Visualization and Manipulation of Actin Cytoskeleton with Small-Molecular Probes Takeru Takagi, Tasuku Ueno, Keisuke Ikawa, Daisuke Asanuma, Yusuke Nomura, Shin-nosuke Uno, Toru Komatsu, Mako Kamiya, Kenjiro Hanaoka, Chika Okimura, Yoshiaki Iwadate, Kenzo Hirose, Tetsuo Nagano, Kaoru Sugimura, Yasuteru Urano
Submitted date: 08/05/2020 • Posted date: 11/05/2020 Licence: CC BY-NC-ND 4.0 Citation information: Takagi, Takeru; Ueno, Tasuku; Ikawa, Keisuke; Asanuma, Daisuke; Nomura, Yusuke; Uno, Shin-nosuke; et al. (2020): Visualization and Manipulation of Actin Cytoskeleton with Small-Molecular Probes. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12272024.v1
Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. Here, in order to dissect the complex mechanisms of actin-related cellular functions, we introduce two powerful tools based on a new class of actin-binding small molecule: one enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green-light-controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.
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probes
Takeru Takagi1, Tasuku Ueno*1, Keisuke Ikawa4, Daisuke Asanuma2, 5, Yusuke
Nomura1, Shin-nosuke Uno2, Toru Komatsu1, Mako Kamiya2, Kenjiro Hanaoka1,
Chika Okimura6, Yoshiaki Iwadate6, Kenzo Hirose2, 7, Tetsuo Nagano3, Kaoru
Sugimura4, and Yasuteru Urano*1, 2, 8
1Graduate School of Pharmaceutical Sciences, 2Graduate School of Medicine, and 3Drug Discovery Initiative, The University of Tokyo, Tokyo 113-0033, Japan
4Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University,
Kyoto, 606-8501, Japan
6Faculty of Science, Yamaguchi University, Yamaguchi, 753-8512, Japan.
7International Research Center for Neurointelligence, The University of Tokyo, 113-
0033 Tokyo, Japan
8CREST (Japan) Agency for Medical Research and Development (AMED), Tokyo 100-
0004, Japan
Abstract
Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates
mechanical forces in the cell. Here, in order to dissect the complex mechanisms of
actin-related cellular functions, we introduce two powerful tools based on a new
class of actin-binding small molecule: one enables visualization of the actin
cytoskeleton, including super-resolution imaging, and the other enables highly
specific green-light-controlled fragmentation of actin filaments, affording
unprecedented control of the actin cytoskeleton and its force network in living cells.
Actin filaments are major components of the cytoskeleton in eukaryotic cells,
functioning to maintain the shape and internal framework of cells, and to provide
the cells with a driving force for shape change and movement1. Natural actin-binding
small-molecular inhibitors have long been recognized as valuable tools for
dissecting the mechanisms of actin-related cellular functions2. Actin-binding small
molecules have also been utilized as platforms for functional molecules; for example,
fluorescent phalloidin conjugate was originally developed in 19793, and is still the
gold standard for labeling endogenous actin filaments in fixed samples. Nevertheless,
there is still a need to develop improved fluorescent actin-binding molecular
conjugates for actin visualization, especially for application to real-time imaging in
living cells and organisms4, 5.
Here, we report that HMRef, a simple rhodol derivative bearing a hydroxymethyl
group, provides a new, powerful tool for actin labeling in live cells (Fig. 1a, 1b).
HMRef was originally developed as a fluorophore for in vivo tumor imaging probes6.
Unexpectedly, we found that HMRef, which is highly fluorescent in aqueous media
and is membrane-permeable, can clearly visualize the actin cytoskeleton, despite
having no structural similarity to any of the known actin-binding natural products,
such as phalloidin or jasplakinolide2. In order to elucidate the mechanism
underlying the visualization of cellular actin fibers by HMRef, we first examined the
in vitro interaction of HMRef with isolated F-actin/G-actin by means of fluorescence
polarization (FP) assay. As expected, the FP signal was increased when HMRef was
incubated with isolated F-actin, i.e., HMRef has intrinsic binding affinity for F-actin,
and does not require assistance from actin-binding proteins (Fig. 1c). The FP signal
increase was negligible in the presence of monomeric actin, suggesting specific
binding of HMRef to polymeric actin. Secondly, we conducted competitive binding
assay with HMRef and natural F-actin stabilizers using fixed HeLa cells
(Supplementary Fig. 1). Both phalloidin and jasplakinolide significantly and dose-
dependently decreased the HMRef fluorescence response to actin in fixed cells,
suggesting that their binding sites are likely to overlap, at least partially, with the
HMRef binding site. HMRef may bind relatively weakly to F-actin as the natural
products could compete with HMRef at lower concentrations. We also found that
HMRef has little effect on actin polymerization or stability under our experimental
conditions (Supplementary Fig. 2), which is consistent with a relatively weak
interaction. Finally, we designed and synthesized a series of HMRef derivatives and
applied them to live and fixed cells (Supplementary Fig. 3,4, Supplementary table
1, and Supplementary Notes 1, 2). Most of the HMRef derivatives also bound to F-
actin in fixed cells, but to a lesser extent. Of the 9 derivatives tested, HMRef seems to
be the most suitable fluorescent probe for imaging the actin cytoskeleton in living
cells.
We next confirmed that HMRef is suitable for super-resolution imaging techniques,
STED7, 8 or SRRF,9 and indeed, we obtained highly detailed actin staining images (Fig.
1d, 1e and supplementary Fig. 5). Actin is one of the most highly conserved
proteins throughout evolution, and so it was not unexpected that HMRef stains actin
in multiple cell lines derived from various species (Fig. 1f and supplementary Fig.
6a-c), including Vero and Cos7 cells, for which SiR-actin does not work well5. Cell
viability was not altered under the imaging conditions, but we found that the cell
shape was highly disrupted at a tenfold higher concentration of HMRef
(Supplementary Fig. 7). We also applied HMRef for imaging primary-cultured cells
and tissues. HMRef clearly visualized retrograde actin flow in migrating fish
keratocytes (Supplementary Fig. 8, and Supplementary Movie 1)10, as well as the
contractile ring dynamics of cleavage furrow ingression during cell division in
Drosophila wing disc (Fig. 1g, 1h). These results indicate that HMRef is a powerful
tool to visualize the actin cytoskeleton of various cells in living cells and organisms
in real time, without affecting the cell behaviors.
In the following, we utilized HMRef as a F-actin-binding scaffold to develop a
functional probe for F-actin manipulation. A technique that can ablate the actin
filament network with single-cell resolution would have great potential to better
define the forces involved in adhesion and migration of multicellular assemblies.
Although actin-targeting natural product inhibitors are useful tools in actin-related
cell biology2, their spatial resolution is limited. One approach to circumvent this
issue is the use of chromophore-assisted light inactivation (CALI), in which a suitable
ligand serves to direct a chromophore specifically to the protein of interest, followed
by light-induced release of reactive oxygen species to trigger spatiotemporally
controlled inactivation of target molecules in situ 11 12.
Aiming to apply this strategy to actin, we have developed a new small-molecular
CALI probe, namely GLIFin (Green Light-mediated Inactivator of F-actin, Fig. 2a) by
iodinating the xanthene core of HMRef in order to increase its singlet oxygen
generation by making use of the so-called internal heavy-atom effect.13, 14 We
confirmed the singlet oxygen-generating ability (Fig. 2b) and the F-actin-binding
ability (Supplementary Fig. 9) of GLIFin, and then tested the ability of GLIFin to
induce degradation of the actin cytoskeleton in cells upon laser irradiation (Fig. 2c,
2d). As expected, GLIFin mediated photoinactivation of the actin filaments of cells in
a light-power- and GLIFin-concentration-dependent manner (Supplementary Fig.
10), while causing no apparent damage to microtubules, which lie adjacent to actin
filaments15, at least under our experimental condition (Supplementary Fig. 11). F-
actin fragmentation was not induced by either HMRef, actin-binding fluorophore
(i.e., a much less efficient photosensitizer), or EosinY, a non F-actin-directing
photosensitizer (Fig 2d). It is noteworthy that actin fragmentation proceeded in the
dark on a time scale of an hour after irradiation (Supplementary Fig. 12 and
Supplementary note 3). As the path length of singlet oxygen is as short as <100 nm
in cells16, GLIFin could achieve single-cell-level spatial resolution for actin
fragmentation (Fig. 2e). We found that F-actin filaments recovered after a day (Fig.
2f), and cell viability was unaffected by GLIFin-mediated photoinactivation in the
concentration range of 300 nM or less (Supplementary Fig. 13).
We next applied GLIFin to epithelial cell monolayers (Fig. 2g and Supplementary
Fig. 14) where cell-cell interaction involving the actin cytoskeleton at adherens
junctions and actin-extracellular matrix (ECM) interplay mediate coordinative cell
movement and morphogenesis17. Migration measurements showed a severe
decrease in migration speed, in a light- and GLIFin-dependent manner (Fig. 2h),
without loss of cell viability (Supplementary Fig. 15). Notably, the effect of GLIFin-
mediated inactivation was relatively long-lasting, and the migration rate was
restored only after ~12 hr (Supplementary Notes 4). Interestingly, a limited
forward movement of cells into open space was frequently observed in non-
irradiated areas, where the cells formed a kind of “boundary layer”, even though the
space in front of actin-inactivated cells was still available (Fig. 2i). These
observations might reflect disorder of long-range interactions via the intracellular
actin network. Finally, we confirmed the applicability of GLIFin-mediated
inactivation to an in vivo model, the wing disk of Drosophila larvae (Supplementary
Fig. 16). Compared to the selective removal of cells by laser ablation, GLIFin-
mediated photoinactivation offers the advantage of low invasiveness. Specifically,
GLIFin allows flexible disruption of intracellular force transmission while leaving
cells alive and adhesive. We believe it will be a powerful tool in a variety of fields,
including the study of supracellular organization to generate force between leader
and follower cells during the cooperative movement of groups of cells18.
In present study, we have developed a new class of live cell visualization and
manipulation probes for F-actin that are membrane-permeable and highly selective
for F-actin at the single-cell level of resolution. We believe they complement existing
visualization/manipulation techniques in our endeavor toward comprehensive
understanding of actin function.
1. Pollard, T.D. & Cooper, J.A. Science 326, 1208-1212 (2009).
2. Peterson, J.R. & Mitchison, T.J. Cell Chem. Biol. 9, 1275-1285 (2002).
3. Wulf, E., Deboben, A., Bautz, F.A., Faulstich, H. & Wieland, T. Proc. Natl. Acad.
Sci. USA 76, 4498-4502 (1979).
4. Riedl, J. et al. Nat. Methods 5, 605-607 (2008).
5. Lukinavicius, G. et al. Nat. Methods 11, 731-733 (2014).
6. Asanuma, D. et al. Nat. Commun. 6, 6463 (2015).
7. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Proc. Natl. Acad. Sci. USA
97, 8206-8210 (2000).
8. Hell, S.W. & Wichmann, J. Opt. Lett. 19, 780-782 (1994).
9. Gustafsson, N. et al. Nat. Commun. 7, 12471 (2016).
10. Lee, J., Ishihara, A., Theriot, J.A. & Jacobson, K. Nature 362, 167-171 (1993).
11. Jay, D.G. Proc. Natl. Acad. Sci. USA 85, 5454-5458 (1988).
12. Jacobson, K., Rajfur, Z., Vitriol, E. & Hahn, K. Trends Cell Biol. 18, 443-450
(2008).
13. McClure, D.S. J. Chem. Phys. 17, 905-913 (1949).
14. Gorman, A. et al. J. Am. Chem. Soc. 126, 10619-10631 (2004).
15. C.Rodriguez, O. et al. Nat. Cell Biol. 5, 599-609 (2003).
16. Moan, J. J. Photochem. Photobiol. B, Biol. 6. 343-344 (1990).
17. Ladoux, B. & Mege, R.M. Nat. Rev. Mol. Cell Biol. 18, 743-757 (2017).
18. Vitorino, P. & Meyer, T. Genes Dev. 22, 3268-3281 (2008).
Figure 1. HMRef as a fluorescent probe for F-actin
(a) Structure of HMRef. (b) Confocal imaging of fixed and permeabilized HeLa cells
stained with HMRef (green) and Alexa FluorTM 647 phalloidin (red). Fixed cells were
incubated with 2U/mL Alexa FluorTM 647 phalloidin in DPBS for 30 min, and then
stained with 500 nM HMRef in DPBS for 2 hr. Scale bar, 20 μm. (c) Evaluation of F-
/G-actin-binding ability of HMRef by means of fluorescence polarization. Values are
mean ± S.D., n = 2 (F-actin, 1 nM) or 3 (others). (d) Stimulated emission depletion
(“STED”) imaging of living COS-7 cells stained with 1 μM HMRef. A confocal image
without STED (“confocal”) is shown for comparison. Scale bar, 20 μm. (e) Magnified
images and line profiles of HMRef-stained COS-7 cells. Line profiles show the
fluorescence signal along the long axes in the insets, and the fitted Gaussian curves
are also shown. Scale bar, 2 μm. (f) Confocal images of HMRef-loaded cells. 500 nM
HMRef, 30 min. Scale bar, 10 μm. (g) Schematic illustration of Drosophila wing disc.
In (h), the posterior-dorsal region of the wing disc is imaged (red square). (h) Live
imaging of Drosophila wing imaginal discs incubated with 500 nM HMRef. Selected
snapshots from a movie showing HMRef (green in top panels, gray in bottom panels)
and E-cad-mTagRFP (red in top panels, gray in middle panels). HMRef labels F-actin
along the cell-cell junction and the contractile ring during cytokinesis (cyan
arrowheads). Magenta asterisks indicate a dividing cell and its sister cells. Scale bar,
10 μm.
Figure 2. GLIFin (Green Light-mediated Inactivator of F-actin), a small
molecular CALI probe for F-actin
(a) Structure of GLIFin. (b) Representative luminescence spectra of 1O2 generated in
response to 508 nm laser illumination. ΦΔ - the quantum yield of 1O2 generation. Dye
concentration, 5 μM in PBS. (c) Schematic illustration of the protocol for F-actin
manipulation upon laser irradiation. (d) Effect of GLiFin-mediated light inactivation
of F-actin. Cells were incubated with growth medium containing 300 nM
probes/vehicle for 1 hr, followed by illumination with green light (27.0 mW/cm2 at
BP515-569 nm, 1 min), and F-actin was visualized with Alexa FluorTM 647. Scale bar:
10 μm (e) F-actin inactivation with single-cell resolution. Inactivation of a pre-
defined localized area (green dashed line) was done by illumination with an argon
laser (24.1 mW/cm2 at 514 nm, 1 min). After 1 hr, images were acquired. The green,
red and yellow windows show enlarged views of the indicated region. Scale bar, 100
μm. (f) Time-dependent recovery of F-actin inactivated with GLIFin. Cells were
incubated with growth medium containing 300 nM GLIFin for 1 hr, followed by
illumination with green light (22.6, 23.5 mW/cm2 at 514 nm, 1 min), and incubated,
fixed and repeatedly irradiated at the indicated time points. F-actin was visualized
with Alexa FluorTM 647. Scale bar, 20 μm. (g) Actin cytoskeleton of cells from locally
irradiated MDCK monolayer migration assay. Locally irradiated (24.1 mW/cm2)
MDCK cells were incubated for 12 hr, fixed and permeabilized, and then stained with
Alexa FluorTM 647 phalloidin. Scale bar, 200 μm. (h) Time-dependent gap closure.
Values are mean ± S.D., n = 2 (GLIFin 300 nM, light (-)) or 3 (others). (i) Asymmetric
locomotion in a cell monolayer triggered by local inactivation. An MDCK monolayer
was prepared as described above, followed by local light irradiation (24.1 mW/cm2,
indicated area) for 1 min. Scale bar, 200 μm.
Supplementary information
Supplementary Note 1-4.
Acknowledgement
This research was supported in part by AMED under grant number JP19gm0710008
(to Y.U.), by Japan Science and Technology Agency (PRESTO, JPMJPR17P1 to D.A.),
by MEXT/JSPS KAKENHI grants JP16H02606 and JP19H05632 (to Y.U.), 16H06574,
26750369, 17K14511 (to T.U.), 17H04764(to D.A.), 19H04935 (to Y.I.), and by JSPS
Core-to-Core Program, A. Advanced Research Networks (JPJSCCA20170007 to Y.U.).
Methods
Cell lines and culture.
All cell lines were grown in DMEM, RPMI 1640, or F-12 containing 10% fetal bovine
serum (FBS), 100 μg/ml penicillin and 100 μg/ml streptomycin (all reagents were
purchased from Life Technologies). All cell lines were maintained at 37 oC in 5% CO2.
Details of the cell sources and culture media are given in Supplementary note 4.
Imaging of the actin cytoskeleton stained with HMRef derivatives and/or
fluorescence-labeled phalloidin in fixed cells.
Cells were plated on glass-bottomed eight-chamber plates (Ibidi, 80826) and
incubated with growth medium for one day. The cells were washed with PBS 3 times,
then fixed and permeabilized with PBS containing 4% HCHO and 0.1% Triton-X for
10 min. PBS was removed, and the fixed cells were washed with PBS 3 times and
incubated in PBS containing 0.66% MeOH and 2 U mL Alexa FluorTM 647 phalloidin
(Thermo Fisher Scientific, A22287) for 30 min. For co-staining with HMRef
derivatives, the Alexa FluorTM 647 solution was replaced with PBS containing 500
nM or 1 μM HMRef derivatives. Unless otherwise mentioned, fluorescence images
were acquired with a confocal fluorescence microscope (TCS SP8, Leica) equipped
with a multi-wavelength argon and He-Ne laser, and an objective lens (HCX PL APO
CS 40x/1.25 Oil, Leica). The excitation and emission wavelengths were 488 nm/510-
550 nm for HMRef derivatives, and 633 nm/661-750 nm for Alexa FluorTM 647
phalloidin.
Fluorescence Polarization analysis.19
Actin (1 mg) from rabbit muscle (Sigma-Aldrich, A2522-1MG) was dissolved in 1 mL
of general actin buffer (Cytoskeleton Inc. Cat. # BSA01-010; ‘G-buffer’). The actin
solution was left on ice for 1 hr for depolymerization. Then 100 μL of actin
polymerization buffer (Cytoskeleton Inc. Cat. # BSA02-001; ‘P-buffer’) and 200 nmol
ATP in 2.0 μL H2O were added and mixed. After 2 hr of incubation for polymerization,
a dilution series of the F-actin solution was prepared. To each solution, HMRef (final
conc., 100 nM) was added and aliquots of the mixtures were pipetted into sample
tubes. The fluorescence polarization was measured using a BEACONTM 2000 (Pan
Vera).
Pyrene-labeled actin polymerization assay.
Polymerization assay was carried out as previously described5. G-buffer (5 mM
Tris-HCl (pH 8.0), 0.2 mM CaCl2 and 0.2 mM ATP) and P-buffer (100 mM Tris HCl, 20
mM MgCl2, 500 mM KCl, 10 mM ATP, 50 mM guanidine carbonate pH 7.5) were
prepared according to the manufacturer’s protocol. Briefly, a stock solution of 20
mg/mL (465 M) pyrene-labeled G-actin (Cytoskeleton Inc., cat. # AP05-A) was
46.5-fold diluted with G-buffer, then centrifuged for 15 min at 15000 rpm, 4oC, and
the supernatant was collected, providing 10 M working solution. The working
solution was left on ice for 1 hr for depolymerization and pipetted into wells of a
black 384-well assay plate (10 L/well) (Greiner Bio-one, cat 784900). 1 L of probe
solution in DMSO was added mixed, and incubated for 10 min, then 1 L of P-buffer
and 20 nmol ATP in 0.2 μL H2O were added and mixed. The time course of the
fluorescence (Ex/Em = 365 nm/407 nm) was measured using a Multilabel plate
reader (EnVision 2103, PerkinElmer).
Pyrene-labeled actin depolymerization assay.
Depolymerization assay was carried out as previously described5. Briefly, a 10 M
working solution of pyrene-labeled G-actin was prepared and pipetted into wells of
a black 96-well assay plate (40 L/well). 10 L of P-buffer and 20 nmol ATP in 0.2
μL H2O were added and mixed. The actin solution was incubated for 2 hr for
polymerization. Then, 1 L of probe solution in DMSO was added and mixed. After
incubation for 5 min, the actin solution was 5-fold diluted with 160 L G-buffer. The
time course of the fluorescence (Ex/Em = 365 nm/407 nm) was measured using a
Multilabel plate reader (EnVision 2103, PerkinElmer).
STED microscopy.7,8
COS-7 cells were cultured on glass-bottomed dishes (Matsunami Glass D11531H) at
37 °C in 5% CO2 in DMEM (Wako 045-30285) with 10% fetal bovine serum (SIGMA
172012), 2% L-glutamine solution (Wako 073-05391), 1% sodium pyruvate
solution (Wako 190-14881), and 1% penicillin-streptomycin mixed solution
(Nacalai 26253-84). The cells were gently washed twice with HEPES-buffered saline,
pH 7.4 (HBS) (25 mM HEPES, 115 mM NaCl, 2.5 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2,
25 mM glucose), and incubated at ambient temperature for 30 min in the dark in
HBS containing 1 M HMRef. Imaging was performed on a TCS SP8 STED 3X
microscope (Leica Microsystems) including a pulsed white-light laser for excitation,
a 592-nm depletion laser for STED, and a HyD detector. HMRef-stained cells were
observed with a 100x oil immersion objective (HC PL APO CS2 100x/1.40 OIL) in a
field of view of 8,192 x 8,192 pixels with a pixel size of 10 nm x 10 nm. The excitation
and emission wavelengths were 488 nm and 500-570 nm, respectively.
Live-cell imaging with HMRef derivatives.
Cells were plated on eight-chamber plates (Ibidi, 80826) and incubated for a day
before imaging, unless otherwise mentioned. Cells were incubated in growth
medium containing indicated concentrations of HMRef derivatives for 30 min, and
differential interference contrast (DIC) and fluorescence images were acquired with
a confocal fluorescence microscope (TCS SP8, Leica) equipped with an argon laser
and an objective lens (HCX PL APO CS 40x/1.25 Oil, Leica). The excitation and
emission wavelengths were 488 nm/510-550 nm for HMRef derivatives and 594
nm/615-750 nm for RFP. Scale bar, 100 m.
SRRF imaging.9
Dual-color SRRF imaging was performed on a spinning-disc confocal microscope.
HMRef and AlexaFluor647-phalloidin excitation was conducted with a 488 nm/150
mW diode laser (LM-488-150 Andor) and a 637 nm/140 mW diode laser (LM-637-
140, Andor), respectively. The two lasers were fiber-coupled (7 line laser combiner,
multi-mode x2, single-mode x1, LC-ILE-700-M2-S1, Andor) to a spinning disk
confocal unit (CR-DFLY505; Andor) equipped with a multi-band dichroic mirror
(DFly laser Dichroic for 405/488/561/640). The fluorescence was processed with
appropriate filter sets for HMRef (TR-DFLY-F525-050, Andor) and AlexaFluor647
(TR-DFLY-F700-075, Andor) to capture fluorescence images with a CCD camera
(iXion Life 888, Andor), driven by Fusion software (ver 2.0, Andor). Images were
taken using a 60× objective (APON60XOTIRF, NA:1.49, Olympus) mounted on an
inverted microscope (IX83, Olympus) equipped with Z-drift compensator (IX3-ZDC2,
Olympus).
CCK8 assay
HeLa cells were seeded in a plastic-bottomed 96-well plate (Greiner Bio-One,
655090) at a density of 7.6 x 104 cells per well. After 24 hr, the medium was
aspirated and replaced with fresh medium containing various concentrations of
probes (adjusted by diluting 10 mM DMSO stock solution). After incubation for ~20
hr, the medium was aspirated and replaced with medium containing 5% Cell
Counting Kit-8 (Dojindo, CK04). After further incubation for 1 hr, the absorbance at
405 nm was measured using a plate reader (EnVision 2103 Multilabel Reader,
PerkinElmer), to determine the cell viability. Values from the wells containing cells
without probe and without photoirradiation were taken as representing 100% living
cells, and values from wells without cells were taken as representing 100% dead
cells.
For GLiFin treatment, the cells were stained with GLiFin for 1 hr, followed by light
irradiation through a rodscope from a Xe light source, MAX301 (BP515-569 nm) for
1 min. The medium was replaced with 200 μL/well of fresh medium. After 20 hr,
CCK8 assay was performed as described above.
HMRef imaging of Fish Keratocytes
Keratocytes of Central American cichlids (Hypsophrys nicaraguensis) were cultured
in culture medium (Leibovitz’s medium: L-15, L5520: Sigma-Aldrich, St Louis, MO)
supplemented with 10% fetal calf serum (Nichirei, Tokyo, Japan) and
antibiotic/antimycotic solution (09366-44: Nacalai Tesque, Kyoto, Japan) as
previously described20. All methods were carried out in accordance with national
guidelines and the Regulation on Animal Experimentation at Yamaguchi University.
All experimental protocols were approved by Yamaguchi University Animal Use
Committee. Cells were treated with 0.5 g/L trypsin and 0.53 mM EDTA (trypsin-
EDTA, 32778-34: Nacalai Tesque) for 30–60 s to separate any cell-cell adhesions. The
single keratocytes ware treated with the culture medium containing 250 nM HMRef
for 10 min. Then, the medium was replaced with the culture medium containing
no probe. The migrating keratocytes were observed using an inverted microscope
(Ti; Nikon, Tokyo, Japan) equipped with a laser confocal scanner unit (CSU-X1;
Yokogawa, Tokyo, Japan) with a 100× objective lens (CFI Apo TIRF 100×H/1.49;
Nikon, Tokyo, Japan). The fluorescence images were detected using an EM CCD
camera (DU897; Andor, Belfast, UK).
HMRef time-lapse imaging of Drosophila wing disc
Drosophila melanogaster larvae expressing E-cadherin-mTagRFP21 were dissected
in Schneider’s medium (Thermo Fisher 21720024) containing 5% FBS (biowest,
s1810). The wing discs were cultured in Schneider’s medium in the presence of 500
nM HMRef on a 35 mm glass-based dish (IWAKI 3911-035). After incubation for 1
hr, time-lapse imaging was performed with an inverted confocal microscope (A1R;
Nikon) equipped with a 60×/NA1.2 Plan Apochromat water-immersion objective.
The excitation and emission wavelengths were 488 nm/500-550 nm for HMRef, and
561 nm/570-620 nm for E-cadherin-mTagRFP. Images were taken at 5 min interval
for 65 min at ~25 .
Image processing was performed by using ImageJ. Briefly, the HMRef and E-
cadherin signals on the adherens junction plane were extracted by using a custom-
made macro. The background signal was subtracted using the “subtract background”
command (r = 50) for the HMRef image.
UV-vis Absorption and fluorescence spectroscopy.
UV−visible absorption spectra were obtained on a Shimadzu UV-1800. Fluorescence
spectra were acquired with a Hitachi F7000. The slit width was 1 nm for both
excitation and emission. The photomultiplier voltage was 400 V. Relative
fluorescence quantum yields were obtained by comparing the area under the
emission spectra of the test samples with standard samples and were calculated
according to the following equation
ΦX / Φst = [Ast / AX][nX 2 / nst 2][DX / Dst]
where st = standard; x = sample; A = absorbance at the excitation wavelength; n =
refractive index; and D = area under the fluorescence spectra on an energy scale.
Optical properties of probes (1 μM) were examined in 0.1 M sodium phosphate
buffer containing 0.1% DMSO as a cosolvent. For determination of fluorescence
quantum efficiency (Φfl), fluorescein in 0.1 M aqueous NaOH (Φfl = 0.85) was used
as a standard22.
Singlet oxygen detection by near-infrared spectroscopy
Singlet oxygen was detected by measuring 1O2 luminescence at around 1270 nm
upon laser irradiation, using a near-infrared emission spectrometer (Fluorolog-3,
Horiba, Japan.). Probe solution (PBS containing 0.1% DMSO as a cosolvent) was
excited with monochromatic light (508 nm) and luminescence was recorded
between 1220-1340 nm in 5 nm steps. To calculate the quantum yield of 1O2
generation, the luminescence signal was integrated for 7 seconds for each
wavelength. The quantum yield was calculated by using Rose bengal in PBS as a
reference (0.75)23.
GLIFin-Mediated light inactivation of F-actin.
Cells were prepared as described above. They were incubated in growth medium
containing GLIFin for 1 hr, followed by light irradiation by using BP515-569 nm light
from a Xe light source, MAX301 (Asahi Spectra Co., Ltd., for global irradiation) or a
TCS SP8 (Leica, 514 nm, for local irradiation). In experiments involving an
incubation time of over 1 hr after irradiation, the medium was replaced with fresh
medium.
After GLIFin-mediated light inactivation of F-actin as described above, cells were
washed with PBS 3 times, then fixed and permeabilized with PBS containing 4%
HCHO and 0.1% Triton-X. After 10 min, the solution was aspirated. The fixed cells
were washed with PBS 3 times and blocked with 1% bovine serum albumin
(BSA)/PBS. After 30 min, the blocking solution was aspirated, and the fixed cells
were incubated in PBS containing 1% BSA, 0.66% MeOH, 2 U/mL Alexa FluorTM 647
phalloidin (Thermo Fisher Scientific, A22287), 3 μg mL anti-alpha tubulin antibody
conjugated with FITC (abcam, ab64503), and 3 μg/mL DAPI (Invitrogen, D1306) at
ambient temperature for 1 hr. The PBS was aspirated and replaced with fresh PBS.
Fluorescence imaging was done at 405 nm/430-465 nm for DAPI, 488 nm/510-550
nm for FITC (tubulin), and 633 nm/661-750 nm for Alexa 647 (F-actin).
GLIFin manipulation of Drosophila wing cells
The wing disc was dissected and mounted as described above and incubated with
Schneider’s medium containing 1 μM GLIFin and 5% FBS for 1 hr prior to the light
irradiation. To perform the light-mediated inactivation experiment, the wing disc
was irradiated for 1.5 min with 488-nm laser at 5% power. Non-irradiated wing
discs were used as a control. The control and irradiated wing discs were observed
at 5 min before and at 3.5 hr after the irradiation. To examine the effect of GLIFin
manipulation on the F-actin intensity, the wing discs were fixed at room
temperature for 30 min in PBS containing 4% paraformaldehyde. After washing
with PBS containing 0.1% Triton X-100, these preparations were incubated
overnight with Alexa FluorTM 647 phalloidin (1/1000, Thermo Fisher A22287). The
E-cadherin and phalloidin signals on the adherens junction plane were extracted as
described above.
Epithelial cell sheet migration assay.
MDCK cells (4.0 × 105 cells / mL) were plated on both sides of eight-chamber plates
(Ibidi, 80826 or 80206-G500) separated with 25 Culture-Inserts 2-well (Ibidi,
80209) and incubated in growth medium for one day. After removal of the medium
from the cells, GLiFin-mediated light inactivation of F-actin was carried out. DIC
images were captured with a confocal fluorescence microscope (TCS SP8, Leica).
Fluorescence images were acquired as described above (also see Supplementary
Fig 18).
Live-dead staining.
Live-dead staining was carried out according to the manufacturer’s protocol.
Briefly, cells were incubated in PBS containing 0.15% DMSO, 2 μM Calcein-AM
(Thermo Fisher Scientific, L3224), 2 μM ethidium homodimer-1 (Thermo Fisher
Scientific, L3224), and 2 μg/mL DAPI (Invitrogen, D1306). Fluorescence images,
(488 nm/ 510-570 nm for live, 561 nm/650-750 nm for dead) were acquired with
a confocal fluorescence microscope (TCS SP8, Leica) equipped with an argon laser
and an objective lens (10x/0.40 dry, Leica).
References
19. Lea, W.A. & Simeonov, A. Expert Opin. Drug Discov. 6, 17-32 (2011).
20. Okimura, C., Taniguchi, A., Nonaka, S. & Iwadate, Y. Sci. Rep. 8, 10615 (2018).
21. Pinheiro, D. et al. Nature 545, 103-107 (2017).
22. Paeker, C.A. & Rees, W.T. Analyst 85, 587-600 (1960).
.
probes
Takeru Takagi1, Tasuku Ueno*1, Keisuke Ikawa4, Daisuke Asanuma2, 5, Yusuke
Nomura1, Shin-nosuke Uno2, Toru Komatsu1, Mako Kamiya2, Kenjiro Hanaoka1,
Chika Okimura6, Yoshiaki Iwadate6, Kenzo Hirose2, 7, Tetsuo Nagano3, Kaoru
Sugimura4, and Yasuteru Urano*1, 2, 8
1Graduate School of Pharmaceutical Sciences, 2Graduate School of Medicine, and 3Drug Discovery Initiative, The University of Tokyo, Tokyo 113-0033, Japan 4Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto,
606-8501, Japan 5JST PRESTO, Tokyo, 102-0075, Japan 6Faculty of Science, Yamaguchi University, Yamaguchi, 753-8512, Japan. 7International Research Center for Neurointelligence, The University of Tokyo, 113-
0033 Tokyo, Japan 8CREST (Japan) Agency for Medical Research and Development (AMED), Tokyo 100-
0004, Japan
visualization probes 21
Note 2. Synthesis of HMRef derivatives. 23
Note 3. Consideration of the mechanism of manipulation with GLIFin. 35
Note 4. Information on cell lines. 36
References 37
Supplementary Figure 1. Competitive binding assay of HMRef vs. actin-binding
natural products (phalloidin and jasplakinolide).
(a, b) Fixed and permeabilized HeLa cells were stained with 1 μM HMRef in DPBS in
the presence of phalloidin (a) or jasplakinolide (b) for 1 hr. Scale bar; 20 μm.
3
assay.
(a) In vitro pyrene-labeled actin polymerization assay. (b) Rate constants obtained
from (a) by fitting the fluorescence increase based on a “plateau followed by
exponential increase”5. Values are mean ± S.D. N = 3. (c) In vitro pyrene-labeled actin
depolymerization assay. (d) Fraction of F-actin in (c), evaluated in terms of
normalized fluorescence intensity at 120 min. Values are mean ± S.D. N = 3 or 4. The
fluorescence intensity was normalized to the initial fluorescence intensity. Probe
concentrations; 1 μM.
The structure of HMRef and eight HMRef derivatives are shown.
5
Supplementary Figure 4 (1/2). Confocal images of live or fixed HeLa cells
loaded with HMRef derivatives.
(Live) HeLa cells were incubated with growth medium containing 500 nM (2) or 1
μM (others) HMRef derivatives shown in Supplementary Figure 3 for 1 hr. Scale
bar; 20 μm. (Fixed) Fixed and permeabilized HeLa cells were stained with Alexa
FluorTM 647 phalloidin, and incubated with 500 nM (2) or 1 μM (others) HMRef
derivatives for 2 hr. Scale bar; 20 μm.
6
Supplementary Figure 4 (2/2). Confocal images of live or fixed HeLa cells
loaded with HMRef derivatives.
(Live) HeLa cells were incubated with growth medium containing 1 μM (others)
HMRef derivatives shown in Supplementary Figure 3 for 1 hr. Scale bar; 20 μm.
(Fixed) Fixed and permeabilized HeLa cells were stained with Alexa FluorTM 647
phalloidin, and incubated with 1 μM (others) HMRef derivatives for 2 hr. Scale bar;
20 μm.
fluctuations (SRRF) imaging.
(a, b) Confocal (a) and super-resolution radical fluctuations (SRRF) (b) images of
HeLa cells stained with 1 μM HMRef. The SRRF image was reconstructed from 100
raw confocal images. HeLa cells were incubated with 1 μM HMRef in growth medium
for 1 hr, and images were captured with an Andor Dragonfly confocal microscopy
system. (c, d) Enlargements of the region outlined by the boxes in (a) and (b) are
shown in (c) and (d), respectively.
8
Supplementary Figure 6. HMRef staining in various cell lines.
(a) Confocal images of various cell lines stained with HMRef. Cells were stained with
500 nM HMRef for 30 min. Scale bar, 10 μm. (b) Confocal imaging of fixed and
permeabilized cells stained with HMRef (green) and Alexa FluorTM 647 phalloidin
(Red). HMRef, 0.5 μM in DPBS for 2 hr. Scale bar, 10 μm. (c) Wide-field (enlarged)
fluorescence images of various cell lines stained with HMRef (500 nM, 30 min). Scale
bar, 50 μm.
Supplementary Figure 7. Cytotoxicity of HMRef.
(a) Wide-field fluorescence images of SKOV3-RFP cells stained with the indicated
concentrations of HMRef in growth medium containing 0.5% DMSO and incubated
for 2 hr. Scale bar, 50 μm. (b) Average cell size in (a). Values are mean ± S.E. n =
42 (control = 0 μM) or 39 (HMRef 10 μM). Statistical significance was calculated
by application of the two-tailed paired Student’s t-test, *** : P < 0.001. (c) Cell
viability assay. HeLa cells were cultured with growth medium containing various
concentrations of HMRef for 21 hr, and cell viability was determined by means of a
Cell Counting Kit-8 (4.7% v/v). A cell viability of 100% was assigned to the positive
control (vehicle (0.1% DMSO-treated cells). Values are mean ± S.E. (N = 8). P value:
one-way analysis of variance (ANOVA).
10
Supplementary Figure 8. Observation of actin dynamics in a migrating
keratocyte.
(a) A typical image among 25 cells observed. Scale bar, 10 µm. (b) Kymographs
constructed from image strips, with width 8 pixels (= 1 µm) (yellow rectangles
labeled in (a)) from consecutive images taken at 2-s intervals. Movements of actin
flow are indicated with a yellow arrow. Also see Supplementary Movie 1.
Supplementary Figure 9. In cellulo evaluation of the F-actin-binding ability of
GLIFin.
Competitive binding assay of HMRef vs. GLIFin. HeLa cells were incubated with
HMRef (500 nM) in the presence of various concentrations of GLIFin (indicated) for
1 hr, and confocal images were captured. Scale bar, 20 μm.
11
mediated light inactivation of F-actin.
HeLa cells were incubated with GLIFin (indicated) in growth medium for 1 hr,
followed by light irradiation (indicated; 515-569 nm) for 1 min, incubated for 1 hr,
and then fixed and permeabilized. F-actin was visualized with Alexa FluorTM 647
phalloidin. Scale bar, 50 μm.
12
GLiFin-mediated light inactivation of F-actin.
Confocal images of F-actin, microtubules and nuclei. HeLa cells were incubated
with GLIFin (200 nM, 1 hr), followed by light irradiation (27.0 mW/cm2 at 515-569
nm for 1 min), then incubated for 1 hr, and subsequently stained with 2 U/mL Alexa
FluorTM 647 phalloidin (red), 3 μg/mL FITC-labeled tubulin antibody (green), and 3
μg/mL DAPI (blue). Scale bar, 20 μm.
13
GLIFin-mediated light inactivation.
(a) Schematic illustration of the protocol of F-actin manipulation. (b) Confocal
images of HeLa F-actin visualized with 2 U/mL Alexa FluorTM 647 phalloidin. HeLa
cells were incubated with or without 300 nM GLIFin in growth medium for 1 hr, then
irradiated (18.9 mW/cm2 at 515-569 nm) for 1 min, incubated, fixed at the indicated
time point, and permeabilized.
Supplementary Figure 13. Viability assay for GLIFin-treated cells.
(a) HeLa cells cultured on a 96-well microplate were incubated with probes
(photosensitizer, indicated) for 1 hr, followed by *light irradiation (22.0 mW/cm2 at
515-569 nm) for 1 min, and incubation for 20 hr. After addition of CCK8 (4.7% v/v)
and further incubation for 1 hr, the absorbance at 405 nm was measured with a
microwell plate reader. The cell count in the positive control (vehicle (0.1% DMSO)-
treated cells) was taken as representing 100% viability. Values are mean ± S.E. (N =
4). *only at ‘light (+)’ conditions. (b) Extracted data comparing non-stimulated cells
and cells irradiated after incubation with GLIFin (indicated). Statistical significance
of differences was calculated by application of the two-tailed paired Student’s t-test.
** : P < 0.01, *** : P < 0.001.
monolayer cells.
(a) Schematic illustration of the protocol for making ‘actin graffiti’. MDCK cells in the
light green area were irradiated (24.1 mW/cm2) through pre-positioned masks for
1 min. Scale bar 200 μm. (b) Obtained actin graffiti. After irradiation, the cells were
incubated for 4.5 hr, fixed and permeabilized, and then stained with 2 U/mL Alexa
FluorTM 647 phalloidin. A ‘face’ pattern is shown. (c) Other examples: ‘play’, ‘stop’,
‘pause’, and ‘fast forward’ patterns.
16
after sheet migration assay.
The cell monolayer sheet used in Fig. 2i was subjected to live-dead staining. The cells
were incubated with 2 μM Calcein-AM (green live cell marker) and 2 μM ethidium
homodimer-1 (red dead cell marker) for 1 hr, and then images were captured.
Similar results were obtained in 3 independent experiments. Scale bar, 200 μm.
17
and the loss of phalloidin signal in Drosophila wing disc.
(a) Live imaging of non-irradiated and irradiated wing discs (upper panels and
bottom panels, respectively) expressing E-cad-mTagRFP at 5 min before and at 3.5
hr after irradiation. Bottom-right: Light-induced activation of GLIFin resulted in
enlargement of the cell area. (b) Fixed imaging of non-irradiated and irradiated
wing discs (upper panels and bottom panels, respectively). Images of E-cad-
mTagRFP (gray in left panels and red in right panels) and phalloidin (gray in middle
panels and blue in right panels) are shown. The phalloidin signal was under the
detection limit in the irradiated wing disc. Scale bar, 10 μm.
18
imaging with HMRef due to environment-sensitive change of the
spirocyclization equilibrium.
(a) Enlarged image of Figure 1b ‘HMRef’. Scale bar. 10 μm. (b) Transverse profiles
of locations corresponding to the region lined in (a). (c) Magnified image of
Supplementary Figure 4 ‘2 Fixed-HMRef derivatives’. Scale bar. 10 μm. (d)
Transverse profiles of locations corresponding to the region outlined in (c).
Comparison of environmental-sensitive structural change of HMRef (e) and 2 (f).
Absorbance (g) and fluorescence (h) spectra of 1 μM HMRef in 100 mM PBS (pH 7.4)
and 1,4-dioxane mixture containing 0.1% DMSO as a co-solvent. Excitation
wavelength was 430 nm. Absorbance (i) and fluorescence (j) spectra of 1 μM 2 in
100 mM PBS (pH 7.4) and 1,4-dioxane mixture containing 0.1% DMSO as a co-
solvent. Excitation wavelength was 430 nm.
19
experiment.
MDCK cells were seeded to culture-inserts 2 well (ibidi, ib80209) on glass-bottomed
dish and incubated for 1 day. After the cells have grown to confluency, epithelial
monolayer migration was prompted by lifting off the confinement. The cell
monolayer was treated with DMSO (0.10 %) or GLIFin (indicated) for 1 hr, irradiated
light for 1 min, and incubated for 1 hr, and then the media was replaced into fresh
growth medium. Cell images were acquired from 1 hr after light irradiation and
subsequently every 3 hr.
Supplementary Table 1. Colocalization analysis of HMRef derivatives and F-
actin (phalloidin).
* - For the whole co-staining images of ‘Supplementary Figure 4’, Pearson’s
correlation coefficient and Spearman’s correlation coefficient were quantified using
ImageJ plugins “Coloc2” without setting the threshold. **- For determination of
relative fluorescence quantum efficiency (Φfl), fluorescein in 0.1 M NaOH aq. (Φfl =
0.85) was used as a standard. N.D. – Not determined.
Supplementary Movie 1.
A typical migrating keratocyte loaded with HMRef. The movie depicts the same cell
as that shown in Supplementary Figure 8 and is shown 40 times faster than real
time.
21
cytoskeleton visualization probes
In F-actin co-staining imaging experiments, F-actin binding of Alexa-labelled
phalloidin (2 U/mL) was significantly decreased in the presence of either 1 M
HMRef or 1 M 2, while other derivatives at this concentration did not effectively
block F-actin binding of Alexa-labeled phalloidin (2 U/mL). Given that a co-staining
fluorescence image could be obtained by using 500 nM HMRef or 2 with 2 U/mL
Alexa-labeled phalloidin, it is likely that the IC50 values of those compounds under
these conditions are in the submicromolar range. Thus, it seems plausible that the
hydroxymethyl group is not required for high-affinity actin binding.
It is also interesting to note that HMRef and 2 provide slightly different F-actin
images, albeit with similar affinity (Supplementary Fig. 17a-d). HMRef shows
bright fluorescence on F-actin with little background or off-target fluorescence,
whereas 2 has a relatively higher background, leading to a loss of image contrast.
We consider that the low background of HMRef is at least partially due to the
environmental sensitivity of the dye. As shown in Supplementary Fig. 17g, 17h,
the absorbance and fluorescence of HMRef were highly sensitive to solvent polarity
due to the effect on the spirocyclic equilibrium, resulting in deconjugation of the
rhodol fluorophore (Supplementary Fig. 17e, 17f). On the other hand, 2 is highly
fluorescent in 0-100% dioxane/H2O (Supplementary figure 17i, 17j). In cells,
lipophilic dyes such as HMRef and 2 can accumulate in the endomembrane, but
HMRef, though not 2, would exist in non-fluorescent and colorless spirocyclic form
in the hydrophobic internal membrane, which reduces off-target fluorescence.
Summary of structure-activity relationship for F-actin binding of HMRef derivatives,
see below.
22
In viable cells, many of the derivatives did not stain F-actin well, even though their
F-actin binding was confirmed in fixed cells. This is probably because of
inappropriate cellular distribution, i.e. small-molecular dyes accumulate in the
endomembrane and/or cellular compartment, and their distribution to the cytosol,
where F-actin filaments are formed, is limited. Among the compounds tested in
this study, HMRef was the best for actin staining of viable cells.
• Me substitution does not decrease F-actin binding (H ~ Me), yet significantly affects cellular distribution in viable cells.
• Rhodamine replacement slightly reduces F-actin binding, and significantly affects cellular distribution in viable cells.
• Electron-withdrawing groups increase F-actin binding. (CH2CF3 > CH2CH2CF3 >> Et)
• Bulky group increases F-actin binding, carboxylate group nearly abolishes F-actin binding. (CH2OH ~ Me >> H >> COO-)
Benzene moiety
Xanthene moiety
Materials and general information.
Reagents and solvents were of the best grade available, supplied by Tokyo Chemical
Industries, Wako Pure Chemical, Sigma-Aldrich, and Kanto Chemical Co., and were
used without further purification. NMR spectra were recorded on a JEOL JNM-LA300
instrument at 300 MHz for 1H NMR and at 75 MHz for 13C NMR, a JEOL JNM-LA400
instrument at 400 MHz for 1H NMR and at 100 MHz for 13C NMR or a JEOL JNM-
ECZ400S instrument at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. All
chemical shifts (δ) are reported in ppm relative to internal standard
tetramethylsilane (δ = 0.0 ppm), or relative to the signals of residual solvent CDCl3
(7.26 ppm for 1H, 77.16 ppm for 13C), CD3OD (3.31 ppm for 1H, 49.00 ppm for 13C),
or acetone-d6 (2.04 ppm for 1H). Coupling constants are given in Hz. Mass spectra
(MS) were measured with a JEOL JMS-T100LC AccuToF (ESI). Preparative HPLC was
performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an
HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2015 or
FP-2025, JASCO) , or a SNAP Ultra 25 g (Biotage) using an IsoleraTM One (Biotage).
UPLC analyses were performed on a Waters Acquity UPLC (H class)/QDa quadrupole
MS analyzer or Acquity UPLC (H class)/Xevo TQD quadrupole MS/MS analyzer
equipped with an Acquity UPLC BEH C18 column (Waters). Eluent C (H2O
containing 0.1% formic acid) and eluent D (80% acetonitrile and 20% H2O
containing 0.1 % formic acid) were used for UPLC analyses.
Abbreviations
24
HMRef, 5 (= HMRpf), and 6 (= HMRet) were synthesized as previously described6.
Synthesis of 1
Scheme S1. Synthetic route of compound 1.
a) Benzyl bromide, K2CO3, DMF, rt, 85%, b)1) NaBH4, THF, MeOH, rt, 2) Tf2O, pyridine,
CH2Cl2, rt, 74% in 2 steps. c) 1) 2,2,2-trifluoroethylamine hydrochloride, Cs2CO3,
xantphos, Pd2(dba)3, toluene, microwave, 100oC, 2) chloranil, CH2Cl2, rt ,3) 10%Pd/C,
CH2Cl2, MeOH, 4) chloranil, CH2Cl2, rt, 50% in 4 steps.
Compound 1a
mL round-bottomed flask equipped with a magnetic
stirring bar was charged with PhTG (170 mg, 0.59
mmol). To this flask, anhydrous DMF 15 mL, K2CO3 (98
mg, 0.71 mmol) and benzyl bromide (84 μL, 0.71
mmol) were added under vigorous stirring. The
reaction mixture was stirred overnight at ambient temperature, then poured into
100 mL water, and extracted with AcOEt 3 times. The combined organic layer was
washed with water and brine, and dried over anhydrous Na2SO4. The solution was
concentrated in vacuo to dryness. The residue was purified by chromatography over
silica gel using dichloromethane/methanol (97/3) as the eluent to give 1a (190 mg,
85%) as an orange solid. 1 H-NMR (400 MHz, chloroform-D) δ 7.57-7.55 (m, 3H),
7.42-7.32 (m, 7H), 7.15 (d, J = 9.1 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 7.01 (d, J = 2.7 Hz,
1H), 6.84 (dd, J = 8.9, 2.5 Hz, 1H), 6.57 (dd, J = 9.8, 2.1 Hz, 1H), 6.43 (d, J = 1.8 Hz, 1H),
5.18 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 70.7, 101.4, 105.9, 113.7, 114.7, 118.2,
127.5, 128.5, 128.8, 129.4, 129.5, 129.8, 139.0, 130.8, 132.9, 135.5, 149.2, 154.6,
158.9, 163.2, 185.6. HRMS (ESI+) m/z calcd for C12H19O3, [M+H]+, 379.13342; found
379.13261 (-0.81 mDa).
magnetic stirring bar was charged with 1a (190 mg,
0.50 mmol). Anhydrous THF 30 mL was added, and
then NaBH4 (76 mg, 2.0 mmol) was added under
vigorous stirring. Next, MeOH 10 mL was slowly
added and the reaction mixture was stirred for 2 hr
at ambient temperature. The resulting solution was neutralized with 2 N HCl aq. 100
mL and extracted with CH2Cl2 twice. The combined organic layer was washed with
brine, dried over anhydrous Na2SO4 and concentrated in vacuo to dryness. The
residue was purified by column chromatography over silica gel using
dichloromethane/methanol (95/5) as the eluent to give a colorless amorphous solid
(this product is light-sensitive and gradually oxidizes in air) together with some by-
product. The amorphous solid was taken up in anhydrous CH2Cl2 and charged into a
50 mL two-necked, round-bottomed flask equipped with a magnetic stirring bar and
a septum. The solution was stirred at -94 °C in a cooling bath with liquid N2/acetone
for 5 min. Next, Tf2O (123 L, 0.72 mmol) and dry pyridine (59 L, 0.72 mmol)
were sequentially added via a syringe. The reaction flask was wrapped with
aluminum foil and stirring was continued overnight on the cooling bath, which was
allowed to warm gradually to room temperature. The reaction mixture was diluted
with CH2Cl2 100 mL, washed with NH4Cl aq and brine, and dried over anhydrous
Na2SO4. The organic layer was concentrated in vacuo and the residue was purified
by column chromatography over silica gel using n-hexane/dichloromethane (6/4)
as the eluent to give 1b as a colorless solid (190 mg, 74% in 2 steps). 1H NMR (400
MHz, CDCl3): 5.05 (s, 2H), 5.19 (s, 1H), 6.66 (dd, J = 2.5, 8.3 Hz,1H), 6.74 (d, J = 2.4
Hz, 1H), 6.90 (m, 2H), 7.05 (d, J = 2.0 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 7.15-7.24 (m,
3H), 7.26-7.44 (m, 7H). 13C NMR (100 MHz, CDCl3): 43.4, 70.2, 102.3, 109.8, 111.7,
115.9, 120.4 (q, J = 315Hz); 123.5, 125.1, 127.0, 127.4, 128.1, 128.5, 128.6, 128.9,
130.4, 131.2, 136.6, 145.7, 148.3, 150.9, 151.6, 158.8. MS (EI+): m/z 312, (M+).
26
stirring bar was charged with Pd2(dba)3 (38 mg,
20 mol%), xantphos (64 mg, 30 mol%), and
Cs2CO3 (748 mg, 2.5 mmol). The reaction vessel
was flushed with argon gas, and a solution of 1b
(190 mg, 0.38 mmol) in anhydrous toluene was
added. Under vigorous stirring, 2,2,2-trifluoroethylamine hydrochloride salt (250
mg, 1.85 mmol) was added to the vessel at once, and the mixture was stirred in a
microwave reactor at 100 °C for 20 hr. The resulting solution was diluted with AcOEt
(100 mL), washed with distilled water and brine, and dried over anhydrous Na2SO4.
The organic layer was concentrated in vacuo, and the residue was dissolved in
anhydrous CH2Cl2 30 mL. Chloranil (183mg) was added, and the mixture was stirred
for 1 hr at ambient temperature. The reaction solution was diluted with CH2Cl2 100
mL and filtered. The filtrate was washed with brine 1 x 50 mL and concentrated in
vacuo to dryness. The residue was subjected to column chromatography on silica gel
using dichloromethane/methanol (92/8) as the eluent. The obtained orange solid
was dissolved in 10 mL CH2Cl2 and 20 mL MeOH, and the solution was loaded into a
50 mL round-bottomed flask equipped with a magnetic stir bar. After addition of a
spatula tip amount of 10% Pd/C, the mixture was stirred under a hydrogen
atmosphere at ambient temperature for 3 hr. After the reaction, the mixture was
filtered, and the filtrate was concentrated in vacuo. The crude intermediate was
dissolved in anhydrous CH2Cl2 50 mL and then chloranil (90 mg, 0.37 mmol) was
added. The mixture was stirred vigorously at ambient temperature for 3 hr, then
diluted with CH2Cl2 100 mL, and filtered. The filtrate was washed with water and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo to dryness. The
residue was purified by column chromatography over silica gel using
dichloromethane/methanol (97/3) as the eluent to afford 1 as a dark red solid (40
mg, 50% in 4 steps). 1H NMR (400 MHz, CD3OD): 4.01 (q, J = 9.5 Hz, 2H), 6.50 (d, J
= 2.2 Hz, 1H), 6.60 (dd, J = 2.2 Hz, 9.5 Hz, 1H), 6.83 (dd, J = 2.2 Hz, 9.5Hz, 1H), 6.95
(d, J = 2.2 Hz, 1H), 7.19 (d, J = 9.5 Hz, 1H), 7.22 (d, J = 9.5 Hz, 1H), 7.42-7.45 (m, 2H),
7.63-6.65 (m, 3H). 13C NMR (100 MHz, CD3OD): 43.4 (q, J = 32.1 Hz), 96.1, 102.6,
114.9, 117.6, 120.3, 123.4, 126.2, 128.7, 129.4, 130.2, 131.6, 132.0, 131.6, 132.0,
132.2, 157.6, 158.7, 159.2, 171.3. HRMS (ESI+): m/z calcd for C21H15F3NO2, [M+H]+,
370.10549; found, 370.10852 (+3.03 mDa). UPLC (eluent; C/D = 95/5 to 5/95 in 4
min): tR = 2.5 min.
2,2,2-trifluoroethylamine hydrochloride,
3% in 2 steps.
described24. To a suspension of TG (79.4 mg, 0263
mmol) in CH2Cl2 (20 μL), triethylamine (100 μL,
0.717 mmol) was added. The reaction solution
was stirred at 0 oC for 10 min, then a solution of
trifluoromethanesulfonic acid anhydride (125 μL,
0.743 mmol) in dichloromethane (3 mL) was slowly added. The resulting mixture
was stirred for 30 min, then concentrated, and the crude residue was roughly
purified by column chromatography over silica gel using
dichloromethane/methanol (90/10) as the eluent. The obtained compound (49.8
mg, 0.115 mmol), Pd2(dba)3.CHCl3 (8.9 mg, 0.00860 mmol), xantphos (10.0 mg,
0.0172 mmol), Cs2CO3 (152.5 mg, 0.468 mmol) and 2,2,2-trifluoroethylamine
hydrochloride salt (62.4 mg, 0.460 mmol) were dissolved in toluene (10 mL) under
an argon atmosphere. The resulting mixture was stirred at 100 oC for 20 hr, then
allowed to cool to room temperature, diluted with dichloromethane, and filtered
through a pad of Celite. The filter cake was washed with dichloromethane. The
combined filtrate and washing were concentrated and the residue was purified by
column chromatography over silica gel using dichloromethane/methanol (95/5) as
the eluent. Further purification by HPLC (eluent, 8% acetonitrile /0.1% TFA aq. (0
min) to 80% acetonitrile /0.1% TFA aq. (15 min); flow rate = 5.0 mL/min) afforded
2 (3.07 mg, 3% in 2 steps) as a red powder. 1H NMR(300 MHz, CD3OD) : 2.05 (s,
3H), 4.34 (q, 2H, J = 8.8 Hz ), 7.10 (dd, 1H J = 2.2, 8.8 Hz), 7.16 (dd, 1H, J = 2.2, 8.8 Hz),
7.23 (d, 1H, J = 2.2 Hz), 7.27-7.31 (m, 2H), 7.35-7.42 (m, 2H), 7.48-7.60 (m, 3H).
HRMS (ESI+) : Calcd. for [M+H]+, 384.12114; found, 414.11845 (-2.68 mDa). UPLC
(eluent; C/D = 95/5 to 5/95 in 4 min): tR = 2.6 min.
28
2,2,2-trifluoroethylamine hydrochloride,
Compound 3
mmol) in dichloromethane (30 mL) was added
triethylamine (181 mL, 1.30 mmol). The reaction
solution was stirred at 0 oC for 10 min, then a
solution of trifluoromethanesulfonic acid
anhydride (110 mL, 0.653 mmol) in
dichloromethane (3 mL) was slowly added. The resulting mixture was stirred for 30
min, then concentrated, and the crude residue was subjected to column
chromatography on silica gel using dichloromethane/methanol (95/5) as the eluent.
A part of the obtained compound (15.6 mg from 66.0 mg, yellow oil),
Pd2(dba)3.CHCl3 (3.5 mg, 0.00338 mmol), xantphos (3.9 mg, 0.0674 mmol), Cs2CO3
(54.7 mg, 0.168 mmol) and 2,2,2-trifluoroethylamine hydrochloride salt (22.8 mg,
0.168 mmol) were dissolved in toluene (10 mL) under an argon atmosphere. The
resulting mixture was stirred at 100 oC for 20 hr, then allowed to cool to room
temperature, diluted with dichloromethane, and filtered through a pad of Celite. The
filter cake was washed with dichloromethane. The combined filtrate and washing
were concentrated and the residue was purified by HPLC (eluent, 8% acetonitrile
/0.1% TFA aq. (0 min) to 80% acetonitrile /0.1% TFA aq. (15 min); flow rate = 5.0
mL/min) to give 3 (0.45 mg, 0.55% in 2 steps) as a red powder. 1H NMR(300
MHz,ACETN-d6) : δ 4.05 (q, 2H, J = 9.0 Hz), 6.57-6.62 (m, 4H), 6.70-6.75 (m, 2H), 7.28
(d, 1H, J = 8.1 Hz), 7.70-7.83 (m, 2H), 7.97 (d, 1H, J = 7.2 Hz). HRMS (ESI+) : Calcd. for
[M+H]+,414.09532; found, 414.09992 (+4.60 mDa). UPLC (eluent; C/D = 95/5 to
5/95 in 4 min): tR = 2.8 min.
29
Scheme S4. Synthetic route of compound 4.
a) 1) H2O, microwave, 210oC, 2) MOMCl, K2CO3, DMF, rt, 3) TBSCl, imidazole, DMF, rt,
12% in 3 steps, b) 1) MOMCl, K2CO3, DMF, rt, 2) DIBAL-H, THF, rt, 3) TBSCl, imidazole,
DMF, rt, 81% in 3 steps, c) 1) sec-BuLi, THF, -78oC, 2) 4b, rt, 3) H2O, rt, 4) TBAF, THF,
80oC, 31% in 4 steps. d) 1) Tf2O, pyridine, CH2Cl2, rt, 2) 2,2,2-trifluoroethylamine
hydrochloride, Cs2CO3, xantphos, Pd2(dba)3.CHCl3, toluene, 80oC, 3) TFA, CH2Cl2, rt,
19% in 3 steps.
19.7 mmol) was diluted with 13 mL H2O and
stirred at 210oC for 3 hr. The reaction mixture
was cooled to ambient temperature and
filtered. The residue was washed with ice-cooled H2O and hexane and then dried in
a vacuum oven. The dried residue (4.68 g) and K2CO3 (5.12 g, 37.1 mmol) were
dissolved in N,N-dimethylformamide (20 mL). To the stirred solution, chloromethyl
methyl ether (1.43 mL, 19.0 mmol) was added and stirring was continued for 24 hr.
The reaction mixture was diluted with H2O (80 mL) and extracted with ethyl acetate
twice. The organic layer was washed with H2O 5 times, dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure. The residue was washed
with ice-cooled H2O, n-hexane, and ethyl acetate and then dried in a vacuum oven. A
part of the residue (366 mg from 1.10 g, flesh-colored solid) and imidazole (180 mg,
2.65 mmol) were dissolved in N,N-dimethylformamide (2 mL). To the stirred
solution, tert-butyldimethylchlorosilane (240 mg, 1.59 mmol) was added and
stirring was continued under an argon atmosphere at ambient temperature for 2 hr.
The reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate.
The organic layer was washed with brine, dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The crude residue was purified
30
by column chromatography on silica gel (dichloromethane : n-hexane = 80 : 20 to
dichloromethane : methanol = 99 : 1) to give 4a (309 mg, 12 % in 3 steps). 1H-NMR
(400 MHz, chloroform-D) δ 8.25-8.14 (m, 2H), 7.04 (d, J = 2.3 Hz, 1H), 6.99 (dd, J =
8.9, 2.5 Hz, 1H), 6.87-6.79 (m, 2H), 5.26 (s, 2H), 3.49 (s, 3H), 0.99 (s, 9H), 0.26 (s, 6H) 13C-NMR (100 MHz, chloroform-D) δ 175.7 162.2 161.5 157.8 128.3 117.7 116.6
116.5 113.9 107.4 103.1 94.4 77.3 56.5 25.6 18.4 -4.3
Compound 4b
5.89 mmol) and K2CO3 (3.80 g, 28.1 mmol) in N,N-
dimethylformamide (20 mL), chloromethyl methyl ether (1.40
mL, 18.6 mmol) was added. The reaction mixture was stirred
for 23 hr, diluted with H2O (80 mL), and extracted with ethyl
acetate twice. The organic layer was washed with H2O 5 times, dried over anhydrous
sodium sulfate, filtered, and concentrated under reduced pressure. The residue was
taken up in dichloromethane (20 mL) and the solution was cooled to 0oC. It was
stirred for 10 min, then diisobutylaluminium hydride (16 mL, 1 mol/L in hexane, 16
mmol) was added, and stirring was continued for 2 hr. The reaction was quenched
with saturated Rochelle salt solution (80 mL) and the whole was extracted with
dichloromethane 3 times. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The
residue and imidazole (1.20g, 17.6 mmol) were taken up in N,N-dimethylformamide
(20 mL), then tert-butyldimethylchlorosilane (2.40 g, 15.8 mmol) was added. The
reaction mixture was stirred for 19 hr under argon atmosphere, diluted with
saturated NaHCO3 solution (80 mL), and extracted with ethyl acetate twice. The
organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered,
and concentrated under reduced pressure. The crude residue was purified by
column chromatography on silica gel (n-hexane) to give 4b as a colorless oil (1.45 g,
81%). 1H-NMR (400 MHz, CHLOROFORM-D) δ 7.41-7.31 (m, 2H), 6.92 (dd, J = 8.1,
1.3 Hz, 1H), 4.70 (s, 2H), 2.32 (s, 3H), 0.97 (s, 9H), 0.13 (s, 6H) 13C-NMR (100 MHz,
CHLOROFORM-D) δ136.9 137.2 131.8 129.0 128.5 117.8 64.7 26.1 21.3 18.5 -5.2
31
Compound 4c
To a stirred solution of 4b (462 mg, 1.47 mmol) in
THF (10 mL) at -78oC, sec-butyllithium (2.5 mL, 1.0
mol/L in THF, 2.50 mmol) was added. The reaction
mixture was stirred for 10 min, then 4a (110 mg,
0.286 mmol) diluted in THF (5 mL) was slowly
added, and stirring was continued at ambient
temperature under an argon atmosphere for 1 hr.
The reaction was quenched with H2O (25 mL) and extracted with ethyl acetate twice.
The organic layer was washed with brine, dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was diluted with
THF (20 mL). To the stirred solution, tetra-n-butylammonium fluoride (4.5 mL, 1
mol/L in THF, 4.50 mmol) was added. The reaction mixture was stirred at 80oC for
2 hr, then diluted with ethyl acetate (80 mL), and washed with brine. The organic
layer was concentrated under reduced pressure. The crude residue was purified by
HPLC (eluent, 10% acetonitrile/0.1% TFA aq. (0 min) to 80% acetonitrile/0.1% TFA
aq. (15 min) to 100% acetonitrile/0.1% TFA (45 min)) to give 4c (33.1 mg, 31%) as
a yellow powder. 1H-NMR (400 MHz, chloroform-D) δ 7.15 (s, 1H), 7.07 (d, J = 7.8 Hz,
1H), 6.94-6.81 (m, 3H), 6.77 (d, J = 7.8 Hz, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.64 (s, 1H),
6.51 (d, J = 8.2 Hz, 1H), 5.22 (s, 2H), 5.17 (s, 2H), 3.46 (s, 3H), 2.41 (s, 3H) 13C-NMR
(100 MHz, chloroform-D) δ 157.8 157.0 151.6 141.9 139.4 138.1 130.1 129.9 129.5
123.8 121.1 118.2 116.5 112.5 112.0 103.3 102.8 94.4 83.9 77.4 71.7 56.2 21.5
HRMS (ESI+) Calcd. for [M+H]+, 377.13890; found, 377.13463 (-4.27 mDa)
Compound 4
mmol) and pyridine (0.150 mL, 1.86 mmol) in
dichloromethane (10 mL), a solution of
trifluoromethanesulfonic anhydride (0.100 mL,
slowly added at 0oC. The reaction mixture was
stirred at ambient temperature for 3 hr, and
subjected to column chromatography on silica gel (dichloromethane). The crude
product (29.0 mg, pale yellow oil), trifluoroethylamine hydrochloride (45.2 mg,
0.335 mmol), cesium carbonate (146.0 mg, 0.448 mmol),
tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct (10.5 mg, 0.0101
32
mmol), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (11.4 mg, 0.0197
mmol) were dissolved in anhydrous toluene (10 mL). The reaction mixture was
stirred at 80oC under an argon atmosphere for 40 hr, then diluted with ethyl acetate
(40 mL), washed with brine and H2O, dried over anhydrous sodium sulfate, and
filtered. The filtrate was concentrated under reduced pressure. The residue was
diluted with dichloromethane (4 mL) and acidified with trifluoroacetic acid (1 mL).
The resulting solution was stirred at ambient temperature for 20 hr, then H2O (9
mL) was added and dichloromethane was evaporated off under reduced pressure.
The remaining solution was purified by HPLC (eluent, 10% acetonitrile/0.1% TFA
aq. (0 min) to 80% acetonitrile/0.1% TFA aq. (15 min) to 100% acetonitrile/0.1%
TFA (45 min)) to give 4 (4.6 mg, 19%) as a red powder. 1H-NMR (400 MHz,
methanol-D + NaOD in D2O) δ 7.18 (s, 1H), 7.08 (d, J = 7.2 Hz, 1H), 6.70 (d, J = 7.6 Hz,
1H), 6.62 (d, J = 8.0 Hz, 1H), 6.48 (d, J = 8.8 Hz, 1H), 6.44 (d, J = 2.8 Hz, 1H), 6.39-6.36
(m, 2H), 6.32 (dd, J = 8.8 Hz, 2.0 Hz), 5.09 (s, 1H), 3.81 (q, J = 9.2 Hz, 2H), 2.41 (s, 3H), 13C-NMR (100 MHz, methanol-D + NaOD in D2O) δ 170.2 153.7 153.6 149.8 143.5
140.8 138.9 130.9 130.9 130.1 124.8 121.9 117.3 115.6 111.2 109.9 105.3 99.6 86.7
71.6 46.2 21.4 HRMS (ESI+) Calcd. for [M+H]+, 414.13170; found, 414.13145 (-0.25
mDa). UPLC (eluent; C/D = 95/5 to 5/95 in 4 min): tR = 2.6 min.
33
compound 7.
benzophenone imine, Cs2CO3, xantphos,
aq. , THF, rt, 37% in 3 steps.
Compound 7
mmol) in dichloromethane (10mL), pyridine
(28.2 mL, 0.350 mmol) was added. The
reaction mixture was stirred at 0 oC for 10 min,
then a solution of trifluoromethanesulfonic
acid anhydride (29.4 mL, 0.175 mmol) in
dichloromethane (3 mL) was added dropwise. The resulting mixture was stirred for
30 min, then concentrated, and the residue was subjected to column
chromatography over silica gel using dichloromethane as the eluent. The product
(35.9 mg, white solid), Pd2(dba)3.CHCl3 (8.7 mg, 0.00826 mmol), xantphos (6.3 mg,
0.0109 mmol) and Cs2CO3 (290 mg, 0.890 mmol) were dissolved in toluene (10 mL)
under an argon atmosphere. Benzophenone imine (51 mL, 0.304 mmol) was added,
and the resulting mixture was stirred at 100oC for 20 hr, then allowed to cool to room
temperature, diluted with dichloromethane, and filtered through a pad of Celite. The
filter cake was washed with dichloromethane. The combined filtrate and washing
were concentrated and the residue was dissolved in THF (10 mL). To this solution
was added 2N HCl aq. (1 mL), and the mixture was stirred at ambient temperature
for 1 hr, then concentrated. The residue was purified by HPLC (eluent, 8%
acetonitrile/0.1% TFA aq. (0 min) to 80% acetonitrile/0.1% TFA aq. (15 min); flow
rate = 5.0 mL/min) to give 7 (14.2 mg, 37% in 3 steps) as a red powder. 1H NMR (300
MHz, CD3OD+NaOD in D2O) : d 3.84 (q, 2H, J = 8.8), 5.21 (s, 2H) , 6.42-6.47 (m, 3H),
6.53 (d, 1H, J = 2.2 Hz), 6.58-6.65 (m, 2H), 6.83 (d, 1H, J = 7.3 Hz), 7.28-7.42 (m, 3H) :
Calcd for [M+H]+, 399.13204; found, 399.12810 (-3.93mmu). UPLC (eluent; C/D =
95/5 to 5/95 in 4 min): tR = 2.3 min.
34
compound 8
a) 2,2,2-trifluoroethylamine
hydrochloride, Cs2CO3.CHCl3,
Cs2CO3 (152.5 mg, 0.468 mmol) and
2,2,2-trifluoroethylamine hydrochloride salt (62.4 mg, 0.460 mmol) were dissolved
in toluene (10 mL) under an argon atmosphere. The resulting mixture was stirred at
100 oC for 20 hr, then allowed to cool to room temperature, diluted with
dichloromethane, and filtered through a pad of Celite. The filter cake was washed
with dichloromethane. The combined filtrate and washing were concentrated and
the residue was purified by column chromatography over NH silica using
dichloromethane/methanol (95/5) as the eluent. Further purification was
implemented by HPLC (eluent, 8% acetonitrile /0.1% TFA aq. (0 min) to 80%
acetonitrile /0.1% TFA aq. (15 min); flow rate = 5.0 mL/min) to give 8 (9.0 mg, 16%)
as a red powder. 1H NMR(300 MHz, CD3OD) : d 4.24 (q, 4H, J = 9.6, h), 7.01 (dd, 2H, J
= 2.1, 9.0 Hz), 7.15-7.21 (m, 4H) , 7.43 (dd, 1H ,J = 1.5, 7.5 Hz), 7.84(m, 2H), 8.35 (dd,
1H, J = 1.5, 7.2Hz). 13C NMR(75 MHz, CD3OD) : d 45.46(q, J = 33.8Hz), 97.5, 116.2,
117.6, 124.8, 127.5, 131.1, 131.7, 132.2, 132.3, 132.6, 134.0, 135.7, 159.6, 160.0,
168.2. HRMS (ESI+) : Calcd for [M+H]+, 495.11434; found, 495.11300 (-1.33 mDa).
UPLC (eluent; C/D = 95/5 to 5/95 in 4 min): tR = 2.8 min.
35
a) I2, HIO3, ethanol, H2O, rt, 31%.
GLIFin
mL). To the stirred solution, iodic acid (11.7 mg,
0.0665 mmol) in H2O (1 mL) was slowly added at
ambient temperature for 10 min. The reaction
mixture was stirred for 10 min, diluted with
ethyl acetate (45 mL), washed with brine for 3 times and with H2O twice, dried over
anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced
pressure. The residue was purified by HPLC (eluent, 20 % acetonitrile /0.08 % TFA
aq. (3 column volume) to 100 % acetonitrile (9 column volume)), desalinated with
Sep-Pak Vac 35cc (10 g) C18 Cartridges (Waters), and taken up in methanol. This
solution was concentrated under reduced pressure to give GLIFin (12.5 mg, 31 %)
as a red solid. 1H-NMR (400 MHz, methanol-D4) δ 7.35 (q, J = 7.0 Hz, 2H), 7.25 (t, J =
7.1 Hz, 1H), 6.82 (d, J = 7.3 Hz, 1H), 6.60 (d, J = 8.7 Hz, 1H), 6.55 (d, J = 2.3 Hz, 1H),
6.43 (d, J = 8.7 Hz, 1H), 6.39 (dd, J = 8.7, 2.3 Hz, 1H), 6.34 (d, J = 8.7 Hz, 1H), 5.30-5.13
(2H), 3.82 (q, J = 9.3 Hz, 2H). 13C-NMR (100 MHz, methanol-D4) δ 168.6 152.4
150.7 148.6 144.8 139.2 129.4 128.5 127.9 127.6 123.8 120.3 114.3 114.1 109.9
109.0 98.4 85.6 78.9 70.4 44.7(q, J = 33.4 Hz) HRMS (ESI+) Calcd. for [M+H]+,
526.01270; found, 526.01273 (+0.03 mDa). UPLC (eluent; C/D = 95/5 to 5/95 in 4
min): tR = 3.0 min.
Note 3. Consideration of the mechanism of manipulation with GLIFin
Unlike direct physical cutting of actin in laser ablation, GLIFin-mediated actin
fragmentation is a relatively slow, time-dependent process (Supplementary Fig.
12), which may suggest the involvement of an enzymatic process involving the
oxidatively modified actin molecule. It is not clear whether this fragmentation is
due to acceleration of the depolymerization of oxidized filaments, or a result of
36
inhibition of the actin polymerization process, or both. Nevertheless, our results
show that GLIFin can mediate selective photoinactivation of actin filaments. Also,
given that the effect of GLIFin-mediated inactivation was relatively long-lasting,
and the decreased migration rate took at least 12 hr to recover (Fig. 2h), it seems
plausible that the damaged actin molecules cannot be re-used for actin filament
network assembly, and that accumulation of newly expressed actin molecules is
required for recovery of cell motility.
Note 4. Information on cell lines.
HeLa cells, purchased from American Type Culture Collection (ATCC), were cultured
in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 11885-084) containing 10%
fetal bovine serum (FBS), 100 μg/ml penicillin and 100 μg/ml streptomycin (all
reagents were obtained from Life Technologies). U2OS cells, obtained from ATCC,
were cultured in DMEM containing 10% FBS and 100 μg/ml penicillin and 100
μg/ml streptomycin. HK-2 cells were a gift from Dr. Masayuki Murata (The University
of Tokyo, Department of Life Sciences); they were cultured in Ham’s F-12 Nutrient
Mix (F-12. Gibco, 11765-054) containing 10% FBS and 100 μg/ml penicillin and 100
μg/ml streptomycin. HEK293T cells, obtained from DSMZ, were cultured in DMEM
containing 10% FBS and 100 μg/ml penicillin and 100 μg/ml streptomycin, and
293T (ACC 635). African green monkey kidney normal cell line Vero (JCRB0111)
cells were obtained from the Japanese Collection of Research Bioresources (JCRB)
Cell Bank; they were cultured in DMEM containing 10% FBS and 100 μg/ml
penicillin and 100 μg/ml streptomycin. Cos-7 cells, obtained from RIKEN Cell Bank,
were cultured in DMEM containing 10% FBS and 100 μg/ml penicillin and 100
μg/ml streptomycin. Colon-26 cells, obtained from RIKEN Cell Bank, were cultured
in RPMI 1640 (Gibco, 11875-093) containing 10% FBS and 100 μg/ml penicillin and
100 μg/ml streptomycin. NIH/3T3 cells, purchased from ATCC, were cultured in
DMEM containing 10% FBS and 100 μg/ml penicillin and 100 μg/ml streptomycin.
NRK-52e cells, obtained from ATCC, were cultured in DMEM containing 10% FBS
and 100 μg/ml penicillin and 100 μg/ml streptomycin. PC-12 cells, obtained from
RIKEN Cell Bank, were cultured in RPMI 1640 (Gibco, 11875-093) containing 10%
FBS and 100 μg/ml penicillin and 100 μg/ml streptomycin. MDCK cells, obtained
from ATCC, were cultured in DMEM containing 10% FBS and 100 μg/ml penicillin
and 100 μg/ml streptomycin. CHO-K1 cells, purchased from ATCC, were cultured in
37
F-12 containing 10% FBS and 100 μg/ml penicillin and 100 μg/ml streptomycin.
SKOV3 cells, purchased from ATCC, were cultured in DMEM containing 10% FBS and
100 μg/ml penicillin and 100 μg/ml streptomycin. SKOV3 cells stably expressing
RFP were prepared as previously described26.
References
24 Urano, Y. et al. J. Am. Chem. Soc. 127, 4888-4894 (2005).
25 Grimm, J. B. & Lavis, L. D. Org. Lett. 13, 6354-6357 (2011).
26 Iwatate, R. J., Kamiya, M. & Urano, Y. Bioconjug. Chem. 22, 1696-1703 (2016).
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