The cepafungins are a class of highly potent and selective eukaryotic proteasome inhibitor natural products with potential to treat refractory multiple myeloma and other cancers. The structure-activity relationship of the cepafungins is not fully understood. This account chronicles the development of a chemoenzymatic approach to cepafungin I. A failed initial route involving derivatization of pipecolic acid prompted us to examine the biosynthetic pathway for the production of 4-hydroxylysine, which culminated in the development of a 9-step synthesis of cepafungin I. An alkyne-tagged analog enabled chemoproteomic studies of cepafungin and comparison of its effects on global protein expression in human multiple myeloma cells to the clinical drug bortezomib. A preliminary series of analogs elucidated critical determinants of potency in proteasome inhibition. Herein we report the chemoenzymatic syntheses of 13 additional analogs of cepafungin I guided by a proteasome-bound crystal structure, 5 of which are more potent than the natural product. Enzymatic strategies enabled the facile synthesis of oxidized amino acids in the macrocycle warhead as well as the tail fragment. Additional analogs were prepared by chemical methods to further explore the SAR at other regions of the scaffold. These studies reveal the criticality of the macrocyclic L-lysine oxidation regio-/stereochemistry introduced in the natural product biosynthesis relative to other possible lysine oxidation patterns found in nature. The lead analog was found to have seven-fold greater proteasome b5 subunit inhibitory activity and has been evaluated against several multiple myeloma and mantle cell lymphoma cell lines in comparison to the clinical drug bortezomib.