The total synthesis of plusbacin A(3) (1) has been accomplished wing a solvent-dependent diastereodivergent Joullie-Ugi three-component reaction (JU-3CR) as a key Step. Two trans-3-hydroxy-L-proline residues were constructed by combining the JU-3CR with a convertible isocyanide strategy Subsequent peptide coupling and macrolactamization afforded plusbacin A(3). Investigating the antibacterial activity of 1 compared with that of its dideoxy analogue revealed that the tlirco-beta- hydroxyaspartic acid residues are essential for antibacterial aCtivity. Notably, there is a low potential for the development of resistance in S. aureus against plusbacin A(3).