Metabolic Engineering with Bioorthogonal Chemical Reporters

描述（由申请人提供）：本项目的总体目标是应用我们实验室开发的一种称为代谢寡糖工程的技术，用于与胚胎发育和癌症相关的糖的整体变化的体内成像。“糖类”是细胞在一定的时间、空间和环境条件下产生的多糖的总和。糖的组成和分布的变化与胚胎发生和癌症进展有关。我们寻求开发化学工具成像动态细胞表面糖在活的有机体。在最后一个授权期内，我们证明了糖的三个重要部分-唾液化聚糖，粘蛋白型o -聚糖和聚焦化聚糖，可以用其生物合成前体的叠氮类似物进行代谢标记。叠氮化物作为化学报告物，用磷化氢探针进行Staudinger连接可视化。我们用n-叠氮酰基甘露胺（ManNAz）进行代谢标记，然后依次注射生物素化膦和荧光链亲和素偶联物，对健康小鼠的唾液酸进行了无创成像。为了直接标记叠氮糖，我们设计了具有多种光谱性质的荧光磷化氢探针。为了提高聚糖成像的灵敏度和时间分辨率，我们开发了一种新的生物正交反应，其动力学比Staudinger连接更快：菌株促进叠氮化物和环辛烷的环加成（“cu -无点击化学”）。我们使用了一种二氟化环星（DIFO）来成像活细胞和发育中的斑马鱼糖的时空变化。在下一个资助期，我们计划在这些发现的基础上实现四个具体目标。首先，我们将扩展我们在斑马鱼发育过程中糖糖转化的分析（目的1）。我们将在不同的发展阶段对糖聚糖的新部门（例如，唾液化聚糖，聚焦化聚糖，糖胺聚糖和n -聚糖）进行成像。此外，我们将干扰某些糖基转移酶的表达，并通过体内成像监测血糖的伴随变化。我们将开发具有改进的药代动力学和荧光特性的新型环新生代成像试剂（目标2）。我们将使用新的磷化氢和环辛基探针对小鼠肿瘤模型中的多糖进行成像（目的3）。最后，我们将开发新的生物正交反应，以扩大化学报告方法的范围（目标4）。

DESCRIPTION (provided by applicant): The broad objective of this project is to apply a technique developed in our lab termed metabolic oligosaccharide engineering to in vivo imaging of global changes in the glycome associated with embryonic development and cancer. The "glycome" is the totality of glycans that cells produce under specified conditions of time, space and environment. Changes in the glycome's composition and distribution are associated with embryogenesis and cancer progression. We seek to develop chemical tools for imaging the dynamic cell-surface glycome in living organisms. In the last granting period, we demonstrated that three important sectors of the glycome - sialylated glycans, mucin-type O-glycans and fucosylated glycans, can be metabolically labeled with azido analogs of their biosynthetic precursors. The azide served as a chemical reporter that was visualized by Staudinger ligation with phosphine probes. We performed non-invasive imaging of sialic acids in healthy mice by metabolic labeling with N-azidoacetylmannosamine (ManNAz) followed by sequential injection of biotinylated phosphine and fluorescent streptavidin conjugates. For direct labeling of azidosugars, we designed fluorescent phosphine probes with a variety of spectral properties. In order to improve the sensitivity and time resolution of glycan imaging, we developed a new bioorthogonal reaction with faster kinetics than the Staudinger ligation: the strain-promoted cycloaddition of azides and cyclooctynes ("Cu-free click chemistry"). We employed a difluorinated cyclooctyne (DIFO) to image spatiotemporal changes in the glycomes of live cells and developing zebrafish. In the next granting period we plan to build upon these discoveries with four specific aims. First, we will expand our analysis of glycomic transformations during zebrafish development (Aim 1). We will image new sectors of the glycome (e.g., sialylated glycans, fucosylated glycans, glycosaminoglycans and N-glycans) at various stages of development. In addition, we will perturb the expression of certain glycosyltransferases and monitor concomitant changes in the glycome by in vivo imaging. We will develop new cyclooctyne imaging reagents with improved pharmacokinetic and fluorogenic properties (Aim 2). With the use of new phosphine and cyclooctyne probes, we will image glycans in mouse tumor models (Aim 3). Finally, we will develop new bioorthogonal reactions to expand the scope of the chemical reporter method (Aim 4). PUBLIC HEALTH RELEVANCE: All human cells are coated with complex sugar molecules termed "glycans". Each type of cell in the human body has its own collection of these glycans coating the cell surface. When cells transform from an embryonic state to a mature state, or from a healthy state to a cancerous state, the collection of glycans changes its makeup. The goal of this project is to develop tools from the field of chemistry that can help researchers and physicians monitor the changes in cell- surface glycans inside the body using imaging techniques. These chemical tools could be useful for cancer detection and diagnosis.