Bioconjugate Chem. 2002, 13, 1309−1313
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Glycosaminoglycan-Mimetic Biomaterials. 3. Glycopolymers Prepared from Alkene-Derivatized Mono- and Disaccharide-Based Glycomonomers Subramanian Baskaran,‡ Daniel Grande,‡ Xue-Long Sun,‡ Avner Yayon,§ and Elliot L. Chaikof*,†,‡ Laboratory for Biomolecular Materials Research, Department of Surgery and Biomedical Engineering, Emory University, Atlanta, Georgia 30322, School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, and Weizmann Institute of Science, Rehovot, Israel. Received May 3, 2002; Revised Manuscript Received August 11, 2002
Mono- and disaccharide-containing glycopolymers were synthesized by two different free-radical processes, and their ability to act as heparan sulfate glycomimetics in promoting the binding of Fibroblast Growth Factor-2 (FGF-2) to its receptor (FGFR-1) was evaluated using an in vitro cellbased assay. Cyanoxyl (•OCtN)-mediated polymerization of acrylamide with alkene-derivatized monoand disaccharides including sulfated or nonsulfated N-acetyl-D-glucosamine is described. The results of this approach are compared to those obtained via the classical ammonium peroxodisulfate (APS)/ N,N,N′,N′-tetramethylethylenediamine (TMEDA) initiating system and confirm the capacity of cyanoxyl-mediated polymerization to generate a variety of glycopolymers with high saccharide contents and low polydispersity indexes. In vitro assays demonstrate that specific glycopolymers can potentiate FGF-2/FGFR-1 binding interactions.
INTRODUCTION
Heparan sulfates (HS) are a large family of polydisperse anionic polysaccharides encountered both in the extracellular matrix and on cell surfaces that bind numerous proteins of biological interest. For example, fibroblast growth factor-2 (FGF-2) binds to heparan sulfate, which stabilizes and protects it against inactivation and facilitates its binding to the FGF receptor-1 (FGFR-1) (1). Moreover, heparan sulfates are required for the oligomerization of the FGF-2/FGFR-1 complex that is necessary for effective transmembrane signaling and a biological response (2). As a consequence of modulating the FGF-2/FGFR-1 signaling pathway, heparan sulfates have a significant functional role in a wide array of physiological processes, including cell proliferation and migration, as well as angiogenesis, wound healing, and inflammatory responses (3-5). Thus, the development of synthetic biopolymers that incorporate some of the biological functionality of heparan sulfate, particularly with respect to FGF-2, carries potential significance in a variety of areas, including drug delivery and tissue engineering (6). Heparan sulfates are structurally complex and consist on average of 20 or more repeating disaccharides units composed of a hexauronic acid (D-glucuronic or L-iduronic acid) attached in 1f4 fashion to N-acetyl-D-glucosamine with varying patterns of sulfation (7). While smaller oligosaccharide sequences have been identified that are responsible for some of the unique biological activities * Address correspondence to Elliot L. Chaikof, M.D., Ph.D., 1639 Pierce Drive - 5105 WMB, Emory University, Atlanta, GA 30322. Phone: (404) 727-8413. Fax: (404) 727-3660. Email:
[email protected]. † Georgia Institute of Technology. ‡ Emory University School of Medicine. § Weizmann Institute of Science.
of heparan sulfate (8, 9), optimal binding of FGF-2 to FGFR-1 requires heparan sulfate-derived oligosaccharides containing eight or more sugar residues (10). Thus, the generation of heparan sulfate-mimetic compounds that modulate FGF-2-dependent responses remains a challenging endeavor, despite reported advances in solution- and solid-phase methods of carbohydrate synthesis. As an alternative approach, efforts in our group have been recently directed at the investigation of ‘glycopolymer’-based strategies in which pendant saccharides are incorporated into a polymer backbone. The observation of enhanced protein binding affinity derived from multivalent oligosaccharide ligands suggested that monomers carrying this or other related saccharides as pendant groups could be used to generate glycopolymers with FGF-2-related bioactivity. Optimization of glycopolymer properties requires the generation of biomolecular architectures that exhibit low fluctuations both in polymer size and in composition. As a consequence, we are currently examining the applicability of cyanoxyl (•OCtN)-mediated free-radical polymerization in the synthesis of model glycopolymers from unprotected glycomonomers. Notably, this polymerization technique can be conducted in aqueous solution, is tolerant of a broad range of functional groups (OH, NH2, COOH, etc.), and can be applied to the synthesis of block and graft copolymers with reported polydispersities below 1.5 (11, 12). In this report, we describe the preparation of alkene-derivatized mono- and disaccharide glycomonomers. Cyanoxyl-mediated and classical freeradical polymerization techniques were then exploited to create a wide variety of water-soluble glycopolymers with high monosaccharide contents and low polydispersity indexes. Finally, the ability of test glycopolymers to potentiate the binding of FGF-2 to FGFR-1 is examined in preliminary bioactivity studies.
10.1021/bc0255485 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/05/2002
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Figure 1. Alkenyl mono- and disaccharide-based glycomonomers used for the synthesis of corresponding glycopolymers. EXPERIMENTAL SECTION
Materials. All solvents and reagents were purchased from commercial sources and were used as received, unless otherwise noted. Heparin was obtained from Hepar Industries and is polydisperse with a range of molecular weight between 5 and 30 kD (Franklin, OH). Deionized water with a resistivity of 18 MΩ‚cm was used as solvent in all polymerization reactions. Methods. All reactions were performed in flame-dried glassware under an atmosphere of dry argon. The reaction medium solutions were evaporated under reduced pressure with a rotary evaporator, and the residue was chromatographed on a silica gel (230-400 mesh) column. Analytical thin-layer chromatography (TLC) was performed on Whatman silica gel aluminum-backed plates of 250 µm thickness on which spots were visualized with UV light or charring the plate before and/or after dipping in a H2SO4-EtOH mixture. Melting point (mp) measurements were performed with a Thomas-Hoover melting point apparatus in open capillary tubes and were uncorrected. Mass spectra (MS/FAB) were obtained at an ionizing voltage of 70 eV. Optical rotations were determined with a Perkin-Elmer-2 GIMC polarimeter. 1H and 13 C NMR spectra were recorded at room temperature with a Varian INOVA 400 spectrometer (magnetic field strengths of 400 and 100 MHz for 1H and 13C NMR analyses, respectively). In all cases, the sample concentration was 10 mg/mL, and the appropriate deuterated solvent was used as the internal standard. Size-exclusion chromatography (SEC) equipment was comprised of a Waters model 510 HPLC pump, a Waters Ultrahydrogel 250 column, and a Wyatt Technology Optilab 903 refractometer. The eluent consisted of a 0.1 mol/L NaNO3 deionized water solution containing 0.05 wt % sodium azide at a flow rate of 0.7 mL/min. The actual molar masses of the glycopolymer samples were determined from the response of the DAWN F (Wyatt Technology) multiangle laser light-scattering (LLS) detector that was connected to the outlet of the SEC apparatus. Synthesis of Alkene-Derivatized Glycomonomers with Pendant Monosaccharides. Synthesis and detailed characterization of n-pentenyl 2-acetamido-2-deoxyR-D-glucopyranoside (1(C-3)), ω-undecenyl 2-acetamido2-deoxy-R-D-glucopyranoside (1(C-9)), and sulfated derivatives thereof (2(C-3) and 2(C-9), respectively) have been reported in detail elsewhere (Figure 1).13 Synthesis of Alkene-Derivatized Glycomonomers Bearing Lactose. n-Pentenyl O-(β-D-galactopyranosyl)-(1f4)-β-D-glucopyranoside, 3. To a mixture of D-lactose (10 g) and 4-penten-1-ol (large excess, ∼125175 mL) was added a catalytic amount of 10-camphorsulfonic acid (CSA) (∼400 mg), and the mixture was
refluxed at 110 °C for 7 h. The reaction mixture was then cooled and neutralized with triethylamine (E3N), and the excess of alcohol was removed under vacuum. The solid mass was subsequently rinsed with hot petroleum ether to remove the remaining alkenyl alcohol. The residue was purified by column chromatography using a chloroform/ methanol (97/3) mixture to afford the expected product, 3. Yield: 45%; [R]20D ) +20.6° (c 1.8, CH3OH). 1H NMR (CD3OD), δppm: 1.69 (m, 2H, C-CH2-C), 2.12 (m, 2H, CH2-Cd), 3.38-3.88 (m, all other H), 5.07 (m, 2H, CH2d), 5.78 (m, 1H, CHd). 13C NMR (CD3OD), δppm: 28.6, 30.2, 67.6, 69.7, 71.5, 71.8, 74.1, 98.6, 115.1, 137.9. MS/FAB, m/z: 411 (M + 1) (14, 15). n-Pentenyl O-(2,3,4,6-Tetra-O-sulfo-β-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-sulfo-β-D-glucopyranoside Heptasodium Salt, 4. Under argon atmosphere, sulfur trioxide-trimethylamine (SO3-NMe3) complex (4 equiv for each hydroxyl group) was added to the nonsulfated glycomonomer (3) in DMF, and the mixture was stirred at 60 °C for 12 h. The reaction medium was then cooled to 0 °C, and a saturated NaHCO3 aqueous solution was added. The crude mixture was stirred for 1 h and concentrated to a smaller volume that was passed through a diethylaminoethyl (DEAE)-sephacel anionexchange resin column. It was first eluted with a 10 mmol/L sodium phosphate buffer (pH ∼ 7.0), thereby removing the unreacted nonsulfated compound. The sulfated homologue was then eluted with a 1 mol/L NaCl buffer (pH ∼ 7.0) and recovered as a mixture of its heptasodium salt with a NaCl excess. The latter eluate was concentrated, redissolved in a minimum amount of water, and passed through a Trisacryl (Gf05 M grade, Sigma-Aldrich) size-exclusion resin column for isolation of the sulfated compound free of NaCl. Yield: 45%; [R]20D ) +64.7° (c 1.7, H2O). 1H NMR (D2O), δppm: 1.72 (m, 2H, C-CH2-C), 2.21 (m, 2H, CH2-Cd), 3.42-3.92 (m, all other H), 5.09 (m, 2H, CH2d), 5.81 (m, 1H, CHd). MS/ FAB, m/z: 986 (M+ + 1Na+). Statistical Copolymerization of Alkene-Derivatized Glycomonomers and Acrylamide Initiated by ClC6H4NtN+BF4-/NaOCN. In a three-neck flask, 6.03 × 10-5 mol (0.008 g) of p-chloroaniline was reacted with 9.04 × 10-5 mol of HBF4 (actually 0.017 g of 48 wt % aqueous solution), at 0 °C, in 2 mL of water and under argon atmosphere. The diazonium salt ClC6H4NtN+BF4was then generated by adding 7.2 × 10-5 mol (0.005 g) of sodium nitrite (NaNO2) to the reaction medium. After 30 min, a degassed mixture of 6.03 × 10-4 mol (0.225 g) of glycomonomer 1(C-9), 2.41 × 10-3 mol (0.171 g) of acrylamide, and 6.03 × 10-5 mol (0.004 g) of sodium cyanate (NaOCN), dissolved in 1 mL of water/tetrahydrofuran (1/1), was introduced into the flask containing the diazonium salt. The polymerization medium was then
Glycosaminoglycan-Mimetic Biomaterials
heated to 50 °C. The statistical copolymers formed after 1.5 and 16 h of reaction were isolated by precipitation in a 10-fold excess of cold methanol, dried, and weighed so as to determine the conversion. Binding of FGF-2 to FGF Receptor-1 (FGFR-1). High-affinity binding of FGF-2 to CHO cells was performed as described in detail elsewhere (10, 16). Briefly, confluent CHO-pgsA-745-flg cells, which are deficient in heparan sulfates and genetically engineered to express the FGF receptor-1, were incubated with 125 I-FGF-2 and increasing concentrations of test gycopolymer for 90 min at 40 °C. Low-affinity-bound FGF-2 was released from the cell surface by a 5 min incubation with a cold solution containing 1.6 M NaCl and 20 mM HEPES, pH 7.4. High-affinity-bound FGF-2 was determined by a 2 M NaCl and 20 mM acetate buffer, pH 4.0, extraction.
Bioconjugate Chem., Vol. 13, No. 6, 2002 1311 Scheme 1. Cyanoxyl-Mediated Free-Radical Copolymerization of AM with Alkene-Derivatized Unprotected Glycomonomers
RESULTS AND DISCUSSION
1. Synthesis of Glycomonomers Bearing Nonsulfated and Sulfated N-Acetyl-D-glucosamine and Lactose. Three different glycosidation methods have been previously reported for the synthesis of monosaccharides containing spacer side chains. Specifically, FeCl3 has been used as a catalyst for generating monosaccharides derivatized in positions C-1 (17). Similarly, trifluoromethanesulfonic acid (18), as well as 10-camphorsulfonic acid (CSA) (19, 20), has been utilized for the synthesis of spacer-armed glycosides either from glucose or an oxazoline derivative. For our purpose, we resorted to the CSA/reflux method that provided a facile means of yielding nonsulfated and sulfated alkene-derivatized monosaccharides from N-acetyl-D-glucosamine (Figure 1) (15). The reflux of N-acetyl-D-glucosamine with a catalytic amount of CSA and a large excess of either n-pentenyl or ω-undecenyl alcohol provided a mixture of R- and β-anomers (1(C-3) or 1(C-9)), with an average crude yield of 60-70%. The R- and β-anomeric configurations of the separated products were determined on the basis of unique J1,2 coupling constants of 3.6 and 8.0 Hz, respectively. Compounds were separated by column chromatography with a ratio R/β of 3/1 for both C-3 and C-9 glycosides. The preparation of compound 3 has been reported previously by two groups (14, 15). Matsuoka et al. (14) synthesized 3 from bromolactose using HgCN during the glycosidation step with a 40% yield for the n-pentenyl lactose. Likewise, Allen and Danishefsky (15) reported that glycosidation proceeds smoothly using AgCO3 with a 75% yield of the pentenyl glycoside and an overall yield from octaacetyl lactose of ∼65% in three to four steps. We elected to synthesize the n-pentenyl glycosides via CSA reflux methodology, which afforded the n-pentenyl lactoside 3 in a single step with moderate yield (∼45%) from inexpensive and commercially available D-lactose (Figure 1). Chemoselective sulfation of all hydroxyl groups on R-anomers, namely compounds 1(C-3), 1(C-9), and 3, was achieved by reaction with SO3-NMe3 at 60 °C in DMF. Preparative chromatography through successive columns containing diethylaminoethyl (DEAE)-sephacel anionexchange resin and trisacryl size-exclusion resin was required for isolation of the pure sulfated product (Figure 1, compounds 2(C-3), 2(C-9), and 4). 2. Synthesis of ω-Alkenyl Mono- and Disaccharide-Based Glycopolymers. Druliner (11) in the early 1990s and more recently Grande and Gnanou (12) have observed that a certain degree of control can be achieved when polymerizing a broad range of (meth)acrylic monomers, and particularly acrylamide, in the presence of
cyanoxyl (•OCtN) persistent radicals, as moderators of the polymerization reaction. Significantly, these persistent radicals function under milder conditions (25-70 °C) than those required for nitroxide-controlled free-radical polymerization or ATRP processes. Given the potential advantages of cyanoxyl-mediated free-radical polymerization, we investigated the copolymerization of acrylamide with a variety of unprotected alkene-derivatized saccharides (compounds 1-4). Cyanoxyl radicals were readily generated by an electrontransfer reaction between cyanate anions (-OCtN) from a NaOCN aqueous solution, and p-chlorobenzene-diazonium salts (ClC6H4NtN+BF4-) that were previously prepared in situ through a diazotization reaction of p-chloroaniline in water. In addition to cyanoxyl persistent radicals, aryl-type active radicals were simultaneously produced, and only the latter species were able to initiate chain growth (Scheme 1). A series of watersoluble glycopolymers were prepared by varying the nature of the ω-alkenyl glycomonomer (nonsulfated/ sulfated, mono-/disaccharide-based), as well as the initial molar ratio of glycomonomer (GM) to AM in the statistical copolymerization of both comonomers performed at 50 °C (Table 1). These statistical copolymers were isolated by precipitation in a 10-fold excess of methanol and characterized by 1H NMR spectroscopy, as well as by SEC coupled with both refractive index and laser lightscattering detectors (13). The absence of residual comonomers, including glycomonomer, was assessed by 1H NMR. The ratio of the resonance signal intensity from the methyl protons of N-acetyl groups from sugar moieties (2.0 ppm) to that of methine protons from the hydrocarbon skeleton (2.1-2.4 ppm) enabled the determination of saccharide content, for those glycopolymers containing N-acetyl-D-glucosamine residues. As for lactose-based samples, elemental analysis was used to assess carbohydrate composition. Notably, saccharide contents as well as molar masses increased with monomer conversion while polydispersity indexes remained below 1.5. Regardless of the glycomonomer used (mono/disaccharide-based, nonsulfated/sulfated), an initial ratio GM/AM of 1/4, associated with a copolymerization time of 16 h, permitted the access to a copolymer that displayed a saccharide content in close agreement with that expected. Samples exhibiting sugar compositions as high as 69 wt % were thus obtained. Nonetheless, copolymers with higher carbohydrate contents were associated with an increase in the polydispersity index. This may be attributable to some loss of control over the copolymerization process in the presence of increasing concentrations of ω-alkenyl
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Table 1. Statistical Free-Radical Copolymerization of AM with ω-Alkenyl Mono- or Disaccharide-Based Unprotected Glycomonomers Using ClC6H4NtN+BF4-/NaOCN as the Initiating Systema glycomonomer (GM) 1(C-3)
monomer ratio GM/AM (mol) 1/4 1/20
1(C-9)
1/4 1/20
2(C-3)
1/4 1/20
2(C-9) 3 4
1/4 1/20 1/20 1/4
time (h)
yieldb (%)
polymer composition GM/AM (mol)
saccharide content (wt %)
Mn (g/mol)
Mw/Mn SEC
1.5 16 1.5 16 1.5 16 1.5 16 1.5 16 1.5 16 16 16 16 16
10 30 23 30 15 20 21 29 21 35 35 51 26 11 15 30
1/7 1/5 1/90 1/70 1/16 1/6 1/166 1/100 1/10 1/6 1/93 1/58 1/10 1/42 1/30 1/6
38 50 4 5 25 46 3 5 44 55 07 11 45 17 16 69
24100 43000 94000 112100 43400 99300 25800 28200 16100 57300 25400 47200 57200 16300 28800 38000
1.46 1.47 1.17 1.20 1.25 1.45 1.14 1.24 1.13 1.37 1.10 1.29 1.20 1.17 1.31 1.50
a T ) 50 °C, [GM] + [AM] ) 1 mol/L, [I] ) [ClC H NtN+BF -] ) [NaOCN] ) 0.02 mol/L. b Total conversion of comonomers as 0 0 0 6 4 4 0 0 determined by gravimetry.
Table 2. Statistical Free-Radical Copolymerization of AM with ω-Alkenyl Mono- or Disaccharide-Based Unprotected Glycomonomers Using APS/TMEDA as the Initiating Systema glycomonomer (GM)
monomer ratio GM/AM (mol)
time (h)
yieldb (%)
polymer composition GM/AM (mol)
saccharide content (wt %)
Mn (g/mol)
Mw/Mn SEC
1(C-3)
1/4
3
1/4 1/20 1/4 1/20
1.5 16 16 16 16 16
67 73 83 80 50 70
1/14 1/11 1/10 1/48 1/18 1/60
22 28 38 11 43 18
133000 146000 140000 156000 70500 80000
1.63 1.73 1.78 2.08 1.59 1.89
4
a T ) 25 °C, [M] ) [GM] + [AM] ) 1 mol/L, [I] ) [APS] ) [TMEDA] ) 0.02mol/L. b Total conversion of comonomers as determined 0 0 0 0 0 0 by gravimetry.
glycomonomers, considering the innate low chemical reactivity of the vinyl group in these saccharide monomers. The limited reactivity of the vinyl group, particularly in association with disaccharide bearing monomers, may also be a contributory factor in reducing monomer conversion. Despite a 16-h reaction period, overall yields for polymers derived from glycomonomers 3 and 4 ranged from 15 to 30%. In a comparative study, classical free-radical copolymerization of glycomonomers and AM were performed using APS/TMEDA as the initiating system. TMEDA accelerates the homolytic scission of APS yielding sulfate (SO4•-), hemiTMEDA ((CH3)2NCH2CH2(CH3)-NCH2•), and hydroxyl (•OH) radical species. The reaction medium was homogeneous and the polymerization proceeded smoothly at room temperature (Table 2). Utilizing otherwise identical experimental conditions as those used for cyanoxyl-mediated processes ([M]0 ) 1 mol/L, [I]0 ) 2 × 10-2 mol/L), the resulting glycopolymers exhibited lower saccharide contents (maximum: 43 wt %) with higher molar masses and polydispersity indexes (1.6-2.0). Moreover, increasing the polymerization time from 1.5 to 16 h increased the polydispersity index, but otherwise had little influence on either saccharide content or molar mass. The observed increase in polydispersity, however, was offset by significantly higher polymer yields, ranging up to 80% for monomers 3 and 4. In summary, these investigations illustrate the anticipated absence of control over the copolymerization process by employing the classical APS/TMEDA initiating system, while confirming the significant role of cyanoxyl radicals in moderating the polymerization process. 3. Binding of FGF-2 to FGF Receptor-1 (FGFR1). The capacity of selected glycopolymers to promote the binding of FGF-2 to FGFR-1 was defined using CHOpgsA-745-flg cells, which are deficient in heparan sulfates
Table 3. Structure-bFGF Binding Activity Relationships Studies: Summary of Molecular Characteristics Associated with Glycopolymers glycomonomer (GM)
polymer composition GM/AM (mol)
saccharide content (wt %)
Mn (g/mol)
Mw/Mn SEC
code
1(C-3) 2(C-3) 2(C-9) 3 4
1/5 1/6 1/10 1/10 1/18
50 55 45 38 43
43000 57300 57200 140000 70500
1.47 1.37 1.20 1.78 1.59
GP1 GP2 GP3 GP4 GP5
and genetically engineered to express a single FGF receptor subtype, FGFR-1. FGFR-1 is normally expressed on a wide variety of cell types and when activated can initiate cell proliferation and migration and other cell type-specific responses. Given the preliminary nature of these bioactivity studies, limited yields for glycomonomers, and low conversions associated with cyanoxylmediated polymerization for vinyl monomers bearing pendant disaccharides, both classical and cyanoxylmediated polymerization strategies were used to synthesize the glycopolymers available for receptor binding experiments (Table 3). Results of these investigations for polymers bearing mono- and disaccharides are summarized in Figures 2 and 3, respectively. As anticipated, heparin is a strong promotor of FGF-2 binding to FGFR1. Monosaccharide bearing glycopolymers were all of similar saccharide content, nominal molecular weight, and polydispersity (Figure 2). Sulfation-enhanced receptor binding of FGF-2 with a biphasic response observed for polymers (GP2) composed of monomer 2(C-3). The reduction in receptor binding at high glycopolymer concentrations is also characteristic of heparin and is likely related to a sequestration effect, which limits FGF-2 availability. Notably, the use of a longer (C-9) spacer arm (GP3) did not enhance receptor binding.
Glycosaminoglycan-Mimetic Biomaterials
Bioconjugate Chem., Vol. 13, No. 6, 2002 1313
of glycotechnology, including the design of biomaterials for tissue regeneration and wound-healing applications. ACKNOWLEDGMENT
This work was supported by grants from the NIH. The authors acknowledge the Emory University NMR and Mass Spectrometry Centers for their facilities. They are also indebted to Dr. Suri Iyer for kind technical assistance. LITERATURE CITED
Figure 2. bFGF-binding assays for monosaccharide-based glycopolymers ([ heparin, b GP1, 9 GP2, 2 GP3). The data are representative of binding assays performed in duplicate. Free sulfated monomer did not potentiate FGF-2 binding to heparan sulfate-deficient CHO cells.
Figure 3. bFGF-binding assays for disaccharide-based glycopolymers ([ heparin, 9 GP4, 2 GP5). The data are representative of binding assays performed in duplicate. Free sulfated monomer did not potentiate FGF-2 binding to heparan sulfatedeficient CHO cells.
Disaccharide-bearing glycopolymers were all of similar saccharide content and polydispersity (Figure 3). Polymers bearing pendant lactose units (GP4) had little effect on receptor binding. A pronounced effect, however, was observed for polymers that contain sulfated lactose groups (GP5), even at very low polymer concentrations (∼1 µg/mL). Of interest, an inhibitory effect on receptor binding was not observed as polymer concentration was increased. CONCLUSIONS
We have synthesized a series of model mono- and disaccharide-carrying unprotected glycomonomers for the design of heparan sulfate glycomimetics. The present study broadens the family of vinyl monomers that are polymerizable with some degree of control by cyanoxylmediated free-radical polymerization. Both nonsulfated and sulfated alkene-derivatized monosaccharides and disaccharides were polymerized using this approach. While high carbohydrate contents and low polydispersity indexes (1.1 < Mw/Mn < 1.6) can be achieved further optimization of this strategy is required to enhance polymer yield, particularly for monomers bearing disaccharide units. Preliminary bioactivity studies reveal that glycopolymers containing pendant sulfated lactose groups significantly enhance FGF-2 binding to its receptor, FGFR-1, even at low polymer concentrations. We believe that tailored glycopolymers will contribute to the progress
(1) Saksela, O.; Moscatelli, D.; Sommer, A.; Rifkin, D. B. (1988) J. Cell. Biol. 107, 743. (2) Schlessinger, J.; Lax, I.; Lemmon, M. (1995) Cell 83, 357. (3) Clowes, A. W.; Karnovsky, M. J. (1977) Nature 265, 625. (4) Jackson, R. L.; Busch, S. J.; Cardin, A. L. (1991) Physiol. Rev. 71, 481. (5) Linhardt, R. J.; Kerns, R. J.; Vlahov, I. R. (1996) In Biomedical Functions and Biotechnology of Natural and Artificial Polymers, p 45, ATL Press, (6) (a) Jozefowicz, M.; Jozefonvicz, J. (1997) Biomaterials 18, 1633. (b) de Raucourt, E.; Mauray, S.; Chaubet, F.; MaigaRevel O.; Jozefowicz, M.; Fischer, A. M. (1998) J. Biomed. Mater. Res. 41, 49. (c) Blanquaert, F.; Barritault, D.; Caruelle, J. P. (1999) J. Biomed. Mater. Res. 44, 63. (d) LogeartAvramoglou, D.; Jozefonvicz, J. (1999) J. Biomed. Mater. Res. 48, 578. (e) Tardieu, M.; Gamby, C.; Avramoglou, T.; Jozefonvicz, J.; Barritault, D. (1992) J. Cell. Physiol. 150, 194. (f) Desgranges, P.; Caruelle, J. P.; Carpentier, G.; Barritault, D.; Tardieu, M. (2001) J. Biomed. Mater. Res. 58, 1. (7) Gallagher, J. T. (1995) In Glycoimmunology, p 125, Plenum Press, New York. (8) (a) van Boeckel, C. A. A.; Petitou, M. (1993) Angew. Chem., Int. Ed. Engl. 32, 2, 1671. (b) Westerduin, P.; Basten, J. E. M.; Broekhoven, M. A.; De Kimpe, V.; Kuijpers, W. H. A.; Van Boeckel, C. A. A. (1996) Angew. Chem., Int. Ed. Engl. 35, 331. (c) Petitou, M.; Duchaussoy, P.; Driguez, P.-A.; Jaurand, G.; He´rault, J.-P.; Lormeau, J.-C.; van Boeckel, C. A. A.; Herbert, J.-M. (1998) Angew. Chem., Int. Ed. Engl. 37, 3009. (d) Petitou, M.; He´rault, J.-P.; Bernat, A.; Driguez, P.-A.; Duchaussoy, P.; Lormeau, J.-C.; Herbert, J.-M. (1999) Nature 398, 417. (9) (a) Westman, J.; Nilsson, M.; Ornitz, D. M.; Svahn, C.-M. (1995) J. Carbohydr. Chem. 14, 95. (b) Ornitz, D. M.; Herr, A. B.; Nilsson, M.; Westman, J.; Svahn, C.-M.; Waksman, G. (1995) Science, 268, 432. (10) (a) Ornitz, D. M.; Yayon, A.; Flanagan, J. G.; Svahn, C. M.; Levi, E.; Leder, P. (1992) Mol. Cell. Biol., 12, 240. (b) Aviezer, D.; Levy, E.; Safran, M.; Svahn, C.; Buddecke, E.; Schmidt, A.; David, G.; Vlodavsky, I.; Yayon, A. (1994) J. Biol. Chem. 269, 114. (11) Druliner, J. D. (1991) Macromolecules 24, 6079. (12) Gnanou, Y.; Grande, D.; Guerrero, R. (1999) Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 40 (2), 99. (13) (a) Grande, D.; Baskaran, S.; Baskaran, C.; Gnanou, Y.; Chaikof, E. L. (2000) Macromolecules 33, 1123. (b) Grande, D.; Baskaran, S.; Chaikof, E. L. (2001) Macromolecules 34, 1640. (14) Matsuoka, K.; Nishimura, S.-I. (1995) Macromolecules 28, 2961. (15) Allen, J. R.; Danishefsky, S. J. (1999) J. Am. Chem. Soc. 121, 10875 (and further references therein). (16) Yayon, A.; Klagsbrun, M.; Esko, J. D.; Leder, P.; Ornitz, D. M. (1991) Cell 64, 841. (17) Peter, M. G.; Boldt, P. C.; Peterson, S. (1992) Liebigs Ann. Chem. 1275. (18) Udodong, U. E.; Rao, C. S.; Fraser-Reid, B. (1992) Tetrahedron 48, 4713. (19) (a) Nishimura, S.-I.; Matsuoka, K.; Kurita, K. (1990) Macromolecules 23, 4182. (b) Nishimura, S.-I.; Matsuoka, K.; Furuike, T.; Ishii, S.; Kurita, K. (1991) Macromolecules 24, 4236. (20) Konradsson, P.; Roberts, C.; Fraser-Reid, B. (1991) Recl. Trav. Chim. Pays-Bas 110, 23. BC0255485
