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Determination of pKa constants of hypericin in aqueous solution of the anti-allergic hydrotropic drug Cromolyn disodium salt
Accepted Manuscript Research paper Determination of pKa Constants of Hypericin in Aqueous Solution of the AntiAllergic Hydrotropic Drug Cromolyn Disodium Salt Peter Keša, Marián Antalík PII: DOI: Reference:
S0009-2614(17)30286-5 http://dx.doi.org/10.1016/j.cplett.2017.03.059 CPLETT 34659
To appear in:
Chemical Physics Letters
Received Date: Revised Date: Accepted Date:
21 February 2017 19 March 2017 21 March 2017
Please cite this article as: P. Keša, M. Antalík, Determination of pKa Constants of Hypericin in Aqueous Solution of the Anti-Allergic Hydrotropic Drug Cromolyn Disodium Salt, Chemical Physics Letters (2017), doi: http:// dx.doi.org/10.1016/j.cplett.2017.03.059
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Determination of pKa Constants of Hypericin in Aqueous Solution of the Anti-Allergic Hydrotropic Drug Cromolyn Disodium Salt Peter Kešaa , Marián Antalíka,b a
Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Watsonova 47, 040 01 Košice, Slovakia b
Department of Biochemistry, Faculty of Natural Science, Pavol Jozef Šafárik University,
Šrobárova 2, 041 54 Košice, Slovakia
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Abstract In this work we established three from altogether six proton dissociation constants (pKa) of hydroxyl groups of hypericin in its monomeric form. The monomeric state of hypericin (5.0 x 10-6 mol.L-1) in aqueous solution was stabilized by the presence of hydrotropic drug Cromolyn disodium salt (6.0 x 10-2 mol.L-1). Data show that one acid-base transition occurs with the pKa of 7.8 and the other two are characterized by the apparent single pKa of 11.5. The spectral changes of hypericin above pH 13 indicate that the last two hydroxyls are deporotonized at this high pH values.
Keywords: cromolyn, ionization state, hydrotrope, hypericin
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1. Introduction Hypericin (Hyp) is a hydrophobic red photosensitive pigment found in plants of the Hypericum genus [1]. The agent has been the subject of extensive study due to its excellent antiproliferative and apoptotic activities against cancer cells during use in photodynamic therapy [2-7]. Hyp and other compounds isolated from Hypericum, e.g. hyperforin and aristoforin, show a tendency to interact with DNA and with G-quadruplex structures in DNA in particular [8-10]. Cromolyn disodium salt (DSCG) is an interesting and powerful drug which is used mainly in anti-allergic and antirhinitis treatments [11,12], but which has also shown some potential for use as an anti-fibrotic agent in the treatment of liver fibrosis and cirrhosis [13]. The physicalchemical and hydrotropic properties of DSCG are well known, and studies have revealed that the addition of hydrophobic Hyp to an aqueous solution of DSCG allows Hyp to adopt a monomeric form [14]. Hyp is one of the weaker acids and can theoretically possesses six possible macroscopic pKa constants. The position of the hydroxyl groups against each other on the phenanthroperylenequinone skeleton may be a reason for the simplification of four of these pKa constants, the theoretically predicted existence of various mesomer Hyp structures. Broad ambiguities can be found in studies relating to the theoretical and experimental knowledge about the dissociation of the hydroxyl groups of Hyp [15]. The dissociation process of Hyp is also hampered by its very low water solubility; high concentrations of organic solvents in aqueous solutions must be used in order to determine experimental pKa constants, although solvents may also have a significant effect on the observations of the pKa constants of the hydroxyl groups because of inaccuracies in pH measurements. Our previous work has demonstrated that low DSCG concentrations can influence the solubility of Hyp in an aqueous solution and also that the 3
low association constant between them indicates that dissolution is not mediated by the direct binding of Hyp to DSCG but is mediated instead by the hydrotropic effect [14]. The initial monodeprotonation of Hyp was clearly detected, with a pKa constant of 2 [16, 17]. In neutral pH, the hydroxylate groups are located in the bay region with a negative final charge. This negative charge on the Hyp bay region is stabilised by a proton of the neighbouring hydroxyls by hydrogen bonding; the increase in quantity during this bonding may enhance the molecule dipole moment. The change in the polarity of the solvent also contributes to the stability of the isomers [18]. It is highly likely that a negative charge is distributed during the intramolecular proton transfer to the surface due to the OH groups situated on the highly aromatic phenanthroperylenequinone ring [19]. This would suggest that a lowering of the pKa values relative to those of the naphthol and phenol families would be detected for the extensively conjugated biomolecules due to the greater stabilization of the conjugate base [20].
(A)
(B)
Fig. 1. Chemical structure of hypericin (A) and cromolyn disodium salt (B).
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As is described in Ding et al., DSCG has two negative charges on the carboxyl groups and is one of the weaker acids with a pKa constant of 2. Thus the attachment of protons to the DSCG structure renders the molecule practically insoluble in water, and, as a result, it is impossible to use DSCG as a hydrotropic substance in a pH environment of less than 3 [21]. In the present study we would like to introduce and describe a simple and accurate method of Hyp pKa constants in aqueous DSCG solutions, especially ranging at neutral and strong alkaline pH, where Hyp has adopted a monomeric form through the hydrotropic effect.
2. Experimental 2.1 Chemicals Hypericin was isolated from Hypericum perforatum (95% purity, commercially available from Sigma Aldrich); dimethylsulfoxide (DMSO, 99% purity) at spectroscopic grade, Cromolyn sodium salt (DSCG, 95% purity) and NaCl (98% purity, for molecular biology) were all purchased from Sigma Aldrich. All of the other chemicals were of analytical grade and were used without further purification. Deionised water was purified using the Purelab flex system (18M resistivity). All stock solutions were freshly prepared before each measurement. 2.2 UV VIS spectra measurements The absorption spectra were measured using a Jasco V-650 UV-VIS spectrophotometer (Jasco, USA) at room temperature with a 1cm glass cuvette in a wavelength range of 400-700 nm (100 nm/min. scan rate, 0.1 nm resolution) in a total volume of 1500 l. The baseline correction was conducted in the same volume of deionised water. Spectra were recorded with a 0.1 nm
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resolution. The pH curve was fitted by means of the sigmoidal Biphasic Dose Response Function using OriginPro 8 software. 2.3 pH measurement All pH measurements were carried out by using a Hanna HI 9017 pH meter (Hanna Instrument, USA) and a microelectrode HI 1082B (Hanna Instrument, USA) at 20°C.
3. Results and Discussion 3.1 Spectral characteristics of hypericin anion As is shown in figure 2, in both neutral and in a strongly alkaline pH level of ca. 13 is sufficient for the deprotonated (Hyp4-) anionic form of Hyp [22]. The spectra obtained in deionised water and 6 x 10-2 mol.L-1 of DSCG aqueous solution at neutral pH (black solid and red dashed lines) and alkaline pH (green solid and blue dashed lines) are characterised by their distinctive red shift and high dependence on the polarity environment. In a strongly alkaline pH range, we must take into consideration the fact that the starting state is marked by an absence of aggregates and has a characteristic green coloration compared to the neutral red [22]. This indicates that these states may be subject to different behaviours of Hyp within different polarity solvents, but it is impossible to measure these effects. Deprotonated anionic Hyp leads to intramolecular hydrogen bonding in the peri region [23], and the length of the intramolecular hydrogen bond of the phenyl group may also influence the estimated pKa value [15,24-25]. By comparing the absorption maxima (in figure 3) of Hyp in polar solvents and 6 x 10-2 mol.L-1 of DSCG it is possible to suggest that Hyp in DSCG also 6
exists in an anionic form under alkaline pH conditions. All of the observed band shifts are characteristic for each solvent (containing of 5% water), i.e., acetone, acetonitrile, DMSO, ethanol and methanol, and are shown in Figure 3 as a dependence on an absorption shift at an approximately neutral pH. Under these conditions, Hyp shows up in dianionic form [26,27]. Figure 3 depicts the absorbance shifts for solvents between the absorbance values from alkaline pH (black Y-axis) and the absorbance maxima relative to neutral pH (X-axis). The straight line which passes between these values was obtained with linear fitting and shows a slope of 2.7±0.7. This clearly reflects the different effects on the polarity environment and an evident spectral shift in the case of the DSCG aqueous solution may be justified as a different polarity value. The DSCG molecule is likely to have more impact on Hyp in a strongly alkaline environment compared to the neutral pH. This should be especially true considering the ionic-type of interactions and the fact that both molecules bear identical charge. On the other hand, such ionictype of interactions are not likely to be present at lower hydroxyl ion concentration.
Fig. 2. Absorption spectra of Hyp (5 x 10-6 mol.L-1) recorded in neutral deionised water (black solid line), 6 x 10-2 mol.L-1 of DSCG at pH6 (red dash line), 6 x 10-2 mol.L-1 of DSCG at 7
pH8.5 blue dash line) 6 x 10-2 mol.L-1 of DSCG at pH13 (orange dash line), deionised water at pH13 (green) and 6 x 10-2 mol.L-1 of DSCG at pH>14 (purple dash line).
Fig. 3. Comparison of band maximum for each solvent (absorbance spectra are inside), namely: ethanol (black), methanol (red), acetone (intensive blue), acetonitrile (yellow), DMSO (green) and DSCG (blue), obtained from the absorbance spectra at the absorbance maxima of Hyp at approx. neutral and strongly alkaline pH (approx. 13).
3.2 pK constant determination Figure 4 shows the absorption spectra of Hyp in a DSCG aqueous solution with a pH range of 5.95-12.40. The bathochromic shift of the spectral bands is evident following the point at which the solution becomes alkalized. As was also visible in figure 2 (blue spectrum), a final spectrum can be obtained through the gradual titration of small amounts of KOH. A successive diminishing of the peaks at 550 nm and 600 nm was observed as the pH range changed from pH
8
6 to the alkaline range. The absorbance spectra in this case are characterised by the progressive creation of a new peak which is typical for the anionic form of Hyp in strongly alkaline media (a band with a maximum at 630 nm). By fitting a black curve (inset to figure 4) obtained from successive pH titrations and taking the absorbance from the absorption maxima at 630 nm peak, we obtained an estimated pKa2 value of 7.8±0.2 and an estimated pKa3 value of 11.5±0.1 for the Hyp-DSCG aqueous solutions, values which are in accordance with Hyp2- and Hyp4- anionic forms [28]. From the steep slope 1.1±0.2 for our first proton dissociation (pKa2) and steep slope 1.6±0.1 for the second (pKa3), we can confirm that one proton is exchanged within the second pKa2, while at pKa3 a two proton reciprocity is possible, since the total spectral change for the first transition (absorption maximum at 630 nm) is only one third of that of the second transition. We also followed up the determination of the pKa constant from an absorption intensity band at 550 nm minus an absorption minimum of 560 nm. This positive approach is independent of the degree of Hyp aggregation. We obtained a similar pKa value of 7.7±0.8 and 11.5±0.2 for the Hyp-DSCG water solution using this method. Compared to cases in which a single pKa constant is measured, our approach involves two deprotonation transitions with three Hyp exchanged protons and can clearly be summarized in a range of pH 6-13. Only a single pKa value of 11 was determined in 80% ethanol and 80% DMSO aqueous solutions [16,29]. When Hyp is solubilised in a solution with a high concentration (percentage by volume) of ethanol or DMSO, the value of pKa obtained will be higher than the real value [15]. A theoretical pKa constant of around 8 would conform with the recent modelling of this structure of Hyp described by Ansto ter et al. [15]. These findings were based on a new statistical method introduced by Popelier group named AIBLHiCoS (Ab Initio Bond Length High Correlation Subsets) which calculates the length of C–O bonds for the 9
naphthol [15], and also the pKa constant estimated in our study is similar to the value obtained in that work. Owing to the low association constant which has been determined previously [14], this pKa value is adequate for Hyp in isolation and is not valid for the Hyp-DSCG complex. This suggests that DSCG does not in fact bind to Hyp but instead influences the environment polarity change through the hydrotropic effect [14]. A second possible explanation for this difference is that the concentration of the hydrotrope is relatively low, and this is physiologically normal with a similar level of ionic strength after the addition of Hyp. Our estimated pKa2 value is in agreement with the similar pKa value of 8 obtained by Strejckova et al. in the presence of diphytanoyl phosphatidylcholine membranes [30]. Jardon has measured the pKa of Hyp in Brij 35 micelles with two deprotonation steps: pKa ~ 7 and pKa ~ 11 [31]. In contrast, a pKa constant of 12.5 was recorded in reverse micelles [32] and 11.7 in Triton micelles [33] Hyp exists in monomeric form in a DSCG aqueous solution (5 x 10-6 mol.L-1), but the overall starting situation of the molecule in water is different, and Hyp clusters in dimers and aggregates. When measuring the absorbance spectra of dimers, a scattering of light on the surface is detected (Rayleigh scattering), and a subsequent movement of the spectral baseline is recorded. In these terms it is difficult to assess the apparent pKa constant from the absorbance results of the UV-VIS spectral measurements, but with the use of narrow absorbance bands, it is also possible to assess apparent pKa constants in gently scattering environments.
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Fig. 4. Absorption spectra of Hyp (5 x 10-6 mol.L-1) in 6 x 10-2 mol.L-1 of DSCG at pH levels of 5.95 to 12.40 measured in aqueous solutions.
By measuring the Hyp absorbance spectra in an aqueous solution with a relatively high ionic strength containing 1.2 x 10-1 mol.L-1 of KCl (similar to the observations made in the presence of 60 mmol.L-1 DSCG) we gradually reach a fuller understanding of the first pKa2 constant. We can also see the successive effect of the solubilisation (fig. 5) of the aggregated Hyp form by conducting a step by step KOH titration of Hyp. Obtaining measurements under these conditions is by no means an easy task. The procedure of determining the pKa constant for Hyp in a KCl water solution is not wholly reliable because the spectra were not recorded at the same baseline and followed the scattering of light onto the aggregate surfaces. Based on this fact, all of the absorbance spectra were arranged on the same baseline at a single point (585 nm) between two dominant peaks (as shown in fig. 5). The values for both peaks were then deduced by determining the value of the shoulders located between the peaks and subtracting the relevant values from each of them. The titration curve depicted in figure 5 is a cut with pH levels ranging from 5.95 to 8.85, a value range which, as in the case of DSCG, was decisive for our 11
observations. A pKa value of 7.5±0.1 was calculated using the same method as that described above. It was not possible to reproduce a pKa constant of around 11 for Hyp in a KCl aqueous solution. The spectrum of final deprotonated Hyp in water (fig. 2, green line) could not be obtained from successive titration or even after a longer duration of the experiment due to the kinetic-freezing state. The very slow dissociation process and aggregation form of Hyp were still present (fig. 5, brown spectrum) but the anionic form was reached at the same time. The scattering of light which marred this experiment meant that we were unable to determine a pKa value for a further deprotonation step. Accordingly, the results complement the claim that Hyp in KCl aqueous solution at a pH range of 6-13 may be assigned in two dissociation steps.
Fig.5. Absorption spectra of Hyp (5 x 10-6 mol.L-1) in the presence of 1.2 x 10-1 mol.L-1 of KCl aqueous solution. The pH titrating curve (inside) was also determined for the lower pKa constant.
All of the the macroscopic changes in the deprotonated Hyp structure are depicted in fig. 6. Firstly, the low value of the pKa1 is generated through Hyp aromaticity and the resulting ion is 12
stabilized by strong hydrogen bonding from the adjacent hydroxyl group on the bay region. Secondly, the negative charge is broadly delocalized on the surface of the molecule by means of the resonance structure of Hyp [34]. It is possible to assume that the deprotonation of the second (pKa2) -OH group is in the bay region of Hyp with one proton reciprocity. A comparable situation within the big difference in pKa constants is also found in 2,2’-biphenyl. It is true that the first pKa of 2,2’-biphenyl is lower, while the second is higher by several units. As is explained by Jonsson et al., this pKa splitting can be attributed to a stabilizing intramolecular hydrogen bond between –O- and –OH in the monodeprotonated 2,2’-biphenyl [35]. This suggestion is also supported by the fact that the deprotonation of hydroxyl groups on the peri region of Hyp occurs primarily at pH value above 9 [17]. The reason why the first –OH from Hyp deprotonates on the bay region and not at the peri region may be that, a negative charge on the bay region destabilizes the hydrogen bond, thereby facilitating the secession of the proton. We therefore conclude that this deprotonation step (pKa2) can also exist for aggregated Hyp, and this supposition was proven by successive titrations of Hyp in an aqueous solution of 120 mmol.L -1 of KCl. Some authors have supposed that a cofacial dimer of Hyp in water is formed [36-37]. The third deprotonation (two protons reciprocity) occurred in the peri region of the molecule near the methyl groups as a result of the strong influence of the negative charge delocalized in the bay region. Thus, pKa3 is the sum of two very close pKa values. A peculiarity of Hyp is also its ability to form intramolecular hydrogen bonding between –OH groups and carbonyl group (C=O) on the peri region which involves the possibility of intermolecular hydrogen binding within two or more Hyp molecules [38]. A comparison of the pKa for 2,2’-biphenyl (in the case of –OH group on the Hyp bay region) and 4,4’-biphenyl (in the case of –OH groups on the Hyp peri region) revealed a general similarity between the biphenyl derivatives, with a difference of several units separating the results [35]. Moreover, the last deprotonation steps which occurred in strongly alkaline pH 13
conditions are difficult to measure experimentally. Therefore, based on the final spectra of Hyp in the presence of DSCG in a strong alkaline solution (pink dash line) as depicted in fig.2, we suggest that the loss of the last two protons occurs in conditions higher than pH 13 and that this becomes possible mainly due to the symmetric mirror structure of the molecule, a form which is similar to that of 4,4’-biphenol [35]. In this paper, the authors have demonstrated that both of the protons from –OH groups on the 4,4’-biphenol are lost in two successive steps, each one unit lower [35]. It is possible that the loss of the two protons is accompanied by third and fourth deprotonations of Hyp with two proton reciprocity.
Fig. 6. pH dependent equilibrium of Hyp and its anionic forms.
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4. Conclusions Understanding Hyp behaviour in conditions similar to those found in vitro is crucial in improving the efficiency of the drug in photodynamic therapy use. This work follows up on our previous work, in which we introduced the coupling of two unique drugs (DSCG and Hyp) in order to augment their physicochemical properties. DSCG at micromolar concentrations serves as an ideal guest for the formation of Hyp monomers in water through the hydrotropic effect. This effect also allows the determination of pKa constants in individual dissociation steps. In general, the first pKa value of 2 for Hyp is created by a strong conjugative system in the bay region and is stabilised through surface charge distribution. Our results confirm that a second pKa2 value of 7.8 and a third pKa3 constant of 11.5 for Hyp in the presence of hydrotropic agent DSCG can be assigned to additional deprotonation steps, on the bay region, and close to the carbonyl groups in the peri region of Hyp (fig. 6), respectively. Thus, a secession of three protons occurs at this point. However, it is important to point out that a further pKa constants occur at a pH level of approximately 13 or higher. In general, our approach can be also helpful for the solubilisation of others water insoluble compounds e.g. parietin, for which is impossible to validate a first pKa in an aqueous solution [39].
Corresponding author: P. Keša, Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia. Tel.: +421 55 720 2334; fax: +421 55 633 6292. E-mail: [email protected]
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Notes The authors declare no competing financial interest.
Acknowledgement The authors would like to acknowledge the financial support of the VEGA 2-0038-16, APVV-15-0665 and APVV-15-0453 projects of the Slovak Research and Development Agency and also European structural fund ITMS code: 26220120033. The authors are also grateful to Mr. Gavin Cowper for manuscript proofreading in English. References [1]
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Graphical abstract
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Highlights: A monomeric form of hypericin in DSCG aqueous solution was confirmed pK constants at neutral and alkaline pH for hypericin were determined Three individual dissociations steps of hypericin were observed
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S0009-2614(17)30286-5 http://dx.doi.org/10.1016/j.cplett.2017.03.059 CPLETT 34659
To appear in:
Chemical Physics Letters
Received Date: Revised Date: Accepted Date:
21 February 2017 19 March 2017 21 March 2017
Please cite this article as: P. Keša, M. Antalík, Determination of pKa Constants of Hypericin in Aqueous Solution of the Anti-Allergic Hydrotropic Drug Cromolyn Disodium Salt, Chemical Physics Letters (2017), doi: http:// dx.doi.org/10.1016/j.cplett.2017.03.059
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Determination of pKa Constants of Hypericin in Aqueous Solution of the Anti-Allergic Hydrotropic Drug Cromolyn Disodium Salt Peter Kešaa , Marián Antalíka,b a
Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Watsonova 47, 040 01 Košice, Slovakia b
Department of Biochemistry, Faculty of Natural Science, Pavol Jozef Šafárik University,
Šrobárova 2, 041 54 Košice, Slovakia
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Abstract In this work we established three from altogether six proton dissociation constants (pKa) of hydroxyl groups of hypericin in its monomeric form. The monomeric state of hypericin (5.0 x 10-6 mol.L-1) in aqueous solution was stabilized by the presence of hydrotropic drug Cromolyn disodium salt (6.0 x 10-2 mol.L-1). Data show that one acid-base transition occurs with the pKa of 7.8 and the other two are characterized by the apparent single pKa of 11.5. The spectral changes of hypericin above pH 13 indicate that the last two hydroxyls are deporotonized at this high pH values.
Keywords: cromolyn, ionization state, hydrotrope, hypericin
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1. Introduction Hypericin (Hyp) is a hydrophobic red photosensitive pigment found in plants of the Hypericum genus [1]. The agent has been the subject of extensive study due to its excellent antiproliferative and apoptotic activities against cancer cells during use in photodynamic therapy [2-7]. Hyp and other compounds isolated from Hypericum, e.g. hyperforin and aristoforin, show a tendency to interact with DNA and with G-quadruplex structures in DNA in particular [8-10]. Cromolyn disodium salt (DSCG) is an interesting and powerful drug which is used mainly in anti-allergic and antirhinitis treatments [11,12], but which has also shown some potential for use as an anti-fibrotic agent in the treatment of liver fibrosis and cirrhosis [13]. The physicalchemical and hydrotropic properties of DSCG are well known, and studies have revealed that the addition of hydrophobic Hyp to an aqueous solution of DSCG allows Hyp to adopt a monomeric form [14]. Hyp is one of the weaker acids and can theoretically possesses six possible macroscopic pKa constants. The position of the hydroxyl groups against each other on the phenanthroperylenequinone skeleton may be a reason for the simplification of four of these pKa constants, the theoretically predicted existence of various mesomer Hyp structures. Broad ambiguities can be found in studies relating to the theoretical and experimental knowledge about the dissociation of the hydroxyl groups of Hyp [15]. The dissociation process of Hyp is also hampered by its very low water solubility; high concentrations of organic solvents in aqueous solutions must be used in order to determine experimental pKa constants, although solvents may also have a significant effect on the observations of the pKa constants of the hydroxyl groups because of inaccuracies in pH measurements. Our previous work has demonstrated that low DSCG concentrations can influence the solubility of Hyp in an aqueous solution and also that the 3
low association constant between them indicates that dissolution is not mediated by the direct binding of Hyp to DSCG but is mediated instead by the hydrotropic effect [14]. The initial monodeprotonation of Hyp was clearly detected, with a pKa constant of 2 [16, 17]. In neutral pH, the hydroxylate groups are located in the bay region with a negative final charge. This negative charge on the Hyp bay region is stabilised by a proton of the neighbouring hydroxyls by hydrogen bonding; the increase in quantity during this bonding may enhance the molecule dipole moment. The change in the polarity of the solvent also contributes to the stability of the isomers [18]. It is highly likely that a negative charge is distributed during the intramolecular proton transfer to the surface due to the OH groups situated on the highly aromatic phenanthroperylenequinone ring [19]. This would suggest that a lowering of the pKa values relative to those of the naphthol and phenol families would be detected for the extensively conjugated biomolecules due to the greater stabilization of the conjugate base [20].
(A)
(B)
Fig. 1. Chemical structure of hypericin (A) and cromolyn disodium salt (B).
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As is described in Ding et al., DSCG has two negative charges on the carboxyl groups and is one of the weaker acids with a pKa constant of 2. Thus the attachment of protons to the DSCG structure renders the molecule practically insoluble in water, and, as a result, it is impossible to use DSCG as a hydrotropic substance in a pH environment of less than 3 [21]. In the present study we would like to introduce and describe a simple and accurate method of Hyp pKa constants in aqueous DSCG solutions, especially ranging at neutral and strong alkaline pH, where Hyp has adopted a monomeric form through the hydrotropic effect.
2. Experimental 2.1 Chemicals Hypericin was isolated from Hypericum perforatum (95% purity, commercially available from Sigma Aldrich); dimethylsulfoxide (DMSO, 99% purity) at spectroscopic grade, Cromolyn sodium salt (DSCG, 95% purity) and NaCl (98% purity, for molecular biology) were all purchased from Sigma Aldrich. All of the other chemicals were of analytical grade and were used without further purification. Deionised water was purified using the Purelab flex system (18M resistivity). All stock solutions were freshly prepared before each measurement. 2.2 UV VIS spectra measurements The absorption spectra were measured using a Jasco V-650 UV-VIS spectrophotometer (Jasco, USA) at room temperature with a 1cm glass cuvette in a wavelength range of 400-700 nm (100 nm/min. scan rate, 0.1 nm resolution) in a total volume of 1500 l. The baseline correction was conducted in the same volume of deionised water. Spectra were recorded with a 0.1 nm
5
resolution. The pH curve was fitted by means of the sigmoidal Biphasic Dose Response Function using OriginPro 8 software. 2.3 pH measurement All pH measurements were carried out by using a Hanna HI 9017 pH meter (Hanna Instrument, USA) and a microelectrode HI 1082B (Hanna Instrument, USA) at 20°C.
3. Results and Discussion 3.1 Spectral characteristics of hypericin anion As is shown in figure 2, in both neutral and in a strongly alkaline pH level of ca. 13 is sufficient for the deprotonated (Hyp4-) anionic form of Hyp [22]. The spectra obtained in deionised water and 6 x 10-2 mol.L-1 of DSCG aqueous solution at neutral pH (black solid and red dashed lines) and alkaline pH (green solid and blue dashed lines) are characterised by their distinctive red shift and high dependence on the polarity environment. In a strongly alkaline pH range, we must take into consideration the fact that the starting state is marked by an absence of aggregates and has a characteristic green coloration compared to the neutral red [22]. This indicates that these states may be subject to different behaviours of Hyp within different polarity solvents, but it is impossible to measure these effects. Deprotonated anionic Hyp leads to intramolecular hydrogen bonding in the peri region [23], and the length of the intramolecular hydrogen bond of the phenyl group may also influence the estimated pKa value [15,24-25]. By comparing the absorption maxima (in figure 3) of Hyp in polar solvents and 6 x 10-2 mol.L-1 of DSCG it is possible to suggest that Hyp in DSCG also 6
exists in an anionic form under alkaline pH conditions. All of the observed band shifts are characteristic for each solvent (containing of 5% water), i.e., acetone, acetonitrile, DMSO, ethanol and methanol, and are shown in Figure 3 as a dependence on an absorption shift at an approximately neutral pH. Under these conditions, Hyp shows up in dianionic form [26,27]. Figure 3 depicts the absorbance shifts for solvents between the absorbance values from alkaline pH (black Y-axis) and the absorbance maxima relative to neutral pH (X-axis). The straight line which passes between these values was obtained with linear fitting and shows a slope of 2.7±0.7. This clearly reflects the different effects on the polarity environment and an evident spectral shift in the case of the DSCG aqueous solution may be justified as a different polarity value. The DSCG molecule is likely to have more impact on Hyp in a strongly alkaline environment compared to the neutral pH. This should be especially true considering the ionic-type of interactions and the fact that both molecules bear identical charge. On the other hand, such ionictype of interactions are not likely to be present at lower hydroxyl ion concentration.
Fig. 2. Absorption spectra of Hyp (5 x 10-6 mol.L-1) recorded in neutral deionised water (black solid line), 6 x 10-2 mol.L-1 of DSCG at pH6 (red dash line), 6 x 10-2 mol.L-1 of DSCG at 7
pH8.5 blue dash line) 6 x 10-2 mol.L-1 of DSCG at pH13 (orange dash line), deionised water at pH13 (green) and 6 x 10-2 mol.L-1 of DSCG at pH>14 (purple dash line).
Fig. 3. Comparison of band maximum for each solvent (absorbance spectra are inside), namely: ethanol (black), methanol (red), acetone (intensive blue), acetonitrile (yellow), DMSO (green) and DSCG (blue), obtained from the absorbance spectra at the absorbance maxima of Hyp at approx. neutral and strongly alkaline pH (approx. 13).
3.2 pK constant determination Figure 4 shows the absorption spectra of Hyp in a DSCG aqueous solution with a pH range of 5.95-12.40. The bathochromic shift of the spectral bands is evident following the point at which the solution becomes alkalized. As was also visible in figure 2 (blue spectrum), a final spectrum can be obtained through the gradual titration of small amounts of KOH. A successive diminishing of the peaks at 550 nm and 600 nm was observed as the pH range changed from pH
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6 to the alkaline range. The absorbance spectra in this case are characterised by the progressive creation of a new peak which is typical for the anionic form of Hyp in strongly alkaline media (a band with a maximum at 630 nm). By fitting a black curve (inset to figure 4) obtained from successive pH titrations and taking the absorbance from the absorption maxima at 630 nm peak, we obtained an estimated pKa2 value of 7.8±0.2 and an estimated pKa3 value of 11.5±0.1 for the Hyp-DSCG aqueous solutions, values which are in accordance with Hyp2- and Hyp4- anionic forms [28]. From the steep slope 1.1±0.2 for our first proton dissociation (pKa2) and steep slope 1.6±0.1 for the second (pKa3), we can confirm that one proton is exchanged within the second pKa2, while at pKa3 a two proton reciprocity is possible, since the total spectral change for the first transition (absorption maximum at 630 nm) is only one third of that of the second transition. We also followed up the determination of the pKa constant from an absorption intensity band at 550 nm minus an absorption minimum of 560 nm. This positive approach is independent of the degree of Hyp aggregation. We obtained a similar pKa value of 7.7±0.8 and 11.5±0.2 for the Hyp-DSCG water solution using this method. Compared to cases in which a single pKa constant is measured, our approach involves two deprotonation transitions with three Hyp exchanged protons and can clearly be summarized in a range of pH 6-13. Only a single pKa value of 11 was determined in 80% ethanol and 80% DMSO aqueous solutions [16,29]. When Hyp is solubilised in a solution with a high concentration (percentage by volume) of ethanol or DMSO, the value of pKa obtained will be higher than the real value [15]. A theoretical pKa constant of around 8 would conform with the recent modelling of this structure of Hyp described by Ansto ter et al. [15]. These findings were based on a new statistical method introduced by Popelier group named AIBLHiCoS (Ab Initio Bond Length High Correlation Subsets) which calculates the length of C–O bonds for the 9
naphthol [15], and also the pKa constant estimated in our study is similar to the value obtained in that work. Owing to the low association constant which has been determined previously [14], this pKa value is adequate for Hyp in isolation and is not valid for the Hyp-DSCG complex. This suggests that DSCG does not in fact bind to Hyp but instead influences the environment polarity change through the hydrotropic effect [14]. A second possible explanation for this difference is that the concentration of the hydrotrope is relatively low, and this is physiologically normal with a similar level of ionic strength after the addition of Hyp. Our estimated pKa2 value is in agreement with the similar pKa value of 8 obtained by Strejckova et al. in the presence of diphytanoyl phosphatidylcholine membranes [30]. Jardon has measured the pKa of Hyp in Brij 35 micelles with two deprotonation steps: pKa ~ 7 and pKa ~ 11 [31]. In contrast, a pKa constant of 12.5 was recorded in reverse micelles [32] and 11.7 in Triton micelles [33] Hyp exists in monomeric form in a DSCG aqueous solution (5 x 10-6 mol.L-1), but the overall starting situation of the molecule in water is different, and Hyp clusters in dimers and aggregates. When measuring the absorbance spectra of dimers, a scattering of light on the surface is detected (Rayleigh scattering), and a subsequent movement of the spectral baseline is recorded. In these terms it is difficult to assess the apparent pKa constant from the absorbance results of the UV-VIS spectral measurements, but with the use of narrow absorbance bands, it is also possible to assess apparent pKa constants in gently scattering environments.
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Fig. 4. Absorption spectra of Hyp (5 x 10-6 mol.L-1) in 6 x 10-2 mol.L-1 of DSCG at pH levels of 5.95 to 12.40 measured in aqueous solutions.
By measuring the Hyp absorbance spectra in an aqueous solution with a relatively high ionic strength containing 1.2 x 10-1 mol.L-1 of KCl (similar to the observations made in the presence of 60 mmol.L-1 DSCG) we gradually reach a fuller understanding of the first pKa2 constant. We can also see the successive effect of the solubilisation (fig. 5) of the aggregated Hyp form by conducting a step by step KOH titration of Hyp. Obtaining measurements under these conditions is by no means an easy task. The procedure of determining the pKa constant for Hyp in a KCl water solution is not wholly reliable because the spectra were not recorded at the same baseline and followed the scattering of light onto the aggregate surfaces. Based on this fact, all of the absorbance spectra were arranged on the same baseline at a single point (585 nm) between two dominant peaks (as shown in fig. 5). The values for both peaks were then deduced by determining the value of the shoulders located between the peaks and subtracting the relevant values from each of them. The titration curve depicted in figure 5 is a cut with pH levels ranging from 5.95 to 8.85, a value range which, as in the case of DSCG, was decisive for our 11
observations. A pKa value of 7.5±0.1 was calculated using the same method as that described above. It was not possible to reproduce a pKa constant of around 11 for Hyp in a KCl aqueous solution. The spectrum of final deprotonated Hyp in water (fig. 2, green line) could not be obtained from successive titration or even after a longer duration of the experiment due to the kinetic-freezing state. The very slow dissociation process and aggregation form of Hyp were still present (fig. 5, brown spectrum) but the anionic form was reached at the same time. The scattering of light which marred this experiment meant that we were unable to determine a pKa value for a further deprotonation step. Accordingly, the results complement the claim that Hyp in KCl aqueous solution at a pH range of 6-13 may be assigned in two dissociation steps.
Fig.5. Absorption spectra of Hyp (5 x 10-6 mol.L-1) in the presence of 1.2 x 10-1 mol.L-1 of KCl aqueous solution. The pH titrating curve (inside) was also determined for the lower pKa constant.
All of the the macroscopic changes in the deprotonated Hyp structure are depicted in fig. 6. Firstly, the low value of the pKa1 is generated through Hyp aromaticity and the resulting ion is 12
stabilized by strong hydrogen bonding from the adjacent hydroxyl group on the bay region. Secondly, the negative charge is broadly delocalized on the surface of the molecule by means of the resonance structure of Hyp [34]. It is possible to assume that the deprotonation of the second (pKa2) -OH group is in the bay region of Hyp with one proton reciprocity. A comparable situation within the big difference in pKa constants is also found in 2,2’-biphenyl. It is true that the first pKa of 2,2’-biphenyl is lower, while the second is higher by several units. As is explained by Jonsson et al., this pKa splitting can be attributed to a stabilizing intramolecular hydrogen bond between –O- and –OH in the monodeprotonated 2,2’-biphenyl [35]. This suggestion is also supported by the fact that the deprotonation of hydroxyl groups on the peri region of Hyp occurs primarily at pH value above 9 [17]. The reason why the first –OH from Hyp deprotonates on the bay region and not at the peri region may be that, a negative charge on the bay region destabilizes the hydrogen bond, thereby facilitating the secession of the proton. We therefore conclude that this deprotonation step (pKa2) can also exist for aggregated Hyp, and this supposition was proven by successive titrations of Hyp in an aqueous solution of 120 mmol.L -1 of KCl. Some authors have supposed that a cofacial dimer of Hyp in water is formed [36-37]. The third deprotonation (two protons reciprocity) occurred in the peri region of the molecule near the methyl groups as a result of the strong influence of the negative charge delocalized in the bay region. Thus, pKa3 is the sum of two very close pKa values. A peculiarity of Hyp is also its ability to form intramolecular hydrogen bonding between –OH groups and carbonyl group (C=O) on the peri region which involves the possibility of intermolecular hydrogen binding within two or more Hyp molecules [38]. A comparison of the pKa for 2,2’-biphenyl (in the case of –OH group on the Hyp bay region) and 4,4’-biphenyl (in the case of –OH groups on the Hyp peri region) revealed a general similarity between the biphenyl derivatives, with a difference of several units separating the results [35]. Moreover, the last deprotonation steps which occurred in strongly alkaline pH 13
conditions are difficult to measure experimentally. Therefore, based on the final spectra of Hyp in the presence of DSCG in a strong alkaline solution (pink dash line) as depicted in fig.2, we suggest that the loss of the last two protons occurs in conditions higher than pH 13 and that this becomes possible mainly due to the symmetric mirror structure of the molecule, a form which is similar to that of 4,4’-biphenol [35]. In this paper, the authors have demonstrated that both of the protons from –OH groups on the 4,4’-biphenol are lost in two successive steps, each one unit lower [35]. It is possible that the loss of the two protons is accompanied by third and fourth deprotonations of Hyp with two proton reciprocity.
Fig. 6. pH dependent equilibrium of Hyp and its anionic forms.
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4. Conclusions Understanding Hyp behaviour in conditions similar to those found in vitro is crucial in improving the efficiency of the drug in photodynamic therapy use. This work follows up on our previous work, in which we introduced the coupling of two unique drugs (DSCG and Hyp) in order to augment their physicochemical properties. DSCG at micromolar concentrations serves as an ideal guest for the formation of Hyp monomers in water through the hydrotropic effect. This effect also allows the determination of pKa constants in individual dissociation steps. In general, the first pKa value of 2 for Hyp is created by a strong conjugative system in the bay region and is stabilised through surface charge distribution. Our results confirm that a second pKa2 value of 7.8 and a third pKa3 constant of 11.5 for Hyp in the presence of hydrotropic agent DSCG can be assigned to additional deprotonation steps, on the bay region, and close to the carbonyl groups in the peri region of Hyp (fig. 6), respectively. Thus, a secession of three protons occurs at this point. However, it is important to point out that a further pKa constants occur at a pH level of approximately 13 or higher. In general, our approach can be also helpful for the solubilisation of others water insoluble compounds e.g. parietin, for which is impossible to validate a first pKa in an aqueous solution [39].
Corresponding author: P. Keša, Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia. Tel.: +421 55 720 2334; fax: +421 55 633 6292. E-mail: [email protected]
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Notes The authors declare no competing financial interest.
Acknowledgement The authors would like to acknowledge the financial support of the VEGA 2-0038-16, APVV-15-0665 and APVV-15-0453 projects of the Slovak Research and Development Agency and also European structural fund ITMS code: 26220120033. The authors are also grateful to Mr. Gavin Cowper for manuscript proofreading in English. References [1]
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Graphical abstract
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Highlights: A monomeric form of hypericin in DSCG aqueous solution was confirmed pK constants at neutral and alkaline pH for hypericin were determined Three individual dissociations steps of hypericin were observed
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