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A highly convergent and efficient strategy was developed for the chemical synthesis of complex oligosaccharides of Streptococcus pneumoniae type 3 capsular polysaccharides that contain multiple glucuronic acid units. Once the oligoglucosides were efficiently and stereoselectively assembled, the designated glucose units were regioselectively oxidized to glucuronic acid in one step at the final synthetic stage, which helped avoid difficult glycosylations that involved glucuronic acid. The target oligosaccharides had a free amino group at the reducing terminus and varied caps at the non-reducing terminus to enable further modification and structure-activity relationship studies. Immunological evaluations of the oligosaccharide-tetanus toxoid conjugates showed that they elicited robust T-cell-dependent immunoglobulin G antibody responses and that the sugar chain length had a major impact on their immunological properties. In particular, the penta- and hexasaccharides were identified as promising antigens for vaccine development.

ABSTRACT␤-N-Acetylhexosaminidases have attracted interest particularly for oligosaccharide synthesis, but their use remains limited by the rarity of enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, genes of 13 ␤-Nacetylhexosaminidases, including 5 from Bacteroides fragilis ATCC 25285, 5 from Clostridium perfringens ATCC 13124, and 3 from Bifidobacterium bifidum JCM 1254, were cloned and heterogeneously expressed in Escherichia coli. The resulting recombinant enzymes were purified and screened for transglycosylation activity. A ␤-N-acetylhexosaminidase named BbhI, which belongs to glycoside hydrolase family 20 and was obtained from B. bifidum JCM 1254, possesses the bifunctional property of efficiently transferring both GalNAc and GlcNAc residues through ␤1-3 linkage to the Gal residue of lactose. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation activities of BbhI were studied in detail. With the use of 10 mM pNP-␤-GalNAc or 20 mM pNP-␤-GlcNAc as the donor and 400 mM lactose as the acceptor in phosphate buffer (pH 5.8), BbhI synthesized GalNAc␤1-3Gal␤1-4Glc and GlcNAc␤1-3Gal␤1-4Glc at maximal yields of 55.4% at 45°C and 4 h and 44.9% at 55°C and 1.5 h, respectively. The model docking of BbhI with lactose showed the possible molecular basis of strict regioselectivity of ␤1-3 linkage in ␤-N-acetylhexosaminyl lactose synthesis. IMPORTANCEOligosaccharides play a crucial role in many biological events and therefore are promising potential therapeutic agents. However, their use is limited because large-scale production of oligosaccharides is difficult. The chemical synthesis requires multiple protecting group manipulations to control the regio-and stereoselectivity of glycosidic bonds. In comparison, enzymatic synthesis can produce oligosaccharides in one step by using glycosyltransferases and glycosidases. Given the lower price of their glycosyl donor and their broader acceptor specificity, glycosidases are more advantageous than glycosyltransferases for large-scale synthesis. ␤-N-Acetylhexosaminidases have attracted interest particularly for ␤-N-acetylhexosaminyl oligosaccharide synthesis, but their application is affected by having few enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, we describe a microbial ␤-N-acetylhexosaminidase that exhibited strong transglycosylation activity and strict regioselectivity for ␤-N-acetylhexosaminyl lactose synthesis and thus provides a powerful synthetic tool to obtain biologically important GalNAc␤1-3Lac and GlcNAc␤1-3Lac. Oligosaccharides are widely distributed in nature and play a crucial role in many biological events, such as cell structure modulation, cell-cell recognition and communication, and cellmicrobe/toxin interaction and adhesion; therefore, they are promising and important potential therapeutic agents (1-3). However, their use is limited because of the difficulty of large-scale production of oligosaccharides. Their...

Lipoarabinomannan (LAM) is one of the major constituents of the Mycobacterium tuberculosis cell wall and an attractive molecular scaffold for antituberculosis drug and vaccine development. In this paper, a convergent strategy was developed for the synthesis of LAM oligosaccharides with an α-1,2-linked dimannopyranose cap at the nonreducing end. The strategy was highlighted by efficient coupling of separately prepared nonreducing end and reducing end oligosaccharides. Glycosylations were mainly achieved with thioglycoside donors, which gave excellent yields and stereoselectivity even for reactions between complex oligosaccharides. The strategy was utilized to successfully synthesize tetra-, hepta-, and undecasaccharides of LAM from d-arabinose in 10, 15, and 14 longest linear steps and 7.84, 7.50, and 2.59% overall yields, respectively. The resultant oligosaccharides with a free amino group at their reducing end were effectively conjugated with carrier proteins, including bovine serum albumin and keyhole limpet hemocyanin (KLH), via a bifunctional linker. Preliminary immunological studies on the KLH conjugates revealed that they could elicit robust antibody responses in mice and that the antigen structure had some influence on their immunological property, thus verifying the potential of the oligosaccharides for vaccine development and other immunological studies.

Sortase A (SrtA) is a membrane-associated enzyme responsible for the covalent anchoring of many virulent factors of Gram-positive bacteria onto the cell wall. It has been shown that SrtA plays a pivotal role in the pathogenic processes of bacterial infection. Additionally, SrtA is not essential for microbial growth and viability, and its inhibition does not therefore place pressure on bacteria to develop drug-resistant mechanism. As an extracellular membrane enzyme, it can more readily be targeted by drugs relevant to intracellular enzymes. SrtA is thus an excellent target for the design and development of novel anti-virulence drugs against the drug-resistant Gram-positive bacteria that have become a major worldwide health problem. A number of SrtA inhibitors have so far been identified by techniques such as the rational design of substrate mimetic inhibitors based on the structure of the enzyme and enzyme substrates, identification of novel inhibitors among natural products, the discovery and development of SrtA inhibitors via high-throughput, and in silico screening of small molecule libraries followed by structural optimization. The present article reviews the progress made recently in the development of SrtA inhibitors as new antibacterial agents using similar techniques.