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1. Excited State Dynamics of 7-Deazaguanosine and Guanosine 5’-Monophosphate

   Krul, S. E. ; Hoehn, S. J. ; Feierabend, K. ; Crespo-Hernández, C. E. ( January 2021 , ChemRxiv)

   Minor structural modifications to the DNA and RNA nucleobases have a significant effect on their excited state dynamics and electronic relaxation pathways. In this study, the excited state dynamics of 7-deazaguanosine and guanosine 5′-monophosphate are investigated in aqueous solution and in a mixture of methanol and water using femtosecond broadband transient absorption spectroscopy following excitation at 267 nm. The transient spectra are collected using photon densities that ensure no parasitic multiphoton-induced signal from solvated electrons. The data can be fit satisfactorily using a two- or three-component kinetic model. By analyzing the results from steady-state, time-resolved, computational calculations, and the methanol–water mixture, the following general relaxation mechanism is proposed for both molecules, Lb → La → 1πσ*(ICT) → S0, where the 1πσ*(ICT) stands for an intramolecular charge transfer excited singlet state with significant πσ* character. In general, longer lifetimes for internal conversion are obtained for 7-deazaguanosine compared to guanosine 5′-monophosphate. Internal conversion of the 1πσ*(ICT) state to the ground state occurs on a similar time scale of a few picoseconds in both molecules. Collectively, the results demonstrate that substitution of a single nitrogen atom for a methine (C–H) group at position seven of the guanine moiety stabilizes the 1ππ* Lb andmore »La states and alters the topology of their potential energy surfaces in such a way that the relaxation dynamics in 7-deazaguanosine are slowed down compared to those in guanosine 5′-monophosphate but not for the internal conversion of 1πσ*(ICT) state to the ground state.« less
2. Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy

   https://doi.org/10.3390/molecules23092226  

   Tang, Longteng ; Zhu, Liangdong ; Taylor, Miles ; Wang, Yanli ; Remington, S. ; Fang, Chong ( September 2018 , Molecules)

   Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green fluorescent protein (GFP) single-site mutant S205V. This mutation alters the original excited state proton transfer (ESPT) chain. By strategically tuning the Raman pump to different wavelengths (i.e., 801, 539, and 504 nm) to achieve pre-resonance with transient excited state electronic bands, the characteristic Raman modes of the excited protonated (A*) chromophore species and intermediate deprotonated (I*) species can be selectively monitored. The inhomogeneous distribution/population of A* species go through ESPT with a similar ~300 ps time constant, confirming that bridging a water molecule to protein residue T203 in the ESPT chain is the rate-limiting step. Some A* species undergo vibrational cooling through high-frequency motions on the ~190 ps time scale. At early times, a portion of the largely protonated A* species could also undergo vibrational cooling or return to the ground state with a ~80 ps time constant. On the photoproduct side, a ~1330 cm−1 delocalized motion is observed, withmore »dispersive line shapes in both the Stokes and anti-Stokes FSRS with a pre-resonance Raman pump, which indicates strong vibronic coupling, as the mode could facilitate the I* species to reach a relatively stable state (e.g., the main fluorescent state) after conversion from A*. Our findings disentangle the contributions of various vibrational motions active during the ESPT reaction, and offer new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and other analogous autofluorescent proteins.« less
3. Photogrammetry of Ultrafast Excited-State Intramolecular Proton Transfer Pathways in the Fungal Pigment Draconin Red

   https://doi.org/10.3390/molecules28083506  

   Solaris, Janak ; Krueger, Taylor D. ; Chen, Cheng ; Fang, Chong ( April 2023 , Molecules)

   Proton transfer processes of organic molecules are key to charge transport and photoprotection in biological systems. Among them, excited-state intramolecular proton transfer (ESIPT) reactions are characterized by quick and efficient charge transfer within a molecule, resulting in ultrafast proton motions. The ESIPT-facilitated interconversion between two tautomers (PS and PA) comprising the tree fungal pigment Draconin Red in solution was investigated using a combination of targeted femtosecond transient absorption (fs-TA) and excited-state femtosecond stimulated Raman spectroscopy (ES-FSRS) measurements. Transient intensity (population and polarizability) and frequency (structural and cooling) dynamics of –COH rocking and –C=C, –C=O stretching modes following directed stimulation of each tautomer elucidate the excitation-dependent relaxation pathways, particularly the bidirectional ESIPT progression out of the Franck–Condon region to the lower-lying excited state, of the intrinsically heterogeneous chromophore in dichloromethane solvent. A characteristic overall excited-state PS-to-PA transition on the picosecond timescale leads to a unique “W”-shaped excited-state Raman intensity pattern due to dynamic resonance enhancement with the Raman pump–probe pulse pair. The ability to utilize quantum mechanics calculations in conjunction with steady-state electronic absorption and emission spectra to induce disparate excited-state populations in an inhomogeneous mixture of similar tautomers has broad implications for the modeling of potential energy surfaces and delineationmore »of reaction mechanisms in naturally occurring chromophores. Such fundamental insights afforded by in-depth analysis of ultrafast spectroscopic datasets are also beneficial for future development of sustainable materials and optoelectronics.« less
4. Femtosecond photodecarbonylation of photo-ODIBO studied by stimulated Raman spectroscopy and density functional theory

   https://doi.org/10.1039/d1cp03512f  

   Shenje, Learnmore ; Qu, Yingqi ; Popik, Vladimir ; Ullrich, Susanne ( November 2021 , Physical Chemistry Chemical Physics)

   Photo-oxa-dibenzocyclooctyne (Photo-ODIBO) undergoes photodecarbonylation under UV excitation to its bright S 2 state, forming a highly reactive cyclooctyne, ODIBO. Following 321 nm excitation with sub-50 fs actinic pulses, the excited state evolution and cyclopropenone bond cleavage with CO release were characterized using femtosecond stimulated Raman spectroscopy and time-dependent density functional theory Raman calculations. Analysis of the photo-ODIBO S 2 CO Raman band revealed multi-exponential intensity, peak splitting and frequency-shift dynamics. This suggests a stepwise cleavage of the two C–C bonds in the cyclopropenone structure that is completed within <300 fs after excitation. Evidence of intramolecular vibrational relaxation on the S 2 state, concurrent with photodecarbonylation, with dynamics matching previous electronic transient absorption spectroscopy, was also observed. This confirms an excited state, as opposed to ground state, photodecarbonylation mechanism resulting in a vibronically excited photoproduct, ODIBO.
5. Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing

   https://doi.org/10.3390/bios13020218  

   Krueger, Taylor D. ; Tang, Longteng ; Fang, Chong ( February 2023 , Biosensors)

   Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca2+) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca2+-free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca2+-bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading tomore »a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities.« less