A large-scale chemoenzymatic methodology has been developed to prepare a natural and substituted analogues of the Glycophorin A tetrasaccharide, in which the α-2,3-sialic acid is modified at either C5 or C9 either as F, OMe, CH3, glycolyl or N3. The strategy involved a chemical synthesis of a Gal-β-1,3-GalNAc disaccharide in which the galactose remains acetylated to first enable selective α-2,6-sialylation using a one-pot aldolase-NmCSS-Pd2,6-ST enzyme system. Finally, C5 or C9 modifed α-2,3 sialosides were placed at the galactoside moiety using C2 or C6 modified MaNAc derivatives catalyzed by aldolase NmCSS-Pd2,3-ST enzyme combination. The synthesised molecules can be useful in binding studies with a protein of interest or as an entry point to establish a library of analytical samples for mass-spectrometric studies.