Glycodendritic Structures Based on Boltorn Hyperbranched Polymers

David K. Wang , David J. T. Hill , Firas A. Rasoul , Andrew K. Whittaker. Journal of Polymer Science Part A: Polymer Chemistry 2012 50 (6), 1143-1157 ...
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Bioconjugate Chem. 2003, 14, 817−823

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Glycodendritic Structures Based on Boltorn Hyperbranched Polymers and Their Interactions with Lens culinaris Lectin Eva Arce,† Pedro M. Nieto,† Vicente Dı´az,‡ Rossana Garcı´a Castro,§ Antonio Bernad,§,‡ and Javier Rojo*,† Grupo de Carbohidratos, Instituto de Investigaciones Quı´micas, CSIC, Isla de la Cartuja, Ame´rico Vespucio s/n, E-41092 Sevilla, Spain, and Departamento de Inmunologı´a y Oncologı´a and Genetrix S.L., Centro Nacional de Biotecnologı´a, CSIC, Cantoblanco, E- 28049 Madrid, Spain. Received January 20, 2003; Revised Manuscript Received March 4, 2003

Multivalent scaffolds bearing carbohydrates have been prepared to mediate biological processes where carbohydrates are involved. These systems consist of dendritic structures based on Boltorn H20 and H30 hyperbranched polymers to which carbohydrates are linked through a convenient spacer. Mannose has been chosen as a sugar unit to test the viability of this strategy. These glycodendritic compounds have been prepared in a few steps with good yields, showing a high solubility in physiological media and low toxicity. The binding of these dendritic polymers to the mannose-binding lectin Lens culinaris (LCA) was studied using STD-NMR experiments and quantitative precipitation assays. The results demonstrate the existence of a clear interaction between the mannose derivative systems and the Lens lectin where the dendritic scaffold does not have an important role in mannose binding but supplies the necessary multivalence for lectin cluster formation. These glycodendritic structures are able to interact with a receptor, and therefore they can be considered as promising tools for biological studies.

INTRODUCTION

Carbohydrates play important roles in many biological processes as embryogenesis, differentiation, morula compaction, metastasis, etc. They are involved in cell-cell and cell-extracellular matrix adhesion and recognition processes through the interaction with their receptors (proteins and carbohydrates). These interactions show strong divalent cation dependence and low affinity, which is compensated in Nature by multivalent presentation of carbohydrates (1). During the past decade, chemists have focused their efforts on preparing multivalent carbohydrate model systems to study and interfere with biological processes in which carbohydrates are involved. Among the multivalent systems used, the most common are liposomes (2, 3), dendrimers (4), calixarenes (5), cyclodextrins (6), oligomers and polymers (7), and nanoparticles (8). Dendrimers with a high degree of versatility are probably the most developed and popular multivalent systems. Unfortunately, they suffer from disadvantages, namely their tedious and expensive synthesis with several steps of separation and purification. Dendritic hyperbranched polymers, in contrast to linear polymers, present many features in common with dendrimers with the advantage of an easier synthesis with low cost (9). However, unlike dendrimers, hyperbranched polymers have to be considered only as a dendritic scaffolds presenting a polydispersity > 1 while dendrimers are monodisperse (10). * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: + 34 95 448 95 68; Fax: + 34 95 446 0565. † Grupo de Carbohidratos. ‡ Departamento de Inmunologı´a y Oncologı´a. Fax: + 34 91 372 0493. § Genetrix S.L.

An example using successfully dendritic polymers as scaffolds for multivalent presentation of carbohydrates is PAMAM (polyamidoamine) (11); although commercially available, PAMAM is relatively expensive. In contrast, Boltorn polymers are inexpensive, commercially available hyperbranched polymers based on 2,2-bis(hydroxymethyl)propionic acid as a AB2 monomer and 2,2-bis(hydroxymethyl)-1,3-propanediol as central core (12). They are supplied with different degrees of branching (generation) having a different number of primary hydroxyl groups on their surface to be functionalized: Boltorn H20 (16 OH), Boltorn H30 (32 OH), and Boltorn H40 (64 OH). Applications of Boltorn polymers have been mainly focused on their use as coatings and additives for plastics with the only exception of its use as a soluble solid support for oligosaccharides synthesis (13). To our best knowledge, these dendritic polymers have never been considered as scaffolds for multivalent presentation of biologically important ligands and for their potential application to study and intervene in biological processes (14). Here, we present a direct strategy for the preparation of glycodendritic structures based on Boltorn polymers BH20 and BH30 (Figure 1) as a dendritic support functionalized with R-D-mannose (1 and 2, respectively). Mannose oligosaccharide structures are found coating the surface of numerous infection agents such as viruses, bacteria, and fungi. Specific receptors for mannose are expressed on the surface of cells as dendritic cells and macrophages, involved on the immune system and responsible of antigen presentation (15). A natural ligand for these receptors is mannan, a highly branched mannose-containing polysaccharide isolated mainly from Saccharomyces cerevisae. It consists of a R(1-6) linked mannose backbone with branches of R(1-2) and R(1-3) linked mannose (16, 17). This mannosylated compound

10.1021/bc034008k CCC: $25.00 © 2003 American Chemical Society Published on Web 05/29/2003

818 Bioconjugate Chem., Vol. 14, No. 4, 2003

Figure 1. BoltornH20 (BH20) and BoltornH30 (BH30).

presents a high affinity for mannose receptors as legume lectins (18, 19). However, mannan cannot be used as therapeutics in vivo due to its toxicity and its mitogenic effect (20, 21). The design and preparation of highly mannosylated systems as potential tools for blocking the entry of these types of pathogens are of great interest (22-24). Also, the fact that mannose receptors are found in dendritic cells and macrophages can be used to stimulate the immune system through their interactions with compounds presenting antigens (25). To test the binding capabilities of our glycodendritic structures, we have selected the Lens culinaris lectin (LCA) which specifically recognizes glucose and mannose (26). Lectins are a large and diverse family of proteins (many of them are commercially available) presenting carbohydrate recognition domains capable to recognize carbohydrates specifically. For this reason, they are considered as ideal models to study carbohydrate-protein interactions with new neoglycosystems. We have performed two different binding experiments with LCA and glycodendritic structures 1 and 2: a quantitative precipitation assay and an NMR study based on saturation transfer difference (STD) experiments. These experiments have allowed demonstration of the existence of the interaction and determination of the part of the molecule involved in binding process. Finally, cytotoxicity studies were carried out to determine the scope and the future applications of these glycodendritic systems in in vitro and in vivo experiments. EXPERIMENTAL PROCEDURES

Materials. All chemicals were obtained from Aldrich and used without further purification, unless otherwise noted. Lens culinaris lectin (LCA) was purchased from Sigma. Boltorn H20 and Boltorn H30 were supplied by Perstorp Specialty Chemicals. Dendritic structures were purified by dialysis against water, using water/benzoylated cellulose dialysis tubing, flat width 32 mm, MW cutoff 1 kDa obtained from Sigma. 1H and 13C NMR were recorded on Bruker Avance DPX 300, DRX 400, and DRX 500 MHz spectrometers. Chemical shift are in ppm with respect to TMS (Tetramethylsylane) using manufacturer indirect referencing method. 2D experiments (COSY, TOCSY, ROESY, and HMQC) were performed when necessary to assign the dendritic structures spectra. IR

Arce et al.

spectra were recorded on a Bruker Vector 22. Optical rotations were measured with a Perkin-Elmer 341 polarimeter. Preparation of BH20sucOH 5. To a solution of Boltorn H20 (5.0 g, 2.86 mmol) in pyridine (30 mL) were added succinic anhydride (5.44 g, 54.3 mmol, 1.2 equiv per OH) and a catalytic amount of DMAP (ca. 100 mg), and the mixture was stirred overnight at room temperature and concentrated. The residue was dissolved in water and concentrated again. Lyophilization afforded BH20sucOH (10.2 g, quant.) as a white solid. 1H NMR (DMSO-d6, 500 MHz): δ 4.08 (m, 56H, 28 CH2OCO), 2.48 (m, 2 × 32H, 16 OCOCH2CH2COOH), 1.14 (m, 36H, 12 CH3); 1H NMR (CD3CN, 500 MHz): δ 9.3 (broad s, 16H, 16 COOH), 4.23 (m, 56H, 28 CH2OCO), 2.58 (m, 2 × 32H, 16 OCOCH2CH2COOH), 1.25 (m, 36H, 12 CH3); 13C NMR (DMSO-d6, 100 MHz): δ 176.3 (COOH), 176.0 (COO), 174.4 (COO), 67.7 (CH2O), 48.8 (C), 31.5 and 31.3 (OCOCH2CH2COOH), 19.7 (CH3); 13C NMR (CD3CN, 125 MHz): δ 174.1 (COOH), 172.9 (COO), 172.8 (COO), 66.3 (CH2O), 47.3 (C), 29.6 and 29.2 (OCOCH2CH2COOH), 17.9 (CH3); IR (KBr): ν 3415, 1731, 1695, 1470 cm-1. Preparation of BH30sucOH 6. To a solution of Boltorn H30 (3.0 g, 0.88 mmol) in pyridine (30 mL) were added succinic anhydride (3.40 g, 34.0 mmol, 1.2 equiv per OH) and a catalytic amount of DMAP (ca. 100 mg), and the mixture was stirred overnight at room temperature and concentrated. The residue was dissolved in water and concentrated again. Lyophilization afforded BH30sucOH (5.83 g, quant.) as a white solid. 1H NMR (DMSO-d6, 500 MHz): δ 4.12 (m, 120H, 60 CH2O), 2.48 and 2.42 (2m, 2 × 64H, 32 OCOCH2CH2COOH), 1.14 (m, 84H, 28 CH3); 1H NMR (CD3CN, 500 MHz): δ 4.18 (m, 120H, 60 CH2OCO), 2.54 (m, 2 × 64H, 32 OCOCH2CH2COOH), 1.20 (m, 84H, 28 CH3); 13C NMR (CD3CN, 125 MHz): δ 174.2 (COOH), 173.0 (COO), 172.9 (COO), 66.3 (CH2O), 47.4 (C), 29.1 (OCOCH2CH2COOH), 17.9 (CH3); IR (KBr): ν 3415, 1735, 1695, 1475 cm-1. Preparation of BH20sucMan 1. To a solution of 2-aminoethyl-R-D-mannopyranoside (10) (27 mg, 0.123 mmol, 1.2 equiv per OH) and BH20sucOH 5 (20 mg, 6.39 µmol) in DMF:CH2Cl2 1:1 (5 mL) were added DIC (48 µL, 0.307 mmol, 3 equiv per OH) and HOBT (41 mg, 0.307 mmol, 3 equiv per OH). The reaction mixture was stirred at room temperature for 24 h and then evaporated. The residue was washed with Et2O and purified by dialysis to obtain compound 1 (22 mg, 60%) as a white lyophilizate. [R]D ) +17 (c ) 0.9, H2O); 1H NMR (D2O, 300 MHz): δ 4.85 (s, 16H, H1), 4.36-4.19 (m, 56H, Ha), 3.92 (m, 16H, H2), 3.86 (m, 16H, H6), 3.76 (m, 16H, He), 3.74 (m, 16H, H6′), 3.69 (m, 16H, H3 or H4), 3.62 (m, 16H, H3 or H4), 3.61 (m, 16H, H5), 3.55 (m, 16H, He), 3.40 (m, 32H, Hd), 2.67 (m, 32H, Hc), 2.56 (m, 32H, Hb), 1.27 (m, 36H, CH3); 13C NMR (D2O, 75 MHz): δ 182.3 (CO), 181.6 (CO), 175.6 (CO), 100.9 (C1), 74.0 (C5), 71.2 (C3 or C4), 71.0 (C2), 68.0 (C3 or C4), 66.9 (Ca), 66.8 (Ce), 62.1 (C6), 47.8 (C), 40.3 (Cd), 31.5 (Cc), 30.9 (Cb), 18.1 (CH3); IR (KBr): ν 3415, 1635, 1615, 1570 cm-1. Preparation of BH30sucMan 2. To a solution of 2-aminoethyl-R-D-mannopyranoside (10) (26 mg, 0.116 mmol, 1.2 equiv per OH) and BH30sucOH 6 (20 mg, 3.03 µmol) in DMF:CH2Cl2 1:1 (5 mL) were added DIC (46 µL, 0.291 mmol, 3 equiv per OH) and HOBT (40 mg, 0.291 mmol, 3 equiv per OH). The reaction mixture was stirred at room temperature for 24 h, then evaporated. The residue was washed with Et2O and purified by dialysis to obtain compound 2 (27 mg, 68%) as a white lyophilizate. [R]D ) +35 (c ) 0.9, H2O); 1H NMR (D2O, 300 MHz): δ 4.84 (s, 32H, H1), 4.32-4.21 (m, 120H, Ha), 3.92

Glycodendritic Structures Based on Boltorn Polymers

(m, 32H, H2), 3.86 (m, 32H, H6), 3.76-3.57 (m, 192H, He + H6′ + H3 + H4 + H5), 3.40 (m, 64H, Hd), 2.67 (m, 64H, Hb), 2.56 (m, 64H, Hc), 1.27 (m, 84H, CH3); 13C NMR (D2O, 75 MHz): δ 176.2 (CO), 175.4 (CO), 175.3 (CO), 101.0 (C1), 74.2 (C5), 71.9 (C3 or C4), 71.4 (C2), 68.0 (C3 or C4), 67.3 (Ca), 67.2 (Ce), 62.2 (C6), 47.9 (C), 40.3 (Cd), 31.4 (Cc), 30.5 (Cb), 18.4 (CH3); IR (KBr): ν 3415, 1735, 1635, 1615, 1570 cm-1. Preparation of BH20suclinker 3. To a solution of 2-aminoethanol (19 µL, 0.307 mmol, 3 equiv per OH) and BH20sucOH 5 (20 mg, 6.39 µmol) in DMF:CH2Cl2 1:1 (5 mL) were added DIC (48 µL, 0.307 mmol, 3 equiv per OH) and HOBT (41 mg, 0.307 mmol, 3 equiv per OH). The reaction mixture was stirred at room temperature for 24 h, then evaporated. The residue was washed with Et2O and purified by dialysis to obtain compound 3 (17 mg, 71%) as an colorless syrup. 1H NMR (D2O, 400 MHz): δ 4.36-4.22 (m, 56H, Ha), 3.63 (t, 32H, J ) 5.7 Hz, He), 3.31 (t, 32H, J ) 5.7 Hz, Hd), 2.67 (m, 32H, Hb), 2.56 (m, 32H, Hc), 1.27 (m, 36H, CH3); 13C NMR (D2O, 100 MHz): δ 182.1 (CO), 181.3 (CO), 176.2 (CO), 66.9 (Ca), 61.1 (Ce), 42.5 (Cd), 31.1 (Cc), 30.2 (Cb), 18.0 (CH3); IR (KBr): ν 3415, 1735, 1655, 1560 cm-1. Preparation of BH30suclinker 4. To a solution of 2-aminoethanol (44 µL, 0.728 mmol, 3 equiv per OH) and BH30sucOH 6 (50 mg, 7.59 µmol) in DMF:CH2Cl2 1:1 (15 mL) were added DIC (115 µL, 0.728 mmol, 3 equiv per OH) and HOBT (100 mg, 0.728 mmol, 3 equiv per OH). The reaction mixture was stirred at room temperature for 24 h, and then evaporated. The residue was washed with Et2O and purified by dialysis to obtain compound 4 (53 mg, 88%). 1H NMR (D2O, 300 MHz): δ 4.37-4.22 (m, 120H, Ha), 3.63 (t, 64H, J ) 5.1 Hz, He), 3.31 (t, 64H, J ) 5.1 Hz, Hd), 2.68 (m, 64H, Hb), 2.57 (m, 64H, Hc), 1.28 (m, 84H, CH3); 13C NMR (D2O, 100 MHz): δ 182.1 (CO), 181.3 (CO), 175.6 (CO), 67.7 and 67.1 (Ca), 61.4 (Ce), 47.9 (C), 42.8 (Cd), 31.4 (Cc), 30.5 (Cb), 18.3 (CH3); IR (KBr): ν 3415, 1740, 1655, 1560 cm-1. Quantitative Precipitation Assays. Quantitative precipitation assays were carried out by a modification of a previously described method (27). LCA was dissolved in PBS (1 mL, 20 µM) at pH ) 7.4 containing 0.1 mM CaCl2 and 0.5 mM MgCl2 (PBST buffer) to make a final concentration of LCA 25 µM (assuming MW ) 49 kD). The solution was incubated for 5 h a room temperature with different concentrations of the corresponding dendritic structures and controls. After centrifugation at 10000g for 10 min, the supernatant solution was removed and the precipitated was washing two times with cold buffer. This precipitate was dissolved using a 1 M solution of methyl-R-D-mannopyranoside (0.8 mL) in PBST buffer. The final solution was vortexed briefly and after 10 min protein concentration was analyzed measured absorbance at 280 nm using a Perkin-Elmer Lambda 12 UV/vis spectrometer. Measurements are the average of two independent experiments. Data represented in Figure 2 are concentration of precipitation agent against concentration of LCA calculated from the experimental absorbance. STD NMR Experiments. All NMR experiments were performed on a Bruker Avance DRX 500 spectrometer at 273 K. The NMR samples were prepared by dissolving the lectin in D2O solutions of dendritic compounds (1 mM and 0.5 mM for 1 and 2, respectively) yielding a ratio of ligand molecules to lectin binding sites of 50:1. The saturation transfer difference (STD) experiments were recorded using essentially the sequence proposed by Meyer (28). A cascade of soft Gaussian-shaped pulses of 50 ms (with a power level of 80-40 Hz for the corre-

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sponding square shape) was used for the saturation. The saturation times were varied from 0.25 to 2.5 s. Onresonance irradiation was done at 7 or 0 ppm while offresonance at +30 ppm. A short spin-lock period (10-15 ms) was used prior to the acquisition in order to eliminate the remaining protein signals. Cell Lines and Culture Conditions. COS-7 and 293-T cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Inc), supplemented with 10% fetal calf serum (FCS; Gibco-BRL), 2 mM L-glutamine (Merck), streptomycin (0.1 mg/mL, Sigma), penicillin (100 U/ml, Sigma), sodium pyruvate (0.1%, Sigma), and β-mercaptoethanol (50 µM, Sigma). All cell cultures were grown in a humidified 37 °C incubator with 5% CO2 and periodically tested for being mycoplama-free using a specific commercial kit (Gen-Probe, San Diego, CA). Cytotoxicity Studies. Cell cultures were routinely monitored by cell scoring using a haemocytometer chamber after careful trypsinization of the adherent cell monolayer, using the trypan-blue vital dye. Cellular proliferation was determined using the MTT colorimetric assay, essentially as described by Mosmann (29), and by direct cell counting. Briefly, 105 cells/well were seeded into 96-well plates in 100 µL of complete medium and incubated for the indicated time periods, in the presence or absence of the tested glycodendritic structures. After the cell monolayer was washed, a solution (10 µL) of tetrazolium salt (MTT, 25 mg/mL in PBS) was then added to each well and incubated at 37 °C for 4 h. Under these conditions, MTT is reduced by living cells into an insoluble blue formazan product that is collected by centrifugation and dissolved by the addition of DMSO (100 µL) with vigorous shaking. Plates are then read with a multiwell scanning spectrometer at 540 nm. RESULTS AND DICUSSION

Glycodendritic polymers have been prepared linking the carbohydrate moiety to the polymer core through a spacer, giving flexibility and accessibility to the sugar for future biological applications. We have selected commercially available Boltorn H20 and H30 hyperbranched polymers as candidates to prove the viability of this strategy (Figure 1). These polymers present 16 and 32 hydroxy groups, respectively, on their surfaces, providing a multivalency comparable to other systems with biological activity described in the literature (30, 31). The first step of the preparation was the introduction of carboxylic groups on the dendritic core surface. This step gives a dendritic scaffold with carboxylic acid ending groups to be easily functionalized with glycoconjugates via an amide bond. This was achieved by mixing Boltorn H20 or H30 polymers and succinic anhydride in the presence of DMAP and pyridine (Scheme 1). After the mixture was stirred overnight at room temperature, the functionalized polymers were obtained (compounds 5 and 6), as demonstrated by spectroscopic techniques. Signals for carboxylic groups can be observed by IR spectroscopy, and the relationship between signals corresponding to the succinate methylene protons and the methyl groups of the polymer in the 1H NMR spectrum confirmed the number of carboxylic groups introduced. A simple monosaccharide (R-D-mannose) has been selected as carbohydrate unit to test this methodology. More complicated oligosaccharidic structures can be envisaged for future applications. Mannose derivative 10 was prepared in four steps with good yields from the

820 Bioconjugate Chem., Vol. 14, No. 4, 2003 Scheme 1. Preparation of Dendritic Compounds 1-4 from the Hyperbranched Polymer BH20 and BH30a

a (i) Succinic anhydride, DMAP cat., Py, 50 °C, 15 h, 100%; (ii) HOBT, DIC, DMF:CH2Cl2 1:1, 10 or HOCH2CH2NH2 (1.2 equiv per OH), 24 h, 60% (1), 70% (2), 68% (3), or 88% (4).

Scheme 2. Synthesis of Glycoconjugate 10 from Peracetylated Mannose 7a

Arce et al.

acting as a multivalent system of the corresponding antigen. Compounds 1-4 and 10 were incubated with LCA in PBST buffer, and the amount of protein in the precipitate was measured. The absorbance observed when glycoconjugate 10 was used at concentrations up to 0.1 mM was almost zero (data not shown). This monovalent system is not capable to form a cluster with the Lens culinaris lectin even a high concentrations, and therefore no precipitation was detected. For compounds 3 and 4, which present dendritic structures but without saccharidic units, a small absorbance was observed (data not shown) at the higher concentration used (0.1 mM). This could be interpreted on the base of nonspecific interactions between dendritic structures and lectin. Different behavior was observed for dendritic structures 1 and 2 which present 16 and 32 copies of mannose on their surface, respectively. Both of them present a similar profile concentration-dependent lectin precipitation. (Figure 2).

a (iii) HOCH CH Br, BF ‚Et O (5 equiv), CH Cl , 0 °C to room 2 2 3 2 2 2 temperature, 24 h, 73%; (iv) NaN3 (8 equiv), DMF, 50 °C, 15 h, 98%; (v) a) NaOMe, MeOH, rt, 15 min; (b) H2 (1 atm), Pd(C), AcOEt:EtOH 1:1, rt, 20 h, 88%, two steps.

corresponding peracetylated mannose 7 following the synthetic pathway represented in Scheme 2 (32). The spacer was introduced directly in the anomeric position by reaction with 2-bromoethanol in the presence of a large excess of BF3‚Et2O (derivative 8). Further manipulation of this chain was necessary to obtain an adequate functionalization. Substitution of bromine by azide with sodium azide in DMF gave compound 9; deacylation under Zemplen conditions (NaOMe/MeOH) (33) and catalytic hydrogenation of the azide group rendered compound 10 ready to be attached to the dendritic cores 5 and 6. The final step consisted of the introduction of 16 or 32 units of 10 on the functionalized Boltorn H20 or H30 polymers 5 and 6, respectively. This was achieved by reaction of 10 with the carboxylic acid residues of the dendritic structures 5 and 6 in the presence of 1-hydroxybenzotriazole (HOBT) and diisopropylcarbodiimide (DIC) (Scheme 1) giving glycodendritic compounds 1 and 2. Dialysis purification afforded 1 and 2 as white solids after lyophilization. For future biological essays dendritic polymers 3 and 4, presenting 16 and 32 linkers respectively, were prepared as control. Reaction of aminoethanol with activated Boltorn polymers 5 and 6 in the presence of HOBT and DIC gave compounds 3 and 4 which were purified by dialysis. (Scheme 1) All dendritic compounds prepared were characterized spectroscopically. Quantitative Precipitation Assays. Glycodendritic structures 1 and 2 presenting several copies of mannose could form, a priori, clusters of mannose-binding lectins (as LCA) that precipitate. Quantification of the protein on this precipitate may give an idea of the efficiency of these systems based on valence (34). The formation of this precipitate clearly indicates that the ligand used is

Figure 2. Quantitative precipitation assay. Final concentration of LCA precipitated plotted against concentration of glycodendritic compounds 1 and 2.

Starting at a ligand concentration of about 2 µM, there is a remarkable increase in the amount of precipitated protein which continues growing slowly with increasing ligand concentration. The maximum amount of protein precipitate during the experiment corresponds to approximately 30% of the initial amount. No significant differences were found between compounds 1 and 2, indicating that, with these specific model systems, an increase in the number of mannose units from 16 to 32 has no influence on the binding in this particular case. These results show the importance of multivalency for cluster formation and precipitation but may also indicate that a crowded presentation of ligands on the dendritic surface might not have a positive effect on the interaction with LCA. This situation could impede the access of ligands to the protein recognition site, and therefore longer and more flexible linkers can be considered to have an important effect on ligand presentation and to promote better interaction with the protein. STD NMR Experiments. STD experiments have been extensively used for ligand screening (see ref 28) and epitope mapping of molecular interactions at atomic level (35). These experiments are based on the transference of NMR saturation, through spin diffusion, from a large receptor into a small ligand due to transient binding. Thus NMR signals for those ligands interacting with a given receptor are detected. The experiment could be used also for epitope mapping. In this case, the signals corresponding to those protons closer to the receptor are detected. The method is rather robust and less sensitive to binding constant requirements than other NMR experiments as transfer NOE. We have used this methodol-

Glycodendritic Structures Based on Boltorn Polymers

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Figure 5. Percentage of survival for COS-7 and 293T cell lines in the presence of different concentrations of glycodendritic compounds 1 and 2 up to 30 mM based on number of mannose units.

Figure 3. NMR Saturation transfer experiments recorded with 2.5 s of protein saturation time (top) and reference 1H NMR spectra (bottom) for (a) BH20sucMan 1 and (b) BH30sucMan 2.

Figure 4. NMR Saturation transfer experiments for 10 at (a) 250, (b) 357, (c) 510, (d) 1020 ms of saturation time, and (e) reference 1H NMR spectra indicating the absence of interaction between the ethylene linker and the lectin. Arrows indicate linker ethylene moiety signals.

ogy to study the interaction of 1, 2, and 10 with Lens culinaris lectin. Similar qualitative results were found for both hyperbranched polymers and the mannose glycoconjugate 10, indicating that the interaction of these three compounds with LCA takes place in a similar way (Figures 3 and 4). Thus, it can be concluded that attaching the carbohydrate moiety to the three-dimensional scaffold does not alter significantly its protein binding properties and only provides polyvalence. STD technique can also yield valuable structural information identifying the regions of the ligand involved in the interaction with the receptor. We have also tried to map the epitope of the interaction of 1, 2, and 10 with LCA in different experimental conditions. In our case, 1 and 2, as a consequence of their size, are in the NOE spin diffusion limit. Therefore, they should have internal saturation transfer rates comparable with the lectinligand ones. These features do not prevent the STD experiments although could make the observation of selectivity within the ligand difficult. This would explain the detection in the STD experiments of all the protons of the dendrimers including the internal CH2 and CH3 groups of the Boltorn polymer, even at relatively short

saturation times of 1-0.5 s. The selectivity was improved using even shorter saturation times. In these cases, the relative intensity of the linker and polymer signals, compared with the carbohydrate ones, decreased. This result suggests that the interaction occurs mainly through the mannose residue. However, under those conditions, such short saturation times cause very low overall signal intensity. Also the spin-lock period used can induce transference of magnetization among the mannosecoupled protons, obscuring the results. Both effects prevent a deeper analysis of the specific zone of the carbohydrate involved in binding. As expected, better selectivity was observed for the case of glycoconjugate 10, which has a more adequate size. All the mannose protons were clearly detected (Figure 4), indicating that this is the part of the molecule in contact with the lectin. The intensity of the signals from the ethylene moiety (see arrows in Figure 4) of the linker clearly decreased, which means a looser interaction of this part of the molecule with the lectin. This result confirms that the linker moiety does not interact with the protein, and therefore the sugar moiety is responsible for the interaction with LCA. The results from these interaction experiments indicate that the lectin binding properties of the mannose are conserved in the larger glycodendritic structure. Finally, from the STD-NMR experiments it can be concluded that the linker moiety does not interact with the protein. Cytotoxicity Studies. Biological application of these glycodendritic compounds relies first on good solubility in physiological conditions. Compounds 1-4 were completely soluble in aqueous buffers commonly used to perform in vitro studies. Another important issue is the compatibility of these systems with biological targets. We tested their potential toxicity against different cell lines (COS-7 and 293T). For that purpose, after exposure of cell cultures during 24 h to increasing amounts of the above-described systems, an MTT colorimetric assay was performed following the procedure described (ref 29, see Materials and Methods for more details). The results (Figure 5) indicated that compounds 1 and 2 were nontoxic in the whole concentration range tested (up to 3 mM, based on number of mannose units) and for the two cell lines included in the study. Higher concentrations (up to 30 mM) induce a moderate decrease in the cell viability (Figure 5). Similar results were obtained for control compounds 3 and 4 (data not shown). Also, human peripheral blood mononuclear cells (PBMCs) were incubated with the glycodendritic structures and, again, cytotoxicity was not found (data not shown). Therefore, these results strongly suggest that the described glycodendritic compounds can be considered as promising candidates to carry on biological studies.

822 Bioconjugate Chem., Vol. 14, No. 4, 2003 CONCLUSIONS

In summary, we have prepared two glycodendritic structures 1 and 2 presenting 16 and 32 units of mannose, respectively, and two controls 3 and 4 without carbohydrate units based on Boltorn H20 (second generation) and H30 (third generation) hyperbranched polymers. Their solubility and their low toxicity are compatible with their use as potential tools to study biological processes. Results obtained from the interaction studies of these glycodendritic structures and a mannose-binding lectin, Lens culinaris lectin, using STD NMR experiments and quantitative precipitation assays demonstrate that the dendritic scaffold has no influence on the binding capabilities of mannose glycoconjugates, that being an adequate platform. On the other hand, the multivalent presentation of the sugar units is essential for lectin cluster formation, although in this particular case, no differences have been found increasing the number of sugar units from 16 to 32. In other words, the capability of these compounds to interact with biological receptors as lectins has been demonstrated. These results open new possibilities for the design and preparation of new glycodendritic structures based on Boltorn hyperbranched polymers with different spacers and degrees of branching, including biologically important carbohydrates. ACKNOWLEDGMENT

This research was supported by the DGES (grants no. PB 960820 to J.R. and SAF 2001-2262 to A.B.). The authors want to thank Perstorp Specialty Chemicals for the generous gift of Boltorn polymers. We thank also Prof. M. Martı´n-Lomas and Dr. S. Penade´s for scientific and financial support. Supporting Information Available: 1H NMR (300 MHz) spectra in D2O of glycodendritic structures 1 and 2. This material is available free of charge via Internet at http://pubs.acs.org LITERATURE CITED (1) Mammen, M., Choi, S. K., and Whitesides, G. M. (1998) Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew. Chem., Int. Ed. 37, 2754-2794. (2) Brewer, G. J., and Matinyan, N. (1992) Congregation of Gangliosides at the Junction between Two Model Membranes. Biochemistry 31, 1816-1820. (3) Stewart, R. J., and Boggs, J. M. (1993) Carbohydratecarbohydrate Interaction between Galactosylceramide-containing Liposomes and Cerebroside Sulfate-Containing Liposomes: Dependence on the Glycolipid Ceramide Composition. Biochemistry 32, 10666-10674. (4) Ro¨ckendorf, N., and Lindhorst, T. K. (2001) Glycodendrimers. Top. Curr. Chem. 217, 201-238. (5) Dondoni, A., Marra, A., Scherrmann, M. C., Casnati, A., Sansone, F., and Ungaro, R. (1997) Synthesis and Properties of O-Glycosyl Calix[4]arenes (Calixsugars). Chem. Eur. J. 3, 1774-1782. (6) Roy, R., Herna´ndez-Mateo, F., Santoyo-Gonza´lez, F. (2000) Synthesis of Persialylated beta-Cyclodextrins. J. Org. Chem. 65, 8743-8746. (7) Roy, R. (1996) Syntheses and Some Applications of Chemically Defined Multivalent Glycoconjugates. Curr. Opin. Struct. Biol. 6, 692-702. (8) de la Fuente, J. M., Barrientos, A. G., Rojas, T. C., Rojo, J., Can˜ada, J., Ferna´ndez, A., and Penade´s, S. (2001) Gold Glyconanoparticles as Water-soluble Polyvalent Models to Study Carbohydrate Interactions. Angew. Chem., Int. Ed. 40, 2258-2261.

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