Figure 5. The WbpI reaction performed in an NMR tube and examined directly with NMR spectroscopy at 500 MHz (¹H).

The reaction mixture contained 90% H₂O/10% ²H₂O, 20 mM sodium phosphate (pH 6.4), 80 μg of His₆–WbpI, 10 mM UDP-α-D-GlcNAc3NAcA in 200 μl, and was incubated at 37 °C. (a) A plot showing the progress of the His₆–WbpI reaction that converts UDP-α-D-GlcNAc3NAcA (I) into UDP-α-D-ManNAc3NAcA (II) expressed as integrals for anomeric signals compared with time (integrals are normalized to I). The reaction was monitored directly through the acquisition of a proton spectrum at 10 min intervals (128 transients). (b) and (c) Proton spectra for the WbpI reaction showing the anomeric region at 12 min and 124 min respectively. * Represents a minor impurity that was present in the chemically synthesized substrate.