Metabolic Engineering with Bioorthogonal Chemical Reporters

生物正交化学报告基因的代谢工程

• 批准号: 7808050
• 负责人: Carolyn Bertozzi
• 金额: $ 45.91万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808050
• 关键词: Azides; Biological; Cancer Detection; Cancerous; Cell surface; Cells; Chemicals; Chemistry; Collection; Complex; Development; Diagnosis; Drug Kinetics; Embryo; Embryonic Development; Engineering; Environment; Gene Expression Profile; Global Change; Glycocalyx; Glycosaminoglycans; Goals; Grant; Human; Human body; Image; Imaging Device; Imaging Techniques; Injection of therapeutic agent; Kinetics; Label; Life; Ligation; Malignant Neoplasms; Metabolic; Metabolism; Methods; Microinjections; Modeling; Monitor; Mucins; Mus; Nucleotides; Oligosaccharides; Organism; Phosphines; Physicians; Polysaccharides; Property; Reaction; Reagent; Reporter; Research; Research Personnel; Resolution; Sialic Acids; Specific qualifier value; Staging; Streptavidin; Techniques; Time; Zebrafish; analog; cell transformation; cell type; cycloaddition; design; glycosyltransferase; improved; in vivo; public health relevance; response; spatiotemporal; sugar; tool; tumor; tumor progression; zebrafish development

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.

描述(申请人提供)：该项目的总体目标是将我们实验室开发的一项名为代谢低聚糖工程的技术应用于体内成像与胚胎发育和癌症相关的糖类的全球变化。“糖链”是细胞在特定的时间、空间和环境条件下产生的糖链的总和。糖类成分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，用于对活体中动态的细胞表面糖蛋白进行成像。在最后的授予期间，我们证明了糖基唾液酸化多糖的三个重要部分，粘蛋白O-糖化多糖和岩藻糖化多糖，可以用其生物合成前体的叠氮类似物进行代谢性标记。叠氮化合物作为化学报告分子，通过Staudinger与磷化氢探针的连接而被可视化。我们通过N-叠氮乙酰甘露糖胺(ManNAz)代谢标记，然后依次注射生物素化膦和荧光链霉亲和素结合物，在健康小鼠身上进行了唾液酸的非侵入性成像。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膦探针。为了提高葡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，其动力学速度比Staudinger连接反应快：菌种促进的叠氮化物和环辛酮的环加成反应(“无铜点击化学”)。我们使用二氟化环辛炔(Difo)对活细胞和发育中的斑马鱼的糖类的时空变化进行成像。在下一个授权期内，我们计划以这些发现为基础，以四个具体目标为基础。首先，我们将扩大我们对斑马鱼发育过程中血糖变化的分析(目标1)。我们将描绘处于不同发展阶段的糖类的新部门(例如唾液酸聚糖、岩藻糖化聚糖、糖胺聚糖和N-聚糖胺)。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伴随的糖基变化。我们将开发具有更好的药代动力学和荧光性能的新型环辛炔显像剂(目标2)。随着新的磷化氢和环辛炔探针的使用，我们将在小鼠肿瘤模型中成像葡聚糖(目标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)。 与公共健康相关：所有的人类细胞都包裹着被称为“多糖”的复杂糖分子。人体中每种类型的细胞都有自己的覆盖在细胞表面的这些多糖的集合。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症状态时，收集的多糖会改变其组成。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多糖的变化。这些化学工具可能对癌症检测和诊断有用。