Glyco-optimization (OPopS) of aminoglycosides has been performed by replacing the existing sugar moiety with a variety of sugar derivatives. Glycosylation of the 6-position of nebramine provided a library of novel 4,6-linked aminoglycosides (AMGs). Among them, compounds 8b,g,i,l, and 8u with 2"-amino, 2",3"-diamino, 2",4"-diamino, 3",4"-diamino, 3"-amino groups, respectively, showed significant antimicrobial activity against Gram-(+) and -(-) bacteria. Several were particularly potent against Pseudomonus aeruginosa with MICs in the 1-2 microg/mL range.