Revealing the Role of Electronic Effect to Modulate\nthe Photophysics and Z‑Scan Responses of <i>o</i>‑Locked GFP Chromophores

Three\nnovel core green fluorescent protein (GFP) chromophore analogues,\nbased on a doubly locked conformation and variable electronic effects\nby replacing one hydrogen with bromine, iodine, and methyl, respectively,\nhave been synthesized to modulate the push–pull effect. These\nchromophores exhibited intramolecular H-bonding, as evidenced by single-crystal\nX-ray and <sup>1</sup>H NMR studies. The fluorescence quantum yields\n(ϕ<sub>f</sub>) of all of the chromophores were found to be\nmore than an order of magnitude higher (∼0.2) than the unlocked\nchromophores (∼0.01). It was found that the electronic effect\ndid affect the nonradiative rates, as the quantum yields were found\nto vary with respect to different analogues in the same solvents.\nThe effect of the push–pull effect was also evident by a higher\nStokes-shifted emission in the case of the methyl derivative with\nrespect to the bromo- and iodo-analogues. Furthermore, the emission\nspectra of these GFP chromophores were found to show positive solvatochromism,\nwhich was supported by a quantum chemical calculation. A detailed\nstudy, correlating the observed spectral changes with various solvent\nfunctions and supported by computational results, established a facile\nproton transfer, followed by twisted intramolecular charge transfer\n(TICT) to be the major nonradiative channels of these chromophores.\nBesides, a completely novel usage of these chromophores was explored\nfor the first time by studying their third-order nonlinear optical\ncharacteristics in DMSO using a single-beam Z-scan technique. All\nof the chromophores exhibited tunable nonlinear refraction (NLR) and\nnonlinear absorption (NLA) properties that depend upon different substituent\ngroups present in the chromophores. Here, the NLR was due to the effect\nof self-defocusing, whereas the NLA was triggered by reverse saturable\nabsorption, which is attributed to the two-photon absorption (TPA)\nprocess. Surprisingly, the efficiency of the TPA ability of the chromophores\nwas found to be a function of the induced electronic effect. Hence,\nthis work opens a new route for the utility of the <i>ortho</i>-locked GFP chromophores in the field of nonlinear optical applications.