Click-to-Release from trans-Cyclooctenes: Mechanistic Insights and Expansion of Scope from Established Carbamate to Remarkable Ether Cleavage

Article abstract of DOI:10.1002/anie.201800402

The bioorthogonal cleavage of allylic carbamates from trans-cyclooctene (TCO) upon reaction with tetrazine is widely used to release amines. We disclose herein that this reaction can also cleave TCO esters, carbonates, and surprisingly, ethers. Mechanistic studies demonstrated that the elimination is mainly governed by the formation of the rapidly eliminating 1,4-dihydropyridazine tautomer, and less by the nature of the leaving group. In contrast to the widely used p-aminobenzyloxy linker, which affords cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic, and aliphatic TCO ethers were cleaved as efficiently as the carbamate, carbonate, and esters. Bioorthogonal ether release was demonstrated by the rapid uncaging of TCO-masked tyrosine in serum, followed by oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in tyrosine-free medium.

Full text of DOI:10.1002/anie.201800402

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Click-to-Release from trans-cyclooctenes: mechanistic insights
and expansion of scope from established carbamate to
remarkable ether cleavage
Authors: Ron M Versteegen, Wolter ten, Raffaella Rossin, Mark AR de
Geus, Henk M Janssen, and Marc Stefan Robillard
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201800402
Angew. Chem. 10.1002/ange.201800402
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Click-to-Release from trans-cyclooctenes: mechanistic
insights and expansion of scope from established
carbamate to remarkable ether cleavage
R. M. Versteegen, W. ten Hoeve, R. Rossin,M. A. R. de Geus, H. M. Janssen, M. S. Robillard*
[*]
Dr. R. Rossin, Dr. M.S. Robillard
Tagworks Pharmaceuticals
Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands
marc.robillard@tagworkspharma.com
Dr. R.M. Versteegen, Dr. H.M. Janssen
SyMO-Chem, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands
Dr. W. ten Hoeve
Syncom, Kadijk 3, 9747 AT Groningen, The Netherlands
M.A.R. de Geus
Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands
Supporting information for this article is given via a link at the end of the document.
Abstract: The bioorthogonal cleavage of allylic carbamates from trans-cyclooctene (TCO) upon
reaction with tetrazine is widely used to release amines. We disclose here that this reaction can also
cleave TCO esters, carbonates, and, surprisingly, ethers. Mechanistic studies demonstrated that the
elimination is mainly governed by the formation of the rapidly eliminating 1,4-dihydropyridazine
tautomer, and less by the nature of the leaving group. In contrast to the widely used p-aminobenzyloxy
linker, which affords a slow cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic and
aliphatic TCO ethers cleaved as efficiently as the carbamate, carbonate and esters. Bioorthogonal ether
release was demonstrated by the rapid uncaging of TCO-masked tyrosine in serum, followed by
oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in
tyrosine-free medium.
Click chemistry has enabled selective conjugation applications in synthesis, material sciences, chemical
biology, diagnostics and medicine.[1] Recently the field was expanded with ‘click-to-release’ chemistry,
the use of a click reaction to achieve rapid bioorthogonal bond cleavage.[2] We reported that the fastest
click reaction, the inverse-electron-demand Diels-Alder (IEDDA) between trans-cyclooctene (TCO) and
tetrazine derivatives,[3] could be transformed into an IEDDA pyridazine elimination reaction (Scheme 1)
by installing a carbamate-linked doxorubicin on the allylic position of TCO. Following reaction of the
TCO-prodrug with a suitable tetrazine, the resulting 1,4-dihydropyridazine product eliminated the
amine-containing drug and CO2.[2] This new bioorthogonal elimination reaction has since been applied
in in vivo activation of antibody-drug conjugates (ADCs),[4] prodrugs[5] and proteins,[6] and in several in
vitro applications, such as oligonucleotide synthesis and delivery.[1, 7, 8] Furthermore, other organic
bioorthogonal bond cleavage reactions have subsequently been added to the repertoire.[9] In particular,
it was recently shown that the tetrazine-induced cleavage of vinyl ethers[10], which occurs through a
different mechanism than the cleavage of allylic substituted TCO, can be successfully applied in in vitro
uncaging of fluorogenic turn-on probes, biomolecules and prodrugs[9d-f]. The small size of the vinyl cage,
its stability and its complete release upon reaction with tetrazine compare favourably to the uncaging
of allylic substituted TCO, but the reactivity is three orders of magnitude lower.[2, 9d, 9e] While this lower
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reactivity has been effectively exploited in the proximity-induced mRNA detection,[9d] for applications at
low concentrations or with very short time frames reactivity will be key.
The faster IEDDA elimination reaction with TCOs has so far only been shown to release amines by
cleavage of TCO-linked carbamates or thiocarbamates.[2, 11] It would be highly advantageous to be able
to liberate other groups as well, such as hydroxyls and carboxylic acids (Figure 1). This would allow the
uncaging of amino acids other than lysine, for example tyrosine, serine and glutamic acid, opening up
new possibilities for protein activation, and it would enable the unmasking of other biomolecules such
as carbohydrates. In addition to the clear relevance for chemical biology, this could lead to ADC and
prodrug approaches with drugs that do not contain an amendable amine, such as duocarmycins, and
to applications as protective groups in (bio)organic synthesis.
Figure 1. Expanding the scope of the IEDDA pyridazine elimination reaction from the cleavage of carbamates to carbonates,
esters and ethers.
To expand the application scope of this reaction, we set out to further explore the factors that govern
the elimination from the dihydropyridazine and to investigate which bonds other than allylic carbamates
can be cleaved. IEDDA reactions give 4,5-dihydropyridazines, which usually rapidly tautomerize to 1,4-
and 2,5-dihydropyridazines,[12] especially in aqueous conditions (Scheme 1A).[13] In our initial 2013
report we demonstrated that the reaction of 3,6-dimethyl-1,2,4,5-tetrazine (2) with a TCO comprising
an allylic benzylcarbamate (5a) gives rapid benzylamine liberation, likely upon tautomerization of the
4,5-dihydropyridazine IEDDA adduct (6) to the 1,4- and 2,5-dihydropyridazines (7 and 8), followed by
an electron cascade elimination in 1,4-tautomer 7 (Scheme 1B).[2] However, the less likely possibility
that the release occurred from the 4,5- or the 2,5-dihydropyridazine could not be completely ruled out.
A method to modulate the formation rate of the 1,4- and 2,5-dihydropyridazine tautomer would aid the
study and understanding of the release process. It seemed reasonable to expect the tautomerization
rate to differ in the presence of acid.[12] Indeed, when we examined the tautomerization in CDCl3 with
1H NMR using unfunctionalized TCO 1 and tetrazine 2, the 4,5-dihydropyridazine product 3
tautomerized to the 1,4-dihydropyridazine 4 with a half-life of 15 min, while in CDCl3 with 1% formic acid
this was already complete within 2 min (Scheme 1A, Figure 2).
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Scheme 1. A) IEDDA conjugation. B) IEDDA pyridazine elimination of the axial isomers of TCO carbamate, carbonate, esters,
and ethers; racemic TCO mixtures: for clarity, only one of the TCO enantiomers is shown.
Figure 2. A) Tautomerization of the 4,5- (3) to the 1,4-dihydropyridazine tautomer (4) at 20 °C in CDCl3 and in CDCl3 with 1 v%
formic acid-d2. B) 1H NMR spectra for tautomerization in CDCl3 without acid.
The relatively slow tautomerization in CDCl3 and its acid induced acceleration allowed us to closely
study the release reaction between 2 and the previously used model TCO 5a with axially positioned
benzylcarbamate, using 1H NMR and GCMS (Scheme 1B). In CDCl3 without acid, fast consumption of
5a and formation of the 4,5-tautomer (6a) was followed by decrease of 6a (t1/2 ~ 70 min) in conjunction
with the formation of the 2,5-tautomer (8a), free benzylamine (11a) and non-aromatic pyridazine
derivative (9), which converted slowly into (aromatic) pyridazine (10) (Figure 3A). The formation and
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the subsequent slow decrease of 8a did not match the fast formation of benzylamine 11a and the
elimination products 9 and 10, indicating that the 2,5-tautomer cannot release 11a. After 5 h 8a slowly
oxidized to pyridazine 12a, which also cannot liberate 11a, limiting the overall elimination yield to 32%.
Figure 3. Tetrazine (2)-triggered release from TCO 5a at 20 °C in A) CDCl3, B) CDCl3 with 1 v% formic acid-d2, C) dioxane-
d8/D2O sodium phosphate buffer pH=7.4 (3/4, v/v), D) dioxane-d8/D2O (3/4, v/v) with 1% formic acid-d2. TCO 5a (9-23 mM) was
consumed completely within minutes upon reaction with a slight excess of 2.
The identity of 2,5-tautomer 8a was confirmed by COSY 2D NMR of the reaction mixture at 21 h,
showing a coupling between the dihydropyridazine H5 and the H7 adjacent to the carbamate
(Supporting Information Figure S4.1). The cis vicinal coupling constant of 3.2 Hz for the doublet for H5
demonstrated that the axial position of the carbamate was retained, suggesting that 7a is short-lived
and does not readily tautomerize back to 6a and on to 8a as this would have resulted in racemization
of H5 of 8a. We reasoned that addition of 1% formic acid to the experiment with 5a and 2 should lead
to a faster tautomerization to the 1,4- and 2,5-tautomers and possibly from the 2,5- to the 4,5- and on
to 1,4-tautomer and therefore lead to a faster and higher release. Indeed, when this release reaction
was repeated in CDCl3 with 1% formic acid, we observed a very fast and complete formation of 11a,
with a half-life of 4 min. The 4,5-tautomer could not even be detected and only a small amount of 2,5-
tautomer was found, which disappeared in minutes (Figure 3B). It is known that aqueous conditions
can favor the 4,5-to-1,4/2,5-tautomerization of the dihydropyridazine.[13] Accordingly, in dioxane-d8/D2O
sodium phosphate buffer pH=7.4 (1/1) the rapidly formed 4,5-tautomer 6a efficiently converted (t1/2 40
min) to 11a, elimination products 9 and 10 and a low and transient amount of 8a (Figure 3C). Oxidation
to 12a, possibly from both the 4,5- and the 2,5-tautomer, was limited, resulting in a high yield of 11a
(85%), corresponding well with earlier studies.[2, 4, 14] Repeating this experiment in the presence of 1%
formic acid further accelerated the release, affording 100% yield within 5 min (Figure 3D). Very recently,
an elegant study by Carlson et al. demonstrated that the 4,5-tautomer can undergo an intramolecular
cyclization with the carbamate linker, hampering the release.15 However, in the case of 5a, the NMR
measurements indicate that the incomplete release in dioxane-d8/D2O and CDCl3 is only due to the
oxidation of 8a to 12a (Figures S4.1, S4.10).
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The foregoing validates the 1,4-tautomer as the releasing species, which upon formation rapidly
liberates CO2 and amine 11a, giving 9 and subsequently aromatic 10. The alternative wherein the 4,5-
tautomer 6a liberates the benzylamine directly by a (possibly cyclic transition state-mediated) beta
elimination in parallel to the formation of non-releasing 1,4- and 2,5-tautomers is unlikely as the 1,4-
tautomer is not observed. Also, if the 4,5-tautomer can liberate 11a then it is likely that the 2,5-tautomer
should do so as well, as H5 is present in both. The lack of 1,4-tautomer 7a, the relative stability of the
2,5-tautomer 8a, the absence of H5 racemization in 8a, the efficient 4,5- to 1,4-tautomerization for
dihydropyridazine 3, as well as the acid-induced acceleration of both the tautomerization of 3 and the
formation of 11a strongly support the mechanism via the 1,4-dihydropyridazine. The results in Figure 3
further indicate that in favorable conditions the 2,5-tautomer can be converted to the releasing 1,4-
tautomer, presumably via the 4,5-tautomer, thus allowing complete cleavage yields, as also shown in
the recent report by Carlson.[15]
We wanted to know what other types of leaving groups could be released from the TCO with this
mechanism and therefore synthesized TCOs comprising a range of axial allylic substituents: aromatic
carbonate 5b, aromatic and aliphatic esters 5c,d, and aromatic, benzylic, and aliphatic ethers 5e-g
(Scheme 1). TCOs 5b, 5c, 5d and 5f were prepared by reacting the axial isomer of (E)-cyclooct-2-en-
1-ol with, respectively, phenyl chloroformate, benzoic anhydride, 3-phenylpropionyl chloride, and benzyl
bromide. TCOs 5e and 5g were obtained by reaction of (Z)-cyclooct-2-en-1-ol with, respectively, phenol
via a Mitsunobu coupling, or 1-bromo-3-phenylpropane, in both cases followed by photochemical
isomerization and isolation of the axial isomer (see Supporting Information S2). First, all compounds
were reacted with tetrazine 2 in CDCl3 and followed with 1H NMR and GCMS. As seen for 5a, TCOs
5b-g were consumed within several minutes in conjunction with the formation of the cycloaddition
products. Interestingly, compared with carbamate 5a, the carbonate 5b afforded much less formation
of 2,5-tautomer 8b, which diminished within several days, and a faster liberation of the leaving group
11b with a half-life of 4 h, leading to a much higher yield (Scheme 1, Figure 4A, Figure S4.2). The
poorer performance of the carbamate in CDCl3 is possibly due to the basicity of the formed benzylamine
hindering the tautomerization from the 4,5- to the 1,4- and 2,5- dihydropyridazines and from the 2,5- to
the 4,5-dihydropyridazine, giving the oxidative side reaction a chance to occur.
Figure 4. Tetrazine (2)-triggered release in CDCl3 at 20 °C from TCOs 5b (A), 5c (B), 5d (C), 5e (D), 5g (E). TCOs 5 (23 mM)
were consumed completely within minutes upon reaction with a slight excess of 2.
Esters 5c,d gave a rapid and complete release. The biphasic profiles started with a fast elimination up
to ca. 80% with half-lives of minutes, followed by conversion of 2,5-tautomer 8c,d within 8 h, completing
the liberation of 11c,d (Scheme 1, Figure 4B and C, Figures S4.3 and S4.4). The complete release is
likely due to the formation of carboxylic acid, resulting in efficient tautomerization to the 1,4-tautomer
and, possibly, the protonation of the leaving group. Indeed, repeating the experiment with 5d in the
presence of pyridine, led to a slower tautomerization (Figure S4.12).
Given the stability of the ether bond and the poor leaving group nature of the alkoxide we were keen to
investigate the liberation from the three ethers 5e-g. Whereas the tetrazine-induced cleavage of vinyl
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ethers is driven by the aromatization of the pyridazine, in the present allylic system aromatization of the
pyridazine does not drive the elimination, and the alkoxide needs to eliminate as result of an electron
cascade. We were therefore pleased to find that the aromatic 5e afforded a good release with a half-
life of 5 h and a simultaneous decline of the 4,5-tautomer with a modest build-up of the 2,5-tautomer.
The latter converted at a slower rate eventually leading to quantitative formation of phenol (11e) within
4 days (Figure 4D and Figure S4.5). Remarkably, the formation profile is very similar to that of the
aromatic carbonate 5b, even though the carbonate is a much better leaving group and has the
thermodynamic advantage of liberating CO2 in addition to phenol.[16] In fact, most studies regarding the
electron cascade-based cleavage of aromatic ethers vs. carbonates from analogs of the widely used
self-immolative linker p-aminobenzyloxycarbonyl (PABC) report 1 to 3 orders slower rates for the
ether.[16] Surprisingly, whereas the p-aminobenzyloxy linker cannot liberate aliphatic ethers,[17] the
benzylic and aliphatic ethers 5f and 5g gave quantitative release as well, with half-lives of respectively
19 and 22 h (Figure 4E, Figures S4.6 and S4.7). The reduced initial cleavage rates may in part be the
result of differences in pKa of the leaving group leading to slower tautomerization and reduced leaving
group capability. Interestingly, also a transient presence of the 1,4-tautomer was observed for 5f. As
found for the carbamate 5a, when the experiment with aliphatic 5g was repeated in the presence of 1%
formic acid the release was complete within 2 min, possibly due to protonation of the alkoxide in addition
to facilitating tautomerization (Fig. S4.9).
The experiments with TCOs 5 in organic solvents lend further support to the mechanism wherein the
initially formed 4,5-dihydropyridazine intermediate tautomerizes to the 1,4-dihydropyridazine, which
rapidly eliminates, and to the 2,5-tautomer, which cannot eliminate but instead is more slowly converted
to the 1,4-tautomer via the 4,5-tautomer. Furthermore, the TCO traces in Figures 3, 4 and S4.9b indicate
that in these conditions the cycloaddition itself is not the rate determining step of the release, but that
this may be different when the release is performed in acidic conditions (Fig 4B, C). Next, TCOs 5b-g
were evaluated in buffered aqueous conditions. In addition to the relevance for biological applications,
these conditions preclude an effect of the cleaved leaving group on the reaction medium while possibly
stabilizing the developing charge on the dihydropyridazine and the leaving group during the electron
cascade process. To avoid artificially high release yields as result of the 0.1% acid typically used in
HPLC-MS analysis we opted for HPLC-PDA in neutral conditions. The TCOs had a comparable and
high reactivity toward 2 (Figure 5A) leading to their complete consumption within several minutes, in
accordance with calculated half-lives of 100-380 seconds under these conditions (Figure 5 and
Supporting Information S5). The resulting dihydropyridazines exhibited similar biphasic release profiles
with a fast liberation up to ca. 70-85% within 10-30 min, followed by a slower phase. Remarkably, the
ethers 5f and 5g gave only slightly lower maximal yields of ca. 80-84% at 20 h compared to 90% for 5b
and 5c,d. In all cases the maximal yield was only limited by the oxidative side reaction. TCO 5e showed
a lower initial elimination of 58% after 30 min, increasing to 70% at 20 h and 90% at 90 h (Fig. S5.18).
Based on the earlier NMR experiments, the fast initial release phase observed for these reactions in
aqueous conditions is likely governed by the efficient tautomerization in this medium, leading to
conversion of the 4,5-dihydropyridazine to the 2,5- and the rapidly releasing 1,4-tautomer. The second,
slower, elimination phase then reflects the less favored tautomerization of the 2,5- back to the 4,5-
tautomer, allowing formation of more 1,4-tautomer. While it is possible that the nature of the allylic
substituent influences the relative amounts of initially formed 1,4- and 2,5-dihydropyridazine, the results
indicate that most studied TCOs favor the formation of the eliminating 1,4-tautomer over the 2,5-
tautomer. In addition, steric bulk has been shown to affect tautomerization rates[12b] as well as the
release from PABC linkers by perturbing pi-orbital overlap.[16] Therefore, a possible explanation for the
slower elimination from 5e vs. 5f and 5g despite the fact that phenol is a better leaving group, may be
the formation of a higher initial amount of the 2,5-tautomer combined with an increased steric hindrance
in the dihydropyridazine derivatives of 5e, affecting the ability to tautomerize and release. This latter
may also explain the observed slower depletion of the residual 4,5- and 2,5-tautomer derived from of
5e vs. 5g in CDCl3 (Figures 4D, 4E). Notably, in CDCl3, 5e gave a lower amount of 2,5-tautomer than
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5g, which may be due to the unbuffered acidity of released phenol or due to a solvent effect on the
tautomer distribution.[12b]
Figure 5. IEDDA reactions and release in aqueous conditions at 20 °C. A) Second order rate constants for TCOs 5 with 2 in
acetonitrile/PBS (1:3). B) and C) Biphasic fits of the release from TCOs 5 (0.4 mM) in acetonitrile/PBS (1/3) (n=3) triggered by
1.3 equiv 2 measured with HPLC; B) scale 0 - 1 h; C) scale 0 - 20 h.
Given the higher serum stability of the ether-linked TCOs 5e-g vs. the carbonate and ester derivatives
5b-d (Table S5.1, Figures S5.1, S5.2), and the potential of a bioorthogonal ether cleavage reaction we
set out to demonstrate the proof of principle of TCO-ether uncaging in biological conditions using TCO-
masked L-tyrosine 17 as a model system (Fig. 6A). To this end, N-trifluoroacetyl-protection of L-tyrosine
methyl ester 13 was followed by Mitsunobu coupling with (Z)-cyclooct-2-en-1-ol affording cis-
cyclooctene ether 15. Photoisomerization and mild basic deprotection led to the axial TCO-caged L-
tyrosine 17, albeit in low yield due to difficulties in separating the axial from the equatorial and residual
cis isomer (see Supporting Information S2). Attempts to improve this by performing the Mitsunobu on
(E)-cyclooct-2-en-1-ol unfortunately led to the cis-isomer (data not shown). Tyrosine plays a central role
in many proteins and is important in a range of biological processes, such as the synthesis of
neurotransmitters and melanin. For example, L-tyrosine (18) is converted by tyrosinase to L-dopa and
on to L-dopaquinone (19), which is a precursor for melanin (Figure 6B).[18] When TCO-caged 17 was
incubated with tyrosinase in serum at 37°C no oxidation to 19 was observed. However, incubation of
17 with tetrazine 2 gave efficient uncaging as evidenced by enzymatic oxidation of the liberated
substrate tyrosine (18) to L-dopaquinone 19 (Figure 6D). In comparison, when we combined the
analogous vinyl ether-caged tyrosine 20 with bis-pyridyl-tetrazine 21 (100-fold more reactive than 2) we
observed a much slower uncaging and corresponding tyrosine oxidation (Figures 6C,D and S6.1), due
to the 3000-fold lower reactivity of this system (Figure S5.9), and despite the relatively high
concentration of caged tyrosine (0.4 mM). Finally, to demonstrate efficacy in cell culture, we explored
whether the tyrosine-TCO uncaging could be used to chemically control cell growth (Figure 6B,E).
LS174T cells were cultured in tyrosine-free RPMI-1640 medium containing either caged tyrosine,
tetrazine, or both, and the cell growth was compared to that of cells grown in tyrosine-containing
medium. In regular RPMI-1640 medium (containing ca. 110 µM tyrosine) the number of cells increased
1.7- and 3.6-fold at day 2 and 3 respectively. While reducing the tyrosine levels to 55 and 27 µM gave
a slight reduction of cell growth at day 3, removing the tyrosine altogether resulted in complete
cytostasis. Similarly, cell growth was impaired in tyrosine-free medium containing caged tyrosine 17 or
tetrazine 2. However, adding 27 or 55 µM of 2 to the cell cultures with 17 resulted in a dose dependent
triggering of cell growth, reaching levels similar to the controls that were grown in medium with resp. 27
or 55 µM tyrosine.
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Figure 6. Tyrosine uncaging. A) Synthesis of TCO-caged tyrosine 17, a) trifluoroacectic anhydride, DCM; b) triphenylphosphine,
DEAD; c) UV, methyl benzoate; d) KOH, methanol, water. B) green: reaction of 17 with tetrazine 2, affording resp. enzymatic
conversion of the liberated tyrosine into L-dopaquinone, or tyrosine-dependent cell growth; red: corresponding controls without
addition of 2. C) Uncaging of vinyl ether-tyrosine (20) by tetrazine 21 (R = NHC(O)(CH2)3CO2H). D) Tyrosine uncaging (0.4 mM)
in serum, by reaction of 17 with 2 or 20 with 21 followed by enzymatic L-dopaquinone formation. E) LS174T cell growth in regular
RPMI medium, or tyrosine-free RPMI medium combined with levels of 17, 2, 17 + 2, or tyrosine.
In summary, we have demonstrated that the TCO-based ‘click-to-release’ reaction can be extended to
the cleavage of carbonate and ester bonds, and surprisingly also ether bonds. We have found that this
pyridazine elimination reaction is mainly governed by the formation (rate) of the fast-releasing 1,4-
dihydropyridazine tautomer, and less on the nature of the leaving group, allowing even aliphatic ethers
to be cleaved efficiently without being driven by aromatization. In contrast, the p-aminobenzyloxy linker
does not give fragmentation of aliphatic ethers. Our studies indicate also that the 2,5-dihydropyridazine
can contribute to the release, albeit typically at a slower rate, most likely through the less favored
tautomerization back to the 4,5-tautomer. Furthermore, tautomerization and elimination were shown to
accelerate in slightly acidic conditions. One limitation is that while axial benzylic and aliphatic ethers
should be accessible by direct modification of (E)-cyclooct-2-en-1-ol, for aromatic ethers this has so far
failed. Nevertheless, the TCO-ether cleavage occurs efficiently and selectively in biological
environments with a 3-orders of magnitude higher reactivity than the tetrazine-induced cleavage of vinyl
ethers. As such we expect these click-cleavable TCOs to be widely applied in, amongst others,
protective group strategies, materials, linkers for solid-phase synthesis and assays, protein and
carbohydrate uncaging, prodrugs and drug delivery.
Acknowledgements
We thank Tom de Greef (Technical University Eindhoven), Holger Grüll (University of Cologne), and
Sander van Kasteren (Leiden University) for useful discussions, and Jos Versleijen and Taeke van der
Kooi (Syncom) and Thuur van Onzen (Tagworks) for assistance. This research was supported by
NanoNextNL (The Netherlands, Grant 03C.3).
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Keywords: Cleavage Reactions • Click Chemistry • Pyridazine Elimination • Tetrazine • Trans-
Cyclooctene
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10.1002/anie.201800402
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COMMUNICATION
Click-to-release….more. In
Author(s), Corresponding Author(s)*
addition to cleaving carbamates,
the IEDDA pyridazine elimination
reaction can, with the same
efficiency, cleave aromatic,
benzylic, and even aliphatic
ethers, as well as esters and
carbonates. Trans-cyclooctene-
caged tyrosine was rapidly
Page No. – Page No.
Click-to-Release from trans-
cyclooctenes: mechanistic
insights and expansion of scope
from established carbamate to
remarkable ether cleavage
uncaged by reaction with tetrazine
and subsequently oxidized by
tyrosinase, while the caged
tyrosine itself was not a substrate.
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