Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.
Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.
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DOI：
10.1002/anie.200903233
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发表时间：
2009
影响因子：
16.6
通讯作者：
Weissleder, Ralph
中科院分区：
文献类型：
--
作者：
Devaraj, Neal K.;Upadhyay, Rabi;Hatin, Jered B.;Hilderbrand, Scott A.;Weissleder, Ralph
人们对使用生物正交共价化学如“点击”化学来标记位于活细胞或固定细胞上的小分子有相当大的兴趣。[1]这种标记已用于聚糖的可视化、基于活性的蛋白质谱分析、蛋白质的位点特异性标记、DNA和RNA合成的检测、揭示植物中小分子的命运以及蛋白质中翻译后修饰的检测。[2-4]大多数报道的应用依赖于铜催化的叠氮化物-炔环加成，其由于铜的细胞毒性而限于体外应用，或者依赖于优雅的应变促进的叠氮化物-炔环加成，其允许活细胞和体内应用使用，但受到相对缓慢的动力学和环辛炔衍生物通常难以合成的阻碍。[4-5]新的生物正交反应，不需要催化剂，并显示出快速的动力学，因此在细胞水平上的不同的分子成像应用的兴趣。在这份报告中，我们证明了使用反电子需求狄尔斯-阿尔德环加成之间的血清稳定的1，2，4，5四嗪和高度紧张的反式环辛烯共价标记活细胞。这种化学方法已被应用于西妥昔单抗（爱必妥）标记的表皮生长因子受体（EGFR）在A549癌细胞上的预靶向标记。我们发现，四嗪环加成到反式环辛烯标记的细胞是快速的，可以通过增加抗体上的亲二烯体的负载来放大。这导致在一个高度敏感的靶向策略，可用于标记蛋白质，使用纳摩尔浓度的第二剂为短的持续时间。最近，我们和其他人已经探索了应变促进反电子需求狄尔斯-阿尔德环加成与1，2，4，5四嗪生物共轭。[6-7]我们以前已经表明，四嗪环加成到异戊烯可以应用于预靶向成像的活乳腺癌细胞。然而，在20 ℃下，血清中四嗪与异戊烯之间的环加成速率为1.6M-1 sec-1。该速率与先前报道的优化叠氮化物-环辛炔环加成的速率相当，并且需要微摩尔浓度以实现足够的标记。[3-4]基于先前报道的速率常数，我们有兴趣探索四嗪与更紧张的亲双烯体的偶联。[8]更高的速率常数将允许更快和更有效的标记，因此需要更少的标记剂并降低背景信号。最近，Fox及其同事报道了使用高度应变的反式环辛烯进行生物缀合。[6，9]虽然报告的比率令人印象深刻，但四嗪
There is considerable interest in the use of bioorthogonal covalent chemistry such as “click” chemistries to label small molecules located on live or fixed cells.[1] Such labeling has been used for the visualization of glycans, activity based protein profiling, site-specific tagging of proteins, detection of DNA and RNA synthesis, revealing the fate of small molecules in plants, and detection of post-translational modification in proteins.[2-4] Most reported applications rely on either the copper catalyzed azide-alkyne cycloaddition, which is limited to in vitro application due to the cytotoxicity of copper, or the elegant strain-promoted azide-alkyne cycloaddition, which permits live cell and in vivo application use but is hindered by relatively slow kinetics and often difficult synthesis of cyclooctyne derivatives.[4-5] New bioorthogonal reactions that do not require catalyst and show rapid kinetics are therefore of interest for different molecular imaging applications at the cellular level. In this report we demonstrate the use of inverse electron demand Diels-Alder cycloaddition between a serum stable 1, 2, 4, 5 tetrazine and a highly strained trans-cyclooctene to covalently label live cells. This chemistry has been applied to the pretargeted labeling of Cetuximab (Erbitux) tagged epidermal growth factor receptor (EGFR) on A549 cancer cells. We find that the tetrazine cycloaddition to transcyclooctene labeled cells is fast and can be amplified by increasing the loading of dienophile on the antibody. This results in a highly sensitive targeting strategy that can be used to label proteins using nanomolar concentrations of a secondary agent for short durations of time.Recently we and others have explored strain promoted inverse electron demand Diels-Alder cycloadditions with 1, 2, 4, 5 tetrazines for bioconjugation.[6-7] We have previously shown that tetrazine cycloaddition to norbornene can be applied to pretargeted imaging of live breast cancer cells. However, the rate of cycloaddition between the tetrazine and norbornene was 1.6 M-1sec-1 in serum at 20 C. This rate is comparable to previously reported rates for optimized azide-cyclooctyne cycloadditions and requires micromolar concentrations to achieve sufficient labeling.[3-4] Based on previously reported rate constants, we were interested in exploring the coupling of tetrazines with more strained dienophiles.[8] Higher rate constants would allow for faster and more efficient labeling thus requiring less labeling agent and lowering background signal. Recently, Fox and coworkers reported the use of a highly strained transcyclooctene for bioconjugation.[6, 9] Though the rates reported were impressive, the tetrazine
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影响因子：
15
作者：
Blackman, Melissa L.;Royzen, Maksim;Fox, Joseph M.
通讯作者：
Fox, Joseph M.
影响因子：
158.5
作者：
Lynch, TJ;Bell, DW;Haber, DA
通讯作者：
Haber, DA
影响因子：
16.6
作者：
Ning, Xinghai;Guo, Jun;Boons, Geert-Jan
通讯作者：
Boons, Geert-Jan
影响因子：
7.2
作者：
Kaschani, Farnusch;Verhelst, Steven H. L.;van der Hoorn, Renier A. L.
通讯作者：
van der Hoorn, Renier A. L.
影响因子：
11.5
作者：
Barrett, Tristan;Koyama, Yoshinori;Kobayashi, Hisataka
通讯作者：
Kobayashi, Hisataka