High Nucleobase-Solubilizing Ability of Low-Viscous Ionic Liquid

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High Nucleobase-Solubilizing Ability of Low-Viscous Ionic Liquid/Water Mixtures: Measurements and Mechanism Debostuti Ghoshdastidar, Dibbendu Ghosh, and Sanjib Senapati J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07179 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 9, 2016

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The Journal of Physical Chemistry

High Nucleobase-Solubilizing Ability of Low-Viscous Ionic Liquid/Water Mixtures: Measurements and Mechanism Debostuti Ghoshdastidar,a Dibbendu Ghosha and Sanjib Senapati* Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institution of Technology Madras, Chennai 600 036, India. Tel: +91-44-22574122, Fax: +91-44-22574102, e-mail: [email protected]

a

These authors have contributed equally to this work

1 ACS Paragon Plus Environment


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ABSTRACT Research on nucleobases has always been on the forefront owing to their ever-increasing demand in the pharmaceutical, agricultural, and other industries. The applications, however, became limited due to their poor solubility in water. Recently, ionic liquids (ILs) have emerged as promising solvents for nucleobase dissolution, as they exhibit >100-fold increased solubility compared to water. But the high viscosity of ILs remains as a bottleneck in the field. Here, by solubility and viscosity measurements, we show that addition of low-to-moderate quantity of water preserves the high solubilizing capacity of ILs, while reducing the viscosity of the solution by several folds. To understand the mechanism of nucleobase dissolution, molecular dynamics simulations were carried out, which showed that at low concentrations water incorporates into the IL/nucleobase network without much perturbing the nucleobase–IL interactions. At higher concentrations, increasing numbers of IL anion–water hydrogen bonds replace IL–nucleobase interactions, which have been confirmed by 1H- and 13C-NMR chemical shifts of the IL ions.

Keywords: Nucleobase solubility, Ionic liquid/water binary solution, low viscous medium, competing hydrogen bonding


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INTRODUCTION Nucleobases are nitrogenous heterocycles that form the building blocks of nucleic acids. This ubiquitous class of biomolecules encompasses only five canonical members, yet constitutes the entire genetic diversity exhibited by all nucleic acids – DNA and RNA. This is possible due to an array of non-covalent interactions made by nucleobases, ranging from electrostatic to hydrophobic interactions, which also underlie the fidelity of storage, transmission and replication of genetic information.1 Such remarkable interaction abilities of nucleobases have been harnessed by the field of supramolecular chemistry to generate functionalized nanomaterials with potential applications in molecular therapeutics, biomimetics, as coordination polymers and in electronics and photonics.1–5 Moreover, the role of nucleobases as biochemical catalysts and as important precursors in several metabolic pathways has been established.6,7 Among the specific applications of different nucleobases, uracil and its derivatives have gained prominence due to their efficacy as anticancer/antivirial drugs.8,9 Over the past decades, nucleobases have found wide use in the agricultural and chemical industries as well.10 Notwithstanding their diverse applications, nucleobases are poorly soluble in water, with solubilities ranging from 0.1 to 0.7 wt% for the least soluble adenine to the most soluble cytosine.11 The pharmaceutically important uracil also has poor solubility of 0.3 wt% in water. Zielenkiewicz, et al. carried out a series of studies to show that polar interactions made by nucleobases are key to their solubility.12,13 Thus, increased polarity of the solvent medium was expected to enhance nucleobase dissolution. However, nucleobase solubility in water containing polar additives, like organic and inorganic salts, exhibited only slight improvements.14 Over the past few decades, a class of neoteric solvents called ionic liquids (ILs) has gained increasing popularity due to their exceptional solvation abilities.15 ILs are composed of cations and anions with unique molecular structures containing both charged and hydrophobic domains. Inter- and intra-domain interactions within ILs give rise to a network of polar, non-polar and amphiphilic nano-zones that can dissolve a wide range of solutes of diverse polarities.16,17 Furthermore, through careful selection of cationanion combinations, the solvation properties of ILs can be modulated for task-specific applications.18 Hence, this class of “designer” solvents continues to find increasing use in the dissolution of several nearly water-insoluble compounds like cellulose19,20 and chitin,21,22 pharmaceutically active molecules like flavonoids,23 sterols, vitamins,24 and ginkgolide homologues,25 as well as metal-salts,26 gases,27 etc. Among all ILs studied for such unique solvation abilities, 1-alkyl-3-methyl imidazolium acetates ([Rnmim][Ac]) have received particular attention, owing to the exceptional hydrogen



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bonding ability of the acetate anion. Thus, Araújo, et al. carried out a series of studies to solubilize nucleobases in [Rnmim][Ac] ILs and obtained a phenomenal ~100-fold increase in solubilities of adenine, uracil and thymine in [emim][Ac] and [bmim][Ac] relative to their solubilities in water.28– 30

The authors ascribed this high solubility to strong hydrogen bonding between the polar groups on

the nucleobases and the IL ions.28,30 Earlier, Kumar, et al. examined the solubilities of nucleobase derivatives in imidazolium-based ILs containing the closely related trifluoroacetate anion and found manifold increased solubility compared to their solubilities in conventional organic solvents.31,32 Interestingly, ILs containing other commonly used anions, like BF4- and PF6-, showed poor solubility for these nucleobase derivatives, suggesting a prominent role of the anion in base dissolution.31 However, the major drawback of these experiments was that ILs were highly viscous and addition of large amounts of nucleobases increased the viscosity of the solution further. For example, addition of 30 wt% uracil led to ~12-fold rise in viscosity of the solution of [emim][Ac].29 Despite the excellent solubilizing capacity of the ILs, such a high viscosity of IL/nucleobase solutions can hinder their potential applications. ILs are usually hygroscopic, and most of them can uptake significant amount of water. The presence of water dramatically alters the thermodynamic and physico-chemical properties of ILs. For example, densities and viscosities of majority of ILs were found to decrease drastically with increasing concentrations of water.33,34 Hence, addition of water is commonly used as a strategy to lower the viscosities of ILs and thereby extend their applications. However, the solvation properties of ILs can alter considerably in the presence of water. For example, the solubility and stability of several biomolecules are enhanced in IL/water binary mixtures.35–37 Contrary to this the dissolution of gases in ILs is often hindered in the presence of water.38,39 Moreover, the amount of water added also plays a crucial role in the dissolution of solutes in ILs. For example, while a small amount of water can aid in cellulose dissolution, beyond a certain concentration the added water causes precipitation of cellulose from the IL/water mixture.19,40 This has been attributed to the differential nanostructural reorganization of the IL constituents in water.41–43 In this study, we determined the effect of water on the solubility of nucleobases in 1-ethyl-3methylimidazolium acetate ([emim][Ac]) and on the viscosities of the resultant nucleobase/IL/water mixtures. The molecular structures for [emim][Ac] and nucleobases are presented in Figure S1 to aid the discussion. The bases selected for the study, namely uracil and adenine, represent the two structural variants among nucleobases, purines and pyrimidines, respectively. This choice helped us to elucidate the effect of the nucleobase type on its solubility. The choice of the IL was based on the study of Araújo, et al., which suggested that the lower viscosity of [emim][Ac], compared to its longer-chain counterparts, could make it a more suitable IL for nucleobase dissolution.28 Moreover,


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this IL has been widely reported to be a potent solvent for dissolution of several other poorly watersoluble molecules, such as hemicellulosic biomass,20 chitin,22 nucleic acid bases28,30 and gases.38 The results of our study showed that with addition of low-to-moderate concentrations of water (Xw < 0.5), the solubility of nucleobases in [emim][Ac]/water binary mixtures was negligibly affected, while the viscosities of the resultant mixtures decreased significantly. Such [emim][Ac]/water binary mixtures can therefore be considered to be more promising solvents for nucleobase dissolution than the neat IL. However, at higher water concentrations, both solubilities and viscosities showed a more rapid decline. We further carried out a series of molecular dynamics (MD) simulations to deduce the mechanism of nucleobase dissolution in neat [emim][Ac] and its binary mixtures with water. Hydrogen bonding interactions between nucleobases and the IL anion were found to primarily drive base dissolution. When water was added in small amounts, it incorporated into the base/anion network without affecting nucleobase dissolution. At higher dilutions, base–anion interactions were increasingly disrupted by the preferential interaction of the anion with the added water, leading to the observed decrease in solubility. This concentrationdependent effect of water on nucleobase solubility in IL/water mixtures was further confirmed using NMR spectroscopic analyses. Overall, this study shows that [emim][Ac]/water binary mixtures serve as superior solvents for nucleobase dissolution with tunable solvation abilities. MATERIALS AND METHODS Experimental Details. 1-ethyl-3-methylimidazolium acetate ([emim][Ac], purity 97%) and the nucleobases, uracil (2,4-dihydroxypyrimidine, purity >99.0%) and adenine (6-aminopurine, purity >99.0%), were purchased from Sigma-Aldrich and used without further purification. The IL was dried under vacuum at 333 K for 96h with vigorous stirring. The water content of the IL after drying was determined using a Karl Fischer Titrator (Metrohm 870KF Titrino Plus) and was found to be 1327 ppm. All nucleobase/IL/water mixtures were prepared in fresh milli-Q water. Solubility Measurements. The solubilities of both uracil and adenine in neat [emim][Ac] and [emim][Ac]/water binary mixtures were determined. For the binary mixtures, the water mole fractions (Xw) were set at Xw (wt/wt of water/IL) = {0.09 (1.0), 0.17 (2.2), 0.26 (3.6), 0.35 (5.3), 0.44 (7.6), 0.53 (10.6), 0.62 (14.7), 0.71 (20.8), 0.81 (30.8), and 0.90 (49.8)}. Nucleobases were added in a step-wise fashion (~2% wt/wt of IL or IL/water mixture) to the above solutions, with sonication at room temperature in an ultrasonic bath sonicator until complete dissolution. At low water concentrations, clear solutions were obtained upon sonicating for a maximum of 2 – 5 minutes, but longer sonication times of up to 15 minutes were required to dissolve nucleobases in binary mixtures containing higher water content. The solutions were then allowed to stand for 12 –



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24 hours at room temperature to check for precipitation, before proceeding for further nucleobase addition. Upon precipitation, the solubility of the previously obtained clear solution was considered to be the solubility limit of nucleobases in that binary mixture. All measurements were repeated at least three times. Viscosity Measurements. All viscosity measurements were carried out using a Lovis 2000 M/ME microviscometer (Anton Paar GmBH, Austria), which is designed based on Hoeppler's rolling ball principle to determine dynamic, kinematic, relative and intrinsic viscosity of liquids. According to the rolling ball principle, the time required for a standardized ball to fall under gravity through the test sample in an inclined tube can be directly related to the sample viscosity. Measurements on the Lovis 2000 M/ME microviscometer can be performed for viscosities ranging between 0.3 cP and 10,000 cP at temperatures varying from 278 K to 373 K. The viscometer temperature is controlled by built-in Peltier thermostat accessories. The Lovis microcapillaries, used as sample holders, allow the use of sample volumes of less than 1 ml for measurement. To calculate dynamic and kinematic viscosities, the sample's density must be known. The sample density can be determined by an inbuilt densitometer (DSA 5000 M) and the viscosity simultaneously calculated. However, determination of density and viscosity by this procedure consumes a large volume of sample (>5 ml). Conversely, when the sample density is not known, one can calculate the relative viscosity of the solution, which is defined as the viscosity of the solution with respect to the pure solvent. In the present study, we were interested in the relative change in viscosities of IL/nucleobase solutions in the presence of water, rather than the absolute viscosities of the solutions. Hence, we determined the relative viscosity of all IL/nucleobase or IL/water/nucleobase solutions for different concentrations relative to the viscosity of pure water at 298 K. For this purpose, only the density of pure water was provided as an input for viscosity measurement of all samples. A steel ball was allowed to roll through the sample-filled capillary. Three inductive sensors determine the ball's rolling time between defined marks on the capillary. The capillary was mounted on a pivot bearing, which allowed rotation of the tube by 180 degrees, thereby allowing multiple cycles of measurement. The rolling time in each cycle was registered and used to determine the average relative viscosity from a total of 12 cycles. The reproducibility of the measured viscosity is within 0.5%. NMR Measurements. Samples for NMR measurements were prepared by a similar mixing procedure as described above. Measurements were carried out for the neat IL, IL/water mixtures, and solutions of nucleobase in neat IL and in the binary mixtures for water mole fraction up to Xw = 0.53, as base dissolution is reasonably high (>20 wt%) at these water concentrations. Subsequently all solutions were prepared with the amount of dissolved nucleobase fixed at 20 wt% of the neat IL or IL/water mixture. Upon dissolution of nucleobase, the solutions were transferred to


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5 mm NMR tubes. Thereafter, capillary tubes containing D2O were coaxially inserted into the NMR tubes to prevent direct contact between the IL and the deuterated solvent, which may cause structural modifications of the IL and alter its ability to solubilize nucleobases.28,44 All tubes were sealed and capped to prevent contact with atmospheric moisture during scanning. 1H- and 13C-NMR spectra of the samples were recorded on a Bruker AM500 spectrometer operated at room temperature. The peak assignments were carried out using 2D COSY and HSQC analyses. The spectra of uracil and adenine solutions in neat [emim][Ac] are presented in Figures S2 and S3, respectively, for reference. The 1H- and 13C-NMR chemical shifts of neat [emim][Ac], its mixtures with water and with the nucleobases are tabulated in Tables S1–S3. Simulation Details. All atom MD simulations were performed on a series of uracil and adenine nucleobase mixtures in neat [emim][Ac] and [emim][Ac]/water binary systems. For the binary mixtures, representative water mole fractions of Xw = {0.1, 0.3, 0.5, and 0.7} were selected. To obtain the desired water concentration, the number of ion pairs in each system was fixed at 512 and the number of water molecules was adjusted. Appropriate number of nucleobases was then added to the simulation boxes based on the experimentally measured solubilities of the nucleobases in the binary mixtures.

The standard molecular mechanics potential energy function of the following form is used to

describe the inter-atomic interactions in the systems: ,

where the first three terms represent the bonded interactions: harmonic bond stretching, angle bending, and dihedral potential. The fourth term represents the non-bonded interactions that include the 12–6 Lennard-Jones (LJ) potential plus Coulomb potential. All equilibrium parameters, e.g. bond lengths (r0), angles (θ0), and dihedrals (ω) for the IL cation and anion were obtained from ab initio-derived optimized geometries of the IL ion pairs. Geometry optimizations were carried out at B3LYP/6-311++G(d,p) level using the Gaussian09 software.45 Harmonic frequency analyses of the resulting structures showed the absence of imaginary frequencies, indicating that the optimized structures were true minima. Atomic charges play a significant role in defining interactions involving ILs and, hence, were determined at the more accurate MP2/ccPVTZ level via fits to the electrostatic potentials obtained from the calculated wave functions, such that the electrostatic potential values surrounding the ions were well reproduced by the obtained partial atomic charges. The total charges on cation and anion were set at +1 and -1, respectively. This is the most



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conventional protocol used for charge estimation of ILs.46 The remaining parameters for the [emim]+ cation were taken from the work of Wang, et al. on the [Rnmim]+ family of ILs,47 and those for the acetate ([Ac]-) anion were obtained from Senapati and coworkers.48 The interaction potential energy parameters and partial atomic charges for the nucleobases were obtained from the parm99bsc0 parameter set defined for nucleic acids in the AMBER force field.49 All these parameters were developed within the same OPLS/AA framework and hence are very compatible with each other. The widely tested three-point TIP3P model was used to represent interaction potentials involving water, since the OPLS/AA force field as well as the IL parameters used here were parametrized with TIP3P parameters. The initial configurations of the systems comprised of ion pairs, water molecules and nucleobases placed randomly in cubic boxes. The dynamics of these systems are slow owing to strong electrostatic forces between the ions, as well as bulkiness of the nucleobases, and hence special care was taken to ensure the attainment of equilibrium. The starting structures were energy minimized for 2000 steps using the conjugant gradient and steepest descent methods to remove bad contacts. Following this, the temperature was gradually increased to 700 K under constant volume. The system was then cooled step-wise to 298 K with intermediate equilibrations at 700, 600, 500, 400 and 298 K in the isothermal-isobaric ensemble. Temperature annealing along with multiple intermittent equilibrations have been commonly used to augment dynamics of these systems and ensure demixing of the IL ions.16 Production runs of 50 ns were carried out for all systems at 298 K and 1 atm pressure. To enable volume variation, production simulations were performed in the NPT ensemble using the Berendsen thermostat and barostat. Both the thermostat and barostat relaxation times were set to 0.5 ps. The calculation of long-range Coulombic forces was performed employing the full-Ewald summation technique. The real space part of the Ewald sum and Lennard-Jones interactions was cut off at 15 Å. Periodic boundary conditions were employed in all directions. SHAKE was employed to constrain bonds involving hydrogen atoms. All equilibration and production runs were carried out using the Amber12 MD simulation package.49 RESULTS Added water reduces viscosity with little interference on nucleobase solubility of [emim][Ac]. The solubilities of two nucleobases – uracil and adenine – in neat [emim][Ac] and its binary mixtures with water were measured. As shown in Figure 1, about 45 wt% of both nucleobases could be dissolved in neat [emim][Ac]. This implies an ~150-fold increase from their solubility in pure water, which ranges between 0.1 and 0.3 wt%. In an earlier study, Araújo, et al. investigated solutions of nucleobases in neat [emim][Ac] containing 31 wt% and 25 wt% of uracil and adenine,


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respectively.28,30 By employing an extensive dissolution protocol in the current study, it was possible to dissolve larger amounts of nucleobase (see 'Materials and Methods' section for details). Briefly, small quantities of nucleobases (~2% wt/wt) were added in a step-wise fashion to the neat IL, and the solutions were subjected to ultrasonic waves generated in an aqueous bath sonicator. The high frequency ultrasound aids in dissolution by disrupting inter-molecular interactions, and is especially useful when high viscosity of the solvent medium, like ILs, can hinder dissolution. Upon obtaining a clear solution, the process was repeated until further addition of nucleobase led to precipitation. Following this, 1H- and

13

C-NMR spectra of the nucleobase/[emim][Ac] solutions

(Figures S2 and S3, respectively) were determined to ensure the structural integrity of the molecular components. A similar protocol was employed for investigating the solubility of nucleobases in IL/water binary mixtures. In order to prevent evaporation of water during the ultrasonic treatment, the solution tubes were sealed and the water bath was maintained at room temperature using an inbuilt thermostat. In the presence of water, the solubility of nucleobases in [emim][Ac]/water binary mixtures exhibited a slow decline up to Xw = 0.71. Interestingly, in this regime, solubility of adenine in the binary mixtures decreased more rapidly compared to uracil. This corroborates well with earlier observations of relatively lower solubility of adenine both in water and in aqueous solutions of additives, such as amino acids, which were used to promote base dissolution.11,14 Finally, at Xw > 0.71, a drastic decrease in nucleobase solubility was observed. However, even in the presence of such a large quantity of water, both nucleobases exhibited ~10-fold higher solubility in the binary mixtures compared to their solubility in pure water (Figure 1).



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Figure 1: Measured solubilities (wt%, nucleobase/(IL+water)) of uracil (dark bar) and adenine (shaded bar) in [emim][Ac]/water binary mixtures containing varying mole fractions of water (Xw). The error bars shown were computed from at least three solubility measurements. For the binary mixtures Xw were set at {0.09, 0.17, 0.26, 0.35, 0.44, 0.53, 0.62, 0.71, 0.81, and 0.90}. Solubilities of uracil and adenine in water were taken from reference 11. Ionic liquids are highly viscous fluids, and dissolution of 40 wt% of nucleobase in neat [emim][Ac] further increased the viscosity by ~35-fold (from 160 cP to 5666 cP). Notably, earlier Araújo, et al. reported a ~12-fold rise in the viscosity of [emim][Ac] upon dissolution of 30 wt% uracil.29 Such high viscosities may limit the use of these nucleobase/IL solutions despite the significant solubility of nucleobases in the ILs. Small quantities of water are commonly added to ILs for reducing their viscosities and making them more usable. Hence, we determined the effect of addition of water on the viscosities of nucleobase/[emim][Ac] mixtures containing a fixed quantity of nucleobase. For this purpose, we selected a water concentration range of Xw = 0 to 0.53, since these compositions exhibited reasonably high nucleobase solubilities (at least half of the nucleobase solubility in neat IL) in presence of water (see Figure 1). As presented in Table 1, dissolution of 20 wt% uracil or adenine increased the relative viscosities of the [emim][Ac]/nucleobase mixtures (773 and 960 cP, respectively) by up to 6-fold compared to that of the neat IL (160 cP). The measured viscosity values for neat IL and nucleobase/IL mixtures are in good agreement with earlier reports (neat [emim][Ac], 128.5 cP; 20 wt% uracil/[emim][Ac], 683 cP; 20 wt% adenine/[emim][Ac], 823 cP).29 The relatively higher values obtained in the present study are due to the lower water content in the neat IL (1327 ppm versus 1500 ppm in reference 29). With the addition of small amounts of


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water at Xw < 0.35, a significant four-fold reduction in the viscosities of the mixtures was observed. Further, at a water concentration of Xw = 0.53, the viscosities of nucleobase/[emim][Ac]/water solutions exhibited a drastic seven-fold decline compared to the viscosity of nucleobase solution in neat IL. It is worth stating that the viscosities reported here are relative viscosities, measured with respect to the viscosity of pure water at 25 °C (see 'Materials and Methods' section for details). Since we were interested in determining the change in the viscosities of nucleobase/IL solutions upon addition of water, measurement of relative viscosities of the mixtures was sufficient. Table 1: Relative viscosities (cP) of the solutions of uracil and adenine in neat [emim][Ac] and [emim][Ac]/water binary mixtures of different molar compositions at 25°C. The viscosity of neat [emim][Ac] was 160 cP. The nucleobase amount added to all solutions was fixed at 20 wt%, which is the solubility limit of the bases in [emim][Ac]/water mixture of Xw = 0.53 (Figure 1). All viscosities reported here were measured relative to the viscosity of pure water at 25°C. Xw

Viscosity (cP) Uracil

Adenine

0

773.0

960.0

0.09

475.6

487.5

0.17

402.9

378.2

0.26

309.3

339.3

0.35

269.2

244.5

0.44

196.1

177.0

0.53

135.6

127.3

Thus, [emim][Ac]/water binary mixtures containing low-to-moderate amount of water can be considered as superior solvents for nucleobase dissolution, since they largely retain the excellent solvation ability of the neat IL while offering a less-viscous alternative. For example at a concentration of Xw = 0.53, the binary mixture exhibited ~83-fold higher uracil solubility than pure water, yet the resultant solution showed several fold lower viscosity than neat IL/base mixtures. A



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detailed understanding of the dissolution mechanism of nucleobases in these [emim][Ac]/water solutions could further expand the applications of these remarkable binary mixtures as solvents for nucleobases and its derivatives. In the following sections, we give atomistic details of the mechanism of nucleobase dissolution in [emim][Ac]/water binary mixtures obtained from MD simulation studies and subsequently validated by NMR spectroscopic data. Acetate anions play the primarily role in nucleobase dissolution. To elucidate the mechanism of nucleobase dissolution, we performed MD simulations on a series of nucleobase/[emim][Ac]/water mixtures. The compositions of the simulated systems were determined based on the experimentally determined base solubilities, and are presented in Table 2. Microscopic details of nucleobase–IL interactions that aid in dissolution can be extracted from site-site radial distribution functions (RDFs), which describe the probability of finding a selected component around another in the solution. Hence, the distributions of [emim]+ cation and acetate anion around specific sites of uracil and adenine were computed for different IL/water compositions (Figure 2). The higher intensities of acetate distribution around both uracil and adenine indicate that the nucleobases interact primarily with the anionic counterpart of the IL in all systems. Moreover, the first minimum of the nucleobase-anion RDF lies at a distance of < 3.5 Å, suggesting the formation of hydrogen bonds between the acidic nitrogens of the dissolved nucleobases (N1/N3 of uracil and N6/N9 of adenine) and the highly basic carboxylate oxygen of the acetate anion. Another noteworthy feature of Figure 2 is that with increased water content, the anion distribution around nucleobases was affected a little for water concentrations of Xw < 0.5, after which it was increasingly perturbed, as indicated by the diminishing peak intensities of base–anion RDFs for Xw > 0.5. This corroborates well with the significantly high solubilities of nucleobases in [emim][Ac]/water mixtures at low-to-moderate water concentrations, followed by a more rapid decline at higher dilutions. Thus, it can be reiterated that base–anion interactions primarily determine the extent of nucleobase solubility in [emim][Ac]/water binary mixtures. These results are in agreement with the predominant role of IL anions in gas solubility,50,51 microemulsion stability,52 cellulose dissolution,19,20 nucleobase solubility,28 etc. observed by several earlier studies.


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Table 2. Summary of the nucleobase/[emim][Ac]/water mixtures simulated in this study. Xw, NIL, NWAT, NBASE and %wtBASE are mole fraction of water, number of IL ion pairs, number of water molecules, number of nucleobase molecules and percentage weight of nucleobase in the mixtures, respectively. Xw

NIL

NWAT

NBASE

%wtBase

Uracil Adenine 0

512

0

350

290

45

0.1

512

57

311

258

40

0.3

512

219

233

194

30

0.5

512

512

156

129

20

0.7

512

1195

93

78

12

The cation-IL RDFs, on the other hand, exhibit the first minima at ~4 Å, indicative of the hydrogen bonding character of these interactions. This finding is in agreement with earlier NMRbased investigations by Araújo et al., which revealed the plausible presence of hydrogen bonding interactions between uracil and the imidazolium cation of neat [emim][Ac].30 In addition, the prominent peak of [emim]+ distribution around uracil compared to adenine observed here indicates that the IL cation participates more significantly in uracil dissolution. This could be attributed to the ease of interaction between the acidic hydrogen of the cation imidazolium ring (C2-H2) and the exocyclic oxygen atoms of uracil (O2/O4) compared to the less approachable ring nitrogens of adenine (N1/N3/N7). Moreover, unlike anion-base interactions, the intensities of base–cation RDFs exhibit a slight increase with the addition of water at moderate-to-high concentrations (insets to Figure 2). This might be caused by a partial disruption of cation-anion pairing by the added water at these dilutions (Figure S4), allowing more number of cations to approach closer to the nucleobase. Since nucleobase-anion distributions are weakened in the presence of high amount of water, the role played by the IL cations in nucleobase dissolution becomes more evident at these concentrations.



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Figure 2: Site-site radial distribution functions of IL ions around potential interacting sites on (a–d) uracil and (e–i) adenine at different Xw. Panels (a,b) show the distribution of acetate anion around N1 and N3 of uracil and (c,d) of imidazolium cation around O2 and O4 of uracil, respectively. Panels (e,f) show the distribution of acetate anion around N6 and N9 of adenine and (g–i) of imidazolium cation around N3, N1, and N7 of adenine, respectively. The insets highlight persistent anion–base distributions in presence of low-to-moderate amounts of water at Xw < 0.5 (a,e) and increased cation–base interactions at higher dilutions of Xw > 0.5 (c,g). The sites of interaction on the IL include the carboxylate oxygen of the anion and C2 of cation. For atom notations see Figure S1.


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Nucleobase-acetate hydrogen bonding is the key factor in base dissolution. The nucleobase-IL interactions were further probed by computing time-averaged spatial probabilities of anion and cation localization in the three dimensional space around a central nucleobase in neat [emim][Ac]. As shown in Figure 3a, one anion was located near each acidic ring nitrogen of uracil (N1 and N3), and one, or occasionally two, anions near the exocyclic amino nitrogen (N6) of adenine and one near its N9 atom. Hydrogen bonds between the IL ions and the nucleobase atoms were identified based on geometric criteria of a radial distance of less than 3.5 Å between the donor and acceptor atoms and a donor-H-acceptor angle greater than 135°. The carboxylate oxygens of the acetate anion were found within 3.0 Å distance of the nucleobase nitrogens and always subtending an angle of greater than 150°, supporting the possibility of base-anion hydrogen bonding as suggested by RDF analyses above. The acidic imidazolium ring carbon (C2), on the other hand, lay at a larger distance from the carbonyl oxygens (O2 and O4) of uracil and the ring nitrogens (N1, N3 and N7) of adenine, indicating weaker base-cation interactions (Figure S5). Moreover, the donor-H-acceptor angle in base-cation interactions were lesser than 135°, implying a weak hydrogen bonding character. Since nucleobases interact primarily with the acetate anion, we determined the number of hydrogen bonds formed per nucleobase with acetate anions as a function of water concentration, and presented them in Figure 3b. Hydrogen bonds were identified based on the geometric criteria defined above. Following this, the total number of hydrogen bonds formed by the different hydrogen bonding sites on the nucleobases (uracil N1/N3 or adenine N6/N9) was separately counted. These values were normalized by the number of nucleobases in each system, since the nucleobase content decreased as a function of water concentration. Corroborating with the spatial distribution, in neat [emim][Ac], adenine formed more number of hydrogen bonds with the IL anions than uracil. However, while the spatial distribution suggested the proximal localization of two anions near the -N(6)H2 group of adenine, only rarely did the amine group simultaneously hydrogen bond with two anions. Thus, both uracil and adenine formed ~1:2 hydrogen bonded complexes with [emim][Ac], which also explains their comparable solubilities in the neat IL. These observations are in partial agreement with the stoichiometric ratio of nucleobase to [emim][Ac] proposed by Araújo et al in an earlier study.28 While the authors proposed a stoichiometric ratio of 1:2 for uracil dissolution, the adenine to [emim][Ac] stoichiometric ratio was deduced as 1:3 based on the solubility of adenine (~25 wt%) in neat IL. However, the significantly higher solubility of adenine achieved in the current study (~45 wt%) indicates an adenine:acetate stoichiometry of ~1:2. The liquid phase simulation studies of IL/nucleobase solutions further confirmed that each nucleobase indeed hydrogen bonds with ~2 acetate anions.



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Page 16 of 30

Figure 3b also shows that with increasing water concentration, the number of base-anion hydrogen bonds was altered in a site-specific manner. Uracil-N3-anion interactions were affected with the addition of low concentrations of water (Xw < 0.3), followed by reduction in uracil–N1– anion hydrogen bonding only at higher dilutions (Xw > 0.3). Similarly for adenine, N6–acetate interactions started diminishing before N9–acetate hydrogen bonds were affected (more details are presented later). Persistence of base-anion hydrogen bonding at certain nucleobase sites even in the presence of low-to-moderate concentrations of water explains why solubility in these [emim][Ac]/water binary mixtures exhibited a slower decline. At dilutions of Xw > 0.5, all baseanion hydrogen bonding interactions were affected, resulting in a rapid fall in nucleobase solubility. The atomistic details of the mechanism by which base-anion interactions were altered in the presence of water were further investigated and have been described in the following sections.


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Figure 3: (a) Spatial distribution of acetate anions around a central uracil (left panel) and adenine (right panel) nucleobase. The time-averaged distances between the interacting heavy atoms of anion and nucleobase are mentioned in Å unit. The sites of interaction include the carboxylate oxygen of the anion and acidic nitrogens of uracil (N1/N3) and adenine (N6/N9). (b) Effect of water on anion– nucleobase interactions is depicted in terms of number of H-bonds formed per nucleobase site with anion in the presence of increasing mole fractions of water: uracil (left panel) and adenine (right panel). Added water at higher concentration competes with base for the IL acetate anions. Nucleobase–anion interactions, which are critical for base dissolution in ILs, can be perturbed by addition of water, a strong hydrogen-bond-forming species. Hence, we determined the interplay between

these

three

species

for

the

formation

of

hydrogen

bonds

in

the

nucleobase/[emim][Ac]/water mixtures, and its effect on base solubility. As shown in Figure 4, in



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Page 18 of 30

the absence of water, each anion formed a single hydrogen bond with a nucleobase – uracil or adenine. At Xw = 0.1, the added water hydrogen bonded with the acetate anion, while forming minimal interactions with the nucleobases. This is expected, owing to the more favorable water– anion interaction energy of -3.69+0.15 kcal/mol compared to water–nucleobase energy of 0.28+0.06 kcal/mol (for uracil) and -0.22+0.06 kcal/mol (for adenine) at this concentration. Despite the interaction of water with the IL anion, nucleobase–anion interactions at this water mole fraction remain unperturbed, as shown in Figure 4. To understand this interplay further, the electrostatic energies of all interactions involving the IL anion in nucleobase/[emim][Ac]/water mixtures were computed for representative compositions (Table 3). As the table points out, the IL anion always made stronger interactions with the added species, nucleobase or water, than with its associated cation, a property that underlies the excellent solvation abilities of ILs. At Xw = 0.1, the anion– nucleobase interactions are stronger than anion–water interactions (see Table 3), and therefore presence of water does not interfere with nucleobase solubility (as shown in Figure 1). To illustrate this graphically, a time-averaged spatial distribution of water and anion around a central nucleobase is presented in Figure 5a for this concentration. The added small quantity of water was found to interact preferentially with the anion that was hydrogen bonded to uracil-N3/adenine-N6. The preferential interaction with N3 of uracil is presumably due to the flanking oxygens. Similarly, the proximal location of two anions near adenine-N6 makes this an ideal site for maximizing water– anion hydrogen bonding in adenine systems. However, as evident from the distribution, the water assimilates into the IL/base network without perturbing their interactions. The base–acetate hydrogen bond distances remain very similar to that in the neat IL/base mixtures, as shown in Figure 3a.

Figure 4: Anion–nucleobase versus anion–water interactions with increasing concentration of water. Results are shown for the number of H-bonds formed per acetate anion with nucleobase (dark bar) or water (shaded bar) in [emim][Ac]/water mixtures containing (a) uracil and (b) adenine.


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Table 3: Interaction energies (kcal/mol) of the acetate anion with the [emim]+ cation, nucleobases and water in nucleobase/[emim][Ac]/water mixtures at representative water mole fractions (Xw). Xw

ANI-CAT

ANI-BASE

ANI-WAT

URC

ADN

URC

ADN

URC

ADN

0

-3.09+0.10

-3.23+0.11

-11.70+0.38

-10.92+0.44

-

-

0.1

-3.05+0.10

-3.21+0.11

-11.10+0.36

-9.62+0.42

-3.69+0.15

-3.70+0.15

0.5

-2.52+0.10

-2.65+0.10

-5.42+0.23

-4.83+0.27

-30.51+0.72

-31.49+0.73

Upon further addition of water, at Xw > 0.3, an increasing number of water-anion hydrogen bonds are formed that replace base-anion hydrogen bonding (Figure 4). This can also be explained energetically, where Table 3 shows that at Xw = 0.5, water–anion interactions become much stronger than base–anion interactions, and approach towards the water–acetate interaction value of -32.44+0.78 kcal/mol in 1:1 [emim][Ac]/water binary mixture. Figure 5b illustrates such a distribution of anion around nucleobase in presence of water at Xw = 0.5. At this concentration or higher, water molecules incorporate near the acetates that were interacting with uracil-N3 and adenine-N6 sites, leading to a progressive weakening, often total disruption, of the existing base– anion interactions through these sites of the bases. This explains why the hydrogen bonds made by these nucleobase sites with the IL anion start diminishing first upon addition of water (Figure 3b). This disruption in base-anion hydrogen bonding leads to a decrease in nucleobase solubility with increasing addition of water at these concentrations. Moreover, water molecules also start interfering with the base–anion interactions made by other nucleobase sites (uracil-N1 and adenineN9) at Xw > 0.5. Thus, the extent of nucleobase solubility in [emim][Ac]/water binary mixtures depends on the relative strength of base–anion versus water–anion interactions in these systems, which in turn depends on the amount of water added.



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Figure 5: Spatial distribution of anions and water around a central uracil (left panel) and adenine (right

panel)

nucleobase

depicting

nucleobase–anion–water

interactions

in

nucleobase/[emim][Ac]/water mixtures at (a) Xw = 0.1 and (b) Xw = 0.5. The time-averaged distances between the interacting heavy atoms of anion, nucleobase and water are mentioned in Å units. The uracil(N3)–anion and adenine(N6)–anion distances at Xw = 0.5 increase to 5.83 and 5.50 Å, respectively (not mentioned in figure), indicating a loss of base–anion interactions at these sites in the presence of water. The sites of interaction include the carboxylate oxygen of the anion, acidic nitrogens of uracil (N1/N3) and adenine (N6/N9) and oxygen of water.


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NMR data confirms base versus water competition for IL acetate anions. As indicated by the MD simulations results above, in neat nucleobase/[emim][Ac] mixtures, the anion carboxylate oxygen is hydrogen bonded to the acidic nitrogens of nucleobases. Upon addition of water, these hydrogen bonding interactions are altered. Alteration in hydrogen bonding is a manifestation of the change in the electronic environment of the interacting atoms, which can be probed very precisely by NMR spectroscopy. Hence, we attempted to validate our MD results by monitoring the NMR chemical shift of the hydrogen bonded and the surrounding nuclei in the nucleobase solutions. Since it is the acetate anion that primarily hydrogen bonds with both the nucleobases and water in nucleobase/[emim][Ac]/water solutions, we monitored the chemical shifts in the 13C-NMR signals associated with the anion carbons in these mixtures. The electronic structure of the acetate carboxylate carbon (C9) is especially sensitive to the nature of the hydrogen bond, and is strongly deshielded upon hydrogen bond formation leading to a downfield shift in its NMR signal. Consequently, the adjacent methyl carbon (C10) presents an upfield shift. Figures 6 and S6 summarize the 13C-NMR spectra of the acetate of [emim][Ac], containing 20 wt% of uracil and adenine, respectively. The effect of water on the same mixture, i.e. IL with 20 wt% nucleobase, is also included. As shown in the figures, upon addition of base to neat IL, signals associated with the anion carbons present a downfield shift for C9 and an upfield shift for C10, indicating the formation of strong hydrogen bond between acetate and the added base. With increasing water concentration, the shift in the anion signals become even more pronounced, implying that acetate anion forms growing number of hydrogen bonds with the added water, in agreement with our MD results (Figure 4). The increasing hydrogen bonding of anion with nucleobase and water leads to gradual disruption of the anion–cation interactions, which causes shielding of the imidazolium ring protons and consequently an upfield shift in the 1H-NMR signals associated with the cation, as shown in Figure S7. In agreement with earlier studies, the acidic H2 proton of the cation presents the strongest upfield shift followed by the other ring protons, these being the primary sites for anion–cation interactions.53



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Page 22 of 30

Figure 6: Effects of added uracil or uracil and water on

13

C-NMR spectra of [emim][Ac]. The

topmost panel represents the spectra of neat IL. Water concentration was varied from Xw = 0.0 to 0.53. In all solutions the amount of uracil added was 20 wt% of the binary/ternary mixtures. Chemical shift changes associated with acetate carbons, carboxylate C9 and methyl C10, are depicted. To determine the effect of the growing water–anion hydrogen bonding on base–anion interactions as more and more water is added, we calculated the difference between the chemical shifts associated with the acetate C9 signal in [emim][Ac]/water/base mixtures (δIL+water+base) and the corresponding [emim][Ac]/water systems (δIL+water). These chemical shift deviations (i.e. Δδ = δIL+water+base - δIL+water) can serve as estimates of the extent of base–anion hydrogen bonding in presence of water, since base–water interaction is negligibly small (0.1–0.3 wt% base solubility in water). For the neat system, the chemical shift deviation (Δδ) was calculated by deducting the chemical shift in C9-signal in neat IL from that in the presence of 20 wt% nucleobase (Δδ = δIL+base - δIL). The results summarized in Figure 7 show that upon addition of nucleobase to neat [emim][Ac] (i.e. at Xw = 0.0), a large chemical shift deviation was obtained. This implies that the anion forms stronger interactions with the added nucleobase compared to the anion–cation interactions in neat IL, which supports our interaction energy values in Table 3 from MD simulations. When the same 20 wt% nucleobase was dissolved in [emim][Ac]/water binary mixtures, similar chemical shift deviations were observed up to Xw = 0.35, beyond which the magnitude of the deviation progressively decreased with increasing water concentration. This implies that the presence of water did not affect base–anion interactions at low-to-moderate water concentrations, which is in agreement with MD simulation results. This is also in accordance with


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the measured solubility data, where small quantity of water did not interfere with base solubility in IL. Interestingly, with further dilution at Xw = 0.44, the chemical shift deviations approach close to zero. This implies that the hydrogen bonding capacity of the acetate anion reaches its saturation limit at this concentration, and addition of base causes a mere rearrangement of the water-anion hydrogen bonded network, manifesting in the similar chemical shifts of both solutions. The negative chemical shift deviation at Xw = 0.53 indicates weaker base–anion interactions in this mixture. However, the continued base solubility in this binary solution implies the possible contribution of base–IL cation interactions in nucleobase dissolution at this concentration, presumably through nucleobase-imidazolium ring stacking. This can be supported by the increase in cation-nucleobase distributions at higher dilutions obtained from MD simulations (Figure 2). At further dilutions (Xw > 0.53 for 20 wt% nucleobase), when interactions of both IL ions with nucleobase become weaker, the base precipitates. A comment is required here about the small rise observed in the chemical shift deviation in binary mixtures up to Xw = 0.17 for uracil and Xw = 0.35 for adenine relative to neat IL (Figure 7), indicating increased downfield shift of the anion C9 signal in presence of both base and water. This could be caused by enhanced deshielding of the acetate C9 due to base–water interactions that cooperatively withdraw more electrons from the carboxylate anion. Although this effect is present at all concentrations, strong water–anion interactions at higher dilutions mask this effect. Overall, the NMR spectroscopic analyses confirm that presence of water in [emim][Ac] interferes with base–anion interactions in a concentration-dependent manner. Thus, nucleobase solubility in [emim][Ac]/water binary mixtures can be tuned by modulating their water content.



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Figure 7. Nucleobase–anion interactions as measured from the chemical shift deviation of acetate C9 carbon in

13

C-NMR signal of [emim][Ac] upon addition of uracil (closed circles) or adenine

(open circles) in the absence and presence of water. In all solutions the amount of uracil added was 20 wt% of the binary/ternary mixtures. Water concentration was varied from Xw = 0.0 to 0.53. Δδ = δIL+base- δIL for Xw = 0.0 and Δδ = δIL+water+base- δIL+water for all Xw > 0.0.

CONCLUSIONS

In this work, we studied the efficacy of [emim][Ac]/water binary mixtures as solvents for

nucleobases. Binary mixtures containing low-to-moderate concentrations of water displayed comparable base-dissolution abilities as that of neat [emim][Ac], with the added advantage of being low-viscous alternatives. At higher water concentrations, nucleobase solubility in [emim][Ac]/water mixtures was lowered. The mechanism of nucleobase dissolution in neat [emim][Ac] and [emim][Ac]/water binary mixtures was investigated using MD simulation and NMR spectroscopic techniques. Atomistic details obtained from MD simulations revealed that the IL anion played the primary role in aiding base dissolution through the formation of hydrogen bonds. Low-to-moderate amounts of water were incorporated into this hydrogen-bonded network without interfering with base–anion interactions. Upon further dilution, water increasingly competed with the nucleobase for the same solvation sites on the IL anion and disrupted base–anion hydrogen bonding. NMR spectroscopic analyses confirmed that competing interactions between nucleobase and water controlled the extent of base dissolution in IL/water binary mixtures. The scope of this study is twofold. Firstly, dissolution of nucleobases in the low-viscous IL/water solvent medium can further


Page 25 of 30

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expand their applications in science and technology. Secondly, high viscosity will no longer limit the use of other prospective ILs, like longer-chained imidazolium-based or biocompatible cholinium- and guanidinium-based ILs, etc., which may possess even better nucleobase solubilizing abilities.

Acknowledgements The computational facilities provided by High Performance Computing Centre, IIT Madras, are gratefully acknowledged. We are extremely grateful to Mr. Akhil P. Singh and Professor Ramesh L. Gardas for helping us with Karl Fischer titration analyses and viscosity measurements. We are thankful to Dr. Dipak K. Roy and Professor Sundargopal Ghosh for useful discussions.

Supporting Information Available Figure S1 giving the molecular structure and atomic notations of the IL ions and the nucleobases. Figures S2 and S3 giving the

1

13

H-NMR and

C-NMR spectra of

uracil/[emim][Ac] and adenine/[emim][Ac] solutions, respectively. Figures S4 showing the distributions of IL anion around cation head group in [emim][Ac]/water binary mixtures. Figure S5 showing the spatial distribution of IL cation headgroups around uracil and adenine nucleobases in nucleobase/[emim][Ac] mixtures. Figure S6 showing

13

C-NMR spectra of [Ac]

anion in adenine/[emim][Ac]/water mixtures. Figures S7 presenting the 1H-NMR spectra of [emim] cation in uracil/[emim][Ac]/water mixtures. Tables S1–S3 presenting the 1H-NMR and 13

C-NMR chemical shifts of [emim][Ac] in presence of water and uracil and adenine

nucleobases. This information is available free of charge via the Internet at http://pubs.acs.org.



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REFERENCES (1) Sivakova, S.; Rowan, S. J. Nucleobases as Supramolecular Motifs. Chem. Soc. Rev. 2005, 34, 9–21. (2) Li, X.; Kuang, Y.; Xu, B. “Molecular Trinity” for Soft Nanomaterials: Integrating Nucleobases, Amino Acids, and Glycosides to Construct Multifunctional Hydrogelators. Soft Matter 2012, 8, 2801–2806. (3) Ji, S.; Guo, Q.; Yue, Q.; Wang, L.; Wang, H.; Zhao, J.; Dong, R.; Liu, J.; Jia, J. Controlled Synthesis of Pt Nanoparticles Array through Electroreduction of Cisplatin Bound at Nucleobases Terminated Surface and Application into H2O2 Sensing. Biosens. Bioelectron. 2011, 26, 2067– 2073. (4) Amo-Ochoa, P.; Zamora, F. Coordination Polymers with Nucleobases: From Structural Aspects to Potential Applications. Coord. Chem. Rev. 2014, 276, 34–58. (5) Ouchen, F.; Gomez, E.; Joyce, D.; Yaney, P.; Kim, S.; Williams, A.; Steckl, A.; Venkat, N.; Grote, J. Investigation of DNA Nucleobases-Thin Films for Potential Application in Electronics and Photonics. Proc. SPIE 8817. 2013, 8817, 88170C. (6) Banks, J. F.; Whitehouse, C. M. Electrospray Ionization Mass Spectrometry of RNA Nucleobases: Implications for Solution Chemistry and Ion Source Operating Conditions. Int. J. Mass Spectrom. Ion Process. 1997, 162, 163–172. (7) Kath-Schorr, S.; Wilson, T. J.; Li, N.-S.; Lu, J.; Piccirilli, J. A.; Lilley, D. M. J. General AcidBase Catalysis Mediated by Nucleobases in the Hairpin Ribozyme. J. Am. Chem. Soc. 2012, 134, 16717–16724. (8) Malet-Martino, M.; Martino, R. Clinical Studies of Three Oral Prodrugs of 5-Fluorouracil. Oncologist. 2002, 7, 288–323. (9) Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Nucleoside Analogues and Nucleobases in Cancer Treatment. Lancet Oncol. 2002, 3, 415–424. (10) Pasquale, R. J. De; Pasquale, R. J. De. Uracil: A Perspective. Ind. Eng. Chem. Prod. Res. Dev. 1978, 17, 278–286. (11) Herskovits, T. T.; Harrington, J. P. Solution Studies of the Nucleic Acid Bases and Related Model Compounds. Solubility in Aqueous Alcohol and Glycol Solutions. Biochemistry 1972, 11, 4800–4811. (12) Zielenkiewicz, W.; Poznariski, J.; Zielenkiewicz, A. Partial Molar Volumes of Alkylated Uracils — Insight into the Solvation Shell? Part II. J. Solution Chem. 1998, 27, 543–551. (13) Zielenkiewicz, A. Partial Molar Volumes of Aqueous Solutions of Some Halo and Amino Derivatives of Uracil. J. Solution Chem. 2000, 29, 757–769. (14) 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.


Page 27 of 30

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


(15) Rogers, R. D.; Seddon, K. R. Ionic Liquids--Solvents of the Future? Science 2003, 302, 792– 793. (16) Canongia Lopes, J. N.; Costa Gomes, M. F.; Pádua, A. A. H. Nonpolar, Polar, and Associating Solutes in Ionic Liquids. J. Phys. Chem. B Lett. 2006, 110, 16816–16818. (17) Pádua, A. A. H.; Costa Gomes, M. F.; Canongia Lopes, J. N. Molecular Solutes in Ionic Liquid Structural Perspective. Acc. Chem. Res. 2007, 40, 1087–1096. (18) Giernoth, R. Task-Specific Ionic Liquids. Angew. Chem. Int. Ed. 2010, 49, 2834–2839. (19) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. Dissolution of Cellulose with Ionic Liquids. J. Am. Chem. Soc. 2002, 124, 4974–4975. (20) Sun, N.; Rahman, M.; Qin, Y.; Maxim, M. L.; Rodríguez, H.; Rogers, R. D. Complete Dissolution and Partial Delignification of Wood in the Ionic Liquid 1-Ethyl-3-Methylimidazolium Acetate. Green Chem. 2009, 11, 646–655. (21) El Seoud, O. A.; Koschella, A.; Fidale, L. C.; Dorn, S.; Heinze, T. Applications of Ionic Liquids in Carbohydrate Chemistry: A Window of Opportunities. Biomacromol. 2007, 8, 2629– 2647. (22) Qin, Y.; Lu, X.; Sun, N.; Rogers, R. D. Dissolution or Extraction of Crustacean Shells Using Ionic Liquids to Obtain High Molecular Weight Purified Chitin and Direct Production of Chitin Films and Fibers. Green Chem. 2010, 12, 968–971. (23) Guo, Z.; Lue, B.-M.; Thomasen, K.; Meyer, A. S.; Xu, X. Predictions of Flavonoid Solubility in Ionic Liquids by COSMO-RS: Experimental Verification, Structural Elucidation, and Solvation Characterization. Green Chem. 2007, 9, 1362–1373. (24) Cao, Y.; Xing, H.; Yang, Q.; Su, B.; Bao, Z.; Zhang, R.; Yang, Y.; Ren, Q. High performance separation of sparingly aqua-/lipo-soluble bioactive compounds with an ionic liquid-based biphasic system. Green Chem. 2012, 14, 2617–2625. (25) Jin, W.; Yang, Q.; Zhang, Z.; Bao, Z.; Ren, Q.; Yang, Y.; Xing, H. Self-assembly induced solubilization of drug-like molecules in nanostructured ionic liquids. Chem. Commun. 2015, 51, 13170–13173. (26) Wasserscheid, P.; Keim, W. Ionic Liquids – New “Solutions” for Transition Metal Catalysis. Angew. Chem. Int. Ed. 2000, 39, 3772–3789. (27) Hu, Y.-F.; Liu, Z.-C.; Xu, C.-M.; Zhang, X.-M. The Molecular Characteristics Dominating the Solubility of Gases in Ionic Liquids. Chem. Soc. Rev. 2011, 40, 3802–3823. (28) Araújo, J. M. M.; Ferreira, R.; Marrucho, I. M.; Rebelo, L. P. N. Solvation of Nucleobases in 1,3-Dialkylimidazolium Acetate Ionic Liquids: NMR Spectroscopy Insights into the Dissolution Mechanism. J. Phys. Chem. B 2011, 115, 10739–10749. (29) Araújo, J. M. M.; Pereiro, A. B.; Alves, F.; Marrucho, I. M.; Rebelo, L. P. N. Nucleic Acid Bases in 1-Alkyl-3-Methylimidazolium Acetate Ionic Liquids: A Thermophysical and Ionic Conductivity Analysis. J. Chem. Thermodyn. 2013, 57, 1–8.



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Page 28 of 30

(30) Araújo, J. M. M.; Pereiro, A. B.; Canongia Lopes, J. N.; Rebelo, L. P. N.; Marrucho, I. M. Hydrogen-Bonding and the Dissolution Mechanism of Uracil in an Acetate Ionic Liquid: New Insights from NMR Spectroscopy and Quantum Chemical Calculations. J. Phys. Chem. B 2013, 117, 4109–4120. (31) Kumar, V.; Parmar, V. S.; Malhotra, S. V. Enhanced Solubility and Selective Benzoylation of Nucleosides in Novel Ionic Liquid. Tetrahedron Lett. 2007, 48, 809–812. (32) Kumar, V.; Parmar, V. S.; Malhotra, S. V. Structural Modifications of Nucleosides in Ionic Liquids. Biochimie 2010, 92, 1260–1265. (33) Jacquemin, J.; Husson, P.; Padua, A. A. H.; Majer, V. Density and Viscosity of Several Pure and Water-Saturated Ionic Liquids. Green Chem. 2006, 8, 172–180. (34) Seddon, K. R.; Stark, A.; Torres, M.-J. Influence of Chloride, Water, and Organic Solvents on the Physical Properties of Ionic Liquids. Pure Appl. Chem. 2000, 72, 2275–2287. (35) Vijayaraghavan, R.; Izgorodin, A.; Ganesh, V.; Surianarayanan, M.; MacFarlane, D. R. LongTerm Structural and Chemical Stability of DNA in Hydrated Ionic Liquids. Angew. Chem. Int. Ed. 2010, 49, 1631–1633. (36) Chandran, A.; Ghoshdastidar, D.; Senapati, S. Groove Binding Mechanism of Ionic Liquids: A Key Factor in Long-Term Stability of DNA in Hydrated Ionic Liquids? J. Am. Chem. Soc. 2012, 134, 20330–20339. (37) Zhao, H. Methods for Stabilizing and Activating Enzymes in Ionic Liquids - A Review. J. Chem. Technol. Biotechnol. 2010, 85, 891–907. (38) Gurau, G.; Rodríguez, H.; Kelley, S. P.; Janiczek, P.; Kalb, R. S.; Rogers, R. D. Demonstration of Chemisorption of Carbon Dioxide in 1,3-Dialkylimidazolium Acetate Ionic Liquids. Angew. Chem. Int. Ed. 2011, 50, 12024–12026. (39) Stevanovic, S.; Podgoršek, A.; Pádua, A. A. H.; Costa Gomes, M. F. Effect of Water on the Carbon Dioxide Absorption by 1-Alkyl-3-Methylimidazolium Acetate Ionic Liquids. J. Phys. Chem. B 2012, 116, 14416–14425. (40) Mazza, M.; Catana, D. A.; Vaca-Garcia, C.; Cecutti, C. Influence of Water on the Dissolution of Cellulose in Selected Ionic Liquids. Cellulose 2009, 16, 207–215. (41) Jiang, W.; Wang, Y.; Voth, G. A. Molecular Dynamics Simulation of Nanostructural Organization in Ionic Liquid/water Mixtures. J. Phys. Chem. B 2007, 111, 4812–4818. (42) Ghoshdastidar, D.; Senapati, S. Nanostructural Reorganization Manifests in Sui-Generis Density Trend of Imidazolium Acetate/Water Binary Mixtures. J. Phys. Chem. B 2015, 119, 10911– 10920. (43) Tokuda, H.; Ishii, K.; Hasan Susan, A. B.; Tsuzuki, S.; Hayamizu, K.; Watanabe, M. Physicochemical Properties and Structures of Room-Temperature Ionic Liquids. 3.Variation of Cationic Structures Physicochemical Properties and Structures of Room-Temperature Ionic Liquids.J. Phys. Chem. B 2006, 2833–2839.


Page 29 of 30

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(44) Hesse-Ertelt, S.; Heinze, T.; Kosan, B.; Schwikal, K.; Meister, F. Solvent Effects on the NMR Chemical Shifts of Imidazolium-Based Ionic Liquids and Cellulose Therein. Macromol. Symp. 2010, 294, 75–89. (45) Frisch, M. J.; et al. Gaussian 09, revision B.01; Gaussian, Inc.: Wallingford, CT, 2010. (46) Morrow, T. I.; Maginn, E. J. Molecular Dynamics Study of the Ionic Liquid 1-n-Butyl-3Methylimidazolium Hexafluorophosphate. J. Phys. Chem. B 2002, 106, 12807–12813. (47) Liu, Z. P.; Huang, S. P.; Wang, W. C. A Refined Force Field for Molecular Simulation of Imidazolium-Based Ionic Liquids. J. Phys. Chem. B 2004, 108, 12978–12989. (48) Chandran, A.; Prakash, K.; Senapati, S. Structure and Dynamics of Acetate Anion-Based Ionic Liquids from Molecular Dynamics Study. Chem. Phys. 2010, 374, 46–54. (49) Case, D. A.; et al. AMBER 12; University of California, San Francisco: San Francisco, CA, 2012. (50) Anthony, J. L.; Anderson, J. L.; Maginn, E. J.; Brennecke, J. F. Anion Effects on Gas Solubility in Ionic Liquids. J. Phys. Chem. B 2005, 6366–6374. (51) Prasad, B. R.; Senapati, S. Explaining the Differential Solubility of Flue Gas Components in Ionic Liquids from First-Principle Calculations. J. Phys. Chem. B 2009, 113, 4739–4743. (52) Chandran, A.; Prakash, K.; Senapati, S. Self-Assembled Inverted Micelles Stabilize Ionic Liquid Domains in Supercritical CO2. J. Am. Chem. Soc. 2010, 132, 12511–12516. (53) Hall, C. A.; Le, K. A.; Rudaz, C.; Radhi, A.; Lovell, C. S.; Damion, R. A.; Budtova, T.; Ries, M. E. Macroscopic and Microscopic Study of 1-Ethyl-3-Methyl-Imidazolium Acetate-Water Mixtures. J. Phys. Chem. B 2012, 116, 12810–12818.



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TOC Graphic


High Nucleobase-Solubilizing Ability of Low-Viscous Ionic Liquid

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