Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked substances on epoxide-coated glass surfaces
Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked substances on epoxide-coated glass surfaces
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DOI：
10.1002/anie.200462720
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发表时间：
2005-01-01
影响因子：
16.6
通讯作者：
Shin, I
中科院分区：
文献类型：
--
作者：
Lee, MR;Shin, I
调节生物过程的小分子在研究生物分子，特别是蛋白质的功能以及药物开发中是有价值的工具。[1]以发现生物活性分子为目标的努力的一个重要组成部分是高通量筛选。依赖于DNA、蛋白质和碳水化合物微阵列的技术已被广泛用于加速先导化合物的选择，并作为基因组、转录组、蛋白质组和糖学研究的高通量分析工具。[2-4]微阵列平台使有限数量的大量样品能够同时进行评估。同样，小分子微阵列被用作高通量方法来识别选择性与蛋白质结合的物质。[5]这些努力中感兴趣的大多数小分子都具有许多不同的官能团，如羟基(OH)、氨基(NH2)、羧基(CO2H)和磺基(SH)。用于制备小分子微阵列的技术的主要要求是将不同官能化的化合物固定在修饰表面上必须具有高度的选择性。使用高效和化学选择性连接过程的策略通常适用于拥有共价连接的生物感兴趣分子的微阵列的制造。在这里，我们描述了一种新的化学选择性固定化过程，在该过程中，含肼的化合物与包覆在玻璃片上的环氧化物反应。这项新技术已被应用于有效构建化学微阵列，并已用于评估蛋白质与多肽和小分子的结合。在设计一种制备各种化学微阵列的通用方法时，必须满足几个标准。首先，固相合成法必须容易地制备含有特定官能团的各种物质，这些物质用于与改性固体表面的选择性反应。此外，将选择性地与小分子反应的官能团必须容易地结合到固体表面上。最后，当它们从固体载体上释放后，不同结构和官能化的小分子必须在修饰的表面上进行特定位置的共价连接。采用高度化学选择性连接反应的策略符合这些标准。我们研究了一种新的固定化技术，该技术依赖于含肼的小分子与环氧化物涂层玻璃片之间的反应(方案1)。酰肼基团结合到小分子中，同时它们附着在固体载体上，并在固相合成中用作手柄。[6]将涂有胺的玻璃片浸入聚乙二醇二缩水甘油醚(3%溶液在10 mM碳酸氢钠中，pH 8.3)的溶液中，很容易产生环氧化物衍生玻璃片。[7]使用酰肼连接的岩藻糖探针(S-或L-富克-NHNH2，S：短，L：长；方案2)探索固定化过程的最佳条件(pH，时间和浓度)。S-或L-Fuc-NHNH_2的制备是通过将WANG树脂上的醇基转化为对硝基苯碳酸酯来开始的。[8]所得树脂用肼处理得到含肼的树脂，该树脂在NEM、BOP和HOBt存在下与Fmoc保护的L或S锚链偶联。氨基
Small molecules that regulate biological processes serve as valuable tools in studies of the functions of biomolecules, especially proteins, as well as in the development of drugs.[1] An important component of efforts that target the discovery of bioactive molecules is high-throughput screening. Technologies that rely on the use of DNA, protein, and carbohydrate microarrays have been widely employed to accelerate the selection of lead compounds and as high-throughput analytical tools in genomic, transcriptomic, proteomic, and glycomic research.[2–4] Microarray platforms enable the simultaneous assessment of a large number of samples that are available in limited quantities. Similarly, small-molecule microarrays have been used as high-throughput methods to identify substances that selectively bind to proteins.[5] Most of the small molecules of interest in these efforts possess a number of different functional groups, such as hydroxy (OH), amino (NH2), carboxy (CO2H), and sulfanyl groups (SH). The major requirement of techniques used to fabricate small-molecule microarrays is that immobilization of the diversely functionalized compounds to the modified surfaces must be highly selective. Strategies that employ efficient and chemoselective ligation processes would be generally applicable to the fabrication of microarrays that possess covalently linked, biologically interesting molecules. Herein we describe a novel chemoselective immobilization process in which hydrazidecontaining compounds react with epoxides coated on glass slides. This new technique has been applied to the efficient construction of chemical microarrays, which have been used to evaluate protein binding to peptides and small molecules. Several criteria must be met in designing a general method to prepare diverse chemical microarrays. Firstly, the diverse substances containing the specific functional groups used for selective reactions with the modified solid surfaces must be easily prepared by solid-phase synthesis. Also, functional groups that will selectively react with the small molecules must be readily incorporated onto the solid surfaces. Lastly, following their release from a solid support, the diversely structured and functionalized small molecules must undergo site-specific covalent attachment to the modified surfaces. Strategies employing highly chemoselective ligation reactions fit these criteria. We have investigated a novel technique for immobilization, which relies on the use of reactions between hydrazide-containing small molecules and epoxide-coated glass slides (Scheme 1). The hydrazide groups are incorporated into the small molecules while they are attached to a solid support and are used as a handle in their solid-phase synthesis.[6] The epoxide-derivatized glass slides are easily created by immersing amine-coated glass slides into a solution of poly (ethylene glycol) diglycidyl ether (3% solution in 10 mm NaHCO3, pH 8.3).[7]A hydrazide-linked fucose probe (S-or L-Fuc-NHNH2, S: short, L: long; Scheme2) was used to probe optimal conditions (pH, time, and concentration) for the immobilization process. Preparation of S-or L-Fuc-NHNH2 was initiated by transforming alcohol groups on a Wang resin into pnitrophenyl carbonates.[8] The resulting resin was then treated with hydrazine to yield the hydrazide-containing resin, which was coupled with the Fmoc-protected L or S tether in the presence of NEM, BOP, and HOBt. The amino groups