Design of new fluorescent cholesterol and ergosterol analogs: Insights from theory

Abstract

Cholesterol (Chol) and ergosterol (Erg) are abundant and important sterols in the plasma membrane of mammalian and yeast cells, respectively. The effects of Chol and Erg on membrane properties, as well as their intracellular transport, can be studied with use of fluorescence probes mimicking both sterols as closely as possible. In the search for new and efficient Chol and Erg probes, we use a combination of theoretical methods to explore a series of analogs. The optical properties of the analogs (i.e. excitation energies, emission energies and oscillator strengths) are examined using time-dependent density functional theory (TDDFT) and their ability to mimic the effects of Chol and Erg on membranes is investigated with molecular dynamics (MD) simulations of each analog in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. From the set of analogs we find two probes (3a and 3b) to display favorable electronic transition properties as well as strong condensing abilities. These findings can lead to the use of new efficient probes and aid in the understanding of the structural features of Chol and Erg that impart to them their unique effects on lipid membranes.

Live‐cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

“…1, panels A and B in Wüstner, ). This is further supported by an only small increase in dipole moment of DHE during excitation (Smutzer et al ., ), a property which is preserved for polyene sterols with more double bonds, as we have shown previously by electronic structure calculations (Nåbo et al ., ). Accordingly, only low environmental sensitivity of emitted fluorescence is expected for polyene sterols.…”

“…In particular, it could be interesting to aim for introducing a fifth conjugated double bond to further shift the excitation and emission maxima into the visible range. Such a fifth double bond should be introduced between carbons 1 and 2 in ring A – any other position would require removing a methyl group which, according to our previously published results, would negatively influence the cholesterol‐mimicking properties of the probe (Nåbo et al ., ). Extending the conjugated system to five double bonds should also increase the relative stability of the enol tautomeric form due to an increased influence of the resonance effect.…”

“…As the energy difference between electronic ground and excited states grows smaller with increasing number of conjugated bonds, addition of conjugated bond shifts the excitation and emission maxima towards longer wavelengths. Thus, a suitable strategy for improving upon existing intrinsically fluorescent sterol probes is to extend the conjugated system in the steroid ring system, and we have shown in a recent theoretical study that this should preserve the cholesterol-and ergosterollike properties of such analogues (Nåbo et al, 2015). Here, we have synthesized two intrinsically fluorescent sterols with four conjugated double bonds in the ring system and performed characterization of these species, as well as theoretical calculations of their structures, absorption maxima and the energetic difference between the keto and the enol tautomers.…”

Dyes as guests in ordered systems: current understanding and future directions

“…Some equivalent studies have also been carried out for biological systems, combining MD simulations and electronic structure calculations to determine the orientation of probes in a phospholipid membrane, [100,101] and in the context of designing fluorescent probes to mimic the alignment behaviour of biological molecules. [102] A further comparative study of five anthraquinone dyes proposed for use within guest-host systems has provided insights into structure-property relationships of the dyes. [103] Variations in dye colour have been rationalised in terms of the electronic properties of the dye substituents, and reported trends in absorption anisotropy have been rationalised in terms of TDM orientations and molecular aspect ratios, determined from the results of DFT and TD-DFT calculations.…”

Structural design of intrinsically fluorescent oxysterols

“…However, spectroscopic charac- (about half the energy) and can thereby be used for live-cell imaging over extended periods of time [25,28]. Probe 25-OH-3a is inspired by our previous work on cholesterol probes [29,30]. Here, we also consider the keto form of this probe because we found that probe 3a can undergo keto-enol tautomerization [31], and this is probably also the case for 25-OH-3a.…”