Establishment of Molecular Design Strategy to Obtain Activatable Fluorescent Probes for Carboxypeptidases

Article abstract of DOI:10.1021/jacs.7b11014

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the aliphatic carboxamide to aliphatic carboxylate structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.

Full text of DOI:10.1021/jacs.7b11014

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Establishment of Molecular Design Strategy To Obtain Activatable
Fluorescent Probes for Carboxypeptidases
Yugo Kuriki,† Mako Kamiya,‡,§ Hidemasa Kubo,†,∥ Toru Komatsu,† Tasuku Ueno,† Ryo Tachibana,†
Kento Hayashi,† Kenjiro Hanaoka,† Suguru Yamashita,‡ Takeaki Ishizawa,⊥ Norihiro Kokudo,#
and Yasuteru Urano*,†,‡,⊗
†Graduate School of Pharmaceutical Sciences and ‡Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
§PRESTO (Japan) Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi-shi, Saitama 332-0012, Japan
∥Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto,
602-8566, Japan
⊥Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, 3-8-31 Ariake,
Koto-ku, Tokyo 135-8550, Japan
#Department of Surgery, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
⊗CREST (Japan) Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan
S
* Supporting Information
ABSTRACT: Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of
peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first
versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine
to translate the “aliphatic carboxamide to aliphatic carboxylate” structural conversion catalyzed by CPs into dynamic fluorescence
activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to
detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This
design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of
fluorescent probes to study various pathological and biological processes.
analysis,12 absorption spectrometry,13,14 or coupled assay
INTRODUCTION
■
systems.15,16 Their use is almost limited to in vitro study, and
Carboxypeptidases (CPs) are a family of proteases that cleave
one or more amino acids from the C-terminal of peptides or
it is difficult to perform real-time imaging of CP activities.
Fluorescence imaging is a powerful methodology to monitor
proteins,1 and they have key roles in both physiological
enzymatic activities in living cells and animals, being simple,
processes and various diseases. For example, carboxypeptidase
safe, and sensitive, as well as offering high spatiotemporal
A (CPA) and carboxypeptidase B (CPB) produced in the
resolution and signal activatability.17 Various fluorogenic
pancreas are responsible for protein degradation,2 and
enzyme substrates (activatable fluorescent probes for enzymes)
angiotensin converting enzyme (ACE) controls blood pressure
by regulating peptide hormone activity.3,4 Expression levels of
have been devised for real-time monitoring of enzyme activities
of interest18 and have been utilized not only in biological
CPs are varied in various diseases: CPA and CPB in pancreatitis
and pancreatic cancer,5,6 prostate-specific membrane antigen
research but also for medical diagnosis or intraoperative
visualization of diseases19 including cancer,20,21 Parkinson’s
(PSMA) in prostate cancer,7,8 carboxypeptidase N (CPN) in
breast cancer,9 carboxypeptidase M (CPM) in several cancers,10
and cathepsin A in galactosialidosis.11 However, currently
available methods for monitoring CP activities are HPLC
Received: October 16, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b11014
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Journal of the American Chemical Society
Article
disease,22 or pancreatic fistula.23 Thus, it is considered that
activatable fluorescent probes for CPs will offer novel ways of
studying enzymatic functions and disease diagnosis.
Despite their potential utility, only a few activatable
fluorescent probes for CPs have been reported so far.24,25
This is mainly from the difficulty in designing the suitable
substrate analogue of CPs. Since CPs strictly recognize C-
terminal amino acid, major strategies of designing activatable
fluorescent probes for hydrolases such as incorporation of
substrate moiety on an aromatic hydroxy or amino group of the
scaffold fluorophore,26−31 or sandwiching substrate moiety
between a FRET donor−acceptor pair,32,33 are not generally
applicable to the enzyme. To our knowledge, only the currently
available fluorogenic substrate is one utilizing the amino acid on
substrate moiety as a FRET donor or static quencher,24,25 but
this strategy is only applicable to limited numbers of CPs that
can accommodate those special amino acids for fluorescent
readout.
Here, we propose a versatile and straightforward molecular
design strategy for activatable fluorescent probes for CPs that
translates chemical structural conversion by CPs (“aliphatic
carboxamide to aliphatic carboxylate”) directly into dynamic
fluorescence activation. This was attained by utilization of
intramolecular spirocyclization of rhodamines; we discovered
that this molecular mechanism is highly suitable for converting
the reaction of aliphatic carboxamide/carboxylate to robust
fluorescence activation. Based on the versatility of the design,
we succeeded in development of fluorescent probes for
clinically important CPs, carboxypeptidase A (CPA: recogniz-
ing C-terminal bulky amino acid such as phenylalanine) and
carboxypeptidase B (CPB: recognizing C-terminal basic amino
acid such as arginine) only by changing the conjugated amino
acid. Further, we showed that one of these probes is available
for ex vivo detection of pancreatic fluid leakage, a clinically
significant manifestation of pancreatic fistula.
Figure 1. Molecular design strategy for activatable fluorescence probes
for CPs. (a) Chemical structures and reaction scheme of hippuryl
amino acid derivative with CP. (b) Molecular design of activatable
fluorescent probe for CPs. (c) Chemical structures, pKcycl values, and
pH dependency of absorbance of HMRBC-COO− or HMRBC-
CONH-Gly in 0.1 M sodium phosphate buffer at various pH’s. In the
titration curve, normalized absorbance at the wavelength in
parentheses was plotted against pH. HMRBC-COO− (523 nm),
HMRBC-CONH-Gly (513 nm).
xanthene moiety affected the pKcycl value35,36 (Figure S1), and
we set out to utilize the decrease in electron-withdrawing ability
caused by reaction with CPs (aliphatic carboxamide to aliphatic
carboxylate) to induce a shift of the pKcycl value that would
provide a large fluorescence activation. Thus, we incorporated a
substrate amino acid into aliphatic carboxylate arranged on the
nitrogen atom of hydroxymethylrhodamine (Figure 1b).
To confirm the validity of this design strategy, we first
prepared a hydroxymethyl rhodamine with two carboxymethyl
groups on the nitrogen atoms, HMRBC-COO−, and its glycine
analogue HMRBC-CONH-Gly as model substrates of CPs. We
found that the pKcycl value of HMRBC-CONH-Gly was much
lower than that of HMRBC-COO− (9.7 for HMRBC-COO−
and 7.8 for HMRBC-CONH-Gly) (Figure 1c), indicating that
it would be feasible to convert the structural change into a shift
in pKcycl value. However, based on the percentage of the open
form (99% for HMRBC-COO− and 69% for HMRBC-CONH-
Gly at pH 7.4), the activation ratio in the physiological pH
range was still limited to 1.5-fold, due to the relatively high
pKcycl values of the probes, and therefore, we decided to modify
the structure to lower the pKcycl value so that we could obtain
greater dynamic fluorescence activation in the physiological pH
range.
RESULTS AND DISCUSSION
■
Molecular Design for Activatable Fluorescent Probes
for CPs. First, we overviewed the known substrates of CPs to
design the core structure of the probe that can be recognized by
enzyme. It is known that hippuryl amino acid derivatives are
widely used as standard substrates for CPs (Figure 1a), so we
considered that it is desirable to conjugate a substrate amino
acid to aliphatic carboxylate to form aliphatic carboxamide for
versatile design of the probe. Therefore, the major chemical
challenge in designing activatable fluorescent probes for CPs is
how to translate the CP-catalyzed “aliphatic carboxamide to
aliphatic carboxylate” structural conversion into dynamic
fluorescence activation at physiological pH.
To overcome this challenge, we focused on intramolecular
spirocyclization as a mechanism of fluorescence activation.
Spirocyclization of rhodamine or rhodol derivatives is a
powerful technique for designing fluorescent probes, as the
spirocyclized derivative is colorless and nonfluorescent due to
separation of the π-conjugation system of the original
fluorophore. Recently we have developed several fluorescent
probes based on this mechanism by precisely controlling the
pKcycl values of the probes.29,34−36 (pKcycl is the pH at which the
extent of spirocyclization is sufficient to reduce the absorbance
of the compound to one-half of the maximum absorbance
calculated from the pH titration curve.) Here, we focused on
our previous finding that the electron-donating or -withdrawing
ability of the alkyl substituent on the nitrogen atom of the
Adjustment of pKcycl Values To Develop Probes
Capable of Sensing CPA and CPB Activities in the
Physiological pH Range. To shift the pKcycl value to the
acidic side, we adopted two strategies. We previously showed
that the pKcycl value is correlated to the nucleophilicity of the
intramolecular nucleophile and the LUMO energy level of the
xanthene moiety.29,34−37 In other words, raising the nucleo-
philicity of the intramolecular nucleophile or lowering the
LUMO energy level of the xanthene moiety should be effective
B
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for shifting pKcycl to the acidic side. Therefore, we prepared two
candidate scaffolds, AMRBC-COO− (aminomethyl rhodamine
with two carboxymethyl groups) and diClHMRBC-COO−
(dichloro hydroxymethyl rhodamine with two carboxymethyl
groups) (Figure 2a,b), by replacing the intramolecular
Figure 2. Adjustment of pKcycl values to the physiological pH range.
Chemical structures, pKcycl values, and pH dependency of absorbance
of (a) AMRBC derivatives or (b) diClHMRBC derivatives in 0.1 M
sodium phosphate buffer at various pH’s. In the titration curve,
normalized absorbance at wavelength in parentheses was plotted
against pH. AMRBC-COO− (528 nm), AMRBC-CONH-Gly (520
nm), diClHMRBC-COO− (529 nm), or diClHRBC-CONH-Gly (521
nm).
nucleophile with an aminomethyl group, which is considered
to be a stronger nucleophile, or by introducing two chlorines
into the xanthene core, which would lower the LUMO level of
the xanthene moiety, respectively (Figure S2). We also
prepared glycine analogues of these scaffolds, AMRBC-
CONH-Gly and diClHMRBC-CONH-Gly, as model sub-
strates. As expected, the pKcycl values of both derivatives were
lowered compared to those of HMRBCs: 7.5 for AMRBC-
COO−, 6.2 for AMRBC-CONH-Gly, 7.3 for diClHMRBC-
COO−, 5.9 for diClHMRBC-CONH-Gly. Thus, dynamic
fluorescence activation could be achieved with these scaffolds.
Based on the newly developed scaffolds, we next set out to
develop fluorescent probes for carboxypeptidase A (CPA) and
carboxypeptidase B (CPB). As a substrate amino acid, we chose
phenylalanine for CPA and arginine for CPB because CPA
prefers bulky amino acids while CPB prefers basic ones,6 and
we prepared four derivatives, AMRBC-CONH-Phe,
diClHMRBC-CONH-Phe, AMRBC-CONH-Arg, and
diClHMRBC-CONH-Arg. The pKcycl values of AMRBC-
CONH-Phe and AMRBC-CONH-Arg were 6.0 and 5.8, and
those of diClHMRBC-CONH-Phe and diClHMRBC-CONH-
Arg were 5.6 and 5.0, respectively (Figure 3a, b). The
photophysical properties of these compounds are summarized
in the Supporting Information (Figures S3, S4 and Table S1).
The percentage of the open form at the physiological pH of 7.4
was calculated to be 54% for AMRBC-COO−, 6.4% for
AMRBC-CONH-Gly, 4.8% for AMRBC-CONH-Phe, 3.6% for
AMRBC-CONH-Arg, 45% for diClHMRBC-COO−, 3.1% for
diClHMRBC-CONH-Gly, 1.8% for diClHMRBC-CONH-Gly,
and 0.79% for diClHMRBC-CONH-Arg. Although we do not
have a clear explanation for the effects of the different kinds of
amino acids on pKcycl values, the results were favorable from the
viewpoint of suppressing the background signal and obtaining
enhanced fluorescence activation. Thus, though the pKcycl
Figure 3. Enzyme reactions of the probes with CPA or CPB. (a, b)
Reaction scheme of the probes with CPA or CPB and pKcycl values of
the derivatives. (a) AMRBC-CONH-Phe or diClHMRBC-CONH-Phe
was recognized as a substrate by CPA and converted into AMRBC-
COO− or diClHMRBC-COO−, respectively. (b) AMRBC-CONH-Arg
or diClHMRBC-CONH-Arg was recognized as a substrate by CPB
and converted into AMRBC-COO− or diClHMRBC-COO−,
respectively. (c) Fluorescence spectra of 1 μM diClHMRBC-
CONH-Phe or diClHMRBC-CONH-Arg upon enzymatic reaction
and signal-to-noise (S/N) ratio. Reaction was performed in 0.80 mL of
phosphate-buffered saline (pH 7.4) containing 0.01% CHAPS and
0.1% DMSO as a cosolvent using 3 μg proCPA activated by trypsin
before addition or CPB at 25 °C. (Left) diClHMRBC-CONH-Phe
was incubated with CPA for 15 h. (Right) diClHMRBC-CONH-Arg
was incubated with CPB for 2 h. Blue and orange lines indicate the
fluorescence spectra before and after enzymatic reaction, respectively.
(d) diClHRBC-CONH-Gly, diClHMRBC-CONH-Phe, or
diClHMRBC-CONH-Arg was reacted with CPA, CPB or trypsin. A
1 μM probe in phosphate-buffered saline (pH 7.4) containing 0.01%
CHAPS and 0.1% DMSO as a cosolvent was reacted with 200 ng of
enzyme at 25 °C (n = 3). The total assay volume was 20 μL, and the
fluorescence increase was measured with a plate reader. Excitation/
emission wavelengths were 485 nm/535 nm. Error bars represent SD.
values of the probes depended upon the kind of amino acid,
the activation ratio (calculated from the percentages of the
open form of the respective probe and COO− derivative)
appeared to be sufficient for obtaining practically useful
fluorescent probes: 8.4−15-fold in AMRBC and 15−58-fold
in diClHMRBC, despite the low activation ratio of 1.5 fold in
HMRBC.
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We then evaluated the reactivity of AMRBC-CONH-Phe or
diClHMRBC-CONH-Phe with CPA and AMRBC-CONH-Arg
or diClHMRBC-CONH-Arg with CPB. As expected, all of the
probes were recognized as substrates, and a fluorescence
increase was observed. The fluorescence activation was 18-fold
for AMRBC-CONH-Phe or 23-fold for diClHMRBC-CONH-
Phe with CPA, and 23-fold for AMRBC-CONH-Arg or 80-fold
for diClHMRBC-CONH-Arg with CPB (Figure 3c and Figure
S5). As we had expected, diClHMRBC derivatives showed
higher activation ratios because of their smaller pKcycl values.
We also confirmed that the probes were cleaved to AMRBC-
COO− or diClHMRBC-COO− by UPLC analysis (Figure S6),
and the kinetic parameters were similar (Table S2). For further
study, we focused on diClHMRBC derivatives because of their
higher activation ratio, and first, we confirmed the substrate
specificity. Indeed, these derivatives selectively reacted with
their target enzyme among CPA, CPB and trypsin (Figure 3d
and Figure S7; trypsin was examined because it was used for
activating proCPA to CPA). Moreover, we examined the
reactivity of the probes with other five proteases: cathepsin K,
dipeptidylpeptidase-IV, γ-glutamyltranspeptidase, leucine ami-
nopeptidase, and chymotrypsin. The probes did not react with
any of these proteases, as expected, supporting their high
selectivity (Figure S8). These results confirmed the suitability
of the developed scaffolds to obtain activatable fluorescent
probes for various CPs simply by changing the conjugated
amino acid.
Fluorescence Detection of Pancreatic Fistula. Next, we
examined whether CPA or CPB activity in human pancreatic
juice can be detected with diClHMRBC-CONH-Phe or
diClHMRBC-CONH-Arg, aiming at development of diagnosis
tools for pancreatic fistula. Pancreatic fistula is one of the most
serious complications after digestive surgery and can cause
death.38,39 However, it is difficult to prevent because of the
inability to visualize leakage of pancreatic juice during surgery.
Therefore, a fluorescent probe that could detect enzymatic
activity in pancreatic juice would become a useful tool for
detecting pancreatic fistula intraoperatively. For this purpose,
we have so far developed gPhe-HMRG,23 an activatable
fluorescent probe for chymotrypsin, and we succeeded in
detecting chymotrypsin activity in human pancreatic juice. But,
although gPhe-HMRG can be used for imaging pancreatic
fistula, it turned out that chymotrypsin activity varies from
patient to patient. Thus, to achieve more accurate diagnosis of
pancreatic fistula, it would be better to evaluate multiple
enzyme activities in pancreatic juice.
Figure 4. Detection of CPA or CPB activities in pancreatic juice. (a)
Fluorescence increase rates of 1 μM probe and 1.25 μg pancreatic juice
from 8 different patients with or without 100 μM inhibitor, (S)-
benzylsuccinic acid for CPA or MGTA for CPB, in phosphate-buffered
saline (pH 7.4) containing 0.01% CHAPS and 0.1% DMSO as a
cosolvent in the presence of 1.5 μg of trypsin as an activator of
proCPA or proCPB in pancreatic juice. Error bars represent SD (n =
4). (b) Detection of pancreatic juice leakage from cut mouse pancreas
ex vivo. Mouse organs were resected, and the pancreas was cut along
the black dotted lines. 50 μM diClHMRBC-CONH-Arg in phosphate-
buffered saline (pH 7.4) containing 1% DMSO as a cosolvent in the
presence of 100 μg/mL trypsin as an activator of proCPB in pancreatic
juice was applied onto cut mouse pancreas. Fluorescence increase in
the blue square was acquired with a Maestro imaging system. Blue
arrows indicate the area where fluorescence increased. Excitation/
emission wavelengths were 523 nm/560 nm. Abbreviations in the
guide map: St, stomach; In, intestine.
This result shows that we could detect CPA or CPB activities in
pancreatic juice with the developed probes. On the other hand,
no fluorescence increase was observed in the case of abdominal
dropsy, which did not contain pancreatic juice (Figure S10).
Compared to gPhe-HMRG, diClHMRBC-CONH-Arg showed
a faster reaction rate (Figure S11). Interestingly, there was
almost no correlation between chymotrypsin activity and CPA
or CPB activity, in spite of the strong correlation between CPA
and CPB activities (Figure S12). These results support the idea
that our probes for CPs, in combination with gPhe-HMRG,
would enable more accurate diagnosis of pancreatic fistula by
detecting multiple enzyme activities in pancreatic juice.
Finally, in order to examine if diClHMRBC-CONH-Arg
works ex vivo, we tried to detect pancreatic juice leakage from
mouse pancreas. Indeed, when diClHMRBC-CONH-Arg was
applied to cut mouse pancreas, we could visualize pancreatic
juice leakage in terms of a marked fluorescence increase (Figure
4b). On the other hand, no significant fluorescence increase
was seen when the probe was applied before cutting the
pancreas (Figure S13). Further, the fluorescence increase was
So, we first set out to examine whether diClHMRBC-
CONH-Phe and diClHMRBC-CONH-Arg could detect CP
activities in eight samples of pancreatic juice from different
patients. Although the pH of pancreatic juice is around 8.0,
almost all of diClHMRBC-CONH-Phe or diClHMRBC-
CONH-Arg exists as the closed form at this pH (ratio of
open form <1%) and the ratio of the open form of
diClHMRBC-COO− is 18%. This should be sufficient for
detection of CPA or CPB activity in pancreatic juice. In living
systems, CPA and CPB exist as proenzymes in pancreatic juice,
as is the case with chymotrypsin, and they are activated by
trypsin after pancreatic juice is secreted into duodenum (Figure
S9). Thus, we performed reaction of the probes with pancreatic
juice in the presence of trypsin as an activator of proCPA or
proCPB in this study. Marked fluorescence increases were
observed with both probes and were inhibited by the respective
CP inhibitors, (S)-benzylsuccinic acid and MGTA (Figure 4a).
D
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triethylammonium acetate) and eluent F (80% acetonitrile and 20%
H2O containg 100 mM triethylammonium acetate) were used for
HPLC purification. UPLC analyses were performed on a Waters
Acquity UPLC (H class)/QDa quadrupole MS analyzer or Acquity
UPLC (H class)/Xevo TQD quadrupole MS/MS analyzer equipped
with an Acquity UPLC BEH C18 column (Waters). Eluent C (H2O
containing 0.1% formic acid) and eluent D (80% acetonitrile and 20%
H2O containing 0.1% formic acid) were used for UPLC analyses. Gel-
permeation chromatography (GPC) purification was performed on a
recycle preparative HPLC LC-9110 NEXT (Japan Analytical Industry)
equipped with a JAIGEL-2HR column (20 mm × 600 mm, Japan
Analytical Industry).
UV−vis Absorption and fluorescence spectroscopy. UV−
visible spectra were obtained on a Shimadzu UV-1800. Fluorescence
spectroscopic studies were performed on a Hitachi F7000. The slit
width was 5 nm for both excitation and emission. The photomultiplier
voltage was 400 V. Relative fluorescence quantum yields were obtained
by comparing the area under the emission spectra of the test samples
with standard samples and were calculated according to the following
equation
inhibited in the presence of CPB inhibitor even after cutting the
pancreas (Figures S14 and S15). We also examined the
reactivity of the probe toward mouse tissue lysate and serum,
and we found that it is highly selective for pancreas lysate in the
presence of trypsin (Figure S16). These data indicate that
diClHMRBC-CONH-Arg is a promising candidate as an
intraoperative diagnostic tool for pancreatic fistula.
CONCLUSIONS
■
We have designed a scaffold that can translate the CP-catalyzed
“aliphatic carboxamide to aliphatic carboxylate” structural
conversion into dynamic fluorescence activation based on
intramolecular spirocyclization of rhodamine. The spirocyclic
equilibrium of the new scaffolds, diClHMRBC-COO− or
AMRBC-COO−, was precisely adjusted by optimizing the
nucleophilicity of the intramolecular nucleophile and/or the
electrophilicity of the fluorophore. We demonstrated the
validity of this design strategy by developing activatable
fluorescent probes for CPA or CPB simply by changing the
substrate amino acid on the scaffold. In addition to these
results, our preliminary work has confirmed that this design
strategy is also applicable to angiotensin converting enzyme,
which has dipeptidyl carboxypeptidase activity (Figure S17).
Further, we succeeded in detecting CPA and CPB activities in
pancreatic juice with diClHMRBC-CONH-Phe and
diClHMRBC-CONH-Arg, respectively, and we visualized
pancreatic juice leakage in an ex vivo mouse model with
diClHMRBC-CONH-Arg. To our knowledge, this is the first
report of a practical design strategy for activatable fluorescent
probes for CPs; such probes have a variety of potential
applications, for example as medical diagnostic tools and as
research tools for studying the biological roles of CPs, as well as
screening and evaluation of CP inhibitors. It is particularly
noteworthy that the pKcycl value of the scaffold is basically
regulated by the electron-withdrawing ability of the aliphatic
functional group and is highly tunable by modification of the
structure. This means that the scaffold should also be applicable
for other enzymes that induce chemical conversions leading to
reduced electron-withdrawing ability, and we believe that this
platform could also be available to develop activatable
fluorescent probes for various other enzymes that have not so
far been targetable.
Φ /Φ = [Ast/Ax][nx2/nst2][D /D ]
x
st
x
st
where st = standard; x = sample; A = absorbance at the excitation
wavelength; n = refractive index; and D = area under the fluorescence
spectra on an energy scale. Optical properties of probes (1 μM) were
examined in 0.1 M sodium phosphate buffer containing 0.1% DMSO
as a cosolvent. For enzyme reaction, proCPA activated by trypsin
before addition or CPB (3 μg) was added to probes (1 μM) in
phosphate-buffered saline (pH 7.4) containing 0.01% CHAPS and
0.1% DMSO as a cosolvent, and the solution was incubated at 25 °C.
Determination of pKcycl Value. Absorption spectra of com-
pounds were measured in 0.1 M sodium phosphate buffer at various
pH values. For compounds with n acid−base equilibria (n = 1 or 2),
pH profiles of absorbance (Abs) were fitted to the following formula
to determine pKa values
k
c0 + ∑n ck·10k·pH−∑
l=1 pKal
k=1
Abs or FI =
k
1 + ∑nk=1 10k·pH−∑
l=1 pKal
where pKa1 < pKa2 < ··· < pKan; cn = constant.
Enzymes. Recombinant human procarboxypeptidase A (2856-ZN)
was supplied by R&D Systems. Carboxypeptidase B from porcine
pancreas (10103233001) was supplied by Roche. Trypsin (T4799),
human recombinant procathepsin K (SRP6001), leucine amino-
peptidase from porcine kidney (L5006), human recombinant
dipeptidylpeptidase-IV (D3446) and chymotrypsin from bovine
pancreas (C4129) were supplied by Sigma. γ-glutamyltranspeptidase
from bovine kidney (46557003) was supplied by Oriental Yeast Co.
Ltd. Activation of procarboxypeptidase A by trypsin was performed by
diluting procarboxypeptidase A to 100 μg/mL with 1 μg/mL trypsin in
phosphate-buffered saline (pH 7.4) followed by incubation at room
temperature for 1 h. Activation of procathepsin K was performed by
adjusting the pH to 4.0 by adding an equal volume of 100 mM NaOAc
pH 3.9, 10 mM DTT, 5 mM EDTA followed by incubation at room
temperature for 40 min.
EXPERIMENTAL SECTION
■
Materials and Instruments. Reagents and solvents were of the
best grade available, supplied by Tokyo Chemical Industries, Wako
Pure Chemical, Sigma-Aldrich, Dojindo, Kanto Chemical Co.,
Watanabe Chemical Industries, Merck Millipore, and R&D Systems,
and were used without further purification. NMR spectra were
recorded on a JEOL JNM-LA300 instrument at 300 MHz for 1H
NMR and at 75 MHz for 13C NMR, a JEOL JNM-LA400 instrument
1
at 400 MHz for H NMR and at 100 MHz for 13C NMR or a JEOL
Enzyme Assay. Unless otherwise mentioned, enzyme assay was
performed in phosphate-buffered saline (pH 7.4) containing 0.01%
CHAPS at 25 °C. Half area 384-well plates (Corning 3677) were used
for the assay (20 μL reaction volume). Fluorescence was detected with
a plate reader, EnVision 2103 Multilabel Reader (PerkinElmer), with
appropriate filter settings. Activity was calculated according to the
following formula:
JNM-ECZ400S instrument at 400 MHz for 1H NMR and at 100 MHz
for 13C NMR. All chemical shifts (δ) are reported in ppm relative to
internal standard tetramethylsilane (δ = 0.0 ppm), or relative to the
1
signals of residual solvent CDCl3 (7.26 ppm for H, 77.16 ppm for
13C), CD3OD (3.31 ppm for 1H, 49.00 ppm for 13C), acetone-d6 (2.04
ppm for 1H) or DMSO-d6 (2.50 ppm for 1H, 39.52 ppm for 13C), and
coupling constants are given in Hz. Mass spectra (MS) were measured
with a JEOL JMS-T100LC AccuToF (ESI). Preparative HPLC was
performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL
Sciences Inc.) using an HPLC system composed of a pump (PU-2080,
JASCO) and a detector (MD-2015 or FP-2025, JASCO). Eluent A
(H2O containing 0.1% TFA), eluent B (80% acetonitrile and 20% H2O
containing 0.1% TFA), eluent E (H2O containing 100 mM
activity = (fluorescence increase rate)/(fluorescence intensity of
product in enzyme or pancreatic juice − fluorescence
intensity of probe just after addition of enzyme or
pancreatic juice)/(protein concentration)
E
DOI: 10.1021/jacs.7b11014
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Journal of the American Chemical Society
Article
Kinetic Assay. Enzyme reaction for kinetic study was performed in
20 μL of phosphate-buffered saline (pH 7.4) containing 0.01%
CHAPS and 0.1% DMSO as a cosolvent using 200 ng proCPA
activated by trypsin before addition or CPB at 37 °C. Clear 384-well
plates (Nunc 262160) were used for the assay. Initial velocity,
calculated from the change in absorbance at 530 nm, was plotted
against substrate concentration. Absorbance was measured with a plate
reader, SH-9000 (Corona Electric Co. Ltd.). The results were fitted to
the Michaelis−Menten equation for calculating the apparent kinetic
parameters
ACKNOWLEDGMENTS
■
We thank Dr. Takuya Myochin, Dr. Hiroki Ito, Dr. Shinpei
Iwaki, Dr. Yu Kagami, and Dr. Shodai Takahashi for valuable
discussions. This research was supported in part by AMED-
CREST, by JST/PRESTO Grant No. JPMJPR14F8 (to M.K.),
by MEXT/JSPS KAKENHI Grant Nos. JP16H02606 and
JP26111012 (to Y.U.), 16H06574 (to T.U.), by
JP15H05951“Resonance Bio” (to M.K.), by JSPS Core-to-
Core Program, A, Advanced Research Networks (to Y.U.), by
the Project for Cancer Research And Therapeutic Evolution (P-
CREATE) from the Japan Agency for Medical Research and
development, AMED (to Y.U.), by a grant from Hoansha
Foundation (to Y.U.), by a grant from Japan Foundation for
Applied Enzymology (to M.K.), and by a Grant-in-Aid for JSPS
Fellows (16J09996) (to Y.K.).
V = Vmax[S]/(Km+[S])
where V = initial velocity and [S] = substrate concentration.
Collection of Clinical Samples. Collection of pancreatic juice or
abdominal dropsy from patients was conducted with the approval of
the Institutional Ethics Review Board of the University of Tokyo. This
study is registered in the UMIN Clinical Trials Registry (registration
number: UMIN000003654; http://www.umin.ac.jp/ctr/index.htm).
Informed consent was obtained from all patients. After collection,
the samples were stored at −80 °C.
Ex Vivo Imaging of Pancreatic Juice Leakage. All procedures
were approved by the Animal Care and Use Committee of the
University of Tokyo. C57BL/6JJcl mice (female, 8-week-old) were
purchased from CLEA Japan, Inc. Mice were euthanized with
isoflurane, and tissues including liver, stomach, pancreas, intestine,
and spleen were resected. Before or after cutting of the pancreas, 50
μM diClHMRBC-CONH-Arg in phosphate-buffered saline (pH 7.4)
containing 1% DMSO as a cosolvent with 20 μg trypsin as an activator
of proCPB (100 μL) was applied onto the pancreas. Fluorescence
images were captured with a Maestro In-Vivo Imaging System (CRi,
Inc.) equipped with an excitation filter set at 523 nm (503−548 nm)
and a 560 nm emission long path filter.
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* Supporting Information
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AUTHOR INFORMATION
Corresponding Author
*uranokun@m.u-tokyo.ac.jp
ORCID
Yugo Kuriki: 0000-0002-1280-7333
Mako Kamiya: 0000-0002-5592-1849
Toru Komatsu: 0000-0002-9268-6964
Kenjiro Hanaoka: 0000-0003-0797-4038
Yasuteru Urano: 0000-0002-1220-6327
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