BODIPY-tetrazine derivatives as superbright bioorthogonal turn-on probes

Article abstract of DOI:10.1002/anie.201301100

The fastest and the brightest: A new design that intimately connects tetrazine to a BODIPY fluorophore enables exceptionally efficient energy transfer and quenching. Upon reaction of the tetrazine, the brightness of the fluorophore increases more than a thousand-fold, which is a fluorogenic activation up to two orders of magnitude greater than previously described. Copyright

Full text of DOI:10.1002/anie.201301100

Angewandte
Chemie
Communications
Fluorogenic Probes
J. C. T. Carlson, L. G. Meimetis,
S. A. Hilderbrand,
R. Weissleder*
&&&& — &&&&
BODIPY–Tetrazine Derivatives as
Superbright Bioorthogonal Turn-on
Probes
The fastest and the brightest: A new
design that intimately connects tetrazine
to a BODIPY fluorophore enables excep-
tionally efficient energy transfer and
quenching. Upon reaction of the tetra-
zine, the brightness of the fluorophore
increases more than a thousand-fold,
which is a fluorogenic activation up to
two orders of magnitude greater than
previously described.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
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DOI: 10.1002/anie.201301100
Fluorogenic Probes
BODIPY–Tetrazine Derivatives as Superbright Bioorthogonal Turn-on
Probes**
Jonathan C. T. Carlson, Labros G. Meimetis, Scott A. Hilderbrand, and Ralph Weissleder*
Visualizing biomolecular processes has been enhanced by
combining fluorophores with bioorthogonal chemistry, result-
ing in new tools to study the complex biochemical milieu of
living cells and organisms.[1] The resulting probes have been
applied to image glycosylation and phospholipid uptake,[2,3]
cellular proteins,[4–6] and intracellular drug distribution.[7] In
some of these applications, the capacity of in situ chemical
conjugation has been paired with fluorogenic turn-on,
whereby fluorophore emission increases upon reaction with
its bioorthogonal counterpart (“turn-on” probes).[8] This has
the very attractive feature of reducing background fluores-
cence when doing in vivo imaging, potentially allowing real-
time imaging, without washing or clearance steps. For optimal
performance, such a fluorogenic real-time reporter should
1) be catalyst-free (to minimize toxicity and the need for
multiple reaction partners), 2) have fast reaction kinetics (to
allow efficient imaging and temporal resolution), and 3) be
highly fluorescent after turn-on (to maximize signal and
minimize background). Existing methods that exploit azide–
phosphine, azide–alkyne, or inverse-electron-demand Diels–
Alder tetrazine cycloadditions have only partially satisfied
these criteria, resulting in subsets of probes with good
fluorescence turn-on ratios but slow reaction kinetics and
another subset with agreeably fast kinetics but only modest
turn-on. What has been missing however, are exceptionally
bright, fast, and biocompatible (water-soluble, cell-mem-
brane-permeable, nontoxic) probes with fluorescence turn-
on ratios exceeding 10-fold.
fluorophore pairs, which were chosen for their ready synthetic
accessibility, are quenched with moderate efficiency, yielding
turn-on ratios in the order of 10–20-fold after reaction with
dienophile targets.[3–5,8] Although intriguing applications have
been demonstrated, the limited turn-on ratios almost always
result in native background during imaging applications. In
our estimation, a turn-on ratio of 102 would be preferable for
robust utility in cellular imaging applications, and a ratio of
103 may be necessary for low-abundance targets or super-
resolution imaging.[9] Mechanistic observations have sug-
gested that quenching in bichromophoric fluorophore–tetra-
zines occurs through Fçrster resonance energy transfer
(FRET), offering an initial theoretical framework for efforts
to optimize turn-on.[8,10] Although its relatively weak visible-
light absorbance inherently limits the range of tetrazine as
a FRETacceptor, Fçrster theory dictates that energy-transfer
efficiency will be crucially dependent upon interchromophore
distance (varying as r6) and upon transition-dipole alignment,
which are both parameters that can be optimized.[11] As an
alternative way of designing more efficient turn-on probes,
one might consider adapting through-bond energy transfer
(TBET) for fluorescence quenching. With these goals in
mind, we synthesized a series of new bioorthogonal boron
dipyrromethene
(BODIPY)–tetrazine
derivatives
(Scheme 1). These structures enhance spatial donor–acceptor
proximity, provide predictable donor–acceptor transition-
dipole orientation, and afford the possibility of accessing
alternate modes of fluorescence quenching.
Previously described tetrazine-based probes achieve their
fluorogenic turn-on by a unique mechanism, in which the
tetrazine (Tz) chromophore is both quencher and bioorthog-
onal reactant.[8] In published studies, flexibly linked Tz–
In the course of selecting a design for these cassettes, we
noted that a crystal structure of p-cyanophenyl-BODIPY
1 exhibited a 908 rotation between the BODIPY and the
phenyl ring, a conformation that prevents p-system conjuga-
tion.[12] We hypothesized that a tetrazine attached to this
phenyl ring would thus be spectrally decoupled from the
fluorophore and capable of efficient FRET-based quenching.
We therefore utilized recently reported conditions for tran-
sition-metal-catalyzed tetrazine formation to directly install
tetrazines on nitrile-derivatized BODIPY scaffolds
(Scheme 1).[13] On treatment of 1 with hydrazine, formami-
dine acetate, and nickel triflate (50 mol%), with DMF as
a cosolvent, the major product observed was the dipyrro-
methene core of 1. When zinc triflate was used, however, we
observed formation of the desired H-tetrazine 2a. The
fluorescence enhancement observed on reaction of 2a with
trans-cyclooctenol (TCO) in water (Scheme 1) was character-
istically fast and of significantly greater magnitude than for
reported Tz–fluorophore derivatives, with a turn-on ratio of
several hundred fold (Figure 1c). However, it proved chal-
lenging to completely eliminate traces of bright fluorescent
impurities from the reaction products. We therefore turned to
[*] Dr. J. C. T. Carlson,[+] Dr. L. G. Meimetis,[+] Dr. S. A. Hilderbrand,
Prof. R. Weissleder
Center for Systems Biology, Massachusetts General Hospital
185 Cambridge Street, Boston, MA 02114 (USA)
E-mail: rweissleder@mgh.harvard.edu
Prof. R. Weissleder
Harvard Medical School
200 Longwood Avenue, Boston, MA 02115 (USA)
[+] These authors contributed equally to this work.
[**] Part of this work was supported by NHI RO1EB010011 and
2P50A086355. J.C. was supported by a DFCI-MGH Hematology
Oncology Fellowship. We thank Prof. Ralph Mazitschek for many
insightful discussions, Dr. Katy Yang for cell culture assistance, Dr.
Sarit Agasti for the gift of reagents, Alex Zaltsman for microscopy
assistance, and Dr. Eszter Boros for her assistance with NMR
spectroscopy. BODIPY=boron dipyrromethene.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301100.
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the synthesis of methyltetrazine 2b, speculating that
enhanced Tz stability would facilitate purification and precise
quantification.[14] With zinc triflate (50 mol%), hydrazine,
and acetonitrile at 608C, we observed formation of both 2a
and 2b in approximately equal quantities (Scheme 1a). H-
tetrazine formation was unexpected, since it has not been
reported under these reaction conditions, but is likely derived
from the DMF cosolvent. In sharp contrast to the parent
compound 1, both 2a and 2b are modestly soluble in aqueous
solution and almost completely nonfluorescent. The methyl-
tetrazine derivative 2b could be isolated free of any bright
fluorescent impurities (see the Supporting Information).
Reaction of 2b with TCO in acetonitrile and in water allowed
us to measure turn-on ratios of 340 and 900-fold, respectively
(Table 1).
Table 1: Quantum yield and fluorogenic activation.
Probe F w/TCO F w/TCO Fluorescence
in water[a] in MeCN[a] increase in
water[b]
Fluorescence
increase in
MeCN[b]
2b
4b
5b
0.80
0.73
ND[c]
0.23
0.58
0.22
900-fold
1600-fold
ND[c]
340-fold
1100-fold
120-fold
Scheme 1. a) Initial synthesis of BODIPY–p-Tz derivatives (2a, 2b),
schematic of the fluorogenic reaction with trans-cyclooctenol (TCO),
and observed turn-on ratio in water. Reaction conditions: 1.) Zn(OTf)2,
NH2NH2, acetonitrile, 608C, 18 h; 2.) NaNO2; 3.) HCl (1m). b) Syn-
thesis of BODIPY–m-Tz derivatives (4a, 4b) and fluorogenic turn-on in
water; reaction conditions as in (a). c) Equatorial BODIPY–Tz deriva-
tives (5a, 5b) and observed turn-on upon reaction with TCO in
acetonitrile. In (a–c) the turn-on ratios for the methyltetrazines are
given below the fluorescent product.
[a] Quantum yield for the dihydropyridazine product after complete
reaction of the indicated compound with TCO; fluorescein in NaOH
(0.1m, pH 13, F=0.925) was used as the standard. [b] Increase in peak
fluorescence intensity at reaction completion; for experiments in water,
BODIPY-Tz (400 nm), and TCO (1 mm) were used. [c] Compound 5b is
insufficiently soluble in water for this determination.
To explore configurational effects on turn-on efficiency,
we moved the position of the tetrazine from para- to meta- on
the phenyl ring. Structural modeling estimated that this one-
bond shift reduces the interchromophore distance (center to
center) from 8.4 to 7 ꢀ. Condensation of dimethylpyrrole
with 3-formylbenzonitrile, followed by routine oxidation with
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), deproto-
nation with Hꢁnigꢂs base, and boron complexation afforded
the m-cyanophenyl-BODIPY 3 (see the Supporting Informa-
tion). From this precursor, meta-Tz derivatives 4a and 4b
were synthesized by using the same reaction conditions as for
2. Impressively, these compounds exhibit even greater
fluorogenic turn-on ratios, reaching 1100-fold in acetonitrile
and 1600-fold in water for 4b (Table 1). In Figure 1a plots of
the normalized fluorescence emission spectrum of 4b in
acetonitrile before and after reaction with TCO are shown;
a logarithmic scale is necessary to visualize both spectra
simultaneously. The fluorescence time courses for represen-
tative compounds in acetonitrile appear in Figure 1b.
The exceptional fluorogenic turn-on ratios of 2 and 4—as
much as 100-fold greater than of flexibly linked fluorophore–
Tz conjugates—suggested that FRET may not be the sole
quenching mechanism for these compounds. Burgess, Topp,
and co-workers observed efficient through-bond energy
transfer in cassettes that linked anthracene to BODIPY in
a similar scaffold.[15] Coumarin–BODIPY and quinoline–
BODIPY derivatives that exploit TBET have also been
reported.[16] In each of these cases, excitation energy transfers
Figure 1. a) Fluorescence emission spectra for compound 4b in aceto-
nitrile at baseline (black) and after addition of TCO (green); excitation
at 490 nm. b) Normalized fluorescence turn-on after addition of TCO
(240 mm) to a solution of the indicated fluorophore (1 mm) in acetoni-
trile. c) Equimolar solutions of compound 2a (left) and 2a plus TCO
(right) under excitation by a handheld UV lamp. d) 3D model of
compound 4a, illustrating a twisted phenyl linker between the BODIPY
and tetrazine chromophores and the orientation of the donor and
acceptor transition dipoles (gray arrows).
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from the attached donor chromophore into the BODIPY
acceptor, whereas TBET in the reverse direction is the
potential mechanism here. Redox-based quenching, such as
through photoinduced electron transfer (PET) from the
excited BODIPY to the relatively electron-poor tetrazine
ring, was judged unlikely, because the fluorescence emission
intensity of 4b was found to be independent of solvent
polarity (see the Supporting Information).[17]
The architectural hallmark of TBET is a conjugated, but
not coplanar, p system, in which steric factors enforce a twist
in the interchromophore linkage.[18] A three-dimensional
structure of compound 4a (Figure 1d) illustrates these
characteristic traits. Also depicted in the Figure are the
known orientation of the BODIPY transition dipole, and the
likely orientation of the tetrazine S0!S1 (n!p*) absorption
dipole, which is perpendicular to the plane of the Tz ring, as
inferred from the known S0!S1 dipole of s-tetrazine.[19,20]
Limited experimental data on the conformational behavior of
aryl tetrazines indicate a preference for coplanarity, as
depicted, which brings the transition dipoles into parallel
alignment.[19] Several practical features distinguish TBET
from FRET, including insensitivity to spectral overlap (allow-
ing donor chromophores to excite significantly red-shifted
acceptors), decreased dependence on donor acceptor dipole
alignment, and energy transfer kinetics (TBET is substantially
faster, owing to the lack of a strict orientational require-
ment).[18] Instead of ultrafast spectroscopy studies, we chose
to perturb the donor–acceptor geometry of the BODIPY–Tz
derivatives and test the effect on fluorescence quenching.
Compounds 5a and 5b were synthesized starting from 2-
iodo-pentamethyl BODIPY[21] (see the Supporting Informa-
tion). An ortho-methyl group was added to enhance twist (i.e.
minimize conjugation) of the phenyl linker. In these deriva-
tives, the tetrazine and BODIPY transition dipoles are
perpendicular irrespective of intramolecular bond rotation,
a configuration in which Fçrster theory predicts FRET
efficiency to be near zero.[11] Elegant confirmation of this
prediction has been made with nonconjugated anthracene–
porphyrin cassettes.[22] Upon reaction of 5b with TCO in
acetonitrile, we observed a turn-on ratio of 120-fold. This
result stands in sharp contrast to both the Fçrster theory
prediction and the exceptionally efficient transfer observed
for 2 and 4. It is, however, consistent with the results observed
for BODIPY–anthracene TBET cassettes, in which efficient
energy transfer was observed in the perpendicular dipole
configuration, but with slower kinetics than when the
transition dipoles were aligned head to tail.[15] We anticipate
that the tetrazine TBET quenching mechanism can be
extended to other fluorophores and other tetrazines, and
that consideration of transition-dipole orientation will be
critical to the design of optimized turn-on probes.
Figure 2. Biological application of activatable BODIPY. a) Fluorogenic
imaging of EGFR expression on both fixed and live A-431 cells. Cells
were incubated with TCO-conjugated monoclonal antibodies,[23]
washed, and then imaged immediately after the addition of BODIPY–
Tz (100 nm) in phosphate-buffered saline (PBS; see the Supporting
Information). b) Fluorogenic live-cell imaging of nanoparticles internal-
ized by RAW 264.7 cells. The nanoparticles are labeled with both TCO
and with the near-infrared dye VT680,[23] and were imaged in two
channels after addition of BODIPY–Tz (100 nm), demonstrating co-
localization.
In summary, we report the synthesis and characterization
of extraordinarily efficient bioorthogonal turn-on probes. We
expect that these materials will find applications for live-cell
imaging, in pretargeting, and perhaps in vivo imaging, that is,
applications where washing steps are difficult to achieve.
Another unique application of the described materials may be
for superresolution microscopy,[24] to achieve new applica-
tions, faster reconstruction, or to address existing problems
where fluorochrome crowding limits reconstructions.[25]
Finally we anticipate that the described principles will find
applications in the design of blue- and yellow/orange fluo-
rochromes to widen the palette of available colors for in vivo
imaging. Irrespective of the specific design, the described
turn-on probes already have activation ratios that are up to
two orders of magnitude higher than those reported.
Received: February 6, 2013
Revised: March 28, 2013
Published online: && &&, &&&&
Keywords: click chemistry · energy transfer · fluorescent probes ·
.
fluorogenic probes · tetrazines
Having advanced our understanding of the quenching
mechanism, we sought to validate the utility of fluorogenic
BODIPY–tetrazines for biological imaging (Figure 2). Both
extracellular and intracellular TCO-labeled targets were
readily visualized, with excellent signal intensity, very low
background, and with no washing steps required after
addition of the dye solution.
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