Caylobolide B: Structure Revision, Total Synthesis, Biological Characterization, and Discovery of New Analogues

Abstract The unique potential of marine polyhydroxylated macrolides in chemical biology and drug discovery has long been constrained by their structural complexity and limited material availability, frustrating efforts in stereochemical assignment, synthesis, and mechanism‐of‐action elucidation. Here, we establish an integrated workflow, combining chemogenomic profiling, ultra‐high‐resolution NMR, and modular total synthesis, for the comprehensive functional and structural interrogation of this challenging natural product class. Applying this approach to caylobolides, natural products isolated from scarce samples of Okeania sp., we performed structure‐activity relationship studies revealing that acetylation at C29 markedly reduces both cytotoxicity and antifungal activity, pinpointing a key pharmacophore. Mechanistic profiling suggests that these macrolides disrupt membrane integrity, similar to amantelide A. Using natural compound samples, we simultaneously revised the structure of caylobolide B through 1 H, 1D‐selective TOCSY and HSQC NMR, and developed a modular fragment‐based synthesis of these compounds. By providing a unified methodology for genetic sensitivity profiling, precise structure and stereochemistry determination, and modular total synthesis, this work unlocks new opportunities for the discovery and rational design of potent marine‐derived therapeutics.