Design and synthesis of regioisomerically pure unsymmetrical xanthene derivatives for staining live cells and their photochemical properties

Article abstract of DOI:10.1016/j.bmcl.2008.06.065

We have demonstrated the synthesis of regioisomerically pure unsymmetrical xanthene derivatives consisting of three units which can be independently modified to control their physical properties. The photochemical properties of the synthetic unsymmetrical xanthene derivatives were investigated in solution by UV-vis absorption and fluorescence measurements, and their cell imaging properties were examined by confocal laser-scanning microscopy.

Full text of DOI:10.1016/j.bmcl.2008.06.065

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4380–4384
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of regioisomerically pure unsymmetrical xanthene
derivatives for staining live cells and their photochemical properties
Shinichiro Kamino, Hayato Ichikawa, Shun-ichi Wada, Yuka Horio, Yoshihide Usami, Takako Yamaguchi,
*
Toshiki Koda, Aki Harada, Kazusa Shimanuki, Masao Arimoto, Mitsunobu Doi, Yoshikazu Fujita
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have demonstrated the synthesis of regioisomerically pure unsymmetrical xanthene derivatives con-
sisting of three units which can be independently modified to control their physical properties. The pho-
tochemical properties of the synthetic unsymmetrical xanthene derivatives were investigated in solution
by UV–vis absorption and fluorescence measurements, and their cell imaging properties were examined
by confocal laser-scanning microscopy.
Received 12 May 2008
Revised 14 June 2008
Accepted 18 June 2008
Available online 21 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Unsymmetrical xanthene derivatives
Regioisomerically pure
Photochemical properties
Chemical biology
Xanthene derivatives, such as fluoresceins and rhodamines, are
widely employed as molecular probes in chemical biology.1 We
have been interested in the synthesis of chromophores possessing
high potential use in sensing applications. Since the first prepara-
tion of fluorescein in 1871 by von Baeyer,2 many synthetic meth-
ods have been reported.3 However, free modification of the
xanthene skeleton as a chromophore having color diversity has
been little attempted. Unsymmetrical xanthenes, such as commer-
cially available rhodol derivatives, are typically prepared as
mixtures of 5- and 6-isomers with difficulty in their separation
by well-established condensation route.4 The conjugation of these
isomers to other molecules leads to differences in physical proper-
ties, including fluorescence polarity, internal fluorescence quench-
ing, and labeling specificity.5 To solve these problems, we assumed
that xanthene derivatives can be divided into three units—chromo-
phore, phenol, and aromatic—each of which can be independently
modified. The combination of these three units would allow us to
optimize the physical properties of xanthene derivatives for the
desired application. We propose herein a synthetic method for a
series of regioisomeric pure unsymmetrical xanthene derivatives,
which involves combining three different moieties, just like pieces
of a jigsaw puzzle.
Benzophenones are usually used as condensation partners of
phenols for the synthesis of xanthenes.6 This method is based on
the formation of a benzophenone via the Friedel–Crafts acylation
reaction of benzoyl chloride with methoxybenzene derivatives, fol-
lowed by condensation with phenol derivatives to afford the de-
sired unsymmetrical xanthenes. The photochemical properties of
synthesized unsymmetrical xanthene derivatives in ethanol solu-
tion were investigated by UV–vis absorption and fluorescence
measurements. We also prepared isomers of rhodol derivatives,
which are hybrid compounds of rhodamine and fluorescein, and
evaluated their photochemical properties by measuring absolute
fluorescence quantum yield (Uf) and molar extinction coefficient
(e) in ethanol solution. Cell imaging properties of the rhodol deriv-
atives were also tested using human lung carcinoma A549 cell line.
The Friedel–Crafts acylation of aromatic compounds is an
important transformation reaction in organic synthesis.7 However,
* Corresponding author. Tel.: +81 72 690 1063; fax: +81 72 690 1063.
E-mail address: fujitay@gly.oups.ac.jp (Y. Fujita).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.065

S. Kamino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4380–4384
4381
there are few examples of the direct acylation of phenolic com-
pounds,8 in particular, polyphenols, owing to severe side reactions,
such as the Shotten–Baumann reaction.9 In fact, 1,2,4-trihydroxy-
benzene 1 reacted with benzoyl chloride 3 with coexisting AlCl3
to form 40-O-monoacyl derivative 510 in very high proportion with-
out C-acylation. Compound 5 was similarly obtained form the reac-
tion of acetylation compound of 1 with 3. Methylation of 1
selectively gave 1,2,4-trimethoxybenzophenone 6a, whose struc-
ture was supported by HSQC and HMBC analyses. Trimethoxybenz-
ophenone derivatives (6b and 6c) were similarly prepared. These
benzophenone derivatives were deprotected by BBr3 at ꢀ78 °C
and trihydroxybenzophenone derivatives (7a–c) were obtained
after recrystallization. Likewise, amine-conjugated benzophenones
(8 and 9) were similarly synthesized by acylation of 8-methoxyju-
lolidine or 1-(diethylamino)-3-methoxybenzene with benzoyl
chloride derivatives. Single crystals of 8b and 9c suitable for
X-ray diffraction analysis were grown by slow evaporation of eth-
anol and H2O solution.11 X-ray analysis supported the finding that
the acyl group was selectively introduced adjacent to the hydroxyl
group. b-Diketones as benzophenones can exist in keto–enol tauto-
merization. Structural data indicated that both 8b and 9c existed in
the enol form in the solid state. The acylation of 1,6-methoxynaph-
thalene with p-methylbenzoyl chloride gave 4-methylbenzoyl-1,6-
methoxynaphthalene 1012 that was not adequate for the synthesis
of xanthene derivatives. The introduction of 1,6-naphthalenediol
into xanthenes is key for synthesis of long-wave fluorescent com-
pound. Therefore, a selective synthesis involving 2-, 5-, or 7-acyla-
tion of 1,6-naphthalenediol has attracted considerable interest. The
chemistry is shown in Scheme 1. These benzophenone derivatives
would serve as precursors for the synthesis of regioisomerically
pure unsymmetrical xanthene derivatives.
The condensation reactions of synthesized benzophenone
derivatives with phenol analogues were subsequently carried out
(Table 1). Although many condensing agents have been reported,
CH3SO3H is the most popular since it acts as both condensing
and oxidizing agents at low temperature.3c Therefore, the reactions
were conducted in neat CH3SO3H. To synthesize unsymmetrical
xanthene derivatives having pyrocathecol sites, semi-fluorescein
compound 11 was prepared by condensation of 7b with resorcinol.
However, the condensation reactions of 7b–c with 8-hydroxyjulo-
lidine and/or 1-(diethylamino)-3-hydroxybenzene gave no semi-
rhodamine compounds due to the formation of inseparable side
products. We found that 8 and 9 are adequate for the synthesis
of a series of semi-rhodamine derivatives. Condensation at 80–
150°C proceeded smoothly in hot CH3SO3H to give the desired
semi-rhodamine derivatives. All compounds except those having
pyrocathecol sites 12 were simply purified by silica column chro-
matography using mixed CH3OH/CH2Cl2 solvent. However, we
were always bothered by the formation of side products. Lukhta-
nov and co-workers, for the first time, discussed the mechanism
of the reaction of benzophenone with resorcinol by retro-Friedel–
Crafts fragmentation, and developed an innovative two-step syn-
thesis of unsymmetrical 20-carboxyethyl-l-substituted fluoresceins
to solve the problem.6a Using their method, it is necessary for us to
characterize the side products and develop mild synthetic condi-
tions to increase the yield.
The UV–vis absorption and fluorescence emission spectra of
11–17 in ethanol solution were measured (see details in ESI). Uf
Scheme 1. Synthesis of benzophenone derivatives.

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S. Kamino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4380–4384
Table 1
Synthesis of unsymmetrical xanthene derivativesa
Reaction No.
Phenol
Benzophenone
Product
Yield (%)
1
Resorcinol
7b
10
2
1,2,4-Trihydroxybenzene
9c
12
3
Resorcinol
8c
11
4
Resorcinol
8b
52
5
Resorcinol
9a
9
6
Resorcinol
9b
75
7
Resorcinol
9c
43
a
Reagents and conditions: CH3SO3H, 80–150 °C, 2–24 h. The detailed experimental procedure is described in ESI.

S. Kamino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4380–4384
4383
Table 2
Photochemical properties of regioisomers of rhodol derivatives
Compound 14 and 17 at 1
l
M were overlaid on the cells, and after
incubation at 37 °C for 30 min, intracellular localization was exam-
ined by confocal laser-scanning microscopy with irradiation at two
excitation wavelengths (Fig. 1). Compound 14 and 17 were easily
taken up by the cells and essentially accumulated in the cytoplasm,
and dual imaging colors of fluorescein (green) and rhodamine (red)
were observed.
Compound Yield Absorption
maximum (nm)a
e
Emission
Ufa,b,c
(ꢁ104)a maximum
(nm)a
15
16
17
9
75
43
523
523
523
8.5
8.4
9.4
555
557
556
0.706/0.708
0.592/0.602
0.599/0.613
In summary, we have developed the synthesis of regioisomeri-
cally pure unsymmetrical xanthene derivatives and investigated
their photochemical properties. We also observed the easy diffu-
sion of these compounds through live cell membrane and con-
firmed their localization in the cytoplasm. The advantage of this
method is the ease of modification of each unit and their combina-
tion for the desired application. Further efforts are ongoing to ex-
plore multifunctional tools for chemical biology by developing
the three units separately and then combining them.
a
Measured in ethanol.
Excited at 480 nm.
Left values indicate Uf measured in ethanol solution and right values indicate
b
c
Uf after excluding O2 effect by bubbling N2 in ethanol solution.
and
e values of regioisomers of rhodol derivatives, 15–17, were
measured in ethanol solution, respectively (Table 2). The purity
of compound 15–17 was confirmed by 13C NMR and analytic
reversed-phase HPLC. Uf had no effects on singlet oxygen in eth-
anol solution. Uf values of 15 were higher than those of 16 and
17, as shown in Table 2. Urano et al. reported that the high
quantum yield of fluorescein was essential to keep the xanthene
moiety and the benzene moiety orthogonal to each other with
an ortho substituent.12 In this study, the fluorescence properties
of ortho-substituted rhodol were superior to those of the other
isomers. One possible explanation was that the increase of
radiationless transition probability due to steric rotation of the
aromatic unit around the linker led to a reduction of Uf of meta-
and para-substituted rhodols. However, drastic changes of Uf
were not observed compared to that of conventional fluorescein.13
Compound 15 was prepared in low yield due to steric effect. Fur-
ther examination of other rhodol derivatives is required to verify
the photochemical system.
Acknowledgments
We thank Ms. Mihoyo Fujitake and Dr. Katsuhiko Minoura of
Osaka University of Pharmaceutical Sciences for MS and NMR mea-
surements; Dr. Kengo Suzuki of HAMAMATSU PHOTONICS K. K. for
fluorescence quantum yield determination; Dr. Seiji Komeda of
Osaka University of Pharmaceutical Sciences for useful discussion;
Ms. Motoko Sawatani of YAMAZEN Co. for insightful discussion;
and Ms. Chiho Ikeda of Osaka University of Pharmaceutical Sci-
ences for design of graphical abstract.
Supplementary data
Electronic Supplementary Data available: materials, synthesis,
methods, spectroscopic data for novel compounds and supplemen-
tary data. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.06.065.
Synthetic rhodol derivatives 14 and 17 were tested for their
ability to diffuse into human lung carcinoma A549 cells. The purity
of 14 and 17 was determined by analytic reversed-phase HPLC.
Figure 1. Top: Fluorescence images of live cells loaded with 1
M 17. (c) Excited at 488 nm and (d) excited at 543 nm.
lM 14. (a) Excited at 506 nm and (b) excited at 560 nm. Bottom: Fluorescence images of live cells loaded with
1
l

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S. Kamino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4380–4384
6. (a) Lukhtanov, E. A.; Vorobiev, A. V. J. Org. Chem. 2008, 73, 2424; (b) Woodroofe,
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