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An Approach to the High-Throughput Fabrication of Glycopolymer Microarrays through Thiol−Ene Chemistry Kevin Neumann,† Antonio Conde-González,† Matthew Owens, Andrea Venturato, Yichuan Zhang, Jin Geng,* and Mark Bradley* EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, U.K. S Supporting Information *

ABSTRACT: The fabrication of microarrays consisting of well-defined glycopolymers is described. This was achieved by postfunctionalization of an immobilized poly(allyl glycidyl ether) using unprotected thiol-modified carbohydrates through thiol− ene conjugation chemistry. This enabled the fabrication of glycopolymer microarrays in which the density and composition of the carbohydrate moieties varied along each of the polymer chains displayed across the array. These glycopolymer microarrays were applied in the evaluation of multivalent ligand−protein interactions with the determination of surface dissociation constants (KD) with concanavalin A and Ricinus communis agglutinin I for surface immobilized mannose-, glucose-, and galactose-containing glycopolymers and validated in solution with ITC.

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INTRODUCTION It is now well appreciated that carbohydrates, presenting as free oligosaccharides or as glycoconjugates, act to transmit information in a plethora of biological processes.1,2 Indeed, specific interactions between carbohydrates and proteins play pivotal roles in viral and bacterial infection, pathogen invasion, and innate immunity.3−7 Research in this area has led to glycan mimetic anti-infective and anti-inflammatory drugs and with potential in future diagnosis.8 However, the study of the structural diversity and functions of glycans remains a challenging discipline. In nature, protein-binding carbohydrates often display higher order structures with “polydentate” binding helping to circumvent the intrinsically weak binding displayed by monovalent carbohydrate ligands.9,10 Such binding activity enhancements can also be achieved synthetically via the application of so-called multivalent polymers or “glycopolymers” that mimic the ligand presentation properties of native glycoconjugates.11−16 Synthetic glycopolymers have increasingly served as tools for the investigation and understanding of the various parameters that govern multivalent interactions between sugars and their receptors; however, the design principles and mechanisms of action of multivalent interactions between glycans and their binding lectins are still not fully understood.17−21 Glycopolymers have been synthesized previously by either controlled living polymerization strategies using carbohydratebased monomers or carbohydrate decoration of preformed polymers,22 resulting in well-defined macromolecular architec© XXXX American Chemical Society

tures, both linear and globular, as well as through automated glycan assembly on solid support. 14,23−26 In addition, monodisperse glycodendrimers have been constructed displaying multiple complementary carbohydrate ligand binding sites.27,28 Glycopolymers can be studied by several techniques such as surface plasmon resonance and isothermal titration calorimetry to investigate multivalent glycan−receptor interactions.22,29,30 Because of the high number of glycopolymers that can be generated with variations in size and composition, microarray technology has become an essential tool for the investigation of carbohydrate−protein interactions. Previously, a number of chain-end-functionalized glycopolymers have been surfaceimmobilized to yield oriented and density controlled glycopolymer microarrays,31−33 including mucin mimetic glycopolymers immobilized onto azide functionalized surfaces,34 and direct polymerization from a thiol-functionalized surface to generate arrays of glycopolymer brushes.35 Although polymer libraries have been fabricated in situ by inkjet-mediated approaches for the elucidation of biologically relevant surface interactions36−40 including RAFT polymerization,41 the generation of arrays of well-defined glycopolymers for microarray application still remains a challenge. Here, we report a robust strategy for the in situ synthesis of an array of glycopolymers using an inkjet fabrication approach. Received: May 9, 2017 Revised: July 19, 2017

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DOI: 10.1021/acs.macromol.7b00952 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. (a) (i) Bromoethanol, BF·OEt2, DCM; (ii) thioacetic acid, Cs2CO3, DMF; (iii) sodium methanolate, methanol (pH 9); (iv) bromoethanol, BF·OEt2, reflux followed by pyridine/acetic acid anhydride (3/2). (b) Reaction of the allyl moieties of PAGE with the thiol-modified carbohydrate resulting in thioether linked glycopolymers. (c) (i) (Trimethoxylsilyl)propyl isocyanate, THF, 24 h; (ii) PAGE, DMF, 24 h; (iii) thiol sugars (10, 11, 12), DMF, UV (365 nm), 2 h. XPS spectra for PAGE immobilized on a glass surface: (d, e) without glucose; (f, g) with glucose modification.

and bromide displacement with thioacetic acid, and hydrolysis (sodium methoxide) to give the free carbohydrate thiol 12. The thiol−ene reactivity of the thiol-modified sugars with PAGE was demonstrated using 2,2-dimethoxy-2-phenylacetophenone (DMPA) as the radical initiator (1 equiv to polymer) with 2 h of UV irradiation on a passive glass surface showing full conversion (as determined by NMR). In order to fabricate the array, PAGE was successfully covalently immobilized onto the surface of glass slides by attaching the terminal hydroxyl group of the PAGE to isocyanate-modified glass, with functionalization of the isocyanate and immobilization of the PAGE confirmed by IR (the sharp band of the isocyanate disappeared after treating the glass with PAGE, resulting in the characteristic carbamate band (see Supporting Information)). In addition, X-ray photoelectron spectroscopy (XPS) analysis after reaction between the glucose thiol 12 and the immobilized PAGE showed the appearance of electrons correlating to the 1s orbital of the anomeric carbon that has a reported energy value of 288 eV (Figure 1f).49 Next, an array of glycopolymers was printed in which the three thiolated sugar inputs were varied. Initially, the influence of the size of the PAGE in the resulting glycopolymer and its lectin binding properties was explored. Glycopolymers based on PAGE 2500 or 5000 Da were prepared in situ with galactose 11 and varying levels of either glucose 12 or mannose 10. Following incubation with fluorescently labeled lectins, the galactose binding Ricinus communis Agglutinin I (RCA I) and the mannose/glucose selective lectin concanavalin (Con A), binding was quantified via fluorescence quantification of each array spot. Arrays were composed of 312 glycopolymers (104 independent features, 3 replicates) (Table S2). Analysis of the arrays in which the sole variant was the size of the PAGE (2500 or 5000 Da) showed that the longer polymer with higher "valency" (43 repeating carbohydrate units compared to 22 units in total) exhibited high affinity and stronger lectin

Since glycopolymers differ in the nature and density of the sugar moieties presented on the polymer scaffold, we explored the use of photoinitiated thiol−ene chemistry to postfunctionalize a narrow disperse poly(allyl glycidyl ether) (PAGE) scaffold using thiol-modified sugars.42,43 This method thus allows for the generation of glycopolymers with varying sugar moieties presented across a common polymer scaffold with identical chain length and chain length distribution, which is not possible to control by polymerization of glycosylated monomers.44 Using this platform, we identified lectin binding glycopolymers, whose binding affinity was confirmed in scaleup by ITC.

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RESULTS AND DISCUSSION To evaluate combinations of carbohydrate units, thiol−ene chemistry was utilized allowing rapid and efficient polymer modification via inkjet printing, following recent reports of the efficient conjugation of a thiol-modified carbohydrate and a terminal alkene using UV light.45 The carbohydrates (glucose, galactose, and mannose) used in this study were modified to display a thiol, linked to the anomeric position via a C2 spacer to ensure flexibility and accessibility. PAGE was used as a scaffold as it has been shown to be suitable for several postpolymerization modifications, via pendant alkenes, while importantly having a water-soluble backbone,46,47 allowing thiol-modified carbohydrate introduction at high density. The three model thiolated carbohydrates were prepared from α-D-mannose (1), β-D-galactose (2), and βD-glucose (3) via substitution at the anomeric position with bromoethanol (Figure 1) followed by thiol incorporation. For α-D-mannose and β-D-galactose the protocol of Rahimipour was used, starting from pentaacetate-protected carbohydrates 10 and 11.48 For β-D-glucose anomeric substitution was achieved through the reaction of the unprotected carbohydrate with bromoethanol at high temperature, with subsequent acetylation B

DOI: 10.1021/acs.macromol.7b00952 Macromolecules XXXX, XXX, XXX−XXX

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Figure 2. (a) Images of the glycopolymer microarray (poly(Gal/Glc)) incubated with Dylight594-labeled RCA 120 (83 nM). λex/λem = 550 nm/570 nm. (b−e) Fluorescence intensity against the density of the galactose epitope along the polymer chain (n = 3). Scale bar = 1.5 mm. F/1000 is observed fluorescence intensity/1000.

where Fmax is the maximum fluorescence, [P] is the total lectin concentration, and KD is the equilibrium dissociation constant for surface immobilized glycopolymers and lectin (Figure 3a).

binding, and thus the 5000 Da polymer was utilized for all subsequent arrays (Figure S23). Specific binding of lectins was investigated by printing microarrays with galactose 11 and varying levels of either mannose 10 or glucose 12. We examined binding of Dylight 594-RCA I 120, a dimeric, galactose binding lectin, with a clear relationship observed between the amount of lectin bound to the surface and the galactose/glucose composition of the glycopolymer (Figure 2). Figure 2a shows an image of the glycopolymer array (with galactose and glucose immobilized) after incubation with Dylight 594-RCA I 120, with the signal intensity of an individual spot depending on the density of complementary carbohydrate along the polymer (Figure 2b). To quantify glycopolymer−protein interactions on the surface, the dissociation constants (KD) for all glycoside combinations were determined following the method of MacBeath,50 by plotting lectin concentrations against fluorescence intensity of different combinations of printed carbohydrates. The array was used to establish the binding profiles of lectin−glycopolymer affinities onto the surface. When the system reaches equilibrium during the incubation step, the mean fluorescence of features (Fobs) can be described by Fobs =

Fmax[P] KD + [P]

Figure 3. (a) Binding curves for galactose-containing glycopolymer arrays with galactose units at different densities alone the polymer chain. The arrays were incubated with lectin (Dylight594-labeled RCA 120) from 25 nM up to 166 nM. (b) Dissociation binding constant (KD) with the lectin RCA 120 as a function of the number of galactose units per polymer chain.

The KD values obtained are shown in Table S24. With a low density of galactose present in the polymer, for example, 20% and 28%, KD are 657 and 663 nM, respectively, and incubation of the lectin at low concentrations (25, 42, and 56 nM), the binding affinity is low as the amount of galactose reaches a critical density. These binding profiles for RCA 120 clearly showed valency-dependent binding to the glycopolymer with

(1) C

DOI: 10.1021/acs.macromol.7b00952 Macromolecules XXXX, XXX, XXX−XXX

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Figure 4. (a) Glycopolymers with different galactose densities on the polymer binding with Dylight594-RCA 120 (83 nM). (b) Binding map of glycopolymers containing galactose and glucose/mannose with Dylight594-RCA 120 (83 nM).

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CONCLUSIONS In summary, we have developed a microarray platform in which a library of glycopolymers that only differ in their epitope density and composition were prepared in situ on a surface by a highly efficient photoinitiated thiol−ene chemistry. This approach generated a highly dense array of varying sugar epitopes, allowing rapid access to a broad range of glycan units on the glycopolymer and, perhaps most significantly, the ability to carry out the parallel synthesis and screening of up to 1350 different glycopolymers on a single glass chip. The polymer, PAGE, has a water-soluble PEG backbone and thus offers advantages in the context of the preparation of glycopolymers for biological application and postfunctionalization processing. In particular, we observed that lectins can differentially recognize and bind glycans presented on glycopolymers. Further work will be performed using sequence-controlled precision glycopolymers to evaluate the glycan sequence and location along the polymer and their impact on specific lectin binding properties.51 The glycopolymer array system described here is a practical tool for high-throughput analysis and quantification of multivalent ligand−protein interactions on surfaces. It is anticipated that this platform could be used to mimic cell− surface interactions for the elucidation of adhesion mechanisms and subsequent development of multivalent inhibitors.

galactose residues. The relative binding affinity of lectin increased as the number of galactose units increased (Figure 3b). Additionally, the same trend was observed using the mannose-binding lectin, Con A (Figure S22), with enrichment with increasing levels of mannose. As validation, isothermal titration calorimetry (ITC) was used to determine the KD value in solution of a galactose only containing glycopolymer and a galactose/glucose (50/50%) containing glycopolymer giving a measured in a KD of 920 and 3810 nM, respectively (see Figures S19 and S20), showing that the glycopolymers identified from the microarray screen can be transferred to applications in solution. When galactose was printed in the absence of a secondary carbohydrate (i.e., without mixing with glucose or mannose), we observed a significant difference in lectin binding. For example, a polymer containing 20% galactose and 80% allyl ether units gave a 5-fold decrease in binding affinity toward RCA 120 (Figure 4a) compared to a glycopolymer printed with the same amount of galactose but with 80% glucose (Figure 2d). Only a small increase in fluorescence was observed for galactose concentrations below 30%, which was due to the low density of galactose and poor water solubility of the polymer as only a small portion of hydrophobic allyl ether were modified with galactose. Even at high concentrations of galactose, the remaining allyl groups can keep the polymer condensed at the surface, thus prohibiting lectin binding, resulting in a lower fluorescence intensity. A larger microarray with glycopolymers consisting of different combinations of the three carbohydrates was also fabricated (Figure 4). Galactose was printed by mixing with both glucose and mannose, thus forming glycopolymers containing three different sugars along the polymer chains. 1350 glycopolymers (450 independent features, 3 replicates) were obtained in situ and probed with Dylight 594-RCA I 120. The binding of the lectin to the surface was again observed to be valency-dependent. Interestingly, galactose-containing glycopolymers cofunctionalized with mannose gave higher fluorescent intensities than those with glucose, meaning stronger binding with lectin RCA 120 (Figure 4b). These results reveal that glycopolymers with high selectivity and affinity depend not only on the number of binding carbohydrate epitopes present on the polymer backbone but also how the carbohydrates are copresented.

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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/acs.macromol.7b00952. Data analysis and details of synthesis of sugars and polymers and fabrication of microarrays (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jin Geng: 0000-0003-2181-0718 Author Contributions †

K.N. and A.C.-G. contributed equally.

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Macromolecules Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work funded by the European Research Council (Advanced Grant ADREEM ERC-2013-340469). REFERENCES

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