Original Article

Structural analyses of polymorphic transitions of sn -1,3-distearoyl-2-oleoylglycerol (SOS) and sn -1,3-dioleoyl-2-stearoylglycerol (OSO): assessment on steric hindrance of unsaturated and saturated acyl chain interactions

Correspondence to: Junko Yano

	ABSTRACT

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Polymorphic transformations in two saturated-unsaturated mixed acid triacylglycerols, SOS (sn -1,3-distearoyl-2-oleoylglycerol) and OSO (sn -1,3-dioleoyl-2-stearoylglycerol), have been studied by FT-IR spectroscopy using deuterated specimens in which stearoyl chains are fully deuterated. A reversible phase transition between sub and and a series of irreversible transitions (ß'ß (ß₂, ß₁) for SOS and ß'ß for OSO) were studied with an emphasis on the conformational ordering process of stearoyl and oleoyl chains. The sub reversible transition was due to the orientational change of stearoyl chains in the lateral directions from the hexagonal subcell to a perpendicularly packed one. As the first stage of the series of irreversible transitions from to ß, the conformational ordering of saturated chains took place in the transition of SOS and in the ß' transition of OSO; one stearoyl chain in SOS and OSO takes the all-trans conformation and the second stearoyl chain in SOS takes the bent conformation like those observed in the most stable ß-type. As the final stage, the ordering of unsaturated chains occurred in the ß'ß transition both for SOS and OSO. A conversion in the layered structure from bilayer to trilayer was also accompanied by the conformational ordering in the transition of SOS and in the ß'ß transition of OSO.—Yano, J., K. Sato, F. Kaneko, D. M. Small, and D. R. Kodali. Structural analyses of polymorphic transitions of sn -1,3-distearoyl-2-oleoylglycerol (SOS) and sn-1,3-dioleoyl-2-stearoylglycerol (OSO): assessment on steric hindrance of unsaturated and saturated acyl chain interactions. J. Lipid Res. 1999. 40: 140–151.

Supplementary key words: polymorphism, phase behavior, infrared spectroscopy, molecular packing, molecular conformation, deuteration

	INTRODUCTION

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The aliphatic chain–chain interactions are critical factors for the determination of physical properties such as softness and flexibility of the fats and lipids present in biotissues. Their packing conditions depend on chemical natures of fatty acid moieties: chain length, parity (odd or even), unsaturation (cis or trans), etc. Most of the biologically important lipid specimens are composed of saturated and unsaturated acyl chains as revealed in diacylglycerols and phospholipids. Diacylglycerols act as an activator of protein kinase C and a signal transductor in metabolism (1) (2) (3) (4) (5). Phospholipids produce fluidity of membrane lipids, often containing a saturated chain in the sn -glycerol 1 position and an unsaturated chain in the sn -glycerol 2 position (6) (7). Triacylglycerols (TAGs), which are a major metabolic fuel, also consist of saturated and unsaturated acyl chains. Unsaturated acyl moieties have a critical role to prevent the solidification of TAGs; solidification causes lipase activity to be remarkably reduced in poikilothermal animals (8) (9). The presence of these moieties affects melting behavior more effectively than the modification of aliphatic chain length (10).

The aliphatic compounds containing cis -unsaturated chains reveal quite diversified polymorphism due to the structural flexibility of olefinic groups. Concerning the cis -unsaturated fatty acids, their molecular structures have been unveiled by X-ray diffraction crystal structure analyses and vibrational spectroscopy, IR and Raman (11) (12) (13) (14) (15) (16). The molecular structures of cis -unsaturated fatty acid esters of cholesterol were also clarified by the use of X-ray diffraction methods (17) (18) (19). Through those studies, it has become apparent that the cis -olefin group can take various internal-rotation angles in solid states to compensate for the conformational disadvantage, depending on the balance between the acyl chain length and the double bond position. The mixed-acid TAGs (20) (21) (22) (23) and diacylglycerols (24) (25) also exhibited diversified polymorphic behavior, all indicating complicated molecular interactions. The mixing behavior of aliphatic chains is important for understanding the interactions between acyl chains. The miscibility between saturated and unsaturated constituents has been studied in the binary systems of fatty acids and those of TAGs. It was clarified that a saturated constituent was hard to mix with an unsaturated one (26) (27) (28) (29) (30) (31). We were interested also in the lateral packing of a TAG containing both saturated and unsaturated acyl chains, which would relate to a significant question of how the saturated and unsaturated chains are accommodated together in biological tissues and industrial products.

In the previous studies on the mixed-acid TAGs of this type, it has been clarified that the steric hindrance between the saturated and unsaturated acyl chains resulted in various metastable phases and a series of polymorphic transformation to the stable phase. From the studies on three kinds of TAGs, Sat-O-Sat (1,3-disaturated-acyl-2-oleoylglycerol) (20) (21), O-Sat-O (1,3-dioleoyl-2-saturated-acylglycerol) (22), and Sat-Sat-O (1,2-disaturated-acyl-3-oleoylglycerol) (23), the following conclusions were obtained. First, the position and quantity of oleoyl groups drastically change polymorphism. For example, SOS (sn -1,3-distearoyl-2-oleoylglycerol) shows five polymorphs, , , ß', ß₂, and ß₁, while OSO (sn -1,3-dioleoyl-2-stearoylglycerol) shows three polymorphs, , ß', and ß ( Figure 1a, b) (20) (22) (32) (33). Second, there are specific composition ratios for binary systems to form molecular compounds. Concerning this point, the binary systems of SOS/rac -1,2-distearoyl-3-oleoylglycerol (SSO) (23), SOS/OSO (28), sn -1, 3-dipalmitoyl-2-oleoylglycerol (POP)/sn -1,3-dioleoyl-2-palmitoylglycerol (OPO) (31), and POP/rac -1,2-dipalmitoyl-3-oleoylglycerol (PPO) (30) were investigated. Although each TAG component has different polymorphism, a specific chain–chain interaction caused the formation of the molecular compounds at the 1:1 ratio for each combination.

View larger version (32K): [in this window] [in a new window]   Figure 1. Schematic representations of polymorphic transformations in (a) sn -1,3-distearoyl-2-oleoylglycerol (SOS) and (b) sn -1,3-dioleoyl-2-stearoylglycerol (OSO). The models were depicted on the basis of the lamellar distances obtained from the powder X-ray diffraction method and the olefinic group conformations were estimated from the frequencies of the =CH out-of-plane bending mode in IR spectra (20) (22) (32) (33). The phases of SOS and OSO and the ß' phase of OSO pack in bilayer structure, whereas the ß' phase of SOS and the ß phases of SOS and OSO have the trilayer structure. The lamellar thickness of each polymorph is given.

In this study we aimed to clarify the conformation and lateral packing of the saturated and unsaturated acyl chains in each polymorphic form of SOS and OSO. For this purpose we used vibrational spectroscopy, which is a useful method to investigate the complicated polymorphism and intermolecular interactions on a molecular level. The information about subcell structure and molecular conformation of aliphatic chain can be derived. We have already applied infrared absorption spectroscopy for the polymorphic studies on mixed-acid TAGs such as SOS, POP, POS, and on binary systems of POP/PPO and POP/OPO (34) (35). In the present study, we adopted the following tactics.

For molecular conformation, the progression bands due to the ₃ branch (CH₂ wagging) modes of polymethylene segments were used as a probe (36) (37) (38) (39) (40) (41) (42). The frequencies of these progression bands are so sensitive to the length of all-trans hydrocarbon segments that we can derive the information about which acyl chain of a TAG takes a bend conformation (43) (44).

To get the information about the lateral packing of stearoyl and oleoyl groups separately, we synthesized partially deuterated specimens of SOS and OSO where stearoyl chains are fully deuterated. Using these deuterated specimens, we could avoid the problem of the ordinary hydrogenated specimens (45) (46) (47): the overlapping of IR bands due to oleoyl and stearoyl groups. Each group showed IR bands in its own frequency regions due to the mass difference between hydrogen and deuterium.

In this paper, we will demonstrate the utilities of these spectral techniques and show that the conformational ordering of each acyl group is accompanied by an ordering in the lateral packing and/or a structural change in the layer structure.

	MATERIALS AND METHODS

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Samples
Hydrogenated-SOS (purity >99%) was provided by Fuji Oil Co. Ltd. Hydrogenated-OSO (purity >99%) was purchased from Sigma Co. Ltd. sn -1,3-Distearoyl-d₇₀-2-oleoylglycerol and sn -1,3-dioleoyl-2-stearoyl-d₃₅-glycerol were synthesized by adopting the methods described in the literature (22) (48) by using perdeuterated stearic-d₃₅ acid (obtained from Aldrich Chemical Company) having >98 atom % deuterium.

In this paper we have used the words "d-SOS" and "d-OSO" for the deuterated specimens and "h-SOS" and "h-OSO" for the usual hydrogenated specimens.

Preparation of crystal forms
The following procedures were applied to obtain each polymorphic form both for hydrogenated and deuterated specimens.

SOS. Specimens of the and phases were prepared by holding samples at 80°C for 10 min and then quenching to 10°C and 30°C, respectively (20). A film of the ß' phase was made from a chloroform solution on KBr by evaporation method. By holding the ß' specimen at 35°C for 8 h, we obtained a film of ß (20). As for the two ß phases of SOS, we previously concluded that the molecular structures of ß₂ and ß₁ are almost the same, but the crystal symmetry is different between them (34). As the ß₂-type crystal was not obtained from the deuterated specimen in this study, we focused on the most stable ß₁ form of SOS, which is described as ß in this paper. Single crystals of the ß form were grown from an acetonitrile solution by slow cooling.

OSO. A specimen of the phase was prepared by quenching a melt to -10°C. By heating the phases to -5° and 10°C, we obtained specimens of the ß' and ß phases, respectively (22). Single crystals of the ß form were grown from an acetonitrile solution by cooling.

There were a few differences in polymorphic behavior between hydrogenated and deuterated specimens. Each polymorph melted at a slightly lower temperature in a deuterated specimen than in a hydrogenated one, and there was a new metastable phase in d-SOS that melted at a temperature between the melting points of ß' (36.5°C) and ß₂ (41.0°C). In spite of these differences, there were no significant structural differences between hydrogenated and deuterated specimens. Each polymorph of the deuterated specimens exhibited almost the same X-ray diffraction pattern as that of the hydrogenated specimens.

Powder X-ray diffraction measurements
The X-ray diffraction pattern of each polymorphic form was measured using a Rigaku X-ray diffractometer (40 kV, 10 mA) with Cu-K radiation. For the measurement at a low temperature, a Rigaku cryostat was used.

Infrared methods
The IR spectra were taken with a Perkin-Elmer Spectrum 2000 spectrometer attached to a Perkin-Elmer i-Series FT-IR microscope. An Oxford flow type cryostat CF1104 and an Oxford temperature controller ITC-4 were used for the measurements at low temperatures. The polarized IR spectra were measured with a MCT detector and a wire-grid polarizer (Perkin-Elmer PR500). The spectra without mention of the conditions were taken at room temperature.

To determine the directions of transition moments three-dimensionally, we used the following three methods of IR measurement.

Polarized IR transmission method. Using single crystal specimens we measured the polarized IR spectra of the ß phases with the experimental conditions depicted in Figure 2a. The incident IR rays impinge normally to the flat face of the crystal. The long crystal edge is set parallel to the direction of = 90°.

View larger version (29K): [in this window] [in a new window]   Figure 2. Schematic representations of (a) the normal incident transmission, (b) RAS, and (c) ATR. In (a), the direction of IR radiation is normal to the (001) plane of the crystal. The direction of the electric field of the incident radiation is fixed and the sample is rotated about the normal of the (001) plane. In (b) and (c), samples were built up on a substrate as a thin film where the lamellar interfaces are parallel to the substrate. With a strong electric field normal to the metal surface, the IR bands having the transition dipole moments normal to the (001) plane are observed selectively in (b). In (c), reflection spectra were measured with p- or s-polarized incident radiation. With p-polarization, the x and z components of a transition moment are observed, and the y component with s-polarization.

As the other metastable phases such as and ß' had difficulty in growing single crystals for the polarized IR measurement, we made a well-oriented film between two KBr windows. The (001) plane of this film is parallel to the KBr window's faces.

The polarization dependence of the IR spectra gives the information about the projections of transition moments onto the (001) plane, i.e., the information is two-dimensional. Therefore, the RAS (reflection absorption spectroscopy) and ATR (attenuated total reflection) methods were necessary in this study.

RAS. For the RAS measurement, a variable angle reflection accessory (Perkin-Elmer) was used. A thin film of TAG was built on a flat aluminum plate by developing a chloroform solution. By controlling the temperature of the substrate, we made a film of each polymorph where the lamellar interfaces are parallel to the metal surface (Figure 2b). The IR bands whose transition moments are parallel to the chain axis are emphasized with a strong electric field normal to the metal surface (49) (50).

ATR. Polarized ATR spectra were measured using an ATR accessory (PIKE technologies) and a wire-grid polarizer. A Ge plate (wedge angle: 45°) was used as an internal reflection element (IRE). Single crystals or films built on the Ge plate were used as specimens. The x- and z-components of a transition moment are observed with p-polarization, while the y-component is taken with the s-polarization (Figure 2c) (51).

Assignments of ₃ progression bands of SSS ß
To study the conformation state of stearoyl chains in each polymorph of SOS and OSO, the information about the IR bands of tristearoylglycerol (SSS) is important as a reference, in particular the progression bands due to hydrocarbon chains (40) (41) (43).

The spectral pattern in the region of 1380–1150 cm^-1 reflects sensitively the stem length of the trans zigzag chain. Two series of progression bands overlap in this region, one is due to the ₃ branch modes (CH₂ wagging: 1360–1150 cm^-1) and the other is due to the ₇ branch modes (CH₂ twisting-rocking: 1300–1150 cm^-1) (52) (53). The ₃ progression bands appear clearly and can be used as a convenient tool.

Figure 3 shows the IR spectra of the ß phase of SSS with the band assignments determined in the previous study (43). The bands marked with open circles are of the all-trans stearoyl chains at the sn -1 and sn -2 positions, and those with wedges are of the bent stearoyl chain at the sn -3 position whose C(2)–C(3) bond takes a gauche conformation (see top left of Figure 3) (54) (55) (56) (57). The conformational difference has two effects on the ₃ progression bands. First, the polarization of the ₃ bands is changed. Because the CH₂ wagging modes of all-trans hydrocarbon chains have transition moments parallel to the chain axis, the ₃ bands of the straight sn -1,2 chains clearly appear in RAS. On the other hand, the ₃ bands of the sn -3 chain appear both in the transmission spectra and in the RAS spectra, because the ₃ modes of the sn -3 chain couple with the C–O stretching mode and have both the perpendicular and parallel components to the molecular axis. Second, the gauche bond makes these bands shift to higher frequencies as shown in Figure 3, due to the shortened stem length of the all-trans moiety.

View larger version (41K): [in this window] [in a new window]   Figure 3. IR spectra (RAS and normal incident transmission) of SSS ß. The molecular structure of SSS is depicted at top left (54) (55) (56) (57). Open circles and wedges reveal the 3 bands of the sn -1,2 straight and sn -3 bent stearoyl chains, respectively. The frequencies of the 3 bands are also given.

	RESULTS

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–sub reversible transitions in SOS and OSO
The phase of monoacid TAGs exhibits a reversible transition to the sub phase (58), but we did not confirm the existence of the sub phase in the previous studies on SOS and OSO.

Figure 4 shows X-ray diffraction patterns of SOS on cooling. The form exhibited a single peak of 0.42 nm due to the hexagonal subcell. Below -50°C, this hexagonal subcell seemed to approach gradually to a pseudohexagonal packing having a main peak of 0.42 nm and a shoulder of 0.38 nm (58). This lower temperature form corresponds to the sub form. The –sub transition occurred reversibly by temperature changes. We observed this reversible transition also in OSO. The detailed structures of the and sub phases will be discussed in a later section.

View larger version (17K): [in this window] [in a new window]   Figure 4. X-ray diffraction patterns: the changes of XRD patterns during the –sub R transition of SOS. A single peak of 0.42 nm at 15°C is characteristic of the hexagonal packing. As temperature decreased, the hexagonal packing was varied to the pseudohexagonal packing having two peaks of 0.42 and 0.38 nm.

Acyl chain conformation
In order to clarify the conformational changes of each acyl chain during the complex phase transitions of SOS and OSO, we followed the spectral changes in the ₃ progression bands in 1360–1150 cm^-1 using the normal incident transmission, RAS, and ATR methods.

SOS. Figure 5a, b show the RAS and transmission spectra of each polymorph of h-SOS, in comparison with those of SSS ß. The bands marked with open circles in the RAS spectra (Figure 5a) of SSS ß are due to the ₃ modes of the all-trans stearoyl chains, while the bands marked with wedges in the transmission spectra (Figure 5b) of SSS ß are due to the ₃ modes of the bent stearoyl chains. Some bands of the bent chain appear also in the RAS spectra (bands marked with wedges).

View larger version (67K): [in this window] [in a new window]   Figure 5. IR spectra ((a) RAS and (b) normal incident transmission) of h-SOS polymorphs with SSS ß. The relationships between the polarization directions and the crystal morphology in the transmission spectra are depicted in (b). In (a) and (b), open circles and wedges show the 3 bands of the sn -1,2 straight and sn -3 bent stearoyl chains, respectively. In (a), the bands marked with closed circles in h-SOS ß indicate those commonly observed in h-OSO ß.

For all polymorphs of h-SOS, the sharp ₃ progression bands appeared in their transmission and RAS spectra. The transmission spectra of the and sub phases exhibited a series of bands whose frequencies are corresponding to those of the all-trans stearoyl chains. In addition the bands due to the bent stearoyl chain were observed in the RAS and transmission spectra of the and ß phases. Although the ß' phase exhibited only the bands due to the trans chain in the transmission spectra, the bands due to the bent chain were certainly confirmed in its RAS spectra. The difference in the polarization of the bent chain's modes between the ß' phase and the and ß phases is presumably related to the orientation of the esterified C–O bond against the zigzag skeletal plane in the bent chain (44).

The 1360–1150 cm^-1 region of d-SOS provides the information about the conformation of oleoyl group ( Figure 6). There were no sharp oleoyl progression bands in the sub and spectra. Marked spectral changes were observed through the ß' transition and some sharpened bands were observed in the and ß' phases. However, only the ß phase exhibited a series of bands due to the progression bands of oleoyl group; those bands were also observed in the RAS spectra (bands marked with closed circles in Figure 5a).

View larger version (24K): [in this window] [in a new window]   Figure 6. IR spectra of d-SOS in the 1360–1150 cm-1 region. Non-polarized spectra were taken for the sub and phases and polarized spectra were taken for the , ß', and ß phases. The bands marked with asterisks show the specific bands observed only in the deuterated specimens. The frequencies of the main bands were also given.

From the above results we inferred the conformational states of stearoyl and oleoyl groups in each polymorph as follows. In the and sub phases, stearoyl chains mostly take all-trans conformation, yet they contain conformational disorder to a certain extent, while the oleoyl group is conformationally disordered. There is no certain conformational difference between the two stearoyl chains at sn -1 and 3 positions. In the and ß' phases, one of the stearoyl chains changes into a bent conformation, as well as the sn -3 chain of SSS; the other stearoyl chain takes the all-trans form. Judging from the frequencies of the ₃ modes, the stearoyl chain takes a gauche conformation at the C(2)–C(3) bond of the sn -3 chain. The oleoyl group is still in a disordered state. Finally, in the ß phase the oleoyl group takes an ordered conformation.

The ordering of the oleoyl group in the ß form can be confirmed with a strong IR band of =C–H out-of-plane deformation (=C–H) at 688 cm^-1, which suggests an ordered skew- cis -skew' type conformation of the –C–C=C–C– portion (34). This also indicated that both the methyl-side (the chain segment between an olefinic group and a CH₃ group) and glycerol-side oleoyl chains (the segment between an olefinic group and a glycerol group) have the all-trans conformation.

The conclusions derived from IR spectral changes were consistent with the results of the previous study with Raman spectroscopy (59).

OSO. We used the polarized transmission and the polarized ATR methods for the evaluation of acyl chain conformations in each polymorph ( Figure 7 a, b).

View larger version (67K): [in this window] [in a new window]   Figure 7. Comparisons of (a) OSO ß spectra (polarized transmission and ATR) with SSS ß spectrum (RAS) and (b) OSO sub , , and ß' spectra (non-polarized transmission) with SSS ß spectrum (RAS). Open circles show the 3 bands of the sn -1,2 straight stearoyl chains. Closed circles in (a) show the common bands observed in SOS ß (Figure 5a). The frequencies of the main bands were also given. The ß' spectrum was taken at a low temperature to avoid the transition to ß and also to make the peak positions clear.

Contrary to the IR spectra of SOS ß, it seemed that the main spectral features of the transmission spectra in the 1360–1150 cm^-1 region of h-OSO ß came from the two oleoyl groups, as the observed bands totally corresponded to those of d-OSO ß ( Figure 8). On the other hand, the ₃ bands due to the stearoyl group clearly appeared in p-polarized ATR spectra where the modes along the chain axis are intensified, as indicated by open circles in Figure 7a. These frequencies corresponded to those of the all-trans stearoyl chain. Because of experimental difficulties, we could measure ATR spectra only for the ß phase. However, the ₃ bands of the all-trans stearoyl chain were observed also in the transmission spectra of the sub , , and ß' phases (Figure 7b).

View larger version (24K): [in this window] [in a new window]   Figure 8. IR spectra of d-OSO in the 1360–1150 cm-1 region. Non-polarized spectra were taken for the sub , , and ß' phases and polarized spectra were taken for the ß phase. The bands marked with an asterisk show the specific bands observed only in the deuterated specimens.

As to the oleoyl groups, there were no sharp progression bands in the 1360–1150 cm^-1 region of d-OSO and sub phases ( Figure 8). The ß' phase exhibited some spectral changes, such as intensity increase, sharpening, and splitting in the 1280–1250 and 1210–1195 cm^-1 regions. Eventually several sharp progression bands due to oleoyl groups were confirmed in the ß phase.

From the above spectral changes, it was inferred that on the phase transitions from to ß, the ordering of the acyl groups proceeds in the following ways. In the and sub phases, oleoyl chains are disordered but stearoyl chains take the all-trans form at a high ratio. The ordering of oleoyl groups takes place to some extent in the ß' phase, and in the ß phase both stearoyl and oleoyl groups take ordered conformations.

Concerning the conformation around the cis-olefin group, a sharp band of =C–H out-of-plane bending (=C–H) mode appears at 690 cm^-1 in ß (not shown here). We consider that the olefin groups of the two oleoyl chains take the skew-cis-skew' type configuration as in the ß phase of SOS (34). On the other hand, there were no detectable (=C–H) bands in the region of 730–600 cm^-1 of the sub , , and ß' phases, which suggests conformational disordering of the olefin group in these three phases.

Chain-chain interactions in lamella
Infrared CH₂ scissoring and CH₂ rocking regions are good indicators of lateral packing, i.e., subcell packing (60). However, the bands of oleoyl groups overlap with those of stearoyl groups for the usual hydrogenated specimens. To get information of individual groups separately, partial deuteration is a useful method (45) (46) (47). Deuterated acyl groups show the CD₂ scissoring (CD₂) and CD₂ rocking r(CD₂) bands around 1090 and 525 cm^-1, instead of the (CH₂) and r(CH₂) bands around 1460 and 720 cm^-1, respectively. For the d-SOS and d-OSO specimens where only stearoyl groups are fully deuterated, we can monitor the packing of oleoyl and stearoyl groups from the (CH₂) and (CD₂) bands.

In this study, we focused on the spectral changes of the (CH₂) and (CD₂) regions ( Figure 9a, b and Figure 10 a, b), because the frequency of the r(CD₂) mode is too low to be detected with the MCT detector equipped with our spectrometer. Here, the symbols of (CH₂)o and (CD₂)s mean the (CH₂) mode of hydrogenated oleoyl chains and the (CD₂) of deuterated stearoyl chains in partially deuterated specimens.

View larger version (37K): [in this window] [in a new window]   Figure 9. The (CH2) bands of hydrogenated (a) SOS and (b) OSO. Non-polarized spectra were taken for the sub and phases of SOS and OSO and the ß' phase of OSO. For the and ß' phases of SOS, polarized spectra were taken by the well-oriented specimens as depicted in the top left in (a). For the ß phases of SOS and OSO, polarized spectra were taken by the single crystals as depicted in the top right in (a).

View larger version (67K): [in this window] [in a new window]   Figure 10. The (CH2)o and (CD2)s bands of deuterated (a) SOS and (b) OSO. The spectra of each polymorph were measured in the same conditions (temperature and polarization) as stated in Figure 9.

SOS. In the form, a single (CH₂) band appeared at 1467 cm^-1, indicating the hexagonal subcell (Figure 9a). The (CH₂) band split into two components by cooling, and the splitting width increased as temperature decreased. At 87K the two components were observed at 1474 and 1463 cm^-1. In the form of d-SOS, a single band was observed at 1467 cm^-1 for (CH₂)o and at 1089 cm^-1 for (CD₂)s (Figure 10a), corresponding to the hexagonal subcell (34). On the transformation from to sub , the (CD₂)s band split into two components at 1093 and 1084 cm^-1, while no marked changes occurred in the (CH₂)o band. The splitting of the (CD₂)s band can be attributed to the interchain vibrational coupling between laterally adjoining perdeuterated chains: the in-phase and the out-of-phase vibrations of the two chains (59). Such a splitting of the (CH₂) band has been observed in many systems of long-chain molecules where hydrocarbon chains form a perpendicular type subcell to a certain extent. We inferred that the orientational changes of stearoyl chains containing somewhat conformational disorders from the hexagonal to the pseudohexagonal packing take place on the –sub transition.

The previous study by powder X-ray diffraction method revealed that the phase takes a trilayer structure (20), which means that the stearoyl and oleoyl moieties make their own leaflet. The (CH₂) band had two components (Figure 9a) for h-SOS; one strong band at 1472 cm^-1 and another weak band at 1467 cm^-1. Because the former band disappeared in the polarized spectra of d-SOS, we assigned this band to the stearoyl scissoring mode and the latter band to the oleoyl scissoring mode (Figure 10a). A sharp single band of (CD₂)s appeared at 1092 cm^-1, while a broad band of (CH₂)o was observed at 1468 cm^-1. It is suggested that the stearoyl groups form a parallel packing and the oleoyl moiety packs in the hexagonal subcell (60).

In the ß' form, the (CH₂) band had two components, 1473 and 1465 cm^-1 (Figure 9a) and the (CD₂)s mode split into two components at 1091 and 1088 cm^-1 (Figure 10a). Although the polarization directions of the (CD₂)s bands were not clear due to the overlapping of other modes, they showed the following characteristics: the 1092 and 1086 cm^-1 became maximum at the 0' and 90' polarization directions, respectively. These spectral data indicate the O subcell of the stearoyl leaflets (60). This splitting width of the (CD₂)s bands (6.5 cm^-1) was smaller than that of the typical O subcell (7.8 cm^-1) of deuterated n-alkanes (61), suggesting that the packing of the stearoyl chains is not so tight as that of n -alkanes. The glycerol backbone probably hinders stearoyl groups being packed tightly. As to the oleoyl leaflet, the (CH₂)o mode became slightly sharper in ß' than in , but its frequency was still 1468 cm^-1, suggesting a lateral packing similar to the hexagonal subcell in .

In the ß form of h-SOS, the single (CH₂) band appeared at 1470 cm^-1 with a clear polarization nearly parallel to the b-axis (Figure 9a); maximum at = 70° and minimum at = 160°. The (CH₂)o and (CD₂)s bands of d-SOS were also observed as single bands at 1471 and 1092 cm^-1 with a clear polarization (Figure 9a). The (CH₂)o bands appeared at a higher frequency than in the ß' form. The results suggested that both the stearoyl and oleoyl moieties were packed in T// subcell (34).

OSO. In the forms of h-OSO and d-OSO, single methylene scissoring bands appeared as follows: the (CH₂) band of h-OSO at 1467 cm^-1 and the (CH₂)o and (CD₂)s bands of d-OSO at 1467 and 1089 cm^-1 (Figure 9b, Figure 10b), showing the hexagonal packing of acyl chains. In the sub form of h-OSO, three components of the (CH₂) modes were observed: 1474, 1467, and 1463 cm^-1 (Figure 9b). The comparison of these bands with those of d-OSO sub showed that two outer bands at 1474 and 1463 cm^-1 are the scissoring doublet arising from pseudohexagonal packing of stearoyl chains and the central band at 1467 cm^-1 is of the hexagonal packing of oleoyl chains. We concluded that the –sub transition of OSO is associated with the orientational changes of the stearoyl chains from the hexagonal to the pseudohexagonal packing as that of SOS.

Contrary to the trilayer structure of SOS ß', OSO ß' takes the bilayer structure (22). In Figure 9b, the (CH₂) modes of h-OSO ß' had two components: a main component of 1473 cm^-1 and a shoulder of 1464 cm^-1. This asymmetric doublet shows the existence of the O packing, supporting the result of the powder XRD measurement (22). On the other hand, both the (CH₂)o and (CD₂)s modes in the deuterated specimen appeared as single bands at 1470 and 1090 cm^-1, indicating a parallel-type subcell packing (Figure 10b). In the deuterated specimen, the interchain vibrational coupling between laterally adjoining stearoyl and oleoyl chains can be neglected due to the mass difference. Therefore, the above results suggest the following features: 1) the hydrocarbon skeletal planes of the neighboring stearoyl and oleoyl chains are oriented perpendicularly, but 2) the skeletal planes of the nearest neighboring stearoyl chains are located parallel to each other and those of the neighboring oleoyl chains are also parallel between them. The details will be discussed later.

In the ß form, the methylene scissoring modes showed the same clear polarization and positions of bands as those of SOS ß. It suggested that both the stearoyl and oleoyl moieties pack in T// subcell (60).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reversible –sub transition
In the SOS bilayer structure, the oleoyl chain is surrounded by the stearoyl chains. These two chains pack together to form the hexagonal subcell in the phase as illustrated in Figure 11a. As for the molecular structure of SOS , the previous ^13C CP/MAS–NMR studies indicated the following two properties: 1) in the oleoyl chains two carbons of the cis double bond are equivalent and 2) the methyl end packing of stearoyl and oleoyl chains in a molecule are identical (62). On the basis of the assignments of the CH₂ progression bands together with the above results, we infer the molecular structure of as follows: the stearoyl chains mostly form the extended conformation but contain conformational disorder at their methyl terminals, whereas the oleoyl chain contains disordered conformation with high concentration. The thermal fluctuation of the disordered portions in is reduced with a decrease in temperature. Consequently, the orientational anisotropy of the neighboring stearoyl chains occurs at the sub transition due to the thermal contraction. By contrast, the oleoyl chain in sub maintains the disordered orientation of the skeletal planes even at low temperatures. As marked differences were not observed between the and sub spectra except for the methylene scissoring modes of the stearoyl chains, we conclude that the –sub transition corresponds only to the orientational changes of the stearoyl chains.

View larger version (28K): [in this window] [in a new window]   Figure 11. Schematic representations of the sub transitions in SOS (a) and OSO (b) and the acyl chain arrangement of OSO ß' (c). Left side reveals the bilayer structure in which the stearoyl and oleoyl chains are indicated by the solid and dotted lines, respectively. Right side shows the chain arrangement in lamella. Open circles and closed circles show the stearoyl and oleoyl chain positions in the XY plane.

In the OSO bilayer structure, each stearoyl chain is surrounded by the oleoyl chains (Figure 11b). As stated previously, the molecular structure of stearoyl and oleoyl chains in and sub of OSO is basically the same as those of SOS. Although the intermolecular interaction between the nearest neighboring stearoyl chains is weak, the same orientational anisotropy of the nearest stearoyl chains as that of SOS occurred on the sub transition of OSO.

The reversible transitions similar to the –sub transitions in SOS and OSO have been observed in other long-chain molecules; for example, Rotator I–Rotator II transition in n-alkanes (63) (64) (65). These transitions are caused by the orientational changes of the neighboring hydrocarbon chains with low energy of activation. Compared with through ß transitions in SOS and OSO having the monotropic nature (66), the –sub transitions take place reversibly, as well as the Rotator II (hexagonal)–Rotator I (pseudohexagonal) transition of n -alkanes. Such enantiotropic transitions observed in the higher entropy phases may be due to the characteristics common to the highly disordered hydrocarbon chain assemblies. The orientational ordering of the stearoyl chains with decreasing temperature in the sub phases of SOS and OSO results in the reduction of the entropy factor, and thereby the enantiotropic transformation occurs between and sub .

Another interesting phenomenon from the biological view point is that the fluctuation of the oleoyl chains is retained even at lower temperatures due to the steric hindrance of the olefinic bond. This must be an important character of unsaturated acyl moieties, to maintain the fluidity in biomembrane lipids.

Irreversible transition from through ß forms
The layered structures and the subcell packings of the stearoyl and oleoyl moieties in each polymorphic form of SOS and OSO were summarized in Table 1.

In SOS, the to transition is initiated by the stabilization of the stearoyl moieties. Correspondingly, the form having bilayer structure transforms to the form having trilayer structure. The molecular conformation of the stearoyl chains in takes the same ordered conformation as those of more stable ß' and ß forms in which one chain extends to form the all-trans conformation, whereas the other takes a bent conformation in the neighborhood of the ester bond. These stearoyl chains take a parallel type subcell in and change to O subcell in ß', but the oleoyl leaflet maintains the hexagonal packing in and ß'. Although the subcell structure is the same in and ß', the molecular conformations of the oleoyl chain are different, judging from the spectral features of the oleoyl progression bands in the 1350–1150 cm^-1 region (Figure 6). In addition, the NMR studies showed that the methyl-side and the glycerol-side of the oleoyl chain in SOS are not conformationally equivalent in and ß' unlike in (62). We infer that the gauche bonds in the oleoyl chain are concentrated in the vicinity of the methyl-side chain in the and ß' phases and are relatively delocated in the and sub phases. The structural difference of the oleoyl chain between and ß' is probably due to the difference in the concentration of gauche bonds. Finally, the ß'to ß transition corresponds to the stabilization of the oleoyl leaflet where the methyl side-chain and glycerol side-chain take an extended structure with the skew - cis - skew' olefinic conformation (34).

In OSO, the to ß' transition corresponds to the stabilization of the stearoyl moiety. Unlike SOS, it was not accompanied by the conversion of the layered structure. As the OSO molecule contains one stearoyl chain, the steric hindrance between the stearoyl and oleoyl chains by the ordering of the stearoyl moiety may not be significant at the initial stage of the phase transition. In the ß' phase, the stearoyl chain takes the all-trans conformation, whereas the oleoyl chains contain some disordered portions. The sharpened oleoyl bands on the to ß' transition (Figure 8) showed the progress of the conformational ordering in the oleoyl moieties. On the other hand, unclear methyl rocking bands (r(CH₃)o, usually observed in 950–850 cm^-1) suggest the conformational disorder in the methyl end group. We infer that the oleoyl chain conformation in OSO ß' is similar to that in SOS ß': the glycerol side-chain takes an extended conformation, but the methyl side-chain contains a certain disorder. Figure 11c is the schematic representation of the subcell packing of OSO ß'. The skeletal zigzag planes of the neighboring stearoyl and oleoyl chains are oriented perpendicularly. On the other hand, the zigzag planes of the nearest neighboring stearoyl chains and those of the neighboring oleoyl chains are located parallel to each other. The unclear doublet of the (CH₂) modes (Figure 9b) compared with that of SOS ß' (Figure 9a) can be explained by the above subcell structure due to the following two points.

First, when the perpendicular and parallel packings coexist in the crystal unit as depicted in Figure 11c, the former contributes to the band intensity at 1473 and 1464 cm^-1 and the latter at 1473 cm^-1. Therefore, the higher frequency component becomes stronger than the lower one.

Second, when the all-trans stearoyl chains and the partially disordered oleoyl chains are adjacent to each other, it is difficult to construct a tightly packed subcell.

As the acyl chains tightly pack together, the effect of the steric hindrance between the stearoyl and oleoyl chains becomes large; it may result in the conversion of the layered structure from bilayer to trilayer. The final conformation on the polymorphism is basically the same as that of SOS; the ß phase forms the trilayer structure where the stearoyl and oleoyl chains make their own leaflet as illustrated in Figure 1b.

The present findings provide molecular level clarification of steric hindrance between the saturated and unsaturated acyl chain in fats and lipids having layered structures. This steric hindrance results in the occurrence of various metastable phases and complicated polymorphic transformations. In the TAG polymorphism having the monotropic characteristics, the transition from the least stable form through the most stable one can be regarded as a process of the crystal growth in solid state. The saturated acyl chains transform to a stable molecular conformation at the first stage of polymorphism and then the unsaturated moieties are stabilized successively with the changes of the subcell structure. The interfacial energy of the stable polymorph is larger than those of the least stable or metastable forms. Namely, the crystallization rates are always higher in the less stable forms and the formation of the stable molecular conformation from melt is sterically hindered.

Abbreviations: TAG, triacylglycerol; SOS, sn -1,3-distearoyl-2-oleoylglycerol; OSO, sn -1,3-dioleoyl-2-stearoylglycerol; SSS, tristearoylglycerol (C18:0); FT-IR, Fourier transform infrared spectroscopy; ATR, attenuated total reflection; RAS, reflection absorption spectroscopy

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