Langmuir 1989,5, 833-838 analysis has been obtained with a missing-row model for the metal surface and with 0 bonding essentially to four neighboring Cu atoms.20 The differing coordinations for 0 and N at the Cu(100) surface are not inconsistent with the fact that valence considerations cause different structural arrangements for the bulk Cu(1) compounds, (20) Zeng, H. C.; McFarlane, R. A.; Mitchell, K. A. R. Surf. Sci. 1989, 208,Ll.
833
namely, CuzO and Cu3N. In both cases, the structures found by LEED for the chemisorption systems are broadly consistent with bond order-bond length relations applied to the corresponding bulk compounds. Acknowledgment. We are grateful to the Natural Sciences and Engineering Research Council of Canada for ,supporting this research. Registry No. Cu, 7440-50-8; N2, 7727-37-9.
Chain-Substituted Lipids in Monomolecular Films. Effect of Polar Substituents on Molecular Packing F. M. Menger,* S. D. Richardson, M. G. Wood, Jr., and M. J. Sherrod Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received November 14,1988. In Final Form: February 5, 1989 A series of highly purified fatty acids and phospholipids each possessing a polar chain-substituent(hydroxy or keto) at varying locations (carbons 8, 10, 12, and 16 for the fatty acids and carbons 4,6,8, 10, and 12 for the phospholipids) on an 18-carbon chain were synthesized. Pressure-area isotherms revealed how these molecules pack in monomolecular films. Most of the fatty acids and phospholipids exhibited pressure-area curves indicative of "looping" conformations where both the polar substituent and polar head group contact the aqueous subphase. As the pressure was increased,the polar substituentswere forced out of the aqueous interface, and the chains assumed more vertical conformations. Pressure-area isotherms for the hydroxylated fatty acids showed unusually small molecular areas in the condensed state owing to the presence of hydrogen bonding. A phospholipid disubstituted at the 12 position with a keto group gave a molecular area of only 21.6 A2/molecule at 35 dynlcm; this is consistent with two vertical chains, one in the water and one in the air. Isotherms reflected a strong dependence on the position of the polar substituent along the chain. Introduction Over the past few years, there has been a growing interest in developing artificial membranes to serve as controlled drug delivery systems. Designing such synthetic membranes requires an understanding of how the individual molecules pack together. In an effort to learn more about "packing" behavior, we examined a series of synthetic fatty acids and phospholipids bearing diverse substituents on their hydrocarbon chains. Earlier, we published the effects of hydrocarbon branches (methyl, nbutyl, phenyl) on the packing behavior of fatty acids and phospho1ipids.l The present study deals with the effects of polar substituents (hydroxy and keto) at various locations along the chains. Stearic acid derivatives were synthesized with a hydroxy substituent a t the 8-, lo-, and 12-, or 16-positions, and distearoylphosphatidylcholines were furnished with a keto group at the 4-, 6-, 8-, lo-, and 12-positions (Scheme I). Both chains of the phospholipids, or only chain 2, were modified in this manner. Owing to the large number of compounds, it is necessary to adopt a short-hand notation. Thus, when both octadecanoyl chains possess a keto group at C4,the phosphatidylcholine will be designated (1,2)-PC-4K; a lipid having a C8 keto group on only the second chain will be called (2)-PC-8K. Packing properties of the chain-modified lipids in monomolecular films were examined by means of a Wilhelmy-type film balance. (1) Menger, F. M.; Wood,M. G., Jr.; Richardson, S.; Zhou, Q.; Elrington, A. R.; Sherrod, M. J. J. Am. Chem. SOC.1988,110,6191.
0743-7463/89/2405-0833$01.50/0
(2)- PC-8K
Recently, Cadenhead? Tachibana? Zografi? and Nagarajan5 have published pressure-area isotherms of hy(2) Kellner, B. M. J.; Cadenhead,D. A. J . Colloid Interface Sci. 1978, 63,452. ( 3 ) Tachibana,T.; Yoshizumi,T.; Hon, K. Bull. Chem. SOC.Jpn. 1979, 52, 34.
0 1989 American Chemical Society
834 Langmuir, Vol. 5, No. 3, 1989
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c
E Increasing pressure
P c
Figure 1. Schematic representationof the possible conformations for hydroxy-branched fatty acids in the course of their compression. Scheme I
t
16
I
12
t
10
droxy- and keto-substituted fatty acids. To our knowledge, molecular packing of chain-substituted phospholipids has never been investigated systematically.
Results and Discussion All pressure-area isotherms for the hydroxy-branched fatty acids exhibited the same characteristics: a large liftoff area, a transition to a plateau region, and a sharp rise in pressure to a highly condensed state. These characteristics, previously observed for other hydroxy fatty acids,2a were attributed to (a) an initial contact of both polar groups with the aqueous subphase, (b) a transition representing the point at which the hydroxyl group is forced out of the interface, and (c) a tight packing of nearly erect molecules (Figure 1). The pressure-area isotherms for the hydroxylated fatty acids are presented in Figure 2. Two parameters proved useful in characterizing these isotherms: the liftoff area (the point on the isotherm where the curves emerge from the base line) and the transition point (the sharp bend in the curve that begins the plateau region). The liftoff area, AL, physically represents the onset of observable intermolecular forces between adjacent molecules in the monolayer. AL can provide conformational information for the molecules a t this stage in the film compression. The transition point physically depicts an abrupt change from a homogeneous film consisting of molecules having both their polar groups in contact with the water (B in Figure 1)to a film where both B and C are p r e ~ e n t .This ~ transition can be used to characterize the "looped" conformation B, e.g., the minimum area and maximum pressure that this particular configuration can withstand. The isotherms in Figure 2 yield AL values of 108.3,126.9, 145.0, and 140.5 for 8-hydroxystearic acid, 10-hydroxystearic acid, 1Zhydroxystearic acid, and 16-hydroxystearic acid, respectively (Table I). It should be emphasized that observed liftoff areas depend on the sensitivity of the film balance; this is not a serious limitation since we are mainly interested in relative values under constant conditions. SHAWW molecular modeling calculations1indicate that the minimum area for a hydroxystearic acid molecule lying flat on the water surface is about 117 A2/molecule. Thus, above AL the 10-hydroxystearic, 12-hydroxystearic, and (4) Rakshit, A. K.; Zografi, G.; Jalal, I. M.; Gunstone, F. D. J. Colloid Interface Sci. 1981,80,466. ( 5 ) Nagarajan, M. IC.; Shah,J. P.J. Colloid Interface Sci. 1981, 80, I.
8
8
4
4 0 ~
0
50
Area
1
150
100
(A /molecule)
Figure 2. Pressurearea isotherma (23.0"C) for 8-hydroxystmric acid (m), 10-hydroxystearicacid (A),12-hydroxystearicacid (a), and 16-hydroxystearicacid (e). Table I. Liftoff Areas A and Transition Points for HydroxyoctadecanoicAcids and Ketooctadecanoic Acids" transition point name 8-hydroxystearic acid 10-hydroxystearic acid 12-hydroxystearic acid 16-hydroxystearic acid
(1,2)-PC-4K (1,2)-PC-6K (1,2)-PC-8K (2)-PC-8K
(l,P)-PC-lOK (1,2)-PC-l2K
AL
108.3 126.9 145.0 140.5 92.7 134.8 151.2 127.5 192.0 194.6
area 55.8 73.8 90.3 87.0
preesure 12.2 9.0 7.3 6.9
100.3 90.3 82.3 148.4 145.8
5.1 8.0 1.9 3.1
2.7
*All areas in A4/molecde;pressure in dyn/cm. Data taken from Figures 2-4.
16-hydroxystearicacids lie flat on the water surface in the expanded (gaseous) state. Table I reveals the dependence of the transition point area and pressure on the location of the hydroxy substituent. 12-Hydroxystearic acid and 16-hydroxystearic acid show nearly the same area and pressure (ca. 90 A22/molecule and ca. 7 dyn/cm, respectively). But 10-hydroxystearic acid and 8-hydroxystearic acid differ substantially at their transitions (73.8 A2/molecule and 9.0 dyn/cm; 55.8
Langmuir, Vol. 5, No. 3, 1989 835
Chain-Substituted Lipids in Monomolecular Films
0 0
40
80
160
120
Area (klmolecule)
Figure 3. Pressurearea isotherms (23.0 "C) for (1,2)-PC-4K(a), (1,2)-PC-6K(e),and (1,2)-PC-8K(A).
A2/moleculeand 12.2 dyn/cm, respectively). Thus, as the number of carbons between the polar groups diminishes, so does the molecular area. This can be explained by assuming that at hydroxyl positions of 10 or less, the distal hydrocarbon segment rises more or less vertically out of the water. The observed area of the fatty acid, consequently, reflects the area of the "loop" separating the hydroxy and carboxy groups (B in Figure 1). Apparently, when the hydroxyl is at or beyond the 12-posiiton, the molecules lie flatter on the water surface, the distal hydrocarbon segment being too short to be ejected. An area reduction from the theoretical 117 to 90 A2/molecule can be accounted for by buckling of otherwise horizontal chains. Since the transition pressures are higher for the 8- and 10-hydroxystearic acids than for the 12 and 16 isomers, molecules with fewer carbons intervening between carboxyl and hydroxyl can withstand greater pressure before the hydroxyls are forced from the water (B C in Figure 1). In the condensed state (steep portion of the pressurearea curve), the hydroxy acids exhibit extremely small molecular areas. For example, 8-hydroxystearic acid occupies an area of 19.3 A2/molecule at 20 dyn/cm, wheras ita methyl-branched counterpart, 8-methylstearic acid, occupies an area of 31.1 A2/moleculeat the same pressure.' These low areas allow at least two conclusions: (a) The hydroxyl, and not the carboxyl, is forced out of the interface. If the hydroxyl persisted in the water, two chains (one with a carboxyl and one wi€hout) would penetrate the air and require large areas. (b) The hydroxy acids hydrogen bond with each other to reduce molecular areas. Melting points of the bulk fatty acids are, incidentally, consistent with hydrogen bonding in the condensed film; hydroxy fatty acids have melting pointa 5.5-8.5 "C higher than their unbranched counterpart, stearic acid. Keto-substituted phospholipids, with the exception of (1,2)-PC-4K,also appear to form the looping conformations seen with the fatty acids (Figures 3 and 4). Again, the pressure-area curves show a large liftoff area, a transition to a plateau region, and a highly condensed region. (1,2)-PC-4K manifests surface behavior much like its unbranched counterpart, DSPC, in that its liftoff area is not unusually high, and it does not show a transition characteristic of the other looping compounds. Cadenhead2observed similar straight-chain behavior for 2-hydroxyhexadecanoic acid and 3-hydroxyhexadecanoicacid. These compounds lack a sufficiently long "spacer" between their
-
0
200
100 Area (A'/molrculr)
Figure 4. Pressurearea isotherms (23.0 O C ) for (l,P)-PC-lOK
(a),(1,2)-PC-l2K(e),and (2)-PC-8K(A).
O 1
Figure 5. Schematic representation of the poasible conformations for (1,2)-PC-l2Kin the course of its compression.
polar groups to form a stable looped conformation. Very likely, the keto groups and phosphatidylcholine head group serve jointly as the hydrophilic unit. Phospholipid isotherms in Figures 3 and 4 also show that a keto substituent on a single chain, as in (2)-PC-8K, produces a smaller liftoff area than does a keto substituent on both chains, as in (1,2)-PC-8K. From this observation, one might postulate an initial contact of both keto groups of a diketo lipid, in addition to the phosphatidylcholine head group, with the aqueous subphase. However, no additional transitions are present in the diketone isotherms that could prove the existence of a new "tripolar" conformation. In the condensed region of the phospholipid isotherms, the areas (ranging from approximately 22 to 40 A2/molecule at 35 dyn/cm) are smaller than one might expect for a substituted phospholipid. By comparison, DSPC, the unbranched analogue of our keto lipids, occupies an area of approximately 42 A2/molecule at the same pressure and temperature! (1,2)-PC-l2K occupies the smallest area (21.6 A2/molecule at 35 dyn/cm), exhibiting packing behavior much like single-chain molecules. For example, stearic acid, an unbranched single-chain analogue, occupies an area of ca. 18 A2/molecule at 35 dyn/cm.' This extraordinarily small mea (obtained with two lipid solutions prepared separately to preclude a possible weighing error) can be explained by a keto-substituted chain being forced into the aqueous subphase (Figure 5). Keto lipids (1,2)-PC-4K, (1,2)-PC-6K, (1,2)-PC-8K, (1,2)-PC-lOK,and (2)-PC-8K show areas at 35 dyn/cm of (6) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces: Interscience: New York,1966. ( 7 ) Feher, A. I.; Collins, F.D.; Healy, T.W.A u t . J . Chern. 1977,30, 511.
836 Langmuir, Vol. 5, No. 3, 1989
Menger et al.
w
w
Figure 6. Schematic representation of interdigitation. Table 11. Yields for the Synthesis of Substituted Octadecanoic Acidsa position a b, c d e 4-keto 62 43 86 6-keto 71 53 90 47 91 8-keto 73 8-OH 89 95 IO-keto 63 62 85 IO-OH 89 99 12-keto 58 67 75 12-OH 85 90 16-OH 68 70 86 100
Table 111. Yields for the Synthesis of Keto-Substituted PhosDhatidylcholines' name a b (1,2)-PC-4K 55 (1,2)-PC-6K 84 (1,2)-PC-8K 59 (2)-PC-8K 61 (1,2)-PC-lOK 76 (1,2)-PC-I2K 59 "Letters a and b refer to the reactions in Figure 8. Values are percentages. Table IV. Melting Points and Elemental Analyses of Hydroxyoctadecanoic Acids elemental analysis' position mp, "C mp (lit.), "C %C %H 8 78.5-79.0 81.5-81.7' 72.16 12.12 10 77-78 79.2-79.5;' 84-86' 72.07 12.07 81-81.5;d 83-84' 84.5;f 81-828 85;h 82.5' 12 75.5-76.5 76.6-76.9;' 78-798 72.19 12.15 16 77.5-78.0 78.4-78.6' 72.06 12.08
'Letters a-e refer to the reactions in Figure 7. All values are percentages.
28.7,40.3, 33.7, 30.7, and 31.7 A2/molecule, respectively. These areas, although not as small as that of (1,2)-PC-l2K, are less than that of DSPC at the same pressure. One plausible rationale is that the keto groups impose a favorable interdigitation upon the lipids in order to minimize dipole-dipole repulsions (Figure 6). This leads to reduced molecular areas. Finally, the diketo transition points vary from 90.3 A2/molecule and 8.0 dyn/cm for (1,2)-PC-8K to 148.4 A2/molecule and 3.1 dyn/cm for (1,2)-PC-lOK. As with the hydroxy acids, there appears to be a critical position for the polar substituent affecting the orientation of the distal hydrocarbon segment with respect to the water phase. When the keto group is at position 8 or less, the distal hydrocarbon segment rises more or less vertically out of the water. When the keto substituent is at or beyond the 10-position,the molecules lie flatter on the water surface (the distal hydrocarbon segment being too short to be ejected). (1,2)-PC-8Koccupies the smallest area and produces the largest pressure at the transition point owing to loop formation. Apparently, (1,2)-PC-6K,with a larger area and smaller pressure than (1,2)-PC-8K at their transition points, lacks a sufficient number of carbons in its "spacer" segment to form as stable a "loop". In summary, we have synthesized a series of highly purified fatty acids and phospholipids possessing polar chain substituents (hydroxy and keto) at varing locations (carbons 4,6,8,10,12, and 16 of an 18-carbon chain). Pressure-area isotherms provided information on how these molecules pack in monomolecular films. With this information now in hand, we are now able to examine how chain substitution influences transport rates through membranes constructed of the various lipids; results from these experiments will be reported in the future.
Experimental Section Synthesis. Since the synthesis of all hydroxy-subatituted fatty acids, and phospholipids derived from them, followed the same route (Figures 7 and 8), only one particular example will be given here. Isolated yields for each step and for each lipid are recorded in Tables II and III). Intermediates were invariably characterized by IR, *H NMR, and 19CNMR. Fatty acids were, in addition, subjected to elemental analyses and precise mass measurements (Tables IV and V). Structures of phospholipids were affirmed by IR, NMR, and FAE3 mass spectral analysis. Complete spectral data are available upon request.s
Hydroxyoctadecanoic acids:
calcd C, 71.95; H, 12.08.
'Bergstrom, S.; Aulin-Erdtman, G.; Rolander, B.; Stenhagen, E.; (I
Ostling, S. Acta. Chem. Scand. 1952,6,1157. CSaytzeff,M.; Sa@ zeff, C.; Saytzeff, A. J. Prakt. Chem. 1887,35,384. dGeitel, A. C. J. Prakt. Chem. 1888,37,82. eArnaud, A.; Posternak, S. Compt. Rend. 1910,150, 1527. fRobinson, G. M.; Robinson, R. J. Chem. SOC.1927, 175. RTomecko, C. G.; Adams, R. J. Am. Chem. SOC. 1927,49,522. hRadcliffe,L. G.; Gibson, W. J. SOC. Dyers Colourists 1923,39,4. 'Pigulevskii, G. V.; Rubosko, Z. J. J. Gen. Chem. (USSR) 1939, 9,829. jReinger, E.Ber. Deut. Pharm. Ges. 1922, 32,131. Table V. Melting Points, Elemental Analyses, and Precise Masses of Ketooctadecanoic Acids elemental analysis" Dosition mD. "C mD (lit.). "C % C %H massa 72.47 11.48 298.249 4 95-96 96.3-96.5' 976 84.5-85.5 86.6-86.8' 72.36 11.48 298.250 87;f-h86' 86-87] 72.54 11.51 298.251 8 80-81 83.6-83.8' 10 79.5-80.5 72.4-82.6' 72.48 11.44 298.250 76;k*'82'" 82.0-82.8" 740 12 80.5-81.0 81.5-81.9' 72.36 11.47 298.250 81;P 82q 81.0-81.5' "Ketooctadecanoic acids. Calcd: C, 72.44; H, 11.48. Mass calcd for C18Hu03: 298.250. Bergstrom, S.; Aulin-Erdtman, G.; Rolander, B.; Stenhagen, E.; Ostling, S. Acta Chem. Scand. 1952,6, 1157. 'Shukoff, A. A.; Schestakoff, P. J. J. Prakt. Chem. 1903,67, 418. dClutterbuck, P. W. J. Chem. SOC.1924, 125, 2330. 'Clutterbuck, P. W.; Raper, H. S. Biochem. J. 1925, 19, 388. fBougault, J.; Charaux, C. Compt. Rend. 1911, 153, 572. BBougault, J.; Charaux, C. Compt. Rend. 1911, 153, 882. hRobinson, G. M.; Robinson, R. J. Chem. SOC.1925, 127, 179. 'Fieser, L. F.; Szmuszkovicz, J. J. Am. Chem. SOC. 1948, 70,3354. JZellner, J. Monatsh. 1928,50,214. kShukoff,A. A.; Schaestakoff, P. J. J. Prakt. Chem. 1903,67,415.'Baruch, J. Ber. Dtsch. Chem. Ges. 1894,27, 174. '"Myddleton, W.W.; Berchem, R. G.; Barrett, A. W. J. Am. Chem. SOC. 1927,49,2267. "Robinson, G. M.; Rob1926,2208. "Fordyce, C. R.;Johnson, J. R. inson, R. J. Chem. SOC. J. Am. Chem. SOC.1933, 55, 3368. PPerrotte, R. Compt. Rend. 1934,199,358. qThoms, H.; Deckert, W. Ber. deut. pharm. Ges. 1921,31,24. 'Perrotte, R. Compt. Rend. 1935,200,746.
'
Ethyl Hydrogen Suberate (2)?91° Suberic acid, 1 (12 g, 0.069 mol) was added to a 1000-mL flask containing 300 mL of ethanol,
Langmuir, Vol. 5, No. 3, 1989 837
Chain-Substituted Lipids in Monomolecular Films
2
1
(a) EtOH/I-I~SO~/cyclohexane (b) SOC12 (c) [CH3(CH2)912Cd (d) KOH/H20 (e) N a B m E t O H
Figure 7. Synthesis of a keto- and hydroxy-substituted fatty acid.
I
CH-OH
I
CH~--C--(CHZ)~-C
a
.CdCIp
s
P
-
CHz-OH
I
P
I
P
P-(CH~)QCH~
P
CH-+C-(CHZ)B-C-(CH~)QCH~
t
CH2-*P-O-CH2-CH,-N(CH3)3
t
6-
(a) 4/DCC/DMAP/CHC13 (b) Figure 8. Synthesis of keto-substituted phospholipids. 360 mL of water, and 3 mL of concentrated sulfuric acid. This mixture was continuously extracted with cyclohexane for 2 days. The aqueous layer was discarded, the cyclohexane cooled, and any unreacted diacid removed by fitration. In order to separate monoester 2 from diester, the cyclohexane was washed with 200 mL (4X) of 1 M aqueous sodium bicarbonate. After acidification with dilute HCl, the monoester was extracted from the aqueous layer with 100 mL (4X) of ether. The ether layer was dried over MgSO, and stripped, leaving 10.2 g (73%) of 2 as a yellow oil. Ethyl 8-Ketooctadecanoate(3).11 A Grignard solution was prepared under a nitrogen blanket from 1.34 g (0.055 mol) of
(8)Spectra and tables of spectral data appear in the Ph.D. Thesie of
M.G. Wood,Jr., entitled "Syntheaie and Study of New Fatty Acids and
Phoepholipida", 1988. (9) Babler, J. H.;Moy, R. K.Synth. Commun. 1979,9, 669. (10) Swam, S.,Jr.; Oehler, R.; Buswell, R. J. Org. Synth. Collect. Vol.
11 1943, 216.
(11)Cason, J. Chem. Reu. 1947,40,15.
magnesium turnings, 12.2 g (0.055 mol) of 1-bromodecane, and 100 mL of anhydrous ether. Anhydrous cadmium chloride (5.04 g, 0.028 mol) was added to the stirred solution at 0 OC. After an exothermic reaction had subsided, the ethereal solution of ndecylcadmium was heated for 1h at 40 "C. A Gilman test12for Grignard reagent was negative at this point. The ether was then removed by distillation and replaced by 50 mL of dry benzene. Acid chloride (10.1 g, 0.046 mol prepared by refluxing 2 with thionyl chloride) in 50 mL of dry benzene was added dropwise to the stirred reaction mixture cooled in an ice bath. After the addition was complete, the greenish-yellow solution was boiled under reflux for 1 h with constant stirring. At the end of this period, the flask was cooled in ice and the orgasocadmiumcomplex broken up with 50 g ice and 50 mL of 6 N sulfuric acid. The benzene layer was then washed sucoessively with 100 mL of water, 5% aqueous sodium bicarbonate, water, and 10% aqueous sodium (12) Gilman, H.; Schulze, F. J. Am. Chem. Soc. 1925,47,2002.
838 Langmuir, Vol. 5, No. 3, 1989
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chloride. The benzene solution was dried over anhydrous sodium sulfate and stripped to yield a white semisolid which was crystallized twice from petroleum ether. In order to remove n-eicosane (an impurity formed from Grignard coupling), the product was subjected to column chromatography ( 8 2 petroleum ether/ether, 100/200-mesh Silicar silica gel). Compound 3 was obtained in 47% yield, mp 37 OC. 8-Ketooctadecanoic Acid (4).13 Keto ester 3 (3.9 g, 0.012 mol) was added to 20 mL of 95% ethanol and 20 mL of 2.5 N aqueous potassium hydroxide in a 100-mL flask. This solution was stirred for 4.5 h at room temperature. After the ethanol was evaporated, the resulting gel was diluted with 100 mL of water, acidified, and extracted with 100 mL (3X) of ether. The ether solution was dried over anhydrous sodium sulfate and stripped to yield a white powder (91% yield). Compound 4 was recrystallized 3 times from HPLC grade acetone to give a melting point of 80-81 OC. Ethyl 8-Hydroxyoctadecanoate (5).14 Keto ester 3 (4.0 g, 0.012 mol) was added to 100 mL of absolute ethanol (approximately 4% w/v) with stirring at room temperature. To this solution, 1equiv (0.5 g) of sodium borohydride was added (slowly). The reaction mixture was stirred at room temperature for 1.5 h or until the evolution of hydrogen had ceased. Unreacted sodium borohydride was destroyed by adjusting the pH of the mixture to neutrality with glacial acetic acid. After the ethanol and acetic acid were stripped, the remaining yellowish-white semisolid residue was dried under vacuum (25 OC, 1mmHg, 12 h). The resulting beige solid was recrystallized once from methanol/water (7030). Compound 5 was obtained as a white solid (95% yield). %Hydroxyoctadecanoic Acid (6)?3 Compound 5 (3.8 g, 0.012 mol) was added to 20 mL of 95% ethanol and 20 mL of 2.5 N aqueous potassium hydroxide in a 100-mL flask (equipped with a reflux condenser). This solution was stirred and refluxed for 4.5 h. After the ethanol was evaporated, the resulting gel was diluted with 100 mL of water, acidified, and extracted with 100 mL (3X) of ether. The ether solution was dried over anhydrous sodium sulfate and stripped to yield a white solid (89% yield). Compound 6 was purified by recrystallizationfrom HPLC acetone (4X) to give product with a sharp melting point (78.5-79.0 "C) and correct elemental analysis and spectra (Table IV). l,Z-Bis(8-ketostearoyl)-sa -glycero-3-phosphatidylcholine (7)>6 L-a-Glycerophosphatidylcholine/CdClzcomplex (89.6 mg, 0.203 mmol, purchased from Avanti and dried for 24 h at 56 "C and 0.1 mmHg) was mixed with keto fatty acid 4 (239 mg, 0.80 mmol) and 4-(dimethy1amino)pyridine (48 mg, 0.40 mmol) in 2 mL of chloroform (freshly distilled from Pz06). Dicyclohexylcarbodiimide (165 mg, 0.80 mmol) was added whereupon the mixture was stirred for 7 days at room temperature in the dark under a blanket of nitrogen. The entire reaction mixture, including the solids, was placed on a column (5.5 g Silicar silica gel in a 1 X 20 cm column) and the product isolated by chromatography. Eluting solvents consisted of various mixtures of chloroform and methanol (20-mL fractions): volume, mL
9i CHC13
200 100 100 200
90 80 50 20
%
CHSOH 10 20 50 80
The lipid fraction was purified again,this time using the following regime: volume, mL 100 100
200 200
% CHCIS
% CHsOH
90 80
10 20 50 80
50 20
Phospholipid 7 was obtained in a 59% yield. Its purity was judged satisfactory by TLC analysis (silica plate eluted with 65:25:4 (13) Bsrgatrom, S.; Aulin-Erdtman, G.; Rolander, B.; Stenhagen, E.; Oetling, 5.Acta Chem. S c a d . 1952,6, 1157. (14) Roseman, M. A.; Lentz, B. R.; Sears, B.; Gibbes, D.; Thompson, T. E. Chem. Phys. Lipids 1978,21,205. (15) Samuel, N. K. P.; Singh, M.; Yamaguchi, K.; Regen, S. L. J. Am. Chem. SOC.1985,107,42.
CHC13/CH30H/Hz0 and developed with Iz followed by Dragendorff's reagent),18NMR, and FAB mass spectrometry. l-Stearoyl-2-(8-ketostearoy1)-sn -glycero-3-phosphatidylcholine (8)>5 Monostearoyllecithin (236mg,0.45 mmol purfrom Avanti and dried for 24 h at 25 "C and 1 mmHg) and keto fatty acid 4 (596 mg, 2.0 mmol) were dissolved in 15 mL of chloroform (freshly distilled from P205). 4-(Dimethylamino)pyridine (110 mg, 0.9 mmol) and dicyclohexylcarbodiimide (206 mg, 1.0 mmole) were then added, whereupon the mixture was magnetically stirred under nitrogen for 12 h in the dark. The solvent was stripped, and the residue, suspended in about 2 mL of chloroform, was chromatographed twice (as described directly above) to give 222 mg (61%) of phospholipid. Film Balance Operation. Pressure vs area curves were obtained with the aid of a Fromherz film balance17(Mayer Feintechnik, Postfach 2864 3400 Gottingen, West Germany) equipped with a Teflon multicompartment trough and Wilhelmy plate device. The calibration and operation of this instrument have been extensively reported in a previous publication.' A Lauda circulating bath provided thermostating for the balance. A Fuher x-y recorder dowed continuous monitoring of the pressure-area isotherms. Purity of the lipid, aqueous subphase,and solvents is extremely critical to obtaining meaningful film balance results. Thus, all fatty acids were crystallized at least 4 times from HPLC acetone. Phospholipids were chromatographed 2 or 3 times. Water, prepurified by means of three large tanks containing deionizers and charcoal, was directed into a four-cartridge Millipore Milli-Q system including a terminal 0.22-pM membrane filter for removing microscopic organisms and particles. Water resistivity was maintained at 18 M Q.cm. All organic solvents used were HPLC grade. Work was carried out while wearing disposable polyethylene gloves (Fisher) to avoid possible contamination by skin lipids. Monolayers were spread from IO4 M fatty acid or phospholipid in chloroform. From 40 to 100 pL was delivered in at least 10 locations across the water surface with a Hamilton microliter syringe equipped with a Chaney adapter. Typically, the initial enclosed surface area encompassed 90 cm2. At least 20 min was allowed for the spreading solvent to evaporate and for the lipid molecules to distribute themselves evenly throughout the film. The subphase conskied of 0.01 N HaO, in purified water for the fatty acids or purified water for the phospholipids. Pressure-area isotherms were recorded at 23.0 f 0.1 "C and at barrier speeds of approximately 4 A2/molecule per min. Increasing or decreasing the barrier speed by 2 A2/molecule per min had no effect on the isotherms. Alongside the film balance, and within its plexiglass housing, open beakers of water with wicks kept the relative humidity at 70-85%. A high relative humidity is desirable to minimize surface cooling effects due to the evaporative loss of water from the trough.18 The quality of the subphase was tested prior to each run by compressing the surface without a lipid Nm while noting any rise in surface pressure. If an increase was detected, the subphase was removed by aspiration with disposable pipets (previously cleaned with Nochromix/H$OJ, and the trough was cleaned with 20% ether/ethanol, a good solvent for our lipids. Also, the quality of our calibrations was routinely checked by reproducing two trustworthy pressure-area isotherms in the literature: pentadecanoic acid carried out by Pallas and PethicaI8and stearic acid carried out by Feher, Collins, and Healy.?
Acknowledgment. T h i s work was supported by the National Institutes of Health a n d t h e Army Research Office. Registry No. 4, 16694-33-0; 6, 6807-91-6; 7, 116212-70-5; 8, 120262-67-1;(1,2)-PC-4K,116212-68-1;(1,2)-PC-6K,116212-69-2; (1,2)-PC-lOK, 116212-71-6; (1,2)-PC-l2K, 116212-72-7; 10hydroxystearic acid, 638-26-6; 12-hydroxystearicacid, 106-14-9; 16-hydroxystearic acid, 17773-37-4; 4-ketooctadecanoic acid, 16694-30-7;6-ketooctadecanoic acid, 502-71-6; 10-ketoodadecanoic acid, 4158-12-7; 12-ketooctadecanoic acid, 925-44-0. (16) Chakrabarti, P.; Khorana, H. G. Biochemistry 1975,14,5021.
(17)Fromherz, P. Rev. Sci. Instrum. 1975,46,1380. (18)Pallas, N. R.; Pethica, B. A. Langmuir 1985,I, 509.
