Synthesis of Sulfated Colominic Acids and Their Interaction with

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Synthesis of Sulfated Colominic Acids and Their Interaction with Fibroblast Growth Factors Megumi Kunou,† Masako Koizumi,† Kengo Shimizu,† Mitsuo Kawase,‡ and Kenichi Hatanaka*,§ Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4295 Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan; NGK Insulators, Ltd., 1 Maegata-cho, Handa, 467-8530, Japan; and Institute of Industrial Science, University of Tokyo, 7-22-1 Roppongi, Minato-ku, Tokyo, 106-8558, Japan Received February 28, 2000; Revised Manuscript Received June 5, 2000

Colominic acid, an R(2f8)-linked poly(sialic acid), was sulfated and characterized by NMR spectroscopy. During sulfation, the secondary hydroxyl group at C-4 had almost the same reactivity as the primary hydroxyl group at C-9, while the secondary hydroxyl group at C-7 was hardly substituted. Analysis by molecular modeling suggested that the lack of substitution at C-7 was due to a steric hindrance. A mobility shift assay indicated that FGF-2 bound to the sulfated colominic acid. Synthetic sulfated colominic acid potentiated the mitogenic activity of FGFs for fibroblasts in the same manner as heparin. Sulfated colominic acid with a low degree of sulfation was able to potentiate FGF activity. Regardless of the degree of sulfation, sulfated colominic acid-induced cytotoxicity was not observed. It was suggested that the carboxyl groups in sulfated colominic acid cooperate with the sulfate groups to reinforce the interaction with FGFs and to reduce the cytotoxicity of sulfated colominic acids. Introduction Colominic acid (Figure 1), an R(2f8)-linked poly(sialic acid), was originally isolated from capsular Escherichia coli K11 and structurally characterized.2,3 It has been demonstrated that colominic acid is inherently flexible and can adopt a wide range of energetically favorable helices.4,5 Although naturally found as a random coil, colominic acid can exist in a highly ordered local helix in solution.6 FGF-1 and -2 are prototype members of the heparin binding growth factors7 which comprise greater than 18 members. They display more than 80% sequence similarity with an identical three-dimensional structural fold8,9 and heparin binding site.10,11 Heparin or heparan sulfate proteoglycans are essential for the biological activities of FGFs.12 A high content of 2-O-sulfate groups in IdoA residues of heparin/heparan sulfate is required for the activation of both FGF-1 and FGF-2, and for maximal stimulation of FGFs which is achieved over 12 monosaccharide units of polysaccharide sequence.13,14 A high content of 6-O-sulfate groups in GlcNS residues has been found to be required for activation of FGF-1, but not FGF-2.15,16 Therefore, it has been reported that the degree of sulfation, positioning of sulfate groups, and molecular weight of heparin/heparan sulfate affect its interaction with FGFs. * To whom correspondence should be addressed. E-mail: [email protected]. Telephone: +81-3-3402-6231 (ext. 2432), Fax: +81-3-34023067. † Tokyo Institute of Technology. ‡ NGK Insulators, LTD. § University of Tokyo.

Figure 1. Chemical structure of colominic acid.

Heparin and heparan sulfate have carboxyl groups and sulfate groups. The binding of proteins to heparin is usually electrostatic. Sulfate groups establish a higher acidic environment relative to carboxyl groups. In the interaction of sulfated polysaccharides with FGFs, the sulfate groups appear to bind predominantly to basic amino acid residues in the FGFs. Polyanions having only sulfate groups interact strongly with FGFs17,18 and mimic the effects of heparin on the biological activities of FGFs.19,20 Since the carboxyl groups in acidic polysaccharides have a negative charge in physiological environments (pKa of a carboxyl group is approximately 4), it is plausible that carboxyl groups are important to the interaction with FGFs. Bagheri-Yarmand et al. have reported that benzylamidated carboxymethyl-dextran inhibited neovessel formation via competition with heparan sulfate for FGF-2 activation.21 The carboxyl groups in polysaccharides may complement the sulfate groups to significantly contribute to the interaction of sulfated polysaccharide and FGFs. Heparin includes both carboxyl and sulfate groups, while colominic acid includes only carboxyl groups. Recently, Ushijima et al. reported that sulfated colominic acid suppressed neuronal cell death induced by scrapie prion protein and HIV-1 gp120.22 It has been found that nonsulfated glycans are much less effective than the sulfated compounds

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Scheme 1. Synthesis of Sulfated Colominic Acid

Table 1. Results of Sulfation of Colominic Acida,b total ratio of reacn yield 10-4Mn deg of compound -SO3/-OHc time (h) (%)d (Da)e Mw/Mn sulfationf S-col-0.89g S-col-1.88g S-col-0.71h S-col-1.47h S-col-2.06h

1.8 5.9 0.6 1.2 8.0

0.75 1.0 1.5 1.0 1.5

43.8 30.0 41.4 18.6 20.9

1.1 1.4 5.4 5.7 5.2

1.6 1.2 1.2 1.3 1.1

0.89 1.88 0.71 1.47 2.06

a Sulfated colominic acid (S-col). The number at the end of the compound name represents the number of sulfate groups per sugar unit. b Colominic acid (1.0 g) was dissolved in 30 mL of DMF, and the reaction was carried out at 0 °C. c Mole ratio of SO3-pyridine complex to hydroxyl groups per sugar unit. d From Na salt of colominic acid. e Molecular weight determined by GPC (eluent, 0.05 M NaCl; reference, pullulan). f Number of sulfate groups per sugar unit. Calculated from elemental analysis. g 10-4M of starting colominic acid ) 3.7 (Da), M /M ) 1.2. This was n w n later purified by dialysis. h 10-4 × Mn of starting colominic acid ) 5.0 (Da), Mw/Mn ) 1.3. After the reaction, the product was purified by gel filtration (Sephadex LH-20 and G-25).

for inhibition of scrapie prion protein.23 The anti-HIV-1 activity of sulfated colominic acid has been found to be much stronger than that of the nonsulfated colominic acid.24 In this study, colominic acid was sulfated and characterized using spectroscopy. The interaction of FGFs and sulfated colominic acid was investigated, highlighting the role of carboxyl groups in sulfated polysaccharides. Results and Discussion Sulfation of Colominic Acid. The sodium salt of colominic acid was changed to the tri-n-butylammonium salt in order to increase its solubility in organic solvents such as dimethylformamide (DMF). Colominic acid was then sulfated by a SO3-pyridine complex under anhydrous conditions (Scheme 1). The results of the sulfations are summarized in Table 1. Dialysis for the purification of sulfated colominic acid resulted in fragmentation of the polysaccharide, while gel filtration at 4 °C yielded high molecular weight sulfated polysaccharide. Since colominic acid and sulfated colominic acid both decomposed easily in acidic conditions at room temperature (data not shown), they were stored in slightly basic solutions (pH 7-8) at low temperatures (4 °C) to prevent saccharide chain cleavage. Characterization of Sulfated Colominic Acid. The IR spectra of colominic acid and sulfated colominic acids (Scol-0.71 and S-col-2.06 in Table 1) are shown in Figure 2. The intensities of the characteristic bands of the -S(dO)2

Figure 2. IR spectra of colominic acid and sulfated colominc acid on KBr: (a) S-col-2.06; (b) S-col-0.71; (c) colominic acid.

stretch (1230 cm-1 and 1120 cm-1) and the S-O stretch (1000-800 cm-1) in R-O-SO3 increased with increasing degree of sulfation. Sialic acid residues in colominic acid readily underwent interresidue esterification under acidic conditions, forming lactones between the C-1 carboxyl group and the hydroxyl group on C-9 of the adjacent residue.25,26 The characteristic bands of esters or lactones (1750 cm-1) were not observed in the IR spectra of the sulfated colominic acids (Figure 2). Comparison of the obtained NMR spectra of sulfated colominic acid (Figures 3 and 5) with polylactone spectra from literature,27 further confirmed that intermolecular lactone was not formed under the conditions used in this study. One-dimensional 1H NMR, distortionless enhancement by polarization transfer (DEPT), and two-dimensional heteronuclear single quantum coherence (HSQC) spectra of S-col0.71 are shown in Figures 3-5, respectively. The peaks were assigned by using correlation spectroscopy (COSY) spectra and data found in the literature.3,28 The C-9 carbon peak

Sulfated Colominic Acids

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Figure 3. 1H NMR spectrum of S-col-0.71 in D2O at 25 °C. “S-” denotes a shifted peak caused by sulfation of a hydroxyl group on the carbon. “′” denotes a shifted peak caused by sulfation of hydroxyl groups on (an) adjacent carbon (s). An astersik denotes the ratio of the area under the peaks. Figure 5. HSQC spectrum of sulfated colominic acid (S-col-0.71) in D2O at 25 °C. “S-” denotes a shifted peak caused by sulfation of a hydroxyl group on the carbon. “′” denotes a shifted peak caused by sulfation of hydroxyl groups on (an) adjacent carbon (s).

Figure 4. DEPT spectra of sulfated colominic acid (s-col-0.71) in D2O at 25 °C. “S-” denotes a shifted peak caused by sulfation of a hydroxyl group on the carbon. “′” denotes a shifted peak caused by sulfation of hydroxyl groups on (an) adjacent carbon (s).

moved to a lower magnetic field (from 64.6 to 71.1 ppm) upon sulfation of the hydroxyl group (-OH) at C-9, and the C-3 carbon peak moved to a higher magnetic field (from 43.4 to 41.9 ppm) upon sulfation of -OH at C-4 (Figure 4). Complete carbon and proton peak assignments of S-col-0.7 are shown for the first time (Figure 5). In Figure 6, the carbon peaks that were substituted by sulfate groups (C-4 and C-9) shifted to lower magnetic fields (approximately 6-8 ppm), and adjacent carbon atom peaks (C-3 and C-5 for C-4, and C-8 for C-9) shifted to higher magnetic fields (1-3 ppm). 13C NMR spectra of sulfated colominic acids (Figure 6) showed that the hydroxyl group at C-7 was hardly substituted even under highly favorable reaction conditions (i.e. large amount of reagent and long reaction time). The secondary hydroxyl group at C-4 showed similar reactivity as the primary hydroxyl group at C-9, and substantially higher

Figure 6. 13C NMR spectra of colominic acid and sulfated colominic acid in D2O at 30 °C (external standard, CD3CD2CO2D): (A) S-col2.06; (B) S-col-0.71; (C) colominic acid. “S-” denotes a shifted peak caused by sulfation of a hydroxyl group on the carbon. “′” denotes a shifted peak caused by sulfation of hydroxyl groups on (an) adjacent carbon (s).

reactivity than the secondary -OH at C-7. Figure 3 showed that the ratio of the peak area of substituted 4-OH (3′H(eq))

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Figure 7. Supposed structure of colominic acid and sulfated colominic acid (six residues) constituted by molecular modeling using Discover/ Insight II. Blue, red, green, and white are N, O, C, and H atoms, respectively. The number in parentheses represents sequential residue number extending from top to bottom of the reducing terminal: (A) colominic acid, view of helical axis. (B) side view of S-col-2. The S atom is indicated by a yellow ball, the O atom at position-7 by a red ball.

to unsubstituted 4-OH (3H(eq)) was 1 to 2.6. Since 7-OH was rarely sulfated, the unsubstituted 4-OH effectively represents the level of sulfation at 9-OH. As the total degree of sulfation was 0.71, 28% of the 4-OH was sulfated, while 43% of 9-OH was sulfated. Structure of Colominic Acid. The structures of colominic acid and 4,9-di-O-sulfated colominic acid (S-col-2) (six residues) were estimated by Insight II and Discover (MSI, Inc.). Colominic acid has been found to have a helical conformation in 5% aqueous acetone solution.4,5 Dimethylformamide (DMF) was used as a reaction solvent in this study. Since DMF is aprotic and polar, it can be presumed that the 3-dimensional structure of colominic acid in DMF is similar to that in 5% aqueous acetone solution. Therefore, the values of dihedral angles from literature5 were used for the starting structure of colominic acid. Brisson et al. proposed that the predominantly stable helical structure in aqueous solution was n ) 4 (where n ) the number of residues per turn of the helix).5 Similarly, results of this study obtained from molecular modeling indicated that colominic acid (six residues) maintained a helical structure (n ) 4) (Figure 7A). The S-col-2 structure used in the modeling was created by substituting the 4-OH and 9-OH groups of the minimized structure of colominic acid by sulfated groups. Then, S-col-2 was minimized simply, and the resulting S-col-2 structure maintained helicity (Figure 7B). CD spectra of colominic acid and sulfated colminic acid are shown in Figure 8. C-1 carboxyl groups and C-5 N-Ac groups contributed to the dichronic absorption of colominic acid.29 The positive peak of colominic acid centered at 200 nm shifted slightly to a higher wavelength and decreased in intensity with increasing degree of sulfation. The modest decrease in intensity indicated that the surrounding environment of the chromophores was relatively unchanged. Molecular modeling of S-col-2 revealed that the region between each helix turn was expanded due to the bulky substituent (-OSO3-). This

Figure 8. CD spectra of colominic acid and sulfated colominc acid at 20 °C in H2O: (heavy bold line) colominic acid; (medium weight line) S-col-0.71; (light weight line) S-col-1.88. θR: molar ellipticity per redisure (104 deg cm2 dmol-1).

expansion was likely responsible for the observed differences in CD spectra, thus supporting the assumption that the helix structure was maintained throughout sulfation. It was found that each 4-OH is located outside the helix, while the 9-OH and 7-OH are located inside the helix (Figure 7A). The reactivity of the hydroxyl groups was found to be: 9-OH = 4-OH . 7-OH. The internally located 7-OH (a secondary hydroxyl group) was minimally sulfated, and the internally located 9-OH (a primary hydroxyl group) had similar reactivity to the externally located 4-OH (a secondary hydroxyl group). The unexpected high reactivity of 4-OH may have been due to its external location. An external location is beneficial as it allows the -OH to be virtually free of steric hindrance. Moreover, aside from external location, the deoxygenated C-3 may have contributed to decrease steric hindrance of 4-OH (Figure 7A). In the disubstituted unit (i.e., 4, 9-di-O-sulfated unit), 7-OH may not have reacted because of steric hindrance by peripheral


Figure 9. Effect of acidic polysaccharide on FGF activities (37 °C, 5% CO2, 48 h): (0) control; (2) with FGF-1 10 ng/mL; (O) with FGF-2 10 ng/mL.

functional groups specifically the -OSO3- groups at position 9 and N-acetyl groups at position 5 (Figure 7B). Biological Activity of Sulfated Colominic Acid. Sulfated colominic acids potentiated the mitogenic activities of FGFs in the same manner as heparin did (Figure 9). The vertical axis in Figure 9 displays the absorbance at 570 nm (ref 630 nm) measured by an MTT assay, in which the cell numbers could be estimated. Heparin (Mn ) 1.46 × 104, number of sulfate groups per sugar unit was 1.5) strongly potentiated the proliferative activities of both FGFs (Figure 9A). With adequate concentration in the medium, sulfated colominic acid activated the biological activity of FGFs in the same manner as heparin did (Figure 9, parts A, C, and D). The minimum concentration of heparin required for activation of FGFs has been found to be different for FGF-1 and FGF-2 (2-4 µg/mL for FGF-1 and ∼100 ng/mL for FGF-2, on adrenocortical endothelial cells),13 and the binding sites for FGF-1 in heparan sulfate of mammary cells are described as being weaker than those of FGF-2.30 In this study, cell proliferation by FGF-2 responded more abruptly to the amount of sulfated colominic acid and S-col-1.88 was able to activate the mitogenic activities of FGFs in lower concentrations than S-col-0.89 (Figure 9, parts C and D). The results indicated that FGF-2 could be activated by sulfated polysaccharides with lower degrees of sulfation and in lower concentrations relative to FGF-1. Mannopyranan sulfate (Mn ) 9.27 × 104; degree of sulfation, 0.98, MPS0.98) weakly stimulated the mitogenic activity of FGF-2, but did not potentiate FGF-1 activity (Figure 10B). Sulfated dextran (Mn ) 3.69 × 104; degree of sulfation, 0.98, SuD0.98) and carboxymethylated dextran (Mn


) 2.69 × 104; degree of carboxymethylation, 1.22, CMD1.22) did not potentiate the proliferative activity of both FGFs (Figure 10, parts C and A). In general, sulfated polysaccharides with a low degree of substitution (carboxyl and sulfate) slightly stimulated or did not activate mitogenic activities of FGFs. However, S-col-0.89 potentiated the mitogenic activity of both FGFs (Figure 9C). For dextran derivatives, the presence of only carboxyl or only sulfate groups did not stimulate biological activities of FGFs for the human umbilical vein endothelial cell growth.31 On the other hand, derivatized dextrans having sulfate and carboxyl groups have mimicked the effects of heparin on FGFs.20,32,33 It was also reported that carboxyl-reduced heparin fails to potentiate FGF-1.34 Therefore, it can be inferred that the carboxyl groups in sulfated colominic acid contribute to the activation of FGFs. The apparent interaction of FGFs and sulfated colominic acid with a low degree of sulfation is likely due to a unique property of colominic acid in solution. The structure of colominic acid in solution has been shown to be highly flexible.29 It appears that in aqueous solution, the flexible conformation of the main chain is easily adjusted to the specific binding site of FGFs. Sulfated dextran and heparin exhibited slight cytotoxicity toward the fibroblasts in the absence of FGFs (Figures 10C and 9A). Polysaccharides with a high content of sulfate groups but lacking carboxyl groups (e.g., dextran sulfate and mannopyranan sulfate) generally showed strong cytotoxicity.35,36 However, sulfated colominic acid with a high degree of sulfation exhibited no cytotoxicity even at high concentration (Figure 9D). The cytotoxicity of heparin has been found to be lower than that of polysaccharides having only sulfate groups with corresponding degrees of sulfation. Both heparin and sulfated colominic acid have carboxyl groups and sulfate groups. Therefore, it seems that the presence of carboxyl groups in sulfated polysaccharide can effectively reduce the cytotoxicity. Another possibility is since one sugar unit of sulfated colominic acid occupied a larger volume than one of heparin or dextran sulfate, the total anionic charge density of heparin or dextran sulfate was more concentrated than that of sulfated colominic acid. Therefore, the anionic charge density of polysaccharides might also affect the cytotoxicity against fibroblasts. Interaction between Sulfated Colominic Acid and FGF2. A mobility shift assay using polyacrylamide gel electrophoresis was used to show the interaction of sulfated colominic acid with FGF-2 (Figure 11). Proteins were dyed and sulfated polysaccharides were decolorized. In Figure 11, FGF-2 alone migrated toward the cathode (lane 1) and BSA (bovine serum albumin) alone migrated toward the anode (lane 2) because the isoelectric point of FGF-2 is 9.6 and that of BSA is 4.7-4.9. When both substances were loaded together (lane 9), each substance should migrate independently toward its respective attraction mode when there is no interaction. If a complex forms, total electrostatic charge of the resulting complex will affect the gel shift pattern. In Figure 11, the migration of both sulfated polysaccharide and FGF-2 toward the cathode indicates that sulfated polysac-

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Figure 10. Effects of acidic polysaccarides on FGF activities (37 °C, 5% CO2, 48 h): (0) sulfated polysaccharide only; (2) with FGF-1 ng/mL; (O) with FGF-2 10 ng/mL.

Figure 11. Mobility shift binding assay for FGF-2 using polyacrylamide gel electrophoresis. 5 µg of protein and 15 µg of polysaccharide were loaded.

charides bound to FGF-2, and the sulfated polysaccharide and FGF-2 complex carried excess negative charge (lanes 3, 5, 7). N-Sulfoacharan sulfate (consisting of glucosamine-2-sufate 1 f 4 linked to iduronic acid-2-sulfate) has been found to strongly bind to FGF-2, but not potentiate FGF-2 mitogenicity.37 Contrastingly, this study revealed that sulfated colominic acid did not only directly bind to FGF-2, but also potentiated mitogenic activity of FGFs. Molecular Modeling. The heparin binding site of FGF-2 in FGF-2 and heparin-derived tetra- or hexasaccharide complex has been described by X-ray crystallography.10 Moy et al. showed that the conformation of FGF-2 in aqueous solution was similar to its crystal structure.8 A simulated structure of the S-col-2 (three residues) and FGF-2 complex based on the crystal structure10 is shown in Figure 12. Energy minimized S-col-2 (3 residue) manually docked to the heparin binding site of FGF-2. To fit S-col-2 on the binding site, various orientations of S-col-2 were examined. Only one orientation of S-col-2 could dock on the binding site. Therefore, it seems that the suggested structure resulting from molecular modeling may be a possible structure of FGF-2 complex with S-col-2. The binding site of FGF-2 to S-col-2 in Figure 12 was the same as that shown for heparin in the literature.8,10 It appeared that the carboxyl group in S-col-2 could strongly interact with Arg 121 at the high affinity binding site in FGF-

2. Sulfate groups appeared to be located in the vicinity of basic amino residues that were located at the high affinity binding site in FGF-2. This result suggests that S-col-2 was able to interact with FGF-2 in the same manner as heparin does. However, it has been reported that the carboxyl group of the iduronic acid residue in heparin hexasaccharide can interact with Lys 136 at a low affinity binding site in FGF2.10,38 Whereas, the FGF-2 model observed in this study showed the carboxyl group in sulfated colominic acid to be relatively far from Lys 136 (Figure 12). There may be two possible explanations for this. It is plausible that the interaction of Lys 136 and its nearest carboxyl group is functionally insignificant in the interaction of FGF-2 with sulfated polysaccharides. The second possibility depends on the aspect that the high flexibility of the sulfated colominic acid sugar chain29 for conformational change whereby the carboxyl group is rotated approximately 60° toward Lys 136 and this type of conformation change would shorten the distance between the carboxyl group and Lys 136 thus allowing their interaction. Recently, Ogura et al. reported that the heparin binding sites of FGF-1 were quite similar to those observed for FGF2-heparin hexasaccharide complex.9 Therefore, it is expected that not only FGF-2, but also FGF-1 can recognize sulfated colominic acid in a similar manner as heparin. Conclusion Sulfation of colominic acid was successfully carried out by SO3-pyridine complexes in DMF at low temperature. Whereas the secondary hydroxyl group at position-4 easily reacted with the sulfating reagent and showed similar reactivity as the primary hydroxyl group at position-9, the secondary hydroxyl group at position-7 was rarely sulfated. Molecular modeling suggests that the differences among the hydroxyl group for sulfation were due to steric hindrance. It is essential that a specific site on the FGFs bind to heparin for FGF function. In other words, since sulfated colominic acid potentiated proliferative activity of FGFs, it is plausible that it interacted with FGFs in the same manner as heparin. Sulfated colominic acids strongly potentiated the mitogenic activity of FGFs, and did not show any cytotoxicity, even with a high degree of sulfation and at high concentration. It is noteworthy that the sulfated colominic



Figure 12. Supposed binding site of sulfated colominic acid (S-col-2) and FGF-2 constitiuted by molecular modeling using Discover/Insight II. The higher affinity binding sites are magenta, whereas the lower affinity binding sites are yellowish green.

acid with a low degree of sulfation (S-col-0.89) was capable of stimulating mitogenic activity of the FGFs. In addition to sulfate groups, it seemed that the carboxyl groups of sulfated colominic acid were also important in stimulating FGF activity. The results indicate that the carboxyl groups in sulfated polysaccharides played important roles in interacting with FGFs by strengthening mitogenic activity of FGFs, and by weakening the cytotoxicity of sulfated polysaccharides. These findings suggest that sulfated colominic acid may be useful as a nontoxic potentiator of FGFs. Experimental Section Materials and Chemicals. E. coli. colominic acid was kindly provided by NGK Insulators, Ltd.39 Human recombinant FGF-1 and -2 were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Sigma, Nakarai Tesque, Inc. (Kyoto, Japan), and Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sulfation of Colominic Acid. Sodium salt of colominic acid (5.0 g) was dissolved in cold water (250 mL). The solution was adjusted to pH 8 with 1 N NaOH and then slowly stirred for several hours at 4 °C. The viscous solution was passed through an ion exchange column (Amberlite IR120B, H+ type) at 4 °C. The pH of the eluate was immediately adjusted to 5.0 by the addition of 10% tri-n-butylamine in ethanol. The eluate was then washed three times with cold diethyl ether and lyophilized to yield colominic acid tri-n-butylamine salt as a slightly yellowish powder (yield 70.1%). Sulfation was carried out in an Ar atmosphere at 0 °C as recommended in the literature.40,41 Tri-n-butylammonium salt of colominic acid (1.0 g) was dissolved in anhydrous DMF (50 mL). SO3-pyridine complex in anhydrous DMF (50 mL) was then added dropwise to the solution, and the mixture was stirred at 0 °C (cf. Table 1). The reaction was terminated by the addition of cold water (20 mL) followed by immediate pH adjustment to 9.0 with 1 N NaOH. The reaction mixture was added dropwise to a large volume of acetone (2 L) while stirring vigorously. The precipitate was collected by decantation and centrifugation (2500 g) and then dried in vacuo. The resulting white powder was dissolved in a small

amount of H2O and then purified by dialysis or gel filtration (Sephadex LH-20, then Sephadex G-25; Amersham Pharmacia Biotech AB, Uppsala, Sweden) using H2O as an eluent. The sodium salt of sulfated colominic acid was obtained by lyophilization. Structural Analysis of the Polymers. NMR spectra of the polymers were measured at 125 MHz for 13C and DEPT, at 500 MHz for 1H with a JEOL JNM GX-500 spectrometer (JEOL Ltd., Tokyo, Japan). Two-dimensional NMR spectra were measured with a 400 MHz NMR spectrometer, Varian UNITY plus 400 (Varian Associates Inc., Palo Alto, CA). All NMR spectra were recorded in D2O at 25 or 30 °C, using Me3Si(CD2)2CO2Na as the external standard. IR spectra of the polymers were measured with a FTIR5300 spectrometer (JASCO Co., Tokyo, Japan) on KBr disks. The number-averaged molecular weight was determined by a GPC (JASCO PU-980) using a GS-510 column (Showa Denko Co., Tokyo, Japan; eluent 0.05 M NaCl, pullulan as standard). Elemental analyses were performed by the addition of WO3 for more precise detection of SO42-. CD spectra of the polymers were obtained using a J-600 spectrometer with a PTC-348W temperature controller (JASCO Co.). Each sample was dissolved in H2O (0.1 mg/mL) and measured in the spectral range 190-240 nm with an optical path length of 5 mm at 20 °C. Cell Culture. 3T3-L1 fibroblasts (CCL92.1) were subcultured in tissue culture flasks (FALCON 3072, Becton, Dickinson and Co., Franklin Lakes, NJ) at subconfluent cell densities in Eagle’s MEM supplemented with 10% fetal bovine serum (Gibco BRL, Life Technologies, Inc., Rockville, MD), kanamycin, and Lglutamin. Cultures were maintained at 37 °C in a humidified tissue culture incubator in a 5% CO2/95% air environment and were used in passages 4-12. Cell Proliferation Assay. Polysaccharide and FGF were introduced in serum-free Eagle’s MEM supplemented with ITS-A (insulin 10 µg/mL, transferrin 5.5 µg/mL, sodium pyruvate 0.11 mg/mL, and sodium selenite 6.7 ng/mL: Gibco) and bovine serum albumin (0.4 mg/mL). 3T3-L1 fibroblasts were plated at 6000 cells/ well on a tissue culture-treated polystyrene 96 well multiplate (FALCON 3072, Becton Dickinson) and cultured for 48 h. Cell proliferation was measured by the MTT method.42 The colorimetric MTT assay is based on color change arising from cleavage of the tetrazolium ring of MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide). The ring is reduced and then cleaved by

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a dehydrogenase to produce formazan in active mitochondria. It was confirmed that the amount of formazan generated was directly proportional to the cell number (data not shown). Gel Shift Assay. The protein (5 µg) and/or polysaccharide (15 µg) was incubated in 10 µL PBS (-) at 37 °C for 30 min for the binding reaction. Samples were then loaded in a 4% polyacrylamide gel with low ionic strength page and electrophoresed at 50 V in Tris-HCl/acetate electrophoresis buffer (pH 7.9). CCB (Coomassie Brilliant Blue R 250) was used for visualization. Molecular Modeling. Molecular modeling was performed using Discover 95/Insight II software (Molecular Simulations, Inc., San Diego, CA) in IRIX 4D/35 workstations (Silicon Graphics, Inc., Mountain View, CA). The CFF91 force field was used for energy minimization and molecular dynamics. To reduce the strength of intramolecular Coulomb interactions, the distance-dependent dielectric constant (coefficient 5) was introduced. The conjugate gradient method was used for the minimization. For molecular modeling of colominic acid (six residues), the initial values of the dihedral angles were taken from the literature.4,5 In our attempt to find stable, low-energy conformations, the initial structure was subjected to energy minimization and molecular dynamics using a CFF91 force field. The resulting structure maintained the initial helical structures. The S-col-2 structure for modeling was created by substituting the 4-OH and 9-OH groups of the minimized structure of coloninic acid (6 residues) with sulfated groups. Then, S-col-2 was simply minimized. S-col-2 maintained its helical structure. Structural data of the FGF-2 and heparin derivative complex was taken from the Protein Data Bank (entry code 1BFC). Heparin derivative hexasaccharide and H2O were removed from the complex. The energy minimized S-col-2 (three residues) was manually docked to the heparin binding site in FGF-2. Among various orientations, only one orientation of S-Col-2 was fitted to the heparin binding site. Simple minimization of the complex of FGF-2 with of the S-col-2 was carried out under the following conditions: (1) the cell-multipole method was used, and (2) the main chain of FGF-2 was weakly tethered (force constant 20) and the side chains were not constrained.

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BM000011K

Synthesis of Sulfated Colominic Acids and Their Interaction with

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