2 research outputs found
Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells
Density
functional theory (DFT) was applied to study the thermodynamics
and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31GÂ(d)
with the SMD solvation model can reliably predict the Gibbs free energy
changes (Δ<i>G</i>) of thiol-Michael addition reactions
with an error of less than 1 kcal·mol<sup>–1</sup> compared
with the experimental benchmarks. Taking advantage of this computational
model, the first reversible reaction-based fluorescent probe was developed
that can monitor the changes in glutathione levels in single living
cells
Quantitative Imaging of Glutathione in Live Cells Using a Reversible Reaction-Based Ratiometric Fluorescent Probe
Glutathione
(GSH) plays an important role in maintaining redox
homeostasis inside cells. Currently, there are no methods available
to quantitatively assess the GSH concentration in live cells. Live
cell fluorescence imaging revolutionized the field of cell biology
and has become an indispensable tool in current biological studies.
In order to minimize the disturbance to the biological system in live
cell imaging, the probe concentration needs to be significantly lower
than the analyte concentration. Because of this, any irreversible
reaction-based GSH probe can only provide qualitative results within
a short reaction time and will exhibit maximum response regardless
of the GSH concentration if the reaction is completed. A reversible
reaction-based probe with an appropriate equilibrium constant allows
measurement of an analyte at much higher concentrations and, thus,
is a prerequisite for GSH quantification inside cells. In this contribution,
we report the first fluorescent probeî—¸ThiolQuant Green (TQ
Green)î—¸for quantitative imaging of GSH in live cells. Due to
the reversible nature of the reaction between the probe and GSH, we
are able to quantify mM concentrations of GSH with TQ Green concentrations
as low as 20 nM. Furthermore, the GSH concentrations measured using
TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible
and well correlated with the values obtained from cell lysates. TQ
Green imaging can also resolve the changes in GSH concentration in
PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ
Green can be conveniently applied in fluorescence activated cell sorting
(FACS) to measure GSH level changes. Through this study, we not only
demonstrate the importance of reaction reversibility in designing
quantitative reaction-based fluorescent probes but also provide a
practical tool to facilitate redox biology studies