Chemical Probe for Imaging of Polo-like Kinase 4 and Centrioles.

Polo-like kinase (Plk4) is a serine/threonine-protein kinase that is essential for biogenesis of the centriole organelle and is enriched at centrioles. Herein, we introduce Cen-TCO, a chemical probe based on the Plk4 inhibitor centrinone, to image Plk4 and centrioles in live or fixed cultured human cells. Specifically, we established a bio-orthogonal two-step labeling system that enables the Cen-TCO-mediated imaging of Plk4 by STED super-resolution microscopy.

A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and -cyclooctene.

Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, -cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates.

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy.

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding.

Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy.

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization.

A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates.

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein-protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DA ).

Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe.

Recent developments in fluorescence microscopy call for novel small-molecule-based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live-cell single-molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell experiments.

Proximity-Induced Bioorthogonal Chemistry Using Inverse Electron Demand Diels-Alder Reaction.

Bioorthogonal chemistry techniques enable the selective and targeted manipulation of living systems. In order to yield universally applicable techniques, it is of great importance for bioorthogonal reactions to take place rapidly, selectively, and with the formation of only benign side products. One of the reactions that match these criteria well is the inverse electron demand Diels-Alder reaction (DA) between tetrazines and strained dienophiles.

Enzymatic and Site-Specific Ligation of Minimal-Size Tetrazines and Triazines to Proteins for Bioconjugation and Live-Cell Imaging.

Diels-Alder reactions with inverse electron demand (DA) have emerged as an indispensable tool for bioorthogonal labeling and the manipulation of biomolecules. In this context, reactions between tetrazines and strained dienophiles have received attention because of high reaction rates. Current methods for the DA-mediated functionalization of proteins suffer from slow reactivity, impaired stability, isomerization, or elimination of the incorporated strained dienophiles.

A Bifunctional Fluorogenic Rhodamine Probe for Proximity-Induced Bioorthogonal Chemistry.

Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels-Alder reaction (DA ) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions.

Green- to far-red-emitting fluorogenic tetrazine probes - synthetic access and no-wash protein imaging inside living cells.

Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging. 1,2,4,5-Tetrazines are known substrates for the bioorthogonal inverse electron demand Diels-Alder reaction (DA) and tetrazine substituted fluorophores can exhibit fluorogenic properties. Herein, we report the synthesis of a palette of novel fluorogenic tetrazine dyes derived from widely-used fluorophores that cover the entire emission range from green to far-red.

Siderophore inspired tetra- and octadentate antenna ligands for luminescent Eu(III) and Tb(III) complexes.

Following the success of the siderophore-inspired 1,2-hydroxypyridonate (HOPO) and 2-hydroxisophthalamide (IAM) chromophores in Eu(III) and Tb(III) luminescence, we designed three new ligands bearing both chromophores. Syntheses of the octadentate ligands 3,4,3-LI-IAM-1,2-HOPO and 3,4,3-LI-1,2-HOPO-IAM, where the chromophores are attached to different positions in the (LI=linear) spermine backbone, are reported in addition to a tetradentate ligand based on 1,5-diaminopentane. The Gd(III) complexes were prepared and revealed localized triplet states typical for the IAM and HOPO chromophores.