1180 J. Am. Chem. Soc., Vol. 120, No. 6, 1998
Scrimin et al.
On the other hand, in a series of papers15 Moss et al.
highlighted some of the structural parameters that influence
transbilayer mobility in vesicles. Under conditions of slow flip-
flop and the impermeability of the bilayer to reactants, chemical
differentiation of the interior and exterior surfaces of vesicles
made of functional lipids was achieved. For instance, only
phosphate esters residing on the outer layer of vesicular 1,
prepared at pH 5.5, were hydrolyzed when the external pH was
raised to 11.8.16 However, when excess CTACl (cetyltri-
methylammonium chloride) was added to the above, partially-
cleaved vesicles, the remaining fraction of lipids was rapidly
hydrolyzed. It was suggested that CTACl “inserted” into the
vesicles providing regions permeable to OH-, thus facilitating
endovesicular attack. More recently, we have observed17 that
addition of Eu(III) to DPGPNP vesicles also resulted in the
hydrolysis of the outer phosphate groups, whereas addition of
the lipophilic ligand TMED C16 accelerated the hydrolysis of
the remaining 1. The reasons for this behavior were not
analyzed. In the present paper, we specifically address the effect
of additives on vesicular 1 in the presence of Eu(III) and other
lanthanide ions.
Chart 1
Results and Discussion
Vesicle Preparation and Characterization. Unilamellar
vesicles of functional lipid 1 were prepared at pH 7 (0.025 M
HEPES buffer) by sonication (immersion probe) or by extrusion
(0.025 M HEPES buffer, 0.025 M KCl) through polycarbonate
filters (two stacked 100/200 nm filters). Note the different ionic
strengths for the two preparations. The vesicles prepared by
sonication were not stable at higher ionic strength and, hence,
could not be prepared under the same conditions as those used
in extrusion. The two different methodologies gave aggregates
which, although quite different in size, showed very similar
phase transition temperatures, Tc. Aggregate dimensions were
determined by dynamic light scattering, whereas Tc values were
obtained from discontinuities in graphs of fluorescence polariza-
tion (P) Vs temperature by using 1,6-diphenylhexatriene as an
internal probe18 (see Supporting Information) or by differential
scanning calorimetry (DSC). The hydrodynamic diameters and
Tc were 580 ( 80 Å, 38 °C (by fluorescence polarization), for
the sonicated vesicles and 1300 ( 150 Å, 41 °C (by fluorescence
polarization), and 41.8-42 °C (duplicate runs, by DSC) for the
extruded ones. The size distribution of the sonicated vesicles
was slightly broader than that of the extruded ones. However,
the light scattering data could be satisfactorily analyzed with a
Gaussian analysis and showed normalized standard deviations
(or coefficients of variation) of 0.27 and 0.21 for sonicated and
extruded vesicles, respectively. These standard deviations,
considering the low ionic strengths of the solutions, are
consistent with unimodal distributions of the vesicles.19 The
size of the vesicles prepared by sonication and the methodology
of preparation for those obtained by extrusion4 strongly support
the formation of unilamellar vesicles made of a single bilayer
of lipids. Indirect support of the unilamellar composition is
provided by the hydrolysis experiments16 (see also below).
Vesicle preparations were stable for several hours but tended
to grow and eventually precipitate after two or more days.
Lanthanide-Catalyzed Hydrolysis of Vesicular 1. The
above vesicular preparations are fairly stable from the hydrolytic
point of view at pH 7, in line with the hydrolytic inertness of
phosphate diesters at neutral pH. However, upon the addition
of lanthanide salts, 54-70% of the total phosphate20 is cleaved
with rate constants that depend only weakly on the nature of
the lanthanide,21 but more strongly on the mode of preparation
of the aggregate (Table 1). Lipids of the larger aggregates
prepared by extrusion appear to react about 5 times faster than
those of the smaller, sonicated vesicles. However, the percent-
age of cleaved lipid is higher with the sonicated vesicles than
with the extruded ones. This is consistent with the cleavage of
the lipids residing in the outer leaflet of the membrane of both
vesicular preparations. The higher curvature of the bilayers of
the smaller vesicles affects both the distribution of lipids residing
in the outer and inner leaflets of the vesicular membrane and
(12) (a) Gokel, G. W.; Murillo, O. Acc. Chem. Res. 1996, 29, 425. (b)
Murillo, O.; Watanabe, S.; Nakano, A.; Gokel, G. W. J. Am. Chem. Soc.
1995, 117, 7665. (c) Murillo, O.; Suzuki, I.; Abel, E.; Gokel, G. W. J. Am.
Chem. Soc. 1996, 118, 7628.
(13) Xie, Q.; Li, Y.; Gokel, G.; Hernandez, J.; Echegoyen, L. J. Am.
Chem. Soc. 1994, 116, 690.
(14) Scrimin, P.; Veronese, A.; Tecilla, P.; Tonellato, U.; Monaco, V.;
Formaggio, F.; Crisma, M.; Toniolo, C. J. Am. Chem. Soc. 1996, 118, 2505.
(15) (a) Moss, R. A.; Bhattacharya, S.; Chatterjee, S. J. Am. Chem. Soc.
1989, 111, 3680. (b) Moss, R. A.; Fujita, T.; Ganguli, S. Langmuir 1990,
6, 1 197. (c) Moss, R. A.; Ganguli, S.; Okumura, Y.; Fujita T. J. Am. Chem.
Soc. 1990, 112, 6391. (d) Moss, R. A.; Fujita, T. Tetrahedron Lett. 1990,
31, 2377. (e) Moss, R. A.; Fujita, T. Tetrahedron Lett. 1990, 31, 7559. (f)
Moss, R. A.; Fujita, T.; Okumura, Y. Langmuir 1991, 7, 2415. (g) Moss,
R. A.; Fujita, T.; Okumura, Y. Langmuir 1991, 7, 400. (h) Moss, R. A.; Li,
J.-M. J. Am. Chem. Soc. 1992, 114, 9227. (i) Moss, R. A.; Fujita, T.;
Okumura, Y.; Hua, Z.; Mendelsohn, R.; Senak, L. Langmuir 1992, 8, 1731.
(j) Moss, R A. Pure Appl. Chem. 1994, 66, 851.
(19) Ko¨lchens, S.; Ramaswami, V.; Birgenheier, J.; Nett, L.; O’Brien,
D. F. Chem. Phys. Lipids 1993, 65, 1. Note also that the Nicomp 370 model
correlator we have used is equipped with a proprietary Nicomp Distribution
Analysis based on the inversion of the Laplace transform (non-linear least-
squares analysis). This analysis gives information on multimodal distribution.
Applied to the present vesicular systems it indicates a unimodal distribution
of sizes.
(20) The total amount of phosphate present was determined by addition
of excess CTABr to an aliquot of the vesicle solution and following the
cleavage at pH ) 12.16 The final absorbance was then corrected for the
difference in pH.
(16) Moss, R. A.; Swarup, S. J. Am. Chem. Soc. 1986, 108, 5341.
(17) Moss, R. A.; Park, B. D.; Scrimin, P.; Ghirlanda, G. J. Chem. Soc.,
Chem. Commun. 1995, 1627.
(18) (a) Andrich, M. P.; Vanderkooi J. M. Biochemistry 1976, 15, 1257.
(b) Lentz, B. R. Chem. Phys. Lipids 1989, 50, 171.
(21) This is in accord with findings of other authors, see: Morrow, J.
R.; Buttery, L. A.; Berback, K. A. Inorg. Chem. 1992, 31, 16. Breslow, R.;
Huang, D.-L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4080.