抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

“Bioinformatics” 특집을 내면서

The Huisgen 1,3-dipolar cycloaddition reaction of organic azides and alkynes has gained considerable attention in recent years due to the introduction in 2001 of Cu(1) catalysis by Tornoe and Meldal, leading to a major improvement in both rate and regioselectivity of the reaction, as realized independently by the Meldal and the Sharpless laboratories. The great success of the Cu(1) catalyzed reaction is rooted in the fact that it is a virtually quantitative, very robust, insensitive, general, and orthogonal ligation reaction, suitable for even biomolecular ligation and in vivo tagging or as a polymerization reaction for synthesis of long linear polymers. The triazole formed is essentially chemically inert to reactive conditions, e.g. oxidation, reduction, and hydrolysis, and has an intermediate polarity with a dipolar moment of ∼5 D. The basis for the unique properties and rate enhancement for triazole formation under Cu(1) catalysis should be found in the high ∆G of the reaction in combination with the low character of polarity of the dipole of the noncatalyzed thermal reaction, which leads to a considerable activation barrier. In order to understand the reaction in detail, it therefore seems important to spend a moment to consider the structural and mechanistic aspects of the catalysis. The reaction is quite insensitive to reaction conditions as long as Cu(1) is present and may be performed in an aqueous or organic environment both in solution and on solid support.

Cu-catalyzed azide-alkyne cycloaddition.

In studying the evolution of organic chemistry and grasping its essence, one comes quickly to the conclusion that no other type of reaction plays as large a role in shaping this domain of science than carbon-carbon bond-forming reactions. The Grignard, Diels-Alder, and Wittig reactions are but three prominent examples of such processes, and are among those which have undeniably exercised decisive roles in the last century in the emergence of chemical synthesis as we know it today. In the last quarter of the 20th century, a new family of carbon-carbon bond-forming reactions based on transition-metal catalysts evolved as powerful tools in synthesis. Among them, the palladium-catalyzed cross-coupling reactions are the most prominent. In this Review, highlights of a number of selected syntheses are discussed. The examples chosen demonstrate the enormous power of these processes in the art of total synthesis and underscore their future potential in chemical synthesis.

Palladium-catalyzed cross-coupling reactions in total synthesis.

The cluster glycoside effect.

The inhibition of carbohydrate-protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GlcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GlcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 microM (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GlcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 microM being 25,500 times higher than that of GlcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.

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Structural basis of multivalent binding to wheat germ agglutinin.

An improved procedure is described for the efficient and high-yield (76−91%) synthesis of nucleoside diphosphate sugars from the readily available nucleoside 5‘-monophosphomorpholidate and sugar 1-phosphate in the presence of 1H-tetrazole. Comparative kinetic investigations by means of 31P NMR spectroscopy with different additives (1,2,4-triazole, acetic acid, N-hydroxysuccinimide, 4-(dimethylamino)pyridine hydrochloride, perchloric acid) and mass spectrometric analysis suggest that tetrazole acts as an acid and as a nucleophilic catalyst in the pyrophosphate bond formation.

/pdf/1h-tetrazole-as-catalyst-in-phosphomorpholidate-coupling-1nrcffwpcv.pdf

1H-Tetrazole as Catalyst in Phosphomorpholidate Coupling Reactions: Efficient Synthesis of GDP-Fucose, GDP-Mannose, and UDP-Galactose

Protein glycosylation is a complex form of posttranslational modification and has been shown to be crucial for the function of many proteins. Sialic acid is prominently positioned at the outer end of membrane glycoproteins. It plays a critical role for the regulation of a myriad of cellular functions and it forms a shield around the cell. Furthermore, it constantly interacts with the environment of cells and contributes to histocompatibility. This makes studying sialylation an interesting field of research, but monitoring sialic acid in vivo is challenging. While proteins are routinely labeled by genetic methods, such as expression as GFP fusion proteins, comparable methods are not available for secondary gene products, such as glycans of glycoconjugates. Metabolic oligosaccharide engineering (MOE) is a successful new strategy to visualize the localization of glycans in vitro and in vivo. In this approach, cells are cultivated in the presence of non-natural monosaccharide derivatives that carry a chemical reporter group and are nonetheless accepted by the biosynthetic machinery of a cell. For instance, peracetylated N-azidoacetylmannosamine (Ac4ManNAz) is taken up by the cell, deacetylated by cellular esterases, and owing to the promiscuity of the enzymes of sialic acid biosynthesis, is converted into N-azidoacetyl neuraminic acid and incorporated into sialoglycoconjugates. Once presented on the cell surface, the azide-containing sialylated glycan can be visualized through a bioorthogonal ligation reaction. Besides Ac4ManNAz, several monosaccharide derivatives of N-acetylgalactosamine, N-acetylglucosamine, and l-fucose are suitable for MOE providing further insights into the role of cellular structures and functions of glycans in the cell. Currently, mainly Staudinger ligation and azide–alkyne [3+2] cycloaddition (copper-catalyzed or strain-promoted, also known as the click reaction) are applied as ligation reactions in MOE. However, both of them rely on the reaction of azides and thus cannot be used for the concurrent detection of two different metabolically incorporated carbohydrates. A labeling strategy that can be carried out in the presence of azides and alkynes would significantly expand the scope of chemical labeling reactions in living cells and is thus highly desirable. Recently, it was shown that the Diels–Alder reaction with inverse electron demand (DARinv) of 1,2,4,5-tetrazines with strained dienophiles, such as trans-cyclooctenes, cyclobutenes, norbornenes, 13] cyclooctynes, and substituted cyclopropenes, fulfills the requirements of a bioorthogonal ligation reaction and furthermore is orthogonal to the azide–alkyne cycloaddition. However, these cyclic alkenes or kinetically stable tetrazines are expected to be too large for being efficiently metabolized by the sialic acid biosynthetic pathway, starting from the corresponding Nacylmannosamine derivative. In search for smaller dienophiles suitable for MOE, we identified monosubstituted (terminal) alkenes as a new class of chemical reporters. We recently reported the successful application of the DARinv between terminal alkenes and 1,2,4,5-tetrazines in the preparation of carbohydrate microarrays. The fact that terminal alkenes are hardly found in biological systems and are completely absent in proteins makes them a promising reporter group. Herein, we show that ManNAc derivatives containing a terminal alkene in the acyl side chain are metabolically incorporated into cell-surface sialic acids and can subsequently be labeled by the DARinv (Figure 1). Moreover, we demonstrate that double labeling of two differently modified, metabolically incorporated monosaccharides is possible by combining the DARinv with strainpromoted azide–alkyne cycloaddition (SPAAC). As the reaction rate of the DARinv of acyclic olefins with tetrazines is very sensitive to steric hindrance, double bonds with more than one substituent react very slowly. Terminal alkenes, on the other hand, can react rapidly without any further activation. This prompted us to design mannosamine derivatives 2 and 4 (Figure 2) that were synthesized in three steps from mannosamine hydrochloride (see the Supporting Information). Based on previous work by Keppler et al., we expected both derivatives to be accepted by cells with Npentenoylmannosamine 2 (owing to the shorter acyl side chain) being incorporated with higher efficiency. On the other [*] Dipl.-Chem. A. Niederwieser, M. Sc. A.-K. Sp te, M. Sc. C. J ngst, Prof. Dr. V. Wittmann University of Konstanz, Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB) 78457 Konstanz (Germany) E-mail: mail@valentin-wittmann.de

Two‐Color Glycan Labeling of Live Cells by a Combination of Diels–Alder and Click Chemistry

Carbohydrate–protein interactions are involved in a multitude of biological recognition processes Since individual protein–carbohydrate interactions are usually weak, multivalency is often required to achieve biologically relevant binding affinities and selectivities Among the possible mechanisms responsible for binding enhancement by multivalency, the simultaneous attachment of a multivalent ligand to several binding sites of a multivalent receptor (ie chelation) has been proven to have a strong impact This article summarizes recent examples of chelating lectin ligands of different size Covered lectins include the Shiga-like toxin, where the shortest distance between binding sites is ca 9 A, wheat germ agglutinin (WGA) (shortest distance between binding sites 13–14 A), LecA from Pseudomonas aeruginosa (shortest distance 26 A), cholera toxin and heat-labile enterotoxin (shortest distance 31 A), anti-HIV antibody 2G12 (shortest distance 31 A), concanavalin A (ConA) (shortest distance 72 A), RCA120 (shortest distance 100 A), and Erythrina cristagalli (ECL) (shortest distance 100 A) While chelating binding of the discussed ligands is likely, experimental proof, for example by X-ray crystallography, is limited to only a few cases

Valentin Wittmann

Papers

Structural basis of multivalent binding to wheat germ agglutinin.

1H-Tetrazole as Catalyst in Phosphomorpholidate Coupling Reactions: Efficient Synthesis of GDP-Fucose, GDP-Mannose, and UDP-Galactose

Two‐Color Glycan Labeling of Live Cells by a Combination of Diels–Alder and Click Chemistry

Bridging lectin binding sites by multivalent carbohydrates.

One-pot procedure for diazo transfer and azide-alkyne cycloaddition: triazole linkages from amines.