Effect of Hydrophobic Bonding on the Conformational Transition and

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J. Phys. Chem. B 1999, 103, 9306-9313

Effect of Hydrophobic Bonding on the Conformational Transition and Properties of Intramolecular Micelles Formed by Copolymers of Maleic Acid and Styrene A. F. Olea* and B. Acevedo Departamento de Quı´mica, Facultad de Ciencias, UniVersidad de Chile, Casilla 653, Santiago, Chile

F. Martinez Departamento de Quı´mica, Facultad de Ciencias Fı´sicas y Matema´ ticas, UniVersidad de Chile, Santiago, Chile ReceiVed: April 19, 1999; In Final Form: July 18, 1999

Fluorescence probing has been used to study the conformational transition and the properties of the microdomains formed by copolymers of maleic acid with styrene esterified with aliphatic alcohols of variable chain length, MAS-n. The steady-state and time-resolved measurements of pyrene fluorescence show the existence of two different microdomains over the whole range of pH. The pH-induced transition modifies the structure of the intramolecular micelles, but the hydrocarbon interaction prevents the expansion of the chain to the random-coil conformation. The copolymers with octyl or longer alkyl groups do not show a conformational transition. At a low degree of neutralization a change of micellar composition, with increasing side chain length, is detected by pyrene. The quenching of pyrene fluorescence by nitromethane, thallium nitrate, and sodium iodide reveals that the environments provided by MAS-n are less rigid than in poly(methacrylic acid) and that the ionized carboxylic groups are not randomly distributed.

Introduction Synthetic polyelectrolytes have been used as simple models of biological macromolecules in order to understand the solution behavior of biomolecules. Polyelectrolytes with hydrophobic groups attached to the side chain may give rise to intramolecular micelles under appropriate conditions. Such polymers have been called “polysoaps”, and typical examples are the n-dodecyl derivatives of poly(4-vinylpyridine);1 copolymers of maleic acid with alkyl vinyl ethers,2-7 styrene,8-11 or R-methylstyrene.12 These polymers are known to form hydrophobic microdomains as a consequence of a pH-induced conformational transition, and this process has been studied by different measuring techniques. The data suggest that at low degree of neutralization, R, the polyelectrolytes adopt a compact form stabilized by the hydrophobic bonding between the alkyl side chains. At a high degree of neutralization the macromolecule stretches out to an extended form. Thus, in aqueous solution the structure of these polyelectrolytes and their equilibrium properties depend on a balance of two opposite forces, namely, electrostatic repulsion and hydrophobic bonding. A series of alternating copolymers of maleic acid and alkyl vinyl ethers have been studied by Strauss and his collaborators to assess the effect of hydrophobic bonding on macromolecular conformation. The results indicate that the copolymer with ethyl vinyl ether adopt a random coil conformation over the whole range of pH, while the butyl, hexyl, and octyl undergo a transition from compact to extended forms upon increase of the neutralization degree.2-5 Polyelectrolytes with longer hydrocarbon side chains maintain a compact structure even at high pH.6,12,13 A similar pH-induced conformational transition has been observed in an alternating copolymer of maleic acid and styrene.8-11 In this polymer, the compact form is stabilized by

hydrophobic interaction between phenyl groups, and the transition occurs near R1 ) 0.3. Fluorescence probing methods have been used to investigate the microenvironment and conformational transitions of polyelectrolytes in aqueous solutions and to study the dynamics of the transition.14-16 In the present paper a series of monoesters of an alternating copolymer of maleic anhydride, MAS-n with n ) 1-12, has been investigated by steady-state and time-resolved fluorescence measurements. Pyrene was used as a luminescent probe because its photophysical properties are strongly dependent on the media in which is solubilized.17-19 The main purpose of this work was to ascertain the effect of hydrocarbon side chains on the transition and properties of the intramolecular micelles formed by MAS. Experimental Section Materials. Two kinds of poly(co-maleic anhydride-styrene), MAS, were used: one purchased from Aldrich and the other polymerized by using 2,2′-azobisisobutyronitrile as initiator, according to a standard method.20,21 The average molecular weight were 350.000 and 110.000, respectively. The latter value was determined by viscometric measurements and gel permeation chromatography using poly(styrene) samples as standard. PMA of 100000 molecular weight was purchased from Polysciences. The esterification reactions were carried out in tetrahydrofurane solution at 65 °C in presence of 4-dimethylaminopyridine as catalyst. Alcohols from methanol to 1-dodecanol (Aldrich) were used as received. The degree of conversion F was determined by FTIR, measuring the changes in absorption at 1779 cm-1 relative to the absorption at 1602 cm-1, which correspond to the maleic anhydride and styrene residues, respectively.22 The sodium salts were obtained by first dissolving the copolymers in a bicarbonate solution, and then dialyzing and

10.1021/jp991267r CCC: $18.00 © 1999 American Chemical Society Published on Web 10/04/1999

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Figure 1. Fluorescence intensity ratio III/I of pyrene (2 µM) as a function of pH in aqueous polymer solution. 9 PMA, b MAS, 2 MAS-1.

lyophilizing these solutions. Stock solutions were prepared by weighing 1 mg of copolymer salt by 1 mL of solution and adding an exact amount of acid equivalent to the carboxylate concentration. The NaCl concentration was adjusted at 100 mM. Potentiometric titrations were made at 25 °C using an Orion digital ion analyzer model EA 940 equipped with an Orion Ross combination pH electrode. The degree of neutralization, R, of the carboxyl groups is given by

R ) {[NaOH] + [H+] - [OH-]}/[COOH]T where [NaOH], [H+], and [OH-] are the molarities of added titrant, free hydrogen ion, and free hydroxyl ion, respectively, and [COOH]t is the concentration of the carboxyl group. Thus, R ) 1 at complete neutralization of the carboxyl groups. Fluorescence Probing. The concentrations used were 1 g/L and 2 µM for the copolymer and pyrene, respectively. All samples contain NaCl 100 mM and were prepared with deionized water. Fluorescence emission spectra were obtained on a SLM/Aminco SPF-500C spectrophotofluorimeter. The ratio III/I corresponds to the ratio of intensities of peak three (λ ) 384 nm) to peak one (λ ) 373 nm). The fluorescence decay of the singlet excited pyrene was monitored at 400 nm, following excitation with pulses from a PRA Nitromite nitrogen laser, model LN-100, with a 0.12-ns fwhm 70-µJ 337.1-nm pulse. The emission, detected by a Hamamatsu R-1644 microchannel plate, was digitized via a Tektronik 7912HB digitizer. A description of the time-resolved fluorescence system has been given previously.23 Time-dependent fluorescence data were fitted to a double exponential function of the form

I(t)/I(0) ) q exp(-k1t) + (1-q) exp(-k2t) where I(t) and I(0) are the fluorescence intensities at time t and t ) 0, respectively, k1 and k2 are the rate constants for two different exponential decays, and q is the fraction of singlet excited pyrene that decays with the slower rate constant k1. Rate constants for quenching kQ of pyrene fluorescence were obtained by the equation

k ) k0 + kQ [Q]

where k and k0 are the measured rate of decay of excited pyrene with and without quencher, respectively, and [Q] is the quencher concentration. Results and Discussion The kinetics of the esterification reaction of MAS copolymer depends on the structure of the alcohol used.22,24 For linear alcohols the degree of conversion is 0.70-0.85, and for substituted alcohols these values decrease to 0.40-0.50. Therefore, the resultant copolymer is a mixture of repetitive units carrying one or two acidic groups. The degree of polymerization is supposed to be the same for all copolymers. This assumption implies that the MAS copolymer does not degrade with the esterification and/or neutralization reactions and that the change in molecular weight is due only to the esterification reaction. The solubilization of linear MAS-n copolymers in water occurs after a certain number of carboxylic groups have been neutralized. These values of neutralization degree, R(dis), depend on the size of the attached alkyl chain. Thus, copolymers MAS-n with n ) 1-4 are completely soluble at R ) 0.15, while for n ) 6 or higher, this dissolution degree of neutralization increases to 0.23. On the other hand, the copolymers carrying a branched alkyl side chain are soluble over the whole range of pH. Fluorescence of Pyrene in MAS-n Aqueous Solutions. The ratio of the intensities of the vibronic bands of the pyrene fluorescence I3/I1 is widely used as a measure of the polarity in microheterogeneous systems.17,25,26 The change of this ratio has been used to monitor the conformational transition in PMA and maleic copolymers.7,13,27,28 Figure 1 shows the values of I3/I1 as a function of pH for MAS and MAS-1. For comparison we have included the results obtained for PMA. The curves obtained for PMA and MAS are similar to those reported previously, and the shape of these titration curves has been explained as follows. At low pH, where these polymers adopt a compact conformation, pyrene senses a hydrophobic environment. As the neutralization degree increases the macromolecule changes its conformation to the random-coil form. This transition brings about a decrease in the ratio I3/I1 as the environment becomes more polar. The pH of the transition is taken at the mid-point of these plots, and correspond to R ) 0.20 and 0.33, for PMA and MAS, respectively. These values are in good agreement

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Figure 2. Fluorescence intensity ratio III/I of pyrene (2 µM) as a function of pH in MAS-n (1 g/L) solution. (a) 2 MAS-1, 9 MAS-2, b MAS-3, 1 MAS-4. (b) b MAS-6, 9 MAS-8, 2 MAS-10, [ MAS-12.

with those obtained previously by fluorescence probing and other techniques.7,8,14 On the other hand, the MAS-n copolymers show a completely different behavior. For copolymers with n e 6 the ratio I3/I1 increases with increasing pH until it reaches a maximum at pH ) 6-7.5. For MAS-n with longer alkyl side chains (n ) 8-12) I3/I1 remains constant over the whole range of pH, where these polyelectrolytes are soluble (see Figure 2). From Table 1 it can be observed that the initial values of I3/I1 increase with increasing length of the alkyl group attached to the side chain. These results indicate that the compact form becomes more hydrophobic with increasing alkyl side chain size, and practically levels off at n ) 8. The increase of I3/I1 with increasing pH can be rationalized in terms of pyrene distribution between two different non polar media. Neutralization of carboxylic groups makes one of these environments more polar and induces pyrene to migrate into the more hydrophobic microdomain. The latter is formed, even at high values of R, by short range interactions

TABLE 1: Steady-State and Time-Resolved Fluorescence Probing of the Conformational Transition of MAS-n Copolymers copolymers MAS MAS-1 MAS-2 MAS-3 MAS-3s MAS-4 MAS-4s MAS-6 MAS-8 MAS-10 MAS-12 PMA

pH(I)

pH(τ)

RT

(I3/I1)min

(I3/I1)max

4.1 6.3 6.0 6.2 4.4 6.5 4.2 7.7

4.5 6.5 6.8 6.5 4.8 6.7 4.7 7.5

0.37 0.49 0.52 0.58 0.27 0.36 0.15 0.35

4.2

4.8

0.09

0.72 0.64 0.64 0.67 0.68 0.68 0.69 0.88 0.98 1.00 1.05 0.62

0.72 0.75 0.71 0.72 0.70 0.81 0.70 0.94 1.01 1.00 1.07 0.85

of phenyl and alkyl groups located in neighboring units of the polymer. This assumption is confirmed by the time-resolved measurements described below.

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Figure 3. Decays of pyrene fluorescence in MAS-n solutions. (A) MAS-6, (B) MAS-8, (C) MAS-12.

Figure 4. Plot of τ1 (b) and q (9), from the double-exponential fit of fluorescence decay of pyrene in MAS-1 solution, as a function of pH.

Fluorescence Decay of Pyrene. Typical fluorescence decays of pyrene in aerated MAS-n aqueous solutions are shown in Figure 3. In spite of the low pyrene concentration (2 µM) and the high value of the ratio [chain]/[pyrene], used in these experiments, all experimental decays were fitted to a doubleexponential function, independent of pH and MAS-n structure. A biexponential decay is indicative that the fluorescent probe is partitioned between two different environments characterized by the rate constants k1 and k2. Figure 4 shows a plot of the longest lifetime, τ1 (k1 reciprocal value) and the fraction of pyrene decaying with τ1, q, as a function of pH for MAS-1. The position of the sharp changes of both parameters is the same, and quite close to that found in steady-state measurements of the ratio I3/I1. Similar results were found for MAS-n with n e 6. In the case of copolymers with longer alkyl side chains neither of these parameters change with pH. The pH and neutralization degrees at which this transition occurs are given in Table 1. The data indicate that attachment of a linear alkyl

side chain to MAS stabilizes the compact form, increasing the neutralization degree required for the conformational transition takes place. The magnitude of this effect depend on the size and structure of the side chain. Thus, in MAS-n copolymers with n ) 1-3, 50-60% of the carboxyl groups must be ionized before the polymer expand; for n ) 4-6 the transition occurs at R near 0.35, and for the longer alkyl chains there is not transition. In copolymers carrying branched alkyl groups the degree of neutralization for the transition is even lower than that measured in MAS. This follows from a repulsion enhancement that originates in the steric hindrance produced by the hydrocarbon chains. These results are in line with those obtained for copolymers of maleic anhydride and alkyl vinyl ethers.2,5,7 In these systems transitions from a compact to a random coil conformation were observed for the butyl, pentyl, hexyl, and octyl copolymers; the methyl and ethyl copolymers remain in the extended form over the whole range of pH; and the decyl and hexadecyl copolymers

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TABLE 2: Fluorescence Parameters of Pyrene in MAS-n Copolymer Solutions at r ) 0.25a copolymer

I3/I1

τ1

τ2

q

MAS-0 MAS-1 MAS-2 MAS-3 MAS-3s MAS-4 MAS-4s MAS-6 MAS-8 MAS-10 MAS-12

0.72 0.64 0.66 0.68 0.68 0.68 0.69 0.88 0.98 1.00 1.05

292 312 316 285 273 303 308 318 213 229 202

46 91 81 52 61 62 116 82 49 70 62

0.82 0.88 0.79 0.78 0.89 0.82 0.69 0.82 0.32 0.50 0.61

a Emission decays were fitted to the function I(t) ) qexp(-t/τ1) + (1-q)exp(-t/τ2). [pyrene] ) 2 × 10-6 M, [chain] ) 1 × 10-3 M.

TABLE 3: Fluorescence Parameters of Pyrene in MAS-n Copolymer Solutions at r ) 0.60a copolymer

I3/I1

τ1

τ2

q

MAS-0 MAS-1 MAS-2 MAS-3 MAS-3s MAS-4 MAS-4s MAS-6 MAS-8 MAS-10 MAS-12

0.64 0.72 0.68 0.65 0.63 0.80 0.64 0.94 1.01 1.01 1.08

132 217 217 195 126 271 128 320 220 228 212

25 35 52 55 22 53 17 120 56 57 61

0.45 0.59 0.59 0.60 0.59 0.74 0.61 0.80 0.39 0.46 0.63

a Emission decays were fitted to the function I(t) ) qexp(-t/τ1) + (1-q)exp(-t/τ2). [pyrene] ) 2 × 10-6 M, [chain] ) 1 × 10-5 M.

adopt a compact form at all values of R. However, in the MAS-n copolymers the nature of the conformational transition seems to be different. This follows from the analysis of the data obtained by fluorescence probing. Figures 1-3, show that the pH-induced transition is characterized by a decrease of fluorescence intensity, τ1, τ2, and q and by an increase of I3/I1. It is noteworthy that the final values of the emission properties of pyrene are quite different from those measured in water under the same experimental conditions, i.e., τ1 ) 134 ( 6, τ2 ) 29 ( 10 ns, q ) 0.90, and I3/I1 ) 0.57 ( 0.02. This indicates that, in aqueous solutions of MAS-n copolymers, pyrene is sensing two different environments provided by hydrophobic coiling of the macromolecule. These results cannot be explained by a compact to random coil transition where pyrene is assumed to be either associated to the polymer coil or dissolved in the aqueous phase. Instead, we must consider a model where both the initial and final conformations of the polymer contains two different microdomains, between which pyrene is distributed. Neutralization of the chain modifies its conformation, affecting the structure of these hydrophobic microdomains, and the distribution of pyrene. Two different environments were also observed by Strauss and Schlesinger in copolymers of maleic acid and alkyl vinyl ethers tagged with dansyl groups.5 To try to understand the conformational transition of the MAS-n copolymers we have intended the characterization of the initial and final states of these copolymers by steady-state and time-resolved fluorescence experiments. Effect of Side Chain Length on Micelle Structure at Low Neutralization Degree. The photophysical parameters of pyrene were measured at two different neutralization degrees and the results are shown in Tables 2 and 3. The values of τ1, τ2, and q indicate that, in the compact and random coil forms, pyrene is partitioned between two different hydrophobic medium. At

low values of R the polymer is in the compact form, and most of the probe decays with the longest lifetime τ1. The data on Table 2 indicate that the ratio I3/I1 and τ1 depends on the side chain length. Figure 5 shows a plot of τ1 and I3/I1 against the number of carbon atoms in the alkyl side chain. It can be seen that when a side chain length of eight methylene groups is attained, the lifetime drops abruptly from ∼300 to ∼210 ns, and the ratio I3/I1 reaches its maximum value. These unexpected results can be explained by assuming a change in the composition of the intramolecular micelle for the longer alkyl chains. It has been proposed that the compact structure of MAS, formed in the range of low pH, is stabilized by hydrophobic interactions between the phenyl groups, which are buried in this intramolecular micelle.8 However, a comparison of the value of I3/I1 (0.72) measured at low R, with those measured in benzene solvents (0.81-0.96), indicates that pyrene is sensing a more polar medium. Addition of a methyl group to the repetitive unit must disrupt the compact structure, as reflected by a decrease of the ratio I3/I1. For MAS-n copolymers with short side chains (n e 6) the values of I3/I1 increase slightly but remain beneath the MAS value, indicating that the intramolecular micelle is still formed mainly by phenyl groups with just a small amount of alkyl chains. This is a consequence of the absence of contact between the alkyl chains caused by the restraints imposed by the polymer chains.2 The lifetime τ1 is about 300 ns for all of them. As the side chain length increases the alkyl group penetrates deeper into the core of the intramolecular micelle disrupting the benzene-like structure, until a critical length is reached. At this point the interaction between the hydrocarbons chains is strong enough to induce a change in the composition of the micellar structure which becomes more hydrocarbon-like. In other words, for the longer alkyl groups the microdomain is formed mainly by hydrocarbon chains, while for short side chains the micelle is formed almost exclusively by the phenyl groups. This drastic change of composition should be reflected on the I3/I1 values because this ratio in alkane solvents is higher than in benzene solvents. Figure 5 shows that, for MAS-n copolymers, the I3/I1 increases from 0.64 to 1.05 as the alkyl side chain goes from methyl to dodecyl. This maximum value is quite similar to those measured in other copolymers of maleic acid, where the intramolecular micelle is formed exclusively by hydrophobic interactions of long alkyl chains.7,13 On the other hand, the polarity change induced by the transition from a phenyl to a alkane structure, brings about an increase of the oxygen concentration into the micellar core. In aerated solutions the fluorescence lifetime of pyrene is determined by oxygen quenching, which has a rate given by kQ[O2]. The quenching rate constant kQ should be the same for all copolymers because quenching by oxygen is a diffusion-controlled process. Thus, the ratio of τ1 obtained in MAS and MAS-12 is a measure of the relative oxygen solubilities in the micelles formed by these copolymers, which should be similar to those measured in benzene and a hydrocarbon solvent. From the data of Table 2 a ratio of τ1 equal to 1.46 is obtained which compares quite well with 1.62 obtained for the ratio of oxygen concentration in hexane and benzene, i.e., 3.09 and 1.91 mM, respectively.29 Thus, both steady-state and time-resolved results indicate that, in MAS-n copolymers, an abrupt change of micellar structure is produced when a critical side chain length is reached. Effect of Side Chain Length on Micelle Structure at High Neutralization Degree. At high values of R the effect of the side chain size on the microdomain structure is not so clear. In MAS, the low values of I3/I1 and τ1 (identical to that determined in water), suggest that pyrene has been expelled to the aqueous

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Figure 5. Plot of the longest lifetime (b) and the ratio III/I (9) for MAS-n copolymers as a function of the side chain length.

phase. However, 65% of the emission is decaying with τ2 ) 25 ns, which is similar to the lifetime of excimer-forming pyrene in water. These results indicate that most of pyrene is still confined by the polymer in an aqueous microdomain. The same behavior is observed for those MAS-n with branched alkyl side chains. The values of I3/I1, τ1, and τ2, obtained for copolymers with linear alkyl groups, indicate that pyrene is also completely associated to the polymer, but partitioned between two hydrophobic environments. The data of Table 2 shows that I3/I1 and τ1 increases with increasing size of the alkyl group, while τ2 remains almost constant from ethyl to dodecyl. This means that, in the microdomain characterized by τ1, there is an important contribution of the alkyl chains to the hydrophobic bonding that maintain the polymer in this form. At the same time the fraction of pyrene located in this environment increases from 0.59 (MAS-1) to 0.80 (MAS-6), as it would be expected for a partitioning of pyrene between a hydrophobic and a polar medium. For copolymers with the longer alkyl chains there is no change in any of these parameters, indicating that the micellar structure is not affected by the increasing electrostatic repulsion. Thus, the conformational transition monitored by pyrene in aqueous solutions of MAS-n copolymers can be explained as follows. At low values of R pyrene is hosted by the polymer in two environments that differ in composition and polarity. Neutralization of the polymer chain produces an increase of the electrostatic repulsion, and a transition from the compact to the random coil conformation of the polymer. This change of configuration affects both environments and the distribution of pyrene. In the first stages pyrene moves to the environment with a higher content of hydrocarbon chains, as reflected by the increase on I3/I1, and decrease of τ1 and q. Further neutralization of the polymer raises the polarity of this environment, and the ratio I3/I1 decreases again. For butyl and hexyl the hydrocarbon interaction restrains the expansion of this polymer region and I3/I1 remains high. Finally, for MAS-n with n g 8 the interaction between the alkyl chains is so strong that the neutralization does not affect the configuration of the polymer chain. Quenching of Pyrene Fluorescence. As discussed above pyrene is partitioned between two environments in different sections of the polymer chain. In order to get an insight into

TABLE 4: Quenching Rate Constants of Pyrene Fluorescence in Aqueous Solution of MAS-n Copolymersa Tl+

CH3NO2 copolymers MAS-0 MAS-1 MAS-2 MAS-3 MAS-3s MAS-4 MAS-4s MAS-6 MAS-8 MAS-10 MAS-12 water PMA PA-18 a

s-1.

k1

k2

8.2 1.7 2.2 3.0 60 2.8 75 2.8 5.2 6.1 9.9 95 1.5 1.2

140 130 40 19 245 6 280

k1 12 11 13 47 15 53 16 10 13 22 62