METABOLIC OLIGOSACCHARIDE ENGINEERING

描述（由申请人提供）：这是RO 1 GM 58867（“代谢寡糖工程”）的竞争性续展申请。该项目的长期目标是开发代谢寡糖工程，作为聚糖功能基础研究的工具，特别是在人类疾病方面。在上一个授权期，我们发现带有生物正交官能团的非天然糖，如酮和叠氮化物，被细胞代谢并掺入细胞聚糖中。当展示在细胞表面上时，官能化的糖可以通过与外源试剂的共价反应来修饰。我们在糖生物学的许多研究中采用了这种新技术，包括作为聚糖表达模式的函数的细胞表面靶向。该项目的一个组成部分是开发可对活细胞上表达的修饰聚糖进行的生物正交反应。我们开发了一种叠氮化物和膦的反应，称为施陶丁格连接，具有如此高的选择性，可以在培养细胞上进行，而不会损害它们的生理学。我们进一步证明了施陶丁格连接在活体动物中进行，允许在体内用膦探针共价靶向细胞表面叠氮糖。 下一个资助期的第一个主要目标是探索代谢寡糖工程在肿瘤成像和免疫治疗中的应用。这一目标反映在目标1-4中。目的1将定义叠氮糖代谢的范围和结果在实验室小鼠。这包括鉴定在体内用叠氮糖标记的特定糖缀合物和在正常和肿瘤组织中叠氮糖代谢的比较研究。目的2将叠氮糖代谢和施陶丁格连接技术应用于肿瘤糖基化的无创显像。目的3：发展笼化策略以提高叠氮糖标记肿瘤的选择性。目的4概述了一种新的肿瘤疫苗治疗方法，该方法利用非天然糖作为能够打破免疫自身耐受的新抗原。 第二个主要目标是扩大代谢寡糖工程，包括新的碳水化合物途径和新的生物正交化学。这一目标反映在目标5和6中。目的5探索叠氮岩藻糖类似物的代谢。目的6描述了对Huisgen叠氮化物-炔环加成的修改，其可以使反应适合于体内应用。

DESCRIPTION (provided by applicant): This is a competing renewal application of RO1 GM58867 ("Metabolic Oligosaccharide Engineering"). The long-term objective of this project is to develop metabolic oligosaccharide engineering as a tool for fundamental studies of glycan function, particularly with respect to human disease. In the last granting period we discovered that unnatural sugars bearing bioorthogonal functional groups, such as ketones and azides, are metabolized by cells and incorporated into cellular glycans. When displayed on the cell surface, the functionalized sugars can be modified by covalent reactions with exogenous reagents. We employed this new technique in numerous studies of glycobiology, including cell surface targeting as a function of glycan expression pattern. An integral component of the project was the development of bioorthogonal reactions that can be performed on modified glycans expressed on living cells. We developed a reaction of azides and phosphines, termed the Staudinger ligation, with such high selectivity that it can be executed on cultured cells without detriment to their physiology. We further demonstrated that the Staudinger ligation proceeds in living animals, permitting the covalent targeting of cell-surface azidosugars with phosphine probes in vivo. The first major objective of the next granting period is to explore applications of metabolic oligosaccharide engineering to tumor imaging and immunotherapy. This goal is reflected in Aims 1-4. Aim 1 will define the scope and consequences of azidosugar metabolism in laboratory mice. This includes identifying the specific glycoconjugates that are labeled with azidosugars in vivo and comparative studies of azidosugar metabolism in normal and tumor tissues. Aim 2 will apply azidosugar metabolism and the Staudinger ligation to noninvasive imaging of tumor glycosylation. Aim 3 focuses on the development of caging strategies to enhance the selectivity of tumor labeling with azidosugars. Aim 4 outlines a new approach to tumor vaccine therapy that, exploits unnatural sugars as neoantigens capable of breaking immune self-tolerance. The second major objective is to expand metabolic oligosaccharide engineering to encompass new carbohydrate pathways and new bioorthogonal chemistries. This goal is reflected in Aims 5 and 6. Aim 5 will probe the metabolism of azido fucose analogs. Aim 6 describes a modification to the Huisgen azide-alkyne cycloaddition that may render the reaction suitable for in vivo applications.