在本實驗室先前的實驗中，於以 Cu (II) 和 o-ABDI 衍生物進行結晶培養的過程中，意外地生成了環化產物 DHPIO。與其未環化前的前驅物相比，DHPIO 的吸收與螢光光譜皆出現藍移現象，且其分子結構更為剛性，不易扭轉，進而導致螢光量子產率的提升。
根據文獻報導，引入共軛系統可導致光譜產生紅移。因此，本研究設計在分子結構中引入額外的共軛系統，期望能使環化產物的吸收與螢光光譜產生紅移，並進一步提升其螢光量子產率。於是本研究參考文獻及實驗室學長姐既有的合成路徑，成功合成出綠色螢光蛋白衍生物 o-CDHPNMI 及其環化產物 CDHPMIO。因分子結構中的共軛程度及結構鋼性的增加，觀察結果顯示，CDHPMIO 相較於其前驅物 o-CDHPNMI，呈現吸收與螢光光譜紅移的趨勢，且其螢光量子產率仍顯著提升，達到期望的實驗結果。此外，o-CDHPNMI 在短波長激發下，顯現出雙重螢光放射光譜，此現象可能源於激發態下分子結構的扭轉與否所造成之不同構型所致。
並在環化反應條件中額外加入 TEMPO，意外合成出反應中間體 o-CDHPNMI-TEMPO，此一結果證實了環化機制中涉及自由基中間體的存在。

In previous experiments conducted in our laboratory, an unexpected cyclized product, DHPIO, was obtained during crystallization of Cu (II) with o-ABDI derivatives. Compared to its non-cyclized precursor, DHPIO exhibited blue-shifted absorption and fluorescence spectra. Additionally, the increased molecular rigidity due to cyclization suppressed intramolecular rotation, resulting in a significant enhancement of the fluorescence quantum yield.
According to literature reports, the introduction of extended conjugation systems can induce red-shifted optical spectra. Therefore, this study was designed to incorporate additional conjugated moieties into the molecular structure, with the aim of shifting the absorption and emission wavelengths of the cyclized products toward longer wavelengths, while also enhancing their fluorescence quantum yields. Based on previous literature and established synthetic protocols developed by former members of our research group, we successfully synthesized the green fluorescent protein (GFP) derivative o-CDHPNMI and its cyclized product CDHPMIO. Due to the increased molecular conjugation and structural rigidity, CDHPMIO exhibited a red-shift in both absorption and fluorescence spectra compared to its precursor o-CDHPNMI. Moreover, its fluorescence quantum yield was significantly enhanced, achieving the desired experimental outcome.
By introducing TEMPO into the cyclization reaction conditions, the intermediate product o-CDHPNMI-TEMPO was unexpectedly isolated. This result provides clear evidence for the involvement of a radical intermediate in the proposed cyclization mechanism.

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