Ester Cleavage Properties of Synthetic Hydroxybenzotriazoles in

Apr 16, 2016 - (d) Ember, L. R. Chem. ... as a large body of kinetic data is available on the hydrolysis of this substrate .... Data in Table 1 show t...
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Langmuir 2005, 21, 71-78

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Ester Cleavage Properties of Synthetic Hydroxybenzotriazoles in Cationic Monovalent and Gemini Surfactant Micelles Santanu Bhattacharya* and V. Praveen Kumar Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India Received May 8, 2004. In Final Form: August 31, 2004 Four new hydroxybenzotriazole derivatives have been synthesized. Two of them, N-tetradecyl-1-hydroxy1H-benzo[d][1,2,3]triazole-6-carboxamide (2) and N-tetradecyl-1-hydroxy-1H-benzo[d][1,2,3]triazole-7carboxamide (3), possess long alkyl chains, while the other two, 1-hydroxy-1H-benzo[d][1,2,3]triazole-6carboxylic acid (4) and 1-hydroxy-1H-benzo[d][1,2,3]triazole-7-carboxylic acid (5), have carboxylate side chains. These compounds along with their parent unsubstituted 1-hydroxybenzotriazole (HOBt), 1, have been examined for the cleavage of p-nitrophenyl hexanoate (PNPH) and p-nitrophenyl diphenyl phosphate (PNPDPP) in comicelles with monovalent cetyltrimethylammonium bromide (CTABr) and the corresponding bis-cationic gemini surfactants 16-m-16, 2Br- of identical chain length at 25 °C and pH 8.2. The apparent pKa values of the HOBt derivatives in the comicelles of CTABr or 16-4-16 gemini surfactant have been determined from the rate versus pH profiles and were found to be comparable. Catalytic system 4/16-4-16 demonstrated over 2200- and 1650-fold rate enhancements in the hydrolysis of PNPDPP and PNPH, respectively, for identical reactions carried out at pH 8.2 and 25 °C in buffered aqueous media. The secondorder rate constants for such bimolecular reactions were determined employing pseudophase micellar models. Experiments in which excess substrate was taken over HOBt derivatives demonstrated that the catalysts “turned over”; hydrolysis of the putative acylated or phosphorylated HOBt intermediates was rapid in either type of host micelles.

Introduction

Chart 1

Phosphate ester hydrolysis is one of the most fundamental chemical and biochemical reactions.1 Many persistent chemical agents such as paraoxon, parathion, VX or sarin, and so forth (Chart 1) are hydrophobic phosphorus(V) substrates, and their decontamination also involves dephosphorylation or hydrolysis.2 Such compounds are toxic to both target and nontargeted organisms. Paraoxon and parathion, the phosphotriester-based pesticides, are often responsible for the poisoning of agricultural workers.3 The decontamination of such phosphate esters therefore continues to be a major practical concern.4 However, the extreme toxicity of such compounds often mandates that research laboratories employ safer simulant substrates instead of the actual toxic compounds. For instance, p-nitrophenyl diphenyl phosphate (PNPDPP) is a phosphotriester which has been widely employed as a simulant and its hydrolytic reactions have been extensively investigated.5 Since PNPDPP is not watersoluble, aqueous solutions of surfactants (micelles) have been generally employed as a reaction medium for the * Corresponding author and Swarnajayanti Fellow (DST, Government of India); also at the Chemical Biology Unit, JNCASR, Bangalore 560 012, India. Fax: +91-080-23600529. E-mail: sb@ orgchem.iisc.ernet.in (1) Westheimer, F. H. Science 1987, 235, 1173. (2) (a) Seabolt, E. E.; Ford, W. T. Langmuir 2003, 19, 5378. (b) Dubey, D. K.; Gupta, A. K.; Sharma, M.; Prabha, S.; Vaidyanathaswamy, R. Langmuir 2002, 18, 10489. (c) Yang, Y. C. Acc. Chem. Res. 1999, 32, 109 and references therein. (d) Ember, L. R. Chem. Eng. News 1994, Feb 14, 7. (3) Zheng, F.; Zhan, C.-G.; Ornstein, R. L. J. Chem. Soc., Perkin Trans. 2 2001, 2355. (4) Yang, Y.-C. Chem. Ind. (London) 1995, 334. (5) The choice of PNPDPP as a model substrate is also advantageous as a large body of kinetic data is available on the hydrolysis of this substrate for comparison. For instance see: (a) Bunton, C. A.; Robinson, L. J. Org. Chem. 1969, 34, 773. (b) Moss, R. A.; Bose, S.; Ragunathan, K. G.; Jayasuriya, N.; Emge, T. J. Tetrahedron Lett. 1998, 39, 347.

cleavage of organophosphates.6 In such a medium, organic reactants are partitioned into the surfactant aggregates by electrostatic and hydrophobic interactions, and the observed rate accelerations are largely due to the increased (6) (a) Bunton, C. A.; Nome, F.; Quina, F. H.; Romsted, L. S. Acc. Chem. Res. 1991, 24, 357. (b) Bunton, C. A.; Savelli, G. Adv. Phys. Org. Chem. 1986, 22, 213.

10.1021/la048858f CCC: $30.25 © 2005 American Chemical Society Published on Web 12/08/2004

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localization of the reactants and also the typical physical chemical properties of the micellar environment, which are significantly different from those of the bulk solvents.6 Chemical means of achieving efficient degradation of organophosphate esters remains an active area of much research, with attention focused recently on nucleophilic reagents such as peroxides,7 iodosoarene carboxylates,8 4-N,N-dialkylaminopyridines,9 and metallomicelles10 employed primarily in cetyltrimethylammonium (CTA+) micelles as media. In the search of new catalysts, we have now turned our attention to another class of nucleophile, 1-hydroxybenzotriazole (HOBt).11 HOBt has been used as an additive for achieving racemization-free peptide coupling12 and in oligonucleotide synthesis.13 Other related molecules such as N-hydroxysuccinimide,14a 1-hydroxy-7-azabenzotriazole,14b and N-hydroxypthalimide14c have also been used in peptide synthesis. Recently surfactants with novel molecular structures have been reported which upon solubilization in water form different types of micellar aggregates.15 Among the new types of surfactants, geminis appear interesting as hosts. Gemini surfactants manifest lower critical micellar concentrations, higher viscoelasticity, and enhanced propensity for lowering the oil-water interfacial tension in comparison to their monovalent single headgroup/single chain counterparts.16 However, most studies on geminis have focused on their specific aggregation behavior and structural properties, with very limited investigations on reaction rates.17 Herein we present the esterolytic properties of HOBt (1) and four of its synthesized derivatives (2-5) (Chart 1) in cationic micellar solutions at pH 8.2. We have examined the reactivities of 1-5 toward dephosphorylation and (7) (a) Bhattacharya, S.; Snehalatha, K. J. Org. Chem. 1997, 62, 2198. (b) Toullec, J.; Moukawim, M. Chem. Commun. 1996, 221. (c) Yang, Y. C.; Szafraniec, L. L.; Beandry, W. T.; Bunton, C. A. J. Org. Chem. 1993, 58, 6964. (8) For a recent review, see: Rojas, H. M.; Moss, R. A. Chem. Rev. 2002, 102, 2497. (9) (a) Bhattacharya, S.; Praveen Kumar, V. J. Org. Chem. 2004, 69, 559. (b) Bhattacharya, S.; Snehalatha, K. Langmuir 1997, 13, 378. (c) Bhattacharya, S.; Snehalatha, K. Langmuir 1995, 11, 4653. (10) (a) Bhattacharya, S.; Snehalatha, K.; Praveen Kumar, V. J. Org. Chem. 2003, 68, 2741. (b) Clandia, S.; Rossi, P.; Felluga, F.; Formaggio, F.; Palumbo, M.; Tecilla, P.; Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 2001, 123, 3169. (c) Yan, J.; Breslow, R. Tetrahedron Lett. 2000, 41, 2059. (11) (a) Konig, W.; Geiger, R. Chem. Ber. 1970, 103, 788. (b) Praveen Kumar, V.; Ganguly, B.; Bhattacharya, S. J. Org. Chem. 2004, in press. (12) (a) Ho, G.-J.; Emerson, K. M.; Mathre, D. J.; Shuman, R. F.; Grabowski, E. J. J. J. Org. Chem. 1995, 60, 3569. (b) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1995, 60, 3561. (13) (a) De Masmaeker, A.; Lebreton, J.; Waldner, A.; Fritsch, V.; Wolf, R. M.; Freir, S. M. Synlett 1993, 733. (b) Lebreton, J.; De Masmaeker, A.; Waldner, A.; Fritsch, V.; Wolf, R. M.; Freir, S. M. Tetrahedron Lett. 1993, 34, 6383. (14) (a) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. Soc. 1964, 86, 1964. (b) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397. (c) Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis; Wiley: New York, 1967; Vol. 1, p 485. (15) (a) Menger, F. M.; Littau, C. A. J. Am. Chem. Soc. 1991, 113, 1451. (b) Zana, R.; Benrraou, M.; Rueff, R. Langmuir 1991, 7, 1072. (c) De, S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. B 1998, 102, 6152. (d) Menger, F. M.; Migulin, V. A. J. Org. Chem. 1999, 64, 8916. (e) Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Angew. Chem., Int. Ed. 2001, 40, 1228. (f) Willemen, H. M.; de Smet, L. C. P. M.; Koudijs, A.; Stuart, M. C. A.; Heikamp, de J.; Ineke, G. A. M.; Marcelis, A. T. M.; Sudholter, E. J. R. Angew. Chem., Int. Ed. 2002, 41, 4275. (g) Jaeger, D. A.; Wang, Y.; Pennington, R. L. Langmuir 2002, 18, 9259. (16) For reviews, see: (a) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39, 1906. (b) Rosen, M. J.; Tracy, D. J. J. Surfactants Deterg. 1998, 1, 547. (c) Zana, R. Curr. Opin. Colloid Interface Sci. 1996, 1, 566. (17) Brinchi, L.; Germani, R.; Goracci, L.; Savelli, G.; Bunton, C. A. Langmuir 2002, 18, 7821.

Bhattacharya and Kumar

deacylation reactions in micellar aggregates of cetyltrimethylammonium bromide and also in the corresponding gemini surfactant systems. We investigated the ester hydrolysis reactions as a function of surfactant concentration with each type of host micelle. Notably the kinetic benefits of these reagents can be potentiated upon their solubilization in aqueous micellar solutions of gemini surfactants such as 16-m-16, 2Br- compared to that of cetyltrimethylammonium bromide (CTABr). The secondorder rate constants of such reactions in micelles were obtained using pseudophase micellar models under saturation conditions. In the presence of a large excess of substrate, these HOBt derivatives rapidly “turn over”, confirming that these are true catalysts, and the true catalytic behavior was also preserved in gemini micelles. Results Catalysts and Substrates. The catalysts in the present study were based on 1-hydroxybenzotriazole, 1. Compounds 2 and 3, which are long-chain derivatives of 1, have been synthesized as outlined in Scheme S1 (Supporting Information). Briefly, 4-chloro-3-nitrobenzoic acid was converted to 4-chloro-3-nitrobenzoyl chloride upon reaction with SOCl2. The acid chloride was then coupled with n-tetradecylamine in the presence of Et3N in dry THF to afford the corresponding amide, 7 (82%), as a yellow solid. This was then treated with ∼10-fold excess of hydrazine hydrate, and the mixture was refluxed in dry EtOH for 20 h to furnish 2 as a light yellow solid (60%). Compound 3, the regio-isomer of 2, was synthesized starting from 3-chloro-2-nitrobenzoic acid employing a similar series of steps as adopted for 2. The carboxylic acid derivatives of 1-hydroxybenzotriazole, 4 and 5, were also prepared from 4-chloro-3-nitrobenzoic acid or 3-chloro2-nitrobenzoic acid, respectively, upon reaction with ∼10fold excess of hydrazine hydrate under refluxing conditions in dry EtOH for 15 h. Acidification of the respective reaction mixtures gave 4 and 5 as white solids in 76% and ∼55% yields, respectively. Spectroscopic and analytical characterizations using IR, 1H NMR, mass spectroscopy, and elemental analysis established their given molecular structures and high purity (Supporting Information). The syntheses of all substrates (PNPDPP and pnitrophenyl hexanoate (PNPH)) and gemini surfactants (16-m-16) have already been reported.18,19 Kinetics. Because we wanted to determine not only the rate constants under pseudo-first-order conditions, kobs, but also the intrinsic activity of each catalyst-mediated hydrolytic reaction on the substrates in micellar conditions, the second-order rate constants, k2, for various catalyst/substrate combinations had to be determined. However, the determination of the k2’s for a given micellebound catalyst and substrate is less obvious. Therefore we first present the rates obtained under pseudo-firstorder conditions and then address the determination of k2 in a micelle-mediated reaction. (a) CTABr Micelles. The kinetics of ester hydrolysis in 0.05 M tris-maleate buffer, pH 8.2 at 25 ( 0.1 °C, was obtained by monitoring the appearance of p-nitrophenoxide spectrophotometrically at 400 nm under the condition of excess HOBt catalyst over the substrate, using [HOBt] ) 2.5 × 10-4 M and [substrate] ) 2.5 × 10-5 M at different CTABr concentrations. The observed pseudofirst-order rate constants (kobs) were determined for the (18) Gulik, W. M.; Geske, D. H. J. Am. Chem. Soc. 1966, 88, 2928. (19) De, S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. 1996, 100, 11664.

Catalytic Cleavage of Esters in Gemini Micelles

Langmuir, Vol. 21, No. 1, 2005 73 Table 1. Rate Constants (kobs, s-1) for the Hydrolysis of PNPH and PNPDPP Catalyzed by Various HOBt Systems in CTABr Micellesa PNPDPP entry

catalyst

1 2 3 4 5 6 7

none CTABr 1 2 3 4 5

PNPH

pKab

103 kobs, s-1 c

kreld

103 kobs, s-1 c

kreld

7.0 6.5 6.7 6.5 6.7

0.007 0.02 3.1 4.5 4.3 5.4 4.8

1 3 470 650 630 780 700

0.01 0.03 5.2 5.8 5.7 6.6 6.0

1 3 550 590 580 670 620

a Conditions: 0.05 M tris-maleate buffer, pH ) 8.2; 25 °C; [substrates] ) 2.5 × 10-5 M, [catalyst] ) 2.5 × 10-4 M, [CTABr] ) 1 × 10-2 M. b See the text for determination of pKa’s. c All kinetic runs were done in triplicate. The precision of the kobs data for the uncatalyzed reactions (entry 1) was within (7%. The values of kobs (entries 2-7) were within 2% of those with freshly prepared solutions. d krel ) kobs/k0, where k0 is the rate constant for the hydrolysis of substrate at pH 8.2 in buffered aqueous media in the absence of CTABr micelles.

Table 2. Kinetic Parameters for the Hydrolysis of PNPDPP and PNPH in 16-4-16 Gemini Surfactant Micellesa PNPDPP entry catalyst pKab 103 kobs, s-1 c

Figure 1. Plots of kobs for the hydrolysis of PNPH (2.5 × 10-5 M) vs different host surfactant concentrations in pH 8.2 trismaleate buffer at 25 °C: (A) by catalysts 2-5 (2.5 × 10-4 M) as a function of [CTABr]; (B) by catalyst 4 (1.25 × 10-4 M) as a function of the concentration of various geminis, 16-m-16. Lines are drawn to show the trend.

hydrolysis of either substrate (PNPH or PNPDPP) at each combination of [CTABr]/[catalyst]. Variations of the kobs as a function of the concentration of CTABr for the hydrolysis of PNPH mediated by each of catalysts 2-5 are shown in Figure 1a. The values of kobs for the cleavage of PNPDPP and PNPH by all catalysts at [CTABr] ) 1 × 10-2 M, when the overall ratio of each catalyst to CTABr is fixed at 1:40, are given in Table 1. The krel reveals the ratio of the hydrolytic rate constants of individual HOBt reagents against a given substrate (kobs) compared to the background cleavage rates in the buffered aqueous media in the absence of catalyst and CTABr (k0). Data in Table 1 show that compounds 1-5 are quite effective catalysts for the cleavages of both PNPDPP and PNPH in that they afford more than 2 orders of magnitude rate acceleration over the background. (b) Reactions in Gemini Micelles. Pseudo-first-order rate constants, kobs, for the hydrolysis of both PNPDPP and PNPH were also determined in gemini micellar media. These were determined at several gemini surfactant (16m-16) concentrations under the condition of excess catalyst over the substrate at pH 8.2 and 25 °C as was done with CTABr micelles. Variations of the kobs as a function of the concentration of 16-m-16, where m ) 2, 3, 4, 5, and 12, for the hydrolysis of PNPH mediated by catalyst 4 are shown in Figure 1b. In each of the gemini surfactant micelles, the values of kobs for the PNPH hydrolysis by 4 increased progressively with the rise in 16-m-16 concentration, till the concentration of the gemini reached ∼2.5 × 10-3 M. For all the gemini micelles, this was true

1 2 3 4 5 6 7 8

none CTABr 16-4-16 1 2 3 4 5

7.0 6.5 6.7 6.5 6.7

0.007 0.02 0.05 6.5 10.4 9.4 15.2 11.3

PNPH kreld

1 3 7.1 975 1510 1377 2207 1656

103 kobs, s-1 c kreld 0.01 0.03 0.11 10.2 14.2 12.5 16.5 15.0

1 3 11 1071 1443 1282 1675 1538

a Conditions: 0.05 M tris-maleate buffer, pH 8.2, 25 °C; [substrate] ) 2.5 × 10-5 M, [catalyst] ) 1.25 × 10-4 M, [16-4-16] ) 2.5 × 10-3 M. b The pKa value for each catalyst was determined in 16-4-16 micelles. The precision of the kobs data for the uncatalyzed reactions (entry 1) was within (7%. c All kinetic runs were done in triplicate, and the values of kobs were within 2% of those with freshly prepared solutions. d krel ) kobs/k0, where k0 is the rate constant for the respective substrate hydrolysis in pH 8.2 buffer in the absence of any surfactant.

although the extents of increase in kobs were different depending on the spacer chain length. Thereafter the kobs values decreased gradually with further increase in the gemini surfactant concentration. Notably, the hydrolysis reactions of both PNPDPP and PNPH, mediated by all HOBt catalysts in gemini surfactant aggregates of 16-4-16, were significantly more rapid than in monovalent CTABr micelles. The relevant data appear in Table 2. As was observed in CTABr micelles, catalyst 4 in 16-4-16 micelles afforded the most efficient combination for the cleavages of both PNPDPP and PNPH. Relative to rates in the buffered aqueous media at pH 8.2, comicellar 4/16-4-16 potentiated the hydrolysis rates of PNPDPP and PNPH by factors of over 2200 and 1650, respectively (Table 2, entry 7). Compound 5 also displayed over 1650- and 1500-fold kinetic advantages in gemini micelles for the cleavage of PNPDPP and PNPH, respectively, over background. We also found that among the gemini surfactants as host micelles, the activities of the catalysts also depend on the gemini spacer chain length. Figure 2 summarizes the influence of spacer length variation (m-value) on the esterolytic rates of PNPDPP by 4 under comparable reaction conditions. A similar trend in the variation of rates as a function of spacer chain length was also observed in gemini micelles for the hydrolysis of the other substrate,

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Bhattacharya and Kumar

Figure 2. Variation of kobs as a function of the spacer chain length (m-value) of 16-m-16 surfactant (2.5 × 10-3 M) for the hydrolysis of PNPDPP by catalyst 4 (1.25 × 10-4 M) in pH 8.2, 0.05 M tris-maleate buffer at 25 °C. The corresponding kobs in CTABr micelles represents the point at the “m-value ) 0”.

PNPH, by 4 (not shown). Interestingly with micelles of various gemini surfactants (16-2-16 to 16-12-16), we observed the maximum of kobs at the identical host surfactant concentration. In other words, at a given ratio of the catalyst to host gemini surfactant, the highest rates were observed under pseudo-first-order conditions irrespective of the spacer chain length. Although variation in spacer chain lengths alters the shapes of gemini micellar aggregates,19,20 these changes do not appear to influence drastically the observed maximum of the rate constants for the ester cleavage reactions. pKa Determination. The N-O- form of HOBt acts as a nucleophilic species in the hydrolysis of carboxylate esters.11 Therefore the reactive forms of reagents 1-5 are the respective N-O- species. Hence we determined the pKa values for each of 1-5 in both types of micellar aggregates (CTABr and gemini). For the determination of pKa values in micellar conditions, the pseudo-first-order rate constants for PNPH or PNPDPP cleavages at 25 °C were determined at several pH values between 6.2 and 8.5 by spectrophotometrically following the release of the p-nitrophenoxide ion at 400 nm. Plots of log kobs versus pH gave discontinuities at definite pH values, which were taken as systemic pKa values for the catalysts in 1 × 10-2 M micellar CTABr solutions. Figure 3a shows the pHrate constant profiles for the cleavage of PNPDPP (2.5 × 10-5 M) by catalysts 2 and 4 (2.5 × 10-4 M) in micellar CTABr (1 × 10-2 M) solutions at 25 °C. The plots of log kobs versus pH showed breaks at pH 6.5 for both catalysts 2 and 4, and accordingly these were taken as the systemic pKa for 2 and 4, respectively, under CTABr micellar conditions. The pKa values were similarly determined for the other catalysts also in CTABr micelles. These values were 6.7 for both 3 and 5 and 7.0 for 1. In Table 1, we record the systemic pKa values of all the catalysts in CTABr micelles. Do gemini micellar aggregates alter the pKa values of the HOBt derivatives further than that in CTABr micelles? To answer this, we also determined the systemic pKa of the HOBt catalysts in cationic gemini micellar aggregates of 16-4-16. Figure 3b shows the pH-rate constant profiles for the cleavage of PNPDPP (2.5 × 10-5 M) by HOBt derivatives 2 or 4 (1.25 × 10-4 M) in host 16-4-16, 2Brgemini surfactant (2.5 × 10-3 M) micelles at 25 °C. The plots of log kobs versus pH show discontinuities at pH 6.5 (20) De, S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. B 1997, 101, 5639.

Figure 3. (A) The log kobs vs pH profiles for the hydrolysis of PNPDPP (2.5 × 10-5 M) by catalyst 2 (2.5 × 10-4 M) (9) and 4 (2.5 × 10-4 M) (b) in CTABr (1 × 10-2 M) micelles. (B) The log kobs vs pH profiles for the hydrolysis of PNPDPP (2.5 × 10-5 M) by 2 (1.25 × 10-4 M) (9) and 4 (1.25 × 10-4 M) (b) in 16-4-16 (2.5 × 10-3 M) gemini micelles.

for both 2 and 4, which were taken as systemic pKa values for both 2 and 4 in cationic gemini 16-4-16 micelles. Intrinsic Reactivity. To understand the reasons for rate enhancements of these esterolytic reactions in gemini aggregates over the CTABr micelles, additional kinetic experiments were performed. This allowed evaluation of the second-order rate constants for the cleavage of PNPH and PNPDPP by various HOBt catalysts in both types of micellar media. Accordingly the kinetic data in CTABr and 16-4-16, 2Br- comicelles were obtained at pH 8.2 and 25 °C for each of 1-5 with solutions containing increasing amounts of catalyst and CTABr with molar ratios relative to the catalyst of 30, 40, and 50 and 16-4-16, 2Br- with molar ratios relative to the catalysts of 20, 25, and 30, respectively, using either PNPH or PNPDPP as the substrate. The corresponding rate-concentration profiles showing saturation behavior appear in Figure 4. Analysis of the curves by fitting the kobs versus [catalyst] data using the Michaelis-Menten equation10a allows the estimation of the klim, that is, the rate constants expected for the substrate being fully incorporated into the micellar aggregates. The analysis of these curves also allows the estimation of the apparent binding constants (Kb) for PNPDPP in different comicelles. Relevant data are summarized in Table 3, which reveal that the binding constants for 1-5 are larger with 16-4-16 gemini micelles in comparison to that with CTABr micelles and explain the source of greater rate enhancements in gemini micelles. Kinetic Studies with Excess Substrates. To examine the real catalytic activities of different HOBt derivatives, experiments in the presence of excess substrate were performed either with PNPDPP or PNPH in both types of micellar media. At pH 8.2 (0.05 M tris-maleate buffer, 25 °C using a [catalyst] ) 2.5 × 10-6 M in CTABr (1 × 10-3

Catalytic Cleavage of Esters in Gemini Micelles

Langmuir, Vol. 21, No. 1, 2005 75

Figure 5. Time-dependent release of p-nitrophenoxide upon hydrolysis of PNPH or PNPDPP by catalyst 2/CTABr in the presence of excess substrate over catalyst. Conditions: 25 ( 0.1 °C, catalyst [2] ) 2.5 × 10-6 M, [CTABr] ) 1.0 × 10-3 M, 0.05 M tris-maleate buffer, pH ) 8.2. The substrate concentrations: [PNPH] ) 6.25 × 10-5 M, [PNPDPP] ) 5.5 × 10-5 M.

Figure 4. Hydrolysis of PNPDPP or PNPH catalyzed by 5 in pH 8.2, 0.05 M tris-maleate buffer at molar ratios 1:30, 1:40, and 1:50 of 5/CTABr indicated. Table 3. Kinetic and Thermodynamic Parameters for the Cleavage of PNPDPP by Comicelles of 1-5 with CTABr or 16-4-16a 16-4-16 103 catalyst 1 2 3 4 5

klim, s-1

8.45 12.8 12.02 16.98 13.58

CTABr 103

Kb, M-1 b

k2, M-1 s-1 b

1672 2053 1667 2947 2375

107.3 155.9 148.1 206.8 167.3

103

klim, s-1

Kb, M-1

103 k2, M-1 s-1

4.58 5.94 5.8 6.72 6.16

885 1081 926 1431 1250

72.1 89.7 88.6 101.4 94.1

a Conditions: Kinetic cleavage experiments at pH 8.2 and 25 °C in 0.05 M tris-maleate buffer using solutions containing increasing amounts of catalyst and CTABr with molar ratios of 1:30, 1:40, and 1:50 or catalyst and 16-4-16, 2Br- with molar ratios of 1:20, 1:25, and 1:30 using PNPDPP as a substrate. b klim and k2 were calculated by fitting with the Michaelis-Menten equation. See the text for details.

M) and 25-fold excess substrate (PNPH) and 22-fold excess PNPDPP with respect to catalyst concentration, we observed a nearly quantitative monoexponential release of p-nitrophenoxide ion with no evidence of “burst” kinetics. Representative plots employing comicellar CTABr/2 against excess substrates PNPDPP and PNPH are shown in Figure 5. To further verify the influence of gemini micelles toward catalysis, we carried out excess substrate experiments in gemini micelles under the following reaction conditions: 25 ( 0.1 °C, 0.05 M tris-maleate buffer, [catalyst] ) 1 × 10-6 M, [16-4-16, 2Br-] ) 2.5 × 10-4 M, [PNPDPP] ) 2.5 × 10-5 M. Even in 16-4-16 micellar media, burst type kinetic profiles were not observed for 2 and other catalysts and only a monoexponential time-course was observed (not shown). Discussion A pKa value of 7.4 for HOBt, 1 in pure water (nonmicellar) has been reported.21 Solubilization of HOBt in (21) Boyle, F. T.; Jones, R. A. Y. J. Chem. Soc., Perkin. Trans. 2 1973, 160.

micellar CTABr lowered the pKa value for 1 to 7.0. Due to the amphiphilic character of 2 and 3, the HOBt ends in these molecules presumably remain projected toward CTA+ headgroups approximately aligning their fatty acyl chains with the CTABr surfactant tails inside comicelles. The net cationic character of comicellar CTABr (1 × 10-2 M)/catalyst (2.5 × 10-4 M) assemblies produces an environment that helps lower the pKa of the N-OH nucleophilic species in the HOBt moieties by at least ∼1 pKa units. The decrease in pKa is also due to an enhanced local pH at the cationic surface of the micelles where hydroxybenzotriazole moieties are located. The carboxylic acid side chains in 4 or 5 should be ionized into their anionic -COO- forms at pH 8.2. This facilitates strong binding of 4 and 5 with the cationic CTABr aggregates at the Stern layer region, via ion-pair interactions, which may be responsible for the effective binding with cationic micelles and the lowering of pKa values of the N-OH to N-O- conversion steps of 4 and 5. The values of kobs for the hydrolysis by catalysts 2-5 increased monotonically with rise in CTABr concentration till [CTABr] reached 1 × 10-2 M (Figure 1a). Thereafter the kobs values decreased progressively with further increase in CTABr concentration. The rate versus [CTABr] profiles obtained with various catalysts are characteristic of micelle-catalyzed reactions.6 Addition of CTABr to the reaction medium in water caused a progressive increase in the rate of ester hydrolysis as micelles formed above the cmc (critical micellar concentration) of CTABr. The variation in rate constants below the cmc is difficult to quantify due to reactant-induced micellization and interaction with nonmicellized surfactants. Subsequent addition of CTABr beyond the cmc caused a decrease in the observed reaction rate possibly due to the decrease in the catalyst concentration per micelle. Although the solubility of 1 in pure buffered aqueous solution was quite low, the solubilization of HOBt in CTABr micelles could be accomplished easily. The attachment of lipophilic amide (C14H29NH-C(O)) tails to HOBt gave 2 and 3, which rendered the former practically insoluble in water. However, they were readily soluble in CTABr micelles. This suggests that 1-3 all bind to micellar aggregates effectively. Since hydrophobic substrates PNPDPP and PNPH also partition into the micellar pseudophase, increased localization of catalysts and substrates led to rate acceleration in the cleavage of both phosphate and carboxylate esters. However, lipidations of HOBt to 2 and 3 led to a ∼1.4-fold kinetic advantage

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Table 4. Hydrodynamic Diameters of the CTABr and Gemini Micellar Aggregates in the Absence and Presence of 4 with [CTABr] ) 10 × 10-2 M and [4] ) 2.5 × 10-4 M in Tris-maleate Buffer at pH 8.2a average size (nm)b surfactant

only host surfactant

in the presence of 4

CTABr 16-3-16 16-4-16 16-5-16 16-12-16

8.0 9.5 10.0 11.5 14.0

11.0 16.0 20.0 19.0 20.0

a Dynamic light scattering (DLS) data were obtained at 25 °C under the conditions described in the text. b Based on an average DLS measurement using three independent samples. The variation was within (0.1 nm.

over 1 for the hydrolysis of PNPDPP. This is not entirely surprising. While lipidation enhances the binding of 2 and 3 to micellar aggregates, the attachment of the lipophilic chain via an electron-withdrawing -CONHgroup results in the mitigation of the nucleophilic potential of the N-OH groups in both 2 and 3. Thus these two factors work in opposite directions and suppress the benefits of improved binding abilities of 2 and 3 with micellar aggregates. The other two catalysts 4 and 5 also partition effectively to the micellar pseudophase via ion pairing with positively charged CTA+ ions in micelles through the carboxylate group of 4 or 5. Ion pairing of 4 and 5 ensures increased localization of these catalysts at the micellar interfaces of cationic CTABr wherein the substrate molecules are also concentrated. This explains why 4 and 5 are better catalysts than 1. Among all the catalytic systems investigated herein, 4/CTABr displayed the best esterolytic activity toward both PNPDPP and PNPH. Thus 4/CTABr cleaved PNPDPP 780 times faster than the same reaction carried out in the buffered aqueous media under identical conditions. Comicellar 5/CTABr was the next most effective catalyst toward both PNPDPP and PNPH. It is interesting to note that both catalysts 4 and 5, although nonlipidated, have been found to be more effective than the lipidated catalysts, 2 and 3. As stated earlier, 2 and 3 associate with micelles through hydrophobic interactions while 4 and 5 form salt bridges with the cationic centers of CTABr molecules within the comicelle. Both types of catalysts display better activity than the parent HOBt, 1. Although the binding of 2-5 to CTABr micelles might be better than that of 1, the electron-withdrawing nature of the carbonyl groups of the carboxamide and carboxylate substituents in 2-5 significantly mitigate their nucleophilic potential of the N-OH form. This is reflected in the manifestation of lower pKa values for 2-5 compared to 1 in micellar conditions (see below). Under the reaction conditions, 2 and 3 associate with CTA+ micelles through hydrophobic interactions. Their N-OH groups are also considerably ionized at pH 8.2 and therefore also bind to CTA+ ions through electrostatic interactions with the quaternary ammonium headgroups. In the same sense, 4 and 5 would turn into their dianionic forms in cationic micelles. Since 4 and 5 are aromatic anions, they can also bring about changes in the sizes of the micellar aggregates. Indeed, the dynamic light scattering data on various HOBt-doped comicelles (Table 4) suggest that the comicelles of CTABr and 4 are considerably larger than the mixed micelles of neutral 2 with CTABr. Similar size increases were also observed in the case of CTABr/5 mixed micelles as opposed to CTABr/3 comicelles. There is evidence in the literature17,22,23a that suggests that the formation of larger aggregates often leads to higher kobs.

It is possible that bulky aromatic anions such as 4 or 5 displace Br- and probably interfacial water leading to growth in aggregate sizes. Ester hydrolysis reactions are known to be facilitated in a reaction environment that is depleted in water.24 Slightly lower reactivities shown by 5 relative to 4 and 3 relative to 2 could be due to steric congestion experienced by the nucleophilic N-OH residue positioned adjacent to the O-carboxylate or carboxamide groups, respectively. Gemini Micelles. The aggregation properties of gemini surfactants, which contain, in order, a long hydrocarbon chain, a polar headgroup, a spacer, a polar headgroup, and a long hydrocarbon chain, have been extensively investigated in recent years.16 The aqueous solutions of such surfactants generally display lower critical micellar concentration and higher viscoelasticity, compared to their more conventional single-chain/single-headgroup monovalent surfactant counterparts. Spontaneous unimolecular reactions such as decarboxylation of 6-nitrobenzoisoxazole-3-carboxylate were studied earlier23 in micellar solutions of gemini surfactant 1,3-bis(N-hexadecyl-N,N-dimethylammonium)-propane dibromide. The dephosphorylation reaction of 2,4-dinitrophenyl phosphate was also examined in micelles of certain gemini surfactants bearing aromatic spacer units in mildly alkaline pH.17 However, the kinetics of bimolecular reactions has not been investigated in gemini micelles. The situation is obviously more complicated for nonspontaneous bimolecular reactions where the distributions of two reacting species (catalyst and substrate) have to be considered. In addition, there are questions regarding the meaning of “concentration” in the micellar pseudophase. The gemini surfactants used in the present study, that is, “16-m-16” possessed two n-C16H33 chains with cationic -NMe2+ headgroups covalently attached by a polymethylene -(CH2)m- spacer of m ) 2, 3, 4, 5, and 12. Although 16-m-16 geminis are quite comparable to CTABr in terms of the hydrocarbon chain length and electrostatic character, the variation in the spacer chain length and conformation in geminis influence their aggregation properties significantly.19,20 For instance 16-m-16, with m ) 2-3, form disklike micelles while the one with m ) 4 forms cylindrical micelles and m ) 5-12 form prolate micelles. Similarly with increase in the spacer chain length, the viscoelasticity of the resulting micelles decreases. These considerations prompted us to examine the kinetic performance of the HOBt catalysts in host gemini micellar media, which also allows us to examine whether spacer chain length variation in geminis (mvalue) leads to any changes in the rate constants of the esterolytic reactions. The superior esterolytic activity in the cationic gemini 16-4-16 micellar medium might reflect activation of the anionic HOBt systems 4 and 5 by ion-pairing to the dual cationic centers of a single 16-4-16 molecule in the micelle. At pH 8.2, both 4 and 5 ionize predominantly into their dianionic forms. Therefore the most effective species for 4 and 5 are dianionic in their active form and, hence, better suited for binding to a bis-cationic gemini surfactant. (22) Cerichelli, G.; Luchetti, L.; Mancini, G.; Savelli, G. Langmuir 1999, 15, 2631. (23) For a review, see: (a) Bunton, C. A.; Minch, M. J.; Hidalgo, J.; Sepulveda, L. J. Am. Chem. Soc. 1973, 95, 3262. (b) Kemp, D. S.; Paul, K. G. J. Am. Chem. Soc. 1975, 97, 7312. (c) Zana, R.; Talmon, Y. Nature 1993, 362, 228. (24) (a) Di Sabato, S.; Jencks, W. P. J. Am. Chem. Soc. 1961, 83, 4395. (b) Bunton, C. A.; Fendler, E. J.; Fendler, J. H. J. Am. Chem. Soc. 1967, 89, 1221. (c) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1966, 88, 1823. (d) Buchwald, S. L.; Friedman, J. M.; Knowles, J. R. J. Am. Chem. Soc. 1984, 106, 4911.

Catalytic Cleavage of Esters in Gemini Micelles

Gemini 16-4-16 offers itself as the most effective medium for such HOBt-mediated esterolytic chemistry among all the gemini surfactant micelles investigated herein. When the spacer chain was lengthened to a -(CH2)12- unit, the reactivity toward ester hydrolysis was considerably reduced, although still the cleavage rates were considerably better in these gemini micelles than in CTABr media. The observation of better rates for the hydrolysis reactions in gemini micelles compared to monovalent surfactant micelles could also be due to the presence of the hydrophobic polymethylene spacer chain between the two headgroups in geminis, which in turn decreases the extent of water penetration at such micellar interfaces. Dephosphorylation and deacylation reactions have been shown to be facilitated by a reduction in the water content in the immediate vicinity where such reactions take place.24 Menger, Romsted, and co-workers have estimated concentrations of H2O and Br- by chemical trapping at the surfaces of cationic gemini micelles.25 It has been demonstrated that the proximity of the gemini headgroups increases anion binding at the expense of binding of H2O. This conclusion is in agreement with our observation and explains why in gemini micellar media, the rates of ester hydrolysis reactions are enhanced over their monovalent micellar counterparts. Interestingly no noticeable difference in pKa values of these HOBt molecules was observed in gemini micellar media as compared to the pKa values obtained in CTABr micelles. Thus the local pH values at the Stern layer region of monovalent CTABr micelles are not much different from that of gemini micelles. However, the cleavage rates were significantly better when the reactions were performed in gemini micelles as compared to that in monovalent CTABr micelles. It may be concluded that the dicationic geminis provide a favorable ester cleavage environment due to depletion of water at the micellar surface where HOBt catalysts react with the ester substrates. The affinity of substrates and catalysts in gemini micelles may also be greater than in monovalent micelles (see below). Intrinsic Reactivity of the Comicellar HOBt Catalysts. Compared to homomicelles, comicelles bring about the problem of dilution of the catalyst in the cosurfactant (CTABr or 16-4-16 in the present study). The issue in micelles is further complicated by the fact that the system can be considered as a separate phase (micellar pseudophase). Bunton and his associates have comprehensively investigated this issue.6 So for the full analysis of the comicellar system it has been necessary to obtain Kb and klim for the determination of reliable second-order rate constants. Accordingly the relative ratio of cosurfactant/ HOBt catalyst does influence the rate of the hydrolytic reaction. Second-order rate constants in enzyme-like reactions can be obtained under saturation conditions as klim/Kb where klim is the rate constant for the fully bound substrate and Kb is the apparent binding constant: Kb

klim

S + Dfm y\z S‚Dfm 98 P where S denotes substrate, Dfm is the comicellar HOBtbased catalyst, and P denotes product upon hydrolysis. Analysis of the curves in Figure 4 by fitting the kobs versus [catalyst] data using the Michaelis-Menten equation10a allows the estimation of the klim, that is, the rate constants expected for the substrate being fully incorporated into the micellar aggregates. These also allow (25) Menger, F. M.; Keiper, J. S.; Mbadugha, B. N. A.; Coren, K. L.; Romsted, L. S. Langmuir 2000, 16, 9095.

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the estimation of the apparent binding constants (Kb) for PNPDPP in different comicelles. The second-order rate constants for these reactions in the micellar pseudophase, k2, were calculated26 using eq 1,

k2 ) klimVM[Dt]m/[Df]m(1 + [H+]/Ka)

(1)

where [Dt]m is the total concentration of the micellized surfactant, [Df]m is the concentration of the catalyst, VM is the molar volume of the Stern layer of the micelle, and Ka is the dissociation constant for the catalyst at pH 8.2 at which the reactions were performed. VM values of 0.37 dm3 mol-1 for CTABr27 and 0.597 dm3 mol-1 for 16-4-16, 2Br- micelles,28 respectively, were used. The term [Dt]m/ [Df]m accounts for the dilution of the catalyst in the CTABr or gemini surfactant comicelles, and the term (1 + [H+]/ Ka) denotes the fraction of the dissociated catalyst at pH 8.2. Comparison of k2 values in Table 3 confirms greater reactivity of the HOBt catalysts in gemini 16-4-16 comicelles over that in CTABr micellar aggregates for both PNPDPP and PNPH. Kinetic Studies with Excess Substrates. In the presence of either type of excess substrate, PNPH or PNPDPP with respect to catalyst, quantitative monoexponential release of p-nitrophenoxide ion was observed (Figure 5). Clearly there was no evidence any of burst kinetics and such plots indicate rapid turnover. Importantly, the catalytic effectiveness was still intact even after the hydrolysis of ∼25-fold excess PNPH. Analysis of the time-dependent formation of p-nitrophenoxide ion for the cleavage of excess substrate revealed pseudo-first-order character of the reaction in all the cases examined. Therefore either the formation of the N-O-acylation or N-O-phosphorylation species or their decay should be the rate-determining step in the esterolytic reaction. The formation of N-O-acylated HOBt has been demonstrated, and its hydrolysis in alkaline medium was reported.29 Based on the observation of a monoexponential timecourse, the following mechanism for the cleavage of esters by HOBt-based catalyst may be proposed. Since there is no accumulation of N-O-acyl species in the reaction media, the hydroxide ion attack on the N-O-acylated species must be fast. Thus, it cleaves the intermediate species fast enough to furnish hexanoate or diphenyl phosphate ion and concomitantly regenerate the catalyst in the reaction media which becomes free to react with a fresh substrate molecule. Thus, formation of O-acylated or O-phosphorylated species is a slow process, which is the ratedetermining step. In the case of gemini micelles (16-4-16) as media, also we did not observe burst type kinetic profiles with 2 and other catalysts. Here, too, the observed turnover was fast and the analysis of the time-dependent formation of p-nitrophenoxide ion for the cleavage of excess substrate revealed perfect pseudo-first-order kinetic behavior. This suggests that the same mechanism operates for the hydrolysis of substrates in both types of micellar aggregates. (26) Fornasier, R.; Tonellato, U. J. Chem. Soc., Faraday Trans. 1980, 76, 1301. (27) (a) Scrimin, P.; Tecilla, P.; Tonellato, U.; Bunton, C. A. Colloids Surf., A 1998, 144, 71. (b) Mancin, F.; Tecilla, P.; Tonellato, U. Langmuir 2000, 16, 227. (28) Wettig, S. D.; Nowak, P.; Verrall, R. E. Langmuir 2002, 18, 5354. (29) (a) McCarthy, D. G.; Hegarty, A. F.; Hathaway, B. J. J. Chem. Soc., Perkin Trans. 2 1977, 224. (b) McCarthy, D. G.; Hegarty, A. F. J. Chem. Soc., Perkin Trans. 2 1977, 231.

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In summary, the above results allow us to draw the following conclusions. The compounds 1-5 manifest truly catalytic ability to induce hydrolysis of reactive, hydrophobic carboxylate ester and neutral phosphotriester substrates under micellar conditions. The observed rate accelerations and the intrinsic activities of these catalysts can be explained using pseudophase models known to estimate second-order rate constants in comicellar media. Anionic HOBt carboxylate catalysts 4 and 5 display the best kinetic advantages in such hydrolytic reactions, and cationic micelles act as good hosts for the cleavages of both types of substrates. However, cationic gemini micelles are significantly more effective as hosts for carrying out such hydrolytic reactions. While the affinity constants of the substrates and the catalysts were larger for geminis than the corresponding parameters in their monovalent counterparts, the depleted hydration of the gemini micellar surfaces may also aid the potentiation of the ester hydrolysis processes. The HOBt catalytic systems were found to be truly catalytic in both types of micellar aggregates. Work is now underway in our laboratory to assess the efficiency of other chemical reactions in gemini micelles. Experimental Section General Methods. UV-visible spectra were recorded on a Shimadzu model 2101 UV-visible recording spectrophotometer. Double-distilled water was used for kinetic studies, and pH measurements were made with Systronics Digital pH-meter 335. Materials. All the reagents and solvents were of the highest grade available commercially and were purified, dried, or freshly distilled as required. PNPDPP was prepared and purified by literature methods.18 Gemini surfactants 16-m-16, 2Br- were synthesized and purified by a reported method19 and were recrystallized several times from EtOAc/hexane solvent mixtures. Kinetic Studies. Reaction mixtures were generated in quartz cuvettes of 1-cm path length. The cuvette was filled with 0.5 mL

Bhattacharya and Kumar of aqueous buffer (0.05 M tris-maleate, pH 8.2) containing a known concentration of the catalytic system. The micellar solution was equilibrated for 10 min in a thermostated cell compartment (Julabo F 25) (25 ( 0.1 °C) of a Shimadzu model UV-visible 2101 spectrophotometer. An appropriate aliquot (2.5 µL) of a stock solution of PNPH or PNPDPP in CH3CN was added using a Hamilton syringe. The reaction mixture was initiated by quick but careful stirring, and the absorbance at 400 nm was recorded as a function of time. The esterolysis followed pseudo-first-order kinetics, and the rate constants were obtained by nonlinear fit of the equation (A∞ - A0)/(A∞ - At) ) ekt where A∞ and At are the absorbances at infinite time and time t, respectively. Values of kobs were within (2% in triplicate runs. Conditions for the determination of rate constant-[surfactant] profiles are also described above, with the results summarized in Figure 1a,b. Conditions for the determination of pH-rate constant profiles are described in the text (above), and the profiles for 2 and 4 in CTABr and gemini 16-4-16, 2Br- micelles appear in Figure 3a,b, respectively. Reactions of excess substrates were carried out, and these micellar reactions followed to >95% completion and showed good first-order kinetics (r > 0.999). Light-Scattering Experiments. Mean hydrodynamic diameters were determined by laser light scattering using a Zetasizer 3000 (Malvern Instruments Ltd., Malvern, U.K.). Light scattering employed a He-Ne laser source at a wavelength of 633 nm, keeping the detector angle at 90°. The data were analyzed using internal instrument software involving a Malvern 7132 digital (16-bit) autocorrelator. A 220 nm latex standard was used for calibration.

Acknowledgment. The work was supported by the Department of Science and Technology. V.P.K. thanks CSIR for a Senior Research Fellowship. Supporting Information Available: Synthetic scheme of 2-5 and characterization of compounds (Scheme S1). This material is available free of charge via the Internet at http://pubs.acs.org. LA048858F