Rational Design of Nile Red Analogs for Sensing in Membranes

Abstract

Development of next-generation fluorescent probes is a key element in the quest for a greater understanding of complex biological environments (e.g., membranes) by bioimaging. Such fluorescence-based techniques rely on specialized small molecules that possess excellent fluorescent properties but also do not perturb the native biological environment in which they reside. Herein we present a theoretical/computational strategy for the design of novel optical probes for sensing in membranes based on the parent chromophore Nile Red. Using a combination of time-dependent density functional theory (TD-DFT) and molecular dynamics (MD), we have studied the optical properties and accommodation in a model membrane of Nile Red and eight analogs. Special attention has been given to the design of probes with improved solvatochromism and two-photon absorption (2PA) without altering the membrane properties. Of the eight studied analogs, two probes were found to possess attractive probe features and are hence suggested to be taken forward to chemical synthesis and experimental exploration.

Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties

“…86 It follows that pull substituents on the northeastern part of Nile Red (ring I) effectively reduce the energy required for excitation. This behavior was predicted in our computational work 20 and is consistent with the presumed stabilizing effect of the electron-withdrawing groups in the excited charge-separated state, such that the transition energy is decreased with respect to Nile Red. The substituents' conjugation with the π system (as in the case of cyano and triflate) may also contribute to the red shift.…”

“…The high two-photon brightness of 2hydroxy derivative 7a, which is due to its large two-photon cross section (σ 2 up to 170 GM), is somewhat puzzling given that this derivative holds a strong electron-donating substituent on the eastern side, which in the context of the few-state model 94,95 ought to diminish the difference and transition dipole moments, and therefore reduce the two-photon transition probability. 20 The molecules are generally 4−5-fold brighter in toluene and chloroform (Φσ 2 = 18.2−78.6) than in methanol (Φσ 2 = 2.9−13.6) due to similar percentwise reductions in the σ 2 and Φ values. The 10-fluoro and 1hydroxy derivatives (compounds 7n and 7c) are special in this regard in that they are inherently dark and therefore unfit for 2PEF-based microscopyespecially the latter.…”

“…In fact, peripheral substitutions appear to have the greatest effect on the photophysical properties according to our preceding computational study. 20 In the present work, we have explored the chemical and optical nature of a new series of substituted 9-diethylbenzo[a]phenoxazin-5-ones. Such new analogues of Nile Red represent an interesting expansion of the fluorescent probe toolbox and for the first time offer a systematic account of the optical consequences of substituents.…”

“…Nile Red (9-diethylbenzo­[ a ]­phenoxazin-5-one) stands out by being electrically neutral and water-insoluble since it features a carbonyl group at the 5-position that readily accepts the electron density. This makes it a strong push–pull chromophore (Scheme b), which underlies its distinct solvatochromicity , and ability to indicate e.g., cellular events in live-cell imaging. − In addition, Nile Red’s inherent lipophilicity, near-infrared (NIR) emission, large Stokes shift, two-photon absorptivity (2PA), , and low background fluorescence , (as the luminosity is quenched in aqueous (aq) media) render it a near-ideal histochemical stain for studying membranes , and lipid droplets in cells. − The increase of its exquisite solvent sensitivity and steep emission when residing in hydrophobic environments has also been employed in various forms of stochastic super-resolution localization microscopy. − …”

The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations

“…Nile red was used as a case study for rational design, through TD-DFT and MD simulations, of probe analogues for sensing in membranes [ 86 ]. All tested analogues showed a preferential location at ~1.0 nm from the center of the bilayer.…”