Bodipy-diacrylate imaging probes for targeted proteins inside live cells

Article abstract of DOI:10.1039/c1cc10362h

A bodipy probe was developed for site-specific labeling of tagged proteins inside live cells which displays a large spectral change upon covalent coupling to the designed peptide that contains two pairs of Arg-Cys.

Full text of DOI:10.1039/c1cc10362h

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 4508–4510
www.rsc.org/chemcomm
COMMUNICATION
Bodipy-diacrylate imaging probes for targeted proteins inside live cellsw
Jae-Jung Lee,a Sung-Chan Lee,a Duanting Zhai,b Young-Hoon Ahn,c Hui Yun Yeo,a
Yee Ling Tana and Young-Tae Chang*ab
Received 19th January 2011, Accepted 16th February 2011
DOI: 10.1039/c1cc10362h
A bodipy probe was developed for site-specific labeling of tagged
proteins inside live cells which displays a large spectral change
upon covalent coupling to the designed peptide that contains two
pairs of Arg-Cys.
Site-specific labeling of target proteins with photophysical
reporter probes allows numerous in vivo studies of protein
functions.1 Although genetic fusion of the fluorescent proteins
(FPs) provides critical advantages, its adverse properties such as
large size (27 kDa) or aggregation often limit the application.2
Fig. 1 Design of a bodipy fluorophore that exhibits a large spectral
Alternatively, a small peptide tag and its binding probe would
provide a less invasive way of protein labeling.3 A pioneering
change upon covalent conjugation to a tag for potential application to
the site-specific labeling of proteins inside live cells.
example utilizing the high affinity between a tetracysteine tag
and a fluorescent biarsenical probe (FlAsH)4 has been used
spectral emission shift upon covalent coupling to a designed
in a number of applications inside live cells with step-wise
improvements.5–7 Development of other peptide tags have been
followed, mostly, in two perspectives: first, by exploiting the
intrinsic affinity between a probe and a peptide tag such as the
D4 tag/Zn-probe8 or IQ-tag,9 and second, by conjugating a probe
to a peptide tag with an assistance of trans-acting enzymes as
shown by the AviTag,10 LAP-tag,11 or ACP/PCP tag.12 However,
application of above systems, except the FlASH, is limited to the
extracellular domain of membrane proteins because of the
cell-impermeability of labeling reagents including the modifying
enzymes. Although a recent report by Uttamapinant et al.13
suggests a clever strategy to label peptide tags inside cells, co-
expression of the modifying enzyme might affect the simplicity of
experimental protocol and deprive the transcription/translational
machinery in cells.
peptide and demonstrate the successful optical imaging of a target
protein labeled by this affinity pair (probe and tag) inside cells.
We designed a bodipy probe based on the disruption of the
extent of conjugation of a fluorescent dye, through covalent
reaction with two thiol groups, which induces a large spectral
emission shift (Fig. 1) as shown by many groups.14,15 We chose a
bodipy fluorophore as it emits strong fluorescence with high
quantum yield, sharp excitation/emission wavelengths, and
relatively facile modification on its structure with predictable
alteration on the excitation/emission.15,16 We envisioned that
attachment of the Michael acceptor to the bodipy core will shift
the excitation and emission significantly to the longer wave-
lengths, while 1,4-addition of thiols to the Michael acceptor will
break the delocalization, shifting the fluorescence back to the
green color of bodipy. Therefore, we incorporated two acrylic
acid groups to the bodipy core. The space between the two acrylic
groups was similar to the distance separating the two reactive
cysteines in the peptide tag. Previously, Girouard et al. reported a
fluorogenic dimaleimide-fluorophore designed to bind to the
dicysteine peptide in a similar manner to our design.14,17 They
showed a successful conjugation of di-functionalized fluorophore
to a dicysteine peptide tag, but labelling on intracellular proteins
is not yet demonstrated.18
Therefore, notwithstanding the toxicity of arsenical probes,
FlAsH still is the most representative peptide-based method
applicable inside cells and thus, new peptide-based labeling
systems with safer probes need to be developed. Here we report
the design of a fluorescent bodipy-derivative that exhibits a large
a Laboratory of Bioimaging Probe Development,
Singapore Bioimaging Consortium, Agency for Science,
Technology and Research (A*STAR), Biopolis, Singapore 138667,
Singapore
The synthesis of bodipy-diacrylate derivatives is outlined
in Scheme 1. Dialdehydes were prepared by Vilsmeier
formylation of 5-phenyldipyrromethanes. The Wittig reaction
of dipyrromethanes with a triphenylphosphorane reagent
afforded the diacrylate compounds, which were characterized
as a symmetrical E isomer (J = 16.0 Hz) by 1H-NMR.
Phenyldipyrromethane acrylates were oxidized by DDQ and
subsequently complexed with BF3ÁOEt2, giving a deep purple
b Department of Chemistry, National University of Singapore 3
Science Drive 3, Singapore 117543, MedChem Program of Life
Sciences Institute, National University of Singapore, Singapore.
E-mail: chmcyt@nus.edu.sg
c Department of Pharmacology and Molecular Sciences, Johns
Hopkins University School of Medicine, Baltimore, MD 21502, USA
w Electronic supplementary information (ESI) available: Methods,
supplemental data, and the 1H-, 13C- NMR spectrum. See DOI:
10.1039/c1cc10362h
c
4508 Chem. Commun., 2011, 47, 4508–4510
This journal is The Royal Society of Chemistry 2011

View Article Online
shows none. Notably, 10-fold excess of N-acetylcysteine (NAC)
induced a small peak at 565 nm. Another control experiment
with high concentration of glutathione (up to 5 mM), mimicking
the intracellular environment of live cells, induced the orange
peak (565 nm) as the major emission and a small green peak
(530 nm) as the minor emission. These results might imply the
potential background signal when the labelling is performed
inside live cells. (Fig. S5, ESIw). In time-dependent monitoring
of fluorescence emission at 530 nm with excitation at 480 nm,
only P1 displayed an exclusive increase of emission (Fig. S6,
ESIw), and therefore, P1 only would enable a selective imaging
at this wavelength channel of fluorescence microscope. This
selectivity was further validated by checking fluorescence
colors under UV irradiation with a hand-held UV lamp
(excitation at 365 nm) (Fig. 2b), and these different colors
lasted even after 24 h (data not shown). After we confirmed the
conjugation product of P1 and 4a through the MALDI-TOF/
MS and LC/MS analysis (Fig. S7, ESIw), we designated amino
acid sequences of the model peptide P1 as the ‘‘RC’’ tag.
To evaluate the labelling efficacy of the RC tag for intracellular
proteins, we further modified the non-cell permeable compound
4a. By changing the acids into esters and also by modifying the
para-position of the phenyl ring, several derivatives were synthe-
sized and tested for cell permeability and low cellular background,
yielding compound 4b as the optimum probe (Fig. 3a). First, we
fused the RC tag to a model protein (monomeric Cherry, a red
fluorescent protein) and analyzed the conjugation of 4b to
the RC-tagged Cherry in gel electrophoresis (SDS-PAGE).
Protein extract of the transfected cells showed an apparent green
fluorescence band resulted from the covalent binding of 4b to
RC-tagged Cherry at the expected molecular weight (34 kDa).
Moreover, we confirmed this spectral change is achieved only
when two cysteine residues are faithfully provided, since muta-
tions on either one or two cysteines in the RC tag completely
disable the spectral change (Fig. 3b and Fig. S8, ESIw). However,
when the RC-tagged Cherry was expressed in cells, the green
fluorescent signal provided by the staining with 4b was marginally
strong to be used for clear optical imaging (Fig. S9, ESIw). In
order to enhance the labeling efficiency, we dimerized the RC tag
and doubled the binding motif (–RCXXRC–) in the tag (RC2)
Scheme 1 Synthetic scheme: (a) POCl3, DMF, then aq. NH4OAc;
(b) PPh3QC(O)OR2, DCM; (c) DDQ, DCM followed by DIEA, BF3OEt2.
solid of bodipy diacrylates. Initially, 4a was evaluated for a
spectral change with model peptides in vitro. The model
peptide P1, based on previous literature,19,20 has two cysteines
at the i and i+4 positions (Fig. 2c) and was proven to form an
a-helix by CD spectroscopy. (Fig. S1 and S2, ESIw).
Additionally, an Arg was paired with each Cys, which
would lower the pKa of thiols and increase the nucleophilicity
in physiological pH.21 Two control peptides are P2, with one
pair of Arg-Cys and P3, having no Arg-cys pairs. Compound
4a in a buffered condition (50 mM HEPES, pH 7.4) has an
absorption maximum at 582 nm and emission at 600 nm with
an orange colour fluorescence (Fig. S3, ESIw).
Addition of P1 (10 mM) induced an immediate spectral change
(reaction rate constant, k = 0.1484 sÀ1, Fig. S3, ESIw), resulting in
a large blue shift of emission to green fluorescence (530 nm,
Fig. 2a), and the Arg residues appear to contribute to the rapid
reaction since control peptide without them induced a slower
reaction (k = 0.0882 sÀ1, Fig. S4, ESIw). By contrast, P2 gave a
slight spectral change with a new emission peak at 565 nm,
possibly due to the single conjugation with Arg-Cys, and P3
Fig. 3 (a) Structure of 4b. (b) RC tagged Cherry and three alanine
mutants. Protein extract from the cells, transfected with the vectors and
stained with 4b, were analyzed in the SDS-PAGE and a-myc western
blotting confirmed the expression of tagged proteins. M: protein size
marker. N: no transfection. (c) Conjugation of 4b to RC2 tagged Cherry
by SDS-PAGE and western blotting. (Gel scan Ex/Em = 488/SP 526 nm).
Fig. 2 Fluorescence responses of 4a incubated with P1, P2, P3 or
NAC in 50 mM HEPES (pH 7.4) with excitation at 480 nm.
(a) Emission spectra after incubation for 40 min at RT. (b) A picture of
4a solutions containing the model peptides under irradiation with a hand-
held UV lamp (ex = 365 nm). (c) Peptide sequences of P1, P2, and P3.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4508–4510 4509

View Article Online
system by utilizing
a
fluorescent bodipy compound that
preferably changes the spectral property when it encounters a
designed peptide containing two pairs of Arg-Cys. Dimeric
peptide tag RC2 and its partner compound 4b would benefit the
current protein labeling methods, especially the optical imaging of
specific target protein inside living cells as demonstrated here.
This system would provide a promising tool for optical
imaging of specific protein due to the following advantageous
properties: the small size of tag (less than 5 kDa, composed of
34 amino acids encoded by RC2), independence from other
enzymes or cofactors, applicability to intracellular proteins
through the cell-permeability of probe, optical confirmation of
proper conjugation from the apparent spectral change, stable
binding be analyzed in SDS-PAGE, and negligible toxicity to
the users when using the bodipy-based reagent. Further efforts
will be focused on the fabrication of the compound structure
to enable a faster labeling and washing.
This work was supported by a Young Investigator Award
(R-143-000-353-101) granted to Y.-T.C. from the National
University of Singapore and intramural funding from the
A*STAR Biomedical Research Council.
Fig. 4 Fluorescence microscopic images of 4b labeling on the RC2
tagged recombinant proteins expressed in 293A cells. Cells transfected
with the respective expression vectors (a–e) were stained with 4b
(1 mM, 15 min, 37 1C) and images were taken in live cells. Filters used
for fluorescence imaging were BF—bright field, D—DAPI, F—FITC,
C—Cy5, M—Merged, scale bars—50 mm, HA—hemmaglutinin tag.
Notes and references
1 J. Zhang, R. E. Campbell, A. Y. Ting and R. Y. Tsien, Nat. Rev.
Mol. Cell Biol., 2002, 3, 906–918.
2 J. Wiedenmann, F. Oswald and G. U. Nienhaus, IUBMB Life,
2009, 61, 1029–1042.
3 I. Chen and A. Y. Ting, Curr. Opin. Biotechnol., 2005, 16, 35–40.
4 B. A. Griffin, S. R. Adams and R. Y. Tsien, Science, 1998, 281,
269–272.
(see the ESIw for the amino acids sequences of RC2). As expected
the RC2 tag produced a stronger fluorescent band in gel (Fig. 3c)
than RC and thus, we focused on this RC2 tag for further live cell
imaging. Combining 4b with the RC2 tag, cell images showed
much stronger green fluorescence than with the RC tag by the
expected spectral change and the green fluorescence overlapped
clearly with the red signal from Cherry inside cells (Fig. 4a and
S10, ESIw). Green fluorescence intensity reached saturation within
15 min and washing/further incubation for 30 min to 1 h in fresh
medium significantly helped the removal of background signal of
4b. In addition, when we confined the expression of RC2 tagged
Cherry to the nucleus, by introducing a nuclear localization signal
(NLS)22 to the expression cassette, the green signal from
compound 4b also was strictly localized to the nucleus in
transfected cells in accordance with the red fluorescence of Cherry
(Fig. S10, ESIw).
5 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, 6063–6076.
6 B. R. Martin, B. N. G. Giepmans, S. R. Adams and R. Y. Tsien,
Nat. Biotechnol., 2005, 23, 1308–1314.
7 C. C. Spagnuolo, R. J. Vermeij and E. A. Jares-Erijman, J. Am.
Chem. Soc., 2006, 128, 12040–12041.
8 A. Ojida, K. Honda, D. Shinmi, S. Kiyonaka, Y. Mori and
I. Hamachi, J. Am. Chem. Soc., 2006, 128, 10452–10459.
9 K. A. Kelly, J. Carson, J. R. McCarthy and R. Weissleder, PLoS
One, 2007, 2, e665.
10 I. Chen, M. Howarth, W. Lin and A. Y. Ting, Nat. Methods, 2005,
2, 99–104.
11 M. Fernandez-Suarez, H. Baruah, L. Martinez-Hernandez,
K. T. Xie, J. M. Baskin, C. R. Bertozzi and A. Y. Ting, Nat.
Biotechnol., 2007, 25, 1483–1487.
12 Z. Zhou, P. Cironi, A. J. Lin, Y. Xu, S. Hrvatin, D. E. Golan,
P. A. Silver, C. T. Walsh and J. Yin, ACS Chem. Biol., 2007, 2, 337–346.
13 C. Uttamapinant, K. A. White, H. Baruah, S. Thompson,
M. Fernandez-Suarez, S. Puthenveetil and A. Y. Ting, Proc. Natl.
Acad. Sci. U. S. A., 2010, 107, 10914–10919.
Finally, we tested the performance of this labeling system with
histone H2B, as a real cellular protein. We linked the ‘‘RC2Ámyc’’
casette to the N-terminus of human H2B and fused Cherry to the
C-terminus of it as a marker to check the expression. Probe 4b
successfully stained the tagged H2B in live cells demonstrating
clear nuclear staining in the transfected cells (Fig. 4b). In the
following experiments of H2B without Cherry, 4b and the RC2
tag provided a reliable labeling to the tagged H2B, which is
specific enough to be recognized without the aid of the traking
marker (Cherry) (Fig. 4c). Additionally, the RC2 tag was
compatible with other peptide tags; combination with other small
peptide tags such as the HA tag, myc tag, or hexa-histidine tag,
barely affected the labeling efficiency (Fig. 4d) and the C-terminal
tagging was also available (Fig. S11, see the lane 4, ESIw). In
summary, we developed a novel peptide-based protein labeling
14 S. Girouard, M. H. Houle, A. Grandbois, J. W. Keillor and
S. W. Michnick, J. Am. Chem. Soc., 2005, 127, 559–566.
15 T. Matsumoto, Y. Urano, T. Shoda, H. Kojima and T. Nagano,
Org. Lett., 2007, 9, 3375–3377.
16 R. P. Haugland, Handbook of fluorescent probes and research
chemicals, Molecular probes, Eugene, OR, 2002.
17 J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G. Skene and
J. W. Keillor, J. Am. Chem. Soc., 2007, 129, 11969–11977.
18 J. Guy, R. Castonguay, N. B. Campos-Reales Pineda, V. Jacquier,
K. Caron, S. W. Michnick and J. W. Keillor, Mol. BioSyst., 2010,
6, 976–987.
19 G. Merutka, W. Shalongo and E. Stellwagen, Biochemistry, 1991,
30, 4245–4248.
20 B. A. Griffin, S. R. Adams and R. Y. Tsien, Science, 1998, 281, 269–272.
21 G. H. Snyder, M. J. Cennerazzo, A. J. Karalis and D. Field,
Biochemistry, 1981, 20, 6509–6519.
22 D. Kalderon, B. L. Roberts, W. D. Richardson and A. E. Smith,
Cell, 1984, 39, 499–509.
c
4510 Chem. Commun., 2011, 47, 4508–4510
This journal is The Royal Society of Chemistry 2011