11244
J. Phys. Chem. B 2008, 112, 11244–11249
Stacking of 2,3,5-Triphenyl-2H-tetrazolium Chloride onto Polyelectrolytes Containing 4-Styrenesulfonate Groups Ignacio Moreno-Villoslada,*,†,‡ Cristian Torres,† Felipe Gonza´lez,† Marcos Soto,† and Hiroyuki Nishide§ Instituto de Química, Facultad de Ciencias, UniVersidad Austral de Chile, Casilla 567, ValdiVia, Chile;, Departamento de Farmacia e Tecnoloxía Farmace´utica, Facultade de Farmacia, UniVersidade de Santiago de Compostela, 15782-Santiago de Compostela, Spain;, Department of Applied Chemistry, School of Science and Engineering, Waseda UniVersity, Tokyo 169-8555, Japan ReceiVed: March 27, 2008; ReVised Manuscript ReceiVed: July 7, 2008
Possible structural aspects are discussed that justify the different resistance to reduction of 2,3,5-triphenyl2H-tetrazolium chloride (TTC) both chemically (by reaction with ascorbic acid (ASC)) and electrochemically, in the presence of different polyelectrolytes such as poly(sodium 4-styrenesulfonate) (PSS), poly(sodium 4-styrenesulfonate-co-sodium maleate) at two different comonomer compositions (P(SS1-co-MA1) and P(SS3co-MA1)), and poly(sodium acrylate-co-sodium maleate) (P(AA1-co-MA1)). Different dissociation constants are found for the complexes between TTC and the different polyelectrolytes by diafiltration (DF). Related to this, spectroscopical differences are also found by 1H NMR and UV-vis spectroscopies. Dynamic light scattering (DLS) showed a higher tendency to undergo intermolecular aggregation for P(SS1-co-MA1) in the presence of TTC, a result that could be related with a higher tendency for TTC to form hydrophobic ion pairs as a consequence of single stacking with the benzene sulfonate groups (BS) of this polyelectrolyte. On the other hand, the lower tendency for PSS to undergo intermolecular aggregation could be attributable to a higher probability to form more hydrophilic adducts by means of double stacking with TTC. 1. Introduction Aromatic-aromatic (ar-ar) interactions take place in the supramolecular assembling of several molecules.1-12 They are found in biological systems such as DNA,13 proteins, and enzymes,14-18 trans-membrane channels,19,20 etc. It is of great interest that both structures and functionalities are determined by the presence of these interactions, and intrinsically related. Thus, the search of synthetic systems whose structures and properties are tuned by ar-ar interactions21-27 has motivated our latest research. In this context, we have shown that the redox behavior of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) can be modified by the presence of water-soluble polyelectrolytes containing benzene sulfonate (BS) groups.27 These were copolymers of 4-styrenesulfonate and maleic acid (MA). These polymers act as protective moieties from the reduction of TTC both chemically by reaction with ascorbic acid (ASC) and electrochemically. Surprisingly, this effect is a function of the linear aromatic density of the polyelectrolytes determined by the relative amount of BS groups with respect to the maleate groups. Apart from spectroscopic techniques, separation techniques are useful to evaluate the binding between molecules. Among these techniques, diafiltration (DF) has emerged as a suitable technique to evaluate the interactions between low molecularweight species (LMWS) and water soluble polymers (WSP).28-38 * Corresponding author. Fax: 56-63-293520. E-mail: imorenovilloslada@ uach.cl. † Instituto de Química, Facultad de Ciencias, Universidad Austral de Chile. ‡ Departamento de Farmacia e Tecnoloxía Farmace ´ utica, Facultade de Farmacia, Universidade de Santiago de Compostela. § Department of Applied Chemistry, School of Science and Engineering, Waseda University.
Figure 1. Molecular structures.
The main magnitudes managed in DF analyses are the filtration factor (F), defined as the ratio between the volume in the filtrate and the constant volume in the diafiltration cell, the concentration in the filtrate of the LMWS under study (cLMWSfiltrate), the concentration of free LMWS in the cell solution (cLMWSfree), the concentration of LMWS reversibly bound to the WSP (cLMWSreVbound), the apparent dissociation constant (KLMWSdiss-WSP), defined as the ratio cLMWSfree/cLMWSreV-bound, the diafiltration parameters km, j, u, and V, and the polymer concentration in mole per liter of monomeric units (cP). km and j parameters (the absolute value of the slope of the curve ln cLMWSfiltrate versus F in the absence and in the presence of the WSP, respectively) are related with
10.1021/jp802668q CCC: $40.75 2008 American Chemical Society Published on Web 08/15/2008
Stacking of 2,3,5-Triphenyl-2H-tetrazolium Chloride
J. Phys. Chem. B, Vol. 112, No. 36, 2008 11245
Figure 2. DF profiles of a 10-3 M TTC solution at pH 8.7 in the absence of NaCl (a), and in the presence of 2 × 10-2 M of NaCl (b); in the absence of any WSP (×) and in the presence of 10-2 M of PSS (9), P(SS3-co-MA1) (2), P(SS1-co-MA1) ([), and P(AA1-co-MA1) (b). Expressions for the linear adjustments are found in Table 1.
TABLE 1: Results for DF of 10-3 M TTC Solutions at pH 8.7 in the Presence of 0.02 M NaCl and Different WSPa experiment
cP (M)
V
u
j
TTC-NaCl PSS-TTC-NaCl P(SS3-co-MA1)-TTC- NaCl P(SS1-co-MA1) -TTC-NaCl P(AA1-co-MA1)-TTC-NaCl
1 × 10-2 1 × 10-2 1 × 10-2 1 × 10-2
1.0 0.9 1.0 0.9 0.9
0.0 0.1 0.0 0.1 0.1
0.08 0.25 0.65 0.86
a
km 0.90
KTTCdiss-WSPb
linear adjustments for the experimental dataa
R2
0.09 ( 0.005 0.35 ( 0.02 2.3 ( 0.3 f∞
y)-0.90x-6.7 y)-0.08x-9.6 y)-0.25x-8.3 y)-0.65x-7.2 y)-0.86x-6.9
1.00 0.99 0.99 1.00 1.00
For linear adjustments: y ) ln〈cTTCfiltrate〉; x ) F; R2 ) linear regression factor. b KTTCdiss-WSP values are calculated following
j kmj diss-wSP e KLMWS e m 1-j k -j
the strength of the interaction, while V and u are related with the amounts of LMWS reversibly or irreversibly bound to the polymer, respectively. By irreversibly bound we consider molecules bound in processes that may be reversible with an apparent dissociation constant that tend to zero at the conditions of the experiment. The aim of this paper is to discuss possible structural aspects that justify the different resistance to TTC reduction in the presence of poly(sodium 4-styrenesulfonate) (PSS), poly(sodium 4-styrenesulfonate-co-sodium maleate) at two different comonomer compositions (P(SS1-co-MA1) and P(SS3-co-MA1)), and poly(sodium acrylate-co-sodium maleate) (P(AA1-co-MA1)), by means of experimental results obtained by DF, 1H NMR and UV-vis spectroscopies, and dynamic light scattering (DLS). 2. Experimental Section 2.1. Reagents. Commercially available PSS (Aldrich, synthesized from the para-substituted monomer), poly(sodium 4-styrenesulfonate-co-sodium maleate) at comonomer compositions 1:1 (P(SS1-co-MA1), Aldrich) and 3:1 (P(SS3-co-MA1), Aldrich), poly(sodium acrylate-co-sodium maleate) at a comonomer composition 1:1 (P(AA1-co-MA1), Aldrich), and 2,3,5triphenyl-2H-tetrazolium chloride (TTC, Merck and TCI) were used to prepare the solutions in deionized distilled water or D2O (Acros, 99.8% D- content). The structures of the different polyelectrolytes and TTC are shown in Figure 1. The pH was adjusted with NaOH and HCl. NaCl (Scharlau) was used to adjust the ionic strength in DF experiments. 2.2. Equipment. The unit used for DF studies consisted of a filtration cell (Amicon 8010, 10 mL capacity) with a magnetic stirrer, a polyethersulfone membrane with a MWCO of 5,000 Dalton (Ultracel PBCC, 25 mm diameter), a reservoir, a selector, and a pressure source. Distilled water was deionized in a
Simplicity Millipore deionizer. The pH was controlled on a Hanna pH211 or Horiba F-15 pH meters. UV-vis measurements were performed in a Heλios γ spectrophotometer. 1H NMR measurements were made in a JNM-Lambda500 (JEOL, 500 MHz) spectrometer. DLS measurements were done in a ZSizer Nano ZS (Malvern Instruments) equipment. 2.3. Procedures. Conventional and well-known procedures have been followed for DF, 1H NMR, UV-vis, and DLS experiments. Particular experimental conditions are provided in the Figure captions. Details for DF procedures can be found elsewhere.36-38 Previous to be used, the polyelectrolytes where fractionated over a polyethersulfone membrane with a MWCO of 10,000 Dalton, and the highest molecular-weight fractions were chosen for DF experiments in order no macromolecule is able to traverse the 5,000 Da DF membrane. 1H NMR experiments were done in D2O, and the pH was adjusted to 7 for PSS and 8.5 for the polyelectrolytes containing maleate units with minimum amounts of NaOH 0.1 M in H2O. We assume that most carboxylic units are negatively charged at this pH. UV-vis measurements were done in quartz cells with 1 cm of path length; decomposition of the spectra in gaussians was done with the Origin50 software. DLS measurements were performed in polystyrene cells with 1 cm of path length. 3. Results and Discussion 3.1. Binding of TTC to the Polyelectrolytes. As a strategy for the development of this discussion, the concentration of the copolymers is chosen so that the concentration of polymeric aromatic groups is constant, and 10 times that of TTC. Polymer concentrations are given in mole of sulfonate or acrylate groups per liter. As TTC is a positively charged molecule, long-range electrostatic interactions are supposed to take place with all the
11246 J. Phys. Chem. B, Vol. 112, No. 36, 2008
Figure 3. 500 MHz 1H NMR spectra in D2O of TTC 10-3 M in the absence of any polyelectrolyte (a) and in the presence of 10-2 M of the following polymers (expressed in BS or AA groups): PSS (b); P(SS3-co-MA1) (c); P(SS1-co-MA1) (d); P(AA1-co-MA1) (e); PSS mixed with 6.6 × 10-3 M of P(AA1-co-MA1) (f).
polyelectrolytes in the absence of any added electrolyte. As longrange electrostatic interactions do not normally produce changes in both 1H NMR and UV-vis spectroscopical measurements, DF has emerged as a suitable technique to evaluate the binding of counterions to polyelectrolytes. It can be seen in Figure 2 the DF profiles for the different polyelectrolytes in the absence of any added electrolyte and in the presence of NaCl 0.02 M. At both conditions, differences are clearly shown considering the binding of TTC to the different polyelectrolytes. In the absence of added NaCl (Figure 2a) the DF profiles in the presence of the polymers do not follow a linear behavior. This lack of linearity is normally found when no electrolyte is added to the solutions and interpreted as a consequence of the change on the predominant counterion:37,39 in the first part of the DF experiment, Na+ ions furnished by the polyelectrolytes should be predominant in solution, and at the end of the DF experiment (at F values higher than 4), TTC is predominant and hence, more sensitive to long-range electrostatic interactions. No analytical treatment is done in this case, and the results can be discussed qualitatively. Compared to PSS, P(SS3-co-MA1) and P(SS1-co-MA1) can be regarded as if some charged segments were added and intercalated into the backbone of a PSS chain, so that the total number of polymer negative charges increases as the maleate residues increase in the copolymers. For P(AA1co-MA1) the total number of polymer charges is equivalent to
Moreno-Villoslada et al. those of the system P(SS1-co-MA1), being the highest among all the different polymers. Thus, long-range electrostatic interactions are expected to be more intense for the copolymers that contain a higher fraction of MA residues. However, DF results clearly show that the binding of TTC to the different polyelectrolytes is correlated with the linear aromatic density, i.e., with the ratio BS/MA, being higher in the order PSS > P(SS3-coMA1) > P(SS1-co-MA1) > P(AA1-co-MA1). This is, then, related to the existence of other forces in addition to long-range electrostatic forces. Although all the solutions exhibit the same concentration of BS groups, the presence of the MA groups is important in the mode of binding of TTC to the polymers. Long-range electrostatic interactions can be quenched in the presence of NaCl, so it allows the evaluation of the short-range electrostatic interactions between the low molecular-weight molecules and the polyelectrolytes. In the presence of NaCl, the constant concentration of ions produces linear DF profiles that can be treated analytically. The results are shown in Figure 2b and in Table 1. The dissociation constants found for the different polyelectrolytes are also correlated to the linear aromatic density being KTTCdiss much lower in the presence of PSS than for the rest of the polymers. Thus, stronger shortrange interactions are held with this polyelectrolyte. On the contrary, for P(AA1-co-MA1), a polymer that does not have any aromatic group, the DF profile at these conditions does not differ much from that of TTC in the absence of the polymers. For this polymer, the interaction is mainly electrostatic and is quenched in the presence of NaCl. 3.2. Spectroscopy. Figure 3 shows the 1H NMR spectra for the interaction of TTC with the different polyelectrolytes in the absence of any added electrolyte. NMR is a useful tool to probe the stacking of aromatic rings. The effect of stacking the rings is to place one ring in the shielding cone of the second, resulting in upfield shifts of 1H resonances for the stacked rings. Besides, broadening of the bands is also normally observed for the stacked molecules. It can be seen that P(AA1-co-MA1) (Figure 3b) does not produce any change on the spectrum of TTC compared to that of the pristine low molecular-weight molecule (Figure 3a). As the relative BS concentration increases with respect to the maleate groups in the copolymers, the TTC bands are shifted upfiled, so that, the highest shifting (around 0.5 ppm) is found for PSS homopolymer (see Figure 3c-e). Although the amount of BS groups is the same for all the systems, the shifting is a function of the linear aromatic density so that it takes higher values as the number of maleate moieties decreases, despite the total amount of charges also decreases in the systems. This is in good agreement with the results found by DF for the binding of TTC to the different polyelectrolytes. In the presence of a mixture of 10-2 M PSS and 6.6 × 10-3 M P(AA1-coMA1) (a concentration such that the total amount of carboxylate groups is equivalent to that of the system P(SS1-co-MA1) 10-2 M), the spectrum of TTC remains equivalent to that of TTC in the presence of PSS (Figure 3f). This may indicate that TTC binds preferentially to PSS than to the carboxylate containing macromolecules. TTC shows two main UV-vis bands, centered at 248 and 300 nm respectively. The BS groups also show two absorption maximum at 227 and 264 nm respectively, which overlay with the absorption maxima of TTC. The intensity of the band at 264 nm is very low compared to those of TTC even in the presence of an excess of polymer of 10 times. Figure 4 shows the corresponding spectra of a 4 × 10-5 M TTC solution in the presence of an excess of the polyelectrolytes. The concentration of the reactants has been chosen in order to obtain accurate
Stacking of 2,3,5-Triphenyl-2H-tetrazolium Chloride
J. Phys. Chem. B, Vol. 112, No. 36, 2008 11247
Figure 4. UV-vis spectra of 4 × 10-5 M TTC at pH 7.5 in the presence of 3 × 10-4 M PSS (a), P(SS3-co-MA1) (b), P(SS1-co-MA1) (c), and P(AA1-co-MA1) (d). The results of the decomposition of the spectra into four gaussians and their respective additions are also shown.
Figure 5. Plot of the UV-vis TTC band centered at 248-254 nm obtained by decomposition of the original spectra of 4 × 10-5 M TTC in the presence of 3 × 10-4 M P(AA1-co-MA1) (a), P(SS1-co-MA1) (b), P(SS3-co-MA1) (c), and PSS (d).
measurements and sufficient binding of TTC to the polyelectrolytes. The resulting spectra have been decomposed into four gaussians showing a good fit with the experimental values. It is interesting to notice that the TTC absorption maximum at 248 nm is shifted to lower energies in the presence of the polyelectrolytes. This shifting is also a function of the linear aromatic density of the polyelectrolytes, as can be seen in Figure 5: while no shift is obtained in the presence of P(AA1-co-MA1), the band is increasingly shifted up to 254 nm in the presence of P(SS1-co-MA1), P(SS3-co-MA1), and PSS. In order to follow this band shifting, similar decompositions have been done for a 4 × 10-5 M solution of TTC in the presence of variable amounts of PSS. It can be seen in Figure 6 that at low PSS
concentration the polymer band centered at 227 nm is shifted to higher energies up to 207 nm. At these conditions it can be assumed that all the BS groups are bound to TTC which is in excess. This shifting decreases for increasing PSS concentration reaching the original value when the PSS/TTC ratio reaches 2. On the other hand, the TTC band is not shifted for low PSS concentrations, and the maximum of absorbance is kept at 248 nm up to a PSS/TTC ratio of 1. Increasing the PSS/TTC ratio from 1 to 3 produces a shift of the TTC band from 248 to 254 nm. For larger excess of PSS, the maximum of absorbance of TTC is kept at 254 nm. 3.3. Different Stacking. Two hypotheses can be formulated to explain the results shown above. For the first one it can be considered that short-range ar-ar interactions take place between the BS groups and TTC in a 1 to 1 stacked complex (see Figure 7a). As TTC is a positively charged molecule, the negative charge of the sulfonate groups is neutralized so that hydrophobic ion pairs are formed. These ion pairs tend to aggregate confining TTC in a hydrophobic environment that is responsible for the higher binding, the spectroscopic changes, and the protective effect of the polymers toward the reduction of TTC in the presence of ASC or electrochemically. As the maleate units increase in the copolymers, the hydrophobicity of the TTC environment decreases, so that the above-mentioned effects are diminished. The second hypothesis can be formulated considering the possibility of double stacking of BS groups onto TTC, as can be seen in Figure 7b. The probability of double stacking with respect to single stacking is higher for PSS, since the BS groups are found at smaller distance from each other than in the copolymers, and no negatively charged maleate groups are intercalated between the aromatic groups. The TTC confined between two parallel BS rings are strongly bound and
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Moreno-Villoslada et al.
Figure 6. Maximum of absorbance of the UV-vis PSS band centered at 207-227 nm (a) and TTC band centered at 248-254 nm (b) obtained by decomposition of the original spectra of 4 × 10-5 M TTC in the presence of variable amounts of PSS.
Figure 7. Possible structures for the single and double stacking of BS groups onto TTC.
less susceptible to reduction in the presence of ASC or electrochemically, while the spectroscopic changes are also justified due to the short-range contacts between the BS groups and TTC. These two hypotheses do not totally exclude each other: the first hypothesis opens the possibility of intra- or intermolecular aggregation due to hydrophobic forces. The formation of a zipper structure could be regarded as a combination of these two hypotheses, for which all the TTC molecules interact with two BS groups, but the overall system have no charge resulting in inter- or intramolecular hydrophobic interactions. This kind of ordered structure could be more probably formed as the amount of maleate groups decreases in the copolymers, avoiding charge repulsion. However, conformational restrictions may cause a disfavored entropic cost. Apart from the situation in which zipper structures are formed, an immediate consequence of the double stacking is that the
adducts present a net negative charge, so that their hydrophobicity is minimized. In this sense, their tendency for aggregation should be lower than in the case of single stacking. Thus, concerning the possibility of intermolecular aggregation, both single stacking and double stacking should produce contrary effects: if only single stacking is held, the polyelectrolytes rich in maleate groups should show a lower tendency to aggregate than the polyelectrolytes poor in maleate groups, since the maleate groups are charged; on the contrary, if double stacking is also held, the polyelectrolytes poor in maleate groups should present a higher probability to undego double stacking versus single stacking and then a lower tendency to aggregate. Intermolecular interactions can be enhanced for highly polymer concentrated solutions. It can be seen in Figure 8 the results found by DLS concerning the appearance of nanoparticles as solutions of 4.6 g/L of the different polyelectrolytes are neutralized with TTC. The neutralization of PSS produce nanoparticle formation in a narrow range of degree of neutralization (around 40%) and precipitation of the system for higher degrees of neutralization (see Figure 8a). A similar situation appears for P(SS3-co-MA1) for which the nanoparticle formation occurs around a 25% of neutralization. This behavior is relevant and may indicate a different stacking of the BS groups onto TTC, since the polymer exhibits a higher tendency to intermolecular aggregation than PSS despite the maleate groups should
Figure 8. Apparent size of nanoparticles formed at pH 8.6 by neutralization with TTC of 4.6 g/L of PSS (9), P(SS3-co-MA1) (2), and P(SS1co-MA1) ([) versus degree of charge neutralization (a) and degree of BS neutralization (b). A size of 1000 nm was used to indicate visible precipitation.
Stacking of 2,3,5-Triphenyl-2H-tetrazolium Chloride tend to stabilize the polymer in solution. The highest tendency for intermolecular aggregation is clearly found for P(SS1-coMA1): at a relatively low degree of neutralization (
