This disclosure provides a method of synthesis of 4′-thioribose NAD+ and analogues thereof, using an efficient chemoenzymatic approach. Also provided are methods of inhibiting the CD38 enzyme and compounds including 4′-thioribose NAD+ and compounds related thereto.

This disclosure provides a method of synthesis of 4′-thioribose NAD+ and analogues thereof, using an efficient chemoenzymatic approach. Also provided are methods of inhibiting the CD38 enzyme and compounds including 4′-thioribose NAD+ and compounds related thereto.

Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry (“MS”), or Ultra Violet (“UV”) detection and/or a combination thereof.

Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry (“MS”), or Ultra Violet (“UV”) detection and/or a combination thereof.

New 2-deoxy-2,3-dehydro-sialic acids and 2,7-anhydro-sialic acids, which are useful as sialidase inhibitors, and enzymatic methods for preparing them are disclosed. The methods include forming a reaction mixture comprising a glycoside acceptor, a sialic acid donor, and a sialyltransferase; maintaining the reaction mixture under conditions sufficient to form a sialoside; and contacting the sialoside with a Streptococcus pneumoniae sialidase to form the sialic acid product. Methods for the inhibition and sialidases and the treatment of cancer and infectious diseases are also disclosed.

New 2-deoxy-2,3-dehydro-sialic acids and 2,7-anhydro-sialic acids, which are useful as sialidase inhibitors, and enzymatic methods for preparing them are disclosed. The methods include forming a reaction mixture comprising a glycoside acceptor, a sialic acid donor, and a sialyltransferase; maintaining the reaction mixture under conditions sufficient to form a sialoside; and contacting the sialoside with a Streptococcus pneumoniae sialidase to form the sialic acid product. Methods for the inhibition and sialidases and the treatment of cancer and infectious diseases are also disclosed.

The invention relates to a method for obtaining an N-acetylglucosamine containing neutral oligosaccharide from a fermentation broth, wherein said oligosaccharide is produced by culturing a genetically modified microorganism capable of producing said oligosaccharide from an internalized carbohydrate precursor, comprising the steps of: i) ultrafiltration (UF), preferably to separate biomass from the broth, ii) nanofiltration (NF), preferably to concentrate said oligosaccharide in the broth and/or reduce an inorganic salt content of the broth, and iii) treating the broth with an ion exchange resin, preferably to remove charged materials, and/or subjecting the broth to chromatography, preferably to remove hydrophobic impurities.

The invention relates to a method for obtaining an N-acetylglucosamine containing neutral oligosaccharide from a fermentation broth, wherein said oligosaccharide is produced by culturing a genetically modified microorganism capable of producing said oligosaccharide from an internalized carbohydrate precursor, comprising the steps of: i) ultrafiltration (UF), preferably to separate biomass from the broth, ii) nanofiltration (NF), preferably to concentrate said oligosaccharide in the broth and/or reduce an inorganic salt content of the broth, and iii) treating the broth with an ion exchange resin, preferably to remove charged materials, and/or subjecting the broth to chromatography, preferably to remove hydrophobic impurities.

Provided are a recombinant yeast having improved ability to produce ginsenoside, which is prepared by overexpressing INO2 and INO4 or deleting OPT1 in a yeast having ability to produce ginsenoside, a method of preparing the yeast, and a method of producing ginsenoside by using the yeast.

A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X-N-X-S/T (SEQ ID NO:1), wherein each X is the same or different and is any natural amino acid other than proline, wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:

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to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X-N-X-S/T (SEQ ID NO:1). Polyisoprenylpyrophosphates used as substrates in the biocatalytic process are also provided, as well as certain glycoconjugates.