Metabolic Engineering with Bioorthogonal Chemical Reporters

描述（由申请人提供）：本项目的主要目标是将我们实验室开发的称为代谢寡糖工程的技术应用于与胚胎发育和癌症相关的糖组整体变化的体内成像。“糖组”是细胞在特定的时间、空间和环境条件下产生的聚糖的总和。糖组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，用于成像活生物体中的动态细胞表面糖组。在上一个授权期，我们证明了糖组的三个重要部分-唾液酸化聚糖、粘蛋白型O-聚糖和岩藻糖基化聚糖可以用其生物合成前体的叠氮基类似物进行代谢标记。叠氮化物充当化学报告物，其通过与膦探针的Staudinger连接而可视化。我们进行了非侵入性成像的唾液酸在健康小鼠的代谢标记N-叠氮乙酰甘露糖胺（ManNAz），然后依次注射生物素化膦和荧光链霉亲和素共轭物。为了直接标记叠氮糖，我们设计了具有多种光谱特性的荧光膦探针。为了提高聚糖成像的灵敏度和时间分辨率，我们开发了一种比Staudinger连接具有更快动力学的新的生物正交反应：叠氮化物和环辛炔的应变促进环加成（“无Cu点击化学”）。我们采用了二氟环辛炔（DIFO）的图像时空变化的活细胞和发育中的斑马鱼的糖。在下一个资助期内，我们计划以这些发现为基础，实现四个具体目标。首先，我们将扩大我们的分析，在斑马鱼发育过程中的糖组学转换（目标1）。我们将对糖组的新部分进行成像（例如，唾液酸化聚糖、岩藻糖基化聚糖、糖胺聚糖和N-聚糖）。此外，我们将扰乱某些糖基转移酶的表达，并通过体内成像监测伴随的糖组变化。我们将开发新的环辛炔显像剂，改善药物动力学和荧光特性（目标2）。通过使用新的膦和环辛炔探针，我们将对小鼠肿瘤模型中的聚糖进行成像（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）。 公共卫生相关性：所有人类细胞都被称为“聚糖”的复杂糖分子包裹。人体中每种类型的细胞都有自己的这些聚糖的集合覆盖在细胞表面。当细胞从胚胎状态转变为成熟状态，或从健康状态转变为癌性状态时，聚糖的聚集改变了其组成。该项目的目标是开发化学领域的工具，帮助研究人员和医生使用成像技术监测体内细胞表面聚糖的变化。这些化学工具可用于癌症检测和诊断。

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.