Brogan Andrew P, Widger William R, Kohn Harold
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
J Org Chem. 2003 Jul 11;68(14):5575-87. doi: 10.1021/jo030020u.
Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. Key aspects of the 1.rho interaction-K(d), stoichiometry for 1.rho binding, and whether 1 and ATP binding induce conformational changes in rho-remain unknown. In this study, the design, synthesis, and characterization of a series of bicyclomycin fluorescent probes (BFP) constructed to sense the 1.rho interaction are described and their use documented. We show that dihydrobicyclomycins with medium-to-large C(5a)-substituents afforded excellent inhibitory activities exceeding those of 1 in the poly(C)-dependent ATPase assay. The utility of BFP in bicyclomycin-rho binding studies was documented through the use of 5a-(phenazin-2-ylmethylsulfanyl)dihydrobicyclomycin (15). Excitation (290 nm) of W381 in wild-type rho in the presence of 15 and ATP led to fluorescence resonance energy transfer (FRET) and gave a K(d) (15) of 9.9 microM. Using ADP in place of ATP or excluding nucleotide did not result in energy transfer, which suggests that ATP binding induced a conformational change in rho. FRET measurements provided an approximate weighted average distance (23 A) between W381 and 15 in the presence of bound ATP. The K(d) value for 15.rho was correlated with ATP binding at the 3 tight ATP binding (K(d)(ATP) = 95 nM) sites in wild-type rho.
双环霉素（1）是一种市售抗生素，其在大肠杆菌中的主要作用位点是转录终止因子rho。1与rho相互作用的关键方面——解离常数（K(d)）、1与rho结合的化学计量，以及1和ATP结合是否会诱导rho的构象变化——仍然未知。在本研究中，描述了一系列为检测1与rho相互作用而构建的双环霉素荧光探针（BFP）的设计、合成和表征，并记录了它们的用途。我们表明，具有中等到大的C(5a)取代基的二氢双环霉素在聚（C）依赖性ATP酶测定中具有优异的抑制活性，超过了1的活性。通过使用5a-（吩嗪-2-基甲基硫烷基）二氢双环霉素（15），证明了BFP在双环霉素-rho结合研究中的效用。在15和ATP存在下，野生型rho中W381的激发（290nm）导致荧光共振能量转移（FRET），并给出9.9 microM的K(d)（15）。使用ADP代替ATP或排除核苷酸不会导致能量转移，这表明ATP结合诱导了rho的构象变化。FRET测量提供了在结合ATP存在下W381与15之间的近似加权平均距离（23 Å）。15与rho的K(d)值与野生型rho中3个紧密ATP结合位点（K(d)(ATP)=95 nM）处的ATP结合相关。
