DESIGN, SYNTHESES, AND EVALUATIONS OF BICYCLOMYCIN-BASED RHO INACTIVATORS

The commercial antibiotic bicyclomycin (1) has been shown to target th e essential transcriptional termination factor rho in Escherichia coli . Little is known, however, about the bicyclomycin binding site in rho . A recent structure-activity relationship study permitted us to desig n modified bicyclomycins that may irreversibly inactivate rho. The fou r compounds selected were C(5a)-(4-azidoanilino)dihydrabicyclomycin (3 ), C(5a)-(3-formylanilino)dihydrobicyclomycin (4), C(5)-norbicyclomyci n C(5)-O-(4-azidobenzoate) (5), and C(5)-norbicyclomycin C(5)-O-(3-for mylbenzoate) (6). In each of these compounds the inactivating unit was placed at the C(5)-C(5a) site in bicyclomycin. In compounds 3 and 5 a n aryl azide moiety was used as; a photoaffinity label whereas in 4 an d 6 an aryl aldehyde group was employed as a reductive amination probe . The synthesis and spectral properties of 3-6 are described. Chemical studies demonstrated that 3 and 4 were stable in D2O and CD3OD (room temperature, 7 d), while 5 and 6 underwent significant change within 1 d. Biochemical investigations showed that 3 and 4 retained appreciabl e inhibitory activities in rho-dependent ATPase and transcription term ination assays. In the ATPase assay, I-50 values for 1, 3, and 4 were 60, 135, and 70 mu M, respectively. Correspondingly, the I-50 values f or 5 and 6 were > 400 and 225 mu M, respectively. In the transcription termination assay, compounds 1, 3, and 4 all prevented (greater than or equal to 97%) the production of rho-dependent transcripts at 40 mu M, whereas little (less than or equal to 15%) inhibition of transcript ion termination was observed for 5 and 6 at this concentration. Antimi crobial evaluation of 3-6 showed that none of the four compounds exhib ited antibiotic activity at 32 mg/mL or less against W3350 E. coli. Th e combined chemical and biochemical studies led to our further evaluat ion of 3 and 4. Photochemical irradiation (254 nm) of 3 in the presenc e of rho led to a 29-32% loss of rho ATPase activity. Attempts to conf irm the irreversible adduction of 3 to rho by electrospray mass spectr ometry were unsuccessful, No higher molecular weight adducts were dete cted. Incubation of rho with 4 at room temperature (4 h) followed by t he addition of NaBH4 led to significant losses (>62%) of rho ATPase ac tivity. Analyses of the 4-rho modified adduct showed appreciable level s of adduction (similar to 40%). Mass spectrometric analyses indicated a molecular weight for the adduct of approximately 47 410, consistent with a modification of a rho lysine residue by 4. Compound 4 was sele cted for additional studies.