STUDIES ON RATIONAL DESIGN PRINCIPLE FOR FLUORESCENCE ON/OFF SWITCHING

荧光成像是目前最强大的技术，可用于连续观察活细胞的动态胞内过程。荧光素被广泛用作各种荧光探针的核心，用于成像重要的生物效应物，如一氧化氮和钙。尽管荧光素衍生物的广泛应用及其应用的重要性，但控制荧光量子产率的机制尚未完全建立。没有这样的机制信息，就没有合理的设计策略来开发新的、实际有用的荧光探针。一种可能控制荧光素衍生物荧光量子产率的合理机制被认为是光诱导电子从电子供体部分转移到杂蒽部分的单线态激发态。由于光致电子转移反应中的荧光量子产率可以根据M…More弧理论进行定量预测，因此上述机理的证实将为荧光探针的合理设计提供定量依据。此外，通过加速由光诱导电子转移产生的自由基离子对中的反向电子转移，可以提高荧光探针的稳定性。然而，基于荧光素的探针中光诱导电子转移的发生尚未确定。我们获得了第一个明确的证据，证明在基于荧光素的探针中发生光诱导电子转移，其中电子供体部分直接与杂蒽部分相连。通过激光闪光光解实验检测荧光素基探针在光照射下自由基离子对的形成，该实验提供了瞬态吸收光谱，显示出由电子供体部分的自由基阳离子和杂蒽自由基阴离子引起的条带。根据马库斯电子转移理论，测定并分析了光致电子转移速率和反电子转移速率。该结果首次为合理设计荧光素探针提供了定量依据，该探针具有高效的荧光开关和高的反复光照射稳定性。我们开发了一种新型的化学探针，用于一氧化氮、高反应性氧和锌离子的生物成像。少

Fluorescence imaging is the most powerful technique currently available for continuous observation of the dynamic intracellular processes of living cells. Fluorescein is widely employed as the core of various fluorescence probes used in imaging important biological effectors, such as nitric oxide and calcium. Despite the extensive use of fluorescein derivatives and the importance of the applications, the mechanism that controls the quantum yield of fluorescence has not been fully established. Without such mechanistic information there can be no rational design strategy to develop new, practically useful fluorescence probes. A plausible mechanism that might control the fluorescence quantum yields of fluorescein derivatives has been suggested to be photoinduced electron transfer from the electron donor moiety to the singlet excited state of the xanthene moiety. Since the fluorescence quantum yields in photoinduced electron transfer reactions may be quantitatively predicted based on the M … More arcus theory of electron transfer, confirmation of the above mechanism would provide a quantitative basis for rational design of fluorescence probes. Further, the stability of fluorescence probes could be increased by accelerating the back electron transfer in the radical ion pair generated by photoinduced electron transfer. However, the occurrence of photoinduced electron transfer in fluorescein-based probes has yet to be established.We obtained the first definitive evidence for the occurrence of photoinduced electron transfer in fluorescein-based probes in which the electron donor moiety is directly linked with the xanthene moiety. Formation of the radical ion pair upon photoirradiation of the fluorescein-based probes was detected by means of laser flash photolysis experiments, which afforded transient absorption spectra showing bands due to the radical cation of the electron donor moiety and the xanthene radical anion. The rates of photoinduced electron transfer and the back electron transfer were determined and analyzed in terms of the Marcus theory of electron transfer. The results provide for the first time a quantitative basis for rational design of fluorescein-based probes with high efficiency in fluorescence ON/OFF switching, as well as the high stability to repeated photoirradiation. We have developed the novel chemical probes for bioimaging of nitric oxide, highly reacrive oxygen species, and zinc ion. Less