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Puzzling Aqueous Solubility of Guanine Obscured by the Formation of Nanoparticles Termeh Darvishzad, Tomasz Lubera, and Stefan S. Kurek J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04327 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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The Journal of Physical Chemistry

Puzzling Aqueous Solubility of Guanine Obscured by the Formation of Nanoparticles Termeh Darvishzad, Tomasz Lubera, Stefan S. Kurek* Department of Biotechnology and Physical Chemistry, Cracow University of Technology, ul. Warszawska 24, 31-155 Krakow, Poland

E-mail address of the corresponding author: [email protected]

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Dissolution of guanine in neutral solutions was obscured by peculiar behavior of guanine, indicating an apparent dependence of solubility on the amount of solid guanine used. Here, we demonstrate that the problem is caused by formation of tiny guanine nanoparticles that tend to grow forming stable particles of ca. 800 nm size. This effect can be minimalized by using small quantities of guanine powder for dissolution. We show also that assuming a constant, independent of pH, concentration of neutral form of guanine, at 25 °C equal 25.4 µM, and applying known pKa values related to its dissociation or protonation, it is possible to calculate the concentrations of all conjugate acids and bases of guanine at the given pH value, and by summing them up, the guanine solubility.


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INTRODUCTION Puzzling behavior of guanine upon dissolution made its solubility determination misleading. It was certain only that its solubility in neutral solutions was negligible. Adding small amounts of water to guanine powder leads to swelling and formation of a thick paste. Its dilution gives a muddy solution. DeVoe and Wasik1 studying guanine solubility noticed that and decided to force water through this paste mixed with silanized diatomaceous silica particles in a liquid chromatography column at a high pressure. They determined the solubility of guanine at 25 °C to be 39 ± 1 µM with ∆solnH equal 49.2 ± 0.6 kJ mol−1. The pH value of the resulting saturated solution was not given. The authors noted that solubility values found in the literature were generally higher. The Handbook of Chemistry and Physics gives also a relatively high value of 45 µM (0.068 mg/L) quoted from the literature2. In 2010, it was reported3 that the concentration of guanine at pH 7 depended on the amount of guanine powder used for solution preparation, reaching the value of even 100 µM. The solutions were prepared by dissolving guanine in citrate buffers at 40 °C and equilibrating them at 25 °C. The method worked faultlessly for other nucleobases and the lowest obtained value of 40 µM was assumed as the most reliable. The authors also mentioned that dissolution of guanine led regularly to a change in pH. Owing to its biological importance, there is a great interest, recently even increasing, in the development of new methods for guanine analysis, mainly electrochemical. In many papers, it was claimed that solutions of concentrations up to 100 µM were used at pH 7, a value exceeding the above mentioned reported values of solubility. In some other, also our own4, the work solutions were prepared by diluting the saturated stock solution, the concentration of which was assumed to be 40 µM. However, some other researchers determined themselves the concentration of their saturated solutions, interestingly, each time obtaining slightly different values, but generally close to 25 µM5. In nature, guanine occurs in the form of nucleosides, in which it is chemically linked to pentoses, either ribose as in guanosine or deoxyribose as in deoxyguanosine. The sugar unit in them could be additionally phosphorylated. The presence of such groups makes these compounds better soluble. However, unsubstituted guanine could also be found in nature. Its tiny crystals are responsible for metallic luster in fish, certain spiders and even for tuning the color of panther chameleons6,7. Tiny crystalline guanine platelets are used for reflecting light in the concave mirror of the scallop eye8. Guanine crystallization is very difficult. The obtained crystal form depends on crystallization conditions, the pH value9, above all. From highly acidic solutions (pH 1-3) guanine monohydrate is obtained, neutral and basic solutions (pH 7-13) give anhydrous guanine crystals, and alkaline solutions (pH 14) allow crystallization of guanine sodium salt. Anhydrous guanine occurs in two crystalline forms, a marginally more stable α-form, and β-form, regarded as more kinetically favored10. So far, only β-form was found in nature. Guanine microcrystals extracted from fish scales or guano are used for their pearly and lustrous 3| ACS Paragon Plus Environment


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effect in cosmetics, but also in other commercial products11. Guanine microcrystals could also be oriented along applied magnetic fields, which makes them very interesting materials for special applications12. Thus, guanine solubility and its pH dependence should be a very important parameter in designing new crystallization methods. Here, we show, how, based on thermodynamics and employing known pKa values, guanine solubilities over a wide range pH values can be calculated, particularly the neutral pH range.

EXPERIMENTAL SECTION Materials. Guanine 98%, and other reagents were purchased from Sigma-Aldrich and used as received. For the preparation of all solutions, Mili-Q water was applied. Instruments. UV spectra were recorded in 10 mm quartz cuvettes using an HP 8453 Spectrophotometer, DLS measurements were done with Zetasizer Nano ZS (Malvern) Instrument. General procedure. A small quantity of guanine (1 ± 0.2 mg, but in preliminary studies 5-100 mg) was added into 10 mL water or buffer solution, then it was sonicated in a laboratory ultrasonic bath (100 W) at 25.0 °C for the total time of 20 mins with removing the mixture from the bath and shaking it every 5 mins. During sonication, finely ground ice was occasionally added to the bath to prevent the temperature to rise by more than 0.5 °C. Afterwards, the mixture was equilibrated at 25.0 °C for 30 mins, and eventually centrifuged in a laboratory centrifuge (16000 g RCF) with its rotor brought to 25 °C just before starting it. After 5 mins centrifuging, the supernatant was transferred to a new tube and centrifuged for another 10 mins. All subsequent measurements were done on the supernatant after this second centrifuging step. Concentration determination. The concentrations were determined from UV spectra of obtained solutions. Guanine stock solution in 5% phosphoric acid was used to prepare standard solutions of guanine in buffer solutions. As can be seen in Fig. 1, guanine in this solution occurs in its monoprotonated form, GuaH+, in concentrated phosphoric acid diprotonated form, GuaH22+, prevails, and in 6.7 buffer solution there is practically only neutral form, Gua, present. As the spectra and absorption coefficients measured by us agreed well with the published data, we used the literature values given in Table 1 to calculate the concentrations. In pH ranges, where more than one guanine species was present, we calculated the ratios of these species from known pKa values at the given pH (vide infra) and based on them ratio-weighed mean absorption coefficients, which we used to calculate the concentration. Table 1. Ultraviolet spectra of guanine and its conjugated acids and bases used for concentration determination Species 2+

GuaH2 GuaH+ Guanine (Gua)

λmax / nm (log ε)

Ref.

236 (4.01); 252 (3.95) 248 (4.03); 271 (3.85) 246 (4.01); 275 (3.89)

13 14 14


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Gua− Gua2−

245 (3.78); 273 (3.87) 221 (4.12); 274 (3.94)

14 14

2+

Figure 1. UV spectra of a solution of guanine in 65% phosphoric acid (GuaH2 ), in 5% phosphoric acid (GuaH+), in pH 6.7 phosphate buffer (Gua), and in pH 10.75 phosphate buffer (Gua−).

RESULTS Dissolution of guanine in neutral solutions is very difficult. We have decided to use sonication at 25 °C, and separation of the resulting suspension by centrifuging it, as described in detail in Experimental Section. The supernatant liquid seemed always to be clear, but UV spectra always showed a slight steady increase in the baseline towards shorter wavelengths, which might indicate the presence of colloidal particles. The DLS confirmed that particles as small as 20 nm in diameter were present. Generally, the concentration of colloidal particles was high and we had to dilute solutions several times to get the size distribution data. The reproducibility was not very good. The fraction and size of smaller particles varied, however, the size of larger particles, of ca. 700-800 nm, was reproducible. Fig. 2 shows a typical DLS picture of a solution in pure water. The smallest particles tend to get bigger and after ca. 10 days the curves shift to larger particle sizes, which can be seen in Fig. 3. Colloidal particles are also formed in acidic solutions. Again, there is a group of small and bigger particles that tend to grow, as shown in Fig. 4. It should be stressed that all the solutions were visibly clear. However, they scattered light, as could be seen by passing a laser beam 5| ACS Paragon Plus Environment


through the solution. As noticed by one of the reviewers, the process may be not aggregation, but rather the growth of the smaller particles. Thus, a sort of Ostwald ripening mechanism may operate here.

Figure 2. Typical size distribution of colloidal particles in a solution of guanine in pure water.

Figure 3. Size distribution of colloidal particles in a solution in 0.1 M pH 7 PBS, directly after preparation and after 14 days.

b)

a) )

)

Figure 4. Size distribution of colloidal particles in a freshly prepared solution of (a) 0.1 M pH 4.0 PBS buffer, and (b) 0.1 M pH 5.5 acetate buffer.

We have also tried passing water through a small glass chromatography column filled with guanine powder. Adding water gave rise to swelling of the powder layer and it was impossible to collect any supernatant liquid. The resulting suspension was relatively stable and did not settle even after a couple of days. A sample of this suspension was centrifuged three times for 20 mins and the pH of supernatant was equal 3.38. The UV spectrum indicated that guanine was present predominantly in its monoprotonated form (GuaH+). The concentration of guanine calculated from UV spectrum of this solution was 55 µM. Most measurements were done in buffer solutions, most often phosphate buffers (PBS), but we used also acetate buffers to check whether the electrolyte type affects the solubility. It appeared that there were no significant differences between the buffers. As it is known that some cations promote the formation of 6| ACS Paragon Plus Environment

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guanine tetrads, and consequently, guanine quadruplexes, and potassium cations are the most efficient promoters of this process15, we checked whether there is a difference between phosphate buffers containing only sodium and those with only potassium cations. As no significant difference was observed*, we used only potassium containing phosphate buffers in subsequent measurements. We have also checked the effect of ionic strength by using 0.5 M sodium chloride and 0.5 M sodium sulfate solutions. In both cases, only a drop of up to 0.5 pH units was noticed, but the concentrations was roughly the same as obtained in buffer solutions of the same final pH. As mentioned above, dissolving guanine in water decreases the pH of the solution. The same concerns to a lesser extent buffer solutions, particularly diluted ones, which was also observed previously3. When using 5 mg guanine and 10 mL water, 0.3 M phosphate buffer is required to keep the pH value constant to 0.01 units. But for 1 mg guanine, 0.1 M buffer solution was sufficient. Sonication may also lead to undesired results. In one case, we used 0.8 g guanine and 10 mL 0.1 M pH 7 PBS, and sonicated this mixture as usual. The UV spectrum of the supernatant after centrifuging was very complex with multiple bands in the UV range, indicating a partial breakdown of guanine. But the use of 1.0 ± 0.2 mg only in 10 mL water or a buffer solution did not lead to any problems. In the given guanine mass range there was no correlation between the deviation from the exact guanine mass and its concentration in solution. Striving to get highly reproducible, accurate results, we repeated the measurements at pH 7 in 0.1 M PBS 20 times and got the mean value of 25.7 µM with the standard deviation ±1.4 µM. Other points in the pH range 4-9 were obtained as average of 3-5 measurements. The obtained results will be discussed below.

DISCUSSION Guanine may undergo dissociation or be protonated, and hence may occur as a neutral molecule, but also as mono- and diprotonated conjugate acids or mono- and dianionic conjugate bases, as shown below. O HN H2N

+

H N

O HN

+

N N H H 2+ GuaH2

H2N

+

H N

O HN

N N H GuaH+

H2N

N

O H N N

–

N H2N

Gua

*

O

N

–

N

N

N Gua−

N H

–

H2N

N

N

Gua2−

In our paper on guanine electrochemistry4 we supposed that there might be a difference in guanine solubility in potassium and sodium phosphate buffers, but repeated measurements done within this work indicated these differences to be not statistically significant.



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2+

Only the most stable tautomers are presented above, based either on NMR spectra (GuaH2 , Gua2−)13 or on quantum chemical calculations16. In the case of cationic and anionic forms, the most stable tautomers are present in excess of 90%, but the neutral 7H tautomer shown is in equilibrium (70:30) with the 9H one16. The dication can be seen only in concentrated acids, like 65% phosphoric acid, 10.85 M sulfuric acid, trifluoroacetic acid and fluorosulfuric acid13. All these forms in solution are in equilibrium with each other, and when solid guanine is present, also with it (Gua denotes neutral guanine in solution, other notations, as above). 2+

Gua(solid) ⇄ Gua ⇄ GuaH+ − H+ ⇄ GuaH2 − 2H+ ⇄ Gua− + H+ ⇄ Gua2− + 2H+

(1)

Accordingly, we can equate chemical potentials: 2+

µ⦵solid = µ(Gua) = µ(GuaH+) − µ(H+) = µ(GuaH2 ) − 2µ(H+) = µ(Gua−) + µ(H+) = µ(Gua2−) + 2µ(H+) = = µ⦵(Gua) + RT ln c(Gua) = µ⦵(GuaH+) + RT ln c(GuaH+) + RT·ln(10)·pH = 2+

2+

= µ⦵( GuaH2 ) + RT ln c(GuaH2 ) + 2RT·ln(10)·pH = = µ⦵(Gua−) + RT ln c(Gua−) − RT·ln(10)·pH = µ⦵(Gua2−) + RT ln c(Gua2−) − 2RT·ln(10)·pH

(2)

Please note that at equilibrium, the values of chemical potentials, and hence, the concentrations of all the species, but the neutral one, are a function of pH. In the presence of solid guanine, the chemical potential, and, accordingly, the concentration of neutral guanine must be constant irrespective of pH. The concentrations of all the other species can then be calculated using known values of pKas, that set the ratios between the respective species and their conjugate bases or acids, and H+ (pH). The solubility of guanine is then equal to the sum of equilibrium concentrations of all the existing forms of guanine in the presence of solid guanine. 2+

Guanine solubility = c(Gua) + c(GuaH+) + c(GuaH2 ) + c(Gua−) + c(Gua2−)

(3)

Table 2. pKa values of acid-base equilibria for guanine and its conjugate acids and bases Notation

Equilibrium 2+

pKa +

+

pKa1 −1.0 ± 0.2 GuaH2 ⇄ GuaH + H pKa2 3.3 ± 0.08 GuaH+ ⇄ Gua + H+ pKa3 9.2 ± 0.08 Gua ⇄ Gua− + H+ pKa4 12.3 ± 0.08 Gua− ⇄ Gua2− + H+ Note: the pKa1 value is a mean of two values based on NMR and UV spectroscopic studies.

Ref. 13 14 14 14

The pKa values listed in Table 2 allow calculating the ratio of concentrations of the acid to its conjugate base as a function of pH, as the example for pKa2 shows:

ܿሺGuaH ା ሻ = 10ሺ୮௄౗మ ି୮ୌሻ ܿሺGuaሻ

(4)


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From the set of all such equations and assuming that the total amount is 100 %, it is possible to determine the fractions of guanine and its ionized species in a solution at various pH values, which is shown in Fig. 5 below. It can be seen that in the middle range, between ca. pH 5 and 7.5 more than 98 % guanine is in its neutral form. The ranges for other forms are much narrower.

Figure 5. Speciation of guanine solution as a function of pH calculated from the pKa values.

Figure 6. pH dependence of guanine solubility. The curve was calculated assuming a constant concentration of neutral guanine equal to 25.4 µM and summing up the concentrations of its ionized forms calculated from the respective pKa values. The inset shows this curve over a wider range of pH.

The concentrations of all ionized forms of guanine are related through the pKa values to the concentration of the neutral form, which should be constant over all the pH range in the presence of solid guanine. Knowing this concentration would allow calculating the concentrations of all the other forms at a given pH, and hence the solubility, which would be the total concentration of all the guanine species present in solution in equilibrium with solid guanine. If there are any other forms existing in significant 9| ACS Paragon Plus Environment


amounts, like dimers or quartets, the deviation between the calculated and experimental values would show it. It appeared that the best least squares fit of the total concentration thus calculated to our experimental points in Fig. 6 gave the value of 25.4 µM for the concentration of neutral guanine. The solubility curve in terms of the total concentration of all guanine forms shown in Fig. 6 was calculated as described above. The inset in this Figure demonstrates how this solubility varies with pH in a wider range. Overlapping this curve onto the experimental points published by other researches3 showed a nearly ideal agreement in the range between pH 1 and 2. This confirms that there are no other guanine forms present in significant amounts, at least in acidic solutions in the range indicated. Getting back to the facts observed in attempts to dissolve guanine in water, the swelling of guanine powder upon adding water can easily be understood, when examining the crystal structure of anhydrous guanine17 or its monohydrate18. Both form sheets consisting of hydrogen bonded guanine molecules. The sheets are stacked and kept together by π-π interactions between offset parallel guanine rings. In guanine monohydrate there are additionally water molecules linking the interacting sheets. In both crystals, the interplanar spacing is equal to ca. 3.3 Å. In guanine monohydrate, N-1 and N-9 atoms are protonated, whereas in the anhydrous crystal, it is N-1 and N-7. The structure of biogenic guanine crystals is just a variant of the anhydrous form10. Guanine is commercially available as an anhydrous, mainly amorphous powder. In such a form, it may be expected that water intercalation into this layered structure would occur leading to swelling and eventually to delamination or exfoliation, like in the case of clay19, or titanium or zirconium phosphates20. Sonication would promote this process. We used a simple low energy 100 W ultrasonic bath, which was enough. Guanine molecules could be pulled out by hydration forces from these delaminated structures. Another intriguing phenomenon is acidification of solution upon guanine dissolution. It is particularly evident in pure water, with pH dropping even below 4. The higher the amount of guanine powder, the lower the final pH and the higher the guanine concentration, which is expected given that at a lower pH value its solubility is much higher. In the case of buffer solutions, the pH value decreased unless the buffer solution concentration was sufficiently high, in the case of PBS, 0.3 M. This phenomenon cannot be explained by the behavior of guanine itself. It is a weak acid of pKa 9.2, as are its minor neutral tautomers, of which the most acidic has the pKa equal 8.7616, so its aqueous solution will not be so acidic, particularly given its low solubility. Moreover, the increased solubility of guanine under these conditions is due to the presence of its protonated form, GuaH+. This means that there should be an extra source of acid in guanine powder itself. Just trace amounts of it would suffice for 100 mg of the powder to change the pH of 10 mL water to 4.0. The formation of the basic oxidation products of guanine, 8-oxoguanine or guanidinohydantoin and spiroiminodihydantoin is accompanied as a rule by the production of two protons per one 2-electron oxidation step16,21. The oxidation products of guanine are also weak acids, comparable 10 | ACS Paragon Plus Environment

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to guanine itself, so only this acidic by-product of oxidation could be the source of protons required to protonate guanine. The oxidation of guanine does not occur upon dissolution. We confirmed it by using argon atmosphere, which did not prevent acidification of the solution. It is plausible that some guanine molecules at the edges of the sheets in the crystal, particularly in its amorphous form, may get oxidized and an acid thus produced remained as an impurity. The amounts of it may be negligible for most guanine applications, but they may affect solubility when a large amount of guanine powder is dissolved in a small amount of water. We should stress that it was also noticed in other studies, where the source of guanine was different. It has already been mentioned in Introduction that in numerous publications on guanine electroanalysis, concentrations as high as 100 µM are used at pH 7. These solutions were generally prepared by dissolving the required amount of guanine. The results show that even at concentrations exceeding the solubility of guanine, the currents were increasing, however, usually showing deviation from linearity at higher concentrations. If the solutions were freshly prepared, they may contain guanine nanoparticles that should also be electroactive and oxidized at about the same potential as single molecules, contributing to the voltammetric current22,23. Their higher masses, however, would make the diffusion coefficients lower, and the expected current response lower, too. It could be concluded from our results that small nanoparticles in such a solution are not stable and would grow, changing the conditions in time, so their presence should be avoided unless their sizes and concentrations are controlled. It is better to work with real solutions, at concentrations not exceeding the solubility that could be calculated with a method described above.

CONCLUSIONS Guanine solubility can be calculated as a total concentration of all guanine species present in solution in equilibrium with solid guanine. Among all the equilibria processes, the equilibrium between solid guanine and its neutral form in solution does not involve protons. Thus, the concentration of the neutral form in solution should be constant over all the pH range, when in contact with solid guanine. Accordingly, the concentrations of all the species formed upon dissociation or protonation can be calculated based on the respective pKa values, and their sum would give guanine solubility at the given pH. The solubility values so obtained agree well with published data in acidic conditions. Dissolution of guanine powder gives rise to the formation of nanoparticles, of 20 to 100 nm size that grow in time to form stable particles of ca. 800 nm size. The presence of these particles is not evident and was apparently the main cause of obtaining too high solubility data. The presence of acidic impurities in commercial guanine powder is the most probable cause of acidification of resulting solutions and a secondary reason behind problems with reproducibility in

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solubility determinations. It is advisable to use appropriately small quantities of guanine for dissolution and not too diluted buffer solutions. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors thank Ms Svitlana Sovinska and Mr Adam Żaba for their help in performing DLS measurements. We are also thankful to Cracow University of Technology for support.

REFERENCES (1) DeVoe, H.; Wasik, S. P., Aqueous Solubilities and Enthalpies of Solution of Adenine and Guanine. J. Solution Chem. 1984, 13, 51–60. (2) Hinz, H.-J., Ed. Thermodynamic Data for Biochemistry and Biotechnology; Springer-Verlag, Berlin, 1986. (3) Hirano, A.; Tokunaga, H.; Tokunaga M., Arakawa, T.; Shiraki, K., The Solubility of Nucleobases in Aqueous Arginine Solutions. Arch. Biochem. Biophys. 2010, 497, 90–96. (4) Darvishzad, T.; Kurek, S. S., Anti- and Pro-oxidative Activity Measured Directly as the Extent of 8Oxoguanine Production. Electrochim. Acta 2017, 240, 466–473. (5) Li, Q.; Batchelor-McAuley, C.; Compton, R. G., Electrochemical Oxidation of Guanine: Electrode Reaction Mechanism and Tailoring Carbon Electrode Surfaces to Switch between Adsorptive and Diffusional Responses. J. Phys. Chem. B 2010, 114, 7423–7428. (6) Tadepalli, S.; Slocik, J. M.; Gupta, M. K.; Naik, R. R.; Singamaneni, S., Bio-Optics and Bio-Inspired Optical Materials. Chem. Rev. 2017, 117, 12705–12763. (7) Gur, D.; Palmer, B. A.; Weiner, S.; Addadi, L., Light Manipulation by Guanine Crystals in Organisms: Biogenic Scatterers, Mirrors, Multilayer Reflectors and Photonic Crystals. Adv. Funct. Mater. 2017, 27, 1603514. (8) Palmer, B. A.; Taylor, G. J.; Brumfeld, V.; Gur, D.; Shemesh, M.; Elad, N.; Osherov, A.; Oron, D.; Weiner, S.; Addadi, L., The Image-forming Mirror in the Eye of the Scallop. Science 2017, 358, 1172–1175. (9) Gur, D.; Pierantoni, M.; Dov, N. E.; Hirsh, A.; Feldman, Y.; Weiner, S.; Addadi, L., Guanine Crystallization in Aqueous Solutions Enables Control over Crystal Size and Polymorphism. Cryst. Growth Des. 2016, 16, 4975−4980. (10) Hirsch, A.; Gur, D.; Polishchuk, I.; Levy, D.; Pokroy, B.; Cruz-Cabeza, A. J.; Addadi, L.; Kronik, L.; Leiserowitz, L., “Guanigma”: The Revised Structure of Biogenic Anhydrous Guanine. Chem. Mater. 2015, 27, 8289–8297. (11) Singh, H. B.; Bharati, K. A., Enumeration of dyes, in: Handbook of Natural Dyes and Pigments; Woodhead Publishing India, 2014, pp. 33–260. (12) Iwasaka, M.; Miyashita, Y.; Mizukawa, Y.; Suzuki, K.; Toyota, T.; Sugawara, T., Biaxial Alignment Control of Guanine Crystals by Diamagnetic Orientation. Appl. Phys. Express 2013, 6, 037002. (13) Benoit, R. L.; Fréchette, M., Protonation of Hypoxanthine, Guanine, Xanthine, and Caffeine. Can. J. Chem. 1985, 63, 3053–3056.


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(14) Mason, S. F., Purine Studies. Part II. The Ultra-violet Absorption Spectra of Some Mono- and Polysubstituted Purines. J. Chem. Soc. 1954, 2071–2081. (15) Zaccaria, F.; Paragi, G.; Fonseca Guerra, C., The Role of Alkali Metal Cations in the Stabilization of Guanine Quadruplexes: Why K+ Is the Best. Phys. Chem. Chem. Phys. 2016, 18, 20895–20904. (16) Verdolino, V.; Cammi, R.; Munk, B. H.; Schlegel, H. B., Calculation of pKa Values of Nucleobases and the Guanine Oxidation Products Guanidinohydantoin and Spiroiminodihydantoin using Density Functional Theory and a Polarizable Continuum Model. J. Phys. Chem. B 2008, 112, 16860–16873. (17) Guille, K.; Clegg, W., Anhydrous Guanine: a Synchrotron Study. Acta Crystallogr. C 2006, 62, O515–O517. (18) Thewalt, U.; Bugg, C. E.; Marsh, R. E., The Crystal Structure of Guanine Monohydrate. Acta Crystallogr. B 1971, 27, 2358–2363. (19) Uzumcu, A. T.; Guney, O.; Okay, O., Highly Stretchable DNA/Clay Hydrogels with Self-Healing Ability. ACS Appl. Mater. Interfaces 2018, 10, 8296–8306. (20) Deshapriya, I. K.; Kim, C. S.; Novak, M. J.; Kumar, C. V., Biofunctionalization of α-Zirconium Phosphate Nanosheets: Toward Rational Control of Enzyme Loading, Affinities, Activities and Structure Retention. ACS Appl. Mater. Interfaces 2014, 6, 9643–9653. (21) Ibañez, D.; Santidrian, A.; Heras, A.; Kalbáč, M.; Colina, A., Study of Adenine and Guanine Oxidation Mechanism by Surface-Enhanced Raman Spectroelectrochemistry. J. Phys. Chem. C 2015, 119, 8191−8198. (22) Li, X.; Lin, C.; Batchelor-McAuley, C.; Laborda, E.; Shao, L.; Compton, R. G., New Insights into Fundamental Electron Transfer from Single Nanoparticle Voltammetry. J. Phys. Chem. Lett., 2016, 7, 1554–1558. (23) Nazmutdinov, R. R.; Zinkicheva, T. T.; Shermukhamedov, S. A.; Zhang, J.; Ulstrup, J., Electrochemistry of Single Molecules and Biomolecules, Molecular Scale Nanostructures, and Low-dimensional Systems. Curr. Op. Electrochem. 2018, 7, 179–187.



Table of Contents Image 40 36 Solubility / µM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

32 28 24

c(neutral guanine) = 25.4 µM const – independent of pH

20 3

4

5

6

7

8 pH 9


Page 14 of 23

Page 15 of 23

12000 +

2+

GuaH

10000

Absorption coefficient


12000

8000 6000 4000 2000

8000 6000 4000 2000 0

12000

12000

Gua

10000 8000 6000 4000 2000

GuaH2

10000

0



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Gua−

10000 8000 6000 4000 2000 0

0 220

270 Wavelength / nm

320

220

ACS Paragon Plus Environment

270 Wavelength / nm

320


40% 35% 30% Percent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

25% 20% 15% 10% 5% 0% 1

10

100 Size (d/nm)

1000

10000


Page 16 of 23

Page 17 of 23

40% 35% 30%

Percent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


25% Freshly prepared

20%

After 14 days

15% 10% 5% 0% 1

10

100 Size (d/nm)

1000

10000


The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


Page 18 of 23

Page 19 of 23

a)

40% 35% 30%

Percent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


25% 20% 15% 10% 5% 0% 1

10

100 Size (d/nm)

1000

10000



ðì9

•

ïñ9 ïì9 îñ9 W Œ vš

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

îì9 íñ9 íì9 ñ9 ì9 í

íì

íìì ^]Ì ~ lvu•

íììì

íìììì


Page 20 of 23

Page 21 of 23

100 2+

GuaH

GuaH2

+

Gua

Gua

80

Percent composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


Gua

−

2−

60 40 20 0 -2

0

2

4

6

pH

8

10


12

14

16


5

40

4

c / mM

38 36 34

c / µM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

3 2

32

1

30

0 0

28

5

pH 10

26 24 22 20 3

4

5

6 pH

7

8

9


Page 22 of 23

Page 23 of 23

40 Solubility / µM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


36 32 28 24

c(neutral guanine) = 25.4 µM const – independent of pH

20 3

4

5

6

7

8 pH 9


Puzzling Aqueous Solubility of Guanine Obscured ... - ACS Publications

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