Bioorthogonal turn-on probes for imaging small molecules inside living cells

Article abstract of DOI:10.1002/anie.200906120

(Figure Presented) Glowing tags: A series of activatable ( urn-on ) tetrazine-conjugated fluorescent probes was developed, which react rapidly in an inverse-electron-demand [4+2] cycloaddition with strained dienophiles such as trans-cyclooctene, thereby strongly increasing the fluorescence intensity (see picture), The novel turn-on probes were applied for intracellular livecell imaging of a microtubuli-binding trans-cyclooctene modified taxol,

Full text of DOI:10.1002/anie.200906120

Angewandte
Chemie
DOI: 10.1002/anie.200906120
Labeling of Live Cells
Bioorthogonal Turn-On Probes for Imaging Small Molecules inside
Living Cells**
Neal K. Devaraj, Scott Hilderbrand, Rabi Upadhyay, Ralph Mazitschek, and Ralph Weissleder*
Bioorthogonal “click” reactions are now widely used in
chemical biology for many applications such as activity-based
protein profiling, monitoring cell proliferation, generating
novel enzyme inhibitors, monitoring the synthesis of newly
formed proteins, identifying protein targets, and studying
glycan processing.[1,2] Arguably, the most fascinating applica-
tions involve using these bioorthogonal chemistries to assem-
ble molecules in the presence of living systems such as live
cells or even whole organisms.[3,4] These latter applications
require that the chemistry does not employ toxic metal
catalysts and maintains kinetics that enable fast reaction to
occur with micromolar concentrations of reagents in a time
span of minutes to hours. To fulfill these criteria, various
“copper-free” click reactions have been reported, such as the
strain-promoted azide–alkyne cycloaddition and the Stau-
dinger ligation, to react with azides on the surface of live cells
both in culture and with in vivo systems such as mice and
zebrafish.[4] However, to date, the application of “click”
reactions in living systems has been largely limited to
extracellular targets.[5] The reasons for this are likely several.
In addition to fulfilling the stability, toxicity, and chemo-
selectivity requirements of “click” chemistry, intracellular
live-cell labeling requires reagents that can pass easily
through biological membranes and kinetics that enable
rapid labeling even with the low concentrations of agent
that make it across the cell membrane. Additionally, a
practical intracellular bioorthogonal coupling scheme would
need to incorporate a mechanism by which the fluorescent tag
increases in fluorescence upon covalent reaction to avoid
visualizing accumulated but unreacted imaging agent (i.e.
“background”). Such activatable “turn-on” probes would
significantly increase the signal-to-background ratio, which is
particularly relevant to imaging targets inside living cells since
a stringent washout of unreacted probe is not possible.
cycloaddition or Staudinger ligation coupling reactions.[6]
Most of these strategies utilize a reactive group intimately
attached to the fluorophore thus necessitating synthesis of
new fluorophore scaffolds or take advantage of a FRET
(fluorescence resonant energy transfer) based activation
requiring appendage of an additional molecule that can act
as an energy-transfer agent. Furthermore, most probes
employing these popular coupling schemes have not been
used to label intracellular targets in live cells. Here we report
a series of activatable “turn-on” tetrazine-linked fluorescent
probes, which react rapidly through an inverse-electron-
demand cycloaddition with strained dienophiles such as trans-
cyclooctene. Upon cycloaddition, the fluorescence intensity
increases substantially, in some cases by approximately 20-
fold. This fluorescence “turn-on” significantly lowers back-
ground signal. We have used these novel probes for live-cell
imaging of a trans-cyclooctene-modified taxol analogue
bound to intracellular tubules. The high reaction rate coupled
with fluorescence activation makes this a nearly ideal method
for revealing target molecules inside living cells.
Recently, we and others have explored conjugation
reactions using inverse-electron-demand Diels–Alder cyclo-
additions between tetrazines and highly strained dienophiles
such as norbornene and trans-cyclooctene.[7–9] We have shown
that a novel asymmetric tetrazine is quite stable in water and
serum and can react with trans-cyclooctene at rates of
approximately 103 mÀ1 sÀ1 at 378C.[9] This extremely high rate
constant allowed the labeling of extracellular targets at low
nanomolar concentrations of tetrazine labeling agent, con-
centrations that are sufficiently low to enable real-time
imaging of probe accummulation. Previous work from our
group relied on tetrazines conjugated to highly charged
carbocyanine-based near-IR-emitting fluorophores. In our
efforts to explore the utility of this reaction for intracellular
labeling, we conjugated 3-(4-benzylamino)-1,2,4,5-tetrazine
to the succinimidyl esters of visible-light-emitting boron-
dipyrromethene (BODIPY) dyes. BODIPY dyes are
uncharged and lipophilic and for these reasons have seen
use in intracellular applications.[10] We also wondered whether
or not visible fluorophores would show electronic interactions
with the tetrazine chromophores, which have absorption
maxima at 500–525 nm. In fact, the tetrazine BODIPY
conjugates (e.g. 1; see Figure 1a) exhibited strongly reduced
fluorescence compared to the parent succinimidyl esters of
the fluorophores. Upon reaction with a strained dienophile
such as trans-cyclooctenol (2) or norbornene the fluorescence
was “switched” back on.
In previous years a number of elegant probes have been
introduced whose fluorescence increases after azide–alkyne
[*] Dr. N. K. Devaraj, Dr. S. Hilderbrand, R. Upadhyay, Dr. R. Mazitschek,
Prof. R. Weissleder
Center for Systems Biology, Massachusetts General Hospital
Richard B. Simches Research Center
185 Cambridge Street, Suite 5.210, Boston, MA 02114 (USA)
Fax: (+1)617-643-6133
E-mail: rweissleder@mgh.harvard.edu
Homepage: http://csb.mgh.harvard.edu
[**] We thank Dr. Justin Ragains for helpful advice and Alex Chudnovsky
for assistance with tissue culture. This research was supported in
part by NIH grants U01-HL080731, T32-CA79443, P50-CA86355,
and RO1-EB010011.
To explore the generality of this phenomenon we reacted
the benzylamino tetrazine with commercially available succi-
nimidyl esters of 7-diethylaminocoumarin-3-carboxylic acid,
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906120.
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fluorescence quenching, which was restored after reaction
with dienophiles. Quenching of the fluorophore by the
tetrazine is wavelength-dependent. Green- and red-emitting
tetrazine dyes showed fluorescence enhancements upon
cycloaddition of approximately 15- to 20-fold in PBS (phos-
phate-buffered saline). In contrast, the shorter-wavelength-
emitting tetrazine–coumarin showed only a threefold en-
hancement. The green- and red-emitting tetrazine dyes also
show strong fluorogenic responses in 100% fetal bovine
serum (see the Supporting Information, Table S2). Near-IR-
emitting dyes such as our previously used carbocyanine
tetrazine–VT680 as well as tetrazine–BODIPY 650-665 (data
not shown) were not quenched, explaining why this phenom-
enon was not observed in previous studies.
We speculate that the mechanism of fluorescence quench-
ing may be due to resonant energy transfer between the
fluorescent chromophore and the tetrazine, which has a
visible absorbance maximum at 515 nm.[8] This would explain
the wavelength dependence of the quenching. Another
possibility could be that the quenching is the result of
photoinduced electron transfer (PET) between the excited
fluorophore and a potential tetrazine acceptor. Tetrazines are
well known to be an electron-poor class of heterocycles, hence
their utility in inverse-electron-demand cycloadditions. The
PET-based mechanism would be reminiscent of the well-
known quenching of fluorophores by electron-poor nitro-
aromatic compounds.[11] The fluorescence signals from the
quenched tetrazine–BODIPY FL and tetrazine–BODIPY
TMR-X conjugates vary by less than 5% between pH 9 and 3,
indicating the quenching mechanism is not pH dependent. It
is important to note that these fluorogenic compounds can be
formed from commercially available fluorophores and that
the tetrazine appears to be a sufficiently strong quencher that
does not require intimate connection to the fluorophore and
can achieve a quenching effect even when separated by
aliphatic spacers. We are currently investigating the mecha-
nism of quenching and designing next-generation tetrazine–
fluorophores that show further enhancements of fluorescence
upon cycloaddition.
Figure 1. a) Tetrazine–BODIPY FL (1) reacts rapidly with trans-cyclo-
octenol (2) in an inverse-electron-demand Diels–Alder cycloaddition to
form isomeric dihydropyrazine products 3 upon extrusion of dinitrogen
and rearrangement. b) Emission spectra of various tetrazine probes
(black lines) and the corresponding dihydropyrazine products (dashed
blue lines). Inset images compare the visible fluorescence emission of
the tetrazine probes (left cuvettes) to their corresponding dihydropyr-
azine products (right cuvettes) under excitation from a handheld UV
lamp.
BODIPY FL, BODIPY TMR-X, Oregon Green 488 (Invi-
trogen), and Vivotag-680 (VT680, Visen Medical). Figure 1b
shows the fluorescence emission spectra of selected dye–
tetrazine conjugates before and after cycloaddition to trans-
cyclooctenol (2). Table 1 lists the photophysical properties of
the dyes before and after reaction. For all dyes with emission
between 400–600 nm conjugation to the tetrazine caused
Although we envision that the featured fluorogenic
probes could have many applications, one use that would
immediately benefit from a fluorogenic probe is the detection
of target molecules inside live cells. This will allow application
for determining the subcellular distribution of pharmaceutical
and metabolic analogues containing
a dienophile tag. To test if the
Table 1: Photophysical properties of the dyes before and after reaction with trans-cyclooctene (TCO).[a]
fluorogenic tetrazines reported
here are relevant for imaging intra-
cellular molecular targets, we chose
Dye
labs
[nm][b]
lem
[nm][b]
Quantum
yield w/o
TCO[c]
Quantum
yield with
TCO[d]
Increase
in fluorescence
dienophile-modified
paclitaxel
tetrazine–coumarin
430
505
495
543
669
480
512
523
573
687
0.01
0.02
0.04
0.02
0.16
0.03
0.24
0.82
0.40
0.16
3.3-fold
15.0-fold
18.5-fold
20.6-fold
1.0-fold
(taxol, 4) as a model system. Taxol
was selected because of its tremen-
dous clinical impact, the large body
of prior work that serves as refer-
ence, and based on its well-studied
ability to stabilize microtubules,
providing us a well-defined intra-
cellular structure to image.[12–15] The
trans-cyclooctene taxol derivative
tetrazine–BODIPY FL
tetrazine–Oregon Green 488
tetrazine–BODIPY TMR-X
tetrazine–VT680
[a] All measurements are in PBS, pH 7.4 (dye concentration 1 mm). Quantum yield measurements are in
triplicate with fluorescein (in water, pH 10), Rhodamine 6G (in EtOH), or Cy5.5 in PBS as standards.
[b] labs and lem are before addition of trans-cyclooctene (10 mm), but there are no significant changes in
these values after trans-cyclooctene addition. [c] Quantum yields for the tetrazine–fluorophore
conjugates. [d] Quantum yields for the dihydropyrazine fluorophore products.
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Figure 2. a) Structure of taxol (4) and trans-cyclooctene-modified taxol
(5, TCO-taxol). b) Comparison of the ability of 10 mm taxol (4), trans-
cyclooctene taxol (5), and a DMSO control to polymerize tubulin in
the absence of GTP (polymerization assayed through absorbance at
350 nm). Note that trans-cyclooctene taxol (5) promotes polymeri-
zation similar to taxol and significantly better than a DMSO control.
c) Microtubule bundles formed in the presence of trans-cyclooctene
taxol (5) treated with tetrazine–BODIPY FL (1) and visualized by
fluorescence microscopy.
Figure 3. a) Confocal microscopy of PtK2 cells after treatment with
1 mm trans-cyclooctene taxol (5) followed by 1 mm tetrazine–BODIPY FL
(1). Scale bar: 30 mm. b) Expansion of the section indicated by the
dashed box reveals that tubular structures are clearly stained. c) Con-
focal microscopy of PTK2 cells after treatment with 1 mm taxol (4)
followed by 1 mm tetrazine–BODIPY FL (1). Scale bar: 50 mm. d) Con-
focal microscopy of PTK2 cells after treatment with 1 mm trans-cyclo-
octene taxol (5) followed by 1 mm tetrazine–VT680. Scale bar: 50 mm.
(5, Figure 2) was synthesized by coupling trans-cyclooctene
succinimidyl carbonate to 7-b-alanyl taxol by reported
procedures.[13] The dienophile was introduced in the C7
position since prior structure–activity relationship studies
have established that modifications at the C7 position do not
significantly affect the biological activity of taxol.[13,15,16] trans-
Cyclooctene taxol (5) rapidly reacts with our tetrazine probes
forming isomeric dihydropyrazine products which can be
detected by LC-MS (see the Supporting Information).
ent. This staining pattern corresponds to immunostaining
using anti-a-tubulin (Figure S2 in the Supporting Informa-
tion). Taxol is known to bind the microtubular networks of
cells and there are several reports of fluorescent taxol
derivatives for imaging microtubular networks.[12,13,15] Control
experiments employing tetrazine–BODIPY FL (1) alone or
with unmodified taxol (4) yielded minimal fluorescence
background signal and demonstrate that there is little non-
specific or background turn-on and that the images result
from the specific tetrazine trans-cyclooctene cycloaddition
reaction (Figure 3c). Furthermore, cells treated with trans-
cyclooctene modified taxol (5) followed by highly charged
non-membrane permeable tetrazine probes such as the
sulfonated tetrazine–VT680 showed very little staining and
an absence of tubular structures, giving further evidence that
tetrazine–BODIPY FL (1) is able to penetrate the cell
membrane and label trans-cyclooctene (2) located within the
cell (Figure 3d).[8,9] Attempts to label cells with fluorescent
tetrazine–taxol conjugates led to nonspecific weak staining
signals (Figure S3 in the Supporting Information).
In conclusion we have developed a robust method for the
bioorthogonal tagging and imaging of targets inside living
cells. Fluorogenic tetrazine probes react specifically and
rapidly with strained dienophiles such as trans-cyclooctene
(2). The ability of these probes to show fluorescence “turn-
on” upon cycloaddition is a major advantage especially for
applications where probe washout is not desired or possible.
We imagine that this method will be useful for applications
requiring the imaging of the intracellular distribution of
tagged small molecules. We are currently exploring imaging
To test the activity of the trans-cyclooctene taxol analogue
(5), we focused on the well-established ability of taxol to
polymerize tubulin in the absence of GTP (guanosine
triphosphate).[17] Optical density measurements at 350 nm
(Figure 2b) were used to determine the degree of tubulin
polymerization after exposure of tubulin monomer to taxol
(4), trans-cyclooctene taxol (5), and a DMSO (dimethyl
sulfoxide) control. Both native taxol (4) and trans-cyclo-
octene taxol (5) induce polymerization compared to a DMSO
control. trans-Cyclooctene taxol induced tubule bundles were
visualized by subsequent staining with tetrazine fluorophore
probes such as tetrazine–BODIPY FL (1), which covalently
couples to the microtubule-bound trans-cyclooctene taxol
molecules, yielding brightly fluorescent tubule structures that
can be imaged by fluorescence microscopy (Figure 2c).
For live-cell studies, PtK2 kangaroo rat kidney cells were
incubated in cell media containing 1 mm trans-cyclooctene
taxol (5) for 1 hour at 378C. PtK2 cells are commonly used in
microtubule studies due to their flattened morphology.[15]
After washing with media three times, the cells were exposed
to media containing 1 mm tetrazine–BODIPY FL (1) for
20 min at room temperature. The cells were then washed and
imaged on a confocal microscope (Figure 3). Intracellular
structures reflecting tubule networks become readily appar-
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Received: October 30, 2009
Revised: January 20, 2010
Published online: March 19, 2010
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