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Ultrafast spectroscopy has advanced our understanding of chemical reaction mechanisms on molecular time scales, guiding the rational design of molecular systems and processes with spatial (atomic, nuclear) and temporal (electronic, vibrational) precisions. Kinetic analysis of ultrafast spectroscopic measurements yields valuable insights into the physical mechanisms that determine the behavior of a wide range of important chemical systems from chromophores and fluorescent proteins to nanosystems. One powerful and widely used method is global analysis, which uses a unified kinetic description of the entire spectrum. Importantly, interpretation of fit parameters to build a mechanistic understanding relies on an accurate estimate of the uncertainty. Software and numerical techniques using global analysis to fit data are well-developed; however, there is much less work on uncertainty analysis of the resulting kinetic parameters. Due to the nonlinear nature of modeling, traditional methods tend to underestimate the uncertainty. Building on previous work, we implement Markov chain Monte Carlo (MCMC) sampling to estimate the parameter uncertainty. We develop a procedure that can be readily included in the existing analysis software and demonstrate it on a series of generated and experimental femtosecond transient absorption data sets. Our results suggest that global analysis, using the simultaneously collected spectral data at many wavelengths, is crucial for accurate parameter estimation. The retrieved parameter uncertainty is well within 10% for a typical spectral data set with reasonable signal-to-noise ratios. 

Flavin adenine dinucleotide (FAD) is prevalent within numerous light‐sensitive protein systems. One of the underexplored areas concerns the different protonation states of the fully oxidized FAD powering many photoreceptors. We implemented an integral electronic and vibrational spectroscopic toolset to investigate FAD at different pH conditions, and uncovered several distinct properties of the anionic FAD. Guided by femtosecond transient absorption (fs‐TA) results, we performed wavelength‐tunable femtosecond stimulated Raman spectroscopy (FSRS) from the ground to excited state to delineate vibrational signatures of a series of cationic, acidic, neutral, and anionic FAD in aqueous solution. The deprotonation of the flavin isoalloxazine polar ring moiety leads to notable electron density redistribution, enhancing the C4═O4’ stretching motion while shifting the absorption band. After 400 and 480 nm excitations, the excited‐state (ES)‐FSRS of anionic FAD reveals a key vibrational peak around 1368 cm⁻¹in S₁, which represents a 21 cm⁻¹blueshift from the 1347 cm⁻¹mode in S₀. Such a site‐specific spectroscopic study of FAD in the physiologically‐relevant water environment by tuning both pH and excitation wavelengths complements the current understanding of correlated FAD electronic and structural dynamics, better bridging the more characterized FAD redox behavior to fundamental insights into photophysical properties of this versatile biological cofactor. 

Fluorescent proteins (FPs) are noninvasive genetically encodable probes that have revolutionized bioimaging and health fields with vivid images and an ever-growing repertoire from jellyfish to sea anemones and corals. Inside the protein matrix, chromophore nonplanarity and flexibility have long been argued to govern the fluorescence efficiency of FPs, yet their fundamental roles and relative importance have been elusive which hinder the rational design of versatile FPs and biosensors. Herein, we tackle this central question by investigating two recently engineered FP-based turn-on chloride (Cl^–) sensors, ChlorON1 and 3, using an ultrafast electronic and vibrational spectroscopic toolset together with advanced multireference simulations for both structure and spectrum. We elucidate that fluorescence enhancement of the chloride-bound ChlorON3 stems from a substantially more twisted chromophore than ChlorON1 via comprehensive simulations starting from the available crystal structure of parent protein (mNeonGreen), also featuring an enhanced radiative pathway due to an adjacent leucine residue in the emissive population. This finding indicates that the commonly stated chromophore planarity is not, but conformational rigidity is, the decisive factor for high fluorescence efficiency. Such mechanistic insights into FPs are generalizable to chromoproteins and other photosensitive biomolecules, which can facilitate the targeted design of brighter and/or tunable biosensors. 

Nitrophenols are environmental pollutants found in waterways and the atmosphere, which can be a significant source of nitrous acid under UV light. In this work, we investigate nitrophenols in aqueous solution upon UV irradiation with a suite of steady-state and ultrafast electronic and vibrational spectroscopies. We find that ortho- and para-nitrophenol are highly sensitive to zinc as a heterogeneous catalyst, accelerating the transformation of nitrophenols with/without UV light. Steady-state absorption, femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations reveal that UV light triggers excited-state intramolecular proton transfer to form an aci-nitro intermediate that is stabilized by zinc. We photoexcite the metastable intermediate and obtain femtosecond transient absorption signatures. The redshifted ground-state absorption and lengthened excited-state lifetime of the aci-nitro intermediate are beneficial for photocatalytic removal with irradiation greater than 400 nm. Furthermore, the water O–H stretching band is tracked by FSRS to unveil a uniquely active role of water during the catalytic reaction of ortho-nitrophenol, rationalizing its reduced photosensitivity in organic solvents such as methanol. These neat treatment strategies are also applied to para- and meta-nitrophenol and nitrophenolate. Our mechanistic insights into the photosensitivity of nitrophenols can inspire more effective cost-efficient treatment strategies for atmospheric aerosols and water systems. 

Photoconvertible fluorescent proteins (pcFPs) have enabled exquisite images of cellular structures due to their genetic encodability and red-shifted emission with high brightness, hence receiving increased traction in the field. However, the red form of Kaede-like pcFPs after photoconversion remains underexplored. We implemented ultrafast electronic and vibrational spectroscopies on the red Kaede chromophore in solution vs the protein pocket of the least-evolved ancestor (LEA, a Kaede-like green-to-red pcFP) to gain crucial insights into the photophysical processes of the chromophore. The measured fluorescence quantum yield (FQY) values were correlated with ultrafast dynamics to reveal that hydrogen-bonding interactions with the solvent can quench the excited-state Kaede in solution. A viscosity-dependent sub-ps decay indicates nonradiative relaxation involving swift chromophore conformational motions. Femtosecond transient absorption and stimulated Raman spectroscopy (FSRS) reveal an additional ∼1 ps decay of the photoconverted red form of LEA that is absent in green LEA before photoconversion. Transient structural dynamics from FSRS elucidate this decay to involve the phenolate and imidazolinone ring twists that are implicated during cis → trans isomerization and on → off photoswitching in phototransformable fluorescent proteins (FPs). Compared to green-emitting species, the FQY of red LEA (∼0.58) and many other red FPs are often reduced, limiting their applications in modern bioimaging techniques. By shining more light on the often overlooked photoconverted form of pcFPs with ultrafast spectroscopies, we envision such essential mechanistic insights to enable a bottom-up approach for rationally improving the brightness of red-emitting LEA and many other controllable bioprobes, including FPs. 

Abstract Hypericin from St. John's wort has been used as a potent photosensitizer, but its working mechanism remains elusive which hinders its rational design for improved functionality. We implement ultrafast spectroscopy and quantum calculations to track the excited‐state dynamics in an intricate hydrogen‐bonding network of hypericin in solution. Using femtosecond transient absorption (fs‐TA), we track excited state intramolecular proton transfer (ESIPT) via a previously unreported blueshift of a long‐wavelength stimulated emission (SE) band with excitation‐dependent dynamics in various solvents, owing to the dominant Q^7,14tautomer that undergoes bidirectional ESIPT. This finding is corroborated by ground‐state femtosecond stimulated Raman spectroscopy (GS‐FSRS) and density functional theory (DFT) calculations. Moreover, contrasting the neutral and anionic forms of hypericin enables us to reveal an intramolecular charge transfer step underlying ESIPT. We demonstrate UV and visible excitations as an integral platform to provide direct insights into the photophysics and origin for phototoxicity of hypericin. Such mechanistic insights into the excited state of hypericin will power its future development and use. 

The Front Cover illustrates ultrafast spectroscopic insights into the photoexcited energy relaxation pathways of St. John's wort-derived fluorescent photosensitizer hypericin in solution. The bidirectional excited-state intramolecular proton transfer (ESIPT) gains prominence after UV excitation with enhanced photoprotection in a “proton pachinko”, whereas visible excitation results in more phototoxicity. More information can be found in the Research Article by C. Fang and co-workers (DOI: 10.1002/chem.202500639). Cover design by S. Johnson and C. Fang. 

Ultrafast electronic and Raman spectroscopies on least evolved ancestor (LEA) fluorescent proteins reveal a distorted photoswitchedoffstate returning toonstate upon photoexcitationviaproton transfer and isomerization, hindering photoconversion. Photoconvertible fluorescent proteins (pcFPs) have greatly advanced life sciences and cellular imaging with sub-diffraction resolution. A subset of Kaede-like pcFPs can reversibly photoswitch and irreversibly photoconvert, which yield intriguing properties for sophisticated bioimaging, yet blinking may complicate image analysis. Many investigations on such pcFPs lack transient information that can dictate their optical properties, especially on ultrafast timescales. We study a family of ancestrally derived pcFPs with varying photoconversion and photoswitching efficiencies based on the least evolved ancestor (LEA). With ultrafast electronic and vibrational spectroscopies that complement steady-state measurements, we dissect the primary events upon near-UV excitation of the native and photoswitched neutral chromophores, which initiates both off → on photoswitching and green-to-red photoconversion. We demonstrate that cis → trans isomerization underlying negative photoswitching occurs the fastest in acidic buffers upon green light irradiation, which forms a distorted neutral off state proportional to the initial green cis anionic population. Femtosecond transient absorption measurements reveal that this dynamic off state rapidly photoswitches back to the bright on state upon near-UV excitation, in contrast to the native form. With various mutants and photoinduced states, we find that off → on photoswitching likely involves excited state proton transfer from the distorted chromophore, which competes with photoconversion. In contrast, femtosecond stimulated Raman spectroscopy (FSRS) of the much less photoswitchable LEA-A69T tracks the efficient nonradiative relaxation of the native cis neutral chromophore. We propose rational design strategies to inhibit off → on photoswitching while improving the photoconversion efficiency of both neutral states. This work is envisioned to inspire more dynamic investigations of diverse photochromic FPs on ultrafast timescales. 

The PEG addition into aqueous electrolytes has an opposite effect on an Fe metal anode compared to a Zn metal anode.