Purification Of Oligosaccharides Research Articles

Purification of oligosaccharides by nanofiltration

Nanofiltration (NF) of a model sugar solution and a commercial galacto-oligosaccharide mixture has been studied with a cross-flow filtration unit in an attempt to investigate the significance of the operating parameters and evaluate a possible purification process. The pressure, feed concentration and filtration temperature effects were studied in experiments carried out in full recycle mode of operation. The rejection factors of the sugar components present in the solutions increased with increasing pressure due to increased solvent flux and also compaction of the membranes (e.g. rejection factor increased from 0.10 to 0.42 for fructose), with this effect being greater for the low molecular weight sugars in the solutions. Increasing the total sugar concentration of the feed caused a decrease to the rejections of the sugars (e.g. from 0.58 to 0.43 for glucose). Increasing the filtration temperature caused a decrease to the observed rejections of the low molecular weight sugars of the solutions (e.g. from 0.72 to 0.48 for fructose). Continuous diafiltration (CD) purification using NF-CA-50 membranes (at 25 °C) and DS-5-DL membranes (at 60 °C), gave yield values of 14–18% for the monosaccharide, 59–89% for the disaccharide and 81–98% for the trisaccharide (oligosaccharide), respectively. The study clearly demonstrates the potential of cross-flow nanofiltration in the purification of oligosaccharide from mixtures containing contaminant monosaccharides.

"Cap-Tag"-Novel Methods for the Rapid Purification of Oligosaccharides Prepared by Automated Solid-Phase Synthesis Financial support from the donors of the Petroleum Research Fund, administered by the ACS (ACS-PRF 34649-G1), Merck (Predoctoral Fellowship for E.R.P.), Boehringer-Ingelheim (Predoctoral Fellowship for E.R.P.), and the NIH (Biotechnology Training Grant for M.C.H.) is gratefully acknowledged. Funding for the MIT-DCIF Inova 501 was provided by the

React or get capped and tagged! Two capping reagents, designated A-Tag and F-Tag, mark unconverted oligosaccharides during automated synthesis for simple separation from the desired products. A-Tagged molecules are removed by an isocyanate scavenger resin after reduction to the amine, and those with an F-tag by filtration through fluorous silica gel. R=oligosaccharide.

“Cap-Tag”—Novel Methods for the Rapid Purification of Oligosaccharides Prepared by Automated Solid-Phase Synthesis

“Cap-Tag”—Novel Methods for the Rapid Purification of Oligosaccharides Prepared by Automated Solid-Phase Synthesis

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides

Heparan sulphates are highly sulphated linear polysaccharides involved in many cellular functions. Their biological properties stem from their ability to interact with a wide range of proteins. An increasing number of studies, using heparan sulphate-derived oligosaccharides, suggest that specific structural features within the polysaccharide are responsible for ligand recognition and regulation. In the present study, we show that strong anion-exchange HPLC alone, a commonly used technique for purification of heparan sulphate-derived oligosaccharides, may not permit the isolation of highly pure heparan sulphate oligosaccharide species. This was determined by PAGE analysis of hexa-, octa- and decasaccharide samples deemed to be pure by strong anion-exchange HPLC. In addition, subtle differences in the positioning of sulphate groups within heparan sulphate hexasaccharides were impossible to detect by strong anion-exchange HPLC. PAGE analysis on the other hand afforded excellent resolution of these structural isomers. The precise positioning of specific sulphate groups has been implicated in determining the specificity of heparan sulphate interactions and biological activities; hence, the purification of oligosaccharide species that differ in this way becomes an important issue. In this study, we have used strong anion-exchange HPLC and PAGE techniques to allow production of the homogeneous heparan sulphate oligosaccharide species that will be required for the detailed study of structure/activity relationships.

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides.

Heparan sulphates are highly sulphated linear polysaccharides involved in many cellular functions. Their biological properties stem from their ability to interact with a wide range of proteins. An increasing number of studies, using heparan sulphate-derived oligosaccharides, suggest that specific structural features within the polysaccharide are responsible for ligand recognition and regulation. In the present study, we show that strong anion-exchange HPLC alone, a commonly used technique for purification of heparan sulphate-derived oligosaccharides, may not permit the isolation of highly pure heparan sulphate oligosaccharide species. This was determined by PAGE analysis of hexa-, octa- and decasaccharide samples deemed to be pure by strong anion-exchange HPLC. In addition, subtle differences in the positioning of sulphate groups within heparan sulphate hexasaccharides were impossible to detect by strong anion-exchange HPLC. PAGE analysis on the other hand afforded excellent resolution of these structural isomers. The precise positioning of specific sulphate groups has been implicated in determining the specificity of heparan sulphate interactions and biological activities; hence, the purification of oligosaccharide species that differ in this way becomes an important issue. In this study, we have used strong anion-exchange HPLC and PAGE techniques to allow production of the homogeneous heparan sulphate oligosaccharide species that will be required for the detailed study of structure/activity relationships.

A Unique Chemoenzymatic Synthesis of α-Galactosyl Epitope Derivatives Containing Free Amino Groups: Efficient Separation and Further Manipulation

A novel chemoenzymatic approach for the synthesis of oligosacchrides containing free amino groups has been developed in which thermophilic glycosidases and a fusion enzyme containing catalytic domains of uridine-5‘-diphospho-galactose 4-epimerase and α(1→3) galactosyltransferase were used. This methodology, in conjunction with a convenient purification procedure employing ion exchange chromatography, facilitates the lengthy and high-cost process of carbohydrate synthesis. The prepared oligosaccharides range from disaccharide lactosamine (1a), trisaccharide α-Gal-(1→3)-β-Gal-(1→4)-GlcNH2 (2), tetrasaccharide β-Gal-(1→4)-β-GlcNH2-(1→3)-β-Gal-(1→4)-β-Glc-N3 (7), to pentasaccharide α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNH2-(1→3)-β-Gal-(1→4)-β-Glc-N3 (15). Compounds 2 and 15 are derivatives of natural α-Gal epitopes. Both of them have shown comparable activities with their natural parent compounds toward human anti-Gal IgG. This method provides a practical approach for the preparation and purification of oligosaccharides containing free amino groups, which can be further derivatized for the enhancement of biological activities.

A general approach to desalting oligosaccharides released from glycoproteins.

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and beta-eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.

HPLC of oligosaccharides in glycobiology.

Carbohydrate chains of glycoproteins are extremely varied and are a wide variety of different biological processes. Determination of their structures is therefore of great interest in both research and clinical fields. HPLC has proved to be the ideal tool for mono- and oligosaccharide purification and analysis. A wide range of adsorbent and solvent systems are available and we describe in this mini-review the main methods used for the separation and detection of oligosaccharides.

Preparation and structural characterization of large heparin-derived oligosaccharides.

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (< 5000) and high-molecular-weight (> 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.

6] Use of lectins in analysis of glycoconjugates

Different lectins are commercially available and used to identify and separate cells, to assay for the activities of glycosyltransferases, to isolate glycoproteins and glycolipids, and to separate and purify oligosaccharides and glycopeptides by affinity chromatography. The technique of serial lectin affinity chromatography allows for the fractionation of oligosaccharides based on their differential affinity for a series of immobilized lectins. The combined use of many lectins is shown to be a powerful method for fractionating and purifying a number of complex oligosaccharides from cell-derived mixtures. The technique of serial lectin affinity chromatography for the purification of oligosaccharides has many advantages. The binding of an oligosaccharide to a particular lectin provide information of the structure of the oligosaccharide. The differential affinity of different oligosaccharides for different lectins can facilitate their rapid separation from each other. When used in conjunction with other techniques, such as gel filtration, high-performance liquid chromatography (HPLC), and paper chromatography, serial lectin affinity chromatography can provide purified oligosaccharides for structural analysis.

Thin-layer chromatography of urinary neutral oligosaccharides: The detection of blood group-related oligosaccharides and screening for lysosomal storage disease.

We devised an improved technique of thin layer chromatography, which permitted the high resolution of urinary neutral oligosaccharides and the qualitative determination of blood group related oligosaccharides as well as oligosaccharides pathologically secreted in lysosomal storage diseases. This procedure can be used inscreening for disorders associated with abnormal excretion of oligosaccharides, as well as in the purification of oligosaccharides.

5] Analysis and purification of oligosaccharides by high-performance liquid affinity chromatography

This chapter focuses on the analysis and purification of oligosaccharides by high-performance liquid affinity chromatography (HPLAC). HPLAC of oligosaccharides offers several advantages as an analytical method. It offers rapid, specific separation, and quantification of oligosaccharides, even in complex mixtures such as urine and serum. Simple procedures for the precise determination of ligand–ligate binding parameters provide a systematic basis for design of columns for specific analytical or preparative applications. The method appears equally well suited to routine assays for clinical studies as well as to research applications, such as analysis of products of glycosyltransferase or glycohydrolase reactions. As hybridoma and molecular cloning technologies make increasing numbers of antibodies and other biologically active proteins available in milligram or gram quantities, it may be profitable to use HPLAC columns heavily loaded with hormones or immunomodulators to search for receptors among complex carbohydrates released as mixtures of free oligosaccharides from surfaces of target cells.

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human α1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.

24] Affinity purification of oligosaccharides using monoclonal antibodies

Publisher Summary Monoclonal antibodies that bind carbohydrate antigens can be linked covalently or noncovalently to solid supports and used for affinity separation of oligosaccharides that contain the target antigen. The method may be generally applied to purification and analysis of naturally occurring free oligosaccharides, complex sugar chains released from glycoconjugates by chemical elimination or endohydrolytic digestion, or oligosaccharide products of specific degradative or biosynthetic reactions. Affinity of antibodies for radiolabeled oligosaccharides can be estimated empirically using a nitrocellulose filter assay. The chapter discusses the materials used in the purification of oligosaccharides. There is also some discussion about the preparation of affinity columns, which includes attachment of antibodies to a solid phase, temperature effects, and flow rates and column designs. Solid-phase immunoabsorbents prepared by binding IgG antibodies via their Fc portions to SPA-Seph have the advantage of greater capacity as compared with columns utilizing chemically activated linker arms.

Purification of oligosaccharides having a free reducing-end from glycopeptide sources

A hydrazinolysis- N-reacetylation procedure, modified by the inclusion of a mild acid-hydrolysis step after N-acetylation, was used to prepare, in overall yields of 60–70%, pure oligosaccharides containing a reducing d-GlcNAc residue from glycopeptide sources. Three types of asparagine-linked glycopeptides were treated: a high-mannose type, a complex-type not containing sialic acid, and a complex-type containing sialic acid, linked both α-(2→3) and α(2→6) to β- d-Gal p residues. After the hydrazinolysis- N-reacetylation procedure, there was often contamination of the reducing oligosaccharides with glycopeptide that remained intact through the procedure, as well as minor oligosaccharide products, altered in the nature of the residue at the reducing end. Oligosaccharides having a reducing d-GlcNAc residue were purified by standard liquid chromatography and high-pressure liquid chromatography (1.c.) 360-MHz 1H-n.m.r. was valuable in establishing common structural reporter signals which enabled major products to be identified at stages during the production of free reducing oligosaccharides, and their purity to be assessed.

The Use of Reverse-Phase Columns for Separation of Unsubstituted Carbohydrates

Abstract Reverse-phase chromatography can be used to separate unsubstituted oligosaccharides using water as the eluent. The retention time of the individual oligosaccharides was found to be dependent on the molecular weight of the oligosaccharide and the type and the anomeric configuration of the linkage. This technique can be used for characterizing polysaccharides based on identification of the oligosaccharide fractions obtained by partial acid hydrolysis or for the isolation and purification of oligosaccharides.

The application of high-performance liquid chromatography to the purification of oligosaccharides containing neutral and acetamido sugars.

Conference Article| December 01 1985 The application of high-performance liquid chromatography to the purification of oligosaccharides containing neutral and acetamido sugars ELIZABETH F. HOUNSELL; ELIZABETH F. HOUNSELL 1Applied Immunochemistry Research Group, M.R.C. Clinical Research Centre, Watford Road, Harrow HA1 3UJ, Middx. U.K. Search for other works by this author on: This Site PubMed Google Scholar NICOLA J. JONES; NICOLA J. JONES 1Applied Immunochemistry Research Group, M.R.C. Clinical Research Centre, Watford Road, Harrow HA1 3UJ, Middx. U.K. Search for other works by this author on: This Site PubMed Google Scholar MARK S. STOLL MARK S. STOLL 1Applied Immunochemistry Research Group, M.R.C. Clinical Research Centre, Watford Road, Harrow HA1 3UJ, Middx. U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1985) 13 (6): 1061–1064. https://doi.org/10.1042/bst0131061 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation ELIZABETH F. HOUNSELL, NICOLA J. JONES, MARK S. STOLL; The application of high-performance liquid chromatography to the purification of oligosaccharides containing neutral and acetamido sugars. Biochem Soc Trans 1 December 1985; 13 (6): 1061–1064. doi: https://doi.org/10.1042/bst0131061 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ODS, silica bonded with octadecyl groups, APS, silica bonded with aminopropyl groups, h.p.t.l.c., high-performance thin-layer chromatography This content is only available as a PDF. © 1985 Biochemical Society1985 Article PDF first page preview Close Modal You do not currently have access to this content.