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Vesicular effect on the reactivity of anthracene derivatives towards singlet molecular oxygen
Journal of Photochemistry
and Photobiology,
B: Biology,
3 (1989)
113 - 122
113
VESICULAR EFFECT ON THE REACTIVITY OF ANTHRACENE DERIVATIVES TOWARDS SINGLET MOLECULAR OXYGEN M. V. ENCINAS, E. LEMP and E. A. LISSI Departamento de Quimica, Facultad de Ciencias, Casilla 5659 Correo 2, Santiago (Chile) (Received April 12,1988;
Universidad
de Santiago de Chile,
accepted August 1, 1988)
Keywords. Surfactant vesicles, .partmentalized systems.
photosensitization,
singlet oxygen,
com-
Summary The effect of dioctadecyldimethylammonium chloride (DODAC) vesicles upon the reactivity of several anthracene derivatives towards O,( ‘A& has been measured. Incorporation of the substrates into the vesicles decreases their consumption rate. The effect depends on both the substrate characteristics and the vesicle size. The most pronounced decreases are observed with those substrates for which deep incorporation of the anthryl group into the vesicles could be expected, i.e., 9,10-dimethylanthracene and 3-(9-anthryl)propionic acid. For these substrates, the reactivity is nearly two times smaller in the large (injected) vesicles than in the small (sonicated) vesicles. The dependence of the bleaching rates on the surfactant concentration allows the evaluation of the substrate distribution between the vesicles and the aqueous solution. For 9-methylanthracene and g-anthracenemethanol, the partition constants are nearly ten times smaller in the large vesicles than in the smaller vesicles.
1. Introduction Photoprocesses in surfactant or phospholipid vesicles have been extensively studied since they are simple models of biological membranes [ 11. Singlet molecular oxygen (‘0,) is generated in different pathological [2] and normal [ 31 biological processes, and its reactions have been extensively investigated [4]. Nevertheless, the reactivity of singlet molecular oxygen towards vesicles with substrates incorporated has only been investigated in a few studies [5 - 71. Furthermore, there are no data on the effect of the substrate intravesicular location and/or the influence of vesicle characteristics on the rate of the process. In the present paper we report a study of the effect of dioctadecyldimethylammonium chloride (DODAC) vesicles loll-1344/89/$3.50
@ Elsevier Sequoia/Printed
in The Netherlands
114
upon the reactivity of several anthracene derivatives towards ‘OZ. The data allow evaluation of both the aromatic partition constants and the effect of the location of the solute on its reactivity towards ‘OZ. Furthermore, by employing small (sonicated) and large (injected) unilammellar vesicles, the effect of their size upon the above-mentioned parameters was also evaluated.
2. Materials and methods DODAC was obtained from Herga Industrias do Brasil and purified by the technique reported by Cuccovia et al. [8]. 9-Anthracenecarboxylic acid and 3-(9-anthryl)propionic acid (Molecular Probes), 9anthracenemethano1, 9,lOdimethylanthracene and 9-methylanthracene (Aldrich) were used without further treatment. Ethyldimethyl[ 3-( 9-anthracenyl)propyl]ammonium bromide was a present from Dr. F. Quina (Universidad de Sao Paulo, Brazil). Its synthesis is in ref. 9. Small vesicles (internal volume, 0.13 1 mol-‘; aggregation number, = 1.5 X 104) were prepared by sonication (10 min, 55 - 60 “C) of aqueous DODAC suspension using the microprobe of a Braunsonic 1510 sonifier at 100 W. Large vesicles (internal volume, 9.0 1 mol-‘; aggregation number, = 4 X 106) were prepared by slow injection (0.25 ml mine’) of a concentrated chloroform solution of the surfactant into 10 ml of water at 73 “C, with chloroform removed by continuous Nz purging during injection. Both methods afford unilammellar vesicles [ 10 - 121. Methylene blue (1.2 X lo-’ M) irradiated at 666 nm was employed as the ‘02 source. It was added from a stock solution to the prepared vesicles. No significant differences were observed when the sensitizer was added prior to the vesicle preparation rather than after. Solutions containing g-anthracenecarboxylic acid and 3-(9-anthryl)propionic acid were prepared at pH 9. The probe-to-surfactant molar ratio was kept below 10e2. All the experiments were performed in air-saturated solutions at 20 “C with fresh solutions. The aromatic consumption (bleaching) was evaluated from the change in the anthracene derivative fluorescence intensity. This technique was selected because of the low concentrations of the substrates employed. No differences were observed when the anthracene derivatives were added prior to the vesicle preparation rather than after. Furthermore, the position and the fine structure of the UV absorption and the emission band of the anthracene derivatives were independent of the substrate consumption.
3. Results and discussion It is well established that anthracene derivatives react with ‘02 to produce 9,10-endoperoxides [13]. The results obtained in this work (total loss of anthracene chromophore without formation of UV-absorbing prod-
115
ucts in the near-UV-visible region) are compatible with this process being the dominant reaction path under all the conditions employed. The consumption of the anthracene derivatives follows first-order kinetics. Typical results are given in Fig. 1. The pseudo-first-order constants k exp given by the slopes of plots such as those shown in Fig. 1 are strongly dependent on the aromatic compound employed, the DODAC concentration and the vesicle preparation method. Typical results are shown in Fig. 2. The results obtained can be explained in terms of a simple “two pseudophases” scheme such as that given by Lee and Rodgers 1141: ‘O?(w) __f
‘02( lip)
A(w) +
A(lip)
A(w) + ‘02(w) -
(2)
k,
A(lip) + ‘Oz(lip) ks 1o2w
(1)
-
products
(3)
products
(4)
k
(5)
02
k’
‘02(lip) -+
O2
(6)
Scheme 1.
6
12 timelminl
Fig. 1. Bleaching of 9-methylanthracene (1.1 X 10-j mM) in DODAC vesicle (injected) suspensions. IO, fluorescence intensity at t = 0; Z, fluorescence intensity at time t. Fluorescence was monitored at 417 nm. 0, water; 0, DODAC 0.1 mM; 0, DODAC 0.25 mM; A, DODAC 1 mM; A, DODAC 3 mM.
116
I
1
ei-+
2.0 [DODAC], (mM)
Fig. 2. Values of kexp for 9-anthracenemethanol 0, injected vesicles; 0, sonicated vesicles.
as a function of DODAC concentration;
with (k + k’) > (k,[A(w)] + kiip[A(lip)]), where w and lip stand for the aqueous and lipidic pseudophases respectively. In the present system, because of the low fraction of the total volume occupied by the lipidic pseudophase (less than 0.3%), it can be considered that the ‘02 lifetime is independent of the surfactant concentration. This is further supported by the fact that for ethyldimethyl[&(Qanthracenyl)propyllammonium bromide, a probe that can be assumed to remain in the water phase because of its charge and the low surfactant concentration employed, the values of k,,, are almost independent of the DODAC concentration (from 0 to 5 mM). Similar conclusions have been reported by Rodgers and Bates [7] for sonicated DODAB vesicles. These results are different from those reported by Dearden [5] in a study of ‘02 reactions in egg yolk lecithin vesicles. Nevertheless, the presence of high intravesicular concentrations of unsaturated phospholipids in their case makes the quantitative interpretation of solute bleaching data more difficult. Taking into account that most of the ‘02 remains in the water pseudophase, Scheme 1 leads to a probe consumption rate given by
-
+
Olfiipklip)[Al
(7)
117
where R, is the ‘02 production rate, a is the ‘02 partition constant between the lipidic and aqueous pseudophase, flip and f, are the fractions of the aromatic associated with the lipidic pseudophase and with the aqueous solution and 12, and kri, stand for the respective bimolecular rate constants in the two reaction media. From eqn. (7), the following equation can be derived: k exP
=
(fwk
+
+
Mlipklip)
Since k and R, are constant parameters, k,, defined by k,, = fwk
+ Olfiiphp
(9)
can be obtained under a variety of experimental conditions. A partition constant can be defined in terms of the molar fraction of the aromatic as K
_
xlip X-N
Thus, the following expression is obtained: K = fii, X 55.6
fwPODW and hence
kw k, - k,, with B=
=B+
k, kw - &lip
55.6B 1 K [DODAC]
(11)
(12)
Plots of k,/(k, - kap) us. l/[DODAC] allow the evaluation of a(klip/ and K. Typical plots are given in Fig. 3. The.iinearity of the plots demonstrates that the data can be adequately represented by eqn. (11). The values of OL(klip/kw) and K obtained are given in Table 1. Table 1 also gives the values of Qklip, calculated by this procedure and the measured k,, and calculated from the value of k,, when flip = 1 (i.e. for 9,10-dimethylanthracene). Owing to the very low solubility of this compound in water, only measurements in the presence of vesicles were performed. k,, values were independent of the surfactant concentration in small vesicles as well as in large vesicles. Values of k,, in the presence of surfactant were also independent of the surfactant concentration (1 - 3 mM) for 3-(9anthryl)propionic acid and for 9-anthracenecarboxylic acid. These results indicate almost complete association with the vesicles over the whole concentration range considered. k,)
118
1.01 0
I
I
3
6 l/[DODAC],
l
(mM-‘)
Fig. 3. Values of k,/( k, - kap) as a function of DODAC concentration plotted according to eqn. (11): 0, 9-methylanthracene in injected vesicles; 0, 9-methylanthracene in sonicated vesicles; 4 9-anthracenemethanol in injected vesicles; A, 9-anthracenemethanol in sonicated vesicles.
TABLE 1 Values of K, k,, kli,/k,
and kli, (T = 20 “C) for the various compounds studied
Compound R,
R
kw (x10’ M-’ s-r)
Vesicle akli,/k,
akli, (~10~ M-‘s-l)
1O-5 K
18 41
CH3
CH3 -
CH3
H
5.8
I S
0.15 0.12
a.4 6.9
CHzOH
H
1.3
I S
0.30 0.20
4.0 2.6
( CH2)3COO-
H
4.8
I S
0.077 0.11
3.7 5.2
coo-
H
0.6
I S
0.15 0.16
0.87 0.94
(CH2)JN(CH3)2(CzHs)Br-I-I
2.5
+ aVesicles obtained by the injection method (external diameter, about 0.5 /+nrr). bVesicles obtained by sonication (external diameter, about 300 A).
4.9 43 0.56 3.8
119
3.1. Values of the partition constant K The values of K obtained conform to the expected pattern. For a given type of vesicle, the value of K is determined by the hydrophobicity of the anthracene substituent. It is interesting to note that the difference between 9-methylanthracene and 9-anthracenemethanol (a factor 8) is similar to the difference observed for 1-methyhraphthalene and 1-naphthylmethanol in cetyltrimethylammonium bromide (CTAB) micelles [ 151. On the other hand, for charged anthracene derivatives, the main factor determining the extent of their association with the vesicles is the sign of the charge. With regard to the values of K for a given substrate in both vesicles, the results of Table 1 show that for both Q-methylanthracene and 9anthracenemethanol the sonicated vesicles (small) have larger solvent capacities (per surfactant molecule) than the large vesicles. Similar differences have been observed for other solutes and have been interpreted in terms of the more “solid” ordering of the chains in the larger vesicles [16 - 181. Nevertheless, the differences obtained in the present work are larger than those reported previously [19]. These differences could be related to the larger distortion associated with the incorporation of the bulky molecules considered in the present study. 3.2. Values of CY(klip/kw) The values obtained for a(kii,/k,) range from 0.077 to 0.3. The fact that these values are considerably smaller than unity implies that incorporation into the vesicles efficiently protects the anthracene group from ‘02 attack. This conclusion differs from that reached from studies of the effect of vesicles in the reaction of 2,3-dimethylindole with ‘02 [7] and stilbene derivatives with ozone [20]. Nevertheless, it should be kept in mind that is determined by the value of (Y. This value could be expected to be &lip larger than unity from the data obtained in micellar solutions [14, 211, and the value of Izii, in the particular reaction media. With regard to these parameters, the reactivity of polycyclic aromatic compounds towards ‘02 is extremely sensitive to the solvent polarity and particularly to the presence of water in the reaction media [22,23], and hence it can be expected that for these systems Fzii,< k,. Another factor that could contribute to a decrease in CuFzii, could be that, owing to the high viscosity of the vesicles, the process becomes diffusion controlled. However, the fact that considerably larger rate constants have been reported for the quenching of vesicle-incorporated probes by molecular oxygen [ 191 strongly argues against this possibility. The values of olkii, for a given aromatic depend on the vesicle considered. For those systems for which rather deep penetration of the aromatic moiety (9,lOdimethylanthracene and 3-( 9anthryl)propionic acid) could be expected, the values are smaller in the injected vesicles, probably reflecting lower oxygen solubility and/or a less polar microenvironment in these more closely packed vesicles. On the other hand, for those compounds in which the aromatic group can be expected to be near the interface (9anthracene-
120
methanol, 9-anthracenecarboxylic acid and 9-methylanthracene), the values are similar or even slightly increased in the larger vesicles, suggesting a more exposed location in these structures. Similar conclusions have been reached with respect to the location of pyrene in both types of vesicle [ 191. In agreement with the previous considerations, smaller values of o(kii,/k,) are obtained for 3-(9-anthryl)propionic acid, a result compatible with a more lipidic location for this substrate. Dearden [5] has reported a value of &ii, = 3.2 X lo8 M-i s-l for the reaction of 9,10-dimethylanthracene with ‘0, in egg-yolk lecithin vesicles. However, in his work it was found that in solutions containing 0.05% eggyolk lecithin nearly 60% of the ‘02 molecules are incorporated into the liposome, suggesting a ‘02 distribution completely different from that considered by us. Values Of &ii, have been also reported for 9,10-dimethylanthracene and 9-methylanthracene in CTAC and CTAB micelles [22]. The values obtained in CTAC, 27 X lO’M_’ s-i and 6.1 X 10’ M-’ s-’ for 9,10-dimethylanthracene and 9-methylanthracene respectively, are considerably larger than those measured in the present work. This difference indicates a lower oxygen solubility and/or a lower intrinsic reactivity in the vesicles than in the micelles. This latter effect can be associated with the more open structure of the micellar aggregates, which provides a more polar environment for the incorporated probes. The difference observed between the two rate constants closely resembles that obtained in the present system. It is also interesting to note that in ethanol (a solvent with properties similar to that of a lipid-water interface) 9,lOdimethylanthracene is 20 times more reactive than 9-methylanthracene [22]. The smaller selectivity observed in micelles and vesicles may be partly due to a less favourable (i.e. more lipidic) location of 9,lOdimethylanthracene in these microphases. In conclusion, the results obtained in the present work show that the reactivity towards ‘02 of a vesicle-incorporated substrate is sensitive to its location and that this can be modified by the nature of the substituents on the substrate.
Acknowledgments We thank DICYT (Universidad de Santiago de Chile) and FONDECYT (Grant 1433) for financial support of this work. Herga Industrias do Brasil is acknowledged for providing the DODAC sample.
References 1 J. H. Fendler, Membrane Mimetic Chemistry, Wiley, New York, 1982, pp. 113 - 183. 2 N. I. Krinsky, Biological roles of singlet oxygen. In H. H. Wasserman and W. A. Murray (ed.), Singlet Oxygen, Academic hess, New York, 1979, pp. 597 - 641.
121 3 C. S. Foote,
4
5 6 7
8
9
10
11
12
13
14
15 16
17
18
19 20 21
Photosensitized oxidation and singlet oxygen: consequence in biological. systems. In W. A. Pryor (ed.), Free Radicals in Biology, Vol. II, Academic Press, New York, 1976, pp. 85 - 133. N. Duran, Singlet oxygen in biological processes. In W. Adam and P. Cilento (eds.), Chemical and Biological Generation of Excited States, Academic Press, New York, 1982, pp. 345 - 369. S. J. Dearden, Kinetics of Os(‘As) photo-oxidation reactions in egg yolk lecithin vesicles, J. Chem. Sot., Faraday Trans. I, 82 (1986) 1627 - 1635. S. J. Dearden, T. F. Hunter and J. Philp, Fatty acid analysis as a function of photooxidation in egg yolk lecithin vesicles, Photochem. PhotobioZ., 41 (1985) 213 - 215. M. A. Rodgers and A. L. Bates, A laser flash kinetic spectrophotometric examination of ,the dynamics of singlet oxygen in unilammellar vesicles, Photochem. PhotobioZ., 35 (1982) 473 - 477. I. M. Cuccovia, R. M. V. Aleixo, R. A. Mortara, P. B. Filho, J. B. S. Bonilha, F. H. Quina and H. Chaimovich, Effect of dialkyldimethylammonium vesicles on the thiolysis of p-nitrophenyl acetate, Tetrahedron Lett., 33 (1979) 3065 - 3068. L. Miola, R. Blotta Abakerli, M. F. Ginani, P. Berci Filho, V. G. Toscano and F. H. Quina, Models for specific counter ion effects on the incorporation of charged amphiphilic substrates into like-charged ionic micelles, J. Phys. Chem., 87 (1983) 4417 - 4425. A. M. Carmona Ribeiro and H. Chaimovich, Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparison with small sonicated vesicles, Biochim. Biophys. Acta, 733 (1983) 172 - 179. R. A. Moss, T. F. Hendrickson, S. Swarup, Y. Hui, L. Marky and K. J. Breslauer, Trans membrane transport of l-anilino-8naphthalenesulfonate in simple cationic surfactant vesicles, Tetrahedron Lett., 25 (1984) 4063 - 4066. K. Kano, A. Romero, B. Djermouni, H. J. Ache and J. H. Fendler, Turbidity, viscosity, fluorescence polarization of 2-methylanthracene and positron annihilation in sonicated dioctadecyldimethylammonium chloride, J. Am. Chem. Sot., 101 (1979) 4030 - 4036. E. J. Corey and W. T. Taylor, A study of the photo-oxidation of organic compounds by externally generated singlet oxygen molecules, J. Am. Chem. Sot., 86 (964) 3881 3882. P. C. Lee and A. J. Rodgers, Singlet oxygen in micellar systems. Distribution equilibria between hydrophobic and hydrophilic compartments, J. Phys. Chem., 87 (1983) 4894 - 4898. E. Abuin, E. A. Lissi, M. Gonzalez and J. Vera, Solubilization of naphthalene derivatives in micellar assemblies, J. Colloid Interface Sci., 98 (1984) 152 - 161. F. Ricchelli and G. Jori, Distribution of porphyrins in the various compartments of unilamellar liposomes of dipalmitoylphosphatidylcholine as probed by fluorescence spectroscopy, Photochem. Photobiol, 44 (1986) 151- 157. K. J. Stelzer and M. A. Gordon, Interactions of pyrethroids with phosphatidylcholine bilayers: comparisons in liposomal systems exhibiting large or small radii of curvature Chem. Biol. Interact., 54 (1985) 105 - 116. S. Kaneshina, H. Kamaya and I. Ueda, Thermodynamic of pressure-anesthetic antagonism on the phase transition of lipid membranes: comparisons in liposomal systems exhibiting large or small radii of curvature, J. Colloid Znterface Sci., 93 (1983) 215 - 224. E. Abuin, E. A. Lissi, D. Aravena, A. Zanocco and M. Macuer, Fluorescence probe study of the effect of size on the properties of dioctadecyldimethylammonium chloride vesicles, J. Colloid Interface Sci., 122 (1988) 201 - 208. D. Aravena and E. A. Lissi, Reactivity of ozone towards micelle incorporated unsaturated compounds, J. Free Radicals Biol. Med., 1 (1985) 327 - 330. I. B. C. Matheson and A. D. King, Solubility of gases in micellar solutions, J. Colloid Interface Sci., 66 (1978) 464 - 469.
122 22 M. A. Rubio, L. Araya, E. B. Abuin and E. A. L&i, O&Z) and O#Ag) processes in microheterogeneous systems, An. Asoc. Quim. Argent., 73 (1985) 301- 309. 23 B. Cazing, J. M. Aubry and J. Rigandy, Is water the best or the worst solvent for [Z + 41 cycloadditions of singlet oxygen to aromatic compounds?, J. Chem. Sot., Chem. Commun., (1986) 952 - 953.
and Photobiology,
B: Biology,
3 (1989)
113 - 122
113
VESICULAR EFFECT ON THE REACTIVITY OF ANTHRACENE DERIVATIVES TOWARDS SINGLET MOLECULAR OXYGEN M. V. ENCINAS, E. LEMP and E. A. LISSI Departamento de Quimica, Facultad de Ciencias, Casilla 5659 Correo 2, Santiago (Chile) (Received April 12,1988;
Universidad
de Santiago de Chile,
accepted August 1, 1988)
Keywords. Surfactant vesicles, .partmentalized systems.
photosensitization,
singlet oxygen,
com-
Summary The effect of dioctadecyldimethylammonium chloride (DODAC) vesicles upon the reactivity of several anthracene derivatives towards O,( ‘A& has been measured. Incorporation of the substrates into the vesicles decreases their consumption rate. The effect depends on both the substrate characteristics and the vesicle size. The most pronounced decreases are observed with those substrates for which deep incorporation of the anthryl group into the vesicles could be expected, i.e., 9,10-dimethylanthracene and 3-(9-anthryl)propionic acid. For these substrates, the reactivity is nearly two times smaller in the large (injected) vesicles than in the small (sonicated) vesicles. The dependence of the bleaching rates on the surfactant concentration allows the evaluation of the substrate distribution between the vesicles and the aqueous solution. For 9-methylanthracene and g-anthracenemethanol, the partition constants are nearly ten times smaller in the large vesicles than in the smaller vesicles.
1. Introduction Photoprocesses in surfactant or phospholipid vesicles have been extensively studied since they are simple models of biological membranes [ 11. Singlet molecular oxygen (‘0,) is generated in different pathological [2] and normal [ 31 biological processes, and its reactions have been extensively investigated [4]. Nevertheless, the reactivity of singlet molecular oxygen towards vesicles with substrates incorporated has only been investigated in a few studies [5 - 71. Furthermore, there are no data on the effect of the substrate intravesicular location and/or the influence of vesicle characteristics on the rate of the process. In the present paper we report a study of the effect of dioctadecyldimethylammonium chloride (DODAC) vesicles loll-1344/89/$3.50
@ Elsevier Sequoia/Printed
in The Netherlands
114
upon the reactivity of several anthracene derivatives towards ‘OZ. The data allow evaluation of both the aromatic partition constants and the effect of the location of the solute on its reactivity towards ‘OZ. Furthermore, by employing small (sonicated) and large (injected) unilammellar vesicles, the effect of their size upon the above-mentioned parameters was also evaluated.
2. Materials and methods DODAC was obtained from Herga Industrias do Brasil and purified by the technique reported by Cuccovia et al. [8]. 9-Anthracenecarboxylic acid and 3-(9-anthryl)propionic acid (Molecular Probes), 9anthracenemethano1, 9,lOdimethylanthracene and 9-methylanthracene (Aldrich) were used without further treatment. Ethyldimethyl[ 3-( 9-anthracenyl)propyl]ammonium bromide was a present from Dr. F. Quina (Universidad de Sao Paulo, Brazil). Its synthesis is in ref. 9. Small vesicles (internal volume, 0.13 1 mol-‘; aggregation number, = 1.5 X 104) were prepared by sonication (10 min, 55 - 60 “C) of aqueous DODAC suspension using the microprobe of a Braunsonic 1510 sonifier at 100 W. Large vesicles (internal volume, 9.0 1 mol-‘; aggregation number, = 4 X 106) were prepared by slow injection (0.25 ml mine’) of a concentrated chloroform solution of the surfactant into 10 ml of water at 73 “C, with chloroform removed by continuous Nz purging during injection. Both methods afford unilammellar vesicles [ 10 - 121. Methylene blue (1.2 X lo-’ M) irradiated at 666 nm was employed as the ‘02 source. It was added from a stock solution to the prepared vesicles. No significant differences were observed when the sensitizer was added prior to the vesicle preparation rather than after. Solutions containing g-anthracenecarboxylic acid and 3-(9-anthryl)propionic acid were prepared at pH 9. The probe-to-surfactant molar ratio was kept below 10e2. All the experiments were performed in air-saturated solutions at 20 “C with fresh solutions. The aromatic consumption (bleaching) was evaluated from the change in the anthracene derivative fluorescence intensity. This technique was selected because of the low concentrations of the substrates employed. No differences were observed when the anthracene derivatives were added prior to the vesicle preparation rather than after. Furthermore, the position and the fine structure of the UV absorption and the emission band of the anthracene derivatives were independent of the substrate consumption.
3. Results and discussion It is well established that anthracene derivatives react with ‘02 to produce 9,10-endoperoxides [13]. The results obtained in this work (total loss of anthracene chromophore without formation of UV-absorbing prod-
115
ucts in the near-UV-visible region) are compatible with this process being the dominant reaction path under all the conditions employed. The consumption of the anthracene derivatives follows first-order kinetics. Typical results are given in Fig. 1. The pseudo-first-order constants k exp given by the slopes of plots such as those shown in Fig. 1 are strongly dependent on the aromatic compound employed, the DODAC concentration and the vesicle preparation method. Typical results are shown in Fig. 2. The results obtained can be explained in terms of a simple “two pseudophases” scheme such as that given by Lee and Rodgers 1141: ‘O?(w) __f
‘02( lip)
A(w) +
A(lip)
A(w) + ‘02(w) -
(2)
k,
A(lip) + ‘Oz(lip) ks 1o2w
(1)
-
products
(3)
products
(4)
k
(5)
02
k’
‘02(lip) -+
O2
(6)
Scheme 1.
6
12 timelminl
Fig. 1. Bleaching of 9-methylanthracene (1.1 X 10-j mM) in DODAC vesicle (injected) suspensions. IO, fluorescence intensity at t = 0; Z, fluorescence intensity at time t. Fluorescence was monitored at 417 nm. 0, water; 0, DODAC 0.1 mM; 0, DODAC 0.25 mM; A, DODAC 1 mM; A, DODAC 3 mM.
116
I
1
ei-+
2.0 [DODAC], (mM)
Fig. 2. Values of kexp for 9-anthracenemethanol 0, injected vesicles; 0, sonicated vesicles.
as a function of DODAC concentration;
with (k + k’) > (k,[A(w)] + kiip[A(lip)]), where w and lip stand for the aqueous and lipidic pseudophases respectively. In the present system, because of the low fraction of the total volume occupied by the lipidic pseudophase (less than 0.3%), it can be considered that the ‘02 lifetime is independent of the surfactant concentration. This is further supported by the fact that for ethyldimethyl[&(Qanthracenyl)propyllammonium bromide, a probe that can be assumed to remain in the water phase because of its charge and the low surfactant concentration employed, the values of k,,, are almost independent of the DODAC concentration (from 0 to 5 mM). Similar conclusions have been reported by Rodgers and Bates [7] for sonicated DODAB vesicles. These results are different from those reported by Dearden [5] in a study of ‘02 reactions in egg yolk lecithin vesicles. Nevertheless, the presence of high intravesicular concentrations of unsaturated phospholipids in their case makes the quantitative interpretation of solute bleaching data more difficult. Taking into account that most of the ‘02 remains in the water pseudophase, Scheme 1 leads to a probe consumption rate given by
-
+
Olfiipklip)[Al
(7)
117
where R, is the ‘02 production rate, a is the ‘02 partition constant between the lipidic and aqueous pseudophase, flip and f, are the fractions of the aromatic associated with the lipidic pseudophase and with the aqueous solution and 12, and kri, stand for the respective bimolecular rate constants in the two reaction media. From eqn. (7), the following equation can be derived: k exP
=
(fwk
+
+
Mlipklip)
Since k and R, are constant parameters, k,, defined by k,, = fwk
+ Olfiiphp
(9)
can be obtained under a variety of experimental conditions. A partition constant can be defined in terms of the molar fraction of the aromatic as K
_
xlip X-N
Thus, the following expression is obtained: K = fii, X 55.6
fwPODW and hence
kw k, - k,, with B=
=B+
k, kw - &lip
55.6B 1 K [DODAC]
(11)
(12)
Plots of k,/(k, - kap) us. l/[DODAC] allow the evaluation of a(klip/ and K. Typical plots are given in Fig. 3. The.iinearity of the plots demonstrates that the data can be adequately represented by eqn. (11). The values of OL(klip/kw) and K obtained are given in Table 1. Table 1 also gives the values of Qklip, calculated by this procedure and the measured k,, and calculated from the value of k,, when flip = 1 (i.e. for 9,10-dimethylanthracene). Owing to the very low solubility of this compound in water, only measurements in the presence of vesicles were performed. k,, values were independent of the surfactant concentration in small vesicles as well as in large vesicles. Values of k,, in the presence of surfactant were also independent of the surfactant concentration (1 - 3 mM) for 3-(9anthryl)propionic acid and for 9-anthracenecarboxylic acid. These results indicate almost complete association with the vesicles over the whole concentration range considered. k,)
118
1.01 0
I
I
3
6 l/[DODAC],
l
(mM-‘)
Fig. 3. Values of k,/( k, - kap) as a function of DODAC concentration plotted according to eqn. (11): 0, 9-methylanthracene in injected vesicles; 0, 9-methylanthracene in sonicated vesicles; 4 9-anthracenemethanol in injected vesicles; A, 9-anthracenemethanol in sonicated vesicles.
TABLE 1 Values of K, k,, kli,/k,
and kli, (T = 20 “C) for the various compounds studied
Compound R,
R
kw (x10’ M-’ s-r)
Vesicle akli,/k,
akli, (~10~ M-‘s-l)
1O-5 K
18 41
CH3
CH3 -
CH3
H
5.8
I S
0.15 0.12
a.4 6.9
CHzOH
H
1.3
I S
0.30 0.20
4.0 2.6
( CH2)3COO-
H
4.8
I S
0.077 0.11
3.7 5.2
coo-
H
0.6
I S
0.15 0.16
0.87 0.94
(CH2)JN(CH3)2(CzHs)Br-I-I
2.5
+ aVesicles obtained by the injection method (external diameter, about 0.5 /+nrr). bVesicles obtained by sonication (external diameter, about 300 A).
4.9 43 0.56 3.8
119
3.1. Values of the partition constant K The values of K obtained conform to the expected pattern. For a given type of vesicle, the value of K is determined by the hydrophobicity of the anthracene substituent. It is interesting to note that the difference between 9-methylanthracene and 9-anthracenemethanol (a factor 8) is similar to the difference observed for 1-methyhraphthalene and 1-naphthylmethanol in cetyltrimethylammonium bromide (CTAB) micelles [ 151. On the other hand, for charged anthracene derivatives, the main factor determining the extent of their association with the vesicles is the sign of the charge. With regard to the values of K for a given substrate in both vesicles, the results of Table 1 show that for both Q-methylanthracene and 9anthracenemethanol the sonicated vesicles (small) have larger solvent capacities (per surfactant molecule) than the large vesicles. Similar differences have been observed for other solutes and have been interpreted in terms of the more “solid” ordering of the chains in the larger vesicles [16 - 181. Nevertheless, the differences obtained in the present work are larger than those reported previously [19]. These differences could be related to the larger distortion associated with the incorporation of the bulky molecules considered in the present study. 3.2. Values of CY(klip/kw) The values obtained for a(kii,/k,) range from 0.077 to 0.3. The fact that these values are considerably smaller than unity implies that incorporation into the vesicles efficiently protects the anthracene group from ‘02 attack. This conclusion differs from that reached from studies of the effect of vesicles in the reaction of 2,3-dimethylindole with ‘02 [7] and stilbene derivatives with ozone [20]. Nevertheless, it should be kept in mind that is determined by the value of (Y. This value could be expected to be &lip larger than unity from the data obtained in micellar solutions [14, 211, and the value of Izii, in the particular reaction media. With regard to these parameters, the reactivity of polycyclic aromatic compounds towards ‘02 is extremely sensitive to the solvent polarity and particularly to the presence of water in the reaction media [22,23], and hence it can be expected that for these systems Fzii,< k,. Another factor that could contribute to a decrease in CuFzii, could be that, owing to the high viscosity of the vesicles, the process becomes diffusion controlled. However, the fact that considerably larger rate constants have been reported for the quenching of vesicle-incorporated probes by molecular oxygen [ 191 strongly argues against this possibility. The values of olkii, for a given aromatic depend on the vesicle considered. For those systems for which rather deep penetration of the aromatic moiety (9,lOdimethylanthracene and 3-( 9anthryl)propionic acid) could be expected, the values are smaller in the injected vesicles, probably reflecting lower oxygen solubility and/or a less polar microenvironment in these more closely packed vesicles. On the other hand, for those compounds in which the aromatic group can be expected to be near the interface (9anthracene-
120
methanol, 9-anthracenecarboxylic acid and 9-methylanthracene), the values are similar or even slightly increased in the larger vesicles, suggesting a more exposed location in these structures. Similar conclusions have been reached with respect to the location of pyrene in both types of vesicle [ 191. In agreement with the previous considerations, smaller values of o(kii,/k,) are obtained for 3-(9-anthryl)propionic acid, a result compatible with a more lipidic location for this substrate. Dearden [5] has reported a value of &ii, = 3.2 X lo8 M-i s-l for the reaction of 9,10-dimethylanthracene with ‘0, in egg-yolk lecithin vesicles. However, in his work it was found that in solutions containing 0.05% eggyolk lecithin nearly 60% of the ‘02 molecules are incorporated into the liposome, suggesting a ‘02 distribution completely different from that considered by us. Values Of &ii, have been also reported for 9,10-dimethylanthracene and 9-methylanthracene in CTAC and CTAB micelles [22]. The values obtained in CTAC, 27 X lO’M_’ s-i and 6.1 X 10’ M-’ s-’ for 9,10-dimethylanthracene and 9-methylanthracene respectively, are considerably larger than those measured in the present work. This difference indicates a lower oxygen solubility and/or a lower intrinsic reactivity in the vesicles than in the micelles. This latter effect can be associated with the more open structure of the micellar aggregates, which provides a more polar environment for the incorporated probes. The difference observed between the two rate constants closely resembles that obtained in the present system. It is also interesting to note that in ethanol (a solvent with properties similar to that of a lipid-water interface) 9,lOdimethylanthracene is 20 times more reactive than 9-methylanthracene [22]. The smaller selectivity observed in micelles and vesicles may be partly due to a less favourable (i.e. more lipidic) location of 9,lOdimethylanthracene in these microphases. In conclusion, the results obtained in the present work show that the reactivity towards ‘02 of a vesicle-incorporated substrate is sensitive to its location and that this can be modified by the nature of the substituents on the substrate.
Acknowledgments We thank DICYT (Universidad de Santiago de Chile) and FONDECYT (Grant 1433) for financial support of this work. Herga Industrias do Brasil is acknowledged for providing the DODAC sample.
References 1 J. H. Fendler, Membrane Mimetic Chemistry, Wiley, New York, 1982, pp. 113 - 183. 2 N. I. Krinsky, Biological roles of singlet oxygen. In H. H. Wasserman and W. A. Murray (ed.), Singlet Oxygen, Academic hess, New York, 1979, pp. 597 - 641.
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