Excited-state Proton Transfer Research Articles

Elucidating photocycle in large Stokes shift red fluorescent proteins: Focus on mKeima.

Large Stokes shift red fluorescent proteins (LSS-RFPs) are genetically encoded and exhibit a significant difference of a few hundreds of nanometers between their excitation and emission peak maxima (i.e., the Stokes shift). These LSS-RFPs (absorbing blue light and emitting red light) feature a unique photocycle responsible for their significant Stokes shift. The photocycle associated with this LSS characteristic in certain RFPs is quite perplexing, hinting at the complex nature of excited-state photophysics. This article provides a brief review on the fundamental mechanisms governing the photocycle of various LSS-RFPs, followed by a discussion on experimental results on mKeima emphasizing its relaxation pathways which garnered attention due to its >200 nm Stokes shift. Corroborating steady-state spectroscopy with computational studies, four different forms of chromophore of mKeima contributing to the cis-trans conformers of the neutral and anionic forms were identified in a recent study. Furthering these findings, in this account a detailed discussion on the photocycle of mKeima, which encompasses sequential excited-state isomerization, proton transfer, and subsequent structural reorganization involving three isomers, leading to an intriguing temperature and pH-dependent dual fluorescence, is explored using broadband femtosecond transient absorption spectroscopy.

Poly(2-oxazoline) Amphiphilicity Tunes the Excited-State Proton Transfer of Pyrenol-based Polyphotoacids.

The ability of light to change the properties of light-responsive polymers opens avenues for targeted release of cargo with a high degree of spatial and temporal control. Recently, we established photoacid polymers as light-switchable macromolecular amphiphiles. In these systems, light-induced excited-state proton transfer (ESPT) causes changes in amphilicity. However, as the intermolecular process itself critically depends on the local environment of the photoacid unit within the polymer, the overall amphiphilicity directly influences ESPT. Thus, understanding the impact of the local environment on the photophysics of photoacidic side chains is key to material design. In this contribution we address both thermodynamic and kinetic aspects of ESPT in oxazoline-based amphiphilic polymers with pyrenol-based photoacid side chains. We will compare the effect of polymer design, i.e. statistical and block arrangements, i.e. in poly[(2-ethyl-2-oxazoline)-co-(1-(6/8-hydroxyperene)sulphonylaziridine)] and poly(2-ethyl-2-oxazoline)-block-poly[(2-ethyl-2-oxazoline)-co-(2-(3-(6-hydroxypyrene)sulphona mide)propyl-2-oxazoline), on the intermolecular proton transfer reaction by combining steady-state and time-resolved absorption and emission spectroscopy. ESPT appears more prominent in the statistical copolymer compared to a block copolymer with overall similar pyrenol loading. We hypothesize that the difference is due to different local chain arrangements adopted by the polymers in the two cases.

Theoretical investigation on the “turn-on” mechanism of a novel fluorescent probe TB-N2H4: Twisted intramolecular charge transfer and excited-state dual proton transfer

Theoretical investigation on the “turn-on” mechanism of a novel fluorescent probe TB-N2H4: Twisted intramolecular charge transfer and excited-state dual proton transfer

Aggregation-Induced Modulation of Ground and Excited State Photophysics of 5-(tert-Butyl)-2-Hydroxy-1,3-Isophthalaldehyde (5-tBHI).

5-(tert-Butyl)-2-hydroxy-1,3-isophthalaldehyde (5-tBHI) is a photochromic material susceptible to either excited state proton transfer or excited state intramolecular proton transfer, depending upon the solvent. However, it has also been found to aggregate in the presence of sodium dodecyl sulfate. In this current study, based on the steady-state and time-resolved spectroscopy, supported by crystallography, quantum chemical density functional theory calculation, and molecular dynamics (MD) simulation, we report on the aggregation of this potential single benzene-based emitter (SBBE) in neat solvents as well as solid phase to modulate its photophysics. It has been found that 5-tBHI forms mixed aggregates of different orders, owing to the presence of both enolic and tautomeric forms, to yield tunable emission, although the emission intensity is quenched. These findings suggest that the intramolecular hydrogen bonding of 5-tBHI not only limits intermolecular interactions but also promotes nonradiative deactivation pathways. Hence, designing and structural engineering, with a focus to suppressing intramolecular hydrogen bonding as well as increasing through space conjugation by replacing the aldehydic moieties with bulky aliphatic or aromatic ketonic groups, can be a plausible approach to yielding improved probes with tunable emission and higher fluorescence quantum yields.

Unraveling the Photoionization Dynamics of Indole in Aqueous and Ethanol Solutions.

The photoionization dynamics of indole, the ultraviolet-B chromophore of tryptophan, were explored in water and ethanol using ultrafast transient absorption spectroscopy with 292, 268, and 200 nm excitation. By studying the femtosecond-to-nanosecond dynamics of indole in two different solvents, a new photophysical model has been generated that explains many previously unsolved facets of indole's complex solution phase photochemistry. Photoionization is only an active pathway for indole in aqueous solution, leading to a reduction in the fluorescence quantum yield in water-rich environments, which is frequently used in biophysical experiments as a key signature of the protein-folded state. Photoionization of indole in aqueous solution was observed for all three pump wavelengths but via two different mechanisms. For 200 nm excitation, electrons are ballistically ejected directly into the bulk solvent. Conversely, 292 and 268 nm excitation populates an admixture of two 1ππ* states, which form a dynamic equilibrium with a tightly bound indole cation and electron-ion pair. The ion pair dissociates on a nanosecond time scale, generating separated solvated electrons and indole cations. The charged species serve as important precursors to triplet indole production and greatly enhance the overall intersystem crossing rate. Our proposed photophysical model for indole in aqueous solution is the most appropriate for describing photoinduced dynamics of tryptophan in polypeptide sequences; tryptophan in aqueous pH 7 solution is zwitterionic, unlike in peptides, and resultantly has a competitive excited state proton transfer pathway that quenches the tryptophan fluorescence.

Substituent Effects in the Cationic Green Fluorescent Protein Chromophore: Ultrafast Excited‐State Proton Transfer or Twisting?

Understanding the structure‐function relationships of the green fluorescent protein (GFP) chromophore is important in rationally developing new molecular tools for biological imaging and beyond. Herein, we systematically modified the GFP chromophore structure with electron‐withdrawing and ‐donating groups (EWGs and EDGs) to investigate the substituent effects on the excited‐state proton transfer (ESPT) and twisting dynamics of the cationic chromophore in solution. With key insights gained from femtosecond transient absorption and stimulated Raman spectroscopy, we reveal that the EWG substitution by –F increases photoacidity in an additive manner and leads to an ultrafast barrierless ESPT by difluorination, while the EDG substitution by –OCH3 also results in ultrafast ESPT despite the weak photoacidity as estimated by the Förster equation. We ascribe the unusually fast kinetics in methoxylated derivatives to the occurrence of a pre‐existing chromophore‐solvent complex that sets up the acceptor site for ESPT. Furthermore, the kinetic competition between ESPT and twisting pathways is crucial for the observation of ESPT in action, particularly for molecules undergoing efficient nonradiative decay in the excited state through torsional motions. Such flexible and highly engineerable molecules can enable more versatile photoswitches and sensors.

Structural dynamics around a hydrogen bond: Investigating the effect of hydrogen bond strengths on the excited state dynamics of carboxylic acid dimers.

The photochemical dynamics of the acetic acid and trifluoro-acetic acid dimers in hexane are studied using time-resolved infrared absorption spectroscopy and abinitio electronic structure calculations. The different hydrogen bond strengths of the two systems lead to changes in the character of the accessed excited states and in the timescales of the initial structural rearrangement that define the early time dynamics following UV excitation. The much stronger hydrogen bonding in the acetic acid dimer stabilizes the system against dissociation. Ground state recovery is mediated by a structural buckling around the hydrogen bond itself with no evidence for excited state proton transfer processes that are usually considered to drive ultrafast relaxation processes in hydrogen bonded systems. The buckling of the ring leads to relaxation through two conical intersections and the eventual reformation of the electronic and vibrational ground states on a few picosecond timescale. In trifluoro-acetic acid, the weaker hydrogen bonding interaction means that the dimer dissociates under similar irradiation conditions. The surrounding solvent cage restricts the full separation of the monomer components, meaning that the dimer is reformed and returns to the ground state structure via a similar buckled structure but over a much longer, ∼100ps, timescale.

Ground-State Tautomerism and Excited-State Proton Transfer in 7-Hydroxy-4-methyl-8-((phenylimino)methyl)-2H-chromen-2-one as a Potential Proton Crane

The tautomerism in the title compound as a potential long-range proton transfer (PT) switch has been studied by using the DFT and TD-DFT approaches. The data show that in aprotic solvents, the enol tautomer dominates, while the increase in the content of the keto tautomer (short-range PT) rises as a function of polarity of the solvent. In ethanol, due to specific solute–solvent stabilization through intermolecular hydrogen bonding, a substantial amount of the keto forms exists in solution. The irradiation leads to two competitive processes in the excited state, namely ESIPT and trans/cis isomerization around the azomethine bond as in other structurally similar Schiff bases. The studied compound is not suitable for bistable tautomeric switching, where long-range PT occurs, due to the difficult enolization of the coumarin carbonyl group.

Theoretical explorations about solvent polarity associated excited state proton transfer behaviour for 2-benzoxazol-2-yl-6-triethylsilanylethynyl-phenol fluorophore

Theoretical explorations about solvent polarity associated excited state proton transfer behaviour for 2-benzoxazol-2-yl-6-triethylsilanylethynyl-phenol fluorophore

Quantum dynamics of excited state proton transfer in green fluorescent protein.

Photoexcitation of green fluorescent protein (GFP) triggers long-range proton transfer along a "wire" of neighboring protein residues, which, in turn, activates its characteristic green fluorescence. The GFP proton wire is one of the simplest, most well-characterized models of biological proton transfer but remains challenging to simulate due to the sensitivity of its energetics to the surrounding protein conformation and the possibility of non-classical behavior associated with the movement of lightweight protons. Using a direct dynamics variational multiconfigurational Gaussian wavepacket method to provide a fully quantum description of both electrons and nuclei, we explore the mechanism of excited state proton transfer in a high-dimensional model of the GFP chromophore cluster over the first two picoseconds following excitation. During our simulation, we observe the sequential starts of two of the three proton transfers along the wire, confirming the predictions of previous studies that the overall process starts from the end of the wire furthest from the fluorescent chromophore and proceeds in a concerted but asynchronous manner. Furthermore, by comparing the full quantum dynamics to a set of classical trajectories, we provide unambiguous evidence that tunneling plays a critical role in facilitating the leading proton transfer.

Synthesis and Photophysical Characterization of a pH-Sensitive Quadracyclic Uridine (qU) Analogue.

Fluorescent base analogues (FBAs) have become useful tools for applications in biophysical chemistry, chemical biology, live-cell imaging, and RNA therapeutics. Herein, two synthetic routes towards a novel FBA of uracil named qU (quadracyclic uracil/uridine) are described. The qU nucleobase bears a tetracyclic fused ring system and is designed to allow for specific Watson-Crick base pairing with adenine. We find that qU absorbs light in the visible region of the spectrum and emits brightly with a quantum yield of 27 % and a dual-band character in a wide pH range. With evidence, among other things, from fluorescence lifetime measurements we suggest that this dual emission feature results from an excited-state proton transfer (ESPT) process. Furthermore, we find that both absorption and emission of qU are highly sensitive to pH. The high brightness in combination with excitation in the visible and pH responsiveness makes qU an interesting native-like nucleic acid label in spectroscopy and microscopy applications in, for example, the field of mRNA and antisense oligonucleotide (ASO) therapeutics.

Light-Triggered Reversible Change in the Electronic Structure of MoO3 Nanosheets via an Excited-State Proton Transfer Mechanism.

Light is an attractive source of energy for regulating stimulus-responsive chemical systems. Here, we use light as a gating source to control the redox state, the localized surface plasmonic resonance (LSPR) peak, and the structure of molybdenum oxide (MoO3) nanosheets, which are important for various applications. However, the light excitation is not that of the MoO3 nanosheets but rather that of pyranine (HPTS) photoacids, which in turn undergo an excited-state proton transfer (ESPT) process. We show that the ESPT process from HPTS to the nanosheets and the intercalation of protons within the MoO3 nanosheets trigger the reduction of the nanosheets and the broadening of the LSPR peak, a process that is reversible, meaning that in the absence of light, the LSPR peak diminishes and the nanosheets return to their oxidized form. We further show that this reversible process is accompanied by a change in the nanosheet size and morphology.

Excited State Vibrational Dynamics Reveals a Photocycle That Enhances the Photostability of the TagRFP-T Fluorescent Protein.

High photostability is a desirable property of fluorescent proteins (FPs) for imaging, yet its molecular basis is poorly understood. We performed ultrafast spectroscopy on TagRFP and its 9-fold more photostable variant TagRFP-T (TagRFP S158T) to characterize their initial photoreactions. We find significant differences in their electronic and vibrational dynamics, including faster excited-state proton transfer and transient changes in the frequency of the v520 mode in the excited electronic state of TagRFP-T. The frequency of v520, which is sensitive to chromophore planarity, downshifts within 0.58 ps and recovers within 0.87 ps. This vibrational mode modulates the distance from the chromophore phenoxy to the side chain of residue N143, which we suggest can trigger cis/trans photoisomerization. In TagRFP, the dynamics of v520 is missing, and this FP therefore lacks an important channel for chromophore isomerization. These dynamics are likely to be a key mechanism differentiating the photostability of the two FPs.

Photochemical initiation of polariton-mediated exciton propagation

Abstract Placing a material inside an optical cavity can enhance transport of excitation energy by hybridizing excitons with confined light modes into polaritons, which have a dispersion that provides these light–matter quasi-particles with low effective masses and very high group velocities. While in experiments, polariton propagation is typically initiated with laser pulses, tuned to be resonant either with the polaritonic branches that are delocalized over many molecules, or with an uncoupled higher-energy electronic excited state that is localized on a single molecule, practical implementations of polariton-mediated exciton transport into devices would require operation under low-intensity incoherent light conditions. Here, we propose to initiate polaritonic exciton transport with a photo-acid, which upon absorption of a photon in a spectral range not strongly reflected by the cavity mirrors, undergoes ultra-fast excited-state proton transfer into a red-shifted excited-state photo-product that can couple collectively with a large number of suitable dye molecules to the modes of the cavity. By means of atomistic molecular dynamics simulations we demonstrate that cascading energy from a photo-excited donor into the strongly coupled acceptor-cavity states via a photo-chemical reaction can indeed induce long-range polariton-mediated exciton transport.

Excited-state antiaromaticity relief drives facile photoprotonation of carbons in aminobiphenyls.

A combined computational and experimental study reveals that ortho-, meta- and para-aminobiphenyl isomers undergo distinctly different photochemical reactions involving proton transfer. Deuterium exchange experiments show that the ortho-isomer undergoes a facile photoprotonation at a carbon atom via excited-state intramolecular proton transfer (ESIPT). The meta-isomer undergoes water-assisted excited-state proton transfer (ESPT) and a photoredox reaction via proton-coupled electron transfer (PCET). The para-isomer undergoes a water-assisted ESPT reaction. All three reactions take place in the singlet excited-state, except for the photoredox process of the meta-isomer, which involves a triplet excited-state. Computations illustrate the important role of excited-state antiaromaticity relief in these photoreactions.

Anomalous proton transfer of a photoacid HPTS in nonaqueous reverse micelles.

The proton transfer reaction is one of the fundamental chemical reactions where the reaction dynamics strongly depend on solvent properties such as acidity or basicity. A photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) shows a sharp decrease of pKa (7.7 → 0.5) upon photoexcitation, and the excited-state proton transfer (ESPT) occurs with ultrafast time constants of 2.5 and 89 ps in bulk aqueous solution. However, the two-step proton transfers via the contact ion pair formation and the proton diffusion are strongly limited inside the nanopools of reverse micelles (RMs). The confinement in small RMs strongly impeded the proton transfer reactions. In this work, we report the ESPT of HPTS confined in methanol-in-oil RMs by steady-state and time-resolved electronic spectroscopy. Interestingly, HPTS shows substantial deprotonation in the excited state only in small RMs, while the ESPT of HPTS does not occur in bulk methanol solution due to the low basicity of aliphatic alcohols. The kinetic analysis of time-resolved fluorescence and transient absorption measurements will compare the proton transfer dynamics of HPTS in the water-in-oil and methanol-in-oil RMs. The ESPT of photoacids, especially in the nonaqueous RMs, can be crucial in understanding many important chemical reactions involving proton transfer in the confined environments of cells and membranes.

Photochromism of phenazine-2,3-diol derivatives through excited state intermolecular proton transfer based on keto-enol tautomerization.

Photochromism through excited-state intermolecular proton transfer (ESInterPT) processes based on keto-enol tautomerization was found in phenazine-2,3-diol PD1 and its monoalkoxy derivative PD2 in a glassy matrix at 77 K: the colorless solutions of enol forms PD1-E and PD2-E at 298 K transformed into orange-colored solutions of keto forms PD1-K and PD2-K upon photoirradiation (λ = 385 nm) at 77 K. Furthermore, this report is the first to achieve the single-crystal X-ray structural analyses of phenazine-2,3-diol PD1 and its monoalkoxy derivative PD2, since the report on the synthesis of PD1 70 years ago. Indeed, it was found that PD1 and PD2 molecules exist in the keto form (PD1-K) and the enol form (PD2-E), respectively, in the crystal, and the neighboring PD1-K and PD2-E molecules are linked by one-dimensional intermolecular NH⋯O and OH⋯N hydrogen bonding, respectively. The fact suggests strongly that for PD1 and PD2, the formation of continuous intermolecular hydrogen bonding in aggregates such as in a glassy matrix at 77 K is involved in the keto-enol tautomerization of phenazine-2,3-diol derivatives based on ESInterPT. More interestingly, the color and the photoabsorption spectrum of the solids obtained by sublimation of crystals of PD2-E are similar to those for the crystals of PD1-K, indicating that the PD2 molecule exists in the keto form (PD2-K) in the solid of the sublimate. Therefore, this study provides a valuable insight for a greater understanding of the keto-enol tautomerization of diazaacene-diol derivatives and their photophysical properties in the solution and in the solid state.

Luminescence turn-on sensor for the selective detection of trace water and methanol based on a Zn(ii) coordination polymer with 2,5-dihydroxyterephthalate.

A highly selective detection of trace water in organic solvents is urgently required for the chemical industry. In this work, the simple sonochemical method was used for producing a luminescent sensor, [Zn(H2dhtp)(2,2'-bpy)(H2O)]n (Zn-CP) (H2dhtp2- = 2,5-dihydroxyterephthalate and 2,2'-bpy = 2,2'-bipyridine). Zn-CP exhibits reversible thermally-induced and methanol-mediated structural transformation. Importantly, Zn-CP has exceptional water sensing performance in both dry methanol and dry ethanol, with high selectivity, wide linear ranges, and a low limit of detection (LOD) of 0.08% (v/v). Upon the incremental addition of water, the luminescent intensities enhanced and shifted, along with the emission color changing from green to greenish yellow. In addition, Zn-CP can detect methanol selectively through turn-on luminescence intensity with LODs of 0.28, 0.52, and 0.35% (v/v) in dry ethanol, dry n-propanol, and dry n-butanol, respectively. The excited-state proton transfer of linker H2dhtp2-via enol-keto tautomerism and collaboration with structural transformation could be attributed to the sensing mechanism.

Excited-state proton transfer based fluorescence in Kaempferol powder and solutions with different concentrations

Kaempferol (KMP) is one of the most common flavonoids, currently being extensively studied for its numerous beneficial health effects. Here we study the fluorescence (FL) emission of KMP powder and of its solutions prepared using different types of solvents (polar and non-polar). In the spectra of KMP powder and KMP solutions with high concentration, the same FL peak with maximum at 1.9 eV is observed. Another FL peak, at higher energy of 2.45 eV, emerges in solutions, its relative intensity increases with decreasing solution concentration. The FL emission of solutions with lowest concentration displays only that peak. To calculate characteristic energies of absorption and emission of KMP molecule in vacuum and in solutions we use time-dependent density functional theory. Comparing the results of computations with measured FL spectra, we associate the FL band at 1.9 eV with the emission due to excited state intramolecular transfer of the proton of –OH5 hydroxyl group. The FL emission at 2.45 eV is related to the –OH3 proton transfer. We measure the FL spectra of KMP powder using two different excitation energies, 3.06 eV and 2.33 eV, and find that its FL spectrum depends on the excitation energy. To understand that dependence, we compare the FL spectra of KMP and Q monohydrate powders. We consider the excited state intermolecular transfer of the proton from –OH3′ hydroxyl group to a neighboring molecule in Q crystal and calculate the energy corresponding to the emission of the resulted anion of Q molecule. The spectral feature at 1.69 eV observed only in the FL spectrum of Q hydrate is attributed to the Q anion FL emission.

Effect of Molecular Dynamics and Internal Water Contact on the Photophysical Properties of Red pH-Sensitive Proteins.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.