Synthesis of linkages-specific sialoside substrates for colorimetric assay of neuraminidases

Abstract

Neuraminidase substrates suitable for analysis of linkage specificity were enzymically synthesized in good yield by linking N-acetylneuraminic acid (Neu p5Ac) to O-6 and O-3 of 4-nitrophenyl β- d-galactopyranoside with β- d-galactoside-α-(2→6)-sialyltransferase and β- d-galactoside-α-(2→3)-sialyltransferase, respectively. By use of these substrates, a convenient colorimetric assay method was developed for the determination of linkage specificity of bacterial and viral neuraminidases. The substrates are incubated with viral or bacterial neuraminidase and subsequently treated with β- d-galactosidase to convert the liberated 4-nitrophenyl β- d-galactopyranoside to 4-nitrophenol. The amount of liberated 4-nitrophenol is equivalent to the amount of Neu p5Ac released from the substrate, thus allowing measurement of neuraminidase activity. The results showed that bacterial and viral neuraminidases can discriminate between these two compounds, making them useful substrates for the rapid determination of neuraminidase linkage specificity.