Institut Curie, CNRS UMR-176, Centre Universitaire d'Orsay, Paris-Sud 91405 Orsay Cedex France.
J Am Chem Soc. 2013 Aug 28;135(34):12697-706. doi: 10.1021/ja404422z. Epub 2013 Aug 20.
We report on the design and synthesis of two-photon fluorescent triphenylamines bearing two or three vinyl branches terminated by a N-methyl benzimidazolium moiety. The new compounds (TP-2Bzim, TP-3Bzim) are light-up fluorescent DNA probes with a long wavelength emission (>580 nm). Compared to their pyridinium models, the TP-Bzim dyes exhibit a remarkable improvement of both their DNA affinity and fluorescence quantum yield, especially for the two-branch derivative (TP-2Bzim: ΦF = 0.54, Ka = 10(7) M(-1)), resulting in a large fluorescence emission turn-on ratio of up to 140. Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ = 1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time. This is attributed to a tight fit of the molecule inside the minor groove of AT-rich DNA which induces geometrical rearrangements in the dye ground state as supported by circular dichroism and molecular modeling data. Consequently, TP-2bzim displays an exceptional two-photon molecular brightness (δ×ΦF = 583 GM), a value unrivalled for a small biofluorophore. These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant, and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.
我们报告了两种二苯乙烯基取代的三苯胺的设计和合成，它们分别带有两个或三个乙烯基支链，末端为 N-甲基苯并咪唑鎓部分。新化合物（TP-2Bzim，TP-3Bzim）是长波长发射（>580nm）的亮场荧光 DNA 探针。与它们的吡啶鎓模型相比，TP-Bzim 染料的 DNA 亲和力和荧光量子产率都有显著提高，特别是对于两个支链衍生物（TP-2Bzim：ΦF=0.54，Ka=10(7)M(-1)），导致荧光发射开比高达 140。同时，TP-2Bzim 在与 DNA 结合时，双光子吸收截面（δ=1080 对游离形式的 110 GM）显著增强。这种 DNA 基质对非线性吸收的影响是首次发现。这归因于分子在富含 AT 的 DNA 小沟内的紧密配合，这导致染料基态的几何重排，圆二色性和分子建模数据支持这一结论。因此，TP-2bzim 显示出异常的双光子分子亮度（δ×ΦF=583 GM），这是一个小生物荧光团无与伦比的值。这些性质使得能够在亚微米浓度下（[TP-2Bzim]=100 nM）在固定细胞中对核 DNA 进行成像，并可视化着丝粒富含 AT 的染色质的超亮焦点。最后，TP-2Bzim 具有高的光稳定性，可穿透活细胞，并且不需要 RNA 酶处理。这种出色的光学和生物学性质的结合使得 TP-2Bzim 成为一种生物探针，超越了最好的 DNA 染色剂，并为研究 DNA 中的进一步非线性光学过程铺平了道路。
