Fluorescent probes for investigating the internalisation and action of bioorthogonal ruthenium catalysts within Gram-positive bacteria.

Autor: Schubert N; Faculty of Chemistry and Biochemistry, Chair of Inorganic Chemistry I - Bioinorganic Chemistry, Ruhr University Bochum Universitätsstraße 150 44801 Bochum Germany nils.metzler-nolte@rub.de., Southwell JW; Department of Chemistry, University of York, Heslington York YO10 5DD UK., Vázquez-Hernández M; Faculty of Biology and Biotechnology, Applied Microbiology, Ruhr University Bochum Universitätsstraße 150 44801 Bochum Germany., Wortmann S; Institut für Physikalische und Theoretische Chemie, Universität Regensburg Universitätsstraße 31 93053 Regensburg Germany., Schloeglmann S; Institut für Physikalische und Theoretische Chemie, Universität Regensburg Universitätsstraße 31 93053 Regensburg Germany., Duhme-Klair AK; Department of Chemistry, University of York, Heslington York YO10 5DD UK., Nuernberger P; Institut für Physikalische und Theoretische Chemie, Universität Regensburg Universitätsstraße 31 93053 Regensburg Germany., Bandow JE; Faculty of Biology and Biotechnology, Applied Microbiology, Ruhr University Bochum Universitätsstraße 150 44801 Bochum Germany., Metzler-Nolte N; Faculty of Chemistry and Biochemistry, Chair of Inorganic Chemistry I - Bioinorganic Chemistry, Ruhr University Bochum Universitätsstraße 150 44801 Bochum Germany nils.metzler-nolte@rub.de.
Jazyk: angličtina
Zdroj: RSC chemical biology [RSC Chem Biol] 2024 Oct 15. Date of Electronic Publication: 2024 Oct 15.
DOI: 10.1039/d4cb00187g
Abstrakt: Bioorthogonal reactions are extremely useful for the chemical modification of biomolecules, and are already well studied in mammalian cells. In contrast, very little attention has been given to the feasibility of such reactions in bacteria. Herein we report modified coumarin dyes for monitoring the internalisation and activity of bioorthogonal catalysts in the Gram-positive bacterial species Bacillus subtilis . Two fluorophores based on 7-aminocoumarin were synthesised and characterised to establish their luminescence properties. The introduction of an allyl carbamate (R 2 N-COOR') group onto the nitrogen atom of two 7-aminocoumarin derivatives with different solubility led to decreased fluorescence emission intensities and remarkable blue-shifts of the emission maxima. Importantly, this allyl carbamate group could be uncaged by the bioorthogonal, organometallic ruthenium catalyst investigated in this work, to yield the fluorescent product under biologically-relevant conditions. The internalisation of this catalyst was confirmed and quantified by ICP-OES analysis. Investigation of the bacterial cytoplasm and extracellular fractions separately, following incubation of the bacteria with the two caged dyes, facilitated their localisation, as well as that of their uncaged form by catalyst addition. In fact, significant differences were observed, as only the more lipophilic dye was located inside the cells and importantly remained there, seemingly avoiding efflux mechanisms. However, the uncaged form of this dye is not retained, and was found predominantly in the extracellular space. Finally, a range of siderophore-conjugated derivatives of the catalyst were investigated for the same transformations. Even though uptake was observed, albeit less significant than for the non-conjugated version, the fact that similar intracellular reaction rates were observed regardless of the iron content of the medium supports the notion that their uptake is independent of the iron transporters utilised by Gram-positive Bacillus subtilis cells.
Competing Interests: The authors declare no conflict of interest.
(This journal is © The Royal Society of Chemistry.)
Databáze: MEDLINE
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