JULY/AUGUST 1996 Volume 7, Number 4 © Copyright 1996 by the American Chemical Society
COMMUNICATIONS Synthesis and Anticoagulant Activity of Sulfated Glucoside-Bearing Polymer Mitsuru Akashi,* Nobuyuki Sakamoto, Kazuya Suzuki, and Akio Kishida Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890, Japan. Received October 23, 1995X
Poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA) sulfate], which contains sulfated glucoside residues, was prepared by the reaction with N,N-dimethylformamide (DMF)/sulfur trioxide (SO3) complex. The degree of sulfation was easily controlled by changing the amount of DMF/SO3 complex added and reaction time. The total human blood clotting time in the presence of poly(GEMA) sulfate was prolonged by increasing the dose or the degree of sulfation of the polymer. The anticoagulant activity of poly(GEMA) sulfate was also compared with that of dextran sulfate, poly(styrenesulfonic acid), poly(vinylsulfuric acid), and heparin. The result suggests that sulfated saccharide residues are essential for endowing anticoagulant activity to synthetic polymer.
It is well-known that heparin has many biological activities such as an anticoagulant activity, a cell growth stimulation activity, an antivirus activity, and a plasma clearing activity. Among them, the anticoagulant activity is the essential activity for heparin. The physiological activity of heparin, however, has not been completely clarified, and it is not so easy to immobilize heparin onto solid polymer surfaces. To solve these problems, various heparinoids, which are synthetic polymers or analogues of naturally occurring heparin, were synthesized and their biological activities studied. From the viewpoint of the synthesis of heparinoid polymers, naturally occurring polysaccharides such as dextran and chitin were sulfated and their anticoagulant activities evaluated (14). Even for synthetic polymers, it has been believed that sulfation is effective for acquisition of anticoagulant * Author to whom correspondence should be addressed (telephone +81-992-85-8320; fax +81-992-55-1229). X Abstract published in Advance ACS Abstracts, May 15, 1996.
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activity (5-7). It has been considered that not only the anionic charge but also the linearity of the polymer backbone, such as a polysaccharide chain, are important in revealing the anticoagulant activity (8). The anticoagulation mechanism of the heparinoid polymers, however, has not been clarified with regard to the importance of monosaccharide units yet. In our previous study, free radical polymerization and copolymerization of glucosyloxyethyl methacrylate (GEMA) were found to give highly water soluble polymers and hydrogels, which have glucoside residue in their side chains, and their properties were reported (9-11). They can be considered as one of the polysaccharide model compounds. In this paper, we report the synthesis of sulfated poly(GEMA) and its anticoagulant activity to determine whether sulfated saccharide residues are important for acquisition of the anticoagulant activity. Scheme 1 illustrates the synthesis of sulfated poly(GEMA). Poly(GEMA) was obtained by free radical polymerization of GEMA in distilled water using ammonium peroxodisulfate (APS) as an initiator (10-12). © 1996 American Chemical Society
394 Bioconjugate Chem., Vol. 7, No. 4, 1996
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Scheme 1. Synthesis of Poly(GEMA) Sulfate
Table 1. Sulfation of Poly(GEMA)a run no.
reaction time (h)
degree of sulfationb
1 2 3 4 5
0.3 1.8 5.0 12.0 24.0
1.91 3.24 3.33 3.42 3.75
Figure 2. Anticoagulant activity of sulfated poly(GEMA), PSS, PVS, DS, and heparin.
a Reaction temperature was room temperature. b Degree of sulfation was estimated by elemental analysis.
Figure 3. Effect of degree of sulfation of poly(GEMA) sulfate on total human blood clotting time.
Figure 1. IR spectra of poly(GEMA) and sulfated poly(GEMA).
The reaction of poly(GEMA) and excess N,N-dimethylformamide (DMF)/sulfur trioxide (SO3) in DMF at room temperature gives poly(GEMA) sulfate in analogy with the sulfation of chitin (3). As shown in Table 1, the degree of sulfation is able to change according to reaction conditions such as reaction time. [The synthesis of poly(2-glucosyloxyethyl methacrylate) sulfate was as follows: Poly(GEMA) (2 g) and SO3 (3 g) were mixed in N,Ndimethylformamide (DMF) (50 g) in molar ratio of 1:3. The mixture was stirred under nitrogen atmosphere, and the temperature of the mixture was not allowed to rise over 25 °C. At the end of the reaction time, the reaction product was neutralized with NaOH (3 g) in a 1:10 water (27 g)-methanol (270 g) mixture solution and purified by dialysis against distilled water for 3 days to remove sodium sulfate. Then, polymer solution was freeze-dried for 4 days. The degree of sulfation was estimated by elemental analysis.] After 24 h, most of the hydroxy groups on the glucoside residue were converted to the sulfate group. The degree of sulfation can be also controlled by the concentration of SO3. Sulfation was confirmed by infrared spectroscopy as shown in Figure 1. Absorptions at 1250 and 810 cm-1 of poly(GEMA) sulfate were assigned to SdO and CsOsS stretch bonds, respectively.
The anticoagulant activity of the resulting poly(GEMA) sulfate was evaluated by total human blood clotting time using the method of Lee-White (13). In the Lee-White test, clotting time was prolonged when coagulant factors were inhibited. The results are shown in Figures 2 and 3. It is clear that poly(GEMA) sulfate prolongs the coagulation time, while, on the other hand, poly(GEMA) shows no effect. Both the concentration and the degree of sulfation of poly(GEMA) sulfate influence the coagulation time. However, the coagulation time in the presence of poly(GEMA) sulfate was not extended longer than that of heparin, which has an anticoagulant activity of 187.5 IU/mg of solid, in the same dose. As shown in Figure 2, poly(styrenesulfonic acid) (PSS) and poly(vinylsulfuric acid) (PVS) , which were purchased from Nacalai Tesque, scarcely prolonged the blood clotting time in this condition. In the case of dextran sulfate (DS), which is a modified polysaccharide, the anticoagulant activity was higher than that of poly(GEMA) sulfate, though the degree of sulfation is about 1.0. As a result, the potency of anticoagulant activity of poly(GEMA) sulfate was between those of synthetic sulfated polymers and sulfated polysaccharide. These results suggest that sulfated saccharide residues play an important role in the acquisition of anticoagulant activity. The activity of heparin is much higher than that of these heparinoid polymers. It is quite difficult to covalently immobilize heparin to polymeric materials for the preparation of antithrombogenic materials, though slow release of heparin from polymer matrix (14, 15) and the physical coating of materials with heparin (16) are considerably easy. It is also difficult to immobilize a polysaccharide such as dextran sulfate from the terminal saccharide unit and to control the chain length of the immobilized polysaccharide. Using GEMA monomer, it is possible to prepare
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a hydrogel by free radical polymerization (11) and to modify polymer surfaces by surface graft polymerization (17). Sulfated poly(GEMA) with the anticoagulant activity, therefore, can be useful as a starting material for antithrombogenic biomaterials. We will compare the antithrombogenicity of poly(GEMA) sulfate with that of thrombomodulin (18, 19), which is protein C activation protein, onto endothelial cell, immobilized materials, or aramid silicone (20) resins which have hydrophobic silicone surfaces. The mechanism of the anticoagulant activity of poly(GEMA) sulfate will be reported in a further publication.
(10) Kitazawa, S., Okumura, M., Kinomura, K., Sakakibara, T.,Nakamae, K., Miyata, T., Akashi, M., and Suzuki, K. (1994) Preparation and some properties glycoside-bearing polymer. In Carbohydrate as Organic Raw Materials (Descotes, G., Ed.) Vol. 2, pp 115-135, VCH, Weinheim.
ACKNOWLEDGMENT
(13) Shapiro, G. A., Huntzinger, S. W., and Wilson, J. E. (1977) Variation among commercial activated partial thromboplastin time reagents in response to heparin. Am. J. Clin. Pathol. 67, 477-480.
We thank Mr. T. Furuzono for experimental support. We are grateful to Professor N. Miyauchi of Kagoshima University for helpful discussions. LITERATURE CITED (1) Mauzac, M., and Jozefonvictz, J. (1984) Anticoagulant activity of dextran derivatives. Part I: Synthesis and characterization. Biomaterials 5, 301-304. (2) Muzzarelli, R. A. A., Tanfani, F., and Emanuelli, M. (1984) Sulfated N-(carboxymethyl)chitosans. Carbohydr. Res. 126, 225-231. (3) Nishimura, S., and Tokura, S. (1987) Preparation and antithrombogenic actives of heparinoid from 6-O-(carboxymethyl)chitin. Int. J. Biol. Macromol. 9, 225-232. (4) Doctor, V. M., Lewis, D., Coleman, M., Kemp, M. T., Marbley, E., and Sauls, V. (1991) Anticoagulant properties of semisynthetic polysaccharide sulfates. Thromb. Res. 64, 413-425. (5) Hatanaka, K., Yoshida, T., Miyahara, S., Sato, T., Ohno, F., and Uryu, T. (1987) Synthesis of new heparinoids with high anticoaglant activity. J. Med. Chem. 30, 810-814. (6) Ito, Y., Iguchi, Y., Kashiwagi, T., and Imanishi, Y. (1991) Synthesis and nonthrombogenicity of polyetherurethaneurea film grafted with poly(sodium vinyl sulfonate). J. Biomed. Mater. Res. 25, 1347-1359. (7) Fougnot, C. (1986) Synthetic heparin-like materials. Dev. Hematol. Immunol. 14, 201-206. (8) Chr. Heuck, C., Schiele, U., Horn, D., Fronda, D., and Ritz, E. (1985) The role of surface charge on the accelerating action of heparin on the antithrombin III-inhibited activity of R-thrombin. J. Biol. Chem. 260, 4598-4603. (9) Kitazawa, S., Okumura, M., Kinomura, K., and Sakakibara, T. (1990) Synthesis and properties of novel vinyl monomers bearing a glycoside residues. Chem. Lett., 1733-1736.
(11) Fukudome, N., Suzuki, K., Yashima, E., and Akashi, M. (1994) Synthesis of nonionic and hydrogels bearing a monosaccharide residue and their properties. J. Appl. Polym. Sci. 52, 1759-1763. (12) Nakamae, K., Miyata, T., Jikihar, A., and Hoffman, A. S. (1994) Formation of poly(glucosyloxyethyl methacrylate)Concanavalin A complex and its glucose-sensitivity. J. Biomater. Sci. Polym. Ed. 6 (1), 79-90.
(14) Akashi, M., Takeda, S., Miyazaki, T., Yashima, E., Miyauchi, N., Maruyama, I., Okadome, T., and Murata, Y. (1989) Antithrombogenic poly(vinyl chloride) with heparin- and/or prostaglandin I2-immobilized in hydrogels. J. Bioact. Compat. Polym. 4, 4-16. (15) Kwon, I. C., Bae, Y. H., and Kim, S. W. (1994) Heparin release from polymer complex. J. Control. Rel. 30, 155-159. (16) Gu, Y. J., Oevern, W., Akkerman, C., Boonstra, P. W., Huyzen, R. J., and Wildevuur, C. R. H. (1993) Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass. Ann. Thorac. Surg. 55, 917-922. (17) Iwata, H., Kishida,A., Suzuki, M., Hata, Y., and Ikada, Y. (1988) Oxidation of polyethylene surface by corona discharge and subsequent graft polymerization. J. Polym. Sci., Polym. Chem. Ed. 26, 3309-3322. (18) Akashi, M., Maruyama, I., Fukudome, N., and Yashima, E. (1992) Immobilization of human thrombomodulin on glass beads and its anticoagulant activity. Bioconjugate Chem. 3, 363-365. (19) Kishida, A., Ueno, Y., Fukudome, N., Yashima, E., Maruyama, I., and Akashi, M. (1994) Immobilization of human thrombomodulin onto poly(ether urethane urea) for developing antithrombogenic blood-contacting materials. Biomaterals 15 (10), 848-852. (20) Furuzono, T., Yashima, E., Kishida, A., Maruyama, I., Matsumoto,T., and Akashi, M. (1993) A novel biomaterial: poly(dimethysiloxane)-polyamide multiblock copolymer I. Synthesis and evaluation of blood compatibility. J. Biomater. Sci. Polym. Ed. 5, 89-98.
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