Cooperativity and Site-Selectivity of Intramolecular Hydrogen Bonds on the Fluorescence Quenching of Modified GFP Chromophores

Deng Hsiang Chang, Chun Lin Ou, Hung Yu Hsu, Guan Jhih Huang, Chen Yi Kao, Yi Hung Liu, Shie Ming Peng, Wei-Guang Diau, Jye Shane Yang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


This paper provides the first example of experimentally characterized hydrogen-bond cooperativity on fluorescence quenching with a modified green fluorescence protein (GFP) chromophore that contains a 6-membered C=N⋯H-O and a 7-membered C=O⋯H-O intramolecular H-bonds. Variable-temperature 1H NMR and electronic absorption and emission spectroscopies were used to elucidate the preference of intra- vs intermolecular H-bonding at different concentrations (1 mM and 10 μM), and X-ray crystal structures provide clues of possible intermolecular H-bonding modes. In the ground state, the 6-membered H-bond is significant but the 7-membered one is rather weak. However, fluorescence quenching is dominated by the 7-membered H-bond, indicating a strengthening of the H-bond in the excited state. The H-bonding effect is more pronounced in more polar solvents, and no intermediates were observed from femtosecond fluorescence decays. The fluorescence quenching is attributed to the occurrence of diabatic excited-state proton transfer. Cooperativity of the two intramolecular H-bonds on spectral shifts and fluorescence quenching is evidenced by comparing with both the single H-bonded and the non-H-bonded counterparts. The H-bond cooperativity does not belong to the conventional patterns of σ- and π-cooperativity but a new type of polarization interactions, which demonstrates the significant interplay of H-bonds for multiple H-bonding systems in the electronically excited states.

Original languageEnglish
Pages (from-to)12431-12443
Number of pages13
JournalJournal of Organic Chemistry
Issue number24
StatePublished - 19 Nov 2015

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