糖类的毛细管电泳及芯片毛细管电泳

 糖类化合物在生物体内发挥多方面的作用。糖研究的复杂性在于其结构的复杂多变。高效毛细管电泳作为一种快速、高效的分离分析手段已广泛应用于糖的研究。芯片毛细管电泳是近几年来发展起来的新的分析技术 ,并已经在生命科学的研究中得到较广泛的应用。就各种糖类化合物的毛细管电泳的分析策略、检测条件及糖类化合物的芯片毛细管电泳进行了阐述 ,共 4 8篇。     

Carbohydrates as the next frontier in pharmaceutical research

Synthetic carbohydrates and glycoconjugates are used to study their roles in biological important processes such as inflammation, cell-cell recognition, immunological response, metastasis, and fertilization. The development of an automated oligosaccharide synthesizer greatly accelerates the assembly of complex, naturally occurring carbohydrates as well as chemically modified oligosaccharide structures and promises to have major impact on the field of glycobiology. Tools such as microarrays, surface plasmon resonance spectroscopy, and fluorescent carbohydrate conjugates to map interactions of carbohydrates in biological systems are presented. Case studies of the successful application of carbohydrates as active agents are discussed, for example, fully synthetic oligosaccharide vaccines to combat tropical diseases (e.g., malaria), bacterial infections (e.g., tuberculosis), viral infections such as HIV, and cancer. Aminoglycosides serve as examples of drugs acting through carbohydrate-nucleic-acid interactions, while heparin works by carbohydrate-protein interactions. A general, modular strategy for the complete stereoselective synthesis of defined heparin oligosaccharides is presented. A carbohydrate-functionalized fluorescent polymer has been shown to detect miniscule amounts of bacteria faster than commonly used methods.     

Carbohydrate–protein interactions and their biosensing applications

Carbohydrate recognition is clearly present throughout nature, playing a major role in the initial attachment of one biological entity to another. The important question is whether these prevalent interactions could provide a real suitable alternative to the use of antibodies or nucleic acid for detection and identification. Currently, examples of carbohydrates being employed in biological detection systems are limited. The challenges of using carbohydrate recognition for detection mainly come from the weak affinity of carbohydrate–protein interactions, the lack of versatile carbohydrate scaffolds with well-defined structures, and the less developed high-information-content, real-time, and label-free assay technology. In this review, we focus on discussing the characteristics of carbohydrate–protein interactions in nature and the methods for carbohydrate immobilization based on surface coupling chemistry in terms of their general applicability for developing carbohydrate- and lectin-based label-free sensors. Furthermore, examples of innovative design of multivalent carbohydrate–protein interactions for sensor applications are given. We limit our review to show the feasibility of carbohydrate and lectin as recognition elements for label-free sensor development in several representative cases to formulate a flexible platform for their use as recognition elements for real-world biosensor applications.     

Carbohydrate arrays for the evaluation of protein binding and enzymatic modification

This paper reports a chemical strategy for preparing carbohydrate arrays and utilizes these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate chips were prepared by the Diels-Alder-mediated immobilization of carbohydrate-cyclopentadiene conjugates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups. Surface plasmon resonance spectroscopy showed that lectins bound specifically to immobilized carbohydrates and that the glycol groups prevented nonspecific protein adsorption. Carbohydrate arrays presenting ten monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of beta-1,4-galactosyltransferase. Finally, a strategy for preparing arrays with carbohydrates generated on solid phase is shown. This surface engineering strategy will permit the preparation and evaluation of carbohydrate arrays that present diverse and complex structures.     

Clinical aspects of altered glycosylation of glycoproteins in cancer

Alteration of the expression of carbohydrate structures is frequently observed in tumor cells. This review summarizes the different changes of O- and N-linked glycoproteins observed in cancer cells, the impact of the tumor-related carbohydrate phenotypes on the clinical outcome of the cancer disease, and the various ways in which carbohydrate structures can interact with different carbohydrate-detecting adhesion molecules, selectins, and sialoadhesins. Various ways of inhibiting the formation of cell adhesion-engaged carbohydrates on the cell surface, or inhibiting the binding are discussed. Carbohydrate structures which are in clinical use as circulating tumor markers and the effect of genotypes on tumor marker concentrations are reviewed.     

Oligodentate glycoconjugates based on calixarenes: methods for the synthesis and biological activity

The review considers principal methods for the preparation of conjugates of carbohydrates with calixarenes and resorcinarenes. The data on the biological activity of glycoconjugates are discussed taking their interaction with the carbohydrate-binding proteins, lectins as an example.     

State-of-the-art of capillary electrophoresis with application to the area of glycoconjugates

Glycoconjugates constitute a major group of biomolecules, which participate in several biological functions and processes. Their carbohydrate components play key roles in determining the properties of glycoconjugates and, therefore, analysis and structural characterization of carbohydrates are essential. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of carbohydrates. In this review the principles of high-performance capillary electrophoresis; mechanisms employed for glycoconjugate analysis as well as the various detection techniques used are summarized.     

Carbohydrate microarrays by microcontact "click" chemistry

Carbohydrate microarrays can be prepared by microcontact printing of carbohydrate alkyne conjugates on azide self-assembled monolayers (SAMs). The carbohydrates are immobilized by a "click" reaction in the contact area between the stamp and the substrate. The immobilized carbohydrates retain their characteristic selectivity toward lectins.     

Recent Progress in Enzymatic Synthesis of Sugar Nucleotides

Glycosylation is one of the most important reactions in nature as it results in the formation of glycoconjugates with diverse biological functions. Sugar nucleotides serve as the natural donor molecules for the biosynthesis of such glycoconjugates and other carbohydrates. Furthermore, these donor molecules are also indispensable building blocks for the enzymatic synthesis of carbohydrates in vitro using Leloir-type glycosyltransferases. Given such importance, the biosynthetic pathways of sugar nucleotides have been exploited, enabling the development of both chemical and enzymatic approaches to produce these molecules. A survey of recent progress in enzymatic synthesis of common mammalian sugar nucleotides as well as their derivatives is thus presented. As a popular strategy, conjugation of sugar nucleotide synthesis with glycosyltransfer reactions and in vivo production of sugar nucleotides are also included.     

Covalent immobilization of carbohydrates on sol-gel-coated microplates

Carbohydrate microarrays have attracted increasing attention in recent years because of their ability to monitor biologically important protein-carbohydrate interactions in a high-throughput manner. Here we have developed an effective approach to immobilizing intact carbohydrates directly on polystyrene microtiter plates coated with amine-functionalized sol-gel monolayers. Lectin binding was monitored by fluorescence spectroscopy using these covalent arrays of carbohydrates that contained six mono- and di-saccharides on the microplates. In addition, binding affinities of lectin to carbohydrates were also quantitatively analyzed by determining IC(50) values of lectin-specific antibody with these arrays. Our results indicate that microplate-based carbohydrate arrays can be efficiently fabricated by covalent immobilization of intact carbohydrates on sol-gel-coated microplates. The microplate-based carbohydrate arrays can be applied for screening of protein-carbohydrate interactions in a high-throughput manner.     

Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates

Carbohydrate analysis has traditionally been viewed as a specialty science, performed only in a few well-established laboratories using conventional carbohydrate analysis technology (e.g. NMR, gas chromatography-mass spectroscopy, high-performance liquid chromatography, capillary electrophoresis) combined with the specialized technical training that has been essential for accurate interpretation of the data. This tradition of specialized laboratories is changing, due primarily to an increase in the number of scientists performing routine carbohydrate analysis. As a result, many scientists who are not trained in traditional carbohydrate analytical techniques now need to be able to perform accurate carbohydrate analysis in their own laboratories. This has created a need for technically simple and inexpensive methods of carbohydrate analysis. In this review, we present application vignettes of a technically simple, yet analytically powerful method called fluorophore-assisted carbohydrate electrophoresis (FACE). FACE can be used for performing routine oligosaccharide profiling, monosaccharide analysis, and sequencing of a variety of carbohydrates.     

Carbohydrate microarrays: an advanced technology for functional studies of glycans

The biological significance of glycans in the post-genomic era requires the development of new technologies to enable functional studies of carbohydrates in a high-throughput manner. Recently, carbohydrate microarrays have been exploited as an advanced technology for this purpose. Efficient immobilization methods for carbohydrate probes on the proper surface are essential for the successful fabrication of carbohydrate microarrays. Up to date, several techniques have been developed to attach simple or complex carbohydrates to a solid surface. The developed glycan microarrays have been applied for functional glycomics, drug discovery, and diagnosis. In this concept article, we discuss the progress of immobilization methods of carbohydrates on solid surfaces, their potential uses for biological research and biomedical applications, and possible solutions for some remaining challenges to improve this new technology.     

The immunological function of iGb3

The mechanistic studies on immune recognition of carbohydrates have been paved by the synergized advances in identifying the precise sugar structures recognized by the immune system, in analyzing the cellular and humoral components bearing the receptors for glycoconjugates, and production of the biological relevant carbohydrate epitopes by synthetic chemistry. In our current studies on natural antigenic glycolipids, we have found that the activation as well as the development of natural killer T cells (NKT) is guided by the information provided by glycolipid metabolism pathways in antigen presenting cells (APC). Based on genetic data and cellular immunological assays, we propose a neutral glycosphingolipid isoglobotrihexosylceramide, iGb3, as one of the candidates recognized by NKT cells under patho-physiological conditions such as cancer and auto-immune disease. New immunotherapy approaches might be explored by interfering with glycolipid metabolism or by directly supplementing rationally designed glycolipids.     

Assembly of a three-dimensional array of glycoconjugates by combinatorial biocatalysis in nonaqueous media

Glycoconjugates can be artificially synthesized by combinatorial biocatalysis. An example is given in this paper describing the construction of glycoconjugates array by using glycosidase and lipase in nonaqueous media. This array was started from glucose, with three aryl alcohols as the aglycone moiety of glycosides and five acids or esters as acyl donors for combinatorial acylation of glycosides, affording a three-dimensional array containing about 30 members with diverse structures. The array would be more abundant if more aglycones and acyl donors with other structures were filled in. Indeed, diverse classes of carbohydrates besides glucose can also be employed for generating diverse glycoconjugates due to their different roles in numerous physiological responses. The composition and distribution of the demonstration glycoconjugates array was detected and evaluated by HPLC-MS with electrospray ionization. And also, the distribution of the artificial array can be adjusted by changing the molar ratio of the auxiliary materials.     

Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging

A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length.     

The degradation pattern of oligomers and polymers from lignocelluloses

Only the carbohydrate portion in lignocelluloses can become a source of food, feed, and chemicals. At different stages of the biological conversion processes, carbohydrates are present as polymers, oligomers or monomers. Carbohydrate samples from ligno-celluloses contain many by-products originating from the material (lignin, extracted substances, silica) or from the conversion processes (enzyme proteins, acids, fermentation by-products). Liquid-chromatography is used with emphasis on performance rather than pressure for highly contaminated samples. Most of the systems used here have an integrated post-column reaction system for the detection of carbohydrates. Monomeric and some oligomeric sugars are analyzed by borate ion-exchange chromatography. As an example of application of the system, the products of the stepwise enzymatic degradation of 4-O- methylglucuronoxylan are identified. Oligomeric sugars are separated by polyacrylamide gel chromatography. This system is used to optimize a pentose-to-ethanol process by thermophilic anaerobic bacteria. Polymers such as cellulose can only be analyzed by liquid chromatography after derivatization. The molecular-weight distribution of carbanilated birchwood celluloses after pretreatment and different stages of enzymatic attack is described. The large through-put of the automated units justifies their high cost.     

Dynamic Kinetic Stereoselective Glycosylation via RhII and Chiral Phosphoric Acid-Cocatalyzed Carbenoid Insertion to the Anomeric OH Bond for the Synthesis of Glycoconjugates

Chemical synthesis of glycoconjugates is essential for studying the biological functions of carbohydrates. We herein report an efficient approach for the stereoselective synthesis of challenging α-linked glycoconjugates via a Rh^II/chiral phosphoric acid (CPA)-cocatalyzed dynamic kinetic anomeric O-alkylation of sugar-derived lactols via carbenoid insertion to the anomeric OH bond. Notably, we observed excellent anomeric selectivity, excellent diastereoselectivity, broad substrate scope, and high efficiency for this glycosylation reaction by exploring various parameters of the cocatalytic system. DFT calculations suggested that the anomeric selectivity was mainly determined by steric interactions between the C2-carbon of the carbohydrate and the phenyl group of the metal carbenoid, while π/π interactions with the C2−OBn substituent on the carbohydrate substrate play a significant role for diastereoselectivity at the newly generated stereogenic center.     

Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes

Carbohydrate microarrays have become very powerful tools to elucidate the molecular basis of carbohydrate-recognition events in a high-throughput manner. This microarray technology has been applied in the rapid analysis of the binding properties of a variety of binding partners such as lectins, antibodies, mammalian cells, pathogens and viruses. In this feature article, methods for the preparation of carbohydrate microarrays and their applications in biological and biomedical research are described.     

Recent developments in sample preparation for chromatographic analysis of carbohydrates

Carbohydrates are a very important group of compounds due to their roles as structural materials, sources of energy, biological functions and environmental analytes; they are characterized by their structural diversity and the high number of isomers they present. While many advances have been made in carbohydrate analysis, the sample preparation remains difficult. This review aims to summarize the most important treatments which have been recently developed to be applied prior to the analysis of carbohydrates by chromatographic techniques. Due to the multiplicity of structures and matrices, many different techniques are required for clean-up, fractionation and derivatization. A number of new techniques which could be potentially adequate for carbohydrate characterization have also been revised.     

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.