J . Phys. Chem. 1990, 94, 7366-7371
7366
the remaining solvent removed by a stream of nitrogen gas. The red-orange residue was recrystallized from petroleum ether (bp 35-60 "C), to produce 0.47 g (88%) of a bright yellow powdery solid: mp 132.1-133.4 "C; 'H N M R (CDC13) 6 7.3-8.3 (m, 8 H), 3.7 (s, 2 H), 2.4 (s, 1 H), 1.7 (s, 3 H) ppm; I3C NMR (CDC13) 6 28.8,48.0, 80.6, 116.6, 122.0, 124.6, 124.8, 125.1, 125.3, 127.3, 127.8, 129.3, 129.5, 133.9, 134.2, 137.0, 150.4ppm; IR 3350, 3060, 2970,2930, 1620, 1430, 1420, 1350, 1260, 1170, 1080,900,880, 850, 800, 770, 760, 740 cm-I; MS m / z (relative abundance) 95 (25), 94 (lo), 1 IO (17), 189 (40), 190 (22), 191 (72), 192 ( l l ) , 202 (16), 213 ( I l ) , 215 (42), 216 (30), 217 (14), 218 (15), 219 (76), 220 (1 5 ) , 234 (loo), 235 (20). Anal. Calcd for C I 7 H l 4 0 : C, 87.14; H, 6.04. Found: C, 86.95; H, 6.08. Synthesis of 2-Methylaceanthrylene. Aluminum oxide (0.5 g, 5 mmol) was added to a suspension of 2-methyl-2-aceanthrenol (0.10 g, 0.43 mmol) in 15.0 mL of toluene in a 25-mL roundbottom flask equipped with a Dean-Stark trap. The mixture was refluxed with magnetic stirring for 2 h. The alumina was removed by filtration and the filtrate rotary evaporated. The red residue was dissolved in heptane and chromatographed on aluminum oxide (neutral) with benzene as the eluent. Rotary evaporation followed
by recrystallization from petroleum ether (bp 35-60 "C) produced 40 mg (43%) of a dark red solid. Vacuum sublimation yielded: mp 88.0-90.0 "C; 'HNMR(CDC13) 6 8.34 (s, 1 H), 8.17 (d, 1 H), 8.05 (d, 1 H), 7.97 (d, 1 H), 7.75 (d, 1 H), 7.50-7.60 (m, 2 H), 7.22 (s, 1 H), 2.53 (s, 3 H);"C N M R (CDCI3) 6 141.6, 138.0, 135.5, 134.4, 130.2, 128.2, 127.9, 126.9, 124.5, 123.9, 123.0, 13.1 ppm; IR 3060, 1620, 1440, 1100,880,850,770,750, 735, 700 cm-I; M S m / z (relative abundance) 95 (lo), 107 (8), 107 (9), 213 (25), 214 (9), 215 (loo), 216 (63), 217 (12); UV-vis, A,, (cyclohexane) 585 nm (c 180), 470 (1400), 419 (4340), 411 (4150), 395 (6050), 378 (5400), 360 (8020), 345 (5400), 255 (47400), 239 (46000). Anal. Calcd for CI7Hi2:C, 94.40; H, 5.60. Found: C, 94.29; H, 5.36. Acknowledgment. The support of the donors of Petroleum Research Fund, administered by the American Chemical Society, and the support of the National Science Foundation with an RUI grant are appreciated. We thank Professor J. Michl for computational support (PPP S C F CI) and N S F and the Keck Foundation for partial support to purchase the VXR-300 and the Cary 23 15.
Pressure Tuning Infrared Spectroscopy of Two Phospholipids and Their Common Fatty Acid W. W. Ley and
H.G.Drickamer*
Materials Research Laboratory and School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801 (Received: August 2, 1989; I n Final Form: March 30, 1990)
Infrared spectra of solid palmitic acid, L-a-dipalmitoylphosphatidylcholine(DPPC), and L-a-dipalmitoylphosphatidicacid (DPPA), are studied at pressures up to 125 kbar to probe the fundamental nature of their molecular vibrations. In palmitic acid spectral changes are observed, which indicate that the stochastic conversion from the cis to the trans isomer occurs with increasing pressure. In palmitic acid and DPPA splitting is observed in the carbonyl region and appears to be related to hydrogen bonding. Correlation splitting in the CH2 bending region increases with pressure and eventually the correlation component becomes stronger than the fundamental and dominates this region of the spectrum. Also a tentative assignment is made for the (Y methylene bending mode, b(C,H,), in DPPA. Introduction
TABLE I: Behavior of Selected Peaks in the IR Spectrum of
Palmitic Acid' Phospholipids are the major components of a wide variety of cell membranes. The study of the molecular dynamics of phosenergy a t pholipids by vibrational spectroscopy is a well-defined field and 5 kbar is summarized in recent reviews.'s2 Many assignments of vi(cm-I) assignment brational peaks have been worked out, and dichroism has been 1408 b(C,H2) used in studies on lipid bilayer systems to determine the orientation of the oscillating dipoles with respect to the membrane ~ u r f a c e . ~ - ~ 1350 w(CH2) mixed with There have also been high pressure infrared investigations involving carboxyl modes 1312 w(CH2) mixed with aqueous dispersions of phospholipids,8-' * but the study of solid, carboxyl modes less hydrated samples" has been more limited. 1280 to w(CH2) In this work the vibrational infrared spectra of solid samples 1200 of L-a-dipalmitoylphosphatidylcholine (DPPC), L-a-dir(CH2)/p(CH2) 1102 palmitoylphosphatidic acid (DPPA), and palmitic acid have been "Symbols for the vibrational ( I ) Amey, R. L.; Chapman, D. In Biomembrane Structure and Function; Chapman, D., Ed.; Macmillan: London, 1983; pp 199-256. (2) Casal, H.L.; Mantsch, H. H. Biochim. Biophys. Acta 1984,779,381. (3) Goni, F. M.; Arrondo, J. L. R. Faraday Discuss. Chem. Soc. 1986.81, 117. (4) Fookson, J . E.; Wallach, D. F. H. Archiu. Biochem. Biophys. 1978, 189, 195. ( 5 ) Brandenburg, K.; Seydel, U. 2.Naturforsch. 1986, I I C , 453. (6) Akutsu, H.; Kyogoku, Y. Chem. Phys. Lipids 1975, 14. 113.
(7) Akutsu, H.; Kyogoku, Y.; Nakahara, H.;Fukuda. K. Chem. Phys. Lipids 1975, 15, 222. ( 8 ) Siminovitch, D. J.; Wong, P. T. T.; Mantsch, H. H . Biophys. J . 1987,
SI, 465. (9) Wong, P. T.T.; Mantsch, H. H. J . Chem. Phys. 1985.83 (7), 3268. (IO) Wong, P. T.T.; Siminovitch, D. J.; Mantsch, H. H. Biochim. Biophys. Acfa 1988, 947, 139. ( 1 1 ) Wong, P. T. T.;Huang, C. Biochemistry 1989, 28, 1259.
0022-3654/90/2094-7366$02.50/0
behavior overall 12 cm-l blue shift at 125 kbar large increase in intensity with 39 cm-l blue shift at 125 kbar small decrease in intensity with 44 cm-' blue shift at 125 kbar decrease in intensity; no longer observable at 125 kbar 30 cm-' blue shift modes are defined in the text.
studied as a function of pressure. The aim of doing so is to gain more understanding of the fundamental nature of the molecular vibrations of phospholipids by examining them in an environment other than the lipid bilayer. Experimental Procedure
All spectra were recorded by a Nicolet 7199 FTIR fitted with a 4X Perkin-Elmer beam condenser to focus the IR radiation on the sample. The samples were held in a diamond anvil cell with type-I1 diamonds and confined by an Inconel gasket;I2 pressure (12) Sherman, W. F.; Stadtmuller, A. A. Experimental Techniques in High Pressure Research; Wiley: New York, 1987.
0 1990 American Chemical Society
Pressure Tuning IR of Phospholipids
The Journal of Physical Chemistry, Vol. 94, No. 19, 1990 7367
1 1
AI-
palmitic acid
1520
cn 1500 1510
1440 1450 1430
Palmitic acid
7 20
0
40
60
80
100
120
140
Pressure (kbar) Figure 3. Energy vs pressure for the methylene bending region of pal-
mitic acid.
1050 1040 0
1700
1500 1300 1100 WAVENUMEERS
7bO
900
Figure 1. Infrared spectra of palmitic acid at various pressures. 17501,
I
I
I
I
,
,
,
,
,
,
,
,
,
t
3c
1700 1690
U
3
1680
Ln L I
Palmitic acid
1740
1030 1020
Q)
1010
E
1000
n
4
-
J
-.- - - - - _ _ _ -
960 1 0
- 0
940
950
-
1660
-
1650
-0
'
I
'
I
20
'
I
40
'
I
60
"
80
'
I
100
'
I
120
'
I
140
Pressure (kbar)
- % I
1670
0 0
Figure 4. Energy vs pressure for the OH out-of-plane vibration in pal-
om%@ '
I
'
I
'
I
mitic acid.
'
I
'
I
'
I
-
was measured by the ruby fluorescence method.I2 The samples were run with cesium chloride as a pressure transmitting medium. Since some amount of pressure is needed to fuse the CsCl surrounding the sample, the lowest pressure obtainable was approximately 5 kbar; 300 to 1000 scans were taken for each spectrum, depending on the strength, and hence S/N ratio, of the interferogram. All spectra taken were found to be. reversible upon release of pressure, and all pressure runs were reproducible. Samples of 99% pure palmitic acid, DPPA, and DPPC were obtained from the Sigma Chemical Company.
Palmitic Acid Results. A series of infrared spectra of palmitic acid are shown in Figure 1. The results for selected peaks are summarized in Figures 2-4 and in Table I.
The carbonyl stretching region for palmitic acid is shown in Figure 2. The u(C=O) vibration extrapolates to a frequency of about 1701 cm-I at atmospheric pressure and splits with increasing pressure. The low energy component of this pair is the less intense and eventually becomes obscured at pressures above 80 kbar. The low energy component has a rapid shift to lower energy (red shift) of about 2 cm-'/kbar for the first 10 kbar. This red shift begins to diminish and the data begin to level off at about 80 kbar. The overall red shift at 80 kbar is 36 cm-l. The high energy component has an initial shift to higher energy (blue shift) of about 0.75 cm-'/kbar, which also levels off at higher pressures. The overall blue shift at 125 kbar is 24 cm-I. The center of mass, which is indicated by the dashed line in Figure 2, has a slight red shift of about 7 cm-', which levels off after 50 kbar. The two dominant peaks in the CH2 bending region, which are observed at 1471 and 1464 cm-' at 5 kbar, are shown in Figure 3. The low energy fundamental, 6(CH2), shows a blue shift of only 2 cm-' at 125 kbar, and the higher energy correlation split band, 6'(CH2), shows a steady blue shift of about 23 cm-' over the same pressure range. The CH2 bend for the CY methylene group, 6(C,H2), is observed at 1408 cm-' at 5 kbar and shifts to 1414 cm-' at 50 kbar. It then
Ley and Drickamer
7368 The Journal of Physical Chemistry, Vol. 94, No. 19, 1990
5,
0
//
J-G,
Cis
Trans
Figure 5. Cis and trans configurations of the carboxylic acid dimer.
splits and the center of mass of the band shifts to 1420 cm-l at 125 kbar. At 5 kbar the CH2 wagging region, w(CH2), 1200-1400 cm-I, is dominated by a pair of strong peaks at 1298 and 1312 cm-l. There is also a weaker peak at 1350 cm-l and a series of peaks of about equal intensity from about 1280 down to 1200 cm-l. As pressure is increased to 125 kbar the peak at 1350 cm-l becomes much more intense and shifts to 1386 cm-'. The peak at 1312 cm-l shifts to 1356 cm-' with a slight decrease in intensity and eventually obscures the peak at 1298 cm-I. The series of equal intensity absorptions from 1280 to 1200 cm-I become weaker with pressure and are no longer observable at 125 kbar. A peak is observed in the C H 2 twisting-rocking region, r (CH2)-p(CH2), at 1 102 cm-I, and shifts blue to 1132 cm-' at 125 kbar. And finally the O H out of plane vibration a(OH), see Figure 4, is observed at 945 cm-I at 5 kbar and has a very large blue shift to 1050 cm-' at 125 kbar. Discussion. At low temperatures Hayashi et a]." have observed splitting in the v(C=O) band in even numbered fatty acids and have attributed it to crystal factor-group splitting, which occurs in the trans configuration, see Figure 5. They observed that this splitting increased with decreasing temperature and attributed it to the increasing population of trans isomers. In other words, the splitting in the carbonyl region is a diagnostic of the trans configuration. The same splitting has been observed in this study with increasing pressure and we postulate that it is also due to the increasing population of the trans isomer. The center of mass and the low energy component of the carbonyl band both shift red. This indicates that there is a strong intermolecular process occurring with increasing pressure; most likely it is hydrogen bonding. The splitting indicates that there are two types of carbonyl environment in the trans configuration: one that is hydrogen bonded, and one that is not. The higher energy component arises from the non-hydrogen-bonded C=O group. The lower energy component arises from the hydrogenbonded C=O group and the red shift of this band indicates that the hydrogen bonding of this c-0group is enhanced by pressure. In other words, the hydrogen bonding becomes increasingly localized with respect to one of the carbonyl groups as a function of pressure. Thii is consistent with the argument of increasing the population of the trans isomer while, of course, decreasing the cis population, since the trans isomer is formed from the cis by double proton transfer.I4 The important processes occurring at the carboxyl region of the acid dimer are regulated by 0-H- - -0 type associations, and the increase in intermolecular interaction caused by increasing pressure has a great effect on the way the system hydrogen bonds. The 1464 and 1471 cm-l bands depicted in Figure 3 have been identified and assigned by Miinch et al.15 as a pure CH2 bend, (13) Hayashi, S.;Umemura, J. J . Chem. Phys. 1975,63, 1732. (14) Umemura, J. J . Chem Phys. 1978.68, 42. (15) Mlinch, W.; Fringeli, U.; Gunthard, Hs. H. Specrrochim. Acto 1977, 33A. 95.
6(CH2),and its correlation split component, 6'(CH2), respectively. The correlation band at 1471 cm-1 shows a 23 cm-I blue shift at 125 kbar, but the pure 6(CH2) band at 1464 cm-' shows only a modest 2 cm-l blue shift. It is obvious from Figure 3 that the correlation splitting continues to increase even at pressures up to 125 kbar. The 1408 cm-I a methylene bend, 6(CaH2), has a relatively modest blue shift of 12 cm-' at 125 kbar. This is a much smaller shift than that of the correlation 6'(CH2) band at 1471 cm-l. Umemura.14 describes the potential energy of this band as having a 0% contribution from the C-O-H bend in the cis configuration and having a 17% contribution from the C U H bend in the trans configuration. As the population of the trans isomer increases with pressure so will the population of 6(C,H2) oscillators with this 17% degree of coupling to the carboxyl moiety via the C-0-H bend. As noted above, the pressure-driven intermolecular process occurring in the carboxyl region of the acid dimer causes the vibrational bands associated with that region to shift to lower energy. It is therefore possible that the blue shift of the 6(C,H2) band is attenuated by its greater coupling to a carboxyl band in the increasingly populated trans configuration. The most convincing evidence for the shift of the isomer population from cis to trans is contained in the CH2 wagging region between 1200 and 1400 cm-l. It has been very clearly s h ~ w n ' ~ J ~ that there are considerable spectral differences between the cis and trans isomers in this region. These differences serve as excellent indicators of which form is predominantly present. In the cis isomer this region is dominated by a series of evenly spaced bands of nearly equal intensity. These bands are predominantly pure CH2 wagging with couplings to the various carboxyl vibrations spread evenly among them.I3J4 In the trans isomer this region is dominated by a pair of strong absorptions centered around 1305 cm-' and the above series of nearly equal intensity bands is extremely weak. In this the couplings to the carboxyl vibrations are concentrated in the three bands at 1298, 1312, and 1350 cm-I; this coupling to the polar end group is responsible for the increased intensity of these three bands at the expense of the rest of the CHI wagging bands. In Figure 1 one can see that the two strong peaks at 1298 and 1312 cm-I coexist with the series of equal intensity bands from 1280 to 1200 cm-I; this indicates the coexistence of the cis and trans isomers. As pressure is increased, the intensity of the 1312 cm-l band remains strong, but the bands in the 1280-1200 cm-I progression fade. This indicates that the trans isomer population is increasing and the cis isomer population is decreasing, i.e., the cis isomers are being converted to trans by pressure. The peak at 1350 cm-' is also plainly visible at 5 kbar and has about the same intensity as the 1200-1280 cm-l region of the wagging progression. As pressure is increased, the intensity of this band increases greatly. This indicates that the effect of pressure is to increase the degree of coupling of this band to the vibrations of the carboxyl group, and, as pointed out by Umemura,14 this is characteristic of the trans configuration. The O H out of plane vibration, a(OH), shifts blue by 105 cm-I at 125 kbar. Large shifts for this band have been observed upon lowering the temperature,I3 and it is well known that this band is very sensitive to intermolecular f ~ r c e s . ~The ~ J ~potential energy di~tribution'~ for this band is 64% torsion about the C-0 single bond and 3 1% 0-H- - -0bending. As pressure is increased, the hydrogen bonding in the acid dimer region becomes stronger and the force constant contribution of the 0-H- - -0bend will become much greater. Therefore the large shift of this band is further evidence of increased hydrogen bonding with increasing pressure. Phospholipids DPPA Results. A series of infrared spectra of DPPA at various pressures are shown in Figure 6. The results for selected peaks are summarized in Figures 7 and 8 and in Table 11. (16) Holland, R. F.; Nielsen, J. R. Acru Crysrullogr. 1963, 16, 902. (17) Umemura, J.; Hayashi, S. Bull. Inst. Chem. Res. Kyoro Uniu. 1974,
52, 5 8 5 .
The Journal of Physical Chemistry, Vol. 94, No. 19, 1990 7369
Pressure Tuning IR of Phospholipids acid, dipalmitoyl
L-a-phosphatidic
dI
a)
D
E
3
1480
$
1470
c
0
3
A A A
1490
- A d
LABA -e &
1460 r
1440
14300
20
40
60
80
100
120
140
Pressure (kbar) Figure 8. Energy vs pressure for the methylene bending region of DPPA.
L-a-phosphatidylcholine.
1700
lSb0
IS00 1300 1100 WWJENUMBERS
.r
dipalmitoyl
7-00
900
Figure 6. Infrared spectra of DPPA at various pressures. 1800 1
v1 L
1780 1790 1770
t
I
I
I
I
1
I
I
I
t
I
I
I
I
I
1
A A
DPPA
Q)
n-
1760
3
1750
$ 0
1740
3
1730
L I
c
1720 1710
AhAAA
[A-
i *& -A%
# AA
Pressure (kbar) Figure 7. Energy vs pressure for the carbonyl stretching region of DPPA. TABLE II: Behavior of Selected Peaks in the IR Spectrum of DPPA' energy at 5 kbar (cm-') assignment behavior 1430 6(C,H2)b 6 cm-I red shift at 125 kbar 1 I79 v,(PO,-) 6 cm-l red shift at 125 kbar 1061 ul(P0,-) 21 cm-' blue shift at 90 kbar "Symbols for the vibrational modes are defined in the text. bTentative assignment.
The peaks in the carbonyl region, which are plotted in Figure 7, show a splitting a t 5 kbar and the growth of a third peak a t 22 kbar. This third peak, which is at higher energy than the first
two, eventually grows in intensity to a point where it obscures the other two. It has an energy of 1742 cm-l a t 22 kbar and shifts to about 1780 cm-I at 125 kbar. The peak observed at 1740 cm-' at 5 kbar has a very slight red shift to 1738 cm" at 20 kbar. It then shifts blue to 1743 cm-l at 62 kbar, where it is obscured by the third peak described above.
19bO
1700
1300 1100 WAVENUflBERS
I500
d
900
700
Figure 9. Infrared spectra of DPPC at various pressures.
The lowest energy peak observed at 1713 cm-1 at 5 kbar shows a more pronounced red shift to 1705 cm-l at 30 kbar. It then shifts blue to 1716 an-l at 73 kbar, where it also becomes obscured by the more intense third peak. At 5 kbar the CHI bending region has the 6'(CHz) and 6(CH2) peaks at 1472 and 1465 cm-l, respectively. The correlation split component, b'(CH2), shifts blue to 1494 cm-l a t 125 kbar, and the fundamental, S(CH2), shifts blue to 1470 cm-l a t 82 kbar, where it is obscured by the 1472 cm-I peak. A peak is observed at 1430 cm-I at 5 kbar, which shows a very slight red shift to 1424 cm-I at 125 kbar. The antisymmetric PO
