ORGANIC
LETTERS
2012
Vol. 14, No. 3
680–683
Fluorescent Coumarin Thiols Measure
Biological Redox Couples
Khalilah G. Reddie,† William H. Humphries,‡ Charlo P. Bain,† Christine K. Payne,‡
Melissa L. Kemp,† and Niren Murthy*,†
Wallace H. Coulter Department of Biomedical Engineering, School of Chemistry and
Biochemistry and the Parker H. Petit Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, Atlanta, Georgia 30032, United States
Received November 19, 2011
ABSTRACT
In this report we present a new chemical probe, 3-HTC, that can reversibly and ratiometrically measure the thiol-disulfide equilibrium of biological
systems. 3-HTC is composed of a coumarin that has a thiolate directly conjugated to its extended aromatic π system while formation of a disulfide
attenuates this conjugation. The fluorescence and absorption properties of 3-HTC are therefore very sensitive to the redox state of its thiol. 3-HTC
reacts reversibly with thiols and disulfides enabling its use to measure dynamic GSH/GSSH ratios in vitro as well as to monitor the reversible
redox status of whole cell lysates.
The thiol-disulfide interchange reaction is a key bio-
chemical transformation that controls cellular processes
such as gene regulation, cell division and differentiation.1
In addition, the disregulation of the thiol-disulfide steady
state is a hallmark of oxidative stress and has been
implicated in numerous diseases, such as atherosclerosis,
obesity, inflammation and aging-related complications.2
Hence, there is great interest in measuring the thiol-
disulfide redox state of biological samples. Although
several small molecule probes have been reported for thiol
detection, they are not reversible and therefore do not
provide a direct report on the thiol-disulfide redox couple.3
Redox sensitive ratiometric green fluorescent proteins
(roGFPs) that can monitor dynamic redox changes have
been developed; however, these probes require genetic
manipulation, which is frequently impossible and also
has several additional complications.4 Currently, chemical
probes that can measure the thiol-disulfide state of living
cells do not exist, presenting a need for novel chemicaltools
that can measure biological thiol-disulfide dynamics.
In this report we present a new chemical probe, termed
3-hetaryl-7-thiol coumarin (3-HTC), that can reversibly
and ratiometrically measure the thiol-disulfide equilibrium
of biological systems. 3-HTC is composed of a coumarin
that has a thiolate directly conjugated to its extended
aromatic π system while formation of a disulfide attenu-
ates this conjugation (Figure 1 and Scheme 1). The fluores-
cence and absorption properties of 3-HTC are therefore
very sensitive to the redox state of its thiol and offer a
† Wallace H. Coulter Department of Biomedical Engineering.
‡ School of Chemistry and Biochemistry and the Parker H. Petit Institute
for Bioengineering and Bioscience.
(1) (a) Antelmann, H.; Helmann, J. D. Antioxid. Redox Signal. 2010,
14, 1049. (b) Matthais, L. J.; Yam, P. T. W; Jiang, X. M.; Vandegraaff,
N.; Li, P.; Poumbourios, P.; Donoghue, N.; Hogg, P. J. Nat. Immunol.
2002, 3, 727. (c) Paulsen, C. E.; Carroll, K. S. ACS Chem. Biol. 2009, 5,
47. (d) Pan, S.; Berk, B. C. Circ. Res. 2007, 100, 213.
(2) (a) Gupta, D; Griendling, K. K.; Taylor, R. W. Oxidative Stress
and Cardiovascular Disease in Diabetes Mellitus Studies on Cardiovas-
cular Disorders; Sauer, H., Shah, A. M., Laurindo, F. R. M., Eds.; Humana
Press: New York, NY, 2010; p 263. (b) Salmon, A. B.; Richardson, A.;
ꢀ
Perez, V. I. Free Radic. Biol. Med. 2010, 48, 642.
(3) (a) Bohndiek, S. E.; Kettunen, M. I.; Hu, D. E.; Kennedy, B. W.;
Boren, J.; Gallagher, F. A.; Brindle, K. M. J. Am. Chem. Soc. 2011, 133,
11795. (b) Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.;
Cho, B. R. J. Am. Chem. Soc. 2011, 133, 11132. (c) Kim, G. J.; Lee, K.;
Kwon, H.; Kim, H. J. Org. Lett. 2011, 13, 2799. (d) Long, L.; Lin, W.;
Chen, B.; Gao, W.; Yuan, L. Chem. Commun. 2011, 47, 893.
(4) For an excellent review on fluorescent protein-based redox probes,
see: Meyer, A. J.; Dick, T. P. Antioxid. Redox Signal. 2010, 13, 621.
r
10.1021/ol203105c
Published on Web 01/20/2012
2012 American Chemical Society