DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins

Abstract

Previously, [1,3]dioxolo[4,5‐f][1,3]benzodioxole (DBD)‐based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10–20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio‐reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP‐binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

Na+ Selective Fluorescent Tools Based on Fluorescence Intensity Enhancements, Lifetime Changes, and on a Ratiometric Response

“…[5d, 7b,c, 8] In af urther step, we measured the fluorescence response towards K + and found as maller influence on the fluorescencei ntensity for 2, 4, 5, 6 and 10 + K + + . We observed for 2 + 1000mm K + aF EF of 1.8, for 4 + 1000 mm K + aF EF of 1.2, for 5 + 1000 mm K + aF EF of 1.1, for 6 + 1000 mm K + aF EF of 1.3 and for 10 + 1000 mm K + aF EF of Scheme4.Synthetic route of the studied Na + responsivefluorescentp robes 1-10:a )CuSO 4 /Na ascorbate, THF/H 2 O, 60 8C, for 4 the 3-azido-7-diethylaminocoumarin, [12a] for 2 and 5 the 3-azido-9-methyl-6,7,8,9-tetrahydro-2H-pyrano [3,2-g]quinolin-2-one, [13] for 6 the 10-azido-2,3,6,7-tetrahydro-1 H,5 H,11Hpyrano[2,3-f]pyrido [3,2,1-ij]quinolin-11-one, [12a] for 7 and 8 the diethyl 2-[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]benzo [1,2-d:4,5-d']bis [1,3]dioxole-4,8-dicarboxylate, [10] for 9 the commercial available 1-ethynylpyrene,and for 10 the 3-ethynyl-7-diethylcoumarin, [12b] respectively. 1.9.…”

“…Both consist of aD BD fluorophore, which exhibits ah igh quantum yield (f f = % 0.60) and an extraordinary fluorescencel ifetime (t f = % 20 ns) in water. [10] These probes 7 and 8 are also PET fluoroionophores, which show FEs at as ingle wavelength and moderate fluorescencel ifetime changes in the presence of Na + . Further,t oo btain ratiometric fluorescentp robes, we synthesized probe 9,w hich consists of aN a + selective ionophore (N-(o-methoxyphenyl)aza-15-crown-5) and ap yrene fluorophore connected by a1 ,2,3-triazole unit.…”

Preparation, Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme

“…Very similar trends in the fluorescence lifetimes of [1,3]dioxolo[4,5- f ][1,3]benzodioxole (DBD) dyes have also been observed [26] and used to probe conformational changes in maltose ATP-binding cassette (ABC) transporter. [27] Specifically, the fluorescence lifetime of a maleimide-substituted DBD dye linked to a cysteine mutant of the MalF subunit of the MalFGK 2 complex increased from 5 ns to 6.3 ns when MalF was bound with vanadate, which locks the enzyme in a conformation that is proposed to generate a more hydrophobic environment [28] around the DBD dye. A similar increase in the fluorescence lifetime of Acr + -Mes in POP-ZA 4 - 5 (Table 2, entry 4) relative to that in solution (entry 3) or on the outside of the protein (POP-Z37- 5 , entry 5) suggests that 5 resides within the hydrophobic POP active site of POP-ZA 4 - 5 .…”

A Fluorescence‐Lifetime‐Based Binding Assay for Class IIa Histone Deacetylases

“…For the synthesis of the DBD ligands, a convergent route, shown in Scheme , was chosen. The DBD amines 6 and 7 were synthesised by the coupling of the known acyl and ester DBD acids 1 and 2 to N ‐Boc‐protected ethylenediamine ( 3 ) followed by deprotection of the intermediates 4 and 5 catalysed by trifluoroacetic acid (TFA) . For the TFMK building blocks, the synthesis of 12 a , b started with commercially available glutaric acid diethyl ester ( 10 a ) and adipic acid monomethyl ester ( 10 b ).…”