWH and enoxaparin in terms of half-life, area under the curve, systemic clearance and volume of distribution was not significantly different when calculated using plasma concentrations determined by anti-IIa or anti-Xa assay. When concentrations determined by Heptest were used, the AUC determined for enoxaparin was approximately 2-fold higher than that determined with the 40% anhydro LMWH.Conclusions: Microchemical changes in the structure of low molecular weight heparin oligosaccharides can induce measurable changes in the biologic activity of LMWHs. While the pharmacokinetic profile does not appear to be altered by an enhanced 1,6-anhydro content, the effect of 1,6-anhydro content on the clinical efficacy and safety of LMWHs is unknown. Such findings may have particular impact on the development of generic LMWHs.