Polymorphism, Structure, and Nucleation of Cholesterol·H2O at Aqueous Interfaces and in Pathological Media: Revisited from a Computational Perspective

Abstract

We revisit the important issues of polymorphism, structure, and nucleation of cholesterol·H₂O using first-principles calculations based on dispersion-augmented density functional theory. For the lesser known monoclinic polymorph, we obtain a fully extended H-bonded network in a structure akin to that of hexagonal ice. We show that the energy of the monoclinic and triclinic polymorphs is similar, strongly suggesting that kinetic and environmental effects play a significant role in determining polymorph nucleation. Furthermore, we find evidence in support of various O-H···O bonding motifs in both polymorphs that may result in hydroxyl disorder. We have been able to explain, via computation, why a single cholesterol bilayer in hydrated membranes always crystallizes in the monoclinic polymorph. We rationalize what we believe is a single-crystal to single-crystal transformation of the monoclinic form on increased interlayer growth beyond that of a single cholesterol bilayer, interleaved by a water bilayer. We show that the ice-like structure is also relevant to the related cholestanol·2H₂O and stigmasterol·H₂O crystals. The structure of stigmasterol hydrate both as a trilayer film at the air-water interface and as a macroscopic crystal further assists us in understanding the polymorphic and thermal behavior of cholesterol·H₂O. Finally, we posit a possible role for one of the sterol esters in the crystallization of cholesterol·H₂O in pathological environments, based on a composite of a crystalline bilayer of cholesteryl palmitate bound epitaxially as a nucleating agent to the monoclinic cholesterol·H₂O form.

Scheme 1. (A) Molecular Structure of Cholesterol; (B) Schematic Representation of a Bilayer Composed of Phosphoglycerolipids (Red Arrowhead) and Cholesterol Molecules (Red Arrow); above a Critical Concentration for Cholesterol, Two-Dimensional (2D) Cholesterol Crystalline Domains are Formed

Figure 1

Packing arrangement of triclinic cholesterol·H₂O, viewed along the: a-axis (A), b-axis (B), and c-axis (C_1,2). In (C_1,2), the packing arrangement is limited to the hydrophilic region indicated in A. The atoms are color-coded in white, H; brown, C; and red, O. The different H-bonded rings in panel C₁ are labeled in grey by r_i and R_i, which refer to tetragons and octagons, respectively; the subscript i = 1···4 designates the unique polygons of each type. The arrangement in all panels, except for C₂, is for the lowest energy pseudopolymorph of the triclinic cholesterol·H₂O. The C₂ panel arrangement is of the hydrophilic region of the disordered mixture of eight H-bonding networks with partial occupation indicated by partial coloring of pertinent atoms in white. Exclusively in C₂, H atoms are colored in grey to avoid confusion with the color code used for partial occupation. OH···O bonds are represented as grey dashed lines. The unit cell is delineated by a black rectangle.

Figure 2

Packing arrangement of the monoclinic cholesterol·H₂O unit cell, viewed along the: a-axis (A), b-axis (B_1,2), and c-axis (C_0,1,2) for the hydrophilic region indicated in (A). For panels (A,B₁,C₀,C₁) the H, C, and O atoms are color-coded in white, brown, and red, respectively. Panels (B₂,C₂) present similar views as (B₁,C₁), respectively, but with different colors representing different symmetry-unrelated cholesterol and water molecules: grey, cholesterol molecule A (mol. A); orange, cholesterol molecule B (mol. B); and blue (W₁) and green (W₂), water molecules. The twofold and twofold screw symmetry axes are shown in black. Given that the exocyclic moieties of the non-symmetry related molecules A and B are part of a pseudo C-centered arrangement (see main text), the row of twofold axes along a are interleaved by pseudo twofold screw axes (indicated by black and white stripes). The hexagonal H-bonded rings in panel C₂ are labeled by R₁ and R₂. The arrangement in all the panels except for C₀ is for the lowest energy pseudopolymorph of the monoclinic cholesterol·H₂O. The C₀ panel arrangement is of the hydrophilic region of the disordered mixture of the three most stable H-bonding networks, with partial occupation indicated by partial coloring of pertinent atoms. Exclusively in C₀, H atoms are colored in grey to avoid confusion with the color code used for partial occupation. OH···O bonds are represented as grey dashed lines. The unit cell is indicated by a black rectangle.

Figure 3

Temperature dependence of the d-spacing of monoclinic cholesterol·H₂O measured by electron diffraction (ED) and grazing incidence X-ray diffraction (GIXD) and compared to calculations at the PBE-TS level of theory.

Figure 4

Views of the H-bonded bilayers of cholesterol·H₂O and cholestanol·2H₂O. Top and side views of (A) Hexagonal H-bonding bilayer in the structure of hexagonal ice, in which the O–H···O bonds are proton disordered and (B) disordered mixture of the three most stable H-bonding networks of monoclinic cholesterol·H₂O. (C) Disordered mixture of the 8 H-bonding networks of triclinic cholesterol·H₂O. (D) Model H-bonded network of cholestanol·2H₂O. Also shown is a drawing of cholestanol·2H₂O crystals taken from the PhD thesis of D. Hodgkin (1937), reproduced by permission of the Hodgkin family. The two crystals are elongated, twinned about the [010] direction, and show mainly the (001) face with minor (100), (010), and (110) side faces. The C and O atoms are color-coded brown and red, respectively, with partial occupation indicated by partial coloring of pertinent atoms. In panels (A–C), H atoms are colored in grey to avoid confusion with the color code used for partial occupation.

Figure 5

Model of the trilayer packing arrangement of stigmasterol hydrate on a water surface, based on an analysis of GIXD measurements thereof^, (Figure S14), on the sigmasterol·H₂O crystal structure (Figure S13B,C), and on the model structure of cholestanol dihydrate (Figures 4D and S12). The trilayer structure viewed along the a-axis incorporates an ordered layer of water molecules whose ice-like hydration structure is probably a double bilayer similar to cholestanol dihydrate. Both structures embody the 10 × 7.5 Ų monoclinic 2₁ motif.

Scheme 2. Cholesteryl Esters

The palmitate derivative has a saturated aliphatic chain (C₁₆H₃₁); the chain of the oleate derivative (C₁₈H₃₃) contains a C=C double bond with a cis configuration; the linoleate has a chain (C₁₈H₃₁) with two C=C double bonds, both with the cis configuration.

Figure 6

Simulation of a composite crystal in which the cholesteryl palmitate interdigitated bilayer is epitaxially bound to monoclinic cholesterol molecules across twofold axes. As a bilayer, cholesteryl palmitate is found to be sufficiently stable and could therefore nucleate the monoclinic crystals of cholesterol·H₂O by epitaxy.