Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization

Article abstract of DOI:10.1021/ja309688m

We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations.

Full text of DOI:10.1021/ja309688m

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Article
Rational Design of Highly Sensitive Fluorescence Probes For Protease
and Glycosidase Based on Precisely Controlled Spirocyclization
Masayo Sakabe, Daisuke Asanuma, Mako Kamiya, Ryu John Iwatate,
Kenjiro Hanaoka, Takuya Terai, Tetsuo Nagano, and Yasuteru Urano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja309688m • Publication Date (Web): 03 Dec 2012
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Rational Design of Highly Sensitive Fluorescence Probes For
Protease and Glycosidase Based on Precisely Controlled
Spirocyclization
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Masayo Sakabe,† Daisuke Asanuma,‡ Mako Kamiya,‡ Ryu J. Iwatate, ‡ Kenjiro Hanaoka,† Takuya
Terai,† Tetsuo Nagano,†* 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. §Basic Research Program, Japan Science and Technology Agency, 3-5 Sanbancho,
Chiyoda-ku, Tokyo 102-0075, Japan.
Supporting Information Placeholder
ABSTRACT: We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing
difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG)
exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open non-spirocyclic
structure. Ac-HMRG is colorless and non-fluorescent, whereas HMRG is strongly fluorescent. Based on these findings, we have
developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the
acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the
non-fluorescent probe by the target enzyme generates a strong fluorescence signal. In order to confirm the validity and flexibility of
our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation
protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All these probes were almost non-fluorescent due to the
formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We
confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and
rapid response (owing to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are
expected to be useful for a range of biological and pathological investigations.
Introduction
obtain strongly fluorescent products, and this results in a
reduction in the speed and efficiency of fluorescence
activation. We recently showed that a spirocyclization design
strategy can be utilized to provide fluorescence OFF/ON
switches for tetramethylrhodamine and diethylrhodol, leading
to probes for hypochlorite7 and β-galactosidase8. However, the
potential molecular targets of these probe designs are strictly
limited in principle to reactive oxygen species and O-directed
glycosidases, respectively. Here, we describe a more general
spirocyclization design strategy, which overcomes the
limitations of the AMC- and RG-based probes, by utilizing the
hydroxymethyl rhodamine green (HMRG) scaffold. This is
based on our finding that, whereas HMRG itself has an open
structure that is highly fluorescent, it predominantly forms a
closed spirocyclic structure that is non-fluorescent when one
of its amino groups is acetylated (Ac-HMRG). Therefore, by
replacing the acetyl group of Ac-HMRG with a suitable
substrate moiety, we can obtain novel probes that show strong
fluorescence activation upon single-step cleavage of the
substrate moiety by the target enzyme. We validated this
strategy by developing fluorescence probes for two N-directed
peptidases, leucine aminopeptidase (LAP) and fibroblast
Proteases catalyze the cleavage of specific peptide bonds of
their substrates at the cell surface, in cytoplasm or within
various cellular compartments, and many of them play
essential roles in physiological functions and also in disease1,2.
For example, alterations in protease activities or secretion of
proteases into extracellular space are thought to be involved in
tumor metastasis and inflammation3. Sensitive and accurate
detection of protease activities in living samples is therefore
essential for research in biology and medicine. Fluorescence
probes for various protease (aminopeptidase) activities have
been developed based on 7-amino-4-methylcoumarin
(AMC)4,5 or Rhodamine Green (RG)6 and used mainly for in
vitro experiments, such as biochemical assays. However, these
probes are generally unsuitable for application in living cells
or in vivo. AMC derivatives require ultraviolet (UV) light
excitation, which is cytotoxic and induces high background
autofluorescence. In contrast, RG-based probes possess
favorable characteristics for biological applications, such as
longer excitation/emission wavelengths and high fluorescence
quantum yield, but these probes normally have two reactive
sites, so that two steps of enzymatic reaction are necessary to
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Absorption
Emission
Maximum
(nm)
Fluorescence
Quantum
Yield
0.81 a,c)
0.27 b,c)
0.044 a,d)
0.02 b,d)
0.042 a,d)
0.01 b,d)
LUMO
activation protein (FAP). To further show the flexibility of the
level e)
Maximum
(nm)
501
pKcycl
strategy, we also developed a highly sensitive fluorescence
probe for O-directed β-galactosidase by using a carbamate
functional unit as the substrate moiety. Our findings indicate
that the present spirocyclization design strategy has a high
degree of generality, and should be suitable to develop probes
for imaging a wide range of target molecules in live cells.
(hartrees)
HMRG
Ac-HMRG
524
8.1
5.3
8.9
6.2
9.3
6.7
-0.2234
-0.2336
-0.2166
-0.2266
-0.2136
-0.2235
495
526
HMDiMeR
Ac-HMDiMeR
HMDiEtR
528
552
499
571
532
555
Ac-HMDiEtR
501
573
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Results and discussion
a) Measured in 0.2 M sodium phosphate buffer, pH 7.4, b) Measured in 0.2 M sodium phosphate
buffer, pH 2.0, c) Relative fluorescence quantum yield determined by using fluorescein (Φfl = 0.85)
Spirocyclization of rhodamine derivatives is a powerful
technique for designing fluorescence probes, as the
spirocyclized derivative is colorless and non-fluorescent due
to separation of the π-conjugation system of the original
fluorophore. Fluorescence probes for metal ions, reactive
oxygen species, pH, and so on have been developed by
utilizing the ring-opening reaction of spirocylized rhodamine
upon reaction with the analyte, as this regenerates the original
d)
e)
as
a
reference,
Absolute fluorescence quantum efficiency,
LUMO energy level of the
corresponding xanthene moiety. Data were calculated at the B3LYP/6-31G level with Gaussian
98W.
we required a hydroxymethyl rhodamine derivative that would
be predominantly present in the spirocyclic form at
physiological pH, thus showing no background fluorescence.
We considered that it might be possible to replace the
substituent on such a derivative with a substrate moiety for a
target enzyme while retaining the closed spirocyclic structure.
Then, specific hydrolysis by the target enzyme might release
the substrate moiety and convert the spirocyclic structure to
the strongly fluorescent open form.
fluorophore, which exhibits strong fluorescence emission9-12
.
Therefore, precise regulation of the equilibrium between the
spirocyclic closed form (colorless and non-fluorescent) and
the open form (strongly fluorescent) might provide a broadly
applicable strategy for controlling the fluorescence. We
previously reported that a series of tetramethylrhodamine
(TMR) derivatives bearing a hydroxymethyl group instead of
the original carboxy group showed unique intramolecular
spirocyclization behavior7. Based on these findings, we aimed
to establish a strategy to design fluorescence probes for a wide
range of proteases, as well as other enzymes. For this purpose,
In order to find a suitable rhodamine derivative, we first
prepared a series of hydroxymethyl rhodamines (HMRs)
bearing a primary aromatic amine on one side of the xanthene
moiety, into which a substrate peptide could be incorporated
via an amide bond. The other side of the xanthene moiety was
designed to have a primary amine (hydroxymethyl rhodamine
green:
HMRG),
N,N-dimethylamine
(hydroxymethyl
dimethylrhodamine: HMDiMeR), or N,N-diethylamine
(hydroxymethyl diethylrhodamine: HMDiEtR) structure, in
order to examine the effect of N-alkyl substitution on the
optical properties (Figure 1a, Scheme S1). We also prepared
the corresponding acetylated derivatives of these three HMRs
(Ac-HMRG, Ac-HMDiMeR, Ac-HMDiEtR), which we
considered to be models of aminopeptidase substrates
(Scheme S2). The optical properties of these HMR derivatives
showed strong pH dependency, as in the case of other
fluorophores bearing a hydroxymethyl group; the absorption
and fluorescence intensity of these HMR derivatives decrease
as the pH is increased due to nucleophilic attack of the
hydroxymethyl group at the C9 position of the xanthene
fluorophore to form the spirocyclic structure (Figure 1b, S1).
In other words, HMRs exist in the open form under acidic and
neutral conditions, but as the spirocyclic closed form under
basic conditions. From pH titration curves, the pKcycl value of
HMRs was calculated to be 8.1 for HMRG, 8.9 for HMDiMeR,
and 9.3 for HMDiEtR (pKcycl is the pH at which the extent of
spirocyclization is sufficent to reduce the absorbance of the
compound to a half of the maximum absorbance) (Table 1).
The pKcycl values varied depending on the N-alkylation and
tended to increase as the N-alkyl chain was extended, but all
these HMRs mainly exist in the open form at the physiological
pH of 7.4 (>95% for HMDiMeR and HMDiEtR, >80% for
HMRG). In contrast, acetylated HMRs exhibited a drastic shift
in pKcycl values towards the acidic side (Figure 1b, S2, Table
1); 5.3 for Ac-HMRG, 6.2 for Ac-HMDiMeR, 6.5 for
Ac-HMDiEtR, meaning that acetylated derivatives are mainly
present in the closed spirocyclic form at physiological pH
(>99% for Ac-HMRG, >90% for Ac-HMDiMeR and
Ac-HMDiEtR). Interestingly, the values shifted in the same
(a)
Acetylation
HMRs: open form
Colored and fluorescent
Ac-HMRs: closed form
Colorless and non-fluorescent
R = H; HMRG
R = Me; HMDiMeR
R = Et; HMDiEtR
R = H; Ac-HMRG
R = Me; Ac-HMDiMeR
R = Et; Ac-HMDiEtR
(b)
1.2
1.2
HMRG
Ac-HMRG
HMRG
Ac-HMRG
HMDiMeR
1
0.8
0.6
0.4
0.2
0
HMDiMeR
1
0.8
0.6
0.4
0.2
0
Ac-HMDiMeR
HMDiEtR
Ac-HMDiMeR
HMDiEtR
Ac-HMDiEtR
Ac-HMDiEtR
2
4
6
8
10
12
2
4
6
8
10
12
pH
pH
Figure 1. Intramolecular spirocyclization behavior of HMRs and
Ac-HMRs. (a) Chemical structures of HMRs (HMRG, HMDiMeR,
HMDiEtR) and the corresponding acetylated derivatives, Ac-HMRs
(Ac-HMRG, Ac-HMDiMeR, Ac-HMDiEtR). (b) pH dependency of
absorbance and fluorescence intensity of HMRs and Ac-HMRs in 0.2 M
sodium phosphate buffer at various pHs. Normalized absorbance and
fluorescence intensity at wavelengths in parenthesis was plotted against
pH. HMRG (501 nm, 501/524 nm), HMDiMeR (528 nm, 258/552 nm),
HMDiEtR (532 nm, 532/555 nm), Ac-HMRG (495 nm, 495/526 nm),
Ac-HMDiMeR (499 nm, 499/571 nm), Ac-HMDiEtR (501 nm, 501/573
nm).
Table 1. Photochemical properties of HMRs and Ac-HMRsa
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order as the values of the parental HMRs. We speculated that
4-methylcoumaryl-7-amide, a fluorescent substrate based on
AMC17) and Leu-pNA (leucyl-p-nitroanilide, colorimetric
substrate18). The kcat/Km value of Leu-HMRG was 4-fold
greater than those of other substrates (Table 3), indicating that
the order of pKcycl values can be explained in terms of the
LUMO energy levels of the fluorophores, because they act as
electrophiles in the spirocyclization reaction. The LUMO
energy levels of the xanthene moieties are summarized in
Table 1, and indeed, the pKcycl values are linearly correlated
with the LUMO energy levels (Figure S3).
Leu-HMRG is
a better substrate for LAP than the
commercially available substrates. These results indicate that
Leu-HMRG is suitable for the sensitive and quantitative
detection of LAP activity.
Next, we evaluated which of these three derivatives exhibits
the best optical properties as a core fluorophore of highly
sensitive fluorescence probes for aminopeptidases. We
calculated the ratio of the open forms between HMRs and
Ac-HMRs, and found that HMRG and Ac-HMRG (52-fold)
show a much greater difference than other derivatives (13-fold
for HMDiMeR and Ac-HMDiMeR, 7-fold for HMDiEtMeR
and Ac-HMDiEtR). Further, while HMDiMeR and HMDiEtR
showed relatively low fluorescence quantum yields (Φfl) and
Next, we examined the feasibility of applying our probe for
diagnostic purposes. As mentioned above, LAP activity in
serum is a clinical marker for diagnosis of liver disease. So,
we conducted enzyme activity measurement in buffer
containing 4% human serum albumin (HSA), the most
abundant plasma protein in human blood, to mimic serum. In
this experiment, we compared the sensitivity of Leu-HMRG
and Leu-MCA. The activation ratio of Leu-HMRG upon
addition of 17.5 mU LAP reached 60-fold at 90 min, whereas
Leu-MCA showed only 2-fold activation (Figure S9). The
reason for the low activation ratio observed with Leu-MCA is
considered to be the strong background fluorescence of
proteins (HSA) resulting from the UV excitation (Figure S10),
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short fluorescence lifetimes (τ) in aqueous solution (Φfl
=
0.044, τ = 0.3 ns for HMDiMeR; Φfl = 0.042, τ = 0.2 ns for
HMDiEtR), HMRG had a high fluorescence quantum yield
(Φfl = 0.81) and a long fluorescence lifetime (τ = 3.8 ns)
(Table S1). Therefore, the pair of HMRG and acetyl-HMRG
seemed to be excellent candidates for producing a strong
dynamic fluorescence change in response to enzymatic
reaction at the physiological pH of 7.4. This result also
demonstrates that it is possible to regulate the fluorescence of
rhodamine by controlling intramolecular spirocyclization at a
single reactive site. Such precise control cannot be achieved
with commercially available RhodamineGreen (RG), as RG
and its acetylated derivative (Ac-RG) both show
pH-independent high absorbance and fluorescence (Figure S4).
Based on the above these findings, we next focused on the
development of a fluorescence probe for aminopeptidase
based on the HMRG scaffold.
and this represents
a critical drawback for diagnostic
application. In fact, it was quite difficult to detect intrinsic
LAP activity in fetal bovine serum with Leu-MCA within a
short incubation time, whereas we could sensitively detect the
activity with Leu-HMRG (Figure 3). These results clearly
indicate the superiority of Leu-HMRG for sensitive detection
of LAP activity in the presence of a large amount of proteins,
such as in serum, and indicate that Leu-HMRG would be
useful tool for diagnostic purposes.
Target enzyme
(a)
In order to confirm the validity of our new strategy based on
HMRG, we chose LAP as a target, since it is well known to be
a clinically significant enzyme that is upregulated in serum of
liver disease patients,13 and it plays important roles in
tumor-cell invasion, metastasis and immune response.14-16
Thus, rapid and sensitive detection of LAP activity in living
samples would have a significant impact in the fields of
biology and medicine. According to our strategy, we designed
Leu-HMRG by incorporating L-leucine into HMRG via an
amide bond (Figure 2a, Scheme S3). The pKcycl value of
Leu-HMRG was calculated to be 4.8 (Table 2, Figure S5),
meaning that it should exist in the colorless and
non-fluorescent closed form at physiological pH, as expected.
Next, we tested the reactivity of Leu-HMRG with LAP. The
fluorescence intensity and absorbance of Leu-HMRG solution
increased drastically following the addition of LAP (Figure
2b), and the fluorescence activation ratio reached more than
400-fold. The fluorescence increase of Leu-HMRG is
dose-dependent and linearly related to the amount of LAP
(Figure S6). The conversion of Leu-HMRG to HMRG by the
enzyme was confirmed by analytical HPLC (Figure S7).
Further, other proteases, such as trypsin, chymotrypsin and
cathepsin B, did not increase the fluorescence of Leu-HMRG,
demonstrating high specificity of the probe for LAP activity
(Figure S8). Moreover, we determined the kinetic parameters
of Leu-HMRG and compared the values with those of other
HMRG-based probes: closed form
HMRG: open form
Colorless & non-fluorescent
Highly fluorescent
=
Leu-HMRG Ac-GlyPro-HMRG
βGal-HMRG
(b)
S/N > 400
Figure 2. Novel design strategy based on HMRG. (a) Enzymatic reaction
of HMRG-based probes with the target enzymes. Leu-HMRG for leucine
aminopeptidase (LAP), Ac-GlyPo-HMRG for fibroblast activation protein
(FAP), βGal-HMRG for β-galactosidase. (b) Absorption and emission
spectra of 1 ꢀM Leu-HMRG at 0, 1, 3, 5, 10, 15, 20, 25 and 30 min after
addition of LAP (0.04 U). Reaction was performed in 0.1 M sodium
phosphate buffer (pH 7.4) at 37 ˚C. Excitation wavelength was 501 nm.
commercial
LAP
substrates,
Leu-MCA
(L-leucine
Table 2. Photochemical properties of HMRG-based probes.
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(M-1cm-1)a)
57,000
270
especially in lysosomes (Figure 4a, Figure S11). In order to
Quantum
Yield
pKcycl
confirm that the obtained signal derives from intracellular
LAP activity and to exclude the possibility that unreacted
substrate merely accumulated in lysosomes, we performed
spectral scanning imaging and inhibitor assay. Spectral
scanning showed that the excitation and emission spectra
observed in lysosomes coincided with those of HMRG, but not
with those of Leu-HMRG, indicating that Leu-HMRG is
efficiently converted to HMRG in live HeLa cells (Figure
S12). We also performed inhibitor assay by pre-treating cells
with 2 ꢀM LAP inhibitor, bestatin methyl ester21. As a result,
the fluorescence increase of Leu-HMRG was clearly blocked
(Figure 4b, S13). These results indicate that we can detect
intracellular LAP activity in live cells with Leu-HMRG. Next,
we compared the sensitivity of Leu-HMRG with that of
bisLeu-RG, a fluorescence probe for LAP activity based on
the RG-scaffold, which requires two-step activation (Figure
S14a, Scheme S4). In living HeLa cells, bisLeu-RG showed
almost no detectable fluorescence, while Leu-HMRG showed
strong fluorescence under the same imaging conditions
(Figure 4c). Similar results were obtained in cell lysate (Figure
S14b). The great difference in sensitivity between the two
probes is considered to result from the hydrolysis reaction;
bisLeu-RG requires two-step hydrolysis for full fluorescence
activation, while Leu-HMRG is fully activated in a single step.
Yoon et al. recently reported a LAP-activatable fluorescence
probe, DCDHF-Leu, but its fluorescence activation ratio is
more than an order of magnitude lower than that of
Leu-HMRG (10-fold vs 400-fold), and it requires fixation
before imaging in cells22. These results show that Leu-HMRG
is highly advantageous for fluorescence detection of LAP
activity both in living cells and in vitro, demonstrating the
validity of our approach.
HMRG
Leu-HMRG
501
496
524
525
527
525
543
536
0.81 a)
0.22 b)
0.23 b)
0.32 b)
0.14
8.1
4.8
Ac-GlyPro-HMRG
βGal-HMRG
HMDERc)
496
340
5.1
494
620
5.4
525
80,000
12,000
11.3
6.9
10
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22
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HMDER- βGalc)
493, 525
0.009
a) Measured in 0.2 M sodium phosphate buffer, pH 7.4. b) Measured in 0.2 M sodium phosphate
buffer, pH 2.0. c) Reference 8.
Table 3. Comparison of kinetic parameters of LAP probes.
Leu-pNA a)
Leu-MCA
103
Leu-HMRG
130
46
13
Km (ꢀM)
Vmax (nM/s)
kcat (s-1)
8.6
6.9
24
19
34
kcat/Km (s-1ꢀM-1)
0.18
0.18
0.75
a) Leu-pNA (leucyl-p-nitroanilide) is a colorimetric substrate for LAP.
(a)
(b)
Leu-MCA
Leu-HMRG
300
200
100
0
150
100
50
30min
15min
10min
5min
3min
1min
30min
15min
10min
5min
3min
1min
before
before
0
450 500 550 600 650 700
350 400 450 500 550 600 650 700
Wavelength (nm)
Wavelength (nm)
Figure 3. Monitoring LAP activity in fetal bovine serum with Leu-HMRG
or commercially available L-leucine 4-methylcoumaryl-7-amide
(Leu-MCA). Assays were performed in 50% heat-inactivated fetal bovine
serum in PBS, containing 1 ꢀM Leu-HMRG (a) or Leu MCA (b).
Excitation wavelength was 501 nm for Leu-HMRG and 380 nm for
Leu-MCA.
Our design strategy should also be applicable to develop
fluorescence probes for other proteases, simply by replacing
the leucine moiety of Leu-HMRG with a group specifically
susceptible to the protease of interest. Since we have
confirmed that HMRG can readily permeate through the cell
membrane and accumulate in acidic organelles of living cells,
whereas unmodified RG cannot pass through the cell
membrane due to its high hydrophilicity (Figure S15), we
expected that HMRG would be available as a scaffold for
probes targeting membrane-anchored enzymes. Based on this
idea, we have already employed the strategy described here to
Leu-HMRG
(a)
(b) Leu-HMRG + inhibitor
(c) bisLeu-RG
160
10
develop
a
fluorescence
probe
targeting
γ-glutamyltranspeptidase (GGT), and used it for cancer
imaging based on GGT activity (Figure S16)23.
In order to further demonstrate the flexibility of our design
strategy, we next focused on different types of targets, i.e.,
dipeptidase and glycosidase. As a dipeptidase, we selected
fibroblast activation protein (FAP), which is a cell-surface
serine protease that is highly expressed in various carcinomas,
such as breast cancer and melanoma24. To develop a probe for
FAP, a substrate peptide (Ac-GlyPro) was conjugated to
HMRG to afford Ac-GlyPro-HMRG (Figure 2a, Scheme S5).
As a glycosidase, we selected β-galactosidase, which is widely
used as a reporter enzyme to examine transcription or to
evaluate transfection efficiency25,26. To develop a probe for
β-galactosidase, a β-galactoside group was coupled to HMRG
via a carbamate linker, affording βGal-HMRG (Figure 2a,
Figure 4. Fluorescence confocal imaging of LAP activity in HeLa cells.
(a) Leu-HMRG, (b) Leu-HMRG with inhibitor, (c) bisLeu-RG. Scale bar
represents 25 ꢀm.
We next applied Leu-HMRG to detect intracellular LAP
activity in living cells. Cytosolic LAP has recently been
reported to play an important role in the degradation of
peptides for MHC class I binding during an immune
response,19 and its expression is significantly increased in
ovarian cancer tissue20. When we incubated HeLa cells with
Leu-HMRG, we observed a strong fluorescence signal,
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applicable to develop probes for a wide range of enzyme
activities.
Scheme S6). Both Ac-GlyPro-HMRG and βGal-HMRG
exhibited almost no fluorescence at physiological pH, owing
to their spirocyclized structure (Table 2, Figure S17, S18.
Upon reaction with FAP or β-galactosidase, respectively, the
absorbance and fluorescence intensity of the probes increased
quickly and dramatically, and conversion of the probes to
HMRG was confirmed by analytical HPLC (Figure S19, S20).
Experimental Procedures
Materials. General chemicals were of the best grade
available, supplied by Tokyo Chemical Industries, Wako Pure
Chemical, Aldrich Chemical Co. and Invitrogen. They were
used without further purification. All solvents were used after
appropriate distillation or purification.
We also compared βGal-HMRG with HMDER-βGal,8
a
previously reported fluorescence probe for β-galactosidase
based on a rhodol scaffold (Figure S21). The pKcycl value was
reduced by more than 1 unit (5.4 for βGal-HMRG, and 6.9 for
HMDER-βGal), indicating improved suppression of the
background signal (Table 2). Consequently, the fluorescence
enhancement upon reaction with the enzyme was larger than
that of HMDER-βGal (440-fold for βGal-HMRG vs 76-fold
for HMDER-βGal). We also observed a marked fluorescence
increase in HEK 293/lacZ cells, while there was no obvious
enhancement in HEK 293 cells, which do not express
β-galactosidase (Figure 5). Thus, βGal-HMRG could visualize
intracellular β-galactosidase activity in living cells, and has
very favorable features for bioimaging. These results indicate
that our design strategy based on precise control of
spirocyclization of HMRG is extremely flexible, and has the
potential to afford highly sensitive probes for a wide range of
target enzymes.
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Instruments. NMR spectra were recorded on a JEOL
1
JNM-LA300 at 300 MHz for H NMR and at 75 MHz for 13C
NMR, a JEOL JNM-LA400 or a Bruker AVANCEⅢ400
1
Nanobay at 400 MHz for H NMR and at 101 MHz for 13C
NMR. Mass spectra (MS) were measured with a JEOL
JMS-T100LC AccuToF for ESI, or with a MicrOTOF
(Bruker). UV-visible spectra were obtained on a Shimadzu
UV-1650 and UV-2450. Fluorescence spectroscopic studies
were performed on a Hitachi F-4500 and F-7000. Lysate assay
on 96-well plates was performed using a plate reader coupled
with a Perkin-Elmer LS-55.
Fluorometric Analysis. The slit width of excitation and
emission was 5 nm for Ac- HMDiMeR and Ac-HMDiEtR, and
2.5 nm for other compounds and kinetic measurements. The
photomultiplier voltage was 700 V. Relative fluorescence
quantum efficiency was obtained by comparing the area under
the emission spectrum of the test sample excited at 490 nm
with that of a solution of fluorescein in 0.1 N NaOH, which
has a quantum efficiency of 0.85. Absolute fluorescence
quantum efficiency was determined with an absolute PL
quantum yield spectrometer, Quantaurus-QY (Hamamatsu
Photonics). Fluorescence lifetime was determined with a
HEK/lacZ
(a)
(b)
HEK
80
5
Figure 5. Fluorescence confocal imaging of HEK 293/lacZ cells (a) or
HEK 293 cells (b) loaded with βGal-HMRG. Scale bar represents 50 ꢀm.
fluorescence
lifetime
spectrometer,
Quantaurus-Tau
(Hamamatsu Photonics).
Conclusion
HPLC analysis. HPLC analysis was performed on an Inertsil
ODS-3 (4.6 x 250 mm) column (GL Sciences Inc.) using an
HPLC system composed of a pump (PU-2080, JASCO) and a
detector (MD-2010 JASCO). During synthetic procedures,
HPLC elution was done with a linear gradient (eluent, 0 min,
20% MeCN/0.1% TFA aq. to 15 min, 100% MeCN/0.1% TFA
aq.; flow rate = 1.0 mL/min). Detection wavelength was 500
nm.
We synthesized a series of rhodamine derivatives bearing a
hydroxymethyl group, and evaluated their intramolecular
spirocyclization behavior. We found that Ac-HMRG exists as
a
closed spirocyclic structure in aqueous solution at
physiological pH, whereas HMRG itself takes an open
non-spirocyclic structure. Thus, we concluded that HMRG
could be a suitable scaffold to produce a large dynamic
fluorescence change via a one-step enzymatic reaction at
physiological pH. On the basis of this result, we developed
one-step-activatable fluorescence probes for sensitive,
selective, and quantitative detection of two aminopeptidases
(Leu-HMRG for LAP and Ac-GlyPro-HMRG for FAP), and a
glycosidase (βGal-HMRG for β-galactosidase). These probes
each exist in the spirocyclic, non-fluorescent form, but are
converted to HMRG by the target enzyme, resulting in strong
fluorescence activation of up to 400-fold. These probes retain
the excellent optical properties of rhodamines, such as
brightness and photostability, and offer great practical
advantages, including high sensitivity and rapid response; they
are expected to be useful for biological and pathological
investigations. We anticipate that our design strategy will be
Preparative HPLC. Preparative HPLC were performed on
an Inertsil ODS-3 (10.0 x 250 mm) column (GL Sciences Inc.)
using an HPLC system composed of a pump (PU-2080,
JASCO) and a detector (MD-2010, JASCO).
Kinetic assay. Various concentrations of the probes
(Leu-HMRG, L-MCA and L-pNA) were dissolved in 0.5 mL
total volume of 0.1 M sodium phosphate buffer, pH 7.4,
containing 1% DMSO as a cosolvent. LAP (0.7 mU) was
added to the solution, and the fluorescence intensity (or
absorbance) was recorded continuously as described above.
Initial reaction velocity was calculated, plotted against probe
concentration, and fitted to a Michaelis Menten curve. The
kinetic parameters were calculated by use of the
Michaelis-Menten equation shown below.
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This research was supported in part by a Grant-in-Aid for
Scientific Research (Specially Promoted Research 22000006 to
V = V max * [S] / (Km + [S])
[S] = substrate concentration
V = initial velocity,
TN), by the Ministry of Education, Culture, Sports, Science and
Technology of Japan (Grants for The Advanced and Innovational
Research Program in Life Sciences, 16370071 and 16659003,
Grants 20117003 and 23249004 to Y. U., and 23113504 to M. K.)
by The Mochida Memorial Foundation for Medical and
Pharmaceutical Research (grants to M. K.), and by The Tokyo
Society of Medical Sciences (grants to M. K.).
Fluorescence confocal microscopy. Fluorescence images
were captured using a Leica Application Suite Advanced
Fluorescence (LAS-AF) instrument with a TCS SP5 and a 60
x objective lens. The light source was a white-light laser. The
excitation wavelength was 501 nm (Leu-HMRG) or 577 nm
(LysoTracker Red) and the emission wavelength was 510-540
nm (Leu-HMRG and βGal-HMRG) or 585-650 nm
(LysoTracker Red). PMT gain: 785 V, offset -0.2%.
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Experiments using living cells. HeLa cells, HEK 293 cells
expressing β-galactosidase (HEK/lacZ) cells or not expressing
β-galactosidase (HEK) were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen Corp.) supplemented
with 10% (v/v) fetal bovine serum (Invitrogen Corp.), 1%
penicillin and 1% streptomycin (Invitrogen Corp.) in a
humidified incubator containing 5% CO2 in air.
Evaluation of fluorescence in living cells. Living cells were
washed with DMEM supplemented with 1% penicillin and
streptomycin three times and incubated at 37 °C with
Leu-HMRG (100 nM) or βGal-HMRG (1 ꢀM) for 30 min.
After incubation, differential interference contrast and
fluorescence images were captured.
Evaluation of dye localization in HeLa cells. HeLa cells
were incubated at 37 °C with 100 nM Leu-HMRG and 10 nM
LysoTracker Red fluorescent lysosome stain for 30 min. After
incubation, differential interference contrast and fluorescence
images were captured by confocal microscopy.
Lysate preparation. HeLa cells were washed twice with 2
mL of DPBS, after reaching 90% confluence. 1 mL of
CelLytic (SIGMA) was added and cells were incubated for 15
min at room temperature on a shaker (180 rpm). The lysed
cells were collected with a scraper and centrifuged for 15 min
at 16,000 g to pellet the cellular debris. The protein-containing
supernatant was aliquoted into chilled test tubes and stored at
-80 °C.
Lysate assay on 96-well plates. Experiments were
performed on black plates (NUNC) at 37 °C. Leu-HMRG or
bisLeu-HMRG was added to 0.1 M sodium phosphate buffer
(pH 7.4). Then, 0.02 mL of cell lysate was added to the well
(total volume 0.2 mL). The plates were incubated for 30 min.
The fluorescence intensity (Ex : 478-492 nm, Em : 523-548
nm) was measured.
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ASSOCIATED CONTENT
Supporting Information. Synthesis and characterization of
compounds, pH profiles, fluorescence imaging, lysate assay,
experimental details.
AUTHOR INFORMATION
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp; uranokun@m.u-tokyo.ac.jp
ACKNOWLEDGEMENT
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Target Enzyme
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HMRG-based probes: closed form
(R = target enzyme substrate moiety)
HMRG: open form
Colorless & non-fluorescent
Highly fluorescent
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