A fluorescent photochromic compound for labeling biomolecules

Article abstract of DOI:10.1039/b713663c

A fluorescent photochromic compound, composed of diarylethene, fluorescein and succinimidyl ester units, was developed for the controllable fluorescent labeling of biomolecules based on a small molecule. The Royal Society of Chemistry.

Full text of DOI:10.1039/b713663c

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A fluorescent photochromic compound for labeling biomolecules{
Nobuaki Soh,a Kenji Yoshida,a Hizuru Nakajima,a Koji Nakano,a Toshihiko Imato,*a
Tuyoshi Fukaminatob and Masahiro Irie*bc
Received (in Cambridge, UK) 5th September 2007, Accepted 2nd October 2007
First published as an Advance Article on the web 12th October 2007
DOI: 10.1039/b713663c
photochromic molecules.8 Photoswitching of fluorescence in
A fluorescent photochromic compound, composed of diaryl-
diarylethene derivatives linked to an appropriate fluorophore has
been demonstrated.9 Such photoswitching of fluorescence is
generally based on the intramolecular energy transfer from the
fluorophore to the diarylethene derivative. In DAE-FL-SE, the
fluorescence would be efficiently quenched along with the photo-
cyclization, since the fluorescent spectrum of the FL unit and the
absorption spectrum of the closed-ring form of the DAE unit are
well overlapped. Thus, DAE-FL-SE, in which a SE unit is
introduced into the DAE-FL for effective binding with proteins, is
expected to function as a novel labeling reagent, which can control
the ‘‘off/on’’ state of fluorescence in the protein of interest
(Scheme 1(b)). In this report, we selected ERK protein and
ethene, fluorescein and succinimidyl ester units, was developed
for the controllable fluorescent labeling of biomolecules based
on a small molecule.
Photochromism is a reversible transformation of chemical species
induced by photoreaction between two forms with different
absorption spectra.1 Photochromic compounds have attracted
much interest because of their potential applications to photonic
devices such as optical memories and display devices.2 On the
other hand, biological applications of them have not yet been
extensively explored although a few preceding works have been
reported.3
Recently, a photochromic fluorescent protein, Dronpa was
isolated by engineering a novel coral protein.4 By tagging the
photochromic protein, a regulated fast nucleocytoplasmic shuttling
of mitogen-activated protein kinase (extracellular signal regulated
kinase; ERK) was successfully observed. Generally, fluorescent
proteins are able to be genetically fused with host proteins to yield
fluorescent chimeras in situ5 and a labeling of such fluorescent
proteins is thus useful to monitor the behavior of a target protein
(protein of interest) using fluorescent microscopy with high
resolution. However, the large size of fluorescent proteins (i.e.
27 kDa for GFP) can perturb the inherent function and
distribution of the target protein.6 Therefore, the development of
new alternative labeling methods for proteins based on smaller
fluorescent molecules is important.7 Especially, a novel small
photocontrollable fluorescent labeling molecule would be a
powerful tool to elucidate the unexplored regulation of fast
protein dynamics in more detail.
Here, we report on a photochromic compound for fluorescent
labeling of proteins. The compound, named DAE-FL-SE, is
composed of a photochromic diarylethene (DAE) unit,
a
fluorescent fluorescein (FL) unit, and an amine-reactive succini-
midyl ester (SE) unit (Scheme 1(a)). Diarylethene derivatives are
known to have excellent thermal stability of two isomers (open-
and closed-ring isomers) and fatigue resistance among several
aDepartment of Applied Chemistry, Graduate School of Engineering,
Kyushu University, 744, Moto-oka, Nishi-ku, Fukuoka, 819-0395,
Japan. E-mail: imato@cstf.kyushu-u.ac.jp; Fax: +81-92-802-2889;
Tel: +81-92-802-2889;
bDepartment of Chemistry and Biochemistry, Graduate School of
Engineering, Kyushu University, 744, Moto-oka, Nishi-ku, Fukuoka,
819-0395, Japan. Fax: +81-92-802-2873; Tel: +81-92-802-2873
cDepartment of Chemistry, Rikkyo University, 3-34-1, Nishi-Ikebukuro,
Toshima-ku, Tokyo, 171-8501, Japan. E-mail: iriem@rikkyo.ac.jp;
Fax: +81-3-3985-2397; Tel: +81-3-3985-2397
Scheme 1 (a) Chemical structure of DAE-FL-SE. (b) Photoswitching of
fluorescence in DAE-FL labeled biomolecule based on the photochromic
reaction.
{ Electronic supplementary information (ESI) available: Experimental
information including analytical data. See DOI: 10.1039/b713663c
5206 | Chem. Commun., 2007, 5206–5208
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investigated the binding ability of DAE-FL-SE with the target
protein and the photoswitching of fluorescence in the DAE-FL-
labeled protein.
irradiation with UV light, a new absorption band appeared at
580 nm, which was bleached after irradiation with visible light.
This absorption band was attributed to the closed-ring isomer of
the diarylethene unit. The photochromic behaviour was observed
reversibly many times. A characteristic fluorescence band was
observed at 520 nm in the open-ring isomer (Fig. 2(b)). Upon
irradiation with UV light, the fluorescence intensity decreased
gradually. Upon irradiation with visible light, the fluorescence
intensity returned fully to the initial intensity.
DAE-FL-SE was synthesized by coupling a boronate diaryl-
ethene with an iodo-phenylated fluorescein derivative and
subsequent esterification using N-hydroxysuccinimide (synthetic
procedures are described in ESI{). The binding ability of DAE-
FL-SE toward the target protein was initially investigated. Fig. 1
shows the result of SDS-PAGE (sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis) for ERK samples, which were
prepared by incubation of ERK with DAE-FL-SE and subsequent
purification by ultrafiltration (see ESI{). As can be seen from the
gel stained by CBB (Coomassie Brilliant Blue) shown in Fig. 1(a),
a protein band with a little higher molecular weight than
control ERK (lane 7) was observed at the position of y80 kDa
in lanes 2–4, corresponding to the concentration of samples.
Simultaneously, the above-mentioned bands at y80 kDa in lanes
2–4 emitted strong fluorescence upon irradiation with 488 nm light
using a molecular imager system, as shown in Fig. 1(b). The results
clearly demonstrated that DAE-FL-SE combined with ERK and
produced the expected DAE-FL-labeled ERK successfully.
Fig. 2 shows the absorption and fluorescence spectral changes of
DAE-FL-labeled ERK upon irradiation with UV (365 nm) and
visible (l . 550 nm) light. In Fig. 2(a), the absorption band at
505 nm is characteristic for the 6-carboxyfluorescein unit. Upon
Because it is difficult to quantitatively evaluate the fluorescence
photoswitching performance of protein-labeled DAE-FL, that of a
model compound (DAE-FL-CO2Et: 16) (see ESI{) was investi-
gated quantitatively. Reversible fluorescence switching was
observed along with photochromic reaction. Upon UV (365 nm)
light irradiation, a new absorption band appeared at 580 nm and
the fluorescence intensity decreased at that time. By irradiating
with visible (l . 550 nm) light, both absorption and fluorescence
spectra were returned to their original state. The fluorescence
intensity at the photostationary state was 39% of the original
intensity of the open-ring isomer. A conversion ratio from the
open- to closed-ring isomer of 16 upon UV light irradiation was
1
estimated from H NMR spectral measurement. Before UV light
irradiation, multiple 1H NMR signals corresponding to two
methyl groups on the thiophene rings were observed at around
2.38 and 2.13 ppm. After UV light irradiation, these signals were
Fig. 1 SDS-PAGE of ERK samples which were reacted with DAE-FL-
SE and purified by ultrafiltration. (a) Proteins in the gel were stained
with CBB. (b) Fluorescent bands in unstained gel were visualized by a
molecular imager system.
Fig. 2 (a) Absorption and (b) fluorescence spectral changes of DAE-FL
labeled ERK protein in PBS buffer–EtOH (7 : 3) upon irradiation with
UV (365 nm) and visible (l . 550 nm) light.
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Systems, Elsevier, Amsterdam, 1990; (c) H. Bouas-Laurent and H. Du¨rr,
Pure Appl. Chem., 2001, 73, 639.
suppressed and new two signals appeared at around 2.30 and
2.04 ppm. The spectral changes in the NMR measurement were
completely reversible and the original spectrum was recovered by
irradiating with visible light. On the basis of the integrated
intensities of the methyl signals, the content of the closed-ring
isomer in the photostationary state upon UV light irradiation was
estimated to be 60%. The value of the conversion is in good
agreement with the quenching ratio of fluorescence intensity at the
photostationary state. This indicates that the closed-ring isomer is
non-fluorescent and the quenching ratio of fluorescence intensity
corresponds to the amount of the closed-ring isomer. It seems that
the results can be also adapted to the protein-labeled DAE-FL
molecule. Therefore, 62% of open-ring isomer of DAE-FL labeled
with ERK protein converted to the closed-ring isomer along with
the photocyclization reaction. The fluorescence quantum yields W
of 16 in ethanol before and after UV light irradiation were 0.71
and 0.14 (at photostationary state), respectively (fluorescein was
used as the standard, W = 0.97). These results indicate that the
fluorescent switching of the DAE-FL-labeled protein can be
accomplished by irradiation of externally-regulated UV/visible
light. Thus, DAE-FL-SE is promising for the controlled labeling of
target proteins which exist in specific areas in single cells such as
photochromic Dronpa. Furthermore, DAE-FL-SE is also applic-
able to various biomolecules containing amino groups.
2 (a) B. L. Feringa, Molecular Switches, Wiley-VCH, Weinheim, 2001; (b)
A. P. de Silva, Electron Transfer in Chemistry, Part 1. Molecular-Level
Electronics, Wiley-VCH, Weinheim, 2001; (c) V. Balzani, M. Venturi and
A. Credi, Molecular Devices and Machines–A Journey into the World,
Wiley-VCH, Weinheim, 2003.
3 (a) M. W. Berns, T. Krasieva, C.-H. Sun, A. Dvornikov and
P. M. Rentzepis, J. Photochem. Photobiol., B, 2004, 75, 51; (b)
A. Koc¸er, M. Walko, W. Meijberg and B. L. Feringa, Science, 2005,
309, 755; (c) S. Loudwig and H. Bayley, J. Am. Chem. Soc., 2006, 128,
12404; (d) I. Willner and S. Rubin, Angew. Chem., Int. Ed. Engl., 1996,
35, 367; (e) T. Sakata, Y. Yan and G. Marriott, Proc. Natl. Acad. Sci.
U. S. A., 2005, 102, 4759–4764; (f) M. Saito, K. Musha, Y. Yokoyama
and Y. Yokoyama, Bull. Chem. Soc. Jpn., 2003, 76, 2423.
4 R. Ando, H. Mizuno and A. Miyawaki, Science, 2004, 306, 1370.
5 (a) R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509; (b) M. Chalfie and
S. R. Kain, GFP: Green Fluorescent Protein Strategies and Applications,
Wiley, New York, 1998.
6 (a) J. Zhang, R. E. Campbell, A. Y. Ting and R. Y. Tsien, Nat. Rev. Mol.
Cell Biol., 2002, 3, 906; (b) B. A. Griffin, S. R. Adams, J. Jones and
R. Y. Tsien, Methods Enzymol., 2000, 327, 565.
7 (a) B. A. Griffin, S. R. Adams and R. Y. Tsien, Science, 1998, 281, 269;
(b) G. Gaietta, T. J. Deerinck, S. R. Adams, J. Bouwer, O. Tour,
D. W. Laird, G. E. Sosinsky, R. Y. Tsien and M. H. Ellisman, Science,
2002, 296, 503; (c) S. R. Adams, R. E. Campbell, L. A. Gross,
B. R. Martin, G. K. Walkup, Y. Yao, J. Llopis and R. Y. Tsien, J. Am.
Chem. Soc., 2002, 124, 6063; (d) B. R. Martin, B. N. G. Giepmans,
S. R. Adams and R. Y. Tsien, Nat. Biotechnol., 2005, 23, 1308; (e) I. Chen
and A. Y. Ting, Curr. Opin. Biotechnol., 2005, 16, 35; (f) A. Keppler,
S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel and K. Johnsson, Nat.
Biotechnol., 2003, 21, 86; (g) E. G. Guignet, R. Hovius and H. Vogel,
Nat. Biotechnol., 2004, 22, 440; (h) N. Soh, D. Seto, K. Nakano and
T. Imato, Mol. BioSyst., 2006, 2, 128; (i) K. Honda, E. Nakata, A. Ojida
and I. Hamachi, Chem. Commun., 2006, 4024; (j) K. M. Marks,
P. D. Braun and G. P. Nolan, Proc. Natl. Acad. Sci. U. S. A., 2004, 101,
9982; (k) B. R. Sculimbrene and B. Imperiali, J. Am. Chem. Soc., 2006,
128, 7346; (l) L. W. Miller, J. Sable, P. Goelet, M. P. Sheetz and
V. W. Cornish, Angew. Chem., Int. Ed., 2004, 43, 1672.
In conclusion, we have synthesized photochromic DAE-FL-SE
composed of diarylethene, fluorescein and succinimidyl ester units
and demonstrated that the novel compound is applicable to the
controllable fluorescent labeling of biomolecules based on small
molecules. It is noteworthy that the SE unit in the DAE-FL-SE
can be easily converted to other functional groups such as an
NTA-Ni2+ unit, which recognizes a repeat of a histidine sequence
(His-tag) with a high selectively. Current efforts are directed
toward applying DAE-FL-SE and relevant fluorescent photo-
chromic compounds for studying dynamics of biomolecules in
living systems.
8 (a) M. Irie, Chem. Rev., 2000, 100, 1685; (b) M. Irie and K. Uchida, Bull.
Chem. Soc. Jpn., 1998, 71, 985; (c) H. Tian and S. Yang, Chem. Soc. Rev.,
2004, 33, 85; (d) S. Takami, S. Kobatake, T. Kawai and M. Irie, Chem.
Lett., 2003, 32, 892.
9 (a) M. Irie, T. Fukaminato, T. Sasaki, N. Tamai and T. Kawai, Nature,
2002, 420, 759; (b) T. Fukaminato, T. Umemoto, Y. Iwata, S. Yokojima,
M. Yoneyama, S. Nakamura and M. Irie, J. Am. Chem. Soc., 2007, 129,
5932; (c) T. A. Golovkova, D. V. Kozlov and D. C. Neckers, J. Org.
Chem., 2005, 70, 5545; (d) L. Giordano, T. M. Jovin, M. Irie and
E. A. Jares-Erijman, J. Am. Chem. Soc., 2002, 124, 7481–7489; (e)
A. de Meijere, L. Zhao, V. N. Belov, M. Bossi, M. Noltemeyer and
S. W. Hell, Chem.–Eur. J., 2007, 13, 2503; (f) T. Kawai, T. Sasaki and
M. Irie, Chem. Commun., 2001, 711; (g) T. B. Norsten and N. R. Branda,
J. Am. Chem. Soc., 2001, 123, 1784; (h) S.-J. Lim, B.-K. An, S. D. Jung,
M.-A. Chung and S. Y. Park, Angew. Chem., Int. Ed., 2004, 43, 6346; (i)
H. Cho and E. Kim, Macromolecules, 2002, 35, 8684.
This work was supported by Industrial Technology Research
Grant Program in 2005 (05A01507a) from New Energy and
Industrial Technology Development Organization (NEDO) of
Japan.
Notes and references
1 (a) G. H. Brown, Photochromism, Wiley-Interscience, New York, 1971;
(b) H. Du¨rr and H. Bouas-Laurent, Photochromism Molecules and
5208 | Chem. Commun., 2007, 5206–5208
This journal is ß The Royal Society of Chemistry 2007