Biomacromolecules 2003, 4, 1264-1268
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Nonnatural Branched Polysaccharides: Synthesis and Properties of Chitin and Chitosan Having Disaccharide Maltose Branches Keisuke Kurita,*,† Hirofumi Akao,† Jin Yang,† and Manabu Shimojoh‡ Department of Applied Chemistry, Faculty of Engineering, Seikei University, Musashino-shi, Tokyo 180-8633, Japan, and Research and Development Department, Toyo Suisan Kaisha, Ltd., Kohnan, Minato-ku, Tokyo 108-8501, Japan Received March 13, 2003; Revised Manuscript Received June 27, 2003
Synthesis and properties of chitin and chitosan derivatives having β-maltoside branches at C-6 have been studied. Chitosan was first transformed into an organosoluble acceptor having a reactive group only at C-6, 3-O-acetyl-2-N-phthaloyl-6-O-trimethylsilylchitosan. Glycosylation with an ortho ester from D-maltose was performed successfully at room temperature in dichloromethane in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst. The degree of substitution could be controlled by the reaction conditions and was up to 0.56. Full deprotection gave chitosan with maltoside branches, and the subsequent N-acetylation resulted in the formation of the corresponding chitin derivative. The introduced disaccharide unit improved hydrophilic properties considerably compared to monosaccharide units as confirmed by high solubility in water and moisture absorption and retention ability. The enzymatic degradability and antimicrobial activity were moderate probably because of the bulky nature of the branches. Introduction A wide variety of linear polysaccharides occur in nature, of which cellulose and chitin are the most abundant and typical structural materials. In contrast, lentinan,1 schizophyllan,2 and pestalotan3 found in mushrooms are branched polysaccharides, β-1,3-glucans having β-1,6-glucoside branches; they are interesting because of their solubility in water, as well as immunoadjuvant activity. These distinctive properties are ascribable to the branched structure, because the corresponding linear β-1,3-glucan is not soluble and has no immunoadjuvant activity. The branched polysaccharides are therefore attractive models for novel types of functional macromolecules owing to their unique physicochemical and biological properties. Branching of easily accessible linear polysaccharides may be a straightforward way to construct tailored biopolymers with desired functions. For developing functional polysaccharides, chitosan should have a much greater potential than chitin and cellulose in view of biological activities and versatility of chemical modifications, because of the presence of free amino groups.4 In the course of our study on chemical modifications and thereby functionalization of chitin and chitosan, we have developed procedures suitable for introducing monosaccharides such as galactose,5 mannose,6 and (N-acetyl)glucosamine7 to synthesize nonnatural branched polysaccharides and revealed the marked influence of the introduced sugar branches on properties including solubility, biodegradability, * To whom correspondence should be addressed. † Seikei University. ‡ Toyo Suisan Kaisha.
and antimicrobial activity. These results prompted us to further examine the possibility of introducing a disaccharide and to discuss the structure-property relationship of the disaccharide-branched polysaccharides. In this paper, we report the introduction of maltose as a typical disaccharide into chitin and chitosan and some properties of the resulting maltoside-branched polysaccharides. Experimental Section General Procedures. IR and UV spectra were recorded on JASCO IRA-700 and Ubest-30 instruments, respectively. NMR spectra were taken with a JEOL JNM-GX270 in DMSO-d6 or D2O. Elemental analysis was performed with a Perkin-Elmer 2400 II. Gel permeation chromatography (GPC) was conducted with a Shimadzu LC-10AD (column, TSK guard column + TSKgel GMPWXL no. PWMXE 0009 + TSKgel GMPWXL no. 0053; solvent, 0.1% aqueous lactic acid; flow rate 1.0 mL/min; standards, pullulan). Chitosan. Chitin was deacetylated with 40% aqueous sodium hydroxide at 110 °C for 4 h three times to give chitosan with a degree of acetylation of 0.0.8 Chitosan Derivative Acceptor. 3-O-Acetyl-2-N-phthaloyl-6-O-trimethylsilylchitosan was prepared from fully deacetylated chitosan in five steps including phthaloylation, tritylation, acetylation, detritylation, and trimethylsilylation according to the reported procedure.6,9 Maltose Ortho Ester Donor. D-Maltose was acetylated with acetic anhydride and sodium acetate to give the peracetylated derivative, mp 158-160 °C (lit.10 159-160 °C), which was then brominated with 30% HBr/acetic acid. The product was treated with excess ethanol in the presence
10.1021/bm034074p CCC: $25.00 © 2003 American Chemical Society Published on Web 08/06/2003
Nonnatural Branched Polysaccharides
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Scheme 1
of 2,6-lutidine, a hindered base necessary for ortho ester formation, and the resulting syrup was crystallized from ethanol/hexane to give 3,6,2′,3′,4′,6′-hexa-O-acetyl-1,2-O(1-ethoxyethylidene)-R-D-maltose as white small crystals, mp 132-134 °C (lit.11 133-134 °C). The overall yield from maltose was 55%. Glycosylation. To a solution of 0.50 g (1.23 mmol of repeating unit) of 3-O-acetyl-2-N-phthaloyl-6-O-trimethylsilylchitosan in 10 mL of dichloromethane were added 2.47 g (3 equiv to pyranose unit) of the maltose ortho ester and 0.022 mL (27 mg, 0.10 equiv to pyranose unit) of trimethylsilyl trifluoromethanesulfonate (TMSOTf). The solution was stirred at room temperature in a nitrogen atmosphere for 72 h and poured into 300 mL of methanol to form a precipitate. The product was collected by centrifugation, washed in 300 mL of methanol overnight, filtered, and dried to give 0.69 g of a light tan powdery material. The degree of substitution (ds) was calculated to be 0.40 from the peak area ratio of 2.87 for acetyl/phthaloyl in the 1H NMR spectrum and to be 0.47 from the C/N value of the elemental analysis (footnote d of Table 1). The yield was 96% on the basis of ds 0.40. IR (KBr): ν 3478 (OH), 1780 (shoulder, imide CdO), 1749 (ester CdO), 1721 (imide CdO), 1231 (C-O-C), and 1150-1000 cm-1 (pyranose). 1H NMR (DMSO-d6): δ 1.6-2.1 (m, COCH3), 3.5-5.4 (m, pyranose H), and 7.8-8.0 ppm (m, phthalimide H). One-Step Deprotection. The glycosylated product (ds 0.40, 400 mg) was dispersed in 70 mL of hydrazine monohydrate, and the mixture was stirred at 80 °C in nitrogen for 18 h. The resulting solution was dialyzed, concentrated, and freeze-dried to give 150 mg (75%) of the fully deprotected product as an off-white powdery material. The Mw was determined to be 59,000 by GPC. IR (KBr): ν 3428 (OH and NH2), 1630 (NH2), and 1150-1000 cm-1 (pyranose). N-Acetylation. To a dispersion of 87 mg of the fully deprotected branched product in 10 mL of methanol was added 0.3 mL of acetic anhydride. The mixture was stirred at room temperature for 24 h and poured into 300 mL of water to give a homogeneous solution. The solution was dialyzed in deionized water for 2 days, concentrated under reduced pressure, and freeze-dried. The yield of the off-white
product was 86 mg (86%). IR (KBr): ν 3406 (OH and NH), 1651 (amide I), 1562 (amide II), and 1150-1000 cm-1 (pyranose). Moisture Absorption and Desorption. Chitin and the derivatives were dried, and the weight changes were measured at 93% relative humidity and then at 32% relative humidity.7 Enzymatic Degradation. Branched chitins and original chitin (degree of deacetylation, 0.10) were treated with lysozyme from egg white in pH 4.50 acetate buffer at 37 °C. After the reaction, a ferricyanide solution was added, and the decrease in the amount of ferricyanide was measured by UV spectroscopy as reported.5,12 Antimicrobial Activity. The antimicrobial activities of chitosan and the branched chitosan were evaluated in terms of the number of colony-forming units by the previously reported method.6,13 Results and Discussion Branching by Glycosylation. To introduce maltose branches into chitin (1) and chitosan (2), 3-O-acetyl-2-Nphthaloyl-chitosan and the derived 6-O-trimethylsilyl derivative (3) are expected to be suitable as acceptors because they allow regioselective glycosylation at C-6. Although these phthaloyl derivatives are soluble in organic solvents, 3 shows particularly high solubility even in low-boiling solvents and was thus chosen for the glycosylation with a donor, an ortho ester of D-mannose (4) (Scheme 1). The acceptor 3 was prepared from fully deacetylated chitosan 2 through consecutive five-step regioselective modification reactions that were quantitative in terms of ds. In our previous study on the glycosylation with galactose and mannose ortho esters,5,6 reaction conditions were examined in detail to reveal that moderate to high degrees of branching (0.3-0.5) were attained with 3-5 equiv of ortho esters in a 24 h reaction. The glycosylation of 3 was thus carried out with 3 or 5 equiv of 4 for 24-72 h. Both 3 and 4 were soluble in dichloromethane, and the glycosylation proceeded in homogeneous solution. After the reaction, the branched product (5) was precipitated in methanol, and the fine precipitate was collected by centrifugation because of
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Table 1. Glycosylation of Chitosan Derivative 3 with Maltose Ortho ester 4a 3 (g)
4/3 (mol/mol)
solvent (mL/g 3)b
time (h)
yield (%)
dsc
0.30 0.50 0.24 0.30 0.10
3 3 3 3 5
20 20 63 67 100
24 48 48 72 72
61 96 57 50 36
0.35 0.40d 0.47 0.51e 0.56
a Catalyst, TMSOTf (0.10 equiv to pyranose); temp, rt. b Amount of dichloromethane per gram of 3. c Degree of substitution calculated from the peak area ratio of acetyl/phthaloyl in 1H NMR in DMSO-d6. d ds ) 0.47 calculated from C/N of elemental analysis. Calcd for (C16H15NO7)0.53(C42H49NO24)0.47‚H2O: C, 52.79; H, 5.18; N, 2.18. Found: C, 52.78; H, 4.91; N, 2.18. e ds ) 0.54 calculated from C/N of elemental analysis. Calcd for (C16H15NO7)0.46(C42H49NO24)0.54‚0.8H2O: C, 52.93; H, 5.17; N, 2.06. Found: C, 52.83; H, 4.97; N, 2.05.
Figure 2. NMR spectra of 5 (ds 0.40) in DMSO-d6 (A), 6 in D2O (B), and 7a in D2O (C).
Figure 1. IR spectra of 3 (A), 5 (ds 0.40) (B), and 7a (C).
the difficulty in direct filtration. The product was obtained as a pale tan powdery material, and the yield was sometimes moderate, as summarized in Table 1, because of the mechanical loss in isolation by centrifugation and filtration. The IR spectrum in Figure 1 shows strong ester bands at around 1745 and 1230 cm-1, indicating the introduction of acetyl-protected maltose branches. The ds was determined from the peak area ratio of acetyl/ phthaloyl (δ 1.6-2.1 and 7.8-8.0 ppm, respectively) in 1H NMR spectra, and a typical spectrum is shown in Figure 2. The ds could also be calculated from the C/N value of the elemental analysis and was close to the ds value based on NMR spectroscopy. Table 1 indicates that the extent of glycosylation reaction was not heavily dependent on the amounts of 4 and solvent. However, the ds value increased with reaction time and reached 0.56 in 72 h reaction. Deprotection and N-Acetylation. Deprotection of 5 was first carried out by two-step reactions to give branched chitosan (6): alkaline hydrolysis with sodium hydroxide or sodium carbonate to remove acetyl groups followed by
hydrazinolysis for dephthaloylation. The Mw of the resulting 6 was, however, 25 000, implying possible degradation during the deprotection process. One-step deprotection with hydrazine was then examined (Scheme 2) and found to be efficient to give 6 of Mw 59 000. This procedure therefore proved to be superior to the twostep deprotection in suppressing main-chain degradation. The IR spectrum of 6 was quite similar to that of chitosan. The complete deprotection was also confirmed by the disappearance of the acetyl and phthaloyl peaks in the 1H NMR spectra as exemplified in Figure 2. The resulting branched chitosan 6 was in turn acetylated with acetic anhydride in methanol to transform 6 into branched chitin (7) (Scheme 2). As a result of N-acetylation, the product showed distinctive amide I and II bands in the IR spectrum. Although these amide bands became relatively weak owing to the incorporation of maltose branches, the spectrum resembles that of chitin (Figure 1). The Nacetylation was also supported by a peak at 2.03 ppm in the NMR spectrum in Figure 2. Molecular Weight. GPC measurements were carried out using pullulan standards. As shown in Table 2, the molecular weights of 6 and the derived 7a were quite similar, and moreover, the Mw/Mn values were low compared to that of chitosan, as observed in other cases of branching.6,7 The Mn and Mw of 6 and 7 appeared lower than those of the starting chitosan 2, though quantitative comparison was difficult because of the structural difference. This suggests the possibility of some degradation during the preparative procedure, but even so it seemed to occur only to a low extent under these reaction conditions. Solubility. Qualitative solubility of the products was examined in large quantities of solvents, and the results are listed in Table 3. The glycosylated products having protective
Biomacromolecules, Vol. 4, No. 5, 2003 1267
Nonnatural Branched Polysaccharides Scheme 2
Table 2. Molecular Weights of Branched Chitosan and Chitina
2 6c 7ad 7be
dsb
Mn
Mw
Mw/Mn
0.40 0.40 0.56
61 000 35 000 22 000 14 000
206 000 59 000 51 000 23 000
3.38 1.69 2.32 1.64
a Determined by GPC in 0.1% aqueous lactic acid with pullulan standards. b Degree of substitution determined by NMR spectroscopy. c Prepared by one-step deprotection. d Prepared by N-acetylation of 6. e Prepared by two-step deprotection followed by N-acetylation.
Table 3. Solubility of Maltoside-Branched Chitosan and Chitina
1 2 5 6 7
H2O
MeOH
CHCl3
DMFb
( + +
( ( (
+ ( (
+ ( (
a + ) soluble; ( ) partially soluble or swelled; - ) insoluble. bN,NDimethylformamide.
groups, 5, showed high solubility in a wide range of organic solvents including low-boiling solvents such as chloroform. The deprotected products, 6 and 7, were readily soluble in pH 7.0 water and swelled considerably in common organic solvents. This solubility behavior is commonly observed with the other branched chitins and chitosans reported thus far.5-7 Hygroscopicity. The water solubility of 7 suggested the enhanced hydrophilic nature, and the hygroscopicity was elucidated in environments of different relative humidities. The influence of the disaccharide on the hygroscopicity was evident in Figure 3, and judging from moisture absorption and retention, 7 was highly hygroscopic as compared even with a monosaccharide-branched chitin. Biodegradability. The importance of biologically degradable polymeric materials is growing in view of biomedical utility, as well as environmental protection, and the biodegradability of the branched chitin was evaluated from the susceptibility to lysozyme. The formation of the reducing ends was followed by titration with ferricyanide, and as a measure of the progress of degradation, the decrease in the absorbance of ferricyanide (∆Absorbance) is illustrated in Figure 4. As shown in this figure, the maltoside-chitin 7 was enzymatically degraded more rapidly than chitin, which would primarily be attributed to the water solubility of 7 and hence the homogeneous reaction conditions. The ds values of 0.40 (7a) and 0.56 (7b) were, however, not significant in degradation. When compared with the galactoside-chitin5 and N-acetylglucosaminide-chitin,7 which were highly biodegradable, 7 was degraded slowly. The low
Figure 3. Moisture absorption (93% relative humidity) and retention (32% relative humidity) behavior of 1 (9, A), galactoside-chitin (ds 0.40) ([, B), and 7a (b, C) at 25 °C.
Figure 4. Lysozyme susceptibility of 1 (9, A), 7a (b, B), 7b (2, C), galactoside-chitin (ds 0.40) ([, D), and N-acetylglucosaminide-chitin (ds 0.45) (1, E).
susceptibility of 7 may therefore be interpreted in terms of the bulkiness of the branches, and the bulky maltoside branches would have reduced the accessibility to the enzyme. Antimicrobial Activity. Chitosan exhibits antimicrobial activity, but it is soluble only in aqueous acidic solution. The branched chitosan 6 was, however, soluble in neutral water and would be interesting as a water-soluble antimicrobial agent. The antimicrobial activity was therefore examined in comparison with those of chitosan and mannoside-chitosan, and the results are summarized in Table 4. Although 6 exhibited substantial antimicrobial activity, this activity was low compared to the activity of the mannosidechitosan. As shown in the table, the linear chitosan 2
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Table 4. Antimicrobial Activity of Branched Chitosans suppression of the growth (%)a mannoside-chitosanb
6
Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Streptococcus mutans Candida albicans
2c
50 ppm
5 ppm
50 ppm
5 ppm
5 ppm
52 ( 3.0 23 ( 2.0 26 ( 2.7 89 ( 5.5 19 ( 1.5 ∼0
20 ( 5.0
90 ( 2.6 39 ( 2.3 26 ( 3.1 >99 36 ( 7.9 ∼0
26 ( 5.4
62 ( 5.0 81 ( 4.7 21 ( 1.4 83 ( 5.5 51 ( 5.1 72 ( 2.7
16 ( 0.7
93 ( 7.9
a Percentage of the colony forming units decreased by the treatment with a 50 or 5 ppm solution of branched chitosans or linear chitosan. b Chitosan having mannoside branches: ds 0.42, Mw 43 000 (data from ref 6). c Data from ref 7.
exhibited higher activity than these branched chitosans, but as previously reported,7 chitosan having glucosamine branches showed even higher activity than the original chitosan. These results confirm the importance of the density of the free amino group, as well as the bulkiness of the branches. Conclusions Acceptor 3 has proved to be a useful organosoluble precursor for regioselective glycosylation with the disaccharide, allowing the introduction of β-maltoside branches at the C-6 position of chitin and chitosan in a well-controlled manner. The disaccharide branch significantly influenced the properties in many respects; for instance, it increased affinity for water more than monosaccharides. The presence of the disaccharide unit, however, somewhat lowered the biodegradability and antimicrobial activity probably because of the bulky nature of the branches. These results indicate that various characteristics could be controlled by the molecular structure of the introduced sugar branches, and diversification of the molecular design would be possible to synthesize tailored polysaccharides with advanced functions. Acknowledgment. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 12650872) from the Ministry of Education, Science and Culture of Japan and by a grant from Towa Shokuhin Kenkyu Shinkoukai.
References and Notes (1) Chihara, G.; Maeda, Y.; Hamuro, J.; Sasaki, T.; Fukuoka, F. Nature 1969, 222, 687. Chihara, G.; Maeda, Y.; Hamuro, J. Int. J. Tissue React. 1982, 4, 207. (2) Mitani, M.; Ariga, T.; Matsuo, T.; Asano, T.; Saito, G. Int. J. Immunopharmacol. 1980, 2, 174. (3) Misaki, A.; Kawaguchi, K.; Miyaji, H.; Nagae, H.; Hokkoku, S.; Kakuta, M.; Sasaki, T. Carbohydr. Res. 1984, 129, 209. (4) Kurita, K. In Desk Reference of Functional Polymers: Syntheses and Applications; Arshady, R., Ed.; American Chemical Society: Washington, DC, 1997; pp 239-259. Kurita, K. Prog. Polym. Sci. 2001, 26, 1921. (5) Kurita, K.; Akao, H.; Kobayashi, M.; Mori, T.; Nishiyama, Y. Polym. Bull. 1997, 39, 543. (6) Kurita, K.; Shimada, K.; Nishiyama, Y.; Shimojoh, M.; Nishimura, S. Macromolecules 1998, 31, 4764. (7) Kurita, K.; Kojima, T.; Nishiyama, Y.; Shimojoh, M. Macromolecules 2000, 33, 4711. (8) Kurita, K.; Mori, S.; Nishiyama, Y.; Harata, M. Polym. Bull. 2002, 48, 159. (9) Nishimura, S.; Kohgo, O.; Kurita, K.; Kuzuhara, H. Macromolecules 1991, 24, 4745. (10) Wolfrom, M. L.; Lederkremer, R. M. J. Org. Chem. 1965, 30, 1560. (11) Heynes, K.; Trautwein, W. P.; Espinosa, F. G. Chem. Ber. 1966, 99, 1183. (12) Kurita, K.; Yoshino, H.; Nishimura, S.; Ishii, S. Carbohydr. Polym. 1993, 20, 239. (13) Shimojoh, M.; Masaki, K.; Kurita, K.; Fukushima, K. Nippon Nogeikagaku Kaisi 1996, 70, 787.
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