Fluorogenic Bifunctional trans-Cyclooctenes as Efficient Tools for Investigating Click-to-Release Kinetics

Article abstract of DOI:10.1002/chem.201905446

The inverse electron demand Diels–Alder pyridazine elimination reaction between tetrazines and allylic substituted trans-cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination of alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate determination of both the click and release rate constants for any tetrazine at physiologically relevant concentrations.

Full text of DOI:10.1002/chem.201905446

A Journal of
Accepted Article
Title: Fluorogenic Bifunctional Trans-cyclooctenes as Efficient Tools for
Investigating Click-to-Release Kinetics
Authors: Mark A.R. de Geus, Elmer Maurits, Alexi J.C. Sarris, Thomas
Hansen, Max S. Kloet, Kiki Kamphorst, Wolter ten Hoeve,
Marc S. Robillard, Andrea Pannwitz, Sylvestre A. Bonnet,
Jeroen D.C. Codée, Dmitri V. Filippov, Herman S. Overkleeft,
and Sander Izaak van Kasteren
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201905446
Link to VoR: http://dx.doi.org/10.1002/chem.201905446
Supported by

Chemistry - A European Journal
10.1002/chem.201905446
FULL PAPER
Fluorogenic Bifunctional Trans-cyclooctenes as Efficient Tools
for Investigating Click-to-Release Kinetics
Mark A. R. de Geus,[a][⁑] Elmer Maurits,[a][⁑] Alexi J.C. Sarris,[a][⁑] Thomas Hansen, Max S. Kloet,
[a]
[a]
[a]
[b]
[c]
[a]
[a]
Kiki Kamphorst, Wolter ten Hoeve, Marc S. Robillard, Andrea Pannwitz, Sylvestre A. Bonnet,
[a]
[a]
[a]
[a]
Jeroen D.C. Codée, Dmitri V. Filippov, Herman S. Overkleeft, and Sander I. van Kasteren*
Abstract: The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans-
cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination on alkylamine substrates
has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for
accurate detection of both the click and release rates for any tetrazine at physiologically relevant concentrations.
Introduction
A
R
O
R
O
NH
NH
R
Bioorthogonal chemistry is broadening in scope to include bond
cleavage reactions alongside known ligation methods,[1–4] with
many new reactions steadily emerging in this arena.[5–12] Of
these, the inverse electron demand Diels-Alder (IEDDA)
pyridazine elimination,[
O
N
O
O
H
O
kIEDDA
N
H
N
N
N
N
- N2
N
N
N
H
13]
=
being the first of the bioorthogonal
krelease
tautomerization + kelimination
N
R
O
N
NH
processes known as “click-to-release” (Figure 1 A). In the
IEDDA decaging sequence, reaction of a tetrazine and a trans-
k
ktautomerization
O
cyclooctene (TCO) bearing
a
leaving group at the allylic
kelimination
N
N
N
N
NH
N
position[
14,15]
results in a 4,5-dihydropyridazine intermediate. This
-
-
CO2
R-NH2
adduct tautomerizes into a 1,4-dihydropyridazine species, which
induces elimination of the allylic payload. The biocompatibility of
the tetrazine and TCO components, combined with their
selectivity and overall deprotection rate have culminated in
various applications, such as the activation of cytotoxic
H
H
B
1
, R =
O
O
H
O
N
(
±)
R
O
(
pro)drugs,[16–18] proteins,[19,20] and immune cells.[21] The
N
,
technique is also compatible with reagents that enable
spatiotemporal control within biological systems.[
In order to successfully apply click-to-release in biology,
the identification of optimized tetrazines is crucial. Altering
tetrazine substituents can have drastic effects on both the rate
and extent of release obtained.[
2 R =
16–18]
N
H
N
O
F
B
OH
O
2
N
N
N
O
O
13,22–24]
O
SO H
Fluorogenic reporters,
3
H
CH3
such as caged coumarin 1[22] (Figure 1 B), are important tools to
characterize tetrazine release-behavior. These are supported by
NMR[13,25] and LC-MS[22–24] analyses. One difficulty associated
with these studies is the profound effect of pH and buffer
concentration on elimination.[23,25] Furthermore, Chen and co-
N
N
O
H
H
N
N
3
(±)
H
O
Figure 1. (A) Overview of the inverse electron demand Diels-Alder (IEDDA)
pyridazine elimination reaction, which is also known as the “click-to-release”-
reaction. (B) Fluorogenic TCOs to determine click-to-release kinetics: TCO-
workers[22] noted that
compared to a caged compound in which the coumarin (aniline
1 displayed a lower decaging rate
coumarin reporters 1 - 2 (by Fan et al.[ and Carlston et al. , respectively)
22]
[23]
and bifunctional TCO-reporter-quencher pair reporter 3 designed in this study.
release) is substituted for Fmoc-lysine ε-amine (alkylamine
release). Carlson et al.[23] described caged reporters which
enabled the detection of primary amine release kinetics by LC-
MS (e.g. Figure 1 B, 2). Compound 2 was also evaluated in a
quenched fluorescence assay[23] which employed a tetrazine
bearing a black-hole-quencher. Click reaction of 2 with 1 mM
black-hole-quencher-tetrazine gave rapid formation of a 4,5-
dihydropyridazine adduct in which the caged MayaFluor is
quenched. Detection of fluorescence thereby provided a direct
measurement of krelease (constituting the sum of ktautomerization and
[
a]
M.A.R. de Geus, E. Maurits, A.J.C. Sarris, T. Hansen, M.S. Kloet,
K. Kamphorst, Dr. A. Pannwitz, Dr. S.A. Bonnet, Dr. J.D.C. Codée,
Dr. D.V. Filippov, Prof. H.S. Overkleeft, Dr. S.I. van Kasteren
Leiden Institute of Chemistry
and The Institute for Chemical Immunology, Leiden University
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: s.i.van.kasteren@lic.leidenuniv.nl
Dr. W. ten Hoeve
[b]
[c]
[⁑]
Syncom
Kadijk 3, 9747 AT Groningen (The Netherlands)
Dr. M.S. Robillard
Tagworks Pharmaceuticals
Geert Grooteplein Zuid 10, 6525 GA Nijmegen (The Netherlands)
These authors contributed equally.
k
elimination) without the influence of cycloaddition (kIEDDA). However,
this method does not allow characterization of (unmodified)
tetrazines at lower concentrations.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905446
FULL PAPER
O
N
O
O
O
O
O
O
N
N
O
O
O
O
H
3C
O
O
O
O
O
CH3
a
e
f
H C
H C
3
3
O
O
O
O
OH
OH
O
1
6 (±)
O
N
OH
(±)
O
1
2 (±)
12 (±)
1
1 (±)
mixture
15
O
g
a
O
O
O
CH3
O
R1
NH
O
c
O
O
CH3
N
CH3
N
O
O
N
b
3
(±)
O
R1
HO
HN
O
d
R2
O
R2
N
= R1
O
O
DABCYL
O
OH
4 (±)
17 (±)
1
3(±)
CH
3
O
O
R2
H
N
N
=
R2
HN
O
O
O
14(±)
HO S
2
R1
EDANS
Scheme 1. Synthesis of bifunctional TCO-reporter-quencher pairs 3 – 4 and CCO-reporter-quencher pair 17 from cyclooctadiene 5. Reagents/conditions for the
o
synthesis of 11 and 12 cyclooctadiene (5) can be found in scheme S1; (a) KOH, MeOH, H
2
O, 4 C, 43% (12), 42% (13); (b) N,N'-disuccinimidyl carbonate, DIPEA,
DMAP, MeCN, rt, 72%; (c) i. EDANS-NH
NH
DABCYL-NH
2
, DIPEA, DMF, rt, 79%; ii. DABCYL-NH
2
2
, DIPEA, DMF, rt, 60%; (d) i. DABCYL-NH , DIPEA, DMF, rt, 96%; ii. EDANS-
o
2
, DIPEA, DMF, rt, 88%; (e) i. KOH, MeOH, H
2
O, 50 C, 79%; ii. N,N'-disuccinimidyl carbonate, DIPEA, MeCN, rt, 66%; (f) hν (CFL), CDCl
3
, rt, 74%; (g) i.
2
, DIPEA, DMF, rt, 60%; ii. EDANS-NH
2
, DIPEA, DMF, rt, 50%.
A method based on fluorescence quenching to rapidly
determine both the cycloaddition rate (kIEDDA) and the alkylamine
release rate (krelease) caused by tetrazines without stringent
concentration requirements would be of great importance. We
hereby report on the design, synthesis and evaluation of
quenched reporter 3 (Figure 1 B) based on the bifunctional TCO
introduced by Robillard and co-workers.[16] By linking a DABCYL
quencher to an EDANS fluorophore via the bifunctional TCO
scaffold, cycloaddition and alkylamine release rates can now be
quantified in a 96-well plate reader format. Pseudo-first order
rate constants were determined for a series of tetrazines and
DFT calculations were employed to probe the differences for the
initial cycloaddition step between monofunctional and
bifunctional TCOs.
O
EDANS
DABCYL
CH3
NH
O
N
H
N
N
N
N
O
O
O
+ N2
3
(±)
Fluorescence
Quenching
DABCYL
NH
O
N
EDANS
kIEDDA
N
N
H
(±)
ktautomerization
EDANS
EDANS
N
O
H
NH
O
O
O
DABCYL
H
+
DABCYL
O
O
N
N
N
NH
N
N
H
H
(±)
(±)
Results and Discussion
k
elimination
oxidation
=
The synthesis of bifunctional TCO reagent 4 was based on the
published route (Scheme 1).[ However, to both simplify and
accelerate the procedure, the initial functionalization of
krelease
EDANS
16]
EDANS
ktautomerization + kelimination
H N
2
NH
O
O
cyclooctadiene
5
into the α-methylated carboxylic acid
6
CO
2
DABCYL
DABCYL
O
O
N
N
(
bromination, cyanide substitution, oxidation, basic hydrolysis
N
N
and methylation) was carried out using crude reaction mixtures
obtained after aqueous workup. Crystallization of iodolactone 7
from EtOH resulted in a yield of 23% over five steps from 5 at
N
N
H
H
Figure 2. Schematic representation of TCO-quenched fluorescence assay.
Bifunctional TCO-reporter-quencher pair 3 does not display fluorescence in its
native state due to fluorescence quenching between the EDANS fluorophore
(±)
(±)
and DABCYL quencher. Upon cycloaddition of
dihydropyridazine adduct may tautomerize into the 1,4-dihydropydazine
intermediate. This species can eliminate CO and the EDANS fluorophore,
a tetrazine, the 4,5-
5
00 mmol scale, without the need for the previously reported
distillations.[
16]
2
thereby disabling the fluorescence quenching and enabling a fluorescent
readout for the elimination step. Alternatively, the 2,5-dihydropyridazine
intermediate is formed which may tautomerize back to the 4,5-
dihydropyridazine adduct or undergo oxidation.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905446
FULL PAPER
Figure 3. (A) Literature tetrazines 18 – 28 investigated in this study. (B) Pseudo-first order reaction rate constants (kobs) determined by treating TCO-reporter-
quencher pair 3 (10 µM) with tetrazines 18 – 28 (100 µM and 400 µM, N = 2) and measuring the fluorescence intensity of released EDANS (ex = 340 nm, em =
4
95 nm). (C) Elimination efficiency observed for TCO-reporter-quencher pair 3 (10 µM) using tetrazines 18 – 28 (100 µM and 400 µM, N = 2, t = 4 h), normalized
to tetrazine 18 (100 µM, t = 1 h, 80%, Figure S5). (D) Fluorescence observed for TCO-reporter-quencher pairs 3 (10 µM) and 14 (10 µM) using tetrazines 18 – 28
100 µM, N = 2, t = 4 h). The fluorescence detected for 14 relative to the corresponding result for 3 is given in percentages. (E) Representative example of a
PCM(H O)-M06-2X/6-31+G(d)-optimized transition state for the reaction of tetrazines (18 in figure) and model axial bifunctional TCO. Bond lengths are in Å. (F)
(
2
Summary of kinetic investigations with TCO-reporter-quencher pair 3, including pseudo-first order reaction rate constants (kobs, for 10 µM 3 and 400/100 µM
tetrazine, Figure 3B), ratio of kobs values (kobs 100µM / kobs 400µM ≈ 1, orange; kobs 100µM / kobs 400µM < 1, transparency increases), estimated rate constants
(krelease and kIEDDA, based on the ratio of kobs values or on a separate experiment at 100 nM 3 with 10 - 100 µM tetrazine, Figure S6) and calculated transition states
between tetrazines 18 - 28 and model mono and bifunctional TCO model compounds.
The transesterification procedure starting from bicyclic
lactone 8 (64 hours, 48% yield)[16] was replaced by a one pot, -
two step procedure to improve overall conversion and reaction
time. Saponification afforded monocyclic carboxylic acid 9,
which was directly methylated to obtain methyl ester 10 in 77%
yield over two steps. Photoisomerization[26] resulted in a 1 : 1.4
mixture of axial isomer 11 (axial hydroxyl, equatorial methyl
ester) and equatorial isomer 12 (equatorial hydroxyl, axial
methyl ester), respectively. The isomeric mixture was treated
(quencher) moieties with complete regioselectivity towards the
carbonate due to the steric hindrance induced by the methyl
group. This was followed by functionalization with the
complementary moiety, obtaining axial TCO-reporter-quencher
pairs 3 and 14. We also synthesized a bifunctional cis-
cyclooctene (CCO) reporter-quencher pair. Attempts to
functionalize carboxylic acid 9 resulted in formation of cyclic
lactone 8. Instead, equatorial isomer 12 was hydrolyzed and
functionalized to obtain equatorial TCO reagent 15, followed by
trans to cis isomerization in the presence of visible light (26 W
CFL bulb, see Figure S1-S3) to obtain bifunctional CCO reagent
16. Functionalization with EDANS and DABCYL occurred with
the same degree of regioselectivity, obtaining CCO-reporter-
quencher pair 17.
o
with potassium hydroxide at 4 C to selectively hydrolyze axial
isomer 11, followed by acid-base extraction to separately obtain
carboxylic acid 13 and ester 12. The bis-NHS functionalization of
1
3 into 4 (three days, 46% yield)[16] was accelerated using
nucleophilic catalysis (DMAP) and hydrolysis of the obtained
product during chromatographic purification was prevented by
employing neutralized silica gel, resulting in a yield of 72%.
Compound 4 reacted with EDANS (fluorophore) or DABCYL
We used 3 to assess a series of tetrazines for their ability
to induce IEDDA pyridazine elimination. Axial TCO-reporter-
quencher pair 3 is fluorogenic by fluorescence quenching until
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905446
FULL PAPER
the EDANS fluorophore is released from the post-ligation
construct (Figure 2) and could thus serve to determine the
overall properties of the deprotection reaction. This was
confirmed by treating 3 and 17 with 3,6-dimethyltetrazine (18,
Figure S4). The reaction between 3 and 18 was further
characterized by quantifying the fluorescence emitted from the
liberated EDANS fluorophore (Figure S5). This experiment gave
determined with 3, density functional theory (DFT) calculations
were performed. The transition state of the IEDDA cycloaddition
step was studied for two model TCOs (mono TCO and bi TCO)
with tetrazines 18, 20 – 22, 24, 26 and 28 (Figure 3E) by the use
2
of PCM(H O)-M06-2X/6-31+G(d) (See the SI). The results reveal
a slight destabilization of the transition state for the cycloaddition
of the bifunctional TCO compared to the monofunctional TCO for
all tetrazines, resulting in a minor increase of the reaction barrier
(Figure 3F). Transition state structures for both model TCOs
were highly similar in terms of geometry (see the SI) and
reactivity trends between tetrazines were maintained. These
results suggest the steric bulk introduced on the bifunctional
TCO does not significantly hamper the initial cycloaddition step.
an EDANS release yield of 80% after 1 hour, which corresponds
with other reports.[
13,23,25]
A panel of tetrazines was selected from
literature (Figure 3 A, 18 - 28), including key entries reported by
Chen,[22] Weissleder[23] and our own recent investigations.[24] The
release of EDANS from TCO-reporter-quencher pair 3 (10 µM)
was measured over time using an excess of tetrazine (100 and
4
00 µM) in a phosphate buffered solution (0.2 M PO 2
DMSO in O, Figure S6). The functional groups of
asymmetric) tetrazines direct the regiochemistry of the initial
Diels-Alder ligation reaction to form the 4,5-dihydropyridazine
adduct. Further tautomerization towards the 1,4-
4
-, 10%
H
2
(
Conclusions
In conclusion, we developed
a new method based on
dihydropyridazine enables fast release whereas tautomerization
to the 2,5-dihydropyridazine results in a slow releasing tautomer
fluorescence quenching to determine both the cycloaddition and
alkylamine release rates of the IEDDA pyridazine elimination in
a 96-well plate reader format. TCO-reporter-quencher pair 3 was
synthesized by functionalization of bifunctional NHS TCO
reagent 4 with an EDANS fluorophore and a DABCYL quencher.
The new method was used to determine click-to-release kinetics
and yields for 3 with tetrazines 18 – 28. For tetrazines 19 – 22,
(
which proceeds via a tautomerization back to the 4,5-
dihydropyidazine). Part of the 2,5-dihydropyridazine population
can also convert to the oxidation product without releasing the
allylic EDANS.[
23,25]
The release rate kobs is therefore the sum of
the rate of these processes, which we fitted with a biphasic
decay trend line. The pseudo-first order reaction rate constants
for the “fast-releasing” adduct (kobs, Figure 3B) and elimination at
the results indicated
a greater concentration dependence
compared to the other tetrazines measured. We were able to
determine both rate parameters (kIEDDA and krelease) for tetrazines
4
hours (Figure 3C, normalized for [18] = 400 µM) were
determined. For several tetrazines (18, 20, 24 and 26) with
comparable kobs values at 100 and 400 µM, the measurements
were repeated at 100 nM 3 and 10 – 100 µM of tetrazine (Figure
S7). We repeated the initial measurements for TCO-reporter-
quencher pair 14 to determine the release of DABCYL over time
1
8, 24 and 26. The DFT-calculations described here, also
indicate similar IEDDA reactivity of both mono and bifunctional
TCOs, suggesting the observations made with probe 3, are
translatable to the deprotection of mono-functionalized TCOs.
Predicting tetrazine behavior in IEDDA pyridazine elimination
remains challenging. We therefore recommend this new
fluorescence assay to rapidly screen tetrazines for click-to-
release potential.
(
Figure 3D, Figure S8-9). In this case, the formed pyridazine
adduct appeared to act as a fluorescence quencher after IEDDA,
limiting the fluorescence emitted by EDANS compared to the
results obtained for 3. These results render 14 unfit for
elimination efficiency analysis.
Within the panel of tetrazines we experimentally examined
at 10 µM 3, tetrazines 19, 21 and 22 displayed reduced kobs
values when switching from 400 µM to 100 µM, indicating that
Acknowledgments
S.I.V.K. was the recipient of an ERC Starting Grant of the
European Research Council (ERC-2014-StG-639005), A.J.C.S.,
D.V.F., H.S.O., S.I.V.K. were funded by the Institute of Chemical
Immunology (NWO Zwaartekracht). The authors thank the
SURFsara for use of the Lisa supercomputer and kindly
acknowledge Mark Somers for technical support.
for these tetrazines the initial cycloaddition step plays
a
significant role in overall reaction rate (kobs ≈ kIEDDA* [tetrazine],
Figure 3F). For tetrazines 18, 23 – 28, the kobs values remained
similar for both concentrations examined, demonstrating that the
overall reaction rate is controlled by the release rate (kobs
≈
k
release, Figure 3F). Additionally, the experiments performed at
100 nM 3 with tetrazines 18, 20, 24 and 26 (10 - 100 µM)
Conflict of interest
allowed us to determine kIEDDA for these tetrazines as well,
because in this concentration range the kobs values decreased
when reducing tetrazine concentrations (kobs ≈ kIEDDA* [tetrazine],
Figure 3F). The total elimination yield for a given tetrazine is
difficult to predict based on the functional groups present and no
clear correlation between release rate and elimination yield can
be determined, highlighting the importance of the kinetic
quantification described here. It should be noted that the 4 hour
timepoint doesn’t always show the absolute endpoint of a given
reaction. In order to rationalize IEDDA cycloaddition rates
Wolter ten Hoeve: employment at Syncom. Marc Robillard:
cofounder of Tagworks Pharmaceuticals.
Keywords: biorthogonal chemistry • click-to-release • trans-
cyclooctenes • tetrazines • quenched fluorescence assay
[1]
J. Li, P. R. Chen, Nat. Chem. Biol. 2016, 12, 129–137.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905446
FULL PAPER
[
2]
K. Neumann, A. Gambardella, M. Bradley, ChemBioChem 2019, 20,
72–876.
[25]
[26]
R. M. Versteegen, W. ten Hoeve, R. Rossin, M. A. R. de Geus, H. M.
Janssen, M. S. Robillard, Angew. Chem. Int. Ed. 2018, 57, 10494–
10499.
8
[3]
[4]
[5]
J. Tu, M. Xu, R. M. Franzini, ChemBioChem 2019, 20, 1615–1627.
N. K. Devaraj, ACS Cent. Sci. 2018, 4, 952–959.
M. Royzen, G. P. A. Yap, J. M. Fox, J. Am. Chem. Soc. 2008, 130,
3760–3761.
J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang, S.
Jia, X. Zhang, P. R. Chen, Nat. Chem. 2014, 6, 352–361.
E. Jiménez-Moreno, Z. Guo, B. L. Oliveira, I. S. Albuquerque, A.
Kitowski, A. Guerreiro, O. Boutureira, T. Rodrigues, G. Jiménez-
Osés, G. J. L. Bernardes, Angew. Chem. Int. Ed. 2016, 1–6.
A. M. Pérez-López, B. Rubio-Ruiz, V. Sebastián, L. Hamilton, C.
Adam, T. L. Bray, S. Irusta, P. M. Brennan, G. Lloyd-Jones, D.
Sieger, J. Santamaría, A. Unciti-Broceta, Angew. Chem. Int. Ed.
[
6]
7]
[
2017, 1–6.
[8]
S. Bernard, D. Audisio, M. Riomet, S. Bregant, A. Sallustrau, L.
Plougastel, E. Decuypere, S. Gabillet, R. A. Kumar, J. Elyian, M. N.
Trinh, O. Koniev, A. Wagner, S. Kolodych, F. Taran, Angew. Chem.
Int. Ed. 2017, 56, 15612–15616.
[
[
[
[
[
9]
J. Tu, M. Xu, S. Parvez, R. T. Peterson, R. M. Franzini, J. Am.
Chem. Soc. 2018, 140, 8410–8414.
10]
11]
12]
13]
S. S. Matikonda, D. L. Orsi, V. Staudacher, I. A. Jenkins, F. Fiedler,
J. Chen, A. B. Gamble, Chem. Sci. 2015, 6, 1212–1218.
L. P. W. M. Lelieveldt, S. Eising, A. Wijen, K. M. Bonger, Org.
Biomol. Chem. 2019, 17, 8816–8821.
H. Wu, S. C. Alexander, S. Jin, N. K. Devaraj, J. Am. Chem. Soc.
2016, 138, 11429–11432.
R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen, M. S.
Robillard, Angew. Chem. Int. Ed. 2013, 52, 14112–14116.
J. Sauer, H. Wiest, Angew. Chem. 1962, 74, 353–353.
[
14]
15]
[
M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc. 2008,
130, 13518–13519.
[16]
[17]
[18]
R. Rossin, S. M. J. van Duijnhoven, W. ten Hoeve, H. M. Janssen, F.
J. M. Hoeben, R. M. Versteegen, M. S. Robillard, Bioconjug. Chem.
2016, 27, 1697–1706.
R. Rossin, R. M. Versteegen, J. Wu, A. Khasanov, H. J. Wessels, E.
J. Steenbergen, W. ten Hoeve, H. M. Janssen, A. H. A. M. van
Onzen, P. J. Hudson, M. S. Robillard, Nat. Commun. 2018, 9, 1484.
J. M. Mejia Oneto, I. Khan, L. Seebald, M. Royzen, ACS Cent. Sci.
2016, 2, 476–482.
[
19]
20]
J. Li, S. Jia, P. R. Chen, Nat. Chem. Biol. 2014, 10, 1003–1005.
G. Zhang, J. Li, R. Xie, X. Fan, Y. Liu, S. Zheng, Y. Ge, P. R. Chen,
ACS Cent. Sci. 2016, 2, 325–331.
[
[21]
A. M. F. van der Gracht, M. A. R. de Geus, M. G. M. Camps, T. J.
Ruckwardt, A. J. C. Sarris, J. Bremmers, E. Maurits, J. B. Pawlak, M.
M. Posthoorn, K. M. Bonger, D. V Filippov, H. S. Overkleeft, M. S.
Robillard, F. Ossendorp, S. I. van Kasteren, ACS Chem. Biol. 2018,
13, 1569–1576.
[22]
X. Fan, Y. Ge, F. Lin, Y. Yang, G. Zhang, W. S. C. Ngai, Z. Lin, S.
Zheng, J. Wang, J. Zhao, J. Li, P. R. Chen, Angew. Chem. Int. Ed.
2
016, 55, 14046–14050.
J. C. T. Carlson, H. Mikula, R. Weissleder, J. Am. Chem. Soc. 2018,
40, 3603–3612.
[
23]
24]
1
[
A. J. C. Sarris, T. Hansen, M. A. R. de Geus, E. Maurits, W.
Doelman, H. S. Overkleeft, J. D. C. Codée, D. V. Filippov, S. I. van
Kasteren, Chem. Eur. J. 2018, 24, 18075–18081.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905446
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
FULL PAPER
Click-to-release kinetics
O
Mark A. R. de Geus, Elmer Maurits,
Alexi J.C. Sarris, Thomas Hansen,
Max S. Kloet, Kiki Kamphorst, Wolter
ten Hoeve, Marc S. Robillard, Andrea
Pannwitz, Sylvestre A. Bonnet,
Jeroen D.C. Codée, Dmitri V. Filippov,
Herman S. Overkleeft, and Sander I.
van Kasteren*
DABCYL
R
R
CH3
EDANS
N
N
N
N
N
H
H N
2
O
O
(±)
Fluorescence
Quenching
NH
+
R
DABCYL
EDANS
O
N
N
N
Page No. – Page No.
H
R
(±)
Fluorogenic Bifunctional Trans-
cyclooctenes as Efficient Tools for
Investigating Click-to-Release
Kinetics
A new fluorogenic trans-cyclooctene probe was designed and
synthesized to determine elimination kinetics of the click-to-release
reaction. This reagent enables accurate detection of the decaging
potential for any tetrazine at physiologically relevant concentrations.
This article is protected by copyright. All rights reserved.