31 research outputs found
Comparative analysis of lanthanide excited state quenching by electronic energy and electron transfer processes
The relative sensitivities of structurally related Eu(III) complexes to quenching by electron and energy transfer processes have been compared. In two sets of 9-coordinate complexes based on 1,4,7-triazacyclononane, the Eu emission lifetime decreased as the number of conjugated sensitising groups and the number of unbound ligand N atoms increased, consistent with photoinduced electron transfer to the excited Eu(III) ion that is suppressed by N-protonation. Quenching of the Eu 5D0 excited state may also occur by electronic energy transfer, and the quenching of a variety of 9-coordinate complexes by a cyanine dye with optimal spectral overlap occurs by an efficient FRET process, defined by a Förster radius (R0) value of 68 Å and characterised by second rate constants in the order of 109 M−1 s−1; these values were insensitive to changes in the ligand structure and to the overall complex hydrophilicity. Quenching of the Eu and Tb excited states by energy transfer to Mn(II) and Cu(II) aqua ions occurred over much shorter distances, with rate constants of around 106 M−1 s−1, owing to the much lower spectral overlap integral. The calculated R0 values were estimated to be between 2.5 to 4 Å in the former case, suggesting the presence of a Dexter energy transfer mechanism that requires much closer contact, consistent with the enhanced sensitivity of the rate of quenching to the degree of steric shielding of the lanthanide ion provided by the ligand
EuroTracker (R) dyes: design, synthesis, structure and photophysical properties of very bright europium complexes and their use in bioassays and cellular optical imaging
The development of the brightest luminescent europium(III) complexes is traced, including analysis of
the C3-symmetric core complex based on a functionalized triazacyclononane and identification of the
most suitable strongly absorbing chromophore. Strategies for the synthesis of the complexes, including
enantiopure analogues, are outlined and opportunities for applications in time-resolved microscopy and
spectral imaging emphasised. Practicable examples are introduced, including selective organelle staining
for cellular optical imaging at 65 nm resolution and the development of new bioassays using time resolved
FRET methods
Comparative analysis of conjugated alkynyl chromophore-triazacyclononane ligands for sensitized emission of europium and terbium
A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors
A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells
In the last two decades, new technologies based on luminescence have been developed to monitor the
organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the
overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However,
there is an increasing interest in developping approaches to conjugate chemical labels to specific residues
of native GPCRs, despite of the low reactivity and the high abundance of such residues.Peer reviewe
A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment.Funding
Agence Nationale de la Recherche
ANR-17-CE11-0046
Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe
CTQ2017-89222-R
Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe
PCI2018-093047
Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe
PID2020-120499RB-I00
Catalan government
2017 SGR 1604
the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement No. 801342 (TecniospringINDUSTRY) and the Government of Catalonia's Agency for Business Competitive-ness (ACCIÓ).
TECSPR19-1-0062Peer reviewe
A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells
Peer reviewe
A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells
Peer reviewe
A MODULAR LIGAND-DIRECTED APPROACH TO LABEL ENDOGENOUS AMINERGIC GPCRS IN LIVE CELLS
In the last two decades, new technologies based on luminescence have been developed
to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These
technologies rely on the overexpression of genetically modified (and/or fluorescently tagged)
receptors of interest. However, there is an increasing interest in developping approaches to
conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and
the high abundance of such residues.
Ligand directed approaches, may offer a solution to this problem. Such approaches
consist in molecular probes that include a selective ligand moiety and a reactive moiety. Upon
ligand binding, the labelling is directed to a nucleophilic amino acid in the vicinity of the binding
pocket. However, this requires the use of non-nucleophilic ligand moieties, which is particularly
difficult since many GPCR ligands contain amines or other nucleophilic functional groups.
In the present work, we developed an innovative ligand-directed toolbox based on a novel
approach. The later uses molecular modules to build fluorescent ligand-directed probes to label
an archetypical aminergic GPCR (D1R). Our molecular probes can be readily prepared before
the labelling reaction from two molecular modules: an activated electrophilic linker which
includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to
a fast and specific chemical reaction, the nucleophilic ligand can barely react with the activated
linker before it is bound to the native target GPCR and the labelling reaction occurs.
Subsequently, the ligand will unbind the GPCR pocket, leaving the receptor fluorescently
labelled and fully functional.
This novel labelling approach allowed us to label endogenous D1 receptor both in
transfected cells and primary cultures of neurons and will pave the way to develop new
reagents and assays to monitor endogenous GPCRs distribution and activity in their native
environment.Peer reviewe
A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells
In the last two decades, new technologies based on luminescence have been developed to monitor the
organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the
overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However,
there is an increasing interest in developping approaches to conjugate chemical labels to specific residues
of native GPCRs, despite of the low reactivity and the high abundance of such residues.Peer reviewe