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.

Alkylated green fluorescent protein chromophores: dynamics in the gas phase and in aqueous solution.

Fluorescent labelling of macromolecular samples, including using the green fluorescent protein (GFP), has revolutionised the field of bioimaging. The ongoing development of fluorescent proteins require a detailed understanding of the photophysics of the biochromophore, and how chemical derivatisation influences the excited state dynamics. Here, we investigate the photophysical properties associated with the S1 state of three alkylated derivatives of the chromophore in GFP, in the gas phase using time-resolved photoelectron imaging, and in water using femtosecond fluorescence upconversion. The gas-phase lifetimes (1.6-10 ps), which are associated with the intrinsic (environment independent) dynamics, are substantially longer than the lifetimes in water (0.06-3 ps), attributed to stabilisation of both twisted intermediate structures and conical intersection seams in the condensed phase. In the gas phase, alkylation on the 3 and 5 positions of the phenyl ring slows the dynamics due to inertial effects, while a 'pre-twist' of the methine bridge through alkylation on the 2 and 6 positions significantly shortens the excited state lifetimes. Formation of a minor, long-lived (≫ 40 ps) excited state population in the gas phase is attributed to intersystem crossing to a triplet state, accessed because of a T1/S1 degeneracy in the so-called P-trap potential energy minimum associated with torsion of the single-bond in the bridging unit connecting to the phenoxide ring. A small amount of intersystem crossing is supported through TD-DFT molecular dynamics trajectories and MS-CASPT2 calculations. No such intersystem crossing occurs in water at T = 300 K or in ethanol at T ≈ 77 K, due to a significantly altered potential energy surface and P-trap geometry.

Turning on Solid-State Fluorescence with Light.

A bioinspired fluorophore that is analogous to the substrate in the bioluminescence of fireflies was prepared and reacts when exposed to weak blue LED light. Upon excitation, this material is photodecarboxylated with a nearly 81-fold enhancement of the solid-state emission, the fluorescence quantum yield of the product in solution is approximately 90 %, and violent disintegrative effects occur as a result of the release of carbon dioxide. Crystallographic and computational results, together with global spectral analysis of the kinetics, confirmed that most of the emission observed in the decay-associated spectra is intrinsic to the product molecule, with only a minor contribution from an excimer through π-π stacking of the molecules in the crystal.

Excited-State Dynamics of Oxyluciferin in Firefly Luciferase.

The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time-resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH-dependent emission to a single chemical species would be an oversimplification.

Conjugates of Benzoxazole and GFP Chromophore with Aggregation-Induced Enhanced Emission: Influence of the Chain Length on the Formation of Particles and on the Dye Uptake by Living Cells.

Six conjugates of benzoxazole and green fluorescent protein chromophore that differ by the length of their alkyl chain (from C1 to C16) are investigated. They exhibit rigidofluorochromism and clear aggregation-induced emission enhancement (AIEE) behavior with emission in the orange-red that is specific to the solid state. A preparation method based on solvent exchange is used to prepare particles. The self-association properties of these molecules depend on the length of the alkyl chain. Microfibers, platelets, and rounded microparticles are successively obtained by increasing the chain length. The same method is used to prepare nanoparticles (NPs) that are fully characterized. In particular, homogeneous populations of stable NPs measuring around 70 nm are obtained with the analogs whose chains contain four to eight carbon atoms. The behavior with respect to living cells is also influenced by the nature of the compounds. Only the dyes with intermediate hydrophobicity are efficiently uptaken by both normal and tumor cells, and fluorescence only originates from dispersed dye molecules. There is no evidence for incorporation of NPs into cells. This work shows that small variations of the chemical structure must be taken into account for making the best use of AIEE compounds in view of precise applications.

pH-Sensitive fluorophores from locked GFP chromophores by a non-alternant analogue of the photochemical meta effect.

We report the synthesis and characterization of a pH-sensitive fluorescence switch based on a conformationally-locked green fluorescent protein (GFP) chromophore. The chromophore differs from difluoroboryl-locked parent by the addition of a titratable alcohol group on the imidazolinone ring. The chromophore is fluorescent at pH ≤ 5, but becomes non-fluorescent at higher pH, where the substituent is ionized. We use a quantum chemical model to show that the mechanism of the fluorescence turn-off is electronically analogous to photochemical meta effects in aryl-containing systems.

Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime.

Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

Hidden photoinduced reactivity of the blue fluorescent protein mKalama1.

Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins, which often results in population of kinetically trapped dark states of various origins, is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however, its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative electronic relaxation. Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range (ca. 300-800 nm); the disappearance of the transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events, followed by deprotonation of the chromophore radical cation to bulk solvent through a novel water-mediated proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent proteins as tracked by its optical transient absorption in the time domain extending up to seconds.

Novel mechanism of bioluminescence: oxidative decarboxylation of a moiety adjacent to the light emitter of Fridericia luciferin.

A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.

Red-shifted fluorescent aminated derivatives of a conformationally locked GFP chromophore.

A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red-shifted spectra, high fluorescence quantum yields and pH-independence in physiological pH range. The intra- and intermolecular mechanisms of radiationless deactivation of ABDI-BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with "infinite" photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.

Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Collapse and recovery of green fluorescent protein chromophore emission through topological effects.

Housed within the 11-stranded ß-barrel of the green fluorescent protein (GFP) is the arylideneimidazolidinone (AMI) chromophore, the component responsible for fluorescence. This class of small-molecule chromophore has drawn significant attention for its remarkable photophysical and photochemical properties, both within the intact protein and after its denaturation. All of the proteins so far isolated that have visible light fluorescence have been found to contain an AMI chromophore. These proteins comprise an extensive rainbow, ranging from GFP, which contains the simplest chromophore, p-hydroxybenzylideneimidazolidinone (p-HOBDI), to proteins having molecules with longer conjugation lengths and a variety of intraprotein interactions. The fluorescence invariably almost vanishes upon removal of the protective ß-barrel. The role of the barrel in hindering internal conversion has been the subject of numerous studies, especially in our laboratories and those of our collaborators. A better understanding of these chromophores has been facilitated by the development of numerous synthetic protocols. These syntheses, which commonly use the Erlenmeyer azlactone method, have evolved in recent years with the development of a [2 + 3] cycloaddition exploited in our laboratory. The synthetic AMI chromophores have allowed delineation of the complex photophysics of GFP and its derivatives. Upon denaturation, AMI chromophores are marked by 4 orders of magnitude of diminution in emission quantum yield (EQY). This result is attributed to internal conversion resulting from conformational freedom in the released chromophore, which is not allowed within the restrictive ß-barrel. To date, the photophysical properties of the AMI chromophore remain elusive and have been attributed to a variety of mechanisms, including cis-trans isomerization, triplet formation, hula twisting, and proton transfer. Advanced studies involving gas-phase behavior, solvent effects, and protonation states have significantly increased our understanding of the chromophore photophysics, but a comprehensive picture is only slowly emerging. Most importantly, mechanisms in structurally defined chromophores may provide clues as to the origin of the "blinking" behavior of the fluorescent proteins themselves. One approach to examining the effect of conformational freedom on rapid internal conversion of the chromophores is to restrict the molecules, both through structural modifications and through adjustments of the supramolecular systems. We thus include here a discussion of studies involving the crystalline state, inclusion within natural protein-binding pockets, complexation with metal ions, and sequestration within synthetic cavities; all of this research affirms the role of restricting conformational freedom in partially restoring the EQY. Additionally, new photochemistry is observed within these restricted systems. Many of the studies carried out in our laboratories show promise for these molecules to be adapted as molecular probes, wherein inclusion turns on the fluorescence and provides a signaling mechanism. In this Account, we present an overview of the AMI chromophores, including synthesis, overall photophysics, and supramolecular behavior. A significant amount of work remains for researchers to fully understand the properties of these chromophores, but important progress achieved thus far in photophysics and photochemistry is underscored here.

Microcrystals with enhanced emission prepared from hydrophobic analogues of the green fluorescent protein chromophore via reprecipitation.

Certain synthetic analogues of the green fluorescent protein (GFP) chromophore are almost nonfluorescent in dilute solutions but are strongly light-emissive in the solid state, thus exhibiting aggregation-induced emission (AIE) behavior. In the present work, two such hydrophobic derivatives of the GFP chromophore known to be fluorescent in the crystalline state (p-hexyloxy- and p-dodecyloxybenzylideneimidazolinone) were used to prepare aqueous suspensions of particles via a mild solvent-exchange reprecipitation (RP) method. This evolution was monitored at various experimental conditions by UV-vis absorption and fluorescence spectroscopy, fluorescence microscopy, as well as electron transmission and scanning microscopy. Both compounds spontaneously produced platelet-like microcrystals, the size and shape of which were influenced by the experimental conditions. The dodecyl derivative also led to the concomitant formation of nanofibers, a tendency reinforced by addition of poly(acrylic acid) to the RP medium. The photoluminescence properties of the solids produced by RP were compared to pristine microcrystalline powders obtained by crystallization in an organic solvent. Significant differences in the emission properties were found and are discussed. This study illustrates the fact that AIE fluorescence is strongly dependent on the nature of the particles and hence on the preparation methods. Being aware of these variations is important for the preparation and subsequent use of AIE-active compounds as fluorescent materials.

Engineering the nanoscale morphology of a quantum dot-fullerene assembly via complementary hydrogen bonding interactions.

We have demonstrated controlled assembly between CdSe quantum dots (QDs) and a fullerene (C60) derivative via complementary three-point hydrogen bonding interactions. The recognition-mediated assembly facilitated an interpenetrated network morphology and hence efficient charge transfer from QD to C60.

Self-assembled benzophenone bis-urea macrocycles facilitate selective oxidations by singlet oxygen.

This manuscript investigates how incorporation of benzophenone, a well-known triplet sensitizer, within a bis-urea macrocycle, which self-assembles into a columnar host, influences its photophysical properties and affects the reactivity of bound guest molecules. We further report the generation of a remarkably stable organic radical. As expected, UV irradiation of the host suspended in oxygenated solvents efficiently generates singlet oxygen similar to the parent benzophenone. In addition, this host can bind guests such as 2-methyl-2-butene and cumene to form stable solid host-guest complexes. Subsequent UV irradiation of these complexes facilitated the selective oxidation of 2-methyl-2-butene into the allylic alcohol, 3-methyl-2-buten-1-ol, at 90% selectivity as well as the selective reaction of cumene to the tertiary alcohol, α,α'-dimethyl benzyl alcohol, at 63% selectivity. However, these products usually arise through radical pathways and are not observed in the presence of benzophenone in solution. In contrast, typical reactions with benzophenone result in the formation of the reactive singlet oxygen that reacts with alkenes to form endoperoxides, diooxetanes, or hydroperoxides, which are not observed in our system. Our results suggest that the confinement, the formation of a stable radical species, and the singlet oxygen photoproduction are responsible for the selective oxidation processes. A greater understanding of the mechanism of this selective oxidation could lead to development of greener oxidants.

Photoinduced dynamics of oxyluciferin analogues: unusual enol "super"photoacidity and evidence for keto-enol isomerization.

The first systematic pico-nanosecond time-resolved spectroscopic study of the firefly emitter oxyluciferin and two of its chemically modified analogues revealed that in the excited state the enol group is more acidic than the phenol group. The 6'-dehydroxylated derivative, in which only the 4-enolic hydroxyl proton is acidic, has an experimentally determined pK(a)* of 0.9 in dimethyl sulfoxide and an estimated pK(a)* of -0.3 in water. Moreover, this compound provided direct evidence that in a nonpolar, basic environment the keto form in the excited state can tautomerize into the enol, which subsequently undergoes excited-state proton transfer (ESPT) to produce enolate ion. This observation presents the first experimental evidence of excited-state keto-enol tautomerization of a firefly fluorophore, and it could be important in resolving the enol-keto conundrum related to the color-tuning mechanism of firefly bioluminescence. The 6'-dehydroxylated form of oxyluciferin adds a very rare case of a stable enol to the family of "super"photoacids.

Conformationally locked chromophores as models of excited-state proton transfer in fluorescent proteins.

Members of the green fluorescent protein (GFP) family form chromophores by modifications of three internal amino acid residues. Previously, many key characteristics of chromophores were studied using model compounds. However, no studies of intermolecular excited-state proton transfer (ESPT) with GFP-like synthetic chromophores have been performed because they either are nonfluorescent or lack an ionizable OH group. In this paper we report the synthesis and photochemical study of two highly fluorescent GFP chromophore analogues: p-HOBDI-BF2 and p-HOPyDI:Zn. Among known fluorescent compounds, p-HOBDI-BF(2) is the closest analogue of the native GFP chromophore. These irrreversibly (p-HOBDI-BF(2)) and reversibly (p-HOPyDI:Zn) locked compounds are the first examples of fully planar GFP chromophores, in which photoisomerization-induced deactivation is suppressed and protolytic photodissociation is observed. The photophysical behavior of p-HOBDI-BF2 and p-HOPyDI:Zn (excited state pK(a)'s, solvatochromism, kinetics, and thermodynamics of proton transfer) reveals their high photoacidity, which makes them good models of intermolecular ESPT in fluorescent proteins. Moreover, p-HOPyDI:Zn is a first example of "super" photoacidity in metal-organic complexes.

Excited-state proton transfer in N-methyl-6-hydroxyquinolinium salts: solvent and temperature effects.

The excited-state proton transfer (ESPT) reaction of the "super"photoacid N-methyl-6-hydroxyquinolinium (MHQ) was studied using both fluorescence upconversion and time-correlated single photon counting (TCSPC) techniques. The ultrafast ESPT kinetics were investigated in various alcohols and water and determined to be solvent-controlled. The ESPT temperature dependence of MHQ was also studied in various alcohols and compared to that observed for another "super"photoacid, 5,8-dicyano-2-naphthol (DCN2). A full set of kinetic and thermodynamic parameters describing the ESPT was obtained. The protolytic photodissociation rate constant for MHQ was higher than that for DCN2, while the ESPT activation energies of MHQ were smaller. These findings are attributed to the approximately 3 orders of magnitude differences in excited-state acidities of MHQ and DCN2.

Hydroxydialkylamino cruciforms: amphoteric materials with unique photophysical properties.

Two amphoteric cruciforms 6 and 7 (XF; 4,4'-[(1E,1'E)-(2,5-bis{[4-(dibutylamino)phenyl]ethynyl}-1,4-phenylene)bis(ethene-2,1-diyl)]diphenol, 4,4'-[{2,5-bis[(E)-4-(dibutylamino)styryl]-1,4-phenylene}bis(ethyne-2,1-diyl)]diphenol) were prepared by a Horner reaction followed by a Sonogashira coupling and subsequent deprotection. The XFs display significant changes in absorption and emission when exposed to trifluoroacetic acid, tetrabutylammonium hydroxide, and metal triflates. The substitution pattern of 6 and 7 leads to spatial separation of the frontier molecular orbitals, which allows the HOMO or LUMO of the XF to be addressed independently by acidic or basic agents. XF 6, which has hydroxyl groups on the styryl axis, displays changes in emission color upon exposure to ten amines in eight different solvents. The change in fluorescence upon the addition of amines was analyzed by linear discriminant analysis. These XFs may have potential in sensor applications for metal cations and amines.