Dual-Self-Restricted GFP Chromophore Analogues with Significantly Enhanced Emission

Abstract

The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP) chromophore to the protein itself makes it impossible to use for applications in molecular and cellular imaging. Here, we developed an efficient methodology to enhance the photoluminescence response of synthetic GFP chromophore analogues by constructing dual-self-restricted chromophores. Single self-restricted chromophores were first generated with 2,5-dimethoxy substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy phenyl to form dual-self-restricted chromophores. These two chromophores showed an obvious solvatofluorochromic color palette across blue to yellow with a maximum emission Stokes shift of 95 nm and dramatically enhanced fluorescence emission in various aprotic solvents, especially in hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken atomic charge translocation over the double-bond bridge and illustrated much higher energy barriers for the Z/E photoisomerization together with larger bond orders compared with the GFP core chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic GFP chromophore analogues in polar solvents while reserved the multicolor emitting function, providing a solid molecular motif for engineering high-performance fluorescent probes.

A Modified Oxazolone Dye Dedicated to Spectroscopy and Optoelectronics

“…During our ongoing work toward the compounds for optoelectronics, we have demonstrated that the 4-(3′-phenyl-2′-propenylidene)-phenylooxazol-5­(4 H )-one reveals the desired nonlinear optical properties to serve as a productive light amplifier, generator of higher harmonics of light, and optical modulator in the sub-microsecond time scale. , Therefore, we decided to modify its structure to obtain an oxazolone dye with desired properties for various applications in spectroscopy, photonics, and optoelectronics. Since one of the most widely applied methods for adjusting the photophysical properties of organic compounds is to play with the nature of the substituents, − we have synthesized an oxazolone dye containing a methoxy group at the ortho position. We hope that the modification influences the electronic distribution within the molecule since the compound reveals the D-π-A with the 5-(4 H )-oxazolone ring constituting an electron-withdrawing moiety and the phenyl ring and methoxy substituent being an electron-donating part.…”

“… 22 , 24 Therefore, we decided to modify its structure to obtain an oxazolone dye with desired properties for various applications in spectroscopy, photonics, and optoelectronics. Since one of the most widely applied methods for adjusting the photophysical properties of organic compounds is to play with the nature of the substituents, 25 − 27 we have synthesized an oxazolone dye containing a methoxy group at the ortho position. We hope that the modification influences the electronic distribution within the molecule since the compound reveals the D-π-A with the 5-(4 H )-oxazolone ring constituting an electron-withdrawing moiety and the phenyl ring and methoxy substituent being an electron-donating part.…”

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore

“…This is in fact not that easy to test, because HBDI in solutions are fraught with side photoreactions such as photoisomerization and, to a much lesser extent, ESPT . However, for neutral HBDI analogues that suppress these side reactions, solvatofluorochromism ranges as wide as 95 nm (∼3700 cm –1 ) have been observed. − In contrast, absorption and emission maxima for GFP mutants that allow for significant A* emission by disrupting the ESPT chain (e.g., S205V, T203V/S205A, and deGFPs) are all fairly close to 400 and 460 nm, respectively, which could be due to the limited sampling of protein environments and/or the relatively poor ability for protein environments to reorganize as compared to simple solvents.…”

A Biomimetic Emitter Inspired from Green Fluorescent Protein

“…In the aggregated state, the emission peak redshifts to around 475 nm with moderate decreased emission intensity, but still detectable under highest water contents (Figure S5). 27 A critical puzzle is the underlining mechanism for enhanced MBP emission. We tried to unravel this mystery by studying the structure−function relationship.…”