Synthesis of lactose-based S-linked sialylmimetics of alpha(2,3)-sialosides.

A new approach toward the synthesis of lactose-based S-linked sialylmimetics of a(2,3)-linked sialosides is described. These compounds, represented by the general structure 3, were prepared from methyl ߭d-lactoside in 11 steps. It was found that the choice of protecting group was crucial to allow the efficient introduction of sulfur at the 3-position of the galactose ring.Office of the Snr Dep Vice Chancellor, Institute for GlycomicsNo Full Tex

Structural studies on the <em>Pseudomonas aeruginosa</em> sialidase-like enzyme PA2794 suggests substrate and mechanistic variations

Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical !-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/ base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site.No Full Tex

Three Streptococcus pneumoniae sialidases: three different products

Streptococcus penumoniae is a major human pathogen responsible for respiratory tract infections, septicemia, and meningitis and continues to produce numerous cases of disease with relatively high mortalities. S. pneumoniae encodes up to three sialidases, NanA, NanB, and NanC, that have been implicated in pathogenesis and are potential drug targets. NanA has been shown to be a promiscuous sialidase, hydrolyzing the removal of Neu5Ac from a variety of glycoconjugates with retention of configuration at the anomeric center, as we confirm by NMR. NanB is an intramolecular trans-sialidase producing 2,7-anhydro-Neu5Ac selectively from α2,3-sialosides. Here, we show that the first product of NanC is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) that can be slowly hydrated by the enzyme to Neu5Ac. We propose that the three pneumococcal sialidases share a common catalytic mechanism up to the final product formation step, and speculate on the roles of the enzymes in the lifecycle of the bacterium

Sialic acid recognition by <em>Vibrio cholerae</em> neuraminidase

Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid from higher order gangliosides to unmask GM1, the receptor for cholera toxin. We previously showed that the structure of VCNA is composed of a central ?-propeller catalytic domain flanked by two lectin-like domains, however the nature of the carbohydrates recognised by these lectin domains has remained unknown. We present here structures of the enzyme in complex with two substrates, ?2,3-sialyllactose and ?-2,6-sialyllactose. Both substrate complexes reveal the ?-anomer of N-acetylneuraminic acid (Neu5Ac, NANA) bound to the Nterminal lectin domain, thereby revealing the role of this domain. The large number of interactions suggest a relatively high binding affinity for sialic acid, which was confirmed by calorimetry, which gave a Kd~30?M. Saturation transfer difference (STD) NMR using a non-hydrolysable substrate, Neu5,9Ac2-2-S-(?-2,6)-GlcNAc?1Me, was also used to map the ligand interactions at the VCNA lectin binding site. It is well known that VCNA can hydrolyse both ?-2,3- and ?-2,6-linked sialic acid substrates. In this study using ?-2,3-sialyllactose co-crystallised with VCNA it was revealed that the inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, DANA) was bound at the catalytic site. This observation supports the notion that VCNA can produce its own inhibitor and has been further confirmed by 1H NMR analysis. The discovery of the sialic acid-binding site in the N-lectin-like domain suggests that this might help target VCNA to sialic acid-rich environments, thereby enhancing the catalytic efficiency of the enzyme.No Full Tex

One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities

Chemoenzymatic synthesis, which combines the flexibility of chemical synthesis and the high selectivity of enzymatic synthesis, is a powerful approach to obtain complex carbohydrates. It is a preferred method for synthesizing sialic acid-containing structures, including those with diverse naturally occurring and non-natural sialic acid forms, different sialyl linkages and different glycans that link to the sialic acid. Starting from N-acetylmannosamine, mannose or their chemically or enzymatically modified derivatives, sialic acid aldolase-catalyzed condensation reaction leads to the formation of sialic acids and their derivatives. These compounds are subsequently activated by a CMP-sialic acid synthetase and transferred to a wide range of suitable acceptors by a suitable sialyltransferase for the formation of sialosides containing natural and non-natural functionalities. The three-enzyme coupled synthesis of sialosides can be carried out in one pot without the isolation of intermediates. The time for synthesis is 4-18 h. Purification and characterization of the product can be completed within 2-3 d.No Full Tex