近年來已有許多綠色螢光蛋白的相關研究，部分研究表示綠色螢光蛋白在酸性環境下會變性，但都是取自天然的GFP模型直接與酸進行作用，且顯少有研究提出在酸性作用下，致使其變性的機制及變性結果。我們的方法則是合成出綠色螢光蛋白發光團類似物，利用甲基化的方法模擬綠色螢光蛋白發光團在酸性環境下受到質子化後的水解反應。藉由比較我們合成出的綠色螢光蛋白類似物p3-Ph和p4-Ph，發現甲基化後五元雜環上amidine carbon，遮蔽效應會減弱，更易受到親核基的攻擊而導致水解。此外，化合物照射波長350 nm之光源後，藉由追蹤核磁共振儀積分值比例並搭配sigma-plot軟體計算出熱異構化速率常數，以及測量吸收、螢光放射光來探討其光物理性質，最後得到化合物的X-ray單晶結構來證實綠色螢光蛋白類似物在酸性環境下會水解的結果。

To simulate other GFP chromophoric analogues under the acidic condition, we used methylation method to synthesize GFP chromophoric analogues p4-Ph and planned to imitate protonated type of GFP simultaneously. In this way, we found that protic solvent or water could easily affected compound p4-Ph. Due to the positively charged nitrogen on p4-Ph, the amidine carbon on five-membered heterocyclic ring became electron deficient and was easily attacked by nucleophile such as H2O. Moreover, we monitored the integration value of 1H NMR and calculated the rate of photo-isomerization and thermal-isomerization with software. Finally, the result of single-crystal X-ray diffraction clarified that p4-Ph would ultimately hydrolyze in water. These results could offer an evidence to previous reports that protonation of GFP derivatives will change its conformation or denaturation as a result of the hydrolysis reaction.

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