catalytic effects of cationic polymeric liposomes on the alkaline hydrolysis of aniomic phenyl...

Makiomol. Chem. 185,157- 165 (1984) 157

Catalytic effects of cationic polymeric liposomes on the alkaline hydrolysis of anionic phenyl esters

Hiromi Kitano, Makoto Katsukawa, Norio Ise*

Department of Polymer Chemistry, Kyoto University, Kyoto, Japan

Klaus Dorn, Helmut Ringsdoif

Institute of Organic Chemistry, Maim University, 6500 Mainz, West Germany

(Date of receipt: July 25, 1983)

SUMMARY: Alkaline hydrolyses of anionic phenyl esters such as 4-acetoxy-3-nitrobenoic acid and

4-butyryloxy-3-nitrobenzoic acid were examined in the presence of cationic and polymeric liposomes, liposomes of low molecular weight compounds, and micelles. All the additives accelerate the reaction due to the hydrophobic interaction between substrates and additives and the electrostatic interaction both between substrates and additives and between OH - and additives. In the Arrhenius plots of the reactions catalyzed by the liposomes, discontinuous regions were observed due to the phase transition of liposomes from the gel state to the liquid crystal state. Activation parameters A H * , AS *, and A V* for these reaction systems were evaluated. Both A S * and A V * values increase upon the addition of cationic liposomes and micelles. These results were attributed to the desolvation of the activated complex by the strong electrostatic affinity to the cationic colloidal additives.

Introduction

Surfaces of cell membranes are the essential regions for many kinds of biological phenomena'), such as energy t r a n ~ f e r ~ . ~ ) and immunity4). Recently, various kinds of biomembrane model compounds have been extensively studied by many research- e r ~ ~ -'). Synthetic compounds with ionic groups and one or two long alkyl chains have been found to form bilayer structures like biomembranes. Using these bilayer forming compounds, morphologies @ and catalytic propertiesg, lo) of membrane model systems have been extensively studied.

One of the difficulties to study biomembrane models in detail is the physical instability of membranes. To solve this problem, some researchers including ourselves have tried to introduce vinyl or diacetylene groups into the bilayer forming compounds and found that the bilayer structure is retained during and after the polymerization processs1 -14,21*37).

In this paper, we report the chemical properties of the polymerized vesicles using them as catalysts in interionic reaction systems. In addition, we expected to obtain informations about polyelectrolyte catalysis. Linear polyelectrolytes and polymer latex particles catalyze many kinds of reactions by electrostatic and/or hydrophobic

0025-1 16X/84/$03.00

158 H. Kitano, M. Katsukawa, N. Ise, H. Ringsdorf, K. Dorn

interactions between substrate and catalyst. We have pointed out that desolvation and/or solvation effects of the substrate and/or the transition state are also very important for the polyelectrolyte catalyses l5 - 1 9 ) . Here we want to elucidate whether such effects are also important in colloidal and spherical polyelectrolyte systems: polymerized vesicles.

Experimental part

Materials

Hexadecyltrimethylammonium bromide (CTABr) from Nakarai Chemicals, Kyoto, was recrystallized from H,O and dried in vacuo. N,N-Didodecyldimethylammonium bromide (DDABr) was prepared by the method of Kunitake et Polymerizable monomer 1 was prepared by the method of Regen et al.,'). 10 g of 11-bromoundecanol (Aldrich, Milwaukee) and 5,6 ml of triethylamine were dissolved in 50 ml of CH,CN in an ice bath. 3.9 ml of meth- acryloyl chloride was slowly added and the solution was stirred overnight at room temperature. CH,CN was evaporated and 50 ml of benzene was added. Precipitated (C,H,),N. HCl was removed by filtration and the filtrate was concentrated by evaporation of the solvent. The oily product was dissolved again in 50 ml of CH,CN and 10 ml of N,N-dimethylhexadecylamine (Tokyo Kasei, Tokyo) was added. The solution was refluxed for 2 h. After the removal of CH,CN by evaporation, the oily product was poured into an excess amount of ethyl acetate. A slightly brown white precipitate was filtered off and purified by repeated precipitation in ethanol-ethyl acetate. The chemical structure of 1 was confirmed by 'H NMR (JEOL-PMX 60, Nihon-Denshi, Tokyo, Japan). Another polymerizable monomer, 2, was prepared as described before2,). An anionic phenyl ester, 4-acetoxy-3-nitrobenzoic acid (3a), was prepared by the method of Overberger et al. 23, 24). A more hydrophobic anionic ester, 4-butyryloxy-3-nitrobenzoic acid (3 b) was prepared by the method of Taniguchi et al. ',). 2,2'-Azodiisobutyronitrile (AIBN) from Nakarai Chemicals was recrystallized from methanol. Dimethyl 2,2'-azodiiso- butyrate (V-601) was obtained from Wako Pure Chemicals, Osaka, Japan. Other reagents were commercially available. Deionized water was distilled before use.

Polymerization of monomer 1

44 mg of monomer 1 was dissolved in 5 ml of H,O. After stirring by a Bortex mixer for 5 min, the solution was ultrasonicated using a Kontes Ultrasonic Cell Disrupter at 23,5 kHz for 15 min at 45 "C under N, atmosphere. 0,4 ml of 0,15 wt.-Vo AIBN aqueous solution was added and the solution was incubated for 6 h at 80°C. Completion of polymerization was confirmed by the disappearance of the signals of a-methyl (2,O ppm) and vinyl (5,6; 6,l ppm) protons in the 'H NMR spectrum.

Polymerization of monomer 2

2 ml of a 1 wt.-% aqueous solution of monomer 2 was ultrasonicated for several minutes at 50°C. After filtration (8 pm Millipore filter) the monomer solution was saturated with N, gas for 30 min, 200 p1 of a 10 mol-% V-601 aqueous solution was added, and N, was further bubbled through the solution for 5 min. The temperature of the solution was kept at 55 "C for 12 h.

Formation of vesicles by monomer and polymer of 1 and 2 was previously confirmed by entrapment experiments of hydrophilic substances such as sucrose and glucose within the colloidal particles of the compounds2'. 22 b).

Catalytic effects of cationic polymeric liposomes. . . 159

Kinetic measurements

The alkaline hydrolysis of 3a and 3 b was followed under pseudo-first order condition ([OH-] s- [substrate], c = /cobs. [substrate] = k2 1 [OH- ] . [substrate], where c, kobs, and k2 are the reaction rate, the pseudo-first order rate constant, and the second-order rate constant, respectively) by observing the increase in product absorbance at 41 8 nm using a high sensitivity spectrophotometer (SM 401, Union Engineering, Hirakata, Osaka, Japan). Reactions at high pressure were followed using a Union High Pressure Spectrophotometer with a Drickamer type reaction cell with sapphire windows 16). Reaction cells were thermostated at 25 0,02 "C using a Neslab RTE-8 waterbath.

DSC measurements

A differential scanning calorimeter (SSC 560, Daini-Seikosha, Tokyo) was used to investigate the transition phenomena of polymerizable monomers and polymers at a scanning rate of 1,5 K/min.

Results and discussion

At first we examined the catalytic effects of cationic vesicles and micelles on the second-order rate constant (k,) of the alkaline hydrolysis of 3a. The results obtained are shown in Fig. 1. All additives accelerate the reaction due to electrostatic interactions both between the anionic substrate and the cationic vesicles or micelles,

Fig. 1. Electrolyte effect on second-order rate constant k2 of the alkaline hydrolysis of 3a at 25°C. [3a] = 5 . mol/l, [NaOH] = 10-3 moI/l, solvent: O,S%

(A) CTABr; (A) DDABr; (W) monomer 1; (0) poly-1; ( 0 ) monomer 2; ( 0 ) poly-2

CH,CN-H,O.

and between OH- and the cationic vesicles or micelles. From Fig. 1 it is apparent that the catalytic effect of 2 is much larger than that of 1 as monomer and as polymer. This might be due to the difference in hydrophobicity (1 has both a hexadecyl and an undecyl group, whereas 2 has two octadecyl groups). Hydrophobic interaction was


previously found by us 24) to be very important in the polyelectrolyte catalyzed alkaline hydrolysis of 3a.

B r-

R

P

O N O *

COOH

30: R=CH3 3 b: R = CH2CH2CH3

In the case of CTABr, the acceleration effect is remarkably increased by the formation of micelles. Except in the low concentration region, CTABr and DDABr show similar catalytic activity to each other.

In the case of 1, both the monomer and the polymer show a deceleration effect in the region of high concentration probably because of the unfavorable dispersion of the reactants in the surface domain of the vesicles; the charge density on the surface of the vesicles is significantly higher as compared with the other additives.

Next we examined the temperature effect on the catalysis by cationic micelles or vesicles. The concentration of the catalysts examined here was 1 - equiv. -1-1 except for monomer 1 and poly-1 ( 5 equiv. - 1-I). At this concentration the acceleration effect of the catalysts on the reaction rate is approximately saturated. So that true reactivity of the substrates with OH- in micelles or vesicles (independent of the binding process of substrate with micelles or vesicles) might be directly reflected in the saturated reaction rate value. The Arrhenius plots are shown in Figs. 2 and 3.

The catalytic effects of DDABr, monomer 2, and poly-2 show discontinuous regions which are considered to reflect phase transition regions of the aliphatic chains of the additives from the gel-state to the liquid crystal-state. At the phase transition of vesicles, the distribution behavior of ionic and/or hydrophobic reactants in the vicinity of the vesicles surface are considered to be strongly influenced, which changes the temperature dependence of the rate of reactions catalyzed by vesicles. Such a


381 3,3 3,5 lo3 .T-'/K-'

1

Fig. 2

Fig. 2. Arrhenius plots of the alkaline hydrolysis of 3 a in the presence of electrolytes. [3a] = 5 . mol/l; [NaOH] = mol/l. (0) none; (0) CTABr; (0) DDABr; (A) monomer 1;

Fig. 3. Arrhenius plots of the alkaline hydrolysis of 3 a in the presence of electrolytes. [3a] = 5 . mol/l, [NaOH] = mol/l. ( 0 ) monomer 2; (0) poly-2

(a )POlY-l

phenomenon is quite familiar in the Arrhenius plots of membrane bound enzyme catalyzed reactions 26). We previously observed such discontinuous regions in the phosphatidylcholine catalyzed coenzyme model reaction2').

DDABr was reported to have a transition point at 20°C from light scattering results2*). It shows a discontinuous region at about 12°C in the catalysis of the decarboxylation of 6-nitrobenzisoxazole-3-carboxylate9~. Phase transition points, which are detected kinetically in the catalytic effect of vesicles, have been often reported to be lower than those observed by a static method, due to the perturbation of the aliphatic chains in vesicles by the added reactants"). As shown in Fig. 2 we also observed a discontinuity, though less clear, in the catalytic effect of DDABr due to the phase transition of DDABr. In Fig. 3 the phase transition of monomer 2 (at about 30 "C) is clearly observed, whereas that of poly-2 (at about 25 "C) is relatively vague, reflecting a less significant change in the molecular structure. In the DSC measurements, monomer 2 shows a phase transition at about 40"C, whereas poly-2 shows it at about 30 "C due to the perturbation effect of polymer chain on the packing of the head groups22b). The enthalpy of the phase transition AH, for poly-2 is smaller than that for monomer 222b) which is consistent with the difference in the catalytic effect. The transition phenomena were also observed by the change in turbidity of the solution and agreed well with the DSC data.

I


- +I m - h l r - o m m

1 ' ; ' + + + +


Monomer 1 shows a vague phase transition at 35 "C in the turbidity measurements which is consistent with the fact that in the presence of monomer 1 above 35 "C there is a larger increase in the reaction rate constant than below as shown in Fig. 2. For the polymer of 1, we could not observe a transition phenomenon in contrast to the polymers of 2. The difference between poly-1 and poly-2 is the position of the polymer chain, which suggests that polymer main chains penetrating into the region of aliphatic chains of vesicles inhibit phase transition of vesicles l 1 c,12).

From Figs. 2 and 3 the activation parameters, AG*, AH", and AS* were evaluated and are given in Tab. 1 .

We also examined the pressure effect on these reaction systems. In Fig. 4 the pressure effect on log(k,/ki) for the hydrolysis of 3a is shown, where ki is the

Fig. 4. Pressure effects on the alkaline hydrolysis of 3s in the presence of various electrolytes. [3a] = 5 . 1 0 - ~ mol/l, [NaOH] = mol/l. ( x ) none; (9) CTABr; (0) DDABr; (A) monomer 1; (A) poly-1; ( 0 ) monomer 2; (0) poly-2 I 1 I

500 1000 1500 P/atrn

reaction rate constant at 1 atm and k, is the reaction rate constant at pressure P. The volume of activation (AV*) was evaluated using Eq. (see Tab. I).

d log (k2 /k$) dP A V * = -2,303RT

AS * and AV* values of the spontaneous reaction are negative, partly because the activated complex is more polar than the reactants (electrostriction) 29) and partly because the activated complex involves bonding between two previously discrete molecular species30). From Tab. 1 it is apparent that almost all AV* and AS* values for vesicles catalyzed reactions are larger (less negative) than those of the correspond- ing spontaneous reaction. These results might be attributed to a desolvation effect of the activated complex by the additives, i.e., the charge number of the activated complex (approximately 2) is more negative than those of the reactants, OH- and esters.

By electrostatic interaction the activated complex is more strongly attracted into the vicinity of the cationic additives than OH- or 3a. By neutralization, the hydrating


P -0-C-0 H

coo-

f:

0-

too-

water around the activated complex is destabilized and as a result the volume change from the initial state to the activated complex is relatively increased as compared with that of the spontaneous reaction. Similar phenomena were previously observed in the aquation of Co(NH3),Br2+ induced by Ag+ in the presence of polyanions'6). In this case, an increase in the A V * value by 81 ml* mol-' was observed in the presence of sodium polystyrenesulfonate. The amount of hydrating water around the reactants examined here (ester and OH-) is less than that around Co(NH3),Br2+ and Agf . So, the relatively small increase in the A V * value observed here is reasonable. In the case of the hydrolysis of 3 b, the amount of hydrophobically hydrating water around the substrate (so called "ice-berg" structured water) is considered to be larger than that around 3a. In the course of activation, this water structure is expected to be destabilized, resulting in an increase in the A V * value because the contribution of the hydrophobic hydration to the partial molar volume is negative3'. 32).

The obtained AV* value for the spontaneous hydrolysis of 3b, however, is the same as that for 3a, probably because the amount of electrostricted water around the molecule of 3b in the initial state is less than that around 3a. By the addition of cationic vesicles and micelles, A V * and A S * values in the hydrolysis of 3b are similarly increased as in the hydrolysis of 3a, which suggests that the desolvation of the activated complex due to neutralization is important in the alkaline hydrolysis of anionic phenyl esters. In the case of CTABr, the micellar structure might be changed by the addition of the hydrophobic 3b and, as a result, A V * might be increased. Recently, we have pointed out the importance of hydrophobic hydration in the alkaline hydrolysis of neutral and cationic phenyl esters33). The driving force of catalysis might be the solvation in the course of activation.

We previously observed an essential contribution of a desolvation effect on the decarboxylation of oxalacetate catalyzed by polymeric amines 34). We have also pointed out the important contribution of desolvation effects in the esterolysis catalyzed by imidazole-containing polymers 35) and found some similarity to enzyme catalysis 36).

In conclusion, polymeric liposomes investigated here catalyze interionic reactions efficiently due to their hydrophobic alkyl chains and cationic charges. The desolvation effect of the activated complex is important in catalysis which is reflected in the larger (less negative) A S * and A V* values as compared with those of the spontaneous reaction. By the appropriate design of a molecule with the polymerizable part, the ionic part, and the hydrophobic part in a favorable place, we may be able to construct


polymeric microspheres which are catalytically active and at the same time physically stable but still have liposomal character.

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catalytic effects of cationic polymeric liposomes on the alkaline hydrolysis of aniomic phenyl...

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