Integrated microfluidics for parallel screening of an in situ click chemistry library
Integrated microfluidics for parallel screening of an in situ click chemistry library
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DOI：
10.1002/anie.200601677
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Tseng, Hsian-Rong
中科院分区：
文献类型：
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作者：
Wang, Jinyi;Sui, Guodong;Tseng, Hsian-Rong
人们对在微流体装置中进行化学反应越来越感兴趣[1-3]，因为它们比宏观反应器具有各种优势，例如减少试剂消耗，高表面积体积比，以及改善对质量和传热的控制。与传统的宏观合成相比，涉及高活性中间体的有机反应[4,5]在微反应器中往往表现出更高的选择性和特异性。然而，在微反应器的发展中仍然存在许多挑战：1)多步合成，其中单个步骤需要改变溶剂，试剂和条件；2)平行筛选，其中使用不同的试剂组合进行类似类型的反应。为改进微反应器的性能，已经投入了大量的努力来开发功能模块。例如，各种阀门[6,7]已被证明可以隔离不同的区域，并防止微芯片中不同反应的交叉污染。在其他地方，已经使用不同的混合模块[8,9]来克服无湍流微流体环境中限制扩散的混合。此外，能够输送和计量流体成分的功能泵[7,10]已成功地与微通道集成在一起。有了这些功能模块，在微反应器中以自动化的方式处理复杂的化学和生物过程变得可行。事实上，集成微反应器已被用于分子成像探针的顺序合成、[9]聚合酶链反应、[11]蛋白结晶、[12]和细胞培养原位点击化学是一种靶标引导合成方法[13-19]，通过1,3偶极环加成在靶标的结合袋内组装互补的叠氮化物和乙炔构建块来发现高亲和力的蛋白质配体。[19-22]与单个片段相比，所得到的配体对目标的结合亲和力要高得多，而且命中的识别就像使用LC-MS等分析仪器检测产物形成一样简单。[19,21]近年来，利用该方法制备了乙酰胆碱酯酶（AchE）、[19,22,23]牛碳酸酐酶II （bCAII）、[21]和HIV蛋白酶[24]的有效抑制剂通常，在原地点击化学实验进行平行使用96孔微量滴定板。不可避免地，实验条件导致大量靶蛋白和试剂的消耗，这阻碍了原位点击化学筛选的广泛应用，特别是当靶蛋白难以获得时。此外，现有方法严重依赖于人工操作，这限制了筛选的吞吐量和保真度。集成微流体为原位点击化学筛选提供了一个极好的实验平台，因为它可以实现并行化和自动化，最重要的是，小型化允许经济地使用目标蛋白质和点击化学试剂。在这里，我们描述了一种新型的微流控化学反应电路（图1），它提供了一个自动化的平台，用于并行筛选32个原位点击化学反应，减少了靶蛋白和试剂的消耗。我们选择了已知的bCAII点击化学系统[21]进行这个概念验证研究。以乙酰苯磺酰胺（1;Kd= 37 Æ6 nm）作为反应支架（锚定分子）筛选20个互补叠氮化物2-21文库。在对照实验中，我们使用活性位点抑制剂乙氧唑胺（22;Kd= 0.15 Æ 0.03 nm）抑制原位…
There is growing interest in performing chemical reactions in microfluidic devices [1–3] because they offer a variety of advantages over macroscopic reactors, such as reduced consumption of reagents, high surface-area-to-volume ratios, and improved control over mass and heat transfer. Organic reactions [4, 5] that involve highly reactive intermediates often exhibit greater selectivities and specificities in microreactors compared to conventional macroscopic synthesis. Many challenges remain, however, in the development of microreactors for 1) multistep syntheses in which the individual steps require a change in solvents, reagents, and conditions, as well as 2) parallel screening in which similar types of reactions are performed using different combinations of reagents. Significant efforts have been devoted to develop functioning modules to improve the performance of microreactors. For example, various valves [6, 7] have been demonstrated to isolate distinct regions and prevent crosscontamination from different reactions in a microchip. Elsewhere, different mixing modules [8, 9] have been utilized to overcome diffusion-limited mixing in the turbulence-free microfluidic environment. Also, functioning pumps [7, 10] that are capable of delivering and metering fluidic components have been successfully integrated with microchannels. With these functioning modules, it becomes feasible to handle complicated chemical and biological processes in microreactors in an automated fashion. In fact, integrated microreactors have been utilized for sequential syntheses of molecular imaging probes,[9] polymerase chain reaction,[11] protein crystallization,[12] and cell culture.[10] Insitu click chemistry is a target-guided synthesis [13–19] method for discovering high-affinity protein ligands by assembling complementary azide and acetylene building blocks inside the binding pockets of the target through 1, 3-dipolar cycloaddition.[19–22] The resulting ligands display much higher binding affinities to the target than the individual fragments, and the identification of a hit is as simple as detecting product formation by using analytical instruments, such as LC-MS.[19, 21] Recently, this approach was employed to prepare potent inhibitors for acetylcholine esterase (AchE),[19, 22, 23] bovine carbonic anhydrase II (bCAII),[21] and HIV protease.[24] Typically, in situ click chemistry experiments are conducted in parallel using 96-well microtiter plates. Inevitably, the experimental conditions result in the consumption of significant amounts of the target proteins and reagents, which hampers the broad application of in situ click chemistry screening, especially when target proteins are difficult to obtain. Moreover, the existing approach relies heavily upon manual operation, which limits screening throughput and fidelity. Integrated microfluidics provides an excellent experimental platform for in situ click chemistry screening because it enables parallelization and automation, and, most importantly, the miniaturization allows an economical use of target proteins and click chemistry reagents. Herein, we describe a new type of microfluidic chemical reaction circuits (Figure 1) that provide an automated platform for the parallel screening of 32 in situ click chemistry reactions, with reduced consumption of target proteins and reagents. We selected the known bCAII click chemistry system [21] for this proof-of-concept study. Acetylenic benzenesulfonamide (1; Kd= 37 Æ6 nm) was used as the reactive scaffold (anchor molecule) for screening a library of 20 complementary azides 2–21. In control experiments, we utilized the active-site inhibitor, ethoxazolamide (22; Kd= 0.15 Æ 0.03 nm), to suppress the in situ …