TY - JOUR A1 - Meyners, Christian A1 - Mertens, Monique A1 - Wessig, Pablo A1 - Meyer-Almes, Franz-Josef T1 - A Fluorescence-Lifetime-Based Binding Assay for Class IIa Histone Deacetylases JF - Chemistry - a European journal N2 - Class IIa histone deacetylases (HDACs) show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, class IIa HDACs like bromodomains have been suggested to act as “Readers” of acetyl marks, whereas enzymatically active HDACs of class I or IIb are called “Erasers” to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small-molecule ligands of class IIa histone deacetylases (HDACs) have gained tremendous attention during the last decade and have been suggested as pharmaceutical targets in several indication areas such as cancer, Huntington's disease and muscular atrophy. Up to now, only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against class IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes that involves a simple mix-and-measure procedure and an extraordinarily robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The principle of the assay is generic and can also be transferred to class I HDAC8. KW - drug discovery KW - enzymes KW - fluorescent probes KW - high-throughput screening KW - hydrolases Y1 - 2017 U6 - https://doi.org/10.1002/chem.201605140 SN - 0947-6539 SN - 1521-3765 VL - 23 IS - 13 SP - 3107 EP - 3116 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Büchner, Dörthe A1 - John, Leonard A1 - Mertens, Monique A1 - Wessig, Pablo T1 - Detection of dsDNA with [1,3]Dioxolo[4,5-f]benzodioxol (DBD) Dyes JF - Chemistry - a European journal N2 - DBD fluorescent dyes have proven to be useful in numerous applications. To widen the range of biological applications, we propose three different types of DBD molecules that have been modified in such a way that DNA interaction becomes probable. After the successful synthesis of all three compounds, we tested their fluorescent properties and their DNA binding abilities. Two of the three probes exhibit an interaction with dsDNA with subsequent fluorescence enhancement. The determined binding constants of the two new DNA dyes are comparable to other minorgroove-binding dyes. Their large Stokes shifts and their long fluorescent lifetimes are outstanding features of these dyes. KW - DNA recognition KW - dyes/pigments KW - fluorescent probes KW - heterocycles KW - scatchard plot Y1 - 2018 U6 - https://doi.org/10.1002/chem.201804057 SN - 0947-6539 SN - 1521-3765 VL - 24 IS - 60 SP - 16183 EP - 16190 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Schwarze, Thomas A1 - Riemer, Janine A1 - Müller, Holger A1 - John, Leonard A1 - Holdt, Hans-Jürgen A1 - Wessig, Pablo T1 - Na+ Selective Fluorescent Tools Based on Fluorescence Intensity Enhancements, Lifetime Changes, and on a Ratiometric Response JF - Chemistry - a European journal N2 - Over the years, we developed highly selective fluorescent probes for K+ in water, which show K+-induced fluorescence intensity enhancements, lifetime changes, or a ratiometric behavior at two emission wavelengths (cf. Scheme 1, K1-K4). In this paper, we introduce selective fluorescent probes for Na+ in water, which also show Na+ induced signal changes, which are analyzed by diverse fluorescence techniques. Initially, we synthesized the fluorescent probes 2, 4, 5, 6 and 10 for a fluorescence analysis by intensity enhancements at one wavelength by varying the Na+ responsive ionophore unit and the fluorophore moiety to adjust different K-d values for an intra- or extracellular Na+ analysis. Thus, we found that 2, 4 and 5 are Na+ selective fluorescent tools, which are able to measure physiologically important Na+ levels at wavelengths higher than 500 nm. Secondly, we developed the fluorescent probes 7 and 8 to analyze precise Na+ levels by fluorescence lifetime changes. Herein, only 8 (K-d=106 mm) is a capable fluorescent tool to measure Na+ levels in blood samples by lifetime changes. Finally, the fluorescent probe 9 was designed to show a Na+ induced ratiometric fluorescence behavior at two emission wavelengths. As desired, 9 (K-d=78 mm) showed a ratiometric fluorescence response towards Na+ ions and is a suitable tool to measure physiologically relevant Na+ levels by the intensity change of two emission wavelengths at 404 nm and 492 nm. KW - crown compounds KW - fluorescence lifetime KW - fluorescent probes KW - ratiometric KW - sodium Y1 - 2019 U6 - https://doi.org/10.1002/chem.201902536 SN - 0947-6539 SN - 1521-3765 VL - 25 IS - 53 SP - 12412 EP - 12422 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Schwarze, Thomas A1 - Mertens, Monique A1 - Mueller, Peter A1 - Riemer, Janine A1 - Wessig, Pablo A1 - Holdt, Hans-Jürgen T1 - Highly K+-Selective Fluorescent Probes for Lifetime Sensing of K+ in Living Cells JF - Chemistry - a European journal N2 - The new K+-selective fluorescent probes 1 and 2 were obtained by Cu-I-catalyzed 1,3-dipolar azide alkyne cycloaddition (CuAAC) reactions of an alkyne-substituted [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ester fluorophore with azido-functionalized N-phenylaza-18-crown-6 ether and N-(o-isopropoxy) phenylaza-18-crown-6 ether, respectively. Probes 1 and 2 allow the detection of K+ in the presence of Na+ in water by fluorescence enhancement (2.2 for 1 at 2000mm K+ and 2.5 for 2 at 160mm K+). Fluorescence lifetime measurements in the absence and presence of K+ revealed bi-exponential decay kinetics with similar lifetimes, however with different proportions changing the averaged fluorescence decay times ((f(av))). For 1 a decrease of (f(av)) from 12.4 to 9.3ns and for 2 an increase from 17.8 to 21.8ns was observed. Variation of the substituent in ortho position of the aniline unit of the N-phenylaza-18-crown-6 host permits the modulation of the K-d value for a certain K+ concentration. For example, substitution of H in 1 by the isopropoxy group (2) decreased the K-d value from >300mm to 10mm. 2 was chosen for studying the efflux of K+ from human red blood cells (RBC). Upon addition of the Ca2+ ionophor ionomycin to a RBC suspension in a buffer containing Ca2+, the fluorescence of 2 slightly rose within 10min, however, after 120min a significant increase was observed. KW - electron transfer KW - fluorescence lifetime KW - fluorescent probes KW - living cells KW - potassium Y1 - 2017 U6 - https://doi.org/10.1002/chem.201704368 SN - 0947-6539 SN - 1521-3765 VL - 23 SP - 17186 EP - 17190 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Wawrzinek, Robert A1 - Ziomkowska, Joanna A1 - Heuveling, Johanna A1 - Mertens, Monique A1 - Herrmann, Andreas A1 - Schneider, Erwin A1 - Wessig, Pablo T1 - DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins JF - CHEMISTRY-A EUROPEAN JOURNAL N2 - 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 (10-20ns), large Stokes shifts (approximate to 100nm), 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. KW - dyes KW - pigments KW - electron transfer KW - fluorescent probes KW - maleimides KW - MalF KW - photoelectron transfer Y1 - 2013 U6 - https://doi.org/10.1002/chem.201302368 SN - 0947-6539 SN - 1521-3765 VL - 19 IS - 51 SP - 17349 EP - 17357 PB - WILEY-V C H VERLAG GMBH CY - WEINHEIM ER -