A library of 2(e),3(a/e)-difluorosialic acids and their C-5 and/or C-9 derivatives were chemoenzymatically synthesized. Pasteurella multocida sialic acid aldolase (PmAldolase), but not its Escherichia coli homolog (EcAldolase), was found to catalyze the formation of C5-azido analog of 3-fluoro(equatorial)-sialic acid. In comparison, both PmAldolase and EcAldolase could catalyze the synthesis of 3-fluoro (axial or equatorial)-sialic acids and their C-9 analogs although PmAldolase was generally more efficient. The chemoenzymatically synthesized 3-fluoro (axial or equatorial)-sialic acid analogs were purified and chemically derivatized to form desired difluorosialic acids and derivatives. Inhibition studies against several bacterial sialidases and a recombinant human cytosolic sialidase hNEU2 indicated that sialidase inhibition was affected by the C-3 fluorine stereochemistry and derivatization at C-5 and/or C-9 of the inhibitor. Opposite to that observed for influenza A virus sialidases and hNEU2, compounds with an axial fluorine at C-3 were better inhibitors (up to 100-fold) against bacterial sialidases compared to their 3Fequatorial counterparts. While C-5-modified compounds were less efficient anti-bacterial sialidase inhibitors, 9-N 3 -modified 2,3-difluoro-Neu5Ac showed increased inhibitory activity against bacterial sialidases. 9-Azido-9-deoxy-2-equatorial-3-axial-difluoro-N-acetylneuraminic acid (2e3aDFNeu5Ac9N 3 ) was identified as an effective inhibitor with a long effective duration selectively against pathogenic bacterial sialidases from Clostridium perfringens (CpNanI) and V. cholerae.

A highly efficient chemoenzymatic method for synthesizing glycosphingolipids using α-Gal pentasaccharyl ceramide as an example is reported here. Enzymatic extension of chemically synthesized lactosyl sphingosine using efficient sequential one-pot multienzyme (OPME) reactions allowed glycosylation be carried out in aqueous solution. Facile C18 cartridge-based quick (<30 minutes) purification proctols were established using minimal amounts of green solvents (CH3CN and H2O). Simple acylation in the last step led to the formation of the target glycosyl ceramide in 4 steps with an overall yield of 57%.

Streptococcus pneumoniae sialidase SpNanB is an intramolecular trans-sialidase (IT-sialidase) and a virulence factor that is essential for streptococcal infection of the upper and lower respiratory tract. SpNanB catalyzes the formation of 2,7-anhydro- N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a potential prebiotic that can be used as the sole carbon source of a common human gut commensal anaerobic bacterium. We report here the development of an efficient one-pot multienzyme (OPME) system for synthesizing 2,7-anhydro-Neu5Ac and its derivatives. Based on a crystal structure analysis, an N-cyclohexyl derivative of 2,7-anhydro-neuraminic acid was designed, synthesized, and shown to be a selective inhibitor against SpNanB and another Streptococcus pneumoniae sialidase SpNanC. This study demonstrates a new strategy of synthesizing 2,7-anhydro-sialic acids in a gram scale and the potential application of their derivatives as selective sialidase inhibitors.