Resolving Electronic Transitions in Synthetic Fluorescent Protein Chromophores by Magnetic Circular Dichroism

The detailed electronic structures of fluorescent chromophores are important for their use in imaging of living cells. A series of green fluorescent protein chromophore derivatives is examined by magnetic circular dichroism (MCD) spectroscopy, which allows the resolution of more bands than plain absorption and fluorescence. Observed spectral patterns are rationalized with the aid of time‐dependent density functional theory (TDDFT) computations and the sum‐over‐state (SOS) formalism, which also reveals a significant dependence of MCD intensities on chromophore conformation. The combination of organic and theoretical chemistry with spectroscopic techniques also appears useful in the rational design of fluorescence labels and understanding of the chromophore's properties. For example, the absorption threshold can be heavily affected by substitution on the phenyl ring but not much on the five‐member ring, and methoxy groups can be used to further tune the electronic levels.