糖肽的化学合成研究进展

糖蛋白在生命活动中扮演着重要的角色,如细胞信号识别、神经调控、细胞间的信息传达及免疫调节等,因此引起了人们的广泛关注。糖肽是位于糖蛋白核心结构区域的由糖基与氨基酸或多肽链以共价键相连而形成的缀合物。含有糖肽的物质与糖蛋白相比,具有多种相似的重要生物功能,同时又没有糖蛋白巨大的分子量和复杂的结构,因此成为研究糖蛋白的重要模型。目前,糖肽合成技术已发展为生化学家探索研究并明确解释糖蛋白科学中相关问题的有利工具,成为目前化学合成领域的研究热点之一。本文综述了近年来在糖肽化学合成方面的研究进展,主要包括天然常见的N-连接糖肽和O-连接糖肽以及不常见的C-连接糖肽和S-连接糖肽的合成,并对糖肽合成的策略,即直线型合成法、汇聚式合成法、直线－汇聚混合法和自然连接合成法进行了简述与评价。     

Glycosylation characterization of Human IgA1 with differential deglycosylation by UPLC-ESI TOF MS

Differential deglycosylation was introduced as an effective technique to characterize glycosylation in glycoprotein containing both N-linked and O-linked glycans at both protein and peptide levels. Human IgA1 was used as a model glycoprotein to demonstrate this technique. The glycans attached to Human IgA1 were removed from their attachment sites by an array of enzymes. After reduction by DTT, the resulting deglycoproteins were analyzed by UPLC-ESI TOF MS to estimate the numbers of N-glycan and O-glycan sites through differential masses. The deglycoproteins and unmodified glycoprotein were further digested to deglycopeptide through trypsin digestion. The glycopeptides and deglycopeptides were identified by UPLC-ESI TOF MS. Two N-glycan and four O-glycan sites were identified and confirmed at peptide levels. These results matched those from deglycoproteins. The N-glycosylation site and N-glycan sequence confirmation were also demonstrated in this study.     

Roles of glycans and glycopeptides in immune system and immune-related diseases

Almost all of the key molecules in organisms involved in the innate and adaptive immune response (including immunoglobulins, cytokines and cytokine receptors, complements, CD molecules, adhesions, T-cell receptors and major histocompatibility complex molecules) are glycoproteins. Besides, foreign antigens, such as many viral envelope proteins, are glycoproteins too. Carbohydrates attached to proteins or peptides are classified by the nature of their linkages to the protein, mostly as either N-linked (N-acetylglucosamine to asparagines) or O-linked (N-acetylgalactosamine to serine or threonine) oligosaccharides. The glycans have three major roles: firstly, the sugars confer stability on the proteins to which they are attached, protecting them from proteases and non-specific protein-protein interactions. Secondly, glycans play key roles in signal transduction, control of cell development and differentiation. Thirdly, specific regions of the oligosaccharide chains provide recognition epitopes, which influence innate and adaptive immune responses. Glycopeptides not only provide specific oligosaccharides, but also have specific information of amino acids sequences. The glycans and glycopeptides not only influence the structure and functions of immune molecules, but also influence the immune response. In addition, changes in glycans or glycopeptides may have a significant role in a variety of human immune-related diseases, such as rheumatoid, autoimmune disease, Wiskott-Aldrich syndrome, infection disease, cancer, etc. In this article, the roles of N-, O-glycans and glycopeptides in immune system and immune-related diseases are discussed. The potential therapeutic significance of the information is also mentioned.     

Characterization of oligosaccharide moieties of rat intestinal phytase

Phytase of Rat Intestine had a large amount of oligosaccharides; The enzyme consisted of two different subunits with the molecular weights of 90 KDa and 70 KDa in its intact form, whereas the apparent molecular weights tumed to 72 KDa and 52 KDa, respectively, after deglycosylation. The treatment with glycopeptidase F reduced the molecular weights from 90 KDa and 70 KDa to 83 KDa and 52 KDa, respectively, while endoglycosidase H caused no change in their molecular weights. These results indicate that the 70 KDa subunit contains only the N-linked oligosaccharide chains, while the 90 KDa subunit contains O-linked oligosaccharides as well as N-linked ones. Enzyme-linked lectin assays suggested that bisectingN-acetyl-D-glucosamine and galactose 1–4N-acetyl-D-glucosamine structures were present and that fucose was included in these oligosaccharide moieties. Sialic acid was not found in either subunit.     

Synthetic glycopeptides from the mucin family as potential tools in cancer immunotherapy

Compared to glycoproteins of healthy cells, glycoproteins of tumor cells are often aberrantly glycosylated. Thus, glycopeptide fragments of surface glycoproteins of tumor cells are of interest as tumor-associated antigens for the distinction between normal and tumor cells. Cancer immunotherapy directed at selectively targeting these tumor-associated glycoprotein structure alterations--deficient glycosylation and, thus, exposure of peptide epitopes which are masked in normal cells--is considered a promising approach for the treatment of cancer. For this purpose, glycoproteins from the mucin family are of particular interest. Mucins belong to a class of heavily O-glycosylated, high-molecular weight glycoproteins present on the surface of many epithelial cells. The mucin core protein consists of numerous tandem repeats rich in serine, threonine and proline. In their tumor-associated forms, epithelial mucins carry cryptic saccharide structures such as T(N)-, T-, sialyl-T(N)- and sialyl-T antigens and more complex oligosaccharides (e.g. Lewis(y)). In contrast to glycoproteins isolated from natural sources, synthetic glycopeptides can be obtained in high purity and with exactly defined structure. In this review, methodologies for the synthesis of mucin-type glycopeptides containing complex tumor-associated antigen structures are described. Due to the low immunogenicity often exhibited by synthetic tumor-associated glycopeptide antigens, their conjugation to carrier proteins or suitable T-cell epitopes is essential for the development of anti-tumor vaccines. The results of immunological evaluations of synthetic (glyco)peptides and oligosaccharides are described. Some of these synthetic vaccines show promising activities inducing proliferation of T-cells and cytotoxic T-cell responses.     

Synthesis and biological evaluation of gallic acid analogs

A novel and efficient synthesis of pyrogallol moiety through a copper(I)-mediated C–O bond forming reaction is described. In particular, syntheses of 3,4,5-trihydroxyphenethyl alcohol and its methyl derivative are reported. Particular attention to dimethyl carbonate as an eco-friendly solvent/reactant has been paid, in order to improve the eco-compatibility of the whole synthetic pathway. Furthermore, the genotoxicity, cytotoxicity and the antioxidant activity of 3,4,5-trihydroxyphenethyl alcohol and its methyl derivative have been investigated.     

长春西汀及其类似物的合成和构效关系研究进展

总结介绍了20世纪90年代以来长春西汀及其类似物的合成及构效关系研究成果。长春西汀是生物碱长春胺的合成衍生物,是目前临床治疗和预防缺血性脑血管疾病的一线药物,近年来研究人员展开了对其衍生物合成路线及生物活性的相关研究,以期进一步阐明该类化合物作用机制和发现活性更好的衍生物。     

RGD多肽—氨基聚乙二醇—乳糖苷糖缀合物的合成

肿瘤细胞和宿主细胞或细胞外基质之间的相互粘连作用，是发生肿瘤转移的重要环节之一。RGD多肽聚乙二醇杂合物和寡糖的聚乙二醇缀合物都具有抗肿瘤转移的作用，RGD多肽－聚乙二醇－寡糖的杂合物也应当具有此功能的潜力。为了探索该类化合物的作用，设计且成功地合成聚乙二醇－RGD多态－寡糖缀合物。     

Effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells

We have investigated the effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells to elucidate the role of carbohydrate in the triggering of T cell function. The inhibitors of glycoprotein processing, especially mannnosidase inhibitors, enhanced the anti CD3-induced production of interleukin-2 (IL-2), which is a cytokine without the linkage sequence of N-linked oligosaccharides. On the other hand, N-methyl-1-deoxynojirimycin (NMdNM, an inhibitor of processing glucosidase 1), 1-deoxynojirimycin (dNM, an inhibitor of processing glucosidase I and II) and bromoconduritol (BCD, an inhibitor of processing glucosidase II) inhibited the secretion of interleukin-4 (IL-4), interferon-gamma (IFN-gamma), or interleukin-5 (IL-5) into culture supernatants of anti CD3-stimulated T cells, which had N-linked oligosaccharides. Mannosidase inhibitors, 1-deoxymannojirimycin (dMAN, an inhibitor of processing mannosidase I) and swainsonine (SWN, an inhibitor of processing mannosidase II) did not inhibit the secretion or production of IL-4, IFN-gamma and IL-5. To confirm the inhibition of N-linked oligosaccharide processing in the cytokines by the above inhibitors, the binding of IFN-gamma to lectins with various sugar-binding specificities was investigated. All inhibitors reduced the binding of IFN-gamma to PHA E4, which had a high affinity to bi- or tri-antennary complex type N-linked oligosaccharides with bisecting N-acetylglucosamine. Similarly, all inhibitors reduced the binding of IFN-gamma to PHA L4, which had high affinity to tri- or tetra-antennary complex type N-linked oligosaccharides with beta1-6-linked branching. SWN and dMAN increased the binding of IFN-gamma to concanavalin A (ConA), which had a high affinity to bi-antennary complex type, hybrid type and high-mannose type N-linked oligosaccharides. These results suggest that the processing inhibitors used here inhibit the N-linked oligosaccharide processing of cytokines, and the inhibition of processing enzyme glucosidases I and II induces a decreased secretion of cytokines with N4-linked oligosaccharides.     

Effect of staurosporine on N-glycosylation and cell adhesion to fibronectin of SW480 human colorectal adenocarcinoma cells.

As N-glycosylation of tumor cell surface proteins affects metastasis of the cells, it was considered that the suppression of metastasis by staurosporine, a protein kinase C inhibitor, is partly caused by changes in N-glycosylation. To examine this possibility, we studied the glycosylation of membrane proteins of SW480 human colorectal adenocarcinoma cells before and after treatment with staurosporine by lectin blot analysis. The results showed that the reactivity of leuko-agglutinating phytohemagglutinin and Datura stramonium agglutinin, both of which bind to highly branched N-linked oligosaccharides characteristic of cancer cells, decreases significantly in the staurosporine-treated cells. In accordance with this, the gene expression of the N-acetylglucosaminyltransferase V, which synthesizes the GlcNAcbeta1-->6 branch of highly branched N-linked oligosaccharides decreased by 30-40% in the drug-treated cells. Since a decrease in the lectin binding was found in several glycoproteins including fibronectin (FN)-receptor, effect of the changes in N-glycosylation of the cells on cell adhesion to FN-matrix was examined. The results showed that the number of cells attached to FN-matrix increases upon treatment of the cells with staurosporine, indicating that the change of N-glycosylation of the FN-receptor promotes cell adhesion to the extracellular matrix, which may lead to the suppression of metastasis of cancer cells.     

男性不育患者精液检查结果分析

目的探讨精液检查在男性不育诊断中的重要性,为男性不育的临床治疗和疗效观察提供依据.方法对270例男性不育患者采用精液分析仪进行精液分析.结果270例男性不育患者中,精液检查指标全部正常61例占22.6%,精液检查中发现有一项或几项异常209例占77.4%,在包括35例从事特殊行业的工人和40例司机的75例男性不育患者中,仅有9例精液检查项目全部正常占12.0%.结论精液检查方便、直观、快捷、经济,能为进一步寻找男性不育的原因提供帮助,为男性不育的诊断和治疗提供依据.     

Aminoacyl-tRNA synthetases: essential and still promising targets for new anti-infective agents

The emergence of resistance to existing antibiotics demands the development of novel antimicrobial agents directed against novel targets. Historically, bacterial cell wall synthesis, protein, and DNA and RNA synthesis have been major targets of very successful classes of antibiotics such as beta-lactams, glycopeptides, macrolides, aminoglycosides, tetracyclines, rifampicins and quinolones. Recently, efforts have been made to develop novel agents against validated targets in these pathways but also against new, previously unexploited targets. The era of genomics has provided insights into novel targets in microbial pathogens. Among the less exploited--but still promising--targets is the family of 20 aminoacyl-tRNA synthetases (aaRSs), which are essential for protein synthesis. These targets have been validated in nature as aaRS inhibition has been shown as the specific mode of action for many natural antimicrobial agents synthesized by bacteria and fungi. Therefore, aaRSs have the potential to be targeted by novel agents either from synthetic or natural sources to yield specific and selective anti-infectives. Numerous high-throughput screening programs aimed at identifying aaRS inhibitors have been performed over the last 20 years. A large number of promising lead compounds have been identified but only a few agents have moved forward into clinical development. This review provides an update on the present strategies to develop novel aaRS inhibitors as anti-infective drugs.     

Recent developments in synthetic oligosaccharide-based bacterial vaccines

Synthetic advances made possible chemical assembly of complex oligosaccharide fragments of polysaccharide domains on the surface of human pathogenic bacteria. These oligosaccharides may be recognized by antibodies raised against high molecular weight, native, polysaccharides. In addition to their antigenicity, synthetic oligosaccharides can also function as haptens in their protein conjugates that can elicit not only oligo- but also polysaccharide-specific IgG antibodies in animal models and in humans. A major milestone in the development of new generation vaccines was the demonstration that protein conjugates of synthetic fragments of the capsular polysaccharide of Haemophilus influenzae type b are as efficacious in preventing childhood meningitis and other diseases as is the corresponding licensed commercial vaccine containing the bacterial polysaccharide. The lessons learnt in this and other endeavors described herein are manifold. For example, they teach us about the significance of the oligosaccharide epitope size, the number of their copies per protein in the conjugate, the possible effect of the spacer on anti-saccharide immune response, and the proper choice of the carrier protein combined with the selection of the animal model. The H. influenzae b story also teaches us that that the synthetic approach can be commercially viable.     

Fractional dynamics pharmacokinetics–pharmacodynamic models

While an increasing number of fractional order integrals and differential equations applications have been reported in the physics, signal processing, engineering and bioengineering literatures, little attention has been paid to this class of models in the pharmacokinetics–pharmacodynamic (PKPD) literature. One of the reasons is computational: while the analytical solution of fractional differential equations is available in special cases, it this turns out that even the simplest PKPD models that can be constructed using fractional calculus do not allow an analytical solution. In this paper, we first introduce new families of PKPD models incorporating fractional order integrals and differential equations, and, second, exemplify and investigate their qualitative behavior. The families represent extensions of frequently used PK link and PD direct and indirect action models, using the tools of fractional calculus. In addition the PD models can be a function of a variable, the active drug, which can smoothly transition from concentration to exposure, to hyper-exposure, according to a fractional integral transformation. To investigate the behavior of the models we propose, we implement numerical algorithms for fractional integration and for the numerical solution of a system of fractional differential equations. For simplicity, in our investigation we concentrate on the pharmacodynamic side of the models, assuming standard (integer order) pharmacokinetics.     

Establishment of a method for mapping of N-linked oligosaccharides and its use to analyze industrially produced recombinant erythropoietin.

A rapid and convenient method for N-linked oligosaccharide structure analysis was developed and applied to the quality examination of commercially manufactured recombinant human erythropoietin (rEPO). Oligosaccharides released from rEPO expressed in Chinese hamster ovary cells were labeled with 2-aminobenzamide, and analyzed by a combination of diethylaminoethyl (DEAE) column HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This newly developed method allowed us to analyze sialylated oligosaccharides directly. The results showed that these oligosaccharides are composed of neutral-mono-di-tri-tetrasialyl oligosaccharides in a ratio of <1:2:13:34:51%, and that the core structure was a mixture of complex type bi-, tri- and tetra-antennary forms with one to three polylactosaminyl chains. Further analysis showed clearly that the N-linked oligosaccharide structure of rEPO has not changed throughout the past ten years of manufacturing by our company. The analysis was carried out using a small quantity (1 nmol) of rEPO, demonstrating the efficacy of the newly developed method. Further, the N-linked oligosaccharides of rEPO manufactured by our company almost coincided with those of the erythropoietin European Pharmacopoeia Biological Reference Preparation (Eur.Ph.BRP), a standard product.     

Altered glycosylation of proteins in cancer: what is the potential for new anti-tumour strategies

It is becoming increasingly apparent that cell surface oligosaccharides play pivotal roles as recognition molecules in a range of cell communication and adhesion processes. Alterations in cellular glycosylation are also associated with diseases, including cancer, and may have functional significance. This paper gives an overview of the complex topic of cellular glycosylation mechanisms and reviews the well-documented alterations in cellular glycosylation of proteins in malignancy. One particular type of cancer-associated glycosylation change, the incomplete synthesis of O-linked glycans, is highlighted, and its possible functional significance in cancer cell metastatic mechanisms is discussed. The significance that cancer-associated changes in glycoprotein glycosylation may have in new approaches to anti-tumour therapies is explored.     

对思想道德修养与法律基础课程案例教学的认识

思想道德修养与法律基础课程是帮助大学生端正思想,提高道德素质和法律素质的一门课程,对于大学生的成长成才有着非常重要的意义。为了提高学生的学习兴趣,增强教学实效,教师在教学改革上做了很多探索和尝试。其中,案例教学法是其中最值得推广的有效方法之一。如何选择好一个教学案例和如何组织好一个案例教学是做好案例教学的关键所在。     

Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner

Herein we report that the novel HIV-inactivating protein cyanovirin-N (CV-N) targets specific, N-linked high-mannose oligosaccharides found on the viral envelope of HIV-1. First, we released the oligosaccharides by PnGase-treatment of HIV-gp120 (containing high-mannose, hybrid-type and complex-type oligosaccharides) or HSV-1 gC (containing only complex-type). Then, in an affinity chromatographic system, we found that CV-N bound to the free oligosaccharides from gp120 but not from gC-1, suggesting that high-mannose oligosaccharides constitute a target structure for CV-N. This was supported by the affinity of CV-N for high-mannose glycans released from gp120 by endo-H as well as high-mannose glycans released from castanospermine-treated HSV-1 gC. Furthermore, free Man-8 or Man-9 oligosaccharides partially inhibited the binding of CV-N to gp120, although neither oligosaccharides smaller than Man-7 nor monosaccharides interfered with CV-N/gp120 interaction, thereby establishing the oligosaccharide-specific affinity of CV-N to high-mannose glycans. This affinity for high-mannose oligosaccharides may explain the broad antiviral activity of CV-N against human and primate immunodeficiency retroviruses as well as certain other viruses that carry these oligosaccharides.     

Nano-LC and HPLC-chip-ESI-MS: an emerging technique for glycobioanalysis

In less than 5 years, an impressive number of methods based on nano-LC and HPLC-chip coupled online to MS were developed and implemented to comprehensively address structural heterogeneity of glycoconjugates and glycans in biological matrices. C18, graphitized carbon and amide-based stationary phases were adapted to nanoflow level and on chip format, leading to improved sensitivity of structural analysis and superior level of information on highly complex glycan and glycoconjugate mixtures. This review offers a summary of the recent progress in the application of nano-LC and HPLC-chip-MS in glycoanalytics of glycopeptides, glycoprotein glycans, glycosaminoglycans, oligosaccharides and glycosphingolipids.     

Recent developments in reversing glycopeptide-resistant pathogens

Recent studies addressing vancomycin-resistance phenomena are surveyed. Besides leading semi-synthetic glycopeptides: BI-397 and LY 333328, other new synthetic derivatives are also presented. Rational design based on the known mode of action is seen in recent research from both industrial and academic groups. Thus, Biosearch Italia SpA worked on new synthetic glycopeptides with a modified binding pocket in order to find drugs active against both vancomycin-sensitive and -resistant strains. Eli Lilly & Co. and research groups at Stanford, Cambridge and Harvard synthesised covalently linked dimers in order to copy the dimerisation and membrane anchoring effects. Both directions proved to be rewarding and some interesting compounds with potent in vitro activity against vancomycin-resistant enterococci (VRE) were found from these investigations. A new mechanistic proposal that can account for the bioactivity of LY 333328 against VRE was summarised. Research on new agents with modes of action different to that of glycopeptide antibiotics have also been successful and recent investigations related to RP-59500, SCH-27899 and U-100766 are presented.