NOVEL CLICK REAGENTS FOR GLYCOPROTEIN ISOLATION AND VISUALIZATION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 叠氮化合物在生物系统中极为罕见，正成为生物缀合的有吸引力的化学手柄。特别地，Cu（I）催化的叠氮化物与末端炔的1，3-偶极环化以得到稳定的三唑已被用于标记各种生物分子，包括蛋白质、核酸、脂质和过氧化物。环加成反应也被用于基于活性的蛋白质分析、酶活性监测以及微阵列和小分子文库的化学合成。将叠氮化物安装到生物分子中的有吸引力的方法是基于代谢标记，由此使用细胞的生物合成机制将含叠氮化物的生物合成前体掺入生物分子中。这种方法已被用于用各种反应性探针标记生命系统的蛋白质、聚糖和脂质。这些探针可以促进糖选择性糖蛋白的作图和鉴定糖基化位点。炔探针也已用于叠氮化物修饰的生物分子的细胞表面成像，并且特别有吸引力的方法涉及通过[3+2]环加成从非荧光前体产生荧光探针。尽管有许多有吸引力的特征，铜催化的环加成的主要缺点是金属催化剂的细胞毒性，排除了其中细胞必须保持活力的应用。因此，有很大的需求，为发展的CuI自由[3+2]环加成。在这方面，炔可以通过环张力活化，例如，将炔限制在八元环内产生18 kcal/mol的张力，其中大部分在与叠氮化物的[3+2]环加成后以过渡态释放。因此，环辛炔与叠氮化物在室温下反应而不需要催化剂。应变促进的环加成反应已被用于标记生物分子，而没有可观察到的细胞毒性。然而，由于反应速度缓慢，这一办法的范围受到限制。在辛炔环上引入吸电子基团可以提高应变促进的环加成反应速率。 然而，这种类型的修饰可能使炔易于亲核攻击。施陶丁格连接与膦试剂提供了最有吸引力的试剂用于细胞表面标记的叠氮化物。

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.