General carbohydrate method

General Carbohydrate Methods

METHODS IN Carbohydrate Chemistry

Roy L. Whistler

Department of Biochemistry, Purdue University, Lafayette, Indiana

James N. BeMiller

Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois

Table of Contents

Cover image

Title page

Methods in Carbohydrate Chemistry

Copyright

Contributors to Volume VI

Preface

Outline of Volumes I–V

Errata and Additions

Section I: Separation and Analysis

Chromatography

Chapter 1: Gas–Liquid Chromatography of Trimethylsilyl Derivatives: Analysis of Corn Syrup

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Introduction

Procedure

Discussion

Chapter 2: Gas Chromatographic Estimation of Carbohydrates in Glycosphingolipids

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Introduction

Procedure

Chapter 3: Gas–Liquid Chromatography of Trimethylsilyl Ethers of Cyclitols

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Introduction

Procedure

Chapter 4: Gas–Liquid Chromatography of Alditol Acetates

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Introduction

Procedure

Chapter 5: Gas–Liquid Chromatography of Methylated Sugars

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Introduction

Procedure

Chapter 6: Qualitative Thin-Layer Chromatography

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Introduction

Procedure

Chapter 7: Quantitative Thin-Layer Chromatography

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Introduction

Procedure

Chapter 8: Preparative Thin-Layer Chromatography

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Introduction

Procedure

Chapter 9: Partition Chromatography on Ion-Exchange Resins

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Introduction

Procedure

Chemical, Physical, and Biochemical Methods

Chapter 10: Determination of Carbonyl Groups with Sodium Cyanide: Total Carbonyls in Starch; Total Carbonyls, Ketones and Aldehydes in Cellulose

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Introduction

Procedure

Chapter 11: Determination of Carbonyl Groups in Oxidized Cellulose: Oximation, Oxime Hydrolysis, and Chlorous Acid-Sodium Borohydride Methods

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Introduction

Procedures

Chapter 12: Direct Spectrophotometric Determination of Iodate following Periodate Oxidation of α-Glycol Groups: Quantitative Removal of Iodate and Periodate by Ion-Exchange Materials or by Solvent Extraction

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Introduction

Procedures

Chapter 13: Determination of Formic Acid in the Periodate Oxidation of Carbohydrates

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Introduction

Procedures

Chapter 14: Determination of Starch with Glucoamylase

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Introduction

Procedure

Discussion

Chapter 15: Use of Concanavalin A for Structural Studies

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Introduction

Procedures

Section II: Preparation of Mono- and Polysaccharides and Their Derivatives

Monosaccharides

Chapter 16: β-D-Allose: From D-Glucose by Oxidation of 1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose and Reduction of 1,2:5,6-Di-O-isopropylidene-α-D-ribo-hexofuranos-3-ulose

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Introduction

Procedure

Derivatives

Chapter 17: D-Gulose: Sodium Borohydride Reduction of 3-O-Acetyl-1,2:5,6-di-O-isopropylidene-α-D-erythro-hex-3-enofuranose

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Introduction

Procedure

Chapter 18: Aldohexofuranoses: Reduction of Aldono-1,4-lactones with Bis(3-methyl-2-butyl)borane

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Introduction

Procedure

Chapter 19: Migration of Epoxide Rings and Stereoselective Ring Opening of Acetoxyepoxides: Methyl 2,3-Anhydro-6-O-triphenylmethyl-α-D-gulopyranoside, Methyl 3-O-Acetyl-α-D-gulopyranoside, Methyl 4-O-Acetyl-α-D-arabinopyranoside, 3,4-Anhydro-α-D-arabinopyranoside, 3,5-Anhydro-1,2-O-isopropylidene-α-D-glucofuranose, and Methyl 3,5-Anhydro-β-D-xylofuranoside

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Introduction

Procedures

Chapter 20: Acetoxonium Ion Rearrangements: α-D-Idopyranose Pentaacetate, 6-Bromo-6-deoxy-α-D-idopyranose Tetraacetate, α-D-Talopyranose Pentaacetate, neo-Inositol

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Introduction

Procedures

Chapter 21: Application of the Wittig Reaction to the Synthesis of Higher Sugars: D-threo-L-galacto- and D-threo-L-ido-Octonic Acid

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Introduction

Procedures

Polysaccharides

Chapter 22: Isolation of Polysaccharides from Gram-Negative Bacteria

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Introduction

Procedure

Chapter 23: Lipopolysaccharides: Preparation from Gram-Negative Bacteria

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Introduction

Procedure

Chapter 24: Teichoic Acids

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Introduction

Procedures

Deoxy Sugars

Chapter 25: 5-Deoxy-D-xylo-hexose: Lithium Aluminum Hydride Reduction of a Tosyloxy Group

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Introduction

Procedure

Derivatives

Chapter 26: 6-Deoxy-D-glucose (D-Quinovose): Lithium Aluminum Hydride Reduction of Methyl 6-Chloro-6-deoxy-α-D-glucopyranoside

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Introduction

Procedure

Derivatives

Deoxyhalo Sugars

Chapter 27: Bromodeoxy Sugars from Epoxides: Methyl 5-O-Acetyl-3-bromo-3-deoxy-α-D-arabinofuranoside, Methyl 5-O-Acetyl-2-bromo-2-deoxy-β-D-xylofuranoside, and Methyl 5-O-Acetyl-3-bromo-3-deoxy-β-D-arabinofuranoside

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Introduction

Procedure

Derivatives

Chapter 28: Methyl 4-O-Benzoyl-6-bromo-6-deoxyhexopyranosides

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Introduction

Procedures

Chapter 29: Chlorodeoxy Sugars via (Chloromethylene)-dimethyliminium Chloride Reactions: 6-Chloro-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose and 6-Chloro-6-deoxy-1,2:3,5-di-O-isopropylidene-α-D-glucofuranose

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Introduction

Procedure

Chapter 30: 6-Chloro-6-deoxy-α-D-glucose: From Methyl α-D-Glucopyranoside by Reaction with Methanesulfonyl Chloride in N,N-Dimethylformamide

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Introduction

Procedure

Derivatives

Chapter 31: Deoxyfluoro Sugars via Displacement of Sulfonyloxy Groups with Tetrabutylammonium Fluoride

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Introduction

Procedure

Derivative

Chapter 32: Deoxyfluoro Sugars from Epoxides: 3-Deoxy-3-fluoro-D-xylose from Methyl 2,3-Anhydro-β-D-ribofuranoside

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Introduction

Procedure

Derivative

Chapter 33: Substitution of Hydroxyl Groups with Iodine: Methyl 5,6-Dideoxy-5-iodo-2,3-O-isopropylidene-α-L-talofuranoside and -β-D-allofuranoside and Methyl 4,6-Dideoxy-4,6-diiodo-3-O-methyl-2-O-p-tolylsulfonyl-α-D-galactopyranoside

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Introduction

Procedure

Aminodeoxy Sugars

Chapter 34: Regeneration of Amino Functions from Acetamidodeoxy Sugars

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Introduction

Procedures

Chapter 35: Displacement of the p-Tolylsulfonyloxy Group in 1,2:5,6-Di-O-isopropylidene-3-O-p-tolylsulfonyl-α-D-glucofuranose: 3-Azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-allofuranose

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Introduction

Procedure

Chapter 36: Amino Sugars via Reduction of Azides: Derivatives of 3-Amino-3-deoxy-D-glueose and 2-Amino-2-deoxy-D-altrose

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Introduction

Procedure

Chapter 37: Synthesis of Amino Sugars with Retention of Configuration: 4-Dimethylamino-2,3,4,6-tetradeoxy-D-erythro-hexose (Isomycamine)

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Introduction

Procedure

Chapter 38: Amino Sugars via Displacement of Sulfonyloxy Groups with Hydrazine: Methyl 4-Amino-4,6-dideoxy-2,3-O-isopropylidene-α-L-talopyranoside and Methyl 2,6-Diacetamido-2,6-dideoxy-3,4-O-isopropylidene-α-D-allopyranoside

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Introduction

Procedure

Chapter 39: Synthesis of Amino Sugars via Oximes: Methyl 4-Amino-4,6-dideoxy-α-D-allopyranoside

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Introduction

Procedure

Chapter 40: Amino Sugars via Anhydro Ring Opening with Ammonia: Ammonolysis of Methyl 2,3-Anhydro-4,6-O-benzylidene-α-D-allopyranoside

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Introduction

Procedures

Chapter 41: Amino Sugars via Nitroolefins: 3-Amino-2,3-dideoxy-D-arabino-hexose Hydrochloride

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Introduction

Procedure

Chapter 42: Methyl 3-Deoxy-3-nitrohexopyranosides: From Methyl Glycosides Through Nitromethane Cyclization

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Introduction

Procedure

Chapter 43: Amino Sugars and Amino Cyclitols via Cyclization of Dialdehydes with Nitromethane: 1,4-Diamino-1,4-dideoxy-neo-inositol, Methyl 3-Amino-3-deoxy-β-D-gluco- and β-D-galactopyranoside, and Methyl 3-Amino-3-deoxy-α-D-mannopyranoside Hydrochloride

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Introduction

Procedures

Chapter 44: Synthetic Approaches to cis-Diamino Sugars: Neighboring Group Reactions—N-3 and N-5 Closures

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Introduction

Procedure

Chapter 45: 2,3-Diamino-2,3-dideoxy-α-D-glucose: A trans-Diamino Sugar from Benzyl 2-Acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside by Double Inversion at C-3 via Neighboring Group Participation

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Introduction

Procedure

Chapter 46: 2,6-Diamino-2,6-dideoxy-α-D-galactose: From Methyl 2,6-Dibenzamido-2,6-dideoxy-4-O-methylsulfonyl-3-O-methyl-β-D-glucopyranoside by Inversion at C-4 via Neighboring Group Participation

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Introduction

Procedure

Derivative

Chapter 47: 2,6-Diamino-2,6-dideoxy-β-D-mannose: From Methyl 2-Benzamido-4,6-O-benzylidene-2-deoxy-α-D-altropyranoside by Inversion at C-3 via Neighboring Group Participation

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Introduction

Procedure

Derivative

Chapter 48: Preparation of Aminomercapto Furanose Sugars from Dithiocarbamoyl Derivatives

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Introduction

Procedure

Chapter 49: 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl Chloride

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Introduction

Procedure

Thio Sugars

Chapter 50: 5-Thio-α-D-glucopyranose: Via Conversion of a Terminal Oxirane Ring to a Terminal Thiirane Ring

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Introduction

Procedure

Derivative

Chapter 51: 4-Thio-D-ribofuranose: From L-Lyxose via Displacement of a p-Tolylsulfonyloxy Group with Thioacetate Anion

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Introduction

Procedure

Derivative

Unsaturated Sugars

Chapter 52: UNSATURATED SUGARS: Unsaturated Sugars via Cyclic Thionocarbonates, Cyclic Orthoformates, and Disulfonic Ester Intermediates: 5,6-Dideoxy-1,2-O-isopropylidene-α-D-xylo-hex-5-enofuranose, 3-O-Benzyl-5,6-dideoxy-1,2-O-isopropylidene-α-D-xylo-hex-5-enofuranose, and Methyl 4,6-O-Benzylidene-2,3-dideoxy-α-D-erythro-hex-2-enopyranoside

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Introduction

Procedures

Chapter 53: 2,3-Unsaturated 3-Nitro Glycosides: Methyl 4,6-O-Benzylidene-2,3-dideoxy-3-nitro-β-D-erythro- and β-D-threo-hex-2-enopyranosides

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Introduction

Procedure

Chapter 54: Modified Synthesis of 1-Deoxyald-1-enopyranose (2-Hydroxyglycal) Esters: Conversion of 1,2-Unsaturated Pyranoid Compounds into 2,3-Unsaturated Glycopyranosyl Derivatives

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Introduction

Procedures

Section III: Oxidation

Chapter 55: Oxidation of Carbohydrates by the Sulfoxide–Carbodiimide and Related Methods: Oxidation with Dicyclohexylcarbodiimide–DMSO, Diisopropylcarbodiimide–DMSO, Acetic Anhydride–DMSO, and Phosphorus Pentaoxide–DMSO

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Introduction

Procedures

Chapter 56: Oxidation of Carbohydrates with Dimethyl Sulfoxide–Acetic Anhydride: Methyl α-D-erythro-Pentopyranosid-3-ulose

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Introduction

Procedure

Chapter 57: Synthesis of a Ketose from a Partially Benzylated Aldose: D-threo-Pentulose from 2,3,5-Tri-O-benzyl-D-arabinofuranose

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Introduction

Procedure

Chapter 58: Oxidation of Carbohydrates with Dimethyl Sulfoxide–Phosphorus Pentaoxide

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Introduction

Procedure

Chapter 59: Oxidation with Ruthenium Dioxide and Hypochlorite: Methyl 6-Deoxy-2,3-O-isopropylidene-α-D-ribo-hexopyranosid-4-ulose

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Introduction

Procedure

Chapter 60: Selective Catalytic Oxidations of Carbohydrates

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Introduction

Procedure

Chapter 61: Unsaturated Glycopyranosiduloses: 2(S)-Methoxy-4-benzoyloxy-6(R)-benzoyloxymethyl-5,6-dihydro-2H-pyran-5-one and 2(S)-Benzoyloxymethyl-4-benzoyloxy-6(S)-methoxy-5,6-dihydro-2H-pyran-5-one

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Introduction

Procedures

Chapter 62: Oxidation of Polysaccharides with Lead Tetraacetate in Dimethyl Sulfoxide

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Introduction

Procedure

Section IV: Acyclic Sugars

Chapter 63: 1,1-Bis(acylamido)-1-deoxyalditols

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Introduction

Procedure

Section V: Etherification

Chapter 64: Methylation of Carbohydrates with Methylsulfinyl Anion and Methyl Iodide in Dimethyl Sulfoxide: Methylation of Aerobacter aerogenes A3(S1) Capsular Polysaccharide and 3-O-α-D-Glucopyranosyluronic acid-D-mannose

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Introduction

Procedures

Chapter 65: Methylation with Diazomethane–Boron Trifluoride Etherate: l,3,4,6-Tetra-O-acetyl-2-O-methyl-β-D-mannopyranose and 2-O-Methyl-D-mannose

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Introduction

Procedure

Chapter 66: Benzyl Ethers: Formation and Removal: Tri-O-benzylamylose and Methyl 4-O-Benzyl-β-D-glucopyranoside: Debenzylation via Sodium in Liquid Ammonia and via Bromination

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Introduction

Procedure

Chapter 67: 2,3,4,6-Tetra-O-benzyl-α-D-glucopyranose

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Introduction

Procedure

Derivative

Chapter 68: Alkylation of Monosaccharides Using Sodium Hydride

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Introduction

Procedure

Chapter 69: C-4 Substitution of Methyl D-Glucosides and Malto-oligosaccharides

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Introduction

Chapter 70: O-Carboxymethylpachyman

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Introduction

Procedure

Section VI: Esterification

Phosphate Esters

Chapter 71: PHOSPHATE ESTERS: Glycosyl Phosphates

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Introduction

Procedure

Chapter 72: L-glycero-Tetrulose (L-Erythrulose) 1-Phosphate

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Introduction

Procedure

Chapter 73: D-αltro-Heptulose (D-Sedoheptulose) 7-Phosphate: Condensation of 2-Nitroethanol and D-Ribose 5-Phosphate

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Introduction

Procedure

Chapter 74: 1L-myo-Inositol 1-Phosphate

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Introduction

Procedure

Chapter 75: Phosphorylation of Starch and Cellulose with an Amine Salt of Tetrapolyphosphoric Acid

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Introduction

Procedure

Other Esters

Chapter 76: OTHER ESTERS: 1,2,3,4-Tetra-O-acetyl-β-D-glucopyranose and Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranoside: Sugar Derivatives with a Free Primary Hydroxyl Group

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Introduction

Procedure

Chapter 77: Use of Xanthates in Synthetic Carbohydrate Chemistry: Thionocarbonate, Chlorothioformate, Dithiocarbonate, Trithiocarbonate, and S-(Methylthio)carbonyl Derivatives

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Introduction

Procedure

Chapter 78: Applications of Phenylboronic Acid in Carbohydrate Chemistry

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Introduction

Procedures

Chapter 79: Sulfation of Polysaccharides

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Introduction

Procedure

Section VII: Nucleosides and Nucleotides

Chapter 80: Isolation of Sugar Nucleotides

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Introduction

Procedure

Chapter 81: Pyrimidine Nucleosides by the Trimethylsilyl Method: Anomeric 1-(D-Ribofuranosyl)thymines and 2-β-D-Ribo-furanosyl-αs-triazine-3,5(2H,4H)-dione (6-Azauridine)

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Introduction

Procedure

Chapter 82: Purine Nucleosides by the Trimethylsilyl Method: 3-Ribofuranosyluric Acid and 2-Chloro-1-(β-D-ribofuranosyl)-5,6-dimethylbenzimidazole

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Introduction

Procedure

Chapter 83: The Hilbert–Johnson Synthesis of Pyrimidine Nucleosides: 1-(2-Deoxy-β-D-lyxo-hexopyranosyl)thymine

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Introduction

Procedure

Chapter 84: Selective Phosphorylation of Ribonucleosides: Sodium Salt of Guanosine 5′-Phosphate by the Cyanoethyl Dihydrogen Phosphate and Phosphoryl Chloride Methods

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Introduction

Procedures

Section VIII: Glycosides

Chapter 85: Cerebrosides, Isolation

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Introduction

Procedure

Chapter 86: Gangliosides, Isolation

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Introduction

Procedure

Chapter 87: Use of Mercuric Cyanide and Mercuric Bromide in the Koenigs-Knorr Reaction: Alkyl β-D-Glucopyranosides, 2-Acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-glucose, and 2-O-α-L-Fucopyranosyl-D-galactose

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Introduction

Procedure

Chapter 88: Synthesis of Oligosaccharides by the Orthoester Method

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Introduction

Procedures

Chapter 89: cis-1,2-Glycosides: Isopropyl 2,3,4,6-Tetra-O-acetyl-α-D-glueopyranoside, Cholesteryl 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranoside, and L-Menthyl 2,3,4-Tri-O-acetyl-6-O-p-tolylsulfonyl-α-D-glucopyranoside

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Introduction

Procedure

Section IX: Radioactively Labeled Sugars

Chapter 90: Preparation of Tritiated Sugars: D-Glucose-5–3H, Dihydroxyacetone-1S-3H 3-Phosphate, D-Galactose-4–3H, 2-Deoxy-D-arabino-hexose-2–3H, Heparin-3H, and 1L-chiro-Inositol-3H

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Introduction

Procedures

Chapter 91: Degradation and Counting of Tritium-Containing Sugars

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Introduction

Procedure

Section X: Physical Methods

Chapter 92: Determination of Molecular Weights by Osmometry

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Procedure

Chapter 93: Conformational Analysis via Nuclear Magnetic Resonance Spectroscopy

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Introduction

Preparation of Solutions

Instrumentation

Confirmation of Spectral Assignments

Interpretation of Coupling Constants

Long-Range Coupling Constants

Chapter 94: Mass Spectrometry of Carbohydrates

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Instrumentation

Sugar Derivatives for Mass Spectrometry

Volatilization

Mass Spectra Interpretation Principles

Fragmentation of Carbohydrate Derivatives

Analytical Application

Glossary

Author Index

Subject Index

Methods in Carbohydrate Chemistry

Volume I: Analysis and Preparation of Sugars

Volume II: Reactions of Carbohydrates

Volume III: Cellulose

Volume IV: Starch

Volume V: General Polysaccharides

Volume VI: General Carbohydrate Methods

Copyright

COPYRIGHT © 1972, BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED

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PRINTED IN THE UNITED STATES OF AMERICA

Contributors to Volume VI

Article numbers are shown in parentheses following the names of the contributors.

G.A. ADAMS(23),     Biochemistry Laboratory, National Research Council of Canada, Ottawa, Ontario, Canada

A.R. ARCHIBALD(24),     Microbiological Chemistry Research Laboratory, Department of Organic Chemistry, The University of Newcastle upon Tyne, Newcastle upon Tyne, England

HANS H. BAER(41, 42, 53),     Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada

C.E. BALLOU(72),     Department of Biochemistry, University of California, Berkeley, California

J.E.G. BARNETT(74, 90, 91),     Department of Physiology and Biochemistry, University of Southampton, Southampton, England

J.N. BEMILLER(6, 7, 8, 66, 69),,     Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois

C.T. BISHOP(62),     Biochemistry Laboratory, National Research Council of Canada, Ottawa, Ontario, Canada

MIROSLAV BOBEK*(51),     Department of Biochemistry, Purdue University, Lafayette, Indiana

A.F. BOCHKOV(88),     N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow, USSR

J.S. BRIMACOMBE(68),     Department of Chemistry, The University, Dundee, Scotland

K.M. BROBST(1),     A. E. Staley Manufacturing Co., Decatur, Illinois

C.P. BRYANT†(37, 39, 59),     Department of Chemistry, Wayne State University, Detroit, Michigan

J.G. BUCHANAN(19),     Department of Chemistry, Heriot-Watt University, Edinburgh, Scotland

KAREL ČAPEK(40),     Laboratory of Monosaccharides, Institute of Chemical Technology, Prague, Czechoslovakia

O.S. CHIZHOV(94),     N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow, USSR

H.E. CONRAD(22, 64),     Department of Biochemistry, University of Illinois, Urbana, Illinois

BRUCE COXON(93),     Institute for Materials Research, National Bureau of Standards, Washington, D.C.

J.O. DEFERRARI(63, 65),     Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

B.A. DMITRIEV(21),     N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow, USSR

W.M. DOANE(77),     Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois

LANDIS W. DONER(35, 76),     Department of Biochemistry, Purdue University, Lafayette, Indiana

MICHAEL E. EVANS(26, 30),     U.S. Army Laboratories, Natick, Massachusetts

R.J. FERRIER*(54, 78),     Department of Chemistry, Birkbeck College (University of London), London, England

HEWITT G. FLETCHER, JR.(57, 67),     National Institutes of Health, Bethesda, Maryland

H.M. FLOWERS(85, 87),     Department of Biophysics, The Weizmann Institute of Science, Rehovoth, Israel

A.B. FOSTER(31),     Chester Beatty Research Institute, London, England

YASUO FUJIMOTO(84),     Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd., Machida-shi, Tokyo, Japan

S.G. GERO(74),     Institut de Chimie des Substances Naturelles, C. N. R. S., Gif-sur-Yvette, France

VICTOR GINSBURG(80),     National Institutes of Health, Bethesda, Maryland

C.P.J. GLAUDEMANS(67),     National Institutes of Health, Bethesda, Maryland

I.J. GOLDSTEIN(15),     Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan

LEON GOODMAN(48),     Department of Chemistry, University of Rhode Island, Kingston, Rhode Island

E.G. GROS(65),     Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

A. GUILBOT(14),     Station de Biochimie et de Physico-chimie des Céréales et de leurs Derivés, Institut National de la Recherche Agronomique, Le Noyer Lambert, Massy, France

S. HANESSIAN(28, 29, 34),     Department of Chemistry, University of Montreal, Montreal, Quebec, Canada

R. HEMS(31),     Chester Beatty Research Institute, London, England

KURT HEYNS(60),     Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany

DEREK HORTON(49, 52),     Department of Chemistry, The Ohio State University, Columbus, Ohio

T.L. HULLAR(44),     Department of Medicinal Chemistry, School of Pharmacy, State University of New York at Buffalo, Buffalo, New York

JIŘí JARÝ(38, 40),     Laboratory of Monosaccharides, Institute of Chemical Technology, Prague, Czechoslovakia

G.H. JONES(55),     Institute of Molecular Biology, Syntex Research, Palo Alto, California

H.G. JONES(5),     Pulp and Paper Research Institute of Canada, Pointe Claire, P.Q., Canada

NAOKI KASHIMURA(58),     Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan

J.F. KENNEDY(13),     Department of Chemistry, University of Birmingham, Birmingham, United Kingdom

J.X. KHYM(12),     Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee

N.K. KOCHETKOV(21, 33, 88, 94),     N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow, USSR

PETER KÖLL(60),     Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany

MARTIN KOSIK†(76),     Department of Biochemistry, Purdue University, Lafayette, Indiana

S.H. KRUSE(27),     Life Sciences Division, Stanford Research Institute, Menlo Park, California

W.C. LAKE(50),     Department of Biochemistry, Purdue University, Lafayette, Indiana

R.U. LEMIEUX(89),     Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

LEON M. LERNER(18),     Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, New York

MENACHEM LEWIN(10),     Institute for Fibers and Forest Products Research, Jerusalem, Israel

F.W. LICHTENTHALER(43, 61),     Institut für Organische Chemie, Technische Hochschule Darmstadt, Darmstadt, Germany

BENGT LINDBERG(56),     Department of Organic Chemistry, Stockholm University, Stockholm, Sweden

F. LOEWUS(3),     Department of Biology, State University of New York at Buffalo, Buffalo, New York

DONALD L. MACDONALD(71),     Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon

BRUCE A. MCFADDEN(73),     Department of Chemistry, Washington State University, Pullman, Washington

CHRISTIANE MERCIER(14),     Station de Biochimie et de Physico-chimie des Céréales et de leurs Derivés, Institut National de la Recherche Agronomique, Le Noyer Lambert, Massy, France

D. MERCIER(74),     Institut de Chimie des Substances Naturelles, C.N.R.S., Gif-sur-Yvette, France

WOLFGANG MEYER ZU RECKENDORF(17, 45, 46, 47),     Institut für Pharmazeutische Chemie der Universität Münster, Münster, Germany

J.G. MOFFATT(55),     Institute of Molecular Biology, Syntex Research, Palo Alto, California

T.L. NAGABHUSHAN(89),     Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

T. NEILSON(44),     Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada

T. NISHIMURA(81, 82),     Central Research Laboratories, Sankyo Company, Ltd., Tokyo, Japan

KONOSHIN ONODERA(58),     Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan

W.G. OVEREND(25),     Department of Chemistry, Birkbeck College (University of London), London, England

FREDERICK W. PARRISH(26, 30),     U.S. Army Laboratories, Natick, Massachusetts

MIKULÁŠ PAŠTEKA(11),     Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Czechoslovakia

HANS PAULSEN(20),     Institut für Organische Chemie der Universität Hamburg, Hamburg, Germany

YECHIEL RABINSOHN(57),     Department of Chemistry, The Weizmann Institute of Science, Rehovoth, Israel

E.J. REIST(27),     Life Sciences Division, Stanford Research Institute, Menlo Park, California

A.C. RICHARDSON(36),     Department of Chemistry, Queen Elizabeth College, (University of London), London, England

OLOF SAMUELSON(9),     Department of Engineering Chemistry, Chalmers Tekniska Högskola, Göteborg, Sweden

R.H. SHAH(3),     Department of Biology, State University of New York at Buffalo, Buffalo, New York

J.H. SLONEKER(4),     Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois

C.L. STEVENS(37, 39, 59),     Department of Chemistry, Wayne State University, Detroit, Michigan

J.D. STEVENS(16),     School of Chemistry, University of New South Wales, Kensington, Australia

B.A. STONE(70),     Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Australia

L. SVENNERHOLM(86),     Department of Neurochemistry, Psychiatric Research Centre, University of Göteborg, Göteborg, Sweden

CHARLES C. SWEELEY(2),     Department of Biochemistry, Michigan State University, East Lansing, Michigan

ROBERT V.P. TAO(2),     Department of Biochemistry, Michigan State University, East Lansing, Michigan

MASAYUKI TERANISHI(84),     Tokyo Research Laboratory, Kyowa Hakko Hogyo Co., Ltd., Machida-shi, Tokyo, Japan

I.M.E. THIEL(63, 65),     Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

P. THIVEND*(14),     Station de Biochimie et de Physico-chimie des Céréales et de leurs Derivés, Institut National de la Recherche Agronomique, Le Noyer Lambert, Massy, France

J. KENETH THOMSON(52),     Department of Chemistry, The Ohio State University, Columbus, Ohio

CHARLES G. TINDALL, JR.(52),     Department of Chemistry, The Ohio State University, Columbus, Ohio

GORDON A. TOWLE(75, 92),     The Copenhagen Pectin Factory, Ltd., Lille Skensved, Denmark

A.I. Usov(33),     N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow, USSR

ROY L. WHISTLER(35, 50, 51, 75, 76, 79),     Department of Biochemistry, Purdue University, Lafayette, Indiana

R.E. WING(6, 7, 8, 66, 69),     Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois

JOHN A. WRIGHT(32),     Division of Biological Chemistry, Sloan-Kettering Institute for Cancer Research, Sloan-Kettering Division of Cornell University Medical College, New York, New York

ALENA ZOBÁČOVÁ(38),     Laboratory of Monosaccharides, Institute of Chemical Technology, Prague, Czechoslovakia

W. WERNER ZORBACH(83),     Department of Chemistry and Chemical Engineering, Michigan Technological University, Houghton, Michigan

*Present address: Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York

†Present address: The Lubrizol Corporation, Cleveland, Ohio

*Present address: Department of Chemistry, Victoria University, Wellington, New Zealand

†Present address: Faculty of Chemical Technology, Slovak Technical University, Bratislava, Janska, Czechoslovakia

*Present address: Station de Recherches sur l’Elevage des Ruminants, Institut National de la Recherche Agronomique, Theix prés Clermont-Ferrand, France

Preface

When I originated the concept of a multivolume treatise to describe useful methods in carbohydrate chemistry, I had no idea that it would become an open-end work. For a long time our laboratories had collected useful procedures in analytical and preparative carbohydrate chemistry. Some of them were mere duplications of excellent working procedures of others, while some were modifications and improvements which we had developed from our own experiences. These were grouped together in a Procedure Book, widely used by graduate students and finally by other laboratories. From this a request arose to compile a set of reliable methods, so complete and clearly descriptive that chemists and biochemists would find the volumes routine and serviceable laboratory aids. Properly, the books should save the carbohydrate expert valuable time from searching through a voluminous literature, and should relieve the nonspecialist in carbohydrates of the difficult problem of deciding which of several procedures is best to use. In seeking contributors, investigators who originated the method or who had detailed knowledge of the method were sought. Their ready and enthusiastic response has been most gratifying. Because the response to the five volumes already published has been so favorable, the work will continue on an open-end basis.

This volume is comprised of a collection of methods selected from the entire field of carbohydrate chemistry. Future volumes will be more selective in approach.

It is my warm pleasure to have my former coworker and the previous Assistant Editor of this work, Professor James N. BeMiller, join me as coeditor of this and the succeeding volumes. It is hoped that workers will continue to recommend, and to offer, methods for inclusion in these volumes.

ROY L. WHISTLER

Outline of Volumes I–V

Outline of Volume I

ANALYSIS AND PREPARATION OF SUGARS

Section I. General Methods Carbohydrate Laboratory Techniques; Chromatography

Section II. Monosaccharides Trioses; Tetroses; Pentoses; Hexoses; Heptoses; Deoxy Sugars; Amino Sugars; Branched-Chain Sugars; C¹⁴-Labeled Sugars; Inososes

Section III. Oligosaccharides Reducing Oligosaccharides; Non-Reducing Oligosaccharides

Section IV. Analyses

Section V. Color Reactions of Carbohydrates

Section VI. Physical Measurements

Outline of Volume II

REACTIONS OF CARBOHYDRATES

Section I. General Considerations

Section II. Oxidation Products Aldonic Acids; Uronic Acids; Aldaric Acids; Hexulosonic Acids; Esterification

Section III. Reduction Products

Section IV. Derivatives of Nitrogen Bases Glycosylamines; Phenylhydrazine Derivatives

Section V. Etherification Methyl Ethers; Benzyl Ethers; Triphenylmethyl Ethers; Sugar Anhydrides; Anhydroalditols; Diketohexose Dianhydrides; Dealkylation

Section VI. Esterification Acetates; Acylglycosyl Halides; Benzoates; Carbanilates; Carbonates; Sulfonates; Nitrates; Phosphates; Sulfates

Section VII. Acetalation Cyclic Acetal Derivatives; Glycosidation; Other Acetals

Section VIII. Unsaturated Sugars

Section IX. Aldosuloses (Osones)

Section X. Acyclic Monosaccharides

Section XI. Thiosugars

Section XII. Configurational Inversion

Section XIII. The Oxo Reaction

Section XIV. Grignard and Friedel-Crafts Reactions

Section XV. Saccharinic Acids

Section XVI. Determination of Isotopic Carbon Distribution in Aldoses

Section XVII. Selected Methods in Carbohydrate Chemistry Found in Other Collections

Outline of Volume III

CELLULOSE

Section I. Preparation of Cellulose

Section II. Chemical Analysis

Section III. Physical Analysis

Section IV. Degradation of Cellulose

Section V. Cellulose Esters: Preparation, Properties, Reactions, and Analysis

Section VI. Cellulose Ethers: Preparation, Properties, Reactions, and Analysis

Section VII. Microscopy

Section VIII. Laboratory Equipment

Section IX. Preparation and Analysis of C¹⁴-Labeled Cellulose

Outline of Volume IV

STARCH

Section I. Preparation of Starch and Starch Fractions Whole Starch; Starch Fractions

Section II. Chemical Analyses Whole Starch and Modified Starches; Starch Fractions; Starch Hydrolyzates

Section III. Physical Analyses Whole Starch and Modified Starches; Starch Pastes; Starch Fractions; Starch Hydrolyzates

Section IV. Microscopy

Section V. Starch Degradations

Section VI. Starch Derivatives and Modifications Reactivity; Esters; Ethers; Oxidation

Outline of Volume V

GENERAL POLYSACCHARIDES

Section I. General Isolation Procedures

Section II. Polysaccharide Preparations

Section III. Chemical Analyses

Section IV. Physical Analyses

Section V. Molecular Weight Determinations

Section VI. Structural Methods

Section VII. Derivatives Oxidation and Reduction; Esterification and Deacylation; Etherification

Section VIII. Selected Methods Found in Other Collections

Errata and Additions

Volume I

p. 8, line 3 from bottom. Cross reference to Vol. I [37].

p. 37, lines 2 and 3. This material is no longer manufactured by the Westvaco Chemical Division of the Food Machinery and Chemical Corp. but by the Waverly Chemical Co., Inc., Mamaroneck, N.Y. The Waverly material, however, may have too alkaline a surface and should then be treated to modify this property; see M. L. Wolfrom, R. M. de Lederkremer, and L. E. Anderson, Anal. Chem., 35, 1357 (1963).

p. 57, Table IV, entry 18. For "gala read galacto."

p. 80, Procedure. Preparation of the calcium salt prior to addition of ferric sulfate and barium acetate is beneficial. A second addition of hydrogen peroxide in the manner described in Vol. I [20] is also useful. Probably, the two most important points are the temperature control (no purple color is obtained if the temperature goes above 45°) and deionization with the ion-exchange resins. The use of a conductivity meter as mentioned in Vol. I [20] is invaluable. If the ion count is too high, a second resin treatment is necessary.

p. 94, subtitle. For epimerization read isomerization.

p. 98, subtitle. For epimerization read isomerization.

p. 171, Introduction, line 2. For "meso-glycero-gulo-heptitol" read D-glycero-D-galacto-heptitol."

p. 175, subtitle. For epimerization read isomerization.

p. 176, Derivative. This compound, here described as the α-hexacetate of D-manno-heptulose, has been reported by E. Zissis, L. C. Stewart, and N. K. Richtmyer [J. Amer. Chem. Soc., 79, 2593 (1957)] to be the pentaacetate.

p. 176, Derivative. At the end of the first sentence insert Solution of the pure sugar (pulverized to pass through a 60- or 80-mesh screen) in the acetylating mixture at 0° required 6 to 7 days. No agitation was used. The solution remained colorless during this period and the 48-hr. standing period at 0° which followed. Presence of color in the acetylating mix may indicate the presence of small amounts of plant residues.

p. 178, Procedure, line 3. For "2-Deoxy-N-phenyl-D-ribosylamine read 2-Deoxy-N-phenyl-D-erythro-pentosylamine."

p. 199, Procedure, line 2. For Three g. read Five g.

p. 217, First heading. For 1-Amino-1-deoxy-D-lyxose read Lyxosylamine.

p. 257, Label structures I and II.

p. 291, line 4. Read "D-epi-inosose-2."

p. 312, reference 1. Read "R. M. McCready and E. A. McComb, J. Agr. Food Chem., 1, 1165 (1953)."

p. 312, reference 2. Read "L. R. MacDonnell, E. F. Jansen, and H. Line weaver, Arch. Biochem., 6, 389 (1945)."

p. 351, Methyl 4,6-O-Benzylidene-α-D-glucopyranoside. Cross reference to Vol. I [30].

Volume II

p. 299, line 2. For thionyl read sulfuryl.

p. 333, Procedure, 2nd paragraph, line 1. For α-D(β-L) read α-D(α-L).

p. 333, Procedure, 2nd paragraph, line 4. For α-D(β-L) read β-D(β-L).

p. 334, third paragraph, line 3. For "α-D-poly-O-acylglycosyl read poly-O-acyl-α-D-glycosyl."

p. 335, Structure (I), For "R = o-NO2 · C6H4— read Ar = o-NO2 · C6H4—."

p. 346, last paragraph, line 2. For α-D(β-L) read α-D(α-L).

p. 388, first heading. For "Benzyl 2-O-Methylsulfonyl-β-D-arabinopyranoside (V) read Benzyl 3,4-O-Isopropylidene-β-D-arabinopyranoside (III)."

p. 418, last heading. For "6-Deoxy-1,2-O-isopropylidene-α-D-xylo-hexofuranosid-5-ulose read 6-Deoxy-1,2-O-isopropylidene-α-D-xylo-hexofuranos-5-ulose."

p. 483, subtitle. For D-Glucose read D-Galactose.

p. 514, Line 7. Read "Kojic Acid R. Bentley, ref. 2, p. 238."

Volume III

Foreword. The editors regret that an acknowledgment to Dr. T. N. Kleinert in the Foreword erroneously gave his address as Division of Industrial and Cellulose Chemistry, McGill University. Dr. Kleinert, formerly of the Pulp and Paper Research Institute of Canada, is now retired.

p. 53, third line from bottom. For oxidation read oximation.

p. 135, Preparation of the Chromatographic Column. The preparation can be scaled up by using a column of 15 × 125 cm. and increasing the sample load by a factor of 10. After analysis of the effluent, the tetra- and hexasaccharide fractions are pooled as are the tri- and pentasaccharide fractions. Each of these is then rechromatographed separately. In this way, the original column can be grossly overloaded, but final products of good purity are obtainable in gram lots (K. W. King, personal communication).

p. 135, line 7. For 1.91 read 1.19.

p. 137, lines 4–6. Five to fifteen percent contamination with stearic acid occurs beyond the tetrasaccharide. A single recrystallization is not enough to remove all the stearic acid; however, this can be done by extracting the liquid fraction concentrates with petroleum ether prior to recrystallization until all stearic acid is removed (K. W. King, personal communication).

p. 137, footnote 4. Frequently precipitation occurs immediately on elution for the tetrasaccharide and up. This results in deposition of oligosaccharides on the delivery tip of the column itself which must be cleaned off immediately after elution of each fraction to prevent contamination of subsequent peaks (K. W. King, personal communication).

p. 139, Introduction, line 2. For Celluloytic read Cellulolytic.

p. 141, Cellulase Assay. The reader is warned that this is only one kind of a cellulase assay and is not valid for many systems. This assay does not yield zero order kinetics with many cellulases. This system will also detect many enzymes having no action on native cellulose.

p. 258, line 13. For "O-methylsulfonycellu- read O-methylsulfonylcellu-."

p. 271, Introduction. Add The methylation of cotton cellulose with diazomethane is described in Vol. II [41].

p. 369, next to last line. For Pfleider read Pfleiderer.

p. 371, Pressure Vessels, line 2. For Telflon read Teflon.

Volume IV

p. xii. Read "30a. Inherent Viscosity of Alkaline Starch Solutions Raymond R. Myers and Robert J. Smith

p. 320, Teflon. For Tetrafluoroethylene read A polymer of tetrafluoroethylene.

Volume V

p. 174, line 1. For 5 read 50.

p. 289, line 7 from bottom. For [31] read [30]."

Section I

Separation and Analysis

Chromatography

[1]

Gas–Liquid Chromatography of Trimethylsilyl Derivatives

Analysis of Corn Syrup

BY

K.M. BROBST,     A. E. Staley Manufacturing Co., Decatur, Illinois

Publisher Summary

This chapter discusses the use of gas–liquid chromatography (GLC) technique for the separation of trimethylsilyl ether (TMS) derivatives of carbohydrates. The technique uses a dual-column gas chromatograph equipped with flame ionization detectors at the high temperatures, required for the elution of the TMS derivatives of oligosaccharides. The elution of the internal standard between dextrose and maltose is especially convenient as this position is not competitive with any saccharide from a starch hydrolyzate and can be used with a wide variety of column lengths and liquid phases. Also, the internal standard is eluted just before sucrose and, thus, can be used in the analysis of sucrose–-corn syrup mixtures. The method can also be applied successfully in the study of the distribution of substituents groups such as hydroxyethyl in modified polysaccharides.

Introduction

Separation of trimethylsilyl ether (TMS) derivatives of carbohydrates by gas–liquid chromatography (glc) is now a well established technique and has been used several years for the analysis of complex sugar mixtures such as corn syrups and polysaccharide hydrolyzates in general. Since the detailed paper of Sweeley, Bentley, Makita, and Wells (1), several review papers on this subject have appeared; two such are those by Bishop (2) and Sloneker (3). Also the book by Pierce, Silylation Organic Compounds (4), devotes a comprehensive chapter to the silylation of carbohydrates. Attendant to the development of silylation procedures has been the appearance of new liquid phases and instrumentation to improve the resolution of complex mixtures and to extend the technique to the separation of heptasaccharides (5).

A number of procedures have been described for the trimethylsilylation of carbohydrates including several variations of the reagents of Sweeley and coworkers that require dry samples. Further improvements to accommodate moderate amounts of water were described by Brobst and Lott (6) who determined the mono- through tetrasaccharides of corn syrup. Later came the development of N-(trimethylsilyl)-imidazole and its mixture with pyridine to give a single reagent commercially available as Tri-Sil′Z′ (Pierce Chemical Co., Rockford, Illinois). Its use, as described by Brittain (7), shows it to be applicable to wet sugars and to possess excellent solubility properties.

The method to be described here is that of Brobst and Lott (6) which has been used since 1964 for the trimethylsilylation of carbohydrates and, in particular, corn syrups and other complex sugar mixtures. In addition to being tolerant of up to 40 mg of water, the large excess of reagent greatly increases the stability of silylated sugars so that calibration mixtures are stable for several months, a desirable feature to conserve scarce supplies of reference oligosaccharides.

Procedure

Apparatus and Standards

A dual column gas chromatograph equipped with flame ionization detectors is preferred for use at the high temperatures required for the elution of the TMS derivatives of oligosaccharides, for example, a Hewlett-Packard Series 5750 (Hewlett-Packard Co., Palo Alto, California) or its equivalent. An integrator should be a part of the recording system.

National Bureau of Standards dextrose and maltose hydrate specially purified by column or preparative scale paper chromatography are used for calibration. Higher sugars may be obtained from corn syrups by preparative scale paper chromatography.

Preparation of TMS Derivative

Commercial corn syrups of varying saccharide composition (15–99 dextrose equivalent) usually contain not over 25% water and may be sampled directly without further water removal. Generally, the sample size need not exceed 100 mg of dry substance and must not contain more than 40 mg of water. The sample is weighed accurately into a 16 × 125-mm test tube with a Teflon-lined screw cap together with 6–7 mg of phenyl β-D-glucopyranoside as the internal standard and 1.0 ml of ACS reagent-grade pyridine. The sample is dissolved in the pyridine by allowing the mixture to stand overnight at ∼25° or by warming it in a bath at 60° for 15 min, keeping the tube tightly capped during the solution operation. Then 0.9 ml of hexamethyldisilazane¹ followed by 0.1 ml of 99% trifluoroacetic acid (Pierce Chemical Co.) is added. The tube is capped, shaken vigorously for 30 sec, and allowed to stand 30 min with occasional shaking and with intermittent release of gas pressure. Properly prepared derivatives should be clear to the point of brilliance. If the solution is not clear, it should be warmed for 5–10 min at 60°. No precipitate is formed during this method of derivatization. The sample is now ready for injection into the gas chromatograph.

Chromatographic Conditions

Several liquid phases are available for the separation of TMS compounds; SE-52, SE-30, JXR, OV-1, OV-101, and OV-17 have all been used, and all show reasonable temperature stability at ∼ 350°.

-in stainless steel packed with 60–80-mesh silanized Chromosorb W containing 3% by weight of liquid phase OV-17; column temperature programmed from 150° to 325° at 10°/min and the temperature program started immediately after sample injection; injection port and detector block at 300°; carrier gas, helium, 40 psi, 50 ml/min, hydrogen 12 psi, 30 ml/min, air 500 ml/min; recorder, 1 mV, chart speed 15 in/hr, range 1000, attenuation 16, integrator equipped.

GLC Separation

Using a Hamilton CR 700 syringe (Hamilton Co., Whittier, California), a 5 μl aliquot of the derivatized sample is injected into the gas chromatograph, and the temperature programmer is started immediately. The column temperature is held at the upper limit until the tetrasaccharide derivative is eluted; then the column temperature is returned to 150°. Areas of the internal standard and saccharide peaks are recorded.

Calibration and Calculations

Each of the various pure sugars (8–10 mg) and the internal standard (6–7 mg) are accurately weighed into a 16 × 125-mm test tube and dissolved in pyridine and derivatized as previously described. A 5 μl aliquot is injected into the gas chromatograph; the TMS derivatives are separated, and the areas of the internal standard and saccharide peaks are recorded. From the sample weights and peak areas, the detector response (K) value for each sugar is determined, and the sugar percentages on a dry substance basis are calculated. As an example, maltose will be used to illustrate the calculation of a K value and its use in the analysis of an unknown.

The amount of maltose in an unknown is then calculated from the maltose peak area, the internal standard peak area, and the K value:

The percent maltose is calculated on an anhydrous maltose basis and also on a sample dry substance basis.

Discussion

The separations achieved by this method are best shown by an actual chromatogram (Fig. 1) where the procedure was applied to a 43 D.E. (dextrose equivalent) corn syrup. In addition to the identified major components, several minor components, peaks 1, 2, 8, and 9, are readily discernible. Peak 1 is levoglucosan (1,6-anhydro-β-D-glucopyranose), a characteristic component of acid-converted syrups (8), but which is not sharply separated by some liquid phases from peak 2, the so-called γ-isomer of D-glucose. Peak 9 is largely isomaltose but may contain gentiobiose which elutes in the leading edge of isomaltose. Peak 8 has not been positively identified although indirect evidence based on the elution time of several disaccharides of D-glucose known to be present in acid-converted corn syrups (9) indicates that the silyl derivative of cellobiose is eluted at this position.

FIG. 1 Chromatogram of TMS derivative of 43 D.E. corn syrup.

The elution of the internal standard between dextrose and maltose is especially convenient as this position is not competitive with any saccharide from a starch hydrolyzate and can be used with a wide variety of column lengths and liquid phases. Also, the internal standard is eluted just before sucrose and, thus, can be used in the analysis of sucrose–corn syrup mixtures.

The precision of the method has been discussed (6,10,11). Usually the relative standard deviation for major components is in the 1–4% range. Satisfactory results for maltose in corn syrup were obtained in a collaborative test by member companies of the Corn Industries Research Foundation, Inc., and the method was incorporated into the Standard Analytical Methods Manual of the member companies. Composition data compare favorably with that obtained by paper chromatography, and results for dextrose in corn syrups were the same as those by the glucose oxidase method (12).

The method has found application to a variety of complex sugar mixtures in our own laboratories and elsewhere (5,8,11,13). The usual eclecticism of gas chromatographers is well illustrated by the work of Beadle (5) who extended the method to the determination of maltoheptaose in corn syrups. Certain confectionery products such as hard candy may be analyzed directly for D-fructose and sucrose plus the usual carbohydrates from corn syrup. The major carbohydrate components of soybeans—sucrose, raffinose, and stachyose—are readily separated. The method has also shown considerable promise in the study of the distribution of substituents groups, such as hydroxyethyl, in modified polysaccharides (14).

References

1. Sweeley, C.C., Bentley, R., Makita, M., Wells, W.W. J. Amer. Chem. Soc. 1963; 85:2497.

2. Bishop, C.T. Advan. Carbohydrate Chem. 1964; 19:95.

3. Sloneker, J.H.Szymanski, H.A., eds. Biomedical Applications of Gas Chromatography,; 2. Plenum Press, New York, N.Y., 1968:87.

4. A. E. Pierce, Silylation of Organic Compounds, Pierce Chemical Co., P.O. Box 117, Rockford, Illinois 61105.

5. Beadle, J.B. J. Agr. Food Chem. 1969; 17:904.

6. Brobst, K.M., Lott, C.E., Jr. Cereal Chem. 1966; 43:35.

7. Brittain, G. The Development of a Reagent for Gas Chromatography,. In: American Laboratory. Greens Farms, Connecticut: American Laboratory Publishing Company; May 1969:57. [06436].

8. Kheiri, M.S.A., Birch, G.G. Cereal Chem. 1969; 46:400.

9. Ough, L.D. Anal. Chem. 1962; 34:660.

10. Brobst, K.M., Lott, C.E., Jr. Amer. Soc. Brew. Chem., Proc. 1966; 71.

11. Marinelli, L., Whitney, D. J. Inst. Brew. 1967; 73:35.

12. Brady, J.T., Zagorski, J.A. J. Ass. Off. Anal. Chem. 1969; 52:556.

13. Otter, G.E., Taylor, L. J. Inst. Brew. 1967; 73:570.

14. Lott, C.E., Jr., Brobst, K.M. Anal. Chem. 1966; 38:1767.

¹A special purified grade available from Pierce Chemical Co., P.O. Box 117, Rockford, Illinois 61105.

[2]

Gas Chromatographic Estimation of Carbohydrates in Glycosphingolipids

BY

CHARLES C. SWEELEY and ROBERT V.P. TAO,     Department of Biochemistry, Michigan State University, East Lansing, Michigan

Publisher Summary

This chapter discusses the gas-chromatographic estimation of carbohydrates in glycosphingolipids. Glycosphingolipids are composed of a hydrophobic lipid moiety called ceramide to which simple sugars or oligosaccharides are attached by glycosidic bonds between the reducing end of the carbohydrate unit and the primary hydroxyl group of the ceramide. Glycosphingolipids are common constituents in tissues of many mammalian species. They can be grouped into several families according to structural similarities of the carbohydrate unit. Glycerophosphatides can be removed by alkali-catalyzed methanolysis of the crude polar lipid fraction obtained by chromatographic purification on silicic acid columns; alternatively, the total lipid fraction can be subjected to alkali-catalyzed methanolysis before column chromatography.

Introduction

The glycosphingolipids are composed of a hydrophobic lipid moiety, called ceramide, to which simple sugars or oligosaccharides are attached by glycosidic bonds between the reducing end of the carbohydrate unit and the primary hydroxyl group of the ceramide. These lipids usually contain a mixture of fatty acids, ranging from C16 to C26 in chain length, that are linked in the ceramides by amide bonds with sphingosine and other long-chain sphingolipid bases.

Glycosphingolipids are common constituents in tissues of many mammalian species. They can be grouped into several families according to structural similarities of the carbohydrate unit. Three of the most common types are the neutral glycosphingolipids, the blood group-active glycosphingolipids, and the acidic glycosphingolipids. The neutral glycosphingolipids are related to globoside, 2-acetamido-2-deoxy-β-D-galactosyl-(l → 3)-β-D-galactosyl-(1 → 4)-β-D-galactosyl-(1 → 4)-β-D-glucosyl ceramide,¹ the major glycosphingolipid in several mammalian erythrocytes (1). The blood group-active glycosphingolipids have complex oligosaccharide groups containing glucose, galactose, fucose, and N-acetylgalactosamine or N-acetylglucosamine. The gangliosides are acidic glycosphingolipids that contain one or more sialic acid residues (N-acetylneuraminic or N-glycolylneuraminic acid). The simplest ganglioside is N-acetylneuraminyl-(2 → 3)-β-D-galactosyl-(1 → 4)-β-D-glucosyl ceramide (2,3); more complex types contain N-acetylgalactosamine or N-acetylglucosamine and may have two or three sialic acid residues (4). Sulfatides are acidic glycosphingolipids that have a sulfate ester group on C-3 of one of the sugar units. The chemistry of these families of glycosphingolipids has been reviewed recently (4,5).

The glycosphingolipids are generally isolated from biological sources along with most other lipids by extraction with 2:1 v/v chloroform–methanol (6). Glycerophosphatides can be removed by alkali-catalyzed methanolysis of the crude polar lipid fraction obtained by chromatographic purification on silicic acid columns (7,8); alternatively, the total lipid fraction can be subjected to alkali-catalyzed methanolysis before column chromatography (9,10). Individual glycosphingolipids can then be separated by chromatography on silicic acid (11–13), Flor (7,14), or DEAE-cellulose (9,15). Small amounts of the neutral glycosphingolipids have been purified by preparative thin-layer chromatography on silica gel (8,9).

Quantitative estimation of glycosphingolipids can be based on weights, if convenient, or on a variety of colorimetric determinations, such as those for sphingosine (16), total ester after acid-catalyzed methanolysis (17), and carbohydrate by the anthrone procedure (18, Vol. I [115]) or the phenol-sulfuric acid method (19, Vol. I [115]). Recently, it has become possible to determine some details of the carbohydrate structure by direct mass spectrometry of the poly-O-trimethylsilyl derivative of the intact glycosphingolipid (20).

Detailed information about the nature and relative molar amounts of the sugar constituents in a glycosphingolipid can be obtained quickly and accurately by gas chromatography of the methyl glycosides released from the lipid by acid-catalyzed methanolysis. The procedure also provides a reliable quantitative method for the estimation of glycosphingolipids at nanomolar levels. After methanolysis, amino- sugars are converted to N-acetyl derivatives and the individual components are determined by gas chromatography of the O-trimethylsilyl derivatives. Recent reviews have described the use of the procedure for analyses of glycolipids (21,22) and glycoproteins (23,24).

Procedure

Reagent-grade organic solvents can be used for routine applications, but they should be carefully redistilled for analyses of less than 50 nanomoles of lipid. A stock solution of 0.75 N hydrogen chloride in anhydrous methanol can be stored at room temperature for periods up to 2 weeks. The stock solution of internal standard consists of 36.4 mg of mannitol in 100 ml of methanol containing 1% water. Pyridine should be redistilled from barium oxide before use, and must be kept anhydrous after the distillation.

A mixture of the glycosphingolipid sample (up to 1 mg), 100 μl of mannitol stock solution (0.20 μmoles) and 3 ml of 0.75 N methanolic hydrogen chloride is heated for 20–24 hr at 75°–80° in a small culture tube with a Teflon-lined screw-cap. The solution is then cooled to ∼25° and about 200 mg of silver carbonate is added to neutralize the hydrogen chloride. After 15 min, 0.2 ml of acetic anhydride is added, and the mixture is allowed to stand at ∼25° for 18 hr to convert methyl glycosides of aminosugars to N-acetyl derivatives. The precipitate is removed by centrifugation (1500 g) in a small clinical centrifuge for several min, and the supernatant solution is transferred to a glass-stoppered, 15-ml centrifuge tube. A drop of water is added, and the methyl esters of fatty acids are removed by three extractions with equal volumes of hexane. The aqueous methanol solution is then evaporated to dryness under a stream of nitrogen.

The residue of methyl glycosides is dissolved in 35 μl of a freshly prepared 5:2:1 v/v mixture of dry pyridine, hexamethyldisilazane, and trimethylchlorosilane.² After 15 min at ∼25°, the trimethylsilylation reaction is completed, and an aliquot of 2 μl of the cloudy mixture is injected into the gas chromatographic column.

in id) packed with 3% SE-30 (or 3% OV-1) on 100–200 mesh, acid-washed, silanized diatomaceous earth.³ Alternatively, the separation can be made by linear temperature programmed analysis on the same column, with an initial temperature of 160° and a programming rate of 2°/min to an upper temperature of 230°. When a hydrogen flame ionization detector is used for the analysis, an amplifier setting corresponding to 1.6 × 10−10 amperes full-scale deflection on the recorder (10 mV) gives suitable peaks for the internal standard and components of the sample.⁴

An identification of fucose, galactose, glucose, galactosamine, glucosamine, sialic acid; and inositol can be made by comparison between the observed retention times relative to that of mannitol and those given in Table I. The yield of each component can be calculated from the total area produced by the various anomeric forms of a given sugar, using the area produced by the known amount of mannitol for comparison. Factors to correct for differences in molecular weights of the trimethylsilyl derivatives are given in Table I. For example, using the ratio of 1.25 observed for the area of trimethylsilyl mannitol to those of trimethylsilyl methyl glucosides with equal amounts (mass) of glucose and mannitol, the yield of glucose from gas chromatographic data is calculated by the equation:

TABLE I

Relative Retention Behavior of Trimethylsilyl Methyl Glycosides

aThe retention times are relative to the time for hexa-O-trimethylsilylmannitol, which was 15 ± 1 min on 3% SE-30 at 160° (isothermal) with 48 ml/min nitrogen flow rate.

Samples that contain sialic acid are analyzed by gas chromatography with temperature programming as shown in Fig. 1. With a higher carrier gas flow rate than that used in the example, it may be necessary to begin the analysis at 140° rather than 160° to avoid having fucose peaks on the steep initial slope of the solvent peak.

FIG. 1 Analysis of O-trimethylsilyl derivatives of methyl glycosides on 3% SE-30, programmed from 160° to 230° at 2°/min with a carrier gas flow rate of 10 ml/min (nitrogen). Peaks correspond to the following sugars: fucose (1,2,3); galactose (4,5,6); glucose (7,8); mannitol (9); N-acetylgalactosamine (10,12); N-acetylglucosamine (11, 13, 14); and N-acetylneuraminic acid (15).

References

1. Yamakawa, T., Nishimura, S., Kamimura, M. Jap. J. Exp. Med. 1965; 35:201.

2. Klenk, E., Padberg, G. Z. Physiol. Chem. 1962; 327:249.

3. Handa, S., Yamakawa, T. Jap. J. Exp. Med. 1964; 34:293.

4. Wiegandt, H. Angew. Chem., Int. Ed. Engl. 1968; 7:87.

5. Sweeley, C.C., Dawson, G.Jamieson G.A., Greenwalt T.J., eds. Red Cell Membrane, Structure and Function,. Lippincott: Philadelphia, Pa., 1969; 172–227.

6. Radin, N.S. Methods Enzymol. 1969; 14:245.

7. Miras, C.J., Mantzos, J.D., Levis, G.M. Biochem. J. 1966; 98:782.

8. Vance, D.E., Sweeley, C.C. J. Lipid Res. 1967; 8:621.

9. Svennerholm, E., Svennerholm, L. Biochim. Biophys. Acta. 1963; 70:432.

10. Hakomori, S., Murakami, W.T. Proc. Nat. Acad. Sci. U.S. 1968; 59:254.

11. Yamakawa, T., Irie, R., Iwanaga, M. J. Biochem. (Tokyo). 1960; 48:490.

12. Sweeley, C.C. Methods Enzymol. 1969; 14:255.

13. Taketomi, T., Kawamura, N. J. Biochem. (Tokyo). 1969; 66:165.

14. Kawanami, J. J. Biochem. (Tokyo). 1968; 64:625.

15. Rouser, G., Kritchevsky, G., Yamamoto, A., Simon, G., Galli, C., Bauman, A.J. Methods Enzymol. 1969; 14:272.

16. Lauter, C.J., Trams, E.G. J. Lipid Res. 1962; 3:136.

17. Sweeley, C.C. J. Lipid Res. 1963; 4:402.

18. Radin, N.S., Lavin, F.B., Brown, J.R. J. Biol. Chem 1955; 217:789. J. Biol. Chem. 1956; 219:977. [see also].

19. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. Anal. Chem. 1956; 28:350.

20. Sweeley, C.C., Dawson, G. Biochem. Biophys. Res. Commun. 1969; 37:6.

21. Sweeley, C.C., Vance, D.E.Marinetti, G.V., eds. Lipid Chromatographic Analysis,; I. Marcel Dekker, New York, N.Y., 1967:465–495.

22. Vance, D.E., Sweeley, C.C.Olson R.E., ed. Methods in Medical Research,. Medical Research Publishers: Chicago, Ill., 1970; 123–130.

23. Clamp, J.R., Dawson, G., Hough, L. Biochim. Biophys. Acta. 1967; 148:342.

24. Dawson, G., Clamp, J.R.Olson R.E., ed. Methods in Medical Research,. Medical Research Publishers: Chicago, Ill., 1970; 131–136.

25. Shapiro, D., Flowers, H.M. J. Amer. Chem. Soc. 1959; 81:2023.

26. Shapiro, D., Rachaman, E.S., Rabinsohn, Y., Diver-Haber, A. Chem. Phys. Lipids. 1966; 1:54.

¹Whether all the sugar units in these glycosphingolipids are pyranosides has not yet been determined; in the cases of D-glucosyl ceramide and lactosyl ceramide, the pyranoside forms were proved by comparison of the natural product with synthetic material (see refs. 25,26).

²The mixture of reagents for trimethylsilylation is stable for extended periods in the absence of moisture. Sealed ampoules containing this mixture are available from several commercial houses.

³Commercially prepared column packings from Applied Science Laboratories, State College, Pa., and Supelco, Inc., Bellfonte, Pa., were used for these analyses.

⁴The conditions given in the procedure are suitable for 25 to 250 nmoles of glycosphingolipid. With 2–25 nmoles, only 25 μl of stock mannitol solution should be used and the gas chromatography amplifier setting should correspond to 4 × 10−11 amperes. With 250–1000 nmoles, 150 μl of stock mannitol solution are added and the amplifier settings correspond to 3.2 × 10−10 amperes. Within each of these ranges, further control is possible by injecting different aliquots of trimethylsilylation mixture into the gas chromatograph.

[3]

Gas–Liquid Chromatography of Trimethylsilyl Ethers of Cyclitols

BY

F. LOEWUS and R.H. SHAH,     Department of Biology, State University of New York at Buffalo, Buffalo, New York

Publisher Summary

This chapter discusses the use of gas–liquid chromatography (GLC) for the preparation of volatile derivatives of cyclitols through trimethylsilylation. Because of the low solubility of some cyclitols, the amount of pyridine included in the trimethylsilylating reagent can be increased. This mixture may be stored over long periods of time if precaution is taken to exclude moisture and to use a bottle cap containing an inert liner such as polyethylene or Teflon. The trimethylsilylating reagent in question is generally useful for all cyclitols. Occasionally, the reaction time must be extended, as in the case of neo-inositol that has 24–48 hours as its reaction time.

Introduction

A simple procedure for the preparation of volatile derivatives of cyclitols for gas–liquid chromatography (glc) is trimethylsilylation. Sweeley and coworkers (1) first used this method to separate myoinositol and chiro-inositol.¹ Subsequently it was found (3,4) that hexa-kistrimethylsilyl ethers of all structural isomers of inositol can be resolved by glc by judicious use of stationary phases. The method presented here supplements other techniques, such as paper chromatography of free cyclitols (5,6), paper electrophoresis of borate complexes (7), and glc of acetylated inositols (8).

The hexakistrimethylsilyl ether of myo-inositol is used to identify myo-inositol and to determine it quantitatively (9–). This derivative has also been recommended and used as a reference compound for the separation and identification of other carbohydrate-derived trimethylsilyl ethers (15–18).

Procedure

1,2,3,4,5,6,-Hexakis-O-(trimethyleilyl)-myo-inositol (II)

myo-inositol (I) is carefully purified by recrystallization from water (solubility 14 g/100 ml at 25°). To a dry portion (1.8 g) of I suspended in 50 ml of redistilled, dry pyridine (p. 10) is added 20 ml of hexamethyldisilazane and 7 ml of trimethylchlorosilane, and the mixture is gently stirred while heating at 50°. After 1 hr, 25 ml of pyridine is added and the solution is stirred an additional 3 hr at ∼25°. The white precipitate is removed by centrifugation or filtration, and the cleared solution is evaporated under diminished pressure to a syrup. The syrup is dissolved in 20 ml of warm n-hexane, and the solution is freed of further traces of white precipitate that might have formed during removal of excess reagent and evaporated under diminished pressure. The product (II) crystallizes as solvent is removed; yield is almost quantitative. Crude crystalline II is further purified by sublimation (100°/0.1 torr); m.p. 118°–119° (16).

1,2,3,4,5,6-Hexakis-O-(trimethylsilyl)-scyllo-inositol

The procedure used to prepare II is used for preparation of the corresponding scyllo isomer. The final crystalline product melts at 179°–180° (16).

2,4,6/3,5-Pentakis-O-(trimethylsilyl)-2,4,6/3,5-pentahydroxyhexanone

The procedure used to prepare II is used to make this derivative from the corresponding ketone. A crystalline product is obtained that melts at 98° (16).

Preparation of Trimethylsilyl Ethers of Cyclitols for Gas Chromatography (see also this Vol. [1])

The procedure of Sweeley and coworkers (1) can be used to prepare small samples for gas chromatography. Due to the low solubility of some cyclitols, the amount of pyridine included in the trimethylsilylating reagent is increased. Pyridine, hexamethyldisilazane, and trimethylchlorosilane in the proportions 17:2:1 v/v are recommended (3). This mixture may be stored over long periods of time if precaution is taken to exclude moisture and to use a bottle cap containing an inert liner such as polyethylene or Teflon.

To prepare a sample for gas chromatography, trimethylsilylating reagent is added to a vial containing dry sample such that the final concentration of unsubstituted cyclitol will be 0.1–1 mg/ml. The reaction proceeds rapidly at ∼ 25° but a minimum of 1 hr is recommended unless the kinetics are known from previous runs. All reactions should be run in capped vials with inert liners in the cap. If the trimethylsilylating reagent and pyridine are removed by evaporation and replaced with n-heptane after the reaction is complete, tailing in the solvent peak of the gas chromatogram is virtually eliminated. Samples transferred to n-heptane may be stored for long periods of time if precautions are taken to reduce evaporation and exposure to moisture.

Use of dimethyl sulfoxide (DMSO) instead of pyridine in the trimethylsilylating reagent permits sample injection about 10 min after combining reactants (9). However, trimethylsilyl ethers of cyclitols prepared in DMSO slowly decompose, and storage in DMSO is not recommended.

The trimethylsilylating reagent recommended here is generally useful for all cyclitols. Occasionally the reaction time must be extended, as in the case of neo-inositol which requires 24–48 hr (3). Modifications which apply both to use of the procedure outlined here and to other silylating reagents are collected into a single sourcebook (19).

Quantitative measurement of II is possible by gas chromatography. There is a linear relationship between the mass of II and the area of the resulting peak when the column is packed with 3% SE-30 (3). The present authors find that 3% JXR (specially prepared SE-30 from Applied Science Lab., Inc., State College, Pennsylvania) permits linear measurements to be made between 0.01–10 μg of II.

Gas Chromatography of Trimethylsilyl Ethers of Cyclitols

Retention times relative to hexakistrimethylsilyl-myo-inositol (II) of structural isomers of inositol on several types of stationary phase material are listed in Table I. Recent experience of the present authors with two silicone-based stationary phases, XE-60 and OV-1, suggest that these substances are particularly useful in cyclitol assays. XE-60, a copolymer of dimethyl and cyanoethylmethyl silicones, separates 6 of the structural isomers of inositol (muco, chiro, scyllo, epi, myo, and cis in order of appearance) and resolves the other two (allo and neo) as a single peak ahead of muco-inositol in less than 4 min. Naturally occurring compounds such as scyllo-inositol, dambonitol and myo-inosose-2 which do not separate on XE-60 are readily resolved on OV-1. The short retention times obtained with XE-60 allow sharp resolution and reduce tailing.

TABLE I

Relative Retention Times of Structural Isomers of Inositol (Hexakis-O-trimethylsilyl Ethers)

aRare inositols used to test separations on XE-60 and OV-1 were kindly donated by Professor L. Anderson, University of Wisconsin.

bConditions used: 3% XE-60 on Supelcoport (80/100 mesh); glass column, 6 ft × 4 mm id; N2 flow, 42 ml/min.

cConditions used: 3% OV-1 on Gas Chrom Q (100/120 mesh); glass column, 6 ft × 4 mm id; N2 flow, 45 ml/min.

OV-1 also separates 6 of the structural isomers of inositol (allo, neo, muco, chiro, cis, and myo in order of appearance) and resolves the other 2 (epi and scyllo) as a single peak between chiro-inositol and cis-inositol. For the separation of inositol and inositol glycosides (or related oligosaccharides), the greater thermal stability of OV-1 is a decided advantage, permitting temperatures up to 350°. At 275°, trimethylsilyl ethers of galactinol (1-L-O-α-D-galactopyranosyl-myo-inositol) and raffinose have retention times (relative to II) of 7 and 19, respectively, on the OV-1 columns used to secure data given in Tables I and II.

TABLE II

Relative Retention Times of Inositol Derivatives (Trimethylsilyl Ethers)

aRare compounds used in this table were kindly provided by Professor L. Anderson, University of Wisconsin.

bConditions used in obtaining data in this table are those given in footnote b, Table I.

cConditions used in obtaining data in this table are those given in footnote c, Table I.

Sherman and Goodwin (13,20) found that the tritium foil electron capture detector is several times more sensitive to 2,4,6/3,5-pentakis-O-(trimethylsilyl)-2,4,6/3,5-pentahydroxy-cyclohexanone than to hexakis-O-trimethylsilyl ethers of scyllo-inositol and myo-inositol. They make use of this difference to measure micromolar quantities of the ketone in animal tissues. Their method should find wide application in instances where low levels of this important, naturally occurring intermediate must be measured.

Ueno and coworkers (21) have separated 7 of the structural isomers of inositol (cis omitted) as the hexakis-O-trifluoroacetyl esters by gas chromatography on silicone columns (SE-30, SE-52, and QF-1). Unlike the per(trimethylsilyl) ether, this per(trifluoroacetyl) ester of myoinositol appears ahead of both anomers of D-glucose. Trifluoroacetyl derivatives slowly degrade silicone columns and care should be exercised that such columns are not used interchangeably to separate trimethylsilyl and trifluoroacetyl-substituted cyclitols.

Table II lists a number of inositol derivatives including the naturally occurring O-methyl ethers of inositol together with retention times (relative to II) of their trimethylsilyl ethers. Ononitol and sequoyitol are not resolved