Fluorescent and Chemiluminescent Cyclooctyne Probes for Imaging Glycosylation

描述（由申请人提供）：癌细胞显示出通常与癌症行为相关的异常糖基化细胞表面。然而，癌细胞为什么以及何时表现出异常糖基化的问题在很大程度上仍然没有答案，因为目前的分析工具无法观察到快速的糖基化变化。我计划开发一种成像平台，能够以空间和时间分辨率揭示癌症糖基化。所提出的策略将使用环辛炔探针（无铜点击反应）与非天然细胞表面叠氮化物标记的糖的化学反应，以将可视化剂附着到癌细胞上。新探针的创新在于叠氮化物成像的灵敏度和特异性; 探针被设计成仅在与叠氮化物反应后形成显色剂。这将增加成像灵敏度，因为脱靶结合和残留探针不会导致背景噪声。两代环辛炔探针将开发两种不同的可视化剂：荧光智能探针（目标1）和荧光探针（目标2）。荧光环辛炔探针将用于定量中国仓鼠卵巢细胞表面上的唾液酸含量（目的3）。荧光环辛炔探针将用于测量具有抗癌转移剂潜力的已知唾液酸转移酶抑制剂的有效性。该项目的长期目标是开发一种成像平台，能够阐明糖基化如何影响模型生物中的癌症行为。

DESCRIPTION (provided by applicant): Cancer cells display unusually glycosylated cell surfaces that are often correlated to cancer behavior. However, the question of why and when cancer cells exhibit aberrant glycosylation remains largely unanswered because current analytical tools cannot observe rapid glycosylation changes. I plan to develop an imaging platform able to reveal cancer glycosylation with both spatial and temporal resolution. The proposed strategy will use the chemical reaction of a cyclooctyne probe (copper-free click reaction) with an unnatural cell surface azide-labeled sugar to attach a visualizing agent to the cancer cell. The innovation of the new probes is sensitivity and specificity for azide imaging; the probes were designed to only form the visualizing agent after reaction with azide. This will increase imaging sensitivity because off-target binding and residual probe will not contribute to background noise. Two generations of cyclooctyne probes will be developed with two distinct visualizing agents: a fluorescent smart probe (Aim 1) and a chemiluminescent probe (Aim 2). The fluorescent cyclooctyne probe will be used to quantify sialic acid content on the surface of Chinese hamster ovary cells (Aim 3). The chemiluminescent cyclooctyne probe will be used to measure the effectiveness of a known sialyl transferase inhibitor with potential as an anti-cancer metastasis agent. The long-term goal of this project is to develop an imaging platform capable of elucidating how glycosylation affects cancer behavior in model organisms.