Scheme 1. Structures of studied donor substrate ligands: pNP-Neu5Ac (1), pNP-Oct5Ac (2), pNP-Hept5Ac (3) and pNP-Neu5,9Ac₂ (4).

Figure 1. Time-dependent ¹H NMR experiments of pNP-Neu5Ac (1) in the presence of TSia. Hydrolysis rates were detected by following the decrease of the pNP signals from pNP-Neu5Ac 1 over time.

Figure 2. (a) TSia activity in the presence pNP-Neu5Ac 1 donor substrate and the absence or presence of methyl β-d-galactoside acceptor. Diamonds denote the overall conversion in the absence of acceptor, asterisks the overall conversion in the presence of acceptor, triangles the transfer rate in the presence of acceptor and squares denote the hydrolysis rate in the presence of acceptor. In the presence of acceptor, the overall conversion rate is reduced. Transfer and hydrolysis rate are in the same range, indicating that about 50% of the conversion is still hydrolysis. (b) TSia activity in the presence of different donor substrate analogues and the absence and presence of methyl β-d-galactoside acceptor. Filled squares denote the conversion rate of pNP-Oct5Ac (2) in the absence of acceptor, open squares the conversion rate of pNP-Oct5Ac in the presence of acceptor, filled triangles the conversion rate of pNP-Hept5Ac (3) in the absence of acceptor, open triangles the conversion rate of pNP-Hept5Ac in the presence of acceptor, filled circles the conversion rate of pNP-Neu5,9Ac₂ (4) in the absence of acceptor and open circles denote the conversion rate of pNP-Neu5,9Ac₂ 4 in the presence of acceptor. In contrast to the ‘natural’ donor, pNP-Neu5Ac shows the analogues pNP-Oct5Ac 2 and pNP-Hept5Ac 3 shows a significant increased TSia activity in the presence of acceptor and pNP-Neu5,9Ac₂ 4 shows a slightly increased activity in the presence of acceptor. (c) Transfer and hydrolysis rate in the presence of different donor substrate analouges in the presence of methyl-β-d-galactoside acceptor. Filled squares denote the hydrolysis rate of pNP-Oct5Ac (2), open squares the transfer rate of pNP-Oct5Ac, filled triangles the hydrolysis rate of pNP-Hept5Ac (3), open triangles the transfer rate of pNP-Hept5Ac, filled circles the hydrolysis rate of pNP-Neu5,9Ac₂ (4) and open circles denote the transfer rate of pNP-Neu5,9Ac₂. For pNP-Oct5Ac 2 and pNP-Hept5Ac 3, the hydrolysis rate is significantly higher than the transfer rate, whereas for pNP-Neu5,9Ac₂ 4, the hydrolysis rate is lower than the transfer rate.

Figure 3. Reference (top) and STD NMR (bottom) spectrum of pNP-Neu5Ac (1) in the presence of TSia. Only hydrolysed pNP leads to significant STD signals, whereas from pNP-Neu5Ac and Neu5Ac alone, no signals are detectable. The signal at 1.7 ppm is an artifact due to direct irradiation of the NAc group.

Figure 4. STD spectra of pNP-Oct5Ac 2 (top), pNP-Hept5Ac 3 (middle) and pNP-Neu5,9Ac₂ 4 (bottom). All theses ligands bind to TSia with the pNP moiety dominating the binding. For the sugar moiety, no sizeable STD signals were detected (the signals in the range between 1 and 4.5 ppm are mostly due to direct irradiation; for a discussion see text). In addition signals from hydrolyzed pNP are visible.