Polysaccharides

9.08

Polysaccharides

E-H Song, J Shang, and DM Ratner, University of Washington, Seattle, WA, USA © 2012 Elsevier B.V. All rights reserved.

9.08.1 9.08.2 9.08.2.1 9.08.2.1.1 9.08.2.1.2 9.08.2.1.3 9.08.2.1.4 9.08.3 9.08.3.1 9.08.3.1.1 9.08.3.1.2 9.08.3.1.3 9.08.3.1.4 9.08.3.2 9.08.3.2.1 9.08.3.2.2 9.08.3.3 9.08.3.3.1 9.08.3.3.2 9.08.3.4 9.08.3.4.1 9.08.3.4.2 9.08.3.4.3 9.08.4 References

Introduction The Chemistry of Carbohydrates Accessing Carbohydrates and Their Polymers through Isolation and Synthesis Isolation and characterization of carbohydrate and polysaccharides from natural sources Enzymatic synthesis Chemical synthesis Chemoenzymatic synthesis Glycopolymers Glucans Starch Cellulose Dextran β-(1,3)-Glucan N-Acetylglucosamine Polymers – Chitin/Chitosan Chemical structure and properties Applications Glycosaminoglycans Chemical structure and properties Applications Other Polysaccharides Agar/agarose Alginate Bacterial polysaccharides Conclusions

9.08.1 Introduction Carbohydrates and polysaccharides are the most abundant biopolymers on earth and are ubiquitous in living systems. Glycans, in the form of glycopolymers, glycoproteins, glycoli­ pids, glycosaminoglycans (GAGs), proteoglycans, and other glycoconjugates, are known to play numerous biological roles related to energy storage and metabolism, protein and lipid modification, cellular structure, signal transduction, as well as mediators of cell–cell interactions and host–pathogen interac­ tions.1 Due to the biological significance of this class of biomolecules, there is growing interest in securing access to these unique biopolymers for a broad range of biomedical and materials science applications. Polysaccharides, derived from a variety of common saccharide monomers (Table 1), can be readily obtained from animal, plant, insect, microbial, and algal sources, representing the most common sources of commercial material. However, access to well-defined and pure carbohydrate polymers can be limited by the inherent struc­ tural complexity and heterogeneity of the natural polymers. Over the past few decades considerable time and effort has been expended to engineer tools for obtaining pure polymeric carbohydrate moieties for investigation, requiring the com­ bined efforts of organic, biological, and polymer chemists. Currently, a variety of natural and synthetic glycans are becom­ ing available in sufficient quantity and purity to find use in a broad range of studies into the glycobiology associated with infectious diseases and cancer, developing vaccines and Polymer Science: A Comprehensive Reference, Volume 9

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targeting drug delivery, pioneering renewable energy sources and biofuels, as well as environmentally friendly and biode­ gradable polymers for commercial applications. This chapter will provide not only a brief overview of the physical properties of the polysaccharides, but also show the recent growth of their applications in biomedicine and industrial manufacturing.

9.08.2 The Chemistry of Carbohydrates An appreciation of polysaccharide chemistry requires a basic level of understanding of carbohydrate chemistry, including the chemistry of the carbohydrate monomer (the monosac­ charide) and the glycosidic linkage between individual monomers. Like their amino acid and nucleic acid counter­ parts, structural and functional diversity of polysaccharide is obtained by stringing together a limited number of monomeric precursors to form a plethora of high molecular weight oligo­ mers and polymers. These carbohydrate polymers are classified as monosaccharides, oligosaccharides, or polysaccharides, based on the number of repeating monomeric units coupled together via a series of glycosidic linkages (Figure 1). The basic monomeric unit of the polysaccharide polymer, the monosaccharide, exists in solution in equilibrium between the open chain and cyclized forms. As the open chain form (Figure 2), monosaccharides are represented by two main classes, the aldoses and ketoses. As the names imply, the aldoses contain a terminal aldehyde functionality while the

doi:10.1016/B978-0-444-53349-4.00246-6

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Polysaccharides

Table 1

Representative examples of the most common natural monosaccharides

Name

Abbreviation

Glucose

Glc

Mannose

Man

Galactose

Gal

N-Acetylglucosamine

GlcNAc

N-Acetylgalactosamine

GalNAc

Fucose

Fuc

Xylose

Xyl

Sialic acid

SA

Glucuronic acid

GlcA

Iduronic acid

IdoA

ketoses display an internal carbonyl group on a nonterminal carbon. Dissolved in an aqueous environment, monosaccharides exist primarily in the cyclic or hemiacetal form, which is more stable in solution than the open chain form. If an aldohexose is the precursor of cyclic hemiacetal, it forms a six-membered pyranoside (Figure 3); if a ketohexose or aldopentose is the precursor of the cyclic hemiacetal, a five-membered furanoside is formed.

Structure

Symbol

In addition to forming the basic sugar ring, cyclization of the hemiacetal also establishes the anomeric configurations of the carbohydrate. Anomericity is represented by either the α-anomer (axial OH-group at C-1) or the β-anomer (equatorial OH-group at C-1). In solution, monosaccharides exist in equi­ librium between the open chain form, α-/β-furanose and α-/β-pyranose. The anomeric ratio at equilibrium depends lar­ gely on the monosaccharide. For example, α-/β-ratio of glucopyranoside at room temperature is � 1/2. Because the

Polysaccharides

Monosaccharide

n =1

Disaccharide

Oligosaccharide

n =2

n = 3–20

139

Polysaccharide

n = 20<

Figure 1 Carbohydrates are classified based on the length of repeating monosaccharide units; for the most part, synthetic carbohydrate chemistry is focused on oligosaccharide synthesis.

Glucose (Aldohexose)

Fructose (Ketohexose)

Figure 2 Two classes of open chain monosaccharides, an aldose (aldohexose) and ketose (ketohexose), formed by the oxidation of a polyhydroxy alcohol.

anomers are diastereomers, each anomer possesses unique physical properties, such as specific optical rotation values [α] and distinct chemical shift values for both proton and carbon nuclear magnetic resonance (NMR). Unlike the peptide and phosphodiester bonds of proteins and nucleic acids, the glycosidic bond between monosacchar­ ides can produce two distinct stereochemical outcomes, depending on the configuration at anomeric carbon (C-1). Therefore, two identical monosaccharides coupled by a

glycosidic linkage can result in two distinct diastereomeric products (Figure 4). The anomericity of the glycosidic bond leads to significant differences in both chemical and physical properties. For example, maltose consists of two α-linked glu­ coses, and its polymeric form, starch, is partially soluble in water. In contrast, cellobiose, two β-linked glucoses, forms the basic repeat of cellulose, which is highly insoluble in water. In addition to the significant physical differences between these two products, there are profound biological implications of glycosidic bond anomericity. For instance, the human gut easily digests starch. However, cellulose, due to the absence of specific hydrolytic enzymes in humans, is indigestible. Beyond the stereochemical implications of the glycosidic linkage, there are other differences between the polysaccharides and other biopolymers. In contrast to the linear protein and nucleic acid polymers, carbohydrates are capable of producing both linear and branched polysaccharides due to the multiple hydroxyl groups in monosaccharide unit, which are capable of forming glycosidic bonds (Figure 5). For instance, amylose represents a linear homopolysaccharide composed of repeating glucose units (5000 � 500 000 units). In contrast, the branched glucose homopolysaccharide, glycogen, contains both α(1–4) and α(1–6) glycosidic linkages and up to 100 000 glucose units. Branching, combined with the stereochemistry of the glyco­ sidic bond, facilitates immense structural diversity within the polysaccharides. As such, carbohydrates possess many times the information density, per unit monomer, of the major bio­ polymers. This quality is exploited by biological systems, which often display glycans as antigens or ligands for specific signal­ ing processes. For instance, the blood group (ABO, Figure 5) antigens2 are a simple family of branched oligosaccharides that

α-Anomer [α] = +150°

β-Anomer [β] = +23° Figure 3 Cyclization and formation of the hemiacetal ring form of D-(+)-mannose from the open chain aldohexose.

140

Polysaccharides

α(1–4') Glycosidic bond

β(1–4') Glycosidic bond

Maltose

Cellobiose

[4-O-(α-D-Glucopyranosyl)-β-D-Glucopyranose]

[4-O-(β-D-Glucopyranosyl)-α-D-Glucopyranose]

Figure 4 The stereochemical significance of the glycosidic linkage, as illustrated by the biochemically distinct glucose disaccharides, maltose and cellobiose.

A

B α(1–3)

α(1–2) α(1–6') linkage

α(1–4') linkage

H(O) α(1–3)

α(1–2)

α(1–2)

β(1–3) β(1–3)

Glycogen

Type-l ABO blood group antigens

Figure 5 Illustration of a branched carbohydrate polymer vs. discrete oligosaccharide moieties. Specific glycosidic linkages result in the structural and biological diversity of carbohydrates.

illustrate how glycosidic linkage and branching can lead to highly specific biological function.

9.08.2.1 Accessing Carbohydrates and Their Polymers through Isolation and Synthesis Although carbohydrates such as cellulose, starch, and sucrose have been used since ancient times, the isolation of discrete carbohydrate species from honey and plant sources only became practical in the late eighteenth century and it would be another century before the chemistry of the carbohydrate would be unraveled. In the 1890s, a German chemist, Hermann Emil Fischer (Nobel Prize for chemistry, 1902), determined the structure of glucose and synthesized it and several other monosaccharides from glycerol (Table 2). This established the field of carbohydrate chemistry and opened the possibility of procuring pure quantities of defined glycans and glycopolymers for research and biomedical applications. However, to this day, access to significant amounts of pure oligosaccharide and polysaccharide is often hindered by the complex nature of the carbohydrate structure. The need for biologically active carbohydrates and glycopo­ lymers has never been greater. The burgeoning fields of glycobiology and carbohydrate chemistry have rapidly grown to encompass a variety of exciting biomedical applications,4 including diagnostic tools for infectious disease, cancer, and HIV vaccine development. This demand for material has forced biochemists and synthetic organic chemists to develop the chemical tools – including isolation, enzymatic synthesis, and chemical synthesis – to obtain access to greater varieties and quantities of pure carbohydrate and polysaccharide structures.

9.08.2.1.1 Isolation and characterization of carbohydrate and polysaccharides from natural sources Due to the unique chemistries of carbohydrates, glycans, in the form of glycoconjugates and polysaccharides, from natural sources can be isolated and purified. This represents a key step in procuring material for characterization and use in bio­ medical applications. Besides a limited number of pure glycopolymers (e.g., cellulose, amylose, chitin, and some gly­ cosylaminoglycans), most glycans are found in natural sources as bioconjugates and must be cleaved from a peptide or lipid by enzymatic or chemical means. As such, isolated carbohy­ drates from natural sources are available only in limited quantities; have significant compositional microheterogeneity; often include impurities such as fatty acids, peptides, or pro­ teins; and frequently exist in both neutral and charged forms. Due to these challenges, carbohydrates and polysaccharides frequently require a multistep process including chromato­ graphic separation5 and mass spectrometric analysis6 for both isolation and purification (Figure 6). However, the structural diversity and complexity of oligosaccharides often overwhelm current separation techniques, resulting in considerable hetero­ geneity within isolated samples. To this day, the challenge of procuring significant quantities of homogenous oligo- and polysaccharide material remains one of the major challenges to the field of glycobiology and hinders the use of carbohydrate-based materials in biomedical research.

9.08.2.1.2

Enzymatic synthesis

Enzymatic synthesis of oligosaccharides7 has long been recog­ nized as a desirable and efficient means of accessing complex carbohydrate structures. This is largely due to the fact that

Polysaccharides

Table 2

141

The history of carbohydrate polymers and the development of carbohydrate chemistry3

Year

Discoveries

�34000 BCE �6000–4000 BCE �4000–3000 BCE 1747

Evidence of flax-based cellulose used in primitive textiles Cotton is domesticated for the production of cellulose in Central America and the Indus Valley Starch is used as an adhesive for the production of papyrus in ancient Egypt Glucose is first isolated from raisins by the German pharmacist A. Marggraf, as described in eine Art Zucke, which means ‘a type of sugar’ The French chemist Jean Dumas names glucose derived from the Greek glycos, which means ‘sweet’ The chemical formula of cellulose, isolated from plant cell walls, is determined by Anselme Payen, a French chemist Emil Fischer, a German chemist, characterizes the structure of glucose, mannose, fructose, and arabinose Emil Fischer synthesizes glucose, fructose, and mannose The nature of carbohydrate-based biopolymers is first described by Emil Fischer Emil Fischer receives the nobel prize in chemistry “in recognition of the extraordinary services he has rendered by his work on sugar and purine syntheses” McLean and Howell isolate Heparin for use as an anticoagulant Haworth receives the Nobel prize in chemistry for his work in carbohydrates, including describing the pyranose ring structure of the monosaccharides N-sulfation of heparin identified

1838 1838 1888 1890 1892–1900 1902 1916 1937 1948

Robyt, J.F. Essentials of Carbohydrate Chemistry; Springer, Heidelberg, 1998.

n

Enzymatic or chemical degradation

Purification and analysis

Characterization

HPLC

Extraction

Purification or derivatization

Analysis by HPLC or electrophoresis

Mass

m /z

Characterization by mass (glycomics)

Figure 6 Isolation of glycans from heterogeneous glycoconjugates. HPLC, high-performance liquid chromatography.

enzymatic synthesis does not require multistep chemical synth­ eses, elaborate protecting group manipulation, stereochemical control of glycosidic bond formation, and deprotection strate­ gies. By contrast, enzymatic synthesis can leverage the extraordinary specificity of natural glycosyltransferases8 and gly­ cosidases9 that can manage the formation or the cleavage of glycosidic linkages with remarkable regio- and stereoselectivity. Enzymatic synthesis of polysaccharides can dramatically reduce the synthetic burden of accessing complex glycans. This is accomplished by relying on natural glycosyl donor and acceptor precursors that can be used in place of synthetically derived intermediates (Figure 7). In addition to glycosyltransferases, which form glycosidic linkages, glycosidases can be used to specifically break or form glycosides through reverse hydrolysis or transglycosylation10 (Figure 7(b)). While enzymes remain a promising alternative to isolating saccharides from natural sources, the paucity of readily available glycotransferases and nucleotide phosphate sugar donors has limited utilization of this enzymatic approach to a relatively small number of sacchar­ ide targets. Efforts by the Consortium for Functional Glycomics (CFG) and others continue to advance the use of glycosyltran­ ferases as a key component in expanding our access to enzymatically derived glycan libraries.11 In addition, many

carbohydrate-modifying enzymes have found wide use in a variety of commercial applications, including ethanol produc­ tion12 as well as the food13 and paper industries.14

9.08.2.1.3

Chemical synthesis

Arguably, chemical synthesis is the most important source of pure and structurally defined oligosaccharide for biomedical research.4 However, carbohydrates remain one of the most challenging targets for organic synthesis, due in large part to the difficulty of controlling the stereochemistry of the glycosidic bond and differ­ entiating hydroxyl groups to control branching of the linkages. Synthetic carbohydrate chemistry is rooted in a tradition that dates back over 100 years, to the first glycosylation reac­ tions reported by Koenigs and Knorr.15 Glycosylation is at the heart of carbohydrate synthesis, as it is the process of linking multiple carbohydrates to each other or to other biomolecules. At the heart of the glycosylation reaction is the formation of a glycosidic bond between a glycosyl donor, the electrophilic saccharide containing the anomeric center of the new linkage, and a nucleophilic acceptor to form a cyclic acetal. The path of a typical reaction begins with the activation of the glycosyl donor, resulting in the departure of a leaving group at the anomeric position and generation of an electrophilic cyclic

142

Polysaccharides

(a) Glycosyltransferase Glycosyltransferase Glycosyl product

Acceptor – Protein – Lipids – Mono-, oligosaccharide O

HO

O Nucleoside O P O n O– Glycosyl donors

n=

1

2

CMP-sialic acid

GDP-fucose GDP-mannose

Nucleotide

2 UDP-galactose UDP-N-acetlygalactosamine UDP-N-acetlyglucosamine UDP-glucose UDP-glucuronic acid UDP-xylose

(b) Glycosidase O

HO

O

HO

OR

+

OH

HOR

Glycosyl hydrolase H 2O Enzyme (glycosidase)

Enzyme HO

OR'

Transglycosidase

O

HO

O

O

HO

OR

O

O

OR'

Figure 7 Enzyme-catalyzed glycoside synthesis via (a) a glycosyltransferase and (b) glycosidase (glycosyl hydrolase and transglycosidase). CMP, Cytidine Monopphosphate; GDP, Guanosine Diphosphate; UDP, Uridine Diphosphate.

(a) Generic glycosylation scheme O Cyclic oxocarbenium ion with nucleophilic attack by acceptor Glycosyl donor Activation

O R'O

LG

–LG

R'O β-Anomer

O+ HO

RO

OR''

RO

R''

O

R'O RO

R'O

OR'' α-Anomer (b) Generic glycosylation scheme with neighboring group participation Cyclic oxocarbenium ion with nucleophilic attack by acceptor

Glycosyl donor Activation

O

O

O HO

RO R'O

LG

–LG

O O⊕ X R'

R''

OR''

RO R'O β-Anomer

Figure 8 The chemical glycosylation reaction often proceeds via a cyclic oxocarbenium ion that can produce either the α- or β-anomeric products. Stereochemical control of the glycosylation reaction as illustrated (a) without neighboring group participation (R′ = benzyl, etc.) and (b) with neighboring group participation (R′ = acetyl, benzoyl, etc.). LG, leaving group.

oxocarbenium intermediate (Figure 8(a)). This species is subsequently reacted with a nucleophilic acceptor, usually a hydroxyl (illustrated as R–OH), generating a glycosidic linkage. As previously described, the glycosylation reaction can yield two distinct stereochemical products, the α- and β-anomers.

Controlling the stereochemical outcome of the glycosylation reaction is essential for the biological activity of the resulting glycan. To overcome this challenge, carbohydrate chemists utilize a variety of synthetic methods to influence the anomeric selectivity of the glycosylation reaction.16 A common technique

Polysaccharides

to produce stereoselective glycosylation reactions is to utilize a ‘participating group’ (Figure 8(b)). effectively blocking a face of the sugar from nucleophilic attack by the acceptor during glycosylation. A variety of other factors can also influence the outcome of a glycosylation. These include the choice of solvent, use of protecting groups, type of the anomeric leaving group, reactivity of the activator, matched versus mismatched donor/ acceptor pairs,17 and anomeric and exoanomeric effects. In addition to controlling the anomericity of the glycosyla­ tion reaction, significant attention must be paid to the position of the linkage being formed on the glycosyl acceptor, as the typical hexose monosaccharide possesses five hydroxyl groups with similar reactivities. Therefore, coupling two monosacchar­ ides can form up to 10 positional and anomeric isomers of the disaccharide product. To address this, carbohydrate chemists make extensive use of temporary protecting groups that mask the chemical functionality of individual hydroxyls on the monosaccharide core, while permitting selective deprotection to yield exposed hydroxyls for coupling (Figure 9). The choice of protecting groups can have a profound effect on the reactiv­ ity of both glycosyl donor and acceptor. Poorly selected protecting groups can complicate an otherwise straightforward synthesis. In contrast to DNA and peptide oligomers, which are readily synthesized commercially, the unique structural properties and diversity of glycosidic linkages have dramatically slowed the progress toward the facile synthesis of libraries of discrete oligosaccharides. In particular, the arduous selection of protect­ ing groups and properly paired glycosyl donors/promoters (Table 3) are still essential steps toward the successful construc­ tion of a desired oligosaccharide with high yield and purity.61 Also, due to the difficulty in assuring absolute stereochemical control over the glycosylation process, certain coupling reac­ tions have significant losses associated with the formation of undesired side products or anomeric mixtures, which must be carefully purified prior to advancing with the synthesis. Presently, large-scale commercial production of made-to-order glycans remains a highly specialized industrial niche, and some carbohydrate structures remain out of the reach of organic synthesis.

9.08.2.1.3(i) Automated chemical synthesis of the oligosaccharides Solution-phase synthesis of biomolecules, including the oligo­ saccharides, has been immensely successful at constructing a variety of complex synthetic targets; however, chemical synth­ esis remains time and labor intensive. Purification of products and intermediates, typically by chromatography, is also one of the most challenging steps during the synthesis of large struc­ tures. By eliminating these purification steps, the automated multistep synthesis paradigm has made a significant impact on the rapid synthesis of biopolymers. Ideally suited for automa­ tion, solid-phase synthesis was originally exploited for the automated synthesis of peptides62 and, two decades later, oli­ gonucleotides.63 The great success of commercial peptide and DNA synthesis has been due in large part to these automated techniques, which made DNA and protein oligomers readily available to customers at low cost and with a short turnaround. It goes without saying that the variety of biomedical uses for synthetic peptides and nucleic acids have been greatly facili­ tated by the ease of access to the materials afforded by automated synthesis. Due to the complexity of carbohydrate synthesis, progress toward the automated synthesis of oligosaccharide lags signifi­ cantly behind that of automated DNA and peptide synthesis. However, significant effort is being invested toward addressing the demand for more rapid chemical means of producing glycans. Over the past decade, several potential solutions have been explored, including one-pot synthesis,64 automated solid-phase synthesis,65 and automated fluorous-phase solu­ tion synthesis.66 All of these methods offer significant advantages in time and labor over conventional solution-phase multi-pot synthesis (Figure 10). Programmable reactivity-based one-pot chemical synthesis of oligosaccharides represents a novel solution-phase approach to dramatically reducing the complexity of multistep chemical syntheses (Figure 10).67 The method is based on an extensive database of differentially protected glycosyl donors sorted according to relative reactivity. The difference in relative reac­ tivity between donors enables the assembly of linear and branched oligosaccharides in a one-pot sequential series of glycosylation reactions followed by a single isolation and

OR4 HO R2O

143

HO O

R3O R2O

OX OR1

O OX OR1

A

D OR4 R3O R2O C

O OX OR1 B OR4

OR4 R3O HO

O OX OR1

R3O R2O

O OX OH

Figure 9 Synthetic carbohydrate chemistry requires elaborate use of orthogonal protecting group strategies to selectively protect and reveal specific hydroxyl groups for subsequent modification or glycosylation. R1–4, protecting groups; A–D, deprotection conditions.

144

Polysaccharides

Table 3

Representative glycosyl donors and promoters used for chemical glycosylation reactions61

Type of glycosyl donors

Promoters

References

Glycosyl bromide

I2/K2CO3 InCl3 Tri(1-pyrrolidino)phosphine oxide Trimethylsilyl iodide (TMSI)/Ph3P=O Activated carbon fiber (ACF) LiClO4 TfOH SnCl2 or SnCl4/AgB(C6F5)4 HB(C6F5)4 Sulfated ZrO2 Potassium examethyldisilazane (KHMDS), (18)-crown-6 Tetrabutylammonium iodide (TBAI), Diisopropylethylamine (DIPEA) I2 N-iodosuccinimide (NIS)/Sn(OTf)2 or Cu(OTf)2 N-iodosuccinimide (NIS)/HClO4-silica N-iodosuccinimide (NIS)/TrB(C6F5)4 Ipy2BF4/HOTf Interhalogen compounds (ICl or IBr) (IX)/AgOTf N-bromosuccinimide (NBS)/Bi(OTf)3 N-iodosuccinimide (NIS)/TMSOTf AgOTf MeOTf Cu(OTf)2 N-iodosuccinimide (NIS)/Yb(OTf)2 N-iodosuccinimide (NIS)/BF3.Et2O TMSOTf TBSOTf Yb(OTF)3 Yb(OTF)3 Tf2O/2,6-di-tert-butyl-4-methylpyridine (DTBMP) TMSOTf tBuOK

18 19 20 21 22 23, 24 25 26, 27 28 29 30 31, 32 33, 34 35 36, 37 38 39 40–42 43 44 45 46 47 48, 49 50 51 52 53 54 55–57 58, 59 60

Glycosyl acetate Glycosyl fluoride

Glycosyl iodide

Thioglycoside

Glycosyl thioimidate

n-Pentenylorthoester Trichloroacetimidate

Trifluoroacetimidate Carboxybenzyl glycoside Glycosyl phosphate Nitroglycal

Zhu, X.; Schmidt, R. Angew. Chem. Int. Ed. 2009, 48, 1900.

TMSI, Trimethylsilyl iodide; KHMDS, Potassium examethyldisilazane; TBAI, Tetrabutylammonium iodide; DIPEA, Diisopropylethylamine;

NIS, N-iodosuccinimide; IX, Interhalogen compounds (ICl or IBr); NBS, N-bromosuccinimide; DTBMP, 2,6-di-tert-butyl-4-methylpyridine.

purification of the final product. This represents a considerable saving in terms of repeated chromatographic isolation of inter­ mediate products, as would be required by a traditional multi-pot synthesis. This one-pot approach is amenable to the synthesis of oligosaccharides of moderate complexity, as recently demonstrated by the synthesis of a linker­ functionalized hexasaccharide (fucosyl GM1) with 44% yield.68 However, it should be noted that one-pot carbohydrate synthesis, as with most synthetic carbohydrate chemistry, requires advanced synthetic products, in the form of differen­ tially protected glycosyl donors. Often, protecting group strategies and donor synthesis represent the bulk of the syn­ thetic steps in the synthesis of a given oligosaccharide.16 Drawing from the playbook of automated solid-phase peptide and DNA synthesis, the synthetic carbohydrate chemistry com­ munity has also turned to the solid phase to reduce the burden of preparing complex oligosaccharides. Analogous to the one-pot approach, solid-phase synthesis permits the synthesis of oligosac­ charides without the need for purifying intermediate structures (Figure 10). Additionally, solid-phase carbohydrate synthesis can leverage existing automated peptide synthesis platforms based on polystyrene resins (e.g., Merrifield resin) using chemically cleava­ ble linkers.69 These modified peptide synthesizers can be readily

adapted for running glycosylation reactions, typically by cooling the reaction vessel, and this approach has been used successfully to prepare a variety of complex glycans, including tumor antigens and the blood group determinants.70 The solid-phase oligosaccharide synthesizer currently remains the only method being tested explicitly for the purpose of introducing automated commercial glycan synthesis. It has been reported that this platform is designed to theoretically be capable of producing 90% of the known mammalian carbohy­ drates using only 65 glycosyl donor building blocks.71 However, these claims still need to be tested. In addition, solid-phase carbohydrate synthesis requires significant quantities of syn­ thetic glycosyl donors to drive the glycosylation reaction to completion on the solid phase. While the strategy of using excess reagent to increase yield on the solid phase has proven cost-effective for peptide and DNA synthesis, the value of differ­ entially protected glycosyl donors represents a significant challenge to adopting this approach for carbohydrate synthesis. While efforts are ongoing to solve the challenge of auto­ mated solid-phase synthesis, parallel studies are underway to develop new techniques to speed solution-phase carbohydrate synthesis, particularly for situations when the aforementioned one-pot approach is not applicable. One exciting technique,

Polysaccharides

145

(a)

(b)

(c)

Solid support or fluorous tag

Figure 10 Examples of chemical synthesis of oligosaccharides: (a) conventional multi-pot synthesis, (b) programmable reactivity-based one-pot synthesis, and (c) automated synthesis based on either solid-phase resins or fluorous tags.

known as fluorous synthesis,72 promises to integrate automa­ tion with solution-phase carbohydrate synthesis.73 The fluorous approach was originally developed for the facile separation of fluorous-tagged molecules (C8F17) from their nonfluorous counterparts.74 Used in place of a solid-phase support resin, fluorous synthesis enables the production of linear and branched oligosaccharides in solution using the differential solubility of short fluorous tags.75 This method reduces the extensive labor for the purification of the desired product, and fluorous-tagged carbohydrates can be directly applied onto fluorous-based microarrays for protein, antibody, and enzyme screening.76 The field of synthetic carbohydrate chemistry is in a contin­ ued state of reinvention, as high demand from the biomedical community for pure synthetic glycans drives the development of new technologies to speed up the synthesis of complex oligosaccharide products. One-pot synthesis, along with auto­ mated solid-phase and fluorous synthesis are only three examples of the many techniques currently in development with the goal of lowering the barriers of access to pure carbo­ hydrate for biological studies.

9.08.2.1.4

Chemoenzymatic synthesis

Advances in both the enzymatic and chemical syntheses of carbohydrates are being leveraged through a synergic chemoen­ zymatic approach that draws upon the strengths of both techniques to produce complex glycans and glycoconju­ gates.77,78 The chemoenzymatic approach can follow one of two routes: (1) the chemical modification of carbohydrate intermediates after enzymatic synthesis or (2) enzymatic mod­ ification of carbohydrate intermediates after chemical synthesis (illustrated in Figure 11). This hybrid methodology represents a versatile synthetic tool for producing diverse glycans

including glycopeptides, glycoproteins, glycolipids, and other complex oligosaccharides, including nonnatural linkages or chemically modified glycans. This chemoenzymatic approach is widely used for the ela­ boration of monosaccharides and anomeric linkages that are especially challenging to synthesize via chemical means. One such category of glycans notoriously difficult to produce synthetically is the sialosides. Sialic acid contains not only an anomeric carbon hindered by an electron-withdrawing carbo­ nyl group, but also a deoxy carbon at C-2. As illustrated in Figure 12, synthetic precursors can be transformed to activated sialic acid derivatives by sialic aldolase and used as an enzy­ matic substrate to glycosylate, a glycosyl acceptor, via a sialic acid transferase-catalyzed reaction.79 Notably, iterative process using multiple enzymes in one-pot enables the generation of diverse glycan libraries for screening glycan-binding proteins (GBRs).80

9.08.3 Glycopolymers The glycopolymers consist of a large family of polysaccharides derived from animals, plants, and microorganisms (Table 4). Due to the diversity of their chemical structures as well as physical and biological properties, glycopolymers have found a variety of applications in biomedicine, pharmaceutical research, and other industrial domains.

9.08.3.1

Glucans

As mentioned previously, the polysaccharides are defined as polymers containing between 30 and 103–4 monosaccharide units. Each polysaccharide can be classified as either homopo­ lysaccharide, composed of repeating units of the same

146

Polysaccharides

Chemoenzymatic synthesis

Chemical synthesis

Enzymatic synthesis

Glycotransferase

1. Chemical modification 2. Deprotection

Figure 11 Chemoenzymatic synthesis leverages a hybrid chemical/enzymatic scheme for elaboration of the glycan product. HO OH

1. Chemical modification

O HO

COO−

OH

2. Sialic aldolase

O AcHN HO

HO OH

COO−

OLG Glycotransferase

O AcHN

OH

HO

Sialic acid

O HO

O O

OR

OH

OR

Figure 12 Chemoenzymatic synthesis of sialic acid derivatives. LG, leaving group. Table 4 Representative examples of the most common polysaccharides found in nature Source

Polysaccharides

Animal Algae Plant Microbial

Chitin/chitosan, GAGs, hyaluronan, glycogen Insulin, laminaran, carrageenan, agar, alginate, agarose Starch, cellulose, pectin, galactomannans, glucomannans Dextran, cyclodextrin, xanthan, gellan (gellan family), curdlan

monosaccharide, or heteropolysaccharide, consisting of more than one monosaccharide building block. As the name sug­ gests, the glucans are a class of polysaccharides composed of glucose. By varying the branching and glycosidic linkages, glu­ cose can be built into a variety of glucans, each with distinct properties and potential applications (Figure 13).

9.08.3.1.1

Starch

9.08.3.1.1(i) Chemical structure and properties Starch is produced by all green plants and contains two different components: 18–28% amylose and 72–82% amylopectin. Amylose is a linear molecule composed of α(1,4)-D­ glucopyranosyl units, slightly branched by α(1,6)-linkages. Amylopectin is a highly branched component in starch. It is formed mainly by α(1,4)-D-glucopyranosyl units but branched through α(1,6) bonds (Figure 14). On average, amylopectin has 12–20 glucose units between branches. Depending on the bota­ nical origin, the molecular weights of amylose and amylopectin are 105–106 and 107–109, respectively.81 Starch is biosynthesized as semicrystalline granules with varying polymorphic types and degrees of crystallinity. The diameters of the microscopic granules in starch range from 2 to 100 μm, and the crystallinity is 15–45%.82 Due to this semi­ crystalline structure, starch is insoluble in cold water. However, when water is heated, starch becomes soluble. The granules in starch swell and burst, the semicrystalline structure is lost, and the smaller amylose molecules start leaching out of the granule, forming a network that holds water and increasing the mix­ ture’s viscosity. This process is called starch gelatinization.

9.08.3.1.1(ii) Applications Due to the gelling ability and biodegradability of starch, it has been widely used in a variety of applications. For example, in the food industry, starch is a major gelling agent used to make jells and jellies. As a biodegradable material, starch is manufactured into ‘green’ plastics, bags, and packaging materials.83 To over­ come the water sensitivity and brittleness of starch materials, nanosized fillers have been integrated with starch to improve the overall mechanical properties of these plastic materials.84 Within the pharmaceutical industry, starch has been used as a bulking agent, binder, disintegrant, and thickening agent.85 Drugs coated with starch capsules can withstand the harsh conditions in the upper gastrointestinal tract and efficiently be delivered to the colon for site-specific treatment.86,87 In the field of tissue engi­ neering, starch has been fabricated as cell-supporting scaffolds to improve cell growth and bone and cartilage regeneration.88,89 In the development of sustainable energy sources, starch from crops has become the main carbon source of the first-generation biofuels. Bioethanol can be generated from starch by enzyme hydrolysis and fermentation. While the jury is still out regarding the long-term environmental benefits of starch-derived biofuel, compared to conventional fossil fuels, greenhouse gas emissions are reduced by 12%, even including the production and combustion of bioethanol.90 In addition, bioethanol readily adapts to the existing internal combustion engine and can be easily blended with petroleum-based fuels. Annual production is estimated to be around 50 billion liters.91 However, there remain significant concerns about the impact of starch-based crop biofuel production on the food supply.

Polysaccharides

Cellulose

β(1–4) O

O

147

O n

O

Amylose

α(1–4) O O n

O

Starch

O O α(1–6) Amylopectin

O O

Glucans

α(1–4) O

Dextran

O O

O

O

α(1–3)

n

O α(1–6)

O n

O β(1–6)

β-Glucan

O

β(1–3) O

O n

Figure 13 Examples of glucose-based polysaccharides (glucans).

n

n Amylose

Amylopectin

Figure 14 Chemical structure of starch, specifically, amylose and amylopectin.

9.08.3.1.2

Cellulose

9.08.3.1.2(i) Chemical structure and properties Cellulose, one of the most abundant natural polysaccharides, is a linear polymer consisting of β(1–4)-D-glucose unit (Figure 15). It is produced by plants, algae, tunicates, and

some bacteria, primarily as a structural polymer. The number of glucose units in cellulose ranges from 500 to 5000, depending on the source of the polysaccharide. Due to the β-acetal linkages and intramolecular hydrogen bonding, cellulose forms crystalline microfibrils with high stiffness and rigidity.

148

Polysaccharides

Figure 15 Chemical structure of cellulose.

It is insoluble in water and most other common solvents. It exhibits superior mechanical properties, which are essential as structural reinforcing components in plants. In addition, the β-acetal linkage in cellulose makes it indigestible in humans. 9.08.3.1.2(ii) Applications The wood, paper, and textile industries dominate in the com­ mercial use of cellulosic materials. However, new applications continue to be advanced, including chromatography, filtration, and electronic displays.92 In biomedicine, cellulose and its derivatives have been widely used as biomaterials for wound care, tissue regeneration, and encapsulation of drugs. Due to its high mechanical strength and remarkable physical properties, microbial cellulose has been explored in would healing appli­ cations.93 Cellulose is manufactured as temporary grafts for a variety of dermal injuries, such as severe burns, facial peeling, and chronic ulcers.94 To repair damaged tissues, injectable hydrogels based on cellulose have demonstrated the potential for in vivo use for the delivery of autologous chondrocytes in articular cartilage defects.95 In drug delivery, cellulose deriva­ tives exhibit robust performance as binders and coatings. Multilayered tablet with cellulose ethers, such as hydroxypro­ pyl methylcellulose (HPMC) and ethylcellulose (EC), is effective at protecting pharmaceuticals from moisture and oxy­ gen and also in modulating drug release.96,97 Because of its intrinsic mechanical properties and biode­ gradability, cellulose has become a promising ‘green’ polymer for advanced materials science. For instance, all-cellulose nano­ composites have been developed through the combination of cellulose nanofibrils and a regenerated cellulose matrix.98 The mechanical properties of this natural self-reinforced material are comparable to synthetic composites. More importantly, this material meets the demand for sustainable nanocomposites. In addition to materials science, cellulose is also a major focus of green energy development. Due to the abundance of cellulose in nonfood plants, it has been used extensively for the produc­ tion of second-generation biofuels. Compared to the first-generation starch-derived biofuels, cellulose-based bio­ fuels do not compete with food crops and can offer better engine performance.91 At present, cost-effective depolymeriza­ tion methods for the digestion of cellulose biomass remains a key challenge in the commercialization of these biofuels.

9.08.3.1.3

Dextran

9.08.3.1.3(i) Chemical structure and properties Dextran consists of α(1,6)-linked glucan with side chains attached to the C-3 position of the backbone (Figure 16). It is synthesized from sucrose by certain lactic acid bacteria, such as Leuconostoc mesenteroides. The molecular weight of natural dex­ tran has a wide range (9–200 � 106). From periodate99 and methylation analysis,100 the degree of branching is estimated to be 5%.101 Most of the side chains contain one or two glucose units. Dextran exhibits an extendable coil structure in solution, due mainly to its flexible α(1,6)-glycosidic linkage.102 It

n Figure 16 Chemical structure of dextran.

has high solubility in water and many organic solvents, such as methyl sulfoxide, formamide, ethylene glycol, glycerol, 4-methylmorpholine-4-oxide, and hexamethylphosphoramide. 9.08.3.1.3(ii) Applications Dissolved in normal saline with a concentration of about 6%, dextran exhibits similar colloid osmotic pressure and viscosity as human blood. Therefore, dextran has been used as plasma volume expander for several decades.103 Dextran 40 and 70, with molecular weight of 40 and 70 kDa, respectively, are pre­ scribed for the treatment of shock or impending shock due to hemorrhage, burns, or trauma.104,105 Dextrans have found wide application in gel filtration tech­ nology. Dextran can be readily cross-linked and manufactured in bead form and used for gel filtration columns. Sephadex® represents one of the many commercialized dextran-based fil­ tration gels that are sold with a variety of porosities and bead sizes that possess characteristic molecular weight fractionation ranges. For instance, Sephadex® G-200, which has the highest porosity, is used to fractionate proteins with molecular weight from 4 to 80 kDa, whereas Sephadex G-25 is specific to separate peptides smaller than 5 kDa.106–108 In addition to neutral dextran gels, quaternized and carboxymethyl derivatives of dextrans are also sold commercially for cation- and anion-exchange chromatography.109 Due to its hydrophilic nature, dextrans have also been used to conjugate bioactive substances (e.g., drugs, enzymes, hor­ mones, and antibodies) to prolong circulation lifetimes, increase stability in vivo, or depress antigenicity.110–113 For example, dextran nanoparticles have been conjugated with insulin for oral delivery. These nanocarriers can protect insulin from degradation in the gut and modulate release profiles.114 In addition, as a hydrophilic and functionalizable polymer, dextrans are used extensively for surface modification in bio­ sensor fabrication. Dextran layers on sensor chips can effectively minimize nonspecific adsorption of analyte and facilitate surface immobilization of ligand, subsequently increasing the sensitivity of biosensors.115

9.08.3.1.4

β-(1,3)-Glucan

9.08.3.1.4(i) Chemical structure and properties β(1,3)-Glucans are glucose polymers mainly linked by β-1,3-glycosidic bonds (Figure 17). β(1,3)-Glucans are often

Polysaccharides

149

believed to be due to the immunomodulatory properties of the glucan, as studies have shown that β(1,3)-Glucans do not attack cancer cells directly, but stimulate maturation, differen­ tiation, and proliferation of the immunocompetent cells involved in host defense mechanisms.118

9.08.3.2 n Figure 17 Chemical structure of β(1,3)-glucan.

branched, in which side chains are attached to the backbone through 1,6-linkage.116 β(1,3)-Glucans typically display a triple helical structure and dissolve in alkaline solutions. However, if the pH of the solution is above 12, the polymer will undergo an irreversible transition from triple helix to coil.117 β(1,3)-Glucans exhibit strong immune-modulating effects.118 These activities are mainly due to the ability of β(1,3)-Glucans to activate cellular and humoral components of the host immune system.119,120 9.08.3.1.4(ii) Applications β(1,3)-Glucans are regarded as biological response modifiers. The immunomodulating action of these polysaccharides is viewed as particularly promising for applications in the preven­ tion of infection and tumor metastasis. For instance, β(1,3)-Glucans have been used as anti-infective agents to pre­ vent infections by bacteria, fungi, and viruses; and numerous studies have shown that treatment with β(1,3)-Glucans can effectively reduce mortality in animal studies.121–123 The mechanism of this anti-infective activity is thought to lie in the binding of β(1-3)-Glucans by macrophage receptors, which leads to improved internalization of bacteria and increased cytoplasmic vacuolization.119 In addition to antimicrobial applications, β(1,3)-Glucans have been explored as antitumor agents for cancer therapeu­ tics.124 It is believed that the activity of these polysaccharides is related to their triple helical conformation,125 as denaturation of the polysaccharide leads to the loss of tumor inhibition properties. However, the precise mechanism of any observed antitumor activity is poorly understood. These effects are

9.08.3.2.1

N-Acetylglucosamine Polymers – Chitin/Chitosan Chemical structure and properties

Chitin, the second most abundant natural polysaccharide, after cellulose, is a linear polymer composed of repeating β(1,4)-N-acetylglucosamine units (Figure 18). Chitin exists in the shells of arthropods such as crabs, shrimps, and insects and is also produced by fungi and bacteria. Chitosan, the partially N-deacetylated analog of chitin, is a heteropoly­ saccharide consisting of D-glucosamine and N-acetyl­ D-glucosamine. The presence of free amino groups makes chitosan a natural cationic polymer and presents chemical functionality for facile derivatization of the chitosan polymer. Both chitin and chitosan are rigid and crystalline polymers, contributing to their strength and insolubility in water at neutral pH. In acidic conditions, chitosan can be dissolved due to the protonation of free amino groups, while chitin is insoluble. The molecular weight of chitin and chitosan can be as high as 106 Da.126

9.08.3.2.2

Applications

Due to the availability, biocompatibility, biodegradability, nontoxicity, and adsorption properties of chitin and chitosan, they have found extensive use in food processing, water pur­ ification, biotechnology, and other industries (Figure 19). Chitosan and its derivatives are excellent antimicrobial agents in food processing and packaging. These antimicrobial properties are believed to stem from the cationic nature of chitosan, which can interact with the cell wall components of microorganisms, leading to the leakage of intracellular com­ ponents, shrinkage of cell membrane, and microbial death.126,127 Chitosan-based materials have shown promising antimicrobial activity against a variety of microorganisms, including both Gram-positive and Gram-negative bacteria and fungi.128,129 In addition to food safety, chitosan has found use in environmental remediation and water

Chitin

n

Chitosan

n Figure 18 Chemical structures of chitin and chitosan (R = H or Ac (Ac < 50%)).

150

Polysaccharides

Food

Water engineering

Chitin/chitosan

Photography Paper finishing Cosmetics Solid-state batteries

Biomedical applications – Drug delivery – Artificial skin – Ophthalmology

Figure 19 Applications of chitin and chitosan in biomedicine and other industries.

purification. Chitosan is used as a nontoxic flocculent and chelating agent for the removal of toxic metals.130 Interactions of the cationic chitosan with organic and inor­ ganic species in solution result in rapid aggregation and sedimentation. For the removal of metal ions, the mechanism is thought to be the formation of dative bonds between amino nitrogen on chitosan and transition metal ions.131 In biomedicine, chitin and chitosan have found multiple applications, including as drug delivery vehicles, wound dres­ sing materials, and tissue engineering matrices. In drug delivery,132 chitosan has been of interest due to its ability to interact with cell membranes, increase cellular permeability, and increase residence time of drugs in the gastrointestinal tract.133 Positively charged chitosan can be used in gel forma­ tion with acidic drugs to protect bioactive molecules from the gastric environment and enhance dissolution.134,135 Chitosan possesses an interesting characteristic in vivo: due to the lack of specific hydrolytic enzymes for chitosan in the small intestine, orally delivered chitosan survives passage into the colon. Therefore, chitosan-based drug carriers are being explored for the treatment of colon diseases.136 In wound healing, hydrogels made from chitosan can protect the wound from bacterial infection, maintain a hydrated local environment, and reduce scarring.137 Chitosans can also be used to incorporate growth factors for local delivery and speeding up the healing process. As such, chitosan and its derivatives have been used extensively as porous scaffolds for the regeneration of various tissues or organs, including skin, bone, cartilage, liver, nerve, and blood vessel.138

9.08.3.3 9.08.3.3.1

Glycosaminoglycans Chemical structure and properties

GAGs are a diverse class of linear polysaccharides consisting of repeating disaccharide units that contain at least one deoxya­ mino sugar. GAGs represent a large number of polymers with significant chemical and structural differences that arise from the patterns of disaccharide building blocks (e.g., N-acetylglucosamine (GlcNAc)–glucuronic acid (GlcA) and uronic acid–glucosamine), variable sulfation (4-, 6-, or 3­ O-sulfation of the sugar rings), and overall chain length.139 GAGs play a vital role on many cell surfaces and in connective tissues and extracellular matrix (ECM). They can regulate many biological processes through their interactions with numerous effector proteins. The diversity of GAG structures and their biological roles are too numerous to detail in this chapter;1

however, the structure and functions of a series of representa­ tive GAGs are illustrated in Table 5.

9.08.3.3.2

Applications

GAGs have been widely employed as therapeutic agents due to their significant bioactivity and biological roles in tumor metastasis, tissue remodeling, neuronal development, blood clotting, and allergic response. Among the GAGs, heparin and heparan sulfate (HS) have been extensively studied for their capability to modulate biological activities through recognition of a wide range of heparin-binding proteins. For example, heparins in the lung tissue can interact with chemokines and modulate neutrophil migration and bacterial clearance from the lungs.164 A pentasaccharide sequence in heparin interacts with the serine protease inhibitor antithrombin and exhibits anticoagulant activity,165 and a synthetic pentasaccharide based on this heparin sequence has been commercialized for the prevention of deep-vein thrombosis and pulmonary embolism.166 Heparin’s use as an anticoagulant dates back to the early 1900s,167 making it one of the oldest and most widely used carbohydrate-based drugs.168 To this day, heparin continues to dominate the anticoagulant drug market, with greater than $4 billion in sales.169 Although a variety of synthetic, recombi­ nant, and chemoenzymatic methods have been explored in order to access pure heparin GAGs, these approaches have met only limited success in improving access to chemically defined GAGs for academic and clinical research. Given the limitations in accessing pure GAGs, porcine intestine continues to be used to produce large quantities of the heparins used clinically as anticoagulants. Unfortunately, due to the poor implementation of rigorous analytical tools for the quality assurance of heparin products, a significant number of deaths were reported in the United States in 2008, when pharmaceutical-grade heparin was contaminated with a semi­ synthetic oversulfated chondroitin sulfate.170 Beyond their use as anticoagulants, GAGs represent an important component in native ECM, due to their ability to promote cell adhesion, migration, proliferation, and differen­ tiation. Because of these biological roles, GAGs have become promising materials for constructing tissue scaffolds. Hyaluronic acid, chondroitin sulfate, and other types of GAGs have been integrated with chitosan or collagen as cell culture matrices and applied in the regeneration of cartilage tissues,68 differentiation of stem cells,171 and reconstruction of neural cell.117

Polysaccharides

Table 5

151

Structure of representative GAGs and their function140 Structure (R = H or SO3 −)

GAGs

Examples of biological functions Anticoagulant141 Stabilization of mast cell tryptases142 Modulation of mast cell chymases143 Regulation of the inflammatory response144 Remodeling of the airway wall in asthma145 Interaction with cytokines, chemokines, and interleukins146–148

Morphogenesis, development, and organogenesis141 Coreceptors for receptor tyrosinekinases149

Heparin

Heparan sulfate (HS)

Chondroitin sulfate

Prevention of inflammation150 Immune modulation151 Maintenance of the structure and function of cartilage150 Cartilage shock-absorbing properties150 Regulation of cell adhesion to the ECM150

Dermatan sulfate

Collagen organization152 Regulation of Transforming growth factor or Tumor growth factor (TGF)-β activity153 Stabilization of the basement membrane152 Regulation of cell–cell and cell–matrix interactions153

Keratan sulfate

Tissue hydration154 Antiadhesive to cell attachment155 Abundant airway secretion156

Hyaluronic acid

Stabilization of connective tissue157 Organization of ECM157 Hydration and water homeostasis157 Receptor-mediated signaling158 Morphogenesis and tissue homeostasis159,160 Regulation of the inflammatory response161 Tissue remodeling162 Cellular migration and phagocytosis163

Reprinted with permission from Critical Care, published by BioMed Central Ltd.

9.08.3.4 9.08.3.4.1

Other Polysaccharides Agar/agarose

9.08.3.4.1(i) Chemical structure and properties Agar, a linear polysaccharide extracted from agarophyte red algae, contains two fractions: neutral agarose and anionic agar­ opectin. Agarose is made up of alternating (1–4)-linked 3,6­ anhydro-α-L-galactopyranose and (1–3)-linked β-D-galactose units. Agaropectin has the same backbone as agarose but con­ tains many anioinic groups such as sulfate, pyruvate, and glycuronate (Figure 20). Agar is soluble in boiling water but insoluble at lower tem­ peratures, forming gels at 30–40 °C. Agar can adopt a single or

n Figure 20 Chemical structure of agarose.

double helical conformation in a gel, creating a three-dimensional (3D) network that easily holds water molecules. The mechanism of gelation is based on the aggregation of helixes and subsequent phase separation.172 The gelling property of agar depends on its composition and degree of sulfation. Generally, agar with higher agarose content (neutral form of polymer) and low degree of sulfation can form better and stronger gels. 9.08.3.4.1(ii) Applications Agar is the most commonly used growth medium for micro­ organisms. Due to the ease with which agar can be transported (dry, dissolved, and gelled), it is ubiquitous in the modern-day laboratory. Solid agar plates can support microbial growth when supplemented with appropriate nutrients or be used for antibiotic selection. Agar media is essential for the study of microorganisms and molecular biology and is widely used in the culture and detection of pathogens from contaminated food and water.173 In addition, due to its porous 3D frame­ work, agar is frequently used in biomolecular separation and purification. Agar is one of the most common basic media for

152

Polysaccharides

gel electrophoresis, gel bead chromatography, and size exclu­ sion chromatography.174–176 In addition to its use as a solid growth media, agar has been fabricated in different forms (e.g., microspheres and films) to encapsulate molecules for sustained-drug delivery177 or immo­ bilize proteins for tissue engineering.178 Due to the gelation property of agar, it is most often used as a hydrogel. To realize sustained-drug delivery, agar hydrogels have been modified by integrating other biopolymers to form an interpenetrating net­ work. This network structure of agar hydrogels is able to improve the mechanical property of the drug delivery systems and also extend the drug release profile.179 In tissue engineer­ ing, agar hydrogels with high porosity have shown promising results to promote cell adhesion and proliferation.180

9.08.3.4.2

Alginate

9.08.3.4.2(i) Chemical structure and properties Alginates are anionic polysaccharides that can be extracted from brown algae181 or produced by soil bacteria.182 They are linear block copolymers composed of 1,4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues and can be found with three block types: alternating M and G, GG, or MM. Depending on the source of alginates, these three blocks are present in varying amount (Figure 21). Alginates can form gels by reaction with divalent cations such as Ca2+. The gelation and cross-linking of alginate is mainly achieved by the exchange of sodium ions from the glucuronic acids (GlcAs) with divalent cation in solution.181 Divalent cations bind to the α-L-guluronic acid blocks in a highly cooperative manner and induce dimerization of alginate chains.183 This gela­ tion process is reversible. Alginate gels can return to soluble state after removing divalent cations in solution. As anionic polymers, alginates are able to electrostatically interact with cationic poly­ mers, resulting in the formation of electrostatic complex. At stoichiometric ratio, the complex is typically insoluble in water. 9.08.3.4.2(ii) Applications Because of the unique gelling properties of alginates, they have been used as food thickeners, dental molds, and adsorption agents for removal of radioactive toxins (e.g., Pb197–200 and Hg197).184 In pharmaceutical and biomedical science, algi­ nates are well adapted for cell culture, tissue engineering, and drug delivery. Hybrid materials composed of alginates and cationic polymers such as chitosan are often engineered into porous scaffolds for regeneration of cartilage and bone tis­ sues.185 Alginate–chitosan complex scaffolds have improved mechanical properties and elicit better cell adhesion compared to the scaffold composed of chitosan alone.186 These complex materials are also fabricated as microbeads for entrapment of drugs.187 The process of alginate–chitosan microbead forma­ tion and drug loading can be accomplished in physiological conditions and is therefore amenable to protein-based drugs that would otherwise be susceptible to denaturation. In

addition, the interactions between alginate and chitosan rein­ force the microbeads and facilitate sustained release of drugs. In wound healing applications, alginate products have been commercialized as hemostatic materials or wound dres­ sings.117 These calcium alginate dressings can adsorb exudates from the wound area up to 20 times their weight. They are biocompatible and easily removed without damaging healthy tissues.

9.08.3.4.3

Bacterial polysaccharides

Bacteria produce a variety of polysaccharides that are both structurally diverse and medically relevant. Capsular polysac­ charides (CPS) and/or lipopolysaccharides (LPSs) are the major components on the surface of bacteria. The CPS are composed of either homopolysaccharides, such as the α(2–8)-linked sialic acid homopolymer in Neisseria meningitidis and Escherichia coli K1, or complex polysaccharides consisting of two to six sugar residues.188 CPS are present in both Gram-negative and Gram-positive bacteria. They are water soluble, are commonly acidic, and have molecular weights on the order of 100–1000 kDa. These polysaccharides provide bacteria several benefits: they prevent bacteria from desicca­ tion, promote the adherence of bacteria to surfaces and other bacteria, confer resistance to various phages, and complement in the vertebrate host.189 Most CPS can elicit an immune response; thus vaccines based on these polysaccharides have been widely used to prevent pathogen infections.190 For exam­ ple, polysaccharide vaccines composed of CPS from 23 of the most common pneumococcal serotypes have been developed to prevent 85–90% of the pneumococcal infections.191 LPS is present on the surface of Gram-negative bacteria and consists of three parts: O-polysaccharide, a core oligosaccharide, and a lipid A anchor.188 The O-polysaccharide, also known as the O-antigen, is composed of repeating units of five to eight mono­ saccharides and is highly diverse among different species of bacteria; over 60 different monosaccharides and 30 noncarbohy­ drate elements have been reported in O-antigens.192 In addition to being antigenic, the O-antigen can serve as a bacteriophage receptor as well as modulate the activation of the alternative complement pathway. The core oligosaccharide of LPS typically contains 2-keto-3-deoxyoctonate, heptose, glucose, galactose, and N-acetylglucosamine. Lipid A consists of a diglucosamine carbohydrate backbone with a β(1–6) linkage. The core lipid A diglycosamine is phosphorylated on the 1 and 4 positions and modified by fatty acyl chains attached by either ester or amide bonds. LPS is essential for the viability of all Gram-negative bacteria.193 LPS maintains the structural integrity of bacteria and protects the bacterial outer membrane from attack. LPSs are well-known immunostimulators as they are recognized by pat­ tern recognition receptors of the innate immune system and induce costimulatory molecules required for antigen presenta­ tion to the acquired immune systems. Because of these immunostimulatory properties, LPS has been employed in the development of vaccines against a variety of pathogens.194,195

9.08.4 Conclusions n Figure 21 Chemical structure of alginate.

Carbohydrates and polysaccharides represent an extensive family of biopolymers that possess a variety of physical and biological properties. Formerly an underappreciated – some

Polysaccharides say neglected – dimension of biochemistry, the past few dec­ ades have witnessed a resurgence of interest surrounding the biological roles played by the glycans and glycoconjugates. Spurred by the growth of glycobiology research and the emer­ gence of the nascent field of glycomics, carbohydrates are garnering attention for their potential applications as pharma­ ceuticals, biomaterials, drug delivery agents, ‘green’ materials, and biofuels. However, the heterogeneity and structural diver­ sity of natural polysaccharides continue to frustrate access to sufficient quantities of pure and well-characterized material for study. For the field to continue to grow, new synthetic and analytical methods are required. While carbohydrate synthesis no longer represents the ‘new frontier’ of organic chemistry, synthetic access to this important class of biomolecules remains elusive; however, new chemical methods are slowly beginning to provide the much needed material for biomedical and other research applications. If the past 10 years have been any indication, the future for carbohydrate- and polysaccharide-based technologies looks promising, as we con­ tinue our 8000-year tradition of leveraging nature’s most abundant biopolymer.

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Biographical Sketches Eun-Ho Song received his BS and MS in chemistry from Sungkyunkwan University, Korea, in 1999 and 2001, respectively. Following graduation, he worked at Samsung-Biomedical Research Institute, Korea, as a research scientist. Dr. Song received his MS in organic chemistry from Florida State University in 2005, followed by a PhD in carbohydrate chemistry from Iowa State University in 2009. He is currently a senior fellow in the Center for Intracellular Delivery of Biologics and in the Department of Bioengineering, University of Washington. His research focuses on carbohydrate-based polymeric drug carriers for targeted delivery of therapeutics.

Jing Shang received her BS degree in materials science and engineering from Tongji University, China, in 2005 and her MS degree in polymer chemistry and physics from Fudan University, China, in 2008. Ms. Shang is currently a graduate student working in Dr. Daniel M. Ratner’s lab in the Department of Bioengineering, University of Washington. Her research focuses on developing label-free biosensors for the study of carbohydrate-mediated host–pathogen interactions.

Daniel M. Ratner trained in carbohydrate chemistry and microarray fabrication at the Massachusetts Institute of Technology with Professor Peter Seeberger, where he received his PhD in synthetic organic chemistry. He subsequently joined the Section of Infectious Diseases at Boston Medical Center/Boston University as an NRSA Postdoctoral Fellow with the Training Program in Host–Pathogen Interactions. In September 2007, Dr. Ratner became a member of the faculty of the University of Washington as an assistant professor in the Department of Bioengineering. The Ratner laboratory is developing new chemical and biophysical tools to probe carbohydrate-mediated interaction using Surface Plasmon Resonance (SPR) and silicon photonic biosensors.