Mini-Review

Chitin and Chitosan: Functional Biopolymers from Marine Crustaceans

Keisuke Kurita
Department of Materials and Life Science, Seikei University, Musashino-shi, Tokyo 180-8633, Japan Received: 10 July 2005 / Accepted: 22 September 2005 / Published online: 17 March 2006

Abstract Chitin and chitosan, typical marine polysaccharides as well as abundant biomass resources, are attracting a great deal of attention because of their distinctive biological and physicochemical characteristics. To fully explore the high potential of these specialty biopolymers, basic and application researches are being made extensively. This review deals with the fundamental aspects of chitin and chitosan such as the preparation of chitin and chitosan, crystallography, extent of N-acetylation, and some properties. Recent progress of their chemistry is then discussed, focusing on elemental modication reactions including acylation, alkylation, Schiff base formation and reductive alkylation, carboxyalkylation, phthaloylation, silylation, tosylation, quaternary salt formation, and sulfation and thiolation. Keywords: Chitin polysaccharide marine biopolymers biomass resource biological activity chemical modication chitosan biomedical application unutilized biomass resource

Introduction Quite a few kinds of polysaccharides occur in nature in a broad range of structures and forms, and most of them are considered to work as structural materials or suppliers of water and energy, though their functions may not have been fully comprehended. Since polysaccharides have peculiar structures and properties, quite different from those of synthetic polymers, they are considered promising biopolymers for developing desirable advanced functions. Among numerous polysaccharides, cellulose and chitin are produced in the largest amounts, estiCorrespondence to: Keisuke Kurita; E-mail: kurita@st.seikei.ac.jp

mated to be around 1011 tons each per year, and actually, they are the most abundant organic compounds on earth. Cellulose (polymer of b-(1Y4)-linked D-glucopyranose) and chitin (polymer of b-(1Y4)-linked 2acetamido-2-deoxy- D -glucopyranose) are found mainly in the plant and animal kingdoms, respectively, as substantial materials to protect the organisms (Scheme 1). They are structurally similar to each other; chitin has an acetamido group at C-2 in place of the hydroxy group in cellulose (Scheme 2). As an essential polymeric material from plants, cellulose has found many applications. Although chitin is readily available in quantity from the shells of marine crustaceans such as crabs and shrimps, it has long been regarded as just a structural material without noteworthy biological functions. Because of the lack of appreciation of the superb characteristics along with the relatively laborious isolation process, only limited attention has been paid to chitin, in contrast to the comprehensive studies hitherto done on cellulose. Chitin thus remains an almost unutilized biomass resource. Chitin and its deacetylated form, chitosan, are, however, attracting increasingly more attention recently as the inherent biological and physicochemical characteristics are being better understood (Muzzarelli, 1977; Japanese Society for Chitin and Chitosan, 1988, 1990; Goosen, 1997). The (acetyl)amino groups on the polysaccharide backbone undoubtedly play crucial roles in exhibiting various unique properties. The notable properties of chitin and chitosan include non-toxicity, lm- and berforming properties, adsorption of metal ions, coagulation of suspensions or solutes, and distinctive rum et biological activities (Uragami et al., 2001; Va al., 2003; Boucher et al., 2004). These properties are quite advantageous to open a way to new biotechnological applications. Chitin and chitosan are thus regarded as biofunctional polymers having much higher potential than cellulose in many elds and 203

DOI: 10.1007/s10126-005-0097-5 & Volume 8, 203226 (2006) & * Springer Science+Business Media, Inc. 2006

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Scheme 1. Chemical structures of cellulose and chitin.

important not only as abundant resources but also as a novel type of polymeric materials that are environmentally benign. Furthermore, the presence of (acetyl)amino groups would be highly benecial for chemical modications to construct sophisticated molecular architectures. Special emphasis has thus been put on the structural transformations of chitin and chitosan to explore their full potential (Roberts, 1992a; Kurita, 1994). As a consequence, basic research on the efcient and controlled chemical modication reactions is advancing notably (Kurita, 1996, 1997a,b, 2001; Lim and Hudson, 2003; Uragami and Tokura, in press), and the resultant derivatives with welldened structures will enable detailed discussion on the structureproperty relationship. Studies on their applications are also encouraging, and several products have been put on the market. Based on the rapidly growing interests in these amino polysaccharides, some results and experimental procedures have been compiled into handbooks (Japanese Society for Chitin and Chitosan, 1991, 1995; Muzzarelli and Peter, 1997). This review focuses on the fundamental aspects of chitin and chitosan and the recent progress of their chemistry with a view to the development of novel intelligent materials. Preparation of Chitin and Chitosan Sources. Although chitin distributes widely in nature (Fungi, Algae, Protozoa, Cnidaria, Aschelminthes, Endoprocta, Bryozoa, Phoronida, Brachiopoda, Echiurida, Annelida, Mollusca, Onychophora,

Arthropoda, Chaetognatha, Pogonophora, and Tunicata), arthropod shells (exoskeletons) are the most easily accessible sources of chitin. From a practical viewpoint, the shells of marine crustaceans such as crabs and shrimps are convenient; they are available as waste from the seafood processing industry and used for commercial production of chitin. The shells contain 1540% chitin (a-chitin; see the section on Crystallography), proteins (2040%) and calcium carbonate (2050%) being two other major components. Pigments and other metal salts are only minor components. b-Chitin is less common than a-chitin, but this allomorph is obtained in a certain amount from squid pens. b-Chitin also occurs in Aprodite chaetae, lorica of sessile ciliate, pogonophore tubes, and diatom spines. It is attractive as another form of chitin having characteristics noticeably different from those of a-chitin, and understanding of the chemistry is steadily progressing (Kurita, 1997c). Other possible sources of chitin production include krill, clams, oysters, insects, and fungi. Table 1 lists the rough estimates of chitin and calcium carbonate contents. Cell walls of some fungi (Zygomycetes) contain chitosan and will be potential sources of chitosan. Chitin. Chitin exists in tightly bound complexes with other substances in the cuticles of crabs and shrimps, and some portions of polypeptides are suggested to be linked covalently to a small number of the C-2 amino groups. Above all, chitin is not

Table 1. Contents of Chitin and Calcium Carbonate

Source Crab cuticle Shrimp cuticle Krill cuticle Squid pen Clam/oyster shell Insect cuticle Fungi cell wall

Chitin (%) 1530 3040 2030 2040 36 525 1025

CaCO3 (%) 4050 2030 2025 Negligible 8590 Negligible Negligible

Scheme 2. Repeating unit of chitin.

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Table 2. Estimates of Worldwide Production of Chitin Derivatives for the Year 2000

Derivative Production (tons) Chitosan Oligosaccharides Glucosamine Total

a

Yield from chitin (%) 75 50 60

Chitin as starting material (tons) 2,667 1,000 6,667 10,334 (1,300)a (1,000)a (5,500)a (7,800)a

2,000 500 4,000

Chitin used for dietary supplements.

soluble in ordinary solvents, and this makes the solvent extraction method inapplicable to the isolation of chitin. However, chitin is fairly stable under mild acidic and basic conditions and thus obtained as the residue remaining after decomposition of the other components with acid and alkali (Bade, 1997; No and Meyers, 1997). Shells are rst treated with dilute hydrochloric acid at room temperature to remove metal salts, primarily calcium carbonate. The decalcied shells are ground and heated at about 100-C in 12 mol/L of sodium hydroxide to decompose proteins and pigments. To ensure removal of these organic substances, the alkaline treatment is repeated a few more times. If necessary, the product is again treated with dilute hydrochloric acid and alkali. On drying, a-chitin is obtained as almost colorless to off-white akes or powdery materials. The yield is 3035% based on dried shrimp shells (Sannan et al., 1976). Starting from crab shells, the yield is usually lower than that from shrimp shells because of the higher content of calcium carbonate. Chitin isolated in this manner contains some free amino groups besides acetamido groups, and the degree of Nacetylation is 0.900.95. Practically, chitin is used almost solely as a raw material for the production of chitosan, oligosaccharides, and glucosamine. Judging from the worldwide productions of these derivatives in year 2000, chitin is estimated to be produced in an amount of about 10,000 tons industrially, as summarized in Table 2 (Sandford, 2003). Proteases may be used to remove proteins under mild conditions without using alkalis. Pseudomonas maltophilia, which is highly proteolytic but neither chitinolytic nor chitin-deacetylating, is effective for removing proteins from decalcied

shrimp cuticle chips (Shimahara et al., 1984). It seems, however, sometimes not easy to accomplish thorough removal, probably because of the heterogeneous and mild reaction conditions. Squid pens are composed almost exclusively of b-chitin and proteins, and unlike crustacean shells, they contain only a very small amount of calcium carbonate. Furthermore, the molecular packing of b-chitin is less tight than that of ordinary a-chitin, and b-chitin can be isolated under similar, but milder conditions. When the b-chitin is pulverized with an ultracentrifugal mill, a white uffy cottonlike material is obtained. During the isolation procedure, some N-acetyl groups are removed, and the resulting chitin has a degree of N-acetylation of around 0.90. The pens of Ommastrephes bartrami gave 35 wt% b-chitin along with 58 wt% proteins (Kurita et al., 1993a; Kurita, 1997d).

Water-Soluble Chitin. When chitin is treated with concentrated aqueous sodium hydroxide and mixed with crushed ice, an alkali chitin solution forms. In the alkaline homogeneous solution, Ndeacetylation proceeds smoothly, and a product with about 50% deacetylation is soluble even in neutral water (Scheme 3) (Sannan et al., 1975, 1976). Higher or lower deacetylations fail to lead to complete solubilization, and only the products with degrees of N-acetylation of 0.450.55 are water-soluble. This solubility is probably ascribable to the random distribution of the N-acetylglucosamine and glucosamine residues along the main chain, as suggested from X-ray diffraction studies. Random N-acetylation of chitosan to a degree of about 0.5 is an alternative way to the water-soluble chitin (Kurita et al., 1991b).

Scheme 3. Preparation of the water-soluble chitin (p $ q) by partial deacetylation.

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Scheme 4. Deacetylation of chitin to chitosan.

Chitosan. Chitosan is prepared by deacetylating a-chitin with 4050% aqueous alkali, sodium hydroxide in most cases, at 120150 -C under heterogeneous conditions (Scheme 4) (No and Meyers, 1997). The degrees of N-acetylation of the products are 0.050.30 in general. Complete deacetylation is possible by repeating the alkaline treatment (Domard and Rinaudo, 1983; Mima et al., 1983). b-Chitin from squid pens is more susceptible to the deacetylation reaction because of the relatively weak intermolecular forces and, moreover, tends to give chitosan in a light tan to brown color under similar conditions. To suppress discoloration, milder reaction conditions, for example, with 40% sodium hydroxide at 80-C, are preferred (Kurita et al., 1993a). The deacetylation processes of a- and b-chitins were compared recently (Lamarque et al., 2004, 2005). Some fungi contain chitosan, which is considered to be formed by the action of chitin-deacetylases on chitin, and chitosan was isolated from their cell walls (McGahren et al., 1984). This procedure to produce chitosan is more costly than the conventional chitin deacetylation process at the present stage. However, chitosan can be isolated without harsh treatment with strong alkalis, and this approach may become important in the future. Colloidal Chitin and Chitosan. Since chitin is not soluble in water, nely powdered chitin called colloidal chitin is conveniently used as a substrate to assay chitin-degrading enzymes and as a carbon and nitrogen source for chitin-digesting microorganisms. Colloidal chitin is prepared by dissolving chitin in hot concentrated hydrochloric acid followed by pouring the solution into water (Shimahara and

Takiguchi, 1988). The molecular weight decreases as a result of the main chain degradation with hydrochloric acid. Once dried, the colloidal chitin cannot be dispersed effectively in water, and it should be stored as an aqueous dispersion in a refrigerator. When a chitosan solution in dilute hydrochloric acid is neutralized with aqueous sodium hydroxide with vigorous stirring, colloidal chitosan is obtained as a dispersion. The dispersed chitosan is used for assaying chitosanases (Yabuki et al., 1988). Oligomers. The glycosidic linkages of the chitin and chitosan main chains are cleaved by acidic hydrolysis, typically with hydrochloric acid, giving chitin and chitosan with lowered molecular weights. Nitrous acid effectively lowers the molecular weight of chitosan and is useful for preparing low molecular weight chitosan (Hirano et al., 1985; Sashiwa et al., 1993). The reaction is clean and rapid at room temperature or 0-C. It involves deamination and scission of the main chain to form a 2,5-anhydromannofuranose unit at the reducing end of the cleaved chitosan (Scheme 5). Treatment of chitin or chitosan with concentrated hydrochloric acid at elevated temperatures results in thorough hydrolysis to give the monomeric sugar glucosamine. Under less severe conditions, chitin and chitosan afford a series of oligomers of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), respectively. Size-exclusion chromatography of a chitosan hydrolysate was reported to separate oligomers up to (GlcN)15 (Domard and Cartier, 1989). Glucosamine oligomers can be converted into the corresponding N-acetylglucosamine oligomers on N-acetylation. These two series of oligomers up to hexamers or heptamers are commercially available. Acetolysis of chitin with acetic anhydride in concentrated sulfuric acid gives peracetylated N-acetylglucosamine oligomers. This is a conventional process for preparing the dimer, N , N 0 diacetylchitobiose [(GlcNAc)2], which is used as a starting material for synthesizing some disaccharide derivatives, but the yield from the ordinary a-chitin

Scheme 5. Degradation of chitosan main chain with nitrous acid.

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is poor. The yield was improved to 16% with colloidal chitin (Nishimura et al., 1989). Starting from b-chitin without any pretreatment, the dimer could be obtained in 17% yield (Kurita et al., 1993b). Chitin oligomers are also prepared by enzymatic hydrolysis of chitin. Certain chitin-digesting bacteria are especially useful for preparing specic oligomers; (GlcNAc)2, for instance, was obtained in 4050% yield under appropriate conditions (Takiguchi and Shimahara, 1988). A continuous membrane process using a chitinase from Streptomyces griseus was efcient and reproducible to manufacture the dimer, and the yield was 36% after purication as the peracetate form (Matsuoka et al., 2000). Degradation of chitosan with a chitosanase produced the dimer to pentamer (Izume and Ohtakara, 1987). A cellulase of Trichoderma viride cleaved chitosan, resulting in the formation of the hexamer to octamer (Muraki et al., 1993). Transglycosylation is another means to prepare chitin oligomers. Certain chitinolytic enzymes such as chitinases and lysozyme express considerable transglycosylation activities, catalyzing the coupling reaction of small oligosaccharides to larger ones. With a chitinase, for example, (GlcNAc)4 gave (GlcNAc)6 and (GlcNAc)2, and with lysozyme, (GlcNAc)2 gave (GlcNAc)6 and (GlcNAc)7 (Usui et al., 1990). Crystallography Chitin is generally classied into two forms depending on the crystalline structure: a- and b-chitins. The most abundant and easily accessible form is a-chitin. Detailed structural analysis has shown that the chitin molecules align in an antiparallel fashion in a-chitin that allows maximum intermolecular hydrogen bonding (Blackwell et al., 1980). The chitin molecules are, however, parallel in b-chitin, which is responsible for the weaker intermolecular forces (Gardner and Blackwell, 1975). b-Chitin obtained from squid pens is semicrystalline, but almost completely crystalline b-chitin was isolated from tubes of Tevnia jerichonana, a deepsea organism (Gaill et al., 1992). Some characteristics of b-chitin such as high afnity for solvents and high reactivity are attributable to its loose crystalline structure. The b-axis of b-chitin widened on forming crystalline inclusion complexes with small polar molecules such as water, alcohols, and amines (Blackwell, 1969; Ro ssle et al., 2003; Noishiki et al., 2004). Dissolution or swelling converts b-chitin to a-chitin, but not a-chitin to b-chitin, suggesting that b-chitin is a metastable form biosynthesized by

a mechanism different from that for a-chitin (Rudall and Kenchington, 1973). Even in the solid state, aqueous hydrochloric acid caused transformation of b-chitin into a-chitin (Saito et al., 1997). Chitosan is also crystalline, but the structure is different from that of either a- or b-chitin as evidenced by the X-ray diffraction patterns. The crystal structures have been rened for the anhydrous and hydrated forms (Yui et al., 1994; Okuyama et al., 1997). Electron diffractometry is also a powerful tool for studying molecular and crystal structures of chitosan (Mazeau et al., 1994). Extent of N-Acetylation The amino groups of native chitin are present mostly in acetylated form, but some may have protein chains or may be present as the free amine. Moreover, deacetylation inevitably takes place during the isolation process with acid and alkali. Chitin samples therefore contain broadly different amounts of N-acetyl groups, depending on their origin and isolation procedure. In general, the degrees of N-acetylation of a-chitin from crab or shrimp shells and b-chitin from squid pens are in the range 0.900.95. To prepare chitin with a uniform structure, that is, poly(N-acetylglucosamine), selective N-acetylation of the water-soluble chitin (Kurita et al., 1977) or b-chitin (Kurita et al., 1994) is necessary. In chitosan, the N-acetyl content also depends on the deacetylation conditions and the chitin sources. Although there is no commonly accepted specication for dening chitosan in terms of the acetyl content, samples with degrees of N-acetylation below 0.3 are commonly referred to as chitosan, since they are readily soluble in dilute acetic acid. The extent of acetylation is one of the most important factors inuencing the properties of chitin and chitosan. Varieties of methods have thus been elaborated for assessing the degree of acetylation, and each method has advantages and disadvantages (Roberts, 1992a). Acidbase and colloid titrations, conductometric titration, and infrared (IR) and H-nuclear magnetic resonance (H-NMR) spectroscopies are relatively easy and reliable. In the IR spectroscopic determination, the absorbance ratio of A1550/A2878 was rst suggested (Sannan et al., 1978), and several other ratios such as A1655/ A3450, A1655/A2867, and A1655/A3450 have also been rum reported (Roberts, 1992a). H-NMR in D2O (Va et al., 1991) or in CD3CO2D/D2O (Hirai et al., 1991) is another method for chitosan. C-NMR (Li et al., 1997) and N-NMR (Yu et al., 1999) are applicable to

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the determination of the acetyl content in the solid state. Properties Solubility. Chitin is not soluble in common solvents because of the strong intermolecular hydrogen bonding, and it is soluble only in special solvents such as hexauoroacetone and N , N dimethylacetamide (DMAc) containing 58% LiCl (Rutherford and Austin, 1978). Recently, a new solvent system was reported; methanol saturated with calcium chloride dihydrate, which is a solvent for nylons, dissolved chitin (Tamura et al., 2004). It is rather surprising to note that a-chitin showed better solubility than b-chitin in this solvent system. Addition of a small amount of water to the solution precipitated highly swollen gels, which is interesting as a new type of smooth hydrogel. Unlike a-chitin, b-chitin is soluble in formic acid and swells in water considerably. b-Chitin can thus be fabricated into paper and non-woven sheets using an aqueous dispersion of highly swollen b-chitin. Chitosan is insoluble in either organic solvents or water. However, it is soluble in aqueous acids owing to the presence of free amino groups. Some organic acids such as formic, acetic, lactic, pyruvic, and oxalic acids are frequently used for dissolution. Mineral acids such as hydrochloric and nitric acids also give chitosan solutions, but phosphoric and sulfuric acids are not suitable. A partially deacetylated chitin with about 50% deacetylation prepared under homogeneous conditions is soluble in water as described in the section on Water-Soluble Chitin. However, the sample is only partially soluble in water at room temperature, leaving highly swollen transparent gels, and it is necessary to treat the sample with crushed ice for complete dissolution (Sannan et al., 1976). Coagulation. Chitosan is a polycation polymer effective in coagulation and dewatering of activated sludge and used in wastewater treatment. Though chitosan is more expensive than widely used synthetic polycation polymers such as a series of acrylamide copolymers, it is biodegradable and less toxic and thus a friendly coagulating agent in view of environmental protection (Thome et al., 1997). Wastewater from food processing plants can be occulated with chitosan, and the process is becoming of practical importance for recovering proteins and thereby producing livestock feed. Adsorption and Chelation. Chitin and chitosan adsorb dyes, aromatic hydrocarbons, and proteins

(Roberts, 1992c). They also adsorb metal cations such as copper, mercury, cadmium, iron, manganese, nickel, zinc, lead, and silver, but chitosan exhibits higher afnity due to the free amino groups (Muzzarelli, 1973; Roberts, 1992b). Chitosan collects even highly toxic organomercury compounds and is a candidate for adsorbents to remove toxic heavy metals from industrial wastewater. The metal adsorption capacity is inuenced markedly by the degree of acetylation; it generally increases with a decrease in the acetyl content. However, the samples prepared by deacetylating chitin in homogeneous solution showed maxima at around 50% deacetylation (Kurita et al., 1979). These results indicate that the adsorption capacity is inuenced not only by the content of the free amino groups, but also by hydrophilicity of the adsorbent molecules. Biological Properties. Chitin and chitosan are non-toxic, biocompatible, and biodegradable. Unlike cellulose, they are amino polysaccharides and show interesting biological, physiological, and pharmacological properties. Their notable bioactivities include promotion of wound healing, hemostatic activity, immune enhancement, hypolipidemic activity, mucoadhesion, eliciting biological responses, and antimicrobial activity. Chitosan is also promising as a supporting polymer for gene delivery, cell culture, and tissue engineering. In medical practice, non-woven chitin fabrics and chitin threads are used as articial skins and sutures with the advantages of wound healing, biocompatibility, and biodegradability. Chitin and chitosan evidently promote wound healing and enable highquality cosmetic restoration (Nishimura, 2001; Degim et al., 2002). Because of the remarkable hemostatic activity, chitosan bandages are being used by paramedics in the U.S. forces in Iraq and Afghanistan to stop bleeding instantaneously. Cotton-like bers and sponges of chitin and chitosan are now commonly used by veterinarians in Japan as wound dressings or lling agents for animals (Minami et al., 1992, 2001). Certain chitin and chitosan oligomers have physiological functions including the induction of phytoalexins, antimicrobial activity, and immune enhancing activity. For example, the hexamers (GlcNAc)6 and (GlcN)6 exhibited growth inhibitory effects against tumor cells such as sarcoma-180, MM-46, and Meth-A solid tumors (Tokoro et al., 1988a,b). They are soluble in water and easily injected intravenously. Though detailed studies are still underway to elucidate the biological or physiological functions of these oligomers, they seem to

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Scheme 6. Full acetylation of chitin in methanesulfonic acid.

be serviceable in the elds of medicine, food, cosmetics, and agriculture. The macrophage activation behavior of chitosan is interesting, and the inuence of the extent of N-acetylation on the activity was examined using partially deacetylated chitins. The activity was dependent on the degree of N-acetylation, and the highest activation was observed at 0.3, suggesting that deacetylated chitins would be potential immunoadjuvants (Nishimura et al., 1987). Unlike inactive cellulose, chitin and chitosan as dietary bers exhibit hypolipidemic activity, as conrmed by the reduced cholesterol and triglyceride levels in blood plasma (serum) and liver of rats (Nagyvary et al., 1979). Chitosan was much more effective than chitin (Sugano et al., 1980, 1988). Chitosan and its derivatives may thus be of medicinal importance (Muzzarelli and De Vincenzi, 1997). Chitosan is a static or cidal agent of microbial growth, useful in medicine, agriculture, food, and household goods manufacturing (Allan and Hadwiger, 1979), and the applications and mode of action have been reviewed (Lim and Hudson, 2003; Rabea et al., 2003). It suppresses the growth of bacteria such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus, probably because of binding to the cell surface (Hadwiger et al., 1984; Uchida et al., 1989). Chitosan also shows antifungal activity as observed in the inhibition of the growth of Fusarium solani, a typical plant pathogenic fungus. In the eld of agriculture, chitin and chitosan are expected to be fungus control agents. The addition of chitin (or crab and shrimp shells) and chitosan to soil, and the spraying of chitosan solutions on vegetables, were claimed in patents to be effective for cultivation, and several forms of powders and solutions are produced commercially. Because of the virucidal and fungicidal activities of chitosan, it sometimes prevents plant diseases and

improves crops. Seed coating with chitosan enhanced the germination ratio and growth rate, leading to increased crops (Hirano et al., 1990; Freepons, 1997).

Chemical Modications Structural modications of chitin and chitosan will promote utilization of these specialty polysaccharides in various elds. Because of the strong intermolecular forces and hence low afnity for ordinary organic solvents appropriate for carrying out chemical reactions, chitin and chitosan are much less accessible to potential reactants than cellulose. In addition to the lack of solubility, multifunctionality and low reactivity are also responsible for the difculty in performing the reactions. Therefore, chemical modications of chitin and chitosan are complicated and not easy to control in general. The reactions under heterogeneous conditions are usually accompanied by problems including poor extents of reaction, difculty in regioselective substitution, structurally ununiformly products, and partial degradation due to severe reaction conditions. To discuss the structureproperty relationship and thereby to develop novel types of polysaccharide-based intelligent materials with specic functions, it is a requisite to establish preparative procedures for derivatives with well-dened structures. Great efforts have thus focused on the development of efcient modication reactions in well-controlled ways under mild conditions, and some signicant reactions are discussed below.

Acylation. Though a-chitin is resistant toward acetylation, full substitution was achieved with acetic anhydride in methanesulfonic acid (Scheme 6)

Scheme 7. Selective N-acetylation of the free amino groups in b-chitin.

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(Nishi et al., 1979). The reaction mixture was heterogeneous initially, but the acetylated product went into solution as the degree of acetylation increased. The solvent is, however, strongly acidic, and main chain degradation resulted. b-Chitin is also insoluble in common solvents, but it swells to some extent in many solvents. Selective N-acetylation of the free amino groups present in b-chitin was possible with acetic anhydride in methanol to give structurally uniform chitin, poly(N-acetylglucosamine) (Scheme 7) (Kurita et al., 1994). Fully acetylated chitin was prepared with acetic anhydride in pyridine under mild conditions. These results indicate the high potential of b-chitin as a versatile starting material for chemical modications. Acylation of chitosan has been reported with various kinds of acylating agents, but the reactions are not regioselective partly because of the heterogeneous reaction conditions. Chitosan is soluble in aqueous acetic acid, and the addition of a similar amount of methanol still keeps chitosan in solution. The acylations with acid anhydrides in a mixture of aqueous acetic acid and methanol proceed almost selectively at the amino group, but O-acylation also takes place to low extents generally. Under these conditions, the acylated products precipitate or form swollen gels because of the reduced solubility (Hirano and Ohe, 1975; Moore and Roberts, 1980). When chitosan was treated with acyl chlorides in a mixture of pyridine and chloroform at reux, highly acylated chitosans were obtained (Fujii et al., 1980), and NMR spectroscopy was useful for the structural analysis of the products (Zong et al., 2000). N,O-Acylated chitosans were prepared with acyl chlorides in methanesulfonic acid (Sashiwa et al., 2002b,c), and the derivatives having 4-chloro-

butyl and decanoyl groups showed higher fungicidal activities than chitosan (Badawy et al., 2004). Modication reactions of water-soluble chitin (50% deacetylated chitin) can be conducted in aqueous solutions or in organic solvents in a swollen state under mild conditions, and selective N-acetylation is possible (Kurita et al., 1977). The free amino group of the water-soluble chitin initiated the ring opening graft copolymerization of a-amino acid N-carboxy anhydrides (NCAs) to introduce peptide side chains (Scheme 8) (Kurita et al., 1988b; Nishiyama et al., 1998). The graft copolymerization of g-methyl L-glutamate NCA was, for example, attained at 0-C in aqueous ethyl acetate to give chitin/polypeptide conjugates, chitin-graft-poly(g-methyl L-glutamate)s, with high grafting efciencies.

Alkylation. Hydroxyethyl-chitin or glycol-chitin is prepared by treating alkali chitin with ethylene oxide. It is soluble in water and a common substrate for assaying chitinolytic enzymes. However, the structure is considered complicated because of the side reactions such as N-deacetylation of chitin and the graft copolymerization of ethylene oxide under strongly alkaline conditions. In a similar manner, propylene oxide gives hydroxypropyl-chitin. Hydroxypropylation of chitosan with propylene oxide is controlled by the pH of the solution. The substitution reaction occurred preferentially at the amino groups or hydroxyl groups under neutral or alkaline conditions, respectively (Scheme 9) (Maresch et al., 1989; Lang et al., 1997a). Hydroxyalkylations with 2-chloroethanol and 3-chloropropane-1,2-diol were also reported (Tokura et al., 1983). With N, N-diethylaminoethyl chloride, diethylaminoethylchitin was derived (Kurita et al., 1990).

Scheme 8. Graft copolymerization of amino acid NCAs onto the water-soluble chitin.

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Scheme 9. Selective hydroxypropylation of chitosan.

Schiff Base Formation and Reductive N-Alkylation.

With Reducing Sugars. Condensation of the free

amino group of chitosan with aldehydes or ketones affords Schiff bases, and the reaction proceeds smoothly in a mixed solvent of aqueous acetic acid and methanol (Scheme 10). Though the reaction is homogeneous in the initial stage, gel formation is frequently observed because of the poor solubility of the resultant Schiff bases (Hirano, 1978). A derivative of decanal showed glass transition because of the long side chains (Kurita et al., 1988a). The imine linkage C=N is fairly stable under neutral and basic conditions but hydrolyzes in acidic solutions. The Schiff base formation can therefore be used for the protection of the amino functionality of chitosan in chemically modifying the remaining hydroxy groups (Moore and Roberts, 1982). Glutaraldehyde, a difunctional aldehyde, is a common crosslinking agent for chitosan. Reduction of the Schiff bases of chitosan with sodium cyanoborohydride or sodium borohydride is a convenient way for regioselective N-substitution. Sugar branches were introduced at the amino group by reductive alkylation, using diverse reducing sugars such as glucose, galactose, N-acetylglucosamine, lactose, and cellobiose (Scheme 11) (Hall and Yalpani, 1980; Yalpani and Hall, 1984). The branched products were soluble in water and/or dilute acids, and the solutions showed interesting rheological behavior (Yalpani et al., 1983).

With a trisaccharide having a 2,5-anhydromannofuranose unit prepared by degrading partially deacetylated chitin with nitrous acid, branched derivatives with degrees of substitution 0.070.40 (Scheme 12) were obtained (Tmmeraas et al., 2002). The apparent pKa values of the unsubstituted free amine and substituted amine were determined to be 6.56.9 and 5.05.2. To improve the chelating ability of chitosan, a galactose derivative having an iminodiacetic acid unit was used for the alkylation similarly (Holme and Hall, 1991a).

Scheme 10. Schiff base formation from chitosan and an

aldehyde.

With Formylated Sugars. In the above cases, the reducing sugars react directly to form open-chain moieties, but when sugar derivatives having an aldehyde group in the aglycone part are used, sugar groups are incorporated without opening the ring. For example, chitosan derivatives carrying glucose or galactose through C10 spacer arms were reported (Scheme 13) (Holme and Hall, 1991b). The solutions of the products in aqueous acetic acid became gels when heated to 50-C, but the gels reverted to solutions on cooling to room temperature. Formylmethyl glycosides of glucose, galactose, lactose, and N-acetylglucosamine gave N-branched chitosan derivatives with C2 spacer arms (Hall and Holme, 1986). The viscosity and pseudoplasticity of aqueous solutions of these derivatives increased with a decrease in the degree of substitution (Holme and Hall, 1992; Holme et al., 1992). Branched products prepared in a similar manner with some monosaccharide derivatives appeared to sensitize the canine polymorphonuclear leukocyte cells by a priming mechanism as evaluated in terms of the active oxygen generation (Morimoto et al., 2001). Sialic acid, a biologically important sugar, was also incorporated into chitosan by reductive alkyl-

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Scheme 11. Reductive alkylation of chitosan with lactose.

Scheme 12. Introduction of trisaccharide branches into chitosan.

Scheme 13. Chitosan/galactose conjugate with a C10 spacer arm.

Scheme 14. Preparation of a chitosan derivative having sialic acid branches.

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Scheme 15. Introduction of b-cyclodextrin branches into chitosan.

ation with a formyl derivative of the acid (Scheme 14), and the products were characterized by interaction with lectins (Sashiwa et al., 2000). Sialic acid dendrimers provided another form of chitosan/sialic acid conjugates that might be interesting because of a high local concentration of the clustering sugar moieties (Sashiwa et al., 2001, 2002a).

With Cyclodextrins. Controlled oxidation of b-cyclodextrin transformed one of the C-6 hydroxymethyl groups into a formyl group, and the product allowed reductive alkylation to give a chitosan/ cyclodextrin conjugate (Yalpani and Hall, 1984). Allylation of a- and b-cyclodextrins and the subsequent ozonolysis afforded formylmethylated cyclo-

Scheme 16. Preparation of two chitosan derivatives for hostguest complexation.

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Scheme 17. O-Carboxymethylation of chitin.

Acetylation of N -alkylated chitosans afforded N-alkylated chitins having enhanced afnity for solvents (Kurita et al., 2002b). N-Adamantylethylchitosan was physically crosslinked by b-cyclodextrin dimers in solution (Lecourt et al., 2004).

dextrins, which were useful for preparing chitosans having pendant cyclodextrins (Scheme 15). The levels of hostguest complexations of the derivatives with p-nitrophenoxide in aqueous solutions were similar to those of the free cyclodextrins (Tanida et al., 1998; Nishiki et al., 2000). Acylation of an aminated b-cyclodextrin with uronic acid gave a cyclodextrin having a reducing sugar residue, which was capable of coupling with chitosan by the reductive alkylation technique (Scheme 16). The uronic acid-mediated chitosan derivative exhibited high afnity for tert-butylbenzoic acid and catechin as model guests, suggesting the possibility of application for encapsulation and ly-Velty delivery of biologically active species (Auze and Rinaudo, 2001). The chitosan/cyclodextrin derivative interacted with N-adamantylethyl-chitosan, which was also prepared by reductive alkylation of chitosan with 1-adamantaneacetaldehyde (Scheme 16). As observed via a viscosity increase, supramolecular assemblies took place between these two kinds of chitosan derivatives to form a network structure caused by the physical cross ly-Velty linking by hostguest complexation (Auze and Rinaudo, 2002).
With Nonsugars. Wide varieties of aldehydes have been used for the reductive alkylation of chitosan. With formaldehyde to hexanal, for instance, chitosans bearing N-methyl to N-hexyl groups were prepared (Muzzarelli et al., 1983). A water-soluble chitosan derivative having polyethylene glycol chains was obtained similarly (Sugimoto et al., 1998). A low extent of N-dodecylation of chitosan brought about hydrophobic aggregation of the product in aqueous solution (Philippova et al., 2001). 2-Pyridinecarboxaldehyde gave N-pyridylmethylchitosan, which selectively collected some metal cations such as palladium, platinum, and mercury (Baba and Hirakawa, 1992; Baba et al., 1998). N-

Carboxyalkylation. As in the carboxymethylation of cellulose, the reaction of chitin proceeds with monochloroacetic acid in the presence of a strong alkali such as sodium hydroxide (Scheme 17). Using 2-propanol as a cosolvent facilitates the reaction (Trujillo, 1968). The preferential substitution position is the C-6 hydroxy group as conrmed by the main chain hydrolysis of carboxymethylated chitin (Miyazaki and Matsushima, 1968). In the course of the reaction, N-deacetylation takes place as a side reaction to give amphoteric polymers having carboxyl and free amino groups. The carboxymethylated chitin is soluble in aqueous alkali. Chitosan gives N,O-carboxymethylated derivatives under similar conditions. N-Carboxymethylation of chitosan is effected through the Schiff base formation with glyoxylic acid and the reduction with sodium cyanoborohydride (Scheme 18) (Muzzarelli et al., 1982). This method, based on reductive alkylation, results in regioselective carboxymethylation of the amino group. Similarly, several other N-carboxyalkylated chitosans were prepared from carboxylic acids having an aldehyde or keto group such as pyruvic, b-hydroxypyruvic, 2-ketoglutaric, 2-oxopropionic, and levulinic acids (Muzzarelli, 1986; Muzzarelli et al., 1989; Shigemasa et al., 1995; Rinaudo et al., 2001). The resulting carboxyalkylated derivatives may nd applications as cosmetic ingredients, biomedical materials, and fungistatic agents (Biagini et al., 1991; Muzzarelli et al., 1993, 2001). Cyclic carboxylic anhydrides such as succinic and maleic anhydrides are often used to obtain amic acid derivatives that are easily soluble in aqueous alkali. N-Phthaloylation
Phthaloylation of Chitosan. Phthaloylation of the

water-soluble chitin, 50% deacetylated chitin, with phthalic anhydride afforded an N-substituted

Scheme 18. N-Carboxymethylation of chitosan by reductive alkylation.

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Scheme 19. Phthaloylation of chitosan in DMF and DMF/water.

derivative soluble in dimethyl sulfoxide (DMSO) (Kurita et al., 1982). When fully deacetylated chitosan was phthaloylated, the product showed higher solubility; it was readily soluble in aprotic polar organic solvents such as pyridine, DMSO, DMAc, and N,N-dimethylformamide (DMF) (Nishimura et al., 1990). The reaction, typically carried out with three equivalents of phthalic anhydride in DMF at 120130-C, was, however, found to involve partial O -phthaloylation in addition to the expected

N-phthaloylation, and the degree of substitution was up to 1.5. The O-phthaloyl groups could be replaced by the triphenylmethyl group or removed by transesterication leading to N-phthaloylated chitosan with a degree of substitution of 1.0 (Kurita et al., 2000b). O-Phthaloylation was completely suppressed by conducting the reaction in DMF containing 5% water to give regiospecically substituted 2-N-phthaloyl-chtiosan in one-step (Scheme 19) (Kurita et al., 2002a).

Scheme 20. Typical modication reactions of phthaloylated chitosan.

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Scheme 21. Lentinan isolated from Lentinus.

Chemical Modications of Phthaloylated Chitosan. With the phthaloylated chitosan, either N,

O-phthaloylated or N-phthaloylated chitosan, as a key intermediate, various modication reactions take place regioselectively as exemplied in Scheme 20 (Nishimura et al., 1991; Kurita et al., 1998). All these transformations proceed smoothly in solution in organic solvents and are quantitative in terms of the substitution degree, in contrast to the conventional sluggish reactions of chitin and chitosan. Phthaloylated chitosan is thus a suitable precursor for a wide variety of site-specic and quantitative modication reactions to construct well-dened molecular environments on chitin and chitosan and is the rst example that has enabled perfect discrimination of the three kinds of functional groups in chemical reactions. Of many kinds of possible modication reactions of phthaloylated chitosan, glycosylation to introduce sugar branches at C-6 is particularly important in view of the biological activities of some naturally occurring branched polysaccharides such as lentinan and schizophyllan isolated from mushrooms (Scheme 21). Acetylation of 6-O-triphenylmethyl-2-N-phthaloyl-chitosan followed by detriphenylmethylation gives an organosoluble derivative having a reactive

group only at C-6. Trimethylsilylation of the C-6 hydroxy group is effective to further enhance the solubility, and the derivative is soluble even in lowboiling solvents such as dichloromethane and 1,2-dichloroethane. As shown in Scheme 22, the silylated derivative underwent facile mannosylation with a mannose orthoester in 1,2-dichloroethane solution at room temperature. The branched products were deprotected with hydrazine to give a-mannose-branched chitosans, which were subsequently N-acetylated to a-mannose-branched chitins (Kurita et al., 1998). In a similar manner, branched chitins and chitosans having b-galactose (Kurita et al., 1997a) and b-maltose (Kurita et al., 2003; Yang et al., 2004) were synthesized. When an oxazoline derived from glucosamine was used as a glycosylation donor, branched amino polysaccharides composed of the same sugar units in both the backbones and branches were formed: chitin and chitosan derivatives having N-acetyl-b-glucosamine and b-glucosamine branches, respectively (Scheme 23) (Kurita et al., 2000a). The non-natural branched chitins and chitosans shown above were readily soluble in neutral water, unlike the insoluble linear chitin and chitosan and showed high capacities of moisture absorption and

Scheme 22. Synthesis of mannose-branched chitosan and chitin.

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Scheme 23. Synthesis of glucosamine-branched chitosan and N-acetylglucosamine-branched chitin.

retention. They were biodegradable, and the degradation rate was regulated by the branching extent; the susceptibility to lysozyme decreased with an increase in the degree of branching. The glucosamine-branched chitosan is especially noteworthy in that it exhibited higher antimicrobial activity than the original linear chitosan. These non-natural branched amino polysaccharides are thus interesting as new types of water-soluble bioactive polymeric materials.
Chemical Modications of Triphenylmethylated Chitosan. Deprotection of 6- O -triphenylmethyl-

2-N-phthaloyl-chitosan with hydrazine produces 6-O-triphenylmethyl-chitosan, which is indispensable for performing chemical modications of the amino group because of the solubility in common organic solvents. A typical reaction is the coupling with the peptide Acyl-Tyr-Ile-Gly-Ser-Arg-bAla-OH, where bAla is a spacer and Tyr-Ile-Gly-Ser-Arg (YIGSR) is a partial sequence of laminin that is a

cell adhesion protein known to be involved in the invasion and metastasis of tumor cells. The reaction proceeded efciently in homogeneous DMAc solution with diphenylphosphoryl azide as the coupling agent. Removal of the triphenylmethyl group gave rise to a chitosanYIGSR conjugate (Scheme 24) (Nishiyama et al., 2000). A similar conjugate was also obtained by the coupling of N-Gly-chitosan with the peptide (Hojo et al., 2000). The conjugates exhibited potent inhibitory activities on experimental lung metastasis with B16BL6 melanoma cells in mice, higher than that of the parent pentapeptide YIGSR. The enhanced activity will be attributable to the protection effect by chitosan against enzymatic digestion of the peptide in vivo, supporting the high potential of chitosan as a carrier for pharmacologically active species. Owing to the organo-solubility and protection of the C-6 hydroxy group, this derivative is also capable of other substitutions at the amino group efciently in solution in a controlled manner. For

Scheme 24. Preparation of a chitosan/peptide conjugate as an antitumor agent.

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Scheme 25. Full trimethylsilylation of chitin.

example, chitosans having poly(ethylene glycol) (Ouchi et al., 1998) and betaine (Holappa et al., 2004) branches were prepared by acylation with a poly(ethylene glycol) monocarboxylic acid and chlorobetainyl chloride, respectively. Triphenylmethylated chitosan has thus proved to be a versatile precursor for the selective modications.

Silylation. O-Trimethylsilylation of chitin is another approach to the effective interference with the strong intermolecular hydrogen bonding. In the reaction of chitin with hexamethyldisilazane in formamide at 70-C, the degree of substitution was 0.6 under the conditions where cellulose gave a fully substituted product (Harmon et al., 1973), indicating the lower reactivity of chitin. With a mixture of hexamethyldisilazane and chlorotrimethylsilane in pyridine, however, b-chitin was easily trimethylsilylated at 70-C, and full substitution was effected in 16 h (Scheme 25) (Kurita et al., 1999). Starting from a-chitin, it took about 96 h for completion. The resulting trimethylsilylated chitin with a degree of silylation above 1.5 was readily soluble in acetone and pyridine and swelled in other solvents. The Si-O linkage was labile and cleaved easily with aqueous acetic acid to regenerate chitin. These characteristics have made possible easy preparation of chitin lms; silylated chitin lms cast from an acetone solution were converted into chitin lms on brief steeping in aqueous acetic acid at room temperature. In addition to the remarkable solubility, silylated chitin exhibited much higher reactivity than the original chitin as evaluated by the triphenylmethylation of the C-6 primary hydroxyl group (Scheme 26) and the acetylation of the C-3 and C-6 hydroxy groups. Silylated chitin is thus a promising starting material for chemical modications (Kurita et al., 2005).

Another important modication reaction of silylated chitin is glycosylation. Although practically no reaction occurred between chitin and an oxazoline derived from glucosamine even at elevated temperatures, the glycosylation of silylated chitin with the oxazoline proceeded smoothly in 1,2-dichloroethane at 80-C, giving rise to branched chitin derivatives with varied substitution degrees. The products were transformed into branched chitosans by alkaline hydrolysis or into branched chitins by transesterication. This preparative procedure is short and simple to provide non-natural branched amino polysaccharides compared to the method based on phthaloyl-chitosan (see the section on Chemical Modications of Phthaloylated Chitosan). Chitosan was trimethylsilylated less easily than chitin, and a degree of substitution was up to 2.9 under appropriate conditions. The highly silylated chitosan was almost soluble in pyridine and showed signicant reactivity as conrmed by acetylation, suggesting the possibility of diverse chemical modications of chitosan (Kurita et al., 2004). Tosylation. Incorporation of bulky groups into chitin and chitosan markedly improves the afnity for organic solvents, and p-toluenesulfonyl (tosyl) is one of the proper substituents for preparing a soluble chitin derivative having considerable reactivity. Although the tosylation of chitin in pyridine was slow even at 100-C, it proceeded smoothly in a two-phase mixture of an aqueous alkali chitin solution and tosyl chloride in chloroform at 0 -C by the interfacial reaction technique (Scheme 27). The substitution degree reached 1.0 with excess tosyl chloride and then leveled off, most likely due to the preferential substitution at the less hindered C-6 position (Kurita et al., 1991a). The tosylation reaction of chitin also proceeded in DMAc/LiCl solution in the presence of triethylamine (Zou and Khor, 2005).

Scheme 26. Quantitative triphenylmethylation of trimethylsilylated chitin.

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Scheme 27. Tosylation of chitin by the interfacial conden-

sation technique.

The tosylated chitin became soluble in common solvents when the degree of substitution was above 0.4, and it is a convenient intermediate for further modications in homogeneous solution. It could be transformed to iodo-chitin with sodium iodide and to deoxy-chitin with sodium borohydride (Kurita et al., 1992). Ethyl benzoate, diethyl malonate, and diethylphosphite moieties were also introduced recently (Zou and Khor, 2005). Quaternary Salt Formation. The free amino groups of chitosan are protonated in aqueous acidic media, and some acids such as acetic, lactic, and hydrochloric acids are frequently used to prepare chitosan solutions. N,N-Dimethyl-chitosan obtained by reductive alkylation gave the N,N,N-trimethylated derivative on treatment with methyl iodide (Muzzarelli and Tanfani, 1985). With excess methyl iodide and sodium hydroxide, the amino group of chitosan was directly trimethylated, and the cationized chitosan was soluble in water in a wide pH range (Domard et al., 1986). Ammonium groups were introduced as side chains by the reaction with quaternized epoxides such as glycidyltrimethylammonium chloride. The product is an analogue of cationized cellulose, which is a common polycation component of shampoos (Lang et al., 1997b). On mixing solutions of chitosan and polyanions, polyelectrolyte complexes precipitate. As polyanion sources, dextran sulfate, chitosan sulfate, carboxymethyl-dextran, carboxymethyl-pullulan, car-

boxymethyl-cellulose, carboxymethyl-chitin, carboxymethyl-chitosan, alginic acid, hyaluronan, poly (aspartic acid), poly(glutamic acid), lignosulfonate, keratin derivatives, DNA, and poly(acrylic acid) have been used. The compositions and properties of the complexes are heavily dependent on the preparative conditions including the concentration, molar ratio, pH, and order of mixing. The resulting chitosan-based polyelectrolyte complexes are attracting increasing interest as biomedical materials; they may be useful for the repair of damaged blood vessels (Thierry et al., rd et al., 2003; 2003a,b), gene delivery (Ko pping-Ho gga Danielsen et al., 2004), anticoagulation and procoagulation (Serizawa et al., 2000, 2002), cartilage tissue engineering (Iwasaki et al., 2004), and controlled release (Hu et al., 2002; Etienne et al., 2005). Sulfation and Thiolation. Direct sulfation of chitin and chitosan is possible with various sulfating agents, but it is difcult to synthesize well-dened derivatives. Starting from triphenylmethyl-chitosan and triphenylmethyl-chitin, however, regioselectively sulfated products were synthesized to examine the effect of the substitution position on anti-HIV and blood anticoagulant activities. The products exhibited potent antiretroviral activity with a high inhibitory effect on the infection of AIDS virus in vitro (Scheme 28) (Nishimura et al., 1998). Acylation of triphenylmethyl-chitosan with palmitoyl chloride gave an N,O-diacylated or N,N,O-triacylated product depending on the reaction conditions. Subsequent detriphenylmethylation and sulfation afforded 6-Osulfated amphiphilic polysaccharides that were capable of forming Langmuir monolayers with a high collapse pressure (Nishimura et al., 1993). Sulfated N-decanoyl- and N-myristoyl-chitosans increased the rigidity of lipid membranes, probably because of the anchoring of their alkyl chains into the bilayer structure of the membranes (Nonaka et al., 2002).

Scheme 28. Preparation of regioselectively sulfated chitosan and chitin.

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Scheme 29. Immobilization of BSA on thiol-bearing chitosan.

The thiol-bearing chitin and chitosan are promising intermediates for modications, because the reactive thiol group is available for attaching various substances under mild conditions, wich is especially useful for conjugation with biologically active species. Acylation of chitosan with S-protected mercaptoacetic acid or treatment of tosyl-chitin with potassium thioacetate followed by deprotection gave thiol derivatives (Kurita et al., 1993c). They were used for enzyme immobilization (Kurita et al., 1997b) and graft copolymerization (Kurita et al., 1996). Ring-opening addition of 2-iminothiolane to the amino group of chitosan gave a thiol-bearing chitosan derivative that effectively underwent conjugation with a bovine serum albumin (BSA) derivative (Scheme 29) (Masuko et al., 2005). Concluding Remarks Although chitin was isolated in 1811 for the rst time from mushrooms by Braconnot, about 30 years
Table 3. Estimated Chitosan Use for the Year 2000 (Tons)

earlier than cellulose, it remained an unused biomass resource for a long time, in sharp contrast to cellulose. However, interest in this abundant biopolymer increased enormously in recent years. The research activity for chitin is now surprisingly high worldwide in both academia and industry as evidenced by the rapid increase in the numbers of relevant research papers and patents. Consequently, basic and applied researches have made great progress. Chitin-related products, particularly chitosan and the derivatives, are beginning to nd applications in a wide range of elds as a new type of functional material. Table 3 shows estimates of recent chitosan use (Sandford, 2003). Major elds of the applications include dietary supplements, coagulants for water treatment, food preservatives, oligosaccharides, agriculture, ingredients for cosmetics and toiletries, and bacteriostatic fabrics. Glucosamine, the monomeric unit of chitosan, has proved clinically efcacious against osteoarthritis and is consumed in the largest amount among the

Market Nutraceuticals (dietary supplements) Flocculation (water treatment) Foods (preservation) Oligosaccharides Agriculture Cosmetics Textiles (hygienic) Pulp and paper Feed Medical devices Total

North America 500 125 0 0 25 25 0 25 10 1 711

Europe 125 25 0 0 0 25 0 0 0 1 176

Asia 250 200 125 150 75 50 50 25 25 1 951

Other 125 50 25 0 25 0 0 0 10 0 235

Total 1000 400 150 150 125 100 50 50 45 3 2073

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