10.1002/anie.202002391
Angewandte Chemie International Edition
COMMUNICATION
bacteria comprising potentially multiresistant strains of the
clinically highly relevant ESKAPE panel. This permitted the first
demonstration of fluorescence lifetime imaging (FLIM) to trace
enzymatic activity in live bacteria with lanthanide luminescent
probes. Taken together these features illustrate that this type of
probe concept is an attractive option for future analytical
applications in medical diagnostics.
Acknowledgements
Figure 3. Fluorescence lifetime imaging of live E. coli bacteria incubated with
probe 12 (20 μM) for 4 h at 37°C. (a) Fluorescence intensity image (b) lifetime
map. λex = 375 nm, fluorescence intensities and lifetimes were collected
through a 641/75 nm long-pass edge filter.
We would like to thank Dr. Edgar Specker and the NMR core
facility of the FMP for their excellent support on compound
characterization and the FMP Screening Unit for access to a plate
reader. We thank Dr. Uwe Grether and Dr. Raffael Koller for
helpful discussions and Dr. Barth van Rossum for preparing
graphical artwork. This work was supported by the Sino-German
research project (GZ 1271).
were selected. The bacteria were lysed by sonication and the
lysate was incubated with probe 12 with or without dicoumarin
inhibitor for 2 h before recording the emission signal at 550 nm.
As shown in Fig. 2 B, our probe was strongly activated in K.
pneumoniae, A. baumannii, E. cloacae and E. coli while the
activation was less pronounced in the other three strains. Co-
incubation with the NTR-inhibitor significantly reduced probe
activation in the former species, indicating selective intracellular
activation by NTR, while showing little to no effect in the latter
ones. These differences could be attributed to different
expression levels as well as species differences of NTR enzymes
amongst the bacterial strains, as sequence alignments of those
enzymes showed low conservation amongst the bacterial strains
as proposed in preceding work.[10d]
We advanced to test whether we could detect NTR in live
bacteria. We were pleased to get similar results in K. pneumoniae,
A. baumannii, E. cloacae and E. coli to those obtained with the
lysates. While probe 12 was highly activated in all four strains, co-
incubation with NTR-inhibitor dicoumarin led to a drastic decrease
in activation (Figure 2 C). The activation of probe 12 was further
investigated by fluorescence lifetime imaging (FLIM) in live E coli.
Figure 3 shows the long-lived emission fluorescence signals with
an average lifetime of 31 µs. These results suggest that probe 12
was readily taken up by the bacterial cells and triggered by
intracellular NTRs. To the best of our knowledge, this is the first
example of FLIM applied to image enzymatic activity in live
bacteria with lanthanide luminescent probes. Interestingly, we
observed that in contrast to caged probe 12, which was readily
taken up by bacterial cells, the activated reference 10 was not
able to permeate into and label bacterial cells (Figure S11).
Moreover, in the FLIM experiments activated probe 10 remained
localized in the bacterial intracellular compartments and was not
cleared to the extracellular medium, e.g. by an efflux mechanism,
as observed for a previous enzyme-activatable lanthanide
probe.[4] Collectively, these findings suggest a synergistically
enhanced localization, detection specificity and contrast due to
intracellular enrichment of the activated probe 10.
Keywords: Bacterial Imaging • Enzymes • Lanthanides •
Luminescence • Nitroreductase
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In summary, we have developed the first luminescent turn-on
probe for the highly selective and sensitive detection of
nitroreductase. Installation of a new type of analyte-responsive
carbostyril forming switch was enabled through the key
Buchwald-Hartwig type synthetic transformation constituting a
robust modular approach for the facile access to similar probes.
Due to its effective intracellular enrichment our probe enables the
simple detection and imaging of nitroreductase activity in live
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