Abstract-The C5 00 -OH group in neomycin B was glycosylated with a variety of mono-and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity and interaction with the aminoglycosides modifying enzyme APH(3 0 )-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3 0 )-IIIa