Chemo-Enzymatic Synthesis of Branched Glycopolymer Brushes as

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Chemo-Enzymatic Synthesis of Branched Glycopolymer Brushes as an Artificial Glycocalyx for Lectin Specific Binding Yuzhen Wang, Lei Gu, Fanli Xu, Fengxue Xin, Jiangfeng Ma, Min Jiang, and Yan Fang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03704 • Publication Date (Web): 08 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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Chemo-Enzymatic Synthesis of Branched Glycopolymer Brushes as an Artificial Glycocalyx for Lectin Specific Binding Yuzhen Wang1, Lei Gu1, Fanli Xu1, Fengxue Xin1,2, Jiangfeng Ma1,2, Min Jiang1,2, Yan Fang1,2* 1

State Key Laboratory of Materials-Oriented Chemical Engineering, College of

Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing 211816, People’s Republic of China. 2 Jiangsu

National Synergetic Innovation Center for Advanced Materials (SICAM),

Nanjing Tech University, Nanjing 211816, People’s Republic of China.

* To whom correspondence may be addressed: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Artificial glycocalyx fabricated by carbohydrates is of interest because it provides a platform to simulate the cell membranes those widely exists in the nature, thus enable extensive applications to be implantable in bioengineering. Here, we present a green strategy combining two polymerization techniques, surface-initiated atom transfer radical polymerization (SI-ATRP) and enzyme-catalyzed elongation (ECE) of polysaccharide, for fabricating a densely packed branched glycopolymer brushes on the gold surface as artificial glycocalyx. In this strategy, SI-ATRP is first performed to graft a linear polymer chain for anchoring maltose, which can be used as enzyme acceptor for dextransucrase (DSase). Under the DSase, branched polysaccharide is efficiently formed through elongation of sucrose substrate. Undoubtedly, enzymatic transglycosylation has unique advantages, such as green, regio- and stereo-selective, etc. The process of DSase-catalyzed polysaccharide is well monitored by quartz crystal microbalance (QCM), and the grafting density of the glycopolymer brushes is estimated on 0.7 chain nm-2 with 23.0 nm dry thickness. The polysaccharide brushes display a branched structure consisting of α-D-glucose residues with 5% of α, 1–3-linked shorter chain branches, and the branched structure is well characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), FT-IR/mirror reflection (FT-IR/MR), water contact angle (WCA) analysis and atomic force microscopy (AFM). Compared with the linear maltose-anchored brushes, the branched glycopolymer analogue prepared here shows high specific binding capacity of concanavalin A (Con A) recognition, which should be of use in biomedical application.


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Introduction A carbohydrate-enriched coating that covers the outside of many cells and bacteria is referred to the glycocalyx. It has shown that the glycocalyx can not only shield the cell wall from direct exposure to invaders, but regulate molecular recognition of the cell with surrounding environment.1~3 Among these actual functions, the specific interaction between carbohydrate and protein is proven to be a necessary prerequisite for cellular processes.4~6 Concomitantly, investigation of the carbohydrate-protein interaction is a fundamental scientific issue for understanding the biological function of glycocalyx and, based thereon, developing their in-service performance in glycotechnology. Among the various strategies to investigate the carbohydrate-protein interaction, constructing artificial glycocalyx on implantable devices to simulate glycocalyx in the nature is considered to be the most effective tool.7~12 In this regard, synthetic polymers brushes bearing carbohydrate residues (glycopolymers) represent an effective method to construct glycocalyx-like biomimetic surfaces.13 This is because the affinity binding capacity to lectins of the glycopolymers brushes can be easily tuned by adjustment of the structure of the displayed carbohydrate ligands as well as the flexibility of polymer chains.14~15 Based on this idea, a large number of glycopolymers have been synthesized in the past decade.16~17 Additionally, compared with the carbohydrate-ended self-assembled monolayers (SAMs), glycopolymers brushes with three-dimensional (3D) structure can significantly improve the protein binding capacity because of the “glycocluster effect”. Hence, there is a need for more facile and straightforward means for conveniently constructing 3D artificial glycocalyx brushes with high proteins binding capacity to mimic the multivalent carbohydrate-protein interaction. Presently, the most commonly used method for fabricating glycopolymer brushes is based on surface-initiated atom transfer radical polymerization (SI-ATRP) of glycosylated


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monomers.13 Despite the SI-ATRP of glycosylated monomers is a powerful tool to construct glycopolymer brushes, the challenges of using the precious glycosylated monomers have held back the development of the artificial glycocalyx field. Another drawback of the glycocalyx produced by this method is the lack of the 3D structure.18 Herein, we have demonstrated a strategy combining two polymerization techniques for fabricating a densely packed branched glycopolymer brushes as artificial glycocalyx, i.e., SI-ATRP and enzyme-catalyzed elongation (ECE) of polysaccharide (Scheme 1). In this strategy, a densely packed linear poly(oligo(ethylene glycol) methacrylate) (POEGMA) chains were first grafted on the gold surface by SI-ATRP, followed maltoses were anchored to the POEGMA brushes through glycosidic bond. The resulting POEGMA brushes with end-capped maltoses can thus be used as acceptor for dextransucrase (DSase), under which branched glycopolymer brushes were efficiently formed through elongation of sucrose substrate. Taking advantage of SI-ATRP and ECE, the branched glycopolymer brushes with 95% (1→6)-linked and 5% of (1→3)-linked polysaccharide branches are well fabricated. Furthermore, we intend to describe the use of this well-defined 3D glycopolymer brushes for affinity adsorption of concanavalin A (Con A), thereby enhancing the possibility that using such artificial glycocalyx for applications in glycotechnology.


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HO

OH OH

OH OH O

HO OH OH O O OH HO OH HO OH O HO OH O OH OH O O O OH HO O O OH O

O CH C O C2H5O

HOHO

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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H3C CH

Br Br

Br Br Br

5

OH

O

OH OH

HO

BF3.Et2O

SI-ATRP

(a)

OH OH O

O HO

ECE (c)

(b)

(d)

Scheme 1 Scheme for fabrication of the densely packed branched glycopolymer brushes on the gold surface (a) Br modified surface, (b) POEGMA brushes, (c) end capped maltose POEGMA brushes, (d) branched glycopolymer brushes.

Experimental Details Materials. Q-sense Inc. provided QCM gold substrates (QSX 301, diameter of 14 mm, gold sputtered on the silica wafers). oligo(ethylene glycol) methacrylate (OEGMA, M ≈ 360,

99%),

11-mercapto-1-undecanol

(MUD),

2,2’-bipyridyl

(BPY,

99%),

2-bromo-2-methyl-propionyl bromide (BMPB), Con A, bovine serum albumin (BSA) and ricinus communis agglutinin (RCA120) were used as received from Sigma-Aldrich (China). -D-maltose Octaacetate (MOA, 99%) was purchased from J&K Chemical (China). Dextransucrase (DSase) was recombinant overexpresed from E. coli in the lab (see the experimental section in the Supporting Information, Figure S1~S5). Copper (I) chloride (CuCl) , Copper (II) bromide (CuBr2), triethylamine (TEA), ethanol, acetic acid, sodium acetate, sodium chloride (NaCl), calcium chloride (CaCl2), manganese chloride (MnCl2), sodium methoxide, dichloromethane and all the other chemicals were purchased from Sinopharm (China). Shanghai ling-feng chemical reagent Inc. (China) provided BF3·Et2O. Water used in all experiments was deionized and ultrafiltrated to 18 Mcm using an


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ELGA lab water system (France). Acetic acid-sodium acetate buffer solution (50.0 mM pH 5.5, 0.15 M NaCl and 1 mM CaCl2 ) was used for the enzymatic elongation reaction. Phosphate-buffered saline solution (PBS, 0.1 M, pH 7.3, 0.1 mM CaCl2, 0.1 mM MnCl2, and 0.1 M NaCl) was used for affinity adsorption of Con A. PBS (0.1 M, pH 7.3, 0.1 mM CaCl2 and 0.1 M NaCl) was used for adsorption of RCA120 and BSA. Surface preparation. The QCM chips were immersed in a mixtures of ammonia (28%), hydrogen peroxide (30%), and ultra-pure water (v/v/v=1:1:5) at 60 oC for 10 min. Then they were rinsed with water, and dried under a steady stream of nitrogen gas for further use. Initiator immobilization. First, the cleaned chips were immersed in MUD ethanol solution (1 Mm) and incubated overnight at room temperature to construct SAM. Then, the gold chips modified with the SAM were put into TEA (0.06 M)/dichloromethane (10 mL) mixtrures, and then BMPB (0.67 mL) was added dropwise in 6 min at room temperature. After that, the chips were taken out and washed with dichloromethane, ethanol and water and dried under ultrapure nitrogen flow. SI-ATRP of OEGMA. Gold chips modified with initiator were first placed into OEGMA (10.0 mL) solution. Simultaneously, CuCl (0.30 mmol), CuBr2 (0.06 mmol), and BPY (0.60 mmol) were dissolved in 10.0 mL of degassed methanol/water mixture (3:2 v/v) solution. Then the two mixtures were transferred into a 50 mL Schlenck flask and purged with nitrogen at room temperature for a predetermined time. After that, the chips were taken out and washed with water overnight and dried under ultrapure nitrogen flow. Anchoring maltose acceptor. MOA was dissolved in dichloromethane and oxygen was removed from the solution by nitrogen bubbling. The POEGMA grafted gold chips and catalyst BF3·Et2O were both added into the above solution for 24h reaction at ambient temperature.

Then

the

modified

chips


were

immersed

into

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sodium methoxide-ethanol mixtrues (5 mg/mL) for 90 min deacetylation reaction at ambient temperature. After that, the substrates were taken out and washed with water and dried under ultrapure nitrogen flow. Enzyme catalyzed elongation of the polysaccharide. The maltose acceptor functionalized

substrates

were

immerged

in

a

50.0

mM

acetic acid-sodium

acetate buffer solution containing DSase (a fixed concentration). The immersed substrates were incubated for 2 hours at 25 oC in a shaking incubator. Then, the substrates were thoroughly rinsed with ultra-pure water, and then immediately immerged in fresh 50.0 mM acetic acid-sodium acetate buffer solution containing sucrose (fixed concentrations). The substrates were incubated for a fixed time at 25 oC in a shaking incubator. After reaction, the substrates were thoroughly rinsed with ultra-pure water, dried with steady stream of ultra-pure nitrogen gas, and stored in vacuo. Characterization. The thicknesses of the polymer brushes were measured by ellipsometry. The measurement was carried out on a MD-2000I spectroscopic ellipsometer (J.A. Woollam, USA) at an incident angle of 60°, 65°, 70° in a wavelength range of 800-1000 nm. A refractive index of 1.45 and a cauchy model were assigned to the studied polymer brushes. Thickness data was obtained on a different place of the surface and reported as mean standard deviation. A refractive index of 1.45 and a cauchy model were

assigned

to the studied polymer brushes FT-IR/mirror reflection (FT-IR/MR) measurements were undertaken by a Nicolet FT-IR spectrometer (Thermo Electron Co., USA) equipped with a mirror reflection (MR) accessory. Thirty-two scans were acquired for each spectrum at a resolution of 4.0 cm-1. X-ray photoelectron spectroscopy (XPS) was prformed on a PHI-5000C ESCA system (Perkin-Elmer, USA) with Al Kα excitation radiation (1486.6 eV). The pressure


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was maintained at 10-6 Pain the chamber . All

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spectra were referenced to the C1s

hydrocarbon peak at 284.6 eV to compensate for the surface charging effect. QCM measurement was carried out using a Q-SENSE E1 system (Q-SENSE, Sweden). The sensor crystals used were 5 MHz, AT-cut, polished quartz discs (chips) with electrodes deposited on both sides (Q-SENSE). The resonance frequency (f) was measured simultaneously at four odd harmonics (5, 15, 25, 35 MHz). The values reported throughout for f and D was measured at the chosen fifth harmonicsat 25 oC. Raw data were analyzed with Origin Pro 8.0 (Origin-Lab, USA) and Q-Tools software (Q-SENSE). Monitoring of DSase-catalyzed elongation by QCM. A stable baseline signal was established by flowing a 50.0 mM acetic acid/sodium acetate buffer solution at a rate of 25 L/min through the sensor. Then, a significant decrease occurs for QCM frequency when DSase solutionawas injected into the channel.

The frequency showed continuous

decrease during the injection of sucrose solution. After the enzymatic polymerization for a fixed time at 30 oC, buffer solution was injected again the channel again to get the final baseline signal. Monitoring of protein adsorption by QCM. A stable baseline signal was established by flowing PBS buffer at a rate of 25 L/min through the chips. Different protein solutions (20 g/mL) of BSA, RCA120, and Con A were injected into the channel, respectively. After adsorption for a fixed time at 25 oC, PBS buffer was injected to the channel again to get the stable baseline signal. The adsorbed amounts of proteins can be calculated from the QCM curves.

Results and discussion Fabrication of branched glycopolymer brushes. The whole process for constructing the branched glycopolymer brushes is schematically depicted in Scheme 1. We first construct


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POEGMA brushes with identical thickness (20.0 nm) and high graft density

(  0.7

chain nm-2) on gold surface by SI-ATRP (see Table S1~S2, Eq. S1 and Figure S6 in Supporting Information).19 Then, BF3·Et2O- catalyzed etherification reaction between the OH groups of POEGMA and MOA OH was used to anchor the substrate acceptor (maltose) of DSase. After deacetylation of MOA, the maltose-anchored POEGMA brushes are prepared, followed the DSase-catalyzed elongation of the polysaccharide (dextran) is carried out from the maltose substrate. We finanly obtain a

densely packed

branched glycopolymer brushes with 0.7 chain nm-2 graft density. Figure 1a shows typical frequency changes of the maltose functionalized QCM chip as a function of time in response to the addition of DSase (80 nM) and sucrose (10 mM). The first decrease of frequency (mass increase to 105.5 ng·cm-2) indicates recognition and binding of enzyme to the maltose acceptor (enzyme/maltose complex formation). Obviously, the decrease of frequency depends on the enzyme concentration (Figure S7a and Eq. S2 ~ S5 in Supporting Information), from which the rate constant of enzyme binding (kon = 7.41  10-4 M-1s-1) and the rate constant of enzyme dissociation (koff = 1.22  10-3 s-1) can be well calculated (Figure S7b in Supporting Information).


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40

0.12

(a) DSase

buffer Sucrose Sucrose

buffer

buffer buffer

-40

0.04

buffer

-60

0.00

-80

20

0.08

-1

0 -20

(c)

0/Hzs

Frequency (Hz)

20

0

(b)

2000 4000 6000 8000 10000 12000

Time (s)

20

30

(d)

40

-40 -60

25mM

buffer

-80

0

1000 2000 3000 4000 5000 6000

Time (s)

-1

5mM 10mM 15mM 20mM

30

-1

buffer 0

-100 -1000

10

Sucrose (mM)

0 /Hz s

-20

0

50

Sucrose

0

Frequency (Hz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20 10 50

100

-1

150 -1

Sucrose (M )

200

Figure 1. (a) QCM curves for DSase catalyzed elongation on POEGMA (□) and the maltose-anchored POEGMA brushes (○); (b) Influence of sucrose concentration for the enzymatic elongation on the maltose-anchored POEGMA brushes; (c) Dependence of the initial elongation rate (ν0) on sucrose concentration; (d) Reciprocal plot of ν0 against sucrose concentration (The amount of bound DSase is 0.78 pmol∙cm-2). As shown in Figure 1a, the frequency sequentially decreases after the careful addition of sucrose (10 mM) into the enzyme solution (mass increased to 150.7 ng·cm-2). This decrease indicate the DSase-catalyzed elongation of polysaccharide (dextran), which can be certificated by the fact that the control surface (QCM chip modified with POEGMA brushes) is washed to zero in frequency (Figure 1a). Figure 1b shows the influnce of different sucrose concentrations on the dextran elongation process. The amount of polymerized dextran increases from 95.4 to 329.4 ng·cm-2 when the sucrose concentration increases from 5 to 25 mM. Importantly, the Michealis-Menten reaction can be used to


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simply describe the enzymatic elongation process (Supporting Information, Eq.S6 ~ S8).20~21 That is, the initial elongation rate (0) also increases with the sucrose concentrations increase(Figure 1c). This is reasonable because there are more substrate molecules can access to the active site of the bound enzyme. Figure 1d shows the reciprocal plot of 0 against sucrose concentration. We can obtain the dissociation constant of lactose (km) and the catalytic elongation constant (kcat) from the slope and intercept of the plot, respectively. In our case, the kcat (1.50  10-2 s-1) and km (28.53 mM) indicates that DSase can quickly proceed to elongation after binding to the maltose acceptors, and consequently form the enzyme/maltose complex, since the decomposition rate of the enzyme/maltose complex is smaller (koff = 1.22  10-3 s-1) than an order of magnitude of kcat. This feature is also exemplified by other enzymatic elongation.22~24 Characterization of the branched glycopolymer brushes. The dry thickness (DT) of the grafted brushes is measured by ellipsometry and the results are shown in Table 1. A slight increase of the thickness on the surface after DSase catalyzed elongation indicates the formation of corresponding quantities of the branched glycopolymers. The thickness of the branched glycopolymer brushes gradually increases with the reaction time, and it increase to ~ 23.0 nm after 72 h enzymatic elongation. However, an obvious decline of elongation rate is observed. We speculate that this may be ascribed to the increasingly space hindrance of side chains that hinder further elongation of the polysaccharide. To confirm this hypothesis, the hydrodynamic thickness (HT) of the linear and branched glycopolymer brushes is measured by QCM (Table1). For the linear maltose-anchored polymer chains, the ratio between the HT and the DT is 2.4, while this ratio decreases to 1.5 for the densely packed branched structure after 72 h enzymatic elongation. This may be because the larger steric hindrance of the branched brushes than for the linear ones that leads to higher packing of the linear brushes in the dry state (Supporting Information,


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Scheme S1). For the linear polymer, its main chain will significantly be stretched upon transition into hydrated state due to the “mushroom” structure on the surface,13, 25~26 while the branched glycopolymer brushes are, to some extent, kept in a partially collapsed conformation after water absorption. Moreover, the roughness of the brushes surfaces show obvious changes as measured by AFM, which also indicate the branched glycopolymer brushes have been successfully fabricated by DSase-catalyzed elongation (Table S3 in Supporting Information). Table 1. Dry thickness and hydrodynamic thickness of the maltose-anchored POEGMA brushes

(maltose-a-POEGMA)

and

dextran-anchored

POEGMA

brushes

(dextran-a-POEGMA) maltose-a-

dextran-a-

dextran-a-

dextran-a-

-POEGMA (nm)

-POEGMAb (nm)

-POEGMAc (nm)

-POEGMAd (nm)

DT

20.2 ± 0.1

21.7 ± 0.1

22.8 ± 0.1

23.2 ± 0.1

HT

48.5 ± 0.1

38.9 ± 0.1

36.5 ± 0.1

34.7 ± 0.1

Ra

2.4

1.8

1.6

1.5

aR

= HT/DT; b,c,denzymatic elongation time is fixed at 24h, 48h, and 72 h, respectively.

Through FT-IR/MR spectra, we determined that the chemical composition of the surface corresponded to the expectation that the glycopolymer brush surfaces (Figure 2). Compared to the ATRP initiator modified gold surface, the obvious absorption peak at 1725 cm-1 (C=O stretching vibration), 1165/1080 cm-1 (C-O-C stretching vibrations) and 3400 cm-1 (O-H stretching vibration) indicates the POEGMA brush surfaces was successfully grafted onto the gold surface (Figure 2a and 2b). The decrease of the O-H stretching vibration of brushes, intense absorption at 1755 cm-1 (C=O stretching vibration) and an emerging band around 1235 cm-1 (C–O stretching vibration of –OC2H5) provide evidence for the successful MOA immobilization. The disappearance of absorption at 1755 and 1235 cm-1 after deprotection again proves that the maltose acceptors have been anchored to the POEGMA brushes (Figure 2d). The increased peak intensity of the O-H


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stretching vibration after DSase-catalyzed elongation comes from OH groups of the branched glycopolymers in Figure 2e. The carbohydrate-related modes were in line with the standard data obtained from the spectral database. [IR spectra of maltose, and dextran (KBr disk) provided by the Spectral Database for Organic Compounds (SDBS) were used for comparison.]

C-O-C

O=C-O

-OH

C-O (e) (d) (c) (b) (a) 4000

3200

2400

1600

-1

800

Wavenumber (cm )

Figure

2.

FT-IR/MS

spectra

of

(a)

Br-SAM,

(b)

POEGMA

brushes,

(c)

MOA-a-POEGMA, (d) maltose-a-POEGMA, (e) dextran-a-POEGMA. 8.0k O1s 6.0k C1s

CPS

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4.0k (d) (c) 2.0k (b) (a) 0.0 600

500

400

300

200

Binding energy (eV) Figure 3. Survey XPS spectra of: (a) POEGMA brushes, (b) MOA-a-POEGMA, (c) maltose-a-POEGMA, (d) dextran-a-POEGMA.


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Then, we analyzed the chemical composition of the branched glycopolymer brushes ausing XPS. The amount of oxygen increases with the binding of carbohydrate moieties (Figure 3). Since the deprotection of MOA involves the replacement of –OC2H5 by -OH groups, the oxygen content of maltose-a-POEGMA is larger than that of MOA-a-POEGMA. Moreover, the experimental O/C value for the branched glycopolymer brushes decreased slighter than the theoretical O/C value (Table S4 in Supporting Information). This may because of the minor impurities on the surface. To fully distinguish the specific functional groups of the glycopolymer brushes, spectra with high resolution corresponding to C1s are shown in Figure 4. The C1s high-resolution spectrum for POEGMA brush surfaces are fitted with three peaks: 284.6  0.1, 286.4  0.1, and 288.8  0.1 eV corresponding to C-H/C-C/C-S, C-O-X, C=O, respectively. There are four components in the C1s core-level XPS spectra. The binding energy values are 284.7, 286.2, 288.0 and 288.8 eV corresponding to C-H, C-O, O-C-O and C=O, respectively. The 288.8 eV of O-C-O reveals a specific acetal component of carbohydrate.27~28 The peak at 286.4 eV for maltose-a-POEGMA is larger than that of MOA-a-POEGMA resulted from the C–OH of the carbohydrate ligand.

9.0k

(a)

9.0k

C-C/C-H/C-S

6.0k

C-C/C-H/C-S

6.0k C-O-X

3.0k

C-O-X O-C-O O-C=O

3.0k

O-C=O

0.0 292

(b)

CPS

CPS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.0 290

288

286

284

Binding Energy (ev)

282

280

292

290


288

286

284

Binding Energy (ev)

282

280

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40k

40k

(c)

30k

CPS

O-C-O

20k

10k

O-C=O

O-C-O C-O-X

20k

C-C/C-H/C-S

O-C=O 10k

0 292

(d)

30k

C-O-X C-C/C-H/C-S

CPS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 290

288

286

284

Binding Energy (ev)

282

280

292

290

288

286

284

282

280

Binding Energy (ev)

Figure 4. High-resolution XPS spectra of C1s: (a) POEGMA brushes, (b) MOA-a-POEGMA brushes, (c) maltose-a-POEGMA brushes, (d) dextran-a-POEGMA brushes. (Peak area (%)：(a) C-C/C-H/C-S: 63.61%, C-O-X: 29.95%, O-C=O: 6.44%; (b) C-C/C-H/C-S: 68.93%, C-O-X: 8.92%, O-C-O: 9.18%, O-C=O: 12.97%; (c) C-C/C-H/C-S: 52.91%, C-O-X: 28.95%, O-C-O: 8.03%, O-C=O: 10.10%; (d) C-C/C-H/C-S: 24.07%, C-O-X: 41.13%, O-C-O: 15.85%, O-C=O: 18.94%) Besides, we also use TOF-SIMS spectra to certificate the structure of the glycopolymer brushes (Supporting Information, Scheme S2, Figure S8a and Figure S8b). The classical (CH2)xO fragment ions as (CH2)2O and (CH2)4O are observed, indicating the PEG brush and MUD SAMs.29 The values at 435, 509, 773, 789 and 426, 590 m/z are typical fragments of MOA-a-POEGMA and maltose-a-POEGMA, respectively.30~31 The fragments of dextran-a-POEGMA at 648 and 901 m/z provides further information of a branched glycopolymer brushes. Finally, we also use WCA to show the changes of surface after each step reaction(Supporting Information Figure S9). The ATRP initiator layer surface is more hydrophobic (WCA = 67) than the POEGMA brush surface (WCA = 44). The WCA of the MOA-a-POEGMA brush increases to 82  2 due to the hydrophobicity of the acetylated glycosides residues. After deacetylation, the maltose-a-POEGMA brush shows


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high hydrophilicity with a WCA of 42. After enzymatic elongation, the WCA of the dextran-a-POEGMA surfaces decreases to 32,

which is ascribed to the increased

amount of the OH groups of the polysaccharides and the aliphatic carbon moieties.32 Protein adsorption on the glycopolymer brush surfaces. We use QCM to measure the protein adsorption on different polymer brush surfaces in real time. Three kinds of protein BSA, RCA120 and Con A are used in this work (Supporting Information, Figure S10~S11). The POEGMA brush surface is highly protein-resistant and the remained proteins after washing with PBS are lower than 3.0 ngcm-2. While Maltose-a-POEGMA and dextran-a-POEGMA brush surface exhibits specific adsorption towards Con A but with high protein resistance to BSA and RCA120 (the nonspecific adsorption is lower than 3.0 ngcm-2). We can see that about 753.3 and 1441.3 ngcm-2 of Con A are adsorbed on the maltose-a-POEGMA and dextran-a-POEGMA brush surfaces, respectively. More than 50 % of the adsorbed Con A remains on the surface after washing with PBS. The adsorption kinetic studies of Con A (0.1 mg/mL)on the two brush surfaces are shown in Figure 5. The adsorbed amount of Con A increase sharply at the beginning and then leveled off due to the approaching saturation values on the both glycopolymer brushes. The binding capacity of Con A on the maltose-a-POEGMA brush is about 1918.5 ngcm-2, while binding capacity increases to 2826.8 ngcm-2 for the dextran-a-POEGMA brush. This significantly enhanced binding capacity of the Con A can be attributed to the well-matched distance between the carbohydrate ligands and the recognition sites of protein as well as the increased glycopolymer side chains.33~34


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3k 2

m (ng/cm )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2k

1k

0

0

25

50

75

Concentration (mg/mL)

100

Figure 5. Influnce of Con A concentration on the adsorbed amount of Con A at 25 oC (□: maltose-a-POEGMA brush, ○: dextran-a-POEGMA brush). Here, we choose both Langmuir and Freundlich models to give quantitative analysis of the adsorption isotherms. The calculated data are listed in Table 2. Langmuir model has a typical assumption that there is only a monolayer adsorption between the adsorbed proteins and the surfaces.35 Freundlich model is more sophisticated which has the assumption that there is multilayer adsorption between the adsorbed proteins and the surfaces.36 Here, we found that the Freundlich model can desceib the adsorption behaviors more satisfactorily (Supporting Information, Figure S12~S13). This suggests that the adsorption of Con A on the both glycopolymer brushes surfaces is multilayer behavoir. Evidently, the multilayer behavior of

Con Aadsorption can be attributed to the flexible

spacer (-(CH2CH2O)5-) of POEGMA since the chain mobility can premote lectin binding.37 In addition, compared with the linear maltose-a-POEGMA (1.87  104 M-1), the adsorption equilibrium constant (Ka) value increases significantly for the branched dextran-a-POEGMA brush (3.99  104 M-1). we can determined that there’s an enhancement affinity between Con A and the branched glycopolymer brushes.


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Table 2. Adsorption behaviors of Con A on the glycopolymer brush surfaces. Sample

Langmuir adsorptiona R2

Log Kf

n

R2

2939.19

0.949

2.20

0.55

0.982

3676.34

0.957

2.68

0.40

0.973

Ka (

Qe

104M-1)

(ng/cm2)

maltose-a-POEGMA

1.87

dextran-a-POEGMA

3.99

aData

Freundlich adsorptionb

were calculated by equation [C]/Q = [C]/Qe + 1/Qe × 1/Ka, (Q is the measured amounts of

adsorbed Con A, Qe is the theoretical equilibrium amounts of adsorbed Con A, [C] is the equilibrium concentration of Con A, and Ka is the value of adsorption equilibrium constant). bData were calculated by equation log Q = log Kf + n log[C], (Kf and n are the Freundlich characteristic constants indicating adsorption capacity and adsorption intensity, respectively).

Conclusions We have demonstrated herein a novel strategy combining surface-initiated atom transfer radical polymerization (ATRP) followed by enzyme-catalyzed elongation (ECE) of polysaccharide to fabricate a densely packed branched glycopolymer brushes as artificial glycocalyx. This approach is enabled by the choice of POEGMA as polymer brushes, which were grafted on the gold surface by SI-ATRP. This −OH-riched POEGMA brush in the presence of the catalyst, serves as acceptor for subsequent anchoring maltose. The resulting end-capped maltoses can thus be used as acceptor for dextransucrase, under which branched polysaccharides were efficiently formed through elongation of sucrose substrate. Using this strategy, the branched artificial glycocalyx with a grafting density of 0.7 chain nm-2 with a thickness of ~23.0 nm was obtained. The prepared branched glycopolymer brushes displayed high binding capacity to Con A, which is expected to have potential applications in further investigating the carbohydrate-protein interactions and glycotechnology applications. Such studies are currently underway in our laboratory. ASSOCIATED CONTENT Supporting Information


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Langmuir

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac. xx. Experiment details of enzyme recombinant, glycopolymer brushes construction and protein adsorption and characterization of the glycopolymer brushes.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no conflict of interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21805135), the Jiangsu Province Natural Science Foundation for Youths (BK20180712), and Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals Foundation (JSBEM2016010).

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TOC

A strategy combining surface-initiated atom transfer radical polymerization (SI-ATRP) and enzyme-catalyzed elongation (ECE) techniques for fabricating a densely packed branched glycopolymer brushes as artificial glycocalyx.


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Langmuir

TOC

A strategy combining surface-initiated atom transfer radical polymerization (SI-ATRP) and enzyme-catalyzed elongation (ECE) techniques for fabricating a densely packed branched glycopolymer brushes as artificial glycocalyx.


Chemo-Enzymatic Synthesis of Branched Glycopolymer Brushes as

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