Development of xanthene dyes containing arylacetylenes: The role of acetylene linker and substituents on the aryl group

Article abstract of DOI:10.1016/j.tet.2018.05.019

Fluorescent dyes possessing a variety of arylacetylenes at the 9-position of a xanthene skeleton were synthesized and their optical properties were investigated. The π system effectively expanded over the xanthene skeleton and the aryl group through the triple bond. Starting from the emission wavelength (λ_em) of 9-methyl xanthene 20 in basic DMSO solution at 536 nm, the emission wavelengths gradually shifted to the red region for methylacetylene 17 (λ_em = 600 nm), phenylacetylene 5 (λ_em = 636 nm), and p-CF₃-phenylacetylene 11 (λ_em = 660 nm). On the basis of these data, we estimated the substituent effects for the red shift on the emission wavelength and rationally explained the results by DFT calculations. Furthermore, potential applicability of these fluorescent dyes to cell staining was exemplified.

Full text of DOI:10.1016/j.tet.2018.05.019

Accepted Manuscript
Development of xanthene dyes containing arylacetylenes: The role of acetylene linker
and substituents on the aryl group
Ryo Hirata, Ayame Torii, Kenichi Kawano, Shiroh Futaki, Ayumi Imayoshi, Kazunori
Tsubaki
PII:
S0040-4020(18)30538-6
10.1016/j.tet.2018.05.019
TET 29526
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 March 2018
Revised Date: 2 May 2018
Accepted Date: 7 May 2018
Please cite this article as: Hirata R, Torii A, Kawano K, Futaki S, Imayoshi A, Tsubaki K, Development of
xanthene dyes containing arylacetylenes: The role of acetylene linker and substituents on the aryl group,
Tetrahedron (2018), doi: 10.1016/j.tet.2018.05.019.
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ACCEPTED MANUSCRIPT
Development of xanthene dyes containing arylacetylenes:
- The role of acetylene linker and substituents on the aryl group -
Ryo Hirata,a Ayame Torii,a Kenichi Kawano,b Shiroh Futaki,b Ayumi Imayoshi,a Kazunori Tsubaki a*
a. Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo,
Sakyo-ku, Kyoto 606-8522, Japan.
b. Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Graphical Abstract
em = 636 nm
O
= 34%
em = 536 nm
= >98%
O
O
O
64 nm
O
36 nm
O
O
O
O
24 nm
em = 600 nm
= 61%
O
O
em = 660 nm
= 24%
O
Keywords: xanthene dyes; fluorescein; phenylacetylene
Abstract
Fluorescent dyes possessing a variety of arylacetylenes at the 9-position of a xanthene
skeleton were synthesized and their optical properties were investigated. The π system effectively
expanded over the xanthene skeleton and the aryl group through the triple bond. Starting from the
emission wavelength (λem) of 9-methyl xanthene 20 in basic DMSO solution at 536 nm, the emission
wavelengths gradually shifted to the red region for methylacetylene 17 (λem = 600 nm),
phenylacetylene 5 (λem = 636 nm), and p-CF3-phenylacetylene 11 (λem = 660 nm). On the basis of
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these data, we estimated the substituent effects for the red shift on the emission wavelength and
rationally explained the results by DFT calculations. Furthermore, potential applicability of these
fluorescent dyes to cell staining was exemplified.
Introduction
Xanthene-based fluorescent dyes represented by fluorescein (1) 1 have been widely used in
the fields of molecular biology and medicinal chemistry.2 Fluorescein and/or xanthene-based
fluorescent dyes usually show emission at approximately 510 nm owing to the use of the xanthene
skeleton as a common fluorophore. 3 To avoid overlap with the auto fluorescence of living organisms
such as tryptophan and heme, many attempts have been reported to shift the emission wavelength of
fluorescent dyes into the red or near infrared regions. In particular, the conversion of the oxygen at
4
5
the 10-position of xanthenes to silicon or phosphorus has been shown to be effective for these
purposes and widely investigated.
We have also been studying fluorescent dyes that show emission in the red/near infrared
region based on xanthene frameworks by alternative approaches (Figure 1). First, we synthesized
dinaphthofluorescein 2, which features an expanded π-conjugate system, by the simple addition of
benzene rings to both sides of fluorescein.6 Compound 2 showed emission at 790 nm as expected;
however, this molecule featured quite a low solubility in organic solvents and water with a low
fluorescence quantum yield (Φ ≤ 0.1%). On the basis of these results, we have tried to develop new
fluorescent molecules that have a molecular weight comparable to that of fluorescein, but show
absorption and emission at longer wavelengths than those of fluorescein. We recently developed
7
8
V-shaped xanthene dyes 3 and 4 , in which a xanthene skeleton and an aryl group are connected
with an ether linkage, and the aryl group is incorporated into the π-system of the xanthene skeleton.
The V-shaped 3 showed emission at wavelengths of 550 nm, reflecting a 40 nm red shift compared
with that of fluorescein.
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Figure 1. Structures of fluorescent dyes 1–4.
In this manuscript, we will demonstrate an alternative approach, which involves
connecting the aryl group to the xanthene skeleton through a triple bond, to achieve large red shifts
in both the absorption and emission wavelengths. Fluorescein and its derivatives generally consist of
a xanthene skeleton and an aryl group at 9-position, which although orthogonal to xanthene system
does subtly interact. However, the acetylene linkages between the two moieties are in the same plane
and expansion of the π system might be expected. Although, four similar fluorescent dyes
connecting aryl groups and (pseudo)xanthene skeletons with acetylene moieties have been reported
9
in patents and papers 10, only phenyl and mesityl groups were investigated as the aryl groups
(Figure 2). We believe that more extensive research on different substituents effects on the aryl
groups and the role of acetylene linker could be useful, together with computational studies. In this
study, we synthesized a variety of acetylene bridged xanthene dyes and revealed their photophysical
properties, including substituent effects on the aryl group and solvatochromism, for applications to
imaging of living cells.
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HO
O
O
HO
O
O
5
6
Cl
N
Cl
N
N
Si
N
7
8
em = 705 nm, = 14%
em = 738 nm, = 7%
Figure 2. Four reported (pseudo)xanthene dyes with arylacetylene moieties.
Syntheses of acetylene bridged xanthene dyes 5 and 10–17
First, the acetylene bridged xanthene dyes 5 and 10–17 were synthesized according to a
reported procedure (Scheme 1, method A).9 Thus, the arylacetylenes were treated with n-BuLi to
prepare corresponding lithium acetylides. These were then reacted with TBS protected xanthone 9 11
to give intermediate A, followed by removal of the two TBS groups under HF-pyridine conditions to
afford the desired acetylene bridged xanthene dyes 5, 10, and 11 12 in yields of 67%, 83%, and 21%,
respectively. However, in the case of a combination of lithium p-F-phenylacetylide and 9, a trace
amount of desired 12 was obtained, and deprotected xanthone 18 and TBS acetylene 19 were
isolated in yields of 78% and 23%, respectively. Thus, the lithium acetylide predominantly attacked
the silicon atoms of the protecting TBS group. These data indicate that the nucleophilicity of lithium
acetylide possessing an electron withdrawing substituent on the aryl group was reduced, and its soft
character was increased. To improve the selectivity and reactivity of lithium acetylide, anhydrous
cerium chloride was added to the solution to generate the corresponding cerium acetylide in situ,
according to Imamoto’s procedure.13 The cerium acetylide reacted smoothly with the carbonyl group
on compound 9, and the following deprotection of the two TBS groups of the phenolic hydroxy
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groups was performed under acetic acid conditions to afford the desired acetylene bridged xanthenes
11–17 in moderate to high yields (Method B). Owing to the low yield of the 2-pyridiyl acetylene
compound 15 (35%) obtained with the use of method B, a combination of LDA and CeCl3 was used
to obtain 15 in 65% yield (Method C). The yields of the products and the applied synthetic methods
are summarized in Table 1.
Scheme 1. Synthesis of acetylene linked dyes 5 and 10-17.
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Table 1. Yields and applied methods.
product
R
entry
Method
yield (%)
1
2
5
10
11
C6H5
A
A
A
B
A
B
B
B
B
C
B
B
67
p-OMe-C6H4
p-CF3-C6H4
83
3
21
4
53
tracea
5
12
p-F-C6H4
6
60
7
13
14
15
m-F-C6H4
o-F-C6H4
2-pyridyl
69
8
68
9
35
10
11
12
65
16
17
TMS
Me
58
65
18
19
were isolated as by-products.
a) Compounds
O
and
F
HO
O
OH
TBS
19
(23%)
18
(78%)
Optical properties of the acetylene bridged xanthene dyes 5, and 10–17
With the nine xanthene dyes (5, and 10–17) in hand, we investigated their optical
properties. First, the simplest compound 5 was selected as a representative of the nine compounds,
the pH response of compound 5 was investigated. Titration UV absorption spectra of compound 5
are shown in Figure 3a. The UV-vis spectrum of compound 5 showed sharp absorption at 608 nm
under basic conditions (10 equiv. of tetrabutylammonium hydroxide) in DMSO. As acetic acid was
added to the solution, the absorption at 608 nm gradually decreased and absorptions at 369 and 504
nm increased with isosbestic points at 386 and 536 nm.
These data suggested that compound 5 featured two forms (anionic and neutral forms)
depending on the solution pH. The fluorescent quantum yields of compound 5 were measured under
neutral (Φ = 4 %) and basic (Φ = 34 %) conditions. The sharp absorption of the UV-vis spectrum
and high fluorescent quantum yield indicated that compound 5 showed higher performance under
basic conditions. Therefore, the optical properties of other compounds 10–17 were examined under
basic conditions.
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Figure 3. UV-vis titration of compound 5 with acid, and UV-vis absorption and FL spectra of
anionic 5: (a) UV-vis absorption spectra for conversion of neutral 5 to corresponding anionic
material. (b) UV-vis absorption and fluorescence spectra of anionic 5. Conditions: [5] = 1.0 × 10−5 M
in DMSO at 25 °C.
Figure 3b shows the absorption and fluorescence spectra of the basic compound 5.
Measurements were performed in the presence of 10 equiv. of TBAOH in DMSO solution. The
absorption and fluorescence spectra mirrored each other. The maximum emission wavelength (λem)
was 636 nm and the fluorescence quantum yield (Φ) was 34%, with a Stokes shift of 28 nm. Spectral
features of compounds 10–17 are shown in the Supplementary Material.
The optical properties (maximum absorption wavelength (λabs), molar absorption
coefficient (ε), excitation wavelength (λex), maximum emission wavelength (λem), fluorescence
quantum yield (Φ), and the Stokes shift of the synthesized compounds 5, and 10–17, together with
those of 6-hydroxy-9-methyl-3H-xanthene-3-one (20) 14,15 as a reference compound, are summarized
in Table 2.
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Table 2. Optical properties of compounds 5, 10–17 and 20 (anionic form) in DMSO 25 °C.
Compound
5 (p-H)
Stokes shift (nm)
λabs (nm)
608
ε (λabs.
)
λex (nm)
607
λem (nm)
636
Φ (%)a
34
27,000
61,000
40,000
60,000
45,000
53,000
38,000
72,000
76,000
79,000
36,000
28
25
38
29
33
31
36
21
20
14
43
10 (p-OMe)
11 (p-CF3)
12 (p-F)
602
601
627
71
622
620
660
24
607
607
636
55
13 (m-F)
14 (o-F)
614
615
647
34
615
615
646
38
15 (2-pyr)
16 (TMS)
17 (Me)
620
620
656
32
602
602
623
52
580
579
600
61
522
520
536
>98
20
11 (p-CF3) b
552
552
595
5
a
Based on a fluorescein in 0.1 M NaOH aq (Φ = 98%). b The UV-vis absorption and FL spectra were measured in 1 mM aqueous NaOH
solution.
On the basis of the maximum emission wavelengths of the compounds in Table 2, the
effects of substituents on increasing the emission wavelength were estimated. Thus,
6-hydroxy-9-methyl-3H-xanthene-3-one (20), which simply had a methyl group at the 9-position
showed an emission wavelength at 536 nm, whereas compound 17, possessing a methylacetylene
group, showed emission at 600 nm. Therefore, the substituent effect of the acetylene bond was
calculated to be approximately 64 nm. Furthermore, when methylacetylene of compound 17 was
converted to phenylacetylene 5, the emission wavelength shifted from 600 to 636 nm, such that the
substituent effect of the phenyl group was estimated to be approximately 36 nm. Notably, the
substituent effect of the acetylene bond was greater than that of the phenyl group on the xanthene
derivatives.
Next, we considered the substituent effects on the terminal aromatic rings. In Figure 4a,
the normalized fluorescent spectra of the selected compounds are shown. The λem of compound 5 (H
at the para position), compound 10 (p-OMe), and compound 11 (p-CF3) were 636, 627, and 660 nm,
respectively. Thus, introducing an electron donating substituent caused a blue shift of the emission
wavelength, and addition of an electron withdrawing substituent caused a red shift. Furthermore, in a
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comparison of the regioisomeric compounds 12 (p-F), 13 (m-F), and 14 (o-F), the λem of compound
12 (p-F) was comparable to that of compound 5 (p-H). Therefore, introducing fluorine at the para
position showed no substituent effect on the emission wavelength. The maximum emission
wavelengths of 13 (m-F) and 14 (o-F) were at 647 and 646 nm, respectively. These data indicate that
the fluorine at meta and ortho positions acted as an electron withdrawing substituent.
The relationship between the maximum emission wavelengths and Hammett’s substituent
constants (σ) 16, 17 is shown in Figure 4b. A quite high correlation (R = 0.98) was observed between
them, suggesting that it is possible to predict the emission wavelength of compounds by applying
this relationship.
(b) Hammett ( ) - em plot
(a) FL spectra
627 660
y = －15.473 + 0.02434x
10
5
12
13
11
R = 0.98324
11
0.4
13
0.2
0
12
5
－0.2
10
630
600
700
800
640
em (nm)
650
Wavelength (nm)
5
10 16
-
(c) Photographs of dyes and
16
10
12
5
13
14
15
15
11
16
10
12
5
13
14
11
Figure 4. (a) Normalized fluorescent spectra of compounds 5 and 10–13. (b) Relationship between
the maximum emission wavelengths and Hammett’s substituent constants. (c) Photographs of dyes
(1.0 × 10−5 M in DMSO) obtained under white light (top) and 365 nm light (bottom).
Pictures of compounds 5, and 10–16 under white light and black light (365 nm) are also
shown in Figure 4c. Pink to blue coloration were observed under white light, and red emission was
found under 365 nm irradiation.
Next, we investigated the solvatochromism of compound 11 (p-CF3), which showed the
longest emission wavelength among the nine compounds. The normalized FL spectra of compound
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11 in various solvents under basic conditions are depicted in Figure 5a. The λem in FL spectra were
shifted to longer wavelength in the order: MeOH, chloroform, acetonitrile, acetone, DMSO, and
THF. A quite high correlation (R = 0.97) was confirmed between the maximum emission wavelength
18
of compound 11 in various solvents and the acceptor number (Figure 5b). The acceptor number
(AN) is one solvent parameter that indicates the electron acceptability of the solvent. Thus, the
solvatochromism of compound 11 can be explained by solvation. In MeOH, which has a high AN,
the anionic compound 11 in the ground state was solvated by hydrogen bonding and the HOMO was
stabilized. Therefore, the HOMO-LUMO gap increased and the UV-vis absorption and FL spectra
were shifted to shorter wavelengths. Conversely, in non-protic and low AN solvents, such as
acetonitrile, acetone, DMSO, and THF, the stabilization of anionic species of compound 11 in the
ground state was low. Thus, negligible shifts were observed in the UV-vis and FL spectra.
Photographs of the solutions under white light and 365 nm irradiation are shown in Figure 5c. The
color of the solution under white light changed from magenta to blue, and red emission was detected
for all solutions under 365 nm irradiation. The optical properties of compound 11 in various solvents
are summarized in Table 3. The solvatochromism regarding the maximum emission wavelength
ranged from 610 nm (MeOH) to 672 nm (THF), quantum yields were approximately 13–24%, and
the Stokes shift was in the range of 38–44 nm.
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(b) AN - em plot
(a) FL of anionic 11
610 672
MeOH
CHCl3
MeCN
Acetone
DMSO
THF
y = 352.64－0.51175x
R = 0.97481
40
30
20
10
MeOH
DMSO
CHCl3
MeCN
Acetone
THF
600
700
800
600
620
640
em (nm)
660
Wavelength (nm)
(c) Photographs of compound 11 in various solvents
MeOH CHCl3 MeCN acetone DMSO THF
Figure 5. (a) Normalized emission spectra of compound 11 in various solvents, (b) relationship
between λem and acceptor number (AN), (c) photographs of compound 11 (1.0 × 10−5 M in various
solvents) obtained under white light (top) and 365 nm light (bottom).
Table 3. Optical properties of anionic compound 11 in various solvents at 25 °C.
Solvent
MeOH
CHCl3
MeCN
Acetone
DMSO
THF
Stokes shift (nm)
λabs (nm)
571
λex (nm)
570
λem (nm)
610
Φ (%)a
22
39
41
43
39
38
44
598
599
639
24
610
612
653
19
621
619
660
17
622
620
660
24
607/628
629
672
13
a Based on a fluorescein in 0.1 M NaOH aq (Φ = 98%).
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Computational studies of the xanthene dyes 19
DFT calculations at the B3LYP/6-31+G(d,p) level of theory indicated that the most stable
conformation of compound 5 featured a perfect planner structure between the xanthene skeleton and
the aryl group owing to no steric repulsion between the two moieties apart from the acetylene linker
(Figure 6). The dihedral angle composed of four carbons (showed in blue in figure 6) was estimated
to be 0.0°. Therefore, orbital interactions were expected between the xanthene skeleton and the aryl
group through the acetylene linkage.
Figure 6. Most stable conformation of compound 5 obtained by DFT calculations at the
B3LYP/6-31+G(d,p) level.; Front view (left) and side view (right).
Figure 7 shows the HOMOs, LUMOs, and energy gaps for the anionic forms of 20, 17, 10,
5, and 11, respectively, with the observed and calculated absorption wavelengths.
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OMe
H
CF3
Me
Me
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
anionic 20
anionic 17
anionic 10
anionic 5
anionic 11
－2.16 eV
LUMO
－2.51 eV
－2.64 eV
－2.71 eV
－2.85 eV
Ecal (eV)
2.96 eV
2.66 eV
2.53 eV
2.47 eV
2.36 eV
abs.cal (nm)
(nm)
445 nm
522 nm
491 nm
580 nm
519 nm
602 nm
531 nm
608 nm
560 nm
622 nm
abs.exp
HOMO
－5.12 eV
－5.17 eV
－5.16 eV
－5.18 eV
－5.21 eV
Figure 7. Energy diagrams and HOMOs-LUMOs for the anionic 20, 17, 10, 5 and 11 in DMSO
obtained by DFT calculations at the B3LYP/6-31+G(d,p) level and observed and estimated λabs.
Results of all calculations show that the HOMO is located on the xanthene skeleton, and
the LUMO is spread over the whole molecule. Therefore, the arylacetylene moiety and/or
substituents on the aryl group exert a greater influence on the level of LUMO than that of the
HOMO. Comparing compound 20 with compound 17, the LUMO is stabilized by approximately
0.35 eV through the addition of the acetylene moiety. A comparison of compounds 17 and 5
indicates that the LUMO is stabilized by approximately 0.20 eV through the incorporation of a
benzene ring. Moreover, we examined the substituent effects of the aryl group, compounds 10, 5,
and 11. On the basis of the LUMO level of compound 5, we found that the OMe group on the para
position of compound 10 contributes to an increase of the LUMO level, conversely, the CF3 group of
compound 11 decreases the LUMO level. Therefore, the HOMO-LUMO gap increases in the
compound 10 and decreases in compound 11.
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Next, we discuss the role acetylene and arylacetylene moieties in extending the π
conjugation system. The measured absorption wavelength of compound 20 was 522 nm and that of
compound 17 was 580 nm. Therefore, one acetylene moiety exerted a 58 nm shift of the absorption
wavelength through π expansion. The calculated wavelengths for compounds 20 and 17 were 445
and 491 nm, respectively. The change in wavelength by the addition of an acetylene moiety was
estimated to be 46 nm. Thus, the effect on π-extension by one acetylene moiety estimated by DFT
calculation agreed well with the actual measurement. The role of the aryl group on π-extension is
considered. The measured absorption wavelength of compound 17 was 580 nm and that of
compound 5 was 608 nm. The degree of the wavelength shift exerted by one benzene ring is 28 nm.
The calculated wavelengths of compounds 17 and 5 were 491 nm and 531 nm, respectively.
Therefore, the change of the wavelength owing to the addition of one benzene ring was calculated to
be 40 nm, which also matches with the actual measurement results. Notably, the effect of one
acetylene moiety on the wavelength was the same or greater than that of one benzene ring, in both
the actual and calculated measurements.
Confocal laser scanning microscopic observation of cells treated with compounds 10 and 11
Finally, cell staining experiments were performed for with a view to potential applications in
bioimaging. Compound 10 (p-OMe), which had the highest quantum yield among the nine
compounds, and compound 11 (p-CF3), which emitted the longest wavelength, were chosen and
applied for bioimaging. HeLa cells were incubated with compounds 10 and 11 (10 µM) in phosphate
buffered saline (PBS) for 15 min, and then fluorescence signals in living cells were analyzed by
confocal laser scanning microscopy (excitation 559 nm; emission 570-670 nm), respectively. The
differential interference contrast (DIC), fluorescence and merged images are shown in Figure 8. It
looks likely that both compounds stained cell membranes and membrane compartments in the cells.
Of course further studies are needed, these results suggested the possibility of use of these
compounds for cell staining.
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Figure 8. (a) DIC image of HeLa cells treated with compound 10, (b) fluorescence image of
compound 10 with 559 nm, (c) merged image of (a) and (b). (d) DIC image of HeLa cells treated
with compound 11, (e) fluorescence image of compound 11 with 559 nm, (f) merged images of (d)
and (e).
Conclusion
We report the syntheses of xanthene fluorescent dyes possessing arylacetylenes and examine their
properties and relationships among their structural features and red shifts of their emission
wavelengths. For the synthesis, we proposed an improvement to the reactivity of the acetylide
toward the carbonyl group of xanthone with the use of hard ceric chloride. Synthetic compounds
having arylacetylenes showed emission wavelengths greater than 600 nm under basic conditions in
DMSO. We found that the absorption and emission wavelength were blue shifted by the introduction
of an electron donating substituent on the aryl group, and shifted in a red direction by the addition of
an electron withdrawing substituent. These behaviors were clarified by DFT calculations, which
showed that the substituent groups mainly affected the LUMO level. Potential applicability of the
xanthene fluorescent dyes to cell staining was exemplified using compounds 10 and 11. These
compounds could be adjusted to more suitable and desirable emission regions for fluorescence
microscopy measurements through the use of different substituents on the aryl groups.
15

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Experimental
General procedure for the preparation of compounds 5 and 10–17.
Method A. A solution of n-BuLi (1.64 M n-hexane solution; 2.38 mL, 3.9 mmol) was added
dropwise to a solution of ethynylbenzene (0.51 mL, 4.6 mmol) in dry toluene (4 mL) under N2
atmosphere at −78 °C. The reaction mixture was stirred for 30 min at 0 °C, and a solution of
3,6-bis(tert-butyldimethylsilyloxy)-9H-xanthene-9-one (9, 600 mg, 1.31 mmol) in dry toluene (8
mL) was subsequently added dropwise. The reaction mixture was stirred for 4 h at room temperature,
and evaporated to dryness under a reduced pressure. The residue was dissolved in THF (7 mL), and a
solution of HF-pyridine (13 M THF solution; 0.5 mL, 6.6 mmol) was added. The reaction mixture
was stirred for 1.5 h at room temperature, and evaporated to dryness under a reduced pressure. The
residue was washed with water and methanol, and the undissolved solid was collected by filtration to
afford compound 5 (274 mg, 67% yield) as a dark red solid.
Compound 5 : 67% yield from compound 9 and ethynylbenzene. Dark red solid. Mp
>300 °C. IR (KBr) 3060, 3043, 2200, 1828, 1795, 1747, 1698, 1635, 1590, 1506, 1457, 1417, 1380,
1315, 1270, 1205, 1178, 1103 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 8.8 Hz, 2H), 7.92
(m, 2H), 7.58 (m, 3H), 6.95–6.65 (br, 2H), 6.64–6.19 (br, 2H). 13C NMR (100 MHz, DMSO-d6) δ
132.6, 131.0, 130.1, 129.0, 128.8, 120.4, 107.8, 103.3, 82.1 (4 signals were not identified in spite of
13,824 number of scans). HRMS (FT-ICR-MS) calcd for C21H13O3 (M + H)+ 313.0859, Found
313.0859.
Compound 10 : 83% yield from compound 9 and 4-ethynylanisole. Red solid. Mp >300 °C.
IR (KBr) 3465, 3432, 3396, 3060, 2919, 2848, 2593, 2559, 2186, 1633, 1598, 1511, 1490, 1467,
1428, 1380, 1340, 1299, 1259, 1209, 1172, 1105, 1041, 1016 cm−1. 1H NMR (400 MHz, DMSO-d6)
δ 7.97 (d, J = 9.2 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 9.2 Hz, 2H),
6.52–6.37 (br, 2H), 3.87 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 161.4, 134.7, 130.2, 129.5,
114.8, 112.2, 109.1, 103.2, 81.8, 55.5 (4 signals were not identified in spite of 24,576 number of
scans). HRMS (FT-ICR-MS) calcd for C22H15O4 (M + H)+ 343.0965, Found 343.0968.
Compound 19 : 23% yield from compound 9 and 1-ethynyl-4-fluorobenzene. Yellow oil.
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IR (neat) 3050, 2954, 2931, 2886, 2857, 2711, 2159, 2123, 1995, 1889, 1648, 1600, 1504, 1471,
1
1407, 1361, 1294, 1251, 1234, 1213, 1155, 1091, 1052, 1008. H NMR (400 MHz, CDCl3) δ 7.45
13
(dd, J = 8.8, 5.6 Hz, 2H), 6.99 (t, J = 8.8 Hz, 2H), 0.99 (s, 9H), 0.18 (s, 6H). C NMR (100 MHz,
CDCl3) δ 162.6 (d, J = 248 Hz), 133.9 (d, J = 8.0 Hz), 119.4 (d, J = 4.0 Hz), 115.4 (d, J = 22.0 Hz),
104.6, 92.1 (d, J = 1.0 Hz), 26.1, 16.7, −4.6. HRMS (FT-ICR-MS) calcd for C14H20FSi (M + H)+
235.1313, Found 235.1315.
Method B. A solution of n-BuLi (1.55 M n-hexane solution; 0.60 mL, 0.93 mmol) was added
dropwise to a solution of 1-ethynyl-4-(trifluoromethyl)benzene (0.16 mL, 1.1 mmol) in dry THF (4
mL) under N2 atmosphere at 0 °C. After stirring for 30 min at 0 °C, this solution was added to a
suspension of cerium (III) chloride (247 mg, 1.0 mmol) in dry THF (2 mL) at −78 °C, and the whole
mixture
was
stirred
for
further
30
min
at
−78
°C.
A
solution
of
3,6-bis(tert-butyldimethylsilyloxy)-9H-xanthene-9-one (9, 140 mg, 0.31 mmol) in dry THF (2 mL)
was subsequently added dropwise, and the reaction mixture was stirred for 1.5 h at 0 °C. The
reaction mixture was quenched by sat. NH4Cl aq. solution (5 mL), diluted with ethyl acetate (5 mL),
and filtered through celite. The filtrate was extracted with ethyl acetate. The organic layer was
washed sequentially with sat. NH4Cl aq. solution, water (three times) and brine, and evaporated to
dryness under a reduced pressure. The residue was dissolved in THF (2 mL) and water (2 mL), and
acetic acid (2 mL) was added. After stirring for 10 min at room temperature, the undissolved solid
was collected by filtration, and washed with water, chloroform, and methanol to afford compound 11
(63.0 mg, 53% yield) as a red solid.
Compound 11 : 53% yield from compound 9 and 1-ethynyl-4-(trifluoromethyl)benzene.
Red solid. Mp 274–276 °C (decomp.). IR (KBr) 3064, 2925, 2757, 2597, 2202, 1635, 1616, 1563,
1
1492, 1465, 1428, 1380, 1322, 1276, 1209, 1174, 1105, 1068, 1016 cm−1. H NMR (400 MHz,
DMSO-d6) δ 8.15 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 9.2 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H), 6.87–6.67
(br, 2H), 6.66–6.28 (br, 2H). 13C NMR (100 MHz, DMSO-d6) δ 133.4, 130.5, 130.2, 128.1, 127.4,
125.8, 125.7, 124.6, 105.3 (some signals were not identified in spite of 14,848 number of scans).
17

ACCEPTED MANUSCRIPT
HRMS (FT-ICR-MS) calcd for C22H10F3O3 (M − H) − 379.0588, Found 379.0586.
Compound 12 : 60% yield from compound 9 and 1-ethynyl-4-fluorobenzene. Red brown
solid. Mp >300 °C. IR (KBr) 3064, 2923, 2595, 2568, 2198, 1889, 1828, 1795, 1747, 1735, 1635,
1614, 1596, 1508, 1490, 1461, 1428, 1378, 1338, 1276, 1228, 1209, 1155, 1126, 1105, 1039 cm−1.
1H NMR (400 MHz, DMSO-d6) δ 8.02 (dd, J = 8.8, 5.2 Hz, 2H), 8.00 (d, J = 9.2 Hz, 2H), 7.42 (t, J
= 8.8 Hz, 2H), 6.82–6.64 (br, 2H), 6.59–6.32 (br, 2H). 13C NMR (100 MHz, DMSO-d6) δ 135.4 (d, J
= 9.0 Hz), 130.2, 128.7, 116.4 (d, J = 23.0 Hz) (9 signals were not identified in spite of 18,227
number of scans). HRMS (FT-ICR-MS) calcd for C21H12FO3 (M + H)+ 331.0765, Found 331.0767.
Compound 13 : 69% yield from compound 9 and 1-ethynyl-3-fluorobenzene. Dark red
solid. Mp >300 °C. IR (KBr) 3432, 3396, 3064, 2767, 2474, 2200, 1735, 1637, 1598, 1579, 1509,
1455, 1415, 1382, 1313, 1267, 1201, 1155, 1108, 1043, 1000 cm−1. 1H NMR (400 MHz, DMSO-d6)
δ 8.02 (d, J = 9.2 Hz, 2H), 7.89 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.60 (m, 1H), 7.46 (dt,
J = 8.8, 2.0 Hz, 1H), 6.96–6.63 (br, 2H), 6.62–6.11 (br, 2H). 13C NMR (100 MHz, DMSO-d6) δ
131.2 (d, J = 8.0 Hz), 129.1 (d, J = 3.0 Hz), 128.4, 119.3 (d, J = 23.0 Hz), 118.3 (d, J = 21.0 Hz),
82.6 (9 signals were not identified in spite of 19,456 number of scans). HRMS (FT-ICR-MS) calcd
for C21H12FO3 (M + H)+ 331.0765, Found 331.0767.
Compound 14 : 68% yield from compound 9 and 1-ethynyl-2-fluorobenzene. Purple solid.
Mp 280–281 °C (decomp.). IR (KBr) 3058, 3039, 2474, 2204, 1828, 1795, 1749, 1700, 1635, 1589,
1536, 1502, 1457, 1419, 1380, 1336, 1272, 1209, 1176, 1103 cm−1. 1H NMR (400 MHz, DMSO-d6)
δ 8.00 (t, J = 8.0 Hz, 1H), 7.89 (d, J = 9.2 Hz, 2H), 7.66 (m, 1H), 7.48 (t, J = 8.8 Hz, 1H), 7.40 (t, J
= 8.0 Hz, 1H), 6.82–6.59 (br, 2H), 6.58–6.19 (br, 2H). 13C NMR (100 MHz, DMSO-d6) No
meaningful signal was observed in spite of 21,504 number of scans. HRMS (FT-ICR-MS) calcd for
C21H12FO3 (M + H)+ 331.0765, Found 331.0767.
Compound 16 : 58% yield from compound 9 and trimethylsilylacetylene. Red brown solid.
Mp 243–244 °C (decomp.). IR (KBr) 2958, 2896, 2767, 2584, 2460, 1828, 1747, 1683, 1635, 1563,
1
1517, 1455, 1417, 1380, 1324, 1270, 1249, 1197, 1159, 1108, 1063 cm−1. H NMR (400 MHz,
DMSO-d6) δ 7.74 (d, J = 9.2 Hz, 2H), 6.73 (d, J = 9.2 Hz, 2H), 6.54–6.29 (br, 2H), 0.37 (s, 9H). 13C
18

ACCEPTED MANUSCRIPT
NMR (100 MHz, DMSO-d6) δ 129.8, 128.1, 115.7, 103.3, 96.2, −0.56 (4 signals were not identified).
HRMS (FT-ICR-MS) calcd for C18H16O3SiNa (M + Na)+ 331.0761, Found 331.0769.
Compound 17 : 65% yield from compound 9 and propyne. Red solid. Mp 176–177 °C
(decomp.). IR (KBr) 2503, 2219, 1828, 1747, 1698, 1635, 1585, 1544, 1504, 1457, 1417, 1380, 1334,
1
1268, 1207, 1159, 1095. H NMR (400 MHz, DMSO-d6) δ 11.4–10.8 (br, 1H), 7.82 (d, J = 9.2 Hz,
2H), 7.18–5.61 (br, 4H), 2.39 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 130.1, 108.5, 72.7, 5.07 (6
signals were not identified in spite of 13,312 number of scans). HRMS (FT-ICR-MS) calcd for
C16H11O3 (M + H)+ 251.0703, Found 251.0703.
Method C. Compound 15 : A solution of n-BuLi (1.55 M n-hexane solution; 0.60 mL, 0.93 mmol)
was added dropwise to a solution of diisopropylamine (0.22 mL, 1.6 mmol) in dry THF (2 mL)
under N2 atmosphere at 0 °C, and the reaction mixture was stirred for 20 min at 0 °C.
2-Ethynylpyridine (0.11 mL, 1.1 mmol) was then added dropwise, and the reaction mixture was
stirred for an additional 30 min at 0 °C. This solution was added to a suspension of cerium (III)
chloride (247 mg, 1.0 mmol) in dry THF (2 mL) at −78 °C, and the whole mixture was stirred for
further 30 min at −78 °C. A solution of 3,6-bis(tert-butyldimethylsilyloxy)-9H-xanthene-9-one (9,
140 mg, 0.31 mmol) in dry THF (2 mL) was subsequently added dropwise, and the reaction mixture
was stirred for 1.5 h at 0 °C. The reaction mixture was quenched by sat. NH4Cl aq. solution (5 mL),
diluted with ethyl acetate (5 mL), and filtered through celite. The filtrate was extracted with ethyl
acetate. The organic layer was washed sequentially with sat. NH4Cl aq. solution, water (three times)
and brine, and evaporated to dryness under a reduced pressure. The residue was dissolved in THF (2
mL) and water (2 mL), and acetic acid (2 mL) was added. After stirring for 10 min at room
temperature, the undissolved solid was collected by filtration, and washed with water and
chloroform to afford compound 15 (62.7 mg, 65% yield) as a purple solid. Mp 185–186 °C
(decomp.). IR (KBr) 3432, 3394, 3052, 2483, 2208, 1828, 1795, 1747, 1639, 1585, 1558, 1504, 1459,
1436, 1419, 1382, 1334, 1315, 1268, 1207, 1176, 1101 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 8.75
(dt, J = 4.8, 1.6 Hz, 1H), 8.06 (dt, J = 7.6, 1.2 Hz, 1H), 8.00 (td, J = 7.6, 1.6 Hz, 1H), 7.94 (d, J = 9.2
19

ACCEPTED MANUSCRIPT
Hz, 2H), 7.58 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H), 6.77 (d, J = 9.2 Hz, 2H), 6.61–6.36 (br, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 150.7, 137.2, 128.9, 127.8, 125.2 (9 signals were not identified in spite of
13,312 number of scans). HRMS (FT-ICR-MS) calcd for C20H12NO3 (M + H)+ 314.0812, Found
314.0812.
Treatment of cells with compounds 10 and 11.
HeLa cells (2 × 105 cells/dish) were seeded in 35 mm (
ϕ) glass based dishes (Iwaki) in
α-minimum essential medium supplemented with 10% (v/v) heat-inactivated bovine serum, and
cultured for 24 h (80-90% confluence) at 37°C in a humidified incubator supplied with 5% CO2 gas.
The medium was removed and the cells were washed twice with PBS(+) (137 mM NaCl, 8.1 mM
Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.33 mM MgCl2, and 0.90 mM CaCl2, pH 7.4) and
treated with compounds 10 and 11 (final concentration 10 µM) in PBS(+) for 15 min at room
temperature, respectively. The intracellular distribution of the each compound was analyzed without
fixing the cells using a confocal scanning laser system (FV1000, Olympus) consisting of an inverted
microscope (IX81, Olympus) equipped with a 40× UPlanSApo objective (dry, NA 0.95) (Ex. 559
nm; Em. 570–670 nm).
Acknowledgment
This study was supported in part by KAKENHI (Nos. 26293005 and 15K14931). This study was
carried out using the Fourier transform ion cyclotron resonance mass spectrometer in the Joint
Usage/Research Center at the Institute for Chemical Research, Kyoto University.
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