Unraveling the contributions to the spectral shape of flexible dyes in solution: insights on the absorption spectrum of an oxyluciferin analogue.

We present a computational investigation of the absorption spectrum in water of 5,5-spirocyclopropyl-oxyluciferin (5,5-CprOxyLH), an analogue of the emitter compound responsible for the bioluminescence in fireflies. Several factors participate in determining the 5,5-CprOxyLH's spectral shape: (i) the contribution of the four close-energy excited states, which show significant non-adiabatic couplings, (ii) the flexible molecular structure and (iii) the specific interactions established with the surrounding environment, which strongly couple the protic solvent dynamics with the dye's spectral response. To tackle the challenge to capture and dissect the role of all these effects we preliminarily investigate the role of non-adiabatic couplings with quantum dynamics simulations and a linear vibronic coupling model in the gas phase. Then, we account for both the molecular flexibility and solvent interactions by resorting to a mixed quantum classical protocol, named Adiabatic Molecular Dynamics generalized Vertical Gradient (Ad-MD|gVG), which is built on a method recently proposed by some of us. It is rooted in the partition between stiff degrees of freedom of the dye, accounted for at the vibronic level within the harmonic approximation, and flexible degrees of freedom of the solute (and of the solvent), described classically through a sampling based on Molecular Dynamics (MD). Ad-MD|gVG avoids spurious effects arising in the excited state Hessians due to non-adiabatic couplings, and can therefore be applied to account for the contributions of the first four excited states to the 5,5-CprOxyLH absorption spectrum. The final simulated spectrum is in very good agreement with the experiment, especially when the MD is driven by a refined quantum-mechanically derived force-field. More importantly, the origin of each separate contribution to the spectral shape is appropriately accounted for, paving the way to future applications of the method to more complex systems or alternative spectroscopies, as emission or circular dichroism.

Spectrochemistry of Firefly Bioluminescence.

The chemical reactions underlying the emission of light in fireflies and other bioluminescent beetles are some of the most thoroughly studied processes by scientists worldwide. Despite these remarkable efforts, fierce academic arguments continue around even some of the most fundamental aspects of the reaction mechanism behind the beetle bioluminescence. In an attempt to reach a consensus, we made an exhaustive search of the available literature and compiled the key discoveries on the fluorescence and chemiluminescence spectrochemistry of the emitting molecule, the firefly oxyluciferin, and its chemical analogues reported over the past 50+ years. The factors that affect the light emission, including intermolecular interactions, solvent polarity, and electronic effects, were analyzed in the context of both the reaction mechanism and the different colors of light emitted by different luciferases. The collective data points toward a combined emission of multiple coexistent forms of oxyluciferin as the most probable explanation for the variation in color of the emitted light. We also highlight realistic research directions to eventually address some of the remaining questions related to firefly bioluminescence. It is our hope that this extensive compilation of data and detailed analysis will not only consolidate the existing body of knowledge on this important phenomenon but will also aid in reaching a wider consensus on some of the mechanistic details of firefly bioluminescence.

Identification of new alpha-synuclein fibrillogenesis inhibitor using in silico structure-based virtual screening.

Abnormal aggregation and accumulation of alpha-synuclein (αSN) in existing neurons is associated with Parkinson's disease (PD) as one of the age-related neurodegenerative disorders. Inhibition of αSN fibrillogenesis could be considered as a solution for PD diseases treatment. Here, virtual screening (VS) approach was used to investigate available ligands in PubChem library with structural similarity with Dihydromyricetin (DHM) (as a recently introduced suitable candidate for designing of novel antiPD drugs) against aggregation of αSN chains. Primary screening identified 314 promising molecules for αSN monomer, which were further analyzed in details by their binding energy and binding modes through molecular docking method. Evidently, the compound with PubChem ID of 100968625 displayed the lowest free binding energy with ΔG0 = -7.1 kcal.mol-1 and was selected for further analysis using molecular dynamics (MD) simulation method. Analysis of MD trajectories showed that molecules of the selected ligand interact with αSN trimer via H-bond interaction and destabilize the compact structure of αSN trimer. Further, prompt in vivo testing to validate the antiPD inhibition efficiency by this molecule can save lives.

Multiconfigurational Quantum Chemistry Determinations of Absorption Cross Sections (σ) in the Gas Phase and Molar Extinction Coefficients (ε) in Aqueous Solution and Air-Water Interface.

Theoretical determinations of absorption cross sections (σ) in the gas phase and molar extinction coefficients (ε) in condensed phases (water solution, interfaces or surfaces, protein or nucleic acids embeddings, etc.) are of interest when rates of photochemical processes, J = ∫ ϕ(λ) σ(λ) I(λ) dλ, are needed, where ϕ(λ) and I(λ) are the quantum yield of the process and the irradiance of the light source, respectively, as functions of the wavelength λ. Efficient computational strategies based on single-reference quantum-chemistry methods have been developed enabling determinations of line shapes or, in some cases, achieving rovibrational resolution. Developments are however lacking for strongly correlated problems, with many excited states, high-order excitations, and/or near degeneracies between states of the same and different spin multiplicities. In this work, we define and compare the performance of distinct computational strategies using multiconfigurational quantum chemistry, nuclear sampling of the chromophore (by means of molecular dynamics, ab initio molecular dynamics, or Wigner sampling), and conformational and statistical sampling of the environment (by means of molecular dynamics). A new mathematical approach revisiting previous absolute orientation algorithms is also developed to improve alignments of geometries. These approaches are benchmarked through the nπ* band of acrolein not only in the gas phase and water solution but also in a gas-phase/water interface, a common situation for instance in atmospheric chemistry. Subsequently, the best strategy is used to compute the absorption band for the adduct formed upon addition of an OH radical to the C6 position of uracil and compared with the available experimental data. Overall, quantum Wigner sampling of the chromophore with molecular dynamics sampling of the environment with CASPT2 electronic-structure determinations arise as a powerful methodology to predict meaningful σ(λ) and ε(λ) band line shapes with accurate absolute intensities.

Frontiers in Multiscale Modeling of Photoreceptor Proteins.

This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments.

The role of CO2 detachment in fungal bioluminescence: thermally vs. excited state induced pathways.

Different fungi lineages are known to emit light on Earth, mainly in tropical climates. Although the preparation of bioluminescent cell-free extracts allowed one to characterize the enzymatic requirements, the molecular mechanism underlying luminescence is still largely unknown and is based on the experimental putative assumption that a high-energy intermediate should be formed by reaction with O2 and formation of an endoperoxide. Here, we aim at determining, through state-of-the-art multiconfigurational quantum chemistry, the full mechanistic landscape leading from the endoperoxide to the emitting species, envisaging different possible pathways and proposing their viability. Especially, thermal CO2 detachment followed by excited-state peroxide opening (thermal-chemiluminescence) can compete with a parallel pathway, i.e., first excited-state endoperoxide opening, followed by CO2 detachment on the same excited-state (excited state-chemiluminescence). Clear differences in the energy supplies, as well as the possibility to directly populate the emitting species from the intersection seam between ground and excited states, land credence to a kinetically efficient thermal-chemiluminescent pathway, establishing for the first time a detailed description of fungal bioluminescence.

Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Bioluminescent Nanoluciferase-Furimamide Complex: A Theoretical Study on Different Protonation States.

Luminescence of furimamide is 150 times brighter than oxidized luciferins in firefly and renilla luciferase. However, we do not have a clear understanding of the structure, function, and dynamic behavior of the nanoluciferase-furimamide complex. Here, for the first time, the absorption and emission properties of eight different possible light emitter forms of furimamide were investigated using the time-dependent density functional theory (TD-DFT) method in the gas phase and aqueous solution. The emission oscillator strengths in the gas phase showed that emission transition may be forbidden for some forms, and fluorescence would not occur. Besides, the charge transfer (CT) as well as the orbitals involved in the transitions were analyzed. Furthermore, molecular docking results showed that furimamide is situated inside the central cavity (ß-barrel) of nanoluciferase. Analysis of the trajectory of molecular dynamics (MD) simulations suggested a less compact structure of protein in the presence of furimamide in comparison to its apo form. The quantum mechanical/molecular mechanical (QM/MM) spectroscopic properties of one form in the binding site of nanoluciferase were investigated. The evolution of the excited states (ESs) of furimamide in the binding pocket of the protein confirmed that after photoexcitation and during the relaxation of the system, a crossing point between the first two singlet ESs exists. Thus, the initially populated S2 (a π→π* transition) becomes the first singlet excited state.

Fungal Light Emitter: Understanding Its Chemical Nature and pH-Dependent Emission in Water Solution.

Fungal bioluminescence is a fascinating natural process, standing out for the continuous conversion of chemical energy into light. The structure of fungal oxyluciferin (light emitter) was proposed in 2017, being different and more complex than other oxyluciferins. The complexity of fungal oxyluciferin arises from diverse equilibria such as keto/enol tautomerization or deprotonation equilibria of four titratable groups. For this reason, still some crucial details of its structure remain unexplored. To obtain further structural information, a combined experimental and computational study of natural and three synthetic fungal oxyluciferin analogues has been performed. Here, we state the most stable chemical form of fungal oxyluciferin regarding its keto and enol tautomers, in the ground and excited states. We propose the (3Z,5E)-6-(3,4-dihydroxyphenyl)-4-hydroxy-2-oxohexa-3,5-dienoic acid form as the light emitter (fluorescent state) in water solution. Moreover, we show that chemical modifications on fungal oxyluciferin can affect the relative stability of the conformers. Furthermore, we show the clear effect of pH on emission. General conclusions about the role of these titratable groups in emission modulation have been drawn, such as the key role of dihydroxyphenyl deprotonation. This study is key to further analyze the properties of fungal bioluminescence and propose novel synthetic analogues.

Light emission colour modulation study of oxyluciferin synthetic analogues via QM and QM/MM approaches.

Firefly oxyluciferin is the chemical product of bioluminescence responsible for light emission. Experiments have already shown that different analogues of natural oxyluciferin, exhibit different emission colours. In particular, the structure of natural oxyluciferin has been modified by atom or group substitutions. However, a rationalization of the origin of the bioluminescence emission colour modulation of these analogues has still not been reported. For these reasons, the aim of this study is to explain the influence of structural modifications within the natural oxyluciferin on the colour modulation of bioluminescence. To do this, natural firefly oxyluciferin and three synthetic analogues whose experimental bioluminescence spectra are red- and blue-shifted compared to the natural one were studied. The absorption and emission transition energies have been calculated at the Time-dependent density functional theory (TD-DFT) level using both quantum mechanics (QM) and quantum mechanics/molecular mechanics (QM/MM) methods. Moreover, the solvent (water using the PCM model) and the protein surrounding effect have also been considered. The predicted emission spectra are in quite good agreement with the available experimental spectra, validating the methodology followed in this study. In particular, it was demonstrated that using the QM/MM approach, and considering explicitly the protein environment, the experimental bioluminescence spectra can be reproduced. Furthermore, this study shows that the substitution within the oxyluciferin structure causes a change of its electronic distribution and energies of the HOMO and LUMO orbitals involved in the vertical transitions, leading to different light emission colours. This work will promote future studies focused on luciferin mutations guided by the prediction of their bioluminescence emission spectra.

Molecular Simulations with in-deMon2k QM/MM, a Tutorial-Review.

deMon2k is a readily available program specialized in Density Functional Theory (DFT) simulations within the framework of Auxiliary DFT. This article is intended as a tutorial-review of the capabilities of the program for molecular simulations involving ground and excited electronic states. The program implements an additive QM/MM (quantum mechanics/molecular mechanics) module relying either on non-polarizable or polarizable force fields. QM/MM methodologies available in deMon2k include ground-state geometry optimizations, ground-state Born-Oppenheimer molecular dynamics simulations, Ehrenfest non-adiabatic molecular dynamics simulations, and attosecond electron dynamics. In addition several electric and magnetic properties can be computed with QM/MM. We review the framework implemented in the program, including the most recently implemented options (link atoms, implicit continuum for remote environments, metadynamics, etc.), together with six applicative examples. The applications involve (i) a reactivity study of a cyclic organic molecule in water; (ii) the establishment of free-energy profiles for nucleophilic-substitution reactions by the umbrella sampling method; (iii) the construction of two-dimensional free energy maps by metadynamics simulations; (iv) the simulation of UV-visible absorption spectra of a solvated chromophore molecule; (v) the simulation of a free energy profile for an electron transfer reaction within Marcus theory; and (vi) the simulation of fragmentation of a peptide after collision with a high-energy proton.

Effect of Protein Conformation and AMP Protonation State on Fireflies' Bioluminescent Emission.

The emitted color in fireflies' bioluminescent systems depends on the beetle species the system is extracted from and on different external factors (pH, temperature…) among others. Controlling the energy of the emitted light (i.e., color) is of crucial interest for the use of such bioluminescent systems. For instance, in the biomedical field, red emitted light is desirable because of its larger tissue penetration and lower energies. In order to investigate the influence of the protein environment and the AMP protonation state on the emitted color, the emission spectra of the phenolate-keto and phenolate-enol oxyluciferin forms have been simulated by means of MD simulations and QM/MM calculations, considering: two different protein conformations (with an open or closed C-terminal domain with respect to the N-terminal) and two protonation states of AMP. The results show that the emission spectra when considering the protein characterized by a closed conformation are blue-shifted compared to the open conformation. Moreover, the complete deprotonation of AMP phosphate group (AMP2-) can also lead to a blue-shift of the emission spectra but only when considering the closed protein conformation (open form is not sensitive to changes of AMP protonation state). These findings can be reasoned by the different interactions (hydrogen-bonds) found between oxyluciferin and the surrounding (protein, AMP and water molecules). This study gets partial insight into the possible origin of the emitted color modulation by changes of the pH or luciferase conformations.

The role of solvation models on the computed absorption and emission spectra: the case of fireflies oxyluciferin.

Surrounding effects are crucial to successfully simulate the absorption and emission spectra of molecular systems. In this work we test different solvation models to compute transition energies and to simulate the spectra of oxyluciferin responsible for the light emission in fireflies and its derivatives. We demonstrate that, within the PCM model, the IBSF formalism is suitable for computing the transition energies of the oxyluciferin chemical forms characterized by a charge transfer character. On the other hand, the LR approach could be used for the chemical forms where an almost negligible charge transfer takes place. Moreover, we demonstrate that explicit solvation models, applied by QM/MM calculations, are needed to accurately reproduce the experimental shape of the spectra. Finally, the vibrationally resolved spectra using a solvation model (implicit or microsolvation) is computed. Some noticeable differences arise when considering the implicit solvation with respect to gas phase vibrational spectra, while small changes were found when explicit water molecules within a microsolvated model are considered.

Beetle luciferases with naturally red- and blue-shifted emission.

The different colors of light emitted by bioluminescent beetles that use an identical substrate and chemiexcitation reaction sequence to generate light remain a challenging and controversial mechanistic conundrum. The crystal structures of two beetle luciferases with red- and blue-shifted light relative to the green yellow light of the common firefly species provide direct insight into the molecular origin of the bioluminescence color. The structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The only known naturally red-emitting luciferase from the glow-worm Phrixothrix hirtus exists as tetramers and octamers. Structural and computational analyses reveal varying aperture between the two domains enclosing the active site. Mutagenesis analysis identified two conserved loops that contribute to the color of the emitted light. These results are expected to advance comparative computational studies into the conformational landscape of the luciferase reaction sequence.

Modeling Chemical Reactions by QM/MM Calculations: The Case of the Tautomerization in Fireflies Bioluminescent Systems.

In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modeling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods.

Rationalisation of the optical signatures of nor-dihydroxanthene-hemicyanine fused near-infrared fluorophores by first-principle tools.

Using a computational approach combining the Time-Dependent Density Functional Theory (TD-DFT) and the second-order Coupled Cluster (CC2) approaches, we investigate the spectral properties of a large panel of nor-dihydroxanthene (DHX)-hemicyanine fused dyes. First we compare the theoretical and experimental 0-0 energies for a set of 14 known synthetic compounds and show that a remarkable agreement between theory and experiment is obtained when a suitable environmental model is selected. In addition, we obtain vibrationally-resolved spectra for several compounds and theory also accurately reproduces the experimental band shapes. We show that the electronic transitions in nor-DHX-based fluorophores are associated with small variations of the dipole moments but large oscillator strengths. Using various chemical strategies, we design a series of compounds with red-shifted 0-0 energies.

QM/MM Study of the Formation of the Dioxetanone Ring in Fireflies through a Superoxide Ion.

The bioluminescence emission from fireflies is an astounding tool to mark and view cells. However, the bioluminescent mechanism is not completely deciphered, limiting the comprehension of key processes. We use a theoretical approach to study for the first time the arrival of a dioxygen molecule inside the fireflies protein and one path of the formation of the dioxetanone ring, the high-energy intermediate precursor of the bioluminescence. To describe this reaction step, a joint approach combining classical molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations is used. The formation of the dioxetanone ring has been studied for both singlet and triplet states with the help of MS-CASPT2 calculations. We also emphasize the role played by the proteinic environment in the formation of the dioxetanone ring. The results obtained shed some light on an important reaction step and give new insights concerning the bioluminescence in fireflies.

Chemi- and Bioluminescence of Cyclic Peroxides.

Bioluminescence is a phenomenon that has fascinated mankind for centuries. Today the phenomenon and its sibling, chemiluminescence, have impacted society with a number of useful applications in fields like analytical chemistry and medicine, just to mention two. In this review, a molecular-orbital perspective is adopted to explain the chemistry behind chemiexcitation in both chemi- and bioluminescence. First, the uncatalyzed thermal dissociation of 1,2-dioxetane is presented and analyzed to explain, for example, the preference for triplet excited product states and increased yield with larger nonreactive substituents. The catalyzed fragmentation reaction and related details are then exemplified with substituted 1,2-dioxetanone species. In particular, the preference for singlet excited product states in that case is explained. The review also examines the diversity of specific solutions both in Nature and in artificial systems and the difficulties in identifying the emitting species and unraveling the color modulation process. The related subject of excited-state chemistry without light absorption is finally discussed. The content of this review should be an inspiration to human design of new molecular systems expressing unique light-emitting properties. An appendix describing the state-of-the-art experimental and theoretical methods used to study the phenomena serves as a complement.

Simulation and Analysis of the Spectroscopic Properties of Oxyluciferin and Its Analogues in Water.

Firefly bioluminescence is a quite efficient process largely used for numerous applications. However, some fundamental photochemical properties of the light emitter are still to be analyzed. Indeed, the light emitter, oxyluciferin, can be in six different forms due to interexchange reactions. In this work, we present the simulation of the absorption and emission spectra of the possible natural oxyluciferin forms in water and some of their analogues considering both the solvent/oxyluciferin interactions and the dynamical effects by using MD simulations and QM/MM methods. On the one hand, the absorption band shapes have been rationalized by analyzing the electronic nature of the transitions involved. On the other hand, the simulated and experimental emission spectra have been compared. In this case, an ultrafast excited state proton transfer (ESPT) occurs in oxyluciferin and its analogues, which impairs the detection of the emission from the protonated state by steady-state fluorescence spectroscopy. Transient absorption spectroscopy was used to evidence this ultrafast ESPT and rationalize the comparison between simulated and experimental steady-state emission spectra. Finally, this work shows the suitability of the studied oxyluciferin analogues to mimic the corresponding natural forms in water solution, as an elegant way to block the desired interexchange reactions allowing the study of each oxyluciferin form separately.

Mechanistic investigations of the 2-coumaranone chemiluminescence.

2-Coumaranones are evolving as a new, efficient, versatile, and synthetically accessible platform for the next generation chemiluminescent probes. Despite the favorable quantum yields, the exact mechanism of their chemiluminescence remains elusive. Here, we analyze the details of the mechanism of the 2-coumaranone chemiluminescence using a combination of experimental and computational methods. By using EPR spectroscopy we show that superoxide radical anions are involved in the reactions, in support of the hypothesis that the mechanism includes a single electron transfer step. The decomposition of the high-energy intermediate, 1,2-dioxetanone, is described in the ground state and in the first three excited singlet states, and indicates that there is at least one conical intersection, which is crucial for generation of excited-state molecules. A peroxy anion that is generated was found to be able to undergo a side reaction that leads to the same (isolated) product as in the light-generating reaction. These results demonstrate the applicability of 2-coumaranones as a model system for several bioluminescence reactions and may lead to the design of new 2-coumaranone derivatives with superior emission characteristics for bioanalytical applications.