Activatable fluorescent probes for hydrolase enzymes based on coumarin-hemicyanine hybrid fluorophores with large Stokes shifts

Article abstract of DOI:10.1039/d0cc00559b

We show that the equilibrium of intramolecular spirocyclization of coumarin-hemicyanine hybrid fluorophores can be finely tuned by means of chemical modifications. We used this scaffold to develop activatable fluorescent probes with large Stokes shifts for γ-glutamyltranspeptidase and esterase.

Full text of DOI:10.1039/d0cc00559b

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DOI: 10.1039/D0CC00559B
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Activatable fluorescence probes for hydrolase enzymes
based on coumarin-hemicyanine hybrid fluorophore with
large Stokes shift
Hiroyoshi Fujiokaa,†, Shin-nosuke Uno , Mako Kamiya , Ryosuke Kojima , Kai Johnsson
and Yasuteru Urano
a,b†
c
c
*,b,d
*,a,c,e
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We show that the equilibrium of intramolecular oxygen species7-9, cyanide10, 11, hydrogen sulphide12, 13, and
spirocyclization of coumarin-hemicyanine
hybrid sulfur dioxide14, 15, as well as in a reagent inducing reductive
1
6
fluorophores can be finely tuned by means of chemical stress . In this work, we set out to develop new
modifications. We used this scaffold to develop activatable fluorescence probes with large Stokes shifts, targeting
fluorescence probes with large Stokes shifts for - hydrolases, based on intramolecular spirocyclization
glutamyltranspeptidase and esterase.
reaction of CHC hybrid fluorophores.
Intramolecular spirocyclization reaction is a ground-state
equilibrium between a spirocyclic form (closed form) and
an intact fluorophore form (open form) which has been
successfully utilized as a fluorescence switching mechanism
of rhodamine-based fluorescence probes17-20. It has also
Hydrolase enzymes play crucial roles in many biological
processes and in diseases such as cancer development and
metastasis1-3. Thus, visualizing these activities is important
for understanding the functions of hydrolases in biological
and pathophysiological contexts. Although various types of
fluorescence probes for detecting hydrolase activities have
been developed based on a series of fluorophores with
emission in the visible to near-infrared (NIR) wavelengths,4
the relatively small Stokes shifts of the fluorophores
sometimes result in severe overlapping between the
excitation and emission spectra, leading to poor signal-to-
noise ratios. Thus, in general, a larger Stokes shift is
advantageous to achieve sensitive detection.
been reported that CHC hybrid fluorophore bearing p-
nitrophenol exists in spirocyclic form, and has been used as
a
photoactivatable fluorophore for super-resolution
21
imaging , due to its ability to be converted to an open
fluorescent form upon photoirradiation, followed by the
thermal reversion to the original spirocyclic form. We
speculated that the spirocyclic behavior of the CHC hybrid
fluorophore might be altered by means of chemical
The coumarin-hemicyanine (CHC) fluorophore is a hybrid
structure of coumarin and hemicyanine5, 6, which shows the
distinct photophysical properties such as red-shifted
fluorescence emission wavelength (> 620 nm), relatively
large Stokes shift (>60 nm), and suitability for ratiometric
measurement. Thus, CHC hybrid fluorophore has been
utilized as the core fluorophore of probes targeting reactive
a)
R3
5
R3
5
4
pKcycl
4
3
6
- H+
3
6
2
7
2
7
1N
N 1
+ H+
X
R2
R2
N
O
O
N
O
O
R1
XH
R1
Open form
Closed form
b)
0
0
.15
400
300
pH 2.0
pH 2.0
pH 3.0
pH 4.0
pH 5.0
pH 6.0
pH 7.0
pH 7.4
pH 8.0
pH 9.0
pH 10.0
pH 11.0
pH 3.0
pH 4.0
pH 5.0
pH 6.0
pH 7.0
a. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: uranokun@m.u-tokyo.ac.jp
0.1
pH 7.4
pH 8.0
pH 9.0
pH 10.0
2
1
00
00
0
b.
Institute of Chemical Sciences and Engineering (ISIC), École Polytechnique
.05
0
pH 11.0
Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
c.
Tokyo 113-0033, Japan.
Department of Chemical Biology, Max Planck Institute for Medical Research,
d.
300 400 500 600 700
Wavelength (nm)
550 600 650 700 750 800
Wavelength (nm)
Jahnstrasse 29, 69120 Heidelberg, Germany. E-mail: johnsson@mr.mpg.de
CREST (Japan) Agency for Medical Research and Development (AMED), 1-7-1
e.
Fig. 1 (a) Chemical structures of the developed coumarin-hemicyanine (CHC)
hybrid fluorophores bearing intramolecular nucleophiles, which undergo
thermally equilibrating intramolecular spirocyclization in aqueous solution
equilibrium constant: pKcycl). (b) Absorption and fluorescence spectra of 2 µM
solutions of CHC-1 at various pH values in 0.2 M sodium phosphate buffer.
Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan
†
†
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: Synthetic procedures
(
and characterization of new compounds, and optical properties details. See
DOI: 10.1039/x0xx00000x
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modification. Firstly, we prepared CHC-1, which has a spirocyclic form, changes in the absorbance of VCieHwCAr-ti1clewOnelirnee
hydroxyethyl (HE) group at the indolenium N1 atom as a monitored (Figure 2). The distinctive a
D
b
O
s
I
o
:
r
10
b
.
a
10
n
3
c
9
e
/D
a
0
t
C
5
C
7
00
6
5
n
59B
m
potential intramolecular nucleophile (Figure 1a, Scheme S1). derived from the -conjugated CHC hybrid decreased after
CHC-1 exhibited absorption/emission maxima at 576/660 addition of NaOH aq, and subsequent addition of HCl aq.
nm with a Stokes shift of 84 nm in aqueous buffer at pH 2.0. resulted in complete recovery of the absorbance,
Ratiometric changes in absorption spectra were observed confirming the reversibility of the spirocyclization. Next, we
as the pH changed from acidic to alkaline: the absorbance at determined the equilibrium constant of intramolecular
5
76 nm decreases while that at around 400 nm increases. spirocyclization, pKcycl, which is defined as the pH at which
The appearance of blue-shifted absorption at around 400 the absorbance of the compound decreases to a half of the
2
2
nm at alkaline pH strongly suggests the generation of an maximum absorbance as a result of spirocyclization . The
intramolecularly spirocyclized structure with a divided - pKcycl value of CHC-1 was calculated to be 6.5 from the pH
conjugation of the fluorophore (Figure 1b, Table 1). titration curve, indicating that CHC-1 exists predominantly
According to previous reports, nucleophiles such as cyanide in its spirocyclic form at the physiological pH of 7.4 (Figure
or phosphine can intermolecularly attack the CHC hybrid S2c).
fluorophore at the indolenium C2 atom or at the carbon-
Further, we examined whether the pKcycl values of CHC
carbon double bond of the hemicyanine unit10, 16, and a p- hybrids can be modulated by changing the nucleophilicity of
nitrophenyl group intramolecularly attacks the indolenium the intramolecular nucleophile; for this purpose, we
2
1
C2 atom ; both reactions result in a significant blue-shift of prepared CHC-2 and CHC-3 bearing an aminoethyl (AE) and
the optical properties. In order to identify the structure of a mercaptoethyl (ME) group, respectively (Table 1, Schemes
1
the spirocyclic form of CHC-1, H-NMR spectrum of CHC-1 S2, S3). CHC-2 showed pH-dependent optical properties
was measured after washing with NaOH aq. to accelerate similar to those of CHC-1, but its pKcycl value was shifted to
spirocycle formation. We observed two distinct proton the more acidic side (pKcycl = 5.4, Figure S2a,c). In contrast,
peaks with a large coupling constant (15.9 Hz) which CHC-3 showed little absorbance at around 600 nm and
derives from the carbon-carbon double bond of the almost constant absorbance at 400 nm at all pH values
hemicyanine unit, providing direct evidence of nucleophilic examined, suggesting that it mainly exists in its spirocyclic
attack of HE group at the indolenium C2 atom of CHC-1 form throughout this pH range (Figure S2b,c). These results
(
Figure S1).
are in accordance with our previous findings with
In order to test the reversibility of the pH-dependent rhodamine scaffolds that the spirocyclic structure is
transition between the -extended open form and the increasingly stabilized as the nucleophilicity of the
intramolecular nucleophile increases, shifting the pKcycl
value to the acidic side23, 24
.
PBS
PBS
0
.3
.25
.2
.15
.1
PBS
Next, we examined whether the pKcycl values of CHC
hybrids can also be modulated by the electron-
withdrawing/donating properties of substituents on the
hybrid structure, in order to identify a suitable scaffold for
developing fluorescence probes for hydrolases. As target
hydrolases, we first focused on aminopeptidases, a family of
proteases that cleave the N-terminal amino acid of peptides
or proteins. Thus, we prepared CHC-4 with an amino group
at the indolenium C5 atom and CHC-6 with an amino group
in the coumarin unit (Table 1, Scheme S4, S5). We also
+
NaOH
+ NaOH + HCl
PBS
0
0
0
+
+
NaOH
HCl
0
0
.05
0
3
00 400 500 600 700 800
Wavelength (nm)
Fig. 2 Reversibility of the pH-dependent intramolecular spirocyclization in
aqueous solution. 1 N NaOH aq (3 µL) was added to 2 µM CHC-1 solution in
PBS. Then 1 N HCl aq (3 µL) was added.
Table 1 Photophysical properties of the CHC hybrid fluorophore a
abs[nm]b em[nm]b
Φfl b
NR R
XH
R3
 [nm]
pKcycl
1
2
OH
NH2
NEt2
H
H
576
596
-
660
662
-
84
66
-
6.5
5.4
< 2
7.6
7.3
7.5
6.8
8.1
6.4
0.040c
0.021c
CHC-1
CHC-2
CHC-3
CHC-4
CHC-5
CHC-6
CHC-7
CHC-8
CHC-9
R3
NEt2
NEt2
NEt2
NEt2
NH2
SH
OH
OH
OH
OH
OH
OH
H
NH2
-
N
0.018c
0.037c
0.026d
0.010e
0.024c
0.025c
590
586
520
385
600
596
664
668
616
585
666
668
74
82
96
200
66
72
R2
N
O
O
NHCOMe
R1
XH
H
H
NHCOMe
NEt2
CO H
2
NEt2
CONHMe
aAbsorption maximum (abs), fluorescence emission maximum (em), Stokes shift (), equilibrium constant for intramolecular spirocyclization (pKcycl) and
b

fl of the open form measured in 0.2 M sodium phosphate buffer (pH 2.0). c-eExcitation wavelength was 570 nm, 520nm,
c
d
absolute quantum yield (fl).
and 400 nm.
e
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prepared their acetylated derivatives (CHC-5 and CHC-7) as S5c). We also confirmed that gGlu-CHC was hyVdierwoAlryticsleeOdnlbinye
models of aminopeptidase substrates. We observed little GGT to CHC-6 as a fluorescent hydro
difference in pKcycl values between CHC-4/CHC-5 (7.6 and shows a large Stokes shift of 96 nm (Figure S5d). Further,
.3, respectively, Figure S3), and the fluorescence of the when we applied gGlu-CHC to human lung carcinoma cell
D
l
O
ysI:
i
1
s
0.
p
10
r
3
o
9
d
/D
u
0
c
C
t,C0
w
0
h
55ic
9B
h
7
2
7
open form of CHC-4 is quenched in the physiological pH line A549, which highly expresses GGT , we observed
range, suggesting that CHC-4 is not a suitable candidate strong fluorescence activation, which was suppressed by
scaffold for fluorogenic probes for aminopeptidases. In co-incubation with a GGT inhibitor (Figure S6). Further, we
contrast, we observed a modest difference in pKcycl values saw no marked fluorescence activation in H226 cells, which
between CHC-6/CHC-7 (7.5 and 6.8, respectively, Figure have low GGT expression. These results indicate that gGlu-
S4); this implies that 20% of CHC-7 and 56% of CHC-6 exist CHC works as a fluorogenic probe with a large Stokes shift
in the open form at pH 7.4 giving an expected fold change to visualize GGT activity in live cells.
due to the spirocyclization of 2.8. This is not sufficiently
Next, we examined whether a larger change in the pKcycl
large for obtaining highly fluorogenic probes for value of the CHC hybrid fluorophore could be obtained by
aminopeptidases. However, we noticed that significant targeting other kinds of hydrolase. Specifically, we focused
fluorescence activation at pH 7.4 could be obtained by on carboxypeptidase and esterase, which hydrolyse an
utilizing the blue-shift in the absorption of the open form electron-withdrawing amide or ester moiety to carboxylate.
upon amidation (from CHC-6 to CHC-7), together with We prepared CHC-8 with a carboxylic group at the
some contribution from a change in the fluorescence indolenium C5 atom, and examined how the spirocyclic
quantum yield of the open forms (fl = 0.026 for CHC-6 and behaviour changes upon amidation of this carboxylic group
0
.010 for CHC-7). Thus, we next prepared gGlu-CHC by (CHC-9) (Table 1, Scheme S7). The pKcycl values of CHC-8
replacing the acetyl group of CHC-7 with a -glutamyl and CHC-9 were calculated to be 8.1 and 6.4, respectively
moiety as a probe for -glutamyltranspeptidase (GGT), (Figure S7), meaning that 83% of CHC-8 and 9% of CHC-9
which is known to be overexpressed in various types of exist in the open form at pH 7.4 and so the expected fold
cancer and so is of interest as a biomarker enzyme for change due to the spirocyclization is more than 9. These
fluorescence imaging of cancer (Figure S5, Scheme S6)25, 26
.
results indicate that changes in the electron-withdrawing
gGlu-CHC showed similar optical properties to those of ability of the substituent at the indolenium C5 atom (from
CHC-7 (pKcycl = 6.9), and the fluorescence intensity of gGlu- amide to carboxylate) successfully induced a sufficient shift
CHC increased dramatically upon reaction with GGT (Figure of the pKcycl value (pKcycl = 1.7). Based on this finding, we
designed and synthesized a probe targeted for esterase,
a)
CHC-AM, by converting the carboxy group of CHC-8 to
acetoxymethyl (AM) ester (Figure 3a, Scheme S8). Since the
ester moiety has a strong electron-withdrawing ability,
comparable to that of the amide moiety according to the
Hammett equation, we expected that the AM ester
b)
c)
derivative would show a sufficiently low pKcycl value. As
expected, CHC-AM showed similar optical properties to
those of CHC-9, and its pKcycl value was calculated to be 6.2,
meaning that only 6% of CHC-AM exists in its open form at
pH 7.4, and the expected fold change due to the
spirocyclization is as large as 13 (Figure S8a). We confirmed
that CHC-AM was hydrolysed by esterase to produce CHC-8
as a fluorescent hydrolysis product with a large Stokes shift
of 66 nm, resulting in fluorescence activation of up to 14-
fold (Figure 3b, Figure S8b). We also examined whether we
could detect esterase activity in a ratiometric manner by
using two excitation and emission wavelengths (Figure S9),
but the fluorescence of the coumarin moiety was also
increased by the hydrolysis of the AM group, probably due
to quenching of the coumarin units of CHC-AM by the
indoline moiety, and this might limit the sensitivity in
ratiometric application of this probe.
0
0
0
0
0
.05
.04
.03
.02
.01
2000
120
00
Before
After
Before
After
1
1
1
500
000
8
0
60
4
0
5
00
20
0
0
3
0
00 400 500 600 700
Wavelength (nm)
550 600 650 700 750 800
Wavelength (nm)
0
200 400 600 800 1000
Time (sec)
CellTrackerTM Green
Merge
CHC-AM
5
0
255
0
0
Fig. 3 (a) A newly developed fluorescence probe for esterase based on the
coumarin-hemicyanine hybrid fluorophore, CHC-AM. (b) Absorption (left) and
fluorescence (middle) spectra of 1 µM CHC-AM before (blue) and after (red)
reaction with 10 units of esterase. (right) Time-dependent changes in the
fluorescence intensity of CHC-AM upon addition of esterase. Spectra were
measured in 0.2 M sodium phosphate buffer (pH 7.4) containing 0.1% DMSO
as a cosolvent. The excitation and emission wavelengths were 560 nm and
Finally, we applied CHC-AM to A549 cells, and observed
fluorescence activation in cells upon hydrolysis of AM ester
by the ubiquitous intracellular esterase (Figure 3c). Thus,
we successfully designed CHC-AM as a cell-permeable
esterase probe based on the CHC hybrid fluorophore by
using intramolecular spirocyclization as a fluorescence
6
80 nm, respectively. (c) Fluorescence confocal microscopy imaging of live
A549 cells. A549 cells were incubated with 1 µM CHC-AM and 1 µM
CellTrackerTM Green CMFDA containing 0.2% DMSO as a cosolvent for 1 h.
Excitation and detection wavelengths were 552 nm and 650-700 nm for CHC-
AM, and 495 nm and 505-540 nm for CellTrackerTM Green CMFDA. Scale bar:
5
0 µm.
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W. Sun, S. Guo, C. Hu, J. Fan and X. Peng, Chemical
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7
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.
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Conclusions
We have prepared a series of coumarin-hemicyanine
0.
(
CHC) hybrid fluorophores, and demonstrated that their
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optical properties can be controlled by means of
intramolecular spirocyclization. By making use of the shift
in spirocyclization behaviour upon esterification of the
carboxy group at the indolenium C5 atom, we developed an
activatable fluorescence probe for esterase, which shows
strong fluorescence activation upon reaction with esterase.
We also developed an activatable fluorescence probe for
GGT by utilizing the significant blue-shift in absorption
1
1
3.
4.
upon amidation of the amino group of the coumarin unit, 15.
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1
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7.
8.
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scaffold will be applicable to develop a range of activatable
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1
9.
This research was supported in part by AMED under grant
Number JP19gm0710008 (to Y.U.), by JST/PRESTO grant 20.
JPMJPR14F8 (to M. K.), by MEXT/JSPS KAKENHI grants
JP16H02606, JP26111012, JP19H05632 (to Y.U.) and
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JP15H05951 “Resonance Bio”, JP19H02826, JP19K22242
to M. K.), by JSPS Core-to-Core Program, A. Advanced
(
Research Networks, by Astellas Foundation for Research on
Metabolic Disorders (to M. K.), by Japan Foundation for
Applied Enzymology (to M. K.). S.U. was supported by Japan
Society for the Promotion of Science as JSPS research fellow.
1
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Conflicts of interest
There are no conflicts to declare
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