In this Communication, we present the synthesis and use of Ru(bpy)2(bpy-CCH)2+, a versatile synthon for the construction of more sophisticated dyads by means of click chemistry. The resulting ...chromophore–acceptor or −donor complexes have been studied by flash photolysis and are shown to undergo efficient electron transfer to/from the chromophore. Additionally, the photophysical and chemical properties of the original chromophore remain intact, making it a very useful component for the preparation of visible-light-active dyads.
Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is ...performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths.
Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this ...pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.
Trehalose-based probes are useful tools that allow the detection of the mycomembrane of mycobacteria through the metabolic labeling approach. Trehalose analogues conjugated to fluorescent probes can ...be used, and other probes are functionalized with a bioorthogonal chemical reporter for a two-step labeling approach. The synthesis of such trehalose-based probes mainly relies on the desymmetrization of natural trehalose using a large number of regioselective protection-deprotection steps to differentiate the eight hydroxyl groups. Herein, in order to avoid these time-consuming steps, we reinvestigated our previously reported tandem protocol mediated by FeCl
·6H
O, with the aim of modifying the ratio of the products to allow the challenging desymmetrization of the
-symmetrical disaccharide trehalose. We demonstrate the usefulness of this method in providing easy access to trehalose analogues with a bioorthogonal moiety or a fluorophore in C-2, and also present their use in a one-step and two-step labeling approach, either of which can be used to study the mycomembrane in live mycobacteria.
SUMMARY
Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans ...because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live‐cell labeling. It consists of the incorporation of a carbohydrate containing a bio‐orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip‐polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long‐term dynamics of labeled extracellular polysaccharides.
Significance Statement
This paper reports a protocol for the metabolic‐click mediated dynamic imaging of a pollen tube, a rapidly elongating plant cell. This method results in the fluorescent labelling of cell wall polysaccharides in living cells, allowing the study of their long‐term dynamics.
Hydrogen peroxide (H
O
) is responsible for numerous damages when overproduced, and its detection is crucial for a better understanding of H
O
-mediated signaling in physiological and pathological ...processes. For this purpose, various "off-on" small fluorescent probes relying on a boronate trigger have been prepared, and this design has also been involved in the development of H
O
-activated prodrugs or theranostic tools. However, this design suffers from slow kinetics, preventing activation by H
O
with a short response time. Therefore, faster H
O
-reactive groups are awaited. To address this issue, we have successfully developed and characterized a prototypic borinic-based fluorescent probe containing a coumarin scaffold. We determined its in vitro kinetic constants toward H
O
-promoted oxidation. We measured 1.9 × 10
m
⋅s
as a second-order rate constant, which is 10,000-fold faster than its well-established boronic counterpart (1.8 m
⋅s
). This improved reactivity was also effective in a cellular context, rendering borinic acids an advantageous trigger for H
O
-mediated release of effectors such as fluorescent moieties.
In plants, 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan‐II (RG‐II). Incubation of 4‐day‐old light‐grown Arabidopsis ...seedlings or tobacco BY‐2 cells with 8‐azido 8‐deoxy Kdo (Kdo‐N₃) followed by coupling to an alkyne‐containing fluorescent probe resulted in the specific in muro labelling of RG‐II through a copper‐catalysed azide–alkyne cycloaddition reaction. CMP‐Kdo synthetase inhibition and competition assays showing that Kdo and D‐Ara, a precursor of Kdo, but not L‐Ara, inhibit incorporation of Kdo‐N₃ demonstrated that incorporation of Kdo‐N₃ occurs in RG‐II through the endogenous biosynthetic machinery of the cell. Co‐localisation of Kdo‐N₃ labelling with the cellulose‐binding dye calcofluor white demonstrated that RG‐II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo‐N₃ and an alkynated derivative of L‐fucose that incorporates into rhamnogalacturonan I, co‐localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click‐mediated labelling with Kdo‐N₃ provides an efficient method to study the synthesis and redistribution of RG‐II during root growth.
In the present work we describe the synthesis and study of a RuII–FeII chromophore–catalyst assembly designed to perform the light-induced activation of an iron bound water molecule and subsequent ...photo-driven oxidation of a substrate. Using a series of spectroscopic techniques, we demonstrate that excitation of the chromophore unit with 450 nm light, in the presence of a sacrificial electron acceptor, triggers a cascade of electron transfers leading to the formation of a high valent iron(IV)–oxo center from an iron(II)-bound water molecule. The activity of this catalytic center is illustrated by the oxidation of triphenyl phosphine.
Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence ...of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture‐based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O‐antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.
Tracking a killer: Almost 40 years after its first identified outbreak in Philadelphia, Legionella pneumophila remains difficult to track. Metabolic lipopolysaccharide labeling with a specific monosaccharide allows detection and identification of living representatives of this dangerous pathogen. Notably other Legionella species are not labeled using this method.
Phenylenediamine-catalyzed click chemistry leads to the efficient, practical, and column-free preparation of neoglycoconjugates from unprotected glucosyl azide, in pure water when aglycon solubility ...permits.
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