In Vitro Glycorandomization of Natural Products

描述（由申请人提供）：天然产物糖附着物决定了母体天然产物的靶向性、生物活性和/或药理学，天然产物糖基化模式的改变已被验证用于基于天然产物的疗法的生成和/或优化。然而，目前还缺乏简单有效的方法来改变天然产物中附加的糖。该研究的第一阶段（RO1 AI52218, 1-5年）导致了一种非常成功的酶促策略的开发和实施，该策略将天然产物糖与各种d -糖阵列交换，并提供了关于两个关键但鲜为人知的酶类（糖激酶和糖-1-磷酸核苷酸转移酶）的基本信息。该项目第二阶段（6-10年级）旨在将该项目扩展到复杂的天然产物l -糖苷。选择模型GT来进行独特的研究，以探索糖基转移酶（GT）的机制，特别关注我们最近发现的GT催化反应的易可逆性。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-磷酸胸苷基转移酶（RmlA）和一组作用于大环内酯类（MegD1， TylCV和OleD）或非核糖体肽（GtfD和GtfE）的GTs——都是自然界中普遍存在的碳水化合物加工反应的模型，与人类疾病有关。在本研究的靶向天然产物支架的众多优点中，大环内酯和非核糖体肽的差异糖基化产生的代谢物具有明显独特的抗生素，抗病毒和抗寄生虫特性。总之，本文所述的研究将扩展我们对必需碳水化合物修饰酶催化反应的理解，并提供前所未有的独特生物活性天然产物库，这些天然产物库不易通过传统的有机合成或体内途径工程获得。

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.