ARTICLE pubs.acs.org/Langmuir
Polymers Containing Hydroxamate Groups: Nanoreactors for Hydrolysis of Phosphoryl Esters Renata S. Mello,† Elisa S. Orth,‡ Watson Loh,† Haidi D. Fiedler,‡ and Faruk Nome*,‡ †
Instituto Nacional de Ci^encia e Tecnologia (INCT) de Catalise, Instituto de Química, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6154, CEP 13083-970, Campinas, S~ao Paulo (SP), Brazil ‡ Instituto Nacional de Ci^encia e Tecnologia (INCT) de Catalise, Departamento de Química, Universidade Federal de Santa Catarina (UFSC), CEP 88040-900 Florianopolis, Santa Catarina (SC), Brazil
bS Supporting Information ABSTRACT: A polyhydroxamicalkanoate (PHA) polymer containing the functional groups hydroxamic acid and carboxylic acid with the ability to accelerate dephosphorylation reactions is proposed. The methodology used to prepare this polymer favored the position of the two functional groups next to each other, which allows for the cooperativity between these groups. This cooperative effect has an important role when one wants to mimic enzymes. The catalytic effect promoted by the polymer was evaluated on the cleavage of the bis(2,4-dinitrophenyl) phosphate (BDNPP) and diethyl 2,4-dinitrophenyl phosphate (DEDNPP) esters. Indeed, PHA was very efficient and promiscuous because it increased the rate of both reactions by a factor of up to 106-fold. Isothermal titration calorimetry (ITC) experiments showed that the presence of PHA aids the formation of cetyltrimethylammonium bromide (CTABr) micelles. Thus, the effect of the cationic surfactant CTABr on the dephosphorylation of BDNPP by PHA was also investigated, and it was observed that, when CTABr is added to PHA, the reaction is ca. 15-fold faster compared to the reaction when only PHA is present.
’ INTRODUCTION Natural enzymes are able to accelerate the rate of hydrolysis of phosphate esters, and such reactions are extensively studied because they are involved in metabolic processes that are still not fully understood at a molecular level.1 The catalytic effect of enzymes is impressive (1061020-fold, comparing rate constants for the reactions in the presence and absence of enzymes), and a substantial improvement of our understanding can be attributed to studies of their ability to stabilize the transition state of a reaction (with cooperative effects in the active site) and to a substantial improvement of quantitative theoretical model studies of enzymatic reactions, specially with the hybrid quantum mechanics/molecular mechanics (QM/MM) method and a microscopic approach for studies of electrostatic effects in proteins, which allows for the mapping of energetics and dynamics of enzymatic reactions.1,2 Many models of the active sites of enzymes have been developed to better understand their mechanism of action and its effect, for example, on the hydrolysis of esters. The existing models are relatively small compared to natural enzymes,3,4 although models based on macromolecules have been proposed,5,6 which still show low rate acceleration, turnover, and specificity compared to natural enzymes.7 Klotz et al. introduced the term synzymes for synthetic enzymes when dodecyl groups and methyleneimidazole side chains were attached to a polyethyleneimine framework and the macromolecule was able to catalyze hydrolysis of uncharged nitrophenyl esters.8 Another class of potential polymeric-based catalysts are ionenes, which are cationic polymers with quaternized nitrogen atoms r 2011 American Chemical Society
in the polymer backbone separated by different numbers (x, y) of methylene groups. We reported previously the alkaline hydrolysis of benzoic anhydride6 and 4-nitrophenyl benzoate9 mediated by ionenes functionalized with reactive groups, thus behaving like a nanoreactor. Results provide evidence that functionalized ionenes, such as poly[(dimethyl)-2-hydroxy propanodiyl chloride] (2-OH-33R1), are nearly 24% more catalytically efficient than a nonfunctionalized ionene (poly[(dipropyliminium)-1, 3-propanediyl bromide], 33R3), thus suggesting that the alkoxide group in the ionene backbone participates effectively in the reaction. Rate enhancements observed for these reactions in the presence of ionenes compared to the spontaneous reactions are up to 25-fold.
Although ionenes are interesting catalytic homogeneous nanoreactors, higher rate enhancements are usually desired when projecting enzymatic properties on a macromolecular model. Therefore, in addition to a macromolecular structure, a suitable Received: September 1, 2011 Revised: November 1, 2011 Published: November 07, 2011 15112
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Langmuir Scheme 1
catalyst or model reaction relies on the nucleophilic effectiveness of reactants. It is known that α-nucleophiles, such as hydroxylamine,10,11 hydrazine,12 and hydroxamic acid,3,13,14 which have unshared electron pairs on the atom adjacent to the nucleophilic center, can accelerate dephosphorylation reactions.15,16 Indeed, reactions of benzohydroxamate (BHO) with bis(2,4-dinitrophenyl)phosphate (BDNPP)3 are extremely fast, where observed rate constant enhancements of up to 105-fold have been reported. We are interested in evaluating the reaction of phosphate esters with nucleophilic groups anchored on the backbone of macromolecules, such as polymers. Thus, the polymer polyacrylamide was functionalized with hydroxamate groups along with carboxylate groups, which can additionally assist by general base catalysis, similar to several enzymatic processes. The functionalized polyhydroxamicalkanoate (PHA) polymer was reacted with the diester BDNPP and the triester 2,4-dinitrophenyl diethyl phosphate (DEDNPP) (Scheme 1).
’ EXPERIMENTAL SECTION Materials. The phosphate esters BDNPP and DEDNPP were synthesized as described previously elsewhere by conventional methods using POCl3.17,18 The PHA polymer containing the hydroxamic acid and carboxylic acid groups was prepared as described by Domb and coworkers19 and modified as follows: 5.0 g (3.0 mol) of polyacrylamide (Mw = 1500 g mol1, Scientific Polymer) was dissolved in 20 mL of water and 5.2 g (75.0 mmol) of hydroxylamine chloridrate dissolved in 25 mL of water was added to the polymer solution. After 2 h of reaction, a solution of NaOH (75.0 mmol) was added and the reaction mixture was stirred for 48 h at 25 °C. Then, concentrated HCl was added to the solution until the pH was around 1.0, followed by the addition of methanol, yielding a white precipitate. The mixture was kept in the freezer for ca. 24 h, and the precipitate was then filtered and washed several times with methanol. The precipitate was dried in an oven and then crushed to powder. All other reagents were of the highest purity available and were used as purchased. The polymer was characterized by the mass technique [electrospray ionization (ESI)] and potentiometric titrations. Potentiometric Titration. Potentiometric titrations were carried out in an automated Metrohm system (713 pHmeter and 765 Dosimat) in a 150.0 mL thermostatted cell, under N2 at 25 °C. A solution containing 15 mL of 0.013 M PHA and polyacrylamide (PAA) was acidified with 1.0 mL of 0.1 M HCl and titrated with small increments of 0.1008 M KOH, which was CO2-free. Ionic strength was maintained constant with 0.1 M KCl. The program BEST7 was used to determine the equilibrium constants and also to quantify the percentage of the ionic groups on the polymer.20 Kinetics. Reactions followed spectrophotometrically were started by adding 10.0 μL of stock solution of the substrate (4 103 M BDNPP and 0.01 M DEDNPP in acetonitrile) in water to 3.0 mL of the aqueous reaction mixture, containing a large excess of the nucleophile, assuring strictly first-order kinetics for the initial nucleophilic attack upon the substrate. Solutions were self-buffered with PHA and were prepared by addition of aqueous KOH to aqueous PHA.
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Scheme 2
Reactions were followed by the appearance of 2,4-dinitrophenol (DNP) at 400 nm on a diode-array spectrophotometer with a thermostatted cell holder maintained at 25 °C. Observed first-order rate constants (kobs) were calculated from linear plots of ln(A∞ At) against time for at least 90% of the reaction using an iterative least-squares program; correlation coefficients were >0.999 for all kinetic runs. Mass Spectrometry (MS). ESIMS spectrum were obtained by a mass spectrometer system consisting of a hybrid triple quadrupole/ linear ion-trap mass spectrometer QTrap 3200 (Applied Biosystems/ MDS Sciex, Concord, Canada) coupled to a Harvard Pump 11 Plus (Harvard Apparatus, Holliston, MA) for sample infusion. A sample of 0.1 g/mL PHA in MetOH/H2O (50%) was used for the analysis. Isothermal Titration Calorimetry (ITC). ITC experiments were performed in a VP-ITC (MicroCal, Northampton, MA) isothermal titration calorimeter with a sample cell of 1.43 mL. The surfactant solution was consecutively injected with a gastight syringe that also acts as a stirrer (at 550 rpm). Injection volumes varied between 5 and 10 μL, with an interval between each injection of 600 s. The volume of the cell is kept constant during the experiments because of an overflow of solution, and this is taken into account during calculations of the actual cell surfactant concentrations. The observed enthalpy changes (ΔHobs) during both surfactant dilution (polymer dilution heat effects are negligible) and surfactantpolymer interaction are reported as a function of the final surfactant concentration.
’ RESULTS AND DISCUSSION Synthesis and Characterization. In the synthesis of PHA, the amide groups of PAA are converted to carboxylate and hydroxamate groups, as depicted in Scheme 2, with neighboring reactive groups. Accordingly, the hydroxamate group formed attacks the nearby amide group, leading to a carboxylate group. The process involved in the formation of the carboxylate group was reported by Blodgett and co-workers,21 where it was observed that, in some peptides containing the hydroxamic acid residue, this group attacks, forming tetrahedral intermediates that break down, cleaving the peptide chain. As described in the Experimental Section, the polymer evaluated was obtained after 3 days of reacting hydroxylamine and PAA, although other reaction times were also evaluated and did seem to influence the percentage of functionalization of PAA (i.e., higher reaction time and higher hydroxamate formation). To mimic enzymatic active sites, which are quite specific in their localization in the enzyme environment, we were interested in studying polymers with low/partial functionalization. Nevertheless, their reactivity in dephosphorylation reactions were not 15113
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Table 1. Values of the Dissociation Constants for PHA and Conversion Percentage Calculated Using BEST7
Figure 1. ESIMS () spectrum of PHA.
Figure 2. Potentiometric pH titration curves of (2) 16 g/L PHA and (9) 16 g/L PAA. The line corresponds to the calculated curve obtained using BEST7. Calculated dissociation constants are given in Table 1.
significantly enhanced; therefore, the synthesis methodology was optimized to 3 days and used in all reaction evaluated here. Mass analysis employing ESI was used to characterize PHA and is shown in Figure 1, which shows a molecular weight of 1600 g/mol by the ESI technique. These results are interesting, because they provide evidence that the functionalization of PAA maintains the initial polymer molecular weight. Moreover, the distribution behavior shows a difference of 71 g/mol, which corresponds to the mean value of the molecular weights of the monomers of PHA. Potentiometric titration was employed to determine the degree of the conversion of the synthesized PHA polymer as well as the dissociation constants and the percentage of hydroxamic acid and carboxylic acid groups of the polymer studied. A typical titration curve of 16 g/L PHA (approximately 0.013 M) along with the theoretical fit calculated using the program BEST720 is given in Figure 2. The titration curve for PAA, the nonfunctionalized polymer, is also shown in Figure 2 for comparison. Results show a good consistency between the experimental data and the calculated curve for PHA and provide evidence of the presence of the functional hydroxamic and carboylate groups, while for PAA, there is no titratable groups, other than the highly basic amine from the acrylamide group.
pKa1 (carboxylic acid)
5.95 ( 0.05
pKa2 (hydroxamic acid)
9.46 ( 0.05
percentage of total conversion of PHA to PAA (%)
12.8
percentage of hydroxamic acid groups (%)
59.1
percentage of carboxylic acid groups (%)
40.9
Table 1 presents the calculated constants, which refer to the equilibriums assigned in Scheme 3 and the percentage of the functionalized groups present in the polymer. The degree of the conversion of the synthesized PHA polymer as well as the percentage of hydroxamic acid and carboxylic acid groups present were calculated considering the mass weight and the molar mass of the functional groups on the polymer backbone, combined with the calculated concentration of the species given by the program BEST7. Table 1 shows typical pKa values of 5.95 and 9.46 for the carboxylic acid and hydroxamic acid groups, respectively. Authors22 have reported a pKa value of 4.76 for the acrylic acid, while here, we determined a slightly higher pKa value. Indeed, through potentiomentric titration of poly(carboxylic acid) of several molecular weights, it was observed that the increase in electrostatic repulsion between the charged monomers along the polymer chain has an important role in the acidbase properties of the polyelectrolyte; the decrease of Ka with the increase of the ionization degree was noticed, which was attributed to the presence of vicinal charges that is responsible to make ionization more difficult.22 For the hydroxamic acid group, similar pKa values have been reported; e.g., benzohydroxamic and acetohydroxamic acids have pKa values of 8.88 and 9.39, respectively.23 Results also show a conversion of nearly 13% from PAA to PHA, with a percentage of the functional groups of approximately 5.2 and 7.8% for the carboxylate and hydroxamate groups, respectively. These percentages were calculated considering the concentrations of hydroxamate and carboylate groups that the program BEST7 used and compared to the initial concentration of the polymer titrated. The molecular weight of the PHA polymer is very similar to that of small PAA, because only three monomeric units were effectively modified (ca. 22 units in each polymeric chain). Moreover, the molecular weights of the monomers of PHA (71 g/mol) are not very different from those of the carboxylate (77 g/mol) and hydroxamate (86 g/mol) groups. Insofar, the polymer was prepared with a low conversion from acrylamide groups to hydroxamic and carboxylic groups, thus aiming to mimic an enzymatic active site, by comprising a few functional groups on the polymer backbone. Kinetic Studies with PHA. The effect of PHA in the dephosphorylation reaction of BDNPP and DEDNPP was evaluated, and the pH rate profile in water is presented in Figure 3. Results show that, at higher pH (pH 911), in the presence of the reactive polymer, the reaction rate is accelerated over 103- and 104-fold for BDNPP and DEDNPP, respectively, when compared to the spontaneous hydrolysis. Interestingly, these enhancements are also observed at lower pH (pH 5.58.5), for both substrates. The data in Figure 3 are consistent with Scheme 4 and eq 1, where ko is the rate constant for the spontaneous reaction, kOH is that for the reaction with OH, kN, kM, and kD are rate constants for reactions with the neutral, monoprotonated, and deprotonated forms of PHA, respectively, χCOOH and χCOO correspond to the molar fraction of carboxylic acid groups in their neutral and anionic 15114
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Scheme 3
Figure 3. pH rate profile for the reaction of 0.013 M PHA with (A) (9) BDNPP and (B) (b) DEDNPP at 25 °C. Reaction with H2O is also shown for comparison, with (0) BDNPP18 and (O) DEDNPP (experimental data, see the Supporting Information).
Scheme 4
Table 2. Values of the Parameters Obtained from Fitting Data in Figure 3 with eq 1, for the Reaction of PHA with DEDNPP and BDNPPa BDNPP constants k0 (s1) s )
kN (M1 s1) 1 1
kM (M
s )
1 1
kD (M pKa1b pKa2b
k/[X]
1.9 107
1 1
kOH (M
k/[PHA]
DEDNPP
s )
k/[PHA] 8.0 106
3
2.9 10
0.42
1.8 104
7.0 104
3
3.0 10
2
2.7 10
k/[X]
0.057
5.5 102
1.05
0.36
1.15
15.2
5.95 9.46
a
forms, respectively, and χNHOH and χNHO correspond to the molar fraction of hydroxamic acid groups in their neutral and anionic forms, respectively (Scheme 4). kobs ¼ k0 þ kOH ½OH þ ðkN χCOOH χNHOH þ kM χCOO χNHOH þ kD χCOO χNHO Þ½PHA
ð1Þ
Values of the individual rate constants obtained from eq 1, according to Scheme 4, are presented in Table 2. The secondorder rate constants are given as a function of the overall PHA concentration (0.013 M) and as a function of the hydroxamate group concentration, considering the percentage calculated by potentiometric titrations (7.8% of the monomeric units in PHA). Results show that the neutral species of PHA accelerates the dephosphorylation reaction nearly 13-fold, as compared to the reaction in water (k0), and these increments reach up to 200and 1800-fold for the monoprotonated and deprotonated species
Rate constants are given as a function of the total polymer concentration (k/[PHA]) (0.013 M) and as a function of the concentration of carboxylate or hydroxamate groups (k/[X]) depending upon the pH region, considering the conversion percentage (9.83 104 M for hydroxamate and 6.79 104 M for carboxylate). b Values were obtained by potentiometric titration at 25 °C.
of PHA, respectively. These increments are increased considerably when only the reactive hydroxamate groups are considered, leading to 105- and 106-fold enhancements for the monoprotonated and deprotonated species of PHA, respectively. Comparing reactions of PHA to the analogous reactions with the reactive α-nucleophile hydroxylamine (BDNPP, k2 = 0.0039 M1 s1; DEDNPP, k2 = 0.265 M1 s1)12,17 shows that the rate enhancements reported here, considering the local concentration of the reactive groups, are consistent with the higher basicity of hydroxamic acids and possibly some local polarity effects. Moreover, the observed rate constants are similar to that observed for the 15115
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Figure 5. Proposed mechanism for the basic catalysis of the carboxylate group on the cleavage of BDNPP by PHA. Figure 4. Comparison of the observed second-order rate constants (k2PHA/k2BHO ) as a function of pH for the cleavage reaction of BDNPP.3 The constants were normalized according to the molarity of the functional groups present in the reaction.
reaction of BHO with BDNPP (k2 = 0.261 M1 s1)3 and significantly higher than that calculated for the reaction of BHO with DEDNPP (k2 = 2.45 M1 s1). In general, the reaction of the PHA polymer with the triester DEDNPP proceeds at a faster rate than that of BDNPP, probably as a result of charge-repulsive effects because BDNPP is negatively charged, as is PHA. This observation is consistent with the even smaller effect observed in the reaction of α-nucleophiles with dianionic phosphate monoesters.11,12 Further analysis of Table 2 shows that, even for the neutral and monoprotonated species of PHA, where the hydroxamic acid is protonated, thus virtually impossible to attack nucleophilically, there are significant rate enhancements. This can be attributed to a synergetic assistance by the carboxylic acid groups, which can act as general base catalysts, enabling hydroxamate to attack, as illustrated in Figure 5. There is also the possibility of a nucleophilic attack by carboxylate (path A of Figure 5). To verify this hypothesis, PAA was tested in the dephosphorylation reactions presented here and no reactivity was observed, for over 33 h. However, this fact might not indicate that path A does not occur. Because in the pH that the experiment was performed (pH 10.0), PAA is entirely deprotonated, it could be repelling the substrate. On the other hand, the PHA probably does not repel the substrates because only some units are negatively charged. An interesting comparison is presented in Figure 4, where pH profiles are presented for the reaction of BDNPP with PHA and BHO and rate constants are normalized, i.e., given as a function of the hydroxamic acid group concentration in each molecule (considering a functional group of ∼74 g/mol). Results show that, at higher pH, where hydroxamate is the predominant ionic species, PHA is slightly more reactive than BHO in the reaction with BDNPP, as previously depicted. The behavior is particularly interesting in the range of pH 78, where normalized rate constants (k2; Figure 4) for PHA are nearly 12-fold higher than for BHO, showing the importance of the carboxylate group in the reaction evaluated, at milder conditions, such as pH 7. The overall mechanism suggested for the dephosphorylation reactions with PHA should depend upon the pH; therefore, in the pH range where the monoprotonated species is predominant, the mechanism can occur through attack of the carboxylate group on BDNPP (path A of Figure 5) and/or through a basic catalysis with the oxygen of the carboxylate group that assists the attack of the hydroxamate on BDNPP, enhancing its nucleophilicity
Figure 6. Logarithmic plot of the rate constants against nucleophilic pKa values for reactions with BDNPP. Rate constants and pKa values are statistically corrected, with (9) nucleophiles from the literature12 and (0) BHO and PHA and PHA2, monoprotonated and deprotonated forms, respectively. The lines are drawn to guide the eye.
(path B of Figure 5). At higher pH, hydroxamate should directly attack the substrate, which may or not be assisted by an intramolecular general base catalysis by the adjacent carboxylate group. To obtain further insight on the nature of intermediates and products of the reactions of PHA with the different phosphate esters, mass techniques were carried out, although because of multiple fragmentations of the polymer and possible intermediates, it was difficult to assign structures. Previous studies with BDNPP and BHO provided evidence by mass and nuclear magnetic resonance (NMR) techniques that the nucleophilic attack on phosphorus gives an unstable intermediate that undergoes a Lossen rearrangement to urea, amine, isocyanate, and carbamyl hydroxamate. Thus, BHO behaves as a self-destructive molecular scissor because it reacts and loses its nucleophilic ability.3 This mechanism was also depicted in reactions of BDNPP with lauryl hydroxamate.13 Therefore, PHA may react by a similar nucleophilic attack on the phosphorus atom, which, after reacting, will lose its reactivity, thus also behaving as a selfdestructive scissor. Additionally, a Br€onsted plot is presented for the reaction of BDNPP with different nucleophiles,12 considering exclusively attack via the phosphorus atom (kBHOP), although for PHA, the overall second-order constant is plotted for the deprotonated 15116
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Figure 7. ITC curves for titration of 1.37 102 M CTABr into (b) 1.27 103 M PHA and (2) H2O at 25 °C and pH 10.
(PHA2) and monoprotonated (PHA) species. Data in Figure 6 show that the reactivity of PHA is similar to highly reactive nucleophiles, such as BHO, hydroxylamine (NH2OH), and its methylated derivatives. Interaction between PHA and Surfactants. Because of the relatively hydrophilic nature of polymers, such as PHA, it is also of interest to evaluate the effect of micelles on the hydrolysis reactions. We initially studied the interaction of PHA with different surfactants using ITC. The interactions of PHA with the cationic cetyltrimethylammonium bromide (CTABr), zwitterionic SB3-14, and anionic sodium dodecyl sulfate (SDS) surfactants was evaluated by calorimetric techniques (ITC), which indicate a low interaction of PHA with SDS and SB3-14 (see the Supporting Information), while for CTABr, there is a significant interaction, as shown below. When the surfactant CTABr is titrated into the PHA solution, one observes a different profile curve compared to the dilution curve of CTABr in water (Figure 7), which might indicate some kind of interaction between the surfactant and PHA. Although the enthalpy variation is positive for the dilution process of CTABr, the addition of the surfactant into the PHA solution was less endothermic, indicating that the interaction between CTABr and PHA is exothermic. As the surfactant concentration passes a critical value, the so-called critical aggregation concentration (cac), a change in the curve can be observed, and it is generally attributed to the cooperative formation of small surfactant aggregates along the polymer chain.20 This aggregation is assumed to occur facilitated by the presence of polymer [cac values are always smaller than critical micelle concentration (cmc) values], and the extent to which it is affected by the polymer can be estimated by the ratio of cac/cmc values; the smaller the value, the more intense the interaction.24,25 Finally, as more CTABr is added to PHA solution, the curve tends to merge into the dilution curve because micelles added are only being diluted with no further polymersurfactant interaction. To determine the cac value for the PHA/CTABr system, a diluted CTABr solution (4.11 103 M) was added to the PHA solution (Figure 8) and a value of 1.1 104 M was found, nearly 10 times smaller than the cmc of CTABr. This association might be attributed to the charge effect promoted by the electrostatic attraction between the positively charged surfactant monomers and the negatively charged polymer chain (region I of Figure 9). After the cac, there is an increase in the enthalpy, which probably indicates that all charges in the polymer are being neutralized at this concentration of CTABr (2 104 M), considering that ca. 13% of the polymer is deprotonated at pH 10.0.
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Figure 8. ITC curves for titration of CTABr (4.11 103 M) into 1.27 103 M PHA at 25 °C and pH 10.0. The line is drawn to guide the eye.
This small increase in enthalpy might be occurring because the added CTABr monomers are interacting with the neutral portion of the polymer and, consequently, water molecules solvating the polymer are removed (region II of Figure 9). After a given concentration, the added surfactant does not further interact with the polymer and, as mentioned before, the curve tends to merge into the dilution curve because micelles added are only being diluted (region III of Figure 9). One way to compare how spontaneous the association between CTABr and PHA is is to determine the Gibbs free energy variation for the interaction. Gibbs free energy changes for the micellization processes of the surfactant in the absence of the polymer (ΔGmic) and the formation of the polymersurfactant aggregates (ΔGagg) can be expressed through eqs 2 and 324 ΔGmic ¼ RT lnðcmcÞ
ð2Þ
ΔGagg ¼ RT lnðcacÞ
ð3Þ
where mic is related to the micellization process and agg is related to the formation of the polymersurfactant aggregate. Thus, Gibbs free energy related to the polymersurfactant interaction can be calculated through eq 4 based on eqs 2 and 324 ΔGps ¼ ΔGagg ΔGic ¼ RT lnðcac=cmcÞ
ð4Þ
where ps is related to the polymersurfactant interaction. The value observed for ΔGps for the interaction of PHA and CTABr at 25 °C is 5.6 kJ mol1, while for SDS, the value is 0.71 kJ mol1 and, for SB3-14, there is no interaction (see the Supporting Information). These data suggest that, if CTABr is added in the PHA solution to study the cleavage reaction of BDNPP, a favorable interaction might occur because of the ability of the surfactant to concentrate both the organic phosphate diester and the PHA polymer. Effect of Micelles on the Cleavage of BDNPP by PHA. The effect of the cationic surfactant CTABr on the ester cleavage by PHA was also evaluated, because it is well-known that micelles have the ability to interact with polymers and concentrate reactants, resulting in the increase of rate reactions, depending upon the type of micelles and reactants involved.26 The effect of the CTABr concentration on the reaction of PHA with CTABr is presented in Figure 10, and it shows that saturation occurs at higher surfactant concentrations. The experimental data were fitted with a Langmuir isotherm that gives an association constant for the substrate BDNPP on the micellar/ polymer domain of Ks = 310 M1. 15117
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Figure 9. Ilustration of the interaction between the PHA polymer and the surfactant CTABr, at different CTABr concentrations.
Scheme 5
Figure 10. CTABr concentration profile on the reaction of 0.013 M PHA with BDNPP at 25 °C and pH 10.
the rate constant shows that ion binding of BDNPP to CTABr micelles concentrates the dianionic phosphate diester in a nanoreactor, which allows for an efficient reaction with the anionic PHA polymer. The micellar effect shown in Figure 11 can be treated in terms of equilibrium distributions of BDNPP and PHA between the aqueous and micellar pseudo-phases (Scheme 5), where subscripts “m” and “w” indicate micellar and aqueous pseudo-phases, respectively. As shown in previous studies,13 at 0.01 M CTABr, both BDNPP and PHA should be fully incorporated in the micelar phase. Therefore, we can neglect reactions in water and in the absence of PHA and fit data in the presence of CTABr (Figure 11), with eq 5, analogous to eq 1, which relates the kinetic parameters (kMm and kDm) in the micelar pseudo-phase. The fraction of the ionic species of PHA in the micelar phase (χCOOmχNHOHm and χCOOmχNHOm) is related to two additional apparent pKa values. Note that kNm was not considered, because it did not show any significant effect in the fit. kobs m ¼ ðkM m χCOO m χNHOH m þ kD m χCOO m χNHO m Þ½PHAm
Figure 11. pH profile for the reaction of BDNPP with 0.013 M PHA in the (2) presence and (9) absence of 0.01 M CTABr and the cleavage reaction of BDNPP in (0) water at 25 °C.
The pH profile for the reaction of BDNPP with PHA in the presence and in the absence of CTABr (Figure 11) shows that the reaction is ca. 15 times faster when the surfactant is present compared to the reaction in the presence of only PHA. Considering that the reaction of BDNPP with PHA is 250 times faster than the reaction in water, the reaction in the presence of both PHA and CTABr occurs 3750 times faster than the reaction in water. Thus, the reaction time needed to complete the reaction (assumed as five half-lives) is reduced from 9 months in water to less than 10 min when both PHA and CTABr are present. This observed increase in
ð5Þ
Fitting of the pH rate profile for the reaction of PHA with BDNPP in the presence of CTABr gave kMm = 6.0 103 M1 s1 and kDm = 0.34 M1 s1 (per [PHA]). The calculated apparent pKa values are 6.52 and 9.25. These results show that the ratio for the second-order constant for the reaction in the presence (kPHACTABr) and absence (kPHA) of CTABr, kPHACTABr/ kPHA, is 2- and 13-fold higher, for the monoprotonated (kMm) and deprotonated species (kDm), respectively. In the concentration used in the kinetics experiments, CTABr exists largely as an aggregate and the observed rate constant increments are largely related to the ability of the micelles to place the reagents in close proximity to the catalytic groups in the polymer and are typical of micelar catalysis.9 Panel II of Figure 12 illustrates this ability of CTABr to concentrate the substrate. The effect of the zwitterionic SB3-14 and anionic SDS surfactants on the cleavage of BDNPP in the presence of PHA was also evaluated and shows that SDS has almost no effect on the PHA reactivity toward BDNPP, while SB3-14, a formally 15118
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Figure 12. Illustration of the possible localization of the substrate in a solution containing (I) only PHA and (II) PHA and the surfactant CTABr.
neutral surfactant, slightly inhibits the reaction of PHA with BDNPP (see the Supporting Information). These observations agree with calorimetry results (ITC) obtained, which indicate a low interaction of PHA with SDS and SB3-14.
’ CONCLUSION A PHA polymer containing the functional groups hydroxamic acid and carboxylic acid was obtained, with cooperativity effects between the neighboring reactive groups. PHA showed the ability to accelerate dephosphorylation reactions, and the catalytic effect promoted was evaluated on the cleavage of BDNPP and DEDNPP. Rate enhancements promoted by PHA are up to 106-fold. The effect of surfactants on the dephosphorylation of BDNPP by PHA was also investigated. It was observed that, when CTABr is added to PHA, the reaction is 15-fold faster compared to the reaction when only PHA is present, which is due to the ability of micelles to concentrate the reactants. ’ ASSOCIATED CONTENT
bS
Supporting Information. Kinetic data for all of the reactions and ITC results for the PHA/SDS and SB3-14 systems. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
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dx.doi.org/10.1021/la203437j |Langmuir 2011, 27, 15112–15119
