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. [1] 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. [2] 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 (Ac 4 ManNAz) 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. [3] Once presented on the cell surface, the azide-containing sialylated glycan can be visualized through a bioorthogonal ligation reaction. [4] Besides Ac 4 ManNAz, several monosaccharide derivatives of N-acetylgalactosamine, [5] N-acetylglucosamine, [6] and l-fucose [7] are suitable for MOE providing further insights into the role of cellular structures and functions of glycans in the cell.Currently, mainly Staudinger ligation [3] and azide-alkyne [3+2] cycloaddition (copper-catalyzed [8] or strain-promoted, [9] 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 [10] with strained dienophiles, such as trans-cyclooctenes, [11] cyclobutenes, [12] norbornenes, [11d,f, 13] cyclooctynes, [11d,f] and substituted cyclopropenes, [14] 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 [15] 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 identifi...

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.

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 (i.e. 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 Å, wheat germ agglutinin (WGA) (shortest distance between binding sites 13-14 Å), LecA from Pseudomonas aeruginosa (shortest distance 26 Å), cholera toxin and heat-labile enterotoxin (shortest distance 31 Å), anti-HIV antibody 2G12 (shortest distance 31 Å), concanavalin A (ConA) (shortest distance 72 Å), RCA120 (shortest distance 100 Å), and Erythrina cristagalli (ECL) (shortest distance 100 Å). While chelating binding of the discussed ligands is likely, experimental proof, for example by X-ray crystallography, is limited to only a few cases.

We identified a pathway in Bacillus subtilis that is used for recovery of N-acetylglucosamine (GlcNAc)-Nacetylmuramic acid (MurNAc) peptides (muropeptides) derived from the peptidoglycan of the cell wall. This pathway is encoded by a cluster of six genes, the first three of which are orthologs of Escherichia coli genes involved in N-acetylmuramic acid dissimilation and encode a MurNAc-6-phosphate etherase (MurQ), a MurNAc-6-phosphate-specific transcriptional regulator (MurR), and a MurNAc-specific phosphotransferase system (MurP). Here we characterized two other genes of this cluster. The first gene was shown to encode a cell wall-associated ␤-N-acetylglucosaminidase (NagZ, formerly YbbD) that cleaves the terminal nonreducing N-acetylglucosamine of muropeptides and also accepts chromogenic or fluorogenic ␤-N-acetylglucosaminides. The second gene was shown to encode an amidase (AmiE, formerly YbbE) that hydrolyzes the N-acetylmuramyl-L-Ala bond of MurNAc peptides but not this bond of muropeptides. Hence, AmiE requires NagZ, and in conjunction these enzymes liberate MurNAc by sequential hydrolysis of muropeptides. NagZ expression was induced at late exponential phase, and it was 6-fold higher in stationary phase. NagZ is noncovalently associated with lysozyme-degradable particulate material and can be released from it with salt. A nagZ mutant accumulates muropeptides in the spent medium and displays a lytic phenotype in late stationary phase. The evidence for a muropeptide catabolic pathway presented here is the first evidence for cell wall recovery in a Gram-positive organism, and this pathway is distinct from the cell wall recycling pathway of E. coli and other Gram-negative bacteria.Bacteria are covered by an exoskeleton-like cell wall that protects the fragile membrane-enclosed cell, the protoplast, and withstands the high internal pressure of the cell, the turgor pressure (27). Despite its stabilizing function, the cell wall is not rigid and static but is highly flexible. It undergoes continuous resynthesis, remodeling, and degradation (turnover) in which a substantial amount of the murein (peptidoglycan), the stabilizing component of the bacterial cell wall, is released during logarithmic growth (5, 47). The Gram-positive model organism Bacillus subtilis was shown to release about 50% of its murein into the medium in one generation during growth (9, 42, 43). Gram-negative bacteria have an outer membrane that keeps most of the cell wall turnover products in the periplasmic space, but Gram-positive bacteria lack such a membrane barrier and therefore cannot retain their turnover products. So far, more than 30 peptidoglycan hydrolases of B. subtilis have been characterized or identified as candidate autolysins on the basis of amino acid sequence identity (50), but the nature of the turnover products remains ambiguous. Moreover, a cell wall recycling-recovery pathway has not been identified in this organism or in any other Gram-positive bacterium so far.In contrast, cell wall turnover and recycling in the Gramneg...

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 31 P 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.Complex carbohydrates and their conjugates are involved in various types of biochemical recognition processes. 1 Glycosyltransferase-catalyzed synthesis of these important structures is attractive since these reactions proceed regio-and stereoselectively in aqueous media without requiring complicated manipulations of protecting groups. 2,3 As glycosyl donors, the glycosyltransferases of the Leloir pathway 4,5 in mammalian systems employ primarily eight sugar nucleotides: UDP-Glc, UDPGlcNAc, UDP-Gal, UDP-GalNAc, GDP-Man, GDP-Fuc, UDP-GlcUA, and CMP-NeuAc. For an efficient use of glycosyltransferases in the synthesis of oligosaccharides a practicable (high yield) preparation of these cosubstrates is demanded.Most of the chemical syntheses of sugar diphosphate nucleosides 6,7 involve the coupling of a glycosyl phosphate 1 with an activated nucleoside monophosphate (NMP) (Scheme 1). [8][9][10][11][12][13][14] Of the commonly used activated NMP derivatives, phosphoramidates such as phosphorimidazolidates 8-10 and especially phosphomorpholidates 2 11-14 are the most popular, the latter being introduced in 1959 by Moffatt and Khorana. 15,16 Nevertheless, the reaction between a sugar 1-phosphate and an NMP-morpholidate is very slow (reaction times of 5 days are usual), and yields rarely exeed 70%. GDP-Fuc, in particular, is obtained in only 20-50% yield. [17][18][19][20][21][22] Recently, a new approach, involving the reaction of glycosyl bromides with UDP and GDP, was reported. 23 In the enzymatic preparation of sugar diphosphate nucleosides, a glycosyl phosphate is reacted with a nucleoside triphosphate (NTP), catalyzed by a nucleoside diphosphate sugar pyrophosphorylase (Scheme 1). 10,[24][25][26][27][28][29]