1OOORequirements for StructuralDetermination of a Carbohydrate-Identification of sugars.-Stereochemistry of each sugar.-Types of linkages.-Types of ring structures.-Anomeric configuration of each sugar.-Sequence of the different sugar residues.ABCBCMB8020April 11, 20062Methods to Determine Carbohydrate StructureThe structural characterization of carbohydrates & glycoconjugates is challenging due to the complexity & diversity of carbohydrate structure. To completely characterize an oligo- orpolysaccharide the following must be done/determined:31. Isolate “pure” oligo-, polysaaccharide, glycoconjugate2. Cleave the carbohydrate(s) from the aglycone3. Determine glycosyl residue compositionMethods to Determine Carbohydrate Structure44. Determine absolute configuration (i.e. D or L) of each glycosyl residue (specific rotation; c.d. absorption; enzymatic susceptibility; derivatization and M.S.; NMR)Methods to Determine Carbohydrate Structure5Methods to Determine Carbohydrate Structure5. Determine glycosyl linkage(s) (methylation analysis; GC-MS; GLC-MS)66. Establish ring form (i.e. furanose or pyranose) of each glycosyl residue (methylation analysis ± ethylation)Methods to Determine Carbohydrate Structure7Methods to Determine Carbohydrate Structure7. Determine sequence of the glycosyl residues (multiple approaches)8Methods to Determine Carbohydrate Structure8. Determine the anomeric configuration (i.e. α or β) of each glycosyl residue (NMR; enzyme susceptibility)9Methods to Determine Carbohydrate Structure9. Determine points of attachment of any non-carbohydrate substituents10. Determine higher levels of structure, when applicable10Organic(lipids)proteinsAqueousCarbohydrates & nucleic acidsGlycolipids & glycoproteins are more difficult to purify. Specific enzymes are often used to cleave the carbohydrate from the aglycone.Oligo- and polysaccharides can be further purified by standard chromatography (i.e. ion exchange, size exclusion, HPLC, preparative electrophoresis, electrofocusing, etc. The preparation of a “purified” carbohydrate or glycoconjugate may be challenging.Carbohydrates can be separated from proteins & lipids by aqueous:organic extractionPurification of Carbohydrates11Detection of CarbohydratesColorimetric Assays for Carbohydrates. Often done in microtitre plates & compared to standard curves. Sensitivity in the nanomole range. General Carbohydrate AssayPhenol-Sulfuric Acid AssaySample + 5% phenol + H2SO4. Read OD at 490 nm. Not good for amino and acetylated sugars. (Dubois,M. et al, 1956, Anal. Chem. 28:350-356)Ferricyanide AssayHydrolyze samples in 2N HCl for 2 hr at 100°C. Treat with reducing agents. Read OD at 690 nm. (Park & Johnson, 1949, J.Biol. Chem. 181:149)12Reducing sugar assaysPara-Hydroxybenzoic Acid Hydrazide (PAHBAH) AssayAdd reagent, boil 10 min. Read OD 410 nm. (Lever, 1972, Anal. Biochem. 47:248)Nelson-Somogyi AssayHeat samples, add reducing agent, heat. Read absorbance at 500 nm. (Nelson, 1944, J.Biol. Chem 153:375-380; Somogyi, 1952, J. Biol. Chem 195:19-23;Spiro, 1966, Methods Enzymol. 7:3-26; Green et al., 1989, Anal. Biochem. 182:197-199)13Uronic AcidsMeta-hydroxybiphenyl Assay for uronic acidsHydrolyze in boiling H2SO45 min, add colorimetric reagent. Read at 520 nm. (Blumenkrantz & Asboe-Hansen, 1973, Anal. Biochem. 54:484; York et al., 1985, Methods in Enzymology 118:3-40, see pg. 26)Orcinol Assay for pentoses & uronic acidsAdd colorimetric reagent & HCL, boil 20 min. Read OD 665 nm. (Dische, 1953, J.Bio.Chem 204:983)Hexose AssaysAnthrone AssaySamples + 0.2% anthrone in H2SO4, Boil. Read OD at 620 nm. Color may vary with type of sugar. (Dische, A., 1962, In Methods Carbohydr. Chem. 1:478-512)14KDO AssayThiobarbituric Acid (TBA) Assay for KDO (and Sialic Acids)Treat with periodate followed by colorimetric reagent. Read OD 549 nm. (Aminoff, 1961, Biochem. J. 81:384; Uchida, 1977, J. Biochem, 82:1425)Sialic Acid AssaysResorcinol Assay for Sialic Acid Add colorimetric reagent in HCl, boil 15 min, add methylbutanol, centrifuge. Read upper phase at OD 450nm & 580 nm. Correct for hexose (OD 450). (Svennerholm, 1959, Biochim. Biophys. Acta 24:604-611; Beeley, 1985, In Laboaratroytechniuqes in biochm. & Mol. Biol., Vol 16, Elsevier)15Other types of carbohydrate detection*Electrochemical Detection (DIONEX) (good sensitivity)*Refractive Index (poor sensitivity)*If carbohydrate is derivatized: UV or fluorescence*Radioactive16Glycosyl Residue Composition AnalysisDetermination of the molar ratio of the constituent monosaccharidesBasic steps:1. Cleave glycosidic bonds (e.g. hydrolysis or methanolysis or solvolysis by hydrogen fluoride). Note: glycosidic bonds differ in their susceptibility to acid hydrolysis: depends on monosaccharide and on position and anomeric configuration of glycosic linkage.172. Separate monosaccharides. Four common methods are:A. Preparation of Alditol acetates & GC (can not distinguish uronic acids)B. Preparation of Trimethylsily (TMS) methylglycosides& GC Useful to detect uronic and amino sugars. Spectrum more complex than alditol acetate: get α & βanomer and maybe pyranose & furanose)C. Separate by high pH anion exchange chromatography (HPAEC)D. Monosaccharide composition analysis by Carbohydrate Electrophoresis (FACETM, Fluorophore-Assisted Carbohydrate Electrophoresis)18Preparation of alditol acetates & GC (composition analysis)GlucitolReduced monosaccharide(NaBD4+ NH4OH)Remove NaBD4& borate with methanol acetic acidAdd acetic anhydride + baseSeparate by GCNOTE: can not specifically detect acidic sugars with this protocol!volatile alditolacetateGlucoseHydrolyzed monosaccharide19OOHHOOOHHOOHHOHOOHOH+NaBD4CHOHOHHOHOOHOHDAc2O/Pyr.CHDOAcCOAcCHCOAcCHOAcCH2OAcAcOPreparation of Alditol Acetates20510MinutesDetector ResponseRhaFucRibAraXylManGalGlcInos.HepGLC Profile of Alditol Acetates(Supelco SP2330 Column)21+OOHHOHOOHOCH3HOOHHOOOHCH3OH(CH3)3SiXOOHHOHOOHOCH3+OOTMSTMSOOTMSOCH3TMSO++OOTMSTMSOOTMSTMSOOCH3(COH)O(COOCH3)(COOCH3)(COOCH3)(COOCH3)Preparation of Trimethylsilyl (TMS)Methyl Glycosides+furanose formsPreparation of Trimethylsilyl (TMS) Methyl GlycosidesThis methods does allow detection of acidic sugars2210 20MinutesDetector ResponseGLC of TMS Methyl GlycosidesAraRhaFucXylGlcAGalAManGalGalAGalGlcGlcAGlcHepHepInos.(J&W Scientific DB1 column)23Separate by high pH anion exchange chromatography (HPAEC)Chromatography often done on a DIONEX HPLC system using a anion exchange column (e.g. CarboPacPA1) with Pulsed