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Principles Of Fluorescence Spectroscopy

Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular “core” dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescence-based technologies in chemical biology elicits a necessity to understand the major classes of small-molecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores and highlight classic and contemporary examples in which utility has been built upon these scaffolds.

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Bright Ideas for Chemical Biology

Dimeric and oligomeric surfactants. Behavior at interfaces and in aqueous solution: a review.

Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules.

For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids.

The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings.

Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review.

This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

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Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

https://escholarship.org/content/qt4393381q/qt4393381q.pdf?t=oevwsx

Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence

Bis(quaternary ammonium halide) surfactants (gemini surfactants) having, variously, diethyl ether, monohydroxypropyl, and dihydroxybutyl spacer groups have been investigated by surface tension, interfacial tension, and steady-state fluorescence techniques. The critical micelle concentration (cmc) and area per molecule (Amin) are shown to deviate from the expected patterns of behavior as the number of carbon atoms in the alkyl chain (n) increases beyond a certain maximum. This aberrant behavior is observed at the hydrocarbon/water as well as the aqueous/air interface. The unexpected values of the physicochemical parameters at long alkyl chain length have been interpreted on the basis of a concentration region in which submicellar or multilayer structures are forming. Fluorescence measurements provide confirmation of cmc values by an alternative technique. Comparison of the fluorescence emission maxima profiles of the gemini surfactants with those of their monoquaternary analogues demonstrates that there is...

Aberrant Aggregation Behavior in Cationic Gemini Surfactants Investigated by Surface Tension, Interfacial Tension, and Fluorescence Methods

A fluorescent phospholipid derivative, the fluoresceinthiocarbamyl adduct of a natural phosphatidylethanolamine, has been synthesized and incorporated into sonicated single-bilayer vesicles of egg lecithin and dipalmitoyllecithin. The surface location of this probe has been confirmed by using extrinsic fluorescence quenching studies together with steady-state emission anisotropy measurements. Electronic excitation energy transfer between 1,6-diphenyl-1,3,5-hexatriene incorporated within the hydrophobic core of the bilayer and the novel derivative has been investigated to estimate the depth within the bilayer at which the former is located. Efficiencies have been measured for two different phospholipids, egg lecithin and dipalmitoyllecithin, in the latter case both above and below the phospholipid phase transition, with and without added cholesterol. The observed dependence of the transfer efficiency on the acceptor concentration was compared with that calculated according to Forster theory applied to random two-dimensional distributions of donor and acceptor molecules in parallel planes for various interplanar separations, taking into account orientational effects. The Forster R0 of about 45 A for this donor-acceptor pair is particularly well suited to such studies since it is of the order of the width of the bilayer. The experiments showed that energy-transfer spectroscopy can provide useful quantitative information as to the transverse location of diphenylhexatriene in homogeneous phospholipid bilayers and may also reflect lateral partitioning of donor or of both donor and acceptor into different phases in systems exhibiting phase separations.

Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer.

The aggregation behavior of four series of bis(quaternary ammonium halide) surfactants (gemini surfactants) having diethyl ether, dihydroxybutyl, monohydroxypropyl, and dimethylene phenylene spacer groups has been studied using steady-state and time-resolved fluorescence spectroscopy. Aggregation numbers were determined using the time-resolved single photon counting method with pyrene as the probe. At certain surfactant concentrations, aggregation numbers of 2, expressed as gemini molecules per micelle, were obtained in all four series when the number of carbon atoms in the alkyl chain length (n) increased beyond a maximum. These long-chain geminis also have a critical micelle concentration (cmc) greater than expected on the basis of plots of log cmc vs n for the shorter chain homologues. This deviation has been attributed to the formation of premicellar aggregates in the surfactant concentration region between the observed and the expected cmc values. The aggregation numbers obtained here indeed point to...

Fluorescence Study of Premicellar Aggregation in Cationic Gemini Surfactants

The two-state excited-state proton-transfer process for d-equilenin [d-3-hydroxyestra-1,3,-5(10),6,8-pentaen-17-one] and dihydroequilenin is found to depend both on pH and on proton acceptor concentration. Both the protonated and deprotonated forms of the excited molecule are fluorescent. As is the case for 2-naphthol, the excited-state pKa (pKa*) is substantially lower than the ground-state pKa. Fluorescence decay studies have been performed as a function of emission wavelength in aqueous solutions at pH 6.9 in the presence of acetate anion (0.1 M). At this pH, both back-reaction from the excited-state and ground-state heterogeneity are minimal. A monoexponential decay is found in the blue region of the spectrum and a biexponential decay on the red edge. The lifetimes measured across both regions are constant, with a negative preexponential term, characteristic of an excited-state reaction, evident at longer wavelengths. Decay-associated spectra (DAS), the preexponential terms associated with the measured lifetimes, have been acquired for these aqueous solutions. Equilenin and dihydroequilenin are found to adsorb to dimyristoyllecithin (DML) vesicles. Rates for excited-state proton transfer are greatly reduced when dihydroequilenin adsorbs to vesicles. The accessibility of the bound probe to acetate as a proton acceptor depends on the cholesterol content of the vesicles.

Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles.

Individual fluorescence spectra for species in a heterogeneous system can be determined by using differences between the rotational correlation times of those components. Each spectrum derived is associated with a particular fluorescence anisotropy decay function; hence, they are anisotropy decay associated spectra (ADAS). We have previously shown [Knutson, J. R., Walbridge, D. G., & Brand, L. (1982) Biochemistry 21, 4671-4679] that a system containing different decay functions for total intensity can be resolved into constituent decay-associated spectra. ADAS extends the technique into the realm of fluorescence polarization, making use of the often disparate Brownian rotations found in heterogeneous biochemical systems. In this paper, we present the basic theory for ADAS in various heterogeneous systems and then present an example of ADAS resolving a binary mixture of macromolecules into "fast-rotor" (smaller or more mobile) and "slow-rotor" (larger or less mobile) components. They correctly superimpose spectra taken for the unmixed components. In the companion paper [Davenport, L., Knutson, J. R., & Brand, L. (1986) Biochemistry (following paper in this issue)], a specific application to a problem of importance of lipid biochemistry--e.g., the origin of the membrane probe order parameter in lipid bilayers--is presented, demonstrating the role rotational heterogeneity may play in biochemical fluorescence.

Lesley Davenport

Papers

Aberrant Aggregation Behavior in Cationic Gemini Surfactants Investigated by Surface Tension, Interfacial Tension, and Fluorescence Methods

Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer.

Fluorescence Study of Premicellar Aggregation in Cationic Gemini Surfactants

Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles.

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 1. Theory and applications.