A Ratiometric Fluorescent Probe for Imaging of the Activity of Methionine Sulfoxide Reductase A in Cells

Article abstract of DOI:10.1002/anie.201605833

Methionine sulfoxide reductase A (MsrA) is an enzyme involved in redox balance and signaling, and its aberrant activity is implicated in a number of diseases (for example, Alzheimer's disease and cancer). Since there is no simple small molecule tool to monitor MsrA activity in real time in vivo, we aimed at developing one. We have designed a BODIPY-based probe called (S)-Sulfox-1, which is equipped with a reactive sulfoxide moiety. Upon reduction with a model MsrA (E. coli), it exhibits a bathochromic shift in the fluorescence maximum. This feature was utilized for the real-time ratiometric fluorescent imaging of MsrA activity in E. coli cells. Significantly, our probe is capable of capturing natural variations of the enzyme activity in vivo.

Full text of DOI:10.1002/anie.201605833

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201605833
German Edition: DOI: 10.1002/ange.201605833
Bioimaging
A Ratiometric Fluorescent Probe for Imaging of the Activity of
Methionine Sulfoxide Reductase A in Cells
ˇ
ˇ
Nikolai Makukhin, Vyacheslav Tretyachenko, Jackob Moskovitz, and Jirꢀ Mꢀsek*
Abstract: Methionine sulfoxide reductase A (MsrA) is an
enzyme involved in redox balance and signaling, and its
aberrant activity is implicated in a number of diseases (for
example, Alzheimerꢁs disease and cancer). Since there is no
simple small molecule tool to monitor MsrA activity in real
time in vivo, we aimed at developing one. We have designed
a BODIPY-based probe called (S)-Sulfox-1, which is equipped
with a reactive sulfoxide moiety. Upon reduction with a model
MsrA (E. coli), it exhibits a bathochromic shift in the
fluorescence maximum. This feature was utilized for the real-
time ratiometric fluorescent imaging of MsrA activity in E. coli
Figure 1. Representation of the reversible oxidation of methionine
residues in proteins.
cells. Significantly, our probe is capable of capturing natural
variations of the enzyme activity in vivo.
M
ethionine sulfoxide reductases (Msrs) are important
enzymes, which are conserved throughout the tree of life
and are responsible for the reduction of free and protein-
bound methionine sulfoxide back to methionine.[1,2] As
methionine sulfoxide has a chiral center at the sulfur atom,
organismal oxidation of methionine to methionine sulfoxide
caused by reactive oxygen species can lead to a mixture of
(R)- and (S)-epimers. Nature has evolved two distinct classes
of Msrs: MsrA, which reduces the (S)-epimer, and MsrB,
which reduces the (R)-epimer (Figure 1).[3,4]
an increasing number of examples have been described in
which the generation of methionine sulfoxide is a gain-of-
function posttranslational modification[14–16] At the heart of
this important regulatory mechanism lie Msrs that have been
shown to play a significant role in the development of
neurological disorders (Alzheimerꢀs disease and Parkinsonꢀs
disease) and cancer.[17–19] It is therefore of great importance to
have a simple and robust tool to monitor Msr activity in real
time both in vitro and in vivo. Despite the utility of current
probes that are based on radio or fluorescently labeled
methionine sulfoxide,[20,21] they can only capture Msr activity
in vitro. Recently, an elegant molecular biology tool for
in vivo imaging of methionine sulfoxide has been de-
scribed.[22] Complementary to this fluorescent protein-based
sensor would be a small molecule fluorescent probe for Msr,
which would provide direct information on Msr activity. As it
is mainly MsrsA that were previously associated with health
disorders and aging, we aimed at targeting the activity of this
particular class of enzymes. Herein, we describe the develop-
ment of ratiometric fluorescent reporter (S)-Sulfox-1 that
enables monitoring MsrA activity in real time and capturing
natural variations in the activity of this enzyme in vivo.
MsrA has been shown to reduce methyl p-tolyl sulfoxide
in an enantioselective fashion.[23] The reduction of sulfoxide to
sulfide provides a significant change of the electronic nature
of the functional groups, which has also been shown to alter
the spectral characteristics of various chromophores.[24–27]
Thus, we have decided to attach the methyl phenyl sulfoxide
moiety to a suitable fluorophore to construct the fluorescent
reporter. We have opted for the BODIPY fluorophore
because it is very bright, stable, environmentally insensitive,
accessible, and modular.[28,29] Aryl substituents in the posi-
tions next to the nitrogens of the BODIPY core cause
a bathochromic shift in fluorescence as the conjugated system
is extended.[30,31] Moreover, functional groups on the aryl
The importance of this class of enzymes has been
demonstrated by knockout studies of MsrA in a number of
organisms (bacteria, yeast, and mice), in which it has been
associated with an increased susceptibility to oxidative
stress.[5–8] On the other hand, overexpression of MsrA
increases resistance to oxidative stress in cells, plants, and
Drosophila.[9–12] Notably, overexpression of MsrA in Droso-
phila almost doubles its lifespan. Interestingly, the over-
expression of MsrB in the Drosophila animal model had no
effect on its aging.[13] It is clear that a large variety of cellular
proteins are deactivated by methionine oxidation; however,
ˇ
[*] Dr. N. Makukhin, Dr. J. Mꢀsek
Department of Organic Chemistry, Faculty of Science
Charles University in Prague
Hlavova 2030/8, 12843 Prague 2 (Czech Republic)
E-mail: misek@natur.cuni.cz
V. Tretyachenko
Department of Biochemistry, Faculty of Science
Charles University in Prague
Hlavova 2030/8, 12843 Prague 2 (Czech Republic)
Dr. J. Moskovitz
Department of Pharmacology and Toxicology, School of Pharmacy,
University of Kansas
Lawrence, KS 66045 (USA)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201605833.
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substituent can further affect the emission maxima, with
electron donating groups increasing the red shift.[32] We
hypothesized that reduction of the methyl phenyl sulfoxide
moiety attached to the BODIPY core would red-shift the
emission maximum and thus serve as a ratiometric probe for
MsrA. The ratiometric nature of the probe would be ideal for
in vivo imaging because the disadvantages of intensity probes,
such as uneven cellular distribution and background fluores-
cence are inherently canceled out.
The (S)-Sulfox-1 probe was synthesized starting with 3-
chloro-BODIPY 1, which was prepared by an established
procedure (Scheme 1).[32] The fluorine atoms were then
Figure 2. a) A sensing mechanism of (S)-Sulfox-1. b) Michaelis–
Menten kinetics for MsrA with both (S)- (&) and (R)- (^) enantio-
mers of Sulfox-1 and the natural substrate (S)-methionine-(R,S)-
sulfoxide (*) measured in 50 mm sodium phopsphate buffer with
20 mm DTT, pH 7.5, at 378C. c) Changes in the emission profile of (S)-
Sulfox-1 (40 mm) upon reduction with MsrA (2 mm) for 70 min at 208C
(excitation at 510 nm). The inset shows changes in the emission ratio
Scheme 1. Synthesis of (S)-Sulfox-1 and sulfide 5.
F542/F576 as a function of time.
substituted with triethyleneglycol arms to increase the
solubility and brightness in aqueous environments. The
synthesis was completed by a Suzuki coupling with (S)-
boronate 3, which was prepared through an asymmetric
oxidation/borylation sequence. The (R)- isomer of Sulfox-
1 was prepared in the same way (see the Supporting
Information), and sulfide 5 was obtained by a Suzuki coupling
of 3-chloro-BODIPY 2 with commercial boronic acid 4.
The spectroscopic characteristics of (S)-Sulfox-1 and
sulfide 5 were assessed in phosphate buffer at pH 7.5. (S)-
Sulfox-1 showed a maximum absorption at 509 nm (e = 3.6 ꢁ
104 mÀ1 cmÀ1) and a strong emission band at 541 nm with
a quantum yield of 0.6. In contrast, the absorption maximum
of sulfide 5 was at 527 nm (e = 2.9 ꢁ 104 mÀ1 cmÀ1) and the
emission was red-shifted by 25 nm, which resulted in a max-
imum at a wavelength of 566 nm and a quantum yield of 0.5.
The spectroscopic data gave us confidence that (S)-Sulfox-
1 could be used as a ratiometric fluorescent probe. Next,
recombinant E. coli MsrA was expressed and purified as
a fusion protein with a GST tag, and its activity was tested
against both the enantiomers of Sulfox-1 and the natural
substrate methionine sulfoxide. (S)-Sulfox-1 proved to be
a good substrate for MsrA and the proposed sensing
mechanism was confirmed by HPLC-MS analysis (Figure 2a
and the Supporting Information, Figure S8). Michaelis–
Menten kinetics for (S)-Sulfox-1 gave kcat = 0.75 sÀ1 and
KM = 913 mm, which are values similar to those for the natural
substrate methionine sulfoxide (kcat = 0.37 sÀ1 and KM =
700 mm) (Figure 2b). On the other hand, under the assay
conditions, no conversion was observed for the (R)-isomer of
Sulfox-1 beyond a minor background reaction with DTT. The
ability of (S)-Sulfox-1 to monitor MsrA activity in real time is
demonstrated by the observed emission ratio at 542 nm and
576 nm (F542/F576). During the course of the reaction, that ratio
was found to change from 3.8 to 0.3, a 13-fold difference
(Figure 2c). Reports have indicated that certain sulfoxides
can be racemized photochemically.[33,34] However, in our case,
(S)-Sulfox-1 showed neither measurable racemization nor
photodegradation during extended exposure (1 h) to 365 nm
UV light (hand lamp, 8 W) in phosphate buffer in an NMR
tube.
To show the utility of (S)-Sulfox-1 for in vivo imaging of
MsrA activity, an E. coli strain with an artificially elevated
level of the MsrA that was used for the recombinant
preparation of the MsrA enzyme was utilized. The IPTG-
induced cells were compared with the cells grown in the
presence of glucose to maximize catabolite repression of the
IPTG inducible promoter. After a 5 min staining of bacterial
cells with (S)-Sulfox-1, the IPTG-induced cell population
showed a substantial change in the fluorescence as compared
with the uninduced cell population (Supporting Information,
Figure S11). No change in the fluorescence ratio F535/F570 was
observed for (R)-Sulfox-1.
The next goal was to capture the natural variation of the
MsrA activity in vivo. As previously reported, MsrA activity
increases in E. coli cells with the growth phase, as measured in
the cell lysate.[35] Thus, E. coli (BL21) cells were grown to
OD600 = 0.5 and OD600 = 3.5, and these two populations were
subjected to our staining protocol with (S)-Sulfox-1. After a 1-
h incubation, the population of cells grown to OD600 = 3.5
2
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soluble over a wide range of concentrations, and sensitive
enough to capture natural variations in the activity of MsrA in
cells. Since MsrA is implicated in a number of serious illnesses
and is an important player in the redox balance and signaling,
we believe our (S)-Sulfox-1 probe will provide a simple
platform for monitoring MsrA activity in vivo and thus help
uncover previously untraceable molecular biology relation-
ships of these enzymes.
Acknowledgements
ˇ
We thank Dr. J. Janda and O. Sebesta for helping us with the
ˇ
´
confocal fluorescent microscopy and Prof. P. Kocovsky for
corrections of the manuscript.
Keywords: chirality · fluorescent probes · oxidoreductases ·
photophysics · redox chemistry
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Received: June 16, 2016
Revised: August 8, 2016
Published online: && &&, &&&&
4
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Communications
Bioimaging
Oxidative stress? Relax, we got a probe
for that: Aberrant activity of methionine
sulfoxide reductase A (MsrA) is impli-
cated in a number of diseases. A fluo-
rescent probe enabling real-time imaging
of the activity of MsrA is reported. The
probe is bright, water-soluble, photosta-
ble, and sensitive enough to capture
natural variations of MsrA in cells.
N. Makukhin, V. Tretyachenko,
ˇ
J. Moskovitz, J. Mꢁsek*
&&&& — &&&&
A Ratiometric Fluorescent Probe for
Imaging of the Activity of Methionine
Sulfoxide Reductase A in Cells
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
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