Langmuir 1991, 7, 1935-1943
1935
Vesicular and Monolayer Properties of Diastereomeric Surfactants David A. Jaeger,',? Witold Subotkowski,+Jamshid Mohebalian,t Yasmin M. Sayed,? Bharat J. Sanya1,t Jonathan Heath,t and Edward M. Amett*** Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071, and Department of Chemistry, Duke University, Durham, North Carolina 27706 Received September 24, 1990.In Final Form: April 1 , 1991
Three diastereomeric, quaternary ammonium surfactants, [3-(~-4,~-5-dihexadecyl-r-2-methyl-l,3-dioxolan-2-yl)propyl]trimethylammoniumbromide (la) and its t-4J-5- (lb) and c-4,t-5- (IC)isomers, were prepared and the properties of their vesicles and monolayers compared. The vesicles were characterized by dynamic laser light scattering, differential scanning calorimetry, and [14C]sucrose entrapment and release studies. Clear differences among the three systems were found in the latter two studies. The phase transition temperature order for both sonicated and vortexed vesicles was la > IC > lb, and for sonicated vesicles the permeabilityorder was l b > IC> la. The three surfactants also displayed different monolayer characteristics. The degrees of expansion in the surface pressure-area isotherms, the monolayer stability limits, and the propensities of the films to spread from their crystals followed the same order: l b > IC > la. Overall, the results suggest that in both vesicular and monolayer form l a has the tightest and l b the loosest surfactant packing. Introduction There have been relatively few studies of the dependence of vesicular' and monolayer2 properties on surfactant stereochemistry. Herein we report a study of diastereomeric surfactants la, lb, and IC that centered around the following question: How, if at all, is their diastereomeric nature expressed in vesicular and monolayer form? If the full potential of vesicles as membrane models and drug delivery agents3is to be realized, it is important to delineate the influence on their properties of such subtle features as the stereochemistry of their constituent surfactants. Differences among sizes, phase transition temperatures, and permeabilities have been found in previous studies' of vesicles derived from diastereomeric Surfactants, each of which contained a head group bearing a phosphocholine or hydroxyl unit. To our knowledge, the present study is the first of this type to be performed with relatively simple quaternary ammonium surfactants. There have been only three previous studies of the monolayer properties of diastereomeric In two of these involving meso and dl double-chain diacids, uniformly the monolayers of the former compressed more easily than those of the latter.2a$bIn the third study, the monolayers of two single-chain quaternary ammonium surfactants behaved
identically, but compressed more easily than that of a 1:l mixture of the two.2c
la
lb
1c
Results
Syntheses. The syntheses of la, lb, and IC are summarized in Scheme I. Alkyne 2 was reduced to trans alkene 3, which was converted into meso diol 5 through epoxide 4. Ketalization of 5 with 5-bromo-2-pentanone gave a mixture of diastereomers 6a and 6b, which was separated by flash chromatography on silica gel. The individual bromo ketals were then transformed into l a and lb. After the reduction of 2 to cis alkene 7, the + University of Wyoming. preparation of surfactant ICparalleled those of la and lb t Duke University. as illustrated. Although these syntheses appear to be (1) Forexamples,see(a)Singer,M.A.;Jain,M.K.;Sable,H.Z.;Pownall, straightforward, several steps were complicated by the H. J.; Mantulin, W. W.; Lister, M. D.; Hancock, A. J. Biochim. Biophys. extreme hydrophobicity of the intermediates. For exActa 1983,731,373. (b) Wisner, D. A,; Rosario-Jansen, T.; Tsai, M.-D. J. Am. Chem. SOC.1986,108,8061. (c) Tsai, T.-C.; Jiang, R.-T.; Tsai, ample, a well-established procedure4 for the anti-hydroxM.-D. Biochemistry 1984,23,5564. (d) Jarrell, H. C.; Wand, A. J.; Giylation of alkenes simply did not work. Only after its ziewicz, J. B.; Smith, I. C. P. Biochim. Biophys. Acta 1987,897,69. (e) modification were the conversions of alkenes 3 and 7 into Iwamoto, K.; Sunamoto, J.; Inoue, K.; Endo, T., Nojima, S. Biochim. Biophys. Acta 1982,691,44. (0 Endo, T.; Inoue, K.; Nojima, S. J. Biothe corresponding diols achieved. chem. 1982, 92, 953. (9) Arnett, E. M.; Gold, J. M. J. Am. Chem. SOC. The stereochemical assignments for la and lb were 1982,104,636. (2) (a) Harvey, N.; Rose, P.; Porter, N. A.; Huff, J. B.; Arnett, E. M. based on their NOE difference 'H NMR spectra (see the J. Am. Chem. SOC.1988,110,4395. (b) Amett, E. M.; Harvey, N.; Rose, Experimental Section), and that for IC followed from its P. L. Langmuir 1988,4,1049. (c) Jaeger, D. A,; Mohebalian, J.; Rose, P. synthesis. The stereochemical assignments were also L. Langmuir 1990,6,547. (d) Harvey, N. G.; Rose, P. L.; Mirajovsky, D.; consistent with comparisons of the 'H and 13C NMR Amett, E. M. J. Am. Chem. SOC.1990, 112, 3547. (e) Stewart, M. V.; Arnett, E. M. In Topics in Stereochemistry; Allinger, N. L., Eliel, E. L., chemical shift patterns for la and lb and for 6a and 6b Wilen, S. H., Eds.; Wiley: New York, 1982; Vol. 13, p 195, and references therein. (3) Knight, C. .G., .Ed.Liposomes: From Physical Structure to Therapeutic Appkcations; Elsevier/North Holland: Amsterdam, 1981.
(4) Swern, D.; Billen, G. N.; Scanlan, J. T. J. Am. Chem. SOC.1946,68, 1504.
0 1991 American Chemical Society
1936 Langmuir, Vol. 7, No. 9, 1991
Jaeger et al.
Scheme I*
3
2 Br(CH,),
,
(
O
4
F
*'.
Br(CH2)3,(ox
+
Me
0
$
H C16H3,
Q
H
Me
C16H33
0
la
+
lb
H
C16H33 C16H33
6a
f
6b
0
OH OH
8
9
I
/ \
C16H33.
7
B r V 3 - l z ) 3 , ( ~ ~ c 1H6 H m
***
Me
H
-
I
e
f
1c
C16H33
6c a Key: (a) Li, EtNH2, 10 "C; (b) HzOz, H20, HCOzH, 80 (60) "C; (c) HCOzH, H20, Ca&, reflux; (d) KOH, EtOH, reflux; (e) Br(CHz)&OMe, p-MeCaH4SOaH, CeHs (Dean-Stark); (0 MesN, MeOH, 25 "C; (g) Hz (60 psi), Pd-BaSO4, quinoline, EtOH-hexane, 25 "C.
Table I. DLLS Measurements of HomoPeneous and Mixed Vesicles. ~~
system
la lb IC 10
lC/10 IC
10
lc/lO
a
vesicle preparation sonicated sonicated sonicated sonicated sonicatedb vortexed vortexed vortexed
~
population 1 diameter, nm vol ?6 19fl 62 f 3 62 f 6 24 f 1 62 f 5 20 f 1 19f3 68 f 1 77 f 18 57 f 5 62 f 16
18f3 20f6 19 f 4
population 2 diameter, nm vol % 60 f 6 34 f 2 34 f 6 77 f 13 55 f 9 35 f 5 61 f 12 30 f 3 294 f 55 188 f 20 231 f 29
*
14f1 17f3 12f2
population 3 diameter, nm vol % 168 f 17 4 f 1 241 19 4f2 177 f 14 3fl 210 f 13 3f2 1352 f 443 1231 f 243 1136 f 60
68f2 63 a 70 5
*
The limits of error are average deviations for 2 3 runs with different samples. Variable results were obtained; see the Experimental Section.
and with the TLC characteristics of the latter pair using arguments employed previously for related diastereomers.% Vesicle Preparation and Characterization. Homogeneous vesicles of 1and dioctadecyldimethylammonium bromide (10, DODAB) and their mixed vesicles (25 mol 76 1) were prepared by sonicating or vortexing the surfactant/surfactant mixture in a pH 7.5 Tris buffer (at ca. 55 "C for 1, or ca. 65 "C for 10 and 1/10 mixtures). They were characterized by dynamic laser light scattering (DLLS),differentialscanningcalorimetry (DSC),and [14C]sucrose entrapment and release studies.
DLLS. Homogeneousvesicles of surfactants 1prepared by sonication were analyzed by DLLS (90"scattering angle) at 23 "C. The results are summarized in Table I. For each system, distribution analysis of the autocorrelation function established the presence of three populations grouped at the indicated hydrodynamic diameters. Populations 1 and 2 correspond to small unilamellar vesicles
(SUVS).~No physical significance can be attributed to population 3. It probably corresponds to a few large particles that were grouped together as part of the trimodal distribution by the mathematical analysis of the light scattering data. A representative histogram of hydrodynamic diameter vs relative volume (mass) for surfactant la is given in Figure 1. Vesicles of 10 and lc/lO prepared by sonication, and those of IC, 10, and lc/lO prepared by vortexing were analyzed as above. The results are summarized in Table I. As indicated, sonicated IC/ 10 gave results that varied from sample to sample (see the Experimental Section). Each vortexed system contained three populations. Populations 2 and 3 correspond to multilamellar vesicles (MLVS),~ and minor population 1 corresponds to SUVs. It should be noted that there can be significant intraparticle scattering for particles larger than ca. 200 nm, resulting in partial destructive interferencea6 Thus for the vortexed systems, which contain significant popula( 5 ) Fendler, J. H. Membrane Mimetic Chemistry;Wiley-Interscience: New York, 1982; Chapter 6. (6) Berne, B. J.; Pecora, R. Dynamic Light Scattering; Wiley-Interscience: New York, 1976; Chapter 8.
Langmuir, Vol. 7, No. 9, 1991 1937
Properties of Diastereomeric Surfactants 12
1
l
1
I
24 27 31
111 126 148
170 106 226 260 300 346 0
20
40
SO
80
100
Relatlve Volume
Figure 1. DLLS histogram of hydrodynamic diameter vs relative volume for sonicated la. tions of vesicles larger than 200 nm, the DLLS results, obtained at a single scattering angle, must be viewed with caution. However, a t a minimum they give a reasonable qualitative representation of the vesicle size distributions. DSC. Thermograms were obtained for sonicated and vortexed homogeneous vesicles of surfactants 1; each contains a single transition. The former are given in Figure 2a, and the latter (not shown) are comparable, except for their greater endothermic nature. The phase transition temperatures (T,)and calorimetric enthalpies ( h H c d )are summarized in Table 11. A t its T,, a vesicle bilayer undergoes a transition from the gel to the less-ordered liquid crystalline state, corresponding to conformational changes of the alkyl group^.^ In a previous study, 1 was obtained as a 1:1:2 mixture of la, lb, and IC, which gave a T,value of 26 f 2 OC in hydrated formss A DSC study of mixed vesicles of 1 and 10 was also performed. Thermograms of sonicated and vortexed 10 and 1/10 are given in Figures 2b and 3, respectively, and the T,and AHcd values are summarized in Table 11. Entrapment and Release Studies. Sonicated homogeneous vesicles of 1 and 10 were prepared in the presence of [14C]sucrose,and nonentrapped material was removed by gel filtration chromatography. The profiles for release of [14C]sucroseat pH 7.5 (Le., without surfactant cleavage by hydrolysis2c) are given in Figure 4. Sephadex G-50 (medium) was used for gel filtration chromatography, which has an exclusion limit of molecular weight 30 000. Since even the smallest SUV has a molecular weight far in excess of this value, no fractionation of the vesicle populations occurred. Therefore, each release profile represents composite results for all of the vesicles present in a sample.9 Each of the profiles for la, lb, and ICexhibits an initial rate, followed by a slower rate of [W]sucrose release. These features may reflect the presence of different vesicle sizes1° and/or different intrinsic permeability characteristicsll for these vesicles. They are also consistent with the (7) Reference 3, p 273. (8) Jaeger, D. A.; Chou, P. K.; Bolikal, D.; Ok, D.; Kim, K. Y.; Huff, J. B.; Yi, E.; Porter, N. A. J. Am. Chem. SOC.1988,110,5123. (9) For each surfactant, a fraction of the [14C]sucrcee release may in fact reflect ita desorption from the outside vesicle surface (CarmonaRibeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983,733,172). (IO) (a) Johnson, S.M.; Bangham, A. D. Biochim.Biophys. Acta 1969, 193,82. (b) Schwartz, G.; Robert, C. H. Biophys. J. 1990,58,577. (11) Stefely, J.; Markowitz, M. A.; Regen, S. L. J. Am. Chem. SOC. 1988,110, 7463.
presence of MLVs,12but the DLLS data of Table I indicate that each of the sonicated systems contains two major populations of SUVs and no more than ca. 4 % by volume (mass) of MLVs. Qualitatively it is evident that the overall permeability order is lb > l c > la. Since the release kinetics were not well behaved, as is evident from Figure 4, quantitation of the initial release rates was limited to calculationll of onepoint first-order rate constants (kl) at time = 4 h: la, 0.80 x 10-5 s-l; lb, 1.1 x s-1; IC, 0.95 x s-l. For s-l for 10 a t t = 4 h. comparison, k l = 6.5 X Monolayer Studies. All monolayer experiments were performed on a subphase buffered at pH 7.5 to avoid surfactant hydrolysis. The surface pressure (dvs area (A) isotherms for surfactants la, lb, and ICat 25 "C are given in Figure 5. The arrows on the curves indicate the directions of the compression and expansion cycles. Although the films are similar in their packing behaviors, the curves are distinct from one another with respect to experimental error (*2 A2/moleculeat the 95% confidence limit). Surfactant lb forms the most expanded film and, like the slightly more condensed film of IC, has identical compression and expansion cycles. Surfactant l a forms the most condensed film and shows hysteresis upon expansion. There is also a kink in the compression cycle of la at high surface pressure (ca. 34 dyn/cm), which is absent in the other two isotherms. The stability limits of the three diastereomeric films, as well as their equilibrium spreading pressures (ESPs), are given in Table 111. The monolayer stability limits and the relative propensities of the films to spread from their crystals (ESPvalues) follow the same order as the degrees of expansion in the ?r/A curves: lb > IC > la. Discussion The results of Table I indicate that by DLLS the sonicated homogeneous vesicles of 1 are very similar. Each system contained major and minor SUV populations with hydrodynamic diameters of 19-24 and 55-77 nm and vol % values of 62 and 34-35, respectively. However, the data suggest that the SUVs of lb are slightly larger than those of la and IC. In a related study, homogeneous micelles of l la and l l b were identical by DLLS but different than their mixed micelles.2c Sonicated 10 contained three vesicle populations comparable to those of sonicated 1. The vortexed vesicles of IC, 10, and lc/10 had similar sizes and vol % values.
llb
The sonicated homogeneous vesicles of 1 gave different
T,values by DSC (Figure 2a). The AHcd value for lb was less than the almost identical values for la and IC. The vortexed homogeneous vesicles gave T,values essentially the same as those for the sonicated vesicles but larger, almost identical A H d values. Often MLVs compared to SUVs give greater endothermic transitions and T,values.13 The fact that la and lb displayed the highest and lowest T,values, respectively, suggests that within the bilayers (12) Dorn, K.; Klingbiel, R. T.;Specht, D. P.; Tyminski, P. N.; Ringdorf, H.; OBrien, D. F. J. Am. Chem. Soc. 1984,106, 1627. (13) For example, see Ringsdorf, H.; Schlarb, B.; Tyminski, P. N.; O'Brien, D. F. Macromolecules 1988,21, 671.
1938 Langmuir, Vol. 7, No. 9,1991
Jaeger et al. 20
15
10
5
I
I
1
20
10
I
I
4.0
30
I
I
50
60
0 10
20
30
40
50
60
Temperature, 'C
Figure 2. DSC thermograms for (a) sonicated 1 and (b) sonicated and vortexed 10. Table 11. DSC Measurements of Homogeneous and Mixed Vesicles. sonicated vortexed system la lb IC
T,,O C
" I *
34.2 f 0.2 28.3 f 0.3 30.5 f 0.1
23.3 f 0.5 17.8 f 0.5 24.4 f 0.5
Tct O C 34.6 f 0.1 28.7 h 0.1 30.8h 0.1
37 f 1 36 f 1 33 k 3
42.6k0.1 38.4f0.1 39.0h0.1
59f 2 52f 1 43f 1
42.9f 0.1 42.0f 0.1 42.4 f 0.1
64 f 1 66 f 1 65 f 1
AHdb
10'
la/10 lb/10 lc/10
The limits of error are average deviations for 1 2 runs. Units:
kJ/mol and J/g for homogeneous and mixed systems, respectively. Both the sonicated and vortexed samples gave multiple transitions: see Figure 2b and the Discussion.
of surfactants 1 the former has the tightest/most organized and the latter the loosest/least organized hydrocarbon chain packing. The DSC thermogram of sonicated 10 contains multiple transitions from 32 to 57 "C and that of vortexed 10, transitions at 33, 46, and 52 "C (Figure 2b). Note that within the complex thermogram of the sonicated system there are shoulders at 33 and 52 "C and a peak at 46 "C, corresponding to transitions in the thermogram of the vortexed system. Except for the transition at 33 "C in the latter thermogram, these results are generally consistent with those of Kodama, Kunitake, and co-workers.14 In particular, the DSC characteristics of 10-water mixtures can be complex, reflecting the existence of two gel and two liquid crystalline states.l*a Two features of the thermograms of the sonicated 1/10 mixtures of Figure 3a are noteworthy. First, each contains a single broad endotherm and is thus simpler than the thermogram of sonicated homogeneous 10. Therefore, mixed vesicles are indeed formed, as opposed to separate homogeneous vesicles of 1 and 10. Second, the la/10 system has a higher Tcvalue than the similar lb/10 and lc/16 systems. Thus, of the three mixed systems, la/10 has the most organized hydrocarbon chain packing, as does homogeneous l a in comparison with lb and IC as noted above. ~~~
~
(14) (a) Kodama, M.; Kunitake, T.; Seki, S. J. Phys. Chem. 1990,94,
1550. (b)Okahata, Y.;Ando, R.; Kunitake, T. Ber. Bunsen-Ges. Phys. Chem. 1Ydl,85, 789.
The thermograms of the vortexed 1/10 mixtures of Figure 3b are quite similar. The Tcvalues for lb/10 and lc/10 are greater, and that for la/10 the same, relative to those for the sonicated systems of Figure 3a. Overall, the order of Tcvalues for both the sonicated and vortexed mixed vesicles is the same as that for the homogeneous vesicles. Thus the diastereomeric nature of the surfactants is expressed in the mixed as well as in the homogeneous vesicles. The greater differences among the thermograms of the sonicated compared to the vortexed mixed systems are perhaps surprising given the fact that the bilayers of SUVs are less ordered than those of MLVs.15 On this basis, sonicated vesicles might be expected to be less sensitive to stereochemical differences among guests. The results of the entrapment and release studies indicate that sonicated homogeneous 1 and 10 contain closed bilayer structures. Also, it is apparent that the dissimilarities in physical properties among vesicular la, lb, and IC,as detected by DSC, are manifested in different membrane permeabilities to [14C]sucrose. The permeability order is consistent with the hydrocarbon chain packing order suggested by the T,values, i.e., la has the tightest bilayer packing and the least permeability, l b the loosest packing and the greatest permeability, and IC intermediate packing and permeability. Note that the permeability of vesicular 10 is considerably greater than those of vesicular la, lb, and IC. For the diastereomeric surfactants the relative degrees of molecular aggregation in monolayer form are consistent with the results obtained in vesicular form. Surfactant la displayed the tightest monolayer packing, whereas lb displayed the loosest packing. The kink in the isotherm for la noted above occurred at a surface pressure well above the film's stability limit (Table 111) and probably represents the collapse of the film to some threedimensional state, rather than a phase transition between monolayer states. The most striking difference in monolayer behavior among the three surfactants occurred when the films were allowed to equilibrate with their crystals (ESPs, Table 111). In this case, the differences among the short-range intermolecular forces in the solid state, wherein chiral (15) Sheetz, M. P.; Chan, S. I. Biochemistry 1972, 1 1 , 4573.
Properties of Diastereomeric Surfactants
Langmuir, Vol. 7, No. 9, 1991 1939 24
1P
18
12
6
20
10
50
4-0
30
60
20
10
40
30
50
60
Temperalure, 'C
Figure 3. DSC thermograms for (a) sonicated 1/10 and (b) vortexed 1/10.
ALleCtlle
4 0
10
20
30
40
50
time, h
Figure 4. Release of [14C]sucrosefrom sonicated vesicles of la (- - -), lb (-), IC (-), and 10 (- -) at ca. 2 3 O C . discrimination is expected to be greatest,2dcan manifest themselves in the degrees to which the monolayers spread spontaneously. In surfactant la, with respect to the 1,3-dioxolane ring, the quaternary ammonium head group is trans to both hexadecyl chains (trans,trans stereochemistry). In lb,the head group is cis to both alkyl chains (&,cis) and in IC, cis to one and trans to another (cis,trans). As noted above, la in monolayer form gave the most condensed film and in vesicular form the greatest T, value and the least permeability to [ l4C]sucrose. These facts suggest that the monolayer and bilayer of la contain the tightest surfactant packing. Thus it is evident that of the three stereochemical relationships of head group and alkyl chains within surfactants 1, that of la is accommodated best by a monolayer/homogeneous vesicle bilayer. It is interesting to speculate about the dispositions of the surfactant head groups of 1 within the bilayer membranes and monolayers. There are two limiting orientations of the l,&dioxolane ring with respect to an aggregate/air-water interface: parallel and perpendicular. With the former arrangement, the hexadecyl chains and head group of la can readily extend into their preferred
Figure 5. Surface pressure-area isothermsfor the compression and expansion cycles of la, lb, and IC on a pH 7.5 Tris-HC1 buffer subphase at 25 "C compressed at a rate of 19.24 (A2/ molecule)/min. Table 111. Monolayer ESPs and Stability Limits ESP, dyn/cm stability limit,O dyn/cm
surfactant la lb IC
*
13.34 2.55 33.12 k 0.90 27.17 dc 3.13
-
-7 10 -8
4 A film is judged stable if halting barrier compression results in a decay of surface pressure of no more than 0.1 (dyn/cm)/min.
microenvironments, bilayer/air and water, respectively. For lb, either the head group or the two alkyl chains, but not all three, can extend directly into the preferred microenvironment without looping from one face of the ring to the other. For IC, the head group and one of the hexadecyl chains can extend directly into the preferred microenvironments, but the other hexadecyl chain must loop. In each of surfactants 1, with the dioxolane ring perpendicular to the surface, the head group and the two hexadecyl chains can extend directly into their preferred microenvironments. Overall, it is likely that the stereochemical differences among surfactants 1 are expressed to the greatest degree with the dioxolane ring parallel to the aggregate/air-water interface. The limiting molecular areas of 80-100 A2/molecule for la-c in Figure 5 are consistent with such an orientation.
1940 Langmuir, Vol. 7,No.9,1991 As noted above, there have been other reports of different bilayer properties for diastereomeric surfactants. For example, Singer and co-workers found different T, values and permeability characteristics for vesicles of diastereomeric cyclopentanoid analogues of 1,2-dipalmitoylsn-g1ycero-3-phosphocho1ine.'~Tsai and co-workers reported different T, values for two diastereomeric 1,2dipalmitoyl-sn-glycero-3-thiophosphocholines in multilamellar form,lbbut the same value for their SUVs, which, however, had different sizes.lC Jarrell and co-workersld and Sunamoto, Nojima, and co-workersle*freported different T,values for glyceroglycolipids with diastereomeric head groups. Fuhrhop and co-workers found different structural characteristics for lipid bilayer fibers of diastereomeric N-octylaldonamidesa16 These examples involve surfactants bearing head groups with hydroxyl or phosphocholine units that can undergo intermolecular intera~ti0ns.l~ Perhaps intermolecular ion-dipole interactions are possible between the quaternary ammonium and ketal groups of 1. There have been previous studies, similar to that above with 1 and 10, of the effects of single- and double-chain surfactants,18 including amphiphilic drugs,19on the phase transitions of vesicle membrane hosts. In general, the interest in mixed-surfactant membranes derives from the fact that biological membranes are composed of complex combinations of surfactants and other components, rather than a single surfactant.20 In a study of membrane hosts containing diastereomeric guests, van Deenen and coworkers found different effects of cholesterol and epicholesterol on the permeabilities and T,values of phospholipid vesicles.21 Similarly, Arnett and Gold noted different effects of cholestanol and epicholestanol on phospholipid vesicles.1g Some of the above results and discussion contrast with those of previous studies. For 10 on sonication, Kunitake and co-workers reported the formation of lamella and perhaps a small amount of vesicles,22whereas Moss and Fujita reported that of a single population of vesicles (hydrodynamic diameter = 29 nm).23 Also, Pansu and c o - ~ o r k e r snoted ~ ~ that closed vesicles (hydrodynamic diameter = 160 nm) are only a minor fraction of sonicated suspensions of n-(C18H37)2N+MezCl-(DODAC),a closely related compound. However, Deguchi and Mino reported that DODAC forms MLVs and SUVs on sonication for short and long times, respectively.= Carmona-Ribeiro and Chaimovich reported that [14C]sucrosedoes not permeate sonicated DODAC vesicles.9 But Fendler and co-workers noted that sonicated DODAC vesicles (diameter = 100150 nm) are permeable to ["CI~erine.~~ Thus there is disagreement about the permeability of DODAC/ 10 vesicles with respect to small organic molecules, and even (16) Fuhrhop,J.-H.; Schnieder,P.; Boekema, E.; Helfrich,J.Am. Chem. SOC.1988,110,2861. (17) Viti, V.; Minetti, M. Chem. Phys. Lipids 1981,28, 215. (18) For example, me (a) Muller, K.; Lipka, G.;Lohner, K.; Laggner, P. Thermochim. Acta 1985,94,187. (b) Mabrey, S.; Sturtevant, J. M. R o c . Natl. Acad. Sci. U.S.A. 1976, 73, 3862. (19) For example, see Kurech, B.; Lullmann, H.; Mohr, K. Biochem. Pharmacol. 1983,32, 2589. (20) McElhaney, R. N. Chem. Phys. Lipids 1982,30,229. (21) (a) Demel, R. A.; Bruckdorfer, K. R.; van Deenen, L. L. M. Biochim. Biophys. Acta 1972,255,321. (b) de Kruyff, B.; Demel, R. A,; van Deenen, L. L. M. Biochim. Biophys. Acta 1972,255,331. (22) Kunitake, T.; Okahata,Y.; Yasunami, S. Chem. Lett. 1981,1397. (23) Moss,R. A.; Fujita, T. Tetrahedron Lett. 1990,31, 2377. (24) (a) Pansu, R. B.; Arrio, B.; Roncin, J.; Faure, J. J. Phys. Chem. 1990,94,796. (b) Pansu, R. B.; Lan, L.; Faure, J.; Roncin, J.; Arrio, B. New. J . Chem. 1990,14, 105. (25) Deguchi, K.; Mino, J. J. Colloid Interface Sei. 1978, 65, 155. (26) Tran, C. D.;Klahn, P. L.; Romero, A.; Fendler, J. H. J. Am. Chem. SOC.1978, 100, 1622.
Jaeger et al.
about their formation. As suggested by Harada and coworker~,~' the discrepancies may derive from different vesicle preparation and characterization protocols, e.g., sonication power and time, solvent (pure water or buffers of different composition), electron microscopy sample preparation. Summary Distinct differences have been detected among the vesicles of diastereomeric, quaternary ammonium surfactants la-c by DSC and [14C]sucrose entrapment and release studies. The three surfactants also displayed different monolayer characteristics. A priori, one might expect the expression of stereochemical differences among surfactants to be more likely within monolayers than vesicles due to tighter surfactant packing within the former.2d Experimental Section General Procedures and Materials. 'H (270 and 400 MHz) and 13C (67.8 MHz) NMR spectra were recorded in CDCl3with Me4Si and CDCl3 (center line at 77.00 relative to Me&) as internal standards, respectively. Only the NOE difference 'H NMR spectra were obtained at the higher field. High-resolution mass spectra were obtained at the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant No. CHE 8211164). Silica gel (Merck 9385,60 A, 230-400 mesh) was used for flash chromatography. TLC analyses of surfactants and nonsurfactants were performed on 0.25" aluminum oxide (Merck 5731-3) and 0.25mm silica gel plates (Merck 5714-3) with 5 vol % EtOH in CHC13 and 3 vol % EtOAc in hexane as eluants, respectively. Surfactant 10 (Kodak) was recrystallized from EtOAc, and CHCls (spectral grade) was stored over NazC03. Two pH 7.5 Tris buffers were used; that for the monolayer studies is described below. All other studies were performed with a buffer prepared from 433 mg (3.57 mmol) of tris(hydroxymethy1)aminomethane (Aldrich), 222 mL of 0.01084 N hydrochloric acid, 14.5 mg (0.0390 mmol) of ethylenediaminetetraacetic acid, disodium salt dihydrate (J. T. Baker), and 275 mL of H20. Sonication was performed with Branson 2200 (125 W)and 3200 (150 W) ultrasonic cleaners, and vortexing was performed with a Thermolyne M16715 mixer. After the solution from an extraction was dried (Nap904 or MgSO4) solvent was removed by rotary evaporation. All melting points are uncorrected. Elemental analysis were performed by Atlantic Microlab, Atlanta, GA. trans-17-Tetratriacontene(3). EtNH2 was collected at -78 "C after distillation from 70 wt % aqueous EtNHz (Aldrich) and passage through a column of NaOH pellets. To 500 mL of stirred EtNHz at 8 "C under N2 was added 450 mg (64.8 mmol) of freshly cut Li containing ca. 1% Na (Aldrich). After the metal completely dissolved, a solution of 1.00 g (2.11 mmol) of alkyne 28 in 50 mL of anhydrous Et20 was added dropwise, and the mixture was stirred for 24 h. Then 100mL of ice-cold 1:l (v/v) MeOHHzO was added, and the mixture was extracted with three 50-mL portions of hexane. The combined extracta yielded 0.947 g of crude product that was recrystallized twice from 1:l EtOH-hexane (5 "C) to give 0.928 g (92%) of 3: mp 43-45 "C; lH NMR 6 5.39 (m, 2 H,CH=CH), 1.88 (m, 4 H, CHzCH), 1.25 ( ~ , 5 H, 6 (CH2)14),0.88 (t,6 H, CHs); 13C NMRG 130.36,32.64,31.93,29.70,29.37,29.18,22.71,14.15; (27) Harada, S.; Takada, Y.; Yasunaga, T.J. Colloid Interface Sci. 1984, 101, 524.
Properties of Diastereomeric Surfactants IR (KBr) 2955 (m), 2916 (s), 2849 (s), 1472 (m), 1462 (m), 962 (w), 730 (m), 719 cm-l (m). Anal. Calcd for C34Hs8: C, 85.63; H, 14.37. Found: C, 85.53; H, 14.38. By 13C NMR analysis this material contained no cis isomer 7. trans-2,3-Dihexadecyloxirane(4). A mixture of 12.50 g (26.26 mmol) of 3,37.5 mL of 88%HCOzH, and 32.0 mL of 30% H202-H20 (31.3 mmol) was stirred at 80 "C for 48 h with the addition of 10 mL of 30% H202-H20 every 12 h. Then the reaction mixture was extracted with four 100-mLportions of hexane, and the combined extracts, after washing with 100 mL of H20, gave 12.05 g of crude epoxide. This material was recrystallized from 1:lEtOHhexane (5 "C) to yield 11.66 g (90%) of 4: mp 65-67 "C; lH NMR 6 2.66 (m, 2 H, CHO), 1.17-1.58 (m + s at 1.25, 60 H, (CH2)15),0.88 (t, 6 H, CH3); '3C NMR 6 58.95,32.18, 31.93,29.70,29.59,29.48,29.37,26.06,22.71,14.12;IR (KBr) 2957 (m), 2918 (s), 2850 (s), 1473 (m), 1464 (m), 899 (w), 877 (m),856 (w), 720 (m), 707 cm-' (m). Anal. Calcd for C34H680: C, 82.85; H, 13.91. Found: C, 82.93; H, 13.88. meso-17,18-Tetratriacontanediol(5). A mixture of 2.00 g (4.06 mmol) of 4,60 mL of 88% HCOzH, 6 mL of HzO, and 75 mL of CsHbMe was refluxed for 24 h, followed by rotary evaporation of solvent. Then a mixture of the residue and 150 mL of 3 N KOH in EtOH was refluxed for 2 h, followed by the addition of 200 mL of H2O and rotary evaporation of EtOH. The resultant white precipitate was collected, washed successively with H20, 5 % hydrochloric acid, and H2O until the pH of the wash was 7, and then dried and recrystallized from CaH6 (25 "C) to give 1.28 g (62%) of 5: mp 119-121 "C; lH NMR (35 "C) 6 3.60 (m, 2 H, CHOH), 1.73 (d, J = 4.8 Hz, 2 H, CHOH), 1.20-1.52 (m + s at 1.26,60 H, (CH2)15),0.88 (t,6 H, CH3); IR (KBr) 3260 (m),2918 (s), 2849 (s), 1471 (m), 1465 (m), 1074(m),720 (m),668cm-l (w). Anal. Calcd for C~H7002: C, 79.93; H, 13.81. Found: C, 79.75; H, 13.43. r-2-Methyl-2-( 3-bromopropyl)-c-4,c-5-dihexadecyl1,3-dioxolane(6a)and r-2-Methyl-2-(3-bromopropyl)t-4,t-5-dihexadecyl-1,3-dioxolane (6b). By a literature procedure,285.00 g (9.80 mmol) of 5 and 1.74 g (10.6 mmol) of 5-brom0-2-pentanone~~ gave 5.58 g (87%) of a mixture of 6a and 6b that was separated by flash chromatography. Typically, 1.2 g of the mixture was chromatographed on a 5 cm (i.d.) X 23 cm column of silica gel with 3 vol % EtOH in hexane as eluant and the collection of 56 fractions of ca. 10 mL each. From this, 480 mg of 6a (Rf= 0.26) as a viscous oil and 700 mg of a mixture of the two were obtained. The mixture was chromatographed again under the same conditions to give 313 mg of 6b (Rf= 0.20) as a viscous oil. For 6a: 'H NMR 6 4.01 (m, 2 H, CHO), 3.43 (t, J = 6.7 Hz, 2 H, CHzBr), 1.95 (m, 2 H, CH2CH2Br),1.75 (m, 2 H, CH2CH2CH2Br),1.48 (m, 4 H, CHzCO), 1.38,1.25 (2 s, 59 H, CH3C02 and (CH2)14, respectively), 0.88 (t, 6 H, CH3); 13C NMR 6 108.26, 78.36, 37.86 (CH2CH2CH2Br), 33.97,31.93,29.70,29.37,28.31,26.44(CH3C02),26.25, 22.69, 14.12; IR (neat) 2919 (s), 2849 (s), 1467 (s), 1378 (m), 1290 (w), 1267 (w), 1202 (m), 1102 (m), 1026 (w), 908 (m), 704 cm-l (m). E1 HRMS calcd for (M - CH3) 641.4873 and 643.4852, found 641.4889 and 643.4877; calcd for C36H7102 (M - CH2CH2CH2Br) 535.5455, found 535.5475; M was not observed. For 6b: lH NMR 6 4.06 (m, 2 H, CHO), 3.44 (t, J = 6.6 Hz, 2 H, CHzBr), 2.00 (m,2 H, CH2CH2Br), 1.88 (m, 2 H, CH2CHzCHzBr), 1.45 (m, 4 H, CH2CO), 1.30 and 1.26 (2 s, 59 H, CH3C02 and (CH2)14,respectively), 0.88 (t, 6 H, CH3); (28) Jaeger, D. A.; Martin, C. A.; Golich, T. G. J . Org. Chem. 1984,49, 4545. (29) Jlger, H.; Keymer, R. Arch. Pharm. ( Weinheim) 1960,293,896. Cannon, G. W.; Ellis, R. C.; Leal, J. R. In Organic Syntheses;Wiley: New York, 1963; Collect. Vol. IV, p 697.
Langmuir, Vol. 7, No. 9,1991 1941
13C NMR 6 107.99, 77.73, 39.41 (CH2CH2CH2Br), 34.05, 31.93, 29.70, 29.37, 27.69, 26.38, 24.10 (CHSCO~), 22.71, 14.12; IR (neat) 2919 (s), 2849 (s), 1467 (s), 1373 (m), 1296 (w), 1259 (m), 1252 (m), 1099 (m), 903 (w), 719 cm-l (m). E1 HRMS calcd for C38H7479/s1Br02(M - CH3) 641.4873 and 643.4852, found 641.4864 and 643.4836; calcd for C36H7102 (M - CH2CH2CH2Br)535.5455, found 535.5467; M was not observed. [3-(c-4,c-5-Dihexadecyl-r-2-methyl-l,3-dioxolan-2yl)propyl]trimethylammonium Bromide (la). By the procedure used below for IC, 510 mg (0.777 mmol) of 6a gave 450 mg (81% ) of la: mp 130-135 "C; lH NMR 6 4.00 (m, 2 H, CHO), 3.59 (m, 2 H, CH2N),3.50 (s,9 H, (CH&N), 1.84 (m, 2 H, CH2CH2N), 1.65 (m, 2 H, CH~CHZCH~N), 1.48 (m, 4 H, CHzCO), 1.39,1.26 (2 s, 59 H, CH3C02 and (CH2)14,respectively), 0.88 (t, 6 H, CH3);13CNMR 6 107.66, 78.69, 66.89, 53.32, 35.74 (CH~CHZCH~N), 31.90, 29.67, 29.54,29.35,26.52 (CHSCO~), 26.19,22.66,18.72,14.12;IR (KBr) 2959 (m), 2918 (s), 2851 (s), 1468 (s), 1462 (m), 1381 (w), 1212 (m), 1064 (m), 721 (w), 660 cm-' (w). In NOE difference lH NMR experiments, irradiation of the signal for CHO gave signals for CHzCHzCHzN and CHzCO but not for CHsC02. Anal. Calcd for C42HsBrN02: C, 70.35; H, 12.09. Found: C, 70.13; H, 12.07. This analysis was obtained after la was dried at 75 "C. [3 4t-4&5-Dihexadecyl-r-2-methyl-1,3-dioxolan-2y1)propylltrimethylammonium Bromide (lb). By the procedure used for IC, 630 mg (0.960 mmol) of 6b gave 570 mg (83%) of lb: mp 138-141 "C; 'H NMR 6 4.07 (m, 2 H, CHO), 3.57 (m, 2 H, CH2N), 3.50 (s,9 H, (CH3)3N),1.86 (m,2 H, CHZCH~N), 1.72 (m, 2 H, CH2CH&H2N), 1.39 (m, 4 H, CHzCO), 1.31,1.25 (2 s, 59 H, CH3C02and (CH2)14, respectively) 0.88 (t, 6 H, CH3); 13CNMR 6 107.23,77.22, 67.16, 53.30, 36.31 (CHZCH~CH~N), 31.90, 29.92, 29.67, 29.35,26.44,23.86 (CHSCO~), 22.66,17.77,14.10; IR (KBr) 2944 (w), 2913 (s), 2846 (s), 1487 (m), 1474 (s), 1461 (e), 1384 (w), 1240 (m), 1128 (w), 1099 (w), 1035 (m), 952 (w), 727 (w), 718 (w), 667 cm-l (m). In NOE difference lH NMR experiments, irradiation of the signal for CHO gave signals for CHzCO and CH3C02,but not for CH2CH2CH2N. Anal. Calcd for C42H&rN0~0.5HzO: C, 69.48; H, 12.08. Found: C, 69.35; H, 12.32. This analysis was obtained after lb was dried a t 75 "C. cis-17-Tetratriacontene(7). A stirred mixture of 5.00 g (10.5mmol) of 2,16175mg of 5% Pd on Bas04 (Aldrich), 175 mg of quinoline (Aldrich), 125 mL of EtOH, and 125 mL of hexane was held under H2 at 60 psi and 25 "C until no further H2 was consumed (ca. 1h). The catalyst was removed by filtration and the solvent rotary evaporated to yield crude alkene that was recrystallized two times from 1:l EtOH-hexane (5 "C) to give 4.91 g (98%) of 7: mp 43-45 "C; lH NMR 6 5.35 (m, 2 H, CH=CH), 2.00 (m, 4 H, CHzCH), 1.28 (s, 56 H, (CH2)14), 0.89 (t,6 H, CH3); l3C NMR 6 129.90,31.93,29.78,29.70,29.56,29.37,27.23, 22.69, 14.12; IR (KBr) 3001 (w), 2954 (w), 2916 (s), 2849 (s), 1472 (m), 1462 (m), 730 (m), 719 (m), 647 cm-' (w). Anal. Calcd for CaHm: C, 85.63; H, 14.37. Found C, 85.63; H, 14.34. By 13C NMR analysis this material contained 110% 3. cis-2,3-Dihexadecyloxirane(8). With the procedure for 4 and a reaction temperature of 60 "C, 12.50 g (26.26 mmol) of 7 gave 11.63 g (89%) of crude epoxide that contained ca. 10% Of 4 by lH NMR analysis. This material was recrystallized 4 times from 1:l EtOH-hexane (5 "C) to give pure 8: mp 71-72 OC; lH NMR 6 2.91 (m, 2 H, CHO), 1.48 (m, 4 H, CHzCO), 1.25 (s,56 H, (CH2)14), 0.89 (t, 6 H, CH3); 13C NMR 6 57.24,31.93,29.70,29.56,29.37,
1942 Langmuir, Vol. 7, No. 9,1991
Jaeger et al.
Subsequently, the sample was held at the temperature 27.85, 26.60, 22.69, 14.12; IR (KBr) 2955 (m), 2918 (s), used for sonication or vortexing for 30 min, and at 25 "C 2850 (s), 1473 (m), 1464 (m), 844 (m),730 (m), 720 (w),668 for 30 min, followed by filtration through a Millipore Millex : 82.85; H, 13.91. cm-l (w). Anal. Calcd for C M H ~ OC, HV1 filter unit (contains a 0.45-pm Durapore membrane) Found: C, 83.00; H, 13.65. or a l-pm polycarbonate filter (Nuclepore 110410), re(&)-17,18-Tetratriacontanediol(9). A mixture of 5.00 spectively, into a 6 mm X 50 mm culture tube (Kimble g (10.2 mmol) of 8,150 mL of 88% HCOzH, 15mL of HzO, 73500-650). Immediately thereafter, the tube was inserted and 200 mL of C6H6 was refluxed for 12 h. The crude diol, into the particle sizer and the run begun. Use of a 2-pm from the same workup procedure as used for 5, was repolycarbonate filter (Nuclepore 110411)in some runs gave crystallized from C6H6 (25 "C) to give 3.25 g (63%) of 9: the same results. mp 94-95 "C; 'H NMR (35 "C) 6 3.41 (m, 2 H, CHOH), As noted in the Resulb and in Table I, sonicated lc/10 1.91 (d, J = 4.6 Hz, 2 H, CHOH), 1.20-1.57 (m + s a t 1.26, gave vesicle sizes and volume percent values that varied 60 H, (CHz)16),0.88 (t,6 H, CH3);IR (KBr) 3339 (m), 2949 from sample to sample. Results for two representative (m), 2917 (s), 2846 (s), 1469 (s), 1118 (w), 1076 (w), 719 runs are as follows: a, population 1, 17 nm (64 vol 76); (m),665 cm-l (w). Anal. Calcd for C34H7002: C, 79.93; H, population 2,48 nm (34 vol %); population 3,158 nm (2 13.81. Found: C, 80.01; H, 14.08. vol %). b, 1,45 nm (69 vol %); 2, 162 nm (24 vol %); 3, r-2-Methyl-2-(3-bromopropyl)-c-4,t-5-dihexadecyl-468 nm (7 vol %). l,&dioxolane (6c). By a literature procedure,28 5.00 g DSC. Calorimetry of 0.58-g portions of sonicated and (9.80 mmol) of 9 and 1.74 g (10.6 mmol) of 5-bromo-2vortexed surfactant samples as prepared below was pentanone gave crude 6c. This material was flash chroperformed on a Hart Scientific Model 7708 differential matographed on a 5 cm i.d. X 15 cm column of silica gel scanning calorimeter. Scans were made from 5 to 75 "C with 3.5 vol 5% EtOAc in hexane as eluant to give 5.73 g and from 75 to 5 "C at 1 "C/min. Each transition was (89%) of 6c as a viscous oil: lH NMR 6 3.60 (m, 1 H, reversible as evidenced by the same T,value for successive CHO), 3.52 (m, 1 H, CHO), 3.43 (t,J = 6.9 Hz, 2 H, CHZup-scans. The values of AHcd were determined by Br), 1.98 (m, 2 H, CHzCHzBr), 1.76 (m, 2 H, CHZCHZintegration of the thermogram^.^^ CHzBr),1.50 (m,4 H, CHzCO), 1.33,1.25 (2 s, 59 H, CH3COz A solution of ca. 6.0 mg of 1, 10, or a 1/10 mixture (25 and (CH2)14, respectively), 0.88 (t, 6 H, CH3); 13C NMR mol % 1)in CHC13 was rotary evaporated and the residue 6 108.67,81.75,80.83,38.81 (CHzCHzCHzBr),34.08,33.29, dried for 12 h (25 "C, 0.1 mmHg). Then 1.00 mL of the 32.67,31.93, 29.70, 29.59, 29.51, 29.35, 27.61,26.17, 25.84 pH 7.5 Tris buffer was added, and the system was either (CH~COZ), 22.69,14.10; IR (neat) 2987 (m), 2957 (m),2923 sonicated for 30 min or vortexed for 4 min (ca. 55 "C for (s), 2850 (s), 1465 (m), 1458 (m), 1375 (m), 1294 (w), 1247 1, and ca. 65 "C for 10 and 1/10 mixtures). The vortexed (w), 1196 (m), 1102 cm-l (m). E1 HRMS calcd for samples were analyzed after cooling to 25 "C. The sonC38H7479/81Br02(M - CH3) 641.4873 and 643.4852, found icated samples were held for 30 min at the sonication tem641.4878 and 643.4847; calcd for C36H7102 (M - CHzCH2perature and then analyzed after cooling to 25 "C. CHzBr) 535.5455, found 535.5449; M was not observed. Entrapment and Release Studies. a. Vesicle Prepa[3-(04,t-5-Dihexadecyl-r-2-methyl-l,3-dioxolan-2-ration. A solution of 6.0 mg of surfactant in 0.80 mL of yl)propyl]trimethylammonium Bromide (IC). By a CHC13 was rotary evaporated in a l-mL ampule at 30-40 "C. The resultant thin film was dried for 12 h (25 "C, 0.1 literature procedure,28 5.00 g (7.62 mmol) of 6c was mmHg), and then 0.60 mL of the pH 7.5 Tris buffer converted into 5.30 g of crude IC. This material was chrocontaining 2-20 pCi of [14C]sucrose(ICN Radiochemimatographed on a 5 cm i.d. X 10 cm column of neutral cals, 360 mCi/mmol) was added. The ampule was flushed alumina (J.T. Baker 0537) packed in CHCl3 with elution with NZand sealed, and the solution was sonicated for 30 by 300 mL of CHC13, followed by 150 mL of 1:l MeOHmin at ca. 55 "C and then held for 30 min at the same CHCl3. Nine 50-mL fractions were collected, and fractions temperature. After 10 min at 25 "C, the vesicle solution 3-5 yielded 5.00 g (92 % ) of IC, which was recrystallized was subjected to gel filtration chromatography. from EtOAc (5 "C) to give 3.96 g of IC: mp 140-148 "C; b. Gel Filtration Chromatography, A fresh column lH NMR 6 3.41-3.71 (m + s a t 3.49,13 H, ( C H h N , CHZN, prepared as follows was used for the chromatography of CHO), 1.85 (m, 2 H, CHzCHzN), 1.73 (m, 2 H, CHZCHZeach sample. Sephadex G-50-150 (Sigma) was allowed to CH2N), 1.49 (m, 4 H, CHZCO),1.33, 1.25 (2 s, 59 H, CH3swell overnight in the pH 7.5 Tris buffer, degassed for 8 COz and (CH2)14, respectively), 0.88 (t, 6 H, CH3); 13C h at 20 mmHg, and added as a slurry to a 1cm X 30 cm NMR 6 107.96,82.16,80.70,67.02,53.27,36.06(CHzCHzCHzN),33.48,32.56,31.88,29.65,29.32,26.25,26.11,25.76, column. The column was washed with 200 mL of the Tris buffer 1 h before the chromatography of a sample. 22.63, 17.55, 14.07; IR (KBr) 2953 (m), 2918 (s), 2851 (s), The column outflow was attached to an ISCO Retriever 1483 (m), 1466 (s), 1377 (m), 1291 (w), 1241 (m), 1211 (w), I11 fraction collector equipped with an ISCO Model UA-5 1135 (w), 1095 (w), 1069 (m), 973 (w),966 (w), 910 (m),722 absorbance monitor (254nm), and fractions were collected cm-1 (m). Anal. Calcd for C42H~BrN02:C, 70.35; H, in 5-mL test tubes (40 drops (ca. 2 mL) per tube). The 12.09. Found: C, 70.10, H, 12.20. This analysis was void volume of the column (11.8mL, end of sixth fraction) obtained after IC was dried at 55 "C. was determined by chromatography of 0.2 mL of a 1% DLLS. Measurements were made at 23 "C on a Nicomp (w/w) solution of blue dextran (Sigma, average molecular Model 370 submicrometer particle sizer (90" scattering weight 2 000 000) in the pH 7.5 Tris buffer. A 100-pL angle). Data were analyzed by the Nicomp distribution sample from each fraction was analyzed by liquid scinanalysis procedure to give histograms of relative volume tillation counting (LSC) to determine the elution profile vs hydrodynamic diameter. for [14C]sucrose. The elution of entrapped [ 14C]sucrose A solution of 1.0-1.5 mg of surfactant/surfactant corresponded to that of vesicles at the void volume, as mixture (25 mol 96 1) in CHCl3 was rotary evaporated, determined by the apparent absorption at 254 nm due to and the residue dried for 12 h (25 "C, 0.1 mmHg). Then light scattering. 1.00 mL of the pH 7.5 Tris buffer was added, and the system was either sonicated for 30 min or vortexed for 4 (30)(a) McElhaney, R.N.Chem. Phys. Lipids 1982,30,229. (b) Takahashi, K.;Sturtevant, J. M. Biochemistry 1981,20,6185. min(ca.55"Cfor l,andca.65"Cfor lOandl/lOmixtures).
Properties of Diastereomeric Surfactants
Langmuir, Vol. 7, No. 9, 1991 1943
c. Dialysis of Vesicles ContainingEntrapped [l4C]Sucrose. A 1.00-mL sample from the vesicle fraction containing the maximum amount of [14C]sucrose was placed into one compartment of a separation cell (Spectrum, 1 mL, No. 132374) fitted with a 33-mm, 1200014000 molecular weight cutoff dialysis membrane disk (Spectrum Spectra/Por 2, No. 132482). To the second compartment, 1.00 mL of the pH 7.5 Tris buffer was added. Then the cell was placed in a wrist shaker and gently shaken at ca. 23 "C. At appropriate times 100-pL samples were withdrawn from the sample and buffer compartments. Only the former were analyzed by LSC. The dialysis membrane disk was soaked in HPLC-grade H2O for 12 h and then in the Tris buffer for 2 h prior to dialysis. d. LSC. The 100-pL sample was added to 2.5 mL of Aqualyte Plus LSC cocktail (J. T. Baker) in a LSC vial (Kimble No. 58502) and counted with a Beckman LS 5000 TD liquid scintillation counter. The time for each counting was 2 min, and each sample was counted 3 times in a cycle, with a total of 10 cycles. The average counts-per-minute value for a sample was determined by averaging the values for the individual cycles after deleting the minimum and maximum values. The results for la-c and 10 are summarized in Figure 4 and represent the averages of at least two runs for each surfactant. For [14C]sucrose alone, izl (see Results) = 21 X s-l. The limit of error for each value of kl is estimated to be f15%. Monolayer Studies. a. Preparation and Purification of Materials. Procedures for the cleaning of glassware and the preparation of surfactant spreading solutions have been described in detail.31 Solutions typically consisted of 3-4 mg of surfactant dissolved in 9:l (v/v) purified hexanes-Et0H in hand-calibrated 25-mL volumetric flasks. The solutions were kept thermally equilibrated in a desiccator in an atmosphere of hexanes. The experiments were performed on a subphase consisting of purified H2O (Millipore R04, plus double distillation) buffered at pH 7.5 with [tris(hydroxymethyl)aminolmethane (Schwartz/Mann Biotech, ultra pure) and concentrated hydrochloric acid (Mallinckrodt AR). The former was recrystallized 3 times from purified H20, and the latter was used without further purification. b. Spreading Solutions. Appropriate volumes of the surfactant solutions were spread onto the subphase to provide a monolayer of 2.2015 X 10l6molecules a t an initial
area of 5.28 X 1018A2,or 240 A2/molecule. After a 15-min period, monolayer compression was begun. c. Langmuir Film Balance Techniques. The Langmuir film balance used in this study detects surface pressure with a floating barrier/torsion head system and is sensitive to changes as small as 0.005 dyn/cm. Ita construction, cleaning, calibration, and technical specifications have been described in detaiL31 The film balance was housed in a Puffer-Hubbard UniTherm cabinet. The subphase temperature was maintained at 25.1 f 0.1 "C by running H2O from a constant temperature bath through a serpentine glass coil placed in the subphase. The subphase was replaced every 12 h to minimize changes in buffer concentration due to evaporation. The monolayer films were compressed at a rate of 19.24 (A2/molecule)/min. Each isotherm was reproduced at least 5 times. The stability of a film was checked by stepwise compression, holding the film at constant area for at least 1min while monitoring the loss of surface pressure. The film was judged stable if the surface pressure decrease was less than 0.1 (dyn/cm)/min. The validity of the film balance calibration was checked every 12 h with the wellknown stearic acid isotherm.32 d. Equilibrium Spreading Pressures. ESPs of the surfactants were measured on the pH 7.5 buffer subphase at 25.0 "C by du Nouy ring tensiometry using either a Fisher Autotensiomat or a manual Cenco du Nouy Tensiometer (No 70535). Prior to each experiment the ring was cleaned by flaming, and the surface tension of the freshly aspirated subphase was taken. Surfactant crystals (0.5-1.0 mg) were then introduced onto the subphase surface in a 6.8-cm (diameter) Teflon dish (Autotensiomat) or a 6.5-cm (diameter) Pyrex T-cup (tensiometer). The crystals were allowed to equilibrate for a minimum of 1 2 h, with successive experiments performed for 18and 24-h periods. Equilibration was assumed when readings increased by no more than 0.2 dyn/cm in a 6-h period. Each experiment was repeated a t least 3 times with fresh subphase and crystals. e. Data Analysis. All reported data were analyzed a t the 95% confidence limit by using the Student t for 5-10 repetitions for II/A isotherms and 3-6 repetitions for ESPs.
(31) Arnett, E. M.;Chao, J.; Kinzig, B.; Stewart, M.; Thompson, 0.; Verbiar, R. J. Am. Chem. SOC.1982,104, 389.
(32) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley: New York, 1966; p 220.
Acknowledgment is made to the National Cancer Institute, DHHS (PHS Grant No. CA45769-Ol),the U:S. Army Research Office, and the State of Wyoming Eagles for support of this research.
