Biomacromolecules 2002, 3, 998-1005
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Galactosylated N-Vinylpyrrolidone-Maleic Acid Copolymers: Synthesis, Characterization, and Interaction with Lectins Rachel Auze´ ly-Velty,*,† Mariana Cristea,‡ and Marguerite Rinaudo† Centre de Recherches sur les Macromole´ cules Ve´ ge´ tales, BP53, 38041 Grenoble cedex 9, France, and Romanian Academy “Petru Poni” Institute of Macromolecular Chemistry, 6600 Iasi, Romania Received March 25, 2002; Revised Manuscript Received June 4, 2002
Water-soluble artificial glycoconjugate polymers were synthesized from poly(N-vinylpyrrolidone-co-maleic anhydride) by amidation with an amine-containing galactose derivative. The glycopolymers having different galactose contents were fully characterized in terms of chemical structure by NMR and potentiometric titrations, and their aqueous behavior was studied by viscometric measurements. Their specific binding properties were examined by enzyme-linked lectin assays using RCA120 lectin. Whatever the glycopolymer, the grafted galactoses were shown to behave similarly to free galactose. Introduction Cell surface carbohydrates from glycoproteins and glycolipids play essential roles as recognition sites between cells or cells and microorganisms.1 These recognition mechanisms are essentially based on specific interactions between the oligosaccharide chains and soluble or membrane proteins. This communication network involving carbohydrates occurs in a wide variety of important biological phenomena such as cell growth, inflammation, cancer, viral, and bacterial infections. The crucial role of carbohydrates as information molecules has sparked the synthesis of suitable glycomimetics, in particular glycopolymers, as artificial antigens, potent inhibitors, cell-specific culture substrata, and drug-targeting delivery systems.2 Glycoconjugate polymers can be obtained by polymerization of sugar-carrying monomers. An alternative synthetic method consists of the chemical modification of preformed polymers using carbohydrate-containing reagents. The latter methodology is generally advantageous as it requires fewer reaction steps than the polymerization method and it allows easy control of the number of sugars along the polymeric chain. However incomplete reactions with the functional groups along the polymer chain may lead to polymers with not well-defined structures. In this paper, we describe the controlled synthesis of new glycopolymers (1) based on the direct coupling of a lactose derivative with poly(N-vinylpyrrolidone-co-maleic anhydride) (P(NVPMAn)). The specific interaction of these glycopolymers with different sugar contents toward RCA120 lectin, which specifically recognizes terminal β-D-galactosyl residues, is discussed. * To whom correspondence may be addressed: rachel.auzely@ cermav.cnrs.fr. Tel: + 33 4 76 03 76 70. Fax: +33 4 76 54 72 03. † Centre de Recherches sur les Macromole ´ cules Ve´ge´tales. ‡ Romanian Academy “Petru Poni” Institute of Macromolecular Chemistry.
Experimental Section Materials and Methods. Lactose, Amberlite IR-120, 1,4diaminobutane, and anhydrous dimethyl sulfoxide (DMSO) were purchased from Fluka. Iodine and silver carbonate were obtained from Prolabo. Biotin-labeled Ricinus communis agglutinin (RCA120), extrAvidin peroxidase conjugate, and o-phenylenediamine dihydrochloride (OPD) peroxidase substrate were obtained from Sigma. Polyacrylamide-bound β-Dgalactose was purchased from Syntesome. Other chemicals and solvents were reagent grade and were used without further purification. 1H NMR experiments were performed using Bruker AC300, DRX500, and Varian Unity Plus 500 spectrometers operating at 300 and 500 MHz. 13C NMR spectra were acquired with inverse-gated decoupling on Bruker DRX400 and AC300 spectrometers operating at 100 and 75 MHz, respectively. Chemical shifts are given relative to external tetramethylsilane (TMS ) 0 ppm), and calibration was performed using the signal of the residual protons or
10.1021/bm020035m CCC: $22.00 © 2002 American Chemical Society Published on Web 07/11/2002
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Galactosylated Copolymers Scheme 1a
a
Reagents and conditions: (i) 0.1 M NaOH, room temperature, 12 h; (ii) Amberlite IR120 (H+).
carbons of the solvent as a secondary reference. Deuterium oxide was obtained from SDS (Vitry, France). Details concerning experimental conditions are given in the figure captions. Fast atom bombardment (FAB) mass spectra were measured on a Nermag R 1010C mass spectrometer. The primary beam consisted of Xe atoms, the samples were dissolved in glycerol, and the positive ions were separated and accelerated over a potential of 9 kV. The steric exclusion chromatography (SEC) analyses were performed using a Waters GPCV 2000 chromatograph equipped with a viscometer, a multiangle laser light scattering (MALLS) DSP-F from Wyatt with two Shodex columns: OH pack 802 and 803. The eluent was a 0.1 M NaNO3 solution. For weightaverage molecular weight (Mw) calculations from light scattering, the value of the refractive index increment dn/dc was determined in 0.1 M NaNO3 using a Brice Phoenix differential refractometer (λ ) 546 nm) and was equal to 0.147 ( 0.005 mL/g. Fourier transform infrared spectroscopy (FTIR) measurements were performed on a FT-IR 1720 X from Perkin-Elmer using KBr pellets. For potentiometric titrations, pH measurements were performed with a Knick 646 pHmeter equipped with a glass microelectrode from Bioblock Scientific. The concentration, Cp, of the copolymers (concentration of the carboxylic acid functions) was 0.0015 M for titration with 0.1 M NaOH. The potentiometric data were used to determine the apparent pKa values of the carboxylic acid functions according to the following relations taking into account the presence of two different carboxylic acid groups per comonomer:3 pKa1 ) pH - log[R/(1 - R)]
(1)
pKa2 ) pH - log[(R - 1)/(2 - R)]
(2)
Viscometric measurements were carried out at 25 ( 0.1 °C using a Fica capillary viscometer with a 0.58 mm capillary. The intrinsic viscosities were obtained by extrapolating the reduced viscosity data to zero polymer concentration using the Huggins equation as described below: ηsp/C ) [η] + k′[η]2C
(3)
where k′ is the Huggins constant. Synthesis. Poly(N-Vinylpyrrolidone-co-maleic anhydride). The parent copolymer of maleic anhydride and N-vinylpyrrolidone was prepared following methods described in the literature.4 For characterization, the copolymer was hydrolyzed with aqueous 0.1 M NaOH at room temperature for 12 h (pH of the solution: 9-9.5), filtered on Sartorius (Goettingen, Germany) membranes (pore size: 0.2 µm), and then purified
by ultrafiltration though an ultramembrane Amicon PM10. The resulting sodium maleate copolymer 2b was transformed to the acidic form to give 2c by passing though an ion-exchange resin column (Amberlite IR-120 (H+)) (Scheme 1). N-(4-Aminobutyl)-O-β-D-galactopyranosyl-(1f4)-D-gluconamide, 3. To a solution of lactose (3.42 g, 0.01 mol) in water (40 mL) at room temperature, an iodine suspension (0.1 M, 200 mL) and a potassium hydroxide solution (0.1 M, 600 mL) were slowly and successively added for 3-4 h. After the color of iodine disappeared, the mixture was passed through an ion-exchange resin column (Amberlite IR-120 (H+)). The eluate was then stirred with silver carbonate (15 g) overnight. After filtration of the suspension, the filtrate was passed again through a column of Amberlite IR-120 (H+). The acidic eluate was then freeze-dried to give D-lactonolactone 5 (3 g, 0.0088 mol). To a solution of 1,4diaminobutane (9.24 g, 0.105 mol) in DMSO (85 mL), a solution of D-lactonolactone 5 (1 g, 0.003 mol) in DMSO (28 mL) was added dropwise for 3 h. After being stirred overnight at room temperature, the reaction mixture was concentrated under reduced pressure, and the residual oil was poured into acetone (200 mL). The white precipitate was filtered, washed with acetone, and dried to give 3 (0.804 g, 63%). 1 H NMR (D2O, 500 MHz) δ/ppm: 4.44 (H-1′, d, J1′,2′ ) 7.5 Hz); 4.27 (H-2, d, J2,3 ) 2.5 Hz); 4.05 (H-3, t, J ) 3.5 Hz); 3.86 (H-4, t, J ) 4 Hz); 3.83-3.58 (H-4′, H-5, H-5′, H-6a, H-6b, H-6′a, H-6′b); 3.55 (H-3′, dd, J3′,4′ ) 3.5 Hz, J3′,2′ ) 9.5 Hz); 3.45 (H-2′, dd); 2.85, 2.80 (CONHCH2CH2CH2CH2NH2, 2t, J ) 7.5 and 6.5 Hz); 1.55 (CONHCH2 CH2CH2CH2NH2, m). 13 C NMR (D2O, 75 MHz) δ/ppm: 174.5 (CONH); 104.0 (C-1′); 81.5 (C-4); 75.9 (C-5′); 73.1, 73.0, 72.9, 71.9, 71.6, 70.9 (C-2, C-2′, C-3, C-3′, C-5); 69.1 (C-4′); 62.5 (C-6); 61.6 (C-6′); 39.5, 39.2 (CONHCH2CH2CH2CH2NH2); 26.4, 26.15 (CONHCH2CH2CH2CH2NH2). IR (KBr pellet) ν (cm-1): 1645 (amide I), 1550 (amide II). FABMS: m/z 429 [M + H]+. General Procedure for the Synthesis of Glycopolymers 1a-c. To a solution of copolymer 2a dried over P2O5 (0.07 M (with respect to anhydride functions)) in anhydrous DMSO, a solution of N-(4-aminobutyl)-O-β-D-galactopyranosyl-(1f4)-D-gluconamide (3) (0.03 M; 0.05, 0.30, or 0.60 equiv) in anhydrous DMSO was added. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. After evaporation of the solvent, the residual oil was diluted with deionized water, and a 0.1 M NaOH solution was added (until pH 9-9.5). After being stirred for 10 h at room temperature, the solution was filtered on
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Sartorius (Goettingen, Germany) membranes (0.2 µm pore size), then diafiltrated with deionized water through an ultramembrane Amicon PM10. The diafiltration was stopped when the filtrate conductivity was lower than 10 µS and the glycopolymer was recovered by freeze-drying. Enzyme-Linked Lectin Assay (ELLA). All incubations were carried out at 37 °C. Except for the washing after the first step (coating of the plates with the ligand), after each incubation, the plates were washed three times with 0.01 M phosphate saline buffer (pH 7.4) containing 0.1% (v/v) Tween 20 (PBST, 200 µL). Nunc microtitration plates were coated overnight with polyacrylamide-bound β-D-galactose (100 µL of a 5 µg/mL solution in carbonate-bicarbonate buffer pH 9.4). Excess polyacrylamide was removed by one washing with PBS (200 µL). Coated wells were then blocked with 3% w/v BSA in the phosphate saline buffer PBS (150 µL) for 1 h. After two washings with PBS (200 µL), the wells were filled with serial dilutions of biotin-labeled RCA120 from 0.1 to 5 µg/mL (100 µL) for 1 h. The plates were washed and extrAvidin peroxidase conjugate (100 µL of a solution in PBS at a dilution of 1/2000) was added. After an incubation period of 1 h and washing, OPD peroxidase substrate (100 µL of a solution in milli-Q water with 0.02% hydrogen peroxide) was added. The reaction was stopped after 20 min by adding a 4 M H2SO4 solution (25 µL), and the absorbance was measured at 490 nm with a Dynatech MR5000 apparatus. The concentration of lectin giving an absorbance of 1, i.e., 1 µg/mL, was used for inhibition experiments. Inhibition Experiments. The microtitration plates were coated overnight with polyacrylamide-bound β-D-galactose (100 µL of a 5 µg/mL solution in carbonate-bicarbonate buffer pH 9.4). The plates were then washed and blocked with BSA as described above. Serial dilutions of each inhibitor, i.e., N-(4-acetamidobutyl)-O-β-D-galactopyranosyl(1f4)-D-gluconamide 6 (obtained by full acetylation of 3 followed by de-O-acetylation under Zemple´n conditions) as reference monovalent ligand and P(NVP-NaM) 2b, P(NVPNaM) 2b/N-(4-acetamidobutyl)-O-β-D-galactopyranosyl(1f4)-D-gluconamide, glycopolymers 1b,c in PBST containing 0.3% w/v BSA (300 µL) were incubated with biotinlabeled RCA120 (300 µL of a 2 µg/mL solution in PBST containing 0.3% w/v BSA) for 1 h. The solutions (100 µL) were then transferred to the galactose coated plates which were incubated for an additional hour. The plates were washed, and extrAvidin peroxidase conjugate (100 µL) was added as described above. After 1 h of incubation and washing, the OPD peroxidase substrate was added. Color development was stopped after a 20 min period, and the absorbance was measured. The percent inhibitions were calculated as follows: % inhibition ) (A(no inhibitor) - A(with inhibitor))/A(no inhibitor) × 100 (4) Each assay was carried out in duplicate. Results and Discussion 1. Synthesis of Maleic Copolymers Bearing Pendant Galactosyl Moieties. The general synthetic pathway to
Auze´ ly-Velty et al. Scheme 2. Synthetic Route to Glycopolymers 1
glycopolymers involved (i) the preparation and characterization of poly(N-vinylpyrrolidone-co-maleic anhydride) (P(NVPMAn)) copolymer 2a, (ii) the synthesis of a lactose derivative possessing a reactive amino endgroup 3, and (iii) the coupling of the latter with the copolymer (Scheme 2). (a) Synthesis and Characterization of Maleic Anhydride Copolymer 2a. Copolymer 2a was prepared by radical copolymerization of N-vinylpyrrolidone (VP) and maleic anhydride (MAn).4 Owing to the participation as an independent monomer in the reaction process of the chargetransfer complex formed from VP and MAn, a 1:1 alternating copolymer is formed. The 1:1 composition of our copolymer was however checked by quantitative 13C NMR and potentiometric titration after hydrolysis of the maleic anhydride groups. As shown by Figure 1, integration of the NMR signals arising from sodium maleate (NaM) and VP moieties of P(NVP-NaM) 2b gives a 1:1 ratio between NaM and VP. The curve relating the pH with the degree of neutralization R of the copolymer transformed to the acidic form (2c), using 0.1 M NaOH as titrant, shows only one inflection corresponding to half the neutralization of the polymer (see Figure 2). When an external salt (1 M NaCl, 0.005 M CaCl2, 0.01 M CaCl2) is added to screen electrostatic interactions,5,6 a two-step neutralization curve is obtained with transitions at R ) 1 and 2 (Figure 2). This differenciation between the two carboxylic functions may be due to the position of -COOH on the two neighboring carbons with a large inductive effect as observed with low molecular weight diacids. Thus, using a supporting electrolyte, the composition of copolymer 2 could be determined by potentiometric titration. In our study, the best results were obtained with 0.005 M CaCl2. The value of the mass per equivalent for the sodium form (P(NVP-NaM)) was 0.00712 g/equiv which is in good agreement with the theoritical value (0.00732 g/equiv). This study thus allowed confirmation of the 1:1 composition of our copolymer. In a second step, the macromolecular parameters of the copolymer, i.e., the weight-average molecular weight Mw and the intrinsic viscosity [η], were determined using SEC and viscometry, respectively. The characteristics of the copolymer in the polycarboxylate form (P(NVP-NaM)) are given in Table 1. Finally before starting on the chemical modification of the polymer, it was important to ascertain the presence of almost all the anhydride functions. Indeed an extensive hydrolysis of these groups may affect the efficiency of the coupling reaction which is based on the nucleophilic attack of an amine on the anhydride functions. In the IR spectrum of 2a, absorption bands at 1780 and 1850 cm-1 corresponding to the carbonyl stretching of the anhydride group could be observed. In addition, a band assigned to the carboxylic
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Galactosylated Copolymers
Figure 1.
13C
NMR spectrum (75 MHz, 25 °C, 100 mg/mL in D2O) of P(NVP-NaM) copolymer.
Figure 2. Titration curves (R ) [NaOH]/Cp) of P(NVP-MAc) copolymer with 0.1 M NaOH as titrant at Cp ) 0.0015 M, together with the first derivatives in (A) pure water, (B) 1 M NaCl, (C) 0.005 M CaCl2, and (D) 0.01 M CaCl2. Table 1. Macromolecular Parameters for P(NVP-NaM) Copolymer 2b
Mw (g/mol) polydispersity index (Mw/Mn) [η] (mL/g) in 0.1 M NaCl at 25 °C [η] (mL/g) in 0.5 M NaCl at 25 °C
31000 ( 500 1.7 ( 0.2 46.3 ( 1 41.2 ( 1
acid group appeared at 1720 cm-1, indicating that the initial copolymer was partly hydrolyzed. Nevertheless, the anhydride functions could be easily re-formed after drying the copolymer in a vacuum over P2O5 at 110 °C for half a day. (b) Synthesis of Lactose Derivative 3 Possessing a Reactive Amino Endgroup. Lactose 4 was oxidized by
hypoiodite according to a literature method (Scheme 3).7 The resulting lactonolactone 5 was then reacted with 1,4-diaminobutane in large excess affording N-(4-aminobutyl)-O-β-Dgalactopyranosyl-(1f4)-D-gluconamide, 3. Thus, lactose could be easily functionalized without protecting the hydroxyl groups to allow galactosylation of copolymer 2a through the D-gluconamide unit as a hydrophilic spacer. Purification of compound 3 could be achieved by treatment with a cation-exchange resin. However we showed that purification on this level was not necessary since byproducts such as unreacted lactonolactone 5 can be removed by ultrafiltration in the final step of the synthesis of the glycopolymer.
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Figure 3. Scheme
13C
Auze´ ly-Velty et al.
NMR spectrum (75 MHz, 25 °C, 100 mg/mL in D2O) of glycopolymer 1b.
3a
Table 2. Degree of Substitution and Weight-Average Molecular Weight (from SEC) for P(NVP-NaM) Copolymers Bearing Pendant Galactoses degree of substitution (%)
a Reagents and conditions: (i) I , KOH, H O, room temperature; (ii) 2 2 Amberlite IR120 (H+); (iii) Ag2CO3, H2O, room temperature, 12 h; (iv) Amberlite IR120 (H+); (v) 1,4-diaminobutane, DMSO, 12 h.
(c) Coupling of Lactose Derivative 3 with P(NVP-AM) Copolymer. Covalent attachment of compound 3 to the copolymer 2a was carried out by the formation of amide linkages. The reaction based on the nucleophilic attack of the amino endgroup of 3 on the anhydride functions of 2a proceeded smoothly at room temperature in anhydrous dimethyl sulfoxide without degradation of the polymer backbone. The reaction was performed using a different molar ratio of 3/P(NVP-MAn) monomole (0.05/1, 0.30/1, 0.60/1) in order to evaluate effects of the sugar content on the solution properties of glycopolymers. After a treatment with aqueous NaOH, an ultrafiltration process, and freezedrying, copolymers bearing pendant galactose moieties 1 were isolated in 53-71% yields. 2. Characterization of Maleic Copolymers Bearing Pendant Galactosyl Moieties. The chemical structure of glycopolymers 1 was ascertained by 13C NMR spectroscopy. As an example, Figure 3 displays the 13C NMR spectrum performed in D2O on glycopolymer 1b having on average 25 galactoses every 100 maleic comonomers. The presence of the galactosyl residues on the polymer backbone was clearly evidenced by the NMR signal at 104 ppm assigned to the anomeric CII-1 resonance of β-D-galactopyranoside and the other ten NMR signals at 82-61 ppm assigned to the CI and CII-2,3,4,5,6 resonances of the galactosyl and gluconamide moieties. The galactose contents relative to the maleic comonomer units (degrees of substitution, DS) were determined by 13C NMR analysis, and potentiometric titration (Table 2). The plots of pKa1 and pKa2 versus R for the glycopolymers are found to show no sign of abnormal behavior, by comparison with those of the initial copolymer 2c. In 0.01 M CaCl2, the apparent pKa values for 2c and 1b are nearly
compound
RMN 13C
potentiometric titration
Mw (g/mol)
1a 1b 1c
3(1 25 ( 2 50 ( 2
3 ( 0.3a 30 ( 3b
32000 ( 1 220000c 210000c
a Performed in 0.005 M CaCl . b Performed in 0.01 M CaCl . c Apparent 2 2 value due to aggregates.
Figure 4. Variation of pKa1 and pKa2 of copolymer 2c and glycopolymer 1b during neutralization of the carboxylic acid functions in 0.01 M CaCl2 at Cp ) 0.0015 M.
independent of R within a large range and one obtains the intrinsic pK0: pK01 ∼ 4-4.3 and pK02 ∼ 6.3 (see Figure 4). These pK0 values are in agreement with those obtained for alternating copolymers of maleic acid and propene in the presence of CaCl2.6 The potentiometric experiment not only could provide information on the degree of conversion but also allowed confirmation of the formation of “hemiamides”. The neutralization in the presence of calcium chloride shows clearly two inflection points corresponding to equal amounts of NaOH added before chemical modification (see Figure 2). But, for the neutralization of the glycopolymer 1b (Figure 5), the second inflection point occurs for a lower volume of NaOH added (V2) compared to the first one (V1). The ratio V2/V1 is found to be 0.70,
Galactosylated Copolymers
Figure 5. First derivative of the titration curve of glycopolymer 1b with 0.1 M NaOH as titrant at Cp ) 0.0015 M in 0.01 M CaCl2.
which indicates that some carboxylic acid groups (30% on the basis of the maleic monomers) are not titrated due to
Biomacromolecules, Vol. 3, No. 5, 2002 1003
chemical functionalization. This result indicates that only one of the two carboxylic groups per maleic monomer is modified. The weight-average molecular weight Mw for glycopolymers 1-c was measured by SEC, and the results are shown in Table 2. It can be noticed that the weight-average molecular weights Mw are very high for higher carbohydrate contents. In fact, the multimodal molecular weight distributions in SEC data (see Figure 6) suggest the presence of aggregates as a result of hydrogen bonding between the sugar moieties. Figure 7 shows the reduced viscosity ηsp/C as a function of concentration C (g/mL) for glycopolymers 1b,c and P(NVP-NaM) 2b in 0.1 M NaCl. Salt was added in order to limit the electrostatic repulsions of the polyelectrolyte chains. It is interesting to observe a decrease of the intrinsic viscosity value as the sugar content increases (Table 3). Indeed, the number of free carboxylate groups decreases whereas the number of OH-rich substituents favoring hydrogen bonds
Figure 6. Molecular weight distribution obtained from SEC for glycopolymer 1b. The molecular weights at the top of the peaks are indicated by an arrow. M (g/mol) ) 65000 and 260000.
Figure 7. Reduced viscosities of P(NVP-NaM) copolymer and glycopolymers 1b,c at 25 °C in 0.1 M NaCl.
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Auze´ ly-Velty et al.
Table 3. Intrinsic Viscosity and Huggins Constant in 0.1 NaCl at 25 °C for P(NVP-NaM) Copolymer 2b and Glycopolymers 1b,c 0.1 M NaCl polymer
[η] (mL/g)
k′
P(NVP-NaM), 2b 1b 1c
46.3 ( 1 45.5 ( 1 29 ( 1
0.35 0.18 0.31
Figure 8. Inhibition of binding of RCA120 to polyacrylamide-bound β-D-galactose by N-(4-acetamidobutyl)-O-β-D-galactopyranosyl-(1f4)D-gluconamide, 6, and glycopolymers 1b,c. Comparison of the binding properties of grafted galactose (a) with free galactose in the absence and in the presence of copolymer 2b and (b) as a function of the degree of substitution.
increases with increasing DS, resulting in a more compact conformation of the polymer chains. 3. Binding Studies of Glycopolymers 1 to RCA120 Lectin. The enzyme-linked lectin assay (ELLA) evaluates the ability of a soluble ligand to inhibit the adhesion of a multivalent lectin to a reference ligand noncovalently immobilized on the surface of a microtitration plate. The lectin is labeled with peroxidase allowing the spectrometric analysis of binding. Polyacrylamide-bound β-D-galactose (PAA-Galβ) was used as a coating antigen in microtitration plates and extrAvidin peroxidase conjugate/biotin-labeled Ricinus communis agglutinin (RCA120) (which specifically recognizes terminal β-D-galactosyl residues) were used for detection. The inhibition data of N-(4-acetamidobutyl)-O-β-D-galactopyranosyl-(1f4)-D-gluconamide, 6, as reference monovalent ligand and glycopolymers 1b,c are shown in Figure 8. The concentration of bound galactose for glycopolymers 1b,c
was derived from 13C NMR analysis and potentiometric titration. The results demonstrate that glycopolymers 1b,c inhibit the binding of RCA lectin to PAA-Galβ at a galactose concentration close to that of the reference ligand. Thus, bound galactoses behave similarly to the reference free ligand. From these inhibition experiments, it appears that no significant benefit of multivalency is observed for glycopolymers contrary to results obtained from mannosylated polymers and dendrimers.8 Multivalent carbohydrateprotein interactions depend closely on the geometry, conformation, and valency of glycoclusters. The loss of the glucosyl residue as a result of the chemical derivation of lactose to be grafted on the polymer might explain this result. Indeed, in previous studies on interactions between lactose/ galactose and specific lectins, it has been shown that the affinity enhancement provided by the use of “cluster” galactosides and lactosides is more efficient when the saccharide residue is lactose although the monovalent analogues possess the same binding affinity toward the lectin.9 This demonstrates that the glucosyl residue plays an important role in the improvement of the binding affinity of multivalent galactosides. Nevertheless, it can be noticed that the grafting of galactose on the polymer backbone does not impair the affinity of the sugar to the lectin. In conclusion, a series of water-soluble N-vinylpyrrolidone-maleic acid copolymers with pendant galactose moieties were efficiently synthesized by graft conjugation of a nucleophilic galactose derivative to the copolymer of maleic anhydride. The glycopolymers were fully characterized by NMR, potentiometric titrations, SEC, and viscometric measurements. Moreover, they showed good inhibitory properties against model RCA120 lectin in solid-phase enzyme-linked assays. Experiments dealing with the potential applications of these glycopolymers and derivatives for drug targeting are currently under investigation. Other sugar moieties can also be grafted to control the specific carbohydrate-protein interactions. Acknowledgment. The authors acknowledge the Re´gion Rhoˆne-Alpes (France) for its financial support and the grant for M. Cristea (Project No. 0814363). They gratefully acknowledge D. Cade and T. Hamaide for the synthesis of the parent copolymer of maleic anhydride and N-vinylpyrrolidone. They appreciate useful discussions, comments, and suggestions by H. Driguez. They thank C. Breton, V. Chazalet, and C. Gautier for experimental support and helpful discussions for ELLA tests. They also thank C. Bosso for the mass spectrometry measurements. References and Notes (1) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Dwek, R. A. Chem. ReV. 1996, 96, 683. (2) For reviews, see: (a) Magnusson, G.; Chernyak, A. Ya.; Kihlberg, J.; Kononov, L. O. In Neoglycoconjugates; preparation and applications; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, CA, 1994; p 53. (b) Tropper F. D.; Romanowska, A.; Roy, R. In Methods in Enzymology; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, CA, 1994; Vol. 242, p 257. (c) Okada, M. Prog. Polym. Sci. 2001, 26, 67. (d) Roy, R. In Carbohydrate Chemistry; Boons, G.-J., Ed.; Thomson Science: London, U.K., 1998; Chapter 1. (e) Roy, R. Top. Curr. Chem. 1997, 187, 242. (f) Roy, R. Trends Glycosci. Glycotechnol. 1996, 8, 79.
Galactosylated Copolymers (3) Katchalsky, K.; Spitnik, P. J. Polym. Sci. 1947, 2, 432. (4) (a) Nagasawa, M.; Price, S. A. J. Am. Chem. Soc. 1960, 80, 5070. (b) Geogiev, G.; Konstantinov, C.; Kabaivanov, V. Macromolecules 1992, 25, 6302. (5) Chitanu, G. C.; Rinaudo, M.; Desbrie`res, J.; Milas, M.; Carpov, A. Langmuir 1999, 15, 4150. (6) Reinhardt, S.; Steinert, V.; Werner, K. Eur. Polym. J. 1996, 32, 935. (7) Schaffer, R.; Isbell, H. S. Methods Carbohydr. Chem. 1963, 2, 11.
Biomacromolecules, Vol. 3, No. 5, 2002 1005 (8) Page´, D.; Zanini, D.; Roy, R. Bioorg., Med. Chem. 1996, 4, 1949. (9) (a) Vargas-Berenguel, A.; Ortega-Caballero, F.; Santoyo-Gonzalez, F.; Garcia-Lopez, J. J.; Gimenez-Martinez, J. J.; Garcia-Fuentes, L.; Ortiz-Salmeron, E. Chem. Eur. J. 2002, 8, 812. (b) Monsigny, M. Personnal communication.
BM020035M
