A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures

The ability of an engineered β-N-acetylhexosaminidase to utilize a reactive oxazoline as donor molecule for transglycosylation reaction to synthesize human milk oligosaccharide backbone structures was studied. The human milk oligosaccharide precursor lacto-N-triose II and three regioisomers could be synthesized using the oxazoline, which was either in situ-generated resulting in a chemo-enzymatic sequential cascade or was used as a purified compound.

The highest observed concentration of overall transglycosylation products in a cascade reaction was 13.7 mM after 18.5 h, whereas the use of purified oxazoline resulted in 25.0 mM of transglycosylation products after 6.5 h. Remarkably, the in situ-generated oxazoline could be used without any further purification and it was shown that the used enzyme tolerated significant amounts of reagents such as triethylamine, which is reported for the first time for an enzyme from the glycoside hydrolase family 20.