Development of a Series of Near-Infrared Dark Quenchers Based on Si-rhodamines and Their Application to Fluorescent Probes

Article abstract of DOI:10.1021/jacs.5b00246

Near-infrared (NIR) fluorescent probes based on the F?rster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers, which are nonfluorescent dyes, as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). Here, we describe Si-rhodamine-based NIR dark quenchers (SiNQs), which show broad absorption covering this region. They are nonfluorescent independently of solvent polarity and pH, probably due to free rotation of the bond between the N atom and the xanthene moiety. SiNQs can easily be structurally modified to tune their water-solubility and absorption spectra, enabling flexible design of appropriate FRET pair for various NIR fluorescent dyes. To demonstrate the usefulness of SiNQs, we designed and synthesized a NIR fluorescent probe for matrix metalloproteinase (MMP) activity using SiNQ780. This probe 1 could detect MMP activity in vitro, in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized, by NIR fluorescence. We believe SiNQs will be useful for the development of a wide range of practical NIR fluorescent probes. (Chemical Equation Presented).

Full text of DOI:10.1021/jacs.5b00246

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Article
Development of a series of near-infrared dark quenchers based
on Si-rhodamines and their application to fluorescent probes
Takuya Myochin, Kenjiro Hanaoka, Shimpei Iwaki, Tasuku Ueno,
Toru Komatsu, Takuya Terai, Tetsuo Nagano, and Yasuteru Urano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b00246 • Publication Date (Web): 12 Mar 2015
Downloaded from http://pubs.acs.org on March 27, 2015
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Development of a series of near-infrared dark quenchers based on
Si-rhodamines and their application to fluorescent probes
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Takuya Myochin,† Kenjiro Hanaoka,*,† Shimpei Iwaki,†,┴ Tasuku Ueno,†,┴ Toru Komatsu,†,¶ Takuya
Terai,† Tetsuo Nagano,‡ and Yasuteru Urano*,†, §,┴
† Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
‡ Open Innovation Center for Drug Discovery, The University of Tokyo, Tokyo 113-0033, Japan
§ Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
¶ PREST, JST, Saitama 332-0012, Japan
┴ CREST, JST, Saitama 332-0012, Japan
ABSTRACT: Near-infrared (NIR) fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have
various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers, which are non-
fluorescent dyes, as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over
780 nm). Here, we describe Si-rhodamine-based NIR dark quenchers (SiNQs), which show broad absorption covering this region.
They are non-fluorescent independently of solvent polarity and pH, probably due to free rotation of the bond between the N atom
and the xanthene moiety. SiNQs can easily be structurally modified to tune their water-solubility and absorption spectra, enabling
flexible design of appropriate FRET pair for various NIR fluorescent dyes. To demonstrate the usefulness of SiNQs, we designed
and synthesized a NIR fluorescent probe for matrix metalloproteinase (MMP) activity using SiNQ780. This probe 1 could detect
MMP activity in vitro, in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized, by NIR fluores-
cence. We believe SiNQs will be useful for the development of a wide range of practical NIR fluorescent probes.
INTRODUCTION
Herein, we present silicon-substituted rhodamine dyes, SiNQs,
as versatile dark quenchers for the development of practical NIR
fluorescent probes.
Near-infrared (NIR) fluorescence imaging is one of the most
powerful in vivo imaging techniques in both life sciences and
medicine,1 and activatable NIR fluorescent probes, which show a
fluorescence change in response to biological events such as en-
zymatic activities, offer particular advantages, including high
tissue transparency and low autofluorescence in the NIR region.
For the molecular design of activatable fluorescent probes, several
fluorescence off/on controlling mechanisms are available, such as
photoinduced electron transfer (PeT), absorption spectral change
and Förster resonance energy transfer (FRET).2-4 Among them,
the FRET-based design strategy can be easily applied to various
fluorophores including NIR fluorescent dyes, unlike PeT or the
absorption spectral change strategy, and FRET-based molecular
design can achieve a low background fluorescence simply by
linking a fluorophore (FRET donor) to a dark quencher, which is
a non-fluorescent dye or a gold nanoparticle (FRET acceptor), via
an enzyme-cleavable peptide linker,5 while FRET is also often
utilized for the ratiometric fluorescence measurement.4d This de-
sign strategy has also been applied to photosensitizers.6
RESULTS AND DISCUSSION
Design and Synthesis of SiNQs. Initially, we focused on
the QSY dark quenchers (Scheme 1). The rhodamine scaffold
intrinsically shows strong fluorescence, but QSYs, which con-
tain the rhodamine scaffold bearing aromatic rings at the N
Scheme 1. Design strategy for novel NIR dark quenchers,
SiNQs.
NIR dark quenchers that have been reported so far include
gold nanoparticle, QSY21, BHQ-3 and IRDye QC-1.5b,7-9 How-
ever, QSY 21 and BHQ-3 do not possess a sufficiently extended
absorption spectrum to quench the fluorescence of Cy7 (over 780
nm), while IRDye QC-1 shows a pH-dependent fluorescence in-
crease below pH 5 (Figure S1). Gold nanoparticle-based quench-
ers are also too large for fast enzymatic reaction. So, there is a
need for NIR dark quenchers that are non-fluorescent within prac-
tical ranges of solvent polarity and pH, and also have sufficiently
broad long-wavelength absorption to quench the fluorescence of
Cy7.
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Scheme 2. Synthesis of SiNQ660 and SiNQ780 a
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aReagents and conditions: (a) Pd(PPh3)4, 1,3-dimethylbarbituric acid, CH2Cl2, 40 °C, 79%; (b) NaNO2, KI, 2 N HCl/acetonitrile, 17%; (c)
N-methyl-p-anisidine, Pd(OAc)2, BINAP, Cs2CO3, toluene, 100 °C, 20%; (d) o-tolylmagnesium bromide, THF, 93%; (e) indoline,
Pd(OAc)2, BINAP, Cs2CO3, toluene, 100 °C, 54%; (f) o-tolylmagnesium bromide, THF, 80 °C, 30%.
atoms at the 3,6 positions of the xanthene moiety, show no fluo-
rescence, irrespective of solvent polarity and pH.10 The fluores-
cence quenching mechanism of QSYs is suggested by some pa-
pers to possibly involve a twisted intramolecular charge transfer
(TICT) state,11 owing to free rotation of the bond between the N
atom and the xanthene moiety, as shown in Scheme 1. We aimed
to apply this fluorescence quenching strategy to far-red to NIR
fluorescent dyes, Si-rhodamines (SiRs),12 in which the O atom at
the 10 position of the xanthene moiety of rhodamines is replaced
with a Si atom. SiRs are rhodamine analogues with far-red to NIR
absorption and fluorescence spectra and are considered amenable
to a variety of structural modifications as required for biological
applications. So, we set out to design and synthesize SiNQs (Si-
rhodamine-based NIR dark quenchers), which contain both the Si-
rhodamine scaffold and aromatic rings directly conjugated to the
N atoms at the 3,6 positions of the xanthene moiety (Scheme 1).
The general synthetic route to SiRs (Scheme S1) was not suit-
able for the introduction of aromatic rings directly at the N atoms
of the xanthene moiety. We therefore established a new general
synthetic route for SiNQs as shown in Scheme 2, i.e., we first
synthesized 1 according to the reported synthetic procedure of
SiRs,13 then deprotected the four allyl groups on the N atoms to
obtain 2. The amino groups of 2 were converted to iodine atoms
(3), and anisidine or indoline was introduced at the 3,6 positions
of xanthone 3 by use of the Buchward-Hartwig reaction. Thus,
compound 3 is a key intermediate for SiNQs. Finally, o-
tolylmagnesium bromide was added to a THF solution of 4 or 6 to
obtain SiNQ660 (5) or SiNQ780 (7), respectively.
SiNQ780 was retained in solvents of various polarities (Figures
S2, S3 and Tables S1, S2). Further, to investigate the fluorescence
quenching mechanism of SiNQs, we synthesized compound 14
(Figure 1C, Scheme S2), in which rotation of the bond between
the N atom and the xanthene moiety is restricted, since rotation of
this bond is considered to be the main factor for the non-
fluorescence of SiNQs.11 The fluorescence quantum yield of 14
was 0.08 (Figure 1C, Table S3), so this compound was fluorescent.
Therefore, we considered that the fluorescence quenching of
SiNQs is mainly caused by molecular rotation around the bond
between the N atom and the xanthene moiety, while the fluores-
cence quenching mechanism cannot still be determined to be the
formation of the TICT state.
TABLE 1. Photophysical Properties of SiNQs a
b
Absmax (nm)
660
Emmax (nm)
n.d.
ΦFL
SiNQ660 (5)
SiNQ780 (7)
<0.001
779
n.d.
<0.001
aAll data were measured in MeOH. bFor determination of the
fluorescence quantum yield (ΦFL), we used Cy5.5 in PBS at pH
7.4 (ΦFL = 0.23) or ICG in DMSO (ΦFL = 0.13) as a fluorescence
standard. n.d. = not detected.
Improvement of the Water-solubility and the Stability of
the Quencher. Next, we examined the photophysical properties
of SiNQ780 in sodium phosphate buffer at various pH values.
However, SiNQ780 was not readily soluble in aqueous solution,
so, we synthesized water-soluble SiNQ780 (wsSiNQ780); i.e., to
improve the water-solubility, the indoline moieties of SiNQ780
were sulfonated. The wsSiNQ780 derivative 1 (R1 = H; R2 = CH3)
was synthesized according to a modification of Scheme 2
(Scheme 3). The product showed good water-solubility and its
absorption spectrum was similar to that of 7 in PBS (pH 7.4), as
we had expected (Figure 2A, S4). However, its absorbance
Photophysical Properties. We next examined the photophys-
ical properties of SiNQs (Table 1, Figure 1A,B). SiNQ660 (5) and
SiNQ780 (7) both showed absorption in the NIR region and their
fluorescence quantum yields were almost zero (less than 0.001 in
MeOH). SiNQ780 showed longer-wavelength absorption (680-
850 nm) than SiNQ660, and its absorption range is suitable for
quenching fluorescence in the Cy7 region via FRET. We also
measured the absorption and emission spectra in various organic
solvents. The fluorescence quenching ability of SiNQ660 and
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Figure 1. Absorption (red) and fluorescence (blue) spectra of SiNQ660 (5) (A), SiNQ780 (7) (B) and 14 (C) in MeOH. The excitation
wavelength was 660 nm, 760 nm or 720 nm, respectively. The chemical structure of 14 is shown in C.
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Scheme 3. Synthesis of wsSiNQ780 Derivative 1, 2 and 3 a
aReagents and conditions: a) ClSO3H, CH2Cl2, 0°C, 74%; b) triisopropyl orthoformate, i-PrOH, 80°C, 71%; c) derivative 1: i) o-
tolylmagnesium bromide, THF, 80°C, ii) 2 N HCl aq., reflux, 32%. Derivative 2 and 3: i) 2-bromo-m-xylene (dericative 2) or 2-bromo-1,3-
dimethoxybenzene (derivative 3), sec-BuLi, THF, −78°C, ii) 2 N HCl aq., reflux, 46% (derivative 2), 41% (derivative 3).
unexpectedly gradually decreased in PBS (pH 7.4) during 1 hr
(Figure 2A). On the other hand, hardly any absorption decrease of
wsSiNQ780 derivative 1 in sodium phosphate buffer at pH 3.0
was observed (Figure 2A). Therefore, we hypothesized that this
absorption decrease of derivative 1 was caused by nucleophilic
attack of a water molecule at the 9 position of the xanthene moie-
ty (see Scheme 3) because the similar phenomenon was previous-
ly observed,13b and we set out to improve the stability of deriva-
tive 1 in aqueous solutions. For this purpose, we introduced two
substituents at the 2’ and 6’ positions of the benzene moiety of
wsSiNQ780; these were expected to prevent the nucleophilic
attack of water through steric hindrance. We synthesized deriva-
tive 2 (R1 = R2 = CH3) and derivative 3 (R1 = R2 = OCH3) of
wsSiNQ780 (Scheme 3) and examined their stability in aqueous
solutions. These compounds showed similar absorption spectra to
derivative 1 (Figure S5). The absorption of these compounds did
not decrease, and these derivatives of wsSiNQ780 showed greater
stability, as we had hoped (Figure 2B). We also measured the
absorption and fluorescence spectra of derivative 3 in sodium
phosphate buffer at various pH values. Derivative 3 showed al-
most no change in absorption spectra and no fluorescence, irre-
spective of the pH of the solution (Figure S6). Thus, we success-
fully developed wsSiNQ780, which has a sufficiently broad ab-
sorption spectrum in the long-wavelength region and showed high
stability in aqueous solutions, with no fluorescence at any pH
value in the range examined.
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Figure 2. (A) Absorption spectral change of derivative 1 in PBS (pH 7.4) (left) and sodium phosphate buffer (pH 3.0) (right) containing
0.1% DMSO as a cosolvent. (B) Absorbance changes of derivatives 1 (circle), 2 (square) and 3 (triangle) at 780 nm in PBS (pH 7.4) con-
taining 0.1% DMSO as a cosolvent.
Scheme 4. Synthesis of 2’,6’-dimethoxy wsSiNQ780 COOH a
aReagents and conditions: a) PPh3, 2-amino-2-methylpropanol, DIEA, CCl4, pyridine, CH3CN, 20%; b) i) 16, 20, sec-BuLi, THF, 60°C, ii)
2 N HCl aq., acetone, reflux, 2 days, 60%.
Application of the Quencher to NIR Fluorescent Probe. To
examine the practical usefulness of wsSiNQ780 derivative 3, we
set out to develop an activatable NIR fluorescent probe for endo-
peptidase activity with wsSiNQ780. We selected matrix metallo-
proteinases (MMPs) as a target. MMPs are a family of zinc metal-
loproteinases that play important roles in degrading extracellular
matrix and in both physiological and pathological processes of
tissue remodelling.14 Activity of MMPs is also involved in em-
bryogenesis, wound healing, inflammation, arthritis, cardiovascu-
lar disease and cancer.14b,15 Among the MMPs, MMP-2, -9 and
membrane-type 1 MMP (MT1-MMP) are related to tumor inva-
sion, metastasis and angiogenesis, and the detection of these activ-
ities can be useful to predict the malignancy of tumors.14a,16 To
obtain the probe, we first synthesized wsSiNQ780 derivative 3
with a carboxy group at the 4’ position of the benzene moiety
(Scheme 4) for conjugation to an enzymatically cleavable peptide.
We also selected DY730 analogue (Figure 3A, Scheme S3),
which has strong fluorescence in the Cy7 region, as a NIR fluo-
rescent dye. A polyethylene glycol (PEG) chain (Scheme S4) was
also attached to the fluorescent probe to obtain a prolonged blood-
circulation time.17 The resulting MMP probe 1, based on the
FRET mechanism, is illustrated in Figure 3A (see also Scheme
S5). It was non-fluorescent (ΦFL <0.001) before the enzymatic
reaction, indicating that wsSiNQ780 worked well as a NIR dark
quencher in the Cy7 region (Figure 3B).
of fluorescence increase in a cuvette. The probe showed a 20-fold
fluorescence increase after incubation with MMP-9 or MT1-MMP
catalytic domain in vitro (Figure 3B, S8), and enzymatic cleavage
of the probe was confirmed by LC-MS analysis (Figure S9). The
absorption spectrum also largely changed before and after the
enzymatic reaction probably due to the stacking between the fluo-
rescent dye and the dark quencher, wsSiNQ780. We also synthe-
sized a control probe, MMP probe 2 (Scheme S6), which has al-
most the same structure as MMP probe 1, but has D-amino acids
in place of the L-amino acids in the MMP-recognition peptide
motif of MMP probe 1. This probe 2 was not cleaved by MT1-
MMP catalytic domain in vitro, as expected (Figure S7).
Live Cell Imaging with the Probe. We next applied MMP
probe 1 to cultured HT-1080 cells, which express MT1-MMP on
the cell surface18 (Figure 3C). The cells were incubated with 1 ꢀM
MMP probe 1 or 2. MMP probe 1 showed a large fluorescence
increase both inside and outside the cells, i.e., the probe was
cleaved outside the cells by MT1-MMP and then the fluorophore
was partially transferred into the cells because of the hydrophobi-
city of the DY730 analogue.19 MMP probe 2 showed little fluo-
rescence increase.
In Vivo Imaging with the Probe. Finally, we applied MMP
probe 1 to in vivo fluorescence imaging of MMP activity in tumor
tissue (Figure 3D,E, Figure S13). MMP probe 1 was intravenous-
ly injected into an HT-1080 tumor-bearing mouse, and the fluo-
rescence change was monitored. The fluorescence intensity of
MMP probe 1 at the tumor was clearly increased at 180 min after
Fluorescence Measurement of the Probe in Cuvette. Next,
we examined whether the probe can detect MMP activity in terms
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cence images of HT-1080 cells exposed to MMP probe 1 or 2 (1
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ꢀM) containing 0.1% DMSO as a cosolvent in HBSS buffer. The
cultured cells were incubated for 6 hrs after addition of the probe.
Scale bar 50 ꢀm. (D) Fluorescence images of an HT-1080 tumor-
bearing nude mouse injected with MMP probe 1 or 2 (100 ꢀM in
100 ꢀL PBS containing 1% DMSO as a cosolvent) via the tail
vein. (E) Time-dependent fluorescence change at the tumor after
tail vein injection of MMP probe 1 or 2 (n = 5). 5 mice were used
in this experiment for each probe. *p<0.05, **p<0.01 Student t
test.
9
the probe injection. The fluorescence intensity of the tumor at 180
min was 14 times larger than that at 1 min after the probe injec-
tion. The fluorescence of the whole body was also increased,
probably due to the prolonged blood retention of the cleaved (or
activated) hydrophobic fluorescent probe, as already mentioned.
On the other hand, the fluorescence intensity of MMP probe 2
was low after its intravenous injection, presumably because it was
hardly cleaved by MMP and other endopeptidases inside the body.
Thus, MMP probe 1 efficiently detected MMP activity of the
tumor in vivo, confirming the practical usefulness of SiNQ780 as
a NIR dark quencher in the Cy7 region.
Modification of the Quenchers. SiNQ780 can also easily be
structurally modified to control both hydrophilicity and hydro-
phobicity, like wsSiNQ780 and SiNQ780 (Figure S10, Scheme
S7), and the indoline moieties of SiNQ780 could also be changed
to tetrahydroquinoline moieties (Figure S11, Table S4, Scheme
S8). Thus, it should be straightforward to tune the chemical prop-
erties and stability of SiNQ780 as required by means of structural
modification.20 Further, for in vivo fluorescence imaging of endo-
peptidase activity, it is important to select an appropriate pair of
fluorescent dye and dark quencher in order to suppress back-
ground fluorescence and to improve enzyme recognition, and the
easy modifiability of SiNQ780 derivatives should allow flexible
design of custom probes for particular purposes.
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CONCLUSION
In summary, we have developed a series of novel dark quenchers,
SiNQs, which are non-fluorescent independently of solvent po-
larity and pH, probably due to free rotation of the bond between
the N atom and the xanthene moiety. This is important, because
dark quenchers like these can work as FRET acceptors in a wide
range of environments. A key advantage of SiNQ780 is that its
absorption in the long-wavelength is sufficient to enable it to
quench Cy7 region fluorescence. We also confirmed that
SiNQ780 could quench the fluorescence of ICG (Figure S12). The
fact that SiNQ780 is non-fluorescent irrespective of pH represents
a significant advantage over the reported NIR dark quencher
IRDye QC-1, which shows strong fluorescence in low pH solu-
tions. Furthermore, NIR fluorescent probes based on absorption
change of dark quenchers have been recently reported.7b,21 This
design strategy enables us to develop fluorescent probes for vari-
ous biological targets, including some that cannot be detected by
using other design strategies. Therefore, SiNQs should be useful
not only as versatile dark quenchers, but also as a scaffold of NIR
fluorescent probes for new biological targets.
EXPERIMANTAL SECTION
Materials and Methods. Reagents and solvents were of the
best grade available, supplied by Tokyo Chemical Industries,
Wako Pure Chemical, Aldrich Chemical Company, Biosearch
Technology, Kanto Chemical Company, Watanabe Chemical
Industry, Applied Bioscience or Invitrogen, and were used with-
out further purification. Saline was purchased from Otsuka Phar-
maceutical Co. Ltd. Mice (BALB/cAJcl-nu/nu) were purchased
from CLEA Japan. Reactions were monitored by means of TLC
with visual observation of the dye spot or by HPLC. All com-
Figure 3. (A) Chemical structure of MMP probe 1. (B) Absorp-
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tion (top) and fluorescence (bottom) spectra of MMP probe 1
(final 1 ꢀM) in TCN buffer containing 0.1% DMSO as a cosol-
vent. MT1-MMP catalytic domain (5 ꢀg) was added to the solu-
tion and incubated for 2 hrs. Ex = 720 nm. (C) DIC and fluores-
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pounds were purified on a silica gel column and by HPLC. All
fluorescent probes were considered pure when a single HPLC
peak was obtained.
solution to the probe solution until the enzymatic reaction was
terminated.
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LC-MS Analysis. 10 ꢀM MMP probe 1 was dissolved in 1
Instruments. 1H or 13C NMR spectra were recorded on a JEOL
JNM-LA300 or JMN-AL400. Mass spectra were measured with a
JEOL JMS-T1000LC mass spectrometer (ESI+ and ESI–). HPLC
purifications and analyses were performed on reversed-phase
columns (GL Sciences (Tokyo, Japan), Inertsil ODS-3 10 mm ×
250 mm for purifications and Inertsil ODS-3 4.6 mm × 250 mm
for analyses) fitted on a Jasco PU-2080 system and eluted with
eluent A (H2O containing 0.1% TFA (v/v)) and eluent B (CH3CN
with 20% H2O containing 0.1% TFA (v/v)). Details of HPLC
conditions are included in the synthesis and characterization data.
Absorption spectra were obtained with a Shimadzu UV-1650
(Tokyo, Japan). Fluorescence spectroscopic studies were per-
formed with a Hitachi F4500 (Tokyo, Japan). The slit widths were
2.5 nm for excitation and 5 nm for emission. The photomultiplier
voltage was 700 V. Peptide synthesis was done on a Biotage Ja-
pan Syro-I (Tokyo, Japan). MPLC purifications were performed
on a Yamazen Smart Flash EPCLC AI-5805 (Tokyo, Japan). Ab-
solute fluorescence quantum yields were determined on a Hama-
matsu Quantaurus QY C11347 (Shizuoka, Japan). LC-MS anal-
yses were performed on an Agilent Technologies Quandrapole
LC/MS 6130 1200 series.
mL TCNB buffer containing 1% DMSO as a cosolvent, and 1 ꢀg
MMP-9 catalytic domain (ENZO Life Science) was added to the
solution. The mixture was then incubated at 37°C for 18 hrs. After
that, the solution was lyophilized and 30 ꢀl H2O was added to the
residue. The solution was analyzed by LC-MS on a reversed-
phase column (GL Sciences (Tokyo, Japan), Inertsil ODS-3 2.1
mm × 250 mm). LC-MS analysis; eluent, a 20 min linear gradient
from 20% to 100% solvent B; flow rate, 0.5 mL/min; detection
wavelength, 680 nm; detection MS, m/z = 904, 958, 1402.
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Live Cell Imaging. HT-1080 cells seeded in an 8-well cham-
ber (Thermo Scientific, USA) were washed with PBS and then
200 ꢀL HBSS was added. The cells were incubated at 37°C for 15
min. After that, MMP probe 1 or 2 (final 1 ꢀM) was added and
incubation was continued at 37°C for 6 hrs. Fluorescence images
were captured without washing out excess probe, using an Olym-
pus IX 71 with a cooled CCD camera (Coolsnap HQ, Olympus)
and a xenon lamp (AH2RX-T, Olympus) with a Cy7 excitation
filter and a Cy7 emission filter.
In Vivo Fluorescence Imaging. All procedures were ap-
proved by the Animal Care and Use Committee of The University
of Tokyo. After tail vein injection of 100 ꢀM MMP probe 1 or 2
in 100 ꢀL PBS containing 1% DMSO as a cosolvent, bright field
and fluorescence images were captured at different time points
with a Maestro In-vivo Imaging System (CRi Inc., Woburn, MA),
with an excitation filter of 671 to 705 nm, an emission filter of
760 to 770 nm and bright field setting. All image analyses were
performed using Image J (NIH, Bethesda, MD, USA).
Cell Lines and Culture Conditions. Human fibrosarcoma
cell line HT-1080 was purchased from the Health Science Re-
search Resource Bank (Osaka, Japan). HT-1080 cells were cul-
tured in EMEM (Eagle’s minimal essential medium) (Wako Pure
Chemical), containing 1% non-essential amino acids (Invitrogen,
Carlsbad, CA), 10% (v/v) fetal bovine albumin (Invitrogen Corp)
and 1% penicillin (100 units/mL)-streptomycin (100 ꢀg/mL)
(Invitrogen Corp). Cells were maintained at 37°C in a humidified
incubator containing 5% CO2.
Mouse Model. All procedures were approved by the Animal
Care and Use Committee of The University of Tokyo. Fluores-
cence imaging with MMP probe 1 or 2 was performed in
BALB/cAJcl-nu/nu mice (6–7 weeks, ♀) bearing a HT-1080
xenograft tumor prepared by injection of two million HT-1080
cells into the left hind leg at 7–14 days before the fluorescence
imaging experiment.
Photophysical Properties and Fluorescence Quantum
Yield. Photophysical properties of dyes were examined in PBS
buffer (pH 7.4), MeOH, DMF, acetonitrile, DMSO or chloroform
containing 0.1% DMSO or 0.1% DMF as a cosolvent, using a
Shimadzu UV-1650 UV-vis spectrometer and a Hitachi F4500
fluorescence spectrometer. For determination of the fluorescence
quantum yield (ΦFL), Cy5.5 in PBS (ΦFL= 0.23)22 or ICG in
DMSO (ΦFL= 0.13)23 was used as a standard, and the results were
calculated according to the following equation (subscript “st”
stands for the reference and “x” for the sample).
ASSOCIATED CONTENT
Supporting Information. Synthesis, experimental details and
characterization of compounds, in vitro assays, LC-MS analysis.
These materials are available free of charge via the internet at
http://pubs.acs.org.
2
2
Φx/Φst = [Ast/Ax][nx /nst ][Dx/Dst]
AUTHOR INFORMATION
Corresponding Author
A: absorbance at the excitation wavelength
n: refractive index
uranokun@m.u-tokyo.ac.jp
khanaoka@mol.f.u-tokyo.ac.jp
Notes
D: area under the fluorescence spectra on an energy scale
The authors declare no competing financial interest.
Stability Assessment of Compounds in Aqueous Solutions.
Stability of SiNQ780 derivatives 1, 2 and 3 was examined in PBS
(pH 7.4) containing 0.1% DMSO as a cosolvent, by using a Shi-
madzu UV-1650 UV-vis spectrometer.
ACKNOWLEDGMENT
This work was supported in parts by MEXT (Specially Promoted
Research Grant Nos. 22000006 to T.N., Grant Nos. 24689003,
24659042 and 26104509 to K.H.), SENTAN, JST to K.H. K.H.
was also supported by The Asahi Glass Foundation, The Uehara
Memorial Foundation, Tokyo Biochemical Research Foundation,
Mochida Memorial Foundation for Medical and Pharmaceutical
Research, The Naito Foundation and Takeda Science Foundation.
T.M. was supported by a Grant-in-Aid for JSPS Fellows.
Enzyme Assay. 5 ꢀg MMP-9 catalytic domain (human)
(ENZO Life Science, USA) in 2 ꢀl TCNB buffer (50 mM Tris, 10
mM CaCl2, 150 mM NaCl, 0.05 % Brij 35) (pH 7.5) or 5 ꢀg
MT1-MMP catalytic domain (human) (Calbiochem, USA) in 2 ꢀl
TCNB buffer was incubated for 15 min at 37°C before the assay.
MMP probe 1 or 2 was used at a final concentration of 1 ꢀM in 1
mL TCN buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH
7.5) containing 0.1% DMSO as a cosolvent at 37°C. Fluorescence
spectra were measured every 10 min after addition of the enzyme
REFERENCES
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Page 7 of 8
Journal of the American Chemical Society
(1) (a) Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580-589. (b)
(22) Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A.
S. Bioconjugate Chem. 1996, 7, 356-362.
(23) Benson, R. C.; Kues, H. A. J. Chem. Eng. Data 1977, 22, 379-
383.
Vahrmeijer, A. L.; Hutteman, M.; van der Vorst, J. R.; van de Velde,
C. J.; Frangioni, J. V. Nat. Rev. Clin. Oncol. 2013, 10, 507-518. (c)
Verbeek, F. P.; van der Vorst, J. R.; Schaafsma, B. E.; Hutteman, M.;
Bonsing, B. A.; van Leeuwen, F. W.; Frangioni, J. V.; van de Velde,
C. J.; Swijnenburg, R. J.; Vahrmeijer, A. L. J. Hepato-Biliary-
Pancreat. Sci. 2012, 19, 626-637. (d) Yuan, L.; Lin, W.; Zheng, K.;
He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622-661.
1
2
3
4
5
6
7
8
9
(2) (a) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi,
K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684-3685.
(b) Yu, F.; Li, P.; Li, G.; Zhao, G.; Chu, T.; Han, K. J. Am. Chem. Soc.
2011, 133, 11030-11033. (c) Yu, F.; Li, P.; Wang, B.; Han, K. L. J.
Am. Chem. Soc. 2013, 135, 7674-7680.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) (a) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J.
Am. Chem. Soc. 2011, 133, 5680-5682. (b) Yuan, L.; Lin, W.; Yang,
Y.; Chen, H. J. Am. Chem. Soc. 2012, 134, 1200-1211. (c) Yuan, L.;
Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.; Zhu, S. J. Am. Chem.
Soc. 2012, 134, 13510-13523.
(4) (a) Blum, G.; Mullins, S. R.; Keren, K.; Fonovic, M.; Jedeszko,
C.; Rice, M. J.; Sloane, B. F.; Bogyo, M. Nat. Chem. Biol. 2005, 1,
203-209. (b) Li, J.; Chen, K.; Liu, H.; Cheng, K.; Yang, M.; Zhang,
J.; Cheng, J. D.; Zhang, Y.; Cheng, Z. Bioconjugate Chem. 2012, 23,
1704-1711. (c) Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H.
Bioconjugate Chem. 2009, 20, 147-154. (d) VanEngelenburg, S. B.;
Palmer, A. E. Curr. Opin. Chem. Biol. 2008, 12, 60-65.
(5) (a) Razkin, J.; Josserand, V.; Boturyn, D.; Jin, Z. H.; Dumy, P.;
Favrot, M.; Coll, J. L.; Texier, I. ChemMedChem 2006, 1, 1069-1072.
(b) Chen, W.-H.; Xu, X.-D.; Jia, H.-Z.; Lei, Q.; Luo, G.-F.; Cheng,
S.-X.; Zhuo, R.-X.; Zhang, X.-Z. Biomaterials, 2013, 34, 8798-8807.
(6) Lovell, J. F.; Chen, J.; Jarvi, M. T.; Cao, W. G.; Allen, A. D.; Liu,
Y.; Tidwell, T. T.; Wilson, B. C.; Zheng, G. J. Phys. Chem. B 2009,
113, 3203-3211.
(7) (a) Stabley, D. R.; Jurchenko, C.; Marshall, S. S.; Salaita, K. S.
Nat. Methods 2012, 9, 64-67. (b) Takahashi, S.; Piao, W.; Matsumura,
Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kamachi, T.; Kohno, M.; Naga-
no, T.; Hanaoka, K. J. Am. Chem. Soc. 2012, 134, 19588-19591.
(8) Johansson, M. K.; Cook, R. M. Chem. Eur. J. 2003, 9, 3466-3471.
(9) Peng, X. X.; Chen, H. X.; Draney, D. R.; Volcheck, W.; Schutz-
Geschwender, A.; Olive, D. M. Anal. Biochem. 2009, 388, 220-228.
(10) Jeon, J.; Lee, K. H.; Rao, J. Chem. Commun. 2012, 48, 10034-
10036.
(11) (a) Vogel, M.; Rettig, W.; Sens, R.; Drexhage, K. H. Chem. Phys.
Lett. 1988, 147, 452-460. (b) Jones II, G.; Jackson, W. R.; Choi, C. J.
Phys. Chem. 1985, 89, 294-300.
(12) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. ACS
Chem. Biol. 2011, 6, 600-608.
(13) (a) Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.;
Nagano, T. Chem. Commun. 2011, 47, 4162-4164. (b) Kushida, Y.;
Hanaoka, K.; Komatsu, T.; Terai, T.; Ueno, T.; Yoshida, K.; Uchiya-
ma, M.; Nagano, T. Bioorg. Med. Chem. Lett. 2012, 22, 3908-3911.
(14) (a) Gialeli, C.; Theocharis, A. D.; Karamanos, N. K. FEBS J.
2011, 278, 16-27. (b) Chakraborti, S.; Mandal, M.; Das, S.; Mandal,
A.; Chakraborti, T. Mol. Cell. Biochem. 2003, 253, 269-285.
(15) (a) Shiomi, T.; Lemaître, V.; D'Armiento, J.; Okada, Y. Pathol.
Int. 2010, 60, 477-496. (b) Egeblad, M.; Werb, Z. Nat. Rev. Cancer
2002, 2, 161-174.
(16) (a) Itoh, Y.; Seiki, M. J. Cell. Physiol. 2006, 206, 1-8. (b) Visse,
R.; Nagase, H. Circ. Res. 2003, 92, 827-839.
(17) Zhu, L.; Xie, J.; Swierczewska, M.; Zhang, F.; Quan, Q.; Ma, Y.;
Fang, X.; Kim, K.; Lee, S.; Chen, X. Theranostics 2011, 1, 18.
(18) Zhao, T.; Harada, H.; Teramura, Y.; Tanaka, S.; Itasaka, S.; Mo-
rinibu, A.; Shinomiya, K.; Zhu, Y.; Hanaoka, H.; Iwata, H.; Saji, H.;
Hiraoka, M. J. Control. Release 2010, 144, 109-114.
(19) Myochin, T.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. J.
Am. Chem. Soc. 2012, 134, 13730-13737.
(20) Kong, Y.; Yao, H.; Ren, H.; Subbian, S.; Cirillo, S. L.; Sacchet-
tini, J. C.; Rao, J.; Cirillo, J. D. Proc. Natl. Acad. Sci. U. S. A. 2010,
107, 12239-12244.
(21) Kiyose, K.; Hanaoka, K.; Oushiki, D.; Nakamura, T.; Kajimura,
M.; Suematsu, M.; Nishimatsu, H.; Yamane, T.; Terai, T.; Hirata, Y.;
Nagano, T. J. Am. Chem. Soc. 2010, 132, 15846-15848.
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Conjugation group
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Improvement of stability
Controlling
water-solubility
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wsSiNQ780
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