Cucurbit[7]uril-Tetramethylrhodamine Conjugate for Direct Sensing and Cellular Imaging
Cucurbit[7]uril-Tetramethylrhodamine Conjugate for Direct Sensing and Cellular Imaging
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DOI：
10.1021/jacs.6b11140
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发表时间：
2016-12-21
影响因子：
15
通讯作者：
Urbach, Adam R.
中科院分区：
文献类型：
--
作者：
Bockus, Andrew T.;Smith, Lauren C.;Urbach, Adam R.
本文描述了一种偶联物（Q7 R）的设计和合成，该偶联物包括合成的主体瓜环[7]-脲（Q7）与荧光染料四甲基罗丹明（TMR）的连接，以及其光学和客体结合性质以及其细胞摄取的表征。Q7 R由单官能化的叠氮丁基-Q7和NHS活化的TMR分两步合成。Q7 R的荧光在客体结合时被淬灭，并且该可观察到的被用于确定平衡解离常数（K-d）值。出乎意料的是，客体与Q7 R和未修饰的Q7结合的K-d值基本相同。因此，Q7 R可以直接报告与Q7的结合，而没有由于缀合的荧光团而导致的能量损失。这一结果证明了一个潜在的一般策略，用于设计的单组分主机指示剂共轭物，灵敏地响应分析物，而不干扰主机的结合特性。Q7 R的独特性质使得能够在3个数量级和低至0.7 nM的浓度下测量K-d值。考虑到Q7所展示的无与伦比的客体范围和结合亲和力，该结果特别相关。活的和固定的HT 22神经元的共聚焦荧光显微镜显示Q7 R的细胞摄取和其在细胞质中的点状定位。Q7 R在浓度高达2.2 μ M时在4天内不改变细胞生长。这些实验证明了Q7 R作为客体结合的直接传感器和作为用于成像应用的细胞可渗透化合物的可行性。
This paper describes the design and synthesis of a conjugate (Q7R) comprising the synthetic host cucurbit[7]-uril (Q7) linked to the fluorescent dye tetramethylrhodamine (TMR), and the characterization of its optical and guest binding properties as well as its cellular uptake. Q7R was synthesized in two steps from monofunctionalized azidobutyl-Q7 and NHS-activated TMR. The fluorescence of Q7R is quenched upon guest binding, and this observable was used to determine equilibrium dissociation constant (K-d) values. Unexpectedly, the K-d values for guests binding to Q7R and to unmodified Q7 were essentially identical. Therefore, Q7R can directly report binding to Q7 without an energetic penalty due to the conjugated fluorophore. This result demonstrates a potentially general strategy for the design of single-component host indicator conjugates that respond sensitively to analytes without perturbing the binding properties of the host. The unique properties of Q7R enabled measurement of K-d values across 3 orders of magnitude and at concentrations as low as 0.7 nM. This result is particularly relevant given the unmatched range of guests and binding affinities demonstrated for Q7. Confocal fluorescence microscopy of live and fixed HT22 neurons revealed the cellular uptake of Q7R and its punctate localization in the cytoplasm. Q7R did not alter cell growth at concentrations up to 2.2,mu M over 4 days. These experiments demonstrate the feasibility of Q7R as a direct sensor for guest binding and as a cell permeable compound for imaging applications.