Synthesis of unsymmetrical Si-rhodamine fluorophores and application to a far-red to near-infrared fluorescence probe for hypoxia

Article abstract of DOI:10.1039/c8cc02451k

Si-Rhodamines are bright fluorophores with red to near-infrared (NIR) emission, and are widely used for fluorescence imaging of biological phenomena. Here, in order to extend the scope of Si-rhodamine fluorophores, we established a versatile synthesis of unsymmetrical Si-rhodamines. To illustrate its value, we used one of these new fluorophores to synthesize a far-red to NIR fluorescence probe for hypoxia, and showed that it can visualize hepatic ischemia in mice in vivo.

Full text of DOI:10.1039/c8cc02451k

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Kagami, W. Piao, T. Myochin, K. Numasawa, Y. Kuriki, T. Ikeno, T. Ueno, T. Komatsu, T. Terai, T. Nagano
and Y. Urano, Chem. Commun., 2018, DOI: 10.1039/C8CC02451K.
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Synthesis of unsymmetrical Si-rhodamine fluorophores and
application to a far-red to near-infrared fluorescence probe for
hypoxia
Kenjiro Hanaoka,*a Yu Kagami,a Wen Piao,a Takuya Myochin,a Koji Numasawa,a Yugo Kuriki,a
Takayuki Ikeno,a Tasuku Ueno,a Toru Komatsu,a Takuya Terai,a Tetsuo Nagano,b and Yasuteru
Urano *a,c,d
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Rsc.li/chemcomm
Si-rhodamines are bright fluorophores with red to near-infrared and emission wavelengths, as well as the HOMO and LUMO
(NIR) emission, and are widely used for fluorescence imaging of energy levels of the fluorophore, which are deeply related to
biological phenomena. Here, in order to extend the scope of Si- the fluorescence off/on switching mechanisms of fluorescent
rhodamine fluorophores, we established a versatile synthesis of probes (e.g. photoinduced electron transfer (PeT),6
unsymmetrical Si-rhodamines. To illustrate its value, we used one intramolecular spirocyclization,7 etc.).8 In addition, it would be
of these new fluorophores to synthesize
a far-red to NIR possible to introduce two different functionalities into both
fluorescence probe for hypoxia, and showed that it can visualize sides of the xanthene ring of unsymmetrical rhodamine
hepatic ischemia in mice in vivo.
scaffolds.9 Therefore, we set out to develop a versatile synthetic
scheme for unsymmetrical Si-rhodamines.
Fluorescence imaging is one of the most powerful techniques
for visualization of biological events in living samples with high
temporal and spatial resolution.1 For fluorescence imaging,
activatable fluorescence probes are indispensable and have
been extensively investigated.2 Among them, red to near-
infrared (NIR) fluorescence probes are of particular interest for
studying biological phenomena inside the body because of the
high tissue transparency and low background in this wavelength
range, and also because of the potential for multicolour imaging
using red-fluorescent probes in combination with widely
available green-fluorescent probes to study cellular events at
the molecular level.1,3 We have previously developed Si-
rhodamines as practical red-to-NIR fluorophores, and they have
found extensive applications, including in vivo imaging,
multicolour imaging and super-resolution microscopy.4,5 Many
symmetrical Si-rhodamines have been developed, but so far,
very few unsymmetrical Si-rhodamines have been reported,
probably due to the lack of suitable synthetic methods.4,5
Nevertheless, unsymmetrical rhodamines are expected to be
very useful as scaffolds of activatable fluorescence probes, since
they would contribute to precise modulation of the absorption
The established synthetic scheme of symmetrical Si-
rhodamines involves homodimerization reaction of two anilines
(Scheme 1).6 Thus, we considered that aniline heterodimers
would be key intermediates for the synthesis of unsymmetrical
Si-rhodamines. Initially, we tried to synthesize aniline
heterodimers by use of the same reaction conditions as
a.Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
b.Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan.
c. Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan.
d.CREST, Japan Agency for Medical Research and Development (AMED), Chiyoda-
ku, Tokyo 100-0004, Japan.
Scheme 1
unsymmetrical Si-rhodamines.
General synthetic scheme of symmetrical and
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Table 1 Heterodimerization reaction of p-hydroxymethyl aniline and
aniline
View Article Online
DOI: 10.1039/C8CC02451K
Isolated yield (%)
Entry
Conditions
H2O/CH3CN, 80˚C, 48 hr
AcOH, 80˚C, 15 min
BF3·OEt2, DCM, 35˚C, 24 hr
BF3·OEt2, DCM, r.t., 24 hr
11
0
67
0
12
0
0
65
94
1
2
3
4
0
employed for symmetrical Si-rhodamines, but the reaction mainly
provided homodimers (see Table S1).
Fig. 1 Synthesized aniline heterodimers. The isolated yields are
shown in parentheses. The reactions were performed at (a) 35oC or
(b) r.t.
Next, we focused on the Lewis acid-activated nucleophilic
substitution reaction of alcohols, and tried to synthesize aniline
heterodimers by condensation between aniline and 4-
hydroxymethylaniline, which can be activated to the aza quinone
methide intermediate by Lewis acid.10-13 First, a formyl group was
introduced into aniline 7 by Vilsmeier reaction, then the formyl group
of the product was reduced with NaBH4 to provide p-hydroxymethyl
aniline (14). We then examined the reaction of 14 with aniline 8
under various conditions. We initially tried a mixture of H2O (proton
donor) and CH3CN as a solvent,11 but the reaction did not proceed
(Table 1, Entry 1). We then examined the conditions used in the
previously reported reaction for aniline homodimers, i.e., 80oC for 15
min in acetic acid (Table 1, Entry 2), but this afforded only aniline
homodimer 11, probably because the hydroxy group of 14 was
acetylated in this reaction and this product rapidly formed the
homodimer 11.12 Finally, we used a Lewis acid,10,13 BF3·OEt2, to
efficiently remove the hydroxyl group of 14. As a result, a moderate
yield of aniline heterodimer 12 was obtained in CH2Cl2 at 35oC for 24
hrs (Table 1, Entry 3). When the reaction temperature was reduced
from 35oC to r.t. in order to prevent side reaction, aniline
heterodimer 12 was successfully obtained in high yield (Table 1,
Entry 4).
To confirm the usefulness of this synthetic scheme, we
synthesized a series of aniline heterodimers (Fig. 1). In the syntheses
of 15 and 24, the temperature was increased to 35oC due to the
relatively low reactivity of the anilines (blue anilines in Fig. 1) as
nucleophiles. Indeed, when we compared the synthetic yields of 12
(Table 1) and 24, which are the same compound, the yield was much
lower in the latter case, probably because of the relatively low
reactivity of 3-bromo-N,N-diallylaniline as a nucleophile. Among
these aniline heterodimers, we selected seven for lithiation with sec-
BuLi, followed by reaction with SiMe2Cl2, and then oxidation with
KMnO4 to provide Si-xanthones. Reaction of the Si-xanthones with o-
tolylmagnesium bromide afforded the corresponding unsymmetrical
Si-rhodamines (Scheme 1). These Si-rhodamines
showed various absorption and emission wavelengths from 620 nm
to 700 nm with relatively high fluorescence quantum yields (Fig. 2
and Fig. 3). Fluorophores 25 and 28 are of particular interest as their
absorption and emission wavelengths are intermediate between
those of symmetrical Si-rhodamines, 2-Me SiR600, 2-Me SiR650 and
2-Me SiR700,14 as are their LUMO energy levels, which are important
for the PeT and intramolecular spirocyclization mechanisms (Fig. S1,
Fig. S2). We calculated the HOMO and LUMO energy levels of these
seven unsymmetrical Si-rhodamines and three previously reported
symmetrical Si-rhodamines at the B3LYP/6-31+G(d) (gas phase) level,
and found that the differences between the HOMO and LUMO
energy levels of the fluorophores, except 31, were highly correlated
with their absorption wavelengths (Fig. S3). The exception in the case
of 31 is probably due to the extension of the π conjugation system.
This result indicates that absorption wavelengths of Si-rhodamines
can be predicted from the calculated HOMO and LUMO energy levels
before synthesis, which would be helpful to obtain fluorophores
optimized for the absorption and emission wavelengths of existing
microscope lasers and emission filters.
Finally, to demonstrate the usefulness of unsymmetrical Si-
rhodamines for the development of novel far-red to NIR fluorescence
probes, we designed and synthesized a NIR fluorescence probe for
hypoxia, which is associated with various diseases.15 In some solid
tumours, the median oxygen concentration has been reported to be
around 4% and locally it may even fall to 0%.16
The hypoxia has various biological consequences, including
stabilization of hypoxia-inducible factor 1α (HIF-1α) and modulation
of HIF-mediated gene expression.17 We have previously developed
azo rhodamine derivatives, MAR and MASR, as hypoxia-sensitive
fluorescence probes and demonstrated that MAR could visualize
retinal hypoxia in a rat artery occlusion model.18 However, from the
viewpoint of in vivo imaging, it would be useful to extend the range
2 | Chem. Commun., 2018, 00, 1-3
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azoSiR640 showed a broad absorbance spectVruiemw ArtwicliethOnlninoe
DOI: 10.1039/C8CC02451K
fluorescence in PBS (Fig. S4, Table S2). A 2,6-dimethylbenzene moiety
was included in the probe structure to prevent nucleophilic attack of
water molecules and/or reduced glutathione (GSH) on the 9-position
of the Si-xanthene moiety of azoSiR640.19 To confirm the stability of
azoSiR640, we examined the reactivity of azoSiR640 with 0-10 mM
GSH, and found that azoSiR640 was stable even in the presence of 10
mM GSH (Fig. S4). In the assay of azoSiR640 with rat liver
microsomes, which contain various bioreductases,
a large
fluorescence increase was observed only under hypoxia and the
fluorescence intensity was increased 43-fold compared with that
under normoxia (Fig. 4b). A small fluorescence decrease was
observed under hypoxia after the maximum fluorescence intensity
was reached, probably due to reduction of the fluorophore, 2,6-diMe
SiR640. However, this phenomenon is not problematic for
fluorescence imaging of hypoxia, because no significant fluorescence
decrease of 2,6-diMe SiR640 was observed in living cells under
hypoxia (Fig. S5). HPLC analyses of reaction mixtures of azoSiR640
with rat liver microsomes also revealed that 2,6-diMe SiR640 was
generated only under hypoxia (Fig. S6).
To examine whether or not this fluorescence probe can detect
hypoxia in living cells, A549 cells were incubated with 100 nM
azoSiR640 under various oxygen concentrations for 6 hr and
fluorescence imaging was performed. A significant fluorescence
increase was observed at 5% oxygen concentration and below (Fig.
4c,d). This fluorescence increase was diminished in a concentration-
dependent manner by the non-specific flavoprotein inhibitor
diphenyliodonium chloride (DPI) (Fig. S7). This result suggests that
the probe is reduced by flavoproteins, including NADPH-
cytochorome P450 reductase, which are thought to be responsible
for the metabolic activation of bioreductive compounds under
hypoxia.20 We also successfully performed real-time live-cell imaging
of hypoxia by placing a cover glass over a cell monolayer18,21 (Fig. S8,
Supplementary movie 1).
Finally, we conducted fluorescence imaging in mice to detect
hypoxia in vivo. AzoSiR640 was administered through an orbital vein,
and then the portal and renal veins were ligated to induce liver and
kidney ischemia. Time-lapse in-vivo NIR fluorescence imaging was
performed (Fig. 4e). Before ligation of the portal and renal veins, no
fluorescence increase was observed at the liver and kidney. On the
other hand, after ligation, strong fluorescence was observed at both
liver and kidney, with increases of 10-fold and 3-fold after 20
minutes, respectively (Fig. S9, Supplementary movie 2). On the other
hand, there was no fluorescence increase in the small intestine,
which was not ligated.
Fig. 2
photophysical properties in 100 mM sodium phosphate buffer (pH
7.4) containing 1% DMSO as co-solvent. Symmetrical Si-
rhodamines, 2-Me SiR600, 2-Me SiR650 and 2-Me SiR700, were
previously reported.14 fl: fluorescence quantum yield. ε: molar
extinction coefficient.
Synthesized unsymmetrical Si-rhodamines and their
a
(a)
(b)
1
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
450
500
550 600 650
700 750
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
Fig. 3 (a,b) Normalized absorption (a) and emission (b) spectra of
reported symmetrical Si-rhodamines (2-Me SiR600, 2-Me SiR650 and
2-Me SiR700) and unsymmetrical Si-rhodamines 25-31. Absorption
and emission spectra were measured in 100 mM sodium phosphate
buffer (pH 7.4) containing 1% DMSO co-solvent.
In summary, we have established a novel synthetic strategy for
unsymmetrical Si-rhodamines via aniline heterodimers. These
unsymmetrical
Si-rhodamine
fluorophores
provide
new
of available excitation and emission wavelengths to achieve deep
tissue penetration and low scattering. For this purpose, we focused
on SiR640 (26) as a scaffold fluorophore, because SiR640 shows NIR
emission and has an amino group on the xanthene moiety (Fig. 2 and
Fig. 3) and this group can be structurally modified to obtain a suitable
functional moiety for fluorescence off/on switching. The designed
probe, azoSiR640 (Fig. 4a), was easily synthesized by azo coupling
reaction with 2,6-diMe SiR640 (see Supporting Information).
opportunities for modulating absorption and emission wavelengths,
as well as HOMO and LUMO energy levels, which are important for
fluorescence off/on switching.22 To illustrate their utility, we
employed the newly synthesized unsymmetrical Si-rhodamine, 2,6-
diMe SiR640, to develop a far-red to NIR fluorescence probe for
hypoxia, azoSiR640. We also found that the calculated HOMO and
LUMO energy levels of Si-rhodamines can be useful to predict the
absorption wavelengths and the HOMO and LUMO energy levels of
fluorophores, which are important determinants of the fluorescence
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off/on switching properties.23 We believe that the synthetic and SENTAN, JST to K.H., who was also supported by Mochida
View Article Online
DOI: 10.1039/C8CC02451K
availability of unsymmetrical Si-rhodamines opens up new Memorial Foundation for Medical and Pharmaceutical
opportunities for expanding the range of applications of red-to-NIR Research.
fluorescence probes.
(a)
Conflicts of interest
There are no conflicts to declare.
azoSiR640 (Φfl <0.001)
Non-fluorescent
2,6-diMe SiR640 (Φfl = 0.18)
Ex/Em = 637/660 nm
(b)
(d)
Fluorescent
Notes and references
70
60
50
40
30
20
10
0
1000
1
B. N. G. Giepmans, S. R. Adams, M. H. Ellisman and R. Y. Tsien,
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Normoxia
800
600
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200
0
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,
685-695.
20%
5%
1%
0.1%
0
600 1200 1800 2400 3000 3600
Time (sec)
Oxygen concentration
T. Ikeno, T. Nagano and K. Hanaoka, Chem. Asian J., 2017, 12
(c)
1435-1446.
O2 concentration
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5%
1%
0.1%
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Fig. 4 Far-red to NIR fluorescence probe for hypoxia, azoSiR640. (a)
NIR fluorescence activation of azoSiR640. azoSiR640 is almost non-
fluorescent, but is reduced by bioreductases under hypoxia to
strongly fluorescent 2,6-diMe SiR640. (b) Time-dependent changes
in the fluorescence intensity of azoSiR640 (1 μM) in the presence of
rat liver microsomes (226 μg/3 mL) under hypoxia or normoxia.
Measurements were performed in a potassium phosphate buffer
(0.1 M; pH 7.4) containing DMSO (0.1%) as a cosolvent. As a cofactor
for reductases, NADPH (50 μM) was added after 5 min (arrowhead).
The excitation and emission wavelengths were 640 and 660 nm. (c)
Fluorescence confocal microscopy images of live A549 cells. A549
cells were incubated with azoSiR640 (100 nM) containing DMSO
(0.1%) as a cosolvent at various oxygen concentrations for 6 hr. (d)
Quantitation of fluorescence intensity at ROI of cells under various
oxygen concentrations. n = 4. * indicates p<0.05 by Student’s t-test.
(e) Fluorescence images of mouse. ICR mouse was injected with 100
μM azoSiR640 in 100 μL saline solution containing 1% DMSO as a
cosolvent from an orbital vein. The portal vein and renal vein were
ligated about 35 min after probe injection. Excitation and emission
wavelengths were 635 nm and 700 nm, respectively. Exposure time:
300 ms.
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This work was supported in part by grants by JSPS KAKENHI
Grant Numbers 16H00823, 16H05099 and 18H04609 to K.H.,
4 | Chem. Commun., 2018, 00, 1-3
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Graphical Abstract
A
versatile synthesis of unsymmetrical Si-rhodamines was
established and applied for development of a hypoxia-sensing far-
red to NIR fluorescence probe.
This journal is © The Royal Society of Chemistry 20xx
Chem. Commun., 2018, 00, 1-3 | 5
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