Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

Article abstract of DOI:10.1016/j.bmcl.2017.05.087

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occas

Full text of DOI:10.1016/j.bmcl.2017.05.087

Accepted Manuscript
Development of an ON/OFF switchable fluorescent probe targeting His tag
fused proteins in living cells
Koyo Okitsu, Takashi Misawa, Takuji Shoda, Masaaki Kurihara, Yosuke
Demizu
PII:
S0960-894X(17)30585-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.087
BMCL 25032
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 April 2017
29 May 2017
30 May 2017
Please cite this article as: Okitsu, K., Misawa, T., Shoda, T., Kurihara, M., Demizu, Y., Development of an ON/
OFF switchable fluorescent probe targeting His tag fused proteins in living cells, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.087
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Development of an ON/OFF switchable fluorescent probe targeting His tag fused
proteins in living cells
Koyo Okitsu, Takashi Misawa*, Takuji Shoda, Masaaki Kurihara, and Yosuke Demizu*
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, Japan
Keywords: cell-penetrating peptide; maleimide; ON/OFF switch; His tag fused protein
*Corresponding author: Tel.: +81-3-3700-1141, Fax: +81-3-3707-6950, E-mail:
misawa@nihs.go.jp, demizu@nihs.go.jp
Abstract
The fluorescent labeling of target proteins is useful for analyzing their functions and
localization in cells, and several fluorescent probes have been developed. However, the
fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally
affects their functions and/or localization in living cells. Therefore, an imaging method
that uses short peptide tags such as hexa-histidine (the His tag) has been attracting
1

increasing attention. Few studies have investigated ON/OFF switchable fluorescent
probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF
switchable probe for imaging targeted intracellular proteins fused with a CH6 tag,
which is composed of one cysteine residue and six histidine residues.
The fluorescent labeling of target proteins is useful for analyzing their functions and
localization in cells, and a number of fluorescent probes have been developed to date.
The green fluorescent protein (GFP) is a representative fluorescent probe and the
genetic fusion of GFP to target proteins is broadly used to visualize the behaviors of
these proteins in cells.1,2 However, the fusion of GFP to a target protein of interest (POI)
sometimes affects its function and/or localization due to the large molecular size of
GFP.3 In order to overcome these issues, several fluorescent labeling methods using
short peptide tags such as the FlAsH4, ReAsH5 tags, and tetraserine motifs6 have recently
been reported. The hexa-histidine tag (His tag) is a well-known short peptide tag that
interacts with metal ions such as copper (Cu), nickel (Ni), and cobalt (Co) through
non-covalent bonds. Therefore, the His tag represents a promising approach to POI
2

labeling because its molecular size has minimal effects7, and it is often utilized in the
affinity purification of genetically modified proteins. Several Ni (II)-NTA
(nickel-nitrilotriacetic acid)-based probes that target His-tagged proteins have been
reported to date.8-10 However, Ni (II)-NTA-based probes for His-tagged proteins are
limited to the labeling of membrane proteins because Ni (II)-NTA itself is impermeable
to the cell membrane. Recent studies reported that a Ni (II)-NTA-based fluorescent
probe bearing a chloroacetamide moiety targeted the CH6 tag, which comprises one
cysteine residue and the His tag, through a covalent bond between a cysteine residue on
the CH6 tag and the chloroacetamide group on the fluorescent probe, thereby increasing
labeling efficiency.11,12
The ON/OFF switching of fluorescence is also important for improving its sensitivity.
An ON/Off switchable probe, which only emits fluorescence when it interacts with POI,
efficiently enables observations of the behavior of POI in living cells without the
washing of excess amounts of the fluorescent probe.13-15 Therefore, ON/OFF switchable
probes have been attracting increasing attention as an effective imaging tool.
We herein developed an ON/OFF switchable imaging method that targets intracellular
3

CH6-tagged POI in living cells. We specifically designed and synthesized fluorescent
probe 1 and the carrier peptide R9-H4A2, as shown in Figure 1. 7-(Diethylamino)
coumarin as a fluorophore was conjugated with three Ni (II)-NTA moieties to strengthen
binding affinity to the His tag16, and with a maleimide moiety not only to form a
covalent bond with the cysteine residue of the CH6 tag, but also to quench fluorescence
by photoinduced electron transfer (PeT).17 We also synthesized the carrier peptide
R9-H4A2, consisting of the representative cell-penetrating peptide nona-arginine (R9)
and H4A2 (Sequence: HAHAHH, H: Histidine, A: Alanine) fragment. The H4A2
moiety interacts with probe 1 through the Ni-NTA moiety and delivers hydrophilic
fluorescent probe 1 into cells by the R9 fragment. Due to the substitution of two
histidines with two alanines, the binding affinity of the H4A2 moiety is weaker than that
of the His tag to POI. Therefore, we expected probe 1 to dissociate from the carrier
peptide and target the CH6 tag in cells.
In the fairly recent past, we have developed the small molecules that mediated
degradation of His-tag fused protein using the same strategy.18 In this report, MV-1,
which induces the ubiquitination of target proteins, was conjugated with three Ni
4

(II)-NTA moieties. The hybrid compound was internalized by the supports of carrier
peptide and formed the covalent bond with cysteine residue of CH6 tagged target
proteins, subsequently the selective degradation of target proteins occurred. These
results indicated that the probe 1 is capable of selectively reacting with CH6 tagged
proteins and visualize the target proteins in living cells.
Figure 1. Chemical structure of fluorescent probe 1 and the sequence of the carrier
peptide R9-H4A2.
The mechanism proposed for the ON/OFF switchable imaging method of CH6-tagged
POI is shown in Figure 2. Fluorescent probe 1 and the carrier peptide R9-H4A2
initially form a complex through interactions between the Ni (II)-NTA and H4A2
5

moieties. The complex then penetrates the cell membrane through the effects of R9.
Fluorescent probe 1 dissociates from the complex and interacts with CH6-tagged POI.
The cysteine residue of the CH6 tag is physically close to the maleimide moiety of 1
and forms a covalent bond. The fluorescence of probe 1 is recovered by the loss of PeT
effects and enables the visualization of CH6-tagged POI (Figure 2).
Fluorescent group
Ni-NTA
CPP
N
O
O
His
O
N
O
His-tag
His tag
His
His
complex
CH6
CH6-tag
CH6 tag
1) delivery
Cell surface
SH
Protein Of
Interest
POI
Cytosol
dissociation
His
quench
N
O
O
fluorescence
2) bind to POI
CH6
N
O
O
CH6
SH
POI
CH6
N
POI
O
O
SH
S
3) imaging of POI
POI
Figure 2. Proposed mechanism for the ON/OFF switchable imaging method.
The synthetic route of fluorescent probe 1 was shown in Scheme 1. H-Glu(OBzl)-OtBu
was treated with tert-butyl 2-bromoacetate to form a NTA moiety and the benzyl group
6

was removed using Pd/C under a H2 atmosphere to give compound 3. Compound 4 was
synthesized
by
the
condensation
of
3
eq.
compound
3
and
1,4,7,10-tetraazacyclododecane. The resulting amino group of 4 was condensed with
6-(carbobenzoxyamino)hexanoic acid [Cbz-Acp(6)-OH]] and the subsequent
deprotection of the Cbz group to give compound 6. Fmoc-azidolysine was coupled with
6 and the Fmoc group was subsequently removed by diethylamine to afford compound 8.
Compound 8 was coupled with 7-(diethylamino)coumarin, the maleimide moiety was
attached by 1,3-dipolar cycloaddition, and all tert-butyl ester groups were then removed
under acidic conditions to afford target compound 10.19 The solution of compound 10
was added to 3 eq. NiCl2 in PBS solution to form the Ni ion chelate 1, and the complex
was then used in assays. The carrier peptide R9-H4A2 was synthesized using the
microwave-assisted solid phase method. 18
7

Scheme 1. (a) tert-Butyl 2-bromoacetate, DIPEA, DMF, 55oC, 16 h; (b) Pd/C, H2,
MeOH, rt. 6 h; (c) 1,4,7,10-Tetraazacyclododecane, EDC, HOBT, DIPEA, DCM, rt., 12
h; (d) Cbz-Acp(6)-OH, HATU, HOAT, DIPEA, DCM, rt., 1 d; (e) Fmoc-azidolysine,
EDC, HOBT, DIPEA, DCM, rt., 20 h; (f) Diethylamine, THF, rt., 6 h; (g)
7-(Diethylamino)coumarin-3-carboxylic acid, EDC, HOBT, DIPEA, DCM, rt., 20 h; (h)
8

N-Propargylmaleimide, CuSO4, sodium ascorbate, rt., 20 h; (i) TFA, DCM, rt., 4 h; (j)
NiCl2, PBS, rt., 2 h.
We initially investigated whether the fluorescence intensity of probe 1 is switchable in
vitro using fluorescence spectra. Probe 1 showed an absorption maxima at 435 nm and
emission at 475 nm. Furthermore, the addition of 6 μM glutathione (GSH) increased
fluorescence intensity, indicating that the maleimide moiety quenches the fluorescence
intensity of probe 1, and the cysteine residue of GSH reacts with the maleimide moiety,
resulting in the regulation of the ON/OFF switch (Figure 3).
800
700
2μM probe 1
600
500
2μM probe 1 +6μM GSH
400
300
200
100
0
450
500
550
600
Wavelength(nm)
Figure 3. Fluorescence intensity of 2 μM of probe 1 with or without 6 μM of GSH.
9

Fluorescent probe 1 is possible to react with thiol groups of other proteins in the cells.
Therefore, we evaluated that the selectivity of probe derivatives 11 to CH6 tagged POI
over 5 mM GSH. We monitored the reaction between compound 11 and CH6 peptide in
the coexistence of 5 mM GSH.20 As shown in Figure 4, compound 11 and CH6 peptide
were detected in 6 min, 20.5 min respectively. Compound 11 reacted with CH6 peptide
with or without 5 mM GSH and the resulting product was detected (lanes 2, 3; retention
time = 18.3 min). On the other hand, the mixture of compound 11 and 5 mM GSH gave
another peak in 17.8 min (Lane 4). These results suggest that the fluorescent probe 1
could preferentially react with CH6 tagged POI over a thiol group of other proteins.
10

Figure 4. The reaction monitoring between compound 11 and CH6 peptide in
coexistence of 5 mM GSH by HPLC. Condition 1: CH6 pep and compound 11 (reaction
time = 0, Lane 1), Condition 2: CH6 pep and compound 11 (Lane 2), Condition 3: CH6
peptide, compound 11, 5mM GSH (Lane 3), Condition 4: Compound 11 and 5 mM
GSH (Lane 4).
We then examined the effects of probe 1 on fluorescence intensity against CH6-tagged
POI-expressing HT1080 cells. HT1080 cells were transfected with and stably expressed
CH6-tagged CRABP2 2×(CH6)-CRABP2), which was prepared as previously reported18.
HT1080 cells constitutively expressing 2×(CH6) -CRABP2 were treated with the
solution of 20 μM of probe 1 and 20 μM of R9-H4A2 at 37 oC for 2.5 h.21 Fluorescence
intensity was measured and quantified using flow cytometry. As shown in Figure 5,
fluorescence intensity against HT1080 cells stably expressing 2×(CH6)-CRABP2 was
2-fold higher than that against HT1080 cells. Moreover, fluorescence intensity without
R9-H4A2 was markedly decreased, indicating that R9-H4A2 played a pivotal role in
the delivery of probe 1 into cells.
11

No expression
2×(CH6)-CRABP2
12
10
8
Concentration;
Probe 1: 20 μM
R9-H4A2: 20 μM
6
4
2
0
probe 1
probe 1
probe 1
probe 1
+
+
R9-H4A2
R9-H4A2
Figure 5. Effects of probe 1 and R9-H4A2 against HT1080 cells and HT1080 cells
stably expressing 2×(CH6)-CRABP2.
We then investigated whether CH6-tagged POI is observed using our method without
washing. HT1080 cells stably expressing 2×(CH6)-CRABP2 were treated with the
o
solution of 20 μM of probe 1 and 20 μM of R9-H4A2 at 37 C for 2.5 h and
fluorescence was observed using confocal laser scanning microscopy.22 An increase in
fluorescence was detected in living cells without washing. The increase observed in
fluorescence against HT1080 cells stably expressing 2×(CH6)-CRABP2 was greater
12

than that against HT1080 cells (Figure 6).
2x(CH6)-CRABP2
20μM probe 1
20μM probe 1
+
20μM R9-H4A2
Figure 6. Confocal laser scanning microscopy analyses. Fluorescent (upper panels) and
phase contrast (lower panels) images are shown. Scale bars represent 10 μm.
We performed a cytotoxicity assay using cell counting kit-8 according to the
13

manufacturer’s instructions.23 The results of the cytotoxicity assay revealed that
fluorescent probe 1 and R9-H4A2 did not exhibit marked cytotoxicity against HT1080
cells at 30 μM. These results suggest that our imaging method is broadly applicable to
CH6-tagged POI (Figure 7). Ni ions are generally cytotoxic; however, the Ni ion of
probe 1 was chelated by the NTA moiety and, thus, was not cytotoxic.
140
120
100
5uM
5 µM
80
60
40
20
0
101u0MµM
20uM
20 µM
30 µM
30uM
probe
+
probe
R9-H4A2
R9-H4A2
Figure 7. Cytotoxicities of probe 1 and R9-H4A2.
In summary, we herein developed an ON/OFF switchable fluorescent probe for
CH6-tagged POI. The fluorescence intensity of probe 1 was increased by the presence
14

of cysteine residues in vitro and in living cells, and was detected using confocal laser
scanning microscopy without washing. Furthermore, synthesized compounds did not
exhibit significant cytotoxicity. These results demonstrate that our method is applicable
to the imaging of several CH6-tagged POI. Further biological analyses are currently
underway.
Acknowledgments
We thank Dr. Naito Mikihiko and Dr. Hattori Takayuki who provided expertise that
greatly assisted this research. This work was supported by the Shorai Foundation for
Science and Technology (Y. D.), a Grant-in-Aid for scientific research (C: 26460169,
17k08385) from the Japan Society for the Promotion of Science (Y. D.), and a
Grant-in-Aid for young scientists (B) from the Japan Society for the Promotion of
Science (T. M.: 15K18905).
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[19] Synthesis of precursor 10: Compound 8 (50 mg, 26 μmol) was dissolved in t-BuOH (4 mL)
and H2O (2 mL) followed by the addition of propargylmaleimide (35 mg, 0.26 mmol), copper
(II) sulfate (6.4 mg, 40 μmol), and sodium ascorbate (7.9 mg, 40 μmol). After continuous
stirring at room temperature for 20 h, the mixture was concentrated in vacuo to give a crude
compound, which was used in the next reaction without further purification.
The above compound was dissolved in DCM (2 mL), followed by the addition of trifluoroacetic
acid (5 mL). The mixture was stirred at room temperature for 4 h. The solvent was then
removed, and the residue was purified by reversed-phase HPLC using a Discovery® Bio Wide
Pore C18 column (25 cm x 21.2 mm). After being purified, the solution was lyophilized to give
15 mg (38%) of compound 10 as a yellow solid. Compound purity was assessed using analytical
HPLC with a Discovery® Bio Wide Pore C18 column (25 cm x 4.6 mm; Retention time = 19.5
min, solvent A: 0.1% TFA/water, solvent B: 0.1% TFA/MeCN, flow rate 1.0 mL/min, gradient:
10-60% gradient of solvent B over 30 min). MS [HR-ESI(+)]: m/z calcd. [M +2H]2+: 777.3176;
found: 777.3199.
[20] Reaction monitoring between compound 11 and CH6 peptide with or without 5 mM GSH
was performed using analytical HPLC with a Discovery® Bio Wide Pore C18 column (25 cm x
4.6 mm; Retention time = 19.5 min, solvent A: 0.1% TFA/water, solvent B: 0.1% TFA/MeCN,
flow rate 1.0 mL/min, gradient: 10-60% gradient of solvent B over 30 min).
[21] Flow cytometry: HT1080 cells were seeded on 6-well dishes at a density of 2.0 x 105
cells/well and cultured in DMEM for 24 h. Cells were treated with compound solution and
incubated for 2.5 h. Cells were then washed with phosphate buffer (PBS) and detached by a
treatment with trypsin-EDTA. The cells collected were pelleted by centrifugation at 3000 rpm
for 5 min and the supernatant was removed. The cells collected were suspended in 500 μL of
PBS and the mean fluorescence intensity of cells was measured using flow cytometry.
[22] Confocal laser microscopy: HT1080 cells were seeded onto glass-bottomed dishes (Greiner
17

Bio-one, Tokyo, Japan) (10,000 cells/well) and incubated overnight in 2 mL of DMEM
containing 10% FBS. Medium was replaced with fresh medium containing 10% FBS, and
compound solution was applied to the wells. After cells had been incubated for 3 h, they were
observed using LSM 710 (Carl Zeiss, Oberkochen, Germany) equipped with a 63X objective
lens (Plan-Apochromat, Carl Zeiss) at an excitation wavelength of 488 nm (Ar laser) for the
compound.
[23] Cytotoxicity; HT1080 cells were seeded on 96-well culture plates (5,000 cells/well) and
incubated for 24 h in DMEM containing 10% FBS. Compound solution in fresh DMEM was
added at each concentration. After 24 h, cell viability was evaluated using cell counting kit-8
(DOJINDO) following the manufacturer’s protocol. Results are presented as the mean and
standard error of 3 samples. The significance of differences was analyzed by the Student’s t-test.
18

fluorescence
CH6
quench
N
O
O
SH
CH6
N
POI
O
O
S
POI
H2O
O
OH2
O
O
Ni2+
O
N
Ni(II)-NTA
O
O
O
O
N
O
O
O
H2O
H2O
O
N
Ni2+
O
N
Fluorophore
O
N
O
N
O
O
O
O
O
N
O
H
N
N
N
H
O
O
O
O
Ni2+
O
H2O
N
N
N
OH2
O
N
O
Maleimide
probe 1