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CARBOHYDRATE-PRESENTING SELF-ASSEMBLED MONOLAYERS: PREPARATION, ANALYSIS, AND APPLICATIONS IN MICROBIOLOGY

Aline Debrassi1, Willem M. de Vos2,3, Han Zuilhof1,4, and Tom Wennekes1

1 Laboratory of Organic Chemistry, Wageningen University, Wageningen, the Netherlands

2 Laboratory of Microbiology, Wageningen University, Wageningen, the Netherlands

3 Department of Bacteriology & Immunology and Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland

4 Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah, Saudi Arabia

1.1 INTRODUCTION

Carbohydrates are a complex class of essential biomolecules that can be considered as the dark matter of the biological universe as they are greatly understudied yet omnipresent in all kingdoms of life and vital to fully understand biological processes. The structurally diverse carbohydrates are present both on the cell surface and inside cells. They decorate the cell surface to form the so-called glycocalyx, a dense and complex layer of carbohydrates unique for every type of cell or organism, and as such are key to many important biological recognition events by interacting with carbohydrate-binding proteins. Carbohydrate-protein interactions play an important role in various biological events occurring at the cell surface, such as bacterial and viral infections [1,2], cancer metastasis [3,4], and immune response [4]. The study of the interactions between carbohydrates and other biomolecules at biological surfaces and interfaces is instrumental in the understanding of these processes and contributing to the development of novel diagnostic methods and medicines. The study of carbohydrates, compared to, for example, nucleic acids and proteins, however, poses unique challenges because their structure is nonlinear and their biosynthesis not template driven. The native glycocalyx is too complex, dense, and dynamic for studying these interactions individually, with the current techniques at our disposal. Therefore, a simplified version is often created by the placement of well-defined, synthetic carbohydrates on a surface, so-called glycoarrays or glycosurfaces, to study specific carbohydrate-protein interactions. These fabricated glycosurfaces can also be more readily incorporated in a sensor or a nanostructure and as such used to elicit, detect, or quantify binding events, for example, in diagnostic devices, molecular imaging, and drug delivery applications. Various approaches have been developed to prepare glycosurfaces, each of them with their advantages and drawbacks, and these approaches will be the main focus of this chapter.

We will start the chapter by presenting an overview of the different methods most commonly used to prepare glycosurfaces. These methods will be discussed divided over three sections that each reflect one of the three distinct approaches used to create glycosurfaces: (i) direct formation of carbohydrate-containing self-assembled monolayers (SAMs), (ii) use of secondary (or tertiary) reactions to install a carbohydrate on a preformed SAM, and (iii) noncovalent immobilization of carbohydrates on a surface. The discussion of the secondary reaction approach (ii) is subdivided into two subsections: one addressing the use of unmodified "natural" carbohydrates and the other, the use of synthetic carbohydrate derivatives with a special emphasis on attachment using so-called "click" chemistry. Next, we will focus on several key surface analysis techniques that can be used to characterize a prepared glycosurface and the type of information that can be obtained from each technique. As previously mentioned, carbohydrate-protein interactions are involved in bacterial pathogenesis and symbiosis. A famous example of carbohydrate-mediated bacterial adhesion is between the gut microbiota and the carbohydrates present on the surface of human intestinal cells. Glycosurfaces can be used for the binding, capture, and sensing of gut bacteria. A representative example of this from our own group is the study of interactions between the mannose-specific adhesin of Lactobacillus plantarum [5]-a lactic acid bacterium present in various probiotic products, fermented foods, and our gut-and fabricated mannose-terminated glycosurfaces (vide infra) [6]. At the end of this chapter, we will discuss several more applications of glycosurfaces in microbiology, focusing on binding, capture, and sensing of bacteria and bacterial toxins and on the multivalency effects that exert a large influence on the interaction between carbohydrates and proteins in biological systems and on fabricated glycosurfaces.

1.2 PREPARATION OF SAMs CONTAINING CARBOHYDRATES

SAMs are ordered molecular assemblies that spontaneously form on a substrate by chemisorption (or strong interaction) of molecules containing a chemical functionality with a strong affinity for the substrate surface. The chemical structure of molecules that are used to prepare a SAM is usually subdivided in its constituting parts; the part that adsorbs on the substrate surface can be called the attaching group, the part on the opposing end of the molecule that ends up at the top of the monolayer is called the end group or terminal group, and the intermediate part is called the chain or backbone [7,8]. In this section, we will present an overview of the recent scientific literature on the preparation and properties of SAMs containing carbohydrates as end groups (Table 1.1).

TABLE 1.1 Approaches Used for the Direct Preparation of Carbohydrate-Presenting SAMs

(a) Thiol on gold, (b) disulfide on gold (monovalent binding), (c) disulfide on gold (multidentate binding), (d) alkene on silicon, (e) alkyne on silicon, (f) phosphonic acid on aluminum oxide, and (g) silane on silica.

One of the most common combinations of substrate and attaching group is the formation of SAMs of thiols on gold (Table 1.1; entry a), and to our knowledge, this was also the first example of a carbohydrate-presenting SAM. In 1996, Spencer and coworkers reported the formation of SAMs on gold surfaces with a thiol-terminated hexasaccharide. The thiol-terminated hexasaccharide, a truncated amylose derivative consisting of six a-1,4-linked glucopyranosides, was assembled on gold surfaces in its protected (peracetylated) and deprotected form. Both protected and deprotected compounds readily formed SAMs on gold, although the kinetics of SAM formation varied, with the deprotected hexasaccharides achieving an SAM with higher density. The protected hexasaccharide was also successfully deprotected on the surface after the SAM formation: however, the degree of deprotection was slightly lower than when conducted in solution before SAM formation [24]. These early studies already indicate that thiol SAMs on gold are best prepared directly with deprotected carbohydrate derivatives in order to circumvent incomplete deprotection of carbohydrates on the surface and degradation of the unstable thiol on gold SAM itself.

Using a similar approach, Russell and coworkers [9] synthesized protected and deprotected thiol-terminated monosaccharides that were assembled as SAMs on gold-coated glass substrates and afterwards assessed for their interaction with a series of lectins. The SAM formed with a thiol-terminated mannose derivative was exposed to concanavalin A (Con A), a lectin known to bind strongly with mannose, and a lectin from Tetragonolobus purpureas, which specifically binds L-fucose. As expected, the mannose-terminated SAM showed selective interaction with Con A, demonstrating that carbohydrate-presenting SAMs can be used to study interactions between carbohydrates and proteins as a simplified version of natural cell surfaces [9].

Houseman and Mrksich [18] were the first to prepare mixed SAMs that consisted of various ratios of a carbohydrate and oligoethylene glycol end group, in which the latter was incorporated to minimize nonspecific interactions. The authors prepared SAMs using N-acetylglucosamine and tri(ethylene glycol) with thiol attaching groups and studied the effect of the concentration of N-acetylglucosamine in the monolayer on an enzymatic reaction [18]. Later in this chapter, we will further discuss the strategy of using molecules to "dilute" the amount of carbohydrate on a surface and thereby tune the carbohydrate presentation and concentration (multivalency effect and optimization of density; page 50).

The relatively easy preparation of thiol SAMs on gold and high tolerance for additional functional groups, including carbohydrate hydroxyls, have made it a popular method to immobilize also other carbohydrates with various levels of complexity: monosaccharides (mannose [10-14], glucose [15-17,32], galactose [13,16,17,37], xylose [17], rhamnose [17]), disaccharides (lactose [15], maltose [17,19], dimethylated maltose [17]) [20,22,23], and oligosaccharides (GM1 pentasaccharide [15], globotriose [21], maltotriose [17]) [37].

A general drawback of SAMs created by the adsorption of thiols on gold is their relative limited stability. In order to increase the stability of SAMs on gold, some research groups have prepared SAMs with molecules that can form multiple bonding interactions with the substrate (multidentate...