Streamlined chemoenzymatic total synthesis of prioritized ganglioside cancer antigens.

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacßSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.

Synthesis and evaluation of triazole linked glycosylated 18ß-glycyrrhetinic acid derivatives as anticancer agents.

A series of glycosyl triazol linked 18ß-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a-h) with propargyl ester of 18ß-glycyrrhetinic acid (GA) (2 and 3) following the concept of 'Click chemistry'. The synthesized triazole derivatives were de-O-acetylated to furnish compounds (7a-h and 8a-c) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7g and compound 8c showed promising anticancer activities.

Removal of benzylidene acetal and benzyl ether in carbohydrate derivatives using triethylsilane and Pd/C.

Clean deprotection of carbohydrate derivatives containing benzylidene acetals and benzyl ethers was achieved under catalytic transfer hydrogenation conditions by using a combination of triethylsilane and 10% Pd/C in CH(3)OH at room temperature. A variety of carbohydrate diol derivatives were prepared from their benzylidene derivatives in excellent yield.

Appel-reagent-mediated transformation of glycosyl hemiacetal derivatives into thioglycosides and glycosyl thiols.

A series of glycosyl hemiacetal derivatives have been transformed into thioglycosides and glycosyl thiols in a one-pot two-step reaction sequence mediated by Appel reagent (carbon tetrabromide and triphenylphosphine). 1,2-trans-Thioglycosides and ß-glycosyl thiol derivatives were stereoselectively formed by the reaction of the in situ generated glycosyl bromides with thiols and sodium carbonotrithioate. The reaction conditions are reasonably simple and yields were very good.

A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides.

Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1-4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.

Engineer P. multocida Heparosan Synthase 2 (PmHS2) for Size-Controlled Synthesis of Longer Heparosan Oligosaccharides.

Pasteurella multocida heparosan synthase 2 (PmHS2) is a dual-function polysaccharide synthase having both α1-4-N-acetylglucosaminyltransferase (α1-4-GlcNAcT) and ß1-4-glucuronyltransferase (ß1-4-GlcAT) activities located in two separate catalytic domains. We found that removing PmHS2 N-terminal 80-amino acid residues improved enzyme stability and expression level while retaining its substrate promiscuity. We also identified the reverse glycosylation activities of PmHS2 which complicated its application in size-controlled synthesis of oligosaccharides longer than hexasaccharide. Engineered Δ80PmHS2 single-function-glycosyltransferase mutants Δ80PmHS2_D291N (α1-4-GlcNAcT lacking both forward and reverse ß1-4-GlcAT activities) and Δ80PmHS2_D569N (ß1-4-GlcAT lacking both forward and reverse α1-4-GlcNAcT activities) were designed and showed to minimize side product formation. They were successfully used in a sequential one-pot multienzyme (OPME) platform for size-controlled high-yield production of oligosaccharides up to decasaccharide. The study draws attention to the consideration of reverse glycosylation activities of glycosyltransferases, including polysaccharide synthases, when applying them in the synthesis of oligosaccharides and polysaccharides. The mutagenesis strategy has the potential to be extended to other multifunctional polysaccharide synthases with reverse glycosylation activities to generate catalysts with improved synthetic efficiency.

Regioselective One-Pot Multienzyme (OPME) Chemoenzymatic Strategies for Systematic Synthesis of Sialyl Core 2 Glycans.

O-GalNAc glycans or mucin-type glycans are common protein post-translational modifications in eukaryotes. Core 2 O-GalNAc glycans are branched structures that are broadly distributed in glycoproteins and mucins of all types of cells. To better understand their biological roles, it is important to obtain structurally defined Core 2 O-GalNAc glycans. We present here regioselective one-pot multienzyme (OPME) chemoenzymatic strategies to systematically access a diverse array of sialyl Core 2 glycans. Regioselectivity can be achieved by using OPME systems containing a glycosyltransferase with restricted acceptor specificity or by differentiating the branches using altered glycosylation sequences. This work provides a general regioselective strategy to access diverse Core 2 O-GalNAc glycans which can be extended for the synthesis of other complex branched glycans.

A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid.

A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.

Synthesis of the Heptasaccharide Repeating Unit of the Cell Wall O-Polysaccharide of Enterotoxigenic Escherichia coli O139.

Enterotoxigenic Escherichia coli (ETEC) like the O139 strain are mostly responsible for traveler's diarrhea and causes diseases in pigs, cattle, and poultry. A convenient synthetic strategy was developed for the synthesis of the heptasaccharide repeating unit of the cell wall lipopolysaccharide of the E. coli O139 strain. The p-methoxybenzyl (PMB) group was used as a temporary protecting group which was removed in situ under the glycosylation conditions by changing the reaction temperature during the synthesis of the target compound. All glycosylation steps gave high yields with good stereoselectivity. A (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-mediated selective oxidation of the primary hydroxyl group was carried out using a biphasic reaction condition at the late stage of the synthesis. Such synthetic oligosaccharides could later be effectively conjugated with proteins to prepare glycoconjugate derivatives as vaccine candidates.

Oxidative stress plays major role in mediating apoptosis in filarial nematode Setaria cervi in the presence of trans-stilbene derivatives.

Lymphatic filariasis, affecting around 120 million people in 80 countries worldwide, is an extremely painful disease and caused permanent and long term disability. Owing to its alarming prevalence there is immediate need for development of new therapeutics. A series of trans-stilbene derivatives were synthesized using aqueous reaction condition showing potential as antifilarial agents demonstrated in vitro. MTT reduction assay and dye exclusion test were performed to evaluate the micro and macrofilaricidal potential of these compounds. Amid 20 trans-stilbene derivatives together with Resveratrol (RSV), a multifunctional natural product was screened; nine compounds (28, 29, 33, 35, 36, 38, 39, 41 and 42) have showed promising micro and macrofilaricidal activities and four of them (28, 39, 41 and 42) showed better effectiveness than RSV. In the treated parasites apoptosis was established by DNA laddering, in situ DNA fragmentation and FACS analysis. The generation of ROS in the treated parasites was indicated by the depletion in the level of GSH, GR and GST activity and elevation of SOD, catalase, GPx activity and superoxide anion and H2O2 level. Along with the ROS generation and oxidative stress, the decreased expression of anti-apoptotic ced-9 gene and increased expression of nematode specific pro-apoptotic genes, egl-1, ced-4 and ced-3 at the level of transcription and translation level; the up-regulation of caspase-3 activity and involvement of caspase-8,9,3, cytochrome-c and PARP were also observed and which denotes the probable existence of both extrinsic and intrinsic pathways apoptosis in parasitic nematodes. This observation is reported first time and thus it confirmed the mode of action and effectiveness of the compounds. Further, the comparative bioavailability-pharmacokinetics studies showed that compound 28 possesses comparable properties with Ivermectin. This study will certainly intensify our understanding of the pharmacological importance of trans-stilbenes as an anti-filarial agent.

Efficient synthesis of the tetrasaccharide repeating unit of the O-antigen of Escherichia coli O174 strain.

The tetrasaccharide repeating unit of the O-antigen of Escherichia coli O174 strain was synthesized applying sequential glycosylations of suitably functionalized monosaccharide intermediates. Activation of glycosyl trichloroacetimidate derivatives using nitrosyl tetrafluoroborate (NOBF4) has been used during the synthesis. The glycosylation steps were high yielding with satisfactory stereo outcome.

Facile synthesis of the pentasaccharide repeating unit of the cell wall O-antigen of Escherichia coli 19ab.

A straightforward synthesis of the pentasaccharide with all α-linked glycosyl linkages corresponding to the cell wall O-antigen of Escherichia coli 19ab has been achieved in excellent yield using sequential glycosylations of monosaccharide intermediates. The structure of the target compound and its synthetic intermediates were unambiguously characterized using their spectral analysis. A generalized glycosylation condition has been used in all glycosylations and minimum number of protecting group manipulations were involved during the synthesis of the target compound.