The biosynthesis of N-N bond-containing natural products

项目总结 微生物天然产物具有复杂的化学结构和强大的生物活性，是一种 重要的毒品来源。虽然这些分子几十年来一直吸引着合成化学家和药物化学家， 最近，构建天然产品支架的潜在生物合成途径是 被公认为新型酶的重要储存库。发现新的酶化学和生物合成 战略拓展了我们对自然综合能力的基本理解。这也是迈向以下目标的关键第一步 这一基础知识的应用，对合成化学家有启发作用。长的- 这项拟议研究的术语目标是确定催化以前未被认识的微生物酶。 化学变化。我们设想通过研究天然的生物合成来发现这种酶。 含有重要的分子结构和未知生物合成来源的官能团的产品。一个 这类结构基序的重要类别是含有氮-氮(N-N)键的官能团， 在200种最畅销的药物中，有9%的药物含有化学物质。含有反应性N-N键的官能团， 包括重氮基和N-亚硝基，是生物活性小分子的重要组成部分，包括 链脲佐菌素(Zanosar®)，临床上用于治疗转移性胰腺癌。它们也是独特的 启用化学试剂，重氮化合物介导许多重要和强大的化学物质 合成化学、生物催化和双正交化学中的转化。通过反应性N-N键 虽然存在于微生物天然产物中，但人们对它们的生物合成来源知之甚少。因此，总体上， 本应用的目的是发现和鉴定构建重氮和N-亚硝基的酶。 含有代谢物的。我们实验室和其他实验室的初步结果发现了生物合成基因簇 负责构建多种含重氮和N-亚硝基的天然产物，包括链脲佐菌素 以及其他正在进行临床试验的分子。这些发现为我们的三个互补奠定了基础 具体目标：1)鉴定和表征负责构建重氮基团的生物合成酶 2)生物合成酶的鉴定和性质 负责构建天然产物链脲佐菌素和丙氨酸氨基的N-亚硝基；3)访问 其他重氮和N-亚硝基生物合成酶和天然产物通过表征隐蔽的基因簇。 通过利用基因组时代微生物天然产物的巨大结构多样性，我们将迅速 发现并表征生物合成转化，以填补我们目前对酶的知识的关键空白 化学能力。最后，我们为研究活性N-的生物合成制定了工作流程。 含N键的官能团也可以很容易地概括为在 微生物天然产物。

PROJECT SUMMARY Microbial natural products possess complex chemical structures as well as potent biological activity and are an important source of drugs. While these molecules have captivated synthetic and medicinal chemists for decades, more recently the underlying biosynthetic pathways that construct natural product scaffolds have been recognized as important reservoirs of novel enzymes. Uncovering new enzymatic chemistry and biosynthetic strategies expands our basic understanding of Nature’s synthetic capabilities. It is also a critical first step toward applications of this fundamental knowledge and can serve as an inspiration for synthetic chemists. The long- term goal of the proposed research is to identify microbial enzymes that catalyze previously unappreciated chemical transformations. We envision discovering such enzymes by studying the biosynthesis of natural products containing important molecular architecture and functional groups of unknown biosynthetic origin. An important class of such structural motifs are functional groups containing a nitrogen-nitrogen (N–N) bond, a chemical linkage found in 9% of the 200 best-selling drugs. Reactive N–N bond-containing functional groups, including diazo and N-nitroso groups, are a critical part of biologically active small molecules including streptozotocin (Zanosar®), a clinically used treatment for metastatic pancreatic cancer. They are also uniquely enabling chemical reagents, with diazo compounds mediating many important and powerful chemical transformations in synthetic chemistry, biocatalysis, and biorthogonal chemistry. Though reactive N–N bonds are present in microbial natural products, their biosynthetic origins are poorly understood. Thus, the overall objective of this application is to discover and characterize enzymes that construct diazo- and N-nitroso- containing metabolites. Preliminary results from our lab and others have uncovered biosynthetic gene clusters responsible for constructing multiple diazo- and N-nitroso-containing natural products, including streptozotocin and other molecules that have been in clinical trials. These findings set the stage for our three complementary specific aims: 1) identify and characterize the biosynthetic enzymes responsible for constructing the diazo groups of the natural products cremeomycin and kinamycin; 2) identify and characterize the biosynthetic enzymes responsible for constructing the N-nitroso groups of the natural products streptozotocin and alanosine; 3) access additional diazo and N-nitroso biosynthetic enzymes and natural products by characterizing cryptic gene clusters. By leveraging the tremendous structural diversity of microbial natural products in the genomic era, we will rapidly discover and characterize biosynthetic transformations that fill critical gaps in our current knowledge of enzymatic chemistry capabilities. Finally, the workflow we have formulated for investigating the biosynthesis of reactive N– N bond-containing functional groups will also be readily generalizable to additional structural motifs found in microbial natural products.