Luminescent peptide labeling based on a histidine-binding iridium(III) complex for cell penetration and intracellular targeting studies.
Luminescent peptide labeling based on a histidine-binding iridium(III) complex for cell penetration and intracellular targeting studies.
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DOI：
10.1002/chem.201100568
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发表时间：
2011-07
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影响因子：
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通讯作者：
Xiaobo Wang;Junli Jia;Zezhu Huang;Ming Zhou;H. Fei
中科院分区：
文献类型：
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作者：
Xiaobo Wang;Junli Jia;Zezhu Huang;Ming Zhou;H. Fei
生物分子的荧光或发光标记允许高灵敏度的成像技术用于各种体外和体内研究以用于检测和追踪目的，因此，它已成为基础生命科学和生物医学研究的许多领域中的关键实践。对于荧光标记的每个特定情况，首先需要考虑发光体的选择。除了最常用的有机染料外，过渡金属配合物染料还表现出许多优良的上级物理化学性质，如较高的光稳定性、较大的斯托克斯位移和较高的量子效率，是生命科学研究中极具吸引力的一类发光材料。[1]例如，最近对发光铱ACHTUNGTRENNUNG（III）络合物的研究已经发现了这些染料在化学传感器、[2]生物标记、[3，4]活细胞染色[5-9]和体内肿瘤成像中的应用的新兴基础。[10]其次，共轭化学的选择也很重要。在标记多肽的情况下，通过N-末端的胺基或赖氨酸残基或半胱氨酸残基的巯基进行共价交联是最常规的方法。例如，几项研究已经报道了通过使用基于羧胺的共价化学用于细胞成像的过渡金属络合物标记的多肽。[11-12]另一种类型的标记技术利用了通过组氨酸侧链的咪唑基团上的自由电子对与金属离子的基于配位的连接。因为组氨酸以比例如赖氨酸相对较低的频率出现在蛋白质中，所以多肽的位点特异性荧光标记可以通过将通常长度为六个残基的多组氨酸标签序列工程化到靶多肽中来实现，如在若干研究中所证明的。[13，14]在最近的一项工作中，Ma等人将环金属化铱ACHTUNGTRENNUNG（III）溶剂配合物表征为富含组氨酸的蛋白质的选择性发光探针，并将其作为染料应用于十二烷基硫酸钠聚丙烯酰胺凝胶（SDS-PAGE）中的蛋白质。[15]在此，我们进一步探索了这种组氨酸特异性铱ACHTUNGTRENNUNG（III）复合物[IrACHTUNGTRENNUNG（ppy）2-ACHTUNGTRENNUNG（CH 3CN）2] OTf（1，ppy= 2-苯基吡啶，OTf=三氟甲磺酸酯）在细胞成像研究中用于发光、位点特异性肽标记的优势。首次阐明了组氨酸与1的配位机理和反应动力学，从而建立了发光肽标记的一般方法。类似于传统的罗丹明标记，我们发现，发光，铱ACHTUNGTRENNUNG（III）标记，细胞穿透肽（CPPs）表现出细胞质和囊泡染色。此外，我们证明了一种双功能肽，位点特异性标记的铱ACHTUNGTRENNUNG（III）发光体，既可以穿透细胞膜，有效地靶向线粒体。
Fluorescent or luminescent labeling of biomolecules allows highly sensitive imaging techniques to be utilized in various in vitro and in vivo studies for detecting and tracing purposes, and thus, it has become a key practice in many fields of basic life sciences and biomedical research. For every specific case of fluorescent labeling, the choice of luminophores needs to be considered first. Besides the most conventional and commonly used organic dyes, transitionmetal-complex dyes represent an attractive family of luminescent materials for life science research, because they display many superior physicochemical properties, such as higher photostabilities, larger Stokes shifts, and higher quantum efficiencies.[1] For example, recent studies on luminescent iridiumACHTUNGTRENNUNG (III) complexes have found emerging grounds for applications of these dyes in chemosensors,[2] biolabeling,[3, 4] live cell staining,[5–9] and in vivo tumor imaging.[10] Secondly, the choice of conjugation chemistry is also important to consider. In the case of labeling polypeptides, covalent cross-linking through the amine group of the N-terminus or a lysine residue, or the thiol group of a cysteine residue are the most conventional methods. For example, several studies have reported transition-metal-complex-labeled polypeptides for cell imaging by using carboxylaminebased covalent chemistry.[11–12] Another type of labeling technique takes advantage of the coordination-based attachment to metal ions through the pair of free electrons on the imidazole group of histidine side chains. Because histidine appears in proteins with a relatively lower frequency than, for example, lysine, site-specific fluorescent labeling of polypeptides can be achieved by engineering polyhistidine tag sequences, often of six residues in length, into target polypeptides, as demonstrated in several studies.[13, 14]In a recent work, Ma et al. characterized a cyclometalated iridiumACHTUNGTRENNUNG (III) solvento complex as a selective luminogenic probe for histidine-rich proteins and applied it as a dye to the proteins in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE).[15] Herein, we further explore the advantage of this histidine-specific iridiumACHTUNGTRENNUNG (III) complex,[IrACHTUNGTRENNUNG (ppy) 2-ACHTUNGTRENNUNG (CH3CN) 2] OTf (1, ppy= 2-phenylpyridine, OTf= triflate) for luminescent, site-specific, peptide labeling in cell-imaging studies. We first elucidated the coordination mechanism and reaction kinetics between histidine and 1, and thus established general methods for luminescent peptide labeling. Similarly to conventional rhodamine labeling, we found that luminescent, iridiumACHTUNGTRENNUNG (III)-labeled, cell-penetrating peptides (CPPs) exhibits cytoplasmic and vesicular staining. Furthermore, we demonstrate that a dual-functional peptide, sitespecifically labeled with an iridiumACHTUNGTRENNUNG (III) luminophore, could both penetrate cell membranes and effectively target mitochondria.