HCID 131718406, compound → HCID 129681278, compound

4-Methylumbelliferyl-N-acetylneuraminic acid (commercially available), 100 mg is incubated with 150 units of sialidase-L in 25 ml of 50 mM sodium acetate buffer, pH 5.5 at 37° C. for 17 hours. The reaction is terminated by the addition of an equal volume of ethanol and the precipitate is removed by centrifugation. The clear supernatant is evaporated to dryness, dissolved in 30 ml of chloroform/methanol (2:1 ν/ν) and subjected to Folch partitioning (Folch, J. Lees, M. and Sloan Stanley, G. H. (1957) J. Biol. Chem. 226, 479-509), by vortexing with 6 ml of water. The 2,7-anhydro-N-acetylneuraminic acid is recovered in the aqueous phase. The organic phase is washed with 8 ml of water. The aqueous phase and the water wash are combined and evaporated to dryness, dissolved in 8 ml of water and applied to a Bio-Gel p-6 column (2.5×90 cm) equilibrated with water. The column is eluted with water at 20 ml per hour wherein 5-ml fractions are collected. Three microliter aliquots of each fraction are analyzed by thin-layer chromatography (solvent:n-butanol/acetic acid/water (2:1:1 ν/ν) and resorcinol spray to locate the product). Fractions containing 2,7-anhydro-N-acetylneuraminic acid are pooled, lyophilized, dissolved in 20 ml of water and applied to a Dowex-1×8 column (formate form, 2×50 cm) equilibrated with water. After washing the column with 800 ml of water to remove the unadsorbed material, the column is eluted with 500 ml of 0.2M formic acid followed by 400 ml of 0.4M formic acid at a flow rate of 40 ml per hour; 10-ml fractions are collected. Under this condition 2,7-anhydro-N-acetylneuraminic acid is eluted by 0.4M formic acid and the eluate is evaporated to dryness to obtain 15 mg of pure 2,7-anhydro-N-acetylneuraminic acid. The above scheme can be proportionally scaled up for large scale preparation of 2,7-anhydro-N-acetylneuraminic acid.

1. customat 37° C.
2. customfor 17 hours
3. customThe reaction is terminated by the addition of an equal volume of ethanol
4. customthe precipitate is removed by centrifugation
5. customThe clear supernatant is evaporated to dryness
6. dissolutiondissolved in 30 ml of chloroform/methanol (2:1 ν/ν)
7. custompartitioning (Folch, J. Lees, M. and Sloan Stanley, G. H. (1957) J. Biol. Chem. 226, 479-509)
8. customThe 2,7-anhydro-N-acetylneuraminic acid is recovered in the aqueous phase
9. washThe organic phase is washed with 8 ml of water
10. washThe aqueous phase and the water wash
11. customevaporated to dryness
12. dissolutiondissolved in 8 ml of water
13. washThe column is eluted with water at 20 ml per hour wherein 5-ml fractions
14. customare collected
15. additionFractions containing 2,7-anhydro-N-acetylneuraminic acid
16. dissolutiondissolved in 20 ml of water
17. washAfter washing the column with 800 ml of water
18. customto remove the unadsorbed material
19. washthe column is eluted with 500 ml of 0.2M formic acid
20. custom10-ml fractions are collected
21. washUnder this condition 2,7-anhydro-N-acetylneuraminic acid is eluted by 0.4M formic acid
22. customthe eluate is evaporated to dryness

ORD record

ord-8752b29f79b641abb12897e2353dab19

Source dataset

uspto-grants-1994_05

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05312747