美学者开发出一类新的荧光探针

　　

生物通报道，新墨西哥州立大学（NMSU）的研究人员近日设计并合成了一种小分子的荧光染料，它在细胞吸收、膜通透性和成像分辨率上有所改善。这种染料有望协助阐明各种分子进程中的微妙分子机制。该研究成果发表在近期的《Journal of the American Chemical Society》杂志上。

新墨西哥州立大学的生物化学教授Jeffrey Arterburn领导的研究小组从2-肼吡啶（HPY）中合成了这种染料，2-肼吡啶常用于药理应用中，让药物与靶向抗体偶联。由于HPY染料很少，化学上稳定，容易与目标分子偶联，并有着可调的成像性质，因此它们在小分子探针的开发上有着独特的优势。

基于此，Arterburn教授和他的同事开发出HPY结合物骨架的多种衍生物，并将它们的光物理和结构性质与目前市场上常用的荧光染料结合物（如FITC、BODIPY和Alexa Fluor 488）进行了比较。这些比较让他们进一步优化了HPY染料的核心结构。

为了评估HPY染料作为成像试剂的效率，研究小组将HPY染料与有丝分裂纺锤体驱动蛋白（KSP）的选择性抑制剂结合，并与Alexa Fluor 488比较了Hela细胞中有丝分裂抑制和染料分布的量。

研究结果表明，HPY-KSP结合物与未结合的KSP抑制剂相比，抑制剂活性降低了5倍，而Alexa Fluor 488-KSP结合物则表现出抑制剂活性的23倍降低。这两种结合物之间抑制剂活性的差异主要源于HPY结合物的体积小和中性电荷。HPY结合物能够通过简单的渗透进入细胞。相比之下，较大的Alexa Fluor结合物则仍然被细胞的内吞小室而隔离。

Arterburn教授谈到：“HPY染料发色团中包含的原子数量比其他传统的染料（如荧光素）要少。而且它的三氮骨架实现了亲脂性和水溶性的良好平衡。”

这篇研究强调了染料结构对性能的影响，并提供了一个适当的替代物来改善性能。此外，HPY染料核心结构具有多功能的化学性质，通过支架修饰以及其他活性基团的掺入，有望开发出新的荧光探针结合物。（生物通 薄荷）

原文摘要：

Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes

J. Am. Chem. Soc., 2011, 133 (17), pp 6780–6790

摘要：
A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.