Mechanism-Based Fluorogenic trans-Cyclooctene–Tetrazine Cycloaddition

Article abstract of DOI:10.1002/anie.201610491

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron-demand Diels–Alder reaction of 1,2,4,5-tetrazines with particular trans-cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4-dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5-tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell-labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.

Full text of DOI:10.1002/anie.201610491

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201610491
German Edition: DOI: 10.1002/ange.201610491
Imaging Agents
Mechanism-Based Fluorogenic trans-Cyclooctene–Tetrazine
Cycloaddition
+
+
ˇ
´
Arcadio Vꢀzquez , Rastislav Dzijak , Martin Dracꢁnsky, Robert Rampmaier, Sebastian J. Siegl,
and Milan Vrabel*
Abstract: The development of fluorogenic reactions which
lead to the formation of fluorescent products from two
nonfluorescent starting materials is highly desirable, but
challenging. Reported herein is a new concept of fluorescent
product formation upon the inverse electron-demand Diels–
Alder reaction of 1,2,4,5-tetrazines with particular trans-cyclo-
octene (TCO) isomers. In sharp contrast to known fluorogenic
reagents the presented chemistry leads to the rapid formation of
unprecedented fluorescent 1,4-dihydropyridazines so that the
fluorophore is built directly upon the chemical reaction.
Attachment of an extra fluorophore moiety is therefore not
needed. The photochemical properties of the resulting dyes can
be easily tuned by changing the substitution pattern of the
starting 1,2,4,5-tetrazine. We support the claim with NMR
measurements and rationalize the data by computational study.
Cell-labeling experiments were performed to demonstrate the
potential of the fluorogenic reaction for bioimaging.
kinetics, became extremely popular as a robust transforma-
tion for biomolecule labeling.[4] The inherent photophysical
properties of the tetrazine heterocyclic core recently led to
the development of fluorescent turn-on probes where the
fluorescence of the attached fluorophore moiety is quenched
through energy transfer and is restored upon reaction with the
dienophile.[5] Although the advantages of such probes are
obvious, the attached fluorophore makes the synthesis
unnecessarily complex and in addition, the intrinsic instability
of some tetrazines under biological conditions[6] may poten-
tially impair their structure and lead to undesired background
signals. In contrast, fluorogenic probes in which the fluoro-
phore is built during the labeling reaction provide a system
with enhanced performance. Such probes, which are activated
in the course of the labeling reaction, are extremely scarce
and, as such, difficult to design. One example represent
fluorescent pyrazolines which are built upon the 1,3-dipolar
cycloaddition of nitrile imines with various alkenes.[7]
Recently, a conceptually similar fluorogenic styrene–tetrazine
cycloaddition was reported.[8] Herein, we disclose the unusual
reactivity of particular trans-cyclooctenes (TCOs) in combi-
nation with 1,2,4,5-tetrazines which enables the rapid for-
mation of tunable fluorescent products without the need for
attachment of an extra fluorophore moiety. Although the
IEDDA reaction was first applied to biomolecule labeling in
2008,[4a,b] and has since been extensively used in many
applications,[4d] to the best of our knowledge this phenomenon
has remained unreported.
Our study began with a startling discovery that the two
TCO isomers which are formed during their photochemical
synthesis[9] react with simple dipyridyltetrazine (diPyTet)
differently. In particular, the axial TCO isomer 1, assigned as
the rel-(1R-4E-pS), reacted to produce with diPyTet fluores-
cent product, while the equatorial isomer 2, assigned as rel-
(1R-4E-pR), did not (Figure 1). Analysis of the reaction
mixtures by HPLC-MS showed that the axial TCO isomer
leads to formation of the expected dihydropyridazine product
while the equatorial isomer produced a more polar product
having a mass greater than the expected product by 18. We
assigned this signal to the product of an addition of a water
molecule. In addition, when we performed the same reaction
using a series of other trans-cyclooctenes[10] we got similar
results.[11] This data further underlined the unique perfor-
mance of the axial TCO isomer 1.
F
luorescent probes are an indispensable tool in biological
research where they are used to investigate biological
processes and to interrogate biomolecules involved in
them.[1] Of particular importance are probes that become
fluorescent upon binding to or reacting with the biological
target of interest. These so-called fluorogenic probes enable
rapid imaging of biomolecules with excellent signal-to-noise
ratio.[2] Recently, the combination of bioorthogonal reactions
with fluorogenic reagents enabled application of a two-step
labeling protocol for this purpose. The first step involves the
introduction of a chemical reporter group to the biomolecule
of interest, which is subsequently used to attach a fluorophore
tag by means of a selective bioorthogonal chemical reaction.[3]
Among other chemical ligations, the inverse electron-demand
Diels–Alder reaction (IEDDA) of 1,2,4,5-tetrazines with
strained dienophiles, owing in particular to its impressive
[*] Dr. A. Vꢀzquez,[+] Dr. R. Dzijak,[+] Dr. M. Dracꢁnsky,
MSc. R. Rampmaier, MSc. S. J. Siegl, Dr. M. Vrabel
Institute of Organic Chemistry and Biochemistry of the Czech
Academy of Sciences
ˇ
´
Flemingovo nꢀm. 2, 166 10 Prague (Czech Republic)
E-mail: vrabel@uochb.cas.cz
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201610491.
To explain the differing behavior of axial and equatorial
TCOs in forming fluorescent products, we monitored the
reaction progress of 1 and 2 with diphenyltetrazine by NMR
spectroscopy and performed a detailed analysis of the
reaction mixtures (Scheme 1). The experimental data were
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Two regioisomers of 2B and 2C were observed. The
conformational analysis of 2A did not suggest any interaction
between the bridgehead hydrogen atoms and the TCO
hydroxy group, even in high-energy conformers, and the
reaction barrier of the intramolecular proton transfer in 2A is
at least 10 kcalmolÀ1 higher than in 1A.[11]
To study the influence of various substituents on the
photophysical properties of the click products we synthesized
a series of tetrazines utilizing the Heck cross-coupling
methodology developed by Devaraj and co-workers,[5c] and
performed the click reaction with 1.[11] We found that the
emission maxima strongly depended on the substitution
pattern (Table 1; see Tables S1 and S2). In general, the
presence of an electron-withdrawing substituent in the
olefinic region of the click products leads to a red shift in
emission maxima. The same substituents were found to
decrease the fluorescence quantum yield. All compounds
have exceptionally large Stokes shifts (up to l = 240 nm) and
show excellent fluorescence turn-on properties (up to 90-fold
increase) except for the nitro substituent which abolished the
fluorescence completely.[11] We also found that the “non-
conjugated” aryl substituent (phenyl, pyridyl, or thiophene)
did not influence the photophysical properties significantly. In
fact, this substituent is not needed for the fluorescence turn-
on and tetrazines substituted with only a methyl group in this
position are fluorescent as well (data not shown). Our data
demonstrate that the photophysical properties of the click
products can be nicely tuned by simply changing the
substituents of the starting tetrazine, thus giving instant
access to a new type of fluorophores whose emissions range
from l = 480 to 605 nm.
Figure 1. The reaction between two TCO isomers 1 and 2 and diPyTet
leads to two different products. The observed product masses are
shown over the HPLC signal.
Table 1: Photophysical properties of selected click products.
Scheme 1. The product distribution arising from the reaction of
diphenyltetrazine with the TCOs 1 and 2 as confirmed by NMR
analysis. Upper right: the transition-state connecting 1A and 1C
(N-protonated form).
R1
R2
lAbs/lEm
Stokes Ffl/emax
Intensity
increase
[a]
[c]
shift[b]
complemented with in silico conformational analysis and
density functional theory (DFT) modelling of the transition-
state structures.
312/480
336/505
357/478
336/539
336/494
336/505
168
169
121
203
128
169
0.12/19
11-fold
73-fold
91-fold
47-fold
61-fold
41-fold
The reaction of 1 with diphenyltetrazine leads to the very
fast formation of a single final product (1C) and no
intermediate could be detected (Scheme 1). We hypothesize
that the initial Diels–Alder reaction leads to the 4,5-dihy-
dropyridazine intermediate 1A, which isomerizes to 1C by
intramolecular hydrogen atom migration[12] with participation
of the TCO hydroxy group. The conformational analysis of
1A shows that the hydroxy oxygen atom is very close (2.1 ꢀ
and 2.0 ꢀ for neutral and N-protonated molecule, respec-
tively) to H8 in a low-energy conformer of 1A and a modest
energy barrier of 15 kcalmolÀ1 for the hydrogen atom
migration to the oxygen atom was found by DFT calculation
(Scheme 1, upper right; see Figure S32 in the Supporting
Information). In contrast, the equatorial TCO 2 leads to fast
formation of the IEDDA product 2A followed by very slow
hydration and dehydration reactions leading first to the
intermediates 2B and then to final products 2C (Scheme 1).
0.14/18
0.14/36
0.08/26
0.20/13
0.17/11
362/605
344/539
243
195
0.01/30
0.09/19
9-fold
42-fold
[a] Absorption and emission maxima were measured in CH3CN con-
taining 5% H2O and are given in nm. [b] Given in nm. [c] Quantum yields
were determined by using quinine sulfate in 0.5m H2SO4 as standard
(F=0.55), extinction coefficients emax are in 103 mÀ1 cmÀ1
.
2
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The fluorescent products are sufficiently stable when
completely disappear. We believe that the mechanistic studies
incubated in phosphate buffered saline or fetal bovine serum,
and time-course HPLC-MS analysis revealed the correspond-
ing fully aromatic pyridazines as major side-products formed
under these reaction conditions over the course of time (see
Figures S19–S22).
provided here could be helpful in the design and development
of new TCO derivatives which can be further modified
without perturbing their ability to form fluorescent products
upon reaction with tetrazines.
To examine if the fluorogenic properties of the reaction
will be preserved under biologically relevant conditions we
synthesized the two probes depicted in Figure 3, which target
the tetrazine moiety to different subcellular compartments.
We chose Taxol because of its ability to bind and stabilize
microtubules.[14] Additionally, we synthesized a triphenylphos-
phonium-derived tetrazine probe (TPP-Tet) which is routed
to mitochondria.[15] We incubated live U2OS cancer cells with
either the TPP-Tet or Taxol-Tet probe and subsequently
added 1 to initiate the reaction. We found that 1 is cell
permeable and in combination with the superb kinetics of the
IEDDA reaction led to expeditious fluorescent cell labeling
after only a few minutes (see Figures S26 and S27).[11] This
experiment demonstrates that the formation of the fluores-
cent 1,4-dihydropyridazine product can be used for efficient
and fast fluorogenic labeling of, for example, cellular com-
partments in live cells.
Kinetic measurements were performed to investigate the
influence of the substituents on the reactivity of the tetrazines.
The presence of electron-withdrawing substituents leads to
higher reaction rates in agreement with the inverse electron-
demand nature of the cycloaddition. All tetrazines reacted
with 1 faster than with 2, with second-order rate constants
ranging from 8 to 90mÀ1 sÀ1 (measured in CH3CN/H2O = 4:1;
see Table S3). As expected, further increase in reactivity was
observed by increasing the water content (see Figure S18).[13]
We next studied the reaction on a model peptide
(KYHWYGYTPQNVI). The w-amino group of the N-
terminal lysine was used to attach the tetrazine moiety by
employing active ester chemistry.[11] We next added the two
TCO isomers separately to the resin and examined the beads
under a fluorescent stereomicroscope. The experiment con-
firmed that addition of 1 leads to the rapid formation of the
fluorescent product while resin beads treated with 2 only
slowly start to fluoresce after a couple of hours (Figure 2).
In conclusion, we report on the discovery that the
conformation of the TCO dienophile directly influences the
formation of products in the IEDDA reaction of 1,2,4,5-
tetrazines. We found that among the dienophiles tested, only
the axial TCO isomer 1 leads to the rapid formation of
Figure 2. The fluorogenic click reaction was performed by using the
tetrazine-modified peptide shown above which was incubated with a) 1
or b) 2. The pictures were captured at indicated time points and colors
were adjusted using Las AF Lite program.
We also conducted the experiment in reverse order. This
means that the peptide was first modified with various TCOs
and then incubated with the respective tetrazine. Although
this experiment validated the superior performance of the
axial isomer, the difference compared to the equatorial
isomer was not so significant.[11] We also found that by
attaching the TCO moiety through an electron-withdrawing
group (such as the carbamate) precluded the fluorogenic
nature of the reaction. These findings fit to our theory about
the involvement of the hydroxy substituent in the tautome-
rization of the dihydropyridazine product postulated from the
computational study. However, these data also indicate that
by attaching various substituents to the hydroxy group of the
TCO the fluorogenic properties can be altered or may
Figure 3. A) Structures of microtubule-selective Taxol-Tet probe and
mitochondria-selective TPP-Tet probe. B) Confocal microscope images
of U2OS cells treated with 5 mm TPP-Tet or Taxol-Tet probe. The
nucleus was stained with DRAQ5 dye. a,d) negative controls (without
1); b,e) merged channel images (after addition of TCO 1); c,f) zoom
of colocalization experiments using Mitotracker and Tubulin tracker,
respectively. The images were acquired using l=405 nm excitation for
click products (emission window l=450–550 nm). DRAQ5 and Mito-
tracker: ex. l=633 nm, emission l=667–748 nm. Tubulin tracker: ex.
l=561 nm, emission l=600–650 nm.
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This work was supported by the Czech Science Foundation
(P207/15-06020Y and 15-11223S) and by the European
Research Council (ERC) under the European Unionꢁs
Horizon 2020 research and innovation programme (grants
agreement No 677465). We thank M. Downey, Dr. U. Jahn
and Prof. M. Hocek for critical reading of the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Keywords: bioorthogonal chemistry · cycloaddition ·
click reactions · heterocycles · imaging agents
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Manuscript received: October 26, 2016
Revised: November 29, 2016
Final Article published: && &&, &&&&
4
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Angewandte
Communications
Chemie
Communications
Imaging Agents
ˇ
´
A. Vꢁzquez, R. Dzijak, M. Dracꢂnsky,
R. Rampmaier, S. J. Siegl,
M. Vrabel*
&&&& — &&&&
Click-in color: A conceptually new
zines with particular trans-cyclooctene
Mechanism-Based Fluorogenic trans-
Cyclooctene–Tetrazine Cycloaddition
approach enables the formation of fluo-
rescent 1,4-dihydropyridazine products
upon reaction of various 1,2,4,5-tetra-
isomers. The chemistry was applied for
rapid fluorogenic labeling of subcellular
compartments in live cells.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5
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