Construction of A Chemical Library of Unnatural Natural Products by Using Biosynthetic Enzymes

1.角鲨烯环化酶。(1)利用酸性酸环杆菌的角鲨烯-藿烯环化酶，将新合成的C_<35>聚苯二烯转化为非天然的六环类聚苯二烯。这是首次证明了角鲨烯环化酶具有构建非自然六环骨架的非凡能力。此外，甲基延伸角鲨烯类似物被酶转化为非天然的五环新型聚戊烯类化合物。(2) Pisum sativum β-amyrin合成酶将22,23-二氢-2,3-氧化角鲨烯转化为bacchar12 -en-3 - β-ol；首次证明了酶促形成六元d环的巴卡烯骨架。值得注意的是，反马尔可夫六元d环的形成并不依赖于终端π电子的参与。此外，重组酶还将24,30-bisnor-2,3-氧化角鲨烯转化为29,30-bisnor-β-amyrin.2。植物聚酮合成酶（pks）。(1)利用丙二酰辅酶a和4-香豆酰辅酶a的类似物研究了植物聚酮合成酶的特异性，植物聚酮合成酶提供了一种非天然的C_6-C_5芳香聚酮。在查尔酮合成酶（CHS）反应中，我们发现聚酮链延伸反应的起始分子和延伸单元都可以同时被非生理底物取代。(2)苯扎丙酮合成酶（BAS）最显著的特征之一是缺乏活性位点Phe215；我们观察到BAS I214L/L215F突变体以pH依赖的方式表现出查尔酮形成活性，这支持了BAS中Phe 215的缺失导致聚酮链延伸在双酮阶段中断的假设。这些结果证实了Phe215在聚酮形成反应中的关键作用，为了解植物III型pks的结构-功能关系提供了结构基础。少

1.Squalene cyclizing enzymes.(1)Newly synthesized C_<35> polyprene was enzymatically converted to unnatural hexacyclic polyprenoid by squalene : hopene cyclase from Alicyclobacillus acidocaldarius. This is the first demonstration of the remarkable ability of the squalene cyclizing enzyme to perform construction of unnatural hexacyclic skeleton. Further, methylidene-extended squalene analogues were converted to unnatural pentacyclic novel polyprenoides by the enzyme. (2)Recombinant β-amyrin synthase from Pisum sativum converted 22,23-dihydro-2,3-oxidosqualene into bacchar-12-en-3β-ol ; the first demonstration of the enzymatic formation of the baccharene skeleton with a six-membered D-ring. It is remarkable that the formation of the anti-Markovnikov six-membered D-ring did not depend on he participation of the termianl π-electrons. Furthermore, the recombinant enzyme also converted 24,30-bisnor-2,3-oxidosqualene into 29,30-bisnor-β-amyrin.2.Plant polyketide synthases (PKSs).(1)Substrate … More specificities of plant polyketide synthases (PKSs) were investigated using analogs of malonyl-CoA and 4-coumaroyl-CoA, plant PKSs afforded an unnatural C_6-C_5 aromatic polyketide. In the chalcone synthase (CHS) reaction, it was shown that both the starter molecule and the extension unit of the poyketide chain elongation reaction could be simultaneously replaced with non-physiological substrates.(2)One of the most characteristic features of benzalacetone synthase (BAS) is that it lacks active site Phe215 ; the residues ^<214>LF conserved in type III PKSs are uniquely replaced by IL. Our observation that BAS I214L/L215F mutant exhibited chalcone forming activity in pH dependent manner supported a hypothesis that the absence of Phe 215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. These results confirmed the critical role of Phe215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs. Less