Atomic resolution structure analysis of beta' polymorph crystal of a triacylglycerol: 1,2-dipalmitoyl-3-myristoyl-sn-glycerol.

The crystal structure of the beta'-2 form of a mixed chain triacylglycerol (TAG), 1,2-dipalmitoyl-3-myristoyl-sn-glycerol (PPM), was determined to a final reliability factor of 0.074. This work is the first to resolve the atomic-level structure of the beta' polymorph, which is of the highest functionality among multiple polymorphs in asymmetric TAG. In particular, fat crystals present in food emulsions are in beta', whose transformation into beta causes deterioration in their physical properties. beta'-2, one of the two beta' forms of PPM, forms a monoclinic unit cell with a space group of C2; Z = 8, a = 16.534(5) A, b = 7.537(2) A, c = 81.626(9) A; beta = 90.28(2) degrees, V = 10171(3) A(3), density = 1.018 g/cm(3), and mu = 4.96 cm(-1). The following characteristics were obtained: 1) two asymmetric units, named A and B, form a hybrid-type orthorhombic perpendicular subcell; 2) the two asymmetric units reveal different glycerol conformations: trans for sn-1 palmitic acid and sn-2 palmitic acid, but gauche for sn-3 myristic acid in A; and trans for sn-2 palmitic acid and sn-3 myristic acid, but gauche for sn-1 palmitic acid in B; 3) a unit lamellae reveals a four-chain-length structure consisting of two double-layer leaflets; 4) the two double-layer leaflets are combined end-by-end in a unit lamellae; and 5) the chain axes are alternatively inclined against the lamellar interface. -- Sato, K., M. Goto, J. Yano, K. Honda, D. R. Kodali, and D. M. Small. Atomic resolution structure analysis of beta' polymorph crystal of a triacylglycerol: 1,2-dipalmitoyl-3-myristoyl-sn-glycerol. J. Lipid Res. 2001. 42: 338--345.

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.

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 alpha and alpha and a series of irreversible transitions (alpha-->gamma-->beta'-->beta (beta2, beta1) for SOS and alpha-->beta'-->beta for OSO) were studied with an emphasis on the conformational ordering process of stearoyl and oleoyl chains. The alpha-->sub alpha 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 alpha to beta, the conformational ordering of saturated chains took place in the alpha-->gamma transition of SOS and in the alpha-->beta' 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 beta-type. As the final stage, the ordering of unsaturated chains occurred in the beta'-->beta 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 alpha-->gamma transition of SOS and in the beta'-->beta transition of OSO.

Single crystal structure of a mixed-chain triacylglycerol: 1,2-dipalmitoyl-3-acetyl-sn-glycerol.

The mixed chain triacylglycerol 1,2-dipalmitoyl-3-acetyl-sn-glycerol was synthesized and its crystal structure was determined to a final reliability factor (R) of 0.11. Two molecules are present in the monoclinic unit cell: space group P2(1); a = 5.375(1), b = 8.286(2), c = 42.96(1) A; beta = 93.30(2) degrees, V = 1910 A3, rho = 1.065 g/cm3, and mu = 5.7 cm-1. The structure is a trilayer: a bilayer of palmitate chains packed in the beta mode (T parallel) and an interdigitated monolayer of acetates. The glycerol backbone and acetate extend roughly linearly from the sn-1 chain. The sn-2 chain bends around the C-2 carbon to lie next to the sn-1 chain. Analysis of the torsion angles indicate that the glycerol conformation of 1,2-dipalmitoyl-3-acetyl-sn-glycerol is markedly different from single acid triacylglycerols and from 1,2-diacyl-sn-glycerols but very similar to 1,2-dimyristoyl-sn-glycero-3-phosphocholine.

Debenzylation and detritylation by bromodimethylborane: synthesis of mono-acid or mixed-acid 1,2- or 2,3-diacyl-sn-glycerols.

A novel method of deprotecting primary alcohols protected with either benzyl or trityl groups by using bromodimethylborane under mild reaction conditions (dichloromethane, -20 to 5 degrees C) has been applied to the synthesis of optically pure mono-acid or mixed-acid 1,2- or 2,3-diacyl-sn-glycerols. This method was particularly useful for the synthesis of long saturated acyl (C12 to C24) as well as unsaturated diacyl-sn-glycerols since little or no acyl migration occurred during deprotection. Diacylation of 3-benzyl-sn-glycerol or 1-benzyl-sn-glycerol followed by bromodimethylborane debenzylation gave mono-acid 1,2- or 2,3-diacyl-sn-glycerols, respectively. The mixed-acid 1,2- or 2,3-diacyl-sn-glycerols were prepared from 1-acyl-sn-glycerols or 3-acyl-sn-glycerols, respectively, by tritylation, acylation with a different fatty acid, followed by detritylation with bromodimethylborane.

The interfacial conformation and transbilayer movement of diacylglycerols in phospholipid bilayers.

The interaction of diacylglycerols, primarily 1,2-dilauroyl-sn-glycerol (1,2-DLG), with egg phosphatidylcholine (PC) bilayers was studied by NMR spectroscopy and other physical techniques. In the low proportions used (less than or equal to 20 mol % with respect to total lipid), 1,2-DLG formed bilayers with PC with no hexagonal phase separation, as assessed by light, polarizing and electron microscopy, and 31P and 13C NMR spectroscopy. The 13C-carbonyl chemical shift of 90% [13C]carbonyl 1,2-DLG was monitored in small unilamellar vesicles as a function of relative DLG content (1.5-20%) and temperature (10-55 degrees C). The chemically inequivalent sn-1 and sn-2 carbonyls gave a single, narrow resonance in vesicles, in contrast to neat 1,2-DLG and 1,2-DLG in organic solvents, whose spectra showed two well-separated carbonyl resonances. The chemical shift of 1,2-DLG in PC shows that the carbonyl groups are proximal to the aqueous interface, necessitating orientation of the DLG molecule along the normal to the bilayer. Both carbonyl groups are H-bonded to H2O, but the secondary ester (sn-2) carbonyl is relatively more hydrated than the primary ester (sn-1) carbonyl. The 13C-carbonyl chemical shift data further suggest that the interfacial conformation resembles that of crystalline and liquid crystalline lamellar 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine and certain PCs, in which the glycerol backbone is perpendicular to the bilayer plane. This conformation is different from that of crystalline 1,2-dilauroyl-sn-glycerol, in which the glycerol backbone is parallel to the bilayer plane. Between 1.5 and 8% DLG in vesicles, the chemical shift of the 1,2-DLG carbonyl at a given temperature was constant. However, above 8% DLG the chemical shift at each temperature increased with increasing DLG concentration, suggesting increased hydration at higher DLG content. At low temperatures 13C NMR spectra of vesicles with the highest proportions of 1,2-DLG studied (15 and 20%) showed two DLG carbonyl resonances, which most likely represent 1,2-DLG on outer and inner leaflets of the vesicle bilayer. The two peaks collapsed into a single resonance by 38 degrees C, at which temperature the two environments equilibrate with a rate constant of approximately 60 s-1 (t1/2 approximately 10 ms). Thus, transbilayer movement of DLG is extremely fast compared with phospholipids. In vesicles the 1,3-isomer of DLG exhibited a narrow carbonyl peak slightly downfield from that of 1,2-DLG. Acyl chain migration from 1,2-DLG to 1,3-DLG was monitored directly in the vesicle by time-dependent NMR measurements.

Structure and polymorphism of saturated monoacid 1,2-diacyl-sn-glycerols.

The 1,2-diacyl-sn-glycerols (1,2-DGs) are the predominant naturally occurring isomer found in cell membranes, lipid droplets, and lipoproteins. They are involved in the metabolism of monoacylglycerols, triacylglycerols, and phospholipids. The 1,2-DGs participate in the activation of protein kinase C, in phosphorylation of target proteins, and in transduction of extracellular signals into the cell. We have undertaken a study of the physical properties of a homologous series of synthetic optically active diacylglycerols. Stereospecific 1,2-diacyl-sn-glycerols were synthesized with saturated fatty acyl chains of 12, 16, 18, 22, and 24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The solvent-crystallized form for all the 1,2-DGs packs in the orthorhombic perpendicular subcell (beta') and melts with a single sharp endotherm to an isotropic liquid. On quenching, the C12, C16 and C18 compounds pack in a hexagonal subcell (alpha), whereas the C22 and C24 pack in a pseudohexagonal subcell (sub-alpha). The sub-alpha phase reversibly converts to the alpha phase. The long spacings of these compounds in both the alpha and beta' phases increase with chain length. In the alpha and beta' phases, the acyl chain tilts were found to be 90 degrees and 62 degrees from the basal methyl plane. The polymorphic behavior of 1,2-diacyl-sn-glycerol is quite different from that of the corresponding monoacid saturated 1,3-diacylglycerols which form two beta phases with triclinic parallel subcells.

Polymorphic behavior of 1,2-dipalmitoyl-3-lauroyl(PP12)- and 3-myristoyl(PP14)-sn-glycerols.

The polymorphic behavior and molecular packing in different polymorphic forms of stereospecific triacylglycerols, 1,2-dipalmitoyl-3-lauroyl-sn-glycerol (PP12) and 1,2-dipalmitoyl-3-myristoyl-sn-glycerol (PP14) were examined by X-ray diffraction, differential scanning calorimetry, infrared and Raman spectroscopy techniques. The molecular packing and the polymorphic behavior of the metastable forms of these two compounds are very similar. In both compounds the isotropic liquid, on quenching, crystallizes into a hexagonally packed alpha-phase. The long spacing periodicity of the alpha-phase indicates a tilted bilayer structure to compensate the voids created by the short acyl chains. Upon heating, the alpha-phase converts into an orthorhombic perpendicular (O perpendicular) beta'2-phase. The beta' 2-phase, on further heating, exothermally converts to beta' 1-phase with slightly different O perpendicular subcell. On incubation of PP12 near the melting temperature of beta' 1-phase, there is a slow (hours) conversion to a beta-phase with triclinic parallel (T//) packing. However the beta' 1-phase of PP14 is the most stable structure and the beta-phase is absent. The thermodynamic parameters of the O perpendicular packings of these compounds compared to those of the higher homologue, tripalmitoylglycerol, suggest that the O perpendicular subcell is more stable in PP12 and PP14. The X-ray diffraction long spacings indicate that all the polymorphic forms of these compounds pack in a bilayer structure. The vibrational spectra confirm the lateral chain packing and inter- and intra-molecular order/disorder in the various polymorphic forms. The Raman and infrared spectra further indicate perturbation in the carbonyl and the end methyl plane regions of the beta'-phases.

Acyl migration in 1,2-dipalmitoyl-sn-glycerol.

The acyl migration of 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) to 1,3-dipalmitoylglycerol (1,3-DPG) in different states, neat, in the presence of egg yolk lecithin (sonicated and unsonicated) and on silica gel was studied. The isomerization was quantitated by scanning densitometry of charred TLC plates, at different temperatures and for varying periods of time. At equilibrium the amount of 1,3-DPG was found to be 56%. The rates of initial isomerization, and the time required to isomerize to half the equilibrium quantity (i.e., t1/2 eq. = 1,3-DPG 28%) under the above conditions was estimated. In the case of neat melt at 74 degrees C and in an organic solvent the time required to t1/2 eq. is 18 h and a few days, respectively. However, at 62 degrees C in the presence of a polar solvent (sodium phosphate buffer at pH 7.0) the t1/2 eq. is 1-2 h. On dry silica gel (TLC plate) at 24 degrees C the t1/2 eq. is reached in less than 1 h.

The effect of triacyl-sn-glycerol structure on the metabolism of chylomicrons and triacylglycerol-rich emulsions in the rat.

A systematic study was undertaken to observe the effects of dietary (dioleoyl) triacyl-sn-glycerol structure on chylomicron composition and metabolism. First studied was a series of 1,2-dioleoyl-3-(saturated)acyl-sn-glycerols, where the fatty acid esterified at the 3-position was varied from 14 to 24 carbons. Next a series of 1,3-dioleoyl-2-acyl glycerols was studied, with various fatty acids esterified at the glycerol 2-position. These stereospecific triacyl-sn-glycerols were fed to donor rats and lymph chylomicrons were isolated, analyzed, and reinjected into recipient rats to study their disappearance from plasma and delivery to tissues. As shown by their compositions, chylomicrons obtained after feeding triacylglycerols containing all sn-3 fatty acid of chain length greater than 20 carbons were under-represented, possibly due to poorer digestion by lipases, or poorer absorption by the intestine. The 18-carbon saturated chain fatty acid (stearic acid) was equally well represented in chylomicrons whether in the 2- or 3-position of the fed triacylglycerol. The presence of increased amounts of long-chain saturated fatty acids in donor chylomicron triacylglycerols affected the metabolism of chylomicrons injected into the bloodstream of recipient rats. In particular the rate of removal of labeled cholesteryl esters, tracing removal of the partially degraded chylomicron remnants was slowed by the saturated chains, with palmitic acid and the 20-carbon fatty acid, arachidic acid, showing the most severe effects. There were clear differences in the removal from plasma of injected lymph chylomicrons derived from fed triacylglycerols containing stearic acid in either the 2- or 3-position, with evidence for remnants from the symmetrical triacylglycerols being less rapidly removed from the circulating blood. This effect was investigated further by injected model emulsions of chylomicrons, where the 2-position was substituted with saturated or transunsaturated acyl chains. Quantitation of removal from the blood stream of these model lipoproteins confirmed that a saturated or transunsaturated long chain fatty acid at the 2-position of the emulsion triacylglycerols slowed remnant removal from the blood. In some cases, with both lymph chylomicron and with emulsions, the lipolytic step mediated by lipoprotein lipase was also slowed.

Structure and polymorphism of 18-carbon fatty acyl triacylglycerols: effect of unsaturation and substitution in the 2-position.

The polymorphic behavior of symmetric diacid triacylglycerols (TGs), 1,3-dioleoyl-2-stearoyl (OSO), 2-elaidoyl (OEO), and 2-vaccinoyl (OVO) glycerols were studied by differential scanning colorimetry (DSC) and X-ray diffraction and compared with the corresponding monoacid TGs triolein (OOO), tristearin (SSS), trielaidin (EEE), and trivaccinin (VVV). The monoacid TGs formed a bilayered structure in all the polymorphic forms. On quenching from the melt, the diacid TGs OEO and OVO formed a bilayered (D = 45 A) beta'-phase with the exception of OSO, which formed a hexagonally packed bilayered (D = 52 A) alpha-phase. At -7 degrees C, the alpha-phase of OSO quickly transformed to a bilayered (D = 45 A) beta'-phase. Incubation at the beta'-phase melting temperature transformed OVO, OEO, and OSO into a trilayered (D = 65 A) beta-phase, where the 1,3-dioleoyl chains are segregated from the vaccinoyl, elaidoyl, or stearoyl chains into alternating layers. In summary, when all the acyl chains in a TG are the same (saturated, cis or trans unsaturated), the stable beta-phase packs into a bilayered structure. However, when the 1- and 3-acyl chains are cis unsaturated (bent) and the 2-acyl chain is either saturated or trans-unsaturated (straight), a bilayered beta'-phase can form, but transforms to a stable trilayered beta-phase, where the 2-acyl chains form a layer between two different layers of 1,3-oleoyl chains.

Improved method for the synthesis of 1- or 3-acyl-sn-glycerols.

Optically active 1- or 3-acyl-sn-glycerols were synthesized from 2,3- or 1,2-isopropylidene-sn-glycerols, respectively. The 2,3- or 1,2-isopropylidene-sn-glycerols were condensed with appropriate long saturated or unsaturated fatty acids and the resulting acyl isopropylidene compounds were treated with dimethylboronbromide at - 50 degrees C to give the title compounds. The ketal cleavage of acyl isopropylidene-sn-glycerols by dimethylboronbromide to produce the long 1- or 3-acyl-sn-glycerols was effective and gave good yields (70-90%). The reaction conditions were mild and there was no acyl migration, as shown by optical rotation of the monoacyl-sn-glycerols. The synthesis of 2,3-isopropylidene-sn-glycerol was improved to give an overall yield of 40% from L-arabinose. L-Arabinose was first converted to its 1,1'-diethylmercapto derivative and then condensed with 2-methoxypropene to yield 1,1'-diethyl-mercapto-4,5-isopropylidene-L-arabinose. Oxidation of this compound with sodium periodate followed by reduction with sodium borohydride under alkaline conditions yielded 2,3-isopropylidene-sn-glycerol [alpha]22D = -14.90 degrees, neat (Lit. 8 [alpha]22D = -14.5 degrees, neat; 14 [alpha]25D = -10.8 degrees; methanol C, 16.9). The optical purity of isopropylidene-sn-glycerols was determined as benzoyl derivatives on a high performance liquid chromatographic column packed with a chiral stationary phase.

Structure and polymorphism of 1,2-dioleoyl-3-acyl-sn-glycerols. Three- and six-layered structures.

Triacylglycerols, which usually contain at least one unsaturated fatty acid, are the most important forms of stored biological lipids in teleosts, mammals, and most plants. Since the physical properties of such mixed-chain triacylglycerols are poorly understood, a systematic study of such compounds has been initiated. Stereospecific 1,2-dioleoyl-3-acyl-sn-glycerols were synthesized with even carbon saturated fatty acyl chains of 14-24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The thermal behavior revealed from one to four major polymorphic transitions depending upon saturated chain length. Plots of enthalpy of fusion and entropy vs. carbon number for melting of the most stable polymorph were linear throughout the series with slopes of 1.0 kcal/mol per carbon atom and 2.6 cal/(mol K) per carbon atom, respectively. These slopes indicate that the saturated chains are packed in a well-ordered tightly packed lattice. When the compounds were rapidly cooled to 5 degrees C, X-ray powder diffraction revealed strong beta' (ca. 3.8 and 4.2 A) reflections and weak beta (ca. 4.6 A) reflections. The beta subcell reflections intensified when the compounds were heated to within 5 degrees C of the melting temperature of the highest melting polymorph. Evidence of an alpha phase was not seen on 30-min X-ray exposures for any of the compounds. In the proposed packing arrangement the saturated and unsaturated chains are segregated into layers. The stable form of all compounds exhibits a triple layer packing mode in which a bilayer of oleoyl chains is segregated from an interdigitated layer of saturated chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and polymorphism of 3-acyl-sn-glycerols.

3-Acyl-sn-glycerols with even-numbered saturated fatty acyl chains from decanoate to lignocerate were synthesized. Successful hydrolysis of the long acyl chain intermediate 1,2-isopropylidene-3-acyl-sn-glycerols from stearate to lignocerate was accomplished by applying the compounds to silica gel and exposing them to hydrogen chloride gas at -75 degrees C. The purity of the compounds was checked by boric acid impregnated thin-layer chromatography, 13C NMR, and reverse-phase high-pressure liquid chromatography. Differential scanning calorimetry and X-ray diffraction techniques were used to study the polymorphism of the compounds. In the beta phase obtained from solvent of crystallization, the acyl chain packing was in a two-dimensional oblique lattice with specific chain-chain interactions with a tilt angle of 55.4 degrees from the bilayer plane. The thickness of the region containing two glycerol head groups was 12.7 A. The phase transition enthalpy of melting for the beta phase was 1.06 kcal/mol of CH2. On being cooled these compounds crystallized reversibly to an unstable alpha phase, which on being further cooled underwent a second crystallization to a beta or beta' phase. The thermodynamic parameters and long spacings of these compounds in both beta and alpha phases were linear, indicating isostructural packing in each phase. The enthalpy of the melting transition of the alpha phase was 0.69 kcal/mol of CH2. In this phase, the chains were packed in a hexagonal lattice with nonspecific chain-chain interactions. The thickness of the head-group region (12.2 A) and the tilt angle (55 degrees) of the acyl chains in the alpha phase were very similar to those in the beta phase.