Katarzyna A. Solanko

Single crystals of aspirin form II: crystallisation and stability

Single crystals of aspirin form II can be obtained by crystallisation of aspirin in the presence of aspirin anhydride in organic solvents such as acetonitrile or tetrahydrofuran. The crystals are stable under ambient conditions for months and do not show any phase transition on application of isotropic pressure up to 2.2 GPa.

How cholesterol interacts with proteins and lipids during its intracellular transport

Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein-lipid and protein-protein interactions. Similarly, membrane lipids and their physico-chemical properties directly affect cholesterol partitioning and thereby contribute to the highly heterogeneous intracellular cholesterol distribution. Movement of cholesterol in cells is mediated by vesicle trafficking along the endocytic and secretory pathways as well as by non-vesicular sterol exchange between organelles. In this article, we will review recent progress in elucidating sterol-lipid and sterol-protein interactions contributing to proper sterol transport in living cells. We outline recent biophysical models of cholesterol distribution and dynamics in membranes and explain how such models are related to sterol flux between organelles. An overview of various sterol-transfer proteins is given, and the physico-chemical principles of their function in non-vesicular sterol transport are explained. We also discuss selected experimental approaches for characterization of sterol-protein interactions and for monitoring intracellular sterol transport. Finally, we review recent work on the molecular mechanisms underlying lipoprotein-mediated cholesterol import into mammalian cells and describe the process of cellular cholesterol efflux. Overall, we emphasize how specific protein-lipid and protein-protein interactions help overcoming the extremely low water solubility of cholesterol, thereby controlling intracellular cholesterol movement. This article is part of a Special Issue entitled: Lipid-protein interactions.

Crystal architecture and physicochemical properties of felodipine solvates

Solvates of the calcium-channel blocking agent felodipine with three structurally related high-boiling point solvents, dimethylacetamide (DMAA), dimethylethyleneurea (DMEU) and tetramethylurea (TMU), are described. The solvates can be formed either by solution crystallisation or solvent-drop mechanical grinding. In all of the crystal structures, the solvent molecule accepts an N–H⋯O hydrogen bond from felodipine. Analysis of the conformational preferences of the felodipine molecule in the crystal structures shows that it adopts a higher-energy conformation in the solvate with TMU. Hot-stage microscopy shows that the solvates decompose below the boiling point of the solvent, then the solvent condenses and dissolves the desolvated felodipine. The decomposition temperature of the solvate is correlated with the van der Waals molecular volume for the solvent molecule within the crystal structure. Measurements of the aqueous dissolution rate show that the concentration of felodipine during the first hour is increased by 4–6 times for dissolution of the solvates compared to pure felodipine.

Cocrystals of the antiandrogenic drug bicalutamide: screening, crystal structures, formation thermodynamics and lattice energies

Two new cocrystals of the non-steroidal anti-androgen drug bicalutamide (Bic) are reported with benzamide (BZA) and salicylamide (2OHBZA), both in a 1 :  1 molar ratio. X-ray crystal structure analysis shows that both cocrystals contain a folded molecular conformation of bicalutamide, similar to that seen in polymorph II of the pure drug. Calculations of intermolecular interaction energies using the PIXEL approach indicate closely comparable total lattice energies for [Bic + BZA] and [Bic + 2OHBZA]. The structures are dominated by dispersion interactions, with a significant contribution also from the coulombic interactions, particularly in [Bic + BZA]. The main difference between the two cocrystals is seen for the bicalutamide–cocrystal former interaction energy, which is calculated to be slightly more stabilizing in [Bic + BZA]. The melting temperatures of the cocrystals (132 °C for [Bic + BZA] and 157 °C for [Bic + 2OHBZA]) are significantly lower than that of the pure API (193 °C). In general, the melting temperatures of all known bicalutamide cocrystals are shown to increase with an increase of the total van der Waals volume (Vvdw) of the molecules in the asymmetric unit of the crystal. The thermodynamic functions of the cocrystal formation were estimated from the solubility of the cocrystals and the corresponding solubility of the pure compounds in chloroform at various temperatures. In both cases, the Gibbs energy of formation was found to be small: −3.4 kJ mol–1 for [Bic + BZA] and −2.2 kJ mol−1 for [Bic + 2OHBZA]. The most significant contribution to the Gibbs energy is provided by the exothermic enthalpy of formation. However, the cocrystal formation is accompanied by a considerable decrease of the system entropy, which diminishes the overall driving force of the process. Both cocrystals demonstrated a classical “spring and parachute” behavior during aqueous dissolution, providing an increased concentration level of Bic compared to that of the parent drug for several hours.

Polymorphism of felodipine co-crystals with 4,4′-bipyridine

The calcium-channel blocking agent felodipine (Fel) forms co-crystals with 4,4′-bipyridine (BP) with 1 : 1 and 2 : 1 molar ratios. The [Fel + BP] (1 : 1) co-crystal exists in two polymorphic forms. Differential scanning calorimetry and solution calorimetry show that form I of the [Fel + BP] (1 : 1) co-crystal is the most thermodynamically stable phase. The difference in the crystal lattice energies between different polymorphs of the co-crystal is found to be comparable with that between the polymorphic forms of pure felodipine. The enthalpies of formation of the co-crystals are small, which indicates that the packing energy gain originates from only weak van der Waals interactions. Analysis of Hirshfeld surfaces of the felodipine molecule shows a similar distribution of intermolecular contacts in the co-crystals and pure felodipine.

Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular Cholesterol Transport.

Cholesterol transport between cellular organelles comprised vesicular trafficking and nonvesicular exchange; these processes are often studied by quantitative fluorescence microscopy. A major challenge for using this approach is producing analogs of cholesterol with suitable brightness and structural and chemical properties comparable with those of cholesterol. This review surveys currently used fluorescent sterols with respect to their behavior in model membranes, their photophysical properties, as well as their transport and metabolism in cells. In the first part, several intrinsically fluorescent sterols, such as dehydroergosterol or cholestatrienol, are discussed. These polyene sterols (P-sterols) contain three conjugated double bonds in the steroid ring system, giving them slight fluorescence in ultraviolet light. We discuss the properties of P-sterols relative to cholesterol, outline their chemical synthesis, and explain how to image them in living cells and organisms. In particular, we show that P-sterol esters inserted into low-density lipoprotein can be tracked in the fibroblasts of Niemann–Pick disease using high-resolution deconvolution microscopy. We also describe fluorophore-tagged cholesterol probes, such as BODIPY-, NBD-, Dansyl-, or Pyrene-tagged cholesterol, and eventual esters of these analogs. Finally, we survey the latest developments in the synthesis and use of alkyne cholesterol analogs to be labeled with fluorophores by click chemistry and discuss the potential of all approaches for future applications.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol‐auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE‐containing cells were exposed to membrane‐impermeant collisional quenchers (spin‐labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass‐bead lysis or repeated freeze‐thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.

Diversity of felodipine solvates: structure and physicochemical properties

Solvates of the calcium-channel blocking agent felodipine with three structurally related common organic solvents, acetone (ATN), dimethyl sulfoxide (DMSO) and acetophenone (APN), are described. A relationship between the felodipine packing arrangement in all known solvates and the van der Waals volume of the solvent molecule is established. Intermolecular interaction energies in the crystals are examined using the PIXEL approach in order to rationalize the difference between alternative molecule packing arrangements. DSC studies show that the desolvation onset temperatures of the solvates are closely comparable, despite the large difference in the boiling points of the solvent molecules. The enthalpies of formation derived from the calorimetric data for the solvates are also found to be similar, despite the difference in the van der Waals volume of the solvent molecules.

Experimental verification of a subtle low-temperature phase transition suggested by DFT-D energy minimisation

Energy minimisation of 2,6-bis(2,4-dichlorobenzylidene)cyclohexanone using dispersion-corrected Density Functional Theory (DFT-D) suggested a subtle low-temperature phase transition that has been verified by experiment. The results demonstrate that DFT-D calculations are sufficiently reliable to guide experimental studies towards targets most likely to exhibit interesting temperature-dependent structural change.

Influence of impurities on the crystallisation of 5-X-aspirin and 5-X-aspirin anhydride polymorphs (X = Cl, Br, Me)

Crystallisation of 5-X-aspirin (X = Cl, Br) by ambient evaporation from organic solvents commonly yields a mixture of polymorphs I and II for X = Cl, but only polymorph I for X = Br. Addition of 5-X-aspirin anhydride (5–20 mol %) leads to sole production of polymorph II for X = Cl, and new production of polymorph II for X = Br. Slurry experiments show that polymorph I transforms to polymorph II for X = Cl, while polymorph II transforms to polymorph I for Br. Thus, addition of the anhydride during crystallisation apparently accelerates the I → II transformation for X = Cl, and inhibits the II → I transformation for X = Br. 5-X-Aspirin anhydride can be produced as a by-product during heating of 5-X-aspirin in organic solvents, or during synthesis from 5-X-salicylic acid, and the duration of the heating step in such a synthesis can therefore change the polymorphic form of the synthesised product. The 5-X-aspirin anhydrides (X = Cl, Br, Me) are also polymorphic. Crystallisation of the pure compound from organic solvents yields only polymorph I, while polymorph II can be obtained by heating in ethanol or acetone. For X = Cl or Br, the heating step produces significant quantities of 5-X-aspirinin situ, while for X = Me, it is necessary also to add 5-X-aspirin (5–20 mol %) to obtain polymorph II.