Aminoglycosides are among the most commonly used broadspectrum antibiotics. [1] The biological activity relies on their high affinity for the major groove of bacterial 16S rRNA, [2] thereby impeding protein synthesis. A number of aminoglycosides also display antiviral activity owing to specific interactions with viral RNA. [3] The rapid emergence of aminoglycoside resistance in the treatment of infections, however, is a serious threat. [4] In clinical isolates of aminoglycoside-resistant strains, the most frequently observed cause of resistance is the expression of N-acetyltransferases. [5] For example, aminoglycoside 6'-N-acetyltransferase type Ii (AAC(6')-Ii) is chromosomally encoded in Enterococcus faecium, which is one of the leading causes of hospitalacquired infections. [6] Studies by Wright and co-workers suggest that catalysis by AAC(6')-Ii occurs through an ordered bi-bi mechanism in which acetyl coenzyme A (Ac-CoA) must bind the enzyme before the aminoglycoside. [5, 7] Next, attack of the aminoglycoside 6'-NH 2 at the thioester of Ac-CoA is believed to generate a tetrahedral intermediate that collapses to yield an 6'-N-acetylaminoglycoside and CoA. Although crystal structures have been reported for complexes of AAC(6')-Ii with either Ac-CoA or CoA, crystallization experiments of enzyme-aminoglycoside complexes have not been successful. [8] It was envisaged that either 6'-N-(S-CoA)aminoglycoside derivatives or bisubstrates (Scheme 1) would facilitate the study of this important class of enzymes. Bisubstrate analogues have exhibited inhibition of serotonin