J . Am. Chem. SOC.1993, 115, 1705-171 1 timum values of the parameters of this equation to fit the conductance kinetic data, Le., the values of [HX],. and ti. Because of interdependencies, all five parameters k , , k2,k3,to, and [HX], could not be accurately determined simultaneously; therefore, calculations were done with fixed values assigned for either one or both of k2 and k3. The program can also be used to calculate first-order rate coefficients where the parameters are k3, to,and [HX], with k , and k2 set to 0. It was determined from calculations on synthetic data generated with probable values of k l , k2, k3, to, and [HX], that, when k3 is small but kinetically significant, setting k2 to its true value and k3 to 0 will result in a small systematic wave in the plot of the resistance residuals. Error plots with such systematic trends were observed for 2b in 80E and 90E, see Figure 4 in the supplementary material. The fit of the data from 2b in 95E as described above produced a wave in the opposite sense which was shown to be due to the presence of a fraction of 1% pemsyl chloride impurity. These
1705
data were analyzed using a Simplex algorithm, which had been developed previously in our l a b o r a t ~ r y .It~ ~was applied in this case to fit conductance data from the solvolysis of a mixture of two components: the ester 2b reacting according to the kinetic scheme described above and the chloride reacting by a simple first-order process. The number of variables derived from the least-squares treatment was reduced to four by fixing k2 and the solvolysis rate constant for pemsyl chloride a t the independently observed values and constraining k3 by fixingf,,, a t 0.968, the value found to give optimum fits to all kinetic runs. Supplementary Material Available: Figures 3,4, and 5, showing the trends in the errors in the calculated resistances (“resistance residuals”) over the time span of the solvolysis of 2a and 3b in 90E and 2b in 80E using the simple first-order rate law and the rate law given in the Appendix for Scheme I (4 pages). Ordering information is given on any current masthead page.
Ultrathin Monolayer Lipid Membranes from a New Family of Crown Ether-Based Bola-Amphiphiles Servando Muiioz, Jesus Mallen, Akio Nakano, Zhihong Chen, Isabelle Gay, Luis Echegoyen, and George W. Gokel* Contribution from the Department of Chemistry, University of Miami, Coral Gables, Florida 331 24. Received April 23, 1992. Revised Manuscript Received October 23, 1992
Abstract: Twelve novel a$-bis(N-azacrown ether) compounds have been prepared, characterized, and converted into a previously unknown type of niosome. Four are bis( 15-crown-5) derivatives having the following spacer chains: (CH2)12( l ) , (CH2)16 (2), CO(CH2)20C0(3), and (CH2)22(4). The eight bis(aza-18-crown-6) derivatives have the following spacers: (CH2)lo (51, C O ( C H ~ ) I O C (61, O (CH2)12 (71, CO(CH2)&0 (81, (CH2)16 (91, (CH2122 (lo), (cH2)120(CH2)12(11), and CWCH2)11S(CH2)12S(CH2)11C0 (12). Aggregation studies of 1 and 7, employing transmission electron microscopy as well as dynamic and static light scattering, demonstrate that these compounds form a novel class of spherical monolayer lipid membrane vesicles when dispersed in water. Debye light-scattering profiles obtained from a suspension of large (=200 nm diameter) vesicles indicate a relative refractive index near 1. Dynamic turbidimetry in acidic media on a suspension of bola-amphisomes formed from 1, suggested that the contribution of micelle-vesicle equilibria to the bolyte aggregation state is negligible. In neutral or slightly alkaline pH at 35 OC, the vesicles grew irreversibly to yield large, probably multilamellar, aggregates. In acidic media at pH 2, the bola-amphisomes do not coalesce, even at 65 OC. On the basis of the observations presented here, bola-amphiphiles having a hydrocarbon span of 10-12 carbon atoms aggregate in aqueous media into vesicles. When the aliphatic backbone incorporates 16 or more carbon atoms, micelles are formed.
Introduction Nearly a decade ago, Fuhrhop and co-workers reported the synthesis and self-assembly properties of several hydrophobic derivatives of succinic acid in which two polar headgroups are linked covalently by a hydrophobic, saturated hydrocarbon skeleton (Figure l ) . ’ Fuhrhop called these surfactant molecules bolaamphiphiles-the name derives from the South American slingshot comprised of two leather balls attached to a string. Bolas are designed to tangle around the legs of cattle and thereby immobilize them. Their molecular counterparts (bola-amphiphiles) often remain extended when dispersed in water and form monolayer lipid membrane vesicles or bola-amphisomes. The covalent chains of bola-amphisome monomers need not interdigitate in the bilayer midplane as do normal amphiphiles. A covalent span of 12 carbons affords an “ultrathin” membrane with a width of less than 20 A compared to common biological bilayer membranes that have thicknesses ranging from about 30 to 100 A.2 The monomers normally involved in the formation ( 1 ) Fuhrhop, J.-H.; Bach. R. In Advances in Supramolecular Chemistry; Gokel, G. W., Ed; Vol. 2, JAI Press: Greenwich, CT, 1992; Vol. 2, pp 25-63. (2) Yeagle, P. The Membranes of Cells; Academic Press: New York, 1987.
of bilayer membranes interdigitate along the membrane’s midplanee3 When the extent of interlaminar overlap becomes small, some of the surfactant monomers protrude from the membrane and are readily exchanged by intercolloidal collision^.^*^ This process facilitates coalescence of the vesicles to yield larger, heterogeneous, multilamellar assemblies. In the case of a covalently linked monolayer membrane, the frequency of intervesicular fusion is reduced because both vertical diffusion and intervesicular exchange of surfactant monomers are inhibited. Covalently linked dipolar surfactants are found in the membrane of thermophilic archaebacteria (unicellular organisms that live in boiling water under conditions of high ionic strength and low pH6). Such bacteria maintain an intracellular pH of about 6.5 when the external p H is 1.5. Gliozzi et a].’ isolated from the (3) Kim, J. T.; Mattai, J.; Shipley, G. G. Biochemistry 1987, 26, 6592. (4) Fendler, J. H. Membrane Mimetic Chemistry;J. Wiley and Sons: New York, 1982. ( 5 ) Guillaume, B.C. R.; Yogev, D.; Fendler, J. H. J. Phys. Chem. 1991, 95, 7489. (6) (a) Gliozzi, A.;
Paoli, G.; Rolandi, R.; de Rosa, R.; Gambacorta, A.
J . Elecrroanal. Chem. 1982. 1 4 1 . 591. (7) Gliozzi, A.; Rolandi, R.;de Rosa, M.; Gambacorta, A. J. Membrane Biol. 1983, 75, 45.
0002-7863/93/1515-1705%04.00/0 , 0 1993 American Chemical Society I
,
Muiior et al.
1706 J. Am. Chem. SOC.,Vol. 115, No. 5. 1993 Scheme I
0
b
NEt3, benzene, 0-5 OC,48h at RT
v Figure 1. Bilayer (left) and monolayer lipid membranes
I LIAIH4, THF, 24h, A Figure 2. Archaebacteria membrane.
membrane of Caldariella acidophila the bola-amphiphathic surfactant shown in Figure 2. They noted that the survival of these bacteria depends in part on the strength of their monolayer cell walls: the covalent link between the polar headgroups precludes vertical dissociation of the membrane, while weak intermonomeric van der Waals attractive forces inhibit lateral lysis. The headgroups probably strengthen the membrane against both transversal and longitudinal lysis because of their contribution to the formation of intermonomeric, hydrogen-bonded domains. Because of their ability to withstand physicochemical stress, monolayer membranes may be useful in such applications as the construction of molecular energy transducers and energy storage devices and perhaps in drug delivery applications. The latter is related to our development of redox-switched vesicles based on the ferrocene nucleus.* Indeed, we have extensive experience in the formation of crown-based membrane systems? We now report the synthesis of 13 novel bola-amphiphiles and present a detailed study of the first bola-amphisome formation from such crown ether-based structures.
Results and Discussion Syntheses. The bola-amphiphile compounds reported in this paper are N-substituted derivatives either of aza-15-crown-5 or aza-18-crown-6. The nitrogen atoms of the crown ethers are spanned by a variety of spacer units such as dodecane (-(CH2),2-) and hexadecanedioyl (-CO(CH2)14CO-), making all of the bola-amphiphiles either bis(tertiary amines) or bis(amides). Among these, the aggregation behavior of compounds 1 and 7 was studied most thoroughly. 1,12-Bis(aza-15-crown-5)dodecane (1) and 1,12-bis(aza-18-crown-6)dodecane (7)were prepared from the dodecanedioic acid and the appropriate crownlo by acylation by dodecanedioyl chloride of either aza- 15-crown-5 or aza- 18crown-6, followed by lithium aluminum hydride reduction of the amide. The double acylation was effected in 70% yield in both cases, and reduction afforded 76% of 1 and 72% of 7. The sequence and structures are shown in Scheme I. The oily products were purified by chromatography and were fully characterized. Compounds 1-12 are shown in Table I. Aqueolro M-Assembly. Bola-amphiphiles 1,5, and 7 aggregate in aqueous media to yield vesicles (Table I). The compounds contain 10-12-carbon backbone spans that are terminated by aza- 15-crown-5 or aza- 18-crown-6. The diamidic precursor 6 to (8) Medina, J. C.; Gay, I.; Chen, 2.; Echegoyen, L.; Gokel. G. W. J. Am. Chem. SOC.1991, 113, 365. (9) (a) Echegoyen, L. E.; Hernandez, J. C.; Kaifer, A,; Gokel, G. W.; Echegoyen, L. J. Chem. SOC.,Chem. Commun. 1988, 836. (b) Echegoyen, L. E.; Portugal, L.; Miller, S. R.; Hernandez, J. C.; Echegoyen, L.; Gokel, G. W. Tetrahedron Lett. 1988, 4065. (c) Fasoli, H.; Echegoyen, L. E.; Hernandez, J. C.; Gokel, G. W.; Echegoyen, L. J. Chem. SOC.,Chem. Commun. 1989, 578. (d) Gokel, G. W.; Echegoyen, L. E. Adu. Bioorg. Chem. Frontiers 1990. I, 116. (e) Muiioz, S.; Malltn, J. V.;Nakano, A,; Chen, 2.; Gay, 1.; Echegoyen, L.; Gokel, G. W. J. Chem. Soc., Chem. Commun. 1992, 520.
(IO) Schultz, R. A.; White, B. D.; Dishong, D. M.; Arnold, K. A,; Gokel, G. W. J. Am. Chem. SOC.1985, 107, 6659.
Table I. Novel Bis(crown) Bola-Amphiphiles compd ring yield no. size spacer chain (%) 1 2 3 4 5
15 15 15 15 18 18 18 18 18 18 18 18
6 7
8 9 10 11 12
(cH2)12 (cH2)16 CO(CH2)zoCO (CH2122 (CH2)10 CO(CH2)loCO (CH2)12 CO(CH2)IPCO (cH2)16 (CH2122 (CH2)120(CHz)iz CO(CH2)IIS(CH2)12S(CH2)IlCO
76 78 69 73 45 79 72 72 70 71
61 57
mp
aggn statea vesicle oil 65-66 micelle 76-78 micelle 55-56 micelle oil vesicle oil micelle vesicle oil oil micelle 48-49 micelle 58-59 micelle micelle oil wax vesicle (OC)
'Determined by dynamic light scattering and/or transmission electron microscopy. Table 11. Aggregation Properties of Bola-Amphiphilic Derivatives of Crown Ethers at 25 OC' diameter, Ab dynamic transmission compd no. turbidimetry electron microscopy 1
7
730 1200
760 1180
'2-5 mM bolyte in water, dispersed for 30 min at 10 'C, using an ultrasonic cell disruptor equipped with a titanium microtip and operated at 40 W. bThe uncertainty in the measured diameter is *30%
7 forms micelles. Likewise, diamides 3, 6, and 8 form micelles. This suggests that all diamidic bola-amphiphiles may do so. Compound 12 is an exception to this possible generalization, but we feel that compound 12 is a special case because it contains a chain that is overall far longer than any other compound and it includes two heteroatoms. Otherwise, those compounds having backbones containing 16 carbon atoms or more appear to favor micelle formation. This suggests that as the length of the spine increases, the surfactant monomers become folded about the midplane of the covalent span forming structures that favor micelles. When the 1,12-bis(aza- 15-crown-5)dodecane bola-amphiphile 1 was dispersed in water by ultrasonic irradiation a t 10 'C, an almost optically transparent suspension of aggregates was formed. The hydrodynamic diameters of these particles measured by dynamic turbidimetry is about 730 A (Table 11). Electron transmission micrographs (TEM) of the amphisomes (treated with Os04) revealed the presence of unilamellar vesicles of apparent diameter 760 A. Dynamic light-scattering on a similarly prepared suspension of 7 indicated the presence of organizates having hydrodynamic diameters of 1200 A. As above, T E M confirmed
J . Am. Chem. SOC.,Vol. 115, No. 5, 1993 1707
A New Family of Crown Ether-Based Bola-Amphiphiles Table 111. Debye Light-Scattering Profile for 2.19 X IO-) M 1 in Water at 25 O C o d turbidance‘ standard correlation A. nm A-* (Xl0”l obsd calcdd deviation coefficient fO.00125 0.9970 6.25 0.0881 0.0865 4000.0775 0.0773 424 5.56 4.94 0.0676 0.0691 450 4.45 0.0610 0.0626 474 5 00 4.00 0.0568 0.0566 0.05 15 0.0518 524 3.64 0.0464 0.0473 550 3.30 0.0457 0.0437 574 3.03 0.0409 0.0404 600 2.78 0.003 5W infinity ODispersed in water for 30 min, at 10 OC using a bath sonicator. The typical vesicle diameter measured by dynamic light scattering is 200 f 60 nm. ‘Addition of 50 mM sodium dodecyl sulfate lyses the vesicles and shows that the electronic absorption by the macrocyclic polyether headgroup is negligible at A 2 400 nm. dThe abbreviation ‘calcd turbidance” is that estimated according to a linear least-squares simulation of the data.
