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Combining bioengineering with chemical synthesis has enabled an efficient method for producing Δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of Δ7-dafachronic acid.

Molecular dynamics simulations of bilayer systems consisting of varying proportions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), and intrinsically fluorescent Chol analogues dehydroergosterol (DHE) or cholestatrienol (CTL) were carried out to study in detail the extent to which these fluorescent probes mimic Chol's behavior (location, orientation, dynamics) in membranes as well as their effect on host bilayer structure and dynamics (namely their ability to induce membrane ordering in comparison with Chol). Control properties of POPC and POPC/Chol bilayers agree well with published experimental and simulation work. Both probes and Chol share similar structural and dynamical properties within the bilayers. Additionally, the fluorescent sterols induce membrane ordering to a similar (slightly lower) extent to that of Chol. These findings combined demonstrate that the two studied fluorescent sterols are adequate analogues of Chol, and may be used with advantage over side-chain labeled sterols. The small structural differences between the three studied sterols are responsible for the slight variations in the calculated properties, with CTL presenting a more similar behavior to Chol (correlating with its larger structural similarity to Chol) compared to DHE.

The transverse motion of molecules from one leaflet to the other of a lipid bilayer, or flip-flop, represents a putative mechanism for their transmembrane transport and may contribute to the asymmetric distribution of components in biomembranes. However, a clear understanding of this process is still missing. The scarce knowledge derives from the difficulty of experimental determination. Because of its slow rate on the molecular time scale, flip-flop is challenging also for computational techniques. Here, we report a study of the passive transbilayer diffusion of steroids, based on a kinetic model derived from the analysis of their free energy surface, as a function of their position and orientation in the bilayer. An implicit membrane description is used, where the anisotropy and the nonuniformity of the bilayer environment are taken into account in terms of the gradients of density, dielectric permittivity, acyl chain order parameters, and lateral pressure. The flip-flop rates are determined by solving the Master Equation that governs the time evolution of the system, with transition rates between free energy minima evaluated according to the Kramers theory. Considering various steroids (cholesterol, lanosterol, ketosterone, 5-cholestene, 25-hydroxycholesterol, and testosterone), we can discuss how differences in molecular shape and polarity affect the pathway and the rate of flip-flop in a liquid crystalline 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer, at low steroid concentration. We predict time scales ranging from microseconds to milliseconds, strongly affected by the presence of polar substituents and by their position in the molecular skeleton.

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.

Derivatives of 3beta-amino-5-cholestene (3beta-cholesterylamine) are of substantial interest as cellular probes and have potential medicinal applications. However, existing syntheses of 3beta-amino-5-cholestene are of limited preparative utility. We report here a practical method for the stereoselective preparation of 3beta-amino-5-cholestene, 3beta-chloro-5-cholestene, 3beta-bromo-5-cholestene, and 3beta-iodo-5-cholestene from inexpensive cholesterol. A sequential i-steroid/retro-i-steroid rearrangement promoted by boron trifluoride etherate and trimethylsilyl azide converted cholest-5-en-3beta-ol methanesulfonate to 3beta-azido-cholest-5-ene with retention of configuration in 93% yield.

The effect of a large increase in mass and extension of substituents on the thermal rearrangement of a 1,2-divinylcyclobutane system has been investigated by the introduction of two 3-cholest-4-ene units. The ratio of two types of exit channel, fragmentation and stereomutation (Fr/St), from a widely accepted diradical intermediary (caldera) has increased significantly relative to the ratio in a simpler system. We believe this to be the first example of the influence of a ponderal effect on the ratio of two competing processes in a thermal rearrangement. The internal torsional rotations necessary prior to reclosure for the realization of stereomutation have been markedly depressed. One of the three stereoisomeric cyclobutanes reacts substantially more slowly than the other two. In its structure, determined by X-ray diffraction, the two cholestenyl wings are folded closely together within van der Waals radii. The slower reaction may reasonably be ascribed to a relative lowering of heat of formation in this ground-state conformation.

The role of the membrane lipid composition and the individual Trp residues in the conformational rearrangement of gramicidin A along the folding pathway to its channel conformation has been examined in phospholipid bilayers by means of previously described size-exclusion high-performance liquid chromatography HPLC-based strategy (Bañó et al. (1991) Biochemistry 30, 886). It has been demonstrated that the chemical composition of the membrane influences the transition rate of the peptide rearrangement from double-stranded dimers to beta-helical monomers. The chemical modification of Trp residues, or its substitution by the more hydrophobic residues phenylalanine or naphthylalanine, stabilized the double-stranded dimer conformation in model membranes. This effect was more notable as the number of Trp-substituted residues increased (tetra > tri > di > mono), and it was also influenced by the specific position of the substituted amino acid residue in the sequence, in the order Trp-9 approximately Trp-13 > Trp-11 > Trp-15. Moreover, it was verified that nearly a full contingent of indoles (Trp-13, -11, and -9) is necessary to induce a quantitative conversion from double-stranded dimers to single-stranded monomers, although Trp-9 and Trp-13 seemed to be key residues for the stabilization of the beta-helical monomeric conformation of gramicidin A. The conformation adopted for monomeric Trp --> Phe substitution analogues in lipid vesicles resulted in CD spectra similar to the typical single-stranded beta6.3-helical conformation of gramicidin A. However, the Trp --> Phe substitution analogues showed decreased antibiotic activity as the number of Trp decreased.

The fluorescent analog NBD-phosphatidylethanolamine and the analogs of cholesterol NBD-cholesterol and cholestatrienol were used to study the distribution of these lipids within the plasma membrane bilayer of human platelets. The probes were incorporated into platelets using phosphatidylcholine donor vesicles. The distribution of NBD lipid and of cholestatrienol in the platelet plasma membrane bilayer was followed by quenching with dithionite and TNBS, respectively. The t1/2 of cholestatrienol incorporation into platelet membranes was 39 min, and approximately 65% of the probe was quenched by addition of TNBS. When platelets were exposed to collagen or to ADP, a portion of the probe became inaccessible to quenching. Within 2 min of stimulation by collagen (10 micrograms/mL), the percentage of cholestatrienol fluorescence quenched by TNBS decreased to 45%. The fluorescent probe was not found to be associated either with the intracellular membranes or in the extracellular media after collagen stimulation. Similar data were obtained with NBD-cholesterol, but the decrease in accessibility of this probe to quenching was considerably slower. The redistribution of endogenous membrane cholesterol was also measured using cholesterol oxidase. Exposure of platelets to collagen decreased the accessibility of endogenous membrane cholesterol to enzymatic oxidation with cholesterol oxidase. Taken together, the foregoing observations are consistent with the stimulus-dependent translocation of cholesterol out of the outer monolayer. Coincident with the redistribution of cholesterol is the reciprocal movement of NBD-phosphatidylethanolamine into the outer monolayer. In the presence of the chaotropic agents urea and guanidine HCl, the movement of cholestatrienol upon collagen stimulation was prevented, but the redistribution of NBD-phosphatidylethanolamine was still detected. We propose that cholesterol translocates to the inner platelet monolayer following collagen stimulation, but the possibility that the sterol moves laterally within the outer membrane monolayer cannot be rigorously excluded.