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-nitroplienyl 2-acetamido-2-deoxy-beta-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 NaClO(2) oxidation was studied using a set of 36 fungal beta-N-acetylhexosaminidases from Acremonium, Aspergillus, Penicillium and Talaromyces genera. The aldehyde 2 was readily hydrolysed by all tested beta-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 beta-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 beta-N-acetylhexosaminidase from Talaromyces flavus gave the best yields (37%) of the transglycosylation product 2-acetamido-2-deoxy-beta-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-beta-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.