417

Diversity in the fatty-acid conformation and chain packing of cisunsaturated lipids Fumitoshi Kaneko*, Junko YanotJ; and Kiyotaka Satot
Recent X-ray diffraction and Fourier transform IR spectroscopic studies have unveiled a great diversity in the molecular conformation and chain packing of lipid molecules containing cis-unsaturated fatty-acid chains. Specifically, a dramatic diversity in the olefinic conformation, subcell packing and chain-chain interactions has been clearly revealed by crystal structures of principal cis-monounsaturated fatty acids. The structural diversity is most manifest in oleic acid crystals. These findings were applied to analyses of the complicated structural transformations of diacylglycerols and triacylglycerols containing oleic acid and saturated acid moieties, in which the stabilization of the oleic acid causes the complexity of the transformation and mixing behavior. Although this knowledge has been obtained in the crystalline state, one may assume that the structural diversity of these unsaturated molecules plays a similar role in the lipids of biomembranes, lipoproteins and lipid deposits in which aliphatic chain packing is a critical problem, since some lipid domains can undergo liquid to solid or solid to liquid alterations.
Addresses

T
TAG

Tm
XRD Introduction

trans triacylglycerol melting temperature X-ray diffraction

*Department of Polymer Science, Graduate School of Science, Osaka University,Toyonaka,560-0043, Japan;e-rnail:toshi@chem.sci.osaka-u.ac.jp tFaculty of Applied Biological Science, Hiroshima University,HigashiHiroshima, 739-8528, Japan ~e-mail:jyano@ipc.hiroshima-u.ac.jp ~e-mail: kyosato@ipc.hiroshima-u.ac.jp
Current Opinion in Structural Biology 1998, 8:417-425

http://biomednet.com/elecref/O959440XO0800417 Current Biology Publications ISSN 0959-440X

Abbreviations APA

C
DAG DSC ELA ERA

FT
GOA HM LM MOA OA OPO OSO

PC
PE POA POP POS PPO PSA S

sn-I,2-SLDG sn-I,2-SODG
SOS SR

asclepic acid (cis-7-octadecenoic acid) cis diacylglycerol differential scanning calorimetry elaidic acid (trans-9-octadecenoic acid) erucic acid (cis-13-docosenoic acid) Fourier transform gondoic acid (cis-11 -eicosenoic acid high melting low melting myristoleic acid oleic acid (cis-9-octadecenoic acid) 1,3-dioleoyl-2-palmitoyl-sn-glycerol 1,3-dioleoyl-2-stearoyl-sn-glycerol phosphatidylcholine phosphatidylethanolamine palmitoleic acid 1,3-dipalmitoyl-2-oleoyFsn-glycerol 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol 1,2-dipalmitoyl-3-oleoyl-rac-glycerol petroselinic acid skew 1-stearoyl-2-1inoleoyl-sn-glycerol 1-stearoyF2-oleoyl-sn-glycerol 1,3-stearoyl-2-dioleoyl-sn-glycerol synchrotron radiation

Unsaturated fatty acids are very important lipid molecules that play critical roles in the functional activities of biological organisms [1]. T h e y comprise about one half of all the acyl chains in biomembrane phospholipids, promoting the fluidity and permeability of the membrane through the conformational flexibility of its acyl chains. T h e major factors that influence the physical and chemical properties of unsaturated fats and lipids are the number, position and configuration of the double bonds [2]. Hence, it is highly important to elucidate a molecular-level understanding of the structure/function relationships of the unsaturated fatty acids that are present in membrane lipids, fats and other biotissues. Studies on the molecular ordering and chain packing of unsaturated fatty-acid segments that are covalently linked to the same molecule have important relevance to the molecular-level understanding of the polymorphic transformation of gel to liquid-crystalline lipid phases [2]. It is noteworthy that combinations of fatty-acid moieties in natural lipids are heterogeneous, for example, saturated and unsaturated acids, unsaturated fatty acids with cis-double bonds placed at different positions, short and long chain acids, even and odd numbered carbon fatty acids and so on. Of particular interest are the interactions between saturated and unsaturated fatty acids when they are forced to pack together in the same lamellae leaflet. T h e y pack together by virtue of the interactions between their polar head groups, as observed in many biomembrane phospholipids. In case of membrane phospholipids, for example, two acyl chains are connected to the polar head group, although the saturated fatty acids are bound to stereospccifically numbered-1 (sn-1) glycerol carbon positions whereas the unsaturated acids are bound to sH-2 carbon positions. T h e same situation applies to the oils and fats that are present in plant and fish tissues, in which the three acyl chains connected to the glycerol group are a mixture of unsaturated and saturated fatty acids. One may call them mixed-acid saturated-unsaturated fats and lipids. In mixed-acid fats and lipids, varying interactions between saturated and unsaturated fatty-acid chains influence the structure and stability of the crystalline and liquid-crystalline phases [3]. rio this end, thermal analyses, using a highly sensitive differential scanning calorimetry (DSC) technique, have been conducted on the gel to liquid-crystalline phase transitions of phosphatidylcholine (PC) [4] and phosphatidylethanolamine (PE) [5]; phospholipids that contain sn-I saturated and sn-2 cis-unsaturated fatty-acid chains whose cis-double-bond positions had

418 Lipids

been systematically changed. It was obvious that, both in PC and PE, the transition temperatures were most lowered when the c/s-double bonds were placed in the central portion of the aliphatic chains. There was also a subtle but distinct deviation in the lowering of the transition temperatures between mixed-acid PCs [4] and dimonounsaturated PCs [6]. The latter result indicates the importance of interactions between saturated and unsaturated fatty-acid chains packed in the same leaflet of lamellar phases. A computer simulation of the molecular conformations and packing of a series of sn-I saturated and sit-2 unsaturated mixed-acid diacylglvcerols (DAGs) [7,8] assumed that maximal interactions of the oleic acid (OA) moieties in adjacent rows of molecules are allowed, and the olefinic conformation may be greatly distorted from that previously reported for OA crystals (see below). Despite these quite interesting indications, no precise structural data from single crystal X-ray diffraction (XRD) techniques are available for fats and lipids containing unsaturated fatty-acid moieties, although much data have been accumulated for saturated acid membrane lipids [9,10]. The major reason for this lack of data can be ascribed to difficulties in obtaining single crystals of fats and lipids that contain unsaturated fatty acids whose crystallinity is suitable fi)r XRI) analysis. Progress has, however, recently been made in precisely analyzing the conformational ordering and chain packing of principal ds-monounsaturated fatty acids [11-23,24",25], a saturated-unsaturated mixed-acid DAG [26-28] and cisunsaturated-saturated mixed-acid triacylglycerols (TAGs) [29-34,35",36-38] in single and mixed states. The combined use of single crystal or powder XRD, DSC and NMR and Fourier transform (FT) IR spectroscopy with multiple techniques [391 have enabled the unveiling of the molecular structures of these fatty acids, both in metastable and in the most stable phases in polymorphic systems. This article attempts to review these new findings.

addition to the above four specific subcells, there is hexagonal (H) chain packing, in which the polymethylene chains do not reveal a specific orientation. Instead, they undergo a large amplitude torsional motion with an aliphatic gaurhe conformation, making the H subcell less stable compared to the other four. The molecular conformation is rclevant to the polymethylene chain, olefinic group and glycerol group, which are each influenced by the chemical structure of the fatty-acid moieties, as well as the external thermodynamic conditions. As for the polymethylene chains, o'alls conformers form rigid and stable polymethylene chains whereas gauche conformers form random and less stable conformations. It must be noted here that the concentration of zauoOe conformers is raised in some c/s-unsaturated fatty acids, in particular in the polymethylene-chain segment between the c/s-double bond and the methyl-end group (m chain), keeping the chain segment between the double bond and the polar group (A chain) all-trans. As for the olefinic group, it has two stable conformations - - skeee-cis-ske~oe, (S-C-S) and ske~e,-ciss k ~ ' (S-C-S') - - as shown in Figure lb. Although these two conformations cause the bent geometry of the polymethylcne chain, there are clearly different forms whereby the c0 and A chains are placed in the same plane for the S-C-S' conformation and the two chains are normal to each other in the S-C-S conformation. A third olefinic conformation, trans-cis-trans (T-C-T), is quite unique in that the t~zms conformation is revealed to be close to the ds double bond. T h e chain-length structure comprises a repetitive sequence of the acyl chains involved in unit lamellae along the long-chain axis (as shown in Figure lc). A double chain-length structure is formed when the chemical natures of the three acyl moieties are the same or very similar. On the contrary, when the chemical nature of one or two of the three acyl chains is largely different from the others, such as in saturated-unsaturatcd mixedacid fats and lipids, a triple-chain length structure forms as a result of chain sorting. An interdigitated chainlength structure also exists, in which a partial interdigitation of the chain is terminated within the lamellar interior, making a saw-tooth chain-packing arrangement. Apparently, this structure seems to be unstable because of the presence of voids in the terminal methyl-end packing. This instability is, however, compensated by other factors through polar interactions, as observed in several membrane lipids that have very spacious packing areas in their head groups [10].

Molecular-chain packing, conformation and chain-length structure

'Fo begin with, it is necessary to note the molecular packing, conformation and chain-length structure of these fatty acids [2,3,40]. These are the main factors that characterize the diversity of the molecular structures of unsaturated lipids. Molecular-chain packing is revealed by the subcell structure - - defined as the cross-sectional packing mode of the zigzag aliphatic chain. Ten types of subcell have been revealed in crystalline fats and lipids [2], among which the five subcells shown in Figure la were found to be most predominant in unsaturated lipids. In "|'//, all the polymethylene zigzag planes are parallel and this is thought to be the most dense packing. The Oz subcell consists of zigzag chains that are perpendicular to the planes of its neighbors. 'l\vo subcells, ()}/ and M//, contain zigzag aliphatic chains arranged in a parallel manner in orthorhombic and monoclinic systems, respectively. In

Structures and t r a n s f o r m a t i o n s of m o n o u n s a t u r a t e d fatty acids

The polymorphisra of a series of rnunounsaturated fatty acids, mostly cis types and a single o'ans type (elaidic acid, El,A), has been summarized in Table 1. "l\vo phases, called c( and 7, are commonly present in OA [11,12], erucic acid (ERA) I131, asclepic acid (APA) [1.Sl,

Conformation and chain packing of cis-unsaturated lipids Kaneko, Yano and Sato

419

Figure 1

Subcell .~11 /

7 . H T// M// Oz 0///

Olefinic conformation

i Chain ii length

(b)

ske w-cis-ske w

skew-cis -skew

trans -cis-trans

(S-C-S')
(c)

(S-C-S)

(T-C-T)

\
Double Triple

\ \
Interdigitated
Current Opinion in Structural Biology

Representation of the (a) subcell structures, (b) olefinic conformations and (c) chain-length structures of fatty acids. These were each confirmed in crystalline phases of cis-monounsaturated fatty acids and in triacylglycerols containing oleic acid moieties.

palmitoleic acid (POA) [21] and gondoic acid (GOA) [25]. T h e ot and g phases exhibit reversible transformation, whereby the aliphatic conformation of the m chain transforms from ordered (y) to disordered (o0 upon heating [12]. Yet, the ordered conformation of the A chain does not change during transformation. Another reversible transition exists between oq and gl in ERA, whereby the m chain changes its inclination angles with respect to the lamella interface [22]. T h e s e reversible transitions, accompanied by the displacement of the m chain, are characteristic of cis- unsaturated fatty acids. The significance of the ds-double bond with respect to the occurrence of these transformations may be to divide the m chain and zXchain into two portions. The m chain is disordered in the c~ form and displaced in the oq form as a result of the thermal energy excitation of heating, whereas the A chain is unchanged because of interactions between carboxylic acid groups in dimeric units. The [3 form polymorphs of OA [11], and the low melting (I,M) and high melting (HM) forms of petroselinic acid (PSA) [14] do not exhibit such reversible solid-state phase transitions, most probably as a result of stronger chain packing compared to polymorphs exhibiting g-->cztransitions.

The diversity in cis-olefinic conformations is revealed by the general tendency of C-C bonds that arc adjacent to C=C bonds to have a stable ske-a;'(S = 120 ) or skea~" torsion angle (S'=-120), whereas its bond rotational energy changes slowly by internal rotation. This makes it possible for the olefin group to adopt various conformations that are deviated from the standard S-C-S or S-C-S' conformation. Indeed, the observed torsion angles are distributed in the region from 91 to 175 , which results in the various molecular conformations and subcell structures. For instance, in the case of the S-C-S' conformation, the A and m chains are located on the same side of the C - C = C - C plane and the molecule as a whole adopts a planar configuration. In the case of the S-C-S conformation, the two chains are located on opposite sides of the C - C = C - C plane and the molecule has a twisted configuration, rl'herefore, S-C-S-type molecules do not form high symmetry suhcells and instead form T//subcells (gl and ez1 phases seen in ERA [22,23]). This is contrary to the high symmetry O~/subcell of S-C-S'-type molecules (g phase). By altering the C-C torsion angles towards aorms conformation, the O~/subcell of the S-C-S'type molecule changes into O, the high symmetry subcell, as revealed by the LM form of PSA shown in

420

Lipids

Figure 2 (a)

c/2

c/2

independent molecules per asymmetric unit, the A and m chains form O_t-like and M//subcells, respectively [20]. During the ~'--+0~transition [23], only the m chain alters its subcell structure, from O)/to QL-like. T h e [31 form of OA is of particular interest in that it belongs to a triclinic system containing two independent molecules per asymmetric unit. T h e molecular layer has a unique interdigitated structure in which the methyl and carboxyl terminals together make up the lamellar interface, as shown in Figure 2b [24"]. T h e local conformation of the C - C = C - C portion is a trans-cis-trans ('F-C-T) type for both molecules and the acyl chains form a TII subcell. T h e interdigitated chain-length structure and the T - C - T olefinic conformation of the 131 form of OA, which were confirmed in cis-monounsaturated fatty acids for the first time [2], are the cause of the abnormally low crystal growth rate of the 131 form, it is lower than other forms by a factor of 10-3-10 -~ [24"]. This indicates that the formation of this unique structure may encounter an enormously high activation barrier energy for nucleation and crystal growth. As for trans-type unsaturated fatty acids, only one substance, ELA [41], has been examined due to the rare availability of trans unsaturated fatty acids. No general tendencies can therefore be drawn for trans acids in comparison with c/s-unsaturated acids. T h e overall structural behavior of ELA has no similarity to that of the c/s-acids. Instead, it has many similarities, in terms of subcell structure and the occurrence of polytypic structures, with saturated fatty acids [42]. T h e binary-mixture systems of OA-PSA [161 and OA-APA [16] revealed eutectic phase behavior, but OA-POA and OA-MOA (myristoleic acid) systems showed the formation of molecular compounds [171. Interestingly, a system exhibiting a miscible solid-solution phase has been observed in OA-GOA mixtures in which the two component fatty acids have the same length k chains and also exhibit a 7--+ot transition [251.

(b)
a b c

m chain

A chain

a CurrentOpinion
in

StructuralBiology

Two crystal structures of cis-monounsaturated fatty acids, partly demonstrating how the variation in chain packing, olefinic conformation and chain-length structure is influenced by changing the cis double bond position in the whole acid chain; a, b and c are the unit cell axes. (a) The HM (high melting) phase of petroselinic acid [20], in which two crystallographically independent molecules, A and B, are isolated in an asymmetric unit. The dihedral angles of the C - C bonds next to the cis C=C bond are 91 o and 130 for molecule A and 137 and 119 for molecule B. With these different conformations of cis-olefin group, the two types of molecules form a M//subcell in the methyl-sided chain segment and an O-Iike subcell in the carboxyl-sided chain segment. (b) The [}1 phase of oleic acid [24], in which the asymmetric unit contains two crystallographically different molecules, A and B. The dihedral angles of C - C bonds next to cis C=C bonds are 174 and 1?3 for molecule A and 175 and 175 for molecule B. The molecular layers form a unique interdigitated structure, in which methyl and carboxyl terminals together make up a lamellar interface. Polymethylene chains form two types of T//subcell, which are oriented in directions different from each other.

Structures and transformations of mixed-acid diacylglycerols

Quite interesting properties were unveiled for saturatedunsaturated mixed-acid D A G s l-stearoyl-2-oleoyl-snglycerol (sn-l,2-SODGs) [26], 1-stearoyl-2-1inoleoyl-sn-glycerols (sn-I,2-SLDGs) [27] and sn-I,3-SODG [28]. T h e first may mimic membrane lipids, since the esterification of itss phosphorus radical groups, for example, the sn-3 glycerol position of sn-I,2-SODG, produces phospholipids. Single crystal XRD analysis of sn-I,3-SODG has shown that the stearoyl and oleoyl chains are stacked in respective leaflets of the double chain-length structure, in which the T//subcell formed in the two leaflets [28]. Of particular interest is the combination of the "Pf/subcell of the oleoyl chains and the trans-ske~,-cis-s~'ew-sbea>trans (T-S-C-S-S-'F) olefinic conformation. Although they are not fully identical, this feature shows that there are some similaritities between

Figure 2a [19]. Depending on the flexibility of the olefinic group, it is possible for the A and co chains to make different types of subcells. In a HM phase that contains two

Conformation and chain packing of c/s-unsaturated lipids Kaneko, Yano and Sato

421

Table 1 Molecular properties of polymorphism in principal c/s-monounsaturated fatty acids and elaidic acid.
Fatty acid Form Space group P21/a P21/a P21/a P2 l/a P21/a P21/a Unclear PT Pbca PT P21/a P21/a PT PT A2/a Unclear Olefinic conformation* S-C-S' S-C-T S-C-S" S-C-T 133 , cis, -133 S-C-S' S-C-T Unclear A: 174 , c/s, 173 T-C-T B: 175 , cis, 175 T-C-T 157 , cis, -160 A: 91 o, cis, 137 S-C-S B: 130 , cis, 119 S-C-S 129 , cis, -128 S-C-S' 105 , cis, -172 S-C-T 98 , cis, 95 S-C-S 96 , cis, 102 o S-C-S 118 , trans, -118 S-T-S' Unclear Subcell Form O'// O'//+ O.L-like O'// O'//+ OL-like O'// O'//+ O.L-like //type T// O O-Iike + M// O'// O'//+ O-Iike T// T// O O + / / t y p e -->o~ melting --+o~ melting --mr melting melting melting meltingt melting ->c~ -->cq -~(z 1 melting melting melting Phase transition References Ttr(C) ~ AHtr (kJ/mol) -18.4 2.0 -15.4 13.8 -2.2 13,3 16.0 16.3 7.5 32.1 7.8 39.8 8.8 39.6 48.9 57.9 [21,22] [15] [11,12] [24 ']

Palmitoleic acid (POA) Asclepic acid (APA) Oleic acid (OA)

y (z g (z y o~ [8~ 131 LM HM y (z Y1 c(1 LM HM

Petroselinic acid (PSA)

[14,19,20] 30.5 -1.0 31.2 9.0 34.0 42.8 42.3 47.5 8.8 5.4 8.9 54.0 [13,18,22,23]

Erucic acid (ERA)

Elaidic acid (ELA)

[40]

*S-C-S', skew-cis-skew'; S-C-S, skew-cis-skew; T-C-T, trans-cis-trans; S-C-T, skew-cis-trans. A and B are two asymmetric unit molecules. 1Meltmediated transformation to HM form. *Transition temperature. Transition enthalpy.

the molecular structures of the oleic acid leaflet ofsn-l,3SOD(; and the most stable J31 form of oleic acid, described above. For the oleoyl chains, the closest chain packing expressed in the T// subcell is formed together with the occurrence of the olefinic trans-conformation. In contrast, powder XRD and DSC studies on sn-I,2-SODG in a dry state isolated eight polymorphic forms, in which the subcell structures were converted to o~ from H, [3 from T//and J3' from O, in accordance with structural stabilization after the formation of the c~ form by chilling the neat liquid. Interestingly, the most stable form ofsn-!,2-SODG is [3', in contrast to TAGs, whose [3 form with the q'//subcell is most stable. This means that some peculiar molecular interactions are operating in sJl-I,2-SODG, whereby the oleoyl and stearoyl chains lie in the same leaflet. Hydration appears to partly minimize the diversity of molecular interactions, since the number of individual polymorphic forms is reduced to three water-associated forms (oqv, [3w and Yw). This again indicates that the diversity of the molecularchain packing is at its greatest when the constraint from the polar glycerol head group is intensified by dehydration. For a full understanding of this feature, more detailed structural analysis on s/t-I,2-SODG is necessary.
Structures, transformations and the mixing behavior of mixed-acid triacylglycerols

For this purpose, it is necessary to observe the sequential transformation from the least to the most stable polymorphic form through multiple metastable forms. T h e subcell packing, chain-length structure and aliphatic chain conformation of the saturated as well as the unsaturated moieties are converted in a synchronous manner. Traditional single crystal X R D does not provide satisfactory data on these sequential transformations, since single crystals are rarely available for the least stable and metastable forms. FT-IR spectroscopy with microprobe polarized, reflection-absorption spectra (RAS), attenuated-total reflection (A'I'R) techniques has provided fruitful information. It employs IR bands characteristic of molecular segments that are either C H z scissoring, C H z rocking, polymethylene progression bands containing
Table 2

Vibrational modes that are specifically sensitive to the conformation and chain packing of polymethylene chains.
Functional Description of vibration group (-OH2-) n CH 2 scissoring CH 2 rocking CH 2 wagging progression CH 2 twisting-rocking progression CH 3 asymmetric stretching CH 3 rocking C=C stretching =CH stretching =CH out of plane bending Symbol Wave number (cm-1) - 1470 - 720 1150-1360 1150-1300 -2960 -900 1600-1680 ~3010 650-750

5(CH 2) r(CH 2) w(CH 2) t(CH 2) Vas(CH3) r(OH 3) v(C=C) vas(=CH) y (=CH)

T h e critical relevance of the study of satt, rated-unsaturated mixed-acid TAGs, apart from implications for the food, pharmaceutical and cosmetic industries, to lipid structural biology is based on the fact that complicated molecular interactions are revealed by polymorphic behavior in single as well as in binary-mixture phases.

-C-OH 3 -HC=CH-

422

Lipids

Figure 3

lY, ~2,13,
(a)

~
(b)

[T m] = 23.5C), to the 131 form (Tm = 43.0C) through the ~, (Tm = 35.4C), 13' (Tm = 36.5C) and [32 (Tin =41"0(:) forms. This indicates that the chain-length structure converted from a double (o0 to a triple (~,, 13',13eand 131)form and the subcell structures changed in different manners between the oleic and stearic acid moieties [30,33]. This complicated transformation behavior is caused by the steric hindrance of the stearic and oleic acid chains and also the structural stabilization of the aliphatic-chain packing, methyl-end packing and glycerol groups. Although some differences were seen, 1,3-dipalmitoyl-2-sn-oleoylglycerol (POP) and -palmitoyl-2-oleoyl-3-stearoyl-lzlc-glycerol (POS) revealed the same feature [30,40]. T h e details of the conversion are described below.
form

()

T h e double chain-length structure of the (~ form, verified by a lamellar distance of 4.83 nm as observed by powder XRD long-spacing, indicates the co-existence of the stearoyl and oleoyl moieties in the same leaflet. T h e hexagonal subcell was shown in a single XRD short-spacing spectrum (0.42 nm) and also in single F T I R bands of 8(CH 2) and r(CHz) modes [33]. No specific structure was shown for the olefinic conformation, since no detectable IR band of "y(=CH) was seen and the two carbons adjacent to the cisdouble bonds were equivalent in the N M R spectra [34].
POP-OPO and SOS-OSO compounds POP-PPO and SOS-SSO compounds
Current Opinion in Structural Biology

Structural models of (a) a polymorphic transformation of SOS, (b) a polymorphic transformation of OSO and (c) molecular compounds.

C H z wagging, or a combination mode of C H z twisting, CH z rocking and =CH out of plane bending (Pable 2). In particular, polymethylene progression bands can be employed as reference spectra for assessing the conformation of the awl-chain moieties that arc connected to the different sn-positions of the glycerol carbons, as exemplified in symmetric, saturated mixed-acid TAGs [43",44]. Furthermore, an isotope FT-IR method, in which partial deuteration is performed on either fattyacid moiety, provides the most informative data on chain-chain interactions, as demonstrated for the gel phases of phospholipids [45] and also for 1,3-stearoyl-2dioleoyl-sn-glycerol (SOS) and 1,3-dioleoyl-2-stearoylsn-glycerol (OSO) (J Yano, unpublished data). In addition, time-resolved synchrotron radiation X R D (SRXRD), DSC and solid-state cross polarization and magic angle spinning N M R methods were also quite informative.

~,form T h e long-spacing value of 7.05 nm represents the triple chain-length structure, in which the oleoyl and stearoyl leaflets were separated as a result of chain sorting during the ct--)~' transformation. T h e subcell structures can therefore be different in each leaflet. T h e stearoyl leaflet assumed specific parallel packing and the nleoyl leaflets retained a hexagonal subcell structure. This was verified by FT-IR 8(CD z) and ~i(CH z) bands of SOS containing fnlly deuterated stearoyl and hydrogenated oleoyl chains (hydrogenated SOS), respectively (J $:ano, unpublished data). T h e progression bands of hydrogenated SOS also showed that one stearoyl chain is extended and the other one is twisted. Although no detectable ~'(=CH) IR band was seen for the olefinic group, the two carbons adjacent to the ds-double bond were no longer equivalent in the NMR spectra, which further indicated that the three glycerol carbons are also not equivalent [34]. T h e increase in the longspacing value of each CH z unit, A1 = 0.255 nm/CH z, measured in POE SOS, 1,3-arachinoyl-2-dioleoyl-sn-glycerol (AOA) and 1,3-behenoyl-2-dioleoyl-sn-glycerol (BOB), indicates that the long chains are arranged almost normal to the lamellar interface [30].
[3' form

Single phases
Figure 3a illustrates the typical features of a polymorphic transformation of SOS from the a form (melting temperature

T h e long-spacing value of 7.00 nm represents the triple chain-length structure. T h e stearoyl leaflet assumed a O subcell structure and the hexagonal subcell structure was retained by the oleoyl leaflets, as shown by the FTIR 8(CD z) and 8(CH z) bands of hydrogenated SOS (J Yano, unpublished data). T h e progression bands of

Conformation and chain packing of cis-unsaturated lipids Kaneko, Yano and Sato

423

hydrogenated SOS also showed that one stearoyl chain is extended and the other one is twisted. T h e IR y(=CH) spectrum was seen as a broad band around 700 cm q [33]. T h e N M R spectra showed more clear differences between both the two carbons that are adjacent to the c/s-double bond and the three glycerol carbons [34]. For the 13"form, the long-spacing value ofAl = 0.242 nm/CH 2 assumes that the long chains are inclined against the lamellar interface at an angle of about 70 [30].
Two 18f o r m s

chains was revealed in smeared-out r(CH3)olcic bands and oleoyl progression bands in the region of 1350-1150 cm -1 of OIISl)O|t. Perpendicular-chain packing was observed between neighboring stearoyl and oleoyl chains, whereas the skeletal zigzag planes of neighboring stearoyl-stearoyl and olcoyl-oleoyl chains arc parallel each other, as revealed by 8(CH2) bands of all-hydrogenated OSO and (~(CH2)olcic and ~(CD2)stcaric bands of OHSDO H. Additional properties were obtained by the newest SRXRD study on SOS [35]. Two thermotropic liquid-crystalline phases were isolated during rapid chilling of liquid (19C/min) and rapid transformation (24C/min) through c~ melting (s-melt mediated transformation). Also, the occurrence of the subcell structure was always preceded by lamella formation, as revealed in time-resolved (10 s intervals) small and wide angle SR-XRD spectra.

T h e long-spacing values were 6.7.5 nm and 6.60 nm for the 13z and 131 forms, respectively, indicating a very inclined chain arrangement against the lamellae interface, compared to the other three forms. The subcell structure was T// for both the stearoyl and the oleoyl leaflets in 131, as revealed in polymethylene scissoring bands of all-hydrogenated SOS and also hydrogenated SOS (J Yano, unpublished data). The subcell of 132 is very similar to T//for the two leaflets, but very subtle differences, particularly in the polarization behavior, were detectable [33]. At present, no precise clarification has yet been carried out. The other properties of the two 13 forms seemed to be identical; for example, one stearoyl chain is extended and the other one is twisted (as revealed by the progression bands of hydrogenatcd SOS). The olefinic conformation of the S-C-S'type was shown by the y(=CH) band. The two carbons adjacent to the c/s-double bond were equivalent and the three glycerol carbons were all different [34]. The longspacing value of AI = 0.20 nm/CH 2 assumes that the long chains are inclined against the lamellar interface at about 52 [30]. It may be worthy to compare the results of OSO [29] with those of SOS, because the oleoyl and stearoyl chains are connected to the glycerol carbons in opposite ways in the two TAGs (Figure 3b). For the most stable 13forms of OSO and SOS, many important properties are quite similar, for example, the T//subcell structure, the S-C-S' olefinic conformation and the lamellar distance (6.5 nm) of the triple chain-length structure. The occurrence of the metastable forms are different, however, since no g form appeared in ()SO and the 13' form of OSO was a double chain-length structure [29]. This means that the manner of chain packing of SOS and OSO in their most stabilized structures is identical - - the stearoyl and oleoyl chains are packed in different leaflets to avoid steric hindrance and the most stable chain packing assumes thc T//subcell structure. In the metastable 13' forms, however, SOS 13' is a triple chainlength structure, whereas OSO [3' is a double chain-length structure. This is probably because the steric hindrance in OSO 13' is not substantial. FT-IR studies of the [3' form of all-hydrogenated OSO and partially deuterated OrtSI)O H unveiled the specific chain-chain interactions between the oleoyl and stearoyl acids in the same leaflets (J Yano, unpublished data). The all-trans conformation of the stearoyl chains were revealed by the w(CH z) progression bands. A partially disordered conformation for the oleoyl

Binary mixture phases

In general, three typical phases may occur in binary solid mixtures in which the two components are miscible in all proportions in a liquid state - - solid solution phase, the eutectic phase and compound formation. For TAG mixtures, phase behavior is rather complicated for two main reasons - - p o l y m o r p h i s m and the awl chain compositions attached to the glycerol group [2]. The structural behavior of binary mixtures of SOS-SSO, SOS-OSO, P O P - P P O (rac-l,2-dipalmitoyl-3-oleoylglycerol) and P O P - O P O (1,3dioleoyl-2-sn-oleoylglycerol) [31,32,36-38] each exhibited molecular-compound formation at a 50:50 concentration ratio, probably as a resuh of molecular interactions between oleoyl chains. Interestingly, the two types of molecular compounds both had a double chain-length structure, as illustrated in Figure 3c, despite the fact that the component materials were triple-chain length in their stable states, as described above. DSC studies showed that each molecular compound forms eutectic phases in a juxtaposed manner with the component materials. For the molecular compounds in P O P - O P O and SOS-OSO mixtures, complete chain segregation occurred between the saturated and oleic acid chains, whose 13 form exhibited a T// subcell structure and S-C-S" olefinic conformation [37,38]. The ]3 form of the molecular compound of POP-PPO, however, exhibited a nonspecific olefinic conformation (neither S-C-S' nor S-C-S) indicating that the oleoyl chains are distorted from those revealed in the compounds of P O P - O P O and SOS-OSO [36,38]. A SR-XRD study confirmed that the molecular compound of P O P - P P O revealed an o~--+13'--+13transformation in all the double chain-length structures [36]. It seems reasonable that the formation of the molecular compounds in SOS-OSO and POP-OPO occurs in terms of repulsive chain-chain interactions between the saturated and oleic acid chains, which were observed in mixed acid DAGs and TAGs in their single phases as reviewed above. The occurrence of molecular compounds with double-chain lengths in POP-PPO and SOS-SSO seems unrealistic. This

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Lipids

is partly because the stable forms of the component materials are all triple-chain length, but is mainly because repulsive chain-chain interactions between the saturated and oleic acid chains would also occur in these mixtures, inducing chain segregation. A possible interpretation of this paradox may be that the metastable ~ and [3' forms of the P O P - P P O or S O S - S S O c o m p o u n d s , illustrated in Figure 3c, may need a molecular displacement accompanied by a high energy of activation for chain segregation. This may be accomplished by a flip-flop m o v e m e n t of the saturated moieties across the glycerol group, not by a simple chain m o v e m e n t along the long chain axes, which would instead be realized in the mixtures of S O S - O S O and P O P - O P O . T h e formation of the double-chain length c form may originate from the structures of the neat liquid, in which the oleoyl and saturated acid chains are packed in the same leaflet. This consideration assumes that the formation of the molecular c o m p o u n d s in P O P - P P O and S O S - S S O is due to kinetic hindrance of the chain segregation of the double chain-length structures. By contrast, the P O P - O P O and S O S - O S O compounds are formed by structural stabilization.

Oil and Fats Co., Amagasaki, Japan), S Ucno (Hiroshima Univcrsity), T Arishima (Fuji Oils and Fats Co., lzumisanu, Japan) and K Smith (Unilever Research Colworth Laboratory, Bedford, UK), whose collaborative work enabled the writingof this revicw.The deepest ffratitude should be given to the late ProfessorM Koba.vashi(Osaka University).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. 2. Gennis RB: Biomembranes: Molecular Structure and Function. New York: Springer-Verlag; 1989. SmallDM: The physical chemistry of lipids. From alkanes to phospholipids. In Handbook of Lipid Research, vol 4. Edited by Hanahan DJ. New York: Plenum Press; 1986:233-284. SmallDM: Lateral chain packing in lipids and membranes. J Lipid Res 1984, 25:1490-1500. Wang G, Lin H-N, Li S, Huang C-H: Phosphatidylcholines with sn-1 saturated and sn-2-cis monounsaturated acyl chains. Their melting behavior and structures. J Bio/Chem 1995, 270:2273822746. Huang C-H, Lin H-N, Wang G: Influence of the positions of cis double bonds in the sn-2-acyl chain of phosphatidylethanolamine on the bilayer's melting behavior. J Bio/Chem 1997, 272:2191721926. Barton PG, Gunstone FD: Hydrocarbon chain packing and molecular motion in phospholipid bilayers formed from unsaturated lecithins. Synthesis and properties of sixteen positional isomers of 1,2-dioctadecenoyl-sn-glycerol-3phosphorylcholine. J Biol Chem 1975, 250:4470-4476. Applegate KR, Glomset JA: Effect of acyl chain unsaturation on the packing of model diacylglycerols: a computer modeling study, J Lipid Res 1991,32:1635-1644. Applegate KR, GIomset JA: Effect of acyl chain unsaturation on the packing of model diacylglycerols in simulated monolayers. J Lipid Res 1991, 32:1645-1655. Pascher I: The different conformations of the glycerol region of crystalline acylglycerols. Curr Opin Struct Bio/1996, 6:439-448.

3. 4.

5.

6.

Conclusions
T h e crystal structures of principal cis-monounsaturated fatty acids and the structural behavior of mixed-acid saturated oleic acid DAGs and TAGs highlight the fact that the dramatic diversity of the olefinic conformation, subcell packing and chain-chain interactions is revealed in the polymorphic transformations and mixing-phase behavior of the lipids so far examined. Although much of the experimental work reviewed here concerns the crystalline phases of nonpolar lipids, one may assume that the structural diversity of these unsaturated molecules may play similar roles in the polar lipids of biomembranes, lipoproteins and lipid deposits. Some lipid domains can undergo liquid to solid or solid to liquid state transitions, in which the unsaturated fatty-acid moieties may play a critical role in combination with the polar group constraint. T h e r e are many problems left unsolved both theoretically and experimentally. T h e s e include an energetic consideration of the occurrence and stabilization of the diversified polymorphism of oleic acid, particularly the stabilization of the whole crystal energy in the [8 form, which has extremely different features from other c/s-monounsaturated fatty acids. T h e structural determination of the binary mixture molecular compounds, with specific attention to subcell and methyl-end packing and glycerol conformations, is yet to be carried out. A more detailed analysis of saturated-unsaturated mixed-acid DAGs and phospholipids, with particular use of multiple F'['-IR techniqt, es whose excellent advantages have been demonstrated in this review, also remains.
7.

8.

9.

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Acknowledgements

The authors are highly indebted to DM Small (Boston University), DR Kodali (Cargill Research, Minneapolis, Minnesota), M Suzuki (Nippon

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35. Ueno S, Minato A, Seto H, Amemiya Y, Sato K: Synchrotron radiation X-ray diffraction study of liquid crystal formation and polymorphic crystallization of SOS (sn-1,3-distearoyl-2-oleoyl glycerol). J Phys Chem B 1997, 101:6847-6854. This paper reveals for the first time the occurrence of thermotropic liquidcrystalline phases in SOS using time-resolved synchrotron X-ray diffractometry. 36. Minato A, Ueno S, Smith K, Amemiya Y, Sato K: Thermodynamic and kinetic study on phase behavior of binary mixtures of POP and PPO forming molecular compound systems. J Phys Chem B 199?, 101:3498-3505. 37. Minato A, Ueno S, Yano J, Smith K, Seto H, Amemiya Y, Sato K: Thermal and structural properties of sn-l,3-dipalmitoyi-2oleoylglycerol and sn-l,3-dioleoyl-2-palmitoylglycerol binary mixtures examined with synchrotron radiation X-ray diffraction. J Am Oil Chem Soc 1997, 74:3498-3505. 38. Minato A, Yano J, Ueno S, Smith K, Sato K: FT-IR study on microscopic structures and conformations of POP-PPO and POP-OPO molecular compounds. Chem Phys Lipids 1997, 88:63-71. 39. Kobayashi M: Vibrational spectroscopic aspects of polymorphism and phase transition of fats and fatty acids. In Crystallization and Polymorphism of Fats and Fatty Acids. Edited by Garti N, Sato K. New York: Macel Dekker; 1988:139-18?. 40. Sato K: Polymorphism of pure triacylglycerols and natural fats. In Advances in Applied Lipid Research, vol 2. Edited by Padley F. London: JAI Press Inc; 1996:213-268. 41. Ueno S, Suetake T, Yano J, Suzuki M, Sato K: Structure and polymorphic transformations in elaidic acid (trans-(o9octadecenoic acid). Chem Phys Lipids 1994, 72:27-34. 42. Sato K, Kobayashi M: Structure, stability and crystal growth of polymorphs and polytypes of long-chain aliphatic compounds. In Crystals. Edited by Karl N. Heidelberg: Springer-Verlag; 1991:65-108. 43. YanoJ, Kaneko F, Kobayashi M, Sato K: Structural analyses of triacylglycerol polymorphs with FT-IR techniques. I. Assignments of CH 2 progression bands of saturated monoacid triacylglycerols. J Phys Chem B 1997, 101:8112-8119. See annotation [44"]. 44. YanoJ, Kaneko F, Kobayashi M, Kodali DR, Small DM, Sato K: Structural analyses of triacylglycerol polymorphs with FT-IR techniques. II. ~'l"form of 1,2-dipalmitoyl-3-myristoyl-sn-glycerol (PPM). J Phys Chem B 1997, 101:8120-8128. This paper, together with [43"], describes the assignment of the polymethylene CH 2 progression bands of a series of monoacid triacylglycerols (TAGs) [43"] and the analysis of the molecular structures of saturated mixed-acid TAGs [44]. 45. Mendelsohn R, Liang GL, Strauss HL, Snyder RG: IR spectroscopic determination of gel state miscibility in long-chain phosphatidylcholine mixtures. Biophys J 1995, 69:1987-1998.