Valency-Dependent Affinity of Bioactive Hydroxyapatite-Binding

Jul 26, 2013 - Ahlam Alalwiat , Wen Tang , Selim Gerişlioğlu , Matthew L. Becker ... Shuang Song , Xiaoli Liang , Matthew D. Watson , Jonathan G. Ru...
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Valency-Dependent Affinity of Bioactive Hydroxyapatite-Binding Dendrons Wen Tang,† Yanrui Ma,† Sibai Xie,† Kai Guo,†,‡ Bryan Katzenmeyer,‡ Chrys Wesdemiotis,†,‡ and Matthew L. Becker*,†,§ †

Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States Department of Chemistry, The University of Akron, Ohio 44325, United States § Center for Biomaterials in Medicine, Austen Bioinnovation Institute in Akron, Akron, Ohio 44308, United States ‡

S Supporting Information *

ABSTRACT: Hydroxyapatite (HA)-coated surfaces are used widely as stationary phase for protein and enzyme purification, coatings for dental and orthopedic implants, and composite materials for tissue engineering substrates. More advanced applications are envisioned, but progress has been slowed by the limited ability to controllably functionalize the surface of HA with biomolecules in a translationally relevant manner. Herein we report the synthesis and characterization of a series of multivalent, HA-binding peptide bioconjugates with variable valency and tether length which afford the ability to precisely tune the desired binding behavior. The respective binding affinities of the multivalent constructs to HA surface were characterized by quartz crystal microbalance with dissipation monitoring (QCM-D) techniques, and the relationship between dendron structure and binding affinity was revealed. Tetravalent constructs of HA-binding peptides show a 100-fold enhancement in binding affinity compared to HA-binding peptide sequences reported previously. Both biotin and bone morphogenic protein-2 (BMP-2) derivative peptide were successfully linked to the focal point as initial demonstrations.

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peptide with HA surface.12 This is an attractive strategy for tethering highly potent biologics where free diffusion in the body may require significant doses for efficacy and as a result may have unintended side effects. As an example, recombinant human bone morphogenetic protein-2 (rhBMP-2) obtained approval from the US Food and Drug Administration (FDA) in 2002 in spine surgery, due to its known ability to induce bone formation.13 Since then, BMP-2 has been used clinically for a number of applications, many of which are off label. When released from the BMP-2-loaded collagen sponge and distributed throughout the body, localized rhBMP-2 levels can reach 106 times greater concentration than would ever occur naturally. This concentration spike may lead to adverse events like uncontrolled bone formation, negative effects on surrounding dura and nerves, even risk of new malignancy.14−16 Recently, we reported that the peptide SVSVGMKPSPRP, identified through phage display methodology, bound to HA with chemical and morphological specificity.17 The binding affinity was measured quantitatively to be Kd = 14.1 ± 3.8 μM using surface plasmon resonance imaging (SPRi).18 Although the measured binding affinity surpasses the values of other synthetic HA-binding peptides,19−21 it does not approach the strength or specificity of noncovalent interactions found in

INTRODUCTION Hydroxyapatite (HA), or Ca10(PO4)6(OH)2 is the primary inorganic component in bone and teeth enamel.1 It is also used as a stationary phase in enzyme and protein purification and as a bioactive structural component in tissue engineering scaffolds and composites. The surface properties of HA have been investigated widely due to its ability to improve the ingrowth and fusion of HA-coated metal and ceramic implants to bone.2 Many commercially available orthopedic implants are coated with a thin HA layer.3 The identification of molecules with strong and tunable binding affinity to HA is important as the biomaterials community seeks to utilize biobased conjugates to anchor bioactive species on the HA surface and further enhance the bone mineralization process.4,5 Modular peptides containing HA-binding sequences and bioactive peptide have been reported previously.5−8 Bioconjugates that involve the fusion of proteins/peptides with strong binding affinity to the surface of HA and moieties that have specific interactions with cellular receptors have emerged as a promising strategy. In this strategy the surface-binding motif sequesters the molecule locally, while the bioactive motif triggers requisite biological processes, e.g., cell adhesion,6,9 stem cell differentiation,8 and cell migration.7 It was also shown that bioactive peptides maintain their bioactivity when covalently tethered to the HA-binding peptide using in vitro8,10 and in vivo experiments.11 The bioactive peptide was confined locally near the tether position due to the binding of HA-binding © 2013 American Chemical Society

Received: June 20, 2013 Revised: July 25, 2013 Published: July 26, 2013 3304

dx.doi.org/10.1021/bm400908c | Biomacromolecules 2013, 14, 3304−3313

Biomacromolecules

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performance liquid chromatography (RP-HPLC) was performed on a Akta Purifier HPLC system by using a ZORBA* 300SB-C18 column (5 μm, 9.4*250 mm). The solvent was degassed A: 0.1% trifluoroacetic acid (TFA) in H2O and B: 0.085% trifluoroacetic acid in 95% acetonitrile. The flow rate was 5 mL/min with the pressure around 14 MPa. General Procedures for the Preparation of NO2-(COOTFP)n. To the mixture of the dendron NO2 -(COOH)n (1 equiv), tetrafluorophenol (TFP) (1.2 × n equiv), and 4-(dimethylamino)pyridinium 4-toluenesulfonate (DPTS) (0.1 × n equiv) in anhydrous CH3CN (∼0.5 g/10 mL for compound NO2-(COOH)n), diisopropylcarbodiimide (DIC) (1.2 × n equiv) were added dropwise. The reaction mixture was stirred at room temperature (r.t.) for 16 h. Then it was concentrated in roto vapor and dissolved in CHCl3. The insoluble white solid was filtered. The filtrate was concentrated to obtain the crude product and purified by flash chromatography. NO2-(COOTFP)2 (4). The crude product was purified by flash chromatography using 1:1 CHCl3:hexane (v:v). The product was obtained as white solid (1.1695 g, 90%). 1H NMR (CDCl3, 300 MHz): δ ppm 1.70 (s, 3 H), 2.24−2.43 (m, 2 H), 2.50−2.67 (m, 2 H), 2.77 (m, J = 6.15, 2.93 Hz, 4 H), 7.03 (s, 2 H). 13C NMR (CDCl3, 500 MHz): ppm 168.0, 147.0, 145.1, 141.6, 139.6, 103.7, 103.5, 103.3, 89.1, 33.8, 28.2, 21.7. 19F NMR (CDCl3, 500 MHz): δ ppm −138.8, −153.0. IR (cm−1): ν 3086, 2926, 1786, 1644, 1524, 1488, 1384, 1179, 1107, 953, 841, 714. ESI-MS: Calc. for C20H13F8NO6Na+: m/z 538.1; Found: m/z 537.6. NO2-(COOTFP)4 (11). The crude product was purified with flash chromatography using 6:1 CHCl3:hexane (v:v). The product was obtained as white solid (0.6401 g, 80%).1H NMR (CDCl3, 500 MHz): δ ppm 1.27 (s, 6 H), 1.47 (s, 3 H), 1.94−2.19 (m, 10 H), 2.19−2.32 (m, 2 H), 2.34−2.47 (m, 4 H), 2.64 (s, 8 H), 5.29−5.49 (m, 2 H), 6.83−7.08 (m, 4 H). 13C NMR (CDCl3, 500 MHz): ppm 170.9, 169.4, 147.0, 145.0. 141.5, 139.5, 129.6, 103.3, 90.4, 55.4, 34.4, 33.0, 31.4, 28.3, 23.7, 22.0. 19F NMR (CDCl3, 500 MHz): δ ppm −138.9, −153.1. MALDI-MS: Calc. for C48H39F16N3O12K+: m/z 1192.2; Found: m/z 1192.2. General Procedures for the Preparation of NO2-(N3)n. To the mixture of NO2-(COOTFP)n (1 equiv) and amine (1.1 × n equiv) in CH2Cl2 or CHCl3(1 mmol/10 mL), diisopropylethylamine (DIPEA) (3 × n equiv.) was added. The mixture was stirred at r.t. for 4 h. Then the reaction solution was washed with NH4Cl saturated solution (5 mL × 3), 5 wt % NaHCO3 (5 mL × 3) and then 5 mL of brine. The organic phase was dried with Na2SO4 and concentrated. The crude reaction mixture was purified by flash chromatography. NO2-(N3)2, n = 2 (5). The crude reaction mixture was purified by flash chromatography using a gradient elution of 100: 1 to 100: 3 CH2Cl2/ CH3OH (v:v). The product was obtained as a light yellow viscous liquid (0.8655 g, 98%). 1H NMR (CDCl3, 300 MHz): δ ppm 5.99 (br. s., 2H), 3.68 (t, J = 4.83 Hz, 4H), 3.52−3.60 (m, 4H), 3.42− 3.51 (m, 4 H), 3.38 (t, J = 4.83 Hz, 4H), 2.02−2.49 (m, 8H), 1.54 (s, 3H). 13C NMR (CDCl3, 500 MHz): δ ppm 171.1, 90.3, 69.6, 50.5, 39.2, 34.4, 30.8, 21.9. IR (cm−1): ν 3300, 3085, 2938, 2870, 2361, 2338, 2106, 1651, 1537, 1446, 1391, 1348, 1285, 1124. ESI-MS: Calc. for C16H29N9O6Na+: m/z 444.23; Found: m/z 443.8. NO2-(N3)2, n = 6 (6). The crude product was purified by flash chromatography using a gradient elution of 100: 2 to 100: 5 CH2Cl2/ CH3OH (v:v). The product was obtained as a light yellow viscous liquid (0.4936 g, 98%). 1H NMR (CDCl3, 500 MHz): δ ppm 1.54 (s, 3 H), 2.11−2.25 (m, 6 H), 2.33−2.39 (m, 2 H), 3.34−3.47 (m, 8 H), 3.55 (t, 4 H), 3.66 (m, 36 H), 6.37−6.49 (br. m., 2 H). 13C NMR (CDCl3, 500 MHz): δ ppm 171.0, 90.1, 70.0, 50.2, 39.0, 34.2, 30.2, 21.4. ESI-MS: Calc. for C16H29N9O6Na+: m/z 818.4; Found: m/z 818.5. NO2-(N3)4, n = 2 (12). The crude reaction mixture was purified by chromatography with a multistage elute of 200:1:8 to 200:1:14 CH2Cl2:isopropylamine:methnol (v:v:v). The product was obtained as a white solid (0.0784 g, 84%). 1H NMR (CDCl3, 300 MHz): δ ppm 1.28 (s, 8 H), 1.52 (s, 4 H), 1.83−2.34 (m, 34 H), 3.31−3.49 (m, 22 H), 3.55 (d, J = 4.98 Hz, 11 H), 3.67 (t, J = 4.54 Hz, 11 H), 6.32 (d, J = 4.68 Hz, 5 H), 7.09 (br. s., 3 H). 13C NMR (CDCl3, 300 MHz): δ

some biological systems, such as the binding affinity between biotin and streptavidin (Kd ∼ 10−14 M).22 To further strengthen the binding affinity, a multivalent approach using the HA-binding peptide was pursued that accommodates both biological and synthetic bioactive molecules at the core.23−25 A multivalent strategy enables stronger and more specific interactions than possible in monovalent systems.26,27 Meijer et al. reported the synthesis of an asymmetric polyamide dendron platform28 that mimics the pentavalent bacteriophage utilized in molecular screening applications.29 The phage mimic was proposed for the systematic study on how to maximize the multivalency effect as well as the development of molecular bioconjugates for bioimaging30 and biomedicine.31 In their reports, the binding affinity of synthetic dendrons was correlated with the number of ligand valency.29 The binding affinity of synthetic dendrons was found to increase dramatically as valency increased. However, the synthetic pentavalent dendrons showed about 1000 fold weaker binding than the natural pentavalent phage with the same binding peptide. This difference calls attention to the importance of adding structure parameters that contribute to the binding affinity, such as the length of linkage between branch points. Understanding the relationship between the structure of multivalent ligands and binding affinity is essential to generate conjugates with tunable binding affinity toward substrates of interest. In this work we show that the binding behavior is dependent on valency and tether length, and functionalized the conjugates with biotin and a widely utilized BMP-2 derived peptide, which demonstrate the utility of this scalable and translationally relevant approach.

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MATERIALS AND METHODS

Materials and Equipment. Chemicals and solvents were purchased from either Sigma-Aldrich or Acros. Unless otherwise stated, all solvents used were reagent grade, and all chemicals were used as supplied. 1H and 13C NMR spectra were acquired on a Varian NMRS at 300 and 500 MHz, in deuterated solvents; chemical shifts are listed in ppm relative to CHCl3 as a standard. Alky functionalized HA-binding peptide (>98% in purity) was synthesized by American Peptide Company (Sunnyvale, CA). The synthesis of BMP-2 derivative peptide was performed on a Liberty 1 peptide microwave synthesizer (CEM Cooperation, Matthews, NC) through solid phase peptide synthesis. Fluorescein images were viewed on a CKX41 Microscope (Olympus, Center Valley, PA). Quantification of the adsorption of HA-binding peptide or dendrons onto the HA surface was performed by using a Q-sense E4 system (Biolin Scientific AB, Sweden). ESI-MS spectra were recorded on a Waters Synapt HDMS quadrupole/time-of-flight (Q/ToF) instrument in positive mode. The sprayed solution was prepared by dissolving sample in chloroform/ methanol (1/1). The following ESI parameters were selected: ESI capillary voltage, 3.5 kV; sample cone voltage, 35 V; extraction cone voltage, 3.2 V; desolvation gas flow, 500 L/h (N2); trap collision energy (CE), 6 eV; transfer CE, 4 eV; trap gas flow, 1.5 mL/min (Ar); sample flow rate, 5 μL/min; source temperature, 80 °C; desolvation temperature, 150 °C. Matrix-assisted laser desorption/ionization timeof-flight mass spectra (MALDI-ToF-MS) were recorded on a Bruker Ultraflex III ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with Nd:YAG laser which emits at 355 nm. The matrix used in MALDI-ToF-MS measurements was sinapinic acid (SA, > 99%, Aldrich) and was dissolved in 7:3 (v:v) acetonitrile: (H2O + 0.1% formic acid). Sample was dissolved in water in concentration of 1 mg/ mL. The matrix and sample solution were mixed in the ratio of 2:1 (v:v). Mass spectra were measured in the reflectron mode, and the mass scale was calibrated externally with a PMMA standard at the molecular weight region under consideration. Data analysis was conducted with Bruker’s flexAnalysis software. Reversed-phase high3305

dx.doi.org/10.1021/bm400908c | Biomacromolecules 2013, 14, 3304−3313

Biomacromolecules

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BMP-2-(N3)2, n = 6 (21). The amidation of compound 20 with BMP-2 peptide was similar to the solid phase peptide synthesis. The reaction happened in a peptide synthesis vessel. The peptide grown from the Wang Resin was deprotected in 25% piperidine, and then washed with DMF, DCM, and MeOH three times each to have the amine (1 equiv) as the end group. Then the resin was swelled in DMF. Compound 20 (4 equiv), HOBt (10 equiv.), and DIC (10 equiv.) was added sequentially. The solution was bubbled with N2 for 3 h at r.t. After that, the solution was filtered, and the resin was washed with DMF, DCM, and MeOH three times each. To cleavage, 28.5 mL:0.75 mL:0.75 mL TFA:TIPS:H2O was added, and the system was bubbled with N2 for 45 min at r.t. The filtrate was collected and concentrated until there was about 2 mL of TFA left. The product was precipitated in cold ethyl ether and then washed with cold ethyl ether three times, then dissolved in phosphate buffered saline (PBS) and dialysis against ultrapure water for 12 h twice. After that, the product solution was freeze-dried. White solid was obtained at last. The product was purified by preparative RP-HPLC with a gradient of solution B in solution A (A: 0.1% TFA in H2O, B: 0.085% TFA in 95% acetonitrile). The Gradient was 25−40% in 15 column volume (CV). MALDI-ToF-MS: Calc. for C133H239N31O44+: m/z 2965.7; Found: m/z 2966.2. General Procedure for the Cu-Catalyzed Cyclic Addition. The alky-functionalized HA-binding peptide and azide-functionalized dendron were dissolved in 95:5 10 × PBS buffer: DMSO (v:v) (pH = 7.4). The volume of the solution was determined by the solubility of alky-functionalized HA-binding peptide which is about 1 mg/mL. After freezing and degassing twice, the solution was frozen. CuSO4 and Na ascorbate was added under N2 to make the c(CuSO4) = 10 mmol/ L and c(Na Ascorbate) = 50 mmol/L. Then the solution was melted and stirred at 40 °C for 2 days. After the reaction, the product was washed by using an ultrafiltration device with a membrane (MWCO = 1000 Da), with 0.01 mol/L EDTA solution (pH = 9) three times and then ultrapure water three times. After that, the product was freezedried and purified with RP-HPLC with a gradient of solution B in solution A (A: 0.1% TFA in H2O; B: 0.085% TFA in 95% acetonitrile). NO2-(HA)2, n = 2 (7). Gradient RP-HPLC: 15−30% in 15 CV. The product was white solid (2 mg, 11%). MALDI-ToF-MS: Calc. for C146H244N47O46S2+: m/z 3455.7; Found: m/z 3456.2. NO2-(HA)2, n = 6 (8). Gradient RP-HPLC: 17−30% in 15 CV. The product was white solid (3 mg, 7%). MALDI-ToF-MS: Calc. for C162H276N48O54S2+: m/z 3807.0; Found: m/z 3808.6. NO2-(HA)4, n = 2 (14). Gradient RP-HPLC: 17−40% in 15 CV. The product was white solid (1 mg, 5%). MALDI-ToF MS: Calc. for C300H500N95O92S4+: m/z 7033.6 Da; Found: m/z 7037.1 Da. ESI-MS: [M+7H]7+: Calc. m/z 1006.2; Found m/z 1006.4. NO2-(HA)4, n = 6 (15). Gradient RP-HPLC: 17−40% in 15 CV. The product was white solid (1 mg, 5%). MALDI-ToF-MS: Calc. for C332H564N95O108S4+: m/z 7738.0; Found: m/z 7743.9. ESI-MS: [M +8H]8+: Calcd: m/z 968.6; Found: m/z 968.8. Lys(Biotin)-(HA)2, n = 6 (24). Gradient RP-HPLC: 15−45% in 15 CV. The product was white solid (1 mg, 5%). MALDI-ToF-MS: Calc. for C182H307N51O58S3+: m/z 4232.2; Found: m/z 4232.8. BMP-2-(HA)2, n = 6 (24). Gradient RP-HPLC: 17−65% in 15 CV. The product was white solid (3 mg, 15%). MALDI-ToF-MS: Calc. for C263H444N69O84S2+: m/z 5977.2; Found: m/z 5977.3. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) Measurement. Quantification of the adsorption of HAbinding peptide or dendrons onto the HA surface was performed by using a Q-sense E4 system (Biolin Scientific AB, Sweden). The sensor was excited at 5 MHz as its fundamental frequency. The frequency shift (Δf) and dissipation (ΔD) were measured at 3rd, 5th, 7th, 9th, 11th and 13th overtones. Twenty-five millimolar HEPES buffer pH = 7.40 at 25 °C was used as the flow medium. AT-cut HA-coated QCM sensors were purchased from Biolin Scientific AB (Sweden). The characterization of the HA-coated surface is shown in Figure S1 and Figure S2 (Supporting Information). HA-coated sensors were washed as the following protocol. Sensors were first treated with UV-ozone for 5 min, and then immersed in 20 mL EtOH for 30 min. After that, sensors were washed with milli-Q water and dried under N2. Finally, sensors were treated with UV-ozone for 2 min again. HA-coated

ppm 173.4, 170.9, 90.4, 70.0, 69.6, 55.4, 50.5, 39.2, 34.1, 31.0, 23.6, 22.1. MALDI-ToF-MS: Calc. for C40H71N19O12Na+: m/z 1032.54; Found: m/z 1032.37. NO2-(N3)4, n = 6 (13). The crude reaction mixture was purified by chromatography with a multistage elute of 200:1:8 to 200:1:12 CH2Cl2:isopropylamine:methnol. The product was obtained as a colorless viscous liquid (0.4376 g, 80%). 1H NMR (CDCl3, 500 MHz): δ ppm 1.32 (s, 6 H), 1.54 (s, 3 H), 1.84−2.35 (m, 24 H), 3.32−3.47 (m, 16 H), 3.51−3.58 (m, 8 H), 3.66 (s, 72 H), 6.40−6.51 (br. m., 4 H), 7.20−7.26 (br. m., 2 H). 13C NMR (CDCl3, 300 MHz): δ ppm 173.4, 170.9, 90.5, 70.5, 55.4, 50.7, 39.4, 34.3, 31.0, 23.6, 22.3. MALDI-ToF-MS: Calc. for C72H135N19O28K+: m/z 1752.9; Found: m/ z 1753.3. FmocNH-(N3)2, n = 6 (19). The crude reaction mixture was purified by flash chromatography using a gradient elution of 100: 2 to 100: 4 CH2Cl2/ CH3OH (v:v). The product was obtained as a colorless plastic-like solid (0.6467 g, 89%). 1H NMR (CDCl3, 500 MHz): δ ppm 1.29 (s, 3 H), 1.86−2.12 (m, 4 H), 2.12−2.31 (m, 4 H), 3.34− 3.48 (m, 8 H), 3.54−3.56 (m, 4 H), 3.64 (d, J = 4.16 Hz, 36 H), 4.15− 4.25 (m, 1 H), 4.30−4.47 (m, 2 H), 5.77 (s., 1 H), 6.32 (br. s., 2 H), 7.32 (m, 2 H), 7.37−7.44 (m, 2 H), 7.62 (d, J = 7.58 Hz, 2 H), 7.77 (d, J = 7.58 Hz, 2 H). 13C NMR (CDCl3, 500 MHz): δ ppm 173.0, 154.8, 144.0, 141.3, 127.6, 127.0, 125.1, 119.9, 70.4, 65.6, 54.5, 50.6, 47.4, 39.3, 34.4, 31.0, 23.9. ESI-MS: Calc. for C31H40N9O6Na+: m/z 1010.5; Found: m/z 1010.4. NH2-(N3)2, n = 6 (24). The compound 19 (0.2876g, 0.29 mmol) was dissolved in 5 mL of 60 v% diethylamine in DCM. The solution was stirred at r.t. for 2 h. After that, the solution was concentrated and purified with a multistage elute of 100:1:2 to 100:1:5 CH2Cl2:isopropylamine:methnol (v:v:v). The product was obtained as a light yellow viscous liquid (0.2024 g, 80%). 1H NMR (CDCl3, 500 MHz): δ ppm 1.06 (s, 3 H), 1.70 (m, 4 H), 1.77−2.03 (br. s., 3 H), 2.24 (m, 4 H), 3.33−3.47 (m, 8 H), 3.52−3.56 (t, J = 5 Hz, 4 H), 3.66 (m, 36 H), 6.44−6.57 (br. s., 2 H). 13C NMR (CDCl3, 500 MHz): δ ppm 173.1, 70.2, 50.4, 39.0, 37.6, 31.1, 27.4. COOH-(N3)2, n = 6 (20). To the solution of compound 24 (0.2024 g, 0.264 mmol, 1 equiv) and succinic anhydride (0.1587 g, 1.59 mmol, 6 equiv) in 5 mL of THF, NEt3 (0.0026 g, 0.026 mmol, 0.1 equiv) was added. The solution was stirred at r.t. for 4 h. After that, the solution was concentrated. The residue was dissolved in 5% NaOH solution and washed with CHCl3. NaHSO4 was added to adjust the pH to 1. The aqueous phase was extracted with CHCl3 six times, and the organic phase was collected, dried with anhydrous Na2SO4, and concentrated. The product was purified with flash chromatography using 85:15:5 CHCl3:MeOH:HOAc (v:v:v). The product was obtained as light yellow viscous liquid (0.2194 g, 96%). 1H NMR (CDCl3, 500 MHz): δppm 1.26−1.34 (s, 3 H), 1.84−1.98 (m, 2 H), 2.01−2.13 (m, 2 H), 2.18−2.32 (m, 4 H), 2.34−2.44 (m, 2 H), 2.59− 2.71 (m, 2 H), 3.35−3.46 (m, 8 H), 3.54−3.60 (m, 4 H), 3.66 (m, 36 H), 6.73 (br., 2 H) 6.80 (br., 1 H). 13C NMR (CDCl3, 500 MHz): δ ppm 175.6, 174.8, 171.9, 70.3, 55.6, 50.6, 39.1, 34.4, 31.0, 29.6, 23.7. ESI-MS: Calc. for C36H67N9O15Na+: m/z 888.5; Found: m/z 888.2. Lys(Biotin)-(N3)2, n = 6 (22). The amidation of compound 20 with Lys(Biotin)-Wang resin was the same with the solid phase peptide synthesis. The reaction happened in a peptide synthesis vessel. The Fmoc-Lys(Biotin) Wang resin was deprotected in 25% piperidine, and then washed with dimethylformamide (DMF), dichloromethane (DCM), and methanol (MeOH) three times each to have the amine (1 equiv) as the end group. Then the resin was swelled in DMF for 15 min. Compound 20 (4 equiv), hydroxybenzotriazole (HOBt; 10 equiv), and DIC (10 equiv) were added sequentially. The solution was bubbled with N2 for 3 h at r.t. After that, the solution was filtered, and the resin was washed with DMF, DCM, and MeOH three times each. To cleavage, 28.5 mL:0.75 mL:0.75 mL TFA:TIPS:H2O (TIPS: triisopropylsilane) was added. The system was bubbled with N2 for 45 min at r.t. The filtrate was collected and concentrated. Toluene was added twice as the chase solvent to take out the TFA. The product was a viscous yellow liquid, which was dissolved in DMF. MALDI-ToFMS: Calc. for C133H238N31O44Na+: m/z 1242.6; Found: m/z 1242.9. 3306

dx.doi.org/10.1021/bm400908c | Biomacromolecules 2013, 14, 3304−3313

Biomacromolecules

Article

Scheme 1. Synthesis of NO2-(HA)2 and NO2-(HA)4 Liganda,b

a Reagents and conditions: (a) Triton-B, THF, 40 °C, 24 h, 94%; (b) Raney-Ni, H2, EtOH, 55 °C, 20 h, 98%; (c) HCOOH, r.t., 20 h, 3: 93%, 10: 94%; (d) 2, 3, 5, 6-tetrafluorophenol (TFP), DIC, DPTS, 4: anhydrous CH3CN, r.t., 16 h, 90%, 11: anhydrous Dioxane: CH3CN= 1:1.5 (v:v), r.t., 17 h, 80%; (e) 6: 16, DIPEA, DCM, r.t., 4 h, 98%; 8: 17, DIPEA, CHCl3, r.t., 4 h, 99%; 12: 16, DIPEA, DCM, r.t., 12 h, 84%; 13: 17, DIPEA, DCM, r.t., 12 h, 80%; (f) 5-hexynoic acid-GGGSVSVGMKPSPRP, CuSO4, Na Ascorbate, 95: 5 10 × PBS:DMSO (v:v), 35 °C, 48 h, 7: 11%, 8: 7%, 14: 5%, 15: 5%; (g) HOBt, DIC, anhydrous DMF, r.t., 20 h, 88%. bThe syntheses of compounds 1−3, 9, 10, 16 and 17 were published previously.28,37,40

sensors were mounted in the modules immediately after cleaning. HEPES buffer flowed above the sensors until flat baseline had been achieved at flow rate of 0.150 mL/min. Then the flowing solution was changed to HA-binding peptide/dendron solution in HEPES buffer, at the same flow rate, and after 3 min, the flow was stopped. After the frequency shift did not change, indicating the system reached its equilibrium state, waiting for another 5 min or more, HEPES buffer was introduced again to wash the HA surface. Three independent measurements were done simultaneously. Calculation of Adsorption Area Mass from Frequency Shift in QCM-D. The adsorbed area mass and viscoelastic properties of adhering conjugates were determined by measuring the frequency shift (Δf) and dissipation (ΔD), respectively.32 For a rigid adsorbed film (ΔD < 1 × 10−6 per 10 Hz), the adsorbed area mass (Δm) is proportional to Δf, and calculated by the Sauerbrey equation.33,34 The Sauerbrey equation is Δm = −(C)/(n)Δf n, where C is the mass sensitivity constant with a value of 17.7 ng Hz1− cm−2 for 5 MHz fundamental frequency crystal, n is the frequency overtone number, and n = 7 was chose to calculate the adsorption area mass in this work. As long as the adsorbed mass is small compared to the crystal, sufficiently thin, and has limited viscoelastic coupling with the surrounding medium (ΔD < 1 × 10−6 per 10 Hz), this relationship is valid. Adsorbed area mass measured from QCM includes water contained in the adhering layer. For adsorptions of HA-binding peptide/dendrons onto HA surface, ΔD were below 1 × 10−6 per 10 Hz, and measurements from multiple overtones were close to each

other, as shown in Figure 2A and Figure S3−6, indicating that adsorbed films were rigid, and the effect from content of water was slight. Models Used to Fit the Adsorption Isotherm. To obtain the binding affinity characterized with apparent disassociation constant Kd, software GraphPad Prism 5 was used to fit the adsorption isotherm of HA-binding peptide and dendrons. For HA-binding peptide, the best fit model is single-site specific binding model, Δm = (Bmax × C)/(Kd + C), where Δm is the amount of adsorbed analyte, c is the concentration of the analyte solution, Bmax is the maximum adsorption of analyte onto the surface, and Kd is the apparent dissociation constant. For HA-binding dendrons, the best fit model is the singlesite specific binding hill slope model, Δm = (Bmax × Ch)/(Khd + Ch), where Δm is the amount of adsorbed analyte, c is the concentration of the analyte solution, Bmax is the maximum adsorption of analyte onto the surface, Kd is the apparent dissociation constant, and h is the hill slope. In this model, the hill slope h is a factor used in pharmacologic analysis, when the receptor or ligand is multimeric protein, which leads to positive cooperativity (h > 1) or negative cooperativity (h < 1) in receptor−ligand binding.35 Because the (HA)n dendrons are divalent or tetravalent binding ligands to HA surface, this is a suitable model to analyze the adsorption isotherm. Fluorescein Imaging Experiment. Freshly cleaned HA-coated QCM sensors were immersed in 1 mL of 25 mM HEPES buffer for control, 0.1 mM (Biotin-HA, n = 6) in 25 mM HEPES buffer and 0.01 mM (Biotin-(HA)2, n = 6) in 25 mM HEPES buffer solutions 3307

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Figure 1. Molecular structures and MALDI-ToF-MS characterization of HA-binding peptide functionalized dendrons, biotinylated-(HA)2 and BMP2-(HA)2.

Scheme 2. Synthesis of Biotinylated-(HA)2 Dimer and BMP-2-(HA)2 Dimera,b

a Reagents and conditions: (a) Raney-Ni, H2, EtOH, 55 °C, 18 h, 99%; (b) Fluorenylmethyl succinimidyl carbonate, DIPEA, DCM, r.t., 24 h, 99%; (c) HCOOH, r.t., 12 h, 94%; (d) 2, 3, 5, 6-tetrafluorophenol (TFP), DIC, DPTS, CH3CN:DCM (v:v = 1:1), r.t., 14 h, 92%; (e) 17, DIPEA, CHCl3, r.t., 7 h, 96%; (f) diethylamine, DCM, r.t., 3 h, 91%; (g) succinic anhydride, NEt3, THF, r.t., 5 h, 75%; (h) 21: Wang resinLYLTSIASLETPVSSAKPIK-NH2, HOBt, DIC, DMF, r.t., 3 h; 22: Wang resin-Lys(Biotin)-NH2, HOBt, DIC, DMF, r.t., 3 h; (i) 28.5:0.75:0.75 TFA:TIPS:H2O (v:v:v), r.t., 45 min; (j) CuSO4, Na Ascorbate, 95: 5 10 × PBS DMSO (v:v), r.t., 48 h. bThe synthesis of compounds 1 and 18 was following the previous literatures.40

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Figure 2. QCM-D measurement of HA-binding peptide onto HA-coating sensor. (A) QCM-D signals from different overtones. As indicated, the above liquid was 25 mM HEPES buffer, then HA-binding peptide solution (c = 0.42 mM), and finally 25 mM HEPES buffer again. The decrease of frequency shift is due to the adsorption of HA-binding peptide onto the HA-coated surface. The close signals from different overtones indicating the formed adsorbed layer is rigid. (B) HA-binding peptide adsorption isotherm onto HA-coated surface. The data was fit with single-site specific binding model, which gives Kd equal to 0.25 ± 0.07 mM.

Figure 3. Frequency shift of adsorbed HA-binding dendrons (A) (HA)2-2, (B) (HA)2-6, (C) (HA)4-2, and (D) (HA)4-6 on HA-coated substrate for various concentrations carried out by QCM-D, n = 7. There are three steps for each measurement: baseline (HEPES buffer, flow rate =150 μL/min), adsorption (dendrons in HEPES buffer, flow rate =150 μL/min, flow for 3 min and then stop), and washing (HEPES buffer, flow rate =150 μL/ min). At each concentration, three independent measurements were preformed, and their results were highly consistent. One adsorption plot was shown in the figure for clarification. respectively at r.t. for 1 h. Then the sensors were washed with 1 mL 25 mM HEPES buffer three times. After the sensors were dried under vacuum, they were incubated with 1 ug/mL Alexa Fluora 488Streptavidin (from Invitrogin) at r.t. under dark overnight. The sensors were washed with 1 mL 25 mM HEPES buffer three times again and dried under vacuum. After that, the sensors were observed on fluorescein microscopy (Olympus, Center Valley, PA) under FITC mode with 20-fold magnifications.

binding dendrons is outlined in Scheme 1. Ligand valency, which is determined by the number of HA-binding peptide chains in one molecule, was adjusted using a convergent approach.36 First-generation (G1) dendrons have a ligand valency of 2, while second-generation (G2) dendrons have a value of 4. The length of the flexible linkage as indicated in Figure 1 was readily tuned by selecting oligo(ethylene glycol) with different number of repeating units as the starting materials. The diethylene glycol and hexaethylene glycol provide flexible linkages with extended chain lengths of ca. 22 Å and 35 Å, respectively, as measured by the Avogadro (MMFF94S) software. The synthesis started from a Michael

■

RESULTS AND DISCUSSION Molecular design and synthesis of HA-binding dendrons. The synthetic scheme of the multivalent HA3309

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Figure 4. Adsorption isotherms of HA-binding dendrons (A) (HA)2-2 and (HA)2-6 and (B) (HA)4-2 and (HA)4-6 onto HA-coated substrates. The error bars represent the ± standard deviation from the mean of independent measurements from 3 samples. The solid line represents the fit to a single-site specific binding with hill slope model, Δm = (Bmax × ch)/(Khd + ch),35 where Δm is the amount of adsorbed analyte, c is the concentration of the analyte solution, Bmax is the maximum adsorption of analyte onto the surface, Kd is the apparent dissociation constant, and h is the hill slope.

Table 1. Dissociation Constant (Kd), Hill Slope (h), Saturation Adsorption (Bmax), Enhancement Parameter β and Normalized Enhancement Parameter β/n of the Ligands for HA-Binding Peptidea and Dendronsb Kd (mol/L) HA-binding peptide (HA)2-2 (HA)2-6 (HA)4-2 (HA)4-6

2.50 5.4 1.7 2.7 4.5

± ± ± ± ±

−4

0.07 × 10 0.5 × 10−5 0.6 × 10−5 0.5 × 10−7 1.0 × 10−7

0.65 0.91 1.26 1.33

h

Bmax (ng/cm2)

R2

β = Kmulti/Kmonoc

β/n

-± ± ± ±

79 ± 12 257 ± 85 229 ± 37 227 ± 21 218 ± 28

0.884 0.979 0.977 0.979 0.984

-4.6 15 93 56

-2.3 7.4 46 28

0.10 0.12 0.15 0.15

The data is fitted with one-site specific binding model. bThe data is fitted with one-site specific binding with hill slope model. cKmulti and Kmono are the association constants of multi- and monovalent binding ligands with the HA surface, respectively.

a

addition of tert-butyl acrylate (2 equiv) to nitroethane (1 equiv) with Triton-B as the catalyst. The nitro group was subsequently reduced to amine by Raney nickel catalyzed hydrogenation. Through amidation, G2 dendron was constructed with four tert-butyl protected carboxylic acid groups, which was further deprotected under acidic conditions and activated with TFP. Starting from oligo(ethylene glycols) with precise molecular mass, the asymmetric linkage molecules with one amine end group and one azide end group were synthesized according to methods published previously.37 Under basic conditions, the amine group reacted with the TFP activated ester, leading to a dendron with two or four azide groups. The last step was a copper-catalyzed Huisgen [3 + 2] cycloaddition reaction between the azide groups of the dendron and the alkyne group on the N-terminus of HA-binding peptide. The synthetic route to biotin and BMP-2 peptide functionalized HA-binding dendron is shown in Scheme 2: through on-resin amidation, biotin/BMP-2 peptide was linked to the focal point of the dendron. Then the dendron went through resin cleavage and “click” reaction with HA-binding peptide. Target molecules were purified by RP-HPLC. Their precisely defined structures were characterized by MALDI-ToF-MS, as shown in Figure 1. Apparent Dissociation Constant of HA-Binding Peptide. The binding affinity of the HA-binding peptide with HA surface was characterized with QCM-D as a reference. The apparent dissociation constant, Kd, of HA-binding peptide was 0.25 ± 0.07 mM, as shown in Figure 2B. This Kd value was determined to be ca. 18 times weaker than that measured by SPRi in our previous work.18 Two factors contribute to this discrepancy. First, the HA-coated substrates in the two studies are from different sources. The SPRi chips were fabricated, characterized by X-ray diffraction, and referenced against the standard referenced material (SRM) certified by NIST. The average thickness of HA layer in the SPRi experiments was 20−

30 nm, while that used in company fabricated HA-chips used in the QCM-D measurements was below 10 nm. Efforts to conclusively characterize the polymorphs present were futile, although X-ray photoelectron spectroscopy (XPS) analysis provided measurable indications of carbonate on the HA coated QCM chips. Studies demonstrated that low receptor surface coverage will lead to decreased apparent values of Kd.29 The second factor is on the intrinsic sensitivity difference between SPRi and QCM-D techniques. The typical value of mass sensitivity of SPR is ca. 1 ng/cm2, while that of QCM is 20 ng/ cm2. Frequency Shift of HA-Binding Dendrons in QCM-D Measurement. To see the enhancement of binding affinity due to multivalency, adsorption of HA-binding dendrons onto HA surface was also measured by QCM-D under the same conditions. Figure 3 shows the frequency shift when solutions of HA-binding dendrons (HA)n at different concentrations flow above the HA-coated QCM sensor. Noticeably, the tetravalent ligands, (HA)4-2 and (HA)4-6, less than or equal to a concentration threshold, 200 nM for (HA)4-2, 300 nM for (HA)4-6, (HA)4 dendrons still bind to the surface even when washing with HEPES buffer. The adsorbed (HA)4-6 remained bound to the HA surface (as shown in Figure S8) even when the flow rate of washing solution was increased to 350 μL/min. For HA-binding peptide and (HA)2, a part of adsorbate was washed away at all concentrations in comparison. This was due to the enhanced binding affinity by the tetravalent binding ligand. Besides the strong binding ability of (HA)4, another phenomenon is observed in their QCM-D measurement: a small increase in frequency shift after adsorption happened due to the rearrangement of molecules on the HA surface. It is due to the loss of water trapped in the adsorbed layer. Especially, (HA)4-2 exhibited the largest amount of water loss, revealing 3310

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Table 2. Adsorption Free Energy of HA-Binding Peptide/Dendrons ΔG0multi (kJ)

Kd(mol/L) HA-binding peptide (HA)2-2 (HA)2-6 (HA)4-2 (HA)4-6

2.50 5.4 1.7 2.7 4.5

± ± ± ± ±

−4

0.07 × 10 0.5 × 10−5 0.6 × 10−5 0.5 × 10−7 1.0 × 10−7

−20.5 ± 0.07 −27.2 ± 0.2 −27.2 ± 0.9 −37.5 ± 0.5 −36.2 ± 0.6

ΔG0mono (kJ) −20.5 −20.5 −20.5 −20.5 −20.5

± ± ± ± ±

0.07 0.07 0.07 0.07 0.07

ΔG0interaction (kJ) 16.7 14 44.7 46.0

-± ± ± ±

0.4 1 0.7 0.8

Figure 5. Fluorescence microscopy images of biotinylated HA binding peptide and dendron bound to HA substrates. Cleaned HA-coated QCM sensors were immersed in 1 mL solutions of HEPES buffer (25 mM), biotinylated-HA (0.1 mM in 25 mM HEPES) and biotin-(HA)2 (0.01 mM in 25 mM HEPES) respectively at ambient temperature for 1 h. After thoroughly washing, the sensors was incubated with Alexa Fluora 488Streptavidin (1 ug/mL) at ambient temperature overnight, washed and visualized with fluorescence microscopy. Divalent HA-binding ligands show extensive coverage on the surface of the HA-coated QCM sensor. The scale bar is 50 μm.

summarized in Table 1. The enhancement parameter β,24 which is the ratio of the association constant of multivalent ligand Kmulti to that of monovalent ligand Kmono with the receptor, was also calculated to quantify the multivalency effect. In general, increasing valency leads to a higher binding affinity. The apparent dissociation constant decreases from ca. 250 μmol/L for HA-binding peptide to 50 μmol/L for (HA)2-2, to 250 nmol/L for (HA)4-2. Also, tetravalent (HA)4 dendrons have a higher hill slope value than dimeric (HA)2 dendrons, suggesting a larger positive collective effect in the ligand− receptor binding process. The ligand length effect is counterintuitive in the binding of (HA)2 and (HA)4 dendrons with HA surface. For divalent dendrons, the longer (HA)2-6 shows a nearly 3-fold stronger binding affinity than (HA)2-2, while for tetravalent ligand, the short tether (HA)4-2 shows approximately 2 times higher binding affinity than (HA)4-6. It is difficult to definitively explain the underlying reason for the reversion effect of linkage length in (HA)2 and (HA)4 dendrons because we are unable to visualize how the HA-binding peptide are adsorbed onto HA surface when tethered to the dendron scaffold, and the distribution of binding site on HA substrate is

this molecule formed the most closely packed layer on the HA surface. Adsorption of HA-Binding Dendrons at Different Concentration. Figure S7 shows that the adsorption correlates directly with the concentration of HA-binding peptide and dendrons (HA)n. Starting from low concentration ranges, the adsorbate increased with the concentration of analyte and gradually reached a plateau. At higher concentrations, there was an inflection point in the plot between the adsorbed area mass and the concentration of the analyst. We postulate the formation of adsorbate monolayer on the HA surface dominates the adsorption process. At concentrations higher than the critical value (where the inflection point appears), multiple layer adsorption of the (HA)n contributes to the additional increase in the adsorption area mass. The binding affinity of the (HA)n dendrons with the HA surface was determined from data in the monolayer adsorption range as shown in Figure 4. Apparent Dissociation Constant of HA-Binding Dendrons. Binding affinities characterized with the apparent dissociation constant Kd of HA-binding peptide/dendrons are 3311

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tetravalent constructs of (HA)4-2, which bind specifically with HA surfaces. Tetrameric constructs of (HA)4-2 show a 100-fold enhancement in binding affinity compared with HA-binding peptide. The functional group in the focal point of the dendron is adaptable. Biotin functional groups facilitating bioimaging, and BMP-2-derivative peptides that are being tested for enhanced bioactivity and osteoconductivity, were successfully linked with the HA-targeting dendron. This HA-targeting dendron has a number of potential applications in developing osteoconductive materials.

unclear. However, it is notable that the length of linkage influences the ultimate binding affinity. Also, among all HAbinding dendrons, tetravalent ligand (HA)4-2 shows the highest binding affinity, with Kd equal to 2.7 ± 0.5 × 10−7 mol/L, almost 100-fold enhancement in binding affinity compared with HA-binding peptide. Also in this architecture, each HA-binding peptide is used in the most efficient manner as defined by the highest normalized enhancement parameter β/n. Free Energy Calculation. Assuming that, in the multivalent ligand, each ligand binds with the receptor site simultaneously, the standard binding free energy for multivalent ligand ΔG0multi is 0 ΔGmulti

=

0 nΔGmono

+

0 ΔGinteraction

■

S Supporting Information *

(1)

The characterization of an HA-coated QCM-D sensor, QCM-D signals from overtones, and the effect of adsorption at higher concentration and flow rate on adsorption are all contained in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

where ΔG0mono is the standard binding free energy of the corresponding monovalent ligand, n is the valency, and 0 ΔGinteraction is the energetic consequence of favorable or unfavorable effect of tethering ligands in one molecule.38 Assuming 0 0 ΔGmono = ΔG HA

ASSOCIATED CONTENT

■

(2)

AUTHOR INFORMATION

Notes

and 1 ΔG = −RT ln K a = −RT ln Kd

The authors declare no competing financial interest.

■

0

ACKNOWLEDGMENTS This work was supported through a grant from the National Science Foundation (DMR-1105329) and the Austen Bioinnovation Institute in Akron. We also thank Professor Tom Leeper for assistance with HPLC purification. The authors also acknowledge support from the National Science Foundation (CHE-1012636 to C.W.).

(3)

combining eqs 1−3, ΔG0interaction of each dendron was calculated and listed in Table 2. The positive value of ΔG0interaction is considered mainly due to entropy loss after linking ligands together with dendron scaffold, including translational, rotational, conformational, and solvation-associated entropies.24 As shown in the Table 2, ΔG0interaction increased nearly 20 kJ as the valency changes from 2 to 4. While the free energy released by the adsorption of one ligand is about −20 kJ. A rigid and condensed architecture may be appropriate to minimize the 0 0 ΔGinteraction , decreasing ΔGmulti and resulting in a larger multivalency effect. Versatile Functionality of the HA-Binding Dendron. The main advantage of our strategy is the use of a versatile tethering strategy of functional groups in the focal point of dendron. (HA)2 with either a biotin molecule or a BMP-2derived peptide39 in the focal point was synthesized. Biotinylated-(HA)2 was used to visualize the HA surface using a fluorescently labeled streptavidin conjugate. In Figure 5, under the same condition, with 10-times lower concentration of biotinylated-(HA)2, the detailed morphology of HA surface was shown more clearly compared to biotinylated-HA peptide. We are confident this strategy can be utilized for any streptavidinlabeled protein. In the case of the BMP-2-(HA)2 conjugate, one face of the dendron is able to bind tightly with the HA surface, while the other face containing the BMP-2 peptide may guide cell differentiation to osteoblast to help new bone formation. While further in vitro work remains ongoing, the tethered approach may eliminate or reduce the use of freely diffusing recombinant BMP-2 in spinal fusions, thereby minimizing the potential risks of using BMP-2 peptide in clinic that have recently come to light.

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CONCLUSIONS A series of HA-binding peptide functionalized dendrons were synthesized, and their binding affinities with HA surface were characterized using QCM-D. By tuning the structure of dendron scaffold, an optimized structure is achieved for 3312

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