METABOLIC OLIGOSACCHARIDE ENGINEERING

描述（由申请人提供）：这是RO1 GM58867（“代谢寡糖工程”）的竞争性续期申请。该项目的长期目标是开发代谢寡糖工程，作为糖功能基础研究的工具，特别是在人类疾病方面。在上一个授权期内，我们发现含有生物正交官能团的非天然糖，如酮和叠氮化物，被细胞代谢并合并到细胞聚糖中。当显示在细胞表面时，功能化糖可以通过与外源试剂的共价反应进行修饰。我们将这种新技术应用于糖生物学的许多研究中，包括作为多糖表达模式功能的细胞表面靶向。该项目的一个组成部分是发展生物正交反应，这种反应可以在活细胞上表达的修饰聚糖上进行。我们开发了叠氮化物和磷化氢的反应，称为Staudinger连接，具有如此高的选择性，它可以在培养细胞上执行而不损害其生理。我们进一步证明了Staudinger连接在活体动物中进行，允许在体内用磷化氢探针对细胞表面叠氮糖进行共价靶向。

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.