Polysaccharides - structural diversity and functional diversity

Polysaccharides structural diversity and functional diversity

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Copyright n n 2005 by Marcel Dekker All Rights Reserved.

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Polysaccharides as natural polymers are by far the most abundant renewable resource on the earth with an annual formationrate surpassing the world production rate of synthetic polymers by some orders of magnitude In contrast to petroleum-basedsynthetic polymers, plant polysaccharides are sustainable materials synthesized by the sun’s energy and fully biodegradable inthe original state Thus, with decreasing supply of oil resources polysaccharides, including cellulose, starch, chitin, andhemicelluloses, are expected to play an increasingly important role in industrial use Polysaccharides are designed by nature tocarry out various specific functions Examples comprise structural polymers such as cellulose and chitin, storagepolysaccharides 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 magnificentstructural diversity and functional versatility Their structural and functional properties are often superior to syntheticmaterials as demonstrated, for instance, by the cellulose based cell wall architecture of plants or the function of hyaluronic acid inthe human body.

It has been a true challenge to present state-of-the-art polysaccharide research from different aspects regarding themacromolecular variety, function and structure in just one volume In this book well-known and recognized authors describethe 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, andindustrial aspects are being discussed It is further demonstrated that cellulosic biomass conversion technology permits largescale 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 polysac-charides Some chapters deal with medical and pharmaceutical aspects including medical foods, anticoagulant properties andthe role of polysaccharides in tissue engineering Furthermore, methodical aspects, including characterization by X-rayscattering, 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 findreferences for a deeper insight into a specific field Thus, this book is a very useful tool for scientists of both academia andindustry interested in the fundamental principles of polysaccharide functions and modifications on one hand and novelapplications on the other Having been involved in similar work mainly with industry-related issues of cellulose research formany years, I would like to stress that the presented state of knowledge, as sophisticated as it might seem to be, should not beunderstood as the final stage, but as an invitation to add new knowledge to this field and to explore additional applications ofpolysaccharides I would be delighted, if this monograph challenged and encouraged scientists to deal with polysaccharides asfascinating polymers with a bright future

Hans-Peter-FinkFraunhofer-Institute for Applied Polymer ResearchPotsdam-Golm, Germany

iii

Polysaccharides are the macromolecules that belong to the means components of life Together with nucleic acids andproteins, the polysaccharides determine the functionality and specificity of the species Polysaccharides have received littlesuch promotion even though they are widely distributed throughout nature and have highly organized structure There areimportant molecules involved throughout the body in signal transduction and cell adhesion Polysaccharides can be broadlyclassified 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 tounderstand the functions of polysaccharides, but the diversity and irregularity of polysaccharide chains make the structuralanalysis a formidable task The conformational analysis involves two aspects: (a)the characterization of a single chainconformation and (b)the analysis of the chain assembly of polysaccharides A remarkable progress has been achieved in recentyears with high-resolution, solution- and solid-state-1H- and13C-NMR including cross-polarization-magic-angle-spinningand two-dimensional techniques Specific electron microscopy techniques can visualize single polysaccharide molecules andcan 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 structureelucidation of polysaccharides have demonstrated that these methods can contribute to a ‘‘decision’’ on the actualconformational properties of oligosaccharides and linear polysaccharides

2 Conformation and dynamic aspects of polysaccharide gels The most important aspect of characterization ofpolysaccharide gels seems to clarify their backbone dynamics together with conformations as viewed from their highlyheterogeneous nature Backbone dynamics of polysaccharide gel network can be characterized by means of simplecomparative high-resolution13C NMR measurements by cross-polarization-magic angle spinning (CP-MAS)and dipolardecoupled-magic angle spinning (DD-MAS)techniques

3 Rheological behavior of polysaccharides in aqueous systems Rheology provides precious tools to explore andunderstand the properties of polysaccharides in aqueous systems The rheological behavior of polysaccharides systemsmanifests the underlying structure of the systems In the simplest case, that of polysaccharides solution, viscosity is directlyrelated 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, theirviscoelastic properties are related to supramolecular organization The main types of polysaccharide systems that areencountered 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 presentsthe mechanisms of biosynthesis of bacterial polysaccharides and provides some information on the engineering ofpolysaccharides that will allow in the near future the production of a polysaccharide with a choice chemical structurehaving a set of predictable physical properties This part covers also pertinent areas such as: bacterial and fungalpolysaccharides, cell-wall polysaccharides, production of microbial polysaccharides, industrial gums, and microbialexopolysaccharides of practical importance

The bacterial polysaccharides are described as: production and synthesis, composition and structure, physicalproperties, degradation by polysaccharases and polysaccharide lyases, polysaccharides common to prokaryotes andeukaryotes, 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 theyform independent wall layers which are mobilized when the seed germinates In both hardwood and softwood, hemi-celluloses 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 andpolycation)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; Macromolecularcomplexes 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 naturalpolysaccharides with anticoagulant properties; Structural elucidation of heparan sulfate-like polysaccharides usingminiaturized LC/MS; Enzymatic synthesis of heparan sulfate; Synthetic and natural polysaccharides having biologicalactivities; 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 200sialyl residue Polysialic acids chains are covalently attached to membrane glycoconjugates on cells that range inevolutionary diversity from bacteria to human brains

Proteoglycans, a group of glycoproteins that are invested with covalently bound glycosaminoglycan chains, are one ofthe important classes of molecules in brain development and maturation The glycosaminoglycan chains that defineproteoglycans 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 cubicphases 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 glycosaminoglycansfrom microbial sources)have been of interest to the medical profession

9 Renewable resources Cellulosic biomass includes agricultural (e.g., corn stover and sugarcane bagase)andforestry (e.g., sawdust, thin-nings, and mill wastes)residues, portions of municipal solid waste (e.g., waste paper)andherbaceous (e.g., switch-grass)and woody (e.g., poplar trees)corps They are appropriate materials used as renewableresources 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, describeand 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 ofcellulose and hemicellulose’’ presents a comprehensive overview of the technology and economic status for cellulose andhemicellulose hydrolysis describes the important structural features of cellulosic materials, applications, process steps, andstoichiometry for hydrolysis reactions The chapter then examines biomass structural characteristics that influence cellulosehydrolysis 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 andsome recent applications, which might become of commercial interest in the near future The polysaccharides are utilized asgallants, 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 forbiotechnological 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 preciousmetal recovery

Separatory systems Cellulose and chitosan derivatives are dominating the membrane market due to their favorablestability and their selectivity in gas- and liquid-phase separations

Biosurfactants Numerous microorganisms (candida lipolytica, Acetinobacter calcoaceticus)produce extracellularglycoconjugates 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 parent solid matrix of mixed esters of cellulose with micrometer-sized pores filled with a nemantic liquid crystal

trans-11 Incorporation of the polysaccharides in the synthetic matrix offers on one hand the possibility to obtain a broaderapplication range of the usual polymers and, on the other hand, ways to optimize and control some properties and producenew 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 UnitedStates The editor is grateful to all the collaborators for their precious contributions

Severian Dumitriu

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 A˚kerholm, and Barbara Hinterstoisser

7 Light Scattering from Polysaccharides

11 Computer Modeling of Polysaccharide–Polysaccharide Interactions

Francßois R Taravel, Karim Mazeau, and Igor Tvarosˇka

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 Modification of Hemicelluloses and Gums

Margaretha So¨derqvist Lindblad and Ann-Christine Albertsson

20 Role of Acetyl Substitution in Hardwood Xylan

Maria Gro¨ndahl 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

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¨nnberg

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

Yutaka Abe Process Development Research Center, Lion Corporation, Tokyo, Japan

Margaretha A˚kerholm STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden

Ann-Christine Albertsson Royal Institute of Technology, Stockholm, Sweden

P L Almeida EST/IPS, Setu´bal, Portugal and FCT/UNL, Caparica, Portugal

Eric H Anderson Case Western Reserve University, Cleveland, Ohio, U.S.A

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 IsraelDeaconess 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

Stephen R Decker National Renewable Energy Laboratory, Golden, Colorado, U.S.A.

Wolf-Dieter Deckwer Biochemical Engineering, GBF–National Research Center for Biotechnology, Braunschweig,Germany

Jean-Louis Doublier INRA-Laboratoire de Physico-Chimie des Macromole´cules, Nantes, France

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 ofTechnology, Go¨teborg, Sweden

Roberto Geremia Laboratoire d’Adaptation et de Pathoge´nie des Microorganismes, Joseph Fourier University,Grenoble, France

Richard D Gilbert North Carolina State University, Raleigh, North Carolina, U.S.A

M H Godinho FCT/UNL, Caparica, Portugal

Maria Gro¨ndahl Biopolymer Technology, Department of Materials and Surface Chemistry, Chalmers University ofTechnology, Go¨teborg, Sweden

Kazuaki Harata Biological Information Research Center, National Institute of Advanced Industrial Science andTechnology, 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 BOKU-University of Natural Resources and Applied Life Sciences, Vienna, AustriaSusan Hockfield Yale University School of Medicine, New Haven, Connecticut, U.S.A

Lisbeth Illum IDentity, Nottingham, United Kingdom

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

Tadeusz Janas University of Colorado, Boulder, Colorado, U.S.A

Teresa Janas University of Colorado, Boulder, Colorado, U.S.A and University of Zielona, Go´ra, PolandHenning 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 Otsuma Women’s University, Chiyoda-ku, Tokyo, Japan

John F Kennedy University of Birmingham Research Park and Chembiotech Laboratories, Birmingham, UnitedKingdom

Charles J Knill University of Birmingham Research Park and Chembiotech Laboratories, Birmingham, UnitedKingdom

Tetsuo Kondo Kyushu University, Fukuoka, Japan

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

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 BethIsrael Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A

Naoji Kubota Oita University, Oita, Japan

Miroslaw Lech Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A and Beth IsraelDeaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A

Jacques Lefebvre INRA-Laboratoire de Physico-Chimie des Macromole´cules, Nantes, France

Margaretha So¨derqvist Lindblad Royal Institute of Technology, Stockholm, Sweden

Robert J Linhardt University of Iowa, Iowa City, Iowa, U.S.A

Bruno Lo¨nnberg A˚bo Akademi University, Turku/A˚bo, Finland

Delphine Magnin Sherbrooke University, Sherbrooke, Quebec, Canada

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 Macromole´cules Ve´ge´tales, Grenoble, France

Michel Milas Centre de Recherches sur les Macromole´cules Ve´ge´tales (CERMAV), CNRS, and Joseph FourierUniversity, 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 Queen’s University, Kingston, Ontario, Canada

Kozo Ogawa Osaka Prefecture University, Sakai, Osaka, Japan

Lisbeth Olsson Center for Microbial Biotechnology BioCentrum-DTU, kgs Lyngby, Denmark

R Parker Institute of Food Research, Norwich Research Park, Norwich, United Kingdom

Serge Pe´rez Centre de Recherches sur les Macromole´cules Ve´ge´tales, Grenoble, France

Denis Poncelet ENITIAA, Nantes, France

Valentin I Popa Technical University of Jassy, Jassy, Romania

Marguerite Rinaudo Centre de Recherches sur les Macromole´cules Ve´ge´tales (CERMAV), CNRS, and JosephFourier University, Grenoble, France

S G Ring Institute of Food Research, Norwich Research Park, Norwich, United Kingdom

Christophe Roca Center for Microbial Biotechnology BioCentrum-DTU, kgs Lyngby, Denmark

Robert D Rosenberg Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A and Beth IsraelDeaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A

Wael Sabra Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt

Hazime Saitoˆ Himeji Institute of Technology, Kamigori, Hyogo, Japan and Center for Quantum Life Sciences,Hiroshima University, Higashi-Hiroshima, Japan

Lennart Salme´n STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden

Kei Shimoda Oita University, Oita, Japan

Catherine E Skopec Cornell University, Ithaca, New York, U.S.A

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

Valdir Soldi Federal University of Santa Catarina, Floriano´polis, SC, Brazil

Iuliana Spiridon ‘‘Petru Poni’’ Institute of Macromolecular Chemistry, Jassy, Romania

I W Sutherland University of Edinburgh, Edinburgh, United Kingdom

Franc¸ois R Taravel Centre de Recherches sur les Macromole´cules Ve´ge´tales (CERMAV), CNRS, and JosephFourier University, Grenoble, France

M Thanou Cardiff University, Cardiff, United Kingdom

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

Tadashi Uragami Kansai University, Osaka, Japan

Kjell M Va˚rum Norwegian University of Science and Technology (NTNU), Trondheim, Norway

Christer Viebke The North East Wales Institute, Water Soluble Polymers Group Plas Coch, Wrexham, UnitedKingdom

Liisa Viikari VTT Technical Research Centre of Finland, Finland

Bryan W Wolf Abbott Laboratories, Columbus, Ohio, U.S.A

Charles E Wyman Dartmouth College, Hanover, New Hampshire, U.S.A

Yunyu Yi Queen’s University, Kingston, Ontario, Canada

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

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

polysaccha-ride 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

irregu-larity of polysaccharide chains make it a formidable task

Polysaccharide chains are partly organized but are

consid-ered 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—wa-ter-soluble, digestible amylose and non-wa-glucans—wa-ter-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-D

-linked polyglucan, whereas cellulose is a (1!4)-a-D

-linked 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]

(Fig 1) In later sections, it will be shown that these basicconformations of amylose and cellulose are supposed to

be retained to some extent in aqueous solutions Thedifference in the structure is reflected by the respectivephysiological functions of edible amylose and nonediblecellulose

There are some evidences that the higher-order ture of polysaccharide chains is related to their physiolog-ical function as exemplified by the triple-stranded helix ofscleroglucan, which is known to possess an antitumoractivity Many polysaccharide chains are able to assume

struc-an ordered or quasi-ordered structure such as a stranded helix, but the ordered structure is interrupted bythe irregularity of the primary structure in the polysaccha-ride chains Many polysaccharide chains form gel in solu-tions by assuming an ordered or quasi- ordered chainstructure, which constitutes a cross-linking domain.The conformational analysis of polysaccharide chainsinvolves two aspects: (1) the characterization of a singlechain conformation and (2) the analysis of the chainassembly (suprastructure) of polysaccharides A singlechain conformation of polysaccharides is primarily deter-mined by the chemical structure specified by the types ofsugar residues, sugar linkages, and side groups A singlechain conformation accounts, to some extent, for theformation of suprastructures such as the complexing ca-pability of amylose and the fringed micelle formation ofcellulose

double-Unlike cellulose and amylose, most polysaccharideshave no regular homopolymeric structure, where the reg-ularity is interrupted by the random intrusion of differenttypes of linkage and/or sugar units The introduction of

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

supra-structure often results to gelation

The complexity in characterizing polysaccharide chain

conformation is due to the fact that the interchain

interac-tion 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

spec-ified, the interchain interaction can be analyzed in terms of

the mode of suprastructure composed of several

polysac-charide chains

This review is intended to demonstrate the recent

strategy in the structural and conformational

characteriza-tion of oligosaccharides and polysaccharides Although

various techniques are applied for the structural and

conformational analysis of oligosaccharides and

polysac-charides, the general inability to crystallize excludes the

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 thatare currently applied to the structural and conformationalanalysis of oligosaccharides and polysaccharides: small-angle x-ray scattering (SAXS) [2] and nuclear magneticresonance (NMR) [3]

Molecular modeling by computer is considered tosupplement the analysis by small-angle x-ray scatteringand NMR Although an initial intention of molecularmodeling is to predict physical properties of carbohydrates

a priori [4], the ab initio calculation is limited to a smallmonosaccharide and the semiempirical quantum methodcan be applied for the structural characterization of mol-ecules up to the size of disaccharides Molecular mechanics

or molecular dynamics is an alternative method applied tothe computer modeling of larger carbohydrate molecules,where the motion of constituent atoms is assumed to bedescribed in terms of classical mechanics

In the final chapter, the structural and conformationalaspect is discussed from the chemical point of view Herethe controlled chemical modification of cellulose is treatedand the physicochemical characteristics are discussed bytaking into account the structural change due to chemicalmodification of cellulose

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

II STRATEGY AND METHODS OF ANALYSIS

Because many monosaccharides have a single,

well-estab-lished conformation, the conformational analysis of

oligo-saccharides and polyoligo-saccharides starts from understanding

the energetic relationship when the monosaccharide

resi-dues are linked in a specific way The entire geometry of

oligosaccharides and polysaccharide chains can be

de-scribed 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

con-formation of saccharide chains is found in most cases to

assume nonrandom conformations due to intra- and

inter-chain 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

found in nature The structural analysis of disaccharideimplies the identification, the linking order, the link posi-tion, and the link mode of constituent monosaccharides.Chemical and optical methods are available to determinethe structure of disaccharide, but the recent development inNMR has facilitated the assignment of specific protons orcarbons as well as the conformational determination of theglucosidic linkage as shown in the next section Fourier-transform infrared (FTIR) and laser Raman spectroscopyare also useful tools for the characterization of glycosidicbonds [5]

X-ray and neutron diffraction can be applied to mine the crystal structure and hydrogen bonding of mono-saccharides and disaccharides that form a single crystal Aclassic example will be found in the crystal structureanalysis of h-maltose monohydrate [6](Fig.4), where theearlier structure determination using x-ray diffraction [7]was refined to give a more accurate description of thehydrogen bond structure The x-ray diffraction analysisprovides the most explicit information on structure interms of the precise atomic coordinates The CambridgeStructural Database lists the crystal structure of about 40small oligosaccharides (cyclodextrins are omitted) includ-ing about 10 trisaccharides, 2 tetrasaccharides, and 1hexasaccharide Here a number of crystal structures ofmono-, di-, and trisaccharides were determined from theacetate derivatives because the acetylated derivatives arefound to crystallize more easily than original (untreated)oligosaccharides (1!3)-h-D-glucopyranosyl residues con-sist of a main chain of a medically important class ofpolysaccharides including curdlan, lentinan, schizophyl-lan, scleroglucan, and grifolan, which possess branches atC6 (except for curdlan) Glcph 1!3 Glc disaccharides aresystematically synthesized and the crystal structures aredetermined A first attempt was made by Takeda et al [8]

deter-on 3-O-h-D-glucopyranosyl-h-D-glucopyranose (h-Dinarabiose) ethyl hepta-O-acetyl-h-D-laminarabioside [9],followed by Perezet al [10] on octa-O-acetyl-h-D-laminar-abiose), and by Lamba et al [11] on (methyl hepta-O-acetyl-h-D-laminarabiose) Recently, 3-O-h-D-glucopyra-nosyl-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

-lam-by x-ray diffraction (Fig 5) Table 2 summarizes twodihedral angles, / and w, with respect to the glycosidicbond for (1!3)-h-linked disaccharides, evaluated from thecrystallographic data of laminarabiose and laminarabio-side 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 ofthe substituents, while the angle w is classified in twogroups of around45j and 8j When the intramolecularhydrogen bond is formed between 04V and 05, the angle yassumes a negative value The introduction of acetylgroups prevents the formation of intramolecular hydrogenbonds as seen from the stereoview of the molecular struc-tures of methyl h-D-laminarabioside and methyl hepta-O-acetyl-h-D-laminarabioside in Fig 5 The invariance of

Figure 2 Pyranose and furanose

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(q  r); that is, the

scattered intensity I( q) is given by the Fourier

transforma-tion of the electron density distributransforma-tion in the object:

IðqÞ ¼ V ¼

ðl 04pr2cðrÞ  expðiq rÞdr ð1Þ

Here the magnitude of the scattering vector is given by (4p /k) sin(h / 2) with k and h being the wavelength and thescattering angle, respectively c(r) is a correlation functionrepresenting the average of the product of two electrondensity fluctuations at a distance r The distance distribu-tion function p(r) is defined as

which is characteristic of the shape of the scattering object.The phase difference between scattered rays becomesmore prominent as the scattering angle increases Thus thescattered intensity is maximum at zero scattering angle andproportional to the number of electrons in the object wherethe scattered rays are all in phase The scattered intensitydecreases with increasing scattering angle and diminishes

at a scattering angle of the order of k / D, where k and D

Figure 3 Pyranose-type pentose and hexose

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(q  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 q2at very small angles by expanding the sine

term to yield the particle scattering factor as

PðqÞuIðqÞ=Ið0Þ ¼ 1 13q2

ðl 04pr2cðrÞ  r2dr=2



ðl 04pr2cðrÞdr þ Oðq4Þ

ð3Þ

where the second term on the right side represents the

radius of gyration RG, that is

R2G¼

ðl 0pðrÞ  r2dr=2

ðl 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]:

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

(Rt) of a flat particle by describing approximately thescattering from the cross-section or the thickness in terms

of the exponential form The scattering factor of a rod-likeparticle (a cylinder) consists of two components of theheight and the cross-section as

PcylinderðqÞc p

2Hq exp q 2R2c= 

ð6Þwhere 2H denotes the height of the cylinder The scatteringfactor of a flat particle (a disk) is given by the product oftwo terms of the cross-sectional area and the thickness as

Table 1 Disaccharides in Nature

cellobiouronic acid GlcUAph 1! 4 Glc D pneumoniae

hyalobiuronic acid GlcUAph 1! 3 GlcN hyaluronic acid

p and f denote pyranose and furanose, respectively.

Figure 4 Stereoview of h-maltose monohydrate (FromRef 6.)

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

and the thickness radius of gyration Rtare 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

circumfer-ence A simple Ornstein–Zemike type is generally applied

to the density correlation function for a Gaussian chain:

cðrÞicn

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

isotro-pic system, Eq (8) yields the scattering profile as

3

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:

The particle scattering from a single molecule is in

principle calculated from the coordinates of the constituent

gigj/iðqÞ/jðqÞ

sindijq

dijq

ð12Þ

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

giis an atomic scattering factor dijis 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

/i¼3 sinðR½ iqÞ  ðRiqÞcosðRiqÞ

where RIis the van deer Walls radius of the ith atom If a

molecule is rigid, the distance dijis fixed and Eq (1) is

equivalent to the particle scattering factor of such a ecule that freely moves in space If a molecule (e.g., aflexible polymer molecule) has a large internal freedom,the distance dij fluctuates with time due to the internalmotion of such a molecule In this case, the particlescattering factor should be calculated as an average over

mol-a stmol-atisticmol-al ensemble genermol-ated by the Monte Cmol-arlo cedure [18,19] according to the conditional bond confor-mation probability [20]

pro-When no molecular model is available, the scatteringprofile can be analyzed in terms of a triaxial body model ofhomogeneous density representing the shape of the object[21] or by assuming a suitable pair correlation function forthe electron density distribution in the object [22] Thescattering factor is explicitly calculated for some homoge-neous triaxial bodies including a sphere, an ellipsoid, acylinder, and a prism For example, the scattering factorfor a sphere is given by Eq (14) as

No interdomain (interparticular) interaction is sidered in the above argument, and the scattering is con-sidered to be due solely to an isolated domain (or anisolated particle) When the interdomain (interparticular)interaction becomes dominant, an interference peak willappear at the q range corresponding to the interactiondistance in the scattering profile If the interdomain (inter-particular) interaction is isotropic and spherically symmet-ric, the scattering profile is decomposed into the product oftwo terms of the particle scattering factor P( q) and theinterference SI( q) [16]:

where m0is the volume of the sphere and the hard-sphereinteraction is represented by the sphere of a uniform radius2R The interaction potential b( q) is approximately given

by the Gaussian function when the interaction is softer

[22,23], and Eq (16) is rewritten as

1þ 2A2Mwcexpn2q2 ð18Þwhere 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,

confor-mation, 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-diffu-sion coefficients One of the advantages of NMR in the

structure analysis is its sensitivity to a microscopic

struc-ture within a short-range order in comparison with

small-angle 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

individ-ually 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

struc-ture 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

com-pliments other methods of the structural and

conforma-tional 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

conforma-tion of oligosaccharides and polysaccharides

1 Chemical Shift

Oligosaccharides and polysaccharides show several 1H

NMR signal peaks in the spectrum region between 2 and

6 ppm for protons on the ring The anomeric protons (Hi)have peaks in the region between 4.5 and 5.5 ppm, whereasthe chemical shifts for other protons (H2–H6) ranges from

2 to 4.5 ppm The H1 chemical shift database will provide astarting key to assign the chemical shifts of unknownsamples, although the chemical shift database for oligo-saccharides and polysaccharides are still far from comple-tion with respect to the accumulation and systematization

As the chemical shifts are also sensitive to the tional change, solvent, and temperature, it requires expe-rience and skill to identify the1H NMR peaks for unknownoligosaccharides and polysaccharide samples Varioustwo-dimensional NMR techniques have been developed

conforma-to facilitate the assignment and identification of the ical shifts as described in a later section

chem-The1H NMR chemical shift data are summarized formonosaccharides inTable 3[24] The data are shown formonosaccharides as the components of oligosaccharides inwhich each is linked to an adjacent monosaccharide via aglycosidic bond oriented either below (a) or above (b) theplane of the ring The chemical shift values of monosac-charides will assist the identification of oligosaccharidesand polysaccharides, but the values vary considerably withthe configuration and conformation of samples

2 Relaxation TimeThe spin-lattice relaxation time (T1) and the spin–spinrelaxation time (T2) reflect the conformational changeand the local tumbling motion of oligosaccharides andpolysaccharide chains The relaxation process has beenobserved to understand the structure-dependent molecularmotion, the helix-coil transition, the sol–gel transition, thecrystalline structure, the amorphous structure, the aggre-gation structure, and the hydration structure

The spin-lattice relaxation time T1 is measured withthe repeated p–s–2/p radio frequency (RF) pulse sequence

by the inversion recovery method [25] T1follows Eq (19)derived from Bloch’s equation:

where l denotes a nuclear moment, a is the effective radius

of a spherical molecule, and r is the distance from theobserved nucleus to its magnetic neighbor T1decreases inproportion to g/T and a3 increases with r6 The effectivevolume a3is replaced with the molar volume in the case ofoligosaccharides and polysaccharides in solution T1as afunction of the correlation time indicates the degree ofmolecular motion, and T1takes a minimum at the temper-ature when the relaxation occurs according to the dipole–

dipole interaction [27] The correlation time, sc, is given

approximately by

1H T1varies with the spin diffusion [28] and the value of T1

is much influenced by O2gas

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 T2measurements are suitable especially

for1H 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

oligo-saccharides and polyoligo-saccharides 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

broaden-ing.13C–1H scalar coupling can be removed by the

high-power proton dipolar decoupling (DD) to improve theresolution

A magic angle spinning (MAS) method is employed todiminish the chemical shift anisotropy [32] A sampleplaced in a cylindrical rotor is rotated about an axis making

an angle a with the magnetic field, H0, at 800–5000 Hzbyair The chemical shift Hamiltonian is composed of a time-independent term and a time-dependent term [33] Thetime-dependent term yields side bands at the multiples ofthe rotation rate in the spectrum, but the side bandsdisappear 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 isrotated at the fixed angle a being equal to 54.74j (magicangle) with respect to the magnetic field, the chemical shiftanisotropy vanishes and the time-independent term con-tains only the isotropic chemical shift

Due to long13C T1, a long repetition time is needed toobserve an NMR spectrum with a sufficient S/N ratio and ahigh resolution in solid state experiments The reduction of

T1can be achieved by transferring the energy of13C spins inthe excited state (at a high-spin temperature) to the NMRlattice The energy is transferred from13C spins at a high-spin temperature to1H spins in the cross-polarization (CP)technique [34,35] where the Hartmann–Hahn condition issatisfied The RF pulse sequence of the CP technique formeasuring 13C nuclei is shown in Fig 6a SL denotes apulse for spin locking and DD is a pulse for heteronucleardipolar decoupling The Hartmann–Hahn condition is

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

satisfied by the pulse applied to13C 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

high-resolution 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

oligo-saccharides and polyoligo-saccharides are complicated and

two-dimensional (2-D) NMR technique is commonly applied to

separate the NMR signals on the basis of J coupling The

2-D 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

spectro-scopic 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

response of the nuclear spin system becomes a sional function of two independent times t1and t2 WhenFID is two-dimensionally Fourier-transformed, a two-dimensional spectroscopy is obtained as a function oftwo independent frequencies

two-dimen-The 2-D shift correlated spectroscopy (COSY) forms the connection of nuclei The pulse sequence ofCOSY for 1H nuclei is shown in Fig 6b (p/2)/1 and(p/2)/2 are the first and second pulses, which differ inphase The time interval between two pulses, t1, is anevolution time and t2corresponds to an acquisition time

in-A1H–1H COSY spectrum is represented by a square, whereboth axes correspond to1H chemical shifts The signals inthe spectrum are classified in diagonal peaks and cross-peaks The diagonal peaks are equivalent to the 1-D NMRspectroscopy The cross-peaks appear symmetrically with-respect to the diagonal peaks and correspond to the dif-ference of the chemical shifts of two sites specified on thediagonal line by the two coordinates of respective peakposition

The 2-D homonuclear Hartman–Hahn spectroscopy(HOHAHA) reveals a spin–spin interaction network as atotally correlated spectroscopy that is obtained by chang-ing the duration of the spin-locking application [36] Whenthe Hartman–Hahn condition is satisfied by spin locking,the magnetization transfer takes place by the spin–spincoupling between I and S spins and its degree can beadjusted by the duration of spin locking HomonuclearHartman–Hahn spectroscopy is more sensitive than COSYwith respect to the line resolution, and facilitates theassignment of1H signals along covalent bonds To satisfythe Hartman–Hahn condition over a wide range, a protonbroadband decoupling is introduced by a speciallydesigned pulse sequence Fig 6c shows the pulse sequence

of HOHAHA, where SLyis a spin-locking pulse and smamixing time The MALEV-17 composite pulse [37] appliedduring the mixing time to lock spins over a wide frequencyrange

The nuclear Overhauser effect correlated spectroscopy(NOESY) observes the nuclear Overhauser effect due to themagnetic dipole–dipole interaction between nuclei in ashort distance, and reveals the conformation, configura-tion, and chemical exchange of large molecules [38] Thepulse sequence of NOESY is basically the same as COSYexcept for the additional p/2 pulse after a fixed time smasshown in Fig 6d, where smdenotes a mixing time Thedistance between1H nuclei is determined from the intensity

of cross-peaks, and offers a mean of investigating spatialrelationships between nuclei through NOE The cross-relaxation rate for an I and S spin system, rIS, is a function

of the distance between the I and S spin:

rIS¼c

4t210r6

correla-Figure 6 Timing diagrams for the NMR pulse sequence: (a)

CP, (b) COSY, (c) HOHAHA, and (d) NOESY

about 0.5 nm scdepends on the motility of molecules The

cross-peaks show negative and positive values for xsc< 1

and xsc> 1, respectively When xscc 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

ob-serve NOB of the sample whose molecular weight ranges

from 1000 to 2000 and xscc 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,

pro-vided that a pyranose ring is rigid (Fig 7) The

conforma-tional analysis of a disaccharide thus comprises the

evaluation of a total conformational energy as a function

of a pair / and w / and w can take any value between180jand +180j The most likely conformation is expected tohave the lowest potential energy For example, 38 pairs of /and w evaluated from the crystallographic data of maltoseGlcpa 1!4 Glc are found to lay within the low-energyrange of 2 kcal/mol above the absolute energy minimum onthe energy map provided by molecular mechanical calcu-lation, proving the validity of computer modeling Heremolecular modeling permits to evaluate the range of attain-able conformations in terms of the potential energy at eachpoint specified by a pair of / and w The observed value of /and w will vary among the attainable conformationsaccording to the crystal packing (in the solid state) or thetype of solvent (in the solution) Fig 7 shows the confor-mational 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 energymap (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

molecular mechanics (MM2 or MM3) with the force-field

including bond vibration, bond stretching, angular torsion,

and van der Waals interaction (seeTable 1for the

termi-nology) Here the glucose residue is assumed to be rigid and

replaced with a virtual bond connecting the neighboring

oxygen atoms of the glycosidic linkage (Fig 7) The bond

angle s is fixed, for example, to 110j in the case of amylose so

as to yield a consistent value for the radius of gyration as

observed for high molecular weight amylose

Longer chains are generated by the Monte Carlo

method according to the scheme summarized in Fig 8,

where the assumption is made that the short-range

inter-action between two adjacent residues determines the range

of permissible values of a pair of the dihedral angles / and

w That is, a pair of / and w is provided from the energy

map (Fig 7) according to the occurrence probability P(/,

w) specified by the Boltzmann factor associated with the

potential energy E(/, w) of a disaccharide with a set of /

and w Here P(/, w) is given as

with kBbeing a Boltzmann constant, T an absolute

tem-perature, and c a normalization constant A chain is

con-structed step by step as the geometry of each unit is

determined by a set of / and w when the bond angle s isfixed The effect of excluded volume can be taken intoaccount by excluding the step that places a unit in aspecified vicinity of the space occupied already by the unit

in a previous step

Because the polysaccharide chain undergoes thermalfluctuation in solution, the scattering from the solution isobserved as an average over space and time Assuming theergodicity, several chains are independently generated toconstitute a microcanonical ensemble, and the particlescattering function is then given by an ensemble averageover the scattering calculated from the atomic coordinates

of each generated chain according to Eq (12).Fig 9showsthe scattering profiles averaged over 500 chains of two 1,4-glucans, (1!4)-a-D-glucan (amylose), and (1!4)-h-D-glu-can (cellulose), generated by the scheme represented by Fig

8 with varying the number of glucosidic residues in terms ofthe Kratky plots by plotting q2I( q) against q Here theoccurrence probability for a pair of / and w is provided by

Figure 7 Continued

Figure 8 Flow chart to generate polysaccharide chains andcalculate the scattering factor

the energy map ofFig 7.The snapshots of simulated chains

(DP = 40) are also shown on the right side of the Kratky

plots The simulated amylose chain reveals the wobble

helical conformation with localized highly ordered helical

regions, whereas the cellulose chain seems to have a rather

extended chain structure as expected from the primary

structure The calculated scattering profiles reveal a

pro-nounced peak in Kratky plots at q = 0.2 A˚1for

(1!4)-a-D-glucan of higher degrees of polymerization, whereas

(1!4)-h-D-glucan exhibits a scattering profile typical to a

rigid rod-like molecule The intramolecular hydrogen

bonding is responsible for stabilizing the quasi-helical

chain conformation of (1!4)-a-D-glucan, which yields athicker cross-sectional radius of gyration evaluated asapproximately 5 A˚ from the cross-sectional Guinier plot[Eq (6)] of the simulated scattering profiles for (1!4)-a-D-glucan chains of over 20 glucosidic residues The cross-sectional radius of gyration remains as small as 2.1 A˚ in thecase of (1!4)-h-D-glucan chains, whose extended chainconformation promotes to assume the intermolecular hy-drogen bonding to form non-water-soluble aggregates

Table 4summarizes the radius of gyration of (1!4)-a-Dglucan and (1!4)-h-D-glucan, each calculated from thesimulated profiles and/or estimated from the observed

-Figure 9 Simulated scattering profiles of (1!4)-h-D-glucan (a) and (1!4)-h-D-glucan (b) as a function of the number ofglucosidic residues with snapshots of a simulated structure of respective glucan chains composed of 40 glucosidic residues (astereo figure) on the right

SAXS profiles Although some refinement of the

probabil-ity map is necessary, the simulation accounts, at least

qualitatively, for the DP dependence of the radius of

gyration and the difference in the radius of gyration due

to the glucosidic linkage mode

When the saccharide chain is longer, the excluded

volume effect becomes more serious The excluded volume

effect can be taken into account by considering the

inter-action between the nonbonded units Conventionally, the

repulsive interaction is dealt with in the Monte Carlo

simulation by replacing the chain units (segments) with

hard spheres of a finite radius.Fig 10demonstrates the

snapshots of amylose chain generated by the Monte Carlo

method with and without excluded volume, which is

rep-resented by a sphere of a radius 4 A˚ at the position of each

glycosidic oxygen The excluded volume effect is seen to

expand the chain, but the helical nature of amylosic chains

is retained in both unperturbed and perturbed states

2 Molecular Dynamics

The Monte Carlo method described in the preceding

section is based on the disaccharide conformation energy

map, and no water molecules are taken into account in the

model Although the Monte Carlo method is capable of

simulating longer polysaccharide chains, it does not allow

including the solvation effect directly through

water-medi-ated hydrogen bonds Molecular dynamics (MD)

simula-tions [41] can be applied to the structural studies of various

polysaccharides, where water molecules can be explicitly

included in the simulation However, the MD simulation is

restricted to relatively shorter chains owing to a present

computational capacity The results of the MD simulation

depend on the employed force-filled models such as

Gro-mos [42], Glycam93/99 [43], and Cff91/Cff [44], as well as

on the starting conformation Among the force fields

mentioned above, Gromos and Glycam is composed of a

set of parameters specifically developed for amylose;

how-ever, they differ in the treatment of the exoanomeric effect

on the glycosidic linkages Here Gromos ignores the anomeric effect, while Glycam incorporates the effectthrough the torsional terms determined from the ab initiogeometry optimization at the HF 6-31G* level Cff91 is ageneral-purpose force field for biomolecules, and Cff isexpanded from Cff91 to include the parameters with aproper account of the anomeric effect on the glycosidiclinkages The example of the MD simulations will beshown in the later section

exo-III STRUCTURAL AND CONFORMATIONALANALYSIS OF OLIGOSACCHARIDESAND POLYSACCHARIDES

The structural characterization of simple homoglucans ismainly introduced in this section The structural andmechanical properties of the gels from various marinepolysaccharides, plant polysaccharides, microbial polysac-charides, and animal polysaccharides are reviewed byClark and Ross-Murphy [45] This chapter intends todemonstrate the advanced methods applied for the struc-tural and conformational characterization of oligosaccha-rides and polysaccharides, particularly in solution, bytaking the examples of the homoglucans composed ofdifferent modes of glucosidic linkage

Table 4 Radius of Gyration of (1!4)-a-D-Glucan and

(1!4)-h-D-Glucan Oligomers

(1!4)-h-D-glucan (A˚) (1!4)-a-D-glucan (A˚)

A (1!4)-A-D-Glucan Represented by Amylose

The oligomers of (1!4)-a-D-glucans dissolve well in water

The observed SAXS profiles from maltohexaose and

mal-tooctaose are shown in Fig 11, where no effect of

associ-ation was observed The simulated SAXS profiles are also

shown in Fig 11 to examine the consistency of simulation

with the observed profiles The characteristics of wobbled

helix represented by a pronounced peak in the Kratky plots

become more distinct in maltooctaose than in maltohexaose

as expected from the molecular weight dependence of

simulated SAXS profiles(Fig.9) A good agreement

be-tween simulated and observed SAXS profiles assures that

the simulation can be extended to a longer chain to elucidate

a single chain conformation of amylose Here no adjustableparameter is involved, except for the normalization withrespect to the scattered intensity at q = 0

Amylose is known to assume a double-stranded form) [46,47] or single-stranded helical (V-form) [48] con-formation in a solid state from the analysis of the x-rayfiber diffraction, the x-ray powder, and the electron dif-fraction pattern of single crystals Amylose aqueous solu-tion forms gel by cooling Gelation takes place through theformation of nanocrystallites that serve as cross-linkingdomains Particle scattering from model nanocrystallites iscalculated [49] by assuming nanocrystallites composed ofB-form double helices or single-stranded V helices Themodel nanocrystallite is approximately represented by an

(B-Figure 11 Simulated and observed scattering profiles from maltohexaose (a) and maltooctaose (b) The (B-Figures on the rightshow a snapshot conformation of simulated maltohexaose and maltooctaose, respectively (a stereoview)

elliptical cylinder of 8.32-nm thickness (contains 42–222

duplexes composed of 24 glucosidic residues per strand) or

a parallelepiped of 6.44-nm thickness (contains 120 helices

composed of 24 glucosidic residues per strand) for the

B-form or the V-B-form, respectively

The SAXS profile from amylose aqueous solution

reveals a sharp upturn at q!0 in the Kratky plots (Fig

12) [ln q2I( q) plotted against q] according to the sol–gel

transition This pronounced upturn is ascribed on the

formation of an infinite structure (gel) as expected by the

cascade theory of gelation [50] At higher q regions, two

scattering profiles from sol and gel coincide, indicating that

the local conformation is identical in the sol and gel states

The local conformation is probably represented by a

single-stranded chain simulated by the Monte Carlo method

shown in Fig 9a, considering that single-stranded chains

are present in amorphous region of gel or in solution [51]

The observed profiles are fit to the scattering profile from

simulated (1!4)-a-D-glucan chains (DP = 40) in Fig 12.The Guinier plots for the cross section [Eq (6)] yields thecross-sectional radius of gyration as 0.45 nm in both geland sol The value of 0.45 nm (close to 0.5 nm), which isevaluated for the cross-sectional radius of gyration fromthe model double-stranded helix [52], also corresponds to

an apparent cross-sectional radius of gyration of a single(1!4)-a-D-glucan chain Here the deviation at lower qranges is considered to be due to the presence of double-stranded helices formed by the coupling of two neighboringsingle-stranded helices without significantly disturbing theconformation The SAXS profile from amylose gel wasanalyzed in terms of two components representing nano-crystallites and amorphous region [53], respectively, byassuming that no interference would take place betweentwo components The structure of the amorphous region inamylose gel should be identical to that in the sol state Thusthe excess scattering in the gel state with respect to the solstate mainly resulted from the formation of nanocrystal-lites that function as the cross-linking domain ( junctionzone) The oblate ellipsoid of revolution was found to yieldthe scattering profile fit to the excess scattering (Fig 13),

and its dimension (three semiaxes 12.9  13.1  4–3 nm)approximately corresponds to the nanocrystallite com-posed of 42 B-form duplexes with 24 glucosidic residuesper strand

The molecular dynamics simulation was performed onmaltopentaose with currently available force fields [54].The results are compared with the small-angle x-ray scat-tering observed from maltopentaose in aqueous solution

Fig 14compares the simulated profiles with the observedSAXS profiles, where Fig 14a provides a series of resultssimulated with available force fields, and Fig 14b the

Figure 12 Small-angle x-ray scattering profile from amylose

gel and sol, where closed and open circles denote gel and sol,

respectively Solid lines represent the calculated scattering

profile from simulated (1!4)-h-D-glucan chains of DP =

40

Figure 13 Scattering profile of amylose gel decomposed intotwo components Iexcess denotes the excess scattering fromamylose gel with respect to amylose sol (= IGEL ISOL)

Imodelis the scattering calculated from the oblate ellipsoid ofrevolution (12.9  13.1  4.3 nm), and Ical= ISOL+ Imodel

Monte Carlo results with two probability maps (Monte

Carlo K denotes a rigid map employed inFig 9a)and the

profiles calculated from the atomic coordinates of three

regular helices Both Monte Carlo results show a

satisfac-tory agreement with observed SAXS profiles, where a small

difference due to the glucose geometry was observed at

higher q The results of MD simulations vary with the force

fields, and the Cff91 seems to yield the best fit to an

observed profile Because the helix model of Goldsmith et

al [55] fits satisfactorily well to the observed SAXS profile,

maltopentaose seems to assume a quasi-helical

conforma-tion specified by a radius of 5.38 A˚, a rise of 2.44 A˚, a pitch

of 17.60 A˚, a repeat of 7.2 A˚, / = 105j, and w =135j A

typical conformation of maltopentaose is shown in Fig 15

as simulated with various force fields In fact, the

confor-mation observed by the MS simulation with the Cff91 is

similar to the helix model of Goldsmith et al

B (1!4)-B-D-Glucan Represented by CelluloseCellopentaose cannot be completely dissolved in waterbecause of a strong intermolecular interaction by hydrogenbonding through OH groups on C6 The SAXS from theaqueous solution of cellopentaose (30 mg/mL) exhibits asharp upturn toward lower q due to the formation of largeaggregates (Fig 16).If the aggregation is caused by inter-molecular hydrogen bonding, the aggregates are con-sidered to be formed by the side-by-side stacking ofcellopentaose chains The simulated profile (a solid line in

Fig 16) reflects a chain stiffness of a (1!4)-h-D-glucanchain, but the observed profile significantly deviates fromthe simulated profile at a lower q region The cross-sectionalradius of gyration is estimated as 3.5 A˚ at the inter-mediate q range and as over 70 A˚ at the smaller q range Asingle (1!4)-h-D-glucan chain has the cross-sectional ra-dius of gyration of 2.1 A˚, so that two cellopentaose chainsare considered to form a stable aggregate and some furtheraggregate into a larger cluster The intermolecular hydro-gen bonds can be broken by adding urea in the aqueoussolution of cellopentaose Fig 16b observes a good agree-ment between the observed and simulated SAXS profiles,and the cross-sectional radius of gyration is estimated as 2.1A˚, which is expected for a single (1!4)-A˚-D-glucan chain.Regarding the local conformation of (1!4)-h-D-glu-can chain in the presence of water, the potential use ofmultiple-RELAY-COSY is suggested from the analysis

of complex spin networks of 1H NMR spectra of oligosaccharides where the complete assignment of 1HNMR resonance was achieved for cellotriose [56] Solvent-suppression COSY provides also a useful method to eluci-date the interaction of the hydroxyl groups with water [57].The 1H NMR of methyl h-cellobioside in H2O-acetone-d6 (85 :15) yields sharp signals due to the seven hydroxylgroups at20jC (Fig 17),where all signals are identified[57]

cello-Figure 14 Small-angle x-ray scattering profiles observed

from maltopentaose aqueous solution (open circles) of 20.13

mg/mL at 25jC with simulated profiles (respective curves)

(a) MD results (the radius gyration and force field are shown

in the Figure) and (b) Monte Carlo results and profiles

cal-culated from crystalline regular helices (the radius of gyration

and the source of other data are shown in the Figure)

Figure 15 Stereoviews of the snapshot conformations ofmaltopentaose as simulated by the Monte Carlo method and

MD, including regular amylose helices (a) Regular helix (8.3A˚) [47], (b) regular helix (7.4 A˚) [55], (c) regular helix (5.9 A˚)[48], (d) Monte Carlo/MM3 (7.57 A˚), (e) Glycam93 (8.85 A˚),(f ) Glycam99 (8.08 A˚), (g) modified Glycam93 (7.72 A˚), (h)Cff91 (7.83 A˚), (i) Cff (8.30 A˚), ( j ) Gromos (8.32 A˚) Thevalues in each bracket denote the radius of gyration, whichwas evaluated as 7.4 F 0.2 A˚ from the SAXS profile

The crystal structure of cellulose has been a subject of a

long-standing argument Cellulose is known to have four

different polymorphic crystalline forms classified as

cellu-lose I, II, III, and IV Parallel chain packing is proposed for

native cellulose I [58], and regenerated cellulose II is

supposed to assume antiparallel chain packing [59,60] as

analyzed from the results of x-ray fiber diffraction pattern

Because CP/MAS13C NMR revealed cellulose I as being

composed of the allomorphic mixture of triclinic Ia and

monoclinic Ih, the refinement of cellulose crystal structure

again became a main issue in the cellulose science [61] Here

the multiplicity at C4, C1, or C6 is due to magnetically

nonequivalent sites present in crystalline domain, and is

found to vary its pattern, implying that the ratio of twoallomorphs Ia/Ih differs by the origin of native cellulose[62–64] It is interesting to note that a single microfibril ofnative cellulose is a composite of two crystalline phases, Iaand Ih[65,66]

The crystal structure of cellulose II is considered toconsist of two antiparallel chains of almost identical con-formation packed in a monoclinic unit cell, where thehydroxymethyl group at C6 assumes a tg or a gt confor-mation in the ‘‘up’’ or ‘‘down’’ chain, respectively How-ever, CP/MAS13C NMR exhibits a singlet at 64 ppm forthe C6 resonance from cellulose II polymorph against theexpected doublet to be observed at 64 and 66 ppm from the

Figure 16 Small-angle x-ray scattering profile from cellopentaose in water (a) and in 1 M urea aqueous solution (observed andsimulated as indicated in the Figures) A stereoview of a simulated cellopentaose chain is shown on the right

tg and gt conformations [67] The cellulose II crystalstructure is re-examined [68] from the crystal structure ofcellodextrin oligomers, including h-D-cellotetraose (Fig.18) [69,70] and methyl h-cellotrio side [71] In those cello-dextrin oligomers, all the hydroxymethyl groups (C6–O6bonds) are in the gt position, but the two antiparallel chainsassume a different glucose ring conformation This findingaccounts, at least qualitatively, a singlet for C6 and adoublet for C1 and C4 observed for cellulose II by CP/MAS13C NMR.

C (1!3)-B-D-Glucan(1!3)-h-D-glucan consists of a backbone of a group

of extracellular plant/fungal glucans such as cinerean,curdlan, krestin, laminaran, lentinan, schizophyllan, andscleroglucan, which are known to affect the immune sys-tem as an unspecific modulator [72] Except for curdlan,which is linear (1!3)-h-D-glucan, the (1!3)-h-D-glucanfamily contains some amount of h(1!6) branched D-glucose residues, and assumes a triple-helical conforma-tion Although the structural requirement is not expli-citly understood, the antitumor activity is said to be more

pronounced in lower h(1!6) branched (1!3)-h-D-glucans

with a relatively high molecular mass [73] Those

(1!3)-h-D-glucans form triple-stranded helices of high rigidity

in aqueous solution [74,75], and the TEM image revealed

the macrocyclic species made of multiple triple-stranded

(1!3)-h-D-glucan chains in some cases after a cycle of

de-naturation-renaturation process [76]

Laminaran is produced by Laminaria seaweeds, and

contains a small amount of h(1!6)-branched D-glucose

residues and alkyl groups at reductive ends The

confor-mation of laminara oligosaccharides was characterized in

aqueous solution by the combined method of small-angle

x-ray scattering and Monte Carlo simulation [77] The

conformational energy map of laminarabiose (Fig 7e)

shows four local minima including two global minima

around (/, w) = (0j, 50j) and (/, w) = (30j, 0j) The

crystallographic data of laminarabiose and

laminarabio-side derivatives (except for methyl b-D-laminarabioside

and h-D-laminarabiose) confirm that two dihedral angles

/ and w with respect to the glycosidic bond fall in one of the

global minima in the conformational energy map of

lam-inarabiose (seeTable2) w is twisted by the formation of

intramolecular hydrogen bond between O4V and O5, which

is prevented by introducing acetate substituents The

glob-al minima indicate the helicglob-al conformation of laminaran,

which will be interrupted by the other local minima at (20j,

170j) and (160j, 10j)

Over 500 chains were generated to constitute a

statis-tical ensemble of laminara-oligomers according to the

scheme shown in Fig 8, and the average scattering factor

over the ensemble was calculated to compare with theobserved SAXS profiles The simulated scattering profiles(in terms of the Kratky plots) exhibit characteristic maxima

of helical conformation with increasing degree of ization (Fig 19).Fig 20shows the observed and calculatedSAXS profiles of laminarahexaose together with a snap-shot of a simulated chain Although laminarahexaose is notlong enough to show the characteristics of helical confor-mation, the observed SAXS profile is in good agreementwith the simulated scattering profile The observed profilehas a smooth shoulder at q = 0.2–0.25 A˚1, whereas asimulated profile shows a slight peak at q = 0.2 A˚1due to

polymer-a qupolymer-asi-helicpolymer-al structure The devipolymer-ation of the observedprofile from the simulated one is probably due to hydra-tion, which is not properly taken into account in thesimulation The radius of gyration RG and the cross-sectional radius of gyration RG,c are consistent with therespective values evaluated from observed and simulatedprofiles—7.8 A˚ (RG) and 3.0 A˚ (RG,c) from the observedprofile for laminarahexaose, and 7.7 A˚ (RG) and 3.4 A˚(RG,c) from the simulation

A triple-helical structure has been proposed for

(1!3)-h-D-glucan [78,79] For example, the molecular and crystalstructure of the anhydrous curdlan and its hydrated formwas determined by combined x-ray diffraction from ori-ented curdlan fibers and stereochemical model refinement[80] Here both hydrous and anhydrous forms assume aright-handed triplex (sixfold triple-helical conformation)crystallized in a hexagonal unit cell with the interstrandO2: : :O2 hydrogen bonds Curdlan is believed to assume a

Figure 19 Scattering profiles calculated for laminara-oligosaccharides as a function of the degree of polymerization

single- or triple-helical sevenfold conformation by swelling,

where a chain is expanded along the chain direction to

increase the helix repeat distance to 22.7 A˚ from 17.6 A˚ (in

anhydrous form) or 18.8 A˚ (in hydrous form) Regular or

irregular short-branch substitutions on the main chain O6

hydroxyls seem not to affect the triplex structure as

exem-plified by scleroglucan [81], schizophyllan [75], and

len-tinan [82], which retain a triplex structure even in aqueous

solution It is interesting to note that the dihedral angles /

and w of curdlan polymorphs are similar to those of the

acetylated derivatives of laminarabiose or laminarabioside

(Table 2).Similar / and w values are also evaluated from

the molecular structure of the tetrasacharride (1!6)

branched (1!3)-h-D-glucan [83]

Gel is formed in the aqueous solution of (1!3)-h-Dglucans, but its mechanism seems to differ from linear andbranched species Curdlan low-set gel is prepared byheating a slurry (>0.5% w/v) to above 60jC, and will behigh-set with annealing at 95jC [84] Gelation is suggested

-to proceed with breaking hydrogen bonds -to solubilizecurdlan and reforming intermolecular hydrogen bondssubsequently to consist the junction zones Hydrophobicinteraction promotes the intermolecular association ofcurdlan at elevated temperatures to form stronger high-set gel Thus curdlan gel is supposed to contain bothliquid-like (composed of flexible chains) and solid-like(composed of associated chains) domains.13C NMR wasapplied to curdlan gel where various methods (including

Figure 20 Small-angle x-ray scattering profile from laminarahexaose in water (observed and simulated as indicated in theFigure) A stereoview of a simulated laminarahexaose chain is shown on the right

CP/MAS, broadband coupling, and MAS) were employed

to obtain the signals from the domains of different

molec-ular motions [85] Fig 21 shows the13C NMR spectra of

curdlan hydrate and gel recorded by various methods

Here a conventional high-resolution NMR coupled with

broadband decoupling confirms that the liquid-like

do-main is composed of single-helical chains that are flexible

and undergo free molecular motion The intermediate

domain is also composed of single chains as indicated by

high-power dipolar decoupling with magic angle spinning

(MAS) The CP/MAS spectrum reveals a small amount of

triple helices visible in the solid-like domain as shown by

an arrow (a C5 signal from the triple helix) in Fig 21, but

otherwise gives the characteristics of the swollen sevenfold

helical form of solid curdlan with C3 at 87 ppm and no

peak at 79 ppm Annealing at elevated temperatures results

in the increase of the fraction of anhydrous (in a later stage

hydrous) sixfold helical domains and the decrease of the

swollen form portion [86] The NMR observation indicates

that curdlan undergoes gelation by forming

quasi-cross-linking domain composed of single helical chains

associ-ated hydrophobically after swelling at lower temperatures,

and subsequently, by increasing the triple-helical fraction

at elevated temperatures The triple-helical conformation

of the anhydrous form appears at the early stage ofannealing, and eventually the transformation takes placefrom the anhydrous to the hydrous form

The13C NMR of branched (1!3)-h-D-glucan gel such

as lentinan and schizophyllan shows the characteristicpeaks of the triple helix [87], but the peaks corresponding

to the liquid-like domain disappear by gelation [82] Thusthe gelation of h(1!6) branched (1!3)-h-D-glucans ismainly due to partial association of triple-helical chains.The gelation of schizophyllan is promoted by thepresence of sorbitol [88] where thermoreversible opticallytransparent gel is formed by lowering the temperature.However, the small-angle x-ray scattering (SAXS) profilefrom the schizophyllan/sorbitol system shows less-markedchange by the sol–gel transition The 1.5% aqueous solu-tion of schizophyllan containing 4 M sorbitol is sol at 60jCbut forms transparent gel at 5jC The SAXS profiles fromthe solution at respective temperature were analyzed interms of a modified broken rod model [89], which reads

q2IðqÞcX

ipqwiMLi4J

2ðqRciÞðqRciÞ2 þ const: ð24Þ

Figure 21 13

C NMR spectra of curdlan hydrate (A) and gel (B–D), observed by CP/MAS (A, D), by broadband decoupling (B),and by MAS (C)

where wi, MLi, and Rci denote the weight fraction, the

linear mass density, and the cross-sectional radius of the

rod-like component i, respectively J1(x) is the first-order

Bessel function, and the constant term accounts that the

rod-like components are connected by a free joint The

model takes into account the heterogeneity with respect to

the cross-section The results are shown in Fig 22 in two

types of Guinier plots Schizophyllan assumes a

triple-helical conformation in water, and undergoes no

confor-mational change by decreasing the temperature from 60jC

to 5jC as shown in Fig 22a,b, where the scattering profile

was calculated from the molecular model of schizophyllan

triple helix The cross-sectional radius of gyration of

schizophyllan is estimated as 6.4 A˚ When sorbitol is

added, the cross-sectional Guinier plots yield a smaller

apparent cross-sectional radius (5.1 A˚), which becomes

even smaller (4.6 A˚) by gel formation at a lower

temper-ature (5jC) Here the SAXS scattering profile at 60jC was

fitted with the molecular model of (1!3)-h-D-glucan triple

helices with no side chain (i.e., curdlan-type triple helix)

The SAXS profile at 5jC can be fitted by a modified

broken rod model [Eq (24)] where each component isreplaced with a triple helix and a single coil of theschizophyllan molecular model The atomic radius isreduced to half of the van der Waals radius to accountfor the smaller cross-sectional radius The inclusion of aconstant term is necessary, so that schizophyllan triplehelices are speculated to disentangle into single chains thatact as a free joint Sorbitol breaks intramolecular hydro-gen bonds of schizophyllan triple helices, and solvates thebroken parts to form a cross-linking junction by intermo-lecular hydrogen bonding through sorbitol An apparentsmaller atomic radius observed at 5jC is probably due tosolvated sorbitol reducing the contrast between solventand solute

D Cyclic and Linear (1!2)-B-D-GlucanGram-negative bacteria such as Agrobacterium and Rhizo-bium [90,91] are known to produce a cyclic (1!2)-h-D-glucan referred to as cyclosophoran The DP value (the

Figure 22 Small-angle x-ray scattering profiles from the 1.5% aqueous solution of schizophyllan and schizophyllan/4 Msorbitol at 60jC (a) and 5jC (b) The solid lines represent the scattering profiles calculated according to the molecular model (c)and Eq (23)

degree of polymerization) of cyclosophoran varies from 17

to 24 depending on the bacterial strain; the largest DPreported is 40 Cyclosophoran is thought to act as aregulator of the osmotic balance between the cytoplasmand the periplasmic space for bacteria to adapt the change

in environmental osmolality [92] or to mediate the rium–plant host [93] interactions during the infection of thehost Although the exact physiological role of cyclic (1!2)-

bacte-h-D-glucan is a matter of speculation, its physiologicalfunction is assumed to be closely related to its conforma-tion [94] The conformation of (1!2)-h-D-glucan has beenextensively investigated by computer modeling and13C/1HNMR [95], but the homopolymeric nature and conforma-tional identity of the glucose residues obscure the structuredetermination by NMR

The conformation of cyclic and linear (1!2)-h-Dglucans was investigated by the combination of the MonteCarlo simulation and SAXS [96] Cyclosophoran mixturesproduced by Agrobacterium radiobactor and Rhizobiumfphaseoli were fractionated into nine fractions from DP

-= 17 to 25 (each designated as CS17 to CS25) by performance liquid crystallography (HPLC) Linear(1!2)-h-D-glucans (designated as LS 19 and LS2 1 accord-ing to DP) was prepared by acid hydrolysis of CS21 andsubsequent fractionation by HPLC

high-Small-angle x-ray scattering (SAXS) was observedfrom the aqueous solutions of cyclic glucans (CS17 toCS24) and linear glucans (LS19 and LS21) at 25jC wherethe concentration was varied from 10 to 40 mg/mL (for thecyclic glucans) or from 12.5 to 25 mg/mL (for the linearglucans) The observed range of the magnitude of thescattering vector was from q = 2.50  102A˚1to q =0.375 A˚1, which is equivalent to the Bragg spacing from

251 to 16.8 A˚ The observed SAXS profiles reveal thestructural difference of cyclic and linear (1!2)-h-D-glucanchains in aqueous solution in the region of q > 0.15 A˚1(Fig 23).The radius of gyration, RG, was evaluated fromthe initial slope of the Guinier plots as summarized inTable

5.The cross-sectional radius of gyration Rcwas evaluatedfrom the Guinier plots for cross section [Eq (6)] in the case

of linear (1!2)-h-D-glucan chains or the thickness T fromthe Guinier plots for thickness [Eq (7)] in the case of cyclic(1!2)-h-D-glucan chains

The results indicate that a cyclic (1!2)-h-D-glucanchain assumes the shape of a flat disk and a linear homologthe shape of a cylinder The compact conformation of a cy-clic (1!2)-h-D-glucan chain is confirmed from the smallerradius of gyration in comparison with a correspondinglinear (1!2)-h-D-glucan chain

(1!2)-h-D-glucan chains were generated by the

Mon-te Carlo method, consisMon-tent with the disaccharide mational energy map(Fig 7f) The region of the energywell is specified as the conformation+A for w >20j, or

confor-A for w < 20j according to York [95] The glucoseresidue is assumed to be rigid, and the conformationalenergy map for h-sophorobiose is calculated by the mo-lecular mechanics as a function of the dihedral angles /and w defined as / = h[H1, C1, O, C2V] and w = h[C1, O,C2V, H2V] Nonbonded van der Waals interactions and

Figure 22 Continued

electrostatic interactions due to partial charges are taken

into account in the calculation The occurrence probability

is given by the Boltzmann factor for a pair of (/, w)

normalized with respect to the sum of the Boltzmann

factors for all pairs of (/, w), whereas the bond angle s

at the glycosidic oxygen is fixed at 113.6j Among the

chains generated by the Monte Carlo method, those with

the end-to-end distance less than 1.5 A˚ are collected to

compose an ensemble of cyclic (1!2)-h-D-glucan chains

An ensemble of linear (1!2)-h-D-glucan is composed of

500 chains

The scattering factors are calculated according to Eq.(12) from the atomic coordinates of generated chains in anensemble, with the radii of carbon and oxygen atoms beingtaken to be 1.67 and 1.50 A˚, respectively Here all the O6atoms of the glucose unit are affixed to the pyranose ring at

a gauche–trans (gt) position with respect to the torsionangle h[O5, C5, C6, O6] and the torsion angle h[C4, C5, C6,O6], respectively

Fig 24shows a reasonable agreement of the simulatedscattering profile for cyclic (1!2)-h-D-glucans with thatobserved by SAXS, where the scattering profiles calculated

Figure 23 Small-angle x-ray scattering profiles of cyclic and linear (1!2)-h-D-glucan chains in (a) Guinier plots [ln P( q) plottedagainst q2] and (b) Kratky plots [ q2P( q) plotted against q]

from two elementary models (a rigid ring [97] and a flexible

Gaussian ring [98]) are shown for comparison The particle

scattering factors of two elementary models are

where u and D(x) denote the reduced scattering vector and

the Dawson integral defined as

DðxÞ ¼

ðx 0

The observed SAXS profiles from cyclic (1!2)-h-D

-glucans exhibit a certain chain stiffness in comparison with

the profiles calculated from the elementary models, where

the conformational freedom is suppressed by linking

end-to-end The Monte Carlo simulated scattering profiles

reproduce the observed SAXS profiles reasonably well

except for the deviation at the higher q region The

devia-tion at the higher q region may indicate that the effect of

hydration should be taken into account, as no interference

term due to the solvent–solute interaction is incorporated

in the scattering profile calculation However, the

intro-duction of an apparent scattering unit smaller than 1.67 or

1.50 A˚ (for a carbon atom or an oxygen atom, respectively)

reduces the deviation from the observed profile at u (u

qRG) > 3 without any physical significance

A typical conformation is shown inFig 25with space

filling models, which reveal an irregular doughnut-like ring

with the thickness of 10 A˚ The diameter of the CS21 ring

annulus is about 4–5 A˚; that is, the cavity in a cyclic

(1!2)-h-D-glucan chain seems too small to embrace a relatively

large molecule for the formation of an inclusion complex.All the glucosidic linkage torsion angles are found withinthe region A of the conformational energy map (Fig 7f )with 13 linkages in the region +A and 7 linkages in theregionA where no special mode is observed for arranging+A andA

The Monte Carlo simulation for linear (1!2)-h-Dglucans yields less satisfactory results with respect to thescattering profile (Fig 24) Although the Monte Carlosimulation yields a consistent value of the radius of gyra-tion with an observed one, the deviation in the scatteringprofile becomes apparent at u (u qRG) > 1.3 A goodlinearity observed in the cross-sectional Guinier plots [Eq.(6)] indicates a cylindrical shape of a linear (1!2)-h-D-glucan chain as shown inFig 26with space filling models.The cross-sectional diameter is evaluated as 11.8 A˚ (LS 19)

-Table 5 The Radius of Gyration RG, the Cross-Sectional

Radius of Gyration Rc, and the Thickness T of Cyclic and

Linear (1!2)-h-D-Glucan Chains Evaluated from the

Corresponding Guinier Plots of SAXS Data