In Vitro Glycorandomization of Natural Products

说明(申请人提供)：天然产品糖的结合决定了亲本天然产品的靶向性、生物活性和/或药理作用，天然产品糖基化模式的改变已被证实可用于天然产品为基础的治疗药物的产生和/或优化。然而，目前还缺乏简单有效的方法来改变添加到天然产品中的糖。这项研究的第一阶段(RO1 AI52218，1-5年)导致了一种非常成功的酶策略的开发和实施，以用不同的D-糖阵列交换天然产物糖，并提供了关于两个关键但知之甚少的酶类(异构体糖激酶和糖-1-磷酸核苷酰转移酶)的基本信息。该项目拟议的第二阶段(6-10年)旨在专门将该计划扩展到复杂的天然产物L-糖苷。模型糖基转移酶(GT)已被选择用于探索糖基转移酶(GT)机制的独特研究，尤其是我们最近发现的GT催化反应的易逆性。所选择的酶--大肠杆菌Galk编码的半乳糖激酶(Galk)、沙门氏菌RMLA编码的α-D-葡萄糖-1-磷酸胸苷转移酶(RMLA)以及一系列作用于大环内酯类(MegD1、TylCV和OLED)或非核糖体肽(GtfD和GtfE)的GT都是碳水化合物加工反应的模型，在自然界中普遍存在，与人类疾病有关。在本研究中靶向天然产物支架的许多优点中，大环内酯类和非核糖体肽的差异糖基化提供了具有明显独特的抗生素、抗病毒和抗寄生虫特性的代谢物。总而言之，本文描述的研究将扩大我们对基本碳水化合物修饰酶催化的反应的理解，并提供前所未有的途径，获得通过传统有机合成或体内途径工程难以获得的独特的生物活性天然产物库。

DESCRIPTION (provided by applicant): Natural product sugar attachments dictate the targeting, biological activity and/or pharmacology of the parent natural product and the alteration of natural product glycosylation patterns has been validated for the generation and/or optimization of natural product-based therapeutics. Yet, simple effective methods to alter sugars appended to natural products are currently lacking. The first phase of this study (RO1 AI52218, years 1-5) led to the development and implementation of a remarkably successful enzymatic strategy to exchange natural product sugars with diverse D-sugar arrays and also provided fundamental information regarding two critical, but poorly understood, enzyme classes (anomeric sugar kinases and sugar-1- phosphate nucleotidylyltransferases). The proposed second phase of this project (years 6-10) is designed to specifically expand this program toward complex natural product L-glycosides. Model GTs have been selected to enable unique studies to probe glycosyltransferase (GT) mechanism with a particular focus upon our recent discovery of the facile reversibility of GT-catalyzed reactions. The enzymes selected - E. coli galK-encoded galactokinase (GalK), Salmonella rmlA-encoded alpha-D-glucose-1-phosphate thymidylyltransferase (RmlA) and a set of GTs which act upon macrolides (MegD1, TylCV and OleD) or nonribosomal peptides (GtfD and GtfE) - are all models for carbohydrate processing reactions ubiquitous in nature and relevant to human disease. Among the many advantages of the targeted natural product scaffolds within this study, differential glycosylation of macrolides and nonribosomal peptides present metabolites with markedly unique antibiotic, antiviral and antiparasitic properties. Cumulatively, the studies described herein will extend our understanding of reactions catalyzed by essential carbohydrate modifying enzymes and offer unprecedented access to uniquely bioactive natural product libraries not readily accessible via conventional organic synthesis or in vivo pathway engineering.