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Combined experimental and theoretical study of Coelenterazine chemiluminescence in aqueous solution
Journal of Luminescence 194 (2018) 139–145
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Combined experimental and theoretical study of Coelenterazine chemiluminescence in aqueous solution João M. Lourençoa, Joaquim C.G. Esteves da Silvab,c, Luís Pinto da Silvaa,c,
MARK
⁎
a Chemistry Research Unit (CIQUP), Department of Chemistry and Biochemistry, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal b 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 c LACOMEPHI, Department of Geosciences, Environment and Territorial Planning, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Keywords: Coelenterazine Chemiluminescence Bioluminescence Superoxide Anion Aqueous Solution Luciferase Imidazopyrazinone
Coelenterazine is a common substrate used by marine species in enzyme-catalyzed bioluminescent reactions, in which thermal energy is converted into light-emission. Besides bioluminescence, Coelenterazine is also known to emit chemiluminescence in aprotic solvents. We report here the study of Coelenterazine chemiluminescence in aqueous solution. Water inhibits light-emission even in mixtures with water content as low as 20%. Moreover, we provide convincing spectroscopic evidence that the presence of water affects the ground state (S0) chemical reaction, and not the excited state processes (as chemiexcitation and the fluorescent quantum yield). However, the energetics of the S0 chemical reaction is not affect by addition of water, which points to the inhibition being caused by the reduced lifetime of superoxide anion in water, which is an intermediate in the luminescent reactions of Coelenterazine. This finding indicates that one of the catalytic roles of bioluminescent enzymes is to extend the lifetime of this radical.
1. Introduction Bioluminescence consists on the conversion of thermal energy into excitation energy, thereby leading to light emission [1–4], and has been attracting attention from the research community due to high quantum yields and high signal-to-noise ratio [5–8]. Furthermore, in bioluminescent reactions there is no autofluorescence arising from the background signal [9], the lack of light excitation also eliminates problems regarding light-penetration into biologic tissues (except for emission) [10]. These features make bioluminescent systems very helpful tools in the real-time and noninvasive imaging of target molecules in vivo [11–14], as well as potential excitation sources for self-illuminating systems in photodynamic therapy of cancer [10]. More than 700 genera have been found to produce bioluminescence, in organisms as diverse as the fireflies, fungi, earthworms, fishes and bacteria, among others [15–18]. However, about 80% of all bioluminescent organisms can be found in the ocean [19,20]. Moreover, the majority of the marine organisms react with the same bioluminescent substrate: Coelenterazine (Scheme 1) [3,4,10,21]. The bioluminescent reaction involving Coelenterazine proceeds as follows (Scheme 1)
[22–24]: the first step is the oxygenation of the imidazopyrazinone scaffold of Coelenterazine, which leads to the quick formation of the 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 (Coelenteramide) in its first singlet excited state (S1). Typical Coelenterazine-based bioluminescent reactions involve the presence of an enzyme named luciferase, which catalyzes the oxygenation step of Coelenterazine [3,4]. As Coelenterazine, these luciferases can be found in a great variety of marine organisms, such the decapod shrimp Oplophorus gracilirostris, the anthozoan Renilla reniformis, and of the copecods Gaussian princeps and Metridia longa [3,4]. Currently, the Gaussian and Renilla reniformis luciferases are the ones with the highest number of practical applications [3,4]. It should be noted that the imidazopyrazinone core of Coelenterazine is a common link among marine luminescent substrates, and so, its bioluminescent mechanism is the same for many other marine organisms. In fact, the same imidazopyrazinone core can be found in Cypridina luciferin (present in the sea firefly Cypridina hilgendorfii), dehydrocoelenterazine (present in the squid Symplectoteuthis oualaniensis) and in Watasenia luciferin (present in the squid Watasenia
⁎ Corresponding author at: Chemistry Research Unit (CIQUP), Department of Chemistry and Biochemistry, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. E-mail address: [email protected] (L. Pinto da Silva).
http://dx.doi.org/10.1016/j.jlumin.2017.10.025 Received 11 July 2017; Received in revised form 3 October 2017; Accepted 10 October 2017 Available online 12 October 2017 0022-2313/ © 2017 Elsevier B.V. All rights reserved.
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Scheme 1. Reaction mechanism of Coelenterazine luminescence.
dynamic sensor and not an accumulation one. This means that when the chemiluminescent reaction between the probe and the target ROS species occurs and light is emitted, no more photons can be produced from those reactions. Coelenterazine also has the advantage of exhibiting chemiluminescence independently from cell-derived myeloperoxidase, and by not sensitizing O2- (which limits the occurrence of false-positives) [36]. Given this, the ROS-mediated chemiluminescence indicates that water by itself does not affect negatively the chemiluminescence quantum yield of Coelenterazine, and that chemiluminescence may also occur in aqueous solution (in the presence of 3O2). However, that would mean that the chemiluminescence reaction of Coelenterazine would compete with its bioluminescent reaction (as this last reaction occurs in aqueous solution), which should affect the efficiency of the latter process in its various practical applications (such as bioimaging and bioanalysis). Thus, it is essential to determine if Coelenterazine can also produce chemiluminescence in aqueous solutions (in the absence of ROS), and if so, to determine the efficiency of this process. Herein, we report such study by using a spectroscopic approach to assess the potential of Coelenterazine for chemiluminescence in aqueous solution. The chemiluminescence in DMSO, an aprotic solvent typically used as a model solution for the bioluminescent reaction, was also measured so to serve as comparison. This approach allowed us to obtain valuable insight into the luminescence of Coelenterazine and related imidazopyrazinone-based ones, which can be found in about eight phyla of luminescent organisms.
scintillans) [3,25,26]. Besides bioluminescence, Coelenterazine and related imidazopyrazinone-based substrates are also able to emit chemiluminescence in aprotic solvents (as DMSO and diglyme) without an enzyme, in an identical mechanism to that of the bioluminescent reaction [22–24,27–29]. The most significant difference between chemiluminescence and bioluminescence is the higher quantum yield of the latter process, which is to be expected due to the absence of the catalytic luciferase in chemiluminescence [22–24,27–29]. Due to these similarities, chemiluminescence has been used as a model for the study of the bioluminescence reaction [22–24,27–29]. These studies have found that Coelenterazine presents appreciable chemiluminescence quantum yields (between 0.0021 and 0.005) in DMSO, N,N-dimethylformamide (DMF) and hexamethylphosphoric triamide, in the absence of base [21]. Addition of base and acidic buffers decreased the measured quantum yield [21]. Contrary to aprotic solvents (mainly DMSO) [21–24], the chemiluminescence of Coelenterazine in aqueous solutions has been sparsely studied, despite it being the solvent in which bioluminescence takes place (and consequently, the practical applications of this system). Nevertheless, some studies regarding other imidazopyrazinone-based substrates found the chemiluminescence in water to be extremely weak [30]. One explanation for this behavior was provided by Shimomura, who have found the fluorescence of Cypridina oxyluciferin to be very weak in aqueous solution [31]. Other authors have studied the chemiluminescence of different imidazopyrazinones in water/DMF and methanol/DMF mixtures, and have found that high contents of water greatly decrease the chemiluminescence efficiency [32]. These results indicate that water should affect negatively the chemiluminescence quantum yield of Coelenterazine, which is composed by three parameters: the yield of the S0 reaction; the efficiency of the S0 → S1 chemiexcitation; the fluorescence quantum yield of the chemiluminophore [1,2]. However, this potentially negative effect exerted by water is not in line with the known reactive oxygen species (ROS)-induced Coelenterazine chemiluminescence in water. Besides molecular oxygen (3O2), the oxygenation step of Coelenterazine chemiluminescence can also be triggered by different ROS, such as superoxide anion (O2-) and singlet oxygen (1O2). In fact, Coelenterazine and other imidazopyrazinone molecules have been used with good results as dynamic probes for ROS in biological media (both in vivo and in vitro) [21,33–35]. Contrary to fluorescent ROS sensors, Coelenterazine is a
2. Experimental methods Coelenterazine was purchased from NanoLight™ Technology, and was dissolved in methanol and stored at −20 °C. 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°) and were performed at least in triplicate. The reactions were carried out in pure DMSO or in DMSO/water mixtures. The reaction was initiated by the injection of the DMSO (or DMSO/water mixture) solution (380 μL) into an assay tube containing Coelenterazine (20 μL). The concentration of Coelenterazine in the final solution is of 3.0 μM. The light was integrated and recorded in 0.1 s intervals. Resulting data was analyzed by using the Graphpad software package (Version 7.03 for Windows). UV–Vis spectra were measured 140
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Table 1 Initial velocities (v, in RLU s−1) and light-emission maximum (in RLU) for Coelenterazine chemiluminescence in DMSO and DMSO/water mixtures. Solvent
Light-emission maximum
v
DMSO DMSO/Water (99%/1%) DMSO/Water (95%/5%) DMSO/Water (90%/10%)
33965 ± 2443 30629 ± 1170 12090 ± 1321 5245 ± 518
37676 ± 3520 28787 ± 836 11268 ± 1539 3728 ± 640
with a UV-3100PC Spectrophotometer. Quartz cells were used. Concentrations and volumes were the same as for the chemiluminescent kinetic assays. 3. Results and discussion We have started by studying the chemiluminescence of Coelenterazine in solutions of pure DMSO and of DMSO/water mixtures (99%/1%, 95%/5% and 90%/10%). The chemiluminescent kinetics were followed by the measurement of direct light-emission intensity. The kinetic curves gave rise to initial velocities (v, in relative light units [RLU] s−1) and to the maximum of light intensity (in RLU) [37,38]. These values were obtained by linear fitting [37,38]. The values for light-emission maximum and v, in those solutions, can be found in Table 1. While in pure DMSO the light-emission maximum of chemiluminescence is appreciable (in line with previous results) [21], addition of only limited amounts of water had a strong inhibition effect. Inhibition was promptly seen even in mixtures containing 1% of water, with solutions containing 10% of water presenting light-emission intensities of only 15% of that observed in pure DMSO. Increasing the quantity of water to 20% quenched completely the chemiluminescence produced by Coelenterazine (data not shown). Thus, these results indicate that chemiluminescence should not occur in pure aqueous solutions, in line with experimental studies made for other imidazopyrazinones [30,32]. Having reached this conclusion, is not clear in what way water quenches the emission of chemiluminescence. That is, if water affects the S0 chemical reaction, the chemiexcitation step or the quantum yield of the chemiluminophore. One way to understand the reason behind this effect is to compare the behavior of the light-emission maximum, as a function of water content, with that of v. As v results from the increase of light as a function of time, addition of water automatically results in a decrease of this parameter (Table 1). However, if the decrease of lightintensity results from the fluorescent quantum yield of the emitter or from the yield of S0 → S1 non-radiative crossing, the slope of the correlation of v with water should be smaller than that of the light-emission maximum. This occurs because both the fluorescent quantum yield and S0 → S1 transition are excited state processes that do not have an effect on the speed of the reaction. For the contrary, the activation barriers present in the S0 reaction determine the speed of reaction, and so, if water increases these barriers high enough to inhibit the S0 reaction, it should decrease the light-emission maximum and v in similar magnitudes.
Fig. 2. UV–Vis spectrum of Coelenterazine in DMSO (a) and water (b), at 1, 5 and 10 min. The pH of these solutions was adjusted by addition of phosphate buffer pH 7.4 (75 mM).
The slopes were obtained by exponential fitting of the correlation found between v/light-emission maximum and DMSO/water ratio (Fig. 1), which were of 0.26 ± 0.02 and 0.20 ± 0.01, respectively. The performing of an unpaired t-test showed the difference between the slopes to be statistically significant, with a two-tailed p value of 0.0073 with a 95% confidence interval. Interestingly, the fact that the slope of the correlation between v and DMSO/water ratio is bigger than that of the slope involving the light-emission maximum, indicates that the negative effect exerted by water is stronger on the velocity of the reaction (controlled by the S0 reaction) than in the emission of light (controlled both by the S0 reaction and by excited state processes). Thus, these results indicate that the inhibiting effect of water is caused by affecting the S0 reaction. This can be explained by increasing the activation barrier of one or more steps of the S0 reaction to values high enough to inhibit the S0 reaction yield, and so, decrease the emission of bioluminescence and the speed of reaction. In order to provide further support for this conclusion, we have studied the UV–Vis spectrum of Coelenterazine in DMSO and water solutions (Fig. 2). With UV–Vis spectroscopy we can observe the formation of the products and consumption of reactants during the chemiluminescent reaction, irrespectively of the amount of emitted light. Thus, if the inhibition of chemiluminescence exerted by water is caused by affecting the S0 chemical reaction, thereby meaning that Coelenterazine is not consumed, the UV–Vis spectrum in water should be different from that measured in DMSO (which should be composed by both Coelenterazine and Coelenteramide). If the water-induced inhibition is caused by excited state processes the spectra should be similar, as Coelenterazine is consumed and Coelenteramide produced in both solvents. Significant differences were observed between the UV–Vis spectra of Fig. 1. Correlation between v (a) or light-emission maximum (b) with the percentage of water in DMSO/water mixtures.
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IC (100 μM) alone. Also, the addition of both 5% water and IC (10 μM) to pure DMSO lead to the same decrease obtained when only 5% water is added, about 90%. This similar effect exerted by IC and water on light-emission indicates that both molecules affect the same step of the chemiluminescent reaction. As, due to its nature as a redox indicator, IC is expected to interfere with the oxygenation step, it is safe to conclude that water also interferes with this step. Given these results, we have performed theoretical calculations based on Density Functional Theory (DFT) in order to determine the energetics of the SET reaction between anionic/neutral Coelenterazine and 3O2 (Scheme 2). Each structure was optimized at the ωB97XD/631G(d) level of theory [46], in either DMSO or water by using an implicit solvent model (IEFPCM) [47]. Vibrational frequency calculations made at the same level of theory allowed us to obtain the Gibbs free energy for each structure with thermal corrections (Gtherm). All calculations were made with the Gaussian 09 program package [48]. The ΔGtherm for both SET reactions, in both solvents, is endergonic. Particularly for SET between anionic Coelenterazine and 3O2. This can be considered to be in line with the finding that SET is the rate-determining step of the chemiluminescent reaction of imidazopyrazinonebased compounds [29]. Nevertheless, what was interesting to find is that the SET reaction presents very similar ΔGtherm in both DMSO and water, thereby indicating that water does not quench the emission of chemiluminescence by impairing the rate-determining step of this reaction. As SET is seemingly not affected by water, we have focused on the next step of the chemiluminescent reaction: the recombination of Coelenterazine and O2- radicals to form the dioxetanone intermediate [21,29,44]. As O2- interacts with the imidazopyrazinone core of radical Coelenterazine, we have calculated the atomic Mulliken charge of this moiety in DMSO (−0.12e) and in water (−0.12e), at the ωB97XD/631G(d) level of theory. We have also calculated the Mulliken spin density of this moiety in DMSO (1.00) and water (1.00), at the same level of theory. As both the charge and spin density of the imidazopyrazinone core of radical Coelenterazine are identical in both solvents, it is safe to conclude that water does also not affect the energetic of the radical recombination step. Nevertheless, we have analyzed the Mulliken spin density (in water and DMSO) of carbon atoms present in in the imidazopyrazinone core of radical Coelenterazine (Scheme 3), and which can act as potential sites of interaction with O2- for the radical recombination step [21,49,50]. However, the spin density is very similar between the two solvents, and the carbon atom with the higher spin density (thereby the one most prone to interact with O2-) is the one that leads to the formation of the dioxetanone intermediate [21,49,50]. Thus, this analysis provides further indication that water does not affect the energetic of the oxygenation step. Besides these calculations, we have also determined the theoretical oxidation potential of Coelenterazine, by following the protocol developed by Winget and co-workers [51]:
Coelenterazine in water and DMSO (Fig. 2). In DMSO, two different peaks can be seen: one at ~ 280 nm and other at 340 nm. Interestingly, the intensity of the first peak (~ 280 nm) decreases with increasing time, while the opposite behavior is seen for the second peak (~ 340 nm). This indicates that the first peak can be attributed to Coelenterazine, which is consumed during the chemiluminescent reaction, while the second one corresponds to Coelenteramide, which is the chemiluminescent product. This is supported by previous experimental work. Kudryasheva and co-workers defined the absorption region of Coelenteramide to be between 310 and 380 [39], while Shimomura and Teranishi found that Coelenteramide and analogs possess an excitation maximum at ~ 340 nm in DMSO [40]. Furthermore, Coelenterazine has been found to possess an experimental absorption maximum at 275 nm [41]. For the contrary, the UV–Vis spectrum in water is dominated by a single peak at ~ 265 nm, which intensity decreases very slightly as the time passes by. This peak can be attributed to Coelenterazine, and its limited decreased indicates that the S0 chemical reaction is inhibited in aqueous solution. The data obtained so far indicate that Coelenterazine cannot emit chemiluminescence in aqueous solution (to the contrary of in DMSO and other aprotic solvents), because water inhibits the S0 chemical reaction probably by increasing the activation barrier of one or more of its steps. As referred before, the S0 chemical reaction is composed by two sequential steps: oxygenation of the imidazopyrazinone core, and thermolysis of the dioxetanone intermediate (Scheme 1) [3,4,10,21]. Currently, there is a lack of experimental data regarding the thermolysis step, due to the high instability of the dioxetanone intermediate. However, different theoretical studies have demonstrated that the activation barrier for the thermolysis of imidazopyrazinone-based dioxetanones decreases with increasing polarity of the solvent [42,43]. Given this, the inhibitory effect exerted by water cannot be due to an increase of the activation barrier for dioxetanone thermolysis, as water is more polar than DMSO and other aprotic solvents used to generate chemiluminescence (as diglyme). Thus, it appears that the reason behind this inhibitory effect is related to the oxygenation step, which consists in a single electron transfer (SET) from Coelenterazine to 3O2 [44]. This reaction generates a Coelenterazine radical and O2-, which recombine latter to form the dioxetanone intermediate. Such SET-based mechanism was also found to be involved in firefly bioluminescence [45]. One possibility to explain this phenomenon when thinking of the oxygenation step (which is a redox reaction), is that Coelenterazine might be more easily oxidized in DMSO (and other aprotic solvents) than in water. So, to obtain more insight into this, we have measured the direct light-emission from Coelenterazine chemiluminescence in the presence of Indigo Carmine (IC), a known redox indicator, in both pure DMSO and in a DMSO/water (95%/5%) mixture. The light-intensity maxima obtained are present in Table 2. Addition of IC (100 and 10 μM) to pure DMSO led to significant quenching of light emission, by about 98% and 60%, respectively. This quenching effect provided by IC (particularly at a concentration of 100 μM) is very similar to what is obtained by addition of 5% water to a DMSO solution, which led to a quenching of about 90% of light-emission. Moreover, the addition of both 5% water and IC (100 μM) to pure DMSO led to a decrease in lightemission of 99%, very similar to the decrease of 98% in the presence of
° ∆Gox = IP+∆Gevr , gas +∆∆Gsolv
IP is the adiabatic ionization potential, included with the difference in zero-point vibrational energy. ∆Gevr , gas is the difference in thermal contributions to Coelenterazine free energy deriving from changes in the electronic, vibrational and rotational partition functions upon ionization. ∆∆Gsolv is the difference between the free energy of solvation of anionic and oxidized forms of Coelenterazine. These calculations were made at the ωB97XD/6-31G(d,p) level of theory [46], in either implicit DMSO or water by using the IEFPCM solvent model [47]. The oxidation potential of Coelenterazine in implicit water (5.65 eV) and DMSO (5.63 eV) was very similar, which supports our previous conclusion that water should not inhibit the oxidation of Coelenterazine. The addition of four explicit water molecules (placed near the four heteroatoms of the imidazopyrazinone core, Fig. 3a) even
Table 2 Light-emission maximum (in RLU) for Coelenterazine chemiluminescence in DMSO and DMSO/water mixtures, and with the addition of IC. Solution DMSO DMSO DMSO DMSO DMSO DMSO
+ + + + +
Light-Emission Maximum
IC (10 μM) IC (100 μM) Water (5%) IC (10 μM) + Water (5%) IC (100 μM) + Water (5%)
(1)
67034 ± 4347 26763 ± 2061 1433 ± 355 6451 ± 1506 6420 ± 2437 486 ± 69
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Scheme 2. Schematic representation of the SET reaction between neutral and anionic Coelenterazine, with the corresponding theoretical ΔGtherm in water (DMSO), at the ωB97XD/6-31G(d) level of theory.
of the effect of explicit solvation in water and DMSO, the oxidation potential of Coelenterazine was once again calculated in both solvents, but with a water/DMSO explicit molecule placed near the deprotonated nitrogen heteroatom (Fig. 3). Two different conformations were found for the interaction between DMSO and Coelenterazine (Fig. 3c and d). The resulting potentials were of 3.69 eV (in water) and 3.67 (Fig. 3c)/ 3.66 (Fig. 3d) eV (in DMSO). These values lead us to two different conclusions. One is that explicit solvation does affect the oxidation potential, in which solvation of the deprotonated nitrogen heteroatom leads to a significant decrease in the oxidation potential. The second conclusion, is that addition of explicit solvation does not appear to create a significant difference between the ability of Coelenterazine to be oxidized in water and in DMSO. Nevertheless, as Coelenterazine is a negatively charged molecule and its ionization leads to the formation of a neutral radical, it might be
Scheme 3. Mulliken spin density of key carbon atoms present in the imidazopyrazinone core of radical Coelenterazine in water (DMSO), at the ωB97XD/6–31 G(d) level of theory.
decreased the oxidation potential of Coelenterazine in water to 4.96 eV, which indicates that explicit aqueous solvation does not impair the oxidation of Coelenterazine (to the contrary). For a better comparison
Fig. 3. Geometries optimized at the ωB97XD/6-31G(d,p) level of theory, of Coelenterazine in the presence of four explicit water molecules (a), one explicit water molecule (b), and one explicit DMSO molecule (c and d).
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This solvent-dependent lifetime of O2- is in line with the chemiluminescence efficiency of Coelenterazine. This imidazopyrazinone does not emit chemiluminescence in water (as demonstrated here) and in methanol (a solvent used to prepare stock solutions of Coelenterazine) [22–24,29], which are protic solvents. For the contrary, chemiluminescence is found to be emitted by Coelenterazine (and other imidazopyrazinone-based molecules) with non-negligible efficiencies in different aprotic solvents, such as DMSO, diglyme, DMF and hexamethylphosphoric triamide [21–24,29]. Therefore, this information indicates that the inhibitory effect exerted by water on the chemiluminescence emitted by Coelenterazine is caused by decreasing the lifetime of O2-, an intermediate of this reaction, and not by water inhibiting directly any one of the two steps composing the S0 chemical reaction. So, this conclusion points to one of the catalytic functions of luciferases using Coelenterazines to be extending the lifetime of O2-, inside their active sites. Nevertheless, further study of the chemiluminescent reaction of Coelenterazine in aqueous solutions might be useful for a better characterization of this system.
Table 3 Atomic Mulliken charges of the imidazopyrazinone core of Coelenterazine (in its neutral and anionic forms), either solely in implicit water/DMSO or in the presence of one explicit water/DMSO molecule. The calculations were made at the ωB97XD/6–31 G(d,p) level of theory. Water Anion
DMSO Radical
Implicit Solvation −0.849 −0.143 Explicit Solvation −0.827 −0.121
Anion
Radical
−0.849
−0.143
−0.862 (−0.848)a
−0.168 (−0.150)a
a The values between parenthesis refer to the structure depicted in Fig. 3d, while the other ones refer to the structure depicted in Fig. 3c.
expected that the anion could be more stabilized in more polar solvents as compared to the radical. If that was the case, the oxidation potential of Coelenterazine should be higher in water than in DMSO. So, we have calculated the atomic Mulliken charge of the imidazopyrazinone core of Coelenterazine, its anionic and radical forms, in implicit and explicit water/DMSO (Table 3). These calculations show that the decrease in negative charge due to ET from the imidazopyrazinone core to 3O2 is very similar in all conditions (~0.70e). This might indicate that the imidazopyrazinone core is not significantly affected by differences in polarity between water and DMSO. Besides assessing the theoretical oxidation potential of Coelenterazine, we have also calculated the theoretical reduction potential of 3O2, to form the O2- radical. The reduction potential was obtained by modifying the protocol devised by Winget and co-workers for oxidation potentials [51]: ° ∆Gred = EA+∆Gevr , gas +∆∆Gsolv
4. Conclusion Coelenterazine is common marine chemi- and bioluminescent substrate found in different organisms, and possesses an imidazopyrazinone core that links together molecules present in about eight phyla of luminescent organisms. Moreover, Coelenterazine-based chemi- and bioluminescence has become a powerful tool in real-time bioimaging, bioanalysis and in biomedicine. Herein, we report a combined experimental and theoretical study of Coelenterazine chemiluminescence in aqueous solution. While chemiluminescence has been extensively studied in aprotic media, it has not been sufficiently characterized in aqueous solution, which is a medium where bioluminescence (and its practical applications) occur. Luminescent assays demonstrate that water has a very strong inhibitory effect on chemiluminescence, being able to quench light emission in mixtures with water content as low as 20%. Moreover, this inhibitory effect is caused by affecting the S0 chemical reaction, and not by impairing excited state processes. However, water was not found to have a significant effect on the steps composing the S0 reaction, which means that the inhibitory effect is not caused by affecting directly the chemiluminescent reaction. The inhibition was then attributed to the lifetime of the O2- radical, which is significantly lower in water than in aprotic solvents, in which Coelenterazine has been found to emit chemiluminescence with nonnegligible efficiency. This finding indicates that one of the catalytic functions provided by bioluminescent enzymes is to extend the lifetime of the O2- radical in their active sites.
(2)
In this case, IP was substituted by the adiabatic electron affinity (EA), included with the difference in zero-point vibrational energy. These calculations were made at the ωB97XD/6-31G(d,p) level of theory, in either implicit water/DMSO or with the presence of one explicit water/DMSO molecule. The reduction potential of 3O2 is very similar in implicit water (−2.69 eV) and DMSO (−2.66 eV). The addition of explicit solvent molecules increases the reduction potential of this molecule (−2.96 eV and −2.92 eV, respectively), but does not cause any significant difference between solvents. In conclusion, Coelenterazine does not emit chemiluminescence in aqueous solution (to the contrary to what is observed in aprotic solvents), as water exerts a strong inhibitory effect on the chemiluminescent reaction of this molecule. This effect arises from the inhibition of the S0 chemical reaction, and not by impairing excited state processes (as the chemiexcitation step or the S1 → S0 radiative transition). However, previous theoretical calculations have shown that water decreases the activation barrier for the thermolysis of dioxetanone [42,43], while the present study indicates that water does not affect the energetic of the oxygenation step. Thus, while water inhibits the S0 chemical reaction, which is composed by the oxygenation step and dioxetanone thermolysis, it apparently does not inhibit by itself any one of these steps. Given this, it is possible that the explanation for this process must be found outside of chemiluminescent reaction. One of the possibilities is the lifetime of the O2- radical. The lifetime of this radical is considerably greater in aprotic solvents than in protic and aqueous solutions, due to strong solvation and spontaneous disproportionation in the latter class of solvents [52,53]. In fact, O2- is generally generated in aprotic media in order for studying its reactions with organic and inorganic compounds [54]. O2- disproportionate in water by forming 3 O2, and the hydroxyl and hydroperoxide anions [52,53], as seen below:
2O2− + H2 O → O2 + HO2− + OH−
Acknowledgments This work was made in the framework of project PTDC/QEQ-QFI/ 0289/2014, which is funded with national funds by FCT/MEC (PIDDAC) and 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. Acknowledgement to project POCI-010145-FEDER-006980 funded by FEDER through COMPETE2020 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 Pollutants-Human Interactions (LACOMEPHI) is acknowledged.
Notes
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The authors declare no competing financial interests. 144
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Symplectoteuthis-oualaniensis, Proc. Natl. Acad. Sci. USA 78 (1981) 6719–6723. [27] T. Goto, H. Fukatsu, Cypridina Bioluminescence. 7. Chemiluminescence in micelle solutions – a model system for cypridina bioluminescence, Tetrahedron Lett. 49 (1969) 4299–4302. [28] K. Usami, M. Isobe, Low-temperature photooxygenation of coelenterate luciferin analog synthesis and proof of 1,2-dioxetanone as luminescence intermediate, Tetrahedron 52 (1996) 12061–12090. [29] H. Kondo, T. Igarashi, S. Maki, H. Niwa, H. Ikeda, T. Hirano, Substituent effects on the kinetics for the chemiluminescence reaction of 6-arylimidazo[1,2-a]pyrazine3(7H)-ones (Cypridina luciferin analogues): support for the single electron transfer (SET)-oxygenation mechanism with triplet molecule oxygen, Tetrahedron Lett. 46 (2005) 7701–7704. [30] T. Goto, Chemistry of bioluminescence, Pure Appl. Chem. 17 (1968) 421–441. [31] O. Shimomura, F.H. Johnson, T. Masugi, Cypridina bioluminescence – light-emitting oxyluciferin-luciferase complex, Science 164 (1969) 1299–1300. [32] K. Teranishi, M. Hisamatsu, T. Yamada, Chemiluminescence of 2-methyl-6-arylimidazo[1,2-a]pyrazine-3(7H)-one in protic solvents: electron-donating substituent effect on the formaton of the neutral singlet excited-state molecule, Luminescence 14 (1999) 297–302. [33] L.L. Bronsart, C. Stokes, C.H. Contag, Chemiluminescence imaging of superoxide anion detects beta-cell function and mass, PLoS One 11 (2016) e0146601. [34] L.L. Bronsart, C. Stokes, C.H. Contag, Multimodality imaging of cancer superoxide anion using the small molecule Coelenterazine, Mol. Imaging Biol. 18 (2016) 166–171. [35] L.L. Bronsart, L. Nguyen, A. Habtezion, C.H. Contag, Reactive oxygen species imaging in a mouse model of inflammatory bowel disease, Mol. Imaging Biol. 18 (2016) 473–478. [36] M.M. Tarpey, C.R. White, E. Suarez, G. Richardson, R. Radi, B. Freeman, Chemiluminescent detection of oxidants in vascular tissue – lucigenin but not coelenterazine enhances superoxide formation, Circ. Res. 84 (1999) 1203–1211. [37] L. Pinto da Silva, J.C.G. Esteves da Silva, Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and l-luciferin, Photochem. Photobiol. Sci. 10 (2011) 1039–1045. [38] C. Ribeiro, J.C.G. Esteves da Silva, Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate, Photochem. Photobiol. Sci. 7 (2008) 1085–1090. [39] R. Alieva, N.V. Belogurova, A.S. Petrova, N.S. Kudryasheva, Fluorescence properties of Ca2+-independent discharged obelin and its application prospects, Anal. Bioanal. Chem. 405 (2013) 3351–3358. [40] O. Shimomura, K. Teranishi, Light-emitters involved in the luminescence of coelenterazine, Luminescence 15 (2000) 51–58. [41] E.V. Eremeeva, S.V. Markova, A.H. Westphal, A.J.W.G. Visser, W.J.H. van Berkel, E.S. Vysotski, The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding, FEBS Lett. 583 (2009) 1939–1944. [42] L. Pinto da Silva, C.M. Magalhães, J.C.G. Esteves da Silva, Interstate crossing-induced chemiexcitation mechanism as the basis for imidazopyrazinone bioluminescence, ChemistrySelect 1 (2016) 3343–3356. [43] B.W. Ding, P. Naumov, Y.J. Liu, Mechanistic Insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea firefly) lumophore, J. Chem. Theory Comput. 11 (2015) 591–599. [44] B.W. Ding, Y.J. Liu, Bioluminescence of firefly squid via mechanism of single electron-transfer oxygenation and charge-transfer-induced luminescence, J. Am. Chem. Soc. 139 (2017) 1106–1119. [45] B.R. Branchini, C.E. Behney, T.L. Southworth, D.M. Fontaine, A.M. Gulick, D.J. Vinyard, G.W. Brudvig, Experimental support for a single electron-transfer oxidation mechanism in firefly bioluminescence, J. Am. Chem. Soc. 137 (2015) 7592–7595. [46] J.D. Chai, M. Head-Gordon, Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections, Phys. Chem. Chem. Phys. 10 (2008) 6615–6620. [47] G. Scalmani, M.J. Frisch, Continuous surface change polarizable continuum models of solvation. I. General formalism, J. Chem. Phys. 132 (2010) 114110. [48] M.J. Frisch, et al., Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford CT, 2009. [49] K. Fujimori, H. Nakajima, K. Akutsu, M. Mitani, H. Sawada, M. Nakayama, Chemiluminescence of Cypridina luciferin analogues. Part 1. Effect of pH on rates of spontaneous autoxidation of CLA in aqueous buffer solutions, J. Chem. Soc. Perkin Trans. 2 (1993) 2405–2409. [50] K. Akutsu, H. Nakajima, T. Katoh, S. Kino, K. Fujimori, Chemiluminescence of Cipridina luciferin analogues. Part 2. Kinetic studies on the reaction of 2-methyl-6phenylimidazo[1,2-a]pyrazine-3(7H)-one (CLA) with superoxide: hydroperoxyl radical is an actual active species used to initiate the reaction, J. Chem. Soc. Perkin Trans. 2 (1995) 1699–1706. [51] P. Winget, E.J. Weber, C.J. Cramer, D.G. Truhlar, Computational electrochemistry: aqueous one-electron oxidation potentials for substituted anilines, Phys. Chem. Chem. Phys. 2 (2000) 1231–1239. [52] M. Hayyan, M.A. Hashim, I.M. AlNashef, Superoxide ion: generation and chemical implications, Chem. Rev. 116 (2016) 3029–3085. [53] D.T. Sawyer, J.S. Valentine, How super is superoxide? Acc. Chem. Res. 14 (1981) 393–400. [54] I.B. Afanas’ev, Superoxide Ion: Chemistry and Biological Implications, 1 CRC Press, Boca Raton, FL, 1989.
References [1] I. Navizet, Y.J. Liu, N. Ferré, D. Roca-Sanjuán, R. Lindh, The chemistry of bioluminescence: an analysis of chemical functionalities, ChemPhysChem 12 (2011) 3064–3076. [2] L. Pinto da Silva, J.C.G. Esteves da Silva, Firefly chemiluminescence and bioluminescence: efficient generation of excited states, ChemPhysChem 13 (2012) 2257–2262. [3] Z.M. Kaskova, A.S. Tsarkova, I.V. Yampolsky, 1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine, Chem. Soc. Rev. 45 (2016) 6048–6077. [4] E.P. Coutant, Y.L. Janin, Synthetic routes to coelenterazine and other imidazo[1,2a]pyrazin-3-one luciferins: essential tools for bioluminescence-based investigations, Chem. Eur. J. 21 (2015) 17158–17171. [5] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing Co. Pte. Ltd, Singapore, 2006. [6] J. Vieira, L. Pinto da Silva, J.C.G. Esteves da Silva, Advances in the knowledge of light emission by firefly luciferin and oxyluciferin, J. Photochem. Photobiol. B 117 (2012) 33–39. [7] A. Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence: latest achievements and new horizons, Anal. Bioanal. Chem. 402 (2012) 69–76. [8] A. Roda, M. Guardigli, P. Pasini, M. Mirasoli, E. Michelini, M. Musiani, Bio- and chemiluminescence imaging in analytical chemistry, Anal. Chim. Acta 541 (2005) 25–36. [9] N. Hananya, A.E. Boock, C.R. Bauer, R. Satchi-Fainaro, D. Shabat, Remarkable enhancement of chemiluminescent signal by dioxetane-fluorophore conjugates: turn-on chemiluminescence probes with color modulation for sensing and imaging, J. Am. Chem. Soc. 138 (2016) 13438–13446. [10] C.M. Magalhães, J.C.G. Esteves da Silva, L. Pinto da Silva, Chemiluminescence and bioluminescence as an excitation source in the photodynamic therapy of cancer: a critical review, ChemPhysChem 17 (2016) 2286–2294. [11] M. Cronin, A.R. Akin, K.P. Francis, M. Tangney, In vivo bioluminescence imaging of intratumoral bacteria, Methods Mol. Biol. 1409 (2016) 69–77. [12] K.M. Grinstead, L. Rowe, C.M. Ensor, S. Joel, P. Daftarian, E. Dikici, J.M. Zingg, S. Daunert, Red-shifted aequorin variants incorporating non-canonical amino acids: applications in in vivo imaging, PLoS One 11 (2016) e0158579. [13] M.J. Hsu, J. Prigent, P.E. Dollet, J. Ravau, L. Larbanoix, G. Van Simaeys, A. Bol, S. Goldman, G. Deblandre, M. Najimi, E. Sokal, C. Lombard, Long-term in vivo monitoring of adult-derived human liver stem/progenitor cells by bioluminescence imaging, positron emission tomography, and contrast-enhanced computed tomography, Stem Cells Dev. (2017), http://dx.doi.org/10.1089/scd.2016.0338. [14] T. Wimmer, B. Lorenz, K. Stieger, Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor, Biosens. Bioelectron. 86 (2016) 609–615. [15] B.R. Branchini, C.E. Behney, T.L. Southworth, D.M. Fontaine, A.M. Gulick, D.J. Vinyard, G.W. Brudvig, Experimental support for a single electron-transfer oxidation mechanism in firefly bioluminescence, J. Am. Chem. Soc. 137 (2015) 7592–7595. [16] M.Y. Wang, Y.J. Liu, Theoretical study of dinoflagellate bioluminescence, Photochem. Photobiol. 93 (2017) 511–518. [17] Z.M. Kaskova, F.A. Dorr, V.N. Petushkov, K.V. Purtov, A.S. Tsarkova, N.S. Rodionova, K.S. Mineev, E.B. Guglya, A. Kotlobay, N.S. Baleeva, M.S. Baranov, A.S. Arseniev, J.I. Gitelson, S. Lukyanov, Y. Suzuki, S. Kanie, E. Pinto, P. Di Mascio, H.E. Waldenmaier, T.A. Pereira, R.P. Carvalho, A.G. Oliveira, Y. Oba, E.L. Bastos, C.V. Stevani, I.V. Yampolsky, Mechanism and color modulation of fungal bioluminescence, Sci. Adv. 3 (2017) e1602847. [18] M.A. Dubinnyi, Z.M. Kaskova, N.S. Rodionova, M.S. Baranov, A.Y. Gorokhovatsky, A. Kotlobay, K.M. Solntsev, A.S. Tsarkova, V.N. Petushkov, I.V. Yampolsky, Novel mechanism of bioluminescence: oxidative decarboxylation of a moiety adjacent to the light emitter of fridericia luciferin, Angew. Chem. Int. Ed. 54 (2015) 7065–7067. [19] S.H.D. Haddock, M.A. Moline, J.F. Case, Bioluminescence in the sea, Annu. Rev. Mar. Sci. 2 (2010) 443–493. [20] E.A. Widder, Bioluminescence in the ocean: origins of biological, chemical, and ecological diversity, Science 328 (2010) 704–708. [21] K. Teranishi, Luminescence of imidazo[1,2-a]pyrazin-3(7H)-one compounds, Bioorg. Chem. 35 (2007) 82–111. [22] T. Hirano, Y. Takahashi, H. Kondo, S. Maki, S. Kojima, H. Ikeda, H. Niwa, The reaction mechanism for the high quantum yield of Cypridina (Vargula) bioluminescence supported by the chemiluminescence of 6-aryl-2-methylimidazo[1,2-a] pyrazine-3(7H)-ones (Cypridina luciferin analogues), Photochem. Photobiol. Sci. 7 (2008) 197–207. [23] Y. Takahashi, H. Kondo, S. Maki, H. Niwa, H. Ikeda, T. Hirano, Chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazine-3(7H)-ones in DMSO/TMG and in diglyme/acetate buffer: support for the chemiexcitation process to generate the singlet-excited state of neutral oxyluciferin in a high quantum yield in the Cypridina (Vargula) bioluminescence mechanism, Tetrahedron Lett. 47 (2006) 6057–6061. [24] R. Saito, T. Hirano, S. Maki, H. Niwa, Synthesis and chemiluminescent properties of 6,8-diaryl-2-methylimidazo[1,2-alpha]pyrazine-3(7H)-ones: systematic investigation of substituent effect at para-position of phenyl group at 8-position, J. Photochem. Photobiol. A 293 (2014) 12–25. [25] F.I. Tsuji, ATP-dependent bioluminescence in the firefly squid, Watasenia-scintillans, Proc. Natl. Acad. Sci. USA 82 (1985) 4629–4632. [26] F.I. Tsuji, G.B. Leisman, K+-Na+-triggered bioluminescence in the oceanic squid
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Combined experimental and theoretical study of Coelenterazine chemiluminescence in aqueous solution João M. Lourençoa, Joaquim C.G. Esteves da Silvab,c, Luís Pinto da Silvaa,c,
MARK
⁎
a Chemistry Research Unit (CIQUP), Department of Chemistry and Biochemistry, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal b 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 c LACOMEPHI, Department of Geosciences, Environment and Territorial Planning, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Keywords: Coelenterazine Chemiluminescence Bioluminescence Superoxide Anion Aqueous Solution Luciferase Imidazopyrazinone
Coelenterazine is a common substrate used by marine species in enzyme-catalyzed bioluminescent reactions, in which thermal energy is converted into light-emission. Besides bioluminescence, Coelenterazine is also known to emit chemiluminescence in aprotic solvents. We report here the study of Coelenterazine chemiluminescence in aqueous solution. Water inhibits light-emission even in mixtures with water content as low as 20%. Moreover, we provide convincing spectroscopic evidence that the presence of water affects the ground state (S0) chemical reaction, and not the excited state processes (as chemiexcitation and the fluorescent quantum yield). However, the energetics of the S0 chemical reaction is not affect by addition of water, which points to the inhibition being caused by the reduced lifetime of superoxide anion in water, which is an intermediate in the luminescent reactions of Coelenterazine. This finding indicates that one of the catalytic roles of bioluminescent enzymes is to extend the lifetime of this radical.
1. Introduction Bioluminescence consists on the conversion of thermal energy into excitation energy, thereby leading to light emission [1–4], and has been attracting attention from the research community due to high quantum yields and high signal-to-noise ratio [5–8]. Furthermore, in bioluminescent reactions there is no autofluorescence arising from the background signal [9], the lack of light excitation also eliminates problems regarding light-penetration into biologic tissues (except for emission) [10]. These features make bioluminescent systems very helpful tools in the real-time and noninvasive imaging of target molecules in vivo [11–14], as well as potential excitation sources for self-illuminating systems in photodynamic therapy of cancer [10]. More than 700 genera have been found to produce bioluminescence, in organisms as diverse as the fireflies, fungi, earthworms, fishes and bacteria, among others [15–18]. However, about 80% of all bioluminescent organisms can be found in the ocean [19,20]. Moreover, the majority of the marine organisms react with the same bioluminescent substrate: Coelenterazine (Scheme 1) [3,4,10,21]. The bioluminescent reaction involving Coelenterazine proceeds as follows (Scheme 1)
[22–24]: the first step is the oxygenation of the imidazopyrazinone scaffold of Coelenterazine, which leads to the quick formation of the 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 (Coelenteramide) in its first singlet excited state (S1). Typical Coelenterazine-based bioluminescent reactions involve the presence of an enzyme named luciferase, which catalyzes the oxygenation step of Coelenterazine [3,4]. As Coelenterazine, these luciferases can be found in a great variety of marine organisms, such the decapod shrimp Oplophorus gracilirostris, the anthozoan Renilla reniformis, and of the copecods Gaussian princeps and Metridia longa [3,4]. Currently, the Gaussian and Renilla reniformis luciferases are the ones with the highest number of practical applications [3,4]. It should be noted that the imidazopyrazinone core of Coelenterazine is a common link among marine luminescent substrates, and so, its bioluminescent mechanism is the same for many other marine organisms. In fact, the same imidazopyrazinone core can be found in Cypridina luciferin (present in the sea firefly Cypridina hilgendorfii), dehydrocoelenterazine (present in the squid Symplectoteuthis oualaniensis) and in Watasenia luciferin (present in the squid Watasenia
⁎ Corresponding author at: Chemistry Research Unit (CIQUP), Department of Chemistry and Biochemistry, Faculty of Sciences of University of Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. E-mail address: [email protected] (L. Pinto da Silva).
http://dx.doi.org/10.1016/j.jlumin.2017.10.025 Received 11 July 2017; Received in revised form 3 October 2017; Accepted 10 October 2017 Available online 12 October 2017 0022-2313/ © 2017 Elsevier B.V. All rights reserved.
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Scheme 1. Reaction mechanism of Coelenterazine luminescence.
dynamic sensor and not an accumulation one. This means that when the chemiluminescent reaction between the probe and the target ROS species occurs and light is emitted, no more photons can be produced from those reactions. Coelenterazine also has the advantage of exhibiting chemiluminescence independently from cell-derived myeloperoxidase, and by not sensitizing O2- (which limits the occurrence of false-positives) [36]. Given this, the ROS-mediated chemiluminescence indicates that water by itself does not affect negatively the chemiluminescence quantum yield of Coelenterazine, and that chemiluminescence may also occur in aqueous solution (in the presence of 3O2). However, that would mean that the chemiluminescence reaction of Coelenterazine would compete with its bioluminescent reaction (as this last reaction occurs in aqueous solution), which should affect the efficiency of the latter process in its various practical applications (such as bioimaging and bioanalysis). Thus, it is essential to determine if Coelenterazine can also produce chemiluminescence in aqueous solutions (in the absence of ROS), and if so, to determine the efficiency of this process. Herein, we report such study by using a spectroscopic approach to assess the potential of Coelenterazine for chemiluminescence in aqueous solution. The chemiluminescence in DMSO, an aprotic solvent typically used as a model solution for the bioluminescent reaction, was also measured so to serve as comparison. This approach allowed us to obtain valuable insight into the luminescence of Coelenterazine and related imidazopyrazinone-based ones, which can be found in about eight phyla of luminescent organisms.
scintillans) [3,25,26]. Besides bioluminescence, Coelenterazine and related imidazopyrazinone-based substrates are also able to emit chemiluminescence in aprotic solvents (as DMSO and diglyme) without an enzyme, in an identical mechanism to that of the bioluminescent reaction [22–24,27–29]. The most significant difference between chemiluminescence and bioluminescence is the higher quantum yield of the latter process, which is to be expected due to the absence of the catalytic luciferase in chemiluminescence [22–24,27–29]. Due to these similarities, chemiluminescence has been used as a model for the study of the bioluminescence reaction [22–24,27–29]. These studies have found that Coelenterazine presents appreciable chemiluminescence quantum yields (between 0.0021 and 0.005) in DMSO, N,N-dimethylformamide (DMF) and hexamethylphosphoric triamide, in the absence of base [21]. Addition of base and acidic buffers decreased the measured quantum yield [21]. Contrary to aprotic solvents (mainly DMSO) [21–24], the chemiluminescence of Coelenterazine in aqueous solutions has been sparsely studied, despite it being the solvent in which bioluminescence takes place (and consequently, the practical applications of this system). Nevertheless, some studies regarding other imidazopyrazinone-based substrates found the chemiluminescence in water to be extremely weak [30]. One explanation for this behavior was provided by Shimomura, who have found the fluorescence of Cypridina oxyluciferin to be very weak in aqueous solution [31]. Other authors have studied the chemiluminescence of different imidazopyrazinones in water/DMF and methanol/DMF mixtures, and have found that high contents of water greatly decrease the chemiluminescence efficiency [32]. These results indicate that water should affect negatively the chemiluminescence quantum yield of Coelenterazine, which is composed by three parameters: the yield of the S0 reaction; the efficiency of the S0 → S1 chemiexcitation; the fluorescence quantum yield of the chemiluminophore [1,2]. However, this potentially negative effect exerted by water is not in line with the known reactive oxygen species (ROS)-induced Coelenterazine chemiluminescence in water. Besides molecular oxygen (3O2), the oxygenation step of Coelenterazine chemiluminescence can also be triggered by different ROS, such as superoxide anion (O2-) and singlet oxygen (1O2). In fact, Coelenterazine and other imidazopyrazinone molecules have been used with good results as dynamic probes for ROS in biological media (both in vivo and in vitro) [21,33–35]. Contrary to fluorescent ROS sensors, Coelenterazine is a
2. Experimental methods Coelenterazine was purchased from NanoLight™ Technology, and was dissolved in methanol and stored at −20 °C. 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°) and were performed at least in triplicate. The reactions were carried out in pure DMSO or in DMSO/water mixtures. The reaction was initiated by the injection of the DMSO (or DMSO/water mixture) solution (380 μL) into an assay tube containing Coelenterazine (20 μL). The concentration of Coelenterazine in the final solution is of 3.0 μM. The light was integrated and recorded in 0.1 s intervals. Resulting data was analyzed by using the Graphpad software package (Version 7.03 for Windows). UV–Vis spectra were measured 140
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Table 1 Initial velocities (v, in RLU s−1) and light-emission maximum (in RLU) for Coelenterazine chemiluminescence in DMSO and DMSO/water mixtures. Solvent
Light-emission maximum
v
DMSO DMSO/Water (99%/1%) DMSO/Water (95%/5%) DMSO/Water (90%/10%)
33965 ± 2443 30629 ± 1170 12090 ± 1321 5245 ± 518
37676 ± 3520 28787 ± 836 11268 ± 1539 3728 ± 640
with a UV-3100PC Spectrophotometer. Quartz cells were used. Concentrations and volumes were the same as for the chemiluminescent kinetic assays. 3. Results and discussion We have started by studying the chemiluminescence of Coelenterazine in solutions of pure DMSO and of DMSO/water mixtures (99%/1%, 95%/5% and 90%/10%). The chemiluminescent kinetics were followed by the measurement of direct light-emission intensity. The kinetic curves gave rise to initial velocities (v, in relative light units [RLU] s−1) and to the maximum of light intensity (in RLU) [37,38]. These values were obtained by linear fitting [37,38]. The values for light-emission maximum and v, in those solutions, can be found in Table 1. While in pure DMSO the light-emission maximum of chemiluminescence is appreciable (in line with previous results) [21], addition of only limited amounts of water had a strong inhibition effect. Inhibition was promptly seen even in mixtures containing 1% of water, with solutions containing 10% of water presenting light-emission intensities of only 15% of that observed in pure DMSO. Increasing the quantity of water to 20% quenched completely the chemiluminescence produced by Coelenterazine (data not shown). Thus, these results indicate that chemiluminescence should not occur in pure aqueous solutions, in line with experimental studies made for other imidazopyrazinones [30,32]. Having reached this conclusion, is not clear in what way water quenches the emission of chemiluminescence. That is, if water affects the S0 chemical reaction, the chemiexcitation step or the quantum yield of the chemiluminophore. One way to understand the reason behind this effect is to compare the behavior of the light-emission maximum, as a function of water content, with that of v. As v results from the increase of light as a function of time, addition of water automatically results in a decrease of this parameter (Table 1). However, if the decrease of lightintensity results from the fluorescent quantum yield of the emitter or from the yield of S0 → S1 non-radiative crossing, the slope of the correlation of v with water should be smaller than that of the light-emission maximum. This occurs because both the fluorescent quantum yield and S0 → S1 transition are excited state processes that do not have an effect on the speed of the reaction. For the contrary, the activation barriers present in the S0 reaction determine the speed of reaction, and so, if water increases these barriers high enough to inhibit the S0 reaction, it should decrease the light-emission maximum and v in similar magnitudes.
Fig. 2. UV–Vis spectrum of Coelenterazine in DMSO (a) and water (b), at 1, 5 and 10 min. The pH of these solutions was adjusted by addition of phosphate buffer pH 7.4 (75 mM).
The slopes were obtained by exponential fitting of the correlation found between v/light-emission maximum and DMSO/water ratio (Fig. 1), which were of 0.26 ± 0.02 and 0.20 ± 0.01, respectively. The performing of an unpaired t-test showed the difference between the slopes to be statistically significant, with a two-tailed p value of 0.0073 with a 95% confidence interval. Interestingly, the fact that the slope of the correlation between v and DMSO/water ratio is bigger than that of the slope involving the light-emission maximum, indicates that the negative effect exerted by water is stronger on the velocity of the reaction (controlled by the S0 reaction) than in the emission of light (controlled both by the S0 reaction and by excited state processes). Thus, these results indicate that the inhibiting effect of water is caused by affecting the S0 reaction. This can be explained by increasing the activation barrier of one or more steps of the S0 reaction to values high enough to inhibit the S0 reaction yield, and so, decrease the emission of bioluminescence and the speed of reaction. In order to provide further support for this conclusion, we have studied the UV–Vis spectrum of Coelenterazine in DMSO and water solutions (Fig. 2). With UV–Vis spectroscopy we can observe the formation of the products and consumption of reactants during the chemiluminescent reaction, irrespectively of the amount of emitted light. Thus, if the inhibition of chemiluminescence exerted by water is caused by affecting the S0 chemical reaction, thereby meaning that Coelenterazine is not consumed, the UV–Vis spectrum in water should be different from that measured in DMSO (which should be composed by both Coelenterazine and Coelenteramide). If the water-induced inhibition is caused by excited state processes the spectra should be similar, as Coelenterazine is consumed and Coelenteramide produced in both solvents. Significant differences were observed between the UV–Vis spectra of Fig. 1. Correlation between v (a) or light-emission maximum (b) with the percentage of water in DMSO/water mixtures.
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IC (100 μM) alone. Also, the addition of both 5% water and IC (10 μM) to pure DMSO lead to the same decrease obtained when only 5% water is added, about 90%. This similar effect exerted by IC and water on light-emission indicates that both molecules affect the same step of the chemiluminescent reaction. As, due to its nature as a redox indicator, IC is expected to interfere with the oxygenation step, it is safe to conclude that water also interferes with this step. Given these results, we have performed theoretical calculations based on Density Functional Theory (DFT) in order to determine the energetics of the SET reaction between anionic/neutral Coelenterazine and 3O2 (Scheme 2). Each structure was optimized at the ωB97XD/631G(d) level of theory [46], in either DMSO or water by using an implicit solvent model (IEFPCM) [47]. Vibrational frequency calculations made at the same level of theory allowed us to obtain the Gibbs free energy for each structure with thermal corrections (Gtherm). All calculations were made with the Gaussian 09 program package [48]. The ΔGtherm for both SET reactions, in both solvents, is endergonic. Particularly for SET between anionic Coelenterazine and 3O2. This can be considered to be in line with the finding that SET is the rate-determining step of the chemiluminescent reaction of imidazopyrazinonebased compounds [29]. Nevertheless, what was interesting to find is that the SET reaction presents very similar ΔGtherm in both DMSO and water, thereby indicating that water does not quench the emission of chemiluminescence by impairing the rate-determining step of this reaction. As SET is seemingly not affected by water, we have focused on the next step of the chemiluminescent reaction: the recombination of Coelenterazine and O2- radicals to form the dioxetanone intermediate [21,29,44]. As O2- interacts with the imidazopyrazinone core of radical Coelenterazine, we have calculated the atomic Mulliken charge of this moiety in DMSO (−0.12e) and in water (−0.12e), at the ωB97XD/631G(d) level of theory. We have also calculated the Mulliken spin density of this moiety in DMSO (1.00) and water (1.00), at the same level of theory. As both the charge and spin density of the imidazopyrazinone core of radical Coelenterazine are identical in both solvents, it is safe to conclude that water does also not affect the energetic of the radical recombination step. Nevertheless, we have analyzed the Mulliken spin density (in water and DMSO) of carbon atoms present in in the imidazopyrazinone core of radical Coelenterazine (Scheme 3), and which can act as potential sites of interaction with O2- for the radical recombination step [21,49,50]. However, the spin density is very similar between the two solvents, and the carbon atom with the higher spin density (thereby the one most prone to interact with O2-) is the one that leads to the formation of the dioxetanone intermediate [21,49,50]. Thus, this analysis provides further indication that water does not affect the energetic of the oxygenation step. Besides these calculations, we have also determined the theoretical oxidation potential of Coelenterazine, by following the protocol developed by Winget and co-workers [51]:
Coelenterazine in water and DMSO (Fig. 2). In DMSO, two different peaks can be seen: one at ~ 280 nm and other at 340 nm. Interestingly, the intensity of the first peak (~ 280 nm) decreases with increasing time, while the opposite behavior is seen for the second peak (~ 340 nm). This indicates that the first peak can be attributed to Coelenterazine, which is consumed during the chemiluminescent reaction, while the second one corresponds to Coelenteramide, which is the chemiluminescent product. This is supported by previous experimental work. Kudryasheva and co-workers defined the absorption region of Coelenteramide to be between 310 and 380 [39], while Shimomura and Teranishi found that Coelenteramide and analogs possess an excitation maximum at ~ 340 nm in DMSO [40]. Furthermore, Coelenterazine has been found to possess an experimental absorption maximum at 275 nm [41]. For the contrary, the UV–Vis spectrum in water is dominated by a single peak at ~ 265 nm, which intensity decreases very slightly as the time passes by. This peak can be attributed to Coelenterazine, and its limited decreased indicates that the S0 chemical reaction is inhibited in aqueous solution. The data obtained so far indicate that Coelenterazine cannot emit chemiluminescence in aqueous solution (to the contrary of in DMSO and other aprotic solvents), because water inhibits the S0 chemical reaction probably by increasing the activation barrier of one or more of its steps. As referred before, the S0 chemical reaction is composed by two sequential steps: oxygenation of the imidazopyrazinone core, and thermolysis of the dioxetanone intermediate (Scheme 1) [3,4,10,21]. Currently, there is a lack of experimental data regarding the thermolysis step, due to the high instability of the dioxetanone intermediate. However, different theoretical studies have demonstrated that the activation barrier for the thermolysis of imidazopyrazinone-based dioxetanones decreases with increasing polarity of the solvent [42,43]. Given this, the inhibitory effect exerted by water cannot be due to an increase of the activation barrier for dioxetanone thermolysis, as water is more polar than DMSO and other aprotic solvents used to generate chemiluminescence (as diglyme). Thus, it appears that the reason behind this inhibitory effect is related to the oxygenation step, which consists in a single electron transfer (SET) from Coelenterazine to 3O2 [44]. This reaction generates a Coelenterazine radical and O2-, which recombine latter to form the dioxetanone intermediate. Such SET-based mechanism was also found to be involved in firefly bioluminescence [45]. One possibility to explain this phenomenon when thinking of the oxygenation step (which is a redox reaction), is that Coelenterazine might be more easily oxidized in DMSO (and other aprotic solvents) than in water. So, to obtain more insight into this, we have measured the direct light-emission from Coelenterazine chemiluminescence in the presence of Indigo Carmine (IC), a known redox indicator, in both pure DMSO and in a DMSO/water (95%/5%) mixture. The light-intensity maxima obtained are present in Table 2. Addition of IC (100 and 10 μM) to pure DMSO led to significant quenching of light emission, by about 98% and 60%, respectively. This quenching effect provided by IC (particularly at a concentration of 100 μM) is very similar to what is obtained by addition of 5% water to a DMSO solution, which led to a quenching of about 90% of light-emission. Moreover, the addition of both 5% water and IC (100 μM) to pure DMSO led to a decrease in lightemission of 99%, very similar to the decrease of 98% in the presence of
° ∆Gox = IP+∆Gevr , gas +∆∆Gsolv
IP is the adiabatic ionization potential, included with the difference in zero-point vibrational energy. ∆Gevr , gas is the difference in thermal contributions to Coelenterazine free energy deriving from changes in the electronic, vibrational and rotational partition functions upon ionization. ∆∆Gsolv is the difference between the free energy of solvation of anionic and oxidized forms of Coelenterazine. These calculations were made at the ωB97XD/6-31G(d,p) level of theory [46], in either implicit DMSO or water by using the IEFPCM solvent model [47]. The oxidation potential of Coelenterazine in implicit water (5.65 eV) and DMSO (5.63 eV) was very similar, which supports our previous conclusion that water should not inhibit the oxidation of Coelenterazine. The addition of four explicit water molecules (placed near the four heteroatoms of the imidazopyrazinone core, Fig. 3a) even
Table 2 Light-emission maximum (in RLU) for Coelenterazine chemiluminescence in DMSO and DMSO/water mixtures, and with the addition of IC. Solution DMSO DMSO DMSO DMSO DMSO DMSO
+ + + + +
Light-Emission Maximum
IC (10 μM) IC (100 μM) Water (5%) IC (10 μM) + Water (5%) IC (100 μM) + Water (5%)
(1)
67034 ± 4347 26763 ± 2061 1433 ± 355 6451 ± 1506 6420 ± 2437 486 ± 69
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Scheme 2. Schematic representation of the SET reaction between neutral and anionic Coelenterazine, with the corresponding theoretical ΔGtherm in water (DMSO), at the ωB97XD/6-31G(d) level of theory.
of the effect of explicit solvation in water and DMSO, the oxidation potential of Coelenterazine was once again calculated in both solvents, but with a water/DMSO explicit molecule placed near the deprotonated nitrogen heteroatom (Fig. 3). Two different conformations were found for the interaction between DMSO and Coelenterazine (Fig. 3c and d). The resulting potentials were of 3.69 eV (in water) and 3.67 (Fig. 3c)/ 3.66 (Fig. 3d) eV (in DMSO). These values lead us to two different conclusions. One is that explicit solvation does affect the oxidation potential, in which solvation of the deprotonated nitrogen heteroatom leads to a significant decrease in the oxidation potential. The second conclusion, is that addition of explicit solvation does not appear to create a significant difference between the ability of Coelenterazine to be oxidized in water and in DMSO. Nevertheless, as Coelenterazine is a negatively charged molecule and its ionization leads to the formation of a neutral radical, it might be
Scheme 3. Mulliken spin density of key carbon atoms present in the imidazopyrazinone core of radical Coelenterazine in water (DMSO), at the ωB97XD/6–31 G(d) level of theory.
decreased the oxidation potential of Coelenterazine in water to 4.96 eV, which indicates that explicit aqueous solvation does not impair the oxidation of Coelenterazine (to the contrary). For a better comparison
Fig. 3. Geometries optimized at the ωB97XD/6-31G(d,p) level of theory, of Coelenterazine in the presence of four explicit water molecules (a), one explicit water molecule (b), and one explicit DMSO molecule (c and d).
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This solvent-dependent lifetime of O2- is in line with the chemiluminescence efficiency of Coelenterazine. This imidazopyrazinone does not emit chemiluminescence in water (as demonstrated here) and in methanol (a solvent used to prepare stock solutions of Coelenterazine) [22–24,29], which are protic solvents. For the contrary, chemiluminescence is found to be emitted by Coelenterazine (and other imidazopyrazinone-based molecules) with non-negligible efficiencies in different aprotic solvents, such as DMSO, diglyme, DMF and hexamethylphosphoric triamide [21–24,29]. Therefore, this information indicates that the inhibitory effect exerted by water on the chemiluminescence emitted by Coelenterazine is caused by decreasing the lifetime of O2-, an intermediate of this reaction, and not by water inhibiting directly any one of the two steps composing the S0 chemical reaction. So, this conclusion points to one of the catalytic functions of luciferases using Coelenterazines to be extending the lifetime of O2-, inside their active sites. Nevertheless, further study of the chemiluminescent reaction of Coelenterazine in aqueous solutions might be useful for a better characterization of this system.
Table 3 Atomic Mulliken charges of the imidazopyrazinone core of Coelenterazine (in its neutral and anionic forms), either solely in implicit water/DMSO or in the presence of one explicit water/DMSO molecule. The calculations were made at the ωB97XD/6–31 G(d,p) level of theory. Water Anion
DMSO Radical
Implicit Solvation −0.849 −0.143 Explicit Solvation −0.827 −0.121
Anion
Radical
−0.849
−0.143
−0.862 (−0.848)a
−0.168 (−0.150)a
a The values between parenthesis refer to the structure depicted in Fig. 3d, while the other ones refer to the structure depicted in Fig. 3c.
expected that the anion could be more stabilized in more polar solvents as compared to the radical. If that was the case, the oxidation potential of Coelenterazine should be higher in water than in DMSO. So, we have calculated the atomic Mulliken charge of the imidazopyrazinone core of Coelenterazine, its anionic and radical forms, in implicit and explicit water/DMSO (Table 3). These calculations show that the decrease in negative charge due to ET from the imidazopyrazinone core to 3O2 is very similar in all conditions (~0.70e). This might indicate that the imidazopyrazinone core is not significantly affected by differences in polarity between water and DMSO. Besides assessing the theoretical oxidation potential of Coelenterazine, we have also calculated the theoretical reduction potential of 3O2, to form the O2- radical. The reduction potential was obtained by modifying the protocol devised by Winget and co-workers for oxidation potentials [51]: ° ∆Gred = EA+∆Gevr , gas +∆∆Gsolv
4. Conclusion Coelenterazine is common marine chemi- and bioluminescent substrate found in different organisms, and possesses an imidazopyrazinone core that links together molecules present in about eight phyla of luminescent organisms. Moreover, Coelenterazine-based chemi- and bioluminescence has become a powerful tool in real-time bioimaging, bioanalysis and in biomedicine. Herein, we report a combined experimental and theoretical study of Coelenterazine chemiluminescence in aqueous solution. While chemiluminescence has been extensively studied in aprotic media, it has not been sufficiently characterized in aqueous solution, which is a medium where bioluminescence (and its practical applications) occur. Luminescent assays demonstrate that water has a very strong inhibitory effect on chemiluminescence, being able to quench light emission in mixtures with water content as low as 20%. Moreover, this inhibitory effect is caused by affecting the S0 chemical reaction, and not by impairing excited state processes. However, water was not found to have a significant effect on the steps composing the S0 reaction, which means that the inhibitory effect is not caused by affecting directly the chemiluminescent reaction. The inhibition was then attributed to the lifetime of the O2- radical, which is significantly lower in water than in aprotic solvents, in which Coelenterazine has been found to emit chemiluminescence with nonnegligible efficiency. This finding indicates that one of the catalytic functions provided by bioluminescent enzymes is to extend the lifetime of the O2- radical in their active sites.
(2)
In this case, IP was substituted by the adiabatic electron affinity (EA), included with the difference in zero-point vibrational energy. These calculations were made at the ωB97XD/6-31G(d,p) level of theory, in either implicit water/DMSO or with the presence of one explicit water/DMSO molecule. The reduction potential of 3O2 is very similar in implicit water (−2.69 eV) and DMSO (−2.66 eV). The addition of explicit solvent molecules increases the reduction potential of this molecule (−2.96 eV and −2.92 eV, respectively), but does not cause any significant difference between solvents. In conclusion, Coelenterazine does not emit chemiluminescence in aqueous solution (to the contrary to what is observed in aprotic solvents), as water exerts a strong inhibitory effect on the chemiluminescent reaction of this molecule. This effect arises from the inhibition of the S0 chemical reaction, and not by impairing excited state processes (as the chemiexcitation step or the S1 → S0 radiative transition). However, previous theoretical calculations have shown that water decreases the activation barrier for the thermolysis of dioxetanone [42,43], while the present study indicates that water does not affect the energetic of the oxygenation step. Thus, while water inhibits the S0 chemical reaction, which is composed by the oxygenation step and dioxetanone thermolysis, it apparently does not inhibit by itself any one of these steps. Given this, it is possible that the explanation for this process must be found outside of chemiluminescent reaction. One of the possibilities is the lifetime of the O2- radical. The lifetime of this radical is considerably greater in aprotic solvents than in protic and aqueous solutions, due to strong solvation and spontaneous disproportionation in the latter class of solvents [52,53]. In fact, O2- is generally generated in aprotic media in order for studying its reactions with organic and inorganic compounds [54]. O2- disproportionate in water by forming 3 O2, and the hydroxyl and hydroperoxide anions [52,53], as seen below:
2O2− + H2 O → O2 + HO2− + OH−
Acknowledgments This work was made in the framework of project PTDC/QEQ-QFI/ 0289/2014, which is funded with national funds by FCT/MEC (PIDDAC) and 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. Acknowledgement to project POCI-010145-FEDER-006980 funded by FEDER through COMPETE2020 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 Pollutants-Human Interactions (LACOMEPHI) is acknowledged.
Notes
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The authors declare no competing financial interests. 144
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Symplectoteuthis-oualaniensis, Proc. Natl. Acad. Sci. USA 78 (1981) 6719–6723. [27] T. Goto, H. Fukatsu, Cypridina Bioluminescence. 7. Chemiluminescence in micelle solutions – a model system for cypridina bioluminescence, Tetrahedron Lett. 49 (1969) 4299–4302. [28] K. Usami, M. Isobe, Low-temperature photooxygenation of coelenterate luciferin analog synthesis and proof of 1,2-dioxetanone as luminescence intermediate, Tetrahedron 52 (1996) 12061–12090. [29] H. Kondo, T. Igarashi, S. Maki, H. Niwa, H. Ikeda, T. Hirano, Substituent effects on the kinetics for the chemiluminescence reaction of 6-arylimidazo[1,2-a]pyrazine3(7H)-ones (Cypridina luciferin analogues): support for the single electron transfer (SET)-oxygenation mechanism with triplet molecule oxygen, Tetrahedron Lett. 46 (2005) 7701–7704. [30] T. Goto, Chemistry of bioluminescence, Pure Appl. Chem. 17 (1968) 421–441. [31] O. Shimomura, F.H. Johnson, T. Masugi, Cypridina bioluminescence – light-emitting oxyluciferin-luciferase complex, Science 164 (1969) 1299–1300. [32] K. Teranishi, M. Hisamatsu, T. Yamada, Chemiluminescence of 2-methyl-6-arylimidazo[1,2-a]pyrazine-3(7H)-one in protic solvents: electron-donating substituent effect on the formaton of the neutral singlet excited-state molecule, Luminescence 14 (1999) 297–302. [33] L.L. Bronsart, C. Stokes, C.H. Contag, Chemiluminescence imaging of superoxide anion detects beta-cell function and mass, PLoS One 11 (2016) e0146601. [34] L.L. Bronsart, C. Stokes, C.H. Contag, Multimodality imaging of cancer superoxide anion using the small molecule Coelenterazine, Mol. Imaging Biol. 18 (2016) 166–171. [35] L.L. Bronsart, L. Nguyen, A. Habtezion, C.H. Contag, Reactive oxygen species imaging in a mouse model of inflammatory bowel disease, Mol. Imaging Biol. 18 (2016) 473–478. [36] M.M. Tarpey, C.R. White, E. Suarez, G. Richardson, R. Radi, B. Freeman, Chemiluminescent detection of oxidants in vascular tissue – lucigenin but not coelenterazine enhances superoxide formation, Circ. Res. 84 (1999) 1203–1211. [37] L. Pinto da Silva, J.C.G. Esteves da Silva, Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and l-luciferin, Photochem. Photobiol. Sci. 10 (2011) 1039–1045. [38] C. Ribeiro, J.C.G. Esteves da Silva, Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate, Photochem. Photobiol. Sci. 7 (2008) 1085–1090. [39] R. Alieva, N.V. Belogurova, A.S. Petrova, N.S. Kudryasheva, Fluorescence properties of Ca2+-independent discharged obelin and its application prospects, Anal. Bioanal. Chem. 405 (2013) 3351–3358. [40] O. Shimomura, K. Teranishi, Light-emitters involved in the luminescence of coelenterazine, Luminescence 15 (2000) 51–58. [41] E.V. Eremeeva, S.V. Markova, A.H. Westphal, A.J.W.G. Visser, W.J.H. van Berkel, E.S. Vysotski, The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding, FEBS Lett. 583 (2009) 1939–1944. [42] L. Pinto da Silva, C.M. Magalhães, J.C.G. Esteves da Silva, Interstate crossing-induced chemiexcitation mechanism as the basis for imidazopyrazinone bioluminescence, ChemistrySelect 1 (2016) 3343–3356. [43] B.W. Ding, P. Naumov, Y.J. Liu, Mechanistic Insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea firefly) lumophore, J. Chem. Theory Comput. 11 (2015) 591–599. [44] B.W. Ding, Y.J. Liu, Bioluminescence of firefly squid via mechanism of single electron-transfer oxygenation and charge-transfer-induced luminescence, J. Am. Chem. Soc. 139 (2017) 1106–1119. [45] B.R. Branchini, C.E. Behney, T.L. Southworth, D.M. Fontaine, A.M. Gulick, D.J. Vinyard, G.W. Brudvig, Experimental support for a single electron-transfer oxidation mechanism in firefly bioluminescence, J. Am. Chem. Soc. 137 (2015) 7592–7595. [46] J.D. Chai, M. Head-Gordon, Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections, Phys. Chem. Chem. Phys. 10 (2008) 6615–6620. [47] G. Scalmani, M.J. Frisch, Continuous surface change polarizable continuum models of solvation. I. General formalism, J. Chem. Phys. 132 (2010) 114110. [48] M.J. Frisch, et al., Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford CT, 2009. [49] K. Fujimori, H. Nakajima, K. Akutsu, M. Mitani, H. Sawada, M. Nakayama, Chemiluminescence of Cypridina luciferin analogues. Part 1. Effect of pH on rates of spontaneous autoxidation of CLA in aqueous buffer solutions, J. Chem. Soc. Perkin Trans. 2 (1993) 2405–2409. [50] K. Akutsu, H. Nakajima, T. Katoh, S. Kino, K. Fujimori, Chemiluminescence of Cipridina luciferin analogues. Part 2. Kinetic studies on the reaction of 2-methyl-6phenylimidazo[1,2-a]pyrazine-3(7H)-one (CLA) with superoxide: hydroperoxyl radical is an actual active species used to initiate the reaction, J. Chem. Soc. Perkin Trans. 2 (1995) 1699–1706. [51] P. Winget, E.J. Weber, C.J. Cramer, D.G. Truhlar, Computational electrochemistry: aqueous one-electron oxidation potentials for substituted anilines, Phys. Chem. Chem. Phys. 2 (2000) 1231–1239. [52] M. Hayyan, M.A. Hashim, I.M. AlNashef, Superoxide ion: generation and chemical implications, Chem. Rev. 116 (2016) 3029–3085. [53] D.T. Sawyer, J.S. Valentine, How super is superoxide? Acc. Chem. Res. 14 (1981) 393–400. [54] I.B. Afanas’ev, Superoxide Ion: Chemistry and Biological Implications, 1 CRC Press, Boca Raton, FL, 1989.
References [1] I. Navizet, Y.J. Liu, N. Ferré, D. Roca-Sanjuán, R. Lindh, The chemistry of bioluminescence: an analysis of chemical functionalities, ChemPhysChem 12 (2011) 3064–3076. [2] L. Pinto da Silva, J.C.G. Esteves da Silva, Firefly chemiluminescence and bioluminescence: efficient generation of excited states, ChemPhysChem 13 (2012) 2257–2262. [3] Z.M. Kaskova, A.S. Tsarkova, I.V. Yampolsky, 1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine, Chem. Soc. Rev. 45 (2016) 6048–6077. [4] E.P. Coutant, Y.L. Janin, Synthetic routes to coelenterazine and other imidazo[1,2a]pyrazin-3-one luciferins: essential tools for bioluminescence-based investigations, Chem. Eur. J. 21 (2015) 17158–17171. [5] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing Co. Pte. Ltd, Singapore, 2006. [6] J. Vieira, L. Pinto da Silva, J.C.G. Esteves da Silva, Advances in the knowledge of light emission by firefly luciferin and oxyluciferin, J. Photochem. Photobiol. B 117 (2012) 33–39. [7] A. Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence: latest achievements and new horizons, Anal. Bioanal. Chem. 402 (2012) 69–76. [8] A. Roda, M. Guardigli, P. Pasini, M. Mirasoli, E. Michelini, M. Musiani, Bio- and chemiluminescence imaging in analytical chemistry, Anal. Chim. Acta 541 (2005) 25–36. [9] N. Hananya, A.E. Boock, C.R. Bauer, R. Satchi-Fainaro, D. Shabat, Remarkable enhancement of chemiluminescent signal by dioxetane-fluorophore conjugates: turn-on chemiluminescence probes with color modulation for sensing and imaging, J. Am. Chem. Soc. 138 (2016) 13438–13446. [10] C.M. Magalhães, J.C.G. Esteves da Silva, L. Pinto da Silva, Chemiluminescence and bioluminescence as an excitation source in the photodynamic therapy of cancer: a critical review, ChemPhysChem 17 (2016) 2286–2294. [11] M. Cronin, A.R. Akin, K.P. Francis, M. Tangney, In vivo bioluminescence imaging of intratumoral bacteria, Methods Mol. Biol. 1409 (2016) 69–77. [12] K.M. Grinstead, L. Rowe, C.M. Ensor, S. Joel, P. Daftarian, E. Dikici, J.M. Zingg, S. Daunert, Red-shifted aequorin variants incorporating non-canonical amino acids: applications in in vivo imaging, PLoS One 11 (2016) e0158579. [13] M.J. Hsu, J. Prigent, P.E. Dollet, J. Ravau, L. Larbanoix, G. Van Simaeys, A. Bol, S. Goldman, G. Deblandre, M. Najimi, E. Sokal, C. Lombard, Long-term in vivo monitoring of adult-derived human liver stem/progenitor cells by bioluminescence imaging, positron emission tomography, and contrast-enhanced computed tomography, Stem Cells Dev. (2017), http://dx.doi.org/10.1089/scd.2016.0338. [14] T. Wimmer, B. Lorenz, K. Stieger, Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor, Biosens. Bioelectron. 86 (2016) 609–615. [15] B.R. Branchini, C.E. Behney, T.L. Southworth, D.M. Fontaine, A.M. Gulick, D.J. Vinyard, G.W. Brudvig, Experimental support for a single electron-transfer oxidation mechanism in firefly bioluminescence, J. Am. Chem. Soc. 137 (2015) 7592–7595. [16] M.Y. Wang, Y.J. Liu, Theoretical study of dinoflagellate bioluminescence, Photochem. Photobiol. 93 (2017) 511–518. [17] Z.M. Kaskova, F.A. Dorr, V.N. Petushkov, K.V. Purtov, A.S. Tsarkova, N.S. Rodionova, K.S. Mineev, E.B. Guglya, A. Kotlobay, N.S. Baleeva, M.S. Baranov, A.S. Arseniev, J.I. Gitelson, S. Lukyanov, Y. Suzuki, S. Kanie, E. Pinto, P. Di Mascio, H.E. Waldenmaier, T.A. Pereira, R.P. Carvalho, A.G. Oliveira, Y. Oba, E.L. Bastos, C.V. Stevani, I.V. Yampolsky, Mechanism and color modulation of fungal bioluminescence, Sci. Adv. 3 (2017) e1602847. [18] M.A. Dubinnyi, Z.M. Kaskova, N.S. Rodionova, M.S. Baranov, A.Y. Gorokhovatsky, A. Kotlobay, K.M. Solntsev, A.S. Tsarkova, V.N. Petushkov, I.V. Yampolsky, Novel mechanism of bioluminescence: oxidative decarboxylation of a moiety adjacent to the light emitter of fridericia luciferin, Angew. Chem. Int. Ed. 54 (2015) 7065–7067. [19] S.H.D. Haddock, M.A. Moline, J.F. Case, Bioluminescence in the sea, Annu. Rev. Mar. Sci. 2 (2010) 443–493. [20] E.A. Widder, Bioluminescence in the ocean: origins of biological, chemical, and ecological diversity, Science 328 (2010) 704–708. [21] K. Teranishi, Luminescence of imidazo[1,2-a]pyrazin-3(7H)-one compounds, Bioorg. Chem. 35 (2007) 82–111. [22] T. Hirano, Y. Takahashi, H. Kondo, S. Maki, S. Kojima, H. Ikeda, H. Niwa, The reaction mechanism for the high quantum yield of Cypridina (Vargula) bioluminescence supported by the chemiluminescence of 6-aryl-2-methylimidazo[1,2-a] pyrazine-3(7H)-ones (Cypridina luciferin analogues), Photochem. Photobiol. Sci. 7 (2008) 197–207. [23] Y. Takahashi, H. Kondo, S. Maki, H. Niwa, H. Ikeda, T. Hirano, Chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazine-3(7H)-ones in DMSO/TMG and in diglyme/acetate buffer: support for the chemiexcitation process to generate the singlet-excited state of neutral oxyluciferin in a high quantum yield in the Cypridina (Vargula) bioluminescence mechanism, Tetrahedron Lett. 47 (2006) 6057–6061. [24] R. Saito, T. Hirano, S. Maki, H. Niwa, Synthesis and chemiluminescent properties of 6,8-diaryl-2-methylimidazo[1,2-alpha]pyrazine-3(7H)-ones: systematic investigation of substituent effect at para-position of phenyl group at 8-position, J. Photochem. Photobiol. A 293 (2014) 12–25. [25] F.I. Tsuji, ATP-dependent bioluminescence in the firefly squid, Watasenia-scintillans, Proc. Natl. Acad. Sci. USA 82 (1985) 4629–4632. [26] F.I. Tsuji, G.B. Leisman, K+-Na+-triggered bioluminescence in the oceanic squid
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