Wängler C, Moldenhauer G, Saffrich R, Knapp E-M, Beijer B, Schnölzer M, Wängler B, Eisenhut M, Haberkorn U, Mier W
Universitätsklinikum Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany.
Chemistry. 2008;14(27):8116-30. doi: 10.1002/chem.200800328.
Fluorescent probes are of increasing interest in medicinal and biological applications for the elucidation of the structures and functions of healthy as well as tumour cells. The quality of these investigations is determined by the intensity of the fluorescence signal. High dye/carrier ratios give strong signals. However, these are achieved by the occupation of a high number of derivatisation sites and therefore are accompanied by strong structural alterations of the carrier. Hence, polyvalent substances containing a high number of fluorescent dyes would be favourable because they would allow the introduction of many dyes at one position of the compound to be labelled.A large number of different dyes have been investigated to determine the efficiency of coupling to a dendrimer scaffold and the fluorescence properties of the oligomeric dyes, but compounds that fulfil the requirements of both strong fluorescence signals and reactivities are rare. Herein we describe the synthesis and characterisation of dye oligomers containing dansyl-, 7-nitro-2,1,3-benzoxadiazol-4-yl- (NBD), coumarin-343, 5(6)-carboxyfluorescein and sulforhodamine B2 moieties based on polyamidoamine (PAMAM) dendrimers. The PAMAM dendrimers were synthesised by an improved protocol that yielded highly homogeneous scaffolds with up to 128 conjugation sites. When comparing the fluorescent properties of the dye oligomers it was found that only the dansylated dendrimers met the requirements of enhanced fluorescence signals. The dendrimer containing 16 fluorescent dyes was conjugated to the anti-epidermal-growth-factor receptor (EGFR) antibody hMAb425 as a model compound to show the applicability of the dye multimer compounds. This conjugate revealed a preserved immunoreactivity of 54%.We demonstrate the applicability of the dye oligomers to the efficient and applicable labelling of proteins and other large molecules that enables high dye concentrations and therefore high contrasts in fluorescence applications.
荧光探针在医学和生物学应用中越来越受到关注，用于阐明健康细胞和肿瘤细胞的结构与功能。这些研究的质量取决于荧光信号的强度。高染料/载体比例会产生强信号。然而，这是通过占据大量衍生化位点来实现的，因此会伴随着载体的强烈结构改变。因此，含有大量荧光染料的多价物质将是有利的，因为它们可以在待标记化合物的一个位置引入许多染料。已经研究了大量不同的染料，以确定与树枝状聚合物支架偶联的效率以及寡聚染料的荧光特性，但同时满足强荧光信号和反应活性要求的化合物很少。在此，我们描述了基于聚酰胺胺（PAMAM）树枝状聚合物的含有丹磺酰基、7-硝基-2,1,3-苯并恶二唑-4-基（NBD）、香豆素-343、5(6)-羧基荧光素和磺基罗丹明B2部分的染料寡聚物的合成与表征。PAMAM树枝状聚合物通过改进的方案合成，得到了具有多达128个共轭位点的高度均匀的支架。比较染料寡聚物的荧光特性时发现，只有丹磺酰化树枝状聚合物满足增强荧光信号的要求。将含有16种荧光染料的树枝状聚合物与抗表皮生长因子受体（EGFR）抗体hMAb425偶联作为模型化合物，以展示染料多聚体化合物的适用性。这种偶联物显示出54%的保留免疫反应性。我们证明了染料寡聚物在蛋白质和其他大分子的高效且适用的标记中的适用性，这使得在荧光应用中能够实现高染料浓度并因此获得高对比度。
