Controlling Ligand Spacing on Surface: Polyproline-Based Fluorous

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX

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Controlling Ligand Spacing on Surface: Polyproline-Based Fluorous Microarray as a Tool in Spatial Specificity Analysis and Inhibitor Development for Carbohydrate−Protein Interactions Tse-Hsueh Lin, Cin-Hao Lin, Ying-Jie Liu, Chun Yi Huang, Yen-Cheng Lin, and Sheng-Kai Wang* Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan R.O.C. S Supporting Information *

ABSTRACT: Multivalent carbohydrate−protein interactions are essential for many biological processes. Convenient characterization for multivalent binding property of proteins will aid the development of molecules to manipulate these processes. We exploited the polyproline helix II (PPII) structure as molecular scaffolds to adjust the distances between glycan ligand attachment sites at 9, 18, and 27 Å on a peptide scaffold. Optimized fluorous groups were also introduced to the peptide scaffold for immobilization to the microarray surface through fluorous interaction to control the orientation of the helical scaffolds. Using lectin LecA and antibody 2G12 as model proteins, the binding preference to the 27 Å glycopeptide scaffold, matched the distance of 26 Å between its two galactose binding sites on LecA and 31 Å spacing between oligomannose binding sites on 2G12, respectively. We further demonstrate this microarray system can aid the development of inhibitors by transforming the selected surface-bound scaffold into multivalent ligands in solution. This strategy can be extended to analyze proteins that lacking structural information to speed up the design of potent and selective multivalent ligands. KEYWORDS: Microarray, Polyproline, Molecular Scaffold, Multivalent binding, Carbohydrate−Protein interaction, Fluorous interaction

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INTRODUCTION Carbohydrate−protein interactions play essential recognition roles in biological systems.1 How these individually weak interactions lead to overall significant binding and functions is explained by the concept of multivalent interaction.2−4 In such binding events, the distances between multiple glycan binding sites on protein have to be matched by the glycan spacing (interligand distance)5 on a scaffold to significantly enhance the avidity and specificity. Therefore, suitable multivalent glycoconjugates6−9 including neoglycoproteins,10 glycopeptides,11 glycodendrimers,12,13 glycopolymers,14 glycolipids,15 and glycoclusters16−18 have been created to conform with the distances and the arrangement of carbohydrate binding sites on lectins. Unfortunately, such protein spatial information is usually obtained from structural studies, which are time-consuming and not practical for analyzing all available lectin targets. Thus, a more efficient strategy to determine preferred glycan arrangement (spatial specificity) for a given protein will be very helpful to develop corresponding multivalent ligands. Glycan microarray technology is widely employed in protein analysis19,20 with glycan ligands immobilized on the surface through various chemical approaches.21 However, conventional glycan microarrays are probably not the best tool for characterizing the spatial specificity of proteins because the individual glycan ligands may not be evenly immobilized on surface resulting a collection of interligand distances between neighboring ligands. Although many studies adjusted glycan © XXXX American Chemical Society

density through surface immobilized supportive molecules, such as polymers,22−26 dendrimers,27,28 clusters,29−32 peptides,33−35 proteins,36,37 nucleic acids,38,39 and lipid-based aggregates,40,41 and many exhibit changes of lectin avidity in respond to glycan density, not all of them reflect the spatial information on the protein due to the limitation to define ligand spacing on surface. For example, glycopolymers that do not have uniform and defined conformation likely leaves the distance between ligands unclear. In the case of dendrimer microarrays, glycan ligands densely distributed on surface initiate strong interaction to target proteins. However, the difficulty to point out which glycans are involved in binding and the lack of defined glycan positions may limit the application for this purpose. Unlike the artificial molecules mentioned above, although proteins have more rigid and defined conformations, when protein scaffolds have many ligand conjugation sites available to present glycan, site-selective conjugation will be required, or the mixture of heterogeneous neoglycoproteins will present a combination of different ligand spacing on microarray surface. Therefore, the scaffold structural design plays an important role in positioning glycan ligands, for example using conformational restrained scaffold may provide better control in ligand positions.39,42,43 Herein, we report a Received: August 31, 2017 Accepted: October 30, 2017

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DOI: 10.1021/acsami.7b13200 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces microarray system based on a peptide with stable secondary structure serving as a scaffold to control glycan presentation on surface, which is examined with two model proteins lectin LecA and oligomannose-specific monoclonal antibody 2G1244 to analyze the protein spatial specificity. Moreover, we test if the output from the microarray analysis can lead to a potent inhibitor for the target protein.

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RESULTS AND DISCUSSION Design and Optimization of Fluorous Polyproline Scaffold. Our scaffold design is based on the polyproline helix type II structure (PPII) (Figure 1a), which has an almost 3-fold

Figure 2. Arrangement of scaffolds on surface. Lateral (a) and terminal (b) immobilization. Interscaffold cross-linking (c) can be prevented by dilution with unliganded scaffold (d).

Scheme 1. Synthesis of Fmoc-Fluorous Proline Building Blocks

Figure 1. (a) Polyproline helix II structure side view. (b) 3-fold symmetry of polyproline helix II. (c) Design of fluorous polyproline scaffold for immobilization on fluorous slide. Wavy curve represent fluorous chain, yellow triangle represent ligand. The molecular models were produced by Discovery Studio Visualizer with structural data from ref 46

Scheme 2. Preparation of Cy3-Fluorous Polyproline Peptides to Optimize Fluorous Chain Length

symmetry if viewed through its axis (Figure 1b) and provides about a 9 Å rise for every helical turn contributed from 3 proline residues.45,46 While polyproline has been exploited for many applications,47−49 we took advantage of this structurally well-defined peptide as a scaffold to present two carbohydrate ligands with controlled spacing and direction. In addition, as every third residue in this peptide points to almost the same direction, using these residues to anchor the peptide to the microarray surface may help further stabilize the PPII helix conformation. Meanwhile, multiple anchors on one side of a helix may limit scaffold motion on the surface (Figure 2a, in contrast to terminally immobilized scaffold in Figure 2b) to reduce undesirable interscaffold protein cross-linking. With these concerns, we employed the concept of a fluorous microarray pioneered by the Pohl group50,51 to noncovalently immobilize peptide scaffolds to the microarray surface through fluorous interactions. We prepared fluorous proline building blocks via known intermediates Boc-4-azido-Pro-OtBu 2,52 and followed by reduction to amine53 and acylation with fluorous group (Scheme 1). As fluorous interaction for microarray is governed by the number and length of the fluorous groups,54 we designed three fluorous building blocks 4a−c in order to optimize the fluorous chain length. We prepared nonapeptides 5a−c by SPPS with building blocks 4a−c at the 2, 5, and 8 positions (Scheme 2). Further Cyanine 3 (Cy3) labeling allows the immobilization of peptides 6a−c on fluorous surface to be evaluated quantitatively. To

fully test the strength of the fluorous modification, we washed the surface with buffered detergent solution repeatedly for prolong duration that is much longer than typical microarray experiments. The fluorescent intensity for peptide 6a reduced significantly compared to 6b and 6c (Figure 3). In addition, the spot area for 6a significantly enlarged after the first wash but gradually decreased with further washing, indicating that 6a easily spread to neighboring areas, yet did not withstand repeated washing. In contrast, 6b and 6c only slightly expanded B


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ACS Applied Materials & Interfaces

Glycan Conjugation to Fluorous Polyproline Scaffolds. To prepare the scaffolds with variable ligand spacing, we employed alkyne groups as chemical handles to allow convenient ligand change in conjugation to a common scaffold through Cu(I) catalyzed alkyne−azide cycloaddition (CuAAC) reaction. In this set of peptides, we designed the alkyne groups on proline residues that are one (9), two (10), and three helical turns (11) apart (Scheme 3), corresponding to 9, 18 and 27 Å on the scaffold. With Fmoc-Pro-OH, trans-Fmoc-4-propargyloxy-Pro-OH 855 and building block 4b, we synthesized three dodecapeptides 9, 10, and 11, each with three C5F11 groups at the 4, 7, and 10 positions, while the two propargyloxy groups were located at the (6, 9), (3, 9), and (2, 11) positions, respectively. These scaffolds were further conjugated with βgalactoside 12 or Man4 13 through CuAAC reaction to give glycopeptides 14−19. Microarray Analysis of LecA and 2G12. Our design installed fluorous groups at one side of the polyproline helix to favor the scaffold rest parallel to the surface (Figure 2a). To control the ligand spacing at the intended distance, each immobilized glycopeptide had to be sufficiently separated to prevent interscaffold glycans cross-linked by the protein (Figure 2c and d). Therefore, to properly immobilize them, while keeping the total fluorous peptide concentration the same, we diluted the glycopeptide 14−16 with different ratios of peptide 5b, which has three C5F11 anchors but no glycan ligand. Cy3 labeled LecA were used as a model lectin to test our microarray system due to its availability, well-documented structure and most importantly, as a promising antibacterial target. LecA was identified from Pseudomonas aeruginosa, which is a major cause for life-threatening lung infection in hospital. This lectin binds to galactoside ligands and thereby involved in the biofilm formation and adhesion to epithelial cells, so that inhibition of these processes may control the infection.56 Structurally, LecA is a tetrameric lectin, which has four galactoside binding sites with the closest distance at 26 Å and followed by 80 Å.7 To evaluate LecA binding on the peptide-immobilized surface, we measured surface dissociation constant (KD,surf)57 from protein binding curve (Figure 5), and a set of representative measurements are shown in Table 1. With 1:3 dilution of glycopeptide 14−16 with peptide 5b, strong binding are observed for all three glycopeptides with different ligand spacing. This can be explained by that the neighboring glycopeptides might participate to provide suitable interscaffold ligand spacing to fit LecA (Figure 2c). To diminish this effect, the glycopeptides were further diluted with 5b to a 1:7 ratio, at which the fluorescent intensity for 14 become too low (SI Figure S36) to provide reliable KD,surf and the KD,surf for 16 is several fold stronger than for 15. Clearly, at 1:7 dilutions, glycopeptide 14−16 can differentiate the spatial specificity of LecA. We reason that the glycopeptides are better separated at higher dilution (Figure 2d), so that the neighboring glycopeptide do not efficiently cross-link LecA in an interscaffold manner. Under such circumstances, LecA has distance between the closest binding sites at 26 Å,7 which is best matched by glycopeptide 16 with a designed 27 Å spacing scaffold and followed by glycopeptide 15 (18 Å) as a weaker binder. These results contribute a selective scaffold design for the development of LecA inhibitors against P. aeruginosa infections. Besides LecA, we further focused on monoclonal antibody 2G12, which is a well-studied HIV neutralizing antibody that binds to multiple oligomannose on viral envelope protein

Figure 3. Fluorous groups of peptides 6 were evaluated by prolong washing experiments on fluorous surface. Image (a), fluorescent intensity (b) and fluorescent area (c) of prior and after wash with PBS buffer containing 0.05% Tween 20 are shown.

the spot area to reflect their strong fluorous interaction. These experiments suggest that polyproline peptide with C5F11 and C7F15 groups are comparable in such assay settings but have significant advantages over the C3F7 chain. Because peptides with shorter fluorous groups are easier to prepare and dissolve better in aqueous solution, C5F11 is the optimal group for this microarray system. Next, to confirm whether the fluorous polyproline peptide remained in the PPII structure, which is the basis of our control in ligand spacing, we measured circular dichroism (CD) spectroscopy in the far UV range for peptide 5b, which showed a stable PPII structure in aqueous solution (Figure 4a). We also

Figure 4. Circular dichroism spectra at far UV range for peptide 5b indicate its PPII structure on fluorous surface. (a) peptide 5b in water; (b) peptide 5b immobilized on fluorous quartz beads suspended in water.

performed CD measurement of peptide 5b immobilized to the fluorous surface of suspended quartz beads. Despite the weak signal, the CD spectrum still shows certain characteristics of PPII helix from immobilized peptide 5b on the surface (Figure 4b). These findings suggest our strategy in controlling glycan spacing is valid with the fluorous polyproline scaffolds. C


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ACS Applied Materials & Interfaces Scheme 3. Preparation of Fluorous Glycopeptides

Figure 5. Binding curves of LecA-Cy3 with surface-bound glycopeptide 16 and 5b mixture (a); and 2G12-Cy3 with surface-bound glycopeptide 19 and 5b mixture (b) on the fluorous microarray system.

Table 1. Surface Dissociation Constants of LecA on the Peptide Immobilized Surfaces

Table 2. Surface Dissociation Constants of 2G12 on the Peptide Immobilized Surfaces

KD,surf to peptide on fluorous slide (nM)

a

KD,surf to peptide on fluorous slide (nM)

ratio of 14∼16: 5b

14

15

16

ratio of 17∼19: 5b

17

18

19

1:3 1:7

538 ± 140 −a

513 ± 143 1797 ± 1493

112 ± 28 354 ± 55

1:3 1:7 1:15

17 ± 3 30 ± 7 84 ± 31

16 ± 3 27 ± 5 55 ± 23

10 ± 2 11 ± 2 12 ± 3

fluorescence intensity too low to give reliable estimation.

gp120.44 Therefore, a strong and relatively rigid inhibitor for 2G12 can be used as hapten for vaccine development in order to elicit 2G12-like neutralizing antibodies. Similar binding trend was also observed when labeled 2G12, tested with glycopeptide 17−19 on fluorous microarray. The dilution of the glycopeptides by 5b from 1:3, 1:7 to 1:15 further increase the KD,surf difference between 19, 18, and 17 indicating glycopeptide 19 provided favorable ligands spacing for 2G12 (Table 2). Again, the change in KD,surf in response to glycopeptide dilution can be explained by interscaffold distance on surface. At 1:3 fold of dilution, the liganded peptides are distributed closer so that the ligands from different glycopeptides may participate in multivalent binding even when the designed ligand spacing on the scaffolds do not match 2G12. When the glycopeptides are more diluted, the

interscaffold distance increase beyond the interscaffold crosslinking to 2G12 can efficiently occur, so that the glycopeptides become selective. Therefore, the KD,surf for 17, 18, and 19 were comparable at 1:3 dilution but reach several folds in magnitude at 1:15 dilution to discriminate the glycopeptide interactions. While the distance between binding sites of 2G12 is projected to be 31 Å,7 it is best matched by the 27 Å spacing from the polyproline scaffold of 19, as expected. These findings demonstrate the capability and potential of this fluorous scaffold-surface system: as a significant number of broadly neutralizing monoclonal antibodies have been isolated from HIV patients,58 this system may provide an opportunity to rapidly characterize antibody specificities to develop new designs for HIV vaccines. D


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ACS Applied Materials & Interfaces To demonstrate the observed protein binding to glycoconjugates on surface is due to their glycan ligands rather than the polyproline moiety, we performed LecA and 2G12 binding assay to glycoconjugates 14−19. As these glycopeptides share 3 common scaffolds, the unambiguous binding specificity (LecA only binds 14−16 but not 17−19 and vice versa for 2G12, SI Figure S40 and S41) clearly rule out the possibility for proteins binding to the scaffolds. In addition, we further test glycopeptides with monomeric ligand on our microarray system to better understand how ligands on scaffold interact with proteins. We prepared such glycopeptide by conjugating a single Man4 to peptide 9 and tested 2G12 binding (SI). We found at 1:3 dilution ratio to 5b, the monomeric Man4 glycopeptide has very weak 2G12 binding compared to glycopeptide 19 (SI Figure S42) that its KD,surf cannot be determined. These results, together with the peptide dilution experiments, suggest the interaction between 2G12 and glycopeptides 17−19 on surface is predominately dimeric, so that the difference in their KD,surf may arise from the easiness for the conjugated ligands to initiate bivalent binding to 2G12. Our present work use structurally well-defined biomolecule as scaffold to control the ligand spacing. Another practical scaffold suitable for this purpose is nucleic acid duplex, which uses well-defined double helix structure to support ligands.59−62 PNA/DNA scaffolds have been exploited to control ligand spacing toward receptors,60 antibodies59 and lectins.61 However, the structure of this nucleic acid duplex takes more than 10 base pairs to finish an revolution and extends for about 30 Å. Therefore, to present two ligands in the same direction, the PNA/DNA scaffold can control the spacing at the multiples of 30 Å, comparing to the multiples of 9 Å for PPII. This difference in their secondary structures makes polyproline the preferable scaffold for finer adjustment at ligand spacing. When nucleic acid duplex were applied to the microarray surface, its hybridization can be used for immobilization,31,38,63,64 programmed combination of ligands39 and in several cases, altering ligand spacing. Although flexible spacer or other cluster scaffold were responsible for the spatial adjustment,39,42,43 such microarray designs demonstrated preference from target proteins. In contrast, our fluorous polyproline microarray system relies on PPII structure and may likely be the first example to exploit a scaffold of well-defined peptide secondary structure to control ligand spacing on microarrays. A microarray system based on our scaffold would allow rapid screening a number of proteins to identify the suitable multivalent requirement of the ligands, and also reveal possible cross-reactivity that may occur to other proteins. In addition, because the fluorous interactions are strong and specific, and fluorous surface has been exploited in other analytical platforms; we also expect our fluorous scaffolds to be extended to other surface-based analytical instruments such as quartz crystal microbalance in the future. Development of Inhibitor Based on Microarray Results. Finally, to demonstrate our microarray system can aid the development of multivalent inhibitors, we test 2G12 with soluble format of inhibitor glycopeptides, which have no fluorous groups. Man4-conjugated polyproline peptides 20−22, corresponding to 9, 18, and 27 Å spacing, respectively, were prepared (Scheme 4) and went through surface plasmon resonance analysis with 2G12 immobilized on sensor chip. The measured dissociation constants (KD) and the off-rate (kd) for these glycopeptides in solution have indicated that 22 elicits strongest binding to 2G12 and followed by 21 then 20 (Table

Scheme 4. Preparation of Glycopeptides for 2G12 Binding Assay in Solution

3). It is interesting how the measured KD correlate to the binding of 2G12 with peptide 20−22. In one well-known Table 3. Dissociation Constants of Glycopeptides 20−22 toward Immobilized 2G12 by Surface Plasmon Resonance Assay 20 KD (M) kd (s−1) ka (M−1 s−1)

21 −5

4.4 × 10 1.9 4.3 × 104

22 −5

1.5 × 10 1.1 7.3 × 104

8.8 × 10−6 0.74 8.4 × 104

example, Kitov and Bundle et al. used glycocluster to investigate Shiga-like toxin interaction, in which 40 folds of improvement on IC50 was observed from monovalent to bivalent inhibitor.65 However, this binding enhancement also varies around 8−50 folds depending on the design of the bridging linker.66 While the measured KD for glycopeptide 22 is almost 2 folds better than 21 and 5 folds better than 20, we tested if the enhancement arise from monovalent to divalent binding by using Man9GlcNAc2Asn, which contains Man4 as its D1 arm, as a monovalent ligand. The SPR analysis for this oligosaccharide did not provide significant binding (SI Figure S46), which suggests all three glycopeptides initiate predominately divalent binding to 2G12. Because 2G12 binds Manα1−2Man at the terminal of Man4, we expect the selectivity to be further improved when shorter glycan or linker (currently (CH2)5 group) is used. Despite of the possible binding mode, our results demonstrated the best spacing found from microarray screening also initiate strongest interaction when transformed into soluble bivalent inhibitor 20−22. Therefore, our microarray system is practical in the search for multivalent inhibitors focusing on ligand spacing. The outcome candidate with polyproline scaffold (22 as an example) can provide spatial selectivity through the defined scaffold structure and ligand positions. Moreover, polyproline peptides have several advantages over PNA/DNA scaffolds include (1) finer spacing control from the smaller 9 Å pitch in its helical structure; (2) smaller molecular weight, as PNA/DNA typically requires around 10 base pairs to hybridize into duplex structure and each base pair is much larger than a proline residue; (3) without the negative charge from DNA backbone that may lead to unwanted charge interactions. In addition, unlike many peptide scaffolds, polyproline scaffolds have high proteolytic stability in serum.67,68 All the above properties support the polyproline as a suitable multivalent scaffold for biomedical applications.

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CONCLUSION In this study, we developed a novel microarray system that exploits the unique polyproline helix II structure to control E


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ACS Applied Materials & Interfaces ligand spacing on fluorous surface. This system differs from other microarrays by using well-defined secondary structure of a peptide scaffold to provide fine adjustments. Our analysis on LecA and 2G12 showed selectivity for the matching ligand spacing on polyproline peptide scaffold, suggesting that it is a useful tool for characterizing the ligand spatial specificity of protein. We further used nonfluorous Man4-polyproline glycopeptides in solution to demonstrate the selected glycoconjugate from microarray analysis leads to superior bivalent inhibitor for 2G12. With the rapid screening capability of microarray, it would be convenient to analyze of a large number of proteins, especially those without structural information, to aid the development of selective multivalent inhibitors for target proteins.

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mL) and sonicated to suspend the beads. Then the aqueous solution of peptide 5b (250 μM, 200 μL) was pipetted into the tube to incubate with the fluorous quartz beads at 4 °C for 12 h. After centrifugation, the supernatant solution was carefully removed and the sediment beads were mildly resuspended in 1 mL ultrapure water. After repeating centrifugation and washing process for two times, 3 mL of ultrapure water was added to resuspend the beads for CD measurement, which was performed in the cuvette of 10.0 mm path length with 60 rpm of stirring. Fluorous Slide Preparation. Commercial glass slides (Brand Microscope Slides, Cat. No. 474744) were cleaned and activated by sonicating in Piranha solution for 15 min, washed with ultrapure water and dried in vacuo. A coating solution prepared with 200 mg of 3(perfluorooctanamido)propyl)triethoxysilane in 100 mL of dry dichloromethane, with 0.25 v/v% of acetic acid was added into a tank containing cleaned and activated slides. After reaction for 20 h, the slides were rinsed with methanol and ultrapure water repeatedly, and dried in vacuo. Microarray Experiment: Cy3-Fluorous Peptide Washing Test. Fluorous slides were printed by a robotic system (BIODOT model no. AD1510) with micro spotting pins (ArrayIt 946 Catalog ID: 946MP2B). The Cy3 labeled peptide was dissolved in a solution of 0.005% Tween 20 in PBS with 10% DMSO. The peptide at concentration of 25 μM was printed to slides with relative humidity of 60−70%. The printed slide was incubated for 20 h under humidifying container and scanned. Then the slide was washed with 0.05% Tween 20 in PBS for 3 min on an orbital shaker (60 rpm) and immediately scanned. Further washing and scanning were performed with the same procedures at the designed intervals. Microarray Experiment: LecA and 2G12 Binding to Glycopeptides. The glycopeptides 14−19 were dissolved in 10% (v/v) acetonitrile in ultrapure water and peptide 5b was dissolved in 25% (v/v) acetonitrile in ultrapure water as 25 μM stock solutions. The mixtures of glycopeptide with 5b were prepared by mixing the stock solutions at 1:3 ratio and then serial dilute with an equal volume of 5b. Therefore, the ratio vary from 1:3, 1:7, to 1:15 with the total peptide concentration maintained at 25 μM. A 20 μL portion of the mixture solution at such ratio were concentrated in vacuo and redissolved in 50 μL trifluoroethanol to incubate for 12 h and then lyophilized. The residue was redissolved in 15 μL of printing buffer (20 v/v% DMSO in PBS) right before applying to slide surface with the final total peptide concentration of 33 μM. After printing, the slide was incubated at 60 °C in a humidifying container for 20 h. Right after washing the printed slide with iced PBS for 1 min, the Cy3 labeled protein of different concentrations (serial diluted in pH 7.4, Tris 25 mM, NaCl 150 mM, CaCl2 0.1 mM buffer with 0.005% Tween 20 and 1% BSA to give the final Cy3-protein concentrations at 35.0, 21.0, 12.6, 7.6, 4.5, 2.7, 1.6, and 1.0 μg/mL for LecA-Cy3; serial diluted in pH 7.4 PBS buffer with 0.005% Tween 20 and 1% BSA to give the final Cy3-protein concentrations at 25.0, 15.0, 9.0, 5.4, 3.2, 1.9, 1.2, and 0.7 μg/mL for 2G12-Cy3) were incubated on the slide at 4 °C for 1 h. The slide was washed with 0.05% Tween 20 in PBS (3 min +6 min) and dichloromethane/methanol (1:1 for 15 min) using orbital shaker (60 rpm). Then the slide was air-dried and immediately scanned. Microarray Scanning and Data Processing. The microarray scanning was performed by GenePix 4000B Microarray Scanner with excitation wavelength at 532 nm. The scanning resolution was set at 10 × 10 μm. The raw data was processed in software GenePix Pro 6.0. The surface dissociation constant (KD, surf) was calculated by Graph Pad Prism (ver. 6.01) using equation:

EXPERIMENTAL PROCEDURES

Materials. All chemical reagents and solvents were purchased from Acros and Alfa Aesar. Cy3-NHS was purchased from Lumiprobe; Lectin LecA was purchased from Sigma-Aldrich; Monoclonal antibody 2G12 was purchase from Polymun Scientific Immunbiologische Forschung GmbH. Quartz beads were purchased from Formosa Nitride Application & Material Inc. Glass slides were purchased from Brand GmbH + Co. KG. Methods for Peptide Synthesis and Analysis. The peptides (5a, 5b, 5c, 9, 10, and 11) were prepared by manual solid phase peptide synthesis using Rink amide resin as solid support. Five different types of amino acids were used during coupling (4 equiv, Fmoc-Pro-OH, 4a, 4b, 4c, or 8) according to the designed sequence. HBTU (4 equiv, for Fmoc-Pro-OH and 8) or HATU (4 equiv, for 4a, 4b, and 4c), HOBt (4 equiv), DIPEA (4 equiv) were dissolved in NMP/DMF (v/v 1:9, final concentration of amino acid at 0.2 M) to react with resin at 40 °C for 90 min for each coupling. The Fmoc deprotection was performed with 20% piperidine in DMF × 2, lasted 3 and 10 min, respectively; capping was processed with 50% acetic anhydride in dichloromethane for 10 min × 2 and peptide cleavage was processed with the cleavage cocktail (1 mL, 95% TFA, 2.5% H2O, 2.5% TIS) for 1 h × 2. The resulted solution was concentrated under reduced pressure and purified by HPLC (Agilent Technology, 1260 Infinity) with Zorbax Rx-C8 9.4 mm × 25 cm column. The peptide products were confirmed by MALDI-TOF mass spectrometry (Bruker Daltonics, Autoflex III smartbeam LRF200-CID). Methods for Cy3 Coupling to Peptide. A mixture of Cy3COOH (1.5 equiv), HATU (6 equiv) and triethylamine (6 equiv) in DMF was added to peptide 5 dissolved in DMF (final peptide concentration is 33 mM). After reaction for overnight, the Cy3 labeled peptide was purified by HPLC. Methods for Glycan Conjugation to Peptide. To prepare glycopeptides 14−22, mixture of aqueous CuSO4 (1 mM in final reaction solution), tris(triazolylmethyl)amine ligand69 in DMSO (1 mM in final reaction solution) and aqueous sodium ascorbate (8 mM in final reaction solution) was added into the mixture of peptide 9−11 (typically 2 mM in final reaction solution) and glycan 12 or 13 (3 equiv. according to peptide) in water. After 1 h of reaction, the product was purified by HPLC. Fluorous Quartz Beads Preparation. Commercial quartz beads (ca. 10 g, with diameter of 1 μm) were cleaned and activated by sonication in Piranha solution for 15 min, then washed with ultrapure water and dried in vacuo. A coating solution prepared with 200 mg of (3-(perfluorooctanamido)propyl)triethoxysilane in 100 mL of dry dichloromethane, with 0.25 v/v% of acetic acid was added into the flask containing cleaned and activated quartz beads and stirred vigorously for 20 h. Then the quartz beads were allowed to settle in the still reaction solution, so that the supernatant solution can be carefully removed. The quartz beads were further washed with methanol and ultrapure water repeatedly and dried in vacuo. Circular Dichroism Spectral Measurement of Fluorous Peptide on Quartz Beads. The fluorous coated quartz beads (2.5 mg) in 1 mL microcentrifuge tube were added ultrapure water (0.8

y=

Bmax · x KD,surf + x

x: The concentration of protein, y: the intensity of fluorescent signal, Bmax: Predicted maximum binding, KD, surf: surface dissociation constant. Surface Plasmon Resonance Assay. Surface plasmon resonance experiments were performed on the Biacore T200 at 25 °C using a F


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ACS Applied Materials & Interfaces functionalized CM5 sensor chip. Protein immobilization was performed according to the instrument build-in wizard template. The CM5 sensor chip was activated with a solution containing EDC (0.2 M) and N-hydroxysuccinimide (NHS) (0.05 M). 2G12 (10 μg/ mL) in sodium acetate buffer of pH 4.5 was injected over the activated flow cell at a flow rate of 10 μL/min for 420 s. Then 1 M ethanolamine at pH 8.5 was injected to block the remaining activated groups. Binding assays were performed with HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) as running buffer. Glycopeptides 20−22 were injected onto the surface, with several concentrations of ranging from 625 nM to 10 000 nM at the rate of 10 μL/min diluted in the running buffer. The surface was regenerated by 30 s injection of 3 M MgCl2. The sensorgrams were analyzed by Biacore T200 Evaluation Software, and the kinetic parameters were obtained by fitting curves to 1:1 Langmuir model.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13200. Synthesis and characterization of compounds and peptides; additional data for microarray experiment (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Cin-Hao Lin: 0000-0003-4581-8968 Sheng-Kai Wang: 0000-0002-3827-7983 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial supported from the Ministry of Science and Technology, R.O.C. We thank Prof. Chun-Cheng Lin and Prof. Jia-Cherng Horng for useful discussion. S.-K.W. received funding from the Ministry of Science and Technology, R.O.C. Grant NSC 103-2113-M-007MY2; MOST 104-2119-M-007-020; MOST 105-2113-M-007003; MOST 105-2633-M-007-002; MOST 106-2113-M-007009; MOST 106-2633-M-007-004.

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