Coumarin-based fluorogenic probes for no-wash protein labeling

Article abstract of DOI:10.1002/anie.201408015

A fluorescent protein-labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens com

Full text of DOI:10.1002/anie.201408015

Angewandte
Chemie
DOI: 10.1002/anie.201408015
Bioorthogonal Chemistry
Coumarin-Based Fluorogenic Probes for No-Wash Protein Labeling**
Yingche Chen, Christopher M. Clouthier, Kelvin Tsao, Miroslava Strmiskova, Hugo Lachance,
and Jeffrey W. Keillor*
Abstract: A fluorescent protein-labeling strategy was devel-
oped in which a protein of interest (POI) is genetically tagged
with a short peptide sequence presenting two Cys residues that
can selectively react with synthetic fluorogenic reagents. These
fluorogens comprise a fluorophore and two maleimide groups
that quench fluorescence until they both undergo thiol addition
during the labeling reaction. Novel fluorogens were prepared
and kinetically characterized to demonstrate the importance of
a methoxy substituent on the maleimide in suppressing
reactivity with glutathione, an intracellular thiol, while main-
taining reactivity with the dithiol tag. This system allows the
rapid and specific labeling of intracellular POIs.
and reversibility represent limitations of its application.[7]
More recent work includes the expression of proteins
incorporating unnatural amino acids[8] that can subsequently
undergo bioorthogonal reactions to allow covalent labeling.[9]
However, unnatural amino acid mutagenesis is not yet widely
applicable and is highly dependent on host cell type.
We have developed a complementary strategy for cova-
lent, fluorogenic protein labeling in which the POI is
genetically fused to a short peptide sequence (dC10a tag)
that presents two Cys residues separated by two turns of an a-
helix (ca. 10 ꢂ); the POI is fluorescently labeled upon
covalent reaction of these two Cys residues with comple-
mentary synthetic fluorogenic reagents[10] (Scheme 1). These
V
isualizing and monitoring specific proteins with minimal
disruption of their biological function and distribution is one
of the foremost challenges in chemical biology. Fluorescent
labeling of a specific protein of interest (POI) is a widely used
method for studying expression, localization, and trafficking.
The genetic encoding of fluorescent proteins (FPs) such as
green fluorescent protein (GFP) is the most broadly applied
approach for protein labeling because of its intrinsic specific-
ity.[1] However, there are some limitations of this method,
including the large size of GFP (ca. 30 kDa), which can
perturb the function and the localization of the POI.[2]
Alternative labeling methods have been developed based
on the fusion of the POI to an enzyme tag. These include the
HaloTag, which involves fusion to haloalkane dehalogenase,[3]
the SNAP-tag, which involves fusion to O6-alkylguanine-
DNA-alkyltransferase,[4] and the related CLIP-tag.[5] These
enzymes can then be labeled with excellent specificity by
using functionalized irreversible inhibitors. However, these
ꢀtagsꢁ are also of considerable size (ca. 18–30 kDa) and thus
pose the same risk for steric perturbation of the POI. In one of
the first attempts to reduce the size of this tag, Tsien and co-
workers designed a short b-hairpin tag, the four Cys residues
of which react with fluorogenic bisarsenic agents such as
FlAsH and ReAsH.[6] This small-molecule method has been
used to label specific proteins in living cells, but its toxicity
Scheme 1. Protein labeling with dimaleimide fluorogens.
fluorogenic reagents comprise a fluorophore and a dimalei-
mide moiety, such that their latent fluorescence is quenched
by photoinduced electron transfer (PeT) until both maleimide
groups undergo specific thiol addition reactions.[11] A study of
the effect of spacer length and conformation on the fluores-
cence quenching confirmed that direct linkage of the fluo-
rophore and the dimaleimide scaffold is critical to the
quenching efficiency. Recently, we have reported “spacerless”
dansyl-based probes, the fluorescence of which increases by
approximately 350-fold after reaction with our target peptide
tag.[12]
The chemistry underpinning this fluorogenic addition
reaction (FlARe) technology is based on two properties of the
maleimide group. First, they are known to undergo specific
thiol addition reactions[13] and are widely applied (although
typically nonspecifically) in protein labeling. Second, they are
also known to quench fluorescence in their conjugated form
but not as their thiol adduct products.[10a,11] Thus, a dimalei-
mide fluorogen must undergo two thiol addition reactions
before its latent fluorescence is restored. This fluorogenic
response is selective for a POI tagged with our dC10a tag
because vanishingly few native proteins present two free Cys
residues on their surfaces, approximately 10 ꢂ apart. How-
ever, intracellular labeling could pose a challenge for the
FlARe method since the intracellular concentration of the
tripeptide thiol glutathione (GSH) is in the millimolar range.
In this environment, we were concerned that our dimaleimide
[*] Y. Chen, C. M. Clouthier, K. Tsao, M. Strmiskova, H. Lachance,
Prof. J. W. Keillor
Department of Chemistry, University of Ottawa
10 Marie-Curie, Ottawa, ON K1N 6N5 (Canada)
E-mail: jkeillor@uottawa.ca
[**] We acknowledge the financial support of the Canadian Institutes of
Health Research (CIHR), the Natural Sciences and Engineering
Research Council (NSERC), and the Ottawa Technology Transfer
Network (OTTN). We also thank Prof. R. E. Campbell (University of
Alberta) and Prof. A. E. Pelling (University of Ottawa) for expression
plasmids.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408015.
Angew. Chem. Int. Ed. 2014, 53, 13785 –13788
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
13785

.
Angewandte
Communications
fluorogen could conceivably react with one Cys residue of an
adventitious protein, followed by a reaction with one
equivalent of the ubiquitous GSH, thus leading to a non-
specific fluorescent labeling reaction. Herein, we report
successful efforts to tune the reactivity of our probes; the
resulting improved selectivity makes them suitable for intra-
cellular application.
Our first attempt was based on the design of an
asymmetric dimaleimide moiety, wherein the intrinsic reac-
tivity of one maleimide is greatly reduced, thereby effectively
suppressing its intermolecular reaction. However, in the
reaction with an appropriate dithiol, the reaction of the less
reactive maleimide is intramolecular and is thus efficiently
accelerated by the high effective molarity[14] of the adjacent
second thiol (Scheme 1). Previous work in our group[10a]
suggested that the reactivity of a maleimide group bearing
an electron-donating substituent may be suitably attenuated.
Herein, we report the synthesis and characterization of
a coumarin-based labeling reagent bearing such an asymmet-
ric dimaleimide moiety.
Scheme 2. Kinetics studies of reactions of YC5 and YC9 with test thiol
MPA.
Our synthetic approach was shaped by our long-term goal
of diversification through the coupling of several different
dimaleimide moieties to several different fluorophores. To
that end, the building blocks of our fluorogens consist of the
fluorophore, the aromatic amine scaffold, and the maleic
anhydride. The fluorophore was designed to present a carbox-
ylic acid group, since this is readily available or synthetically
accessible for most fluorophores and enables facile conjuga-
tion to an aromatic amine. For this study, we chose coumarin
as a fluorophore because of its high quantum yield, photo-
stability, and hydrophilicity. Moreover, the excitation wave-
length of the 7-diethylamino derivative is in the visible
spectrum, thus offering the potential to be useful in two-
photon excitation experiments. The maleic anhydrides
required for the desired labeling agents are either commer-
cially available (citraconic anhydride) or synthetically acces-
sible (methoxymaleimide).[15] Finally, for the aromatic scaf-
fold of the dimaleimide moiety, we developed a selective
reduction strategy to reduce two nitro groups, thereby
allowing successive functionalization of the resulting amine
without the requirement for protecting groups. Detailed
synthesis procedures can be found in the Supporting Infor-
mation.
In addition to the asymmetric labeling agent YC9, we also
prepared the symmetric analogue YC5 for comparison
purposes. Maltose-binding protein (MBP) was chosen as
a highly soluble test protein and the dC10a tag was fused to
the C terminus (MBP–dC10a) as described previously.[10b]
Both YC5 and YC9 were found to exhibit negligible back-
ground fluorescence, thus indicating that their dimaleimide
moieties are both capable of effectively quenching coumarin
fluorescence, regardless of the substituents on the pendant
maleimide groups. Furthermore, both fluorogenic reagents
became strongly fluorescent after reaction with the test
protein MBP–dC10a, with acceptable fluorescence enhance-
ment (FE) ratios of 69 for YC5 and 62 for YC9 (see the
Supporting Information).
acid (MPA) as a water-soluble, nucleophilic model thiol
(Scheme 2). Reactions were run in the presence of a large
excess of MPA, thereby making them pseudo-first-order with
respect to the fluorogen. The resulting time-dependent
increase in fluorescence was fit to a model for consecutive
first-order reactions (see the Supporting Information),
thereby providing pseudo-first-order rate constants for both
addition steps. The concentration of MPA was then varied to
allow second-order rate constants to be calculated for both
steps (see the Supporting Information). For YC5, the first
thiol addition is 7 times faster than the second addition,
possibly owing to a steric effect; however, for YC9, the second
addition is 63 times slower than the first (Table 1). This
confirms our assumption that for an asymmetric dimaleimide
moiety with a less reactive maleimide, the second intermo-
lecular thiol addition is significantly slower.
Table 1: Second order rate constants measured for the consecutive thiol
addition reactions of MPA with fluorogens YC5 and YC9 (see Scheme 2).
Fluorogen
k1 [mÀ1 sÀ1
]
k2 [mÀ1 sÀ1
]
YC5
YC9
1.208Æ0.249
2.480Æ0.216
0.1571Æ0.0510
0.0388Æ0.0133
We then tested the selectivity of our fluorogens by
measuring the rate of their fluorogenic reactions with our
test protein MBP–dC10a and GSH. As shown in Figure 1a,
YC5 reacted faster with one equivalent of MBP–dC10a than
with two equivalents of GSH, but the latter reaction was still
appreciable; for YC9 (Figure 1b), the reaction with two
equivalents of GSH was negligible but the reaction with
MBP–dC10a was still rapid. However, when the GSH
concentration was increased to the millimolar range (i.e.,
40 equivalents) to mimic the intracellular environment, the
reaction of YC9 with GSH was still apparent.
Kinetics studies were then performed for the thiol
addition reactions of YC5 and YC9 with 3-mercaptopropionic
Although YC9 did not provide the degree of specificity we
were hoping for, a comparison of the results obtained with
13786 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 13785 –13788

Angewandte
Chemie
to 5 mm GSH was used to probe the specificity. The reaction
of YC20 with GSH was dramatically suppressed even at this
high concentration of GSH, while the reaction with the more
reactive MBP–dC10a was still rapid (Figure 2b).
We then confirmed that fluorogens YC9 and YC20 were
nontoxic by performing an MTT assay (see the Supporting
Information) before testing both fluorogens in live-cell
labeling studies. The red fluorescent protein mNeptune was
chosen as a target protein because its intrinsic fluorescence
serves as a control for the transfection and expression of the
target protein; the target sequence dC10a was genetically
fused to the C terminus (see the Supporting Information).
Human embryonic kidney 293T (HEK293T) cells transfected
with mNeptune–dC10a were incubated with YC9 or YC20
for 30 min and imaged either directly or after washing
(Figure 2c). Cells transfected with pcDNA (empty protein
expression vector) were labeled following the same protocol
to provide a negative control. The fluorescence intensities of
the labeled cells were measured after various washing times
and compared (Figure 2d). Cells transfected with mNeptune–
dC10a showed strong cyan fluorescence with both YC9 and
YC20. For YC9, cells without target protein also showed
slight cyan fluorescence from a nonspecific reaction, but this
background fluorescence could be removed with one washing
step. However, for YC20, no nonspecific labeling was
observed. Cells that did not express the target protein
showed negligible fluorescence, even without any washing
steps. This result is consistent with those from our kinetics
studies, which showed that YC9 still displays some reactivity
with high concentrations of GSH, a reaction that could take
place during an intracellular protein labeling experiment.
However, YC20 is highly selective and as such can be used as
a no-wash labeling agent in live cells. Furthermore, both
fluorogenic reagents provided robust covalent labeling of the
target proteins, the fluorescence of which was stable even
after 3 h of washing (Figure 2d; red bars).
Figure 1. The time-dependent fluorescence increase of 25 mm YC5 (a)
or YC9 (b) treated with one equivalent of test protein MBP–dC10a
(red) or two equivalents of tripeptide thiol GSH (black), or with a large
excess (40 equiv) of GSH (blue). lex =440 nm, lem =485 nm.
YC9 and YC5 showed that the methoxymaleimide moiety
reacted much more slowly with GSH without losing much
reactivity with the dC10a tag. This result suggests the Cys
residues of our dC10a tag may be highly nucleophilic and
relatively insensitive to the attenuated electrophilicity of the
methoxymaleimide. Inspired by these results, we then
designed YC20 (Figure 2a), a fluorogenic labeling agent
The no-wash fluorogenic labeling reagent YC20 was then
used to label cellular proteins localized to different parts of
the cell. Histone H2B, which localized to the nucleus, and
actin, which is located in the cytosol as filaments, were chosen
as test proteins. The target sequence dC10a was cloned to the
C terminus of Histone H2B and to the N terminus of actin
(see the Supporting Information) to demonstrate that the
target sequence can be fused to either terminus of a POI.
HEK293T cells transfected with a plasmid encoding histone-
H2B–dC10a or dC10a–actin were then labeled with YC20 for
45 min and directly imaged by fluorescence microscopy
without any washing steps (Figure 3). For cells expressing
dC10a–actin, filament structures in the cytosol were clearly
visible in the cyan channel of the microscope, thus indicating
that the dC10a–actin was labeled by YC20. For cells
expressing histone-H2B–dC10a, only the nuclei were fluo-
rescently labeled and visible in the cyan channel, thus
indicating that YC20 specifically labeled histone-H2B–
dC10a in these cells. Cells transfected with pcDNA (empty
vector) as a negative control showed negligible fluorescence.
To summarize, we have synthesized a novel protein-
labeling reagent that contains a dimethoxymaleimide moiety
and fluorescently labels POIs bearing our dC10a tag with
Figure 2. a) Structure of YC20. b) The time-dependent fluorescence
increase of 25 mm YC20 upon reaction with 25 mm test protein MBP–
dC10a (red), 50 mm GSH (black), 1 mm GSH (blue), or 5 mm GSH
(pink). lex =440 nm, lem =485 nm. c) Labeling and washing protocol.
d) Fluorescence intensity (meanÆSD) of intracellular labeling of test
protein mNeptune–dC10a with fluorogen YC20 in HEK293T cells
(n=10, triplicates). The red bars show cells transfected with the test
protein and the blue bars show cells transfected with empty (mock)
vector.
bearing two methoxymaleimide groups, in an effort to
completely suppress the reaction with GSH, while relying
on the ideal geometry and enhanced nucleophilicity of the
dC10a tag to maintain reactivity with the POI. The synthesis
of YC20 followed a synthetic scheme similar to that for YC5
(see the Supporting Information).
The novel dimethoxymaleimide fluorogen YC20 exhib-
ited negligible background fluorescence and became strongly
fluorescent after reaction with the test protein MBP–dC10a
(see the Supporting Information). The selectivity of YC20
was then tested by comparing the rate of its fluorescence
increase upon reaction with the test protein MBP–dC10a and
with GSH. To closely mimic the intracellular environment, up
Angew. Chem. Int. Ed. 2014, 53, 13785 –13788
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 13787

.
Angewandte
Communications
[1] a) J. Zhang, R. E. Campbell, A. Y. Ting, R. Y. Tsien, Nat. Rev.
Mol. Cell Biol. 2002, 3, 906 – 918; b) R. Y. Tsien, Annu. Rev.
Biochem. 1998, 67, 509 – 544.
[2] M. Andresen, R. Schmitz-Salue, S. Jakobs, Mol. Biol. Cell 2004,
15, 5616 – 5622.
[3] G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N.
Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohana,
M. Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G.
Vidugiris, J. Zhu, A. Darzins, D. H. Klaubert, R. F. Bulleit, K. V.
Wood, ACS Chem. Biol. 2008, 3, 373 – 382.
[4] A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K.
Johnsson, Nat. Biotechnol. 2003, 21, 86 – 89.
[5] A. Gautier, A. Juillerat, C. Heinis, I. R. Correa, Jr., M. Kinder-
mann, F. Beaufils, K. Johnsson, Chem. Biol. 2008, 15, 128 – 136.
[6] a) B. A. Griffin, S. R. Adams, R. Y. Tsien, Science 1998, 281,
269 – 272; b) B. R. Martin, B. N. Giepmans, S. R. Adams, R. Y.
Tsien, Nat. Biotechnol. 2005, 23, 1308 – 1314.
[7] M. F. Langhorst, S. Genisyuerek, C. A. Stuermer, Histochem.
Cell Biol. 2006, 125, 743 – 747.
[8] L. Wang, P. G. Schultz, Chem. Commun. 2002, 1 – 11.
[9] a) M. Grammel, H. C. Hang, Nat. Chem. Biol. 2013, 9, 475 – 484;
Figure 3. Fluorescence (left column) and corresponding bright-field
(right column) confocal microscopy images for no-wash cell labeling
with YC20: a,b) dC10a–actin-expressing cells treated with YC20.
c,d) Histone-H2B–dC10a-expressing cells treated with YC20.
e,f) pcDNA-transfected cells (negative control) treated with YC20.
Scale bars=10 mm. Cyan fluorescence conditions: Em 482 nm (band-
width: 35 nm), dichromic mirror: 400–457 nm.
´
b) I. Nikic, T. Plass, O. Schraidt, J. Szymanski, J. A. Briggs, C.
Schultz, E. A. Lemke, Angew. Chem. Int. Ed. 2014, 53, 2245 –
2249; Angew. Chem. 2014, 126, 2278 – 2282; c) J. C. Carlson,
L. G. Meimetis, S. A. Hilderbrand, R. Weissleder, Angew. Chem.
Int. Ed. 2013, 52, 6917 – 6920; Angew. Chem. 2013, 125, 7055 –
7058; d) Y. Takaoka, A. Ojida, I. Hamachi, Angew. Chem. Int.
Ed. 2013, 52, 4088 – 4106; Angew. Chem. 2013, 125, 4182 – 4200.
[10] a) S. Girouard, M. H. Houle, A. Grandbois, J. W. Keillor, S. W.
Michnick, J. Am. Chem. Soc. 2005, 127, 559 – 566; b) J. Guy, R.
Castonguay, N. B. C. R. Pineda, V. Jacquier, K. Caron, S. W.
Michnick, J. W. Keillor, Mol. Biosyst. 2010, 6, 976 – 987.
[11] J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G. Skene, J. W.
Keillor, J. Am. Chem. Soc. 2007, 129, 11969 – 11977.
[12] K. Caron, V. Lachapelle, J. W. Keillor, Org. Biomol. Chem. 2011,
9, 185 – 197.
great selectivity in living cells. Kinetic studies clearly show
that the improved selectivity for the dC10a tag over mono-
thiol compounds is due to the attenuated reactivity of the
maleimide groups, such that they react preferentially with the
highly reactive thiols of the dC10a tag. This new labeling
reagent is nontoxic and enables the highly specific labeling of
a POI in live cells with no washing. It thus has significant
potential for general application to labeling specific proteins
in living cells.
[13] A. Russo, E. A. Bump, Methods Biochem. Anal. 1988, 33, 165 –
241.
Received: August 6, 2014
[14] A. J. Kirby, P. W. Lancaster, J. Chem. Soc. Perkin Trans. 2 1972,
1206 – 1214.
Published online: October 14, 2014
[15] M. K. Sahoo, S. B. Mhaske, N. P. Argade, Synthesis 2003, 346 –
349.
Keywords: bioorthogonal chemistry · fluorescent probes ·
glutathione · maleimide · protein labeling
.
13788 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 13785 –13788