Clickable fluorescent dyes for multimodal bioorthogonal imaging

Article abstract of DOI:10.1039/b917119n

Bioorthogonal ligation with functionalized fluorescent dyes enables visualization of nuclei acids, proteins and metabolites in biological systems. Bright and modular azide- and alkyne-functionalized dyes are therefore needed to expand the fluorescence ima

Full text of DOI:10.1039/b917119n

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COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Clickable fluorescent dyes for multimodal bioorthogonal imaging†‡
Lun K. Tsou, Mingzi M. Zhang and Howard C. Hang*
Received 18th August 2009, Accepted 18th September 2009
First published as an Advance Article on the web 26th October 2009
DOI: 10.1039/b917119n
Bioorthogonal ligation with functionalized fluorescent dyes
enables visualization of nuclei acids, proteins and metabolites
in biological systems. Bright and modular azide- and alkyne-
functionalized dyes are therefore needed to expand the
fluorescence imaging capabilities of bioorthogonal ligation
methods. We describe the concise synthesis of clickable
fluorescent dyes based on 2-dicyanomethylene-3-cyano-2,5-
dihydrofuran fluorophores and demonstrate their utility for
multicolor imaging of azide- and alkyne-modified proteins as
well as FRET studies.
Bioorthogonal ligation of fluorescent dyes and affinity tags onto
biomolecules has afforded new opportunities to investigate cellular
pathways with chemical tools.1,2 Notably, the development of
chemical reporters functionalized with azides or alkynes has
enabled the fluorescence imaging of nucleic acid and protein
synthesis as well as posttranslational modifications (PTMs) using
the Staudinger ligation or click chemistry.1 Moreover, chemical
reporters enable dynamic imaging of biomolecules in cells and
in vivo by pulse-labeling in combination with functionalized-
fluorophores with orthogonal spectral properties.3–6 The ability
to separate the biochemical reactivity of mechanism-based probes
from imaging/affinity reagents also allows the detection of specific
enzymatic activities in living cells.7,8 Development of modular fluo-
rescent dyes compatible with bioorthogonal ligation methods will
expand the repertoire of reagents for diverse imaging applications
using mechanism-based probes or chemical reporters. Herein,
we report a concise synthesis of clickable fluorescent dyes based
on 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) flu-
orophores for multimodal imaging applications using Cu(I)-
catalyzed azide-alkyne cycloaddition (CuAAC).2
For fluorescent detection of alkyne- and azide-labeled pro-
teins, we synthesized a new set of clickable fluorescent dyes
based on DCDHF fluorophores, given their photostability
for single-molecule imaging and tunable red-shifted fluores-
cent emission properties.9–11 Condensation of tert-butyl 4-
formylphenylcarbamate12 and 3-cyano-2-dicyanomethylene-4,5,5-
trimethyl-2,5-dihydrofuran13 afforded compound 1 in 72% yield
(Scheme 1). Removal of t-Boc group and alkylation of com-
pound 2 with 1-bromobutyne gave alkynyl-DCDHF derivative
(alk-CyFur) in 72% yield (Scheme 1). Acylation of DCDHF
Scheme 1 Synthesis of clickable CyFur dyes.
fluorophore 2 with alkyl-azide substrates afforded az-CyFur-1
and az-CyFur-2 in 65% and 57% yield, respectively (Scheme 1
and Supporting Information Fig. S1). While alk-CyFur exhibited
absorption (abs)/emission (em) maxima at 580 nm/640 nm,
acylated-DCDHF derivatives (az-CyFur-1 and az-CyFur-2)
yielded abs/em maxima at 470 nm/580 nm (Fig. 1A and Support-
ing Information Fig. S1B and S1C). The differential fluorescence
properties of N-acylated compared to N-alkylated DCDHF
derivatives are consistent with previous studies demonstrating
that capping of the aniline functionality quenches the red-shifted
emission of DCDHF fluorophore 2.9–11 The quantum yields of
all three clickable CyFur dyes are similar to previously described
The Laboratory of Chemical Biology and Microbial Pathogenesis, The
Rockefeller University, New York, NY 10065, USA. E-mail: hhang@mail.
rockefeller.edu
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on protein labelling and chemical proteomics. Guest editors: Ed Tate
and Matthew Bogyo.
‡ Electronic supplementary information (ESI) available: Synthetic meth-
ods, supplementary figures (Fig. S1–Fig. S4) and spectra. See DOI:
10.1039/b917119n
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unreacted 1:1 mixture of az-CyFur-1:alk-CyFur afforded similar
spectral properties to the dyes alone (Fig. 1B and Supporting
Information Fig. S3). These results demonstrate that differential
modification of DCDHR fluorophores provides clickable red-
shifted fluorescent dyes with tunable spectral properties that can
also function as donor and acceptor FRET pairs.
To explore the utility of these clickable CyFur dyes for
imaging azide- and alkyne-modified proteins in vitro and in cells,
we employed azido- and alkynyl-fatty acids chemical reporters
to metabolically label N-myristoylated and S-palmitoylated
proteins.14,15 Jurkat T cell lysates labeled with azido-fatty acids
(az-12, az-15) were subsequently reacted with alk-CyFur or
alk-Rho via the CuAAC and separated by gel-electrophoresis
(Fig. 2). Labeled proteins were visualized by in-gel fluorescence
scanning at various excitation/emission channels to detect CyFur-
and Rho-modified proteins. Fluorescence imaging at 633 nm
excitation and 670 nm emission allowed selective detection of alk-
CyFur-labeled proteins analogous to the profile of fatty-acylated
proteins visualized with alk-Rho (excitation 532 nm/emission 580
nm) (Fig. 2).14 Similarly, Az-CyFur-1 allows selective fluorescent
imaging of alk-12-labeled cell lysates (Supporting Information
Fig. S4). The near-infrared fluorescent properties of alk-CyFur
enabled profiling of azide-modified proteins in gels with minimal
spectral overlap to acylated-CyFur and Rho fluorophores (Fig. 2
and Supporting Information Fig. S4).
The clickable and environmentally-sensitive CyFur dyes also
allow robust fluorescent imaging of azide- and alkyne-labeled
proteins in cells. HeLa cells were metabolically labeled with
az-12 or alk-12, fixed/permeabilized, reacted with alk-CyFur,
az-CyFur-1 or az-Rho and imaged as previously described.14
Az-12 and alk-12-labeled HeLa cells yielded significantly higher
levels of fluorescent labeling with alk-CyFur and az-CyFur-1,
respectively, compared to DMSO control using settings for
fluorescein dyes (excitation 488 nm/emission 560 nm) (Fig. 3A).
In contrast, image settings for red-emitting Cy5 dyes (excitation
630 nm/emission 650 nm) enables visualization of alk-CyFur-
labeled cells with no crosstalk to az-CyFur-labeled cells (Fig. 3B).
CyFur-labeled proteins were concentrated within intracellular
membranes and excluded from the nuclei, which is in accordance
with previous imaging studies of fatty-acylated proteins14,16 and
Fig. 1 Spectral properties of clickable CyFur dyes. (A) Emission spectra
of clickable CyFur dyes. Emission spectra were acquired at excitation of
470 nm and 580 nm for az-CyFur-1 (solid line) and alk-CyFur (dash line),
respectively. (B) Emission spectra of alk-CyFur (¥), az-CyFur-1 (D) and
fluorescent adduct 3 (ꢀ), excitation at 410 nm.
Table 1 Optical properties of clickable CyFur dyes
a
a
b
c
lmax (nm)
lmax (nm)
emax (M-1cm-1)
UF
alk-CyFur
az-CyFur-1
az-CyFur-2
580
470
470
640
580
580
33 933
20 533
12 100
0.0147
0.0067
0.0027
a Spectra were obtained in DMSO. b Measurements were done in
MeOH. Extinction coefficients at 470 nm for az-CyFur-1, az-CyFur-2 and
at 580 nm for alk-CyFur are averaged over three independent experiments.
c Quantum yields referenced against cresyl violet (UF = 0.54 in MeOH).
Alk-CyFur was excited at 580 nm. Both az-CyFur-1 and az-CyFur-2
were excited at 470 nm. Experimental details are described in Supporting
Information.
N-alkylated and quenched DCDHF derivatives (Table 1).9–11 In
accordance with other reported DCDHF-based fluorophores,9–11
the fluorescence emission of CyFur dyes increases significantly in
a more viscous or rigid environment such as glycerol (Supporting
Information Fig. S2). Based on the observed absorption and
emission spectra of az-CyFur-1 and alk-CyFur, dimerization of
these two fluorophores was predicted to undergo Fo¨rster reso-
nance energy transfer (FRET). Indeed, coupling of az-CyFur-1
and alk-CyFur via CuAAC afforded clicked fluorophore 3, which
exhibited FRET between the acylated- and alkylated-DCDHF
derivatives (Fig. 1B). Excitation of fluorophore 3 at 410 nm
resulted in strong fluorescence emission at 640 nm, whereas an
Fig. 2 In-gel fluorescence imaging of azido-fatty acid (az-FA)-modified proteins from metabolically labeled Jurkat T cells after bioorthogonal ligation
with clickable CyFur dyes.
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reporter labeled cells visualized with clickable CyFur dyes yielded
more punctate intracellular membrane structures compared to
Rho dyes, which may be due to solvatochromism for hydrophobic
environments reported for DCDHF fluorophores.9,11
In summary, we have synthesized a new set of clickable fluores-
cent dyes based on the DCDHF fluorophores for bioorthogonal
ligation applications. Synthesis of these clickable CyFur dyes is ef-
ficient, modular and scalable, which enables facile access to azide-
or alkyne-modified fluorophores with different spectral properties
by alkylation or acylation of single common DCDHF fluorescent
precursor. The in-gel fluorescence scanning and cellular imaging
studies of azide- and alkyne-modified fatty-acylated proteins
showcase the utility of the clickable CyFur dyes for imaging
endogenously expressed proteins. The fluorescent property of alk-
CyFur complements previously reported clickable near-infrared
dyes17–19 and should facilitate dual imaging of chemical reporters
as well as in vivo imaging applications in the future. Additionally,
the spectral overlap of the acylated- and alkylated-CyFur dyes
yields useful donor and acceptor pairs for further FRET studies.
The clickable CyFur dyes reported here provide alternative and
readily accessible reagents for multimodal fluorescence imaging
applications using bioorthogonal chemical probes/reporters to
study cellular pathways.
Acknowledgements
The authors thank Dr. Anuradha Raghavan, Guillaume Charron
and Paul Dossa for providing fatty acid chemical reporters and
clickable Rho dyes. L. K. T. acknowledges support from The
Rockefeller University Marie-Jose´e and Henry Kravis Postdoc-
toral Fellowship. M. M. Z. is supported by A*STAR, Singapore.
H. C. H. acknowledges support from The Rockefeller University,
Irma T. Hirschl/Monique Weill-Caulier Trust and Ellison Medical
Foundation.
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(alk-12)-fatty acid labeled HeLa cells after bioorthogonal ligation with
clickable dyes. (A) Imaging with 488 nm excitation and 560 nm emission
long-pass filter. Top panel: az-CyFur-1-labeled proteins. Bottom panel:
alk-CyFur-labeled proteins. (B) Imaging with 633 nm excitation and
646-753 nm emission filter. Top panel: az-CyFur-1-labeled proteins.
Bottom panel: alk-CyFur-labeled proteins. (C) Imaging of az-Rho-labeled
proteins. 543 nm excitation and 560-615 nm emission filter. DAPI imaging
in all panels was performed by 405 nm excitation and 420-480 nm emission
filter.
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