Surfactant Complexes as Catalysts for

Feb 10, 2017 - Eduardo H. Wanderlind , Catiunaiara R. Bittencourt , Alex M. Manfredi , Adriana P. Gerola , Bruno S. Souza , Haidi D. Fiedler , Faruk N...
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Supramolecular Polymer/Surfactant Complexes as Catalysts for Phosphate Transfer Reactions Adriana P. Gerola, Eduardo H. Wanderlind, Yasmin S. Gomes, Luciano A. Giusti, Luis García-Río, René A Nome, Anthony J. Kirby, Haidi D Fiedler, and Faruk Nome ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b00097 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 10, 2017

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Supramolecular Polymer/Surfactant Complexes as Catalysts for Phosphate Transfer Reactions Adriana P. Gerola,† Eduardo H. Wanderlind,† Yasmin S. Gomes,† Luciano A. Giusti,† Luis García Río,‡ René A. Nome,# Anthony J. Kirby,§ Haidi D. Fiedler,† Faruk Nome*,† †

INCT-Catálise, Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil ‡

Departamento de Química Física, Centro de Investigación en Química Biológica y Materiales Moleculares, Universidad de Santiago de Compostela 15782, Santiago de Compostela, Spain

#

Instituto de Química, Universidade Estadual de Campinas, Campinas 13083-970, SP, Brazil

§

Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK *e-mail: [email protected]

KEYWORDS: Supramolecular Complex, Catalysis, Artificial Enzymes, Polymer, Surfactant, Phosphate Ester. ABSTRACT: Designing artificial enzymes with tailored molecular interactions between substrate and active site is of major intellectual and practical significance. We report the improved catalytic efficiency of a supramolecular polymer/surfactant complex comprised of PAIM– a polyacrylic acid derivative with imidazole groups attached to the polymer by amide bonds and the cationic surfactant cetyltrimethylammonium bromide (CTAB). Supramolecular complex formation, at concentrations below the respective cmc's, provides convenient hydrophobic pockets for the reactants close to the multiple catalytic centers; where imidazole and carboxylate groups act as nucleophiles for the degradation of a model phosphate triester, delivering the highly efficient performance of the supramolecular catalysts. Catalytic effects are of the order of thousands for nucleophilic catalysis, and higher by two orders of magnitude for the supramolecular polymer/surfactant complex at pH 9. The reported supramolecular catalytic complexes allow important changes in polarity and, given the presence of functional groups common to a variety of hydrolytic enzymes, could be of general applicability in such reactions.

Introduction Enzymes, as the fundamental biological catalysts, are a source of inspiration for the development of artificial catalytic systems, ranging from simple molecules to complex macromolecular systems such as dendrimers, cyclic hosts, surfactants and polymers.1-15 The chemistry of functional polymeric structures is highly promising, offering well-organized structures with electrostatic and hydrophobic forces designed to allow the incorporation of organic substrates and ionic reagents. Such structures should include potential nanoreactors of high efficiency based on the principles of enzyme mimetics, with active sites close to hydrophobic pockets.16,17 Polymer/surfactant complexes allow the formation of functional supramolecular assemblies which have been widely applied in the development of products for medical applications, cosmetics, foods, detergency, textiles, paints and catalytic systems.18-24 Polymer/surfactant complexation can occur at concentration levels below the critical micelle concentration (cmc) of the surfactant,

thus minimizing the amount of surfactant required for many applications and reducing process costs and environmental impact. Such complexation is accomplished through the interaction of oppositely charged surfactant and polymer components to form charged aggregates maintained by electrostatic interactions. Additional aggregate stability can be reinforced by hydrophobic interactions between surfactant and polymer alkyl chains.25 The organization achieved on complex formation could result in a catalytic effect greater than the sum of the individual polymer and surfactant parts. We recently reported the synthesis and characterization of PAIM, a novel polymer prepared by functionalization of polyacrylic acid, with imidazole moieties linked to the polymer backbone via amide bonds and with carboxyl groups as nearest neighbors (Scheme 1).26 PAIM possesses basic structural features that mimic natural proteins and was shown to catalyze the cleavage of a variety of activated esters. Thus the spontaneous hydrolysis of the model diethyl 2,4-dinitrophenyl phosphate ester (DEDNPP) in the presence of PAIM was 1.1x104 times faster than the uncatalyzed reaction. However, the polymeric catalyst did

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Scheme 1. Functionalization of polyacrylic acid with imidazole groups.

We speculated that PAIM might be improved as a catalyst by the formation of supramolecular polymer/surfactant complexes, which could form new organized domains to promote the incorporation of reagents close to the reactive groups.27 We report the formation of supramolecular complexes of PAIM with the cationic surfactant cetyltrimethylammonium bromide (CTAB). The PAIM/CTAB species distribution, and the size of the supramolecular aggregates were characterized as functions of pH by conductivity, dynamic light scattering and zeta potential measurements. We find significantly enhanced catalytic effects for the supramolecular PAIM/CTAB complexes, which behave as artificial enzymes in dephosphorylation reactions of the model phosphate triester, DEDNPP.

Results and discussion Zeta potential evaluation of PAIM in the absence and presence of CTAB. pKas of 4.02 and 6.7 for the carboxyl and imidazole groups, respectively, were determined by potentiometric titration, with results consistent with a 1:1 ratio of carboxyl to imidazole moieties (Figure S2). The distribution of the different ionic species (Scheme 2) as a function of pH is shown in Figure 1A.

ly positive zeta potential of the polymer decreases with increasing pH due to the charge neutralization promoted by the dissociation of the carboxylic groups, with decreasing [PAIM+] and increasing concentration of the zwitterionic form. The zeta potential of the polymer was zero at pH = 6.18. At pH > 6.18, the zeta potential becomes negative, reaching a plateau at pH 9.0 as a result of the formation of the negatively charged PAIM– polymer, with neutral imidazoles and carboxylate anions. At pH < 4.02 the PAIM+ species predominates, while the the zwitterionic PAIM± species is present in greater proportion in the pH range 4.02 to 6.7. The data illustrated in Figure 1B show that at pH 3 the polymer is mostly PAIM+; at pH 6.0 a mixture of species containing PAIM± (83%) and 17% of PAIM–; at pH 7.0 67% of PAIM– and 33% of PAIM±, while at pH 9 only the negatively charged PAIM– species is present in solution. + 1.0 PAIM

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Figure 1. (A) Species distribution for PAIM and (B) zeta potential variation of PAIM (2x10−4 mol L−1), as a function of pH, at 25°C.

Scheme 2. Species of PAIM as a function of pH, at 25°C. The zeta potential measurements for PAIM (2x10−4 mol L ) as a function of pH (Figure 1B) reflect the mole fractions of the different ionic species, which in turn depend on the pKa values. In the pH region from 3 to 6 the initial−1

Upon addition of CTAB at pH 9.0, the zeta potential of the supramolecular PAIM/CTAB aggregates increases with the surfactant concentration (Figure 2) as the aggregates become positively charged. Note that the PAIM/CTAB aggregate becomes electrically neutral at a CTAB concentration of about 2.4x10−4 mol L−1, close to the PAIM concentration employed. For [CTAB] > 2.4x10−4 mol L−1, the zeta potential becomes positive, reaching a maximum of about +30 mV above the cmc (ca. 9.6x10−4 mol L−1) of the cationic surfactant. The saturation appears to be related to the formation of aggregates containing CTAB micelles and PAIM (see below).

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Figure 2. Zeta potential for supramolecular PAIM/CTAB aggregate at pH 9.0, as a function of CTAB concentration. 25.0 ºC. [PAIM]= 2x10−4 mol L−1. Characterization of supramolecular PAIM/CTAB complexes. The formation and average size of PAIM/CTAB aggregates were investigated further at different pH values by conductometric titration and dynamic light scattering. Figure 3 shows the changes in conductivity observed upon addition of CTAB to aqueous solutions of PAIM (2x10−4 mol L−1) at pH 6, 7 and 9. The conductivity profile of CTAB in the absence of polymer shows a single break at 9.6x10−4 mol L−1, which corresponds to the cmc of CTAB in water (see Figure S3).28,29 In the presence of PAIM at pH values 6, 7 and 9 the conductivity as a function of added surfactant showed two breaks at concentrations below the cmc (Figures 3A, 3C and 3E, Figure S4). The observed breaks in the conductivity versus [CTAB] plots are indicative of the interaction of pre-micellar aggregates and PAIM. The first break is usually defined as the polymer/surfactant Critical Aggregation Concentration (CAC), which is well below the free surfactant cmc. Surfactant concentration values corresponding to the CAC, second break (SB) and the cmc are shown in Table 1 for PAIM/CTAB systems at pH 6, 7 and 9. At pH 3, a break is observed at 2.60x10−4 mol L−1, in a region similar to that of the second break observed at other pH values, and the cmc is observed at 8.8x10−4 mol L−1, slightly smaller than the cmc in pure water, due most probably to the expected salt effect. Table 1. Values of Critical Aggregation Concentration (CAC), Second Break (SB) and Critical Micellar Concentration (cmc) of PAIM/CTAB aggregates, at 25 ºC and different pHs. pH CAC SB cmc (10−4 mol L−1) (10−4 mol L−1) (10−4 mol L−1) Water 9.5 3.0 3.30 8.8 6.0 0.36 2.61 8.8 7.0 0.92 3.80 11.8 9.0 0.46 3.30 9.8 The species distribution and zeta potential of PAIM as a function of pH (Figure 1) can be associated with the con-

ductivity profiles shown in Figure 3. Between pH 6.0 and 7.0, the zeta potential of PAIM is close to zero and the PAIM polymer is zwitterionic, with multiple positive and negative charges. As a consequence, supramolecular polymer/surfactant complexation must be promoted by the interaction of negatively charged carboxylate moieties with surfactant headgroups, with additional van der Waals interactions between hydrocarbon polymer chains and the hydrophobic tail of CTAB. The most favorable situation occurs at pH = 9, where the PAIM polymer shows a negative zeta potential and favors complex formation via both (i) hydrophobic interactions of the surfactant tail with the polymer backbone; and (ii) electrostatic interactions between carboxylate and the CTA+ headgroups, perhaps reinforced by neutral imidazole groups stabilizing the supramolecular complex via πinteractions. At pH 3, the main driving force for polymer/surfactant aggregation should be the interactions between the hydrophobic hydrocarbon chains of polymer and surfactant, reducing the contact area with water. The change observed in the conductivity plot (Table 1), is reinforced by the increase in pyrene excimer formation (see below). Light scattering measurements were employed to obtain hydrodynamic radii (aggregate “particle size” values reported in Figure 3), which formally correspond to a spherical model having the same diffusion coefficient as the PAIM/CTAB aggregates. The particle size numberweighted distribution determined as a function of the surfactant concentrations at pH 6 is shown in Figure 3B. At [CTAB] < CAC, the interactions between individual surfactant molecules and PAIM are limited and we see no significant aggregate in solution, a result which indicates that the surfactants are predominantly present as monomers in the bulk.30 Formation of monodisperse aggregates (size about 135 nm) was observed for [CTAB] > CAC, consistent with binding of CTAB to specific sites in PAIM. This specific binding is expected to be promoted by electrostatic interaction between surfactant headgroups and PAIM carboxylate groups, together with some additional hydrophobic contribution.31 At [CTAB] > SB, there is an appreciable interaction of CTAB molecules with PAIM due to cooperative binding, promoted by earlier surfactant binding to PAIM, which probably causes unwinding of the PAIM structure, allowing the formation of micellelike structures of surfactants in the supramolecular aggregate. For [CTAB] > cmc, the particle size decreases with increasing [CTAB], a result consistent with molecular reorganization of the aggregate and formation of an equilibrium mixture with CTAB micelles containing PAIM molecules. A structural model of the PAIM–/CTA+ interaction is shown in Scheme 3. The PAIM/CTAB system at pH 7 showed a similar profile to that at pH 6 with formation of monodisperse particles at [CTAB] > CAC (Figure 3D). The particle size increased as a function of CTAB concentration up to the cmc, probably reflecting the fact that at pH 7 the mole fraction of imidazolium groups is considerably smaller than at pH 6, decreasing the electrostatic repulsion.

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Figure 3. Specific conductance for CTAB and PAIM solutions at (A) pH 6 (C), pH 7 and (E) pH 9. Diameter of PAIM/CTAB complex at (B) pH 6, (D) pH 7 and (F) pH 9. The measurements were performed at 25.0 ºC and [PAIM]= 2x10−4 mol L−1. At pH 9, PAIM imidazole groups are neutral and carboxylate groups anionic (see Figure 1). Light scattering profiles for PAIM/CTAB complexation indicate larger particle sizes (Figure 3F) at pH 9 than at lower pH. Specifically, for CTAB concentration close to 2.4x10−4 mol L−1, the solution becomes slightly opaque (not shown). Nevertheless, multiple light scattering was not observed, and thus concentration-dependent intensity autocorrelation times and diffusion coefficients could be interpreted in terms of varying particle sizes via the Stokes-Einstein relation. At this CTAB concentration particle sizes of about 900 nm were obtained. As shown in Figure 2, at

this CTAB concentration the zeta potential is close to zero due to charge neutralization of the carboxylate groups by the added surfactant. Thus, PAIM–/CTA+ interactions result in increased hydrophobicity and a consequent increase in the particle size. Upon further increase of CTAB concentration at pH 9, the PAIM/CTAB solution becomes fully translucent again, and the light scattering measurements show decreasing particle size, with a plateau at surfactant concentrations near the cmc. As shown in Figure 2, at pH 9 the positive charge increases and the particles become more hydrophilic upon further increase of CTAB concentration.

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dition of CTAB in the absence of polymer showed a decrease of the II/IIII ratio only for surfactant concentrations above the cmc of CTAB: the observed decrease is a strong indication of the expected decrease of polarity which occurs upon solubilization of pyrene in the cationic micelle. As expected aqueous CTAB solutions with concentrations of surfactant below the cmc showed intensities consistent with an aqueous environment. Addition of surfactant to PAIM solutions shows decreases in the II/IIII intensity ratios at CTAB concentrations well below the cmc, indicating cooperative binding of surfactant and polymer, with pyrene incorporated into hydrophobic sites in the supramolecular PAIM/CTAB aggregates.34 1.6 (A)

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Scheme 3. Structural model of PAIM–/CTA+ complexation. The cartoon shows two steps in the modification of the PAIM structure, first by electrostatic interactions and subsequently by hydrophobic interaction.

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In summary, the size of the supramolecular PAIM/CTAB complex depends on both pH and surfactant concentration. For example, at CTAB = 2.4x10-4 mol L−1 the complex size was 136 nm at pH 6, 182 nm at pH 7 and 887 nm at pH 9 (Figure S5). This indicates the preferential formation of different supramolecular aggregates. Specifically, the PAIM± species predominates at pH 6, with aggregate formation depending on the balance of attractive and repulsive interactions between CTAB and PAIM molecules. Attractive interactions (van der Waals or electrostatic) favor the formation of supramolecular complexes, while charge repulsion limits the aggregate size to relatively small particles.32 On the other hand, the aggregation profile at pH 9 is favored by electrostatic interactions of the cationic surfactant with the anionic polymer backbone. This allows the formation of larger aggregates, especially in conditions where charge neutralization occurs, and the particles become more hydrophobic.33 The polarity of the microenvironment of the PAIM/CTAB supramolecular complex was investigated using pyrene as a fluorescent probe.31 Figure 4A shows the ratios of the intensities of the first to the third vibronic band (II/IIII) as a function of [CTAB], in the presence and absence of PAIM. In aqueous solutions in the presence and absence of PAIM the ratios of intensities are basically identical, indicating a polarity similar to an aqueous environment. Ad-

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Figure 4. (A) Pyrene II/IIII intensity ratio and (B) Pyrene fluorescence intensity ratio of the excimer to monomer (IEx/IM), as a function in both cases of [CTAB] in aqueous solutions in the absence and presence of PAIM at pH 3, 6 and 9 (λexc= 334 nm, [Pyrene] = 5x10−7 mol L−1. [PAIM] = 2x10−4 mol L−1). The cooperative formation of the supramolecular PAIM/CTAB complex was shown to be pH-dependent. In fact, the surfactant concentration needed to observe a decrease in polarity depends on pH and the changes in the II/IIII intensity ratios could be used to measure the binding constants of CTAB monomers to the PAIM polymer as a function of pH. The solid lines in Figure 4A correspond to the fitting of the experimental data with nor-

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Dephosphorylation reaction: Effect of Supramolecular Aggregates. Kinetic studies of the dephosphorylation of diethyl 2,4-dinitrophenyl phosphate (DEDNPP) were performed using UV-visible absorption spectroscopy, following the appearance of 2,4-dinitrophenolate. The rate-constant surfactant-concentration profiles for the reaction of the phosphate triester shown in Figure 5 illustrate the catalytic effects promoted by the formation of different supramolecular PAIM/CTAB aggregates, at different pH values, on the dephosphorylation reaction.

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mal Langmuir binding isotherms (equation and fitting procedures are given in the supplementary information) and allowed us to calculate values of the binding constants of CTAB to PAIM polymer of 1.5 x104 L mol-1 at pH 3, 3.5x104 L mol−1 at pH 6 and 5.9x104 L mol-1 at pH 9, showing a significant increase in binding as a function of pH. Microscopic polarities of the supramolecular PAIM/CTAB aggregates were observed to be pHindependent. At pH 9 and [CTAB] > 2x10-4 mol L-1, the supramolecular aggregate polarity was equivalent to a mixture containing 30% water/ethanol (Figure S6). Whereas at pH 6, the polarity of the pyrene solubilization sites are comparable to a 35% water/ethanol mixture when [CTAB] > 4x10-4 mol L-1. Similarly, at pH 3 and [CTAB] > 8x10-4 mol L-1 the microscopic polarity is equivalent to 30% water/ethanol. These results clearly indicate that the microenvironments of the supramolecular polymer/surfactant aggregates are somewhat hydrophobic. It is interesting that the increase in local concentration of pyrene in the surfactant/polymer complex allows the formation of a pyrene excimer at concentrations well below the cmc of the cationic CTAB surfactant. In fact, the relative concentrations of pyrene excimer and monomer in solution are reflected in the ratio of the fluorescence intensity of the excimer to that of the monomer (IEx/IM ratio in Figure 4B), showing the highly efficient local concentration effect promoted by the supramolecular aggregate.35 As can be seen in Figure 4B, in the absence of PAIM, the formation of the excimer, reflected in the IEx/IM ratio, reaches a maximum when [CTAB] = cmc. For higher surfactant concentrations, there is a decrease in the ratio IEx/IM due to a dilution effect related to the increased number of micelles and decreasing amount of pyrene per micelle. In the presence of the PAIM/CTAB supramolecular aggregate, the maximum value of the IEx/IM ratio was observed at [CTAB] < cmc, in the same region as the observed changes in microscopic polarity. Interestingly, at pH 3, the largest excimer formation suggests the proximity of pyrene moieties within favorable hydrophobic pockets, indicating the formation of small aggregates (which could not be detected by DLS) (Figure S7).35,36 The slightly smaller relative amounts of excimer at pH 6 and especially at 9 may be related to the larger size of the aggregates (Figure 3) since the larger volume of the hydrophobic phase in the aggregates reduces the local concentration of pyrene in the supramolecular complex.

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Figure 5. Rate constants for the dephosphorylation of DEDNPP (5.0x10-5 mol L-1) in aqueous solutions containing [PAIM] = 2.0x10-4 mol L-1, as a function of [CTAB] and pH, at 25.0 ºC. It is important to note that addition of CTAB to aqueous solutions containing PAIM (2x10-4 mol L-1) promotes significant increases in the observed rate constants for the degradation of DEDNPP at concentrations well below the cmc of the cationic surfactant, where CTAB micelles are absent and supramolecular PAIM/CTAB aggregates predominate. The effect depends on pH, and kobs values at pH 3 are similar to those observed in the absence of surfactant. At pH values 6 and 7, the catalytic effect can be observed at [CTAB] > CAC (see Figure S8 and Figure 5), where the supramolecular complex starts to be formed by cooperative interactions. Note that the large increase in catalytic effect is observed for [CTAB] > SB, where the formation of large supramolecular PAIM/CTAB aggregates offers significant hydrophobic microenvironments. The experimental data are consistent with the formation of PAIM±/CTAB aggregates at CTAB concentrations close to the break regions determined by conductivity measurements. The largest increase in the rate constant is observed for PAIM– at pH 9, reaching a plateau between [CTAB] 3.0x10-4 mol L-1 and the cmc, consistent with the formation of supramolecular aggregates with increased catalytic activity. The observed increase in rate constant allows us to calculate an equilibrium constant for the association of CTAB and PAIM and formation of the catalytic supramolecular aggregate of 6.0x103 mol-1 L. The kinetic observation of an additional stage (above the cmc), is consistent with the formation of micelles containing the supramolecular aggregates formed by electrostatic interactions between PAIM– and CTA+. Assuming an aggregation number of 6037 the highest concentration of CTAB micelles used in the kinetic experiments is only 1.67x10-5 mol L-1, significantly lower than the concentration of PAIM. The kinetic profiles at pH 6 and 7 show similar increases in the observed rate constants when [CTAB] > 1.5x10-4 mol L-1, behavior consistent with the conductivity experiments and with the presence of hydrophobic pockets. As expected, the increase in kobs

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at pH 7 is larger than at pH 6, due to the larger mole fraction of neutral imidazole groups (Figure 1A), known to act as powerful nucleophiles in dephosphorylation reactions.26 The catalytic effect at pH 9.0 follows closely the changes in zeta potential of the PAIM–/CTA+ complex (Figure 2), and the largest catalytic effects are observed when the supramolecular polymer/surfactant aggregates show positive zeta potential. Since the increase in zeta potential is due to the incorporation of CTAB in the aggregate, there are increases in both charge and hydrophobicity, compared to PAIM in the absence of CTAB. Both factors favor the incorporation of the substrate anion, and thus catalysis by its neutral imidazole groups.

stants were fitted to Equation 1, based on Scheme 4.

       ,± .  ,±  , . ,  , . ,  , . ,    ( 1) The Scheme considers the various paths for dephosphorylation of DEDNPP in aqueous solution (k0, kOH, kw,PAIM± and kw,PAIM−), plus the dephosphorylation catalyzed by the supramolecular aggregates PAIM±/CTAB (kS,PAIM±) and PAIM–/CTAB (kS,PAIM−). In the presence of CTAB the molar fraction of PAIM in the form of a supramolecular aggregate was calculated using the equilibrium constant for the association of CTAB and PAIM of 6.0x103 L mol-1 (see above).

Effect of pH on the Catalytic Efficiency of the Supramolecular Aggregates. Rate constants for the dephosphorylation reaction of DEDNPP in the presence of the PAIM/CTAB complex are shown as a function of pH in Figure 6, with data for the reaction in water included for comparison. The effect of CTAB in aqueous solution at pH 8.5, in the absence of PAIM and using a [CTAB] = 2.0x10-3 mol L-1 is very small, with a first order rate constant of 2.8x10-5 s-1. PAIM/CTAB > cmc PAIM/CTAB < cmc PAIM H2O

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Figure 6. Rate constant versus pH profiles for the dephosphorylation of DEDNPP (5.0x10-5 mol L-1) in aqueous solutions, in the presence of [PAIM]= 2x10-4 mol L-1 without CTAB and in the presence of [CTAB]= 2x10-3 mol L-1 (PAIM > cmc) at 7.5x10-4 mol L−1 (PAIM < cmc), at 25ºC. The solid lines represent the fit to Equation 1. Experimental data for the reaction in water in the presence of PAIM 2x10-4 mol L-1 are included for comparison. The results are consistent with those shown in Figure 5 and show that in the presence of PAIM there are at least two reaction paths in different pH regions, with a small plateau close to pH 5.0 and a larger one at pH > 8. The shapes of the pH profiles are similar in the presence and absence of CTAB. At pH 5.0, the predominant form in solution includes dissociated carboxylate and protonated imidazolium groups in the supramolecular complex, while at pH 8.0, the negatively charged PAIM- is the main species in solution The observed pH profiles of the first order rate con-

In the absence of CTAB, the reaction is accounted for by contributions from the kw,PAIM− and kw,PAIM± terms and the kinetic pKas of 4.04 and 7.0 for the carboxyl and imidazole groups, respectively, which are consistent with those obtained by titration (see above). The similarities between the profiles, in the presence and absence of CTAB, show that the acid dissociation constants of the carboxyl and imidazole moieties are not affected significantly by the formation of the supramolecular complexes. In fact, the kinetic pKas obtained by fitting the kinetic data at [CTAB] above and below the cmc, of 3.85±0.15 and 6.95±0.15, show only minor variations compared with those in water. The contribution of the aqueous phase reaction is relatively unimportant, and literature values were used for k0 and kOH.19,26 And the molar fractions of the different catalytic species of PAIM were initially assumed to be similar to those in the absence of CTAB. However, allowing the acid dissociation constants to vary did not affect results to a large extent, probably because the catalytic sites are close to the surface of the supramolecular aggregates. The kinetic parameters obtained by fitting the experimental data of Figure 6, at [CTAB] above and below the cmc, are given in Table 2.

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Table 2. Rate constants for the dephosphorylation of DEDNPP in water, PAIM and in the presence of PAIM/CTAB, at 25.0 ºC, [PAIM]= 2x10-4 mol-1L-1. Constants DEDNPP k0, s-1 a 8.00x10-6 kOH, Lmol-1 s-1 a 4.20x10-1 ± -1 -1 kw,PAIM , Lmol s 5.01x10-2 − -1 -1 b 9.03x10-2 kw,PAIM , Lmol s ± -1 -1 1.12c (0.37)d kS,PAIM , Lmol s − -1 -1 9.23c (4.70)d kS,PAIM , Lmol s a Rate constants for dephosphorylation of DEDNPP from the literature19. bkw,PAIM− reported for dephosphorylation of DEDNPP, at pH>8.26 cRate constant at [CTAB] = 2x10-3 mol L -1. dRate constant at [CTAB] = 7.5x10-4 mol L -1. The solvent kinetic isotope effect (kH2O/kD2O = 1.0) for the dephosphorylation of DEDNPP at pH 5.0, in the presence of the PAIM± /CTAB indicates that the carboxylate anion (pKa1= 4.02) acts as a nucleophile. The second reaction path, showing the larger catalytic effect, reaches a plateau above pH 8, where both the carboxylate and imidazole groups (pKa2 = 6.7) of PAIM–/CTAB are largely deprotonated (Scheme 2). The solvent isotope effect (kH2O/kD2O) is also 1.0 at pH 9, substantially smaller than the (kH2O/kD2O) =1.59 value reported for the reaction in the absence of surfactant.26 The observed decrease in isotope effect is consistent with nucleophilic attack by the neutral imidazole groups, indicating that the more hydrophobic environment of the supramolecular complex enhances the nucleophilic over the general-base pathways. The lack of catalytic effect at pH < 4 shows that PAIM+ has no active catalytic sites for the dephosphorylation reaction. Comparisons of the rate constants for the dephosphorylation of DEDNPP in the presence of the various PAIM/CTAB supramolecular aggregates with the rate constant (8.00 x 10-6 s−1) for the spontaneous hydrolysis (Table 2), show significant rate effects. Increases from 6.0x104 to 1.42x105-fold are observed for the reaction of the PAIM±/CTA+ aggregate, at concentrations below and above the cmc, respectively. The value obtained below the cmc corresponds to the catalytic effect obtained with supramolecular aggregates described as premicellar CTAB-PAIM complexes in Scheme 3 and, the value obtained at [CTAB]> cmc corresponds to the effect of the system described as PAIM-CTAB micelles complexes in Scheme 3. The observed catalytic effect is attributed to the surfactant carboxylate anions reacting as nucleophiles, combined with a change in microscopic polarity of the supramolecular complex. A considerably larger effect (6.0x105-fold at [CTAB]=7.5x10-4 mol L-1 and 1.16x106-fold at [CTAB] = 2.0x10-3 mol L-1) is observed at pH 9.0, where PAIM–/CTA+ are the predominant species, and the catalytic effect is presumed to be due primarily to neutral imidazole groups. Imidazole is known to be a good nucleophile for phosphate triesters such as DEDNPP.3,38-40 Compared with the second order rate constant kIm = (1.46±0.08)x10-2 L mol−1 s−1 for the reaction of imidazole with DEDNPP (see Figure S9,) kPAIM−− given in Table 2 is faster by a factor of 632. Similar comparison of the second

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order rate constant kAc = (1.49±0.07)x10-4 L mol−1 s−1 for the reaction of acetate with DEDNPP (see Figure S10) shows that the carboxylate nucleophiles in kPAIM± react 7,500-fold faster than acetate ion. Comparing the rate constants kw,PAIM± and kw,PAIM−, with those for the same species in the presence of surfactant (kS,PAIM± and kS,PAIM−) shows that the supramolecular aggregate promoted catalytic effects of 22-fold and 102-fold for the reactions of the PAIM± and PAIM- species, respectively. These catalytic effects are considerably larger than expected for the polymer and surfactants individually. The observed results for these dephosphorylation reactions show increased catalytic efficiencies for both PAIM±/CTA+ and PAIM–/CTA+ supramolecular aggregates. The formation of the major species of the supramolecular PAIM/CTAB complexes increases the reaction rate by factors of 10 (PAIM±/CTA+) and 100 (PAIM–/CTA+) compared to DEDNPP hydrolysis in the presence of PAIM only (kw,PAIM− in Table 2).26 These further increases in kobs are attributed to the incorporation of the reactant triester in close proximity to the active groups in the supramolecular PAIM/CTAB complex. On addition of CTAB the zeta potential of the supramolecular aggregates becomes increasingly positive (Figure 2) as larger aggregates form, with hydrophobic pockets capable of binding the DEDNPP substrate (Scheme 4). The results show the potential of supramolecular polymer/surfactant complexes as catalysts, and highlight the importance of these complexes in the search for new model systems for enzymatic reactions.

Conclusions The formation of supramolecular complexes of a polymer containing carboxylate and imidazole groups as nearest neighbours (PAIM) with the cationic surfactant CTAB, depends markedly on pH. In the low pH region where PAIM± predominates, imidazolium and carboxylate groups promote the formation of supramolecular aggregates with CTAB and the relatively large polymer/surfactant complexes show remarkable catalytic potential, with an up to 1.4x105–fold increase in the rate of spontaneous hydrolysis. At pH 9, the supramolecular PAIM–/CTA+ complex shows even larger catalytic ability in the dephosphorylation reaction, with a 1.16x106-fold increase in the rate of spontaneous hydrolysis of the phosphate triester. The catalytic efficiencies of the different supramolecular aggregates formed between PAIM and CTAB, as a function of pH, are significantly higher than the sum of the individual contributions of PAIM and CTAB in separate solutions. Organization of the supramolecular aggregates provides solvation sites for the reactant triester within reach of the catalytic centers, where nucleophilic groups catalyze its efficient degradation. The rate constants for the reactions of DEDNPP with PAIM– and PAIM± were "Up to" 632-fold and 7,500-fold faster than the second order rate constants for the reaction of the nucleophiles imidazole and acetate, respectively, assuming a single catalytic site per supramolecular aggregate. These high catalytic efficiencies for supramolecular sys-

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tems using very low concentrations of surfactant and polymer, allow the reactions to proceed under much greener conditions. The reported supramolecular catalytic complexes allow important changes in polarity and, with the presence of functional groups common to a variety of hydrolytic enzymes, could be of general purpose in such reactions. We are currently examining reactions of a variety of phosphate diesters, acyl esters and decarboxylation reactions in the presence of the PAIM/CTAB supramolecular catalysts.

Experimental Section Polyacrylic acid (PAA) with molecular weight 1800 g mol-1, 1-(3-aminopropyl)-imidazole, CTAB and buffers (TRIS, BIS-TRIS, citric acid and sodium bicarbonate) were purchased from Sigma and used as received. All other reagents were of the highest purity available. All solutions were prepared from Milli-Q water. The phosphate triester DEDNPP was synthesized by conventional methods using diethyl phosphorochloridate, as described previously.3,41-43 The polymer (PAIM, MM = 3071±350 g mol−1) containing carboxylic and imidazole groups was prepared as previously described from polyacrylic acid and 1-(3-aminopropyl)-imidazole.26 PAIM Characterization. The composition of branched PAIM was assessed by 1H NMR in D2O at 25 °C using a Bruker AC 200 MHz instrument (Bruker Analytische Messtechnik Gmbh, Rheinstetten, Germany). The 1H NMR spectrum of the PAIM polymer in D2O (Figure S1) showed the functionalization of around 50% of the carboxyl groups with imidazole groups.26 Further, potentiometric titration of PAIM was carried out in an automated Metrohm system (713 pH-meter and 765 Dosimat) in a 50 mL thermostated cell under N2. A solution containing 25 mL of 0.023 g of PAIM at ionic strength 0.1 mol L-1 KCl was acidified up to pH 2.4. The solution was titrated with CO2-free KOH (0.086 mol L-1) and the content of imidazole groups found were consistent with the NMR results. Methods Conductometric Titration. Conductance measurements were carried out using a Metrohm model 712 conductometer, using a 50 mL thermostated cell, at 25.0 ± 0.1 ºC. For titrations in the presence of CTAB (6.16x10−3 mol L−1), the initial volume in the cell was 15 mL. Titrations were performed for determination of the cmc of the surfactant; and were performed in solutions of PAIM (2x10−4 mol L−1) at pH 3, 6, 7 and 9 to evaluate the PAIM/CTAB complex formation. Titrations were carried out in the absence of buffer and the solutions were self-buffered with PAIM and the pH adjusted by addition of aqueous NaOH and HCl to aqueous solutions of PAIM. Dynamic Light Scattering and Zeta potential Measurements. All the size and zeta potentials reported were performed in quintuplicate, at 25.0 ºC, by Dynamics Light Scattering (DLS) measurements using a zeta potential analyzer model ZetaPlus, from Brookhaven. Size estimates of PAIM/CTAB aggregates at pH 6, 7 and 9 were performed using 2-[bis(2-hydroxyethyl) amino]-2(hydroxymethyl)-1,3-propanediol (BIS-TRIS) buffer at pH 6 and 7 and 2-amino-2-hydroxymethyl-propane-1,3-diol

(TRIS) buffer at pH 9. All solutions were previously filtered in a Disposable syringe filter (Chromafil®XtraCA45/25). The zeta potential of PAIM (2x10−4 mol L−1) in aqueous solutions as a function of pH were also measured in the presence of adequate buffers: citric acid (pH 3-5), BIS-TRIS (pH 6-7), TRIS (pH 7.5-9) and sodium bicarbonate (pH 9.5-10.5). The zeta potential of PAIM/CTAB complex was also evaluated at pH 9 (Tris buffer), varying the CTAB concentration in the range of 0 to 2x10−3 mol L−1, keeping constant the concentration of PAIM (2x10−4 mol L−1). Pyrene Fluorescence Measurements. Emission spectra of pyrene (5x10−7 mol L−1) were taken on a Cary Eclipse spectrofluorometer (Varian). Excitation was at 334 nm and emissions were recorded in the 350–600 nm wavelength range. The slit-widths for both excitation and emission were fixed at 5 nm. A concentrated surfactant solution was added stepwise to pure water and buffer solutions of PAIM (2.0x10−4 mol L−1) at pH 3, 6 and 9 and emission spectra were recorded. Polarities of micellar and supramolecular microenvironments were evaluated from ratios of vibronic bands I and III at 373 and 393 nm, respectively. Further, the ratio of the excimer (Ex) to the monomer (M) fluorescence intensity was calculated from the 475 nm to 385 nm signal ratio. The polarities of water/ethanol mixtures (v/v) were evaluated using the pyrene scale for comparison with the colloidal systems. Kinetics. Hydrolysis of DEDNPP were followed by monitoring the appearance of 2,4-dinitrophenolate at 400 nm on a diode-array spectrophotometer with a thermostated cell holder maintained at 25.0 ºC. Reactions were started by adding 10.0 µL stock solution of the substrate (0.01 molL−1 of DEDNPP in acetonitrile) in water to 2.0 mL of the aqueous reaction mixture, containing a large excess of the nucleophile, assuring pseudo-first order kinetics for the nucleophilic attack upon the substrate. Solutions were buffered with citric acid (pH 3-5), BIS-TRIS (pH 6-7), TRIS (pH 7.5-9) and sodium bicarbonate (pH 9.5-10.5). The effects of CTAB concentration on the dephosphorylation rates of DEDNPP in the presence of PAIM (2x10−4 mol L-1) were evaluated at pH 3, 6, 7 and 9. Rates of reactions were measured at constant concentrations of PAIM and [CTAB] = 2x10−3 mol L-1. Observed first-order rate constants were calculated using an iterative least-squares program; correlation coefficients were > 0.999 for all kinetic runs. Solvent kinetic isotope effect measurements were performed for PAIM± at pD(pH) 5.0 and for PAIM− at pD(pH) 9.0. Comparisons were made at [PAIM]= 2.0x10−4 mol L−1 and [CTAB] 2.0x10−3 mol L−1. The pD was corrected by the relation pD = pH + 0.4 at 25 °C.44 Acknowledgements. We thank the Brazilian foundations FAPESC, CNPq (INCT-Catalysis) and CAPES and the Ministerio de Economia y Competitividad of Spain (project CTQ2014-55208-P) and Xunta de Galicia (GR 2007/085) for support of this work. Supporting Information Available: Including experimental data and Figures with NMR, conductivity, light scattering and fluorescence data. This material is available free of charge via the Internet at http://pubs.acs.org.

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