MUTASYNTHETIC AND BIOORGANIC STUDIES OF ANTIBIOTICS

我们建议继续进行含苯二酚的生物合成的工作。 抗生素，并将进一步发展我们对沙罗比星A、激动素的研究 D、链球菌素和萘嘧啶霉素，以及启动 我们认为可能与生物遗传有关的抗生素G7063-2 在我们正在进行的所有四个项目中，我们将合成和 测试我们认为是关键中间体的各种结构 每条生物合成途径。这些以及对G7063-2的研究将 主要使用稳定的同位素13C，2H，15N和18-0，它们将是 由高场核磁共振波谱检测，通常使用自旋耦合或 同位素诱导的位移技术。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氨基酸，我们 将准备每种相关微生物的无细胞提取物，以便 研究每种负责的酶和反应的机制 它们所催化的。其中包括转化莽草酸的酶。 在沙罗比星A生物合成中转化为6-羟基邻氨基苯甲酸 莽草酸合成4-氨基邻氨基苯甲酸和/或 4-氨基-3-羟基邻氨基苯甲酸在链球菌素生物合成中的作用 在激动素D中乙酸酯转化为3-氨基-2-羟基-6-甲基苯甲酸 生物合成。我们还将诱变产生 萘霉素、链球菌素、沙罗比星A和激动霉素D 开发出阻断在每条途径上的突变体。我们预料到 这些突变体将积累新的代谢物，这些代谢物是 抗生素的生物合成，这些化合物中的每一个都将被分离和 以提供有关路径的信息为特征的，否则 得不到的。在更遥远的未来，这些突变体将提供 用于研究这些通道的调节和理解的车辆 它们适应生产有机体发育的方式。

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.