Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394

Solvolysis of Tris-p-nitrophenyl-phosphate in aqueousand reverse micelles

U. Costas-Costasa, C. Bravo-Dıaza, H. Chaimovichb, I.M. Cuccoviab,∗a Departamento de Qu´ımica Fısica, Faculdad de Ciencias, Universidad de Vigo, Vigo, Spain

b Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade de S˜ao Paulo, Av Prof Lineu Prestes 748, S˜ao Paulo, SP 05508-900, Brazil

Received 1 February 2004; accepted 30 April 2004

Abstract

The widespread use of toxic phosphates and phosphonates as insecticides, and their use as chemical weapons, has led to investigation of fastdetoxification and decontamination methods. Micelles, microemulsions, cyclodextrines and liposomes have been used to accelerate phosphateester decomposition by nucleophiles. Here, hydrolysis, methanolysis and hexanolysis of Tris-p-nitrophenyl phosphate (TNPP), a model forr quantitativelya mmoniumb pectively.T n those in thea hexanolysisd lvents.S e( tiod©

K

1

pmwDtppc

pho-s, hasina-oseo-cle-

, mi-beenucle-bic

ed toellesnd

0d

eactive phosphate esters, were studied in homogeneous phase, aqueous and reverse micelles. Kinetic micellar effects werenalyzed using pseudo-phase models. TNPP hydrolysis was catalyzed by cetyltrimethylammonium chloride (CTAC), cetyltrimethylaromide (CTAB), and hexadecylammonium propanesulfonate (HPS), micelles by factors of five, CTAC, and three, CTAB, HPS, reshe calculated rate constants for spontaneous and acetate-catalyzed hydrolysis in the micellar phase were significantly higher thaqueous phase. While in water and in methanol the effect of the acetate cation was negligible, the catalytic efficiency of acetate forepended on the nature of the cation with the K+ salt being ca. 20 times more efficient than the tetraethylammonium salt in non-polar soodium dodecylsulfate, SDS, micelles inhibited TNPP hydrolysis by a factor of eigth. Reverse micelles of CTAB inn-hexanol/isooctan

10:90, v/v) did not catalyze TNPP hydrolysis, but changed the bis-p-nitrophenyl phosphate/hexyl-bis-p-nitrophenylphosphate product raepending of CTAB concentration and water/detergent ratio.2004 Elsevier B.V. All rights reserved.

eywords:Tris-p-nitrophenyl-phosphate; Reverse micelles; CTAB

. Introduction

Phosphate mono- and di-esters are relatively stable andlay a central role in biochemical reactions[1]. Phosphateonoesters are intermediates in a variety of metabolic path-ays and phosphate di-esters are the essential components ofNA and RNA[1]. Phosphotriesters are more reactive than

he corresponding mono- and di-esters and do not partici-ate in living organisms as metabolites[1–3]. Some phos-hotriesters are neurotoxic and irreversibly inhibit acetylholinesterase[4,5].

∗ Corresponding author. Tel.: +55 11 3091 3839; fax: +55 11 3091 2186.E-mail address:imcuccov@quim.iq.usp.br (I.M. Cuccovia).

The widespread use of toxic phosphates and phosnates as insecticides, and their use as chemical weaponled to investigation of fast detoxification and decontamtion methods[6]. Several nucleophiles react and decomporganophosphorous compounds[2]. Spontaneous decompsition of phosphotriesters, as well as their reaction with nuophiles, has been investigated in homogeneous phase[7–11].Biomimetic models such as micelles, reverse micellescroemulsions, cyclodextrines and liposomes, have alsoused to accelerate reactions of phosphate esters with nophiles[7,12–21]. These aggregates solubilize hydrophosubstrates and locally concentrate nucleophiles[12]. Catal-ysis of phosphotriester decomposition has been relatchanges in the water properties at the interface of micand vesicles[22]. Reactions involving phosphotriesters a

927-7757/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2004.04.084

386 U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394

Scheme 1.

nucleophiles have also been studied in the presence of macro-cyclic complexes containing metals. These macrocyclics ac-celerate the alkaline hydrolysis of phosphotriesters and havebeen used as simple models for metal-containing alkalinephosphatases[23–30].

One of the phosphotriesters commonly used in stud-ies of the catalytic properties of macrocyclics is Tris-p-nitrophenylphosphate, TNPP. In spite of the extensive useof TNPP as a model substrate[23–30], TNPP hydrolysis andthe pH effect on reaction rate in homogeneous phase, have notbeen described in detail. The neutral and alkaline hydrolysisof TNPP has been studied in solvent/water mixtures due thelow solubility of this substrate in water, and the mechanismof acetate ion-catalyzed methanolysis have been described(Scheme 1) [31]. Phosphoryl transfer to methanol from TNPPis general base catalyzed by acetate[31].

Here, we report the study of the hydrolysis and alcoholysisof TNPP in homogeneous phase and in aqueous and reversemicelles.

2. Experimental section

2.1. Materials

h P,2m y-d -1-p lene9 r,C A)( tate,N mo-n asl reet iumb e-t ium

dodecylsulfate (SDS) (Sigma) was recrystallized twicefrom methanol by heating at 50◦C and cooling at roomtemperature. No minimum was found in the surface tensionversus [SDS] plot. Sodium and potassium acetate (Baker)were dried at 120◦C for 24 h. Chloroform (Merck) waswashed with water, dried over CaCl2 and distilled beforeuse.

Synthesis ofn-hexyl-bis-p-nitrophenylphosphate, hexyl-bis-NPP: 0.2 g of TNPP, dissolved in 5 mL of chloroform,was added to 5 mL of hexanol. The reaction mixture wasmaintained for 4 days at 65◦C. Chloroform was eliminatedin a rotary evaporator and the remaining hexanol was re-moved in a Speed-Vac. The crude product was dissolved in20 mL of chloroform and extracted (three times) with aque-ous 5% sodium bicarbonate and then with water. The prod-uct was dried (Na2SO4) and the chloroform removed in arotary evaporator. The product was purified by silica (1 g)chromatography eluted with hexane/chloroform (75:25, v/v).Aliquots of 2 mL were collected and the elution of hexyl-bis-NPP followed by HPLC. The pooled product-containingsolution was evaporated and 65 mg of a product were ob-tained (yield ca. 30%).1H NMR of hexyl-bis-NPP wasrecorded using a Varian-Inova, 300 MHz spectrometer.1HNMR of n-hexyl-bis-NPP in CDCl3: δ: 0.88 (3H, t,J =6.9 Hz, CH3); 1.25–1.39 (6H, m, (CH2)3 ), 1.70–1.77 (2H,m7n

ofT ac-e mix-t vap-o se ywa llizedfb7n

Isooctane (Merck),n-dodecane (Aldrich, 99+%),n-exanol (Fluka), Tris-p-nitrophenylphosphate, TNP-[N-morpholino] ethanesulfonic acid, Mes, 3-[N-orpholino] propanesulfonic acid, Mops, Tris [hroxymethyl] aminoethane, TRIS, 4-[2-hydroxyethyl]iperazineethanesulfonic acid, HEPES, polyoxyethy-lauryl ether, C12E9, polyoxyethylene 5-decyl ethe10E5 (Sigma), acetic acid (Synth), triethylamine (TE

Aldrich), potassium acetate, KAc, and sodium aceaAc, were used as received. Hexadecyltrimethylamium chloride (CTAC), 25% aqueous solution (Aldrich) w

yophilized, recrystallized from acetone/methanol (thimes) and dried in vacuum. Hexadecyltrimethylammonromide (CTAB) (Merck) was recrystallized from ac

one/methanol (three times) and dried in vacuum. Sod

, CH2 ), 4.33 (2H, dt,J = 7.9 and 6.6 Hz, CH2O ),.38–7.44 (4H, m,meta-nitro) and 8.25–8.30 (4H, m,ortho-itro).

Methyl-bis-NPP was synthesized dissolving 0.2 gNPP in 25 mL of methanol containing 0.1 g sodiumtate and 0.05 g acetic acid and maintaining the

ure for 2 h at room temperature. The solvent was erated and dicloromethane was added.p-nitrophenol waxtracted with 5% NaHCO3 aqueous solution followed bater. The dichloromethane solution was dried (Na2SO4)nd the solvent evaporated. The product was recrysta

rom HCCl3/CCl4 as described[31]. 1H NMR of methyl-is-NPP in CDCl3: δ: 4.07 (3H, d,J = 11.8 Hz, CH3);.38–7.44 (4H, m,meta-nitro) and 8.25–8.30 (4H, m,ortho-itro).

U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394 387

2.2. Methods

All kinetics were done at 30◦C unless stated and reportedrate constants are the mean of, at least, three independentexperiments differing not more than 5%.

TNPP solvolysis, in micelles or non-aqueous solvents,was initiated by the addition of 0.01 mL of a TNPP so-lution (0.003 M in acetonitrile) to 1.5 mL of the reactionmixture (final concentration of TNPP = 2.0× 10−5 M).The reactions in aqueous solutions were done in 5% ace-tonitrile/water (v/v) using a final concentration of 1.65×10−6 M TNPP in order to avoid precipitation of the triester.If acetonitrile and/or detergent is not added and if the sub-strate concentration is higher the reactions were not firstorder.

Reactions were followed (Varian Cary 3E spectropho-tometer) at 307 or 317 nm when using solvent mixtures orreverse micelles, respectively, at 400 nm at pH higher than7.0 and at 330 nm in water/acetone mixtures in order to avoidinterference of acetone absorption.

Buffer solutions in organic solvents were prepared byweighing the acetate salt and acetic acid at the desiredmolar ratio. Aqueous buffers were prepared by neutraliza-tion of acetic acid with the desired base to the adequatepH.

ara-t in-t i-c i.d.× tedwes andt rn , re-s PPa s int inedw ob-t lpN1 l-b[(

olu-t 0,v alla thes nto gentm re-p ofC izet

Fig. 1. Effect of pH on thek� for TNPP hydrolysis in 5% acetonitrile, [TNPP]= 1.65× 10−6 M. (�) HBr; (©) acetate 0.02 M; (�) borate 0.02 M; (�)Tris–HCl 0.01 M; (�) Mops 0.01 M; (�) Mes 0.01 M.

3. Results

3.1. Reactions in aqueous solution

The observed rate constant,k�, for TNPP hydrolysis didnot change between pH 3–8 and the reaction was OH−-catalyzed above pH 8 (Fig. 1). The hydrolysis of TNPP wasgeneral base catalyzed by several buffers (see below) but, un-der the conditions ofFig. 1, buffer catalysis can be neglected.The values of the rate constants for the water reaction,kw, de-termined in the pH-independent region, and for OH− attack,kOH

2 determined above pH 8 by plottingk� versus [OH−],were consistent with those obtained previously in aqueous orin buffer/acetone at 25◦C (Table 1) [9–11].

Due to the low water solubility, and to ensure thatk� cor-responds to the rate of non-aggregated substrate, TNPP hy-drolysis was also studied in mixtures of HCl/acetone. Thevalues ofk� increased with decreasing acetone (Table 1) andthe value obtained by extrapolation to zero solvent is consis-tent with that obtained in 5% acetonitrile (Table 1). Thus, thevalues ofk� between pH 3 and 7, acetonitrile 5% (v/v), werewithin the range described in the literature[9–11]and reflectthe hydrolysis of monomeric TNPP.

The second-order rate constants for acetate (Ac)- and Mes(N)-catalyzed TNPP hydrolysis,kAc

2w andkN2w, respectively,

c ormo

3

int1 is,uw ew them lyzed

HPLC analysis were performed in a Shimadzu appus equipped with SPD-10A UV–vis detector, C-R6Aegrator, IC-6 AD pump, 20�L sample loop and a Mrosorb C18 (Rainin) reverse phase column (4.6 mm

25 cm; 5 mm particle size). The samples were diluith methanol (1:4, v/v) containing 1× 10−4 M HCl, andluted with methanol/H2O (75:25, v/v) (0.8 mL min−1, pres-ure 140). The compounds were detected at 285 nmhe retention times were 4.2, 5.3, 6.4, and 10.6 min fop-itrophenol, TNPP, methyl-bis-NPP and hexyl-bis-NPPpectively. The concentrations of hexyl-NPP, methyl-Nnd p-nitrophenol were calculated from the peak area

he HPLC chromatograms using standard curves obtaith pure compounds. Product concentrations were

ained as follows: [p-nitrophenol] = area ofp-nitrophenoeak (�V s) × 6.94 × 10−11(�V−1 s−1 M), [methyl-bis-PP] = area of methyl-bis-NPP peak (�V s) × 1.63 ×0−11 (�V−1 s−1 M), [hexyl-bis-NPP] = area of hexyis-NPP peak (�V s) × 2.11 × 10−11 (�V−1 s−1 M),

TNPP] = area of TNPP peak (�V s) × 1.61 × 10−11

�V−1 s−1 M).Reverse micelle solutions were prepared from stock s

ion containing 0.2 M CTAB inn-hexanol/isooctane (10:9/v). In order to solubilize CTAB in this solvent, a smmount of buffer (acetate, Mes or Mops) was added totock solution (0.015 mL/mL of 0.2 M CTAB). This amouf water was included in the calculation of the water/deterolar ratios,ω0. Reverse micelles of CTAB also were pared in HCCl3/dodecane (60:40, v/v). To each mililiterTAB (0.1 M) 0.0075 mL of buffer were added to solubil

he detergent.

alculated from linear plots of concentration of the basic ff the buffer versusk� (not shown), are similar (Table 1).

.2. Micellar catalyzed reactions in aqueous solutions

CTAC and CTAB micelles catalyzed TNPP hydrolysishe pH-independent region (Fig. 2A). Increasing [CTAX] in× 10−3 M HX (X = Br or Cl) accelerated TNPP hydrolysntil a maximum value of the observed rate constant,kMax

ψ ,as reached at high detergent (Fig. 2A). The rate increasith [CTAX] can be attributed to substrate binding toicelles, and to a higher rate constant for the water-cata

388 U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394

Table 1Buffer and pH effects on TNPP hydrolysis and solvolysis

Conditions k� (min−1) kOH2 (M−1 min−1) kAc

2 (M−1 min−1) kN2 (M−1 min−1)

Water, 5% acetonitrile 0.12MeOH 0.0013n-HexOH 0.0011n-HexOH:isooctane (10:90, v/v) 0.000450% acetone/buffer, pH 5.6 0.061a; 0.050b

20% acetone, HCl 0.001 M 0.07530% acetone, HCl 0.001 M 0.05650% acetone, HCl 0.001 M 0.044Water, OH− 2060c; 2040d, 2000e, 2300g

Water, Ac 1.32Water, Mes 1.01n-HexOH:isooctane (10:90, v/v), TEA-Ac 1.99n-HexOH:isooctane (10:90, v/v), KAc 36.7MeOH, KAc or TEA-Ac 7.42MeOH, NaAc 10.8; 3.32f

a Data from Ref.[9], at 25◦C, buffer/acetone (1:1, v/v) [buffer Na+ citrate 0.042 M and Na+ phosphate 0.0116 M, pH 5.6 (theoretical) or 7.25 (measured)].b Our data under the conditions of reference[9].c Data from Ref.[9] at 25◦C.d Reference[10], at 25◦C.e Data from reference[4] in water/acetone (1:1, v/v) at 25◦C.f NaBr, 0.475 M, 25◦C, reference[31].g This work, in 5% acetonitrile.

hydrolysis of TNPP at the micellar interface (Scheme 2). ThekMaxψ /kw ratios for TNPP hydrolysis in CTAB and CTAC

micelles were ca. 3 and 5, respectively (Table 2).The effects of [CTAX] on the observed rate constant for

TNPP hydrolysis,kHyd, were analyzed quantitatively using apseudo-phase model[12,32]. According to this model, TNPPdistributes between the aqueous and the micellar phase witha binding constantKS and reacts in water and in the micel-lar pseudo-phase with rate constantskw andkm, respectively(Scheme 2, Eqs.(1)–(3).

kHyd = kw + kmKSCD

1 +KSCD(1)

KS = Sb

SfCD(2)

Table 2Effect of micelles on the rate constants for TNPP solvolysis

Detergent, buffer kMaxψ

/km km (min−1) km/kw kAc2m (M−1 min−1) kN

2m (M−1 min−1) kAc2m/k

Ac2w kN

2m/kN2w

CTAB, HBra 2.66 0.32a 2.66CTAB, Mesb 12.1b 11.98CTAB, Acc 15.0c 11.36CTACl, HCla 4.83 0.58a 4.83HPS, HCla 2.95 0.35 2.95C12E9, HCl 1.63 0.20 1.63C E , HCl 1.28 0.15 1.28SCC

pKw = 11

= 0.8,

using

CD = CT − CMC (3)

CD is the concentration of micellized detergent, CMC is thecritical micellar concentration andCT is the stoichiomet-ric detergent concentration.S is the analytical concentrationof TNPP and the sub-indicesb and f refer to the analyti-cal concentrations of TNPP bound to the micelles and freein the aqueous phase, respectively. The data ofkHyd versus[CTAX] were fitted using Eq.(1) usingkm andKS as ad-justable parameters (Fig. 2A). A KS of 1000 M−1 was usedand the best-fit value forkm in CTAC was twice that forCTAB (Table 2). As expected for a hydrolysis reaction of aneutral substrate where the rate in the micellar pseudophaseis higher than that in water[12], the calculatedkm valueswere identical to the respective values ofkMax

ψ (Fig. 2A,Table 2).

10 5

DS, HCl 0.12 0.014 0.12TAB, Rev, HBr, hydrolysisd 0.149d 1.31TAB, Rev, HBr, hexanolysisd 0.008d

a Parameters used were:Ks = 1,000 M−1.b α = 0.2;KOH/N = 0.6;KOH/Br = 0.8;V = 0.37 LM−1; KS = 1000 M−1;

0−4 M.c KOH/Br = 0.8;V = 0.37 LM−1; KS = 1,000 M−1; pKw = 13.833,KOH/Acd In n-hexanol/isooctane (10:90, v/v),km corresponds tokHyd

m calculated

3.833,k2N =1.01 M−1 min−1, pKMesa = 6.1;KHN = 150 M−1; CMC = 5×

pKAca = 4.75,KHAc = 5, kAc

2w = 1.32 M−1 s−1.

Eq.(11) (see text).

U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394 389

Fig. 2. Effect of aqueous micelles onk� for TNPP hydrolysis. [TNPP] =6.7 × 10−6 M. (A) (�) CTAB, HBr 1 × 10−3 M and (©) CTAC, HCl 1× 10−3 M; (�) HPS in water; (�) HPS in HCl 1× 10−3 M. (�) C12E9 inwater; (�) C12E9 in HCl 1 × 10−3 M; (�) C10E5 in HCl 1 × 10−3 M; (�)SDS in HCl 1× 10−3 M. Lines calculated using Eq.(1). (B) NaMes buffer0.02 M, pH 5.33.Line calculated using Eq.(4). (C) CTAB 0.1 M and NaMesbuffer 0,02 M, pH 5.0. Line calculated using Eq.(4).

Negatively charged SDS micelles inhibited TNPP hydrol-ysis by a factor of ca. 9 at high [SDS] (Fig. 2A, Table 2). Theopposite effect of micelles of different head groups in a wa-ter reaction can be rationalized in terms of different locationof the substrate in the micelle, water organization near theinterface, high concentration of head group at the interface

Scheme 2.

or stabilization of transition state by the charged interface ofmicellar interface[33–35].

Although rate of TNPP hydrolysis was determined at verylow substrate concentration (see figure legends) rate measure-ments can be affected by substrate aggregation in the aque-ous phase[36]. The non-ionic detergents C10E5 and C12E9completely solubilizes TNPP and the measured reaction rateobserved in both solutions was very similar to that obtainedin aqueous phase (Fig. 2A, Table 2). Therefore we can confi-dently conclude thatkw can be attributed to monomeric TNPPhydrolysis[37].

The effect of zwitterionic micelles was very similar tothat obtained in CTAB (Fig. 2A, Table 2), probably due tothe presence of the positive ammonium group in the structure[33].

The hydrolysis of TNPP in Mes buffer was also catalyzedby CTAB, but thek� versus [CTAB] dependence was strik-ingly different from that without added buffer (Fig. 2A andB). The maximum in the value ofk� (Fig. 2B) is typicalof bimolecular reactions where one substrate is neutral andthe charge of the other reacting specie is that of the micellarcounterion[38]. Increasing [CTAB] rises the binding of thenegatively charged basic form of Mes buffer, N, at the mi-cellar surface but, at higher [CTAB], N is displaced from themicellar surface by [Br−] reaching the rate in water (Fig. 2B)[sBM water([

k

N

B

B

K

K

32,38]. At high detergent concentration the value ofk� wasimilar to that obtained in CTAB in HBr at pH 3.0 (Fig. 2A and). The variation ofk� for TNPP hydrolysis with [CTAB] ines buffer can be described as the sum of reactions withk

Hydψ , Eq. (1)) and Mes catalyzed hydrolysis (Eqs.(4)–(9))

38].

ψ = kHydψ +

[N]TKa[kN2w + (kN

2m/V )

×KSKN/Br(Brb/Brf )]

[(Kw/OHf )(1 +KNHCD)

+Ka(1 +KN/Br(Brb/Brf ))](1 +KSCD)

(4)

T = NHT + N (5)

rb = αCD + Nb + OHb (6)

rf = (1 − α)CD − OHb −Nb (7)

N/Br = NbBrfNfBrb

(8)

NH = NHbNHfCD

(9)

390 U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394

NT is the total concentration of Mes buffer, given by the totalconcentrations of basic and acid forms of Mes (N and NH,respectively).kN

2m is the second-order rate constant for thereaction catalyzed byN in the micellar pseudophase.Ka isthe dissociation constant of Mes (pKa 6.1) [39] and Brb andBrf are given by Eqs.(6) and (7), respectively.KN/Br is theexchange constant betweenN and Br (Eq.(8)) andα is themicellar dissociation degree.KNH is the binding constant ofthe NH to the micelle, (Eq.(9)), Kw is the water ionizationconstant (pKa = 13.833 at 30◦C) [39], OHf is the hydroxideconcentration in the buffered aqueous phase andV is the mo-lar volume of the detergent (taken as 0.37 L mol−1 for CTAB)[22]. The parameters used in the fit of data inFig. 2B withEq. (4) are presented inTable 2. The best-fit value forkN

2mwas ca. 12 times greater thankN

2w (Table 2). Experiments atlow CTAB were not performed and thus the data does notexhibit the theoretically expected maximum[32].

The effect of acetate (Ac), on the hydrolysis of TNPPwas determined at pH 5.0 in CTAB 0.1 M, in the presenceof 0.02 M Mes buffer (Fig. 2C). The values ofk� increasedwith total buffer concentration, [NaAc+HAc]T, where HAcrefers to acetic acid. The data inFig. 2C were fitted usingEqs.(4)–(9)and the parameters inTable 2. The value ofkAc

2mwas ca. 15-fold greater thankAc

2w, and similar tokN2m.

3

andtf with[ Accv -fp ionics Ac-c tate,Te

dh sol-u cw . Oneo ecteda hex-a c,a iono nol[ nn an inm

ix-t c,k anol,r

Fig. 3. (A) Effect of the concentration of acetate salts onk� for TNPPsolvolysis: methanolysis with (�) KAc and (©) TEA-Ac; hexanolysis (inn-hexanol/isooctane (10:90, v/v)) with (�) KAc and (�) TEA-Ac. (B) Ef-fect of percentn-hexanol (v/v), inn-hexanol/isooctane mixtures, onk� forTNPP hexanolysis with: (©) KAc, 0.0034 M, [KAc + AcH]T = 0.0081 M(�) [TEA-Ac] = 0.0104 M, [HAc + TEA-Ac]T = 0.021 M.

3.4. Reactions in reversed micelles

3.4.1. Product compositionThe expected products of TNPP solvolysis in CTAB re-

verse micelles prepared inn-hexanol/isooctane and buffersarep-nitrophenol, bis-p-nitrophenylphosphate, bis-NPP, andhexyl-bis-NPP (Scheme 1). Product composition, determinedafter complete reactions in CTAB reverse micelles at severalmolar water/CTAB ratios,ω0, and buffer concentration, arein Fig. 4A. The % yield of hexyl-bis-NPP formed in CTABreverse micelles depended on detergent concentration andω0, but was only slightly modified by buffer nature or con-centration (Table 3). The % yield of hexyl-bis-NPP in CTAB(0.01 M,ω0 = 4, HCl 0.001 M) was equal to that formed atthe same [detergent] andω0 in 0.02 M acetate buffer, pH 5(Fig. 4A).

In ω0 = 4, the percent yield of hexyl-bis-NPP decreasedfrom 30 to 5% as [CTAB] increased from 0.01 to 0.1 M.At higherω0’s the yields of hexyl-bis-pNPP were 5% andessentially [CTAB] independent (Fig. 4A).

.3. Reactions in organic solvents

TNPP reacts with methanol yielding methyl-bis-NPPhe reaction is catalyzed by Ac[31]. The values ofk�

or the Ac-catalyzed TNPP methanolysis increasedAc] (Fig. 3). The second-order rate constant for Naatalysis,kNaAc

2MeOH, was obtained from the linear plot ofk�

ersus [NaAc] (Table 1). The value ofkNaAc2MeOH was ca. 2–3

olds higher than that determined previously[31]. This dis-arity can be attributed to differences in temperature andtrength (Table 1). The second order rate constants foratalyzed methanolysis with tetraethylammonium aceEA-Ac,kTEA-Ac

2MeOH, and potassium acetate, KAc,kKAc2MeOH, were

qual and 30% lower thankNaAc2MeOH (Fig. 3, Table 1).

The solvent used here for reverse micelles containen-exanol, expected to react with TNPP. Due the limitedbility of NaAc in n-hexanol/isooctane, KAc and TEA-Aere used to study acetate-catalyzed TNPP hexanolysisf the expected products, i.e., hexyl-bis-pNPP, was dett the end of the reaction. The rate constants for TNPPnolysis, determined with and without KAc and TEA-Are inTable 1. k� increased linearly with the concentratf Ac, indicating that in this solvent, as well as in metha

31], Ac is the catalytic species (Fig. 3A). Reaction rates i-hexanol/isooctane were more sensitive to the cation thethanol,kKAc

2Hexbeing 18 times higher thankTEA-Ac2Hex (Table 1).

Changes in the % ofn-hexanol in isooctane/hexanol mures did not affectk� using TEA-Ac. In contrast, using KA� decreased sharply with the increase in the % of hexemaining constant above 30% (Fig. 3B).

U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394 391

Fig. 4. (A) Effect of CTAB reverse micelles on the % yield of hexyl-bis-NPP. TNPP = 4× 10−5 M. In HCl 0.001 M: (©) ω0 = 4; (�) ωo = 17.5; (�)ω0 = 20. In NaAc buffer 0.02 M, pH 5.5: (�) ω0 = 4; (�) ωo = 12.5; (�)ω0 = 26. (B) Effect of CTAB reverse micelles onk� for TNPP solvolysis inn-hexanol/isooctane (10:90 v/v), HBr 2× 10−4 M, at the followingω0: (©)12.5; (�) 13.6; (�) 14.9; (�) 17.7; (�) 20;(�) 25. Lines calculated usingEq. (1). (C) Plot of 1/ωo vs. 1/km. km’s were calculated fitting the data inFig. 4B with Eq.(1).

3.4.2. KineticsThe rate of hydrolysis of TNPP in CTAB reverse micelles

in n-hexanol/isooctane (10:90, v/v), HBr (2× 10−4 M) were[CTAB] andω0 dependent (Fig. 4B). At constantω0, k� in-

Table 3Effect of buffer and pH atω0 = 12.5 on the % yield of hexyl-bis-NPP inreverse micelles of CTAB, 0.05 M inn-hexanol/isooctane (10:90, v/v)

[Buffer]T (M) pH Hexyl-bis-NPP %

AcNa-HAc, 0.01 5.5 10.0AcNa-HAc, 0.05 5.5 13.6Mes, 0.01 6.5 7.5MOPS, 0.01 7.5 8.0HEPES, 0.01 8.5 6.9

creased with [CTAB] to a plateau (Fig. 4B). This pattern sug-gests that the reaction rate is higher in the micelle than in theorganic phase since, as the micelle concentration increases,[TNPP] at the interface also increases. As TNPP is sparinglysoluble in water the relative amount of substrate solubilizedin the water pool can be neglected and its distribution can bedescribed as a partitioning between the organic solvent andthe micellar interface.

In the organic phase the predominant reaction should bethe hexanolysis because the amount of water dissolved inthis solvent is negligible. As CTAB and buffer increases, thesubstrate is transferred to the micellar interface, where it canreact with water and hexanol. Hence, the reaction in CTABreverse micelles containing HBr, can be quantitatively ana-lyzed using Eq.(10).

kψ = kHex + kRevm KSCD

1 +KSCD(10)

kRevm = kHex

m + kHydm (11)

wherekRevm is the sum of the rate constants of hydrolysis,k

Hydm ,

and hexanolysis,kHexm , at the interface andkHex is the rate

constant of hexanolysis in bulk solvent. A very good fit wasobtained at allω0, Fig. 4B, using Eq.(10)and the followingparameters:KS = 5 M−1, CMC = 5× 10−4 M andkHex = 4× −4 −1 Rev fore taiω llarri lysisw e ati df Ath xyl-bw

sl( re-v anicp

lvol-y e in[w -

10 min . km was the only adjustable parameterachω0. The values ofkRev

m obtained from fitting the dan Fig. 4B increased withω0. A linear fit of 1/kRev

m versus0 (Fig. 4C) provided an estimate of the interfacial mice

ate constant at infiniteω0, kLim, of 0.157 min−1. SincekLims the sum of rate constants of hydrolysis and hexanohen TNPP is totally transferred to the micellar interfac

nfinite ω0, the values ofkHydm and kHex

m can be calculaterom Eq.(11)and the product yields under this condition.ighω0 the product yields were 95% bis-NPP and 5% heis-NPP (Fig. 4A) and the calculated values ofkHyd

m andkHexm

ere 0.149 and 8× 10−3 min−1, respectively.The calculatedkHyd

m in CTAB reverse micelles is two timeower thankm in aqueous CTAB micelles and similar tokwTable 2). The calculated rate for hexanolysis in CTABerse micelles is ca. eight times greater than in the orghase (Tables 1 and 2).

The basic form of several buffers also catalyzed the sosis of TNPP in CTAB reverse micelles. The increasCTAB] at severalω0 produced a non-linear increase ink�

ith both NaAc and TEA-Ac (Fig. 5A). As expected for gen

392 U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394

eral base catalysisk� increased linearly with and [Ac + HAc]Tat constant [CTAB] andω0 (Fig. 5B).

The effect of [Mes] on the rate of solvolysis of TNPP atfixed [CTAB] was also dependent onω0 and the rate con-stants were lower with Mes than with acetate buffer at the

Fv=11ff(

same concentrations (Fig. 5C) evidencing that Ac is a bettercatalyst.

In CTAB (0.1 M) reverse micelles prepared in HCCl3/n-dodecane (60:40, v/v) (ω0 = 13.5, Na acetate buffer 0.05 M,pH 5.0) only hydrolysis of TNPP can occur. In this sol-vent the rate of TNPP hydrolysis was slower (k� = 9 ×10−4 min−1) than in CTAB reverse micelles prepared inn-hexanol/isooctane under similar conditions. This effect canbe rationalized by assuming that in this solvent mixture,TNPP distribution (between the organic phase and CTABreverse micelles) favors the organic phase.

4. Discussion

As described for other phosphotriesters, TNPP hydrolysiswas OH- and general base catalysed[2,3,8]. The rate con-stant for water hydrolysis is high when compared with otherphosphate triesters due the presence of threep-nitrophenylgroups[2,3,9].

The rate of hydrolysis was one hundred and three hundredtimes faster than methanolysis and hexanolysis, respectively.The ratio between the second order rate constant for acetatecatalysis and solvolysis (calculated from the observed rateconstant divided by [solvent]) of TNPP are approximately1 3 5 6 ol,r n bee iencyo d byt of

ig. 5. (A) Effect of CTAB reverse micelles inn-hexanol/isooctane (10:90,/v) on k� for TNPP solvolysis. (�)[AcNa + AcH]T = 0.05 M, pH 5.5,ω0

12.5; [AcNa + AcH]T = 0.02 M, pH 5.5 at followingω0: (©) 12.5; (�)7.5; (�) 20; (�) 25; [TEA-Ac + AcH]T 0.02 M, pH5.5,ω0: (©) 12.5; (�)7.5; (♦) 20; (�);25. (B) Effect of [AcNa + AcH]T buffer, pH 5.5, onk�

or TNPP solvolysis, in CTAB 0.05 M atω0 = 12.5. (C) Effect ofω0 on k�

or TNPP solvolysis at pH 5.5 in CTAB 0.05 M in: [AcNa + AcH]T, 0.02 M©). [Mes buffer]T, 0.01M; (�); 0.02 M (�); 0.05 (�) 0.10 M (�).

t v)t alyzedh inedw

or-t eingmh yf de-c and,a orderr (h anicm arentr s thed -e ad airsa ophilicr d fora lytice at int ated.

ousm tantsd l

0 , 2 × 10 and 1× 10 for water, methanol and hexanespectively. Thus, the reactivity of poor nucleophiles canhanced by the presence of adequate catalyst. The efficf the basic catalyst, depending on solvent, was modifie

he cation (Table 1). In water and in methanol the effecthe cation was negligible. Inn-hexanol/isooctane (10:90, v/he apparent second-order rate constant for acetate-catexanolysis with KAc was 18 times higher than that obtaith TEA-Ac.In the Ac-catalyzed TNPP hexanolysis, high prop

ions of n-hexanol decreased the rate, the effect bore pronounced with KAc, which is poorly soluble inn-exanol/isooctane (Fig. 3B). TEA, on the other hand, ma

orm a stable ion pair with acetate in this solvent,reasing the effective concentration of the free bases a consequence, decreasing the apparent secondate constant for Ac catalysis. Under these conditionsn-exanol/isooctane, 10:90, v/v) the capacity of the orgedia to dissociate the ion pair may determine the app

eactivity of the nucleophile, as in other reactions such aecarboxylation of benzisoxazol[40]. It is apparent, howver, that the effect of adding moren-hexanol must haveual effect: while increasing the dissociation of the ion pcetate solvation may compensate and decrease nucleeactivity and hence, as a limit, no difference was observecetate catalysis with both salts. The identity of the catafficiency for both acetate salts in MeOH also suggests th

his solvent both salts are similarly solvated and dissociQuantitative analysis of hydrolysis of TNPP in aque

icelles indicates that the value of the micellar rate consepended of the head group of the micelles[33]. Neutra

U. Costas-Costas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 250 (2004) 385–394 393

detergents did not affect reaction rate, positive and zwitte-rionic micelles accelerated and negatively charged micellesinhibited. These effects can be attributed to several featuresthat distinguish these aggregates such as charge, interfacialwater concentration and reactivity and detailed local struc-ture. The rate limiting step for nucleophilic displacement onphosphorous, in reactive and symmetrical triesters such asTNPP, is generally the attack[2,3,41]. Hydrolysis can involvemore than one water molecule in the reactive complex com-plicating even further a detailed analysis of micellar effects[42–44].

The buffer catalyzed hydrolysis of TNPP was acceleratedin CTAB and adequately described by PPIE theory. A cat-alytic factor of 12 was obtained comparing the second-orderrate constant for Ac reaction in micelle with that in water(Table 2). This value (i.e., 12) is small but significant, sincein most cases micellar rate enhancement derives mostly fromreagent concentration at the interface[12]. The rate constantsin the micellar phase for TNPP hydrolysis in CTAB, CTAC,HPS aqueous micelles were factors of 3, 5 and 3, respec-tively, higher than in water while in SDS micelles the ratewas eight times lower. Neutral detergents did not affect thereaction rate. These factors are large for water reactions inthe micellar surface[33]. It is attractive to speculate that wa-ter reactivity enhancement by CTAB, CTAC, HPS is relatedt (e.g.a ase inp

sis.T nter-f ndingb

d-u con-c her eter-g l-l de-c stanta thato anicp

s ofT oly-s tanti haseoI of theo alsol byB cor-p zed.A y-d anicp

Acknowledgements

This work was partially supported by FAPESP, CNPq,Pronex, CAPES and Pro-Reitoria de Pesquisa da USP, Brasil.Financial support from the following institutions is alsoacknowledged: MCyT(BQU2003-04775-C02-02), FEDER,Xunta de Galicia (PGDIT03TAL30101PR) and Universidadde Vigo, Spain. U. C. C. thanks Xunta de Galicia, Spain, forgraduate research training grant. The authors thank ProfessorC. A. Bunton, of the University of California, Santa Barbara,CA, USA and Dr. H. M. C. Ferraz of Instituto de Quımicada USP for helpful suggestions. M. N. Wandermurer, F. M.Oliveira, L. E. Narimatsu and A. P. Nunes from CentralAnalıtica, Instituto de Quımica, USP are also acknowledged.

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