The Mechanism of Action of (-)-Lomaiviticin A.

(-)-Lomaiviticin A (4) is a complex C-symmetric bacterial metabolite that contains two diazofluorene functional groups. The diazofluorene consists of naphthoquinone, cyclopentadiene, and diazo substituents fused through a σ- and π-bonding network. Additionally, (-)-lomaiviticin A (4) is a potent cytotoxin, with half-maximal inhibitory potency (IC) values in the low nanomolar range against many cancer cell lines. Because of limitations in supply, its mechanism of action had remained a "black box" since its isolation in the early 2000s. In this Account, I describe how studies directed toward the total synthesis of (-)-lomaiviticin A (4) provided a platform to elucidate the emergent properties of this metabolite and thereby connect chemical reactivity with cellular phenotype. We first developed a convergent strategy to prepare the diazofluorene (9 + 10 → 13). We then adapted this chemistry to the synthesis of lomaiviticin aglycon (21/22) and the natural monomeric diazofluorene (-)-kinamycin F (3). The key step in the lomaiviticin aglycon (21/22) synthesis involved the stereoselective oxidative coupling of two monomeric diazofluorenes (2 × 18 → 20) to establish the cojoining carbon-carbon bond of the target. As the absolute stereochemistry of the aglycon and carbohydrate residues of (-)-lomaiviticin A (4) were unknown, we developed a semisynthetic route to the metabolite that proceeds in one step and 42% yield by diazo transfer to the more abundant isolate (-)-lomaiviticin C (6). This allowed us to complete the stereochemical assignment of (-)-lomaiviticin A (4) and provided a renewable source of material. Using this material, we established that the remarkable cytotoxic effects of (-)-lomaiviticin A (4) derive from the induction of highly toxic double-strand breaks (DSBs) in DNA. At the molecular level, 1,7-nucleophilic additions to each electrophilic diazofluorene trigger homolytic decomposition pathways that produce sp radicals at the carbon atoms of each diazo group. These radicals abstract hydrogen atoms from the deoxyribose of DNA, a process known to initiate strand cleavage. NMR spectroscopy and molecular mechanics simulations were used to elucidate the mode of DNA binding. These studies showed that both diazofluorenes of (-)-lomaiviticin A (4) penetrate into the duplex. This mode of non-covalent binding places each diazo carbon atom in close proximity to each DNA strand. Throughout these studies, isolates containing one diazofluorene, such as (-)-lomaiviticin C (6) and (-)-kinamycin C (2), were used as controls. Consistent with our mechanistic model, these compounds do not induce DSBs in DNA and are several orders of magnitude less potent. Reactivity studies suggest that (-)-lomaiviticin A (4) is more electrophilic than simple monomeric diazofluorenes. We attribute this to through-space delocalization of the developing negative charge in the transition state for 1,7-addition. Consistent with this mechanism of action, (-)-lomaiviticin A (4) displays selective low-picomolar potencies toward DNA DSB repair-deficient cell types. The emergent properties of (-)-lomaiviticin A (4) derive from the specific arrangement of diazo, naphthoquinone, cyclopentadiene, and ketone functional groups. These functional groups work together to yield, essentially, a masked vinyl radical that can be exposed under biological conditions. Furthermore, the rotational symmetry of the metabolite, deriving from dimerization, allows it to interact with the antiparallel symmetry of DNA and affect cleavage of the duplex.