Fosfazinomycin and kinamycin are natural products that contain nitrogennitrogen (N-N) bonds but that are otherwise structurally unrelated. Despite their considerable structural differences, their biosynthetic gene clusters share a set of genes predicted to facilitate N-N bond formation. In this study, we show that for both compounds, one of the nitrogen atoms in the N-N bond originates from nitrous acid. Furthermore, we show that for both compounds, an acetylhydrazine biosynthetic synthon is generated first and then funneled via a glutamyl carrier into the respective biosynthetic pathways. Therefore, unlike other pathways to N-N bond-containing natural products wherein the N-N bond is formed directly on a biosynthetic intermediate, during the biosyntheses of fosfazinomycin, kinamycin, and related compounds, the N-N bond is made in an independent pathway that forms a branch of a convergent route to structurally complex natural products.More than 200 natural products containing nitrogen-nitrogen (N-N) bonds have been identified with various bioactivities (Fig. 1a) 1 . For instance, streptozotocin, a nitrosamine-containing compound, exhibits cytotoxic activity and is currently deployed clinically 2 , and valanimycin, featuring an azoxy group, displays both antimicrobial and cytotoxic activities 1 . Other examples are azamerone containing a pyridazine, kutzneride with a piperazic acid 2, 3 , the fosfazinomycins having a phosphonohydrazide 4 , and a group of molecules including cremeomycin, the lomaiviticins and the kinamycins containing diazo groups 1 .

More than 35% of human urinary bladder cancers involve oncogenic H-Ras activation. The goal of this study was to investigate the role of the ERK pathway in mediating apoptotic signals induced by oncogenic H-Ras, FK228 treatment, and exogenous H(2) O(2) treatment to increase Nox-1 elevation, leading to production of intracellular reactive oxygen species (ROS) for inducing apoptosis in human bladder cancer J82 cells. Our study revealed that FK228 combined with exogenous H(2)O(2) cooperatively induced activation of Mek1/2 and Erk1/2 to increase Nox-1 elevation, intracellular ROS production, caspase activation, and cell death. Expression of oncogenic H-Ras significantly increased these FK228- and exogenous H(2)O(2)-induced effects. Oncogenic H-Ras-increased cell susceptibility to FK228 could be alternately achieved by additional treatment with exogenous H(2)O(2). Hence, combined use of FK228 with ROS-generating agents may apply to therapeutic strategies to preferentially kill malignant cells with or without oncogenic H-Ras activation.