Sequence-Defined Glycopolymer Segments Presenting Mannose

Apr 6, 2012 - We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Fu...
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Sequence-Defined Glycopolymer Segments Presenting Mannose: Synthesis and Lectin Binding Affinity Daniela Ponader,† Felix Wojcik,† Figen Beceren-Braun,‡ Jens Dernedde,‡ and Laura Hartmann*,† †

MPI of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin, 12203 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands.

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INTRODUCTION Carbohydrates take part in many biological processes like intercellular recognition and pathogen identification by recognition of carbohydrate binding proteins (lectins).1−4 Because monovalent carbohydrate−receptor interactions are usually weak, nature applies multivalent binding modes to increase binding strength and specificity.5−7 This phenomenon is called the cluster-glycoside effect.8 Many different synthetic architectures such as glycopeptides, globular glycomacromolecules (dendrimers), or linear glycopolymers have been successfully used for the multivalent presentation of carbohydrate ligands mimicking the cluster-glycoside effect.9 It has also been shown in literature that the presenting scaffold has an important influence on the multivalent binding mode.9−15 Binding affinity and selectivity can be controlled through variation of number, density, and spacing of ligands.5,13,16−18 Furthermore, the scaffold itself can contribute to the binding through secondary interactions, for example, hydrophobic interactions, and thus vary the ligand− receptor interactions. Nevertheless, most synthetic multivalent ligands are still optimized empirically, especially systems based on polymeric scaffolds. Because of their inherent polydisperse nature and the limitation in controlling precise positioning of functionalities along the backbone, polymer scaffolds make it especially difficult to correlate their chemical structure with the resulting binding properties. Despite these difficulties, they remain a highly important class of scaffolds regarding the large variability of polymer synthesis: Through polymer−analogue reactions as well as the functionalization of suitable monomers, glycopolymers presenting various saccharide ligands have been synthesized and proved to be able to recognize and bind lectins with high affinities.15,18−23 © 2012 American Chemical Society

Moreover, different architectures and functionalities are accessible, and a large number of polymeric systems is known to be nontoxic and nonimmunogenic.9,12,18,22 These attributes are especially interesting for biomedical applications of glycopolymers because they have high potential as glycopolymeric drugs or drug delivery systems.24,25 The ideal polymeric multivalent scaffold therefore should be monodisperse, biocompatible, and highly functional. An important step toward such controlled polymer systems is the new class of precision polymers.26−30 Previously, we introduced a novel approach toward precision polymers via the solid-phase synthesis of monodisperse, sequence-defined poly(amidoamines) (PAAs) by stepwise addition of diacid and diamine or dimer building blocks.31−34 Here we introduce a new dimer building block presenting an alkyne moiety that allows for the attachment of sugar ligands to the PAA backbone via click chemistry and the synthesis of highly defined multivalent glycopolymer segments. (See Scheme 1.) We synthesized three different structures having the same contour length but differing in their number and spacing of presented mannose ligands. These glycopolymer segments were tested for their lectin binding affinity via surface plasmon resonance (SPR). Because of the monodispersity of the glycopolymer segments, we expect to be able to attribute possible changes in binding affinity between the synthesized polymer segments to the scaffold structure. We anticipate our approach to help further understand the multivalent binding of polymeric Received: March 1, 2012 Revised: April 6, 2012 Published: April 6, 2012 1845

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in 75 mL of dichloromethane (DCM), 24.1 g (24.1 mmol) of succinic anhydride and 6.1 mL of (43.8 mmol) NEt3 were added and stirred for 1 h. The reaction mixture was washed five times with 5% aqueous citric acid and dried over Na2SO4, and solvent was removed under reduced pressure. To the resulting white powder, we added 200 mL of water and the mixture was sonicated 30 min to remove traces of succinic anhydride. This was repeated two times, followed by filtration to give 7.8 g (15.55 mmol, 71%) of a white solid. 1H NMR (400 MHz, DMSO-d6) δ =7.73 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.34−7.27 (m, 4H), 4.32 (d, J = 6.6 Hz, 2H), 4.13 (t, J = 6.6 Hz, 1H), 3.43−3.18 (m, 8H), 2.60−2.50 (m, 4H), 2.46−2.36 (m, 4H), 2.24− 2.15 (m, 1H). 13C NMR (100 MHz, CD3SO): δ = 174.70, 173.48, 172.80, 157.45, 143.83, 141.16, 127.35, 126.72, 124.66, 119.51, 82.59, 68.73, 66.37, 66.14, 45.65, 45.14, 37.46, 36.99, 31.56, 30.12, 30.03, 28.69, 13.96. ESI-MS calcd for C28H31N3O6 [M+H]+ 506.3; found 506.2 [M+H]+. For synthesis of ethylenedioxy building block (EDS) and 2-azidoethylO-α-D-mannopyranoside, see the Supporting Information. Solid-Phase Polymer Synthesis. As resin for solid-phase synthesis, a commercially available trityl-tentagel−OH resin was modified with ethylenediamine (EDA) as linker. (See the Supporting Information.) General Coupling Protocol. After swelling 0.05 mmol (0.23 g) of Trt-EDA resin in DCM, the initial coupling to the EDA linker was performed by dissolving 0.5 mmol of the building block in DMF (1 mL), followed by the addition of 0.48 mmol HBTU and 0.25 mmol HOBT in 1 mL of DMF. DIPEA (1 mmol) was added, and the mixture was shaken for 30 s. This mixture was added to the resin and shaken for 1 h. Completeness of reaction could be qualitatively confirmed by a negative Kaiser test.4,5 Then, the Fmoc protecting group was cleaved by the addition of a solution of 25% piperidine in DMF three times for 5, 10, and 15 min, respectively. Cleavage of Fmoc protecting group could be confirmed by a positive Kaiser test. This was followed by repetitive coupling of building block and Fmoc cleavage as previously described. Capping of N-terminal Site. Before click reaction, the N-terminal site was deprotected with a solution of 25% piperidine in DMF. This was followed by the addition of 2 mL of acetic anhydride (Ac2O). After shaking for 20 min, the resin was washed with DMF and DCM. Cleavage from Resin. Cleavage was performed by the addition of a solution of 30% TFA in DCM and shaking for 0.5 h. The filtrate was added to diethylether. The obtained precipitate was washed once with diethylether and collected. Final products were dissolved in water and lyophilized. PAA-Alkyne Intermediates (See Scheme 3). EDA-[EDS]2[TDS][EDS]2-NHFmoc, 7. RP-HPLC (5%/95% MeCN/H2O→95/5% H2O/MeCN in 60 min): tR = 21.4 min. ESI-MS calcd for C70H109N13O21 [M+2H]2+ 734.5, found 734.8 [M+2H]2+; 490.3 [M+3H]3+, found 490.4 [M+3H]3+. 1H NMR (400 MHz, D2O): δ = 3.7−3.39 (m, 75H), 3.17 (t, J = 5.7 Hz, 4 H), 2.66 (t, J = 6.2 Hz,

Scheme 1. General Overview of Solid Phase Synthesis of Sequence-Defined Glycopolymer Segmentsa

a

First, suitable building blocks are synthesized presenting a spacer unit or a functional moiety in the side chain (a). These building blocks are then coupled on solid phase until the desired chain length and sequence are obtained (b), followed by on-resin modification of the side-chain functionalities introducing sugar ligands (c). The sequence-defined, monodisperse products are obtained after cleavage from solid support.

ligands and to allow for a rational design of new improved systems.

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EXPERIMENTAL PART

Synthesis of Building Blocks. For synthesis of precursors 1−4 (Scheme 2), see the Supporting Information. 1-(Fluorenyl)-3,11-dioxo-7-(pent-4-ynoyl)-2-oxa-4,7,10-triazatetradecan-14-oic acid, 5, TDS. To a solution of 8.9 g (21.9 mmol) of 4

Scheme 2. Building Blocks Synthesis of TDSa

a Synthesis of TDS (5) building block and structure of EDS building block (6). Reagents and conditions: (a) (1) TrtCl, DCM, (2) TFAOEt, THF; 71%; (b) 4-pentynoic acid, EDC, HOBt, NEt3, 50 °C; 68%; (c) (1) K2CO3, H2O/MeOH, (2) FmocCl, K2CO3, H2O/THF; 70%; (d) TFA, DCM, triethylsilane; 90%; and (e) succinic anhydride, NEt3; 71%. (For synthesis of EDS, see the Supporting Information.)

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Scheme 3. Solid-Phase Synthesis of Glycopolymer Segmentsa

a

Schematic representation of solid-phase glycopolymer synthesis: (a) Coupling of building blocks with HBTU, HOBt, and DIPEA in DMF, followed by Fmoc deprotection with a solution of 25% piperidine in DMF. These steps can be repeated for desired repeating numbers of EDS and TDS. (b) Deprotection and capping of N-terminal side: Fmoc deprotection with a solution of 25% piperidine in DMF, followed by capping with Ac2O. (c) On-resin 1,3-dipolar cycloaddition of mannose azides with CuSO4, ascorbate in H2O/DMF. (d) Cleavage from the resin with 20% TFA in DCM giving the final products 10−12.

4 H), 2.58−2.37 (m, 24H), 2.38 (q, J = 1.9 Hz, 1 H), 2.01 (d, J = 1.7 Hz, 3H). EDA-[TDS][EDS]4[TDS]-NHFmoc, 8. RP-HPLC (5%/95% MeCN/ H2O→95/5% H2O/MeCN in 60 min): tR = 21.8 min. ESI-MS calcd for C73H110N14O20 [M+2H]2+ 752.4, found 752.5 [M+2H]2+; [M+3H]3+ 501.9, found 502.0 [M+3H]3+. 1H NMR (400 MHz, D2O): δ = 7.69 (d, J = 7.3 Hz, 2H), 7.48 (br. s, 2H), 7.30 7.20 (m, 4H), 4.36 (d, J = 6.6 Hz, 2H), 4.06 (br. s, 1H), 3.49−1.79 (m, 99H). EDA-[[EDS][TDS]]2[TDS]-NHFmoc, 9. RP-HPLC (5%/95% MeCN/ H2O→95/5% H2O/MeCN in 60 min): tR = 21.4 min. ESI-MS calcd for C76H111N15O19 [M+2H]2+ 769.9, found 770.0 [M+2H]2+; [M+3H]3+ 513.6, found 513.7 [M+3H]3+. 1H NMR (400 MHz, D2O): δ = 7.92 (d, J = 7.1 Hz, 2H), 7.70 (br. s, 2H), 7.51−7.44 (m, 4H,), 4.58 (d, J = 12 Hz, 2H), 4.34 (br. s, 1H), 3.70−3.16 (m, 65H), 2.63−2.36 (m, 38 H). Conjugation of Mannoses on Solid Phase. To 0.01 mmol of tentagel-trityl resin loaded with EDS and TDS was added 1 mL of acetic anhydride, and the solution was shaken for 30 min. We dissolved 0.1 equiv of 2-azidooethyl-mannoside per alkyne group, 20 mol % sodium ascorbate per alkyne group, and 20 mol % CuSO4 per alkyne group in 0.5 mL of DMF and 0.5 mL of water. This mixture was added to the resin and shaken for 4 h. The resin was washed with a 1 M solution of sodium dithiocarbamate in DMF, water, DMF, and DCM. Cleavage was performed as described above. Final Glycopolymer Segments (Scheme 3 and Figure 1). EDA[EDS] 2 [TDS]Man][EDS] 2 -NHFmoc, 10. RP-HPLC (5%/95% MeCN/H2O→30%/70% MeCN/H2O in 60 min): tR = 19.6 min. ESI-MS calcd for C65H116N16O26 [M+2H]2+ 769.4, found 769.4 [M +2H]2+; 513.3 [M+3H]3+, found 513.5 [M+3H]3+. 1H NMR (400 MHz, D2O): δ = 3.69 (br. s, 20H), 3.65−3.62 (m, 21H), 3.54−3.50 (m, 9H), 3.41−3.39 (m, 26H), 3.16 (t, J = 5.7 Hz, 3H), 2.65 (t, J = 6.9 Hz, 3H), 2.58−2.48 (m, 30H), 2.38 (q, J = 1.9 Hz, 1H), 2.02 (d, J = 1.7 Hz, 3H). EDA-[TDS]Man][EDS]4[TDS]Man]-NHFmoc, 11. RP-HPLC (5%/ 95% MeCN/H2O→30%/70% MeCN/H2O in 60 min): tR = 18.3 min.

Figure 1. First set of mono-, di-, and trivalent glycopolymer segments (10, 11, and 12) obtained by solid-phase synthesis. ESI-MS calcd for C76H132N20O31 [M+2H]2+ 911.5; found 911.5 [M+2H]2+, 608.0 [M+3H]3+ found 608.2 [M+3H]3+, 456.2 [M+4H]4+ found 456.4 [M+4H]4+. 1H NMR (400 MHz, D2O): δ = 4.71 (br. s, 2H), 4.13 (br. s, 2H), 3.97 (br. s, 2H), 3.86 (br. s, 2H), 3.77−3.72 (m, 5H), 3.70−3.56 (m, 37H), 3.48−3.39 (m, 14H), 3.39−3.31 (m, 22H), 3.16−2.96 (m, 11H), 2.85 (br. s, 6H), 2.52 (br. s, 2H), 2.53−2.50 (m, 24H), 1.94 (br. s, 3H). EDA-[[[EDS][TDS]Man]2[TDS]Man]-NHFmoc, 12. RP-HPLC (5%/ 95% MeCN/H2O→30%/70% MeCN/H2O in 60 min): tR = 16.6 min. 1847

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ESI-MS calcd for C87H148N24O36 1053.5 [M+2H]2+, found 1053.8 [M +2H]2+; 702.7 [M+3H]3+, found 702.8 [M+3H]3+; 527.3 [M+4H]4+, found 527.5 [M+4H]4+. 1H NMR (400 MHz, D2O): δ = 4.50 (br. s, 10H), 3.96 (br. s, 5H), 3.93 (br. s, 5H), 3.79 (br. s, 4H), 3.70−3.18 (m, 69H), 2.99 (t, J = 6 Hz, 4H), 2.86 (br. s, 12H), 2.50 (br. s, 8H), 2.39−2.32 (m, 22H), 1.77 (d, J = 5 Hz, 3H). Inhibition Studies of Concanavalin A Binding in Surface Plasmon Resonance Measurements. SPR experiments were run with a streptavidin chip modified with biotinylated α-D-mannose-PAA (PAA = poly[N-(2-hydroxyethyl)acrylamide] Mr ≈ 30 kDa, Lectinity Holdings) on flow channel 2 and biotinylated N-acetyllactosaminePAA on flow channel 1 (which served as reference). Binding studies were carried out with HBS-EP (GE Healthcare) running buffer. To evaluate Concanavalin A (Con A) binding of the polymer segments 10−12, 7 (as control), D-mannose, and α-methyl mannose, we incubated 100 nM Con A with the specific substance at final concentrations of 300 nM, 1 μM, 3 μM, 10 μM, and 30 μM in running buffer for 18 min at room temperature. We injected 35 μL of each sample over both flow channels, whereas the binding signal on the reference channel (N-acetyllactosamine-PAA) was subtracted from the D-mannose-PAA flow channel during the binding measurement. Each binding cycle consisted of an association phase for 105 s, followed by a 180 s dissociation phase. The chip was regenerated after each run (60 s) with regeneration buffer consisting of 100 mM glycine, pH 2.5 in water. The response values were calculated by subtraction of the report point at the beginning of the sample injections (0 s) from the report point at the end of the dissociation phase (285 s). The binding signal obtained by the 100 nM Con A solution in running buffer without polymer segment/D-mannose/α-methyl mannose was set to 100% binding. The binding signals of the specific polymer segment/Dmannose/α-methyl mannose were referred to Con A and calculated for relative Con A binding in % of Con A. Each data point represents the mean value (±SEM) of three measurements. IC50 values represent the concentration of polymer segment/D-mannose/α-methyl mannose that results from 50% binding of Con A to α-D-mannose-PAA on the sensor chip. (For a more detailed description, see the Supporting Information.)

selective for primary amines in the presence of a secondary amine.41,42 Such desymmetrization reactions are usually of low yield, but here we were able to optimize this reaction to give 71% of crystalline product after two steps in large quantities (up to 120 g in one batch).34 To introduce the alkyne moiety for later 1,3-dipolar cycloaddition, we modified the unprotected secondary amine by coupling orthogonal protected precursor 1 with 4-pentynoic acid and N-(3-dimethylaminopropyl)-N′ethylcarbodiimide (EDC) in the presence of 1-hydroxybenzotriazole (HOBt) and triethylamine (NEt3). The trifluoracetyl protecting group (NHTFA) was then exchanged to Fmoc to generate building blocks that can later be coupled on solid phase according to standard Fmoc peptide synthesis protocols. This was done by one-pot deprotection in aqueous basic methanol (MeOH) and, as second step, exchange of MeOH to tetrahydrofurane (THF), followed by the addition of Fmoc chloride to give orthogonal protected compound 3. In the final step, a carboxylic unit has to be introduced. Therefore, the primary amine was liberated by cleavage of trityl with TFA in DCM with triethylsilane as trityl scavenger and precipitation from diethylether to give 4. Coupling to succinic anhydride yielded the final alkyne building block 5. The complete synthesis can be carried out on a large scale resulting up to 8 g of final building block TDS with a total yield of 22% in one synthesis. This building block will later serve as conjugation sites for mannose ligands. To allow for specific distances between the mannose ligands in the final glycopolymer segments, we synthesized a second building block presenting a hydrophilic spacer unit. 2,2′(Ethylenedioxy)bis(ethylamine) was used as precursor and modified with Fmoc and a Succinyl rest. Synthesis of such an SPPS-applicable molecule was previously reported by Song et al.43 but here modified. It was monoprotected with Boc anhydride in excess of the diamine.44 The intermediate was isolated by pHdependent extraction, followed by the protection of the second primary amine with Fmoc chloride in basic, aqueous THF. After deprotection of the Boc-moiety, a carboxyl moiety was introduced by reaction with succinic anhydride in the presence of triethylamine to give 6, EDS building block. (See Scheme 2.) The overall yield for EDS building block is more than 40% and can be carried out on a multigram scale giving up to 15 g in one batch. Solid-Phase Polymer Synthesis. After successful synthesis of building blocks TDS and EDS, they were applied for solid-phase synthesis of sequence-defined functional polymer segments on a standard peptide synthesizer. (See Scheme 3.) As solid support, commercially available tentagel trityl−OH resin was used, which had been modified with an EDA linker. The building block coupling on solid phase was performed with 10 equiv building block (EDS or TDS) and 10 equiv O(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and 5 equiv HOBt as coupling reagents in the presence of 20 equiv N,N-diisopropylethylamine (DIPEA) as base in dimethylformamide (DMF). Coupling was monitored by colorimetric Kaiser test as well as UV detection of the Fmoc cleavage adduct. We synthesized three different polymer segments using the alkyne and ethylenedioxy building blocks in solid-phase polymer synthesis keeping the chain length constant while varying the number and spacing of alkyne functionalities along backbones affording oligomer intermediates 7−9. For structure confirmation, 7−9 were cleaved from the resin with 20% TFA in DCM. The products were precipitated in diethylether, dried, redissolved in water, lyophilized, and analyzed with ESI-MS, RP-HPLC, and NMR. ESI-MS spectra showed expected mass-to-charge (m/z) ratios confirming sequence-control

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RESULTS AND DISCUSSION The first step was to synthesize a new building block for solidphase polymer synthesis carrying a functional unit for later modification with carbohydrate ligands. Suitable building blocks need to fulfill a number of requirements: Building block synthesis generally should be feasible on high scale, using inexpensive reagents while avoiding laborious purification procedures. Building block coupling on solid phase should proceed according to standard Fmoc SPPS protocols; therefore, both building blocks were equipped with a carboxylic acid moiety for coupling and an Fmoc-protected amine. As functional unit of the first building block (TDS), an alkyne moiety was introduced to modify the scaffold after solid-phase assembly with mannose azides via 1,3-dipolar cycloaddition. 1,3-Dipolar cycloaddition between azides and alkynes is the most anticipated reaction among the so-called click reactions and has been widely used, for example, for bioconjugation, functionalization of polymers, and so on.35−40 To adjust the spacing between the mannose ligands, we synthesized a second building block introducing a flexible, hydrophilic spacer (EDS). Synthesis of Building Blocks TDS and EDS. The Triple bond functionalized building block (TDS, 5) with a Succinyl rest was synthesized starting from Diethylenetriamine (Scheme 2). This symmetrical diamine was monoprotected with a trityl protecting group (Trt) using excess of commercially available diethylenetriamine in high dilution. This was followed by protection of the second primary amine with ethyl trifluoroacetate (TFAOEt) to give orthogonal protected amine 1. Trt and trifluoroacetic acid (TFA) protecting groups are known to be 1848

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After successful synthesis of this first set of glycopolymer segments, they were tested for their lectin binding affinity via inhibition studies using SPR. Binding Studies with Concanavalin A. The three glycopolymer segments have the same contour length of ∼10 nm with a distance between the sugar units of a maximum distance of ∼10 nm for 11 and ∼7 nm for 12. (These distances were obtained by the addition of bond lengths with the structures presenting an all-trans linear conformation and were calculated by Chem3D Pro.) The molecular weights range between 1500 and 2300 Da depending on the number of attached sugar ligands. Structures 10−12 differ in their number and spacing of presented mannoses. (See Figure 1.) For binding studies, we examined interactions of 10−12 with Con A. Con A is a well-characterized tetrameric lectin presenting four mannose specific binding sites in a distance of 6.5 nm and should serve as model lectin system to compare binding properties of our systems to the binding of other known multivalent scaffolds.47 It has been shown before that scaffolds with a sugar epitope density of one, two, or three ligands on dendrimers and peptides can result in large enhancements of lectin binding affinities due to multivalent binding modes.13,14,48−50 Here we want to investigate the correlation between number and spacing of ligands presented on a flexible, hydrophilic polymer backbone with the resulting binding properties. A competitive binding assay to evaluate the glycopolymer segments 10−12 as ligands for Con A binding determined by SPR was used. (For details, see the Supporting Information.)51 Direct binding measurements of 10−12 to a Con A-functionalized SPR chip surface were not suitable as the polymer segments are relatively small constructs, and direct binding results in only a small change in refractive index difficult to detect. Turbidimetry assay, as previously reported for systems with a larger number of presented mannoses,20,51 did not show measurable effects. For the competitive SPR assay, Con A was injected in a continuous flow over a α-D-mannose modified sensor chip. The obtained binding signal with only Con A in the flow solution was set as 100% binding. The addition of glycopolymer as inhibitor to the Con A flow solution reduces the binding signal if the compound addresses the Con A binding sites. Concentrations of 0.3 up to 100 μM of the glycopolymer segments were measured. The resulting binding signal was calculated relative to the Con A binding signal, thus enabling the determination of IC50 values (concentrations of 50% inhibition) for compounds 10−12. (See Figure 2 and Table 2.) As a control, scaffold 7 with no mannose ligands was tested and did not show any inhibition effect. Therefore, no unspecific interactions between the scaffold and Con A were observed. To control that no degradation of the mannose chip surface occurred during inhibition measurements, we performed control runs with pure Con A after each measurement for the different glycopolymer segments. No loss of response units indicating degradation was observed. All mannosylated structures showed strong inhibition of Con A with IC50 values in the micromolar range. Glycopolymer segment 10, presenting one mannose unit surrounded by four ethylendioxy building blocks, showed an IC50 value of 8 μM (Figure 2) and a relative activity of 94. (See Table 2.) Relative activity to α-methyl-D-mannose is the valencecorrected value comparing the obtained IC50 values to the IC50 of α-methyl-D-mannose as standard glycoside. Surprisingly, the relative activity for 10 is much higher than activities described for other monovalent systems in literature such as in aliphatic systems (rel. value: 3.549) or aromatic systems (rel. values: 8.7,49 39,14 253). Scaffold 10 carrying one mannose ligand is composed

during synthesis. (See Table 1.) Furthermore, the obtained products are monodisperse, as was analyzed by RP-HPLC Table 1. Analytical Details of PAA and Glycopolymer Segments compound 7 8 9 10 11 12

Mwa

ESI-MS m/zb

purity [%]c

(a) alkyne polymers segments 1467.8 734.8 [M+2H]2+ 98 1502.8 752.4 [M+2H]2+ 96 1537.8 770.0 [M+2H]2+ 99 (b) mannosylated polymer segments 1536.8 769.4 [M+2H]2+ 95 1820.9 911.8 [M+2H]2+ 90 2105.1 1053.8 [M+2H]2+ 95

retention time tR [min]d 21.4 21.8 21.4 19.6 18.3 16.6

a Calculated exact mass. bFor complete data, see the Supporting Information. cDetermined by integration of HPLC-UV signal at 214 nm. dRP-HPLC 5/95→95/5 (MeCN/H2O) in 60 min.

traces showing uniform products with retention times (tR) of ∼21 min for structures 7−9 (gradient: 5/95→95/5 MeCN/ H2O in 60 min; see Table 1). The structures can be isolated in high purity of above 95% directly after cleavage from the resin and without further purification (determined by integration of UV signal at 214 nm of RP-HPLC traces). Structures were also confirmed by NMR. (See the Supporting Information for detailed analytical data.) After successful assembly of the alkyne carrying segments, mannose ligands were introduced via click reaction of mannose azides on solid support. The 1,3-dipolar cycloaddition between azides and alkynes is well-known as a reliable conjugation method also suitable for on resin modifications.45 Conjugation of Mannoses to Alkyne Polymer Segments. Azidoethyl mannosides were synthesized according to literature.37 In brief, the hydroxyl groups of mannose were acetylated, the anomeric center was glycosylated with bromoethanol, followed by substitution of bromine with azide and deprotection of hydroxyl groups. (For details, see the Supporting Information.) Before conjugation to the alkyne scaffolds 7−9, the terminal Fmoc group of the polymer segment was cleaved and substituted with an acetyl group using acetic anhydride (Ac2O). A copper(II) source, CuSO4, together with the reducing agent ascorbate (20 mol % each) gave full conversion of all scaffolds applying 10 equiv of mannose-O-ethyl azide in DMF and water for 4 h. Cu(I) catalyzed on-resin 1,3-dipolar cycloaddition using CuI as Cu(I) source has been shown before but did not work for our systems.45 After conjugation, the resin was washed with a solution of diethyldithiocarbamate in DMF to remove excess of copper catalyst, and the final glycopolymer segments were cleaved of the resin by acidic treatment with TFA in DCM, leading to the final structures 10, 11, and 12.46 After lyophilization from water, the glycopolymer segments were analyzed by ESI-MS, RP-HPLC, and NMR. (See Table 1.) ESI-MS spectra showed expected mass-tocharge (m/z) ratios confirming sequence-control during synthesis. (See Table 1.) Monodispersity of the products was confirmed by RP-HPLC showing an expected difference in retention time (tR) with respect to the number of presented mannoses. (See Table 1.) The structures were isolated in high purity of above 90% directly after cleavage from the resin and without further purification (determined by integration of UV signal at 214 nm of RP-HPLC traces). Structures were also confirmed by NMR. (See the Supporting Information for detailed analytical data). 1849

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cross-linking between the Con A molecules is needed such as a direct binding SPR measurements on a Con A-functionalized chip surface or turbidimetry assays did not show any binding. For the trivalent species 12, carrying three mannose ligands in a distance of ∼7 nm, we observed an eight-fold lower IC50 compared with the monovalent system (10). The relative binding activity per mannose compared with α-methyl-D-mannose is increased by 250fold. Such high values have so far been observed only for peptidebased branched scaffolds (rel. value: 18114); other trivalent systems presenting ligands in a similar spacing compared with our scaffold showed much weaker binding (rel. values for dendritic or branched scaffolds: 0.8,53 and 2.6;50 or linear scaffolds: 1.412). This large enhancement in binding affinity can potentially be attributed to the sum of different factors. First, the length of the spacer containing two ethylenedioxy-units between two of the three mannoses is smaller compared with the divalent system 11 (∼7 nm for 11). Therefore, the spacing between the ligands should be closer to the actual distance of 6.5 nm between two binding sites of Con A and thus possibly allows for a more favored chelation binding. Second, the trivalent species can profit from enhanced binding via a statistical effect: In the case of a binding of the mannose located in the middle of the trivalent system, the chance to bind one of the other two mannose ligands is twice as high compared with the divalent system. A synergistic effect of these two factors together with the supporting influence of the ethylenedioxy-spacers, as already seen in the binding of 10, might lead to the observed increase in relative activity by two orders of magnitudes for the trivalent system compared with α-methyl-D-mannose.

Figure 2. Binding of mono-, di-, and trifunctionalized mannose polymer segments to Con A determined by competitive SPR binding assay. Relative Con A binding is plotted against varying concentrations of 7 (◆), 10 (▲), 11 (■), and 12 (●). The concentrations were measured after incubation with Con A, leading to a lowered binding signal. Each data point represents the mean of at least three measurements. The error shown is the standard deviation; some error bars are smaller than the symbols. Data were fit to a four-parameter logistic equation/dose response (with GraphPad Prism 5).50,52

Table 2. Binding Values and Relative Activities Obtained by SPR Measurements IC50 [μM]

compound α-D-mannoseb α-methyl-D-mannoseb 10 11 12

relative activity of of per no. polymer mannose glycopolymer segment per mannose unita mannose segment unit 1 1 1 2 3

8 5 1

6500 750 8 10 3

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CONCLUSIONS In summary, we introduced a new approach to synthesize sequence-defined, monodisperse glycopolymer segments via solid-phase synthesis. New building blocks were synthesized allowing for the sequence-defined positioning of alkyne moieties within monodisperse PAA segments. These segments can then be functionalized via click chemistry directly on solid phase introducing sugar ligands to the scaffold. Three different segments were synthesized, introducing different numbers and positions of mannose ligands within polymer segments of the same chain length. These structures were subject to Con A inhibition SPR studies. Starting from a single mannose ligand on the scaffold up to a trivalent scaffold with three mannose units, a nonlinear correlation of the number of ligands to the resulting binding properties was observed. For the monovalent system, consisting of a very hydrophilic backbone, a strong increase in Con A binding compared with α-methyl-D-mannose and other literature known monovalent systems was shown. We attribute this gain in relative activity to the chemical composition of the scaffold. Its supposedly high hydration due to its composition of mostly ethylenedioxide units allows for the release of water upon binding and thus a gain in entropic energy for the system. For Con A binding of the divalent species, a slight decrease in relative binding was observed, which is attributed to an unfavorable presentation of the two mannoses resulting from a too long linkage. The trivalent glycopolymer segment showed a strong increase in relative binding activity up to a value of 250. This indicates a synergistic combination of a statistical effect, optimal ligand spacing, and chemical composition of the scaffold. Overall, with our new monodisperse, linear glycopolymer segments consisting of very hydrophilic scaffolds, unexpected high binding signals for the monovalent and trivalent systems were obtained. We interpret these findings with a strong dependence of not only

1 96 77 257

IC50 value of α-methyl-D-mannose divided by IC50 value of polymer segment per mannose unit. bFor data curves of α-D-mannose and α-Dmethyl-D-mannose, see the Supporting Information. a

mainly of hydrophilic ethylenedioxy units that are highly hydrated. It could be expected that the hydration shell enhances the binding affinity due to release of water upon binding and the resulting entropic gain.54−56 This effect is known for polargroup displaying lectin binding sites where water acts as “molecular mortar”.4 Glycopolymer segment 11, presenting two mannose units with a spacing of ∼10 nm, reveals a further decrease of IC50 concentration to 5 μM. However, regarding the number of presented mannose units, this system shows a lower relative activity compared with the monovalent scaffold. In general, such a bivalent system can undergo bridging of two binding pockets of Con A. Binding is enhanced because translational and rotational entropic penalties were already brought up by the first binding event and need to be paid only once.57,58 In addition to that the linkage between the two ligands is important. It was shown by crystallographic structures that an optimal distance between the ligands seems to be essential.13,59 A longer distance of only one C−O ether bond led to a six-fold drop in potency before.13 In our system, the spacing between the two mannoses of ∼10 nm might be too long for a favored chelation binding, as the distance between two binding sites of Con A is ∼6.5 nm, and thus binding affinity is not increased. Intermolecular binding between two Con A molecules is not considered for structures 11 and 12 because they are probably too small. Experiments in which 1850

dx.doi.org/10.1021/bm300331z | Biomacromolecules 2012, 13, 1845−1852

Biomacromolecules

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the number of presented mannose units but also much more of the chemical composition of the scaffold together with the spacing between the ligands. All in all, the solid-phase synthesis approach allows for the use of building blocks having desired properties together with a precise positioning and distancing of ligands. This now allows us to synthesize scaffold structures with a diversity of properties, for example, hydrophilic/hydrophobic, flexible/stiff, and longer length, which will also be analyzed regarding their thermodynamic properties (e.g., with ITC measurements) to investigate systematically multivalent effects of polymeric ligands.

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ASSOCIATED CONTENT

S Supporting Information *

Materials and instrumentation, more detailed experimental data, and spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by Emmy Noether Program HA 5950/11; SFB 765 and the Max Planck Society. We thank Simone Mosca and Dr. Jens Weber for their support. We are grateful to Prof. Dr. Peter H. Seeberger and the Max Planck Institute of Colloids and Interfaces for the provision of technical facilities.

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