Journal of the American Chemical Society
Communication
NS1 cells stained with FP1 with 0.25 and 0.5 mM FA. As with
higher concentrations of FA, incubation of NS1 cells with 0.25
and 0.5 mM FA at 37 °C for 3 h afforded a dose-dependent signal
enhancement of 16% and 38%, respectively (Figure S15).
Likewise, flow cytometry analyses of NS1 cells incubated with
0.25 and 0.5 mM resulted in a 42% and 50% fluorescence turn-
on, respectively (Figure S16). Moreover, we anticipate extended
incubation beyond the 3 h time point will result in improved
contrast because a larger fraction of FP1 will have sufficient time
to react with FA. These crucial experiments validate the utility of
FP1 for detection of FA at endogenous levels and, thus, provides
us with unique opportunities to study both normal and
pathological systems.
In summary, we have developed a new fluorescent probe, FP1,
for imaging FA using the 2-aza-Cope sigmatropic rearrangement.
We strategically incorporated a 4-nitrobenzyl dark quencher to
reduce background fluorescence via d-PeT. The ensuing product
features an aldehyde moiety at the C3 indole position which
enhances fluorescence and, thus, the dynamic range through
perturbation of a-PeT quenching. As such, the high sensitivity of
FP1 enabled us to detect FA in vitro at concentrations below
those reported in the brain. In addition, FP1 was successfully
used to image physiological levels of FA in live HEK293TN and
NS1 cells in a dose- and time-dependent manner. Of note, we
developed a new julolidine-based silicon rhodol fluorescent
scaffold which features an absorption maxima centered at 633
nm. This property enabled us to excite FP1 with the 633 nm
HeNe laser in both confocal microscopy and flow cytometry
experiments. Moreover, because FP1 exhibits excellent photo-
stability, we performed a time-lapse imaging experiment where
the reaction between FP1 and FA could be captured in real-time.
Taken together, this study demonstrates that FP1 will find utility
in elucidating the physiological roles of FA in live cells.
Grant (5T32-GM070421). C.A. thanks the Robert C. and
Carolyn J. Springborn Endowment and the UIUC Graduate
College for graduate fellowships.
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* Supporting Information
The Supporting Information is available free of charge on the
Experimental details, including synthesis of 1−5 and FP1
and all data for in vitro and in cellulo experiments are
W
* Web-Enhanced Feature
A time-lapse imaging movie of the reaction between FP1 and FA
in AVI format is available in the online version of the paper.
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AUTHOR INFORMATION
Corresponding Author
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2424.
Author Contributions
†These authors contributed equally.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Profs. Paul Hergenrother (UIUC Department of
Chemistry) and Kai Zhang (UIUC Department of Biochemis-
try) for generously supplying HEK293TN and NS1 cells,
respectively. Furthermore, we thank Dr. Sandy McMasters
(UIUC Cell Media Facility) for her technical expertise. A.R. and
C.A. acknowledge the National Institute of General Medical
Sciences (NIGMS)-NIH Chemistry-Biology Interface Training
D
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX