Wycisk Virginia, Pauli Jutta, Welker Pia, Justies Aileen, Resch-Genger Ute, Haag Rainer, Licha Kai
†Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany.
‡BAM Federal Institute for Material Research and Testing, Division Biophotonics, 12489 Berlin, Germany.
Bioconjug Chem. 2015 Apr 15;26(4):773-81. doi: 10.1021/acs.bioconjchem.5b00097. Epub 2015 Apr 3.
The synthesis of water-soluble dyes, which absorb and emit in the range between 650 and 950 nm and display high extinction coefficients (ε) as well as high fluorescence quantum yields (Φf), is still a demand for optical imaging. We now present a synthetic route for the preparation of a new group of glycerol-substituted cyanine dyes from dendronized indole precursors that have been functionalized as N-hydroxysuccinimide (NHS) esters. High Φf values of up to 0.15 and extinction coefficients of up to 189 000 L mol(-1) cm(-1) were obtained for the pure dyes. Furthermore, conjugates of the new dendronized dyes with the antibody cetuximab (ctx) that were directed against the epidermal growth factor receptor (EGFR) of tumor cells could be prepared with dye to protein ratios between 0.3 and 2.2 to assess their potential as imaging probes. For the first time, ctx conjugates could be achieved without showing a decrease in Φf and with an increasing labeling degree that exceeded the value of the pure dye even at a labeling degree above 2. The incorporation of hydrophilically and sterically demanding dendrimers into cyanines prevented dimer formation after covalent conjugation to the antibody. The binding functionality of the resulting ctx conjugates to the EGFR was successfully demonstrated by cell microscopy studies using EGFR expressing cell lines. In summary, the combination of hydrophilic glycerol dendrons with reactive dye labels has been established for the first time and is a promising approach toward more powerful fluorescent labels with less dimerization.
合成在650至950nm范围内吸收和发射、并显示出高消光系数（ε）以及高荧光量子产率（Φf）的水溶性染料，仍然是光学成像的需求。我们现在提出一种合成路线，用于从已官能化为N-羟基琥珀酰亚胺（NHS）酯的树枝状吲哚前体中制备一组新的甘油取代花青染料。对于纯染料，获得了高达0.15的高Φf值和高达189000 L mol⁻¹ cm⁻¹的消光系数。此外，可以制备新的树枝状染料与针对肿瘤细胞表皮生长因子受体（EGFR）的抗体西妥昔单抗（ctx）的缀合物，染料与蛋白质的比例在0.3至2.2之间，以评估它们作为成像探针的潜力。首次可以实现ctx缀合物，而不会显示Φf降低，并且标记程度增加，甚至在标记程度高于2时也超过了纯染料的值。将具有亲水性和空间需求的树枝状聚合物掺入花青中可防止在与抗体共价缀合后形成二聚体。使用表达EGFR的细胞系通过细胞显微镜研究成功证明了所得ctx缀合物与EGFR的结合功能。总之，首次建立了亲水性甘油树枝体与反应性染料标记的组合，这是朝着具有更少二聚化的更强大荧光标记发展的一种有前途的方法。
