A series of new derivatives of the clinically used aminoglycoside antibiotic paromomycin were designed, synthesized, and their ability to read-through premature stop codon mutations was examined in both in vitro translation system and ex vivo mammalian cultured cells. One of these structures, a pseudo-trisaccharide derivative, showed notably higher stop codon read-through activity in cultured cells compared to those of paromomycin and gentamicin.