Chemical synthesis of 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid glycoside and its use for the fluorometric detection of poorly expressed natural and recombinant sialidases.
When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid (CF3MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF3MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for the in vivo examination of sialidases and for the detection of recombinant sialidase by means of expression cloning.
Engstler
M
M
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
Talhouk
J W
JW
Smith
R E
RE
Schauer
R
R
D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006027:Glycosides / Q000138:chemical synthesis* / Q000737:chemistry; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000302:isolation & purification*; D013379:Substrate Specificity; D013997:Time Factors
C108020:4-trifluoromethylumbelliferyl-N-acetylneuraminic acid glycoside; D005456:Fluorescent Dyes; D006027:Glycosides; D006923:Hymecromone; D009439:Neuraminidase