Rapid evolution of fluorescence imaging techniques in recent years demands fluorophores with enhanced brightness and photostability. However, many existing fluorophores lack sufficient brightness and photostability for single-molecule and live-cell imaging.

Twisted intramolecular charge transfer (TICT), a process involving the twisting of amino substituents, is one of the major non-radiative de-excitation pathways in fluorophores. Traditionally, TICT is prevented via rigidizing flexible amino substituents. However, such substantial modification often leads to cell-impermeable dyes that are not suitable for intracellular imaging in vivo.

In a landmark paper (Nature Methods, 2015, 12, 244–250), Lavis and co-workers show that introducing a four-membered azetidine ring as an electron-donating unit reduces TICT formation rate and affords improved fluorophore brightness and stability, while retaining biological properties of the parent compounds. Yet, this method does not completely suppress TICT formation, especially in highly polar fluorophores.

Dalian Institute of Chemical Physics (DICP) research team led by XU Zhaochao replaced conventional dialkylamino substituents with a three-membered aziridine ring in naphthalimide dyes. And it leads to significantly enhanced brightness and photostability. These results show that the aziridine ring possesses higher TICT resistance than the azetidine ring and other dialkylamino substituents. By incorporating the aziridine ring, the quantum yields of naphthalimide dyes rise from 0 to 43.2% in water, even outperforming its azetidinyl analogue (19.9%).

Researchers replaced conventional dialkylamino substituents with a three-membered aziridine ring in naphthalimide dyes. (Image by LIU Xiaogang)

This simple structural modification permits facile synthesis and further derivatization. Inspired by the improved brightness and enhanced stability in naphthalimide dyes, the researchers then employed the aziridine ring in coumarin, rhodamine, phthalimide and NBD dyes. Quantum chemical calculations show that aziridinyl fluorophores demonstrate high TICT resistance. Experimental data also show that aziridinyl fluorophores exhibit higher brightness and stability than conventional dimethylamino fluorophores.

The researchers also found one plausible mechanism to explain the vulnerabilities of quantum yield to hydrogen bond interactions among different fluorophores. Substantial charge increase at hydrogen bond formation sites in a fluorophore is detrimental to its quantum yield in protonic solvents. Such knowledge on controlling TICT and managing hydrogen bond interactions will inspire the rational development of abundant high-performance dyes across different fluorophore families, thus enabling unprecedented fluorescence imaging applications.

This work has been published in J. Am. Chem. Soc.