In situ synthesis of alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell imaging probes

Article abstract of DOI:10.1002/anie.201400135

In spite of the wide application potential of 1,2,4,5-tetrazines, particularly in live-cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)-3-substituted 6-alkenyl-1,2,4

Full text of DOI:10.1002/anie.201400135

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Chemie
DOI: 10.1002/anie.201400135
Synthetic Methods
In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic
Bioorthogonal Live-Cell Imaging Probes**
ˇ
ˇ
˙
Haoxing Wu, Jun Yang, Jolita Seckute, and Neal K. Devaraj*
Abstract: In spite of the wide application potential of 1,2,4,5-
tetrazines, particularly in live-cell and in vivo imaging, a major
limitation has been the lack of practical synthetic methods.
Here we report the in situ synthesis of (E)-3-substituted 6-
alkenyl-1,2,4,5-tetrazine derivatives through an elimination–
Heck cascade reaction. By using this strategy, we provide 24
examples of p-conjugated tetrazine derivatives that can be
conveniently prepared from tetrazine building blocks and
related halides. These include tetrazine analogs of biological
small molecules, highly conjugated buta-1,3-diene-substituted
tetrazines, and a diverse array of fluorescent probes suitable for
live-cell imaging. These highly conjugated probes show very
strong fluorescence turn-on (up to 400-fold) when reacted with
dienophiles such as cyclopropenes and trans-cyclooctenes, and
we demonstrate their application for live-cell imaging. This
work provides an efficient and practical synthetic methodology
for tetrazine derivatives and will facilitate the application of
conjugated tetrazines, particularly as fluorogenic probes for
live-cell imaging.
bioorthogonal chemistry applications. The technique can also
be used to readily prepare unique p-conjugated 1,2,4,5-
tetrazine derivatives that are either difficult or not possible to
prepare using alternative synthetic strategies, facilitating the
future use of p-conjugated tetrazines as electron-deficient
components in molecular electronics, photovoltaics, and non-
linear optics.[1a,6] Finally, we demonstrate the ability to
synthesize a diverse set of tetrazine fluorogenic probes, both
from xanthene and BODIPY precursors. Due to conjugation
between the alkenyl tetrazine and the fluorescent core, these
dyes show excellent fluorogenic properties after reaction with
dienophiles, with turn-on ratios up to 400-fold. We demon-
strate their suitability for live-cell imaging applications by
detecting dienophile-modified cell surface markers.
Recently we developed a metal-catalyzed one-pot proce-
dure to prepare symmetric and unsymmetric tetrazines from
aliphatic nitriles and anhydrous hydrazine.[7] Nevertheless,
this technique has limitations. Synthesis requires excess
anhydrous hydrazine and heating, conditions that are not
compatible with several functional groups such as carbonyls
and alkyl halides, that are susceptible to either nucleophilic
addition or reduction.[8] It is therefore difficult to directly
introduce 1,2,4,5-tetrazine onto relatively complex molecules
such as fluorophores using this method. Conjugated alkenyl
substituted 1,2,4,5-tetrazines were not obtainable from the
corresponding alkenyl nitriles. Additionally, there is limited
commercial availability of anhydrous hydrazine in Europe
and China due to safety concerns, further encumbering
methods that require anhydrous hydrazine every time a new
tetrazine derivative is synthesized.
T
he chemistry of 1,2,4,5-tetrazines has gained growing
interest in the last decade, owing to their unique physico-
chemical characteristics.[1] Tetrazines have seen expanding
use in chemical biology, material science, natural product
synthesis, coordination chemistry, electrochemistry, photo-
voltaics, and explosives research.[1a,b,2] Of particular interest
has been the use of tetrazines for bioorthogonal live-cell
imaging applications.[1b,c,3] In spite of the application potential
of tetrazines, a major limitation has been the lack of practical
synthetic methods. This has hampered the development of
new fluorescent tetrazine probes, particularly those with
fluorogenic properties.[4,5] To address this problem, herein we
report the in situ synthesis of (E)-3-substituted 6-alkenyl-
1,2,4,5-tetrazine derivatives through an elimination–Heck
cascade reaction. This method enables convenient introduc-
tion of 3-substituted 6-alkenyl-1,2,4,5-tetrazine moieties onto
a diverse array of functional molecules. These include
unnatural nucleotides and amino acids that are relevant to
We envisioned developing a simple tetrazine building
block that was stable, could be easily synthesized, and readily
installed onto complex substrates, including commonly used
fluorescent probes, under mild conditions. In previous studies,
s-dichlorotetrazine
and
s-dithiomethyltetrazine were
regarded as typical tetrazine building blocks and have been
used to prepare numerous functional s-tetrazines by nucleo-
philic displacement.[1a,2f] Related tetrazines undergo SNAr
reactions with carbanions and limited cross-coupling reac-
tions; however, the reactions take place only if the tetrazine is
deactivated by one donating substituent (alkylamino, alkoxy,
or alkylthio), and the desired products are obtained in
moderate yield, greatly restricting the possible tetrazine
derivatives and potential applications.[9] For instance,
although 1,2,4,5-tetrazines have been widely used in bioor-
thogonal reactions, owing to their high reactivity in inverse-
electron-demand Diels–Alder cycloadditions, mono(bis)-al-
kylamino (alkoxy, alkylthio) substituted tetrazine derivatives
are not expected to be rapidly reacting due to the electronic
effects of the electron-donating groups.[10]
[*] Dr. H. X. Wu,[+] Dr. J. Yang,[+] Dr. J. Seckute, Prof. N. K. Devaraj
Department of Chemistry and Biochemistry
University of California, San Diego
ˇ
ˇ
˙
9500 Gilman Drive, La Jolla, CA 92093 (USA)
E-mail: ndevaraj@ucsd.edu
Homepage: http://devarajgroup.ucsd.edu/
[+] These authors contributed equally to this work.
[**] We acknowledge helpful discussions with Prof. Carlos Guerrero.
This work was partially funded by the University of California, San
Diego, and the NIH under grant number K01EB010078.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201400135.
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We hypothesized that 3-substituted 6-vinyl tetrazines
could be versatile tetrazine building blocks readily appended
onto a diverse array of molecules using the Heck coupling
reaction. However, there have been very few reports of
alkenyl-modified tetrazines, and unsymmetric vinyl tetrazines
are unknown, likely due to the previous difficulty in
synthesizing unsymmetric tetrazines as well as the potential
volatility of simple vinyl tetrazines.[11] As mentioned, we
recently disclosed a straightforward route to unsymmetric
tetrazines, and using this technique we synthesized 3-hydroxy-
ethyl-6-methyltetrazine in a one-pot fashion from commer-
cially available starting materials.[7] Mesylation of 3-hydroxy-
ethyl-6-methyltetrazine, to form 1a, followed by elimination
led to 3-methyl-6-vinyl tetrazine. As expected, the resulting
vinyl tetrazine was very volatile and not convenient to isolate.
In contrast, tetrazine 1a is a stable and easily handled pink
powder, which could be stored at À208C for several months
without noticeable decomposition (see Supporting Informa-
tion). We therefore turned to precursor 1a as a substitute of 3-
methyl-6-vinyl tetrazine and explored an in situ elimination–
Heck cascade reaction.[12]
With these optimized conditions in hand, we surveyed the
substrate scope of the in situ elimination–Heck reaction
(Table 2). Substitutions at the 3-position of the alkenyl
tetrazines could be introduced by the synthesis and use of
alternative vinyl tetrazine precursors. In this fashion, tert-
butyl, unsubstituted, phenyl, heterocyclic, and protected-
amine alkenyl tetrazine coupling products could be obtained
Table 2: Substrate scope.[a,b]
We initially screened the reaction conditions for the Heck
cascade reaction of 1a with iodobenzene using common
catalysts, ligands, bases, and solvents. However, using
1.5 equiv iodobenzene, 10% [Pd(PPh3)4]/Et3N/DMF and
heating to 808C for 90 min, led to no observable product
(Table 1, entry 1). This result is in agreement with past
Table 1: Optimization of the reaction conditions.[a]
Entry
Cat., Ligand
Temp., time
Base
Yield [%][d]
1[b]
2[b]
3[b]
10% [Pd(PPh3)4]
10% [Pd(PPh3)4]
10% [Pd2(dba)3],
40% P(o-Tol)3
3% [Pd2(dba)3],À
12% tBu3P+BF4
3% [Pd2(dba)3],
12% ligand 3
808C, 90 min
508C, MW, 30 min
508C, MW, 30 min
I
II
II
0
55
80
4[c]
608C, MW, 40 min
508C, MW, 30 min
II
II
58
99
5[b,c]
[a] All reactions were carried out on a 0.02 mmol scale in 1.5 mL DMF.
Ms=Mesyl, dba=dibenzylideneacetone, MW=microwaves. [b] Iodo-
benzene as starting material. [c] Bromobenzene as starting material.
[d] Yield (isolated) based on 1a, no (Z)-3-methyl-6-styryl-s-tetrazine was
observed.
difficulties in using standard Heck coupling conditions with
tetrazines.[9a] In recent years, microwave irradiation has been
widely used to improve yields in cross-coupling reactions and
we therefore decided to explore the use of microwave
activation for the Heck cascade reaction.[13] After a screening
of catalysts, ligands[14] and bases (Table 1, entries 2–5), we
found [Pd2(dba)3] catalyst at 3% loading and ligand 3 at 12%
enabled the isolation of 2a in nearly quantitative yield from
both iodobenzene and bromobenzene (Table 1, entry 5).
[a] Conducted on 0.02 mmol scale. Reaction time and equivalents of
bromide compounds shown under each product, 3 mol% [Pd2(dba)3],
12 mol% ligand 3, 3 equiv Cy2NMe, microwave 508C. [b] Yield of isolated
product. [c] 5 mol% [Pd2(dba)3], 20 mol% ligand 3. [d] 6 mol% [Pd2-
(dba)3], 24 mol% ligand 3. [e] 10 mol% [Pd2(dba)3], 40 mol% ligand 3.
[f] Iodide as starting material. [g] Microwaves 558C. [h] Microwaves
608C. [i] 6 equiv Cy2NMe. [j] 4 equiv Cy2NMe.
2
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in high yields (2b–2 f). Diversified phenyl bromides possess-
ing sterically bulky, electron-donating, electron-withdrawing
and heterocyclic substituents were also tolerated by the
reaction conditions and gave the corresponding alkenyl
tetrazines in excellent to good yields (2g–2n). Interestingly,
reduction of the double bond of alkenyl tetrazines such as 2a
through hydrogenation was possible, providing a novel route
to unsymmetric alkyl-substituted 1,2,4,5-tetrazines such as 11
(Supporting Information).
Despite significant interest in incorporating electron-
deficient tetrazine heterocycles in conjugated bridges, a road-
block has been the lack of accessible methods to synthesize p-
conjugated tetrazines, particularly alkenyl-substituted tetra-
zines, of which there are very few reported[11,15] and longer
conjugated buta-1,3-diene substituted tetrazines which, to our
knowledge, are unknown in the literature. Remarkably, by
using the Heck cascade reaction, we were also able to readily
synthesize conjugated mono-phenylbutadiene, bistyryl and
biphenylbutadiene substituted s-tetrazines (2o–2q) in mod-
erate to good yield.
derivatives, are arguably the most popular class of fluorescent
probes for cellular imaging and are typically highly soluble in
aqueous solutions. However, to date, highly quenched tetra-
zine xanthene dyes have not been demonstrated.
We hypothesized that the mild in situ Heck reaction could
be used to directly conjugate tetrazines through p-conjuga-
tion to a diverse series of fluorescent dyes, potentially
enabling highly efficient quenching by TBET.[22] To our
delight, we were able to apply our method to synthesize
tetrazine-conjugated xanthene dyes such as 2’,7’-difluoro-
fluorescein (Oregon-Green) tetrazine derivative 2v and
tetramethylrhodamine-tetrazine 2w in 58% and 53% yield,
We next examined the installation of bioorthogonal
tetrazine handles on several biologically relevant and func-
tionally complex molecules such as coumarin, deoxyribose,
and amino acid derivatives.[1c,3–4,16] Under the modified Heck
reaction conditions, these substrates smoothly reacted with 1a
and delivered 2r–2u in 47–83% yield. Deprotection of 2s–2u,
gave unnatural tetrazine-modified deoxyuridine 12, dl-phe-
nylalanine 13 and dl-tryptophan 14 (see Supporting Infor-
mation). We evaluated the stability of (E)-3-substituted 6-
styryl-s-tetrazines 2a–c in aqueous solutions and in the
presence of biologically relevant nucleophiles such as thiols.
Additionally, we measured the reaction kinetics between
alkenyl tetrazines 2a–c and a highly strained trans-cyclo-
octene (TCO) dienophile. Stability and reactivity trends were
consistent with past observations (Supporting Informa-
tion).[17]
A major application of tetrazine ligations has been the
live-cell imaging of dienophile-tagged small molecules,
including proteins, lipids, sugars, and drug analogs.[2b,3a,b,16a,b,18]
These applications are aided by the existence of fluorogenic
tetrazines, which consist of popular fluorophores for cellular
imaging such as xanthene and BODIPY dyes quenched
through energy transfer by a tetrazine handle.[19] Unfortu-
nately, when tetrazines are appended through aliphatic
linkers, fluorescence increases after ligation are typically 10-
fold and background signal from unreacted fluorophore limits
the sensitivity of detection.[4] Fluorogenic tetrazine probes
possessing fluorescence intensity increases greater than 100-
fold after ligation would enable far more sensitive detection
of dienophile targets. Recently, it was demonstrated that
tetrazines could be appended directly onto BODIPY fluo-
rophores resulting in highly fluorogenic probes that were
quenched by through-bond energy transfer (TBET).[5]
Unfortunately, the harsh conditions required for heterocycle
synthesis resulted in low yields and the technique was only
demonstrated for a green emitting BODIPY dye. Conven-
tional BODIPY probes have several drawbacks, including
a small Stokes shift[20] and poor aqueous solubility.[21] In
contrast, xanthene dyes, such as fluorescein and rhodamine
Scheme 1. a) Fluorogenic reaction of 2v with cyclopropene 4 and TCO
along with observed optical properties in phosphate-buffered saline
(PBS) at pH 7.4 (2 mm). Fluorescein (in 0.1m NaOH) was used as the
standard for quantum yield measurement. b) Fluorogenic reaction of
2w with cyclopropene 4 and TCO along with observed optical proper-
ties in EtOH (2 mm). Rhodamine 6G (in EtOH) was used as the
standard for quantum yield measurement. c) Fluorogenic reaction of
2x with cyclopropene 4 and TCO along with observed optical proper-
ties in EtOH (2 mm). Fluorescein (in 0.1m NaOH) was used as the
standard for quantum yield measurement. Note that the reaction
product of tetrazine with TCO can readily aromatize with oxidation.
TCO=trans-cyclooct-4-enol. [a] Only one adduct isomer is shown.
[b] 1 equiv 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was
added to finish the oxidation.
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respectively. Additionally, BODIPY-tetrazine dye 2x could
be synthesized in 83% yield. All fluorophore alkenyl
tetrazines were highly quenched, but became strongly emis-
sive upon reaction with dienophiles (Scheme 1). Oregon-
Green tetrazine derivative 2v showed the highest turn-on
with 135-fold and 400-fold increases in fluorescence intensity
after reaction with a cyclopropene[23] and a TCO, respectively
(Scheme 1a, Figure 1a). The alkenyl tetrazine fluorophores
derivatives of popular xanthene and BODIPY dyes suitable
for live-cell imaging applications. We believe this method-
ology will greatly facilitate the study of 1,2,4,5-tetrazines,
advancing their further application in chemical biology,
material science, electrochemistry, photovoltaics, nonlinear
optics, and particularly live-cell imaging.
Received: January 7, 2014
Revised: February 5, 2014
Published online: && &&, &&&&
Keywords: bioorthogonal reaction · cellular imaging ·
.
cycloaddition · fluorophores · heterocycles
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Figure 1. a) Fluorescence emission spectra for compound 2v in PBS
(gray line) and compound 5 (red line) and 6 (green line) in PBS;
excitation at 480 nm. b) Equimolar solutions of compound 6 and 2v
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2v for 30 min. Scale bar=15 mm.
were stable when stored at À808C for 1 month and remained
stable in solution over 24 h (Supporting Information). Rhod-
amine 2w also showed significant turn-on (up to 76-fold), with
maximimum emission in the red (569–573 nm) after reaction
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building blocks, which can smoothly react with aryl halides in
a mild and high-yielding in situ elimination–Heck cascade
reaction leading to the formation of (E)-3-substituted 6-
alkenyl-1,2,4,5-tetrazines. This method enables convenient
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previously unreported buta-1,3-diene substituted 1,2,4,5-tet-
razines. Moreover, this methodology provides a new strategy
to prepare highly quenched fluorogenic tetrazines, including
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Communications
Synthetic Methods
ˇ
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H. X. Wu, J. Yang, J. Seckute,
N. K. Devaraj*
&&&& — &&&&
In Situ Synthesis of Alkenyl Tetrazines for
Highly Fluorogenic Bioorthogonal Live-
Cell Imaging Probes
Stitching probes together: The in situ
synthesis of (E)-3-substituted 6-alkenyl-
1,2,4,5-tetrazine derivatives through an
elimination–Heck cascade reaction is
reported. 24 examples of p-conjugated
tetrazine derivatives are provided,
including conjugated fluorescent probes
that show drastic fluorescent turn-on
when reacted with dienophiles. Their
suitability for live-cell imaging is demon-
strated.
6
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