Sungjin Park

Construction of Carbohydrate Microarrays by Using One-Step, Direct Immobilizations of Diverse Unmodified Glycans on Solid Surfaces

Carbohydrate microarrays have received great attention as high-throughput analytic tools in studies of carbohydrate-mediated biological processes. Most of the methods employed to fabricate glycan microarrays rely on the immobilization of modified glycans on the properly derivatized surfaces. This immobilization strategy requires the availability of modified glycans whose syntheses in many cases are time-consuming and difficult. We have developed a simple and direct immobilization technique that involves a one-step, site-specific attachment of diverse unmodified glycans to the hydrazide-derivatized glass surface. To demonstrate the generality of this direct immobilization method, we examined its use for the construction of carbohydrate microarrays containing a variety of glycans. The results of protein and cell-binding experiments indicate that the glycan microarrays, prepared by using this methodology, are applicable to the rapid evaluation of glycan-mediated biomolecular interactions and the determination of quantitative binding affinities between carbohydrates and proteins.

An apoptosis-inducing small molecule that binds to heat shock protein 70.

Apoptosis (or programmed cell death) is a fundamental biological process that regulates a variety of normal physiological processes, ranging from development to aging. Damaged or unwanted cells in organisms are removed by the intrinsic and/or extrinsic apoptotic pathways. The intrinsic apoptotic pathway occurs by the release of cytochrome c from mitochondria. The extrinsic apoptotic pathway is caused by the binding of death ligands, such as TNF (tumor necrosis factor), Fas, and TRAIL (TNF-related-apoptosis-inducing ligand), to their corresponding receptors. Although programmed cell death is involved in a number of key biological phenomena, aberrant apoptosis results in diverse human diseases. For example, the dysregulation of apoptosis disrupts tissue homeostasis by prolonging cell survival and contributes to the progression of diverse human tumors. In addition, retarded apoptosis causes the elimination of autoreactive lymphocytes to fail, leading to autoimmunity. Moreover, excessive apoptosis results in cell-loss disorders such as neurodegenerative (Alzheimer,s and Parkinson,s diseases) and cardiovascular diseases. Since apoptosis is involved in both normal physiology and various human diseases, research on apoptosis has become a central area in basic biological studies and in the development of therapeutic agents. Small molecules that either induce or prevent apoptotic cell death have significant potential as therapeutic agents to treat apoptosis-related diseases. In addition, these agents could also be employed to understand the roles that apoptotic regulatory proteins play in biological processes. Herein we describe a novel apoptosis-inducing small molecule which interacts with Hsc70 and Hsp70. Cell-based screening with a small molecule library is an attractive approach to identify bioactive compounds that regulate protein functions in cells or affect processes such as cell differentiation or morphology. We applied this approach to select molecules with apoptosis-inducing activity, using a recently prepared imidazole library on a solid support to identify bioactive compounds that induce interesting cellular events (Scheme 1a). The amine-conjugated diethylene glycol linker was introduced into the library for facile solid-phase synthesis and the identification of target protein(s) by affinity chromatography. To search for molecules that induce apoptosis in cells, 216 imidazole derivatives (1 mm) were incubated with the highly proliferative P19 embryonic carcinoma cell line for 3 h and subsequently treated with a mixture of annexin V-fluorescein (0.5 mgmL ) and propidium iodide (PI, 2 mgmL ) to rapidly screen for apoptosis inducers. The exposure of phosphatidylserine on the outer leaflet of the cell plasma membrane is a key feature of the early stages (2–4 h) of apoptosis. Phosphatidylserine can be detected fluorescently by using annexin Vfluorescein. Propidium iodide (PI) can be used to monitor membrane-perturbed cells which result from the plasma membrane becoming permeable (a feature of necrosis) or late-stage apoptosis. Therefore, the combined use of annexin V-fluorescein and PI allows for the rapid evaluation of apoptosis in cells treated with the compound library. In our screen, compounds that exhibited positive annexin V and negative PI staining in P19 cells after 3 h incubation were selected as inducers of apoptosis. However, compounds that showed positive annexin Vand positive PI staining in the cells were not selected as “hits” because it is possible that the treated cells underwent necrosis rather than apoptosis. One compound apoptozole-linker (Az-linker) showed a high level of positive annexin V and negative PI staining in P19 cells (Scheme 1b). For further studies, Az (without linker) was resynthesized and purified (see the Supporting Information). Scheme 1. Structures of a) an imidazole library (see the Supporting Information for substituents R–R) and b) apoptozole (Az).

Fabrication of carbohydrate chips and their use to probe protein|[ndash]|carbohydrate interactions

Carbohydrate microarrays have received considerable attention as an advanced technology for the rapid analysis of carbohydrate–protein interactions. This protocol provides detailed procedures for the preparation of carbohydrate microarrays by immobilizing hydrazide-conjugated carbohydrates on epoxide-derivatized glass slides. In addition, we describe the use we make of these microarrays in glycomics research. Unlike other techniques that require large amounts of samples and long assay times, carbohydrate microarrays are used to carry out the rapid assessment of a number of carbohydrate-recognition events with tiny amounts of carbohydrate samples. Furthermore, the microarray technology is also utilized for the rapid assay of enzyme activities. We are able to routinely prepare carbohydrate microarrays within 12 h by using hydrazide-conjugated carbohydrates and apply these microarrays for the studies of glycan–protein interactions within 8 h.

Probing Cell-Surface Carbohydrate Binding Proteins with Dual-Modal Glycan-Conjugated Nanoparticles

Dual-modal fluorescent magnetic glyconanoparticles have been prepared and shown to be powerful in probing lectins displayed on pathogenic and mammalian cell surfaces. Blood group H1- and Le(b)-conjugated nanoparticles were found to bind to BabA displaying Helicobacter pylori, and Le(a)- and Le(b)-modified nanoparticles are both recognized by and internalized into DC-SIGN and SIGN-R1 expressing mammalian cells via lectin-mediated endocytosis. In addition, glyconanoparticles block adhesion of H. pylori to mammalian cells, suggesting that they can serve as inhibitors of infection of host cells by this pathogen. It has been also shown that owing to their magnetic properties, glyconanoparticles are useful tools to enrich lectin expressing cells. The combined results indicate that dual-modal glyconanoparticles are biocompatible and that they can be employed in lectin-associated biological studies and biomedical applications.

One-step, acid-mediated method for modification of glass surfaces with N-hydroxysuccinimide esters and its application to the construction of microarrays for studies of biomolecular interactions

Microarray technologies have received considerable attention owing to the fact that they serve as powerful tools for the high-throughput analysis of biomolecular interactions and the identification of bioactive substances that bind to biomolecules. Most of the current methods used to construct microarrays rely on the immobilization of substances on properly derivatized surfaces. Among various functional groups used for this purpose, the N-hydroxysuccinimide (NHS) ester group has been largely employed because it can be readily reacted with amine or hydrazide functionalities in substances of interest. However, the NHS ester group is usually introduced onto the surface of a glass slide by employing inconvenient and time-consuming multistep processes. In recent studies, we have developed an efficient, single step method for derivatization of glass surfaces with NHS ester groups that takes advantage of an acid-mediated reaction of NHS ester functionalized dimethallylsilanes with silanols on the glass surface. Conditions for the surface modification procedure that utilize TfOH rather than Sc(OTf)(3) were found to be superior. Protein and RNA-binding experiments show that glass surfaces modified by employing this method are suitable for efficient immobilization of various substances that are appended by amine, hydrazide, and alcohol functionalities. The microarrays, generated in this way, are applicable to procedures for rapid analysis of protein-protein, protein-glycan, protein-small molecule, and peptide-RNA interactions, as well as for profiling enzyme activities. The newly developed acid-mediated, glass surface modification method should be generally applicable to the preparation of various functional group-modified surfaces.

Fluorescent glycan derivatives: their use for natural glycan microarrays.

Glycan microarrays have become powerful tools in the investigation of biological systems because they enable fast, quantitative, and simultaneous analysis of glycan-protein interactions with small quantities of samples. One hurdle in the construction of glycan microarrays is to obtain a diverse set of glycans for immobilization on surfaces. An article in this issue of ACS Chemical Biology describes the microscale preparation of fluorescently labeled reactive glycans in a cyclic form of the reducing sugar, from natural sources, that can be utilized directly to create natural glycan microarrays for use in functional glycomics.

Carbohydrate Analogue Microarrays for Identification of Lectin-Selective Ligands

Fifty‐five mono‐ and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin‐selective ligands. The microarray study showed that two disaccharide analogues, 28′ and 44′, selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28′ and 44′ selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.

Carbohydrate microarrays for enzymatic reactions and quantification of binding affinities for glycan-protein interactions.

Glycans are involved in a variety of physiological and pathological processes through interactions with proteins. Thus, the molecular basis of glycan-protein interactions provides valuable information on understanding biological phenomena and exploiting more effective carbohydrate-based therapeutic agents and diagnostic tools. Carbohydrate microarray technology has become a powerful tool for evaluating glycan-mediated biological events in a high-throughput manner. This technology is mostly applied for rapid analysis of glycans-protein interactions in the field of functional glycomics. In order to expand application areas of glycan microarrays, we have used carbohydrate microarrays for measurement of binding affinities between glycans and proteins and profiling of glycosyltransferase activities. The glycan microarrays used for these studies are constructed by immobilizing maleimide or hydrazide-conjugated glycans on the thiol or hydrazide-derivatized glass slides, respectively. This protocol describes the fabrication of carbohydrate microarrays and their applications to enzymatic reactions and determination of quantitative binding affinities.

Chemical microarrays constructed by selective attachment of hydrazide-conjugated substances to epoxide surfaces and their applications.

Microarray technology has received considerable attention for rapid analysis of biomolecular interactions and high-throughput screening to identify binding partners. An efficient and selective immobilization technique of substances on the surface is essential for successful construction of microarrays. Although a variety of immobilization methods have been exploited to prepare microarrays over the past decade, a superior technique needs to be developed for diverse applications. Recently, an efficient and simple method that relies on selective reactions between the hydrazide conjugated to substances and the epoxide derivatized on the solid surface was developed to fabricate chemical microarrays. Reactions between hydrazides with epoxides are highly selective in that they take place even in the presence of other potent nucleophiles such as amines and thiols. This technique is utilized to immobilize various substances such as small molecules, carbohydrates, and peptides to glass surfaces. The microarrays constructed by this immobilization method are used to evaluate protein binding to carbohydrates, peptides, and small molecules. In addition, the microarrays are also employed to determine binding affinities between proteins and binding partners as well as profiling of enzyme activities.