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Communications Chemical Modification of Chitosan: Preparation and Lectin Binding Properties of r-Galactosyl-chitosan Conjugates. Potential Inhibitors in Acute Rejection following Xenotransplantation Hitoshi Sashiwa,† Jennifer M. Thompson,† Sanjoy K. Das,† Yoshihiro Shigemasa,‡ Sasmita Tripathy,† and Rene´ Roy*,† Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5, and Faculty of Engineering, Tottori University, Tottori 680-8552, Japan Received March 30, 2000
Water-soluble R-galactosyl-chitosan conjugates were prepared by reductive amination of p-formylphenyl β-melibioside on chitosan in good yield. Strong binding with phytohemagglutinin was demonstrated using Griffonia simplicifolia. Carbohydrates are ubiquitous components of cell wall membranes.1 As such, they can participate in forefront intermolecular and intracellular events. Moreover, carbohydrate residues can serve as cell surface receptors for antibodies, viruses, and bacteria.2 To mimic some of the above interactions, tremendous efforts have been directed at the syntheses of artificial carbohydrate-polymer conjugates (neoglycopolymers).3 Chitosan is a polysaccharide consisting of β-(1-4)-2-amino-2-deoxy-D-glucopyranose (GlcN) repeating unit and includes a small amount of N-acetyl-Dglucosamine (GlcNAc) residues. Recently, a number of interesting biological properties were reported for chitosan.4-7 Chitosan itself is nontoxic7 and biodegradable8 and is thus suitable as macromolecular carrier. Human antibodies against R-galactosyl (Gal) epitope, which occur naturally, are abundant (1-2%) and responsible for acute rejection of xenotransplanted organs from lower animals.9 Therefore, artificial glycopolymers having R-Gal epitope are of much interest from the viewpoint of medical transplantation of pig liver since they can block immune rejection. The present work describes the preparation and lectin binding property of chitosan-melibiose conjugates containing R-Gal residues. To prepare R-Gal-chitosan conjugates, we attempted the direct reductive N-alkylation of chitosan with p-formylphenyl melibioside (2), which contains an exposed R-galactoside epitope. Compound 2 was prepared in 85% yield from peracetylated melibiose R-bromide by an adaptation of a published procedure (Scheme 1).10 The results from the reductive amination are summarized in Scheme 2 and Table 1. The degrees of substitution (DS) were controlled by the * To whom correspondence may be addressed. Tel.: (613)-562 5800, ext 6055. FAX: (613)-562 5170. E-mail:
[email protected]. † University of Ottawa. ‡ Tottori University.
Scheme 1a
a Reagents: i, HOPhpCHO, TBAHS, EtOAc, 1 M Na CO , 85%; ii, 2 3 NaOMe, MeOH, quant.
amount of 2 in two kinds of differently substituted chitosan (a, NH2 ) 0.8; b, NH2 ) 0.96; DP ) 140, Mn ) 24 kDa); see Experimental Section. Approximately 50% of melibioside 2 reacted with the primary amines of chitosan independently from the degree of substitution of chitosan. Unfortunately, all conjugates 3a,b were insoluble in water, although they were soluble in acidic water. To further improve the solubility of 3a,b, the remaining amino groups were transformed into 4a,b by N-succinylation with succinic anhydride in 90100% yields (i, succinic anhydride, AcOH, H2O, MeOH, room temperature, 1 day; ii, 0.5 M NaOH, room temperature, 2 h, dialyzed, lyophilized).11 The chemical structures of the succinylated conjugates are summarized in Table 2. Some succinyl groups were also introduced onto the secondary
10.1021/bm005536r CCC: $19.00 © 2000 American Chemical Society Published on Web 06/02/2000
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Scheme 2a
prepared. Preliminary biological evaluation of these conjugates and others showed strong affinity toward human antiR-galactoside antibodies.12 Experimental Section
a Reagents: i, succinic anhydride, AcOH/H O/MeOH, room tempera2 ture, 1 day; ii, 0.5 M NaOH, room temperature, 2 h.
Table 1. Preparation of Conjugate 3a,b with Aldehyde 2 chitosan compd
mg
NH2
2 (equiv)
3a 3a 3b 3b
100 40 40 40
0.8 0.8 0.96 0.96
0.5 1.0 0.5 1.0
recovery (mg)
DSa
MWb (kDa)
150 90 80 90
0.26 0.46 0.22 0.47
39 51 36 51
a DS was determined from the peak area of aryl protons (δ 7.2-7.5 ppm) and N-acetyl group (δ 2.0 ppm: 0.60 H for 3a) or H-2 GlcN residue (δ 3.2 ppm: 0.96 H for 3b). b MW was determined from the FW × DP based on NMR data and DP ) 140 (Mn ) 24 kDa) for the original chitosan.
Table 2. Chemical Structures and Binding Test of Conjugates 4a,b to Griffonia simplicifolia Lectin starting Chitosan (mole fraction NHAc) a a b b bc
0.20 0.20 0.04 0.04 0.04
mole fraction substitution NH- NH- N(sugar)- MW binding NH2 Sucb Sugar (Suc) (kDa) to lectina 0 0.02 0.21 0 0.46
0.54 0.32 0.53 0.49 0.50
0.20 0.14 0.19 0.43 0
0.06 0.32 0.03 0.04 0
48 60 43 59 30
+ ++ ++ ++ -
a ++, strong band observed; +, weak band; -, no band. b Suc, COCH2CH2CO2Na. c N-Succinylated chitosan.
amines of N-glycosylated moieties of chitosan. All succinylated conjugates 4a,b were freely soluble in neutral water. The protein binding properties of the novel R-Gal-chitosan conjugates were initially evaluated with the plant lectin Griffonia simplicifolia (GSI-B4), which is known to be specific toward R-galactoside residues. Strong or weak immunodiffusion bands were observed for water-soluble conjugates 4a,b. In contrast, N-succinylated chitosan taken as a negative control showed no band against the lectin, thus demonstrating the specificity of the binding toward the R-Gal epitope in the conjugates. In conclusion, water-soluble and lectin binding R-Gal-chitosan conjugates were successfully
Materials and Methods. Two kinds of chitosan (a, NH2 ) 0.8; DP ) 140; Mn ) 24 kDa and its N-deacetylated product,13 b, NH2 ) 0.96) were obtained from Kyowa Tecnos Co. Plant lectin Griffonia simplicifolia (GS-I-B4) was purchased from Sigma Co. The DS of the conjugates were determined by 1H NMR (Bruker AMX 500 MHz). The DS of amino group was determined by a colorimetric method with ninhydrin at 570 nm. Preparation of Conjugate. Chitosan (100 mg of a or b) was dissolved in H2O (10 mL), AcOH (100 mg), and MeOH (40 mL). Various amounts of 2 (Table 1) were added to the solution, which was stirred at room temperature. After 1 h, NaCNBH3 (2-3 equiv/2) was added. After 1 day, the reaction mixture was quenched by precipitation with 5% NaOH (2 mL) and acetone (80 mL). The precipitate was collected by filtration, dispersed with 0.5 M NaOH for 2 h, dialyzed, and lyophilized to give conjugate 3a,b. Selected data for 3a (DS ) 0.26) are as follows: 1H NMR (0.2 M DCl in D2O) δ 2.07 (s, 0.60 H, NHAc), 3.20 (br m, 0.80 H, H-2 of GlcN), 3.5-4.2 (br m, H-2,3,4,5,6 of β-D-glucose (Glc) and R-D-galactose in melibiose, H-2 of GlcNAc, H-3,4,5,6 of GlcN and GlcNAc), 4.60 (br, -NH-CH2-Ph), 4.87 (br, H-1 of β-D-Glc), 5.25 (br, H-1 of R-D-Gal), 7.22 (br, 0.52 H, H-2,6 of Ar), 7.49 (br, 0.52 H, H-3,5 of Ar); 13C NMR δ 25.1 (NHAc), 58.8 (C-2 of GlcN), 63.1 (C-6 of GlcN), 63.9 (C-6 of Glc and Gal), 100.4 (C-1 of β-D-Glc), 101.6 (C-1 of R-D-Gal), 119.6 (C-2 and C-6 of Ar), 127.7 (C-4 of Ar), 135.4 (C-3 and C-5 Ar), 160.2 (C-1 Ar). N-Succinylation of conjugate was performed as described previously.11 Agar gel diffusion experiments were performed in 1% agarose (BDH) containing 2% poly(ethylene glycol) (MW ) 8000, Sigma) in phosphate-buffered saline (PBS) according to a published literature.14 The concentration of conjugates 4a,b was at 1 mg/mL in PBS, and that of the GSI-B4 lectin was 2 mg/mL. The precipitation bands were allowed to form overnight at 4 °C in a humid chamber. Acknowledgment. We are thankful to NSERC (Canada) for financial support and to Kyowa Tecnos Co. (Japan) for a gift of chitosan. References and Notes (1) Sharon, N. In Complex Carbohydrates. Their Chemistry, Biosynthesis, and Functions; Addison-Wesely: Reading, MA, 1975. (2) Goldstein, L. J.; Poretz, R. D. In The Lectins. Properties, Functions, and Applications in Biology and Medicine; Liener, I. E., Sharon, N., Goldstein, L. J., Eds.; Academic Press: Orlando, FL, 1986. (3) (a) Roy, R. Carbohydr. Eur. 1999, 27, 34. (b) Roy, R. In Carbohydrate Chemistry; Boons, G.-J., Ed.; Blackie A&P: London, 1998; pp 243. (c) Roy, R. Top. Curr. Chem. 1997, 183, 241. (4) Minami, S.; Okamoto, Y.; Matsuhashi, A.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y.; Tanigawa, T.; Tanaka, T.; Tokura, S. In AdVances in Chitin and Chitosan; Brine, C. J., Sandford, P. A., Zikakis, J. P., Eds.; Elsevier: London, 1992; p 61.
Communications (5) Nishimura, K.; Nishimura, S.; Nishi, N.; Saiki, I.; Tokura, S.; Azuma, I. Vaccine 1984, 2, 93. (6) Tanigawa, T.; Tanaka, Y.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y. In AdVances in Chitin and Chitosan; Brine, C. J., Sandford, P. A., Zikakis, J. P., Eds.; Elsevier: London, 1992; p 206. (7) Uchida, Y. In Chitin, Chitosan Handbook; Japanese Society of Chitin and Chitosan; Gihodo Co.: Japan, 1995; p 302. (8) (a) Sashiwa, H.; Saimoto, H.; Shigemasa, Y.; Ogawa, R.; Tokura, S. Int. J. Biol. Macromol. 1990, 12, 295. (b) Shigemasa, Y.; Saito, K.; Sashiwa, H.; Saimoto, H. Int. J. Biol. Macromol. 1994, 16, 43. (9) (a) Galili, U. Blood Cells 1988, 14, 205. (b) Cooper, D. K. C.; Oriol, R. In Glycoscience; Gabius, H.-J., Gabius, S., Eds.; Chapman & Hall: Weiheim, 1997; pp 531. (c) Galili, U. Sci. Med. 1998, 5, 28. (d) Wang, J.-Q.; Chen, X.; Zacharek, S. J.; Zhang, W.; Wang, P. G. J. Am. Chem. Soc. 1999, 121, 8174.
Biomacromolecules, Vol. 1, No. 3, 2000 305 (10) Roy, R.; Tropper, D. F.; Romanowska, A.; Letellier, M.; Cousineau, L.; Meunier, S. J.; Boratynski, J. Glycoconjugate J. 1991, 8, 75. (11) Sashiwa, H.; Shigemasa, Y. Carbohydr. Polym. 1999, 39, 127. (12) Roy, R.; Thompson, J. M.; Sashiwa, H.; Das, S. K.; Tripathy, S.; Gabius, H.-J. 67th ACFAS meeting, May 10-14, 1999, Ottawa, ON. (13) Horton, D.; Lineback, D. R. Methods Carbohydr. Chem. 1965, 5, 403. (14) Ouchterlony, O.; Nilsson, L. A. In Handbook of Experimental Immunology; Weir, D. M., Ed.; Blackwell Scientific Publications: Oxford, 1978; Chapter 19.
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