Correlation between Molecular Weight and Branch Structure of

Article pubs.acs.org/Macromolecules

Correlation between Molecular Weight and Branch Structure of Glycopolymers Stars and Their Binding to Lectins Yong Chen,† Megan S. Lord,‡ Alberto Piloni,† and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design, School of Chemistry, University of New South Wales, Sydney NSW 2052, Australia Graduate School of Biomedical Engineering, University of New South Wales, Sydney NSW 2052, Australia

‡

ABSTRACT: It has been hypothesized that maximum binding of carbohydrates can be achieved when the distance between two carbohydrate units are equivalent to two binding sites of a lectin. Therefore, a library of glyco starpolymers was prepared using RAFT polymerization to study the interaction with Concanavalin A (Con A). The starpolymers had a block structure in each arm and were composed of poly(2acryloylethyl-2′,3′,4′,6′-tetra-O-acetyl-α-D-mannopyranoside) (PMEA) as the outer block and the inactive, but water-soluble, poly(2-hydroxyethyl acrylate) (PHEA) as the inner block. The sizes of the starpolymers were determined using dynamic light scattering and hydrodynamic diameters between 3 and 12 nm in buffer solution were measured, which match the distance between two binding pockets of 6.5 nm (Chem. Rev. 2002, 102, 555). Turbidity assay, precipitation assay, surface plasmon resonance and quartz crystal microbalance were employed to investigate the binding of the polymers with Con A. The effect of the molecular weight of PHEA, which is located in the core of the star, was found to contribute to chain stretching allowing the interaction with more Con A molecules. Longer PMEA blocks showed a lower binding efficiency per mannose functionality than shorter blocks. It was found that the initial diameter of the polymer in solution does not play a role in determining the binding efficiency. The measured amount of Con A conjugated per star polymer suggests that during the process the chains stretch significantly to accommodate a maximum amount of Con A.

■

INTRODUCTION Carbohydrates are extensively expressed on surfaces of living cells and micro-organs and are actively involved in many biological processes such as cell−cell recognition and signaling events.1 Crucial is the fact that the interaction between carbohydrates and sugar-binding proteins (lectins) is rather weak and the high strength is only achieved by the simultaneous interaction between lectins and several carbohydrates (cluster glycoside effect).2 To help to understand the interaction between carbohydrates and proteins, glycopolymers were employed.3−5 Glycopolymers are synthetic polymers bearing carbohydrate ligands and their synthesis is now wellestablished allowing the design of various glycopolymer structures where the molecular weight can be controlled. They are promising candidates for studying the binding affinity between multivalent carbohydrate molecules and proteins due to the fact that their molecular structures are easily tailored to promote or suppress binding to proteins. The binding efficiency between glycopolymers and lectins, which are a group of proteins that bind to only one or two specific types of sugar molecules, is significantly affected by the structure of the glycopolymer, specifically the density of sugar moieties and flexibility and length of polymer chains.4 Glycopolymers are of interest as model compounds to understand lectin binding, but they also have practical application as bioactive polymers, for example for drug delivery.6,7 © 2015 American Chemical Society

The multivalency effect using glycopolymers has been studied widely in recent years. Several parameters play a role in determining the strength of the complex such as enthalpy and entropy factors as well as steric stabilization of the complex. Whitesides and co-workers proposed a similar spacing between two ligands and two binding sites is one of the determining parameters to achieve good binding.8 It is therefore not surprising that glycopolymers with flexible carbohydrate pendant groups, which can freely adjust to the position of the binding pockets, can lead to better binding.9−12 This would suggest that stiff polymers would have lower binding unless the distance between two carbohydrates matches that of two binding pockets.13 However, glycopolymers with stiff helical structures were observed to have very efficient lectin binding.14 One aspect that seems to be agreed upon in literature is the epitope density. It is generally accepted that a high sugar density is not required to achieve good binding. Considering the large size of lectins, it is not surprising that a carbohydrate functionality at each repeating unit is not required to achieve the maximum amount of lectin binding.15,16 Statistical copolymers present therefore an optimum balance between Received: August 24, 2014 Revised: December 18, 2014 Published: January 7, 2015 346

DOI: 10.1021/ma501742v Macromolecules 2015, 48, 346−357

Article

Macromolecules Scheme 1. Synthesis Approach to Four-Arm Star Polymers

binding efficiency between Con A and star glycopolymers with various core structures. Some of the stars were homogeneous glycopolymers while others were cores that were prepared using 2-hydroxyethyl acrylate units. An R-group RAFT approach was chosen to synthesize a library of glycopolymers of different molecular weight with the core either carrying PHEA as nonactive polymer or not (Scheme 1).

multivalency of multiple carbohydrate copies and possible accessibility of the lectin11,12,16−18 Another effect that seems to promote binding is the molecular weight. Kiessling and co-workers found an increasing capability of the polymer to inhibit erythrocyte agglutination when the molecular weight increases.19 This initially linear relation though levels off at a certain value with no more changes being observed at higher molecular weights.20 Most studies focus on the lectin binding with linear glycopolymers, surface bound carbohydrates21 or dendrimers.22 Structures such as glycopolymers star polymers have rarely been investigated although they present an intermediate between linear polymers and dendrimers. In a previous study carried out in our group, different sizes of four-armed star glycopolymers carrying glucose moieties were mixed with Con A, a lectin with four binding sites selective to mannose and glucose.23 The results show that the binding efficiency is dependent on the molecular weight of the stars, and the maximum activity is achieved at a medium molecular weight. It is suspected that the low binding efficiency of polymers with higher molecular weights is caused not by the lack of bioactivity but the fact that their size is too large. Glycopolymers close to the center of the core may not have been accessible and glycopolymers at the end of the branches exceed the distance between two binding sites (Scheme 1). This inspired us to explore further the relationship between the structure of a star glycopolymer and the extent of Con A binding to determine if binding to Con A would be the same for a star glycopolymer carrying sugar moieties only on its arms where the distance to the core matches the binding sites of a Con A molecule to the Con A core, compared to a similar star constructed entirely of sugar (Scheme 1). It is therefore proposed to compare the

■

EXPERIMENTAL SECTION

Materials. 1-Butanethiol, carbon disulfide, 1,2,4,5-tetrakis(bromomethy)benzene, AIBN, sodium methoxide (25% w/w solution in methanol), boron trifluoride etherate, α-D-mannose, 2-hydroxyethyl acrylate HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris buffer (tris(hydroxymethyl)amino methane), Concanavalin A (Con A), deuterated chloroform, and dimethyl sulfoxide (DMSOd6) were all purchased from Sigma-Aldrich and used as received. Synthesis. Synthesis of RAFT Agent 1,2,4,5-Tetrakis(butyltrithiomethyl)benzene. A 7 g sample of 1-butanethiol (0.0777 mol) was suspended in 100 mL of water in a 250 mL round-bottom flask and added slowly with 4.36 g (0.0777 mol) KOH. After the suspension merged to one phase, the flask was moved onto an ice bath. Then 7 mL (0.116 mol) of carbon disulfide was added slowly while stirring, then the ice bath was removed and the solution was stirred overnight. The resulting mixture was purified by freeze-drying. After this, 1 g (5 mmol) of the crude product was dissolved in equal-molar methanol/DCM mixture, and 0.45 g (1 mmol) of 1,2,4,5-tetrakis(bromomethy)benzene was dissolved in 15 mL of DCM and added to the solution drop by drop. The solution was stirred for 1 h before adding 20 mL of water and 20 mL of ethyl acetate. The mixture was separated and the aqueous phase washed three times with ethyl acetate. The organic phases were combined, washed with brine, dried with MgSO4, and filtered. The solvent was removed under reduced pressure. The crude product (yellow oil) was purified by flash 347


Article

Macromolecules

(Mn = 3100 g mol−1), 0.2 mg of AIBN (0.0013 mmol), and 219 mg of MEA (0.5064 mmol) were dissolved in 0.25 mL of dioxane. The mixture was transferred to a quartz cuvette, sealed and purged with nitrogen for 50 min. The cuvette was then placed in a FT-IR at 60 °C. The polymerization was monitored until the monomer conversion reached 50%. Deprotection of acetic protective groups was performed as per the deprotection of homogeneous PMEA polymers. Analysis. Turbidimetry Binding Assay via UV−Vis Spectroscopy. Con A was dissolved in 0.01 M HEPES buffer at pH 7.4 to form a 1 g mL−1 solution. Then 0.5 mL of this solution was transferred to a 0.6 mL masked quartz crystal cuvette and placed on the sample holder of a Cary 300 UV−vis spectrophotometer. A baseline was taken after 1 min. The cuvette was taken out and 0.05 mL of 1 mg mL−1 solution of glycopolymer in HEPES buffer (0.01 M, pH = 7.4) was added and mixed thoroughly. The cuvette was immediately placed back into the spectrophotometer and the absorbance at 420 nm was recorded over a period of 20 min. Surface Plasmon Resonance (SPR). The kinetics and extent of interaction between the glycopolymers and ConA were analyzed with a BiaCore 2000 (GE Healthcare) at 25 °C by monitoring the response in Response Units (RU). The buffer used was filtered PBS (pH 7.4). Gold sensor surfaces (GE Healthcare) were coated with star-polymers by injection of 150 μL of 10 μg mL−1 glycopolymer at 5 μL/min (diluted in DPBS). The surfaces were then blocked by injection of 100 μL of 1% w/v BSA in PBS at 5 μL min−1. Con A was injected (50 μL) at three different concentrations (1, 10, and 100 μg mL−1 in DPBS) at 20 μL min−1. Sensograms were analyzed using BIAcore 2000 evaluation software 3.0. Sensograms were fitted with separate differential rate equations for the parts of the binding curve representing association and dissociation to obtain ka, kd and kD values. Additionally, 200 μL of Con A (600 μg mL−1) was injected over each glycopolymer to determine the maximum amount of Con A binding. Precipitation Assay. The assay was performed at 25 °C in Tris− HCl buffer (0.1 M, pH= 7.2, containing 0.15 M NaCl, 1 mM CaCl2 and 1 mM MnCl2) according to a procedure described earlier.25,26 For each glycopolymer, 2 mg of polymer was dissolved in 2 mL buffer solution to yield a stock solution. The stock solution was further diluted to 0.5, 0.25, 0.1, and 0.05 mg mL−1 test solutions. Then 1 mL of each solution was moved into an Eppendorf centrifuge tube and mixed with 500 μL of Con A (9.6 μM in buffer). The mixtures were allowed to sit for 24 h before being centrifuged at 7000 rpm for 10 min. A pipet was used to remove the upper clear solution and the remaining pellet was washed by cold buffer three times. After the last wash, 500 μL of 1-methylmannose solution (0.1 M, in Tris−HCl buffer) was added to dissolve the precipitate. The solutions were diluted using 500 μL Tris−HCl buffer and transferred to a quartz cuvette. A Cary 300 UV−vis spectrophotometer was used to measure the UV absorbance of the solutions at a wavelength of 280 nm. Nuclear Magnetic Resonance (NMR) Spectroscopy. All NMR spectra were acquired using a Bruker Avance III 300 MHz spectrometer. Deuterated chloroform or DMSO was used as a solvent at a temperature of 298 K. Size Exclusion Chromatography (SEC). Molecular weight distributions were determined by SEC with a Shimadzu modular system having N,N-dimethylacetamide (DMAc) (0.03% w/v LiBr, 0.05% BHT stabilizer) at 50 °C with a flow rate of 0.85 mL min−1. The system incorporated a DGU-12A solvent degasser, a LC-10AT pump and a CTO-10A column oven and was equipped with a RID-10A refractive index detector. Polymer Laboratories 5.0 μm bead-size guard column (50 × 7.8 mm) followed by four 300 × 7.8 mm linear PL columns (105, 104, 103, and 500 Å) were used to separate the samples. The system was calibrated using narrow polystyrene standards ranging from 3000 to 106 g mol−1. Dynamic Light Scattering Analysis. The average hydrodynamic diameter (number distribution) in an aqueous solution was obtained using a Malvern Nano-ZS as particle size analyzer (laser, 4 mW, λ = 632 nm; measurement angles of 12.8° and 175°). Samples were filtered to remove dust using a microfilter 0.45 μm prior to the measurements and run at least three times at 25 °C. Polymers were

chromatography using 1:3 ethyl acetate/petroleum spirits as eluent to yield a yellow crystalline. 1 H NMR (300 MHz; CDCl3): 1.88, 2.04, 2.1, 2.17 (15 H, s, Me); 3.8−3.9 (2H, m, H6); 4.02−4.06 (1 H, m, H5); 4.12−4.25 (1H, m, H4); 4.88 (1 H, d, H1); 5.28−5.35 (1 H, m, H2, H3). 13C NMR (300 MHz; CDCl3): 20.45 (5 C, CH3); 62.1, 66.6, 69.3, 77.0 (5C, C2,3,4,5,6); 97.9 (1 C, C1), 161.7 (4C, CO). Synthesis of 2-Acryloylethyl-2′,3′,4′,6′-tetra-O-acetyl-α-D-mannopyranoside (MEA). 1,2,3,4,6-Penta-O-acetyl-α-D-mannose was obtained using procedures described elsewhere.24 Then, 1 g (5.56 mmol) of 1,2,3,4,6-penta-O-acetyl-α-D-mannose and 1.29 g (11.11 mmol) 2-hydroxyethyl acrylate were dissolved in 10 mL of dry dichloromethane. The mixture was placed in an ice bath and 3.9 g (27.78 mmol) of boron trifluoride etherate was added dropwise over 15 min. The mixture was kept in an ice bath for an hour and then continued at room temperature until thin-layer chromatography (TLC) examinations indicated complete consumption of the acetylated mannose. The mixture was poured into 15 mL ice water and the two phases were separated. The aqueous phase was extracted three times with dichloromethane and the organic phases were combined and washed (water, saturated NaHCO3 water solution, water). The resulting solution was concentrated under vacuum and then isolated by silica gel chromatography (20%ethyl acetate, 80% petroleum spirit). 1 H NMR (300 MHz; CDCl3): 1.88 (2H, t, COCH2CH2OOCH); 2.02, 2.04, 2.1, 2.17, (12 H, s, Me); 3.8−3.9(2H, m, H6); 4.02−4.06 (1 H, m, H 5); 4. 12− 4. 25 (1H , m, H4 ); 4 .3 6 (2 H, t , COCH2CH2OOCH); 4.88 (1 H, d, H1); 5.28−5.35 (2 H, m, H2, H3); 5.86−5.9(1H, d, CH2CHOO); 6.42−6.48 (1H, d, CH2CHOO); 6.1−6.2, (1H, t, CH2CHOO). 13CNMR (300 MHz; CDCl3): 20.45 (5 C, CH3); 62.1, 66.6, 69.3, 77.0 (5C, C2,3,4,5,6); 97.9 (1 C, C1), 161.7 (4C, CO) 166; 20.4 (1C, COCH2CH2OOCH); 70.3 (1C, COCH 2 CH 2 OOCH); 131.1 (2C, CH 2 CHOO); 128.1, (1C, CH2CHOO). Synthesis of Star-Shaped PMEA Homopolymers. In a typical synthesis, acetylated MEA (250 mg, 0.579 mmol), 1,2,4,5-tetrakis(butyltrithiomethyl)-benzene 4-arm RAFT agent (1.5 mg, 0.002 mmol), 0.13 mL of AIBN (0.0008 mmol, 1 mg·mL−1 stock solution in dioxane), and 0.2 mL of dioxane were mixed in a 1 mL quartzcrystal cuvette and sealed. The cuvette was deoxygenated by nitrogen for 50 min before being put on a Bruker IFS66/S high end FT-NIR/IR spectrometer. The reaction was monitored at 60 °C until the conversion reached 50%. All homogeneous PMEA polymers were polymerized to 50% conversion, with their chain lengths controlled only by altering the ratio between monomer and RAFT agent. Deprotection of Acetyl Protective Groups. The polymerization was stopped by immersing the cuvette in an ice bath. The mixture of polymer and monomer was diluted with 10 mL of methanol, deoxygenated with nitrogen, and put into an ice bath. 17 mL of 2.5% (w/w) sodium methoxide solution in methanol was slowly added to the mixture. The solutions turn cloudy and small amounts of water were added to ensure good solubility of the polymer. The mixture was allowed to react for a few hours only to avoid esterhydrolysis to the backbone before being transferred into a 1000 Da dialysis membrane tube and dialyzed against water for 24 h. Synthesis of Star-Shaped PHEA-b-PMEA Block Copolymers. In a typical synthesis, HEA (9.0 g, 77.59 mmol), 1,2,4,5-tetrakis(butyltrithiomethyl)-benzene 4-arm RAFT agent (76.6 mg, 0.097 mmol) and AIBN (6.4 mg, 0.039 mmol) were added into a polymerization vial containing 30 mL dioxane and then equally divided into two parts and transferred into two polymerization flasks. The vials were sealed and deoxygenated by nitrogen for 50 min before they were placed into a 60 °C oil bath. Flasks were collected and immersed into ice water after 2 h (conversion= 17.3%, Mn,theo= 17000 g mol−1, Mn,SEC = 31000 g mol−1) and 4 h (conversion= 25.5%, Mn,theo= 24000 g mol−1, Mn,SEC= 47000 g mol−1). The resulting mixtures were then dialyzed against water using 1000 Da molecular weight membranes. The purified PHEA polymers were used as macroRAFT agents to synthesize the PHEA-b-PMEA block copolymers. In a typical synthesis, 105 mg (0.0033 mmol) of PHEA 348


Article

Macromolecules dissolved in PBS buffer (0.01 M, pH = 7.4) to form 1 mg mL−1 solutions. Quartz Crystal Microbalance with Dissipation Monitoring (QCMD). QCM-D (D300, Q Sense AB, Sweden) was used to measure the extent of interaction between Con A and the star glycopolymers. Measurements were performed at 37.0 ± 0.1 °C. Gold QCM-D crystals were mounted in the chamber then frequency (f) and dissipation (D) measurements versus time were recorded for the fundamental f (5 MHz) as well as the third, fifth and seventh overtones. PBS (0.01 M, pH 7.4) was injected into the chamber and allowed to stabilize before being removed and replaced with 0.1 mg mL−1 Con A in PBS and allowed to adsorb to the gold sensor surface for 30 min before the chamber was rinsed using PBS buffer allowing for f and D stabilization. Bovine serum albumin (BSA, 100 μg mL−1) in PBS was exposed to the sensor surface for 30 min to bind to remaining areas of the gold sensor surface. The sensor was rinsed with PBS until stable f and D measurements were achieved and then 0.01 mg mL−1 glycopolymer solution in PBS was added for 30 min, before the chamber was rinsed with PBS and Con A (0.1 mg mL−1 in PBS buffer) added for at least 16 h to reach maximum reaction. The QCM frequency and dissipation measurements were modeled using the Voigt model to obtain mass of ConA bound to each of the polymers. f and D measurements for the fundamental and all overtones were modeled using the Voigt viscoelastic model to estimate layer mass and thickness.

Figure 1. Molecular weight distribution of star-shaped PMEA homopolymers and PHEA-b-PMEA core−shell star polymers, with the number of HEA and MEA repeating units per star polymer.

distribution remained narrow for most samples (Figure 1). A small high molecular weight shoulder suggested some star−star coupling. This observation was more obvious for the low molecular weight polymers in each set of polymers, which used lower monomer to macroRAFT agent ratios. One sample had an unusually high dispersity of Đ= 1.8. It seems that the abovementioned side reactions became more pronounced. It is not clear what is unique about this sample since the conversion is not higher than the other polymerizations. There may be a possibility that the pure glycopolymer structure (this polymer does not have a PHEA core) exerts a certain stiffness that may cause more coupling reactions. The deviation of the measured molecular weight from the calculated value was likely to be caused by the polystyrene calibration of the SEC and has been observed in numerous earlier publications.23,34 The polymers were deprotected using NaOH in methanol, which readily dissolved the polymer. Deprotection led to the precipitation of the final product. To ensure that the reaction was driven to completion, water was added to enhance the solubility of the deprotected starpolymer to enable quantitative cleavage of the acetyl ester (Figure 2). In the absence of water the deprotection is incomplete,35 while water can accelerate the ester hydrolysis that might lead to the loss of mannose. It is therefore crucial to monitor this step carefully by 1H NMR and to quickly transfer the solution to a dialysis tube against water to remove excess sodium methoxide. The molecular weight of the final product decreased as expected while the dispersity Đ remained below 1.3 (Table 1). A set of nine glycopolymer star were then used for further investigations (Table 1). Dynamic light scattering (DLS) is used to determine the hydrodynamic diameter of the star-shaped glycopolymer molecules (Table 1).36 In general, the hydrodynamic diameter of glycopolymers increases as the total number of repeating unit increases, regardless of the composition of polymer (Figure 3A). However, closer inspection of the results and replotting solely against the number of repeating units of the glycopolymer NMEA revealed a strong dependency on the glycopolymer while PHEA does not seem to contribute to the size. This might be caused by the difference in flexibility. PMEA homopolymers are less flexible due to steric hindrance caused by bulky sugar units, generating uncoiled stars, while PHEA may be more flexible or have a less favorable interaction with the buffer solution, thus resulting in a more collapsed chain. PHEA stars alone did indeed show very low hydrodynamic diameters in solution. However, these values could not be added because they were either irreproducible or no signal

■

RESULTS AND DISCUSSION A four arm star polymer with a block structure in each arm was prepared using RAFT polymerization.27 Since the Z-group approach, where the RAFT agent is attached via the Z-group, faces problems such as shielding effects,28,29 the R-group approach was chosen for this task and a RAFT agent was prepared using established procedures.30 According to theory, RAFT reactions via the R-group approach can suffer from star− star coupling and the formation of linear RAFT-terminated polymer chains.31 Since both reactions are a function of the radical concentration, the ratio of RAFT agent to initiator was kept low. Other measures for suppressing the side reactions include using fast propagating monomers, a small number of arms, a high addition rate of the macroradical to the RAFT agent and a high transfer rate to the R-group.31−33 The synthesis of four-arm stars is less likely to be affected by sidereactions. However, the initiator amount was kept at a low ratio to suppress these side reactions. Since detailed kinetic investigations were not the focus of this work, FT-NIR was employed to monitor the reaction of HEA and MEA, and the reaction was stopped when approximately 50−70% monomer conversion was reached. The absorption band of the vinyl functionality at 6180 cm−1 was used as an indicator of monomer consumption, but the precise monomer conversion was measured using 1H NMR. Prior to the glycopolymer synthesis two PHEA star polymers were prepared, which later formed the water-soluble and inactive core of the star-polymer. The well-defined polymers with 138 (34.5 per arm) and 204 (51 per arm) HEA repeating units, coined 138-0 and 204-0, respectively (Figure 1), were then employed as star-shaped macroRAFT agent in the subsequent reaction with MEA. The subsequent polymerization of MEA was carried out in DMAc as solvent with various monomer concentrations to generate star polymers with varying degrees of branch length (Table 1). 1H NMR was employed to determine the exact monomer conversion after the polymerization was stopped at approximately 60% conversion according to FT-NIR (Figure 2). The molecular weight shifts with increasing monomer to RAFT agent ratio were as expected; while the molecular weight 349


Article

Macromolecules

Table 1. Polymerization Conditions for the Polymerization of MEA and SEC Analysis before and after Deprotection and the Hydrodynamic Diameter Dh of the Deprotected Star Polymers in PBS Buffer (0.01 M, pH = 7.4)a before deprotection

after deprotection

sample

[M]/g mol−1

[M]:[RAFT]b

t/h

convnc

NHEA

NMEA

Mn,theod

Mn,SECe

Đ

M′n,theo

M′n,SEC

Đ

Dh/nm

1 (0-78) 2 (0-150) 3 (0-394) PHEA1f (138-0) 4 (138-110) 5 (138-187) 6 (138-360) PHEA2f (204-0) 7 (204-120) 8 (204-243) 9 (204-433)

1 1 1

150 300 600

3 6 6

3.1 5.6 11

1.11 1.08 1.11

47000 68000 116000

33000 46000 42000

1.18 1.14 1.17

3.6 5.6 8.7

150 300 600

1 2 3

0.80 0.81 0.72

204 204 204

27000 40000 56000 1.15 46000 50000 61000 1.15 55000 74000 75000

1.02 1.01 1.30

2 2 2

34000 65000 170000 31000 64000 97000 172000 47000 76000 129000 210000

20000 20000 43000

3 3 6

78 150 394 17000 110 187 360 24000 120 243 433

22000 42000 110000

150 300 600

0 0 0 0 138 138 138

1.21 1.26 1.80

1 1 1

0.52 0.50 0.66 138 0.74 0.62 0.60

1.21 1.17 1.26

57000 91000 144000

58000 79000 50000

1.22 1.23 1.33

6.5 7.5 8.7

a [RAFT]:[AIBN] = 5:2; The number of repeating units N represents the number for the star and not the branch. b[RAFT] concentration relates the concentration of the star-RAFT agent and not the amount of thiocarbonylthio groups. cConversions shown in Table 1 are calculated from NMR spectra of polymerization mixtures by comparing the amount of vinyl residuals and the acetic protective groups on sugar molecules. dTheoretical molecular weight was calculated from linear relationship between molecular weight and conversion. eThe experimental Mn and Đ was measured by SEC using dimethylacetamide (DMAc) as eluent and polystyrene standards. fPolymerization conditions for the PHEA macroRAFT agent are included in the experimental part.

Figure 2. 1H NMR (DMSO-d) of PHEA, PHEA−PMEA, and the glycopolymer after deprotection.

however carried out using linear polymers, often with a statistical distribution of mannose along the polymer chain. Three techniques were employed to study lectin interaction in dependency of the star polymer architecture: Turbidity assay, precipitation assay and QCM-D. Turbidity Assay. Turbidity assays, which monitor the precipitation of the lectin-polymer complex using UV−vis at a wavelength of 420 nm, are a vital tool to evaluate the rate of glycopolymer−Con A clustering. Each Con A molecule possesses four binding sites which are capable of binding to mannose. When mixed with multifunctional mannose-bearing

could be detected probably because of the low scattering intensity of these small stars. The nine mannose-containing polymers were tested for their ability to bind to lectin, in particular Con A, which is known to form strong interaction with mannose.37−39 It is known that parameters such as the chain length or the mannose density are highly influential on the rate of lectin binding or the amount of bound lectin.15,39 Generally, higher sugar density and flexibility of the polymer backbone leads to higher binding capability. To further improve the binding efficiency, the polymer chain should also be long enough to “tether” between two binding sites on the same Con A molecule.40 Most detailed studies were 350


Article

Macromolecules

result seems similar and a tentative increase of the binding rate k with the hydrodynamic diameter could be identified. However, the largest star polymer based on PMEA394, which also had the largest diameter shows comparatively low binding on this scale (Figure 4D). Earlier work showed that the binding efficiency can go through a maximum and medium-sized homo glycopolymer stars were more efficient than large stars.23 However, homopolymers cannot be directly compared to the block copolymer structures in this work, which seemed to have different solution properties. The incorporation of PHEA may provide more flexibility, which allows better adjustment between the glycopolymers and the active binding pockets of the lectin.9,10,13 While the turbidity assay can provide some indication between polymer structure and binding, the results of this assay are subject to many uncertainties. The fast reaction does not allow the confident determination of the k and t1/2 values and the displayed value should only act as a guide. It should be noted here that the solution remained clear when the glycopolymer was mixed with peanut agglutinin (PNA) or bovine serum albumin (BSA), which emphasizes that the observed binding is indeed specific. Surface Plasmon Resonance (SPR). More detailed information about the rate and extent of binding between the glycopolymers and Con A can be obtained using SPR.21 The development of the model of the interaction between mannose and ConA has been described in detail earlier.22 Figure 5 (left panel) shows a typical measurement curve for the immobilization of the glycopolymer on the gold sensor chip (part A) followed by blocking of the surface with BSA (part B) and then binding of Con A to the glycopolymer (parts C and D). The binding of different concentrations of con A (1−100 μg/mL)

Figure 3. Dependency of the hydrodynamic diameter Dh on the total number of repeating units NTotal or the number of glycopolymer repeating units NMEA. The straight lines are only for guidance and should not indicate that a linear relationship is expected.

glycopolymers, Con A molecules act as cross-linkers and form a network with the polymer. The binding between all polymers and Con A took place rapidly reaching half of the maximum turbidity t1/2 within the first 2 min (Figure 4A). A correlation between the molecular size and the t1/2 revealed a linear decrease of t1/2 with increasing molecular size (Figure 4B) suggesting that larger glycopolymer molecules are more efficient in binding to Con A. The rate of reaction, k, was derived from the initial slope of the reaction curves. Since the reaction took place before the cuvette could be placed in the UV−vis spectrometer, the turbidity had to be extrapolated to −30 s, which was the approximate time it took to mix the solution and to start the reaction. This causes a certain uncertainty and the determined k values are therefore subject to large errors. Higher rates of reaction were observed from polymers with a larger molecular size with PHEA204PMEA433 showing the highest binding efficiency. When plotted against the hydrodynamic diameter of the star polymers the

Figure 4. Turbidity assay to monitor binding between the nine polymers listed in Table 1 and Con A. (A) Raw data as obtained using UV−vis spectroscopy; (B) time t1/2 when half of the maximum absorption had been obtained against the hydrodynamic diameter; (C) correlation between number of repeating using and the initial rate of the reaction k; (D) initial rate of reaction k obtained from the slope at t = 0 against the hydrodynamic diameter. 351


Article

Macromolecules

Figure 5. Left: Experimental setup of the SPR assay demonstrated using PHEA138-b-PMEA110 showing the change in response units (RU) with the injection of (A) polymer, (B) BSA, (C) Con A 100 μg/mL, and (D) Con A 600 μg/mL. Right: normalized sensogram for the injection of ConA for three different concentrations of Con A.

Table 2. Binding Data between Glycopolymers and ConA Obtained Using SPRa NHEA

NMEA

NTotal

Dh/nm

ka/M‑1 s‑1

138 138 138 204 204 204

110 187 360 120 243 433

248 325 499 324 447 638

3.6 5.6 8.7 6.5 7.5 8.7

33.2 132 178 156 301 31.8

kd/s‑1 9.07 8.99 19.8 22.7 7.42 11.6

× × × × × ×

10−4 10−4 10−4 10−4 10−4 10−4

KD/nM 27.3 6.81 11.1 14.6 2.47 36.5

Con A/polymer 3.49 3.24 3.73 1.04 0.76 1.02

± ± ± ± ± ±

1.34 0.11 1.22 0.33 0.57 0.66

a Affinity constants were determined using Con A concentrations of 1, 10, and 100 μg/mL while the amount of Con A/polymer was determined after exposure of the glycopolymers to 600 μg/mL Con A.

Figure 6. Amount of Con A bound per polymer (A) and per mannose functionality (C) determined using precipitation assay in correlation with the number of repeating units N (B and D).

were used to determine the kinetics (Figure 5, right panel). As the amount and kinetics of Con A binding to the glycopolymers will be dependent on the surface grafting density, analyses were performed on 1100 ± 75 RU of bound glycopolymers. The

glyocpolymer was immobilized onto the surface and the rate and amount of bound Con A was correlated to the chemical structure. The association constant ka was found to increase in rate with increasing length of the glycopolymer block (Table 352


Article

Macromolecules

Quartz Crystal Microbalance with Dissipation Monitoring. QCM-D offers a quantitative method for measuring the amount of Con A binding to the polymers. The interaction between the glycopolymer and Con A was measured by QCMD and a typical result is depicted in Figure 7 using PMEA78.

2), which in in agreement with the rate constant k obtained using the turbidity assay. Also in agreement with the former assay is the conclusion that longer PHEA blocks seem to accelerate binding to ConA although this trend is less clear since PHEA204-b-PMEA433 gave repeatedly low binding constants, which may be caused by the quality of the sample. The apparent dissociation rate constant kd is expected to decline with increasing length of PMEA, but this trend is only vaguely recognizable. Both rate constants were then used to calculate the equilibrium dissociation constant KD. The values suggest that medium PMEA block lengths such as in PHEA138b-PMEA187 and PHEA204-b-PMEA243 have the highest affinity to ConA. This observation is similar to earlier results showing that large PMEA blocks are not necessarily required and medium-sized polymer can be more efficient.23 Although the obtained values are in the same order of magnitude to mannose-based dendrimers, the obtained values are all slightly higher compared to the measured values using [G4] (Kd = 0.4 nM) and [G2] (Kd = 9.9 nM) dendrimers indicative of better binding of stiffer architectures.22 The amount of bound Con A per polymer could be calculated from the experiments where the glyocpolymers were exposed to a high concentration of Con A showing significant better binding using shorter PHEA block lengths. These values will be discussed in more detail later. Precipitation Assay. The precipitation assay identified the stoichiometry between the glycopolymer and the lectin. For this purpose, Con A and the polymers were mixed and allowed to react for prolonged period (20 h) to enable the maximum interaction to be achieved. The precipitant was separated from the supernatant by centrifugation, followed by the addition of methyl mannose, which can occupy the Con A binding sites in a competitive manner to the glycopolymer allowing the precipitate to redissolve. The clear solution was analyzed for the amount of Con A using UV−vis spectroscopy and the calculated amount was correlated to the amount of glycopolymer in solution. At first glance it seems to be a simple correlation that the star polymers with larger hydrodynamic diameters lead to higher Con A binding. Figure 6A shows the correlation between the diameter of glycopolymer molecules and the amount of Con A the specific solution was able to recruit. There is strong evidence that the amount of Con A recruited is linearly related to the diameter of polymer molecules. The correlation between the amounts of Con A vs polymer (Figure 6B) gives again the impression that polymers with higher overall molecular weight may be more capable to bind Con A, which is in agreement with the correlation between hydrodynamic diameter and the amount of bound Con A. However, it is more crucial to look at the role of each mannose molecule. Therefore, the amount of Con A per mannose was calculated using the theoretical number of repeating units. Disregarding one outlier in Figure 6 C, there does not seem to be a strong correlation between the amount of bound Con A per mannose functionality and the diameter. The diameter therefore does not seem to play a crucial role while the absolute number of repeating units has a more influential part. Maximum binding was achieved with a medium PHEA molecular weight. There was also an indication that a medium PMEA block size was more active than large PMEA blocks. In agreement with earlier results, a medium star size was able to recruit the highest amount of Con A, which supports the idealized binding scheme depicted in Scheme 1.

Figure 7. Example using PMEA78 showing the changes in frequency ( f) and dissipation (D) as measured by the QCM-D for the third overtone for the binding of Con A to the gold sensor surface (5−35 min), followed by rinsing with PBS (35−50 min), blocking the remaining gold surface with BSA (50−80 min), rinsing with PBS (80− 90 min). The polymer was added (90−120 min) followed by rinsing with PBS (120−130 min) and then the addition of Con A (130−500 min).

After the addition of Con A at 5 min, the frequency of oscillation of the crystal decreased by approximately 85 Hz and an increase in dissipation to 6 × 10−6, indicating that Con A bound to the gold sensor surface. The mass of Con A bound to the gold sensor surface was determined by modeling the f and D values using the Voigt viscoelastic model and found to be approximately 1400 ng/cm2, indicating that the gold sensor surface supported a multilayer Con A film. The level of Con A binding to the gold sensor surface was found to be 1680 ± 235 ng/cm2 for all experiments. The mass of Con A attached to the crystal remained approximately the same after being rinsed with buffer at 30 min, which was followed by the addition of BSA which did not cause any change in the f and D values indicating that the Con A had saturated the gold sensor surface. The addition of the glycopolymer resulted in a decrease in f of approximately 5 Hz and an increase in D indicating that the glycopolymer had bound to the immobilized Con A. Modeling of the changes in f and D indicated that approximately 500 ng/ cm2 of polymer bound to the immobilized Con A. Subsequent rinsing by PBS buffer did not remove detectable amount of polymer, indicating effective binding between Con A and glycopolymer. The mass of glycopolymer and Con A bound for each condition is shown in Figure 8B. One Con A molecule was found to recruit on average 1.65 star polymer molecules in this case. Given the density of the immobilized Con A it is probable that a considerable proportion of binding sites were buried within the protein layer and not available to the star polymers. After 132 min, fresh Con A was added resulting in a dramatic decrease in frequency, which kept decreasing for more than 4 h indicating that the interaction between the star polymer and Con A was slow to reach steady state. Some resonance frequency changes were observed at approximately 400 min and may have been due to structural rearrangements in the multilayer film that resulted in the loss of water. It is likely that during that time constant rearrangements take place, which enables maximum binding of Con A, resulting in a tightly 353


Article

Macromolecules

Figure 8. (A) Amount of polymer bound per Con A molecule as determined by QCM-D in dependency of the number of repeating units of the glycopolymer block NMEA and the number of PHEA repeating units in the center of the glycopolymer. . (B) Mass of bound glycopolymer and Con A as determined by QCM-D.

Figure 9. Amount of Con A per glycopolymers measured using QCM-D correlated against the (A) hydrodynamic diameter and (B) against the number of repeating units NHEA and NMEA; (C and D) numbers of Con A per mannose molecules against hydrodynamic diameter (C) and number of repeating units (D).

packed, probably dehydrated, layer. It is calculated that each glycopolymer molecule recruited 0.5 Con A molecule. Considering the hydrodynamic diameter of the star polymer of only 3 nm in buffer solution (Table 1) and the comparatively large size of Con A this result can only be explained with significant stretching of the coiled polymer chains. Stretching of polymer chains is usually entropically unfavorable and it can be seen as a measure of the strength of the binding between mannose and Con A. Figure 8 illustrates the number of polymers recruited by the immobilized layer of Con A. The layer of Con A that was immobilized to the gold sensor surface was able to bind more

polymers when the polymers had a lower molecular weight. While this may at first seem counterintuitive, the result is in agreement with the behavior of linear glycopolymer of varying length.41 Larger star polymers can span over a larger surface area and therefore several Con A binding sites, preventing other polymer molecules in the solution from accessing these binding sites. The effect of the length of PHEA is inconclusive, but there is strong evidence that the PHEA blocks in the center of the star lead to a lower number of polymers absorbed. The flexibility of the PHEA block might allow the starpolymer to be easily stretched over the lectin layer preventing access to the free Con A binding sites. 354


Article

Macromolecules Table 3. Summary of Results Obtained from Precipitation Assay, QCM and SPR NHEA

NMEA

NTotal

Dh/nm

0 0 0 138 138 138 204 204 204

78 150 394 110 187 360 120 243 433

78 150 394 248 325 499 324 447 638

3.1 5.6 11 3.6 5.6 8.7 6.5 7.5 8.7

Con A/polymer molecule Con A/mannose molecule precipitation precipitation 0.49 2.19 3.47 1.77 2.69 10.83 0.64 2.98 3.55

0.006 0.014 0.009 0.016 0.014 0.030 0.005 0.012 0.008

These immobilized starpolymers were then allowed to interact with Con A over the next several hours. The amount of bound Con A onto the glycopolymer surface was subsequently quantified for all polymers (Figure 9) Figure 9 shows the relation between the diameter of glycopolymers and the number of Con A molecules recruited by each polymer molecule. While the general trend is that the amount of Con A recruited per polymer molecule increases with its hydrodynamic diameter, the highest binding capability is achieved by PHEA138-b-PMEA360 with 22 Con A molecules recruited per polymer molecule, followed by PHEA204-bPMEA433, recruiting 8 proteins per star. Con A-polymer binding behaviors obtained using QCM-D showed similar trends in comparison to the results from precipitation assay. However, there are slight deviations, in particular for the polymers with PHEA core (Table 3). Results obtained using QCM-D showed approximately double the amount of Con A bound per polymer, while the results of PMEA are in reasonable agreement. One possible cause of this can be attributed to the different composition and structure of the polymer cores. As found earlier in this study, stars containing PHEA in the core have comparatively lower hydrodynamic diameters than homopolymers with a similar molecular weight. The PHEA block is highly flexible and seems to be in a coiled state in an aqueous, lectin-free solution. Polymers analyzed by the QCM-D may have undergone an elastic stretching process due to the vibration of quartz sensor, where the stars can span beyond their hydrodynamic diameter. In this process, stars with coiled PHEA core may show higher “stretching ability”, which allows them to expand to a further distance and recruit more Con A molecules. The disparity between the amount of ConA per polymer between the QCM-D and SPR measurements is most likely a consequence of the differences between the measurement techniques. QCM-D is a gravimetric technique that measures bound molecules and their associated water, while the SPR is an optical technique that measures proteins without the associated water.42 This indicates that there was a significant amount of bound water in the Con A/polymer layers. Analysis of the dissipation versus frequency profiles (Df plots) for the different glycopolymers indicated for most of the analysis period the amount of Con A that bound to the polymer steadily increased as shown by the steady increase in dissipation and decrease in frequency (Figure 10). During the remainder of the analysis period there was rearrangement of the Con A as shown by decreases in dissipation indicating that the layers were more rigid and therefor contained less bound water. Of note are the different gradients observed during the binding phase for each of the Con A polymer interactions that resulted in quite different Con A/polymer measurements (Table 3).

Con A/polymer molecule QCM-D

Con A/mannose molecule QCM-D

Con A/polymer molecule SPR

0.51 1.43 2.56 3.17 5.44 22.00 1.70 6.52 8.19

0.007 0.010 0.007 0.029 0.029 0.061 0.014 0.027 0.019

3.49 3.24 3.73 1.04 0.76 1.02

Figure 10. Df plot for Con A binding to PHEA138-b-PMEA110, PHEA138-b-PMEA187 and PHEA138-b-PMEA360 as measured by the QCM-D for the 3rd overtone.

This shape Df plot has been seen previously for Con A binding to glycopolymers,41 however the longer time points analyzed in this study show the dynamic nature of the Con A glycopolymer interactions. These numbers again can be expressed in correlation with the number of repeating units (Figure 9B and Table 3), which expresses binding per mannose. Similar to the precipitation assay, the star polymers with less mannose pendant groups are more efficient in capturing Con A. Also in agreement with the precipitation assay is the role of the PHEA block. It seems that a medium PHEA block length enhances binding. The following conclusion can be drawn from these studies: Influence of Diameter. In the Introduction, it was hypothesized that maximum binding may be achieved when the diameter of the star polymer is equivalent to the distance of two binding pockets. This would mean that stars with diameters of roughly 5 nm would have the highest performance. However, there is no clear correlation, if anything, there is a slight decline in activity with the diameter. The reason for the absence of any correlation can be found when looking at the numbers of bound Con A determined using QCM-D. The polymer seems to undergo significant stretching to accommodate more than 20 Con A molecules per star polymer. The initial hydrodynamic diameter is therefore irrelevant highlighting the strength of binding that can lead to significant chain stretching. This is probably the most significant difference to dendrimers. The stiff character of dendrimers allows direct comparison between the distance between two mannose molecules and the distance between two binding pockets. Star polymers in contrast are flexible and can easily change conformations. It could even be speculated that that there is rather a correlation between the flexibility of the backbone and the amount of lectin binding than the size of the starpolymer in an undisturbed conformation. Interestingly, it may seem that the flexibility of the polymer is rather a property that reduces binding. According to the SPR assay, the binding is lower than 355


Article

Macromolecules Notes

the literature values of dendrimers may suggest. This is not overly surprising since earlier results when comparing linear polymers with micelles led to the conclusion that stiffer brushlike polymer enhance binding.43−45 Influence of PMEA. Similar to earlier results, longer polymers are not necessarily more efficient in binding lectins.23 Although a certain amount of mannose is required for good binding, it is not necessary to overload the polymer with carbohydrates. The polymer can stretch to a certain extend to accommodate a number of Con A. More mannose is therefore wasted in the process and does not further support more lectin binding. This result does indeed confirm earlier studies on epitope density highlighting that the sugar concentration needs to be carefully balanced: On one side, sufficient carbohydrates are required to display the multivalency effect; on the other side, the steric demands of the lectin means that carbohydrates should be spaced accordingly. QCM-D results suggest that only every twentieth to hundredth mannose functionality of these star polymers is involved in lectin binding. Influence of PHEA. It has been speculated in earlier studies that the core is not accessible by lectins and therefore these carbohydrates do not take part in the binding process. A neutral core-polymer that spaces the various arms from each other should therefore enhance the binding. All three assays, precipitation assay, SPR and QCM-D, gave similar results, but different order of magnitudes. Medium-sized PHEA have the highest binding efficiency per mannose. PHEA introduces higher flexibility, but it seems that a too high flexibility, as found with a large PHEA core, is counterproductive.

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The authors acknowledge funding from the Australian Research Council (DP130101625).

■

CONCLUSIONS A series of star-shaped block copolymers consisting of PHEA block near the core and PMEA block were successfully synthesized via RAFT polymerization using 1,2,4,5-tetrakis(butyltrithiomethyl)-benzene as the chain transfer agent. Nine star polymers with different block length of PHEA and PMEA were prepared. The binding capability of these glycopolymers was studied using precipitation assay, QCM-D, SPR, and turbidity assay. It was noticeable that all assays gave noticeably different results emphasizing that different assays are subject to different errors, but they also tend to looks at slightly different mechanism. The trends in all assays were found to be similar, but it highlights that these assay are all subjects to potentially large errors. It was found that the highest binding was not achieved when the distance of two mannose functionalities matches the distance between two binding pockets. While this may be true for stiff polymers, most polymers are in a highly coiled state in solution. The presence of Con A led to strong binding with the polymers, which subsequently resulted in chain stretching to accommodate more lectins. In conclusion, to achieve the highest possible binding per mannose functionality it is recommended to space the mannose functionality along the polymer chains taking into account that many polymers are in a coiled state that can be unfolded by lectins. Furthermore, it is recommended to employ stiff polymers when trying to match the hydrodynamic diameter with the binding to Con A.

■

REFERENCES

(1) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102 (2), 555−578. (2) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124 (50), 14922−14933. (3) Pieters, R. J. Org. Biomol Chem. 2009, 7, 2013−2025. (4) Kramer, R. H.; Karpen, J. W. Nature 1998, 395 (6703), 710−713. (5) Fan, E.; Zhang, Z.; Minke, W. E.; Hou, Z.; Verlinde, C. L. M. J.; Hol, W. G. J. J. Am. Chem. Soc. 2000, 122 (11), 2663−2664. (6) Ahmed, M.; Deng, Z.; Liu, S.; Lafrenie, R.; Kumar, A.; Narain, R. Bioconjugate Chem. 2009, 20 (11), 2169−2176. (7) Wu, Y.; Wang, M.; Sprouse, D.; Smith, A. E.; Reineke, T. M. Biomacromolecules 2014, 15 (5), 1716−1726. (8) Mammen, M.; Chio, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37 (20), 2755−2794. (9) Kumar, J.; McDowall, L.; Chen, G. J.; Stenzel, M. H. Polym. Chem. 2011, 2 (8), 1879−1886. (10) Richards, S.-J.; Jones, M. W.; Hunaban, M.; Haddleton, D. M.; Gibson, M. I. Angew. Chem. Inter. Ed. 2012, 51 (31), 7812−7816. (11) Furuike, T.; Nishi, N.; Tokura, S.; Nishimura, S.-I. Macromolecules 1995, 28 (21), 7241−7. (12) Polizzotti, B. D.; Kiick, K. L. Biomacromolecules 2006, 7 (2), 483−490. (13) Hasegawa, T.; Kondoh, S.; Matsuura, K.; Kobayashi, K. Macromolecules 1999, 32 (20), 6595−6603. (14) Liu, S.; Kiick, K. L. Macromolecules 2008, 41 (3), 764−772. (15) Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124 (8), 1615−1619. (16) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124 (50), 14922−14933. (17) Schuster, M. C.; Mortell, K. H.; Hegeman, A. D.; Kiessling, L. L. J. Mol. Catal. A: Chem. 1997, 116 (1−2), 209−216. (18) Tsuchida, A.; Akimoto, S.; Usui, T.; Kobayashi, K. J. Biochem. 1998, 123 (4), 715−721. (19) Strong, L. E.; Kiessling, L. L. J. Am. Chem. Soc. 1999, 121 (26), 6193−6196. (20) Kanai, M.; Mortell, K. H.; Kiessling, L. L. J. Am. Chem. Soc. 1997, 119 (41), 9931−9932. (21) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125 (20), 6140−6148. (22) Munoz, E. M.; Correa, J.; Riguera, R.; Fernandez-Megia, E. J. Am. Chem. Soc. 2013, 135 (16), 5966−5969. (23) Chen, Y.; Chen, G. J.; Stenzel, M. H. Macromolecules 2010, 43 (19), 8109−8114. (24) Watt, J. A.; Williams, S. J. Org. Biomol Chem. 2005, 3 (10), 1982−1992. (25) Khan, M. I.; Mandal, D. K.; Brewer, C. F. Carbohydr. Res. 1991, 213, 69−77. (26) Wolfenden, M. L.; Cloninger, M. J. Bioconjugate Chem. 2006, 17 (4), 958−966. (27) Stenzel, M. H. Macromol. Rapid Commun. 2009, 30 (19), 1603− 1624. (28) Frohlich, M. G.; Vana, P.; Zifferer, G. J. Chem. Phys. 2007, 127 (16), 164906/1−164906/7. (29) Frohlich, M. G.; Nardai, M. M.; Foerster, N.; Vana, P.; Zifferer, G. Polymer 2010, 51 (22), 5122−5134. (30) Mayadunne, R. T. A.; Jeffery, J.; Moad, G.; Rizzardo, E. Macromolecules 2003, 36 (5), 1505−1513. (31) D’Agosto, F. Macromol. Rapid Commun. 2008, 29 (11), 934− 935. (32) Gregory, A.; Stenzel, M. H. Prog. Polym. Sci. 2012, 37 (1), 38− 105.

AUTHOR INFORMATION

Corresponding Author

*(M.H.S.) E-mail: [email protected]. 356


Article

Macromolecules (33) Barner-Kowollik, C.; Davis, T. P.; Stenzel, M. H. Aust. J. Chem. 2006, 59 (10), 719−727. (34) Mori, H.; Ookuma, H.; Endo, T. Macromolecules 2008, 41 (19), 6925−6934. (35) Ambrosi, M.; Batsanov, A. S.; Cameron, N. R.; Davis, B. G.; Howard, J. A. K.; Hunter, R. J. Chem. Soc., Perkin Trans. 1 2002, No. 1, 45−52. (36) Plummer, R.; Hill, D. J. T.; Whittaker, A. K. Macromolecules 2006, 39 (24), 8379−8388. (37) Seto, H.; Ogata, Y.; Murakami, T.; Hoshino, Y.; Miura, Y. ACS Appl. Mater. Interfaces 2012, 4 (1), 411−7. (38) Kanai, M.; Mortell, K. H.; Kiessling, L. L. J. Am. Chem. Soc. 1997, 119 (41), 9931−9932. (39) Gou, Y.; Geng, J.; Richards, S. J.; Burns, J.; Remzi Becer, C.; Haddleton, D. M. J. Polym. Sci. A Polym. Chem. 2013, 51 (12), 2588− 2597. (40) Ting, S. R. S.; Chen, G. J.; Stenzel, M. H. Polym. Chem. 2010, 1 (9), 1392−1412. (41) Gou, Y.; Richards, S.-J.; Haddleton, D. M.; Gibson, M. I. Polym. Chem. 2012, 3 (6), 1634−1640. (42) Lord, M. S.; Stenzel, M. H.; Simmons, A.; Milthorpe, B. K. Biomaterials 2006, 27 (8), 1341−1345. (43) Kumar, J.; Bousquet, A.; Stenzel, M. H. Macromol. Rapid Commun. 2011, 32 (20), 1620−1626. (44) Hetzer, M.; Chen, G. J.; Barner-Kowollik, C.; Stenzel, M. H. Macromol. Biosci. 2010, 10 (2), 119−126. (45) Ting, S. R. S.; Min, E. H.; Zetterlund, P. B.; Stenzel, M. H. Macromolecules 2010, 43 (12), 5211−5221.

357


Correlation between Molecular Weight and Branch Structure of

Recommend Documents