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Molecular Organization in Hydrotrope Assemblies V. Srinivas,† G. A. Rodley,‡ K. Ravikumar,§ Ward T. Robinson,| Mark M. Turnbull,⊥ and D. Balasubramanian*,† Centre for Cellular and Molecular Biology, Hyderabad 500 007, India, Centre for Peace and Conflict Studies, University of Sydney, Sydney, New South Wales, Australia, Indian Institute of Chemical Technology, Hyderabad 500 007, India, Chemistry Department, University of Canterbury, Christchurch, New Zealand, and Department of Chemistry, Clark University, Worcester, Massachusetts 01610-1477 Received September 23, 1996. In Final Form: February 24, 1997X Hydrotropes are a class of compounds that are widely used in chemical, cosmetic, and pharmaceutical industries. Though amphiphilic in character, they have short hydrophobic regions and thus differ from classical surfactants. Yet they display substantial (and often selective) ability to solubilize lipophilic compounds in water. Beyond the minimal hydrotropic concentration, the molecules self-aggregate to produce the operating assembly. We show in this paper, based on crystal structure analysis of several hydrotropes, that these compounds form open-layer assemblies, reminiscent of lamellar liquid crystals consisting of alternating hydrophobic clustering of the nonpolar regions adjacent to ionic or polar regions that are knitted together in a two-dimensional network. Stacking of aromatic rings is not seen. Two types of assemblies are seen, one with a more open and extended hydrophobic layer than the other. We suggest that the solubilizates enter the hydrophobic layers of microunits producing a cooperative and mutual stabilizing effect. The observed open layer structure of hydrotropes might also account for the occasional ability of these compounds to solubilize even better than micelles.
1. Introduction Hydrotropes are a diverse class of substances that, at high concentrations, enhance the solubility of nonpolar compounds in water. Some examples of hydrotropes are the anionic benzoates and benzosulfonates, neutral phenols like catechol and resorcinol, aliphatic glycolsulfates, alicyclic bile salts, and the amino acid proline. Figure 1 shows the diverse compounds that are classified as hydrotropes. Solubilization by hydrotropes is characterized by the relatively high concentrations of the hydrotropes needed and the larger amounts of solute solubilized, compared with that observed for micellar surfactants.1 Hydrotropes have both wide industrial usage and biological relevance. They are used as (a) solubilizing agents in drug formulations,2 (b) disinfectants and antibacterials,3 (c) catalysts in heterogeneous phase chemical reactions,4 (d) agents in the extractive separations of mixtures,5 (e) * Author for correspondence at: Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India; fax, 91 40 671195; phone, 91 40 673487; e-mail,
[email protected]. † Center for Cellular and Molecular Biology. ‡ University of Sydney. § Indian Institute of Chemical Technology. | University of Centerbury. ⊥ Clark University. X Abstract published in Advance ACS Abstracts, May 1, 1997. (1) Balasubramanian, D.; Friberg, S. E. In Surface and Colloid Science; Matijevic, E., Ed.; Plenum Press: New York, 1993; Vol. 15, p 197. (2) Woolfson, A. D.; McCafferty, D. F.; Launchbury, A. P. Int. J. Pharm. 1986, 34, 17. Jain, N. K.; Patel, V. V.; Taneja, L. N. Pharmazie 1988, 43, 254. Nishihata, T.; Rytting, J. H.; Higuchi, T. J. Pharm. Sci. 1981, 70, 71. Nishihata, T.; Rytting, J. H.; Kamada, A.; Higuchi, T.; Routh, M.; Caldwell, L. J. Pharm. Pharmcol. 1983, 35, 148. Osborne, D. W. Colloids Surf. 1988, 30, 13. (3) Neish, A. L. Recl. Trav. Chim. Pays-Bas 1948, 67, 393. (4) Janakiraman, B.; Sharma, M. M. Chem. Eng. Sci. 1985, 40, 2156. Pandit, A.; Sharma, M. M. Chem. Eng. Sci. 1987, 42, 2517. Sane, P. V.; Sharma, M. M. Synth. Commun. 1987, 17, 1331. (5) Gaikar, V. G.; Sharma, M. M. Solvent Extr. Ion Exch. 1986, 4, 839. Mahapatra, A.; Gaikar, V. G.; Sharma, M. M. Sep. Sci. Technol. 1988, 23, 429. Pasciak, J.; Chiwistek, M.; Przybyla, B. Chem. Anal. (Warsaw) 1978, 23, 675.
S0743-7463(96)00922-5 CCC: $14.00
Figure 1. A garland of hydrotropes: aromatics, aliphatics, alicyclics, anionics, cationics, nonionics, and zwitterionics.
agents in the paper and pulp industry,6 (f) fillers and extenders in cleaning and washing formulations, shampoos, and cosmetics,7 and (g) as protein-compatible solutes in biochemistry and cell biology.8 Hydrotropes are generally compounds characterized by short hydrophobic regions that are less prominent than those in surfactants and were indeed studied in some detail quite some time earlier9 than surfactants. Yet the molecular basis of hydrotropy has remained uncertain. (6) Styan, G.-E.; Bramhall, A. E. Pulp Pap. Can. 1979, 80, T25. Bland, D. E. Res. Rev.sAust. C. S. I. R. O., Div. Chem. Technol. 1976, 27. Nelson, P. J. Appita 1978, 31, 437. Bland, D. E.; Skicko, J.; Menshun, M. Appita 1978, 31, 374. Springer, E. L.; Zoch, L. L. Pap. Puu 1979, 61, 815. (7) Raney, K. H.; Miller, C. A. J. Colloid. Interface Sci. 1987, 119, 539. (8) Schobert, B. Naturwissenschaften 1977, 64, 386. (9) Neuberg, C. Biochem. Z. 1916, 76, 107. Neuberg, C. J. Chem. Soc. 1916, 110 (II), 555.
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Figure 2. Concentration-dependent changes in the properties of the hydrotrope NaPTS in water, where γ is the surface tension in dyn cm-1, φ is the enhancement factor in the alkaline hydrolysis of p-ethylnitrobenzoate (EPNB), in the presence of NaPTS, and [EPNB] is the concentration of the solubilized EPNB in g L-1.
Hydrotropy is different from simple phase mixing, or the cosolvency process, and also from salting-in action. Intermolecular complex formation between the hydrotrope and the solubilizate may occur in some instances but is not the general rule. Instead, it appears to be a collective molecular phenomenon that operates above a characteristic concentration of the hydrotrope in water. Above this concentration, which we have termed the minimal hydrotrope concentration10 or MHC, the hydrotrope molecules self-aggregate into a loose noncovalent assembly which offers a microenvironment of lowered polarity that aids the solubilization of the hydrophobic solute molecule. Figure 2 illustrates the hydrotropic properties of sodium p-toluenesulfonate (NaPTS). Solubilization of the sparingly soluble ethyl p-nitrobenzoate (EPNB) increases significantly above 0.35 M NaPTS, which is thus designated as its MHC value. The surface tension of the hydrotrope solution drops to a limiting value of 50 dyn cm-1 beyond this concentration, suggesting self-aggregation of NaPTS. It is also apparent that the surface activity here is mild and weaker than that of classical surfactants (we see this as a general feature of hydrotropes). Also, the enhancement of the rate of the alkaline hydrolysis reaction of EPNB by NaPTS occurs above its MHC value; below 0.35 M NaPTS, the rate of the reaction is the same as that in water. That the self-aggregation of NaPTS occurs above 0.35 M is confirmed by other measurements, namely fluorescence probing, NMR, and electron spin resonance (ESR) spin-label studies.10 Similar behavior is seen with other hydrotropes such as sodium xylenesulfonate (NaXS), the aliphatic molecule sodium n-butyl monoglycolsulfate (NaBMGS), the alicyclic amino acid L-proline,11 and the nonionic hydroxyphenols such as catechol, resorcinol, and 4-methylcatechol.12 While the self-aggregation of hydrotropes is reminiscent of surfactant self-assemblies, there are important differences. Hydrotropes exhibit a higher and often more selective ability to solubilize guest molecules. For example, resorcinol increases the solubility of riboflavin 300fold in water and NaXS increases the solubility of (10) Balasubramanian, D.; Srinivas, V.; Gaikar, V. G.; Sharma, M. M. J. Phys. Chem. 1989, 93, 3865. (11) Srinivas, V.; Balasubramanian, D. Langmuir 1995, 11, 2830. Rajendrakumar, C. S. V.; Reddy, B. V. B.; Reddy, A. R. Biochem. Biophys. Res. Commun. 1994, 201, 957. Taneja, S.; Ahmad, F. Biochem. J. 1994, 303, 147. (12) Srinivas, V.; Sundaram, C. S.; Balasubramanian, D. Indian J. Chem. 1991, 30B, 147.
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nitrobenzene 50-fold.13 Similarly the hydrotropic actions of isomeric o-, m-, and p-hydroxybenzoates differ notably, as also those of 2,4-, 2,5-, and 2,6-dihydroxybenzoates.14 Comparison of the ternary phase diagrams of solubilizate, solubilizer (hydrotrope or the micellar surfactant), and water, done by Friberg and co-workers15 underscores this difference between hydrotropes and micelles. It is in this light that we present and analyze the X-ray crystal structures of the sulfonic acid hydrotropes: I, sodium p-tert-butylbenzenesulfonate dihydrate; II, sodium cumenesulfonate hemihydrate; III, NaPTS (in the hemihydrate form); IV, sodium 3,4-dimethylbenzenesulfonate trihydrate. The intermolecular packing patterns that we see in these structures offer some clues about the mode of action of hydrotropes at the high concentrations at which they function. 2. Experimental Section The compounds used were of the highest purity available from commercial sources and were purified further by recrystallization. (A) Solubilization Experiments. Solid fluorescein diacetate (FDA) was added to the hydrotrope solutions (the concentration of each hydrotrope being kept at 4 times its respective MHC value) and equilibrated for several hours in a constant-temperature shaker. The suspension was centrifuged and the concentration of FDA determined spectrophotometrically at 480 nm, using a Hitachi Model 330 instrument. (B) Methods Used in X-ray Crystal Structure Analysis and Its Parameters. The experimental conditions were as follows: Mo KR radiation, graphite monochromator, 2θmax ) 48°, omega scan. Each structure was solved by direct methods and refined using the Siemens-Shelxtl-Plus program system and Shelxl-93. I: sodium p-butylbenzenesulfonate dihydrate, C10H13O3NaS‚2H2O, M ) 240.23, monoclinic, space group C2/c, a ) 39.985(5) Å, b ) 6.161(1) Å, c ) 10.802(2) Å, β ) 102.12(1)°, V ) 2601.7(7) Å3, Z ) 8, Fcal 1.227 mg/cm3; 1694 independent reflections, 1265 observed reflections with I g 3σ(I), data to parameter ratio 8.4:1; R ) 4.3%, Rw ) 4.7%. II, sodium cumenesulfonate hemihydrate, C9H12O3.5NaS, M ) 231.24, orthorhombic, space group Pbca, a ) 16.051(6) Å, b ) 6.563(2) Å, c ) 40.24(2) Å, V ) 4239(3) Å3, Z ) 16, Fcal 1.449 mg/cm3; 3323 independent reflections, 2007 observed reflections with Fo g 4σ(Fo), data to parameter ratio 7.49:1; R ) 5.5%, Rw ) 13.0%. III: sodium p-toluenesulfonate hemihydrate, C7H8O3.5NaS, M ) 203.18, monoclinic, space group P21/c, a ) 17.81(2) Å, b ) 14.588(11) Å, c ) 6.686(7) Å, β ) 90.45(6)°, V ) 1737(3) Å3, Z ) 8, Fcal 1.544 mg/cm3; 1380 independent reflections, 1103 observed reflections with Fo g 4σ(Fo), data to parameter ratio 4.65:1; R ) 5.8%, Rw ) 14.6%. IV: sodium 3,4-dimethylbenzenesulfonate trihydrate, C8H9O3NaS‚3H2O, M ) 501.52, monoclinic, space group P21/n, a ) 7.128(2) Å, b ) 6.352(2) Å, c ) 24.844(5) Å, β ) 90.28(2)°, V ) 1124.8(5) Å3, Z ) 4, Fcal 2.961 mg/cm3;1474 independent reflections, 1381 observed reflections with I g 3σ(I), data to parameter ratio 9.5:1; R ) 3.7%, Rw ) 4.7%.
3. Results and Discussion A. Two Structural Types. Analysis reveals two types of crystal structures. In type A, represented by I, II, and III, the molecules are packed in a two-dimensional sandwich manner, the aromatic portions associating endto-end to form a broad and extended hydrophobic region (Figures 3-5). The ionic regions are knitted together in a hydrophilic two-dimensional network. In type B, IV has a comparable structure except that rings from adjacent layers intermesh to give a more dense hydrophobic region (Figure 6). This latter structure was also observed for (13) Saleh, A. M.; El-Khordagui, L. K. Int. J. Pharm. 1985, 24, 231. (14) Poochikian, G. K.; Cradock, J. C. J. Pharm. Sci. 1979, 68, 728. (15) Flaim, T.; Friberg, S. E. J. Colloid Interface Sci. 1984, 97, 26. Cox, J. M.; Friberg, S. E. J. Am. Oil Chem. Soc. 1981, 58, 743. Friberg, S. E.; Rananavare, S. B.; Osborne, D. W. J. Colloid Interface Sci. 1986, 109, 487.
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Figure 3. Cell packing for I, sodium p-tert-butylbenzenesulfonate dihydrate.
Figure 4. Stereo diagram of packing for II, sodium cumenesulfonate hemihydrate.
sodium 2,4,5-trimethylbenzenesulfonate (P. J. Steel, unpublished). The two structural types, A and B, correlate with the benzene ring substitution pattern. The mono-parasubstituted compounds form the more open A layer structure, while those with ortho- and or meta-substitution form the B structure. Formation of the latter may be the result of stronger lateral hydrophobic interactions, favored by the presence of the o- or m-methyl groups. This suggests that A-type hydrotropes would show greater solubilizing properties than B-type ones, in lieu of the more open and more extended hydrophobic layer size. In a test of this prediction, we measured the solubility values of fluorescein diacetate in sodium cumenesulfonate, sodium p-toluenesulfonate, and sodium xylenesulfonate and found them to be in the order 20:7.5:4, which is in keeping with the prediction. B. Aromatic Stacking Is Not Prominent. Of significance is the general absence of direct overlay of aromatic rings at the normally observed distance of 3.4 Å. This is in contrast to the suggestion of Saleh and ElKhordagui who thought stacking to be an important feature of hydrotrope aggregation.13 Rather, the rings fill the hydrophobic region in a more twisted, staggered, and intermeshing manner, as shown in the figures for II, III, and IV (Figures 4, 5, and 6). While some overlay of adjacent rings does occur for I (Figure 3) these are
staggered with respect to each other. As a consequence, lateral packing is more compact in all cases, as indicated by the cell-repeat for the relevant axis (corresponding to two intermolecular separations) being less than 6.8 Å. This feature provides an indirect explanation as to why both aromatic ring sulfonates and aliphatic chain ones display comparable solubilizing features (e.g., as observed10 for sodium n-butyl monoglycolsulfate and sodium p-toluenesulfonate). The common feature would be cluster association of the nonpolar organic regions to form a hydrophobic layer that allows association of Na+, SO3-, and water entities to occur, as observed in the structures reported. With aromatic ring stacking not being a crucial factor, the length of the hydrophobic component may be the key factor. The latter could be achieved by a range of both aromatic and aliphatic entities. The ionic association involves coordination of oxygens from the sulfonate group as well as oxygens from water molecules. The precise details vary among the structures studied, but the overall effect is similar. A separate hydrophilic network extending in two dimensions is formed. C. Open Sandwich-Type Dynamic Cluster Arrangement. The overall stabilization of the sandwich arrangement is therefore a combination of favorable ionic network formation, combined with hydrophobic layer clustering of the nonpolar regions. This affords an overall
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Figure 5. Stereo diagram for packing for III, sodium p-toluenesulfonate monohydrate.
Figure 6. Cell packing for IV, sodium 3,4-dimethylbenzenesulfonatetrihydrate, showing the more dense inter-ring packing type.
planar configuration of the hydrophobic and hydrophilic regions. Such an arrangement corresponds to an openedup micelle having greater accessibility to the hydrophobic regions than in a micelle. The arrangement is akin to that of a lamellar liquid crystal.1 The observed solid-state structure is considered to indicate the operative molecular detail for hydrotrope action.1 This relates to the fact that relatively high concentrations of hydrotrope are required to initiate solubilization. Since crystals seed and form from such concentrations, the observed structural detail would reflect the solution state structure existing at that point. Thus, hydrotrope solutions may be deduced to contain dynamic aggregate clusters having essentially the sandwich structure found in the solid state. The characteristic solubilizing properties of hydrotropes may be understood in terms of this configuration. Entities having low water solubility could readily become solubilized by entering the hydrophobic layers of microunits. These would, in turn, stabilize the layered structure, producing a cooperative effect. Direct evidence for such an effect is provided by the sigmoidal character of solubility curves.10-12
Furthermore, the observed open-layer structure is consistent with the (occasionally seen) significantly higher solubilizing feature of hydrotropes than observed for micelles.1 Relevant to this argument is the case of the amino acid proline, which displays hydrotropy at high concentrations and which acts as a “compatible solute” that stabilizes protein structures in water-stressed situations.11 We believe that proline’s ability to do so arises from its ability to form incipient intermolecular network structures of the kind that it adopts in the crystal state.16 A structure of relevance regarding hydrotrope solubilization is that of the adduct, Na9(C7H7O3S)8I3‚2H2O,17 the association of one molecule of NaI3 with eight sodium p-toluene sulfonate molecules. The polarizable triiodide anions are embedded in between opposing layers of aromatic rings of the hydrotrope, with the sodium ions incorporated into the ionic network. This demonstrates (16) Kayushina, R. L.; Vainshstein, B. K. Kristallografiya 1965, 10, 834. (17) Herbstein, F. H.; Reisner, G. M.; Schwotzer, W. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1985, C41, 510.
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the likely mode of solubilization of such species. The significance of the basic sandwich structure for hydrotropes is indicated by retention of essentially the same configuration as observed for the parent hydrotrope (III) in the adduct.17 D. Hydrotropes as Vehicles for Chemical Oscillators. The hydrotropes I, II, and III have been used in the study of binary systems involving the BelousovZhabotinskii (B-Z) chemical oscillator.18,19 These studies have shown that the robustness of batch B-Z oscillators is markedly enhanced by the presence of the hydrotropes, which significantly lengthen the oscillating lifetimes of B-Z systems. What is indicated by these studies is the development of a situation that is more “open” than that existing in the reference no-hydrotrope oscillators. In turn, this suggests compartmentalization of key reacting species in a manner that allows the “flow” of reactants and products.20 The host hydrotrope could provide “reservoir” and “sink” locations for species, although the host system would itself be subject to dynamic change. The B-Z oscillator involves a wide range of molecular entities participating in an extended array of interlinked reactions. These include the nonpolar species, Br2, CO2, and probably O2, as well as various ionic ones. What the hydrotrope/water structure affords is a versatile molecular host for such entities. Aqueous micelles, by contrast, do not show a marked enhancement of B-Z oscillation lifetimes.19,21 This indicates the hydrophilic hydrotrope layer is also significant as a host for ionic B-Z species like BrO3-. It provides a more protected site than available for micelles. It is significant, in relation to the comparison made above with liquid crystal structure, that the B-Z oscillator has also been incorporated into such a structure.22 The liquid crystal system prepared using sodium bis(2ethylhexyl)sulfosuccinate (abbreviated as Aerosol OT or AOT) in isooctane, in which the B-Z oscillator was incorporated, shows similar characteristics to those just mentioned. Additional features of oscillation patterns show the hydrotropes to be more versatile host systems than the AOT liquid crystal. This probably relates to the latter, of necessity, being significantly more concentrated than the hydrotrope solutions. That feature would inhibit spatial dispersion of B-Z species, regarded as significant for extending B-Z oscillation lifetimes.22
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lizate. Layered structures seem preferred by hydrotropes, in contrast to the “oil drop with a polar coat” compact assembly preferred by surfactant micelles. This leads us to suggest that the primary micelles of bile salts such sodium deoxycholate might be better considered as hydrotropes. Again, like bile salts, the aggregation number of hydrotrope assemblies is expected to be small. An additional feature that arises out of the layered structural arrangement of hydrotropes in water is the manner in which they would solubilize guest molecules. Nonpolar molecules would be expected to enter the hydrophobic layers of the hydrotrope assemblies. These would, in turn, stabilize the layered structure, producing a cooperative solubilizing isotherm. The solute imparts stability to the host assembly. This feature is novel, different from what is seen in classical micelles, and is put into use in chemical engineering applications by the Bombay group.4,5 The inclusion of short chain alkanoates as hydrotropes is interesting,23 since octanoate and higher homologs are considered as regular micelles. Likewise, while toluenesulfonate and p-ethylbenzenesulfonate are hydrotropes, the higher member in the series, octylbenzenesulfonate, forms micelles. How long should the alkyl chain be in order that the compound will change over from a hydrotrope into a micellar assembly is an interesting question; it involves a shift in the hydrophile-lipophile balance and also a concomitant change in the pattern of molecular organization from the layered structure into a finite spherical or ellipsoidal packing. This aspect of hydrotropy is currently being addressed in our laboratory. It is also interesting to note that while the study of hydrotropes was pioneered by a biochemist,9 greater appreciation of their role and utility has happened in chemistry and chemical engineering than in biology. Proline, as a protein-compatible hydrotrope, is known to accumulate and help in osmotolerance in cells. We believe that more such compounds will be detected in the near future. Unlike classical detergents or some of the hydrotropes which tend to denature biopolymers, a biocompatible hydrotrope would serve a dual purposessolubilize apolar and amphipolar molecules and keep them in their functionally competent forms. The search for such biocompatible hydrotropes is expected to be rewarding and practically useful as well.
4. Conclusions Hydrotropic substances are regaining importance in the currently blossoming field of self-organizing molecular systems. Their hydrophile-lipophile balance being on the higher side, their water solubility is significantly more than that of traditional surfactants, and yet they provide a sequestered niche or microenvironment for the solubi(18) Balasubramanian, D.; Rodley, G. A. J. Phys. Chem. 1988, 92, 5995. (19) Balasubramanian, D.; Rodley, G. A. Unpublished results. (20) Gonda, I.; Rodley, G. A. J. Phys. Chem. 1990, 94, 1516. (21) Maritato, M.; Nikles, J.; Romsted, L. S.; Tramontin, M. J. Phys. Chem. 1985, 89, 1341. (22) Balasubramanian, D.; Rodley, G. A. J. Phys. Chem. 1991, 95, 5147.
Acknowledgment. D.B. thanks the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India, for an honorary Professorship. Supporting Information Available: Tables of crystal data and structure refinement, atomic coordinates, bond angles and distances (geometric parameters), displacement parameters, and hydrogen atom coordinates for I-IV (32 pages); tables of observed and calculated structure factors for I-IV (24 pages). Ordering information is available on any current masthead page. LA9609229 (23) Danielsson, I.; Stenius, P. J. Colloid Interface Sci. 1971, 37, 264.
