| 研究実績の概要 |
Sialidases are members of a family of exoglycosidases specifically catalyzing the removal of sialic acid residues on the surface of exposed glycoconjugates. A novel fluorescence probe HMRef-S-Neu5Ac has been synthesised by incorporating a self-immolative spacer group between the sialic acid residue and the HMRef fluorophore to prevent hydrolysis via an acid-mediated pathway (i.e. in the absence of the target enzyme sialidase). First, kinetic measurements have shown the probe to have similar properties to that of a commercially available sialidase substrate, 4-MUNANA, demonstrating that the spacer group does not impede substrate recognition. Second, the stabilities of HMRef-S-Neu5Ac and a commercially avaiable 4MU-Neu5Ac were monitored over time at selected pHs of 2, 4, 6, 7.4 and 8. 4MU-Neu5Ac only showed significant instability at pH 2 where over the two-hour period fluorescence increased 5.6-fold. HMRef-S-Neu5Ac showed stability across all pHs within the two-hour experiment time. These experiments demonstrate that this probe is a better probe than 4MU-Neu5Ac due to its superior stability allowing for specificity for sialidase hydrolysis over acidic hydrolysis. The probe has been assessed in prostate cancer cells (LNCaP, DU145, PC3) which are reported to have significant levels of sialidase. Furthermore, some preliminary experiments of bacterial samples using this probe have been very positive and are currently under investigation.
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