Combined Application of Galactose Oxidase and β-N-Acetylhexosaminidase in the Synthesis of Complex Immunoactive N-Acetyl-D-galactosaminides

A high‐yield preparatory procedure for the synthesis of p‐nitrophenyl 2‐acetamido‐2‐deoxy‐β‐D‐galacto‐hexodialdo‐1,5‐pyranoside (2) using the galactose oxidase from Dactylium dendroides in a batch reactor was developed. Enzymatic recognition of this aldehyde and the respective uronic acid 3 obtained by NaClO2 oxidation was studied using a set of 36 fungal β‐N‐acetylhexosaminidases from Acremonium, Aspergillus, Penicillium and Talaromyces genera. The aldehyde 2 was readily hydrolysed by all tested β‐N‐acetylhexosaminidases but neither the uronic acid 3 nor its methyl ester 4 were accepted. Molecular modelling with docking into the active centre of the β‐N‐acetylhexosaminidase from Aspergillus oryzae revealed that the aldehyde 2 is processed as a C‐6 geminal diol by the enzyme. The aldehyde 2 was tested for transglycosylation reactions using GlcNAc as an acceptor. The β‐N‐acetylhexosaminidase from Talaromyces flavus gave the best yields (37%) of the transglycosylation product 2‐acetamido‐2‐deoxy‐β‐D‐ galacto‐hexodialdo‐1,5‐pyranosyl‐(1→4)‐2‐acetamido‐ 2‐deoxy‐D‐glucopyranose, which was oxidised in situ to yield the final product 2‐acetamido‐2‐deoxy‐β‐D‐galactopyranosyluronic acid‐(1→4)‐2‐acetamido‐2‐deoxy‐D‐glucopyranose (6). Compounds 3 and 6 were shown to be high‐affinity ligands for two natural killer cell activation receptors, NKR‐P1A and CD69. For the latter receptor they turned out to be among the best ligands described so far. This increase was obviously due to the presence of a carboxy moiety.