This proposal aims to develop a method that utilizes aminoglycoside acetyltransferases for the in-vitro generation of novel and clinically potent N-acylated aminoglicosides. I propose an efficient and unconventional chemoenzymatic approach for the production of a large variety of N-acylated aminoglycosides. By accomplishing the goals of the proposed research, I will introduce a new and important tool that will provide access to previously unknown and synthetically challenging aminoglycosides. This study may lead to the discovery of new aminoglycosides with superior activity against resistant strains as well explore directions to reduce their toxic side effects. The proposed strategy offers an elegant solution to the following problem: There are no existing general synthetic methodologies for the regio-selective N-acylation of aminoglycosides. Indeed, there are no efficient solutions as to how to chemically modify a specific amine group on an aminoglycoside that contain a series of chemically identical amines. The few existing examples require substantial efforts in both syntheses as well as in cost, and are limited to very specific cases. The three specific aims of the proposed research are: 1) To chemically synthesize coenzyme A (CoA) derivatives as substrates for the chosen AACs. Some CoAs will be utilized to directly produce the desired analogs. Other CoAs will serve as handles for the generation of libraries of new compounds after further derivatization by nucleophilic displacement or click chemistry. 2) We plan to perform biochemical studies on AACs that will target three different positions to be N-acylated on a large number of aminoglycosides. We will study enzyme promiscuity and generate libraries of new compounds using our methodology. 3) We plan to scale-up and test for antibacterial activity some of our novel analogs in search of improved antibacterial activity.