Biomacromolecules 2002, 3, 1065-1070
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Glycosaminoglycan Mimetic Biomaterials. 4. Synthesis of Sulfated Lactose-Based Glycopolymers That Exhibit Anticoagulant Activity Xue-Long Sun,† Daniel Grande,† Subramanian Baskaran,† Stephen R. Hanson,† and Elliot L. Chaikof*,†,‡ Laboratory for Biomolecular Materials Research, Departments of Surgery and Bioengineering, Emory University School of Medicine, Atlanta, Georgia 30322, and the School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30320 Received May 3, 2002; Revised Manuscript Received June 24, 2002
Cyanoxyl persistent radicals can be used as chain-growth moderators of the statistical copolymerization of a variety of monomers. We report herein the preparation of fully sulfated lactose-based glycopolymers by cyanoxyl (•OCtN)-mediated free-radical polymerization of acrylamide derivatized glycomonomers in good yield (60-80%) and low polydispersity (1.1 < Mw/Mn < 1.6). Prolongation of the activated partial thromboplastin time (aPTT) was observed, and structure-activity relationships were defined. Specifically, the anticoagulant effect varied in response to both polymer molecular weight and the density of pendant sulfated lactose units. Nonetheless, measured thrombin times were only modestly prolonged suggesting that the observed anticoagulant effect is not primarily related to direct thrombin inhibition. Introduction In general terms, glycosaminoglycans (GAGs) are involved in a wide range of physiological processes, including cell proliferation and migration, as well as the modulation of angiogenesis and inflammatory responses.1-3 However, many of the specific physiological activities that are mediated by GAGs, including heparin, have not been completely defined due to their diverse and complicated structures and capacity to interact with numerous biologically active proteins. The recent identification that smaller oligosaccharide sequences may be responsible for the unique biological activities of the parent polysaccharide holds the promise of generating relatively small molecule GAG equivalents through a total synthesis strategy.4-8 An alternative glycomimetic strategy has consisted of the design of synthetic glycopolymers that contain a hydrocarbon backbone with biologically active pendant saccharides.9 Indeed, fundamental studies on the synthesis and properties of model glycopolymers have proven to be useful in the characterization of specific biomolecular recognition processes that hold relevance for both pharmaceutical and biomaterial applications.10 Detailed studies have now revealed the precise structural determinants that are responsible for heparin’s anticoagulant effect. However, as a first approximation, both the presence of anionic groups and the linearity of the heparin backbone have been exploited in the generation of synthetic polymers * To whom correspondence may be addressed: 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; e-mail, echaiko@ emory.edu. † Departments of Surgery and Bioengineering, Emory University School of Medicine. ‡ School of Chemical Engineering, Georgia Institute of Technology.
with heparin-like functional characteristics.11,12 Specifically, a number of heparin mimetics exhibiting anticoagulant activity have been developed based on the derivatization of naturally occurring polysaccharides, such as dextran or ancroid, with sulfate or carboxylate groups or through the direct synthesis of sulfated glycopolymers.13,14 In principle, the synthesis of sulfated glycopolymers should provide a high level of control over regiospecific sulfation patterns. However, to date many of the sulfated glycopolymers have been prepared by derivatization after initial polymerization. Initial synthesis of a sulfated glycomonomer followed by polymerization clearly provides a more facile route for creating polymers with higher degrees of control over sulfation patterns. In prior reports, we have noted that cyanoxyl (OCN)-mediated free-radical polymerization and copolymerization of unprotected glycomonomers is a straightforward technique that can be conducted in aqueous solution, is tolerant of a broad range of functional groups, including -OH, -NH2, and -COOH moieties, and has the capacity to yield polymers of low polydispersity.15,16 In this investigation, we describe the synthesis of lactose heptasulfate-based glycomonomers, corresponding homo- and copolymers using cyanoxyl-mediated free-radical polymerization, and a preliminary structure-property analysis of anticoagulant activity. Experimental Section Materials. All solvents and reagents were purchased from commercial sources and were used as received, unless otherwise noted. Deionized water with a resistivity of 18 MΩ‚cm was used as a solvent in all polymerization reactions. Sulfated monosaccharide-based homoglycopolymers and
10.1021/bm025561s CCC: $22.00 © 2002 American Chemical Society Published on Web 07/30/2002
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coglycopolymers were synthesized as previously reported16 from sulfated 2-acrylethyl R/β-glycoside of N-acetylglucosamine with or without acrylamide. Low molecular weight dextran sulfate was purchased from Sigma and used without further purification. Methods. 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 by charring the plate after dipping in 10% H2SO4 in methanol. 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 13C 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 an internal standard. The size-exclusion chromatography (SEC) equipment comprised 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 Sulfated 2-Acrylamidoethyl β-lactoside. 2-Azidoethoxyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galatopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (2). To a cooled (ice-water bath), stirred solution of peracetyl-lactose 1 (5.0 g, 7.37 mmol), 2-azidoethanol (0.770 g, 8.85 mmol, 1.2 equiv) in dichloromethane (20 mL) was added BF3etherate (5.198 g, 36.87 mmol, 5 equiv). The reaction mixture was stirred for 1 h at 0 °C and then for 12 h at room temperature under an Ar atmosphere. The mixture was diluted with chloroform (20 mL), washed with cold water (20 mL) and aqueous sodium hydrogen carbonate (20 mL), and filterd. The filtrate was evaporated to give a residue, which was purified by column chromatography (SiO2) using acetone-n-hexane (2:3) as eluent to afford 2 (4.420 g, 85%).1H NMR (CDCl3) δ: 5.34 (br.d, 1 H, J ) 3.0 Hz), 5.19 (t, 1 H, J ) 9.3 Hz), 5.10 (dd, 1 H, J ) 10.5, 7.8 Hz), 4.94 (dd, 1 H, J ) 10.5, 3.3 Hz), 4.91 (dd, 1 H, J ) 9.6, 7.8 Hz), 4.55 (d, 1 H, J ) 8.1 Hz), 4.48 (d, 1 H, J ) 7.5 Hz), 4.15-4.03 (m, 2 H), 4.00-3.95 (m, 1 H), 3.86 (t, 1 H, J ) 6.6 Hz), 3.81 (t, 1 H, J ) 9.3 Hz), 3.70-3.58 (m, 2 H), 3.50-3.42 (m. 1 H), 3.29-3.22 (m, 1 H). HR-MS (EI): calcd for C28H39O18N3Li, 712.2406; found, 712.2398 [M + Li]+. 2-Azidoethoxyl 4-O-(β-D-galatopyranosyl)-β-D-glucopyranoside (3). To a solution of 2 (4.0 g, 5.68 mmol) in anhydrous methanol (20 mL) at 0 °C was added sodium methoxide (320 mg, 5.68 mmol). The reaction mixture was stirred for 3 h at room temperature. Dowex cation-exchange resin (H form) was added to adjust the pH to 6-7 and filtered. The filtrate was evaporated to give a residue, which was purified by column chromatography (SiO2) using chloroform-methanol (4:1) as eluent to afford 3 (2.240 g,
Sun et al.
96%).1H NMR (CD3OD) δ: 4.35 (d, 1 H, J ) 7.5 Hz), 4.34 (d, 1 H, J ) 7.8 Hz), 4.02 (t, 1 H, J ) 5.1 Hz), 3.98 (dd, 1 H, J ) 5.7, 5.1 Hz), 3.90 (dd, 1 H, J ) 2.4, 9.3 Hz), 3.87 (dd, 1 H, J ) 8.1, 5.1 Hz), 3.81 (br, 1 H), 3.78-3.60 (m, 2 H), 3.62-3.44 (m. 4 H). HR-MS (EI): calcd for C14H26O11N3, 412.1577; found, 412.1567 [M + H] +. 2-N-Acryoyl-aminoethoxyl 4-O-(β-D-galatopyranosyl)β-D-glucopyranoside (5). In the presence of Pt2O (240 mg), 3 (2.0 g, 4.86 mmol) in anhydrous methanol (20 mL) was charged with a hydrogen balloon for 3 h at room temperature. The reaction mixture was filtered, and the filtrate was evaporated to give crude 2-aminoethoxyl 4-O-(β-D-galatopyranosyl)-β-D-glucopyranoside (4). Triethylamine (1 mL) and acryloyl chloride (0.4 mL in 10 mL of chloroform) was added dropwise to a solution of crude 4 in methanol (20 mL) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 2 h to give the product 5 as precipitate (1.515 g, 71%).1H NMR (D2O) δ: 6.30 (dd, 1 H, J ) 17.1, 9.9 Hz), 6.20 (dd, 1 H, J ) 17.1, 1.8 Hz), 5.78 (dd, 1 H, J ) 9.9, 1.8 Hz), 4.51 (d, 1 H, J ) 7.8 Hz), 4.44 (d, 1 H, J ) 7.8 Hz), 4.02-3.94 (m, 2 H), 3.91 (br.d, 1 H, J ) 3.3 Hz), 3.82-3.50 (m, 11 H), 3.34-3.29 (m, 1 H). HR-MS (EI): calcd for C17H29O12NLi, 446.1858; found, 446.1850 [M + Li] +. 2-N-Acryoyl-aminoethoxyl 4-O-(2,3,4,6-tetrasulfoxy-βD-galatopyranosyl)-2,3,6-trisufoxy-β-D-glucopyranoside (6). To a solution of 5 (100 mg, 0.228 mmol) in 4 mL of DMF was added sulfur trioxide-trimethylamine (SO3-NMe3) complex (1.108 g, 7.972 mmol, 5 equiv for each hydroxyl group). The mixture was stirred at 60 °C for 12 h. The reaction medium was then cooled to room temperature and concentrated under reduced pressure to give a residue, which was purified on Sephadex-LH 20 with MeOH to afford 6 (188 mg, 84%). 1H NMR (D2O) δ: 6.28 (dd, 1 H, J ) 17.0, 9.9 Hz), 6.18 (dd, 1 H, J ) 17.0, 1.8 Hz), 5.75 (dd, 1 H, J ) 9.9, 1.8 Hz), 5.08 (d, 1 H, J ) 2.4 Hz), 5.02 (d, 1 H, J ) 6.3 Hz), 4.75 (d, 1 H, J ) 7.8 Hz), 4.66 (dd, 1 H, J ) 8.0, 9.1 Hz), 4.49-4.40 (m, 2 H), 4.80-4.40 (m, 9 H), 3.803.70 (m, 1 H), 3.60-3.40 (m, 3 H). MS/FAB, m/z: 1154.3976 [M + 7Na + 1]+. Homopolymerization of Acylamide Derived Glycomonomers Initiated by ClC6H4NtN+BF4-/NaOCN. In a threeneck flask, 3 mg (2.45 × 10-5 mol) of p-chloroaniline was reacted with 7 mg of HBF4 (48 wt % aqueous solution, 3.67 × 10-5 mol) in 2 mL of water at 0 °C and under an Ar atmosphere. The diazonium salt ClC6H4NtN+BF4- was generated by adding 2 mg (2.93 × 10-5 mol) of NaNO2 to the reaction medium. After 30 min, a degassed mixture of 390 mg (1.22 × 10-3 mol) of glycomonomer 6 and 2 mg (2.45 × 10-5 mol) of sodium cyanate (NaOCN) dissolved in 0.5 mL of water, was introduced into the flask containing the arenediazonium salt. The polymerization solution was then heated to 50 °C for 4 and 16 h. By varying the [M]/[I] ratio and reaction times resulting glycopolymers 7a-e were isolated by precipitation in a 10-fold excess of cold methanol and dried to yield white cotton wool like materials. Comopolymerization of Acylamide Derived Glycomonomers Initiated by ClC6H4NtN+BF4-/NaOCN. In a threeneck flask, 8 mg (6.03 × 10-5 mol) of p-chloroaniline was
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Glycosaminoglycan Mimetic Biomaterials Scheme 1. Synthesis of Nonsulfated and Sulfated 2-Acrylamidoethyl β-Lactosides
reacted with 17 mg of HBF4 (48 wt % aqueous solution, 9.04 × 10-5 mol) in 2 mL of water at 0 °C and under an Ar atmosphere. The diazonium salt ClC6H4NN+BF4- was then generated by adding 5 mg (7.2 × 10-5 mol) of sodium nitrite (NaNO2) to the reaction medium. After 30 min, a degassed mixture of 225 mg (6.03 × 10-4 mol) of glycomonomer 6 (or 5), 171 mg (2.41 × 10-3 mol) of acrylamide, and 4 mg (6.03 × 10-5 mol) of NaOCN dissolved in 1 mL of water was introduced into the flask containing the diazonium salt. The polymerization solution was then heated to 50 °C. The statistical copolymers 8a-c formed after 16 h of reaction were isolated by precipitation in a 10-fold excess of cold methanol and dried. The conversions were determined by weight of the resultant glycopolymer. In Vitro Clotting Assays. The anticoagulant activity of sulfated glycopolymers was initially evaluated by determining an activated partial thromboplastin time (aPTT). Blood was collected in 3.8% sodium citrate solution in a ratio of 9:1. Platelet-poor plasma (PPP) was prepared by centrifugation for 10 min at 4 °C. PPP and sulfated glycopolymers were incubated at 37 °C, and the time to formation of a fibrin network was measured by a fibrometer. The antithrombin activity of selected glycopolymers was determined by measuring the thrombin time (TT), which reflects the amount of time it takes to form a clot when thrombin is added to a sample of plasma or blood. Briefly, 100 µL of plasma was added to 100 µL of Owen’s buffer and prewarmed to 37 °C for 3 min. To this mixture was added 100 µL of human R-thrombin solution (10 U/mL in saline) and either heparin or glycopolymer diluted in saline. Results and Discussion 1. Synthesis of Sulfated 2-Acrylaminoethyl β-Lactoside. As an aglycon group that can be polymerized at a later stage, we chose the acrylamido function, which has been widely used in the synthesis of glycopolymers.17-19 The group was introduced by N-acryloylation of an aminoethyl aglycon, which was generated from the catalytic hydrogenation of 2-azidoethyl aglycon. Glycosylation of lactose peracetate (1) with 2-azidoethanol, which is easily accessible from commercial 2-chloroethanol and sodium,20 in the presence of boron trifluoride etherate gave the β-glycoside 2 (85%). The
anomeric configuration was confirmed by 1H NMR data, in which the J1,2 value of 7.8 Hz indicated the lactoside 2 to be β-glycoside. Saponification of 2 with sodium methoxide in methanol afforded 2-azidoethyl lactoside 3. The catalytic hydrogenation of 3 in methanol gave the corresponding 2-aminoethyl lactoside 4 in 93% yield, which was treated with acryloyl chloride in the presence of triethylamine to give 2-acrylaminoethyl lactoside 5 (71%). The expected signals of the vinyl of acryloyl group were observed in 1H NMR spectrum as a typical AMX spin system at 6.61 (dd, J ) 17.0, 10.5 Hz), 6.32 (dd, J ) 17.0, 2.0 Hz), and 5.71 (dd, J ) 2.0, 10.5 Hz) ppm. O-Sulfonation of 5 with sulfur trioxide-trimethylamine complex in N,N-dimethylformamide (DMF) at 50 °C gave the persulfate 6 in 84% yield. Comparison of the 1H NMR spectra of 5 and sulfated lactose 6 in deuterium oxide (D2O) showed the downfield chemical shifts of the ring protons due to sulfation. 2. Synthesis of Sulfated Lactose-Based Glycopolymers. Prior investigations have confirmed that cyanoxyl (OCN)mediated free-radical polymerization of alkene-derivatized glycomonomers is a convenient tool to produce water-soluble glycopolymers. However, glycopolymer yields were limited using alkene-derivatized glycomonomers. As a consequence, in this report acrylamide monomers, which have higher reactivity levels compared with alkene monomers were investigated. Notably, the synthesis of polyacrylamide-based neoglycoconjugates has been extensively reported. These polymers have been used as diagnostic reagents, such as in the inhibition of hemagglutination by pathogens21 or as solidphase coatings in enzyme-linked immunosorbent assays.22,23 Cyanoxyl radicals were generated by an electron-transfer reaction between cyanate anions (-OCtN), from a NaOCN aqueous solution, and p-chlorobenzenediazonium salts (ClC6H4NtN+BF4-), which were previously prepared in situ through a diazotization reaction of p-chloroaniline in water (Scheme 2). In addition to cyanoxyl persistent radicals, aryltype active radicals were simultaneously produced, and only the latter species is capable of initiating chain growth. As summarized in Table 1, cyanoxyl-mediated homopolymerization of lactose heptasulfate monomers generated the expected sulfated glycopolymers in high conversion yield (60-80%) and with relatively low polydispersity (1.19 < Mw/Mn < 1.62). Polymers of varying molecular weight were
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Table 1. Free-Radical Homopolymerization of Nonsulfated and Sulfated Lactose and Statistical Copolymerization with AM
glycomonomer (GM) 6 6 6 6 6 5 6 6 a
GM/AM (mol) 1/0 1/0 1/0 1/0 1/0 1/7 1/1 1/1
[M]0/[I]0
yield (%)
10 25a 25 50 35 20 10 50
81 55 78 11 34 71 80 67
polymer composition (mol) 1/0 1/0 1/0 1/0 1/0 1/6 1/10 1/11
lactose content (wt %)b
Mn (g/mol)
Mw/Mn SEC
glycopolymer
100 100 100 100 100 49 57 56
2100 7500 38600 51900 114000 9000 9300 33400
1.47 1.19 1.30 1.45 1.62 1.30 1.46 1.47
7a 7b 7c 7d 7e 8a 8b 8c
Polymerization was performed for 4 h, all other reaction were performed for 16 h. b Mass content of saccharide were determined by 1H NMR analysis.
Figure 1.
1H
NMR spectrum of glycopolymer 8a in D2O (300 MHz).
Scheme 2. Cyanoxyl-Mediated Free-Radical Copolymerization of Sulfated Lactose with Acrylamide (AM)
prepared by altering either monomer conversion or the initial ratio of monomer to initiator concentrations ([M]0/[I]0). Similarly, copolymers were prepared by copolymerization of nonsulfated and sulfated lactose monomers with acrylamide in low polydispersity (∼1.47). The resultant glycopolymers were also characterized by NMR spectroscopy. For example, as illustrated in Figure 1, comparison of the integrated signals produced from the chain-end phenyl
protons (H2′,6′ and H3′,5′) with those due to the anomeric protons of lactose (H1′-Lact and H1-Lact) and the backbone protons indicate that the average composition of glycopolymer 8a was 10 lactose units and 60 acrylamide units. 3. Bioactivity Studies of Synthetic Sulfated Glycopolymer. The anticoagulant activity of sulfated glycopolymers was evaluated by the method of Lee-White (Table 3).24 The anticoagulant activity of heparin was significantly higher than that of all synthetic heparinoid polymers. Nonetheless, lactose heptasulfate-based glycopolymers induced a significant prolongation in coagulation time, while a nonsulfated glycopolymer (8a) had no effect. It is noteworthy that sulfated monosaccharide-based homo- (9) and co-glycopolymers (10) (Table 2), which were synthesized as previously described from sulfated 2-acrylethyl R/β-glycoside of N-acetyl-glucosamine,16 showed no activity in this bioassay. Interestingly, heteroglycopolymers 8c and 8b showed higher anticoagulant activity than that of homoglycopolymers 7a-7d, as well as low molecular weight dextran sulfate (LMWDS). These
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Glycosaminoglycan Mimetic Biomaterials Table 2. Sulfated Monosaccharide-Based Acrylic Glycopolymers
monomer ratio GM/AM (mol)
polymer composition (mol)
monosaccharide content (wt %)
Mn (g/mol)
Mw/Mn SEC
1/0 1/0 1/1 1/1 1/4 1/1
1/0 1/0 1/4 1/9 1/9 1/12
100 100 66 47 42 40
9900 20800 48000 5400 115600 21700
1.13 1.49 1.45 1.15 1.57 1.20
Table 3. Anticoagulant Activity of Synthetic Sulfated Glycopolymers
7a 7b 7c 7d 7e 8a 8b 8c 9 10 LMWDS Lovenox Heparin
aPTT (100 s) 298.6 218.8 a 305.6 116.7 a 79.9 100.7 a a 111.1 16.0 5.6
9a 9b 10a 10b 10c 10d
Table 4. Antithrombin Activity of Synthetic Sulfated Glycopolymers
glycopolymer concentration (µg/mL) required to prolong aPTT glycopolymer
glyco polymer
glycopolymer
glycopolymer concn (µg/mL)
thrombin time (s)
100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 2.3
12.6 13.3 14.4 15.6 18.0 19.0 19.0 13.4 14.0 13.5 17.0 19.4 20.0 28.9 36.0 38.5 21.0 23.4 24.4 27.9 26.9 29.0 13.5 13.9 14.9 13.9 14.5 13.5 >300
aPTT (200 s) 812.5 531.2 a 840.3 309.0 a 184.0 246.5 a a 312.5 34.7 13.8
results suggest that anticoagulant activity is dependent upon the presence of sulfated disaccharides. Moreover, modulating the density of these groups by using acrylamide as a comonomer can be used as a means of optimizing anticoagulant activity. Nonetheless, measured thrombin times, as summarized in Table 4, were only modestly prolonged suggesting that the observed anticoagulant effect is not primarily related to direct thrombin inhibition. In principle, the observed prolongation of the PTT may have been due to a selective sequestration of fibrinogen by sulfated glycopolymers or by a potentiating effect on other anticoagulant factors, such as heparin cofactor II. Conclusions We have synthesized a series of sulfated lactose-based glycopolymers as heparin mimetics. Sulfated as well as acrylate-derivatized glycomonomers were polymerized using cyanoxyl-mediated free radical polymerization, which further demonstrates the versatility of this approach for preparing water-soluble glycopolymers in good yield and of relatively low polydispersity (1.1 < Mw/Mn < 1.6). Sulfated lactose and acrylamide-based heteroglycopolyomers were found to have higher anticoagulant activity than nonsulfated lactose
7a
7b
7c
7d
7e
8b
8c
9a
10b
Heparin
glycopolymers or LMDS. The use of selective -OH blocking groups for regiospecific sulfation and carboxylation of lactose monomers may provide an additional approach for future efforts directed at generating polymers with heparin-like activity. Acknowledgment. This work was supported by grants from the NIH. The authors acknowledge the Emory University NMR and Mass Spectrometry Centers for use of their facilities.
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References and Notes (1) Clowes, A. W.; Karnovsky, M. J. Nature 1977, 265, 625. (2) Jackson, R. L.; Busch, S. J.; Cardin, A. L. Physiol. ReV. 1991, 71, 481. (3) Linhardt, R. J.; Kerns, R. J.; Vlahov, I. R. In Biomedical Functions and Biotechnology of Natural and Artificial Polymers; ATL Press: 1996; p 45. (4) Van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. Engl. 1993, 32, 1671. (5) Westerduin, P.; Basten, J. E. M.; Broekhoven, M. A.; De Kimpe, V.; Kuijpers, W. H. A.; Van Boeckel, C. A. A. Angew. Chem., Int. Ed. Engl. 1996, 35, 331. (6) Petitou, M.; Duchaussoy, P.; Driguez, P.-A.; Jaurand, G.; He´rault, J.-P.; Lormeau, J.-C.; Van Boeckel, C. A. A.; Herbert, J.-M. Angew. Chem., Int. Ed. Engl. 1998, 37, 3009. (7) Petitou, M.; He´rault, J.-P.; Bernat, A.; Driguez, P.-A.; Duchaussoy, P.; Lormeau, J.-C.; Herbert, J.-M. Nature 1999, 398, 417. (8) Ornitz, D. M.; Herr, A. B.; Nilsson, M.; Westman, J.; Svahn, C.-M.; Waksman, G. Science 1995, 268, 432. (9) Akashi, M.; Sakamoto, N.; Suzuki, K.; Kishida, A. Bioconjugate Chem. 1996, 7, 393. (10) Miyata, T.; Nakamae, K. Trends Polym. Sci. 1997, 5, 198. (11) Chr. Heuck, C.; Schiele, U.; Fronda, D.; Ritz, E. J. Biol. Chem. 1985, 260, 4598. (12) Bentolila, A.; Uodavsky, I.; Ishai-Michael, R.; Kovalchuk, O.; Haloun, C.; Domb, A. J. J. Med. Chem. 2000, 43, 2591.
Sun et al. (13) Hatanaka, K., Yoshida, T., Miyahara, S., Sato, T., Ohno, F., and Uryu, T. J. Med. Chem. 1987, 30, 810. (14) Ito, Y., Iguchi, Y., Kashiwagi, T., and Imanishi, Y. J. Biomed. Mater. Res. 1991, 25, 1347. (15) Grande, D.; Baskaran, S.; Basakaran, C.; Gnanou, Y.; Chaikof, E. L. Macromolecules 2000, 33, 1123. (16) Grande, D.; Baskaran, S.; Chaikof, E. L. Macromolecules 2001, 34, 1640-1646. (17) Weigel, P. H.; Schnaar, R. L.; Roseman, S.; Lee, Y. C. Methods Enzymol. 1982, 831, 294. (18) Chernyak, A. Y.; Levinsky, A. B.; Kochetkov, N. K. Bioorg. Khim. 1988, 14, 1047. (19) Roy, R.; Tropper, F. D. Glycoconjugate J. 1988, 5, 203. (20) Chernyak, A. Y.; Sharma, G. V. M.; Kononov, L. O.; Kvishna, P. R.; Levinsky, A. B.; Kochetkov, N. K. Carbohydr. Res. 1992, 223, 303. (21) Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 3769. (22) Roy, R.; Tropper, F. D.; Romanowska, A. J. Chem. Soc., Chem. Commun. 1992, 1611. (23) Bovin, N. V. Glycoconjugate J. 1998, 15, 431. (24) Shapiro, G. A., Huntzinger, S. W., and Wilson, J. E. Am. J. Clin. Pathol. 1977, 67, 477.
BM025561S
