Mechanistic Insight into Cypridina Bioluminescence with a Combined

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Mechanistic Insight into Cypridina Bioluminescence with a Combined Experimental and Theoretical Chemiluminescent Approach Luís Pinto da Silva, Rui F.J. Pereira, Carla M. Magalhães, and Joaquim C.G. Esteves da Silva J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06295 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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The Journal of Physical Chemistry

Mechanistic Insight into Cypridina Bioluminescence with a Combined Experimental and Theoretical Chemiluminescent Approach Luís Pinto da Silva,†‡* Rui F.J. Pereira,† Carla M. Magalhães,† Joaquim C.G. Esteves da Silva‡§ †

Chemistry Research Unit (CIQUP), Department of Chemistry and Biochemistry, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. ‡

LACOMEPHI, Department of Geosciences, Environment and Territorial Planning, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. §

Chemistry Research Unit (CIQUP), Department of Geosciences, Environment and Territorial Planning, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. Supporting Information Placeholder ABSTRACT: The bioluminescent reaction of the “sea firefly”

Cypridina hilgendorfii is a prototypical system for marine bioluminescence, as its substrate possesses an imidazopyrazinone core that is a common link among organisms of eight phyla. The elucidation of the mechanism behind Cypridina bioluminescence is essential for future applications in bioimaging, biomedicine and bioanalysis. In this study we have investigated the key step of chemiexcitation with a combined experimental and theoretical approach. The obtained results indicate that neutral dioxetanone is responsible for efficient chemiexcitation, as the thermolysis of this species gives access to a long region of the potential energy surface (PES), where the ground and excited singlet states are degenerated. Contrary to expected, neither Chemically Induced Electron-Exchange Luminescence (CIEEL) nor Charge TransferInitiated Luminescence (CTIL) can be used to explain imidazopyrazinone-based bioluminescence, as there is no clear relationship between electron (ET)/charge (CT) transfer (occurring between the electron-rich moiety and dioxetanone) and chemiexcitation. Attractive electrostatic interactions between the CO2 and oxyluciferin moieties allow neutral dioxetanone to spend time in the PES region of degeneracy, while repulsive interactions for anionic dioxetanone leads to a quicker CO2 detachment.

1. INTRODUCTION Bioluminescence is a beautiful and amazing natural phenomenon in living organisms, which consists on the conversion of thermal energy into light emission.1-4 Bioluminescent reactions have attracted significant attention from the research community due to their high bioluminescent quantum yields,5,6 relative nontoxicity of luciferins, and high signal-to-noise ratio.7,8 Furthermore, bioluminescence does not require light excitation, and so, there is no autofluorescence arising from background signal.9 The lack of light excitation also eliminates the problems associated with light-penetration into biologic tissues, except in light emission.10 These characteristics make these systems a very powerful tool in medical and life sciences, by allowing the real-time and noninvasive imaging of target molecules in vivo.11-14 These systems have also been explored as intracellular excitation sources for self-illuminating photodynamic therapy of cancer.10

Scheme 1 - Reaction mechanism of Cypridina bioluminescence. There are more than 700 genera able to produce bioluminescence, with the most well-known being that of fireflies,2,6,15-19 but being also found in fungi, earthworms, fishes and bacteria, among others.1,3,6,20-22 Nevertheless, about 80% of all luminescent organisms reside the ocean!23,24 Cypridina hilgendorfii, also known as sea firefly, is one of the simplest marine organisms, with a body

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length of only 2-3 mm.25-27 This organism emits bioluminescence with an emission maximum at 465 nm, in a reaction requiring only molecular oxygen, the luciferase enzyme and Cypridina luciferin (Scheme 1).26 Unlike other bioluminescent reactions,28 this system does not require any cofactor, which simplify its use in bioanalysis and bioimaging. Another noteworthy characteristic of this system is its high quantum yield of 0.28.29,30 Due to these characteristics, the Cypridina system is already a powerful and indispensable tool in enzyme immunoassays,31 imaging,32,33 in two-photon fluorescent probing,34, immunohistology,35 energy transfer assays,36 and as a gene reporter,37 among others. The Cypridina bioluminescent reaction has been described as follows (Scheme 1):38-40 first occurs the oxygenation of the imidazopyrazinone scaffold of Cypridina luciferin, which rapidly forms a dioxetanone intermediate; the second step is the thermolysis of dioxetanone, which allows for a thermally-activated ground state (S0) reaction to produce a reaction product (oxyluciferin) in its first singlet excited state (S1). The Cypridina bioluminescent mechanism is the same for many other marine organisms, as the imidazopyrazinone scaffold of Cypridina luciferin is a common link among marine luciferins. In fact, imidazopyrazinone is also present in Coelenterazine, the luciferin molecule of the majority of marine bioluminescent species: decaopod shrimp Oplophorus gracilirostris, anthozoan Renilla reniformis, copecods Gaussia princeps and Metridia longa, jellyfish Aequorea victoria and the hydrozoan Obelia longissimi, among others.3,4,41 Other known imidazopyrazinone-based substrates are Watasenia luciferin (found in the squid Watasenia scintillans),42 and dehydrocoelenterazine (found in the squid Symplectoteuthis oualaniensis).43 Thus, Cypridina bioluminescence can be considered a prototypical system for the majority of marine bioluminescent system, which in turn encompass about 80% of all luminescent organisms.23,24 So, the detailed elucidation of Cypridina bioluminescence will allow to explain the bioluminescence of other marine systems. Particularly of importance is the characterization of the thermolysis reaction of the dioxetanone intermediate, as it is this reaction that allows for chemiexcitation of oxyluciferin.1,2,27 Dioxetanones are responsible for chemiexcitation not only in imidazopyrazinone-based systems, but also for fireflies, earthworms and Latia.1,2 Dioxetanones are also present in chemiluminescent systems, such as luminol, acridinium esters and AMPPD.44,45 The first explanation provided for the efficient generation of S1 states was provided by the Chemically Induced Electron-Exchange Luminescence (CIEEL) mechanism.46,47 This mechanism is based on an electron transfer (ET) from an oxidazable electron-rich moiety to the peroxide with formation of a radical ion pair. Back ET (BET) leads to S1 chemiexcitation due to charge annihilation. However, re-examination of the quantum yields of prime CIEEL examples has put this mechanism into question.48,49 These studies revealed that these systems have quantum yields significantly lower than the ones reported when the CIEEL mechanism was first proposed.46-49 These conclusions were supported by the study of polyacene endoperoxide and two different dioxetanones.49 Given this failure, different authors have tried to modernize the CIEEL mechanism.27,44,50-53 Now, there is neither full ET and BET processes, nor radical ion pairs. Instead, the generation of S1 states is explained by gradual charge transfer (CT) and back CT (BCT) between an ionized electron-rich moiety and the cyclic peroxide, leading to the development of Charge Transfer-Initiated Luminescence (CTIL) mechanisms.27,44,50-53 It should be noted that dioxetanone derivatives, as Cypridina dioxetanone, decompose within two kinds of forms: neutral and anionic (Scheme 1).1,2,27,44,50-56 However, only the thermolysis of the anion has been found to follow the CTIL mechanism, with CT and BCT occurring in concert with O1-O4 and C2-C3 bond breaking, respectively.27,44,50-56 Protonation leads to minimal CT and ET

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processes, with thermolysis proceeding via homolytic cleavage of O1-O4.27,44,50-56 In the case of Cypridina bioluminescence, and related imidazopyrazinone-based systems, this has led to the identification of anionic dioxetanone as the responsible for efficient chemiexcitation.27,51 However, this identification does not correlate so well with experimental and even theoretical results. One study has provided indirect evidence that Cypridina luminescence is the product of chemiexcitation from a neutral dioxetanone.57 Similar conclusions were also obtained for firefly bioluminescence.58 Theoretical calculations using density functional theory (DFT) approaches have attributed a more efficient S1 chemiexcitation pathway to neutral dioxetanones than to anionic ones.27,51,55,56 This results from neutral dioxetanones having access to a large and flat region of the PES where S0 and S1 are degenerate/near-degenerate. This region of the PES is only accessed with limited CT/ET, and not by ionized dioxetanones.27,51,55,56 The presence of this degeneracy region in the thermolysis of neutral dioxetanones, and its absence for anionic ones, has been confirmed by higher-level multireference calculations.50,59,60 Other authors have also attributed the efficient chemiexcitation to neutral dioxetanones instead of anionic ones, based on experimental characterization of chemiluminescent imidazopyrazinones.38-40 Finally, it should be said the proposal of anionic dioxetanone has been based on energetic reasons.27,50-53 That is, after determining that both neutral and anionic dioxetanones lead to chemiexcitation, these authors identified the ones with the lowest activation energy (the anionic species) as the responsible for chemiexcitation. However, this approach is insufficient for several reasons. First of all, no experimental values are available for the thermolysis of imidazopyrazinone-based dioxetanones to serve as reference. Secondly, the calculations presented so far were made outside of the enzyme (a biological catalyst), which means that the obtained energies for each species are not necessarily the same as the ones obtained in bioluminescence. Finally, the activation energies calculated for the neutral species27,51,55,56 are in line with experimental activation energies obtained for other dioxetanes and dioxetanones (about ~20 kcal mol-1).61-63 Given all of this, it is evident that the chemiexcitation mechanism of Cypridina bioluminescence and other imidazopyrazinonebased systems, is very far from understood. Part of this lack of clarity stems from the instability of the dioxetanone molecule, which limits its experimental characterization. Theoretical calculations can circumvent this limitation and provide detailed insight into the thermolysis reaction of dioxetanone. However, the lack of experimental data that can be used as reference difficult the interpretation of the theoretical results. Herein, the chemiexcitation mechanism of Cypridina bioluminescence was characterized by means of a combined experimental and theoretical approach. The energy and kinetic profiles of this reaction were obtained with luminometric and spectroscopic methods. The thermolysis and chemiexcitation mechanisms of Cypridina were investigated with a reliable density functional (DFT). This approach allowed us to rationalize the bioluminescence of the Cypridina system and related imidazopyrazinone-based ones, which can be found in about eight phyla of luminescent organisms. 2. MATERIAL AND METHODS 2.1. Experimental Methods Cypridina luciferin was purchased from NanoLightTM Technology, and was dissolved in methanol and stored at -20ºC. Kinetic chemiluminescent assays were performed in a homemade luminometer using a Hamamatsu HC135-01 photomultiplier tube. All reactions took place at ambient temperature (24-27 ºC) and were performed at least in sextuplicate. All light reactions were carried


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out in DMSO, to which was added acetate buffer pH 5.12, phosphate buffer pH 7.4 (0.075 M) or NaOH (0.1 M). The reaction was initiated by the injection of the DMSO solution (400 µL) into an assay tube containing Cypridina luciferin (30 µL). The concentration of Cypridina luciferin in the final solution is of 4.3 µM. The light was integrated and recorded in 0.1 s intervals. Resulting data were analyzed by nonlinear regression analysis, by using the Graphpad software package (Version 7.03 for Windows). Chemiluminescence and fluorescence spectra were measured with a Horiba Jovin Yvon Fluoromax 4 spectrofluorometer, with an integration time of 0.1 s. For obtaining the chemiluminescence spectra (due to lower intensity), were used slit widths of 29 nm for the emission monochromators. For fluorescence spectra were used slit widths of 5 nm for both the excitation and emission monochromators. Quartz cells were used. The spectra were obtained with a concentration of 12.3 µM for Cypridina luciferin, in solutions with a final volume of 500 µL. 2.2. Theoretical Methods For a compromise between reliable results and available computational power, we have substituted the large and flexible R2 and R3 moieties of Cypridina dioxetanone (seen in Scheme 1), with methyl groups. This type of substitution is reasonable and retain the innate properties of this system, as confirmed before.27,39 S0 geometry optimizations and frequency calculations for the neutral and anionic forms of the model dioxetanones were performed at the ωB97XD/6-31G(d,p) level of theory,64 with a closed-shell (R) approach for the reactants and an open-shell (U) one for transition states (TS). The U approach was used with a broken-symmetry (BS) technology, which mixes the HOMO and LUMO, making an initial guess for a biradical.27,51,55,56 Intrinsic reaction coordinates (IRC) were carried out in order to assess if the obtained TS connect the desired reactants and products.65 The energies of the S0 IRC-obtained structures were re-evaluated by single-point calculations at the ωB97XD/6-31+G(d,p) level of theory. The S1 state was calculated by using the time-dependent (TD) DFT approach,66 at the TD ωB97XD/6-31+G(d,p) level of theory. ωB97XD is a long-range-corrected hybrid exchangecorrelation functional, which provides quite good estimates for π → π*, n → π* local excitation, and CT and Rydberg states.67 Long-range-corrected hybrid exchange-correlation DFT methods have proven to provide accurate qualitative pictures for dioxetanone systems.27,51,55,56 Moreover, ωB97XD has already been used in the study of other imidazopyrazinone-based dioxetanones, which facilitates the comparison of results here obtained with previous work.55,56 Geometry optimizations, frequency and IRC calculations were made in vacuo, while single-point calculations were made in implicit DMSO. This was achieved with the Polarizable Continuum Model using the integral equation formalism variant (IEFPCM).68 All calculations were made with the Gaussian 09 program package.69 3. RESULTS AND DISCUSSION 3.1. Chemiluminescence of Cypridina Luciferin in DMSO While the objective of this work is the characterization of Cypridina bioluminescence, working with enzymes is particularly expensive, complex and time-consuming. Moreover, the use of the luciferase enzyme in the experimental assays would disrupt the comparison with the results obtained in theoretical study of the model Cypridina dioxetanones. To overcome these problems we have studied Cypridina chemiluminescence as a model for the bioluminescent reaction, which is a common and reliable approach.38-40,57,70-72 Cypridina luciferin also chemiluminesces in aprotic solvents (as DMSO) without an enzyme, in an identical

mechanism to that of the bioluminescent reaction.38-40,70-72 The major difference between the chemiluminescent and bioluminescent reactions is that the latter process leads to a higher quantum yield, which is to be expected due to the present of the catalytic luciferase.38-40,70-72 The kinetic assays for Cypridina chemiluminescence were then performed in DMSO, which has been used as a prototypical solution for the study of imidazopyrazinone compounds.38-40,72 The chemiluminescent kinetics were followed by the measurement of direct light-emission intensity, in different pH. The kinetic curves gave rise to rate constants (k, in s-1) and to the plateau of light intensity (in relative light units, RLU),73,74 which corresponds to the value of light intensity at infinite times. These values were obtained by exponential fitting. Table 1 - Rate constants (k, in s-1) and plateau of light intensity (normalized values) of Cypridina chemiluminescence in DMSO, with addition of either phosphate buffer pH 7.4 or NaOH (0.1M). Phosphate pH 7.4

buffer

NaOH (0.1M)

k

0.30 ± 0.06

1.59 ± 0.09

Plateau

1.00 ± 0.16

0.23 ± 0.03

The plateau and k values were initially obtained in neutral (with phosphate buffer pH 7.4) and basic (with 0.1 M NaOH solution) pH, and can be found in Table 1. These results indicate that light emission is five times higher in neutral pH, but the reaction is slower. These results are supported by further assays in acidic and basic media (Figures 1.a and 1.b, and Table S1). Acidic media was achieved by addition of acetate buffer pH 5.12 to DMSO, in different concentrations (0.1%, 0.08% and 0.04%). A basic media was once again achieved with the addition of NaOH to DMSO, in different concentrations (0.01, 0.05 and 0.1 M). It should be noted that the approach of achieving acidic and basic media with different percentage of acetate buffer and concentration of NaOH (respectively), was used to better compare with previous chemiluminescent studies.38-40,72 The plateau values (light intensity at infinite times) decrease significantly with increasing pH. In fact, the plateau at 0.1 M of NaOH is only 5% of the plateau found when acetate buffer (0.1%) was added to DMSO. For the contrary, the reaction rate increases significantly with increasing pH, between 0.65±0.01 s-1 and 1.51±0.10 s-1. Thus, our experiments indicate that the velocity of the chemiluminescent reaction and the light intensity are both pH-dependent. However, these two parameters present an opposite behavior: while the reaction rate is higher with increasing pH, light intensity increases with decreasing pH. The pH-dependence of Cypridina light intensity is not entirely a new finding. It was already reported that appreciable chemiluminescent yields (ca. 10% of the bioluminescent yield) for Cypridina-related imidazopyrazinones were only found in acidic solutions containing acetate buffer, where solutions containing NaOH (0.1 M) presented only negligible chemiluminescence.38-40 However, these measurements were made in different solvents: acidic medium consisted on diglyme (a non-polar solvent) solutions, while basic media consisted on DMSO (a polar solvent) solutions.38-40 Thus, it was not clear if this difference in quantum yields was due to the pH, the polarity of the environment, or to a combination of both factors. While both the reaction rate and the plateau value are both affected by the pH, it is not clear if this pH-dependence is caused by the same mechanism, or if are unrelated processes. One way for the light intensity be affected, without the reaction rate being necessarily altered, is if the light emitting species is not the same in different pH as each species is characterized by a different



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fluorescence quantum yield (ɸF).1,2 Cypridina oxyluciferin, as the respective dioxetanone intermediate, is present in either a neutral and anionic (amide) form (Scheme 1).27,57 To analyze the validity of this hypothesis, we have obtained the chemiluminescent spectra (Figure 1.c) for Cypridina light emission in DMSO solutions at different pH: acetate buffer pH 5.12 (0.68%), phosphate buffer pH 7.4, and NaOH (0.1M). Contrary to our previous hypothesis, the results indicate that it is the same oxyluciferin species to chemiluminesce in different pH. Only one emission peak was found, with an emission wavelength at ~460 nm. The major difference is the emission intensity of each spectra, which decreases significantly with increasing pH (the same trend as for the plateau values found before). It should be noted that the chemiluminescent wavelength maximum here found (~460 nm) is very similar to the one found for Cypridina bioluminescence (465 nm),26 which supports the validity of using chemiluminescence as a model for the corresponding bioluminescent reaction. Moreover, this similarity indicates that it is the same species to emit chemiluminescence and bioluminescence. In order to determine if the chemiluminescence can be attributed to either the neutral or amide Cypridina oxyluciferin, we have calculated the fluorescent emission spectra of the anionic and neutral forms of a Cypridina oxyluciferin derivative (Figure S1.a). This was made by using a state-specific (SS) TD-DFT approach.75 Some authors have stated that for imidazopyrazinone-based compounds, the chemiluminescent state is different than the fluorescent one.76,77 However, subsequent studies showed that the chemiluminescent state is a “dark” state that needs to evolve to a bright fluorescent state.41,78 This is supported by our comparison between the chemiluminescent and fluorescent spectra here performed (Figure S1.b), which show peaks with identical emission maxima. Our calculations indicate that the chemiluminescent species is the neutral species (Table S2). Its theoretical emission energy (2.87 eV) only differs 0.17 eV from the experimental value (2.70 eV), which is well within the type error attributed for TD-DFT calculations (0.20 eV). By its turn, the amide species (2.93 eV) differs from the experimental value by 0.24 eV. Moreover, the oscillator strength of the neutral species is higher than for the amide species (0.29 vs. 0.16). The identification of the neutral species as the chemiluminophore is in line with previous experimental results.57 In summary, our experimental study demonstrated that both the reaction rate and light intensity of Cypridina chemiluminescence are pH-dependent in DMSO. However, while the reaction rate increases with increasing pH, light intensity is inversely proportional to pH. Moreover, the effect of pH on the light intensity is not related to ɸF, as it is the same species (neutral oxyluciferin) to emit in acidic and basic pH. The increase of reaction rate with pH indicates that the activation barrier of the chemiluminescent reaction decreases with increasing pH. As already noted, this chemiluminescent reaction consists mainly of two major steps (oxygenation and thermolysis),38-40 which then determinate the global activation barrier of chemiluminescence. Oxygenation can explain some of the variation of the reaction rate as a function of pH as this step is expected to involve the deprotonation of the imidazopyrazinone scaffold.38-40,51 As the deprotonation should be slower in acidic pH, the reaction rate should be affect. However, while the oxygenation step might affect in this way the speed of the reaction, there is no obvious reason for it to affect the light intensity at infinite times, as a slower reaction should only delay reaching the plateau and not decrease it. Even if an oxygenation step with high energy barrier at acidic pH (and low barrier at basic pH) effectively affected the plateau, it should lead to higher values of light intensity with increasing pH and not to the opposite. Given this, the answer for this problem should be in the other step in the chemiluminescent reaction: the thermolysis of diox-

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etanone.1,2,27,44,50-56 This reaction is the one responsible for chemiexcitation. Moreover, dioxetanone can co-exist in its neutral and anionic forms, and theoretical studies have indicated that these two species are characterized by different activation barriers and chemiexcitation processes.27,44,50-56 Thus, if neutral dioxetanone is responsible for chemiexcitation at acidic pH and the anionic species at basic pH, that could explain the pH-dependent reaction rate and light intensity value.

Figure 1 -Rate constants (k, in s-1) at acidic (by addition of acetate buffer pH 5.12) and basic (by addition of NaOH) pH in DMSO solutions (a). Plateau of light intensity (normalized values) at acidic (by addition of acetate buffer pH 5.12) and basic (by addition of NaOH) pH in DMSO solutions (b). Chemiluminescence spectra in DMSO (c). 3.2. Theoretical Study of the Thermolysis and Chemiexcitation of Cypridina Dioxetanone In order to test this hypothesis we have tried to characterize the thermolysis and chemiexcitation of both neutral and anionic Cypridina dioxetanone. Given that the known instability of dioxetanone has prevented its experimental characterization, we have employed a TD-DFT approach to overcome this problem. Calculations with TD-DFT methods has proven to be a reliable approach for the study of these systems.27,51-56 First we have calculated the activation energies of both the neutral and anionic dioxetanone models described in the Theoretical Methods section (Scheme 1, R2=R3=CH3), which can be found in Table 2. The activation energies were also calculated for two


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more dioxetanone models (Scheme 1; R2=R3=H; R2=H and R3=CH3). As in line with previous studies, neutral and anionic dioxetanones present significantly different activation energies.27,44,50-56 More importantly, the neutral dioxetanone presents a higher activation barrier (~22 kcal mol-1) than its anionic form (~10 kcal mol-1), which is in line with the experimental behavior of the reaction rate, as a function of pH (Figures 1.b and 1.c, and Table S1). Moreover, the activation energies for the neutral species are in line with the activation parameters for other dioxetanones and dioxetanes (~20 kcal mol-1).61-63 The calculation of (S2) values indicate the involvement of a biradical species in the thermolysis reaction of both anionic and neutral dioxetanones.27,44,50-56 Analysis of the behavior of both O1-O4 and C2C3 (Figures S2.a and S2.b) shows that both species decompose via a stepwise-biradical mechanism, as in line with previous reports.27,44,50-56

with what was observed in the study of other imidazopyrazinonebased dioxetanones.27,51,55,56 Having said this, it does appear that is indeed the thermolysis reaction of Cypridina dioxetanone to explain the pH-dependence of the chemiluminescent reaction. While neutral dioxetanone presents the more efficient chemiexcitation pathway, it also presents the higher activation barrier. This is in line with the experimental results here obtained, in which the light intensity is higher at acidic pH and lower at basic pH. By the contrary, the reaction rate is lower at acidic pH and higher at basic pH. These results correlate well with chemiexcitation at acidic pH resulting from the thermolysis of neutral dioxetanone, and at basic pH chemiexcitation being the result of the thermolysis of the anionic species.

Table 2 - Activation energies, with thermal corrections, (in kcal mol-1) for the thermolysis reaction of neutral and anionic model Cypridina dioxetanones (Scheme 1), at the ωB97XD/631+G(d,p) level of theory in DMSO. The R1 substituent is an indole moiety in all models (Scheme 1). Model Molecule

Neutral Species

Anionic Species

R2=R3=H

22.0

10.6

R2=H and R3=CH3

22.9

9.6

R2=R3=CH3

21.3

10.4

In Figures 2.a and 2.b are presented the potential energy curves of S0 and S1 states, as a function of intrinsic reaction coordinates, of model dioxetanone (Scheme 1, R2=R3=CH3). According to the curves, the S1 state of the neutral species becomes closer to the S0 state in the biradical region, generated by O1-O4 elongation. In fact, S1 and S0 appear to become nearly-degenerated, with an energy gap between 10.5 and 12.8 kcal mol-1 during a large portion of the thermolysis reaction (Figure 2.a). It should be noted that previous studies indicate that multi-reference calculations will surely predict smaller energy gaps between S0 and S1 states.27,44,5056,59,60 This results from the role of multi-reference correlation in these systems,27,44,50,51,59,60 which implies that S0 and S1 become nearly degenerate in the biradical region, thus allowing for chemiexcitation. The energetic error present in the biradical region may come from spin contamination in the reference state, introduced by a BS technology.27,51 However, while multireference methods are obviously important to obtain quantitative pictures for these systems, they are too computationally demanding to employ on situations where solvation and more structurally complex systems are involved. Thus, we have employed a longrange-corrected density functional (ωB97XD), which performs well for π → π*, n → π* local excitation, and CT and Rydberg states.67 This type of approach can provide quite good qualitative pictures for dioxetanone-based systems.27,44,51-56 This is the case here, as the potential energy curves of S0 and S1 states are in line with was found for different imidazopyrazinone-based dioxetanones.27,51,55,56 The potential energy curves (Figure 2.b) show a less efficient chemiexcitation pathway for the anionic species. There is no long region where S1 and S0 are nearly-degenerated. In fact, except two reaction points in which the two states differ only by 15.2 and 13.9 kcal mol-1, the S1-S0 energy gap is always higher than ~17 kcal mol-1. This contrasts with the S1-S0 energy gap between 10.5 and 12.8 kcal mol-1 during a large portion of the thermolysis reaction of the neutral species (Figure 2.a). This high gap is in line

Figure 2 - Potential energy curves of S0 and S1 states, as a function of intrinsic reaction coordinates, of model (Scheme 1, R2=R3=CH3) neutral (a) and anionic (b) dioxetanone. These results also provide valuable insight into the bioluminescent mechanism of the Cypridina system (and related imidazopyrazinones). These results point to neutral dioxetanone as the responsible for efficient light emission, and not the anionic species, due to its more efficient chemiexcitation pathway. Moreover, the fact that different reaction rates were found at acidic and basic pH, which can be correlated with the present of neutral and anionic dioxetanone (respectively), indicates that the higher activation barrier for the neutral dioxetanone is not a detriment for the bioluminescent reaction. This indicates that the identification of the anionic species as the responsible for efficient chemiexcitation, made by some authors based on energetic reasons,27,50-53 has limited validity. It should be noted that while our study indicate that neutral dioxetanone is present in acidic pH, bioluminescent assays are generally made in neutral/basic pH. However, enzymatic active sites are considered to be hydrophobic microenvironments (dielectric constants between ~2 and ~4).79,80 As more hydrophobic environments are known to lower acidity, proteins and enzymes can lead to significant shifts in pKa.81-83 Thus, it is



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expected that the active site of firefly luciferase shifts the pKa of Cypridina dioxetanone, and prevents its deprotonation.

Figure 3 - Atomic Mulliken charge of the CO2 and oxyluciferin moieties of anionic (a) and neutral (b) model Cypridina dioxetanone (Scheme 1, R2=R3=CH3), as a function of intrinsic reaction coordinates. The next of this study was then to try to understand the mechanism behind the chemiexcitation step of Cypridina chemi/bioluminescence. As already stated, proposed mechanisms are based on ET (CIEEL)46,47/CT (CTIL)27,44,50-53 from the electronrich moiety to the peroxide, with chemiexcitation resulting from charge annihilation due to BET/BCT steps.27,44,46,47,50-53 Given this, we have analyzed the charge (Figure S3) and electron spin (Figure S4) density transfer between the imidazopyrazinone and dioxetanone moieties, for both neutral and anionic dioxetanone (Scheme 1, R2=R3=CH3). Figure S3.a shows that the thermolysis reaction of anionic dioxetanone proceeds with significant CT, with the transfer of negative charge (-0.67e) from the imidazopyrazinone to dioxetanone. Moreover, with the evolution of the reaction, it can be seen the occurrence of BCT. Analysis of the electron spin density (Figure S4.a) shows an obvious separation in the spin density between the two moieties, which indicates that the biradical species is formed due to ET from the imidazopyrazinone moiety to dioxetanone, as in line with the charge density (Figure S3.a). The return to a non-biradical state is accomplished with BET. Thus, these data indicates that anionic dioxetanone decomposes via a CIEEL/CTIL mechanism, as in line with other theoretical studies for imidazopyrazinone species.27,51,55,56 More interesting is the analysis of charge (Figure S3.b) and electron spin (Figure S4.b) density transfer for the neutral species. As for the anionic species, the thermolysis reaction proceeds by CT (-0.85e) from the imidazopyrazinone moiety to the dioxetanone, followed latter by BCT (Figure S3.b). Analysis of the electron spin density (Figure S4.b) showed that the thermolysis of

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neutral dioxetanone proceeds by ET from the imidazopyrazinone moiety and not by homolytic bond cleavage of O1-O4. These findings are different from what was observed in previous theoretical studies of imidazopyrazinone dioxetanones.27,51,55,56 In those studies, the thermolysis reaction of neutral dioxetanones was shown to proceed via the homolytic bond cleavage of O1-O4 with very limited ET/CT steps. However, in those studies the R1 substituent (Scheme 1) was either -H55,56 or benzyl groups,27,51 while in this work is a indole moiety. As the indole moiety was found to present the higher electron-donating ability among several other electron-rich moieties when acting as the R1 of imidazopyrazinones,38,40 we can attribute the absence of significant ET/CT in the thermolysis of neutral dioxetanone (as seen in 27,51,55,56) due to the insufficient electron-donating ability of those substituents to trigger these ET/CT processes. It should also be noted that it was thought so far that the long region of PES where S1 and S0 are nearly degenerated, could only accessed if the thermolysis reaction proceed by homolytic cleavage of O1-O4 with limited CT/ET.27,51,55,56 This was thought because this region was only found in the thermolysis of neutral dioxetanones, which decomposed by that mechanism.27,51,55,56 However, the finding that a neutral dioxetanone can access that region of nearly-degeneracy even when decomposing with significant CT/ET, indicates that the presence of that region is not linked to the degree of CT/ET between the imidazopyrazinone moiety and dioxetanone. At the first glance, the results here presented might provide support for both CIEEL and CTIL theories. Neutral dioxetanone decomposes with significant ET and CT between the imidazopyrazinone moiety and dioxetanone (followed by ET and BCT), while it allows for efficient chemiexcitation due a long region of the PES where S0 and S1 are nearly-degenerated. However, as said above, this efficient pathway for chemiexcitation can also be found in the thermolysis of neutral dioxetanones that proceed with very limited CT and ET.27,51,55,56 Thus, it appears that this chemiexcitation pathway is not dependent on CT/BCT and/or ET/BET steps, but simply may occur concurrently. Moreover, significant ET/CT (followed by BET/BCT) is also seen for anionic imidazopyrazinone dioxetanones, both in the present and in another studies,27,51,55,56 and these decomposition reactions have resulted always in less efficient chemiexcitation pathways (significantly higher S1-S0 energy gaps) than the one found in the reactions involving their neutral forms.27,51,55,56 Once again, these data point to the chemiexcitation pathway being independent of ET/CT between the imidazopyrazinone moiety and dioxetanone. So, in conclusion, the results presented here (and in other studies)27,51,55,56 do not support the CIEEL and CTIL theories, at least for imidazopyrazinone-based systems. There is no clear relationship between ET/BET and CT/BCT steps and the efficiency of the chemiexcitation pathway, which appears to be explained instead by the access to a long region of the PES where S1 and S0 are nearly-degenerated. Significant ET/CT appears to be only a consequence of the S0 thermolysis reaction under specific conditions, such as deprotonation of the amide group and the electrondonating ability of the substituents of the imidazopyrazinone scaffold, and not the cause for efficient chemiexcitation. Having reached this conclusion, the question remains: why are neutral dioxetanones able to reach these regions of nearlydegeneracy, while anionic dioxetanones are not? From the analysis of the potential energy curves of S0 and S1 states (Figure 2) and of the behavior of both O1-O4 and C2-C3 bonds (Figure S2), it is clear that as the anionic species does not spend time on the region of nearly-degeneracy it releases the CO2 molecule (Scheme 1) sooner than the neutral species. Given this, it is possible that the quicker CO2 detachment is the reason why anionic dioxetanones cannot access the region of nearly-degeneracy. To this end we have measured the CT between the CO2 and oxyluciferin moieties


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as a function of intrinsic reaction coordinates (Figure 3), for both anionic and neutral dioxetanone. For anionic dioxetanone (Figure 3.a), there is a clear CT (-0.41e) from the oxyluciferin moiety to CO2 with O1-O4 bond elongation. However, due probably to anionic dioxetanone having a single negative charge, both moieties still remain negatively charged. This is expected to create electrostatic repulsion between the moieties and accelerate the detachment of CO2. In the case of neutral dioxetanone, there is also a clear CT (-0.49e) from the oxyluciferin moiety to CO2. However, due to the charge neutrality of neutral dioxetanone, this CT step increases significantly the negative charge of CO2 and the positive charge of oxyluciferin. This should then lead to attractive electrostatic interactions and favorable polarization effects between the two moieties, which should stabilize their interaction and slow down the release of CO2. Thus, our calculations support neutral dioxetanone having access to a region of nearlydegeneracy (which accounts for efficient chemiexcitation), due to attractive interactions between the CO2 and oxyluciferin moieties, which slows down the release of CO2 and allows the reaction to spend time in a long region of S1-S0 nearly-degeneracy.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Fluorescent and chemiluminescent spectra. Experimental k and plateau values measured in DMSO with acetate buffer and DMSO. Variation of O1-O4 and C2-C3 bonds during the thermolysis reaction of model Cypridina dioxetanone. Analysis of charge and electron spin density transfer between imidazopyrazinone and dioxetanone moieties. Cartesian coordinates of important structures.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT 4. CONCLUSIONS Cypridina luciferin, the bioluminescent substrate of the sea firefly Cypridina hilgendorfii, possesses an imidazopyrazinone scaffold that links together compounds present in about eight phyla of bioluminescent organisms. Despite of decade’s worth of research, the mechanism responsible for imidazopyrazinone-based bioluminescence is still far from understood, mainly due to the lack of sufficiently detailed experimental and theoretical evidence. Herein, we provided mechanistic insight into Cypridina bioluminescence with a combined experimental and theoretical chemiluminescent investigation. A luminometric and fluorescent spectroscopic approach was used to determine energy and kinetic chemiluminescent profiles in DMSO. We have found that Cypridina chemiluminescence has a pH-dependent behavior: this reaction has a higher reaction rate at higher pH, while the light intensity presents the opposite trend. Despite this, the chemiluminophore is the same (neutral oxyluciferin) at different pH values. Reliable electronic structure calculations at the DFT and TD-DFT level, indicate that this behavior can be explained by the thermolysis of neutral Cypridina dioxetanone at acidic pH, and of the anionic form at basic pH. The neutral species decomposes with a higher activation energy (~20 vs. ~10 kcal mol-1) than the anionic species. However, the thermolysis of the former species leads to a more efficient chemiexcitation pathway. This results from the ability of neutral dioxetanone to access a long region of the PES where S1 and S0 are nearly-degenerated, contrary to the anionic dioxetanone. These results suggest neutral dioxetanone as the responsible for chemiexcitation in the bioluminescent reaction. Further theoretical calculations demonstrated that both ET/BET and CT/BCT steps, between the imidazopyrazinone moiety and dioxetanone, occur during the thermolysis of neutral/anionic Cypridina dioxetanone. However, these steps have no clear relationship with the efficiency of the chemiexcitation process, which indicates that CIEEL and CTIL theories (as they are now formulated) cannot be applied to imidazopyrazinone-based systems. Efficient chemiexcitation by neutral dioxetanone is then explained by attractive electrostatic interactions between the CO2 and oxyluciferin moieties, which allows for the reacting molecules to spend time in a long region of the PES of S1-S0 nearlydegeneracy. For the contrary, repulsive electrostatic interactions between the moieties of anionic dioxetanones leads to a quicker CO2 detachment and prevents the access to the region of nearlydegeneracy.

ASSOCIATED CONTENT

This work was made in the framework of project PTDC/QEQQFI/0289/2014, which is funded with national funds by FCT/MEC (PIDDAC). The project is also co-funded by FEDER through COMPETE-POFC. This work was also made in the framework of the project Sustainable Advanced Materials (NORTE-01-0145-FEDER-000028), funded by FEDER through NORTE2020. Acknowledgment to project POCI-01-0145FEDER-006980 funded by FEDER through COMPETE 2020 is also made. L. Pinto da Silva acknowledges the Post-Doc grant funded by project NORTE-01-0145-FEDER-000028. The Laboratory for Computational Modeling of Environmental PollutantsHuman Interactions (LACOMEPHI) is acknowledged.

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Scheme 1 - Reaction mechanism of Cypridina bioluminescence. 120x177mm (300 x 300 DPI)



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Figure 1 -Rate constants (k, in s-1) at acidic (by addition of acetate buffer pH 5.12) and basic (by addition of NaOH) pH in DMSO solutions (a). Plateau of light intensity (normalized values) at acidic (by addition of acetate buffer pH 5.12) and basic (by addition of NaOH) pH in DMSO solutions (b). Chemiluminescence spectra in DMSO (c). 77x136mm (96 x 96 DPI)


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Figure 2 - Potential energy curves of S0 and S1 states, as a function of intrinsic reaction coordinates, of model (Scheme 1, R2=R3=CH3) neutral (a) and anionic (b) dioxetanone. 123x145mm (96 x 96 DPI)



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Figure 3 - Atomic Mulliken charge of the CO2 and oxyluciferin moieties of anionic (a) and neutral (b) model Cypridina dioxetanone (Scheme 1, R2=R3=CH3), as a function of intrinsic reaction coordi-nates. 114x147mm (96 x 96 DPI)


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Table of Contents Graphic 301x127mm (96 x 96 DPI)


Mechanistic Insight into Cypridina Bioluminescence with a Combined

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