MUTASYNTHETIC AND BIOORGANIC STUDIES OF ANTIBIOTICS

我们建议继续我们的工作，生物合成含醌的 抗生素，并将进一步发展我们的研究， D，链黑菌素和萘啶霉素，以及启动工作， 抗生素G7063 - 2，我们认为它可能与 沙蚕A. 通过我们正在进行的所有四个项目，我们将综合和 测试各种我们认为是关键中间体的结构， 每个生物合成途径。 这些以及对G7063 - 2的研究，将 主要使用稳定同位素13 C、2 H、15 N和18 - 0，它们将 通过高场NMR光谱检测，通常使用自旋耦合或 同位素诱导位移技术。 我们的工作已经揭示了 在这些途径中的三个中存在新的芳香族氨基酸，我们 将制备每种相关微生物的无细胞提取物， 研究每一种负责的酶和反应机制 它们催化的。 这些酶包括将莽草酸 6-羟基邻氨基苯甲酸， 莽草酸至4-氨基邻氨基苯甲酸和/或 4-氨基-3-羟基邻氨基苯甲酸， 在卡那霉素D中将乙酸盐转化为3-氨基-2-羟基-6-甲基苯甲酸 生物合成 我们还将诱变微生物 萘啶霉素、链黑菌素、沙丁胺醇A和卡那霉素D， 来产生阻断这些通路的突变体。 我们预计 这些突变体将积累新的代谢物，这些代谢物是 抗生素生物合成，这些化合物中的每一种都将被分离， 其特征在于提供了关于该途径的信息， 无法获得。 在更遥远的未来，这些突变体将提供 研究这些途径的调节和理解的工具 它们如何适应生产生物体的发展。

We propose to continue our work on the biosynthesis of quinone-containing antibiotics, and will further develop our studies on sarubicin A, kinamycin D, streptonigrin, and naphthyridinomycin, as well as initiate work on antibiotic G7063-2 which we believe may be biogenetically related to sarubicin A. With all four of our ongoing projects we will synthesize and test a variety of structures that we believe to be key intermediates in each biosynthetic pathway. These as well as the study of G7063-2, will primarily use the stable isotopes 13C, 2H, 15N, and 18-0, and they will be detected by high field NMR spectroscopy, often using spin-coupling or isotope-induced shift techniques. Our work has already revealed the presence of new aromatic amino acids in three of these pathways, and we will prepare cell-free extracts of each relevant microorganism in order to study each of the responsible enzymes and the mechanisms of the reactions that they catalyze. These include the enzymes that convert shikimic acid to 6-hydroxyanthranilic acid in sarubicin A biosynthesis, that convert shikimic acid to 4-aminoanthranilic acid and/or 4-amino-3-hydroxyanthranilic acid in streptonigrin biosynthesis, and that convert acetate to 3-amino-2-hydroxy-6-methylbenzoic acid in kinamycin D biosynthesis. We will also mutagenize the microorganisms that produce naphthyridinomycin, streptonigrin, sarubicin A, and kinamycin D, in order to develop mutants blocked in each of these pathways. We anticipate that these mutants will accumulate new metabolites that are intermediates in the antibiotic biosynthesis, and each of these compounds will be isolated and characterized to provide information about the pathway that would otherwise be unobtainable. In the more distant future, these mutants will provide vehicles for studying the regulation of these pathways and understanding the way they fit into the development of the producing organism.