The photophysics of N,N'-bis(salicylidene)-p-phenylenediamine (BSP) is analyzed both theoretically and experimentally. The alternative intramolecular proton-transfer reactions lead to three different tautomers. We performed DFT and TDDFT calculations to analyze the topography of the reactions connecting the three tautomers. Deactivation paths through a Conical Intersection (CI) region are also analyzed to explain the low fluorescence quantum yield of the phototautomers. The complex molecular structure of BSP provides a large number of deactivation paths, almost all of them energetically available following the initial photoexcitation. Femtosecond (fs) time-resolved emission studies in solution and flash photolysis experiments (nano to millisecond regime) were performed to get detailed information on the time domain of the full photocycle. The picture that emerges by combining theoretical and experimental results shows a very fast (less than 100 fs) photoinduced single proton transfer process leading to a phototautomer where a single proton has moved. This species may deactivate through a low-energy CI leading in about 20 ps to a rotameric form in the ground state that has a lifetime of several tens of microseconds in solution. This process competes with another deactivation path taking place prior to the proton-transfer reaction which involves a low-energy CI leading to a rotamer of the enol structure. In the flash photolysis studies, the rotamer of the enol structure was directly identified by the positive transient absorption band in the 250-260 nm and its lifetime in n-hexane (10 ms) is almost 3 orders of magnitude longer than the lifetime of the photochrome (around 40 μs). Our findings do not exclude a double proton transfer reaction in the excited enol form to give a tautomer in less than 100 fs during the first (impulsive) phase of the reaction which reverts back to the photoproducts of the simple proton transfer in 1-3 ps.

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To determine the energetic feasibility of the mechanisms involved in the generation of the fluorescent species in red fluorescent proteins LSSmKate1 and LSSmKate2 developed by Piatkevich et al. (Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5369-5374 and J. Am. Chem. Soc. 2010, 132, 10762-10770), a potential energy scan for the respective reaction coordinates was performed in large cluster models including the surroundings of the chromophores, based on the respective crystallographic structures, using DFT and TDDFT. The predicted absorption wavelengths agree to within 5 nm with experiment, thus confirming the accuracy of the calculational level and modeling done. In both proteins, it was found that the adiabatic electronic state with the largest oscillator strength at the Franck-Condon region was not the one from which fluorescence could occur in the products. A diabatization procedure was used to determine an approximate photoactive state, based on selecting the state with the largest oscillator strength throughout. For LSSmKate1, this led to a rather flat potential energy profile but still did not predict a minimum in the product side. It is suggested that relaxation processes, absent from the model, could bring about such a minimum. LSSmKate2, on the other hand, clearly displays a favorable exoergic process in the photoactive state, and its double-proton transfer can be described as concerted but highly asynchronous, involving a barrier in the transfer of the first proton. In this way, the model provides strong support for the mechanism proposed for LSSmKate2.

Classical molecular dynamics (MD) simulations are carried out for the recently developed TagRFP675 fluorescent protein (FP), which is specifically designed to fully absorb and emit in the near infrared (NIR) region of the electromagnetic spectrum. Since the X-ray data of TagRFP675 reveal that the chromophore exists in both the cis and trans configuration and it can also be neutral (protonated) or anionic (deprotonated) depending on the pH of the media, a total of 8 molecular dynamic simulations have been run to simulate all the possible states of the chromophore. Time-dependent DFT (TDDFT) single point calculations are performed at selected points along the simulation to theoretically mimic the absorption spectrum of the protein. Our simulations compare well (within the expected error of the computational method) with the experimental results. Our theoretical procedure allows for an analysis of the molecular orbitals involved in the lowest energy electronic excitations of the chromophore and, more interestingly, for a full analysis of the H-bond interactions between the chromophore and its surrounding residues and solvent (water) molecules. This study does not support the hypothesis, exclusively based on the analysis of X-ray data, that the isomerization of nearby residues provokes the rearrangement of the hydrogen bonds in the chromophore's immediate environment leading to the observed red shift of the absorption bands at higher pHs. Instead, we attribute this shift mainly to the superposition of bands of the neutral and anionic chromophores that are expected to coexist at almost the full range of pHs experimentally analyzed. An additional factor that could contribute to this shift is the experimentally observed increase of the cis configuration of the chromophore at higher pHs.

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