NOVEL CLICK REAGENTS FOR GLYCOPROTEIN ISOLATION AND VISUALIZATION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 叠氮化合物在生物系统中极其罕见，正在成为生物偶联的诱人的化学处理。特别是，铜(I)催化叠氮化物与末端炔的1，3-偶极环化反应得到稳定的三氮唑，已被用于标记包括蛋白质、核酸、脂类和糖类在内的各种生物分子。环加成也被用于基于活性的蛋白质图谱，酶活性的监测，以及微阵列和小分子文库的化学合成。将叠氮化合物安装到生物分子中的一种有吸引力的方法是基于代谢标记，即利用细胞的生物合成机制将含有叠氮化合物的生物合成前体结合到生物分子中。这种方法已经被用来用各种反应性探针标记生命系统的蛋白质、多糖和脂类。这些探针有助于糖选择性糖蛋白的定位和糖基化位点的识别。炔探针也被用于叠氮修饰生物分子的细胞表面成像，一个特别吸引人的方法涉及通过[3+2]环加成从非荧光前体生成荧光探针。尽管有许多吸引人的特点，铜催化的环加成反应的一个主要缺点是金属催化剂的细胞毒性，这排除了电池必须保持活性的应用。因此，开发无CuI的[3+2]环加成反应是非常有必要的。在这方面，炔可以被环应变激活，例如，将炔限制在八元环内会产生18千卡/摩尔的应变，其中大部分在与叠氮[3+2]环加成时以过渡态释放。因此，环辛炔在室温下与叠氮化合物反应，而不需要催化剂。菌种促进的环加成反应已被用于标记生物分子，但没有明显的细胞毒性。然而，由于反应速度较慢，该方法的范围受到了限制。在辛炔环上添加吸电子基团可以提高应变促进的环加成反应的速率。然而，这种类型的修饰可能会使炔烃容易发生亲核攻击。Staudinger与膦试剂的连接为叠氮化物的细胞表面标记提供了最有吸引力的试剂。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Azides, which are extremely rare in biological systems, are emerging as attractive chemical handles for bioconjugation. In particular, the Cu(I) catalyzed 1,3-dipolar cyclization of azides with terminal alkynes to give stable triazoles has been employed for tagging a variety of biomolecules including proteins, nucleic acids, lipids, and saccharides. The cycloaddition has also been used for activity-based protein profiling, monitoring of enzyme activity, and the chemical synthesis of microarrays and small molecule libraries. An attractive approach for installing azides into biomolecules is based on metabolic labeling whereby an azide-containing biosynthetic precursor is incorporated into biomolecules using the cells' biosynthetic machinery. This approach has been employed for tagging proteins, glycans, and lipids of living systems with a variety of reactive probes. These probes can facilitate the mapping of saccharide-selective glycoproteins and identify glycosylation sites. Alkyne probes have also been used for cell surface imaging of azide-modified biomolecules and a particularly attractive approach involves the generation of a fluorescent probe from a non-fluorescent precursor by a [3+2] cycloaddition. Despite many attractive features, a major disadvantage of the copper-catalyzed cycloaddition is the cellular toxicity of the metal catalyst, precluding applications wherein cells must remain viable. Hence, there is great need for the development of CuI free [3+2] cycloadditions. In this respect, alkynes can be activated by ring strain and, for example, constraining an alkyne within an eight membered ring creates 18 kcal/mol of strain, much of which is released in the transition state upon [3+2] cyclcoaddition with an azide. As a consequence, cyclooctynes react with azides at room temperature without the need of a catalyst. The strain-promoted cycloaddition has been used to label biomolecules without observable cyto-toxicitiy. The scope of the approach has, however, been limited due to the slow rate of reaction. Appending electron-withdrawing groups to the octyne ring can increase the rate of strain-promoted cycloadditions. However, this type of modification may make the alkyne prone to nucleophilic attack. Staudinger ligation with a phosphine reagent offers the most attractive reagent for cell surface labeling of azides.