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.     

Synthesis of heparin oligosaccharides

An efficient preparation of rare 2-O-benzoyl-3-O-benzyl-1,6-anhydro-beta-l-idopyranose from commercially available diacetone alpha-d-glucose in five straightforward steps is described here. With this key building block in hand, the total syntheses of heparin oligosaccharides with three, five, seven, and nine sugar units are successfully carried out.     

Modular synthesis of heparin oligosaccharides

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di- and trisaccharide modules in a fully selective fashion. Installation of the alpha-glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans-uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII-binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.     

Microwave-assisted sulfonation of heparin oligosaccharides

The use of microwaves for the efficient and fast O-sulfonation of heparin oligosaccharide intermediates has been reported for the first time. Experimental problems typically associated with this chemical reaction, such as poor isolated yields and long reaction times, have been avoided with the present method. The efficiency of this protocol was demonstrated by the high-yielding sulfonation of a series of oligosaccharides using SO₃·Me₃N complex. Microwave-assisted sulfonation is expected to greatly facilitate the preparation of heparin oligosaccharides, a crucial step for understanding the role of these complex carbohydrates in biological processes.     

Interaction of oligopeptides with heparin

This heparin affinity chromatography study revealed some very interesting aspects with respect to oligopeptide retention. The first and most surprising effect observed was that oligopeptides with no aromatic groups were not retained on the column while those with aromatic groups were retained. The aromatic interaction was determined to be related to a charge transfer ‐like interaction as electron donating groups on the aromatic moiety increased retention on the column and electron withdrawing groups on the aromatic moiety deceased retention time. Column retention was dependent on oligopeptide size; the tripeptides were not retained while tetrapeptides and the larger hexapeptides were effectively retained if they contained some aromatic functionality. Retention was pH dependent; low pH's of ∼2.5 were most effective while neutral pH was not effective in retaining the oligopeptides on the heparin affinity column. In addition to the aromatic interaction, retention was dependent on the hydrophobicity of the oligopeptides as well as their ability to hydrogen bond as determined by eluent solvent effects on the heparin affinity column. These results have led us design and carry out other experiments to determine more accurately the type and the degree of oligopeptide to heparin interaction.     

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.     

Sodium hydroxide permethylation of heparin disaccharides

Permethylation is a valuable and widely used tool for the mass spectrometry of carbohydrates, improving sensitivity and fragmentation and increasing the amount of information that can be obtained from tandem mass spectrometric experiments. Permethylation of most glycans is easily performed with sodium hydroxide and iodomethane in dimethyl sulfoxide (DMSO). However, permethylation has not been widely used in the mass spectrometry of glycosaminoglycan (GAG) oligosaccharides, partly because it has required the use of the difficult Hakomori method employing the methylsulfinylmethanide ('dimsyl') base, which has to be made in a tedious process. Additionally, the Hakomori method is not as effective as the sodium hydroxide method in making fully methylated derivatives. A further problem in the permethylation of highly sulfated oligosaccharides is their limited solubility in DMSO. This paper describes the use of the triethylammonium counterion to overcome this problem, as well as the application of the sodium hydroxide method to make permethylated heparin disaccharides and their workup to yield fully methylated disaccharides for electrospray ionization mass spectrometry. The ease, speed, and effectiveness of the described methodology should open up permethylation of GAG oligosaccharides to a wider circle of mass spectrometrists and enable them to develop further derivatization schemes in the effort to rapidly elucidate the structure of these important molecules. Permethylation may also provide new ways of separating GAG oligosaccharides in LC/MS, their increased hydrophobicity making them amenable for reversed-phase chromatography without the need for ion pairing reagents.     

Analysis and characterization of heparin impurities

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007?C2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.

Array-based functional screening of heparin glycans

Array methodologies have become powerful tools for interrogation of glycan-protein interactions but have critically lacked the ability to generate cell response data. Here, we report the development of a slide-based array method exemplified by measurement of activation of fibroblast growth factor signaling by heparin saccharides. Heparan sulfate-deficient Swiss 3T3 cells were overlaid onto an aminosilane-coated slide surface onto which heparin saccharides had been spotted and immobilized. The cells were transiently stimulated with FGF2 and immunofluorescence measured to assess downstream ERK1/2 phosphorylation. Activation of this signaling pathway response was restricted to cells exposed to heparin saccharides competent to activate FGF2 signaling. Differential activation of the overlaid cells by different-sized heparin saccharides was demonstrated by quantitative measurement of fluorescence intensity. This "glycobioarray" platform has significant potential as a generic tool for functional glycomics screening.     

Engineered bio-active polysaccharides from heparin

[Image: see text] Heparin, the well-known anticoagulant polysaccharide, is also active in many other biological systems owing to its structural similarity to HS, but usually lacks selectivity because it is more highly sulfated. A series of straightforward chemical reactions (de-O-sulfation, de-N-sulfation and re-N-acetylation), carried out to partial or complete extent, were combined, resulting in a number of modified heparin polysaccharide derivatives with altered properties. These exhibited a range of abilities to promote cell signalling through the FGF/FGFR tyrosine kinase signalling system, in an in vitro cell assay with combinations of FGF-1, -2, -3 and FGFR 1 and 3. One polysaccharide (N-acetylated, 6-O- and 2-O-sulfated heparin), with only a fraction (<10(-3)) of the anticoagulant activity of heparin (200 U . mg(-1)), promoted FGF-2-mediated angiogenesis (10-fold) and therefore had an improved ratio of pro-angiogenic activity to anticoagulant activity in excess of 10(4) compared to heparin. These results demonstrate that heparin-derived polysaccharides can be engineered for selected activities and have potential in a wide range of medical, biotechnological and tissue-engineering applications. Effect of selected engineered heparin polysaccharides on angiogenesis.     

A fluorescent polymeric heparin sensor

Linear copolymers have been developed which carry binding sites tailored for sulfated sugars. All binding monomers are based on the methacrylamide skeleton and ensure statistical radical copolymerization. They are decorated with o-aminomethylphenylboronates for covalent ester formation and/or alkylammonium ions for noncovalent Coulomb attraction. Alcohol sidechains maintain a high water solubility; a dansyl monomer was constructed as a fluorescence label. Statistical copolymerization of comonomer mixtures with optimized ratios was started by AIBN (AIBN=2,2'-azoisobutyronitrile) and furnished water-soluble comonomers with an exceptionally high affinity for glucosaminoglucans. Heparin can be quantitatively detected with an unprecedented 30 nM sensitivity, and a neutral polymer without any ammonium cation is still able to bind the target with almost micromolar affinity. From this unexpected result, we propose a new binding scheme between the boronate and a sulfated ethylene glycol or aminoethanol unit. Although the mechanism of heparin binding involves covalent boronate ester formation, it can be completely reversed by protamine addition, similar to heparin's complex formation with antithrombin III.     

藏红T褪色分光光度法测定微量肝素

在pH 5.02的Britton-Robinson缓冲溶液中,肝素与碱性吩嗪染料藏红T形成离子缔合物,染料发生明显的褪色,体系吸光度的降低与肝素钠质量浓度成正比,0~2μg/mL范围内符合比耳定律,比吸光系数为0.36×103L.g-1.cm-1。研究了表面活性剂和共存物质的影响,表明方法选择性好。用于肝素钠注射液效价的测定。     

Synthesis of heparin saccharides IV. Synthesis of disaccharides possessing the structure of a repeating unit of heparin.

The synthesis of disaccharides possessing the structure of a repeating unit of heparin is reported. 2-Acetamido-2-deoxy-4-O-(methyl α-D-glucopyranosyluronate)-D-glucopyranose ( 1 ) and 2-[1-(benzyloxy)formamido]-2-deoxy-4-O-(methyl α-D-glucopyranosyluronate)-D-glucopyranose ( 2 ) have been prepared by two routes, (a) from D-glucose and D-glucosamine, and (b) from D-glucuronolactone and D-glucosamine.     

Electrochemical methods for the determination of heparin

A review of studies on the determination of heparin in various samples (pharmaceuticals, biological fluids) by electrochemical methods of analysis in 1976–2014 is presented. Heparin is most often determined in pharmaceuticals by polarography using cationic dyes, and in biological samples, by differential pulse methods on non-stationary mercury electrodes. Works on the creation of heparin-selective electrodes coated with a polyvinylchloride membrane with quarternary ammonium salts are most promising; they can, probably, be used for the creation of portable devices for the determination of heparin.     

Differentiation of 3-O-sulfated heparin disaccharide isomers: Identification of structural aspects of the heparin CCL2 binding motif

The presence of 3-O-sulfated glucosamine residues in heparin or heparan sulfate plays a role in binding to antithrombin III and HSV infection. In this study, tandem mass spectrometry was used to differentiate between two heparin disaccharide isomers containing variable sulfate at C6 in a common disaccharide and C3 in a more rare one. The dissociation patterns shown by MS² and MS³ were clearly distinguishable between the isomers, allowing their differentiation and quantitation. Using this technique, we show that an octasaccharide with 11 sulfate groups with high affinity for inflammatory chemokine CCL2 does not contain 3-O-sulfated disaccharides.     

A colorimetric sensing ensemble for heparin

A receptor possessing ammoniums and a novel amino acid with a boronic acid side chain was designed and synthesized. The receptor shows good affinity and selectivity for heparin over similar polysaccharides possessing lower anionic charge density. The affinity for heparin is similar to that for a heparin disaccharide, indicating that disaccharidic units are the likely sites for binding to the receptor. The receptor has a potential use for creating a colorimetric assay for heparin.