Anthony J. Hancock

Epitaxial crystallization of alkaline chain lipids for electron diffraction analysis

Thin microcrystals of a wide variety of polymethylene chain materials, including n-alkanes, linear waxes, glycerides, a detergent, phospholipids and phospholipid analogs based on cyclopentane-1,2,3-triol, are epitaxially grown on naphthalene to give an orientation with long chain axes parallel to the best developed crystal face. These crystals, which represent a different orientation than those grown from solution, facilitate ab initio quantitative crystal structure analysis from electron diffraction intensity data from the projection yielding the most crystallographic information.

The properties of membranes formed from cyclopentanoid analogues of phosphatidylcholine

We have examined the thermal characteristics and barrier properties of vesicles formed from six analogues of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These analogues differ from DPPC in that the glycerol backbone has been replaced by each of the diastereoisomeric cyclopentane-1,2,3-triols. All of these compounds have main gel to liquid-crystal phase transition temperatures within 5 Kelvin of DPPC and four possess comparable enthalpies and entropies of transition. For two of the analogous, however, the values of the enthalpy and entropy of transition are more than double that of DPPC. The permeability characteristics and organization (as measured by diphenylhexatriene fluorescence depolarization) of vesicles formed from these two compounds suggest that their large transition enthalpy and entropy result from either a reorganization of the polar head group region during the transition or interdigitation of the acyl chains of opposing monolayers.

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373-377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues (n = 3 through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by 22Na permeability measurements, differential scanning calorimetry, fluorescence polarization, 31P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with n = 2 or 3 exhibit a sharp transition at 46 degrees C or 49 degrees C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with n greater than 3 exhibit a main transition between 38 and 43 degrees C with enthalpies less than 10 kcal/mol and after prolonged coding (more than 3 days at 4 degrees C) a broad endotherm at about 20 +/- 3 degrees C with enthalpies greater than 4 kcal/mol. These same dispersions display a permeability peak at 20-25 degrees C and a second increase in 22Na efflux in the temperature range 30-40 degrees C. The results of 31P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentano-1-phosphocholine (n = 2) bilayers have restricted rotation below the main phase transition temperature.

Cyclopentanoid analogs of dipalmitoyl phosphatidic acid: effect of backbone geometry on thermotropic properties.

Seven geometrical or positional isomers of dipalmitoyl cyclopentanophosphoric acid (DPCPA) have been synthesized and studied: 1,3/2-1P (I); 1,2/3-1P (II); 1,2/3-3P (III), 1,2,3/0-1P (IV); 1,2,3/0-2P (V); 1,3/2-2P (VI); 1,2/3-2P (VII). When dispersed in 0.1 M Tris-HCl at pH 7.4, I-VII gave thermal transitions (Tc) of 60.0 degrees, 59.0 degrees, 56.8 degrees, 55.3 degrees, 38.3 degrees, 36.8 degrees and 34.0 degrees C, respectively, as measured by differential scanning calorimetry (DSC). When the lipids were dispersed at pH 9.5 in 0.1 M borate, Tc of I-IV decreased, whereas Tc of V-VII increased. In contrast, at pH 1.5 in 0.1 M HCl/KCl, Tc of I-IV decreased slightly, but Tc of V-VII rose markedly. To determine the effect of head group geometry and substitution pattern on acyl chain motion, EPR spectra of 1-palmitoyl, 2-[16-doxylstearoyl]-glycero-3-phosphoric acid in bilayers of DPCPA isomers were acquired. Abrupt spectral changes occurred at temperatures closely correlating with transition temperatures observed by DSC. These results have led to the conclusions that: (i) isomers I-IV containing vicinal acyl chains form bilayers that exhibit structural transitions at temperatures higher than those at which transitions are exhibited by isomers V-VII which have a polar phosphate group interposed between the two chains; (ii) the effects of differences in backbone structure are transmitted down the entire length of the acyl chains; (iii) the orientation of the cyclopentane ring in the isomers I-IV is significantly different from that in isomers V-VII at pH values where the phosphate group is doubly negatively charged.

Glycerol conformation and the crystal structure of lipids. I. An electron diffraction study of tripalmitin and conformationally fixed analogs.

Abstract Transmission electron diffraction is used as a qualitative analytical probe of poly methylene chain packing in three conformationally-restricted analogs of tripalmitin derived from configurational isomers of cyclopentane-1,2,3-triol. The analysis demonstrates that the all trans “tuning-fork” conformation seen in X-ray crystal structure analyses on homologous compounds is also found in microcrystals, in contrast to a proposal by Fringeli et al. (Z. Naturforsch. 27 b, 780 [1972]) that an all cis conformation of the triglyceride exists in solvent-grown microcrystals and in multilayers. The presence of mixed polymorphs is also shown for bulk samples previously examined by X-ray powder diffraction techniques by other workers.

Influence of Molecular Conformation on the Solid State Packing of 1,2-Diglycerides Study of 1,2-Dipalmitin and Some Structural Analogs by Electron Diffraction, X-Ray Diffraction, and Infrared Spectroscopy

Abstract Diffraction studies on natural 1,2-dipalmitin and on analogs, including those based on the configurational isomers of cyclopentane-1,2,3-triol reveal that the 1,2-diglycerides crystallize from solvent with chain methylene packing identical to the monoclinic form of even-chain alkanes. The chains probably are folded back in “hairpin” fashion as found in phospholipid crystal structures. Acyl shifts are observed to occur in the crystalline solid state at room temperature to give the 1,3-diglyceride. Analogs based on the above-mentioned cyclitols show that isomers with adjacent chains trans to the ring (possibly extended chain packing) or with chains cis to the ring (“hair pin”) crystallize readily. Both possibly extended chain configurational isomers have the α-form as well as β′-forms and a β-polymorph. The hairpin isomers each give a β′-polymorph but only the all-cts isomer gives an α-form.

Glycerol Conformation and the Crystal Structure of Lipids II. A Further Study of Tripalmitin and Conformationally Fixed Analogs by Small-Angle X-Ray Diffraction and Reflection Electron Diffraction

Abstract The utility of analogs to glycerol-containing lipids based on the configurational isomers of cyclo-pentane-1,2,3-triol for ab initio crystal structure analysis via electron diffraction is assessed further. Such analogs of tripalmitin are examined with the natural triglyceride via low angle X-ray diffraction. The 1,3/2 (all-trans) and 1,2/3 (cis-trans) analogs give long spacing dimensions some 23 Å greater than found for the β-2 form of the natural compound, consistent with the long spacing observed for a β-3 form. The 1,2,3/0 (all-cis) analog gives a long spacing near that of the a-form of the triglyceride. Reflection electron diffraction measurements on the 1,3/2 and 1,2/3 analogs reveal a chain tilt near 60° for both and untilted chains for 1,2,3/0. A more accurate tilt determination from X-ray long spacings of the homologous series of 1,3/2 pseudotrilaurin to 1,3/2 pseudo-tripalmitin confirm the 67° tilt expected for the β-3 form. Therefore, given the same T,, methylene subcell, the molecular packing is very close to natural triglycerides. The subtle influences of the cyclopentane ring are overcome for 1,3/2 analogs based on stearic and arachidic acids. This emphasizes the utility of these structural analogs for ab initio crystal structure determinations of glycerol containing lipids.

ARTIFICIAL LIPIDS CONTAINING CYCLOPENTANOID BACKBONES

SUMMARY : Two series of analogs of glycerolipids, in which the glycerol backbone is replaced by each of the isomeric cyclopentane-1,2,3-triols have been prepared. Physical studies have been carried out dealing with polymorphic crystalline states, surface properties and the ability to reconstitute lipoproteins.