High Molecular Weight Polyglycerol-Based Multivalent Mannose

Biomacromolecules 2010, 11, 2567–2575

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High Molecular Weight Polyglycerol-Based Multivalent Mannose Conjugates Jayachandran N. Kizhakkedathu,*,†,‡ A. Louise Creagh,§ Rajesh A. Shenoi,† Nicholas A. A. Rossi,† Donald E. Brooks,†,‡ Timmy Chan,† Jonathan Lam,† Srinivasa R. Dandepally,† and Charles A. Haynes*,§ Centre for Blood Research, Department of Pathology and Laboratory Medicine, Michael Smith Laboratories, Department of Chemical and Biological Engineering, and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada Received May 4, 2010; Revised Manuscript Received August 10, 2010

We report the synthesis and characterization of multivalent mannose conjugates based on high molecular weight hyperbranched polyglycerols (HPG). A range of glycoconjugates were synthesized from high molecular weight HPGs (up to 493 kDa) and varying mannose units (22-303 per HPG). Hemagglutination assays using fresh human red blood cells and concanavalin A (Con A) showed that HPG-mannose conjugates exhibited a large enhancement in the relative potency of conjugates (as high as 40000) along with a significant increment in relative activity per sugar (up to 255). The size of the HPG scaffold and the number of mannose residues per HPG were all shown to influence the enhancement of binding interactions with Con A. Isothermal titration calorimetry (ITC) experiments confirmed the enhanced binding affinity and showed that both molecular size and ligand density play important roles. The enhancement in Con A binding to the high molecular weight HPG-mannose conjugates is due to a combination of inter- and intramolecular mannose binding. A few fold increments in the binding constant were obtained over mannose upon covalent attachment to HPG. The binding enhancement is due to the highly favorable entropic contribution to the multiple interactions of Con A to mannose residues on HPG. The high molecular weight HPG-mannose conjugates showed positive cooperativity in binding to Con A. Although carbohydrate density has less of an effect on functional valency of the conjugate compared to the molecular size, it determines the binding affinity.

Introduction Hyperbranched polyglycerols (HPG) received significant attention in recent years due to its excellent biocompatibility1-6 and the availability of a large number of peripheral hydroxyl groups (equal to the degree of polymerization) that can be used as potential sites for modification.5-8 Hydrophobically derivatized HPG has been designed recently as a human serum albumin substitute9 as well as carrier for hydrophobic drug molecules.10 HPGs have been used for the conjugation of drug molecules, peptides, and carbohydrates.11-15 It has also been used as a biocompatible protective layer for synthetic surfaces,16,17 microfluidic devices,18 liposomes,19 and cell surfaces.20,21 Very recently, aldehyde functionalized HPGs have been used as a versatile soluble peptide capturing agent in proteomic analysis.22 Thus, HPGs are particularly attractive for various bioconjugation strategies.5 Multivalent carbohydrate interactions are believed to be responsible for many biological processes.23-29 The importance of carbohydrate-protein/cell binding in various biological events makes them very attractive in the development of therapeutic agents that specifically target certain proteins or cells. However, the low affinity of monovalent carbohydrate ligands to proteins * To whom correspondence should be addressed. Phone: 604-822-7085 (J.N.K.); 604-822-5136 (C.A.H.). Fax: 604-822-7742 (J.N.K.). E-mail: [email protected] (J.N.K.); [email protected] (C.A.H.). † Centre for Blood Research, Department of Pathology and Laboratory Medicine. ‡ Department of Chemistry. § Michael Smith Laboratories, Department of Chemical and Biological Engineering.

and cell receptors has hindered the development of carbohydratebased therapeutic agents. The enhancement of binding affinities can be achieved by presenting the carbohydrates in multivalent arrays, such as in the case of polymer-based glycoconjugates.23,28-34 Carbohydrate-based linear and dendritic polymers have been synthesized by incorporating multiple copies of carbohydrate ligands on the surface of polymers and have shown to increase both the binding affinities and the dissociation constants in the millimolar to micro- and nanomolar range.24,25,27,28,35-50 Such constructs are also shown to inhibit certain biological interactions and it has been demonstrated that multivalent/polyvalent presentation is critical to achieving this goal. However, the mechanism for the binding affinity enhancement of such polymeric or macromolecular ligands is still under investigation.43,51-56 Among the many factors suggested, the heterogeneity or polydispersity of macromolecular ligands has been deemed to be a critical factor. Furthermore, most of the studies have been performed with polyvalent ligands with dimensions that are smaller than those of lectin.55,56 The use of high molecular weight HPG for the decoration of carbohydrate epitopes has many advantages. First, its circulation half-life (t1/2) in mice depends on its molecular weight; for example, t1/2 reaches almost 60 h for HPG having a molecular weight of 540 kDa.3 Thus, conjugates with high vascular retention can possibly be designed. Second, very high molecular weight HPGs (up to ∼1 million) can be synthesized by an overnight experiment, with good control over polydispersity (as low as ∼1.05) and in good yields.8 Third, the flexibility of HPGs compared to dendrimers; the HPG scaffold can potentially orientate or change conformation to make the maximum number

10.1021/bm1004788  2010 American Chemical Society Published on Web 08/30/2010

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of epitopes or ligands available for binding without a large entropic penalty. On the other hand, HPG is less flexible than linear polymers. The conformational freedom that allows a polymer to take up an optimal structure for interaction appears to be a useful property in this regard. The presentation of carbohydrates on such a scaffold is anticipated to enhance its binding to proteins and cells. As shown by Rele et al., branched polymers offer additional factors such as accessibility, mobility, and density, while the organization of carbohydrate epitopes could influence the optimal binding of carbohydrate epitopes to proteins and cell surface.57 In addition, it has been suggested that the orientation of carbohydrate residues on the cell surface plays an important role in receptor function.58 To summarize, these properties make high molecular weight HPG a suitable candidate for the development of scaffolds as multivalent glycoconjugates. In the present work, we report the synthesis and characterization of HPG-glycoconjugates in an effort to understand the effect of assembling carbohydrate units on high molecular weight HPGs and to determine whether such arrangements can produce multivalent conjugates. We used mannose as a model carbohydrate for the synthesis of HPG conjugates and studied their interactions with mannose binding lectin Concanavalin A (Con A) using hemagglutination measurements and isothermal titration calorimetry (ITC) experiments. To the best of our knowledge, this is the first report of glycoconjugates based on high molecular weight HPGs and their evaluation using ITC.

Experimental Section Materials and Methods. Concanavalin A CanaValia ensiformis (Calbiochem), methyl R-D-mannopyranoside, adenosine (99%), N-[2hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), and bovine serum albumin (96%, fatty-acid free; Sigma-Aldrich, Oakville, ON, Canada), tris(hydroxymethyl)-aminomethane (TRIS; Bio-Rad Laboratories, Hercules, CA), sodium phosphate (monobasic anhydrous, NaH2PO4; Sigma-Aldrich, Oakville, ON, Canada), sodium phosphate (dibasic anhydrous, Na2HPO4), and sodium chloride (Fisher Scientific, Fair Lawn, NJ) were commercially obtained. Milli Q water was used for all the experiments. All other reagents were purchased from SigmaAldrich, Oakville, ON. 1H NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer. 13C NMR spectra were acquired on a Bruker Avance spectrometer running at 75 MHz. The molecular weights, molecular weight distribution, and hydrodynamic radius of the polymers were determined by gel permeation chromatography (GPC) using a DAWN-EOS multiangle laser light scattering (MALLS) and Optilab RI detectors equipped with QELLS (Wyatt Technology Inc., CA) in aqueous 0.1 N NaNO3 solution; the details have been described previously.8,9 We adapted a reported procedure for the synthesis, purification, and characterization of alkyl epoxide derivative of R-D-mannopyranoside (4′,5′-epoxypentyl R-D-mannopyranoside).59-62 The structures of the compounds synthesized were confirmed by NMR analysis and purity of the compounds were checked by thin layer chromatography (1H NMR (300 Mz; D2O; 4′,5′-epoxypentyl R-Dmannopyranoside): 1.48-1.82 (m, 4H), 2.49 (qui, 1H, J ) 2.4, 4.8 Hz), 2.75 (t, 1H, J ) 4.4 Hz), 2.96 (m, 1H), 3.41-3.84 (m, 8H), 4.75 (s, 1H)). Conductometric titrations were done on a YSI model 35 conductance meter and 3403 cell with platinum electrode at 25 °C. A syringe pump (Harvard Instruments) was used to inject dilute NaOH at constant flow rate of 0.102 mL/min. For a typical titration, modified HPG (-COOH/-NH2; 10 mg) was dissolved in distilled water and titrated first with 0.05 N HCl followed by back-titration with 0.05 N NaOH. Conductance of the solution was measured at every 30 s. Potassium hydrogen phthalate solution (0.05 N) was used for standardizing sodium hydroxide solution.

Kizhakkedathu et al. Synthesis. Synthesis and Amine Modification of Hyperbranched Polyglycerols. Hyperbranched polyglycerols were synthesized by following a reported procedure.8 The polymer was characterized by GPC-MALLS equipped with QELLS in aqueous conditions for the determination of absolute molecular weight, polydispersity (Mw/Mn), radius of gyration (Rg), and hydrodynamic radius (Rh) and NMR spectroscopy. The degree of branching of high molecular weight HPGs determined by NMR analysis8 was similar to that of low molecular weight HPGs63 and were between 0.5 and 0.6. A two-step modification procedure was used to convert some of the hydroxyl groups of HPGs to primary amine groups. HPG-500k (Mn ) 493 k; 1.5 g, 0.02 mols of hydroxyl groups) was dissolved in water (10 mL) and was cooled to 0 °C. To this, powdered KOH (0.8 g, 0.014 mols) was added and stirred until a homogeneous solution was formed. Acrylonitrile (2 g, 0.0377 mols) was added dropwise to this solution over a period of 10 min. Stirring was continued for 5 h at 0 °C. A precipitate was formed after about 1 h of the reaction. Concentrated H2SO4 (12 g, 0.122 mols, 36 N) was added to the mixture slowly (care should be taken to keep the temperature below 5 °C). The precipitate completely dissolved. Then the reaction mixture was heated at 95 °C for 3 h with stirring, cooled, and diluted with 20 mL of water. The mixture was dialyzed against water (1000 MWCO cellulose membrane) for two days with frequent changes in water (daily five times). A small amount of the dialyzed solution was lyophilized and analyzed by 1H NMR and conductometric titration using NaOH and HCl. To the remaining solution excess ethylene diamine (15 g, 0.25 mols, greater than 25-fold molar excess of carboxyl groups) was added along with EDAC · HCl (3.8 g, 0.02 mol). The reaction mixture was stirred at room temperature for 2 days and then dialyzed (2 days) against water (three times daily change in water) using cellulose membrane 1000 MWCO. The solution was acidified using 0.1 M HCl, dialyzed for 1 day, and then neutralized using 0.1 N NaOH and dialyzed for another 2 days. The product was recovered by freeze-drying the solution to form a yellowish white fluffy solid. The primary amine-modified HPG was characterized by 1H NMR, 13C NMR, conductometric titration using HCl and NaOH, and GPC-MALLS analysis. Other HPG-amines were synthesized using a similar protocol. HPG-acid (D2O): δ 2.4-2.7 (-CH2-COOH, broad peak), 3.3-4.2 (HPG-main chain protons and O-CH2-CH2-COOH). HPG-amine (D2O): δ 1.12 (NH2), 2.30-2.55 (CH2CO2H, CH2CONH-), 2.70-2.80 (CH2NH2), 3.0-3.30 (-CONHCH2-), 3.3-4.2 (HPG-OCH2CH2Oand OCH2CH2CO-). Synthesis of HPG-Mannose Conjugates. HPG-mannose conjugates were prepared by the reaction of epoxide derivative of pent-4enyl R-D-mannopyranoside59-62 with HPG-amines. HPG-500k-amine (0.05 g), 4′,5′-epoxypentyl R-D-mannopyranoside (0.1 g, 3.77 × 10-4 moles) was dissolved in 5 mL water and was stirred at 50 °C for 48 h for the synthesis of HPG-500k-M303. Other conjugates were prepared by changing the HPG-amine and the amount of 4′,5′-epoxypentyl R-Dmannopyranoside used (Table 1S, Supporting Information). After the reaction, the mixture was dialyzed against water for 4 days (MWCO 1000) with frequent changes in water. The product was recovered by lyophilization and characterized by 1H and 13C NMR analyses. The mannose density on HPG was determined from proton NMR integrations of different peaks and the absolute molecular weight of HPG determined by GPC-MALLS. Because signals from mannose protons overlap with the HPG peaks, we used -CH2-CH2- group of the R-substituent (-HN-CH2-CH(OH)-CH2-CH2-CH2-O-) of mannose derivative and signals from HPG backbone for the determination of molar content of mannose. The details of the calculation are given in Supporting Information. All HPG-mannose conjugates had similar NMR spectra except for the change in peak intensity. It was difficult to analyze the polymers using MALDI-TOF due to their high molecular weights. HPG-500k-M303. 1H NMR (300Mz, D2O): δ 1.15 (NH2), 1.3-1.8 (-CH2-CH2-), 1.9-2.1 (-CH2-), 2.35-2.75 (CH2CO2H, CH2CONH-),

Polyglycerol-Based Multivalent Mannose Conjugates


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Scheme 1. Synthesis of HPG-Amines

2.80 (CH2NH2), 3.1-3.30 (-CH2NHCO-), 3.4-4.2 (HPG- OCH2CH2Oand HPG-OCH2CH2CO-, HN-CH2-CH(OH)-). Hemagglutination Experiments. Preparation of Human Red Blood Cell Suspension. Whole blood was collected from consented unmedicated volunteers in EDTA solution. Red blood cells (RBCs) were separated by centrifugation at 1000 × g for 5 min, washed three times with isotonic saline (0.9% NaCl) and a final wash with PBS. RBCs were centrifuged and hematocrit of packed RBCs was measured. RBC suspension (2% hematocrit) containing 0.5% BSA, 0.8% adenosine in PBS was prepared and used for hemagglutination experiments. Hemagglutination Assay and Inhibition Experiments. Concanavalin A (Con A) solution was prepared as per literature protocol.52 Using a 96-well plate (Corning Incorporated, Corning, NY), 100 µL Con A solution was serially diluted and 100 µL of 2% RBC suspension was added to each well and incubated at 37 °C for 2 h. The suspensions from each well were examined on microscope slides using the light microscope (Zeiss Axioskop2 plus, QImaging Retiga, Northern Eclipse 6.0) to determine the extent of agglutination. Experiments were done in duplicates. Inhibition Assay. An inhibition assay was used to determine the minimum concentration of HPG-mannose conjugate or methyl R-Dmannopyranoside required to inhibit the agglutination of red blood cells by Con A. HPG-mannose conjugates and methyl R-D-mannopyranoside were prepared in PBS solution and were serially diluted first starting with 5 mg/mL concentration in a 96-well microtiter plate. To this solution, Con A solution (5 times minimum agglutination concentration) and 2% RBC suspension were added. The plate was incubated at 37 °C for 2 h and the minimum inhibition concentration (MIC) was determined using optical microscopy analysis (Figure S2, Supporting Information). For each HPG-mannose conjugate, methyl R-D-mannopyranoside and a well without mannose or conjugate were used as controls. The experiments were run in duplicates and each experiment was repeated at least twice. Isothermal Titration Calorimetry (ITC) Experiments. Sample Preparation for ITC Experiments. Con A solution at pH 5.2, Con A (25 mg/mL) was prepared by dissolving in acetate buffer (0.1 M sodium acetate pH 5.2, 0.1 M NaCl, 5 mM CaCl2 and 5 mM MnCl2) followed by dialysis against 1 L of the same buffer overnight at 4 °C using a 1000 MWCO membrane (Pierce Slide-A-Lyzer cassette). Methyl R-Dmannopyranoside and HPG-mannose conjugates were prepared in the final dialysate buffer (at pH 7.2 or 5.2) used for Con A preparation. All solutions were degassed at room temperature before loading in the ITC sample cell or injection syringe. Determination of Binding Constants by ITC. Isothermal titration calorimetry (ITC) was performed using a VP-ITC (MicroCal Inc.,

Northampton MA) in 0.1 M sodium acetate pH 5.2, 0.1 M NaCl, 5 mM CaCl2, and 5 mM MnCl2. Typical titrations were performed by injecting consecutive 5-10 µL aliquots of Con A solution (0.16 mM) into the ITC cell (volume ) 1.4 mL) containing HPG-mannose conjugates (0.36 µM). In some cases, Con A concentrations were varied from 0.16-0.84 mM and HPG-mannose conjugate concentrations were varied from 0.36-7.10 µM. A set of titrations was also performed with Con A solution (2-20 µM) in the ITC cell and HPG-mannose conjugates (0.7-10 µM) in the injection syringe. For the Con A-methyl R-D-mannopyranoside system, titrations were performed with the ConA (0.36 mM) in the sample cell and methyl R-D-mannopyranoside (6.2 mM) in the injection syringe, unless otherwise mentioned. The ITC data were corrected for the heat of dilution of the titrant by subtracting mixing enthalpies for 5-10 µL injections of Con A (or methyl R-Dmannopyranoside or HPG-mannose conjugates) solution into HPGmannose conjugates (or Con A)-free buffer. Unless otherwise indicated, at least two independent titration experiments were performed for each system at 25 °C to determine the binding constant of Con A to methyl R-D-mannopyranoside or HPG-mannose conjugates. Binding stoichiometry, N, enthalpy, ∆Hb, entropy, ∆S, and equilibrium association constants, Ka, were determined by fitting the corrected data to a bimolecular interaction model.

Results and Discussion Synthesis of HPG-Amines. Scheme 1 shows synthetic steps involving the modification of hydroxyl groups of high molecular weight hyperbranched polyglycerols (HPGs) to primary amine groups. Three high molecular weight HPGs with wide variation in molecular weights were used (Table 1). The average hydrodynamic radius of the high molecular weight HPGs ranged from 3.9 to 7.3 nm. A two-step modification procedure under aqueous conditions was used for converting the hydroxyl groups of HPG to primary amines. Initially, the hydroxyl groups were converted to nitrile groups and subsequently hydrolyzed to carboxyl groups, which were then reacted with ethylene diamine (25× excess) to form primary amine functionalized HPG. Proton NMR showed characteristic peaks of HPG-COOH and HPGamines. GPC-MALLS of HPG-amine in 0.5 M NaNO3 showed monomodal peak, indicating that the modification was successful without cross-linking. The use of excess amount of ethylene diamine (∼25 times molar excess) in the coupling reaction with HPG-COOH prevented any cross-linking between HPG molecules. More than 90% conversion of carboxyl groups to amine

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Table 1. Characteristics of HPG, HPG-COOH, and HPG Amines sample

Mn (k)

Mw/Mn

Rha (nm)

HPG-COOH

number of carboxyl groupsb

HPG-amine

number of amine groupsc

MW of HPG-amined

HPG-106k HPG-200k HPG-500k

106 208 493

2.0 1.58 1.05

3.9 5.6 7.3

HPG-COOH-106k HPG-COOH-200k HPG-COOH-500k

492 ( 10 683 ( 20 1980 ( 52

HPG-NH2-106k HPG-NH2-200k HPG-NH2-500k

446 ( 12 628 ( 14 1827 ( 38

156800 279600 701300

a Rh: hydrodynamic radius determined by QELLS analysis. b Determined by conductometric titration. c Determined by conductometric titration. Molecular weight (MW) of HPG-amine is theoretically calculated using the equation MW of HPG-amine ) Mn of HPG × (no. of amine groups ×114) (see Scheme 1). d

Figure 1. Effect of number of mannose residues on HPG on the minimum inhibition concentration of mannose HPG conjugates synthesized on HPG-500k.

groups was obtained from the conductometric titrations (Table 1). It was difficult to calculate the conversions from proton NMR due to the overlapping peaks. After conducting several experiments with different reagents and solvents, the current indirect method was chosen for the preparation of HPG-amine. This is due to the insolubility of high molecular weight HPGs and derivatized HPGs in commonly used organic solvents, difficulty in achieving a higher degree of modification of high molecular weight HPG in solvents other than water. Although modification in organic solvents was possible for low molecular weight HPG,15 it was difficult to adapt it for high molecular weight HPGs. We have also investigated the use of N-Boc-ethylenediamine for the coupling reaction with HPGCOOH, but resulted in a gel after reaction with N-Boc-ethylene diamine and freeze-drying. The polymer was insoluble even after attempts to deprotect N-Boc groups. Synthesis of HPG-Mannose Conjugates. The synthetic route for the development of HPG-mannose conjugate from HPG-amine is given in Scheme 2. The conjugation was achieved through a ring-opening reaction of 4′,5′-epoxypentyl R-Dmannopyranoside59 with the primary amine groups on HPGamine in water. The appearance of a new peak between 1.3-1.8 ppm in the 1H NMR spectra of the conjugates due to the -CH2CH2- group of the R-substituent (-HN-CH2-CH(OH)-CH2-CH2CH2-O-) of mannose derivative support the conjugation to HPGamine. Representative 1H NMR spectra of conjugates are given in Figure S1C-E in Supporting Information. 13C NMR spectra of the HPG-mannose conjugates also confirmed the presence of mannose in HPG (Supporting Information, Figure S1F,G). The ratio of mannose derivative to HPG-amine was altered to produce different number of mannose groups per HPG molecule (Table S1). The characteristics of the synthesized conjugates are given in Table 2. The details of the calculation of mannose density on HPG are given in Supporting Information. In the

case of the HPG-500k, the number of mannose residues in the conjugate was varied from 22 to 303 per HPG. In spite of using molar excess of epoxide derivative of pent-4-enyl R-D-mannopyranoside with respect to the amount of amine groups for conjugation, maximum number of mannose groups per HPG obtained was only 303. All the HPG-mannose conjugates were highly soluble in water. Hemagglutination Experiments. Concanavalin A, a mannose binding lectin extensively used in the study of glycoconjugates, was used for evaluating the conjugates described.51,55,56 Con A exists as a homotetramer at the conditions of the experiments (pH ∼ 7.4) and can bind up to four mannose residues. The carbohydrate binding sites are spatially separated by ∼6.5 nm.64,65 The inhibition of Con A induced agglutination of fresh human red blood cells (RBCs) by HPG-mannose conjugates was measured and compared with methyl R-Dmannopyranoside.66-68 Unlike rabbit or mouse RBCs, human RBCs are less responsive to hemagglutination by Con A, so we used adenosine to enhance the agglutination.66,68 The minimum inhibitory concentration (MIC) and relative potency of the HPG-mannose conjugates are compared with methyl R-D-mannopyranoside and are given in Table 2. All the HPG-mannose conjugates showed relatively low MIC compared to methyl R-D-mannopyranoside, ranging from 2.17 to 0.08 µM. The relative potency of the HPG-mannose conjugates varied from 1000 to ∼40000. The corrected activity per mannose for each HPG conjugate (Table 2) showed that when mannose is attached to HPG, its activity increases suggesting cluster glycoside effects.48 The precursor, HPG-amine, showed no inhibition of hemagglutination, suggesting that the mannose units are solely responsible for the inhibition. The measured MIC depended on the molecular weights of the conjugates as well as the number of mannose units per HPG (Table 2). For instance, when the number of mannose residues per HPG was kept constant (e.g., samples HPG-106k-M65 and HPG-500k-M63), the MIC dramatically decreased with an increase in molecular weight of the conjugates, showing the importance of the molecular weight of the scaffold. A relatively larger size of HPG-500k conjugate is facilitating a better interaction with Con A. In other cases, when the molecular weight of the scaffold was constant (samples HPG-500k-M22 to HPG-500k-M303), the increase in the number of mannose residues tremendously decreased the MIC; ∼27 times the number of mannose residues increased from 22 to 303 per HPG (Figure 1). The MIC decreased almost exponentially as the number of mannose units per HPG increases (Figure 1). The large decrease in MIC observed for these HPG conjugates may be explained by a combination of proximity effects (facilitate dynamic equilibrium by increasing probability of binding after one of the mannose ligands dissociate from Con A) and the simultaneous multiple binding (bridging) of mannose units to Con A through bidentate interactions. As the number of mannose residues reached above a threshold level, the steric



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Scheme 2. Synthesis of HPG-Mannose Conjugates

Table 2. Characteristics and Inhibition of Hemagglutination by Mannose Containing HPG Conjugates glycoconjugates

No. of mannose per polymera

minimal inhibitory conc.b [µM]

relative potencyb

enhancement in inhibition per sugar

methyl R-D-mannopyranoside HPG-106k-NH2 HPG-106k-M54 HPG-106k-M65 HPG-200k -NH2 HPG-200k-M47 HPG-500k -NH2 HPG-500k-M22 HPG-500k-M63 HPG-500k-M175 HPG-500k-M222 HPG-500k-M303

0 54 65 0 47 0 22 63 175 222 303

2651 ( 661 no inhibition 1.45 1.41 no inhibition 0.38 no inhibition 2.17 ( 0.44 0.32 ( 0.15 0.15 ( 0.080 0.10 ( 0.060 0.08 ( 0.040

1 no inhibition 1790 1850 no inhibition 9770 no inhibition 1048 11485 ( 3427 39842 ( 11347 35826 ( 12663 36746

1 0 36 31 0 232 0 52 205 255 180 136

a Up to 5% error can be associated with the number of mannose residues calculated from NMR analysis. b Relative potency is calculated by taking ratio of minimal inhibitory concentration (MIC) of conjugates to methyl R-D-mannopyranoside. MICs were calculated based on the molecular weight of the conjugates. MIC of methyl R-D-mannopyranoside was slightly different for each set of measurements; average value is given. MIC of methyl R-D-mannopyranoside from each set is used for the calculation (HPG-106k-M54 and 65; HPG-200k-M47 and HPG-500k-M22 to M303) of relative potency.

factors could be preventing further enhancement of Con A binding, as supported by similar MIC values of HPG-500kM175, M222, and M303. Due to steric constraints and the fact that the carbohydrate binding sites in Con A are spatially separated by ∼6.5 nm,64,65 the bridging of binding sites by Con A is difficult in the case of smaller conjugates.56,69 Instead, several Con A molecules are bound to the conjugate via single interactions to form an extended structure, as has been shown in the case of small multivalent conjugates.53,54 However, as the molecular size of the conjugate increases, multiple interactions of Con A to the conjugate are possible.56,69 Our data showed that conjugates having a molecular mass greater than 200 kDa have this possibility. The large enhancement of MIC for high molecular weight conjugates may be due to combined contributions from these two types of binding interactions (both bridging (bidentate) and single interactions; Table 2). The enhancement in relative activity per sugar of HPGmannose conjugates with increase in molecular weight and number of mannose residues was similar to the PAMAM dendrimer glycoconjugates reported previously.56 A G6 PAMAM dendrimer (MW-101 200 Da) with 178 mannose residues

showed a relative potency of ∼60000 (up to 600 relative activity/ sugar)56 in hemagglutination experiments. The G6 dendrimer was more flexible and larger than the other generation dendrimers, which showed a higher relative potency compared to smaller dendrimers.56,70 In comparison, linear glycopolymers showed a relatively lower increase in activity.68,70 For example, neoglycopolymers synthesized by ROMP showed a relative potency ∼2000 for a 63000 Da (degree of polymerization ) 143) polymer with 143 sugar residues71 compared to single mannose units. Based on these reported observations, the hemagglutination inhibition efficiency of HPG-mannose conjugates (Table 2) are between those of linear and perfectly branched dendrimer structure with close resemblance to dendrimers. However, HPG has the added advantage of excellent biocompatibility1-3 and ease of synthesis.8 Gold nanoparticles with an average size of 20 nm decorated with mannose showed an ∼128-fold increase in relative activity/ sugar,72 and vesicles attached with mannose containing dendritic polymers produced an ∼92-fold increase in relative activity/ sugar73 compared to monomeric sugar units. High molecular weight HPG-mannose conjugates showed much higher enhancement compared to these structures (Table 2).

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Table 3. Summary of ITC Results for Con A-HPG-Mannose Conjugate Titrations at pH 5.2 and 25 °Ca ligand methyl R-D-mannopyranoside HPG-500k-M22 HPG-500k-M175 HPG-500k-M303

N

Ka (M-1)

0.94 ((0.01) NA 36 ((8) 39 ((15)

1.03 ((0.02) × 10 3 ((2) × 104 5.1 ((0.5) × 105 4 ((1) × 105

4

Ka relative to mannose

∆G (kJ/mol)

∆H (kJ/mol)

Τ∆S (kJ/mol)

1 3 50 44

-22.90 ((0.04) -25 ((13) -32.6 ((0.4) -32.2 ((0.8)

-32.2 ((0.4) -23 ((1) -21 ((8) -21 ((2)

-9.2 ((0.4) 2 ((1) 13 ((8) 11 ((1)

a Errors represent the standard deviations of replicate experiments. N represents the number of binding sites per polymer (except for methyl-R-Dmannopyranoside: N represents the number of binding sites per Con A monomer. All calorimetric values are expressed in terms of mannose equivalents.

Thermodynamics of Con A Binding to HPG-Mannose Conjugates. Isothermal titration calorimetry (ITC) has been used extensively to study the molecular basis of interactions between glycoconjugates and proteins.33-35,52-54,74,75 Such studies have been quite useful in elucidating the mechanism and thermodynamics of enhanced binding affinities of multivalent glycosides with lectins. Although, the cluster glycoside effects have been extensively studied for small molecular weight multivalent glycoclusters,48,74 there is a lack of detailed understanding in the case of very high molecular weight glycoconjugates (i.e., larger than lectins). Unlike small molecular weight analogues, steric reasons may not play a significant role in their interaction with lectins. To gain insight into the mechanism of the enhanced hemagglutination inhibition displayed by HPG-mannose conjugates, ITC experiments were performed. Con A exists as a homotetramer at pH 7.2 (the conditions of the hemagglutination experiments), and association of Con A with HPG-mannose conjugates leads to precipitation under these conditions. Therefore, ITC experiments were performed at pH 5.2, where Con A exists as a dimer. Under the latter conditions, the sugar complexes remained soluble during the ITC experiment. Table 3 shows the results for titrations of Con A-methyl mannose and Con A-HPG-mannose conjugates at pH 5.2. HPG-500k-mannose conjugates were selected for a detailed ITC study due to the very low polydispersity of the scaffold (Mw/Mn 1.05). This meant a less heterogeneous (structurally well-defined) conjugate with a wide variation in ligand density could be studied. Three representative HPG-500k-mannose conjugates with wide variation in mannose density are chosen for the ITC study. The results for the interaction of methyl R-Dmannopyranoside with Con A at pH 5.2 are shown in Table 3 and Figure 2 and are in good agreement with literature values.52,53 The result for methyl R-D-mannopyranoside at pH 5.2 is in good agreement for K and ∆H. HPG-500k-M22 shows little improvement in the affinity of methyl R-D-mannopyranoside for Con A. The density of the mannose units on this conjugate is relatively low, such that the probability of multiple contacts between the Con A dimer and the conjugate is very low; it is likely that only single interactions (to form extended structure) take place between Con A and the conjugate as the carbohydrate binding sites on the Con A dimer are spatially separated by approximately 6.5 nm.64 A decrease in enthalpy is observed, due to a more constrained tethering of mannose to the conjugate; this also results in a lower entropic penalty when Con A binds to the mannose units of HPG-500kM22 compared to free methyl-R-D-mannopyranoside. Binding to the latter will result in a greater loss in rotational and translational degrees of freedom and as a result, a greater entropic penalty. This is in agreement with the hemagglutination experiments described previously (Table 2). Figure 3 shows HPG-500k-M175-Con A titrations at pH 5.2; the titrations of HPG-500k-M175 into Con A (Figure 3a) and of Con A into HPG-500k-M175 (Figure 3b) at pH 5.2 result in similar regressed values for N, K, and ∆H; the average values

Figure 2. Raw data (a) and integrated heats (b) for 10 µL injections of 6.2 mM methyl R-D-mannopyranoside into 362 µM Con A: T ) 25 °C, 0.1 M sodium acetate pH 5.2, 0.1 M NaCl, 5 mM CaCl2, and 5 mM MnCl2. The line (b) shows the best fit to a bimolecular interaction model.

are reported in Table 3. Similar results are found for HPG500k-M22 and HPG-500k-M303. HPG-500k-M175 and -M303 show similar enhancements in affinity for Con A when compared to methyl R-D-mannopyranoside. The enthalpy change is similar to that of HPG-500kM22 but is lower than that of the free methyl R-D-mannopyranoside. This is due to constraints on the mannose unit tethered to the conjugate. However, the entropy change is considerably more favorable for the conjugates with higher mannose densities. Due to the increased density of mannose units on HPG-500kM175 and HPG-500k-M303, multiple or bridging Con Aconjugate interactions (bidentate interactions) are possible.56,69 The model used to fit this data assumes that all binding events are equivalent; therefore, average values for K and ∆H are regressed. However, multiple Con A-conjugate interactions, as in the case of HPG-500k-M175 and HPG-500k-M303, can result in an avidity effect. Thus, while the first binding event is thermodynamically similar to that of HPG-500k-M22, due to the proximity of the mannose units and the second binding event is energetically more favorable. This is due to a very low entropic penalty for the second binding event. Therefore, cooperative binding is possible with HPG-500k-M175 and HPG500k-M303. Figure 4 shows a Hill plot for HPG-500k-M175 and for methyl-R-D-mannopyranoside. Raw ITC data was used to calculate parameters for the Hill plot using the method of Dam et al.53,54 As expected, the Hill plot for methyl R-Dmannopyranoside is linear with a slope close to 1.0, indicating noncooperative binding. The Hill plot for HPG-500k-M175 is not linear; the slope increases with increasing saturation of the



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Figure 4. Hill plot of ITC raw data for methyl R-D-mannopyranoside (slope ) 0.99 ((0.01)) and HPG-500k-M175: T ) 25 °C, 0.1 M sodium acetate pH 5.2, 0.1 M NaCl, 5 mM CaCl2, and 5 mM MnCl2. Xf ) concentration of free ligand; Y ) Xb (functional valency of ligand)/ Mt; Xb ) concentration of bound ligand, Mt ) total concentration of macromolecule. Con-A is the macromolecule; HPG-500k-M175 or methyl R-D-mannopyranoside is the ligand.

Figure 3. (a) Raw data (i) and integrated heats (ii) for 10 µL injections of 7.1 µM HPG-500k-M175 into 13.3 µM Con A. T ) 25 °C, 0.1 M sodium acetate pH 5.2, 0.1 M NaCl, 5 mM CaCl2, and 5 mM MnCl2. The line (ii) shows the best fit to a bimolecular interaction model. (b) Raw data (i) and integrated heats (ii) for 10 µL injections of 174 µM Con A into 0.46 µM HPG-500k-M175. T ) 25 °C, 0.1 M sodium acetate pH 5.2, 0.1 M NaCl, 5 mM CaCl2, and 5 mM MnCl2. The line (ii) shows the best fit to a bimolecular interaction model.

binding sites, indicating positive cooperativity. This is consistent with multiple Con A or bridging interactions with a single HPG500k-mannose conjugate of high mannose density. Unlike methyl R-D-mannopyranoside or other small molecular multivalent glycoclusters reported previously,52-54,74 multiple binding or bridging interactions of a Con A to HPG-mannose conjugate are not sterically restricted. Also, due to the large number of mannose residues on the HPG-mannose conjugates, the functional valency of the conjugates does not decrease as the number of binding lectin molecules increases. Both of these factors contribute to the positive cooperative behavior of these large glycoconjugates. Although the bridging interactions are contributing to the enhancement in the affinity, one cannot rule out the interactions of Con A to several HPG-mannose

conjugates. Thus, in the present case, the enhancement in the affinity is due to the combined contributions from these types of interactions. As shown in Table 3, an increase in the number of mannose units (up to 175) on a given molecular weight scaffold (HPG500k), results in an increased affinity for Con A. This is in agreement with hemagglutination experiments that show that the minimum inhibitory concentration remains quite constant for conjugates with more than 175 mannose units. Thus, there appears to be an optimum number of mannose residues required for enhanced affinity; steric hindrance may impede further enhancement of affinity for conjugates with a high mannose density. Very recently Munoz et al. highlighted the importance of the size of the glyco-ligands as well as the density of the surface immbolized lectin.76 They have pointed out that glycodendrimers are not large enough to produce bridging interactions in solution. But when Con A was immbolized on a surface, the size of the conjugate played an important role in enhancing the binding affinity. In the present case, the HPG-mannose conjugate (500k) is larger in size than that of the Con A and it can act as a surface thereby increasing the chances of bidendate binding interactions to Con A. Such bridging interactions occur only when the density of the carbohydrate is sufficiently high (HPG500k-M63, HPG-500k-175, HPG-500k-222, and HPG-500k303; Table 3), which significantly enhances the binding strength. Our hemagglutination as well as ITC results support such enhancement in activity. When the size of the HPG is sufficiently high, the mannose density on HPG determines such interactions and is consistent with the large increase in the relative activity per sugar when the mannose density is increased from low to higher values (Table 2).

Conclusions We have synthesized HPG-mannose conjugates with a wide variation of molecular weight, size, and mannose density from hyperbranched polyglycerols under aqueous conditions. In the

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case of HPG-500k, the number of mannose units per HPG changed from 22 to 303, an approximately 15-fold increment in the carbohydrate density. The HPG-mannose conjugates inhibit concanavalin A induced hemagglutination reactions of fresh human red blood cells. The relative potency of the HPG-mannose conjugates increases with an increase in the number of mannose units as well as with the size of the conjugates, suggesting a cluster glycoside effect. The potency of high molecular weight HPG-mannose conjugates increases ∼40000 times relative to methyl R-D-mannopyranoside. The relative activity per sugar increases up to ∼255 for HPG-500k-based conjugates. We show that both the size and ligand density play important roles in enhancing the binding interactions between Con A and HPG-mannose conjugates. A comparison of the literature reports showed that increase in activity per sugar on HPG is between perfectly branched glycodendrimers and linear glycopolymers. Isothermal titration calorimetry has been used to determine the thermodynamic and molecular basis of the enhancement of binding affinity. A 50-fold increment in binding affinity relative to methyl R-D-mannopyranoside is observed for HPG conjugates, suggesting that multiple binding of Con A to HPG conjugates is responsible for this effect. We have seen that the favorable entropic contribution due to multiple binding or bridging of Con A to high molecular weight HPG-mannose conjugate along with single interactions are the reasons for the enhanced binding. Positive cooperative binding is shown by certain HPG-mannose conjugates unlike methyl R-D-mannopyranoside or other small multivalent glyco-clusters. In the present case, the larger molecular size of the HPG conjugates is responsible for this observation where bridging or multiple binding by the lectin to a single conjugate molecule is not sterically constrained. The carbohydrate density on conjugates determines the binding affinity. These studies will significantly improve the molecular design of glycoconjugates based on high molecular weight HPGs. Acknowledgment. This research was funded by University of British Columbia and Canadian Institutes of Health Research (CIHR). The authors thank the LMB Macromolecular Hub at the UBC Centre for Blood Research for the use of their research facilities; the infrastructure facility is supported by the Canada Foundation for Innovation (CFI) and the Michael Smith Foundation for Health Research (MSFHR). Tong Wu and Joyleene Yu are thanked for assistance with ITC experiments. N.A.A.R. is a recipient of a CIHR/CBS postdoctoral fellowship in Transfusion Science and J.N.K. is a recipient of a CIHR/ CBS new investigator in Transfusion Science. Dr. K. N. Jayaprakash is thanked for critical reading of the manuscript. Supporting Information Available. A table showing reaction conditions, 1H and 13C NMR spectra, optical micrographs showing hemagglutination, and composition calculation are given. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM1004788

High Molecular Weight Polyglycerol-Based Multivalent Mannose

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