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PIGMENT CELL & MELANOMA Research Effect of pH on elementary steps of dopachrome conversion from first-principles calculation Ryo Kishida, Yohei Ushijima, Adhitya G. Saputro and Hideaki Kasai
DOI: 10.1111/pcmr.12256 Volume 27, Issue 5, Pages 734–743 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
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ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 27; 734–743
Effect of pH on elementary steps of dopachrome conversion from first-principles calculation Ryo Kishida1, Yohei Ushijima1, Adhitya G. Saputro1 and Hideaki Kasai1,2 1 Department of Applied Physics, Osaka University, Suita, Osaka, Japan 2 Center for Atomic and Molecular Technologies, Osaka University, Suita, Osaka, Japan
KEYWORDS DHI/DHICA/dopachrome/eumelanin/ first-principles calculation/pH effect
CORRESPONDENCE Hideaki Kasai, e-mail: [email protected]
PUBLICATION DATA Received 11 March 2014, revised and accepted for publication 1 May 2014, published online 7 May 2014 doi: 10.1111/pcmr.12256
Summary Dopachrome conversion, in which dopachrome is converted into 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole2-carboxylic acid (DHICA) upstream of eumelanogenesis, is a key step in determining the DHI/DHICA monomer ratio in eumelanin, which affects the antioxidant activity. Although the ratio of DHI/DHICA formed and the conversion rate can be regulated depending on pH, the mechanism is still unclear. To clarify the mechanism, we carried out first-principles calculations. The results showed the kinetic preference of proton rearrangement to form quinone methide intermediate via b-deprotonation. We also identified possible pathways to DHI/DHICA from the quinone methide. The DHI formation can be achieved by spontaneous decarboxylation after proton rearrangement from carboxyl group to 6-oxygen. a-Deprotonation, which leads to DHICA formation, can also proceed with a significantly reduced activation barrier compared with that of the initial dopachrome. Considering the rate of the proton rearrangements in a given pH, we conclude that the conversion is suppressed at acidic pH.
Introduction Melanin is well known for its diverse protective functions in physiological systems. The major roles of melanin as a pigment are photoprotection and antioxidation of the skin, hair, and eyes. Melanin broadly absorbs UV and visible light, and dissipates more than 99.9% of the absorbed light energy nonradiatively (Meredith and Riesz, 2004). Melanin has also been reported to have a role as a scavenger of epidermal reactive oxygen species such as superoxide (Bustamante et al., 1993), hydroxyl radical (Korytowski and Sarna, 1990), and singlet oxygen (Sarna et al., 1985), corresponding to its antioxidative effect. To
exert such functions properly by pigmentation, animals must regulate melanogenesis. The relationship between melanogenesis and melanosomal pH is important to understand the diversity of human pigmentation. The significant difference in the color between Black and White human skin has been characterized as a consequence of a difference in melanosomal pH; melanosomes from Black skins are regulated to neutral by various ion transporters, whereas those from White skins exhibit acidic pH, probably due to polymorphisms in those ion transporters (Ito and Wakamatsu, 2010; Lamason et al., 2005; Smith et al., 2004; Sulem et al., 2008).
Significance Dopachrome converts into DHI or DHICA, which controls the DHI/DHICA ratio of generated eumelanin. Depending on the ratio, generated eumelanin exhibits different antioxidant activity. The dopachrome conversion rate for each product can be regulated by pH, with conversion rate accelerated by neutral pH. However, how pH affects the conversion is still unclear. Our study identifies the possible reaction scheme, including two proton rearrangements. From the scheme, the importance of neutral pH in the conversion can be attributed to high rate of the proton rearrangements.
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Effect of pH on dopachrome conversion
Natural melanin is a mixture of two chemically distinct pigments, black to brown eumelanin and yellow to reddish-brown pheomelanin (Simon et al., 2009). Ito and Wakamatsu (2008) emphasized that the real melanogenesis should be considered ‘mixed melanogenesis’, in which the two reaction pathways are chemically wellcontrolled, varying the ratio of eumelanin/pheomelanin. Recently, it was shown that lag of eumelanogenesis by knocking out dopachrome tautomerase (Dct) in mice leads to an increase in reactive oxygen species, sunburn cells, and apoptotic cells after UVA exposure (Jiang et al., 2010). These findings indicate the importance of eumelanogenesis for the protective functions of melanin. Biochemical relationships between melanosomal pH and various steps in the mixed melanogenesis have also been investigated, showing that the preferential pH for eumelanogenesis is a neutral pH (Ancans et al., 2001; Fuller et al., 2001; Ito et al., 2013; Thompson et al., 1985). From chemical degradation analysis, it is now generally recognized that eumelanin mainly consists of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and peroxidative cleaved pyrroles as building blocks or monomers (Ito, 1986). Therefore, regulation of the DHI/DHICA ratio in eumelanogenesis defines the main chemical composition of generated eumelanin, determining its main properties. In a recent experiment in which the radical scavenging activity of DHI-melanin and DHICA-melanin were compared, it was revealed that inclusion of DHICA in eumelanin is necessary to exert antioxidation activity (Jiang et al., 2010). Eumelanin can be obtained by the oxidation of certain precursors such as tyrosine or 3,4-dihydroxyphenylalanine (dopa) in the presence of tyrosinase or alternative catalysts. The formation of eumelanin proceeds via a number of intermediates. In particular, dopachrome is a critical intermediate determining the main properties of generated eumelanin. Mason (1948), who first identified dopachrome, demonstrated the subsequent conversion of the molecule into DHI or DHICA (Figure 1). As shown in Figure 1, there are two possible routes in this conversion, the decarboxylative route and the tautomerization route, corresponding to the formation of DHI and DHICA, respectively. Dopachrome conversion spontaneously but slowly proceeds to the formation of DHI, which is achieved non-enzymatically, whereas the conversion into €rner and Pawelek, 1980) in DHICA requires DCT/Dct (Ko mammals. Actually, in vitro experiments have shown highly selective formation of DHI at >95% at the usual pH. Using spectrophotometry and high performance liquid chromatography (HPLC) analysis, Sugumaran et al. (1990) and Sugumaran and Semensi (1991) revealed that dopachrome conversion to DHI or DHICA involves a quinone methide structure as an intermediate. Furthermore, several studies have indicated that, depending on experimental conditions such as pH value and concentration of metal ions, the ratio of DHI/DHICA and the conversion rate can be regulated even if the solution does ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Figure 1. Dopachrome conversion to DHI or DHICA. Labels in this figure denote the position for each carbon and nitrogen atom corresponding to usual chemical nomenclature.
not contain any enzymes (Ito et al., 2013; Mason, 1948; Muneta, 1977; Palumbo et al., 1987; Stravs-Mombelli and Wyler, 1985; Wakamatsu and Ito, 1988). It was found that in strongly acidic solutions (pH 1.3–2.0) dopachrome preferably converts to DHICA rather than DHI (Mason, 1948); furthermore, DHICA can be obtained under a strongly basic condition (pH 13; Stravs-Mombelli and Wyler, 1985; Wakamatsu and Ito, 1988). It was also shown that the reaction rate of dopachrome conversion is greatly promoted at neutral pH (Ito et al., 2013; Muneta, 1977). It should be noted that the reaction rate at pH 7.3 is 4.6-fold greater than that at pH 5.3 (Ito et al., 2013). The above experimental findings may provide important insights into the understanding of dopachrome conversion and eumelanogenesis. However, there has as yet been no detailed knowledge available of the reaction mechanism of dopachrome conversion that would enable us to interpret the experimental results properly. For further development of melanin chemistry and medical applications related with pigmentation, understanding dopachrome conversion at the molecular level is important for a basic knowledge of eumelanogenesis. Despite the importance of pH for dopachrome conversion, the accurate prototropic behaviors in the conversion are difficult to measure experimentally due to the intangible features of protonated/deprotonated structures of the molecule. Therefore, we employ theoretical approaches independent of experimental considerations in this study. To clarify the mechanism and prototropic equilibria of dopachrome conversion, we present theoretical calculations based on density functional theory (DFT) (Hohenberg and Kohn, 1964; Kohn and Sham, 1965). In these calculations, we calculated the total energy for various isomers of dopachrome and intermediates of this conversion. Candidates for the possible reaction schemes can be derived by comparing the stabilities of these 735
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isomers. To determine the actual scheme, we also evaluated the activation barriers for important elementary steps; a-deprotonation, b-deprotonation, and decarboxylation. Our results showed the energetic and kinetic preference of proton rearrangement to form quinone methide intermediate via b-deprotonation. In the next step, the formed quinone methide can proceed via two possible routes. The first starts from proton rearrangement from carboxyl group to 6-oxygen, leading to spontaneous decarboxylation, producing DHI. The quinone methide can also proceed via a-deprotonation to form DHICA, although the activation barrier is relatively high compared with that in DHI formation. Considering the rate of the proton rearrangements in a given pH condition, we conclude that the conversion is suppressed at acidic pH.
Results and Discussion Energetic stability for isomers in the first elementary steps of dopachrome conversion To understand dopachrome conversion, we first considered prototropic tautomers; dopachrome (A) and its tautomers in terms of prototropic rearrangement from carboxyl group or nitrogen to 5/6-oxygen (B–E) as defined in Table 1. The optimized structure of dopachrome (A) and its detailed geometrical parameters are shown in Figure 2 and Supporting Information Table S1, respectively. The structures of other molecules in this work are shown in Supporting Information Figures S1–S3. In Table 1 the thermodynamic stability of each tautomer is compared. As described in the Methods section, we considered the solvent effect using the polarizable continuum model (PCM). Table 1 shows the energetic preferability of dopa-
Figure 2. Optimized structure of dopachrome A (defined in Table 1).
chrome A in aqueous solution. Thus, tautomers B–E would appear to make only minor contributions to the conversion. However, there is still ambiguity. In particular, some of the features of tautomer B differ from those of A and C: an altered structure in carboxyl group and isosurface of the highest occupied molecular orbital (HOMO) (Supporting Information Figures S1 and S4, respectively). These features may imply a critically changed reactivity. To check whether the reaction scheme via structure B can be ignored, we will discuss the possibility of subsequent reactions from tautomer B below. It also should be noted that tautomer C is the most stable structure in vacuo. We emphasize that the natural atomic charges (see Methods section), shown in Supporting Information Table S2, of all structures are zwitter-ionic, with a relatively negative 6-oxygen and positive 1-hydrogen being observed. Therefore, the electronic structures of dopachrome tautomers are similar not to that of o-quinones but to that of iminochromes.
Table 1. Comparison of stability for each tautomer of dopachrome at first step of dopachrome conversion
Tautomera
Protonated sitesb
Energy/kcal∙mol1c
Gibbs Free energy/kcal∙mol1d
Equilibrium compositione
A (vac.) B (vac.) C (vac.) D (vac.) E (vac.) A (aq.) B (aq.) C (aq.) D (aq.) E (aq.)
Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl, Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl,
0.000 11.257 5.518 27.947 Unstablef 18.453 11.808 15.351 0.649 0.328
0.000 11.728 4.753 27.478 Unstablef 18.552 11.368 15.415 0.683 0.498
4.400 2.299 1.000 1.736 0 0.994 8.400 6.055 2.596 1.771
N1 O6 O5 N1 O6 O5
9 104 9 1012 9 1023
9 9 9 9
106 103 1014 1013
a Symbols for dopachrome tautomers at first step of dopachrome conversion. Calculation without and with PCM is respectively denoted as (vac.) and (aq.). b Numbers in this column correspond to the labels in Figure 1 that follow usual chemical nomenclatures. Carboxyl denotes protonation to the carboxylate ion. c The origin is set to the value of A (vac.). d The origin is set to the value of A (vac.). Temperature was set to 309.5 K as a condition of human body. e Equilibrium composition is defined as the mole fraction of each tautomer in equilibrium state, normalized by amount of all tautomers in vacuo or in aqueous solution. These compositions were calculated based on the value of the Gibbs free energies. The activity coefficients were ignored. f Spontaneous proton transfer to O6 was occurred.
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Effect of pH on dopachrome conversion
Energetic stability for isomers in the intermediate steps of dopachrome conversion As candidates for the intermediates of the conversion, we considered possible isomers formed via irreversible processes from structure A, namely the structures after proton rearrangement from a/b-carbon to 5/6-oxygen (A0 , B0 , E0 , F0 ), and their prototropic tautomers formed via proton rearrangement from carboxyl group or nitrogen to 5/6-oxygen (C0 , D0 , G0 , H0 ), as defined in Table 2. Comparison of the energetic stability is shown in Table 2. As shown in Table 2, structure A0 , which has a quinone methide structure, exhibits the lowest free energy of the b-deprotonated structures. This corresponds to the experiment where the quinone methide structure was detected as an intermediate of the conversion (Sugumaran and Semensi, 1991). It also should be noted that stable structure C0 was not found, and that spontaneous decarboxylation from structure C0 during optimization was confirmed. Therefore, we can assume that the DHI formation can be achieved by spontaneous decarboxylation via proton rearrangement from structure A0 to C0 if the quinone methide structure (A0 ) is involved in the actual reaction scheme. Other possibilities will be discussed below. Activation barriers for the first elementary steps in dopachrome conversion The activation barriers for the first elementary steps are evaluated here. As discussed above, irreversible proton
rearrangements are included in the conversion. Thus, the two structures between the proton rearrangement must be connected in terms of reaction coordinate. There are two ways in which protons can be arranged in water: the coherent process, where simultaneous deprotonation and protonation proceed in a concerted fashion, and the incoherent process, where the rearrangement is achieved via the deprotonation from dopachrome as the first step and subsequent proton diffusion to the oxygen. According to experimental and theoretical work, it generally has been concluded that the coherent process is not acceptable for the reaction coordinate of proton migration in real aqueous solution (Agmon, 1995). Therefore, we only consider the incoherent process. Dividing the process, we only evaluate the activation barrier for deprotonation because of the triviality of the subsequent steps. Figure 3 shows the energy profile during a-deprotonation (Figure 3A) and b-deprotonation (Figure 3B) from dopachrome A. As we discuss in Methods section, H2O trimer coordinated structures (Figure 4) were chosen as initial structures for a/b-deprotonation. Each curve in Figure 3 has two distinct regions, a steep zone and a plateau zone. We can see a similarity between the two steep zones, implying that the strengths of the two C–H bonds are almost same. On the other hand, the two plateau zones show different features; a-deprotonation proceeds with an inevitable increase of potential energy, and b-deprotonation exhibits a slightly negative gradient of the curve. Therefore, one can definitely say that it is
Table 2. Comparison of stability for each tautomer of dopachrome after proton rearrangement from a/b-carbon
Tautomera
Deprotonated/Protonated sitesb
Energy/kcal∙mol1c
Gibbs Free energy/kcal∙mol1d
Equilibrium compositione
A0 (vac.) B0 (vac.) C0 (vac.) D0 (vac.) E0 (vac.) F0 (vac.) G0 (vac.) H0 (vac.) A0 (aq.) B0 (aq.) C0 (aq.) D0 (aq.) E0 (aq.) F0 (aq.) G0 (aq.) H0 (aq.)
b-C/O5, N1, Carboxyl b-C/O6, N1, Carboxyl b-C/O5, O6, N1 b-C/O5, O6, Carboxyl a-C/O5, N1, Carboxyl a-C/O6, N1, Carboxyl a-C/O5, O6, N1 a-C/O5, O6, Carboxyl b-C/O5, N1, Carboxyl b-C/O6, N1, Carboxyl b-C/O5, O6, N1 b-C/O5, O6, Carboxyl a-C/O5, N1, Carboxyl a-C/O6, N1, Carboxyl a-C/O5, O6, N1 a-C/O5, O6, Carboxyl
11.252 0.430 Unstablef 6.466 2.311 5.929 1.566 13.889 22.910 14.293 Unstablef 6.218 13.827 19.121 23.517 25.843
9.777 1.036 Unstablef 6.958 3.281 4.319 1.890 13.128 22.331 13.698 Unstablef 5.850 12.986 17.982 22.748 25.180
1.000 2.313 0 1.522 2.586 6.017 2.482 1.000 1.000 7.895 0 2.300 2.411 8.106 1.881 0.981
9 108 9 9 9 9
1012 1012 107 1011
9 107 9 9 9 9
1012 109 106 102
a Symbols for dopachrome tautomers after proton rearrangement from a/b-carbon. Calculation without and with PCM is respectively denoted as (vac.) and (aq.). b Numbers in this column correspond to the labels in Figure 1 that follow usual chemical nomenclatures. Carboxyl denotes protonation to the carboxylate ion. c The origin is set to the value of A (vac.). d The origin is set to the value of A (vac.).Temperature was set to 309.5 K as a condition of human body. e Equilibrium composition is defined as the mole fraction of each tautomer in equilibrium state, normalized by total amount of tautomers deprotonated either from a/b-carbon in vacuo or in aqueous solution. These compositions were calculated based on the value of the Gibbs free energies. The activity coefficients were ignored. f Spontaneous decarboxylation was occurred.
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A
B
Figure 3. Energy profile for a/b-deprotonation of dopachrome A (defined in Table 1), corresponding to (A) and (B), respectively.
A
B
Figure 4. Initial structures for (A) a-deprotonation and (B) b-deprotonation. As an acceptor for dissociating proton, H2O trimer was chosen.
impossible for the proton rearrangement via a-deprotonation to proceed. Thus, we conclude that dopachrome (A) first forms the quinone methide intermediate (A0 ) via b-deprotonation. We were able to determine the exact transition state of the b-deprotonation, which has only one imaginary frequency, and the activation barrier (23.983 kcal/mol). If the solution is acidified, the apparent rate of the b-deprotonation will decrease due to the acceleration of proton recombination to the b-carbon. Thus, we can say that the formation of the quinone methide is a base-catalyzed reaction. The rapid formation of the quinone methide at neutral to weakly basic pH as experimentally reported could be described as the consequence of base-catalyses of hydroxyl ions or buffer ions such as phosphate ions. DHI/DHICA formation in dopachrome conversion From results in the subsection above, b-deprotonation would appear to be preferred to a-deprotonation, corresponding to formation of the quinone methide intermediate. In this section, we show the possible path to form DHI or DHICA from the quinone methide. To form DHI, the quinone methide must undergo decarboxylation via dissociation or rearrangement of the carboxyl proton, whereas the DHICA formation proceeds via a-deprotonation. First, we show the energy profiles during the a-deprotonation from the neutral quinone methide and 738
the decarboxylation from the carboxyl dissociated quinone methide in Figure 5 (A and B, respectively). As shown in Figure 5, the activation barriers for the a-deprotonation and decarboxylation are estimated at 11.353 and 8.960 kcal/mol, respectively. We were also able to determine the exact transition state for decarboxylation with its activation barrier, 8.975 kcal/mol. Although one can see the slightly lower activation barrier of decarboxylation, this is not evidence for the preferability of the DHI formation, especially if the instability of the carboxyl-dissociated quinone methide is considered. For an alternative choice, there is the DHI formation after proton rearrangement from the carboxyl group to 6-oxygen (C0 ). As mentioned above, the proton rearrangement leads to spontaneous decarboxylation. Therefore, the rate-determining step of the DHI formation from the quinone methide can be regarded as the dissociation of the proton from the carboxyl group or diffusion of the proton to the 6-oxygen. Considering the abnormally high mobility of proton in water, we assume that the rate for proton diffusion to the 6-oxygen can be ignored compared with that for proton dissociation from the carboxyl group. Due to the trivial superiority of carboxyl dissociation to a-deprotonation, we can state that the DHI formation dominates the DHICA formation unless carboxyl dissociation is suppressed. We consider that the preferability of the DHI formation corresponds directly to the experiment ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of pH on dopachrome conversion
in which more than 95% of DHI formation was confirmed. If the solution is acidified, the apparent rate of the carboxyl dissociation will decrease due to the acceleration of proton recombination to the carboxylate group. On the other hand, one can assume that the acidification will not contribute significantly to the acceleration of the backward reaction of a-deprotonation because of the relatively high activation barrier (7.564 kcal/mol; estimated from Figure 5A). On the basis of this, we conclude that DHI formation is more suppressed than DHICA formation at acidic pH, which might correspond to the pronounced formation of DHICA at the strongly acidic pH (Mason, 1948). The quinone methide route versus the indolenine route As mentioned in the first subsection of the Results and Discussion section, the reaction pathway that proceeds via unstable isomer B may contribute to the conversion. To confirm whether this possibility can be ignored, we also evaluated the activation barriers for subsequent reaction from structure B. Figure 6A,B, and C (the diamonds) show the energy profiles of tautomer B during a-deprotonation, b-deprotonation, and decarboxylation, respectively. In the case of decarboxylation, we determined the transition state that has only one imaginary
A
frequency, and the activation barrier, 9.250 kcal/mol. However, the diamonds in Figure 6(C) show a curious profile with a shoulder in the curve. In this shoulder, we confirmed a non-physically ‘rapid’ variation of dihedral angle in the bond between the carboxyl C and the a-C. This is an artificial result in which the motion is adiabatically restricted. To correct the activation barrier to reproduce the more reasonable value in the actual dynamics, we used the frozen value of the dihedral angle from before the non-physical variation. The circles in Figure 6(C) show the corrected energy profile during decarboxylation with the frozen coordinate. In the correction, the true activation barrier for decarboxylation is raised to 10.703 kcal/mol. On the basis of these results, the activation barrier for the decarboxylation is relatively lower than the values for a/b-deprotonation. However, we cannot regard these results as direct evidence for the preferability of the decarboxylative route because the rate of the decarboxylation from tautomer B depends not only on the absolute rate of the C–C bond cleavage along decarboxylation, but also on the relative rate constant of the C-C cleavage against the proton dissociation from structure B. To test the stability of tautomer B against the proton dissociation, we calculated the Gibbs free energy change between the associated and dissociated structures using the solvation model density (SMD), in which
B
Figure 5. Energy profile for a-deprotonation and decarboxylation of the quinone methide A0 (defined in Table 2), corresponding to (A) and (B), respectively.
A
B
C
Figure 6. Energy profile for (A) a-deprotonation, (B) b-deprotonation, and (C) decarboxylation of dopachrome tautomer C (defined in Table 1). The small pictures in this figure (C) exhibit a ‘rapid’ variation of dihedral angle in the cleaving C-C bond. The diamonds in this figure (C) show the fully unrestricted relaxed potential energy curve for all degrees of freedom except for the distance of the cleaving C-C bond. The circles show the potential energy curve along the frozen dihedral angle in the cleaving C-C bond preventing ‘rapid’ geometrical change.
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the short-range interactions between the solute and the solvent molecules are taken into account, in addition to the electrostatic interaction evaluated by the PCM (see Methods section) (Marenich et al., 2009). The thermochemical cycle we considered is shown in Figure 7. We used 265.3 kcal/mol as the value of DsG* for the absolute proton hydration free energy that was experimentally obtained at 298.15 K (Donald and Williams, 2010). To correct the value to that at 309.5 K, we considered the effect of temperature change by subtracting D(SDT) = 11.35 kcal/mol [where S is the molar entropy of proton, 39.8 cal/(K∙mol); Donald and Williams, 2010;]. Thus, we obtained the Gibbs free energy change with the carboxyl dissociation (2.816 kcal/mol), corresponding to pKa 1.99. On the other hand, the Gibbs free energy change (with the SMD) for tautomerization from structure A to B is 3.780 kcal/mol. This result clearly shows the stability of the dissociated state compared with structure B. Thus the dopachrome conversion does not appear to start via tautomerization to structure B due to its instability, but rather with the formation of quinone methide via proton rearrangement from b-carbon to 5oxygen. This route agrees with experiments in which it was concluded that the pathway via indolenine, a decarboxylated intermediate, is not acceptable in the dopachrome conversion scheme (Sugumaran and Semensi, 1991). Proposed scheme of dopachrome conversion The picture and energy diagrams of the whole scheme are outlined respectively in Figures 8 and 9. Dopachrome first undergoes proton rearrangement from b-carbon to 5oxygen, producting the quinone methide intermediate. This process is suppressed by acidification due to acceleration of proton recombination to b-carbon. The quinone methide formed can proceed via two possible routes whose rate depends on pH. At neutral pH, the quinone methide prefers to form DHI by spontaneous decarboxylation after proton rearrangement from the carboxyl group to the 6-oxygen. In contrast, an acidic pH prevents the DHI formation by reducing the apparent rate of the carboxyl dissociation. These effects of the acidic pH can lead to the reaction rate of dopachrome conversion being suppressed. Furthermore, the predominant formation of DHICA at the strongly acidic pH can
Figure 8. Proposed scheme of dopachrome conversion.
Figure 9. Energy diagram in dopachrome conversion. Numbers correspond to those of Figure 8.
also be achieved with suppressed carboxyl dissociation and weakly suppressed a-deprotonation. Based on the difference of activation barriers for carboxyl dissociation and a-deprotonation from the quinone methide, we consider that the formation of DHI is kinetically preferable compared with that of DHICA at the usual pH, corresponding qualitatively to the experiments at > 95% DHI formation. Although the rate constant for each elementary step can be calculated based on the transition state theory, such calculations will require additional assumptions and approximations. To omit the non-trivial modeling errors, we focused instead on the qualitative description here. In this study, we were able to derive the possible scheme of dopachrome conversion and to determine the correspondence of the pH effect to the reaction rate from first-principles calculations. Since our approach is based on the universal theory in physics, we expect that the effects of other factors in dopachrome conversion such as metal ions can also be clarified from first-principles calculations as the most transferable methodology.
Methods First-principles calculations based on density functional theory Figure 7. Thermochemical cycle to calculate pKa of dopachrome (DC).
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First-principles calculations were performed based on density functional theory using the Gaussian 09 program (Frisch et al., 2010). For
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of pH on dopachrome conversion Table 3. Comparison of different levels of theory
Table 4. Comparison of stability for each tautomer of dopachrome in the use of CAM-B3LYP
Exchange correlation functional
Basis set
Dipole moment/D
Bond length/ Aa
B3LYP B3LYP B3LYP B3LYP B3LYP CAM-B3LYP CAM-B3LYP CAM-B3LYP CAM-B3LYP CAM-B3LYP
6-31++G 6-31G(d,p) 6-31++G(d,p) 6-311++G(d,p) aug-cc-pVDZ 6-31++G 6-31G(d,p) 6-31++G(d,p) 6-311++G(d,p) aug-cc-pVDZ
10.2237 9.0513 9.6359 9.6230 9.6231 10.0312 8.8565 9.4546 9.4446 9.4685
1.514 1.531 1.521 1.520 1.521 1.506 1.523 1.513 1.512 1.513
Tautomera
Protonated sitesb
Energy/kcal∙mol1c
A (vac.) B (vac.) C (vac.) D (vac.) E (vac.) A (aq.) B (aq.) C (aq.) D (aq.) E (aq.)
Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl, Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl,
0.000 16.502 4.825 3.725 Unstabled 18.246 11.100 14.470 3.523 Unstabled
N1 O6 O5 N1 O6 O5
a
Symbols for dopachrome tautomers. Calculation without and with PCM is respectively denoted as (vac.) and (aq.). b Numbers in this column correspond to the labels in Figure 1 that follow usual chemical nomenclatures. Carboxyl denotes protonation to the carboxylate ion. c The origin is set to the value of A (vac.). d Spontaneous proton transfer to O6 was occurred.
Distance between a-carbon and carboxyl carbon was chosen as the representative of structure.
a
the energetic analyses and determination of optimized structures of dopachrome and intermediates on the pathway being converted into DHI or DHICA, we used the Becke three-parameter hybrid method (Becke, 1993) with the Lee–Yang–Parr correlation functional approximation (Lee et al., 1988) (B3LYP) and 6-31++G(d,p) basis set. To validate the level of theory employed here, we compared various combinations of the exchange correlation functionals and the basis sets. As an alternative exchange correlation functional with B3LYP, we considered the Coulomb-attenuating method (CAM-B3LYP) where the exchange potential is improved by an additional parameter l so that charge transfer interactions such as hydrogen bondings are properly evaluated (Yanai et al., 2004). Basis dependence of the electric dipole moment, as the representative of electronic density, and the distance between a-carbon and carboxyl carbon, as the representative of structure, were investigated by comparing the results from 6-31++G, 6-31G(d,p), 6-31++G(d,p), 6-311++G(d,p), and aug-cc-pVDZ. Table 3 shows these comparisons for dopachrome A (defined in Table 1). As can be seen in Table 3, the polarization functions and the diffuse functions contribute significantly to the electric dipole moment and the bond length, whereas use of the different split valence basis sets or the correlation consistent basis sets only show small contributions. The basis dependence with CAM-B3LYP does not qualitatively differ from that with B3LYP. However, CAM-B3LYP might significantly stabilize highly hydrogenbonded structures, such as structure B (defined in Table 1). We therefore also compared the stability of dopachrome tautomers A, B, C, D, and E (defined in Table 1) with CAM-B3LYP. We tabulate the comparison in Table 4. To make comparison between Tables 1 and 4
A
easier, we also present the results of the use of polarizable continuum model (PCM; described later) in Table 4. Use of CAM-B3LYP instead of B3LYP did not show any remarkable stabilization. Although CAM-B3LYP significantly destabilized the O5-protonated structures (Table 4), these structures had already been evaluated as unstable for B3LYP. From the results summarized in Tables 3 and 4, we can conclude that the B3LYP/6-31++G(d,p) level is sufficient for addressing dopachrome conversion.
Natural population analysis Natural population analysis (Foster and Weinhold, 1980) was used to extract information about atomic charges from calculated electronic density of dopachrome A (defined in Table 1). The result of the analysis is shown in Table S2.
Reaction coordinates The activation barrier for each bond-break can be obtained by calculating the total energies along the intrinsic reaction coordinate. The bond-breaking requires an increase in the distance between the cleaving two atoms. Therefore we only chose one degree of freedom, the distance between two atoms cleaving with the dissociation, and all of the other degrees of freedom were allowed to relax adiabatically. When the reaction coordinate is treated in this
B
Figure 10. Energy profile for (A) a-deprotonation and (B) b-deprotonation of dopachrome A (defined in Table 1) using H2O pentamer as an acceptor for dissociating proton.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Kishida et al. way, the water trimer accepting the dissociating proton is adiabatically accessible during the deprotonations. Although it is non-trivial whether such a treatment is able to reflect the actual dynamics of the deprotonation, we can evaluate the minimum activation energy for the deprotonation as a starting point for discussion. Thus, the true reaction coordinate is projected on a one-dimensional relaxed potential energy surface in this study.
Solvation model To take into account solvent effects, the polarizable continuum model using the integral equation formalism variant was employed (Tomasi et al., 2005). In this model, solvent is considered a continuous medium with a dielectric constant that is same as that of the liquid solvent, and solute is put in a cavity surrounded by the dielectric continuum. In this study, we focused solely on water as the target solvent. It should be noted that the solvent effect evaluated by the PCM changes the most stable tautomer of dopachrome; structure A is greatly stabilized, becoming the most stable tautomer, although structure C is the most stable structure in vacuo (A and C are defined in Table 1). Table 1 shows the comparison of energetic stability for each tautomer with and without the PCM stabilization.
Proton solvation with deprotonation In the case of proton solvation with the deprotonations from dopachrome and the intermediate of the conversion, an assessment that only takes into account polarization of the solvent using the PCM could lead to significant inaccuracies because the formation of a strong chemical bond between the proton and water molecules might affect the transition state. To appropriately reflect such an interaction between the proton and water molecules, we, in addition to taking the PCM, put a water trimer as an acceptor of the dissociating proton from dopachrome and the intermediate of the conversion (Figure 4A, B) only in the evaluation of the activation barrier for deprotonations. In general, a solvated proton in water forms triply coordinated H3O+ (H2O)3 and readily jumps over to other water molecules by forming an H5O2+ dimer structure, triggered by cleaving the hydrogen-bond between water molecules in the first and second hydration shell (Agmon, 1995). In our system, it is expected that the energy required for cleavage of old hydrogen-bond (to accept water molecule) with formation of hydronium ion H3O+ by the deprotonation can be assumed to be sufficiently small to be negligible compared with the energy required for cleavage of the covalent bond of the hydrogen within dopachrome. By contrast, once hydronium ion H3O+ is formed, hydrogen-bonds between H3O+-H2O will be enhanced; this stabilization effect is relatively strong and cannot be neglected. Therefore, the trimer acceptor model that we employed can be regarded as a model that takes into account such strengthening of the hydrogen-bond only in the first hydration shell. To check whether the size of water cluster we used is enough to evaluate the real activation barrier, we evaluated the activation barriers for a/b-deprotonation from dopachrome A using a water pentamer as an acceptor. Figure 10 shows the energy profile along a/b-deprotonation with the five water molecules, demonstrating the similarity with Figure 3. Because of the similarity, we only used the water trimer acceptor model in this work.
Acknowledgements This work is supported in part by: MEXT Grant-in-Aid for Scientific Research on Innovative Areas Program (2203-22104008), Scientific Research Programs (A) (24246013), and Grants for Excellent Graduate Schools (130820-140331) ‘Atomically Controlled Fabrication Technology’; JST ALCA Program ‘Development of Novel Metal-Air Secondary Battery Based on Fast Oxide Ion Conductor Nano Thickness Film’ and Strategic Japan-Croatia Research Cooperative
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Program on Materials Sciences Program ‘Theoretical Modeling and Simulations of the Structural, Electronic, and Dynamical Properties of Surfaces and Nanostructures in Materials Science Research’. Some of the numerical calculations presented here were done using the the computer facilities at the following institutes: CMC (Osaka University), ISSP, KEK, NIFS, and YITP.
References Agmon, N. (1995). The Grotthuss mechanism. Chem. Phys. Lett. 244, 456–462. Ancans, J., Tobin, D.J., Hoogduijn, M.J., Smit, N.P., Wakamatsu, K., and Thody, A.J. (2001). Melanosomal pH controls rate of melanogenesis, eumelanin/pheomelanin ratio and melanosome maturation in melanocytes and melanoma cells. Exp. Cell Res. 268, 26–35. Becke, A.D. (1993). Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652. Bustamante, J., Bredeston, L., Malanga, G., and Mordoh, J. (1993). Role of melanin as a scavenger of active oxygen species. Pigment Cell Res. 6, 348–353. Donald, W.A., and Williams, E.R. (2010). An improved cluster pair correlation method for obtaining the absolute proton hydration energy and enthalpy evaluated with an expanded data set. J. Phys. Chem. B 114, 13189–13200. Foster, J.P., and Weinhold, F. (1980). Natural hybrid orbitals. J. Am. Chem. Soc. 102, 7211–7218. Frisch, M.J., Trucks, G.W., Schlegel, H.B. et al. (2010). Gaussian 09, Revision C. 01. Gaussian, Inc., Wallingford, CT. Fuller, B.B., Spaulding, D.T., and Smith, D.R. (2001). Regulation of catalytic activity of preexisting tyrosinase in black and caucasian human melanocyte cell cultures. Exp. Cell Res. 262, 197–208. Hohenberg, P., and Kohn, W. (1964). Inhomogeneous electron gas. Phys. Rev. 136, B864–B871. Ito, S. (1986). Reexamination of the structure of eumelanin. Biochim. Biophys. Acta 883, 155–161. Ito, S., and Wakamatsu, K. (2008). Chemistry of mixed melanogenesis – pivotal roles of dopaquinone. Pigment Cell Melanoma Res. 84, 582–592. Ito, S., and Wakamatsu, K. (2010). Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation. Pigment Cell Melanoma Res. 24, 63–74. Ito, S., Suzuki, N., Takebayashi, S., Commo, S., and Wakamatsu, K. (2013). Neutral pH and copper ions promote eumelanogenesis after the dopachrome stage. Pigment Cell Melanoma Res. 26, 817–825. Jiang, S., Liu, X., Dai, X., Zhou, Q., Lei, T., Beermann, F., Wakamatsu, K., and Xu, S. (2010). Regulation of DHICA-mediated antioxidation by dopachrome tautomerase: implication for skin photoprotection against UVA radiation. Free Rad. Biol. Med. 48, 1144–1151. Kohn, W., and Sham, L.J. (1965). Self-consistent equations including exchange and correlation effects. Phys. Rev. 140, A1133–A1138. €rner, A.M., and Pawelek, J. (1980). Dopachrome conversion: a Ko possible control point in melanin biosynthesis. J. Invest. Dermatol. 75, 192–195. Korytowski, W., and Sarna, T. (1990). Bleaching of melanin pigments. Role of copper ions and hydrogen peroxide in autooxidation and photooxidation of synthetic dopa-melanin. J. Biol. Chem. 265, 12410–12416. Lamason, R.L., Mohideen, M.A., Mest, J.R. et al. (2005). SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310, 1782–1786. Lee, C., Yang, W., and Parr, R.G. (1988). Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785–789.
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Effect of pH on dopachrome conversion Marenich, A.V., Cramer, C.J., and Truhlar, D.G. (2009). Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. 114, 6378–6396. Mason, H.S. (1948). The chemistry of melanin. III. Mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. J. Biol. Chem. 172, 83–99. Meredith, P., and Riesz, J. (2004). Radiative relaxation quantum yields for synthetic eumalanin. Photochem. Photobiol. 79, 211– 216. Muneta, P. (1977). Enzymatic blackening in potatoes: influence of pH on dopachrome oxidation. Am. Potato J. 54, 387–393. Palumbo, A., d’Ischia, M., Misuraca, G., and Prota, G. (1987). Effect of metal ions on the rearrangement of dopachrome. Biochim. Biophys. Acta 925, 203–209. Sarna, T., Menon, I.A., and Sealy, R.C. (1985). Photosensitization of melanins: a comparative study. Photochem. Photobiol. 42, 529– 532. Simon, J.D., Peles, D., Wakamatsu, K., and Ito, S. (2009). Current challenges in understanding melanogenesis: bridging chemistry, biological control, morphology, and function. Pigment Cell Melanoma Res. 22, 563–579. Smith, D.R., Spaulding, D.T., Glenn, H.M., and Fuller, B.B. (2004). The relationship between Na+/H+ exchanger expression and tyrosinase activity in human melanocytes. Exp. Cell Res. 298, 521–534. Stravs-Mombelli, L., and Wyler, H. (1985). Reinvestigation of formation of dopa-melanin: new aspects of the autoxidation of dopa. In Biological, Molecular and Clinical Aspects of Pigmentation, J. Bagnara, S.N. Klaus, E. Paul, and M. Schartl, eds (Tokyo: University of Tokyo Press), pp. 69–76. Sugumaran, M., and Semensi, V. (1991). Quinone methide as a new intermediate in eumelanin biosynthesis. J. Biol. Chem. 266, 6073– 6078. Sugumaran, M., Dali, H., and Semensi, V. (1990). Formation of a stable quinone methide during tyrosinase-catalysed oxidation of a-methyl dopa methyl ester and its implication in melanin biosynthesis. Bioorg. Chem. 18, 144–153. Sulem, P., Gudbjartsson, D.F., Stacey, S.N. et al. (2008). Two newly identified genetic determinations of pigmentation in Europeans. Nat. Genet. 40, 835–837. Thompson, A., Land, E.J., Chedekel, M.R., Subbarao, K.V., and Truscott, T.G. (1985). A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa) and the cysteinyldopas. Biochim. Biophys. Acta 843, 49–57. Tomasi, J., Mennucci, B., and Cammi, R. (2005). Quantum mechanical continuum solvation models. Chem. Rev. 105, 2999–3093.
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Wakamatsu, K., and Ito, S. (1988). Preparation of eumelanin-related metabolites 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, and their O-methyl derivatives. Anal. Biochem. 170, 335–340. Yanai, T., Tew, D.P., and Handy, N.C. (2004). A new hybrid exchange-correlation functional using the Coulomb-attenuation method (CAM-B3LYP). Chem. Phys. Lett. 393, 51–57.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Optimized structures (with PCM) of dopachrome tautomers at first step of dopachrome conversion. The blue, red, yellow, and purple spheres respectively represent hydrogen, oxygen, carbon, and nitrogen atoms. Labels in this figure denote each atom, but do not correspond to the numbers used in usual chemical nomenclatures. Figure S2. Optimized structures (with PCM) of dopachrome tautomers after proton rearrangement from a/bcarbon. For reference, the transitional structure of C0 is also listed, although this structure is not stable. The blue, red, yellow, and purple spheres respectively represent hydrogen, oxygen, carbon, and nitrogen atoms. Labels in this figure denote each atom, but do not correspond to the numbers used in usual chemical nomenclatures. Figure S3. Optimized structures (with PCM) of DHI and DHICA. The blue, red, yellow, and purple spheres respectively represent hydrogen, oxygen, carbon, and nitrogen atoms. Labels in this figure denote each atom, but do not correspond to the numbers used in usual chemical nomenclatures. Figure S4. HOMO isosurfaces of dopachrome tautomers at first step of dopachrome conversion. Table S1. Geometrical parameters (with PCM) of reactant (dopachrome A), intermediate (quinone methide A0 ), and products (DHI, DHICA) in dopachrome conversion. Table S2. Natural atomic charges (e) (with PCM) of tautomers of dopachrome at first step of dopachrome conversion.
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PIGMENT CELL & MELANOMA Research Effect of pH on elementary steps of dopachrome conversion from first-principles calculation Ryo Kishida, Yohei Ushijima, Adhitya G. Saputro and Hideaki Kasai
DOI: 10.1111/pcmr.12256 Volume 27, Issue 5, Pages 734–743 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
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ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 27; 734–743
Effect of pH on elementary steps of dopachrome conversion from first-principles calculation Ryo Kishida1, Yohei Ushijima1, Adhitya G. Saputro1 and Hideaki Kasai1,2 1 Department of Applied Physics, Osaka University, Suita, Osaka, Japan 2 Center for Atomic and Molecular Technologies, Osaka University, Suita, Osaka, Japan
KEYWORDS DHI/DHICA/dopachrome/eumelanin/ first-principles calculation/pH effect
CORRESPONDENCE Hideaki Kasai, e-mail: [email protected]
PUBLICATION DATA Received 11 March 2014, revised and accepted for publication 1 May 2014, published online 7 May 2014 doi: 10.1111/pcmr.12256
Summary Dopachrome conversion, in which dopachrome is converted into 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole2-carboxylic acid (DHICA) upstream of eumelanogenesis, is a key step in determining the DHI/DHICA monomer ratio in eumelanin, which affects the antioxidant activity. Although the ratio of DHI/DHICA formed and the conversion rate can be regulated depending on pH, the mechanism is still unclear. To clarify the mechanism, we carried out first-principles calculations. The results showed the kinetic preference of proton rearrangement to form quinone methide intermediate via b-deprotonation. We also identified possible pathways to DHI/DHICA from the quinone methide. The DHI formation can be achieved by spontaneous decarboxylation after proton rearrangement from carboxyl group to 6-oxygen. a-Deprotonation, which leads to DHICA formation, can also proceed with a significantly reduced activation barrier compared with that of the initial dopachrome. Considering the rate of the proton rearrangements in a given pH, we conclude that the conversion is suppressed at acidic pH.
Introduction Melanin is well known for its diverse protective functions in physiological systems. The major roles of melanin as a pigment are photoprotection and antioxidation of the skin, hair, and eyes. Melanin broadly absorbs UV and visible light, and dissipates more than 99.9% of the absorbed light energy nonradiatively (Meredith and Riesz, 2004). Melanin has also been reported to have a role as a scavenger of epidermal reactive oxygen species such as superoxide (Bustamante et al., 1993), hydroxyl radical (Korytowski and Sarna, 1990), and singlet oxygen (Sarna et al., 1985), corresponding to its antioxidative effect. To
exert such functions properly by pigmentation, animals must regulate melanogenesis. The relationship between melanogenesis and melanosomal pH is important to understand the diversity of human pigmentation. The significant difference in the color between Black and White human skin has been characterized as a consequence of a difference in melanosomal pH; melanosomes from Black skins are regulated to neutral by various ion transporters, whereas those from White skins exhibit acidic pH, probably due to polymorphisms in those ion transporters (Ito and Wakamatsu, 2010; Lamason et al., 2005; Smith et al., 2004; Sulem et al., 2008).
Significance Dopachrome converts into DHI or DHICA, which controls the DHI/DHICA ratio of generated eumelanin. Depending on the ratio, generated eumelanin exhibits different antioxidant activity. The dopachrome conversion rate for each product can be regulated by pH, with conversion rate accelerated by neutral pH. However, how pH affects the conversion is still unclear. Our study identifies the possible reaction scheme, including two proton rearrangements. From the scheme, the importance of neutral pH in the conversion can be attributed to high rate of the proton rearrangements.
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Effect of pH on dopachrome conversion
Natural melanin is a mixture of two chemically distinct pigments, black to brown eumelanin and yellow to reddish-brown pheomelanin (Simon et al., 2009). Ito and Wakamatsu (2008) emphasized that the real melanogenesis should be considered ‘mixed melanogenesis’, in which the two reaction pathways are chemically wellcontrolled, varying the ratio of eumelanin/pheomelanin. Recently, it was shown that lag of eumelanogenesis by knocking out dopachrome tautomerase (Dct) in mice leads to an increase in reactive oxygen species, sunburn cells, and apoptotic cells after UVA exposure (Jiang et al., 2010). These findings indicate the importance of eumelanogenesis for the protective functions of melanin. Biochemical relationships between melanosomal pH and various steps in the mixed melanogenesis have also been investigated, showing that the preferential pH for eumelanogenesis is a neutral pH (Ancans et al., 2001; Fuller et al., 2001; Ito et al., 2013; Thompson et al., 1985). From chemical degradation analysis, it is now generally recognized that eumelanin mainly consists of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and peroxidative cleaved pyrroles as building blocks or monomers (Ito, 1986). Therefore, regulation of the DHI/DHICA ratio in eumelanogenesis defines the main chemical composition of generated eumelanin, determining its main properties. In a recent experiment in which the radical scavenging activity of DHI-melanin and DHICA-melanin were compared, it was revealed that inclusion of DHICA in eumelanin is necessary to exert antioxidation activity (Jiang et al., 2010). Eumelanin can be obtained by the oxidation of certain precursors such as tyrosine or 3,4-dihydroxyphenylalanine (dopa) in the presence of tyrosinase or alternative catalysts. The formation of eumelanin proceeds via a number of intermediates. In particular, dopachrome is a critical intermediate determining the main properties of generated eumelanin. Mason (1948), who first identified dopachrome, demonstrated the subsequent conversion of the molecule into DHI or DHICA (Figure 1). As shown in Figure 1, there are two possible routes in this conversion, the decarboxylative route and the tautomerization route, corresponding to the formation of DHI and DHICA, respectively. Dopachrome conversion spontaneously but slowly proceeds to the formation of DHI, which is achieved non-enzymatically, whereas the conversion into €rner and Pawelek, 1980) in DHICA requires DCT/Dct (Ko mammals. Actually, in vitro experiments have shown highly selective formation of DHI at >95% at the usual pH. Using spectrophotometry and high performance liquid chromatography (HPLC) analysis, Sugumaran et al. (1990) and Sugumaran and Semensi (1991) revealed that dopachrome conversion to DHI or DHICA involves a quinone methide structure as an intermediate. Furthermore, several studies have indicated that, depending on experimental conditions such as pH value and concentration of metal ions, the ratio of DHI/DHICA and the conversion rate can be regulated even if the solution does ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Figure 1. Dopachrome conversion to DHI or DHICA. Labels in this figure denote the position for each carbon and nitrogen atom corresponding to usual chemical nomenclature.
not contain any enzymes (Ito et al., 2013; Mason, 1948; Muneta, 1977; Palumbo et al., 1987; Stravs-Mombelli and Wyler, 1985; Wakamatsu and Ito, 1988). It was found that in strongly acidic solutions (pH 1.3–2.0) dopachrome preferably converts to DHICA rather than DHI (Mason, 1948); furthermore, DHICA can be obtained under a strongly basic condition (pH 13; Stravs-Mombelli and Wyler, 1985; Wakamatsu and Ito, 1988). It was also shown that the reaction rate of dopachrome conversion is greatly promoted at neutral pH (Ito et al., 2013; Muneta, 1977). It should be noted that the reaction rate at pH 7.3 is 4.6-fold greater than that at pH 5.3 (Ito et al., 2013). The above experimental findings may provide important insights into the understanding of dopachrome conversion and eumelanogenesis. However, there has as yet been no detailed knowledge available of the reaction mechanism of dopachrome conversion that would enable us to interpret the experimental results properly. For further development of melanin chemistry and medical applications related with pigmentation, understanding dopachrome conversion at the molecular level is important for a basic knowledge of eumelanogenesis. Despite the importance of pH for dopachrome conversion, the accurate prototropic behaviors in the conversion are difficult to measure experimentally due to the intangible features of protonated/deprotonated structures of the molecule. Therefore, we employ theoretical approaches independent of experimental considerations in this study. To clarify the mechanism and prototropic equilibria of dopachrome conversion, we present theoretical calculations based on density functional theory (DFT) (Hohenberg and Kohn, 1964; Kohn and Sham, 1965). In these calculations, we calculated the total energy for various isomers of dopachrome and intermediates of this conversion. Candidates for the possible reaction schemes can be derived by comparing the stabilities of these 735
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isomers. To determine the actual scheme, we also evaluated the activation barriers for important elementary steps; a-deprotonation, b-deprotonation, and decarboxylation. Our results showed the energetic and kinetic preference of proton rearrangement to form quinone methide intermediate via b-deprotonation. In the next step, the formed quinone methide can proceed via two possible routes. The first starts from proton rearrangement from carboxyl group to 6-oxygen, leading to spontaneous decarboxylation, producing DHI. The quinone methide can also proceed via a-deprotonation to form DHICA, although the activation barrier is relatively high compared with that in DHI formation. Considering the rate of the proton rearrangements in a given pH condition, we conclude that the conversion is suppressed at acidic pH.
Results and Discussion Energetic stability for isomers in the first elementary steps of dopachrome conversion To understand dopachrome conversion, we first considered prototropic tautomers; dopachrome (A) and its tautomers in terms of prototropic rearrangement from carboxyl group or nitrogen to 5/6-oxygen (B–E) as defined in Table 1. The optimized structure of dopachrome (A) and its detailed geometrical parameters are shown in Figure 2 and Supporting Information Table S1, respectively. The structures of other molecules in this work are shown in Supporting Information Figures S1–S3. In Table 1 the thermodynamic stability of each tautomer is compared. As described in the Methods section, we considered the solvent effect using the polarizable continuum model (PCM). Table 1 shows the energetic preferability of dopa-
Figure 2. Optimized structure of dopachrome A (defined in Table 1).
chrome A in aqueous solution. Thus, tautomers B–E would appear to make only minor contributions to the conversion. However, there is still ambiguity. In particular, some of the features of tautomer B differ from those of A and C: an altered structure in carboxyl group and isosurface of the highest occupied molecular orbital (HOMO) (Supporting Information Figures S1 and S4, respectively). These features may imply a critically changed reactivity. To check whether the reaction scheme via structure B can be ignored, we will discuss the possibility of subsequent reactions from tautomer B below. It also should be noted that tautomer C is the most stable structure in vacuo. We emphasize that the natural atomic charges (see Methods section), shown in Supporting Information Table S2, of all structures are zwitter-ionic, with a relatively negative 6-oxygen and positive 1-hydrogen being observed. Therefore, the electronic structures of dopachrome tautomers are similar not to that of o-quinones but to that of iminochromes.
Table 1. Comparison of stability for each tautomer of dopachrome at first step of dopachrome conversion
Tautomera
Protonated sitesb
Energy/kcal∙mol1c
Gibbs Free energy/kcal∙mol1d
Equilibrium compositione
A (vac.) B (vac.) C (vac.) D (vac.) E (vac.) A (aq.) B (aq.) C (aq.) D (aq.) E (aq.)
Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl, Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl,
0.000 11.257 5.518 27.947 Unstablef 18.453 11.808 15.351 0.649 0.328
0.000 11.728 4.753 27.478 Unstablef 18.552 11.368 15.415 0.683 0.498
4.400 2.299 1.000 1.736 0 0.994 8.400 6.055 2.596 1.771
N1 O6 O5 N1 O6 O5
9 104 9 1012 9 1023
9 9 9 9
106 103 1014 1013
a Symbols for dopachrome tautomers at first step of dopachrome conversion. Calculation without and with PCM is respectively denoted as (vac.) and (aq.). b Numbers in this column correspond to the labels in Figure 1 that follow usual chemical nomenclatures. Carboxyl denotes protonation to the carboxylate ion. c The origin is set to the value of A (vac.). d The origin is set to the value of A (vac.). Temperature was set to 309.5 K as a condition of human body. e Equilibrium composition is defined as the mole fraction of each tautomer in equilibrium state, normalized by amount of all tautomers in vacuo or in aqueous solution. These compositions were calculated based on the value of the Gibbs free energies. The activity coefficients were ignored. f Spontaneous proton transfer to O6 was occurred.
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Effect of pH on dopachrome conversion
Energetic stability for isomers in the intermediate steps of dopachrome conversion As candidates for the intermediates of the conversion, we considered possible isomers formed via irreversible processes from structure A, namely the structures after proton rearrangement from a/b-carbon to 5/6-oxygen (A0 , B0 , E0 , F0 ), and their prototropic tautomers formed via proton rearrangement from carboxyl group or nitrogen to 5/6-oxygen (C0 , D0 , G0 , H0 ), as defined in Table 2. Comparison of the energetic stability is shown in Table 2. As shown in Table 2, structure A0 , which has a quinone methide structure, exhibits the lowest free energy of the b-deprotonated structures. This corresponds to the experiment where the quinone methide structure was detected as an intermediate of the conversion (Sugumaran and Semensi, 1991). It also should be noted that stable structure C0 was not found, and that spontaneous decarboxylation from structure C0 during optimization was confirmed. Therefore, we can assume that the DHI formation can be achieved by spontaneous decarboxylation via proton rearrangement from structure A0 to C0 if the quinone methide structure (A0 ) is involved in the actual reaction scheme. Other possibilities will be discussed below. Activation barriers for the first elementary steps in dopachrome conversion The activation barriers for the first elementary steps are evaluated here. As discussed above, irreversible proton
rearrangements are included in the conversion. Thus, the two structures between the proton rearrangement must be connected in terms of reaction coordinate. There are two ways in which protons can be arranged in water: the coherent process, where simultaneous deprotonation and protonation proceed in a concerted fashion, and the incoherent process, where the rearrangement is achieved via the deprotonation from dopachrome as the first step and subsequent proton diffusion to the oxygen. According to experimental and theoretical work, it generally has been concluded that the coherent process is not acceptable for the reaction coordinate of proton migration in real aqueous solution (Agmon, 1995). Therefore, we only consider the incoherent process. Dividing the process, we only evaluate the activation barrier for deprotonation because of the triviality of the subsequent steps. Figure 3 shows the energy profile during a-deprotonation (Figure 3A) and b-deprotonation (Figure 3B) from dopachrome A. As we discuss in Methods section, H2O trimer coordinated structures (Figure 4) were chosen as initial structures for a/b-deprotonation. Each curve in Figure 3 has two distinct regions, a steep zone and a plateau zone. We can see a similarity between the two steep zones, implying that the strengths of the two C–H bonds are almost same. On the other hand, the two plateau zones show different features; a-deprotonation proceeds with an inevitable increase of potential energy, and b-deprotonation exhibits a slightly negative gradient of the curve. Therefore, one can definitely say that it is
Table 2. Comparison of stability for each tautomer of dopachrome after proton rearrangement from a/b-carbon
Tautomera
Deprotonated/Protonated sitesb
Energy/kcal∙mol1c
Gibbs Free energy/kcal∙mol1d
Equilibrium compositione
A0 (vac.) B0 (vac.) C0 (vac.) D0 (vac.) E0 (vac.) F0 (vac.) G0 (vac.) H0 (vac.) A0 (aq.) B0 (aq.) C0 (aq.) D0 (aq.) E0 (aq.) F0 (aq.) G0 (aq.) H0 (aq.)
b-C/O5, N1, Carboxyl b-C/O6, N1, Carboxyl b-C/O5, O6, N1 b-C/O5, O6, Carboxyl a-C/O5, N1, Carboxyl a-C/O6, N1, Carboxyl a-C/O5, O6, N1 a-C/O5, O6, Carboxyl b-C/O5, N1, Carboxyl b-C/O6, N1, Carboxyl b-C/O5, O6, N1 b-C/O5, O6, Carboxyl a-C/O5, N1, Carboxyl a-C/O6, N1, Carboxyl a-C/O5, O6, N1 a-C/O5, O6, Carboxyl
11.252 0.430 Unstablef 6.466 2.311 5.929 1.566 13.889 22.910 14.293 Unstablef 6.218 13.827 19.121 23.517 25.843
9.777 1.036 Unstablef 6.958 3.281 4.319 1.890 13.128 22.331 13.698 Unstablef 5.850 12.986 17.982 22.748 25.180
1.000 2.313 0 1.522 2.586 6.017 2.482 1.000 1.000 7.895 0 2.300 2.411 8.106 1.881 0.981
9 108 9 9 9 9
1012 1012 107 1011
9 107 9 9 9 9
1012 109 106 102
a Symbols for dopachrome tautomers after proton rearrangement from a/b-carbon. Calculation without and with PCM is respectively denoted as (vac.) and (aq.). b Numbers in this column correspond to the labels in Figure 1 that follow usual chemical nomenclatures. Carboxyl denotes protonation to the carboxylate ion. c The origin is set to the value of A (vac.). d The origin is set to the value of A (vac.).Temperature was set to 309.5 K as a condition of human body. e Equilibrium composition is defined as the mole fraction of each tautomer in equilibrium state, normalized by total amount of tautomers deprotonated either from a/b-carbon in vacuo or in aqueous solution. These compositions were calculated based on the value of the Gibbs free energies. The activity coefficients were ignored. f Spontaneous decarboxylation was occurred.
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A
B
Figure 3. Energy profile for a/b-deprotonation of dopachrome A (defined in Table 1), corresponding to (A) and (B), respectively.
A
B
Figure 4. Initial structures for (A) a-deprotonation and (B) b-deprotonation. As an acceptor for dissociating proton, H2O trimer was chosen.
impossible for the proton rearrangement via a-deprotonation to proceed. Thus, we conclude that dopachrome (A) first forms the quinone methide intermediate (A0 ) via b-deprotonation. We were able to determine the exact transition state of the b-deprotonation, which has only one imaginary frequency, and the activation barrier (23.983 kcal/mol). If the solution is acidified, the apparent rate of the b-deprotonation will decrease due to the acceleration of proton recombination to the b-carbon. Thus, we can say that the formation of the quinone methide is a base-catalyzed reaction. The rapid formation of the quinone methide at neutral to weakly basic pH as experimentally reported could be described as the consequence of base-catalyses of hydroxyl ions or buffer ions such as phosphate ions. DHI/DHICA formation in dopachrome conversion From results in the subsection above, b-deprotonation would appear to be preferred to a-deprotonation, corresponding to formation of the quinone methide intermediate. In this section, we show the possible path to form DHI or DHICA from the quinone methide. To form DHI, the quinone methide must undergo decarboxylation via dissociation or rearrangement of the carboxyl proton, whereas the DHICA formation proceeds via a-deprotonation. First, we show the energy profiles during the a-deprotonation from the neutral quinone methide and 738
the decarboxylation from the carboxyl dissociated quinone methide in Figure 5 (A and B, respectively). As shown in Figure 5, the activation barriers for the a-deprotonation and decarboxylation are estimated at 11.353 and 8.960 kcal/mol, respectively. We were also able to determine the exact transition state for decarboxylation with its activation barrier, 8.975 kcal/mol. Although one can see the slightly lower activation barrier of decarboxylation, this is not evidence for the preferability of the DHI formation, especially if the instability of the carboxyl-dissociated quinone methide is considered. For an alternative choice, there is the DHI formation after proton rearrangement from the carboxyl group to 6-oxygen (C0 ). As mentioned above, the proton rearrangement leads to spontaneous decarboxylation. Therefore, the rate-determining step of the DHI formation from the quinone methide can be regarded as the dissociation of the proton from the carboxyl group or diffusion of the proton to the 6-oxygen. Considering the abnormally high mobility of proton in water, we assume that the rate for proton diffusion to the 6-oxygen can be ignored compared with that for proton dissociation from the carboxyl group. Due to the trivial superiority of carboxyl dissociation to a-deprotonation, we can state that the DHI formation dominates the DHICA formation unless carboxyl dissociation is suppressed. We consider that the preferability of the DHI formation corresponds directly to the experiment ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of pH on dopachrome conversion
in which more than 95% of DHI formation was confirmed. If the solution is acidified, the apparent rate of the carboxyl dissociation will decrease due to the acceleration of proton recombination to the carboxylate group. On the other hand, one can assume that the acidification will not contribute significantly to the acceleration of the backward reaction of a-deprotonation because of the relatively high activation barrier (7.564 kcal/mol; estimated from Figure 5A). On the basis of this, we conclude that DHI formation is more suppressed than DHICA formation at acidic pH, which might correspond to the pronounced formation of DHICA at the strongly acidic pH (Mason, 1948). The quinone methide route versus the indolenine route As mentioned in the first subsection of the Results and Discussion section, the reaction pathway that proceeds via unstable isomer B may contribute to the conversion. To confirm whether this possibility can be ignored, we also evaluated the activation barriers for subsequent reaction from structure B. Figure 6A,B, and C (the diamonds) show the energy profiles of tautomer B during a-deprotonation, b-deprotonation, and decarboxylation, respectively. In the case of decarboxylation, we determined the transition state that has only one imaginary
A
frequency, and the activation barrier, 9.250 kcal/mol. However, the diamonds in Figure 6(C) show a curious profile with a shoulder in the curve. In this shoulder, we confirmed a non-physically ‘rapid’ variation of dihedral angle in the bond between the carboxyl C and the a-C. This is an artificial result in which the motion is adiabatically restricted. To correct the activation barrier to reproduce the more reasonable value in the actual dynamics, we used the frozen value of the dihedral angle from before the non-physical variation. The circles in Figure 6(C) show the corrected energy profile during decarboxylation with the frozen coordinate. In the correction, the true activation barrier for decarboxylation is raised to 10.703 kcal/mol. On the basis of these results, the activation barrier for the decarboxylation is relatively lower than the values for a/b-deprotonation. However, we cannot regard these results as direct evidence for the preferability of the decarboxylative route because the rate of the decarboxylation from tautomer B depends not only on the absolute rate of the C–C bond cleavage along decarboxylation, but also on the relative rate constant of the C-C cleavage against the proton dissociation from structure B. To test the stability of tautomer B against the proton dissociation, we calculated the Gibbs free energy change between the associated and dissociated structures using the solvation model density (SMD), in which
B
Figure 5. Energy profile for a-deprotonation and decarboxylation of the quinone methide A0 (defined in Table 2), corresponding to (A) and (B), respectively.
A
B
C
Figure 6. Energy profile for (A) a-deprotonation, (B) b-deprotonation, and (C) decarboxylation of dopachrome tautomer C (defined in Table 1). The small pictures in this figure (C) exhibit a ‘rapid’ variation of dihedral angle in the cleaving C-C bond. The diamonds in this figure (C) show the fully unrestricted relaxed potential energy curve for all degrees of freedom except for the distance of the cleaving C-C bond. The circles show the potential energy curve along the frozen dihedral angle in the cleaving C-C bond preventing ‘rapid’ geometrical change.
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the short-range interactions between the solute and the solvent molecules are taken into account, in addition to the electrostatic interaction evaluated by the PCM (see Methods section) (Marenich et al., 2009). The thermochemical cycle we considered is shown in Figure 7. We used 265.3 kcal/mol as the value of DsG* for the absolute proton hydration free energy that was experimentally obtained at 298.15 K (Donald and Williams, 2010). To correct the value to that at 309.5 K, we considered the effect of temperature change by subtracting D(SDT) = 11.35 kcal/mol [where S is the molar entropy of proton, 39.8 cal/(K∙mol); Donald and Williams, 2010;]. Thus, we obtained the Gibbs free energy change with the carboxyl dissociation (2.816 kcal/mol), corresponding to pKa 1.99. On the other hand, the Gibbs free energy change (with the SMD) for tautomerization from structure A to B is 3.780 kcal/mol. This result clearly shows the stability of the dissociated state compared with structure B. Thus the dopachrome conversion does not appear to start via tautomerization to structure B due to its instability, but rather with the formation of quinone methide via proton rearrangement from b-carbon to 5oxygen. This route agrees with experiments in which it was concluded that the pathway via indolenine, a decarboxylated intermediate, is not acceptable in the dopachrome conversion scheme (Sugumaran and Semensi, 1991). Proposed scheme of dopachrome conversion The picture and energy diagrams of the whole scheme are outlined respectively in Figures 8 and 9. Dopachrome first undergoes proton rearrangement from b-carbon to 5oxygen, producting the quinone methide intermediate. This process is suppressed by acidification due to acceleration of proton recombination to b-carbon. The quinone methide formed can proceed via two possible routes whose rate depends on pH. At neutral pH, the quinone methide prefers to form DHI by spontaneous decarboxylation after proton rearrangement from the carboxyl group to the 6-oxygen. In contrast, an acidic pH prevents the DHI formation by reducing the apparent rate of the carboxyl dissociation. These effects of the acidic pH can lead to the reaction rate of dopachrome conversion being suppressed. Furthermore, the predominant formation of DHICA at the strongly acidic pH can
Figure 8. Proposed scheme of dopachrome conversion.
Figure 9. Energy diagram in dopachrome conversion. Numbers correspond to those of Figure 8.
also be achieved with suppressed carboxyl dissociation and weakly suppressed a-deprotonation. Based on the difference of activation barriers for carboxyl dissociation and a-deprotonation from the quinone methide, we consider that the formation of DHI is kinetically preferable compared with that of DHICA at the usual pH, corresponding qualitatively to the experiments at > 95% DHI formation. Although the rate constant for each elementary step can be calculated based on the transition state theory, such calculations will require additional assumptions and approximations. To omit the non-trivial modeling errors, we focused instead on the qualitative description here. In this study, we were able to derive the possible scheme of dopachrome conversion and to determine the correspondence of the pH effect to the reaction rate from first-principles calculations. Since our approach is based on the universal theory in physics, we expect that the effects of other factors in dopachrome conversion such as metal ions can also be clarified from first-principles calculations as the most transferable methodology.
Methods First-principles calculations based on density functional theory Figure 7. Thermochemical cycle to calculate pKa of dopachrome (DC).
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First-principles calculations were performed based on density functional theory using the Gaussian 09 program (Frisch et al., 2010). For
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of pH on dopachrome conversion Table 3. Comparison of different levels of theory
Table 4. Comparison of stability for each tautomer of dopachrome in the use of CAM-B3LYP
Exchange correlation functional
Basis set
Dipole moment/D
Bond length/ Aa
B3LYP B3LYP B3LYP B3LYP B3LYP CAM-B3LYP CAM-B3LYP CAM-B3LYP CAM-B3LYP CAM-B3LYP
6-31++G 6-31G(d,p) 6-31++G(d,p) 6-311++G(d,p) aug-cc-pVDZ 6-31++G 6-31G(d,p) 6-31++G(d,p) 6-311++G(d,p) aug-cc-pVDZ
10.2237 9.0513 9.6359 9.6230 9.6231 10.0312 8.8565 9.4546 9.4446 9.4685
1.514 1.531 1.521 1.520 1.521 1.506 1.523 1.513 1.512 1.513
Tautomera
Protonated sitesb
Energy/kcal∙mol1c
A (vac.) B (vac.) C (vac.) D (vac.) E (vac.) A (aq.) B (aq.) C (aq.) D (aq.) E (aq.)
Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl, Carboxyl, N1, O6 Carboxyl, N1, O5 Carboxyl,
0.000 16.502 4.825 3.725 Unstabled 18.246 11.100 14.470 3.523 Unstabled
N1 O6 O5 N1 O6 O5
a
Symbols for dopachrome tautomers. Calculation without and with PCM is respectively denoted as (vac.) and (aq.). b Numbers in this column correspond to the labels in Figure 1 that follow usual chemical nomenclatures. Carboxyl denotes protonation to the carboxylate ion. c The origin is set to the value of A (vac.). d Spontaneous proton transfer to O6 was occurred.
Distance between a-carbon and carboxyl carbon was chosen as the representative of structure.
a
the energetic analyses and determination of optimized structures of dopachrome and intermediates on the pathway being converted into DHI or DHICA, we used the Becke three-parameter hybrid method (Becke, 1993) with the Lee–Yang–Parr correlation functional approximation (Lee et al., 1988) (B3LYP) and 6-31++G(d,p) basis set. To validate the level of theory employed here, we compared various combinations of the exchange correlation functionals and the basis sets. As an alternative exchange correlation functional with B3LYP, we considered the Coulomb-attenuating method (CAM-B3LYP) where the exchange potential is improved by an additional parameter l so that charge transfer interactions such as hydrogen bondings are properly evaluated (Yanai et al., 2004). Basis dependence of the electric dipole moment, as the representative of electronic density, and the distance between a-carbon and carboxyl carbon, as the representative of structure, were investigated by comparing the results from 6-31++G, 6-31G(d,p), 6-31++G(d,p), 6-311++G(d,p), and aug-cc-pVDZ. Table 3 shows these comparisons for dopachrome A (defined in Table 1). As can be seen in Table 3, the polarization functions and the diffuse functions contribute significantly to the electric dipole moment and the bond length, whereas use of the different split valence basis sets or the correlation consistent basis sets only show small contributions. The basis dependence with CAM-B3LYP does not qualitatively differ from that with B3LYP. However, CAM-B3LYP might significantly stabilize highly hydrogenbonded structures, such as structure B (defined in Table 1). We therefore also compared the stability of dopachrome tautomers A, B, C, D, and E (defined in Table 1) with CAM-B3LYP. We tabulate the comparison in Table 4. To make comparison between Tables 1 and 4
A
easier, we also present the results of the use of polarizable continuum model (PCM; described later) in Table 4. Use of CAM-B3LYP instead of B3LYP did not show any remarkable stabilization. Although CAM-B3LYP significantly destabilized the O5-protonated structures (Table 4), these structures had already been evaluated as unstable for B3LYP. From the results summarized in Tables 3 and 4, we can conclude that the B3LYP/6-31++G(d,p) level is sufficient for addressing dopachrome conversion.
Natural population analysis Natural population analysis (Foster and Weinhold, 1980) was used to extract information about atomic charges from calculated electronic density of dopachrome A (defined in Table 1). The result of the analysis is shown in Table S2.
Reaction coordinates The activation barrier for each bond-break can be obtained by calculating the total energies along the intrinsic reaction coordinate. The bond-breaking requires an increase in the distance between the cleaving two atoms. Therefore we only chose one degree of freedom, the distance between two atoms cleaving with the dissociation, and all of the other degrees of freedom were allowed to relax adiabatically. When the reaction coordinate is treated in this
B
Figure 10. Energy profile for (A) a-deprotonation and (B) b-deprotonation of dopachrome A (defined in Table 1) using H2O pentamer as an acceptor for dissociating proton.
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Kishida et al. way, the water trimer accepting the dissociating proton is adiabatically accessible during the deprotonations. Although it is non-trivial whether such a treatment is able to reflect the actual dynamics of the deprotonation, we can evaluate the minimum activation energy for the deprotonation as a starting point for discussion. Thus, the true reaction coordinate is projected on a one-dimensional relaxed potential energy surface in this study.
Solvation model To take into account solvent effects, the polarizable continuum model using the integral equation formalism variant was employed (Tomasi et al., 2005). In this model, solvent is considered a continuous medium with a dielectric constant that is same as that of the liquid solvent, and solute is put in a cavity surrounded by the dielectric continuum. In this study, we focused solely on water as the target solvent. It should be noted that the solvent effect evaluated by the PCM changes the most stable tautomer of dopachrome; structure A is greatly stabilized, becoming the most stable tautomer, although structure C is the most stable structure in vacuo (A and C are defined in Table 1). Table 1 shows the comparison of energetic stability for each tautomer with and without the PCM stabilization.
Proton solvation with deprotonation In the case of proton solvation with the deprotonations from dopachrome and the intermediate of the conversion, an assessment that only takes into account polarization of the solvent using the PCM could lead to significant inaccuracies because the formation of a strong chemical bond between the proton and water molecules might affect the transition state. To appropriately reflect such an interaction between the proton and water molecules, we, in addition to taking the PCM, put a water trimer as an acceptor of the dissociating proton from dopachrome and the intermediate of the conversion (Figure 4A, B) only in the evaluation of the activation barrier for deprotonations. In general, a solvated proton in water forms triply coordinated H3O+ (H2O)3 and readily jumps over to other water molecules by forming an H5O2+ dimer structure, triggered by cleaving the hydrogen-bond between water molecules in the first and second hydration shell (Agmon, 1995). In our system, it is expected that the energy required for cleavage of old hydrogen-bond (to accept water molecule) with formation of hydronium ion H3O+ by the deprotonation can be assumed to be sufficiently small to be negligible compared with the energy required for cleavage of the covalent bond of the hydrogen within dopachrome. By contrast, once hydronium ion H3O+ is formed, hydrogen-bonds between H3O+-H2O will be enhanced; this stabilization effect is relatively strong and cannot be neglected. Therefore, the trimer acceptor model that we employed can be regarded as a model that takes into account such strengthening of the hydrogen-bond only in the first hydration shell. To check whether the size of water cluster we used is enough to evaluate the real activation barrier, we evaluated the activation barriers for a/b-deprotonation from dopachrome A using a water pentamer as an acceptor. Figure 10 shows the energy profile along a/b-deprotonation with the five water molecules, demonstrating the similarity with Figure 3. Because of the similarity, we only used the water trimer acceptor model in this work.
Acknowledgements This work is supported in part by: MEXT Grant-in-Aid for Scientific Research on Innovative Areas Program (2203-22104008), Scientific Research Programs (A) (24246013), and Grants for Excellent Graduate Schools (130820-140331) ‘Atomically Controlled Fabrication Technology’; JST ALCA Program ‘Development of Novel Metal-Air Secondary Battery Based on Fast Oxide Ion Conductor Nano Thickness Film’ and Strategic Japan-Croatia Research Cooperative
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Program on Materials Sciences Program ‘Theoretical Modeling and Simulations of the Structural, Electronic, and Dynamical Properties of Surfaces and Nanostructures in Materials Science Research’. Some of the numerical calculations presented here were done using the the computer facilities at the following institutes: CMC (Osaka University), ISSP, KEK, NIFS, and YITP.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Optimized structures (with PCM) of dopachrome tautomers at first step of dopachrome conversion. The blue, red, yellow, and purple spheres respectively represent hydrogen, oxygen, carbon, and nitrogen atoms. Labels in this figure denote each atom, but do not correspond to the numbers used in usual chemical nomenclatures. Figure S2. Optimized structures (with PCM) of dopachrome tautomers after proton rearrangement from a/bcarbon. For reference, the transitional structure of C0 is also listed, although this structure is not stable. The blue, red, yellow, and purple spheres respectively represent hydrogen, oxygen, carbon, and nitrogen atoms. Labels in this figure denote each atom, but do not correspond to the numbers used in usual chemical nomenclatures. Figure S3. Optimized structures (with PCM) of DHI and DHICA. The blue, red, yellow, and purple spheres respectively represent hydrogen, oxygen, carbon, and nitrogen atoms. Labels in this figure denote each atom, but do not correspond to the numbers used in usual chemical nomenclatures. Figure S4. HOMO isosurfaces of dopachrome tautomers at first step of dopachrome conversion. Table S1. Geometrical parameters (with PCM) of reactant (dopachrome A), intermediate (quinone methide A0 ), and products (DHI, DHICA) in dopachrome conversion. Table S2. Natural atomic charges (e) (with PCM) of tautomers of dopachrome at first step of dopachrome conversion.
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