Total Synthesis and Biosynthesis of Bioactive Substances

描述（由申请人提供）：此修订的资助申请专注于实验研究，以阐明三种生物遗传相关家族的天然产物的生物合成：（1）Paraherquamides，Escherichinines，Malbranchemaides，Marcfortines和Chrysogenamide（单氧代哌嗪）;（2）Stephacidins，Notoamides，Waikialoid和Brevianamides（二氧代哌嗪类）;和（3）桔碱类、桔碱类、环匹胺类和PF 1270生物碱类（脱羰基生物碱类）。目标是利用生物合成酶的全合成、全基因组测序、生物信息学分析、基因组挖掘、功能表达和X射线晶体学的强大协同作用，充分阐明所有三个生物碱家族的相应生物合成途径。I. Paraherquamides，alcoholine，Malbranchemaides，Marcfortine和Chrysogenamide：单氧代哌嗪。我们的实验室已经阐明了这类生物碱是通过一种罕见的生物合成的分子内[4+2]环加成反应构建的，该反应是由Trp-Pro的还原释放引起的（或Trp-Pip）二肽氨基-醛，所述二肽氨基-醛来自NRPS模块，其在反向异戊二烯化时经历环化、脱水通过异构化和分子内Diels-Alder环加成来构建该家族共有的单氧代哌嗪双环[2.2.2]二氮杂辛烷环系统。通过使用同位素标记的中间代谢物的全化学合成，基因组挖掘，生物合成基因簇鉴定和生物合成酶的功能表达，这些复杂的次级代谢物的生物合成途径的关键特征将被实验阐明。在与大卫谢尔曼教授实验室（密歇根大学）的多PI关系和子奖项中，我们积极参与这些生物医学重要生物碱的整个生物合成途径的高分辨率阐明。二. Stephacidins，Notoamides，Waikialoids和Brevianamides：二氧哌嗪。双环[2.2.2]二氮杂辛烷生物碱的二氧代哌嗪家族是通过一个完全的 异戊二烯化二氧代哌嗪底物。我们已经发现，曲霉属的两个直链菌种产生相反的对映体Stephacidn A和Notoamide B。这种迷人的对映体生物发生将由新的合作者，著名的进化遗传学家Martin Kreitman教授使用生物信息学分析进行进一步评估，以确定导致这些复杂生物碱的对映体罕见产生的进化机制。三. Citrinadins，Citrinalins，Cyclopiamines和PF 1270生物碱：脱羰基Alakloids。作为我们对异戊烯化吲哚生物碱的Paraherquamide家族的工作的自然产物，我们建议启动一个新的项目来研究这些结构相关的生物碱的全合成和生物合成，这些生物碱似乎是从生物遗传学上产生的。 双环[2.2.2]二氮杂辛烷前体的还原脱羰基。在这里，我们也将部署强大的协同作用，全合成，全基因组测序，生物信息学分析，基因组挖掘和功能表达的生物合成酶，充分阐明相应的生物合成途径，以所有三个家庭的生物碱。在所有三个子项目中，天然产物和同位素标记的生物合成中间体和探针分子的全合成将用于确认途径转化。在该计划中产生的新化学实体，无论是来自生物来源还是通过化学合成，都将在密歇根大学化学基因组学中心，国家人类基因组研究所，和Sachiko Tsukamoto教授（日本），使用一系列生物化学和细胞-基于与癌症和寄生虫病靶标相关的测定。其他合作者包括：日本熊本大学Sachiko Tsukamoto教授、丹麦技术大学Jens Frisvad教授、芝加哥大学Martin Kreitman教授和密歇根大学Janet Smith教授。

DESCRIPTION (provided by applicant): This revised grant application is focused on experimental studies to elucidate the biosynthesis of three biogenetically related families of natural products: (1) the Paraherquamides, Asperparalines, Malbranchemaides, Marcfortines and Chrysogenamide (the monooxopiperazines); (2) the Stephacidins, Notoamides, Waikialoids and Brevianamides (the dioxopiperazines); and (3) the Citrinadins, Citrinalins, Cyclopiamines and PF1270 alkaloids (the decarbonylated alkaloids). The goals are to exploit the powerful synergies of total synthesis, whole genome sequencing, bioinformatics analysis, genome mining, functional expression and X-ray crystallography of biosynthetic enzymes to fully elucidate the corresponding biosynthetic pathways to all three families of alkaloids. I. Paraherquamides, Asperparalines, Malbranchemaides, Marcfortines and Chrysogenamide: the Monooxopiperazines. Provocative evidence has been elucidated in our laboratory indicating that this class of alkaloids, are constructed by a rare biosynthetic intramolecular [4+2] cycloaddition reaction resulting from the reductive release of a Trp-Pro (or Trp-Pip) dipeptide amino-aldehyde from a NRPS module that upon reverse prenylation, suffers a cascade of cyclization, dehydration, tautomerization and intramolecular Diels-Alder cycloaddition to construct the monooxopiperazine bicyclo[2.2.2]diazaoctane ring system common to this family. Through the use of total chemical synthesis of isotopically labeled intermediate metabolites, genome mining, biosynthetic gene cluster identification and functional expression of biosynthetic enzymes, key features of the biosynthetic pathways to these complex secondary metabolites will be experimentally elucidated. In a multi-PI relationship and sub-award with Prof. David Sherman's laboratory (University of Michigan), we are actively engaged in the high-resolution elucidation of the entire biosynthetic pathway to these biomedically significant alkaloids. II. Stephacidins, Notoamides, Waikialoids and Brevianamides: The Dioxopiperazines. The dioxopiperazine family of bicyclo[2.2.2]diazaoctane alkaloids are constructed by a net oxidative transformation of a fully prenylated dioxopiperazine substrate. We have discovered that two orthologous species of Aspergillus produce the opposite enantiomers of Stephacidn A and Notoamide B. This fascinating enantiodivergent biogenesis will be further evaluated using bioinformatics analysis by a new collaborator, Prof. Martin Kreitman, a renowned evolutionary geneticist, to determine the evolutionary mechanisms that resulted in this rare production of opposite enantiomers of these complex alkaloids. III. Citrinadins, Citrinalins, Cyclopiamines and PF1270 Alkaloids: The Decarbonylated Alakloids. As a natural out-growth of our work on the Paraherquamide family of prenylated indole alkaloids, we propose to initiate a new project to study the total synthesis and biosynthesis of these structurally related alkaloids that appear to have arisen biogenetically from the reductive decarbonylation of bicyclo[2.2.2]diazaoctane progenitors. Here also, we shall deploy the powerful synergies of total synthesis, whole genome sequencing, bioinformatics analysis, genome mining and functional expression of biosynthetic enzymes to fully elucidate the corresponding biosynthetic pathways to all three families of alkaloids. In all three sub-projects, total synthesis of the natural products and isotopically-labeled biosynthetic intermediates and probe molecules will be utilized to confirm pathway transformations. New chemical entities generated in this program, either from the biological sources or through chemical synthesis, will be extensively screened and evaluated for biological activities at the Univ. of Michigan Center for Chemical Genomics, the National Human Genome Research Institute, and to Prof. Sachiko Tsukamoto (Japan) for analysis of biological activity using a series of biochemical and cell-based assays relevant to cancer and parasitic disease targets. Additional collaborators include: Prof. Sachiko Tsukamoto, Kumamoto University, Japan; Prof. Jens Frisvad, Technical University, Denmark; Prof. Martin Kreitman, University of Chicago; and Prof. Janet Smith, University of Michigan.