Page 3 of 4
Journal Name
ChemComm
DOI: 10.1039/C4CC09947H
removed by cytosolic esterases) was detected by LC/HRꢀESIꢀMS transportation stage by an intracellular Cuꢀfree click reaction and
(see ESI†) from the cell extract. This observation suggested that that the unbound probes were completely washed away from the
acetylated azideꢀmannose was firstly taken up into the cytosol before cells.
incorporation into glycoproteins, which is performed in the
In conclusion, we synthesized a novel cyclooctyneꢀbased Cuꢀfree
endoplasmic reticulum (ER) and/or Golgi apparatus.24 Fluorescence clickable fluorescent probe, FCꢀDBCO, and achieved the
signals were observed on the membrane of some cells after 12 hours intracellular specific imaging of azideꢀmodified biomolecules in
(Fig. 3e) and most of cells after 24 hours (Fig. 3f), suggesting the living cells. FCꢀDBCO displayed good cellꢀmembrane permeability
glycoproteins were transferred into the cell membrane during this and any unbound probe was efficiently removed from the cells by a
period. These images clearly indicate that FCꢀDBCO were simple washing step, leading to very low background fluorescence
conjugated to azideꢀmannose and azideꢀsialic acid contained signals. In addition, the fluorescence properties of our probe were
mannoglycoproteins inside of the living cell as well as on the cell similar to those of fluorescein. As such, the probe is highly versatile
membrane by the Cuꢀfree click reaction.
Finally, we investigated the localization of the target azideꢀ available organelle specific trackers. In order to demonstrate the
molecules in the living cell at an organelle level. Ac4ManNAzꢀ application of our probe, the intracellular dynamics of
for coꢀstaining with other fluorescent dyes such as commercially
a
incorporated cells were coꢀstained with FCꢀDBCO and one of four commercially available azide mannose was visualized using FCꢀ
different organelle trackers; ER, lysosome, Golgi apparatus, and DBCO with several organelle trackers. FCꢀDBCO specifically
mitochondria at 6 or 12 hours incubation after the introduction of labeled azideꢀmannose and/or azideꢀmannoglycoproteins at each
Ac4ManNAz. Fluorescence signals of FCꢀDBCO primarily merged transportation stage in the living HeLa cells. We believe FCꢀDBCO
with those of ERꢀtracker and partly with Lysoꢀtracker after 6 hours will be an invaluable tool not only for metabolism imaging in living
incubation. We did not find any merged signals between FCꢀDBCO cells by using a variety of azideꢀmodified bioactive small molecules
and Golgiꢀtracker or Mitoꢀtracker (Fig. 4a). These observations such as lipids, nucleic acids and other metabolites, but also
suggested that fluorescenceꢀlabeled azide mannose was localized in monitoring intracellular dynamics of drugs.
the cytosol just after cell membrane permeation, ER during
This work was supported by the Leading Graduate Program in
incorporation into glycoprotein and then lysosomeꢀlike acid vesicles Science and Engineering, Waseda U. from MEXT, Japan (K. Y.) and
during transportation from the ER to the Golgi apparatus. These Waseda U. Grant for Special Research Project (S. T.).
observations also suggested that 6 hours incubation was insufficient
time for azide mannose to reach the Golgi apparatus. The
fluorescence signals of Mitoꢀtracker were entirely independent from
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Fig. 4 Investigation of the localization of azideꢀmannose and azideꢀ
mannoglycoproteins in living HeLa cells. Confocal images of azideꢀ
mannoseꢀintroduced HeLa cells stained with FCꢀDBCO (left), each organelle
tracker (middle) and merged images (right) are shown. Localization of azideꢀ
mannose after 6 (a) and 12 hours (b) incubation was observed using FCꢀ
DBCO (incubated for 2 hours) and organelle specific tracking dyes (0.5ꢀ1.0
hour incubation) after the wash process (30 min). Endoplasmic reticulum,
lysosome (and lysosomeꢀlike vesicles), Golgi apparatus, and mitochondria
(from top to bottom) were coꢀstained with FCꢀDBCO.
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