Scalable and concise synthesis of dichlorofluorescein derivatives displaying tissue permeation in live zebrafish embryos
Scalable and concise synthesis of dichlorofluorescein derivatives displaying tissue permeation in live zebrafish embryos
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DOI：
10.1002/cbic.200700565
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发表时间：
2008-01-25
期刊：
影响因子：
3.2
通讯作者：
Hukriede, Neil A.
中科院分区：
文献类型：
--
作者：
Koide, Kazunori;Song, Fengling;Hukriede, Neil A.
荧光成像正成为一种越来越重要的技术，用于检查模型生物（如斑马鱼）中生物相关分子的实时定位。这种成像通常需要使用2′，7′-二氯荧光素（DCF，方案1）、2′，7′-二氟荧光素（俄勒冈州绿色）或它们的衍生物之一，因为每一种都提供pH不敏感性、紧凑的尺寸和高量子产率。[1，2]然而，这些化合物中带负电荷的羧基可以与带正电荷的生物分子非特异性地相互作用。此外，羧基可能是负责时，固有的细胞渗透性分子变得不渗透后共轭荧光素。Lindqvist和同事表明，羧基的这些化合物是不是必不可少的荧光发射。[3]这一观察结果最近得到了东京大学长野研究小组的证实，他们用甲基取代了荧光素的羧基，形成了一种化合物东京绿色，这种化合物没有表现出量子产率的损失。[4]宾夕法尼亚州立大学的彼得森小组也进行了类似的甲基取代，这次是用俄勒冈州的绿色[5]来生产宾夕法尼亚州的绿色。[6]在活的人类细胞中，宾夕法尼亚绿色比东京绿色更荧光，这是由于其强大的pH不敏感性。[6]虽然宾夕法尼亚绿色是一种优良的荧光探针，但其适合缀合的衍生物的合成是一个漫长的过程，需要十个线性步骤。[6]在这份报告中，我们描述了可扩展的和简洁的合成两个组织渗透的DCF衍生物，匹兹堡绿色和匹兹堡黄色绿色。匹兹堡黄绿色既具有宾夕法尼亚绿色的良好荧光和溶解性能，又具有可以与靶生物分子缀合的“手柄”。
Fluorescent imaging is becoming an increasingly important technology for examining the realtime localizations of biologically relevant molecules within model organisms such as the zebrafish. Such imaging often requires the use of 2′, 7′-dichlorofluorescein (DCF, Scheme 1), 2′, 7′-difluorofluorescein (Oregon Green), or one of their derivatives, since each offers pH insensitivity, compact size, and high quantum yield.[1, 2] However, the negatively charged carboxyl groups in these compounds can nonspecifically interact with positively charged biomolecules. Furthermore, the carboxyl group might be responsible when inherently cellpermeable molecules become impermeable following conjugation to fluorescein.Lindqvist and co-workers showed that the carboxyl group of these compounds was not essential for fluorescence emission.[3] This observation was recently corroborated by the Nagano group at the University of Tokyo, who replaced fluorescein’s carboxyl group with a methyl group to form a compound, Tokyo Green, that exhibited no loss of quantum yield.[4] A similar methyl substitution was carried out by the Peterson group at The Pennsylvania State University, this time with Oregon Green,[5] to produce Pennsylvania Green.[6] In live human cells, Pennsylvania Green was more fluorescent than Tokyo Green due to its robust pH insensitivity.[6] While Pennsylvania Green is an excellent fluorescent probe, the synthesis of its conjugation-amenable derivatives is a lengthy process, requiring ten linear steps.[6] In this report, we describe the scalable and concise synthesis of two tissue-permeable DCF derivatives, Pittsburgh Green and Pittsburgh Yellowgreen. Pittsburgh Yellowgreen possesses both the favorable fluorescent and solubility properties of Pennsylvania Green and a “handle” that can be conjugated to target biomolecules.