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J. Phys. Chem. 1982, 86,2324-2327
should be useful for analytical purposes in the application of negative chemical ionization analysis and should further add to our understanding of atmospheric ion-molecule reactions resulting from inadvertent or intentional modification of the stratosphere or ionosphere. Several reactions, those of 0 and 02-with NF3 and that of NH2- with 02,were found to be substantially slower than expected and are added to the interesting list of exothermic, yet
slow, ion-molecule reactions. The apparent universal reactivity of 0-and 02-with halogenated molecules suggests that the technique of enhancing the response of electron capture detectors by oxygen doping should be widely applicable. Acknowledgment. This work was supported by the US Department of Energy.
Determination of Acyl Chain Conformation at the Lipid Interface Region: Raman Spectroscopic Study of the Carbonyl Stretching Mode Region of Dipalmitoyl Phosphatidylcholine and Structurally Related Molecules Ernest Mushayakarara and Ira W. Levin' Laboratory of Chemical Physics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethe&, hfaryiand 20205 (Received: December 17, 1981; In Final Form: February 8, 1982)
The two carbonyl stretching modes of 1,2-dipalmitoyl phosphatidylcholine (DPPC) at approximately 1721 and 1739 cm-' reflect the structural inequivalence of the two acyl chains at the lipid interface region. A comparison of the Raman spectra of DPPC to a series of related lipid systems whose carbonyl groups exist in various lipid environments confirms the assignment of the higher and lower frequency components to the sn-1 and sn-2 chain carbonyl groups, respectively. In particular, frequencies for carbonyl groups from about 1727 to 1744 cm-I reflect a nearly straight chain or trans conformation about the C2-C1 bond of the C3C2C1(=O)0 structural unit for the ester group of the sn-1 acyl chain. Frequencies from about 1716 to 1728 cm-' are characteristic of the carbonyl group for the sn-2 chain, which assumes an approximately gauche conformation about the C2-C1 bond (see Figure 1 for bond definitions). The samples used in addition to DPPC for spectral comparisons are (a) ypalmitoyl-L-a-lysophosphatidylcholine(lysolecithin),whose single acyl chain exists in a nearly trans conformation about the C2-C1 bond, (b) 1,2,3-propanetrioltrihexadecanoate (tripalmitin) whose 1-and 2-chains form a single, nearly straight chain unit, while the 3-chain bends analogously to the sn-2 chain of DPPC, and (c) 1,3-dipalmitoylglycerol(1,3-dipalmitin)whose C3C2C1(=O)0structural elements probably assume a nearly trans conformation about the C2-CIbond with one of the carbonyl groups being hydrogen bonded to the hydroxyl group of a neighboring glycerol unit.
Introduction Among the vast array of physical techniques used to probe biological membrane processes, vibrational spectroscopy offers a potentially powerful method for assessing the contributions of bilayer lipids to the structural and dynamical properties of membrane assemblies. In particular, frequency, intensity, and linewidth changes of selected vibrational spectroscopictransitions are extremely sensitive to conformational alterations and packing rearrangements involving either the polar headgroups or the hydrocarbon chain segments of bilayer lipids.lP2 The advantages of using vibrational techniques t o relate the effects of bilayer reorganizations to the dominant lipid-lipid and lipid-protein interactions which define membrane functions stem from several sources. First, the experimental accessibility of numerous spectroscopic transitions originating from various functional groups of the lipid provides information pertinent to localized regions of the bilayer. Second, the spectroscopic data are obtained in a noninvasive manner; that is, inferences regarding bilayer behavior are derived from molecular probes which do not intrinsically perturb the lipid environment. *Reprints may be obtained from Dr. Ira W. Levin, Building 2, Room B1-27, National Institutes of Health, Bethesda, MD 20205.
Although correlations exist between the behavior of the vibrational spectra and the properties of the headgroups and acyl chains comprising the bilayer, spectroscopic probes of the lipid interface region are not as well characterized. The interface region, defined by the -C2C1(= O)OCglyeerol structural unit in Figure 1, acts as a link between the polar and hydrophobic region of the m~lecule.~ Since the conformational inequivalence of the acyl chains of like-chain phosphatidylcholine and phosphatidylethanolamine crystals has been demonstrated by X-ray diffraction techniques?+ an understanding of the spectra reflecting the interface region is important for monitoring the behavior of the individual chains as a function, for example, of either temperature or intermolecular interactions. For the systems exhibiting this conformational inequivalence at the interface region, the sn-2 chain ini(1) R. Mendelsohn, R. Dluhy, T. Taraschi, D. G. Cameron, and H. H. Mantach, Biochemistry, 20, 6699 (1981). (2) S. F. Bush, R. G. Adams, and I. W. Levin, Biochemistry, 19,4429 ( I wn) (3) C. Huang, Lipids, 12, 348 (1977). (4) R. H. Pearson and I. Pascher, Nature (London),281,499 (1979). (5) P. B. Hitchcock, R. Mason, K. M. Thomas, and G. G. Shipley, Proc. Natl. Acad. Sci. U.S.A., 71, 3036 (1947). (6)M. Elder, P.Hitchcock,R. Mason, and G. G. Shipley,h o c . R. SOC. London, Ser. A , 354,157 (1977). \____,_
This article not sub]ect to U.S. Copyright. Published 1982 by the American Chemical Society
The Journal of Physical Chemistry, Vol. 86, No. 13, 1982 2325
Acyl Chain Conformation at Lipid Interface Region
cis about
C2-Cl
bond
w n s about C2-C,
bond
gauche about C2-C,
bond
Figure 1. Definition of conformers about the C2-C1 bond of the lipid hydrocarbon chain. R represents the glycerol backbone.
tially extends in a perpendicular direction to the linear sn-1 chain. A gauche bond at the carbon 2 position then enables the remaining chain segment to pack parallel to the sn-1 chain. In contrast to the gauche conformation assumed by the upper region of the sn-2 chain, the bonding about the ester group of the sn-1 chain is trans about the initial C2-C1 bond (see Figure 1). In addition to the structurally inequivalent ester carbonyl groups, the abrupt bend of the sn-2 chain displaces the two methyl chain termini by about 3.7 A. The structural inequivalence of the two chains near the glycerol backbone is reflected by two distinct carbonyl stretching modes, at approximately 1721 and 1739 cm-l, in both the infrared and Raman spectra of anhydrous lipid assemblie~.~JTwo sets of vibrational assignments for associating the observed spectral features with specific carbonyl groups have been In the present communication we clarify the vibrational assignments for the carbonyl stretching modes by discussing the Raman spectral data for a series of lipid systems whose carbonyl functional groups exist in various molecular environments.
Experimental Section Commercial samples of 1,2-dipalmitoyl-~-a-phosphatidylcholine (DPPC, purity in excess of 99%), y-palmitoyl-L-a-lysophosphatidylcholine(lysolecithin, purity in excess of 99 %), and 1,3-dipalmitoylglycerol(1,3-dipalmitin or 1,3-DPG, 98% pure) were obtained from Sigma Chemical Co. 1,2,3,-Propanetriol trihexadecanoate (tripalmitin, purity in excess of 99%) was purchased from Supelco, Inc. DPPC, tripalmitin, and lysolecithin were used without further purification, since thin-layer chromatography revealed no contamination. 1,3-Dipalmitinwas recrystallized prior to use. Solid samples, dried under vacuum, were sealed in melting point capillaries and subsequently annealed by thermal cycling. Vibrational Raman spectra were recorded with a Spex Ramalog 6 spectrometer equipped with a set of holographic gratings and a Coherent Model CR-12 argon ion laser delivering 200 mW at the sample at 514.5 nm. Spectral resolution varied from 2 to 4 cm-l. Spectral frequencies, calibrated with atomic argon lines and laser plasma lined0 are reported to k2 cm-'. Sample temperatures were maintained by placing the capillaries within a thermostatically controlled mount. A copper-constantan thermocouple, placed adjacent to the capillary within the holder, recorded temperatures. Spectra were acquired with a Nicolet NIC-1180 data system interfaced to the spectrometer. Since the carbonyl spectral features in the 1680-1780-cm-' region exhibit weak Raman scattering, 10-40 signal averaged spectral scans, obtained a t a scan rate of 1 cm-'/s, were required. (7) S. F. Bush, H. Levin, and I. W. Levin, Chem. Phys. Lipids, 27, 101 (198001. .~..., (8) E. Bicknell-Brown, K. G. Brown, and W. B. Person, J.Am. Chem. Soc., 102,5486 (1980). (9)E. Bicknell-Brown and K. G. Brown, Biochem. Biophys. Res. Commun., 94,638 (1980). (10)N. C. Craig and I. W. Levin, Appl. Opt., 33, 475 (1979).
8. LYSO PC
I
iI
t
1.m
1890
1700
1710
1720
im
1740
1750
1760
in0
1780
WAVENUMBER DISPLACEMENT (cm-1)
Figure 2. Raman spectra of carbonyl stretching mode region of anhydrous, polycrystalline liplds: (A) DPPC at 6 OC; (B)Lyso PC at 4 OC; (C) tripalmitin at 1 OC; (D) 1,bDPG at 0 OC.
Results and Discussion Figure 2A displays the Raman spectrum for the carbonyl stretching mode region for anhydrous, polycrystalline DPPC at a temperature of 6 "C. On the basis of the infrared spectra of lysolecithin, a system containing only the sn-1 chain with a trans conformation about the C2-C1 bond, and of arguments concerning the effect of different carbonyl environments upon the carbonyl stretching frequency, Levin and coworkers2Jassigned the infrared and Raman features at -1739 and 1721 cm-' to the carbonyl moieties of the sn-1 and sn-2 chains, respectively, The shoulder at 1743 cm-' in Figure 2A corresponds to the 1743-cm-' infrared feature which was assigned to a chain rotational isomer. From the Raman spectra of polycrystalline, anhydrous DPPC and DPPE (dipalmitoyl phosphatidylethanolamine) Bicknell-Brown and coworker^^^^ reversed the above assignments by associating the 1739 and 1721-cm-' transitions to the sn-2 and sn-1 acyl chains, respectively. These authors based their assignments on an unpublished normal coordinate study of theirs involving bond rotations about the C2-C1 bond and a low-temperature infrared study by Hayashi and Umem~ra.~J'The latter investigation discusses the relative stabilities of cis (11)S. Hayashi and J. Umemura, J. Chem. Phys., 63, 1732 (1975).
2326
The Journal of Physical Chemistry, Vol. 86, No. 13, 1982
and trans configurations for carbonyl groups in crystalline fatty acid dimers. We contend that Hayashi and Umemurals infrared observations for the fatty acids" support the first assignment by Levin and c o - w ~ r k e r scontrary ,~~~ to the view expressed by Bicknell-Brown et alaa We accept the reasoning of Bicknell-Brown et ale8for which the cis and trans conformers formed by hydrogen atom exchange in the fatty acid dimer system provide an analogous frequency pattern to that for the approximately gauche and trans conformers assumed about the ester linkages of the phospholipid systems. Thus, Hayashi and Umemura" assigned infrared bands at 1709 and 1699 cm-' to the trans and cis forms, respectively, as defined by Bicknell-Brown et al.,778who used the bond definitions described in Figure 1. This ordering of frequencies is then consistent with the ordering presented by Levin and cow o r k e r ~for ~ , phospholipids ~ in which the higher frequency mode was associated with the sn-1 chain and an approximate trans conformation about the C2-C1 bond.44 The confusion in ref 8 arose, in part, from the use by Bicknell-Brown et al. of cis and trans bond definitionsa which differed from the scheme presented by Hayashi and Umemura." The Raman spectrum for a single sn-1 chain phospholipid, lysolecithin, shows in Figure 2B one spectral component at 1737 cm-'. Raman spectra for other sample preparations of this anhydrous system display a pronounced shoulder at 1744 cm-'. This latter feature is indicative of a population of rotational isomers involving the entire acyl chain.' In comparison to DPPC the observation of a single, primary component at high frequencies is consistent with the sn-1 chain exhibiting a nearly trans conformation about the ester linkage. This nearly straight chain conformation for an sn-1 lysophosphatidylcholine has been demonstrated by an X-ray crystal analysis.12 Figure 2C displays the Raman spectrum of the carbonyl region for tripalmitin, a three-chain structure. Crystal structures determined by X-ray diffraction for trilaurin and tricaprin, in which the three acyl chains in each system are either all C12 or Cl0, respectively, yield a modified "tuning fork" configuration for the hydrocarbon chains.'*15 For these structures chains 1and 2 form one long, nearly straight chain, while chain 3, which branches from the third glyceryl carbon atom, runs perpendicularly to the two straight chain segments before bending a t the carbon 2 position. The remaining segment of chain 3 then packs parallel to the other chains. Although the crystal structure for tripalmitin has not been determined, the close correspondence of the Raman spectra of the three triglycerides in the carbonyl stretching mode interval16 indicates an analogous packing of the acyl linkages in the three systems. The spectrum in Figure 2C shows the two primary features at 1728 and 1743 cm-', with a shoulder at 1737 cm-l. The ratio of the integrated area of the 1728-cm-' feature, determined from deconvoluted spectra, to the band area for the 1743-cm-' feature is 0.32:0.68 or about 1:2. This intensity relationship indicates a correspondence of the lower 1728-cm-' transition to the gauche configuration assumed about the ester group for chain 3. The greater intensity of the higher 1743-cm-' feature is associated with the two chains whose ester linkages are nearly trans.13-15 The shoulder at 1737 cm-' probably indicates a small popula(12) H. Hauser, I. Pascher, and S. Sundell, J. Mol. Biol., 137, 249 (1980). (13) K. Larsson, Ark. Kemi, 23, 1 (1965). (14) V. Vand and I. P. Bell, Acta Cryst., 4, 465 (1951). (15) L. H. Jensen and A. J. Mabis, Acta Cryst., 21, 770 (1966). (16) E. Mushayakarara and I. W. Levin, unpublished data.
Mushayakarara and Levin
TABLE I: Summary of the Carbonyl Stretching Mode Vibrations of Related Polvcrvstalline LiDids lipid
frequency, cm-'
T,"C chain assignment
DPPCa
1 7 4 3 (sh)= 1739 1721
6
1 1
LysoPCb
1 7 4 4 (sh) 1737
4
1 1
tripalmitinC
1743 1 7 3 7 (sh) 1728
1
1,3f 193 2g
1,3-DPGd
1732 1 7 2 7 (sh) 1708
0
2
1,3 193 hydrogen bonded
a 1,2-Dipalmitoyl phosphatidylcholine. y-PalmitoylL-0-lysophos hatidylcholine. 1,2,3-Propanetriol trihexa1,3-Dipalmitoylglycerol. e sh represents decanoate. shoulder. f 1 and 3 represent straight chains in a modified "tuning fork" structure. g 2 represents bent chain in a modified "tuning fork" structure.
tion of molecules reflecting a slightly different overall chain rotation, at least near the glyceryl carbon atoms. We turn now to the Raman spectrum for 1,3-dipalmitoylglycerol (1,S-DPG) given in Figure 2D. Two primary features are again observed, although the lower transition is broadened and shifted compared to the 1721-cm-' feature for DPPC in Figure 2A. A second primary feature appears at 1732 cm-', along with a shoulder at 1727 cm-'. Although the crystal structure of 1,3-DPG has not been determined, it is useful to consider the known structure for the closely related 1,3-diglyceride of 3-thiadodecanoic acid.17 In this system the carboxyl and glycerol groups form a nearly straight zig-zag chain. The sulfur atoms substituted for the y-methylene groups create a bend in the chains at the 3 positions. Since the X-ray studies of Abrahamsson and Westerdahll8 have shown that sulfur atom substitution does not perturb the straight-chain nature of the remaining hydrocarbon segments running to the chain termini, it is reasonable to assume that the conformations about the C3C2C,(=O)0 structural elements in 1,3-DPG will closely approximate the nearly trans conformer determined by X-ray diffraction for the 1-chain in DMPC dihydrate: dilauroyl phosphatidylethan~lamine,~~~ and 3-dodecyanoyl propanediol-lphosphorylcholine,'2 a deoxy derivative of lysophosphatidylcholine. Finally, the crystal structure for 1,3-diglyceride of 3-thiadodecanoic acid indicates a hydrogen bond between the hydroxyl group on the glycerol unit and one of the carbonyl oxygens from a neighboring chain.17 This hydrogen bonding scheme would probably also occur for 1,3-DPG. From the above structural considerations the broadened and shifted 1708-cm-' component may readily be assigned to a hydrogen-bonded chain carbonyl group. The upper 1732-cm-' transition for 1,3-DPG is then assigned to the ester carbonyl groups participating in the nearly trans conformations for the C,C2Cl(=O)0 units. The latter assignment for 1,3-DPG is consistent with an infrared feature observed at 1731 cm-' in DPPC at liquid nitrogen temperatures ( - 7 7 K) and assigned in DPPC to a rotational isomer involving the entire acyl sn-1 chain.6 That is, although the carbonyl frequency for 1,3-DPG is somewhat lower than the frequencies observed in DPPC, lyso PC and tripalmitin for the 1-chain carbonyl moiety, the (17) K. Larsson, Acta Cryst., 16, 741 (1963).
(18) S. Abrahamsson and A. Westerdahl, Acta Cryst., 16,404 (1963).
J. Phys. Chem. 1982, 86,2327-2336
observed frequency in 1,3-DPG corresponds to that for a chain rotational isomer of the 1-chain in DPPC. A summary of the carbonyl frequencies and assignments for the four lipid systems appears in Table I.
Conclusion A consideration of the Raman spectra of the carbonyl stretching mode regions for a series of phospholipids, di-, and triglyceride molecules indicates that the spectral patterns in the 1700-1750-cm-' region are clearly useful in distinguishing between conformations about the C,C&(=O)O-ester groups for the hydrocarbon chains. In particular, the carbonyl stretching modes for acyl chains with an abrupt bend at the carbon 2 position exhibit a lower vibrational frequency in comparison to the carbonyl frequencies for the ester groups of the straight-chain systems. Thus, frequencies for the carbonyl mode from about 1727 to 1744 cm-l reflect a nearly straight chain segment, while frequencies from about 1716 to 1728 cm-' are characteristic of the bent chain conformation. Although some ambiguity may arise for frequencies occurring at 1728 cm-l, the relative intensities of the transitions for the systems investigated, as shown for tripalmitin, allows a confident assignment to be made. Since the
-
2327
carbonyl groups for the sn-1 and sn-2 chains may be separately identified, we have available an important molecular probe for monitoring effects involving changes in bilayer structure at the membrane interface region. Although the carbonyl features broaden, shift, and tend to merge in hydrated systems, curve deconvolution could be used, in principle, to follow effects involving individual chains.' For example, changes in the carbonyl stretching modes resulting from the combined effects of both cholesterol (at low concentrations) and water suggest that the bent sn-2 chain in hydrated DPPC bilayers assumes a conformation more analogous to the linear sn-1 chain.' In a recent infrared study involving the effects of the basic myelin protein on diipalmitoyl phosphatidylglycerol bilayers, we noted that the lipid chain conformations maintain their conformational inequivalence in both the gel and liquid crystalline states.lQ That is, two distinct features at -1726 and 1742 cm-' are observed in the presence of the protein. In the absence of the protein, a broad, nearly symmetrical feature, which is analogous to that recorded for DPPC, is observed -1735 cm-1.237J9 (19)R. G.Adams and I. W. Levin, unpublished data.
Conformers and Internal Rotation Barrlers in Nitrous Acid Esters. Low-Resolution Microwave Spectroscopic Studies Nancy S. True Department of Chemistry, University of California, Davis, Caiifornis 95616
and Robert K. Bohn' Department of Chemistry and Instltute of Materials Science, Universky of Connecticut, Storrs, Connecticut 06268 (Received April 28, 1981; In Final Form: February 3, 1982)
Low-resolutionmicrowave (LRMW) spectra of 10 alkyl nitrites have been observed and characterized. LRMW spectra are sensitive to internal rotation of asymmetric groups only, and these 10 compounds have from one asymmetric internal rotor (about the N-0 bond in tert-butyl nitrite, O=N-O-C(CH,),) to four (about the N - O , O-C, and two subsequent C - C bonds in n-butyl nitrite and isopentyl nitrite). Eight of the ten compounds are observed in conformers having both syn and anti c o d i a t i o n s of the O = N - O - C chain. The two exceptions (anti only) are the two tertiary nitrites in the study which have the C atom bonded to the nitrite group bonded to no H atoms. tert-Butyl nitrite has one stable conformation,isopropyl, neopentyl, isobutyl, tert-amyl, neohexyl, and isopentyl nitrites have two stable conformations, and n-propyl, n-butyl, and 2-butyl nitrites have at least three stable conformations. Most of these have been assigned. Also, eight of the compounds display spectra of species rotating about the 0-C bond above the internal rotation barrier. These species occur only when configuration is anti, and the observations have allowed the low barriers to internal rotation the O=N-0-C above the 0-C bond to be estimated in each case. The two exceptions are tert-butyl nitrite, whose LRMW spectra are insensitive to internal rotation about the 0-C bond, and 2-butyl nitrite.
Introduction Alkyl nitrites are of interest structurally, biologically, and environmentally. The microwave spectrum of methyl nitrite' reveals the presence of syn and anti rotamers, the syn form being more stable by 314 (22) cm-l.ld Barriers (1)(a) W.D.Gwinn, Second Austin Symposium on Gas Phase Molecular Structure,1968,Abstract M2; (b) P. H. Turner, M. J. Corkill, and A. P. Cox, J.Phys. Chem., 83,1473(1979);(c) K.Endo and Y. Kamura, J. Chem. SOC.Jpn., 729 (1977);(d) P.N.Ghosh, A. Bauder, and HE. H. Gunthard, Chem. Phys., 53,39 (1980). 0022-3654/8212006-2327$0 1.2510
to internal rotation of the methyl group in methyl nitrite are remarkably different, 734 (2) cm-l for the syn formld and 10.1 cm-' for the anti form.lb Recent ab initio Hartree-Fock molecular orbital calculations have reproduced the surprisingly large difference in methyl barriers in the two conformers.2 Three stable rotamers of ethyl nitrite have been identified from microwave spectral data? They (2) F. R. Cordell, J. E. Boggs, and A. Skancke, J. Mol. S t r u t . , 64,57 (1980). (3)P. H.Turner, J. Chem. SOC.,Faraday Trans. 2,75,317 (1979).
0 1982 American Chemical Society
