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.     

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used ¹H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the ¹H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion.     

Chromatographic methods for analysis of aminoglycoside antibiotics.

Aminoglycosides are antimicrobial agents used frequently in treatment of human and animal diseases caused by aerobic, gram-negative bacteria. Because of the toxicity of these compounds, considerable effort has been attributed to analysis of aminoglycoside content in drug preparations, in serum and urine specimen in therapeutic drug monitoring, and in edible animal tissues in residue control. The present review emphasizes the analytical problems associated with aminoglycoside analysis. Screening methods based on microbiological and immunological procedures were briefly discussed. Gas chromatography and especially high-performance liquid chromatography appeared the most widely used chemical methods for the analysis of these compounds. Due to lack of volatility, chromophore, and hydrophility of aminoglycosides, most methods applied derivatization for enhancement of their chromatographic characteristics. The applicability and advantages of the various derivatization procedures were discussed in detail. A wide variety of detection methods, including mass spectrometry have been used. Packed column separation was generally used for gas chromatographic separation. In liquid chromatography, reversed phase, ion pair, ion exchange, and normal phase separation has been employed. Mass spectrometry, as a detection method, was discussed in detail. Extraction procedures from body fluids and tissues were emphasized. The performance and the operational conditions of the methods were described and detailed information of the data was provided also in table format.     

Chromatographic methods for tetracycline analysis in foods.

The tetracyclines have served for decades as an important class of antibiotics in food animal health and production. As such, they have also been a source of concern for residue monitoring authorities around the world. In response to this concern a number of microbial inhibition, immunoassay and bacterial receptor methods have evolved for the detection of this class of compounds in various foods of animal origin. However, these methods often lack specificity and are subject to false positive and false negative results. For these reasons a number of chromatographic methods for the separation and determination of the tetracyclines isolated from foods have been developed that are capable of identifying and quantifying individual tetracycline drugs. We present here an overview of tetracycline analytical methods, including microbial inhibition, immunoassay and receptor technologies for detection, techniques for isolation from food matrices, and thin-layer chromatographic, high-performance liquid chromatographic, gas chromatographic and mass spectrometric procedures for determination of this class of compounds. A discussion of the variables involved in such methodology and a review of method criteria are offered.     

Chromatographic methods for petroleum fractions analysis

The state-of-the-art problem of the analysis of various petroleum fractions by the methods of high performance liquid and capillary gas chromatography, IR-spectroscopy, and mass-spectrometry has been considered. The core principle of high performance liquid chromatography as the principal method for petroleum fractions’ separation has been described. Some methods for the chemical modification of domestic silica have been presented. Methods for aromatic hydrocarbons in benzine, kerosene, and diesel petroleum fractions by the chromatography method have been described.     

Chromatographic and electrophoretic methods used for analysis of milk proteins.

Current knowledge of milk proteins and their behavior in dairy foods is based on early applications of chromatography and electrophoresis. Electrophoretic identification of the number and genetic variety of milk proteins inaugurated a research effort in which chromatographic techniques were successfully applied to the isolation of each milk protein, thus facilitating the characterization and further study of milk and dairy products. This review focuses on recent applications of chromatography for separations and analysis and on analytical applications of electrophoresis.     

Chromatographic methods for the analysis of vancomycin

Four thin-layer chromatographic systems were developed for the separation of vancomycin, related antibiotics and degradation products. Bioautography was suitable for detecting trace amounts of biologically active components. High-performance liquid chromatography was used to examine the composition of vancomycin and other glycopeptide antibiotics and to monitor the stability of vancomycin. Degradation of vancomycin lead to changes in the composition which were not matched by a similar loss of potency.     

Chromatographic methods for the analysis of crude tall-oil

Chromatographic methods currently used for isolation, separation, and identification of the components of crude tall-oil have been reviewed, and their resolution and analytical methodology compared. Sample introduction by cold on-column, rather than vaporizing, injection provides good reproducibility and operational convenience for capillary GC analysis. Size exclusion chromatography for isolation followed by capillary GC-MS for identification, gave the best performance for analysis of acidic and dimeric compounds.     

Chromatographic methods for the analysis of basic neurotransmitters and their acidic metabolites.

Chromatographic techniques for the determination of trace amounts of neurotransmitters were reviewed. The two techniques found to be most useful were GC-MS and the reversed-phase mode of HPLC with an electrochemical or fluorescent detector. For structure determination or unequivocal peak identification, GC-MS is the method of choice. In addition the limits of detection of GC-MS were better than those obtained by HPLC. However for routine analyses, HPLC is now being used in studies of mental illness and other diseases. Good resolution, reproducibility and sensitivity can be obtained without the derivatisation steps required for GC-MS, and catecholamines, serotonin, and their acidic metabolites can be concomitantly determined in one analysis.     

Chromatographic procedures for the isolation of plant steroids.

In this review, we consider the general principles and specific methods for the purification of different classes of phytosteroids which have been isolated from plant sources: brassinosteroids, bufadienolides, cardenolides, cucurbitacins, ecdysteroids, steroidal saponins, steroidal alkaloids, vertebrate-type steroids and withanolides. For each class we give a brief summary of the characteristic structural features, their distribution in the plant world and their biological effects and applications. Most classes are associated with one or a few plant families, e.g., the withanolides with the Solanaceae, but others, e.g., the saponins, are very widespread. Where a compound class has been extensively studied, a large number of analogues are present across a range of species. We discuss the general principles for the isolation of plant steroids. The predominant methods for isolation are solvent extraction/partition followed by column chromatography and thin-layer chromatography/HPLC.     

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.     

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.     

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.     

Chromatographic methods for blood alcohol determination.

This review is focused on the different chromatographic strategies for blood alcohol determination which can be adopted for clinical and/or forensic purposes. Particular attention is paid to gas chromatography and to high-performance liquid chromatography. However, other analytical techniques in common use, such as chemical and enzymic methods, are also briefly presented, together with some, at present unusual or experimental, approaches, such as enzymic reactors and catalytic electrodes, which are suitable for application in column liquid chromatography. Finally, mention is made of the methods for the determination of acetaldehyde, the major ethanol metabolite, and of some proposed markers of chronic alcohol abuse, such as acetaldehyde-protein adducts and carbohydrate-deficient transferrin. In order to give the background of knowledge for the rational choice of an analytical strategy, an updated outline of ethanol metabolism and toxicology is presented, together with basic information for the interpretation of the results. Problems concerning blood sampling and storage are also discussed.     

Chromatographic methods for the analysis of heterocyclic amine food mutagens/carcinogens.

A series of potent heterocyclic amines that are mutagenic and carcinogenic have been discovered that are formed in some heated foods, most notably, meats derived from muscle. Determining the heterocyclic amine content in foods and food products is required for toxicological research, industry quality control, and possibly in the future, regulatory control. The contents of food needs to be determined using reliable analytical techniques. Since heterocyclic amines are present in foods at ng/g levels, a variety of liquid-liquid or solid-phase purification techniques are required, followed by gas or high-performance liquid chromatography. Peak detection has been successful using UV, fluorescence, and mass spectrometric detection, and biological activity using the Ames/Salmonella test. The low levels present require that chromatographic efficiency, and both detector sensitivity and selectivity be optimized. The cartridge solid-phase extraction and high-performance liquid chromatography method have been used to measure the known food-derived heterocyclic amines for several types of food, and to the authors knowledge, this is the only method undergoing intralaboratory comparison and validation. Our analysis of the literature shows that chromatographic analysis of the heterocyclic amines by high-performance liquid chromatography or gas chromatography (with derivatization) is satisfactory for heterocyclic amine analysis in foods although the methods are just now being optimized for routine use. The biggest improvements in speed and accuracy will probably come from improved extraction methods as analysis of complex food samples for heterocyclic amines will always be a challenge.     

Ion exchange chromatographic separation and isolation of oligosaccharides of intact low-molecular-weight heparin for the determination of their anticoagulant and anti-inflammatory properties

It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9?×?250 mm) ion exchange column with sodium chloride gradient elution and UV detection at 232 nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions.

Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions

Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure-function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N-hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure-activity relationship in biological processes.