Red fluorescent scaffold for highly sensitive protease activity probes

Article abstract of DOI:10.1016/j.bmcl.2012.04.114

We have developed a novel red fluorescent dye, 2Me SiR600 (λ_em = 613 nm), in which the O atom of Rhodamine Green at the 10 position of the xanthene moiety is replaced with a Si atom, as a scaffold for probes to detect protease activity with extremely high S/N ratio. As proof of concept, we designed and synthesized probes for caspase-3 activity (Z-DEVD-SiR600) and leucine aminopeptidase activity (Leu-SiR600). Caspase-3-mediated cleavage of Z-DEVD-SiR600 resulted in a large bathochromic shift (93 nm) of the absorption maximum and a 432-fold fluorescence enhancement.

Full text of DOI:10.1016/j.bmcl.2012.04.114

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3908–3911
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Red fluorescent scaffold for highly sensitive protease activity probes
Yu Kushida, Kenjiro Hanaoka, Toru Komatsu, Takuya Terai, Tasuku Ueno, Kengo Yoshida,
⇑
Masanobu Uchiyama, Tetsuo Nagano
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a novel red fluorescent dye, 2Me SiR600 (kem = 613 nm), in which the O atom of Rho-
damine Green at the 10 position of the xanthene moiety is replaced with a Si atom, as a scaffold for
probes to detect protease activity with extremely high S/N ratio. As proof of concept, we designed and
synthesized probes for caspase-3 activity (Z-DEVD-SiR600) and leucine aminopeptidase activity (Leu-
Received 6 April 2012
Revised 24 April 2012
Accepted 25 April 2012
Available online 2 May 2012
SiR600). Caspase-3-mediated cleavage of Z-DEVD-SiR600 resulted in
(93 nm) of the absorption maximum and a 432-fold fluorescence enhancement.
Ó 2012 Elsevier Ltd. All rights reserved.
a large bathochromic shift
Keywords:
Fluorescent probes
Enzymes
Proteases
Proteases have key roles in various physiological processes,
including protein digestion,1 spermatogenesis,2 bacterial killing,3
receptor activation4 and apoptosis.5 Fluorescence probes are useful
tools to detect protease activity, offering high sensitivity, high spa-
tiotemporal resolution and simplicity in use. Therefore, various
fluorescence probes for detecting activities of proteases, such as
caspase,6 calpain,7 cathepsin8 and MMP,9 are employed in biologi-
cal studies.
1) sec-BuLi, THF, -78°C
2)
O
N
Si
N
1
THF, -78°C→r.t.
3) 2N HCl
N
Si
N
Br
There are three main types of activable fluorescence probes for
detecting protease activity. FRET (Förster resonance energy trans-
fer)-based probe6–10 are composed of a fluorophore and a quench-
er, such as DABCYL7 and Disperse red-1,10 linked by a peptide
containing a protease recognition sequence. This design strategy
can be applied to various fluorophores in the UV to near-infrared
regions, but has some disadvantages. For example, the molecular
size of the probes is relatively large, and this design strategy is of-
ten not applicable to monitoring exopeptidase activity. The second
type is spiro-ring-opening reaction-based probes, such as urea-
3
2
y. 95%
Pd(PPh3)4
1,3-dimethylbarbituric acid
NaBH4
MeOH, 0°C
H2N
Si
NH2
CH2Cl2, 35°C
4
y.60% in 2 steps
p-chloranil
H2N
NH2
Si
CH2Cl2, r.t.
y.56%
2Me SiR600 (5)
COO
Cbz-DEVD-OH
HATU
H2N
O
NH2
p-chloranil
CH2Cl2, r.t.;
TFA, r.t.
H2N
Si
NH2
HOBt·H2O
DIPEA
Rhodamine Green
2Me SiR600 (5)
O
4
DMF, r.t.
Z-DEVD
N
H
Si
NH2
Figure 1. Chemical structures of Rhodamine Green and 2Me SiR600.
y. 4% in 3 steps
Z-DEVD-SiR600 (6)
Scheme 1. Synthesis of 2Me SiR600 and Z-DEVD-SiR600.
⇑
Corresponding author. Tel.: +81 3 5841 4850; fax: +81 3 5841 4855.
E-mail address: tlong@mol.f.u-tokyo.ac.jp (T. Nagano).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.114

Y. Kushida et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3908–3911
3909
Table 1
unsubstituted for conjugating peptide substrates, and synthesizing
this kind of compounds is often troublesome. To establish its
usefulness, we designed Z-DEVD-SiR600 (6) as a red fluorescence
probe for caspase-3, in which the caspase-3-cleaveable tetrapep-
tide sequence DEVD is conjugated to the N atom of the xanthene
moiety of 2Me SiR600 (5). 2Me SiR600 was synthesized as shown
in Scheme 1 (see Supplementary data for details). Deprotection
of the allyl groups of 3 was initially unsuccessful, but we found
that reduction of the xanthene ring allowed their removal. So,
the allyl groups were deprotected following reduction of the xan-
thene ring, then 2Me SiR600 was obtained by re-oxidizing the re-
duced compound 4. Similarly, Z-DEVD-SiR600 was synthesized
by conjugating the tetrapeptide, Cbz-DEVD, to the key intermedi-
ate 4, then re-oxidizing it.
Table 1 shows the photophysical properties of 2Me SiR600 and
Z-DEVD-SiR600 in 100 mM sodium phosphate buffer at pH 7.4.
2Me SiR600 emitted a red fluorescence (kem = 613 nm), and also
showed sufficiently large values of molar absorption coefficient
and fluorescence quantum yield for practical use.11 Interestingly,
when Cbz-DEVD peptide was conjugated to the N atom of the xan-
thene moiety of 2Me SiR600, a very large blueshift (93 nm) of the
absorption maximum was observed. We then examined the enzy-
matic reaction of Z-DEVD-SiR600 with caspase-3 (Fig. 2a). As ex-
pected, the probe was recognized by caspase-3 and cleaved to
2Me SiR600, formation of which was confirmed by HPLC analysis
(Fig. S1), resulting in a 432-fold fluorescence increment upon exci-
tation at 593 nm (Fig. 2b). We considered that one of the main
reasons for this extremely large fluorescence enhancement is the
93 nm blueshift of the absorption maximum of Z-DEVD-SiR600
after the enzymatic reaction with caspase-3 (Fig. 2c). The kinetic
parameters of Z-DEVD-SiR600 for caspase-3 are shown in Figure 2d.
The Km value of this probe is comparable to that of the reported
FRET-based probe,16 while the kcat/Km value is 40 times larger than that
of Ac-DEVD-AMC, which is widely used as a standard fluorescent sub-
strate for caspase-3.17
Photophysical properties of 2Me SiR600 and Z-DEVD-SiR600a
b
e
(MÀ1 cmÀ1
)
UFL
kex (nm)
kem (nm)
2Me SiR600
Z-DEVD-SiR600
91,000
13,000
0.38
0.19
593
500
613
600
a
Photophysical properties were measured in 100 mM sodium phosphate buffer
containing 0.1% MeOH as a cosolvent at pH 7.4.
b
For the determination of fluorescence quantum yields, Rhodamine B in EtOH
(UFL = 0.65) was used as a fluorescence standard.
rhodamine11 and HMRG,12 which contain a recognition sequence
at the N atom of the xanthene moiety. Upon protease cleavage of
the substrate peptide moiety, the non-fluorescent rhodamine moi-
ety is converted to the strongly fluorescent open form. However,
because these probes are all based on the Rhodamine Green scaf-
fold (Fig. 1), they are fluorescent only in the green region. The third
type is AMC (7-amino-4-methylcoumarin)-based probes. These
utilize the change of fluorescence emission triggered by cleavage
of a substrate peptide conjugated to AMC.13 But, these probes
usually show high background fluorescence due to the need for
excitation in the UV range.
Thus, though many fluorescence probes for detecting protease
activities are available, they suffer from various limitations, and
in particular, there are few red fluorescent probes for exopeptidase
activity. Such probes are needed, because they would provide an
additional color window for multi-color imaging and because they
have superior characteristics for in vivo imaging (tissues are more
transparent to red light than to green light).14
Here, we describe the development of a novel Rhodamine Green
analog, 2Me SiR600 (Fig. 1), which has a Si atom substituted for the
O atom at the 10 position of the xanthene moiety of Rhodamine
Green, as a fluorescent scaffold for protease probes operating in
the red region. We previously reported the Si-rhodamine dyes with
fully substituted amino groups at xanthene moiety.15 But in this
scaffold, the amino groups of the xanthene moiety should be
Figure 2. (a) Reaction scheme of Z-DEVD-SiR600 with caspase-3, and photographs of 2
l
M Z-DEVD-SiR600 and 2Me SiR600 solutions in 100 mM sodium phosphate buffer
M Z-DEVD-SiR600 before and 10 h after addition of caspase-3 (0.5 g).
M DTT, 10% glycerol, 0.1% CHAPS, 100 mM NaCl and 0.1% DMSO as a co-solvent at 37 °C.
The excitation wavelength was 593 nm. (d) Kinetic parameters of Z-DEVD-SiR600 with caspase-3 (Fig. S2).
upon excitation with an illuminator (>550 nm). (b) Fluorescence and (c) absorption spectra of 2
Reaction was performed in 0.75 ml of 20 mM HEPES buffer (pH 7.4) containing 100
l
l
l

3910
Y. Kushida et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3908–3911
Figure 3. (a) Reaction scheme of Leu-SiR600 with LAP. (b) Fluorescence and (c) absorption spectra of 6
1 unit of LAP is defined as the amount of enzyme that hydrolyzes 1.0 mol of -leucine-p-nitroanilide to
was performed in 0.75 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 0.8% DMSO as a cosolvent at 37 °C. The excitation wavelength was 593 nm.
lM Leu-SiR600 before and 2.5 h after addition of LAP (0.096 unit).
l
L
L-leucine and p-nitroaniline per min at pH 7.2 at 37 °C. The reaction
We also examined the pH-dependency of the absorption spectra
of 2Me SiR600 and Z-DEVD-SiR600 in 100 mM sodium phosphate
buffer at various pH values (Figs. S3 and S4). 2Me SiR600 showed
no absorption spectral change in the pH range examined
(Fig. S3a). On the other hand, Z-DEVD-SiR600 showed a pH-
dependent change of absorbance at 500 nm (Fig. S4a). We think
that the highly electrophilic 9 position of the xanthene moiety
may be attacked by nucleophilic hydroxide ion under basic condi-
tions, and density functional theory (DFT) calculations (B3LYP/6-
31+G(d)) indicated that N-acylation of 2Me SiR600 lowers its
LUMO energy level (Fig. S5). We also observed that the decreased
absorbance of Z-DEVD-SiR600 at pH 7.4 was restored to the level
at pH 3.0 by adding a small drop of 2N HCl aq and this result seems
to support our proposed mechanism of the pH-dependent absor-
bance change (Fig. S4d). The above pH-dependent absorbance de-
crease, however, has no influence on the function of Z-DEVD-
SiR600 as a protease probe (Fig. S6).
photoinduced electron transfer (PeT) mechanism),22 and so should
be available for modification. Further development of the structure
is now under way.
Acknowledgments
This research was supported in part by the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan (Specially
Promoted Research Grant Nos. 22000006 to T.N., and 24659042
to K.H.), SENTAN, JST to K.H. K.H. was also supported by Inoue
Foundation for Science, Takeda Science Foundation, Grant-in-Aid
from the Tokyo Biochemical Research Foundation, Konica Minolta
Science and Technology Foundation, The Asahi Glass Foundation
and Astellas Foundation for Research on Metabolic Disorders.
Supplementary data
To further confirm the potential of 2Me SiR600 as a widely
available red fluorescent scaffold for protease probes, we also syn-
thesized Leu-SiR600 as a fluorescence probe for leucine aminopep-
tidase (LAP). Leu-SiR600 was found to show a large fluorescence
increment (151-fold) upon reaction with LAP (Fig. 3).
Supplementary data (detailed descriptions of materials, general
methods and synthetic procedures. Data on the measurement of
the pH profiles of absorption and fluorescence spectra of 5, pKa plot
of absorbance change of 6, HPLC analyses for reaction product of 6
with caspase-3, kinetic studies of 6, Figs. S1–S6) associated with
this article can be found, in the online version, at http://dx.doi.
org/10.1016/j.bmcl.2012.04.114.
In conclusion, we have developed a Si-substituted Rhodamine
Green analog, 2Me SiR600, as a novel red fluorescent scaffold for
protease probes. As proof of concept, we synthesized Z-DEVD-
SiR600, which showed a 432-fold fluorescence increment upon
reaction with caspase-3, and Leu-SiR600, which showed a 151-fold
fluorescence increment upon reaction with LAP. The molecular size
of Z-DEVD-SiR600 is smaller, and its fluorescence activation is lar-
ger, than those of other FRET-based red fluorescence probes for
proteases.8,18–20 The emission wavelength of 2Me SiR600
(kem = 613 nm) is similar to that of Texas Red dye,21 and to our
knowledge, few protease fluorescent probes emitting at this wave-
length have been reported. We expect that 2Me SiR600-based
probes will be useful for multi-color imaging, because the emission
wavelength of 2Me SiR600 (613 nm) can be well separated from
the green emission of fluorescein (511 nm), in contrast to typical
rhodamines, such as TMR, Rhodamine B and Rhodamine 6G. Final-
ly, we believe 2Me SiR600 offers an easily tunable fluorescent scaf-
fold, because its 2Me benzene moiety is not required for off/on
fluorescence switching (the structure of the benzene moiety is
strictly limited for off/on fluorescence switching based on the
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