Environment-sensitive fluorescent turn-on probes targeting hydrophobic ligand-binding domains for selective protein detection

Article abstract of DOI:10.1002/anie.201302884

Bind and shine: An approach for the selective detection of both enzymes and non-enzymatic proteins using an environment-sensitive fluorescent turn-on probe is described (see scheme). This approach targets the hydrophobic ligand-binding domain of the targe

Full text of DOI:10.1002/anie.201302884

Angewandte
Chemie
Communications
Fluorescent Probes
Y.-D. Zhuang, P.-Y. Chiang, C.-W. Wang,
K.-T. Tan*
&&&& — &&&&
Environment-Sensitive Fluorescent Turn-
On Probes Targeting Hydrophobic
Ligand-Binding Domains for Selective
Protein Detection
Bind and shine: An approach for the
selective detection of both enzymes and
non-enzymatic proteins using an envi-
ronment-sensitive fluorescent turn-on
probe is described (see scheme). This
approach targets the hydrophobic ligand-
binding domain of the target protein to
trigger the fluorescence turn-on and was
shown to be specific for the targeted
protein.
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DOI: 10.1002/anie.201302884
Fluorescent Probes
Environment-Sensitive Fluorescent Turn-On Probes Targeting
Hydrophobic Ligand-Binding Domains for Selective Protein
Detection**
Yu-De Zhuang, Po-Yi Chiang, Chia-Wen Wang, and Kui-Thong Tan*
Protein-specific detection or imaging is important in medical
diagnosis to detect protein biomarkers, as well as in biology to
investigate cellular processes. Small-molecule fluorescent
turn-on probes that can detect specific proteins are partic-
ularly valuable as they allow for sensitive, simple, and specific
detection with high signal-to-background ratios.[1] Currently
most of the small-molecule fluorescent turn-on probes are
designed for monitoring enzyme activities, for example,
glycosidases, proteases, lactamases, and kinases.[2] Typically,
their fluorescence turn-on mechanism is based on an enzy-
matic reaction with the chemical probes to convert a non-
fluorescent substrate into a fluorescent product. On the other
hand, the design of fluorescence probes for non-enzymatic
proteins remains a challenging task. Currently, several
fluorescence turn-on strategies such as hairpin peptide
beacons,[3] aptamers,[4] supramolecular approaches,[5] poly-
mer-conjugated nanoparticles,[6] and ligand-directed affinity
labeling[7] have been reported to detect proteins through non-
enzymatic reactions. Fluorescence signals can be turned-on
upon recognition of the target proteins through charge–
charge interaction, affinity labeling, or recognition of a spe-
cific peptide sequence. Although these fluorescent probes use
novel strategies to detect non-enzymatic proteins, neverthe-
less, they suffer from either low selectivity and small
fluorescent turn-on ratios, or are limited to sensing proteins
on the cell surface. Thus, the development of a more general
strategy to generate protein-specific fluorescent turn-on
probes is necessary.
target protein. The fluorescent probes can be synthesized by
incorporating an environment-sensitive fluorophore with
a protein-specific small-molecule ligand (Figure 1a). Envi-
ronment-sensitive fluorophores have emission properties that
are highly sensitive to their immediate environment.[8]
Typically they exhibit very weak fluorescence in polar and
protic environments but show strong fluorescence and blue-
shifted emission in hydrophobic surroundings. The rationale
behind this new fluorescent probe design is that most of the
ligand-binding sites in proteins are hydrophobic, and hydro-
Herein, we introduce a novel strategy to generate
fluorescent chemical probes for the selective detection of
both enzymes and non-enzymatic proteins. Fluorescence
turn-on can be achieved upon the binding of the fluorescent
probes to the hydrophobic ligand-binding domain of the
[*] Y.-D. Zhuang,[+] P.-Y. Chiang,[+] C.-W. Wang, Prof. Dr. K.-T. Tan
Department of Chemistry, National Tsing Hua University
101 Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan (ROC)
E-mail: kttan@mx.nthu.edu.tw
Figure 1. a) Illustration of the fluorescent turn-on probes for selective
protein detection. The mechanism is based on the binding of the
ligand to a specific hydrophobic site in a protein, whereby the adjacent
hydrophobic environment can cause the environment-sensitive fluoro-
phore to exhibit stronger fluorescence. In the absence of target protein,
the fluorescent probe has low fluorescence. b) Chemical formulas of
fluorescent probes 1, 2, 3, and 4 for hCAII; 5, 6, and 7 for trypsin; 8
for avidin. Probe 1 is conjugated with the SBD fluorophore, while
probes 3 and 4 are conjugated with NBD and dansyl fluorophores,
respectively. R1, R2, and R3 are the ligands for binding with hCAII,
trypsin, and avidin.
Prof. Dr. K.-T. Tan
Frontier Research Center on Fundamental and Applied Sciences of
Matters, National Tsing Hua University
101 Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan (ROC)
[+] These authors contributed equally to this work.
[**] We are grateful to the National Science Council (Grant No.: 100-
2113-M007-018-MY2) and Ministry of Education (Grant No.:
101N2011E1), Taiwan (ROC) for financial support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302884.
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phobic interactions consti-
tute the principal thermo-
dynamic driving force for
the binding of small-mole-
cule ligands to their pro-
teins.[9] Therefore we expect
that the binding of the
ligand to the hydrophobic
ligand-binding domain of
the target protein would
bring the environment-sen-
sitive fluorophore closer to
the hydrophobic pocket of
the protein, and the prox-
imity of the hydrophobic
environment could cause
the fluorophore to emit
stronger fluorescence. In
contrast, in the absence of
target protein, the fluores-
cent probe would remain in
the aqueous buffer and
should exhibit only weak
fluorescence.
Although
many examples have been
reported, such as the con-
jugation of environment-
sensitive fluorophores to
peptides and proteins to
study protein–protein inter-
actions[10] and for the sens-
ing of small molecules,[11]
the application of small-
Figure 2. Detection of hCAII with environment-sensitive sulfonamide probes. a) Fluorescence spectra of 1 mm
1 in the absence (dashed line) and presence of 10 mm hCAII (solid line) in PBS buffer (1% DMSO), and after
addition of 100 mm ethoxzolamide (EZA, dotted line). lex =430 nm. b) Fluorescence response of 2 in the
absence and presence of 10 mm hCAII. lex =448 nm. c) Fluorescence spectra of 3 (1 mm) in the absence and
presence of 10 mm hCAII. lex =460 nm. d) Fluorescence spectra of 4 (1 mm) in the absence and presence of
10 mm hCAII. lex =340 nm.
molecule ligands conjugated to environment-sensitive fluo-
rophores for the selective detection of proteins has not been
described before.
upon the addition of hCAII (Figure 2a). The enhanced
fluorescence of 1 can be quenched again by the addition of
ethoxzolamide, a strongly competitive sulfonamide inhibitor
for hCAII. In addition, such a fluorescence increase was not
observed with the control probe containing no sulfonamide
ligand moiety (Figure S1). These results show that the
fluorescence increase is reversible and triggered only by the
specific recognition of the arylsulfonamide ligand moiety to
the hydrophobic pocket of hCAII.
We also compared the fluorescence spectra of 1 in PBS
buffer alone and in the presence of hCAII. The maximum
emission wavelength of 1 is blue-shifted about 39 nm from
589 nm in PBS buffer to 550 nm when hCAII is added. The
blue-shifted emission spectrum of 1 overlays well with the
spectrum from 1 dissolved in a hydrophobic solvent, aceto-
nitrile (Figure S2). To further ascertain that the fluorescence
enhancement of 1 is due to the hydrophobic environment of
the arylsulfonamide binding site, we synthesized probe 2 by
incorporating an amino acid sarcosine as a linker in between
the arylsulfonamide ligand and the SBD fluorophore. In the
presence of hCAII, 2 exhibits only a twofold increase in
fluorescence (Figure 2b) and there is almost no fluorescence
enhancement when the linker was extended even further (up
to eight atoms; Figure S3). These results indicate that the
blue-shifted emission and fluorescence increase can be
attributed to the close proximity of the SBD fluorophore to
To generate environment-sensitive fluorescent turn-on
probes for the selective detection of proteins, a small and
highly environment-sensitive fluorophore, 4-sulfamonyl-7-
aminobenzoxadiazole (SBD)[12] was incorporated with differ-
ent ligands (Figure 1b). We further found that this fluoro-
phore is a unique and optimal environment-sensitive fluo-
rophore for this probe design because it gives remarkable
fluorescence enhancement upon binding of the ligand to the
target protein. Other environment-sensitive fluorophores,
such as NBD and dansyl, gave no such fluorescence increase.
In addition, SBD can be easily derivatized with different
substituents at the 4-sulfamonyl moiety, which allows for
optimization of the fluorescence enhancement with different
substituents.
To test our fluorescent probe, human carbonic anhydrase
II (hCAII), a monomeric soluble protein that consists of
a hydrophobic binding site for many arylsulfonamide ligands,
was selected as a target protein.[13] For the detection of hCAII,
fluorescent probe 1 was prepared by reacting benzylamine
sulfonamide with dimethyl-4-sulfamonyl-7-fluorobenzoxadia-
zole (DBD-F; Supporting Information, Scheme S1). The
fluorescence was extremely weak when 1 was in aqueous
PBS buffer, but this was dramatically enhanced (by 15-fold)
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the hydrophobic binding domain. When probe 1 was incu-
bated with an increasing concentration of hCAII, a gradual
increase in fluorescence intensity with concentration was seen
and the binding constant for hCAII with probe 1 was 4.0 Æ
0.3 mm (Figure S4).
The choice of an environment-sensitive fluorophore was
crucial for the probes to give high fluorescence enhancement
upon binding to the target protein. To ascertain whether other
environment-sensitive fluorophores could be used in this
probe, we attached the same arylsulfonamide to two well-
known environment-sensitive fluorophores, NBD-Cl and
dansyl-Cl, giving probes 3 and 4. Interestingly 3, which has
a very similar structure to 1, showed no change in fluores-
cence intensity in the presence of hCAII (Figure 2c). This is
interesting because probe 1 and 3 share the same benzox-
adiazole fluorophore skeleton, with the only difference being
that 1 has a dimethyl-4-sulfamonyl moiety and 3 has a nitro
group.[14] We also found that the dimethyl-4-sulfamonyl group
on the benzoxadiazole skeleton is critical for high fluores-
cence enhancement, because probes where the dimethyl-4-
sulfamonyl was changed to sulfonic acid exhibited no
fluorescence enhancement (Figure S5). Currently, the effect
of the dimethyl-4-sulfamonyl group on enhancing the fluo-
rescence in the protein ligand-binding domain is not under-
stood. We postulate that, as compared with the sulfonic and
nitro moieties, the dimethyl-4-sulfamonyl SBD fluorophore is
perhaps more susceptible to the hydrophobic microenviron-
ment around the ligand-binding domain. On the other hand, 4
exhibited only a onefold fluorescence increase upon addition
of hCAII (Figure 2d). These results suggest that the highly
environment-sensitive SBD fluorophore is a good choice for
our fluorescent sensor.
Figure 3. a) Fluorescence responses of probe 5, 6, and 7 (1 mm each)
in the absence and presence of trypsin (100 mm). lex =430 nm and
lem =560 nm (for 5), lem =530 nm (for 6), lem =550 nm (for 7).
b) Fluorescence response of 1 mm probe 8 with 1 mm avidin.
lex =430 nm, lem =569 nm.
To demonstrate the modular nature of our probe for
protein detection, we replaced the arylsulfonamide on the
SBD fluorophore to benzamidine for the detection of trypsin.
It was reported that the binding pocket of benzamidine for
trypsin is also hydrophobic,[15] therefore we expected that its
binding to trypsin would also place the SBD fluorophore in
close proximity to the hydrophobic pocket, causing an
increase in fluorescence. Probe 5, which consists of the
dimethyl substituent, was prepared initially for the detection
of trypsin. In contrast to the large fluorescence enhancement
of 1 with hCAII, probe 5 showed only 2.5-fold fluorescence
increase upon addition of trypsin. On the other hand, probe 6
which consists of a benzylguanine substituent at the 4-
sulfamonyl moiety showed a dramatic 17-fold fluorescence
increase (Figure 3a). Titration of 5 and 6 with an increasing
concentration of trypsin reveals that 6 has a stronger binding
affinity to trypsin (Kd = 10.6 Æ 1.3 mm), while 5 has Kd = 20.6 Æ
2.6 mm (Figure S6). Probe 7, which has a benzyl substituent,
was synthesized to help understand the high fluorescence
enhancement of probe 6. In the presence of trypsin, 7 exhibits
a fluorescence increase of 4.5-fold with a binding affinity of
5.7 Æ 0.8 mm. The stronger binding affinity of 6 and 7 is in
agreement with a previous study, which reported that besides
the binding of the benzamidine group in the trypsin
S1 pocket, a secondary sulfonamide moiety in the molecule
can also interact favorably with the amino acids located in the
S3/S4 hydrophobic pocket.[16] We therefore postulated that
this additional interaction results in the better positioning of
the SBD fluorophore in the trypsin binding pocket with the
larger size of the benzylguanine group providing a more
hydrophobic environment and therefore exhibiting higher
fluorescence enhancement. The fluorescence of 6 can be
quenched by the addition of an excess amount of free
benzamidine (Figure S7), indicating that the fluorescent
increase is produced by the specific recognition of the
benzamidine ligand by the hydrophobic pocket of trypsin.
Similar to the situation with hCAII, two other trypsin probes
constructed by attaching benzamidine to NBD-Cl and dansyl-
Cl do not exhibit fluorescence enhancement upon addition of
trypsin (Figure S8), again indicating that SBD fluorophore is
an optimal environment-sensitive fluorophore for our strat-
egy.
Based on the results for trypsin detection, where three
different substituents introduced at the 4-sulfamonyl moiety
exhibited several fold fluorescence enhancement, we replaced
the dimethyl substituent of probe 1 with benzyl and benzyl-
guanine to see if high fluorescence turn-on could be achieved
in hCAII as well. In the presence of hCAII, both probes
showed fluorescence enhancement of about fivefold (Fig-
ure S9). This indicates that the SBD fluorophore is a versatile
environment-sensitive fluorophore and different substituents
can be tolerated at the 4-sulfamonyl moiety, which could lead
to a variety of applications in diagnostics and imaging of
proteins. For example, an SBD fluorescent probe incorpo-
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rated with a benzylguanine moiety could be site-specifically
conjugated to a self-labeling protein, SNAP-tag,[17] to study
protein ligand interaction in a specific subcellular compart-
ment of living cells.[18]
of proteins at 100 mm were used. At this high protein
concentration, we observed some nonspecific fluorescence
increases from BSA (fourfold), concanavalin A (twofold) and
b-casein (threefold), and virtually no change of fluorescence
intensity for the other proteins (Figure 4b). For avidin probe
8, fluorescence enhancement occurred only when avidin was
added, but not with the other nine non-targeted proteins
(Figure S11). The excellent selectivity of our fluorescent
probes toward their target proteins shows its potential for
clinical diagnostics and cellular imaging for specific detection
of non-enzymatic proteins.
In summary, based on the fact that most ligand-binding
sites are hydrophobic and that the emission properties of
environment-sensitive fluorophores differ between aqueous
and hydrophobic environments, we have established new
fluorescent turn-on probes by using the SBD fluorophore
conjugated to a protein-specific ligand for the detection of
native enzymes and non-enzymatic proteins with diverse
functions and structures. This probe is modular and versatile
as demonstrated by the selective detection of hCAII, trypsin,
and avidin. Because most ligand-binding sites have a hydro-
phobic environment, we expect this approach to be a general
strategy to detect enzymes and non-enzymatic proteins.
Herein, we found that the SBD fluorophore is an ideal
environment-sensitive fluorophore for the current probe
design, because ligands conjugated with NBD or dansyl
fluorophores exhibited no significant fluorescent enhance-
ment.
Finally, fluorescent probe 8 was synthesized for the
detection of avidin, a non-enzymatic protein which consists
of a hydrophobic binding pocket for biotin.[19] In the presence
of avidin, the fluorescence intensity of 8 was enhanced by 4.5-
fold (Figure 3b; Figure S10). The lower fluorescence en-
hancement for 8 was attributed to the ethylene linker between
the SBD fluorophore and biotin, which distances the fluo-
rophore from the hydrophobic biotin binding domain. We
believe that an avidin probe without the linker would give
a better fluorescence increase.
We then proceeded to study the selectivity of our
environment-sensitive fluorescent probe by incubating nine
other non-targeted proteins with 1, 6, and 8. As shown in
Figure 4a, 1 shows exceptional selectivity toward its target
protein hCAII. With the exception of BSA, which shows
a 1.7-fold increase in fluorescence, all the other non-targeted
proteins exhibited no fluorescence change with probe 1 at
10 mm. For a selectivity test of the trypsin probe 6, the same set
As compared with other fluorescent turn-on strategies to
detect non-enzymatic proteins, our fluorescent probes show
remarkable fluorescence enhancement and high protein
selectivity. Preparation of our fluorescent probes requires
only simple synthetic steps and can be generated in large
quantities at low cost (see Supporting Information for
synthetic details). Because these fluorescent probes have
smaller molecular size and neutral charge, we believe that
they could easily diffuse into cells for imaging and to study
various intracellular receptors, for example, the estrogen
receptor.[20] Finally, we believe that this novel fluorescent
turn-on probe will be useful for a wide range of applications,
such as diagnosis and molecular imaging where high fluo-
rescent turn-on ratios and simple detection methods are
required.
Received: April 8, 2013
Revised: May 27, 2013
Published online: && &&, &&&&
Keywords: environment-sensitive fluorophores ·
.
fluorescent probes · non-enzymatic proteins · protein detection ·
protein modifications
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