Polysaccharides - structural diversity and functional diversity

The first edition was published as Polysaccharides: Structural Diversity and Functional Versatility, edited by Severian Dumitriu (Marcel Dekker, Inc.,
1998).
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Copyright 2005 by Marcel Dekker

Foreword

Polysaccharides as natural polymers are by far the most abundant renewable resource on the earth with an annual formation
rate surpassing the world production rate of synthetic polymers by some orders of magnitude. In contrast to petroleum-based
synthetic polymers, plant polysaccharides are sustainable materials synthesized by the sun’s energy and fully biodegradable in
the original state. Thus, with decreasing supply of oil resources polysaccharides, including cellulose, starch, chitin, and
hemicelluloses, are expected to play an increasingly important role in industrial use. Polysaccharides are designed by nature to
carry out various specific functions. Examples comprise structural polymers such as cellulose and chitin, storage
polysaccharides such as starch and glycogen, and gel forming mono- and copolymers such as mucopolysaccharides
(glycosaminoglycans), agar, and pectins. Generally, polysaccharides are highly functional polymers with magnificent
structural diversity and functional versatility. Their structural and functional properties are often superior to synthetic
materials as demonstrated, for instance, by the cellulose based cell wall architecture of plants or the function of hyaluronic acid in
the human body.
It has been a true challenge to present state-of-the-art polysaccharide research from different aspects regarding the
macromolecular variety, function and structure in just one volume. In this book well-known and recognized authors describe
the current state of research in their specific fields of expertise in which many of them have been active for decades. With regard
to cellulose and starch as the most abundant polysaccharides, structure, chemical modification, physical chemistry, and
industrial aspects are being discussed. It is further demonstrated that cellulosic biomass conversion technology permits large
scale sustainable production of basic chemicals and derived products. The focus of other chapters are bacterial polysaccharides,
hemicelluloses, gums, chitin, chitosan, hyaluronan, alginates, proteoglycans, glycolipides, and heparan sulfate-like polysaccharides. Some chapters deal with medical and pharmaceutical aspects including medical foods, anticoagulant properties and
the role of polysaccharides in tissue engineering. Furthermore, methodical aspects, including characterization by X-ray
scattering, spectroscopic methods, light scattering, and rheology are discussed.
In summary, the comprehensive, improved, and expanded second edition of ‘‘Polysaccharides’’ reflects the current state
of knowledge of nearly the entire spectrum of polysaccharides with emphasis on structures, methods of structural analysis,
functions and properties, novel routes of modification, and novel application fields. With each chapter, the reader will find
references for a deeper insight into a specific field. Thus, this book is a very useful tool for scientists of both academia and
industry interested in the fundamental principles of polysaccharide functions and modifications on one hand and novel
applications on the other. Having been involved in similar work mainly with industry-related issues of cellulose research for

many years, I would like to stress that the presented state of knowledge, as sophisticated as it might seem to be, should not be
understood as the final stage, but as an invitation to add new knowledge to this field and to explore additional applications of
polysaccharides. I would be delighted, if this monograph challenged and encouraged scientists to deal with polysaccharides as
fascinating polymers with a bright future.
Hans-Peter-Fink
Fraunhofer-Institute for Applied Polymer Research
Potsdam-Golm, Germany
iii

Copyright 2005 by Marcel Dekker


Preface

Polysaccharides are the macromolecules that belong to the means components of life. Together with nucleic acids and
proteins, the polysaccharides determine the functionality and specificity of the species. Polysaccharides have received little
such promotion even though they are widely distributed throughout nature and have highly organized structure. There are
important molecules involved throughout the body in signal transduction and cell adhesion. Polysaccharides can be broadly
classified into three groups based on their functions, which are closely related to their occurrence in nature: structural,
storage, and gel forming. The first compounds used at the industrial level were the polysaccharides.
This work provides the most complete summary now available of the present knowledge of polysaccharide chemistry.
This book discusses eleven fundamental aspects of polysaccharides:
1. Progress in structural characterization. The structural analysis may offer the most fundamental knowledge to
understand the functions of polysaccharides, but the diversity and irregularity of polysaccharide chains make the structural
analysis a formidable task. The conformational analysis involves two aspects: (a) the characterization of a single chain
conformation and (b) the analysis of the chain assembly of polysaccharides. A remarkable progress has been achieved in recent
years with high-resolution, solution- and solid-state-1H- and 13C-NMR including cross-polarization-magic-angle-spinning
and two-dimensional techniques. Specific electron microscopy techniques can visualize single polysaccharide molecules and
can yield reliable information on their contour length distribution, persistence length and conformational aspects.
Some recent progress reports on computational methods for simulations and calculations associated with structure

elucidation of polysaccharides have demonstrated that these methods can contribute to a ‘‘decision’’ on the actual
conformational properties of oligosaccharides and linear polysaccharides.
2. Conformation and dynamic aspects of polysaccharide gels. The most important aspect of characterization of
polysaccharide gels seems to clarify their backbone dynamics together with conformations as viewed from their highly
heterogeneous nature. Backbone dynamics of polysaccharide gel network can be characterized by means of simple
comparative high-resolution 13C NMR measurements by cross-polarization-magic angle spinning (CP-MAS) and dipolar
decoupled-magic angle spinning (DD-MAS) techniques.
3. Rheological behavior of polysaccharides in aqueous systems. Rheology provides precious tools to explore and
understand the properties of polysaccharides in aqueous systems. The rheological behavior of polysaccharides systems
manifests the underlying structure of the systems. In the simplest case, that of polysaccharides solution, viscosity is directly
related to fundamental molecular properties (molecular conformations, molecular weight and molecular weight distribution,
intramolecular and intermolecular interactions). In the case of more structured polymer systems, gels, for example, their
viscoelastic properties are related to supramolecular organization. The main types of polysaccharide systems that are
encountered in the applications can be distributed schematically in three classes: solutions, gels, and polysaccharide/
polysaccharide (or polysaccharide/protein) mixtures in aqueous media.
4. Biosynthesis, structure, and physical properties of bacterial polysaccharides (exopolysaccharides). This part presents
the mechanisms of biosynthesis of bacterial polysaccharides and provides some information on the engineering of
polysaccharides that will allow in the near future the production of a polysaccharide with a choice chemical structure
having a set of predictable physical properties. This part covers also pertinent areas such as: bacterial and fungal
polysaccharides, cell-wall polysaccharides, production of microbial polysaccharides, industrial gums, and microbial
exopolysaccharides of practical importance.

Copyright 2005 by Marcel Dekker


vi

Preface

The bacterial polysaccharides are described as: production and synthesis, composition and structure, physical

properties, degradation by polysaccharases and polysaccharide lyases, polysaccharides common to prokaryotes and
eukaryotes, biological properties and applications and commercial products.
One chapter is dedicated to the presentation of the order-disorder conformational transition of xanthan gum.
5. Hemicelluloses may function both as framework and matrix substances or reserve substances in seeds, where they
form independent wall layers which are mobilized when the seed germinates. In both hardwood and softwood, hemicelluloses fraction in lignified cell walls represents the matrix substance. This important part of the polysaccharides chemistry
is presented in three chapters: Hemicelluloses: Structure and properties; Chemical modification of hemicelluloses and gums;
Role of acetyl substitution in hardwood xylan.
6. In this edition a particular emphasis is placed on the presentation of the ionic polysaccharides (polyanion and
polycation) in the following chapters: Alginate—A polysaccharide of industrial interest and diverse biological functions;
Characterization and properties of hyaluronic acid (hyaluronan); Structure – property relationship in chitosans; Chitosan as
a delivery system for transmucosal administration of drugs; Pharmaceutical applications of chitosan; Macromolecular
complexes of chitosan.
7. Cellulose and starch are the two polysaccharides which constitute the majority of the polysaccharide production.
They are presented in four chapters: Chemical functionalization of cellulose; The physical chemistry of starch; Starch:
commercial sources and derived products; New development in cellulose technology.
8. The polysaccharides of a major importance in medicine and biology are extensively discussed in nine chapters:
Polysialic acid: structure and properties; Brain proteoglycans; Crystal structures of glycolipids; Synthetic and natural
polysaccharides with anticoagulant properties; Structural elucidation of heparan sulfate-like polysaccharides using
miniaturized LC/MS; Enzymatic synthesis of heparan sulfate; Synthetic and natural polysaccharides having biological
activities; Polysaccharide-based hydrogels in tissue engineering and Medical foods and fructooligosaccharides.
Polysialic acids form a structurally unique group of linear carbohydrate chains with a degree of polymerization up to 200
sialyl residue. Polysialic acids chains are covalently attached to membrane glycoconjugates on cells that range in
evolutionary diversity from bacteria to human brains.
Proteoglycans, a group of glycoproteins that are invested with covalently bound glycosaminoglycan chains, are one of
the important classes of molecules in brain development and maturation. The glycosaminoglycan chains that define
proteoglycans are of four major classes: heparan sulfate; chondroitin sulfate, dermatan sulfate and keratan sulfate.
The glycolipids play roles as the structural holder of membrane proteins suspended in bilayer or bicontinuous cubic
phases and as the key code of the intercellular communication or immune system.
Anticoagulant polysaccharides as heparin, heparan sulfate and nonheparin glycosaminoglycans (dermatan sulfate,
chondroitin sulfates, acharan sulfate, carrageenas, sulfated fucans, sulfated galactan and nonheparin glycosaminoglycans

from microbial sources) have been of interest to the medical profession.
9. Renewable resources. Cellulosic biomass includes agricultural (e.g., corn stover and sugarcane bagase) and
forestry (e.g., sawdust, thin-nings, and mill wastes) residues, portions of municipal solid waste (e.g., waste paper) and
herbaceous (e.g., switch-grass) and woody (e.g., poplar trees) corps. They are appropriate materials used as renewable
resources for the production of building blocks for various industrial chemicals and engineering plastics polysaccharides.
The chapters ‘‘Bioethanol production from lignocellulosic material’’, and Cellulosic biomass-derived products, describe
and evaluate the process for ethanol fuel production. The raw material, hydrolysis, and fermentation are described in detail
as well as the different possibilities to perform these process steps in various process designs. The chapter ‘‘Hydrolysis of
cellulose and hemicellulose’’ presents a comprehensive overview of the technology and economic status for cellulose and
hemicellulose hydrolysis describes the important structural features of cellulosic materials, applications, process steps, and
stoichiometry for hydrolysis reactions. The chapter then examines biomass structural characteristics that influence cellulose
hydrolysis by enzymes, types of cellulose hydrolysis processes, experimental results for enzymatic conversion of cellulose,
and summarizes some of the factors influencing hydrolysis kinetics.
10. New applications of polysaccharides. This section provides a selection of some new developmental products and
some recent applications, which might become of commercial interest in the near future. The polysaccharides are utilized as
gallants, thickeners, film formers, fillers, and delivery systems in pharmaceutical and cosmetic applications.
Immobilization. The use of ionic polysaccharides for the immobilization (enzymes, cells and other biocatalysts for
biotechnological production)
Ligand systems. Chitin, chitosan and other functional polysaccharides have also been widely used for the preparation
of metal chelators. Industrial application ranges from waste water treatment, ion exchange resins, and precious
metal recovery.
Separatory systems. Cellulose and chitosan derivatives are dominating the membrane market due to their favorable
stability and their selectivity in gas- and liquid-phase separations.
Biosurfactants. Numerous microorganisms (candida lipolytica, Acetinobacter calcoaceticus) produce extracellular
glycoconjugates with pronounced capabilities to modify interfacial and surface conditions.
Cellulose derivative composites for electro-optical applications. These studies present an optical cell formed by a transparent solid matrix of mixed esters of cellulose with micrometer-sized pores filled with a nemantic liquid crystal.

Copyright 2005 by Marcel Dekker

Preface

vii

11. Incorporation of the polysaccharides in the synthetic matrix offers on one hand the possibility to obtain a broader
application range of the usual polymers and, on the other hand, ways to optimize and control some properties and produce
new materials with unexpected performance at low cost.
The treatise is truly international with authors now residing in Austria, Brazil, Canada, Denmark, Egypt, Finland,
France, Germany, Greece, Japan, The Netherlands, Norway, Portugal, Romania, Sweden, United Kingdom, and the United
States. The editor is grateful to all the collaborators for their precious contributions.
Severian Dumitriu

Copyright 2005 by Marcel Dekker


Contents

Foreword
Hans-Peter-Fink
Preface
Contributors
1. Progress in Structural Characterization of Functional Polysaccharides
Kanji Kajiwara and Takeaki Miyamoto
2. Conformations, Structures, and Morphologies of Celluloses
Serge Pe´rez and Karim Mazeau
3. Hydrogen Bonds in Cellulose and Cellulose Derivatives
Tetsuo Kondo
4. X-ray Diffraction Study of Polysaccharides
Toshifumi Yui and Kozo Ogawa
5. Recent Developments in Spectroscopic and Chemical Characterization of Cellulose

Rajai H. Atalla and Akira Isogai
6. Two-Dimensional Fourier Transform Infrared Spectroscopy Applied to Cellulose and Paper
˚
Lennart Salme´n, Margaretha Akerholm, and Barbara Hinterstoisser
7. Light Scattering from Polysaccharides
Walther Burchard
8. Advances in Characterization of Polysaccharides in Aqueous Solution and Gel State
M. Rinaudo
9. Conformational and Dynamics Aspects of Polysaccharide Gels by High-Resolution Solid-State NMR
Hazime Saitoˆ
10.

Correlating Structural and Functional Properties of Lignocellulosics and Paper by Fluorescence
Spectroscopy and Chemometrics
Emmanouil S. Avgerinos, Evaggeli Billa, and Emmanuel G. Koukios

Copyright 2005 by Marcel Dekker


x

Contents

11.

Computer Modeling of Polysaccharide–Polysaccharide Interactions
Francois R. Taravel, Karim Mazeau, and Igor Tvarosˇka
ß

12.

Interactions Between Polysaccharides and Polypeptides
Delphine Magnin and Severian Dumitriu

13.

Rheological Behavior of Polysaccharides Aqueous Systems
Jacques Lefebvre and Jean-Louis Doublier

14.

Stability and Degradation of Polysaccharides
Valdir Soldi

15.

Biosynthesis, Structure, and Physical Properties of Some Bacterial Polysaccharides
Roberto Geremia and Marguerite Rinaudo

16.

Microbial Exopolysaccharides
I. W. Sutherland

17.

Order–Disorder Conformational Transition of Xanthan Gum
Christer Viebke

18.

Hemicelluloses: Structure and Properties
Iuliana Spiridon and Valentin I. Popa

19.

Chemical Modication of Hemicelluloses and Gums
Margaretha Soăderqvist Lindblad and Ann-Christine Albertsson

20.

Role of Acetyl Substitution in Hardwood Xylan
Maria Groăndahl and Paul Gatenholm

21.

Alginate—A Polysaccharide of Industrial Interest and Diverse Biological Functions
Wael Sabra and Wolf-Dieter Deckwer

22.

Characterization and Properties of Hyaluronic Acid (Hyaluronan)
Michel Milas and Marguerite Rinaudo

23.

Chemical Functionalization of Cellulose
Thomas Heinze

24.

The Physical Chemistry of Starch
R. Parker and S. G. Ring

25.

Starch: Commercial Sources and Derived Products
Charles J. Knill and John F. Kennedy

26.

Structure–Property Relationship in Chitosans
Kjell M. Va˚rum and Olav Smidsrød

27.

Chitosan as a Delivery System for the Transmucosal Administration of Drugs
Lisbeth Illum and Stanley (Bob) S. Davis

28.

Pharmaceutical Applications of Chitosan and Derivatives
M. Thanou and H. E. Junginger

29.

Macromolecular Complexes of Chitosan
Naoji Kubota and Kei Shimoda

30.

Polysialic Acid: Structure and Properties
Tadeusz Janas and Teresa Janas

Copyright 2005 by Marcel Dekker


Contents

31.

Brain Proteoglycans
Russell T. Matthews and Susan Hockfield

32.

Crystal Structures of Glycolipids
Yutaka Abe and Kazuaki Harata

33.

Synthetic and Natural Polysaccharides with Anticoagulant Properties
Fuming Zhang, Patrick G. Yoder, and Robert J. Linhardt

34.

Structural Elucidation of Heparan Sulfate-Like Polysaccharides Using Miniaturized LC/MS
Balagurunathan Kuberan, Miroslaw Lech, and Robert D. Rosenberg

35.

Enzymatic Synthesis of Heparan Sulfate
Balagurunathan Kuberan, David L. Beeler, and Robert D. Rosenberg

36.

Polysaccharide-Based Hydrogels in Tissue Engineering
Hyunjoon Kong and David J. Mooney

37.

Synthetic and Natural Polysaccharides Having Specific Biological Activities
Takashi Yoshida

38.

Medical Foods and Fructooligosaccharides
Bryan W. Wolf, JoMay Chow, and Keith A. Garleb

39.

Immobilization of Cells in Polysaccharide Gels
Yunyu Yi, Ronald J. Neufeld, and Denis Poncelet

40.

Hydrothermal Degradation and Fractionation of Saccharides and Polysaccharides
Ortwin Bobleter

41.

Cellulosic Biomass-Derived Products
Charles J. Knill and John F. Kennedy

42.

Bioethanol Production from Lignocellulosic Material
Lisbeth Olsson, Henning Jørgensen, Kristian B. R. Krogh, and Christophe Roca

43.

Hydrolysis of Cellulose and Hemicellulose
Charles E. Wyman, Stephen R. Decker, Michael E. Himmel, John W. Brady, Catherine E. Skopec,
and Liisa Viikari

44.

New Development in Cellulose Technology
Bruno Lo
ănnberg

45.

Polysaccharide Surfactants: Structure, Synthesis, and Surface-Active Properties
Roger E. Marchant, Eric H. Anderson, and Junmin Zhu

46.

Structures and Functionalities of Membranes from Polysaccharide Derivatives
Tadashi Uragami

47.

Electro-optical Properties of Cellulose Derivative Composites
J. L. Figueirinhas, P. L. Almeida, and M. H. Godinho

48.

Blends and Composites Based on Cellulose Materials
Georgeta Cazacu and Valentin I. Popa

49.

Preparation and Properties of Cellulosic Bicomponent Fibers
Richard D. Gilbert and John F. Kadla

Copyright 2005 by Marcel Dekker

xi


Contributors

Yutaka Abe

Process Development Research Center, Lion Corporation, Tokyo, Japan

˚
Margaretha Akerholm STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden
Ann-Christine Albertsson

Royal Institute of Technology, Stockholm, Sweden

P. L. Almeida EST/IPS, Setubal, Portugal and FCT/UNL, Caparica, Portugal
´
Case Western Reserve University, Cleveland, Ohio, U.S.A.

Eric H. Anderson
Rajai H. Atalla

USDA Forest Service and University of Wisconsin, Madison, Wisconsin, U.S.A.

Emmanouil S. Avgerinos

National Technical University of Athens, Athens, Greece

David L. Beeler Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
Evaggeli Billa

National Technical University of Athens, Athens, Greece

Ortwin Bobleter University of Innsbruck, Innsbruck, Austria
John W. Brady

Cornell University, Ithaca, New York, U.S.A.

Walther Burchard

Institute of Macromolecular Chemistry, University of Freiburg, Germany

Georgeta Cazacu ‘‘Petru Poni’’ Institute of Macromolecular Chemistry, Iasi, Romania
JoMay Chow

Abbott Laboratories, Columbus, Ohio, U.S.A.

Stanley (Bob) S. Davis University of Nottingham, Nottingham, United Kingdom

Copyright 2005 by Marcel Dekker


xiv

Contributors

Stephen R. Decker National Renewable Energy Laboratory, Golden, Colorado, U.S.A.
Biochemical Engineering, GBF–National Research Center for Biotechnology, Braunschweig,

Wolf-Dieter Deckwer
Germany

´
INRA-Laboratoire de Physico-Chimie des Macromolecules, Nantes, France

Jean-Louis Doublier
Severian Dumitriu

Sherbrooke University, Sherbrooke, Quebec, Canada

J. L. Figueirinhas

CFMC/UL, Lisbon, Portugal

Keith A. Garleb Abbott Laboratories, Columbus, Ohio, U.S.A.
Paul Gatenholm Biopolymer Technology, Department of Materials and Surface Chemistry, Chalmers University of
Technology, Goteborg, Sweden
ă

Roberto Geremia Laboratoire dAdaptation et de Pathogenie des Microorganismes, Joseph Fourier University,
Grenoble, France
Richard D. Gilbert

North Carolina State University, Raleigh, North Carolina, U.S.A.

FCT/UNL, Caparica, Portugal

M. H. Godinho

ă
Maria Grondahl Biopolymer Technology, Department of Materials and Surface Chemistry, Chalmers University of
Technology, Goteborg, Sweden
ă
Kazuaki Harata Biological Information Research Center, National Institute of Advanced Industrial Science and
Technology, Ibaraki, Japan
Thomas Heinze Center of Excellence for Polysaccharide Research at the Friedrich Schiller University of Jena, Jena,
Germany
Michael E. Himmel

National Renewable Energy Laboratory, Golden, Colorado, U.S.A.

Barbara Hinterstoisser
Susan Hockfield

Yale University School of Medicine, New Haven, Connecticut, U.S.A.

IDentity, Nottingham, United Kingdom

Lisbeth Illum
Akira Isogai

BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria

Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, Japan

Tadeusz Janas
Teresa Janas

University of Colorado, Boulder, Colorado, U.S.A.
´
University of Colorado, Boulder, Colorado, U.S.A. and University of Zielona, Gora, Poland

Henning Jørgensen

Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark

H. E. Junginger Leiden University, Leiden, The Netherlands
John F. Kadla North Carolina State University, Raleigh, North Carolina, U.S.A.
Kanji Kajiwara

Copyright 2005 by Marcel Dekker

Otsuma Women’s University, Chiyoda-ku, Tokyo, Japan


Contributors

xv

John F. Kennedy
Kingdom

University of Birmingham Research Park and Chembiotech Laboratories, Birmingham, United

Charles J. Knill
Kingdom

University of Birmingham Research Park and Chembiotech Laboratories, Birmingham, United

Kyushu University, Fukuoka, Japan

Tetsuo Kondo

University of Michigan, Ann Arbor, Michigan, U.S.A.

Hyunjoon Kong

Emmanuel G. Koukios

National Technical University of Athens, Athens, Greece

Kristian B. R. Krogh Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
Balagurunathan Kuberan Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth
Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
Oita University, Oita, Japan

Naoji Kubota

Miroslaw Lech Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
´
Jacques Lefebvre INRA-Laboratoire de Physico-Chimie des Macromolecules, Nantes, France
ă
Margaretha Soderqvist Lindblad

Royal Institute of Technology, Stockholm, Sweden

Robert J. Linhardt University of Iowa, Iowa City, Iowa, U.S.A.
˚
˚
Abo Akademi University, Turku/Abo, Finland

ă
Bruno Lonnberg

Sherbrooke University, Sherbrooke, Quebec, Canada

Delphine Magnin
Roger E. Marchant

Case Western Reserve University, Cleveland, Ohio, U.S.A.

Russell T. Matthews Yale University School of Medicine, New Haven, Connecticut, U.S.A.
Karim Mazeau

´
´ ´
Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France

´
´ ´
Michel Milas Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph Fourier
University, Grenoble, France
Takeaki Miyamoto

National Matsue Polytechnic College, Matsue, Japan

David J. Mooney University of Michigan, Ann Arbor, Michigan, U.S.A.
Ronald J. Neufeld
Kozo Ogawa

Queen’s University, Kingston, Ontario, Canada

Osaka Prefecture University, Sakai, Osaka, Japan

Lisbeth Olsson Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
R. Parker
´
Serge Perez

Copyright 2005 by Marcel Dekker

Institute of Food Research, Norwich Research Park, Norwich, United Kingdom
´
´ ´
Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France


xvi

Contributors

ENITIAA, Nantes, France

Denis Poncelet

Technical University of Jassy, Jassy, Romania

Valentin I. Popa

´
´ ´
Marguerite Rinaudo Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph
Fourier University, Grenoble, France
Institute of Food Research, Norwich Research Park, Norwich, United Kingdom

S. G. Ring

Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark

Christophe Roca

Robert D. Rosenberg Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt

Wael Sabra

ˆ
Hazime Saito Himeji Institute of Technology, Kamigori, Hyogo, Japan and Center for Quantum Life Sciences,
Hiroshima University, Higashi-Hiroshima, Japan
STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden

´
Lennart Salmen

Kei Shimoda Oita University, Oita, Japan
Catherine E. Skopec Cornell University, Ithaca, New York, U.S.A.
Norwegian University of Science and Technology (NTNU), Trondheim, Norway

Olav Smidsrød

´
Valdir Soldi Federal University of Santa Catarina, Florianopolis, SC, Brazil
Iuliana Spiridon ‘‘Petru Poni’’ Institute of Macromolecular Chemistry, Jassy, Romania
University of Edinburgh, Edinburgh, United Kingdom

I. W. Sutherland

´
´ ´

Francois R. Taravel Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph
¸
Fourier University, Grenoble, France
Cardiff University, Cardiff, United Kingdom

M. Thanou

Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia

Igor Tvarosˇ ka

Kansai University, Osaka, Japan

Tadashi Uragami
˚
Kjell M. Varum

Norwegian University of Science and Technology (NTNU), Trondheim, Norway

Christer Viebke
Kingdom

The North East Wales Institute, Water Soluble Polymers Group Plas Coch, Wrexham, United

Liisa Viikari

VTT Technical Research Centre of Finland, Finland

Bryan W. Wolf

Abbott Laboratories, Columbus, Ohio, U.S.A.

Charles E. Wyman
Yunyu Yi

Copyright 2005 by Marcel Dekker

Dartmouth College, Hanover, New Hampshire, U.S.A.

Queen’s University, Kingston, Ontario, Canada


Contributors

xvii

Patrick G. Yoder University of Iowa, Iowa City, Iowa, U.S.A.
Takashi Yoshida

Kitami Institute of Technology, Kitami, Japan

Toshifumi Yui

Miyazaki University, Miyazaki, Japan

Fuming Zhang

University of Iowa, Iowa City, Iowa, U.S.A.

Junmin Zhu

Copyright 2005 by Marcel Dekker

Case Western Reserve University, Cleveland, Ohio, U.S.A.


1
Progress in Structural Characterization of Functional
Polysaccharides
Kanji Kajiwara
Otsuma Women’s University, Chiyoda-ku, Tokyo, Japan

Takeaki Miyamoto
National Matsue Polytechnic College, Matsue, Japan

I. INTRODUCTION
Oligosaccharides and polysaccharides are biopolymers
commonly found in living organisms, and are known to
reveal the physiological functions by forming a specific
conformation. However, our understanding of polysaccharide chains is still in its premature state with respect to their
structure in solid and in solution. Structural analysis may
offer the most fundamental knowledge to understand the
functions of polysaccharides, but the diversity and irregularity of polysaccharide chains make it a formidable task.
Polysaccharide chains are partly organized but are considered to be mostly amorphous. No single crystal was made
from polysaccharides up to now. Thus the crystallographic
analysis of polysaccharide chains has been performed by
either using the small oligosaccharide single crystals or the
x-ray fiber pattern diffraction from drawn polysaccharide
gels.
Although a monosaccharide unit is common to many

polysaccharides, its linkage mode varies and characteristic
functions/properties will appear accordingly. A good
example is demonstrated by simple poly-D-glucans—water-soluble, digestible amylose and non-water-soluble,
nondigestible cellulose. Both amylose and cellulose are
homopolymers composed of glucosidic residues, but they
differ in the mode of linkage. Amylose is a (1!4)-a-Dlinked polyglucan, whereas cellulose is a (1!4)-a-Dlinked polyglucan. The (1!4)-a linkage (amylose) and
the (1!4)-a linkage (cellulose) of D-glucosidic residues
yield a wobbled helix and a stretched zigzag chain,
respectively, by joining the D-glucosidic residues in a
simple manner so as to place the chain on a plane [1]

Copyright 2005 by Marcel Dekker

(Fig. 1). In later sections, it will be shown that these basic
conformations of amylose and cellulose are supposed to
be retained to some extent in aqueous solutions. The
difference in the structure is reflected by the respective
physiological functions of edible amylose and nonedible
cellulose.
There are some evidences that the higher-order structure of polysaccharide chains is related to their physiological function as exemplified by the triple-stranded helix of
scleroglucan, which is known to possess an antitumor
activity. Many polysaccharide chains are able to assume
an ordered or quasi-ordered structure such as a doublestranded helix, but the ordered structure is interrupted by
the irregularity of the primary structure in the polysaccharide chains. Many polysaccharide chains form gel in solutions by assuming an ordered or quasi- ordered chain
structure, which constitutes a cross-linking domain.
The conformational analysis of polysaccharide chains
involves two aspects: (1) the characterization of a single
chain conformation and (2) the analysis of the chain
assembly (suprastructure) of polysaccharides. A single
chain conformation of polysaccharides is primarily determined by the chemical structure specified by the types of

sugar residues, sugar linkages, and side groups. A single
chain conformation accounts, to some extent, for the
formation of suprastructures such as the complexing capability of amylose and the fringed micelle formation of
cellulose.
Unlike cellulose and amylose, most polysaccharides
have no regular homopolymeric structure, where the regularity is interrupted by the random intrusion of different
types of linkage and/or sugar units. The introduction of


2

Kajiwara and Miyamoto

Figure 1 Wobbled helical conformation (a) and stretched zigzag conformation (b), representing the basic conformations of
amylose and cellulose, respectively.

such an irregularity hampers crystallization and promotes
the formation of a suprastructure that is characteristic of
the polysaccharide species. The interchain interaction of
polysaccharides seems to be specific as exemplified by the
suprastructure depending on the chemical structure and
counterions (in the case of polysaccharides possessing
carboxyl or sulfate groups). The formation of the suprastructure often results to gelation.
The complexity in characterizing polysaccharide chain
conformation is due to the fact that the interchain interaction of polysaccharides is so specific that polysaccharide
chains are seldom dispersed in solvent as a single chain.
Thus a first task to understand the structure–function
relationship of polysaccharides is to evaluate the intrinsic
chain (single chain) characteristics free from interchain
interaction. Once the intrinsic chain conformation is specified, the interchain interaction can be analyzed in terms of

the mode of suprastructure composed of several polysaccharide chains.
This review is intended to demonstrate the recent
strategy in the structural and conformational characterization of oligosaccharides and polysaccharides. Although
various techniques are applied for the structural and
conformational analysis of oligosaccharides and polysaccharides, the general inability to crystallize excludes the

Copyright 2005 by Marcel Dekker

potential application of the crystallographic approach,
which has been a main method of the structural analysis
in protein science. Here we will describe two methods that
are currently applied to the structural and conformational
analysis of oligosaccharides and polysaccharides: smallangle x-ray scattering (SAXS) [2] and nuclear magnetic
resonance (NMR) [3].
Molecular modeling by computer is considered to
supplement the analysis by small-angle x-ray scattering
and NMR. Although an initial intention of molecular
modeling is to predict physical properties of carbohydrates
a priori [4], the ab initio calculation is limited to a small
monosaccharide and the semiempirical quantum method
can be applied for the structural characterization of molecules up to the size of disaccharides. Molecular mechanics
or molecular dynamics is an alternative method applied to
the computer modeling of larger carbohydrate molecules,
where the motion of constituent atoms is assumed to be
described in terms of classical mechanics.
In the final chapter, the structural and conformational
aspect is discussed from the chemical point of view. Here
the controlled chemical modification of cellulose is treated
and the physicochemical characteristics are discussed by
taking into account the structural change due to chemical

modification of cellulose.


Progress in Structural Characterization of Functional Polysaccharides

II. STRATEGY AND METHODS OF ANALYSIS
Because many monosaccharides have a single, well-established conformation, the conformational analysis of oligosaccharides and polysaccharides starts from understanding
the energetic relationship when the monosaccharide residues are linked in a specific way. The entire geometry of
oligosaccharides and polysaccharide chains can be described in terms of a set of the pairs of dihedral angles of
rotation about the monosaccharide links. If the rotation is
independent at each monosaccharide link, the chain should
assume a random coil conformation. However, the conformation of saccharide chains is found in most cases to
assume nonrandom conformations due to intra- and interchain interactions that suppress the conformational space
available for the chains linked by independent rotation.
Even the crystal structure is partly retained in solution as in
the case of protein. Thus a single chain conformation may
account, to some extent, for the mode of interactions and
the formation of suprastructures.
This section gives a brief introduction on the structure
of monosaccharides and disaccharides as the basis of the
structural and conformational analysis of oligosaccharides
and polysaccharides. The fundamentals of SAXS and
NMR together with the molecular modeling are also
described.

A. Structure of Monosaccharide
and Disaccharide
A monosaccharide is given by the chemical formula
CnH2nOn, where n = 3–10. Pentose (n = 5) and hexose
(n = 6) are the most abundant in nature, and are

composed of a pyranose or a furanose (Fig. 2) as a basic
ring structure. A pyranose ring has two stable chair form
(C) conformers C1 and 1C where four atoms of O, C2,
C3, and C5 are on the same plane. Fig. 3 lists some of
pyranose-type pentose and hexose, which appear in later
sections, where the abbreviated description is given for
each monosaccharide.
A disaccharide is composed of two monosaccharides
linked by any of the four modes of glycosidic linkages a,aV,
a,hV h,aV or h,hV Table 1 shows some disaccharides
,
-,
-.

Figure 2 Pyranose and furanose.

Copyright 2005 by Marcel Dekker

3

found in nature. The structural analysis of disaccharide
implies the identification, the linking order, the link position, and the link mode of constituent monosaccharides.
Chemical and optical methods are available to determine
the structure of disaccharide, but the recent development in
NMR has facilitated the assignment of specific protons or
carbons as well as the conformational determination of the
glucosidic linkage as shown in the next section. Fouriertransform infrared (FTIR) and laser Raman spectroscopy
are also useful tools for the characterization of glycosidic
bonds [5].
X-ray and neutron diffraction can be applied to determine the crystal structure and hydrogen bonding of monosaccharides and disaccharides that form a single crystal. A

classic example will be found in the crystal structure
analysis of h-maltose monohydrate [6] (Fig. 4), where the
earlier structure determination using x-ray diffraction [7]
was refined to give a more accurate description of the
hydrogen bond structure. The x-ray diffraction analysis
provides the most explicit information on structure in
terms of the precise atomic coordinates. The Cambridge
Structural Database lists the crystal structure of about 40
small oligosaccharides (cyclodextrins are omitted) including about 10 trisaccharides, 2 tetrasaccharides, and 1
hexasaccharide. Here a number of crystal structures of
mono-, di-, and trisaccharides were determined from the
acetate derivatives because the acetylated derivatives are
found to crystallize more easily than original (untreated)
oligosaccharides. (1!3)-h-D-glucopyranosyl residues consist of a main chain of a medically important class of
polysaccharides including curdlan, lentinan, schizophyllan, scleroglucan, and grifolan, which possess branches at
C6 (except for curdlan). Glcph 1!3 Glc disaccharides are
systematically synthesized and the crystal structures are
determined. A first attempt was made by Takeda et al. [8]
on 3-O-h-D-glucopyranosyl-h-D-glucopyranose (h-D-laminarabiose) ethyl hepta-O-acetyl-h-D-laminarabioside [9],
followed by Perez et al. [10] on octa-O-acetyl-h-D-laminarabiose), and by Lamba et al. [11] on (methyl hepta-Oacetyl-h-D-laminarabiose). Recently, 3-O-h-D-glucopyranosyl-h-D-glucopyranoside (methyl h-D-laminarabioside)
[12] and methyl hepta-O-acetyl-h-D-laminarabioside [13]
were prepared, and the crystal structures were determined
by x-ray diffraction (Fig. 5). Table 2 summarizes two
dihedral angles, / and w, with respect to the glycosidic
bond for (1!3)-h-linked disaccharides, evaluated from the
crystallographic data of laminarabiose and laminarabioside derivatives. Here the dihedral angles are taken as / =
h[H(C1) ,C1 ,O1, C3V] and w = h[C1, O1, C3V, H(C3V)].
(See Sec. II.D for the definition of dihedral angles / and w.)
The angle / is almost invariant around 45j regardless of
the substituents, while the angle w is classified in two

groups of around À45j and 8j. When the intramolecular
hydrogen bond is formed between 04V and 05, the angle y
assumes a negative value. The introduction of acetyl
groups prevents the formation of intramolecular hydrogen
bonds as seen from the stereoview of the molecular structures of methyl h-D-laminarabioside and methyl hepta-Oacetyl-h-D-laminarabioside in Fig. 5. The invariance of


4

Kajiwara and Miyamoto

Figure 3 Pyranose-type pentose and hexose.

the angle 4) is attributed to the exo-anomeric effect that
restricts rotation around the bond between an anomeric
carbon atom and a glycosidic oxygen atom [14].

B. Fundamentals of Small-Angle X-Ray
Scattering [2]
Small-angle x-ray scattering is characterized by its small
scattering angle. A scattering process obeys a reciprocal
law that relates the distance r in an ordinary (real) space
with the scattering vector q in a Fourier (scattering) space
by the phase factor defined by exp(Àq Á r); that is, the
scattered intensity I( q) is given by the Fourier transformation of the electron density distribution in the object:
ðl
4pr2 cðrÞ Á expðÀiq rịdr
1ị
Iqị ẳ V ẳ
0

Copyright 2005 by Marcel Dekker

Here the magnitude of the scattering vector is given by (4p /
k) sin(h / 2) with k and h being the wavelength and the
scattering angle, respectively. c(r) is a correlation function
representing the average of the product of two electron
density fluctuations at a distance r. The distance distribution function p(r) is dened as
prị ẳ Vr2 Á cðrÞ

ð2Þ

which is characteristic of the shape of the scattering object.
The phase difference between scattered rays becomes
more prominent as the scattering angle increases. Thus the
scattered intensity is maximum at zero scattering angle and
proportional to the number of electrons in the object where
the scattered rays are all in phase. The scattered intensity
decreases with increasing scattering angle and diminishes
at a scattering angle of the order of k / D, where k and D


Progress in Structural Characterization of Functional Polysaccharides

5

Table 1 Disaccharides in Nature
Mode of linkage

Common name

(1!6) Linkage

(1!3) Linkage

(1!2) Linkage

(1!1) Linkage

Origin

maltose
cellobiose
lactose
xylobiose
chitobiose
cellobiouronic acid
isomaltose
gentiobiose
melibiose
planteobiose
nigerose
laminaribiose
turanose
hyalobiuronic acid
chondrosine
kojibiose
sophorose
sucrose
a,a-trehalose

(1!4) Linkage

Structure
Glcpa 1 ! 4 Glc
Glcph 1 ! 4 Glc
Galph 1 ! 4 Glc
Xylph 1 ! 4 Xyl
GlcNh 1 ! 4 GlcN
GlcUAph 1 ! 4 Glc
Glcpa 1 ! 6 Glc
Glcph 1 ! 6 Glc
Galpa 1 ! 6 Glc
Galpa 1 ! 6 Fruf
Glcpa 1 ! 3 Glc
Glcph 1 ! 3 Glc
Glcpa 1 ! 3 Fruf
GlcUAph 1 ! 3 GlcN
GlcUAph 1 ! 3 GalN
Glcpa 1 ! 2 Glc
Glcph 1 ! 2 Glc
Frufh 1 ! 2 aGlcp
Glcpa 1 ! 1 aGlcp

starch
cellulose
mammal milk
xylan
chitin
D. pneumoniae

amylopectin, etc.
gentianose
raffinose
planteose
mutan
laminaran
meleziose (honey)
hyaluronic acid
chondroitin sulfate
Aspergillus orryzae
Sophora japonica
beet sugar
yeast

p and f denote pyranose and furanose, respectively.

denote the wavelength of an incident beam and the average
diameter of scattering objects. When x-ray is used as an
incident beam (k = 0.154 nm), the limiting scattering angle
to be observed is approximately equal to 0.450 when D =
10 nm, or to 0.0450 when D = 100 nm.
Because the phase factor exp(Àq Á r) can be replaced by
its space average sin qr/qr for the statistically isotropic
system according to Debye [15], Eq. (1) can be expanded in
the series of q2 at very small angles by expanding the sine
term to yield the particle scattering factor as
l
1
4pr2 crị r2 dr=2
PqịuIqị=I0ị ẳ 1 À q2

3
0
ð3Þ
ð

(Rt) of a flat particle by describing approximately the
scattering from the cross-section or the thickness in terms
of the exponential form. The scattering factor of a rod-like
particle (a cylinder) consists of two components of the
height and the cross-section as
À
Á
p
Á exp Àq2 R2 =2
ð6Þ
Pcylinder ðqÞc
c
2Hq
where 2H denotes the height of the cylinder. The scattering
factor of a flat particle (a disk) is given by the product of
two terms of the cross-sectional area and the thickness as
Pdisk qịc

l

4pr2 crịdr ỵ Oq4 ị



0

where the second term on the right side represents the
radius of gyration RG, that is
l
l
prị r2 dr=2
prịdr
4ị
R2 ẳ
G
0



2p
exp q2 R2
t
2
Aq

ð7Þ

where A denotes the cross-sectional area. Equations (6) and
(7) suggest that the cross-sectional radius of gyration Rc

0

in terms of the distance distribution function Eq. (2). The
sine expansion of Eq. (3) is approximately closed in the
exponential form, and the particle scattering factor is

reduced to the Guinier approximation [2,16]:
PðqÞcexpðÀq2 R2 =3Þ
G

ð5Þ

suggesting that the radius of gyration can be evaluated
from the initial slope by plotting ln P( q) against q2 (the
Guinier plot). A similar argument can be applied to
evaluate the radius of gyration corresponding to the
cross-section of a rod-like particle (Rc) or the thickness

Copyright 2005 by Marcel Dekker

Figure 4
Ref. 6.)

Stereoview of h-maltose monohydrate. (From


6

Kajiwara and Miyamoto

Figure 5

Stereoview of methyl h-D-laminarabioside (top) and methyl hepta-O-acetyl-a-D-laminarabioside (bottom).

Table 2 Dihedral Angles of the Glycosidic Linkage for Glcph 1!3 Glc Disaccharides
Compound

Methyl h-D-laminarabioside
h-D-laminarabiose
Methyl hepta-O-acetyl-h-D-laminarabioside
Methyl hepta-O-acetyl-a-D-laminarabioside
Octa-O-acetyl-h-D-laminarabioside
Octa-O-acetyl-a-D-laminarabioside

Copyright 2005 by Marcel Dekker

/ = h[H(C1), C1, O1, C3V]

w = h[C1, O1, C3V, H(C3V)]

43
28
43
43
42
54

À52
À38
5
2
14
11


Progress in Structural Characterization of Functional Polysaccharides

and the thickness radius of gyration Rt are evaluated from
the initial slope of the corresponding Guinier plots: ln
qPcylinder ( q) plotted against q2 or ln q2Pdisk( q) plotted
against q2.
Although the polymeric chain has an approximate
shape as represented by a sphere or an ellipsoid as a whole
in solution, the density distribution is not homogeneous
but decays exponentially from the center to the circumference. A simple Ornstein–Zemike type is generally applied
to the density correlation function for a Gaussian chain:
n
cðrÞic expðÀr=nÞ
r

ð8Þ

where c is a concentration of polymer chains and n is a
correlation length specifying the range of effective density
fluctuation. Introducing in Eq. (1) for a statistically isotropic system, Eq. (8) yields the scattering prole as
Iqịc

cn3
1 ỵ n2 q2

10ị

Equation (10) yields the scattering prole as
cn3

11ị

1 ỵ n2 q2 ị2

which exhibits a faster decay of the scattered intensity with
q.
The particle scattering from a single molecule is in
principle calculated from the coordinates of the constituent
atoms
n
X

g2 /2 qị ỵ 2
i i

iẳ1

n1 n
XX
tẳ1 jẳiỵ1

12ị

3ẵsinRi qị Ri qịcosRi qị
Ri qị3

Rqị3

13ị

where RI is the van deer Walls radius of the ith atom. If a
molecule is rigid, the distance dij is fixed and Eq. (1) is

#2
ð14Þ

where R denotes the radius of a sphere. The observed
scattering profile is compared with that calculated from
an assumed triaxial model of a suitable dimension, which is
supposed to be composed of associated oligosaccharides or
polysaccharide chains.
No interdomain (interparticular) interaction is considered in the above argument, and the scattering is considered to be due solely to an isolated domain (or an
isolated particle). When the interdomain (interparticular)
interaction becomes dominant, an interference peak will
appear at the q range corresponding to the interaction
distance in the scattering profile. If the interdomain (interparticular) interaction is isotropic and spherically symmetric, the scattering profile is decomposed into the product of
two terms of the particle scattering factor P( q) and the
interference SI( q) [16]:
ð15Þ

where the interference term is written as
SI ðqÞc

where q denotes the magnitude of the scattering vector
given by (4p / k) sin(h / 2) with k and h being the wavelength
of the incident beam and scattering angle, respectively, and
gi is an atomic scattering factor. dij is the distance between
the ith and jth atoms, and the form factor for a single atom
/i( q) is assumed to be given by the form factor for a sphere
with a van deer Walls radius of the ith atom

Copyright 2005 by Marcel Dekker

Pqị ẳ / qRị ẳ

3sin Rq Rq cos Rqị

IqịcPqị SI qị

gi gj /i qị/j qị

sindij q

dij q

/i ẳ

"
2

crịiexpr=nị

Iqị ẳ

equivalent to the particle scattering factor of such a molecule that freely moves in space. If a molecule (e.g., a
flexible polymer molecule) has a large internal freedom,
the distance dij fluctuates with time due to the internal
motion of such a molecule. In this case, the particle
scattering factor should be calculated as an average over
a statistical ensemble generated by the Monte Carlo procedure [18,19] according to the conditional bond conformation probability [20].
When no molecular model is available, the scattering
profile can be analyzed in terms of a triaxial body model of

homogeneous density representing the shape of the object
[21] or by assuming a suitable pair correlation function for
the electron density distribution in the object [22]. The
scattering factor is explicitly calculated for some homogeneous triaxial bodies including a sphere, an ellipsoid, a
cylinder, and a prism. For example, the scattering factor
for a sphere is given by Eq. (14) as

ð9Þ

The volume term V in Eq. (1) is replaced by cn3, which
corresponds to the number of units in the correlated
density fluctuation. Debye and Beuche [17] proposed a
correlation function that specifies the density correlation
for a randomly associated system:

IðqÞc

7

1
3=2

1 À ð2pÞ

ðe=m1 ÞbðqÞ

ð16Þ

with e and m1 being a constant close to unity and the average
volume allocated to each interacting domain, respectively.

b( q) represents the interaction potential in the Fourier
(scattering) space. When the interdomain interaction is
given in terms of a hard-sphere repulsion, b( q) is represented by the scattering amplitude of a sphere, Eqs. (13)
and (16) reduce to
SI qịc

1
1 ỵ 8m0 =m1 ịe/2qRị

17ị

where m0 is the volume of the sphere and the hard-sphere
interaction is represented by the sphere of a uniform radius
2R. The interaction potential b( q) is approximately given


8

Kajiwara and Miyamoto

by the Gaussian function when the interaction is softer
[22,23], and Eq. (16) is rewritten as
SI qịc

1


1 ỵ 2A2 Mw c exp Àn2 q2

ð18Þ

where the Gaussian-type interaction potential is specified
by the correlation length n of interaction.

C. Fundamentals of Nuclear Magnetic
Resonance Spectroscopy Applied
to the Conformational Analysis
Nuclear magnetic resonance (NMR) spectroscopy has
been widely employed in the structural analysis and the
conformational dynamics of polymers in solution, gel, or
solid states. However, its application is limited to the
polymers that are not entirely crystalline in general. It
provides information on microscopic chemical structures,
including the primary structure, stereoregularity, conformation, and secondary structure of synthetic polymers,
proteins, and polysaccharides. Various NMR techniques
have also been developed to investigate molecular motion
through relaxation times, correlation times, and self-diffusion coefficients. One of the advantages of NMR in the
structure analysis is its sensitivity to a microscopic structure within a short-range order in comparison with smallangle x-ray scattering. The other advantages of NMR are
that (1) it is a noninvasive method where no probes are
needed; (2) the sample for measurement can be liquid,
solid, or gel; (3) the NMR signals can be assigned individually to the main chain, the side chain, or the functional
group of a sample and yield the structural information on a
specific site; and (4) the molecular motion and dynamic
structure (time-dependent structure) can be observed.
However, NMR has some disadvantages: (1) the spatial
position of atomic groups is not determined accurately; (2)
the information on the long-range and higher-order structure will be lost; and (3) the duration time is long to observe
NMR peaks from polymer samples with a reasonable S/N
ratio and high resolution. Thus NMR spectroscopy compliments other methods of the structural and conformational analysis of polymers, including x-ray diffraction,
light scattering, and small-angle x-ray (neutron) scattering.

A variety of NMR techniques are available for the
structure analysis of oligosaccharides and polysaccharides.
The one-dimensional pulse NMR technique is mainly
applied for the analysis of the saccharide primary structure
in solution state and the determination of relaxation times.
The solid state, high-resolution NIVIR technique can be
applied for the structure analysis of oligosaccharides and
polysaccharides in viscose solution, gel, and solid state. The
two- or three-dimensional techniques are used to determine
the primary and secondary structures and the conformation of oligosaccharides and polysaccharides.
1. Chemical Shift
Oligosaccharides and polysaccharides show several 1H
NMR signal peaks in the spectrum region between 2 and

Copyright 2005 by Marcel Dekker

6 ppm for protons on the ring. The anomeric protons (Hi)
have peaks in the region between 4.5 and 5.5 ppm, whereas
the chemical shifts for other protons (H2–H6) ranges from
2 to 4.5 ppm. The H1 chemical shift database will provide a
starting key to assign the chemical shifts of unknown
samples, although the chemical shift database for oligosaccharides and polysaccharides are still far from completion with respect to the accumulation and systematization.
As the chemical shifts are also sensitive to the conformational change, solvent, and temperature, it requires experience and skill to identify the 1H NMR peaks for unknown
oligosaccharides and polysaccharide samples. Various
two-dimensional NMR techniques have been developed
to facilitate the assignment and identification of the chemical shifts as described in a later section.
The 1H NMR chemical shift data are summarized for
monosaccharides in Table 3 [24]. The data are shown for
monosaccharides as the components of oligosaccharides in
which each is linked to an adjacent monosaccharide via a

glycosidic bond oriented either below (a) or above (b) the
plane of the ring. The chemical shift values of monosaccharides will assist the identification of oligosaccharides
and polysaccharides, but the values vary considerably with
the configuration and conformation of samples.
2. Relaxation Time
The spin-lattice relaxation time (T1) and the spin–spin
relaxation time (T2) reflect the conformational change
and the local tumbling motion of oligosaccharides and
polysaccharide chains. The relaxation process has been
observed to understand the structure-dependent molecular
motion, the helix-coil transition, the sol–gel transition, the
crystalline structure, the amorphous structure, the aggregation structure, and the hydration structure.
The spin-lattice relaxation time T1 is measured with
the repeated p–s–2/p radio frequency (RF) pulse sequence
by the inversion recovery method [25]. T1 follows Eq. (19)
derived from Blochs equation:
lnAl As ị ẳ ln 2Al À s=T1

ð19Þ

where Al and As are the magnitude of the recovering
vector of magnetization evolved by a p/2 RF pulse at time t
= l and s, respectively. T1 is evaluated from the plot of
ln(Al À As) against s. T1 is given in terms of the viscosity g
and temperature T [26] as
1
ẳ
T1



128p3
h2




l4 a3  g 
kT
r6

20ị

where l denotes a nuclear moment, a is the effective radius
of a spherical molecule, and r is the distance from the
observed nucleus to its magnetic neighbor. T1 decreases in
proportion to g/T and a3 increases with r6. The effective
volume a3 is replaced with the molar volume in the case of
oligosaccharides and polysaccharides in solution. T1 as a
function of the correlation time indicates the degree of
molecular motion, and T1 takes a minimum at the temperature when the relaxation occurs according to the dipole–


Progress in Structural Characterization of Functional Polysaccharides

9

Table 3 Chemical Shifts (ppm) of Monosaccharides from Acetone at 2.225 ppm in D2O at 22–27jC
Protons
Monosaccharidea

H1

H2

H3

H4

H5

H6

H7

CH3

NAC

a-D-Glc-(1!
h-D-Glc-(1!
a-D-Man-(1!
h-D-Man-(1!
a-D-Gal-(1!
h-D-Gal-(1!
h-D-GlcNAc-(1!
a-D-GalNAc-(1!
h-D-GalNAc-(1!
a-L-Fuc-(1!
a-L-Rha-(1!

h-D-Xyl-(1!
3-u-Me-a-L-Fuc-(1!
3-u-Me-a-L-Rha-(1!
2,3-di-u-Me-a-L-Rha-(1!
3,6-di-u-Me-h-D-Glc-(1!

5.1
4.4
1.9
4.7
5.2
4.5
4.7
5.2
4.7
5.1
4.9
4.5
4.8
5.0
5.1
4.7

3.56
3.31
3.98
4.04
3.84
3.52
3.75

4.24
3.96
3.69
4.06
3.27
3.70
4.24
3.94
3.34

3.72
3.51
3.83
3.63
3.90
3.67
3.56
3.92
3.87
3.90
3.80
3.43
3.40
3.59
3.52
3.31

3.42
3.41
3.70

3.58
4.02
3.92
3.48
4.00
3.92
3.79
3.46
3.61
–
3.52
3.41
3.51

3.77
3.45
3.70
3.37
4.34
3.71
3.45
4.07
3.65
4.1–4.9b
3.74

3.77
3.74
3.78
3.76

3.69
3.78
3.90
3.79
3.80
–
–
–
–
–
–
3.66

3.87
3.92
3.89
3.93
3.71
3.75
3.67
3.68
3.75
–
–
–
–
–
–
3.78

–
–
–
–
–
–
–
–
1.23
1.28
–
1.32
1.32
1.32
–

–
–
–
–
–
–
2.04
2.04
2.01
–
–
–
–
–

–
–

c

3.89
3.77
3.73
3.51

a
These are average values for nonreducing terminal sugars linked by a glycosidic linkage to the adjacent monosaccharides. Signals for protons at
the ring carbons are shifted downfield when linked by another monosaccharide at the hydroxyl group of that carbon.
b
These signals considerably vary more than other signals due to conformational features.
c
H5ax 3.29; H5eq 3.93.
Source: From Ref. 24.

dipole interaction [27]. The correlation time, sc, is given
approximately by
sc ẳ 4p3 a3 g=3kT

21ị

1

H T1 varies with the spin diffusion [28] and the value of T1
is much influenced by O2 gas.
The T2 experiments are performed to observe the

molecular motion in an extreme narrowing condition [27]
where the viscosity of a sample solution is low and the
motion is fast. The T2 measurements are suitable especially
for 1H nuclei because the problem resulting from the spin
diffusion can be avoided in the T2 experiments. The T2
value is determined by the Carr–Purcell [29]/Meiboom–
Gill [30] (CPMG) method. Here the pulse sequence (p/2)–
s–py–2s–py–2s–py–p. . .. (s is the pulse interval) is used to
avoid the cumulative error due to incorrect pulse lengths.
3. High-Resolution Solid State Nuclear Magnetic
Resonance
A rapid isotropic tumbling molecular motion is restrained
in the viscose solution state or in the solid state of oligosaccharides and polysaccharides. The NMR spectrum
shows a proton dipolar broadening of many kilohertz
due to strong dipole–dipole interaction and a chemical
shift anisotropy as a result of the restraint of the molecular
motion. A high-power, proton-decoupling field [31] is
found to be effective to remove a proton dipolar broadening. 13C–1H scalar coupling can be removed by the high-

Copyright 2005 by Marcel Dekker

power proton dipolar decoupling (DD) to improve the
resolution.
A magic angle spinning (MAS) method is employed to
diminish the chemical shift anisotropy [32]. A sample
placed in a cylindrical rotor is rotated about an axis making
an angle a with the magnetic field, H0, at 800–5000 Hz by
air. The chemical shift Hamiltonian is composed of a timeindependent term and a time-dependent term [33]. The
time-dependent term yields side bands at the multiples of
the rotation rate in the spectrum, but the side bands

disappear at a spinning rate faster than a half of the width
of the chemical shift anisotropy powder pattern observed
in the viscose solution or solid samples. When the sample is
rotated at the fixed angle a being equal to 54.74j (magic
angle) with respect to the magnetic field, the chemical shift
anisotropy vanishes and the time-independent term contains only the isotropic chemical shift.
Due to long 13C T1, a long repetition time is needed to
observe an NMR spectrum with a sufficient S/N ratio and a
high resolution in solid state experiments. The reduction of
T1 can be achieved by transferring the energy of 13C spins in
the excited state (at a high-spin temperature) to the NMR
lattice. The energy is transferred from 13C spins at a highspin temperature to 1H spins in the cross-polarization (CP)
technique [34,35] where the Hartmann–Hahn condition is
satisfied. The RF pulse sequence of the CP technique for
measuring 13C nuclei is shown in Fig. 6a. SL denotes a
pulse for spin locking and DD is a pulse for heteronuclear
dipolar decoupling. The Hartmann–Hahn condition is


10

Figure 6 Timing diagrams for the NMR pulse sequence: (a)
CP, (b) COSY, (c) HOHAHA, and (d) NOESY.

satisfied by the pulse applied to 13C while applying the SL
pulse. The signal created by CP is four times the original
magnetization in an ideal condition. The DD and the MAS
are usually combined with the CP technique to obtain highresolution spectra (CP/MAS).
4. Two-Dimensional Nuclear Magnetic Resonance
In interpreting the NMR spectra, the first step is to identify

signal peaks. As mentioned above, the spectra for oligosaccharides and polysaccharides are complicated and twodimensional (2-D) NMR technique is commonly applied to
separate the NMR signals on the basis of J coupling. The 2D NMR technique yields information on the spin–spin
coupling between heteronuclei, chemical exchange, and the
nuclear Overhauser effect (NOE).
The 2-D NMR technique involves several spectroscopic methods classified by the mode of pulse sequence
(Fig. 6). The response of the nuclear spin system to the RF
pulse is observed as FID (free induction decay) as a
function of a time t2, which is Fourier transformed to yield
an NMR spectrum in the conventional (1-D) spectroscopy.
By applying two RF pulses with a time interval t1, a second
time axis t1 (an evolution time) can be introduced where the

Copyright 2005 by Marcel Dekker

Kajiwara and Miyamoto

response of the nuclear spin system becomes a two-dimensional function of two independent times t1 and t2. When
FID is two-dimensionally Fourier-transformed, a twodimensional spectroscopy is obtained as a function of
two independent frequencies.
The 2-D shift correlated spectroscopy (COSY) informs the connection of nuclei. The pulse sequence of
COSY for 1H nuclei is shown in Fig. 6b. (p/2)/1 and
(p/2)/2 are the first and second pulses, which differ in
phase. The time interval between two pulses, t1, is an
evolution time and t2 corresponds to an acquisition time.
A 1H–1H COSY spectrum is represented by a square, where
both axes correspond to 1H chemical shifts. The signals in
the spectrum are classified in diagonal peaks and crosspeaks. The diagonal peaks are equivalent to the 1-D NMR
spectroscopy. The cross-peaks appear symmetrically withrespect to the diagonal peaks and correspond to the difference of the chemical shifts of two sites specified on the
diagonal line by the two coordinates of respective peak
position.

The 2-D homonuclear Hartman–Hahn spectroscopy
(HOHAHA) reveals a spin–spin interaction network as a
totally correlated spectroscopy that is obtained by changing the duration of the spin-locking application [36]. When
the Hartman–Hahn condition is satisfied by spin locking,
the magnetization transfer takes place by the spin–spin
coupling between I and S spins and its degree can be
adjusted by the duration of spin locking. Homonuclear
Hartman–Hahn spectroscopy is more sensitive than COSY
with respect to the line resolution, and facilitates the
assignment of 1H signals along covalent bonds. To satisfy
the Hartman–Hahn condition over a wide range, a proton
broadband decoupling is introduced by a specially
designed pulse sequence. Fig. 6c shows the pulse sequence
of HOHAHA, where SLy is a spin-locking pulse and sm a
mixing time. The MALEV-17 composite pulse [37] applied
during the mixing time to lock spins over a wide frequency
range.
The nuclear Overhauser effect correlated spectroscopy
(NOESY) observes the nuclear Overhauser effect due to the
magnetic dipole–dipole interaction between nuclei in a
short distance, and reveals the conformation, configuration, and chemical exchange of large molecules [38]. The
pulse sequence of NOESY is basically the same as COSY
except for the additional p/2 pulse after a fixed time sm as
shown in Fig. 6d, where sm denotes a mixing time. The
distance between 1H nuclei is determined from the intensity
of cross-peaks, and offers a mean of investigating spatial
relationships between nuclei through NOE. The crossrelaxation rate for an I and S spin system, rIS, is a function
of the distance between the I and S spin:
c4 t2
rIS ¼

10r6



6sc
À sc
1 ỵ 4x2 s2
c


22ị

where x is the Larmor frequency and the sc is the correlation time of reorientation [39]. rIS is evaluated from the sm
dependence of cross-peak intensities. The spatial information obtained by NOESY is restricted within the distance of


Progress in Structural Characterization of Functional Polysaccharides

about 0.5 nm. sc depends on the motility of molecules. The
cross-peaks show negative and positive values for xsc < 1
and xsc > 1, respectively. When xsc c 1, the cross-peaks
of NOE are not observed. By applying spin locking, a
positive NOB is observed over the wide time scale of
molecular motion. The rotating frame nuclear Overhauser
effect spectroscopy (ROESY) is developed [39,40] to observe NOB of the sample whose molecular weight ranges
from 1000 to 2000 and xsc c 1.

D. Molecular Modeling
1. Monte Carlo Method
Two dihedral angles / and w with respect to the glycosidic

bond determine the conformation of a disaccharide, provided that a pyranose ring is rigid (Fig. 7). The conformational analysis of a disaccharide thus comprises the

11

evaluation of a total conformational energy as a function
of a pair / and w. / and w can take any value between À180j
and +180j. The most likely conformation is expected to
have the lowest potential energy. For example, 38 pairs of /
and w evaluated from the crystallographic data of maltose
Glcpa 1!4 Glc are found to lay within the low-energy
range of 2 kcal/mol above the absolute energy minimum on
the energy map provided by molecular mechanical calculation, proving the validity of computer modeling. Here
molecular modeling permits to evaluate the range of attainable conformations in terms of the potential energy at each
point specified by a pair of / and w. The observed value of /
and w will vary among the attainable conformations
according to the crystal packing (in the solid state) or the
type of solvent (in the solution). Fig. 7 shows the conformational energy map of maltose, cellobiose, xylobiose,
chitobiose, laminaribiose, and sphorobiose calculated by

Figure 7 Definition of two dihedral angles, / and w, to determine the conformation of a disaccharide, and 2-D contour energy
map (the potential energy as a function of two dihedral angles / and w) of (a) maltose, (b) cellobiose, (c) xylobiose, (d)
chitobiose, (e) laminaribiose (s = 112.5j), and (f ) sophorose.

Copyright 2005 by Marcel Dekker


Figure 7 Continued.

Copyright 2005 by Marcel Dekker