Fast and pH-Independent Elimination of trans-Cyclooctene by Using Aminoethyl-Functionalized Tetrazines

Article abstract of DOI:10.1002/chem.201803839

The inverse-electron-demand Diels–Alder/pyridazine elimination tandem reaction, in which the allylic substituent on trans-cyclooctene is eliminated following reaction with tetrazines, is gaining interest as a versatile bioorthogonal process. One potential shortcoming of such currently used reactions is their propensity to proceed faster and more efficiently at lower pH, a feature caused by the nature of the tetrazines used. Here, we present aminoethyl-substituted tetrazines as the first pH-independent reagents showing invariably fast elimination kinetics at all biologically relevant pH values.

Full text of DOI:10.1002/chem.201803839

A Journal of
Accepted Article
Title: Fast and pH independent elimination of trans-cyclooctene using
aminoethyl functionalized tetrazines
Authors: Alexi JC Sarris, Thomas Hansen, Mark AR de Geus, Elmer
Maurits, Ward Doelman, Herman S Overkleeft, Jeroen DC
Codee, Dmitri Filippov, and Sander I van Kasteren
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201803839
Link to VoR: http://dx.doi.org/10.1002/chem.201803839
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Fast and pH independent elimination of trans-cyclooctene using
aminoethyl functionalized tetrazines
Alexi J.C. Sarris, Thomas Hansen, Mark A.R. de Geus, Elmer Maurits, Ward Doelman, Herman S.
Overkleeft, Jeroen D.C. Codée, Dmitri V. Filippov,* and Sander I. van Kasteren*
Abstract: The inverse electron demand Diels-Alder/pyridazine
elimination tandem reaction, in which the allylic substituent on trans-
cyclooctene is eliminated following reaction with tetrazines, is gaining
elimination rates much faster than tetrazine 2[13b]. It was also
discovered that, for tetrazines containing simple alkyl substituents,
elimination rates are very sensitive to the pH of the reaction
medium: while release is completed within an hour at acidic
conditions (tetrazine 2, pH 5.0), it takes several hours under
interest as
a versatile bioorthogonal process. One potential
shortcoming of such currently used reactions is their propensity to run
faster and more efficient at lower pH, a feature caused by the nature
of the tetrazines used. Here we present aminoethyl substituted
tetrazines as the first pH independent reagents showing invariably fast
elimination kinetics at all biologically relevant pH’s.
physiological conditions (tetrazine 2, pH 7.0)[13b]
, as the
elimination step is subject to general acid catalysis.
What is currently lacking are tetrazines that maintain fast
elimination kinetics over the whole biologically relevant pH range
(pH 3.5 – pH 7.5). We postulated that tetrazines 4, 5, 6, and 7
(Figure 1) could serve as pH-independent eliminating tetrazines.
The amine on the aminoethyl substituent, as cationic ammonium
functionality at and below physiological pH, would function as
general acid during the elimination step, potentially improving
both release rate and efficiency.
Here we describe the design, synthesis and characterization of a
new family of tetrazines based on this premise. We show that,
through intramolecular proton delivery, an increase in the
elimination rate by 18-fold compared to the fastest reported
literature tetrazines 1 and 3 could be attained. Furthermore, this
intramolecular proton source renders the reaction pH
independent with minimal change in the reaction rates from pH 3
to pH 7.4.
Introduction
Bioorthogonal chemistry – the execution of selective chemical
conversions within a biological sample – has provided a wealth of
information on a wide variety of biological processes[1]. Initial work
focused on the controlled ligation reactions within biological
systems, used copper-catalyzed Huisgen cycloaddition[2],
Staudinger ligation[3], inverse electron demand Diels-Alder
(IEDDA)[4], and other chemistries[5] to introduce reporter groups,
while minimally impacting the biological processes being studied.
Recently, bioorthogonal reactions have also been used to
unmask functional groups in living systems[6]. The IEDDA-
pyridazine elimination reaction[7] – a “click-to-release” reaction by
which an allylic substituent on trans-cyclooctene (2-TCO, axial
(E)-cyclooct-2-en-1-ol) is eliminated upon rearrangement of the
pyridazine intermediate – has proven particularly favorable in this
regard. It has excellent biocompatibility[9] and low toxicity[10]. In
vivo applications include chemical control of drug release[11]
,
,
control over T-cell activation[8], release of drugs from hydrogels[12]
as well as the control over kinase activity in mice[10]
.
Figure 1. Known tetrazines (1, 2, 3) and aminoethyl tetrazines designed
in this study (4, 5, 6, 7)
Mechanistic insights into this reaction have provided a pathway to
the improvement of this ligation-elimination reaction[13]. Chen and
co-workers, for example, discovered that asymmetric tetrazines
carrying both an electron-donating and electron-withdrawing
substituent showed a significant improvement in the elimination
rates compared to their symmetric counterparts, leading to
improved results of their most prominent tetrazine 1 over 3,6-
dimethyl tetrazine 2 (Figure 1)[13a]. Weissleder and co-workers
used carboxy-functionalized tetrazine 3 (Figure 1) resulting in
Results and Discussion
Design
We hypothesized that the presence of an aminoethyl functionality
could serve as intramolecular catalyst for both the 4,5- to 1,4-
tautomerization and subsequent elimination process (Scheme 1).
Tetrazines can add to 2-TCO in two ways with the aminoethyl
functionality positioned at either the eliminating end (A, ”head-to-
head” adduct), or the non-eliminating end (B, ”head-to-tail”
adduct). In the “head-to-head” adduct a tautomerisation of the
initial 4,5-tautomer may be promoted leading to either eliminating
(C) 1,4-tautomer or non-eliminating (D) 2,5-tautomer. Elimination
(E) may then be driven through the proximity of the intramolecular
catalytic site near both the N-1 of the dihydropyridazine core and
carbamate linkage.
A. J. C. Sarris, T. Hansen, M. A. R. de Geus, E. Maurits, W.
Doelman, Prof. H.S. Overkleeft, Dr. J. D.C. Codée, Dr. D. V.
Filippov, 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@chem.leidenuniv.nl,
filippov@lic.leidenuniv.nl
Supporting information for this article is given via a link at the end of
the document
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To investigate the behavior of tetrazines 4-7 density functional
Optimization was done in combination with a polarizable
theory (DFT) calculations were performed (Figure S1-S4,
continuum model (PCM) using water as solvent parameter
The initial IEDDA reaction proved to favor the “head-to-head”
adduct over the “head-to-tail” adduct for all studied tetrazines
(Figure 2). For example, the “head-to-head” transition state (TS-
1, Figure 2A) for tetrazine 5 proved to be 2.5 kcal/mol lower than
the “head-to-tail” transition state (TS-2, Figure 2B). The
thermodynamic preferences for the other aminoethyl tetrazines 4,
6, 7 appeared to be even greater at 4.4 - 4.6 kcal/mol (Figure 2C).
For the “head-to-head” transition states the cationic ammonium
functionality showed interaction with the carbamate linkage
resulting in an energetically favorable approach.
Supporting Information)[14]
.
The transition state of the
cycloaddition step was evaluated computationally using the
reported transition states for initial guesses.[14] All structures were
optimized with Gaussian 09 using the ωB97XD long-range
corrected hybrid functional and 6-31+G(d) as basis set.
After establishing the theoretically favored geometries of the
transition state in the IEDDA step, leading to the “head-to-head”
adduct, we investigated the lowest energy geometries of the
formed adducts with respect to the feasibility of the intramolecular
proton transfer from the ammonium functionality. Towards this
end we generated a conformer distribution, with Spartan 10
program using molecular mechanics with MMFF94 as force field,
for the formed initial 4,5-tautomer and subsequent 1,4-tautomer
after cycloaddition of a model TCO with tetrazines 2 and 5. All
generated structures were further optimized in the gas-phase with
Gaussian 09 using the ωB97XD functional with 6-311G(d,p) basis
set and optimized with PCM using water as solvent parameter,
providing lowest energy geometries (Figure 3, S3, S4). The
calculations show that tautomerization from initial 4-5-
dihydropyridazine towards eliminating 1,4-dihydropyridazine
appears energetically favorable and shows prominent interaction
between the aminoethyl functionality and the N-1 of the
dihydropyridazine core in both neutral (Figure 3A) and cationic
(Figure 3B) state. The hypothetical proton transfer through a six-
membered-cyclic-transition-state is geometrically feasible in the
kinetically favored “head-to-head” adduct and likely to facilitate
both tautomerization and elimination (Scheme 1). The design
consideration described above, supported by the calculations,
indicated that the tetrazines 4-7 would show fast and pH
independent kinetics for the ”click-to-release” reaction.
Scheme 1. The proposed mechanism for the tautomerisation and the
elimination via intramolecular catalysis by aminoethyl functionality.
Figure 2. ωB97XD/6-31+G(d)-optimized transition states for head-to-head
(TS-1) and head-to-tail (TS-2) reactions of tetrazines with model axial (E)-
cyclooct-2-ene. A) 3-ethylamine-6-methyltetrazine
5
head-to-head
transition state. B) 3-ethylamine-6-methyltetrazine 5 head-to-tail transition
state. C) Transition state energies of TS-1 and TS-2 for tetrazines 4-7 and
2. ∆ꢀꢁ‡ꢂ shown in kcal/mol.
Synthesis
To test this hypothesis, we synthesized a library of amino
functionalized tetrazines (4-17, Scheme 2). Readily accessible N-
Boc-protected aminonitriles 18-20 were prepared in good (20) to
quantitative yields (18, 19) by treatment of the corresponding
aminoalkylated precursors with di-tert-butyl dicarbonate in
presence of an appropriate base. Compounds 18, 19 and 20 were
subsequently converted to the N-Boc protected aminoalkyl
tetrazines 4b-17b according to the well-established method
involving Lewis acid catalyzed condensation of nitriles with
hydrazine followed by oxidation with sodium nitrite under acidic
conditions.[15] Optimization of this two-step synthetic protocol was
required to ensure successful synthesis of each tetrazine (Table
1). At the condensation stage five variables were altered (co-
solvent, catalyst, reaction container, temperature and reaction
time) to obtain the required individually tailored conditions for
each tetrazine. Altering one of these variables generally led to
poor or no product formation at all. As a co-solvent dioxane was
Figure 3. ωB97XD/6-311G(d)-optimized lowest energy geometries of
dihydropyridazine adducts. Proton transfer is geometrically feasible (red
lines). A) “Neutral” Initial 4,5-tautomer (left) and thermodynamically
favorable 1,4-tautomer (right) B) “Cationic” Initial 4,5-tautomer (left) and
thermodynamically favorable 1,4-tautomer (right).
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required for the synthesis of 4b, 5b, 8b, 17b, 13b to ensure
efficient formation of the dihydrotetrazine intermediate. The used
catalysts were limited to Zn(OTf)2, Ni(OTf)2 and ZnI2 due to their
successful use in literature.[15a] Overall Zn(OTf)2 was the preferred
catalyst, however Ni(OTf)2 worked well with in reactions having
Me-CN as the nitrile component (5b, 13b). Condensation step
towards compounds 6b, 9b-11b, 14b, 15b was performed in an
round-bottom flask under inert atmosphere (“Flask”) while the
other reactions were executed in a closed pressure resistant test-
tube (“Tube”). A closed set up prevents the loss of ammonia that
is formed as a side product in the condensation stage and
apparently has a beneficial effect on the solubility of components
present during this stage. Although we did not observe a
consistent improvement of the yields for all tetrazines when
executing the condensation stage in a closed vessel.
The reaction temperature and time were adjusted simultaneously,
thus the condensations that required lower temperatures (20-30
oC) were left to react 3 days while those at higher temperatures
(60-80 cC) were reacted overnight.[15] Oxidation of the formed
dihydropyridazine intermediates was facilitated by transferring the
reaction mixture to a 1:1 solution of AcOH and DCM followed by
addition of solid NaNO2.[15b] For the oxidation of dihydropyridazine
intermediates of tetrazines 4b, 6b and 13b a solution of NaNO2 in
aqueous HCl was used, a standard reagent described in the
literature to oxidize dihydroterazines of different nature.[15a] N-Boc
protected symmetric aminoalkyl tetrazines 16b and 17b were
formed as side products in the synthesis of tetrazines 12b and 6b
respectively.
Scheme 2. Synthesis of tetrazine library. (a) Boc2O, NaOH, H2O (b) Boc2O,
TEA, DCM (c) Boc2O, TEA, DCM. (d) NH2NH2, Zn(OTf)2, formamidine
acetate, then NaNO2 oxidation. (e) NH2NH2, Ni(OTf)2, acetonitrile, then
NaNO2 oxidation. (f) NH2NH2, Zn(OTf)2, 2-cyanopyridine, then NaNO2
oxidation. (g) NH2NH2, Zn(OTf)2, 2-cyanopyrimidine, thzen NaNO2
oxidation. (h) 4M HCl/Dioxane:DCM (1:1, v:v).
The N-Boc protective group could be readily removed under
anhydrous acidic conditions (4M HCl in dioxane) without
decomposition of the tetrazine core yielding the target aminoalkyl
tetrazines 4-17 in quantitative yields.
Determination of Elimination Kinetics
With the library of tetrazines (4-17) in hand we determined the
elimination behavior as compared to the tetrazines from literature,
either “releasing” (1, 2, 25) or “non-releasing” (23, 24) (Figure 4A).
To probe the importance of the intramolecular proton delivery by
the aminoethyl functionality we included N-Boc protected
tetrazines (4b-17b) and tetrazines (8-16) in which the amino
group was expected to be unable to provide the intramolecular
assistance due to its suboptimal position.
To assess the elimination properties of all tetrazines a direct
fluorescence based assay[13a] was chosen in order to monitor the
reaction mixture in real time during the elimination process. This
method was preferred over LC-MS based analysis[13a][13b] as it
allows a rapid measurement of several tetrazines simultaneously,
acquisition of more data (at the initial stage of the reaction) and is
not affected by possible “pseudo-release” artefacts caused by LC-
MS analysis.[13b]
Tetrazine elimination properties were assessed through reaction
with fluorogenic 2-TCO protected 7-amino-4-methylcoumarin 28
(2-TCO-AMC)[13a] and tracking the product AMC 27 by measuring
its characteristic fluorescence at 450 nm (Figure 4B, S5, S6).
Initially we tested the literature tetrazines in both
Table 1. Top: General procedure for the synthesis of N-Boc protected
tetrazines 4b-17b and carboxy functionalized tetrazines 3 and 21. Bottom:
Variations in five experimental parameters used to synthesize tetrazines
4b-17b, 3, and 21.
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DMSO/H2O (1:1, v:v) and PBS (0.25% DMSO) (Figure 4C, right
panel, S8). The elimination properties in PBS were vastly different
compared to DMSO/H2O. Tetrazine 2, for example, was both
faster and more efficient in PBS (Figure 4C, left panel). As a result,
the further focus was on the experiments conducted in PBS. The
elimination efficiency (Eff %) and rate (kelim) of the tetrazine library
was determined by exposing 2-TCO-AMC 28 (5 µM) to 4
equivalents (20 µM) of a tetrazine in PBS (pH 7.4) at 37 oC for 8
days (Figure S7-S13). At various time points, the fluorescence
intensity relative to 2-TCO-AMC 28 (0%) and AMC 27 (100%) was
quantified (Figure S14-S15). The solvent and concentrations
were chosen to be relevant to experiments conducted in biological
systems.
The data obtained was analyzed and the rate (kelim) and efficiency
(Eff%) was determined for each tetrazine. The values for all
tetrazines were plotted as dots in an XY-scatterplot as kelim
(logarithmic scale) vs Eff% (linear scale) (Figure 5A, XY-
scatterplot). Three classes of tetrazines were most conspicious:
14b, 15b, 23, 11 caused slow elimination rates (kelim < 0.20 * 10-5
s-1; Figure 5B). 11b, 2, 5, 17 all displayed intermediate elimination
rates (kelim between 1.5 and 3.0 * 10-5 s-1; Figure 5C). Tetrazines
1, 4, 6, 7 formed a third, most interesting, class of tetrazines
defined by their very fast release kinetics (Figure 5D). While
reference tetrazine 1 is fast (kelim = 8.86 * 10-5 s-1), it is still
significantly slower compared to 4, 6, and 7 (kelim = 15.3, 23.7,
35.9 10-5 s-1 respectively). Release rates for tetrazines 4, 6 and 7
were too fast to be accurately determined by our initial assay and
have therefore been displayed as their recorded minimum rates.
It is noteworthy, that the tetrazines with the protected amino group
(14b, 15b) and the tetrazines with a suboptimal positioned amino
group (11) showed slow elimination rates (Figure 5B). This
behavior proved to be general across the whole test set (Figure
S7-S13) and underscores the importance of the intramolecular
proton delivery for the rate of the “click-to-release” process.
Figure 4. A) Tetrazines 1, 2, 23-25 used as literature references. B)
Fluorescence based assay: Fluorogenic 2-TCO-AMC 28 is used as a
measurement tool to track the release of fluorescent AMC 27 upon
reaction with tetrazines. C) Data obtained in DMSO/H2O (2, left panel) or
PBS (1, 2, 23-25, right panel) as solvent during the fluorescence based
assay.
Figure 5. A) XY-scatterplot of all data obtained in PBS as solvent during
the fluorescence based assay. Each result (tetrazines 1, 2, 23-25, 4b-17b,
4-17) is depicted as a dot based on their elimination efficiency and rate. B)
Tetrazines 14b, 15b, 23 and 11 with slow release properties kelim
< 0.2 * 10-5 s-1. C) Tetrazines 11b, 2, 5 and 17 with intermediate release
properties: Eff% > 60%. D) Tetrazines 1, 4, 6 and 7 with fast release
properties: kelim > 8 * 10-5 s-1.
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Figure 8. A) Rate enhancement of pH independent functional groups
depicted in orange over pH dependent functional groups depicted in blue
and red.
availability of protons in solution enhances the elimination rate.
Lowering the proton concentration by raising pH to 6.0 or 7.4
resulted in large decreases in the elimination rates observed for
most tetrazines (Figure 6B, 6C, S16-S18). At pH 7.4 tetrazines 1-
3, 21, 6b, and 7b showed much slower elimination rates (5-15 *
10-5 s-1, Figure 6C). Aminoethyl tetrazines 6 (kelim = 241 * 10-5 s-1)
and 7 (kelim = 120 * 10-5 s-1) on the other hand showed a minimal
reduction in rate at increased solvent pH (Figure 7), resulting in a
rate 18-27-fold faster at pH 7.4. Substituting amino (6, 7) for
alcohol (1) or carbamate (6b, 7b) functionality reintroduced pH-
dependency in the elimination process (Figure 8). A carboxy
functionality, as seen in tetrazine 3, provided an overall moderate
elimination rate and moderate pH-dependency (Figure 8).
Substitution of carboxy (3) for amino (5) functionality improved
both properties. Furthermore, the fastest tetrazine 6 outperforms
tetrazine 3 by 18-fold in terms of elimination rate. Finally, when
comparing tetrazines 4-7, the data also shows that the elimination
rate is positively affected by electron-withdrawing substituents
(pyridine/pyrimidine) explaining the differences between the
tetrazines, in line with previous findings.[13b]
Figure 6. Proton depletion of solvent. A) Scatter plot of tetrazines at pH =
3. B) Scatter plot of tetrazines at pH = 6. C) Scatter plot of tetrazines at pH
= 7.4. D) Scatter plot of pH dependent tetrazines (6, 7) and pH independent
tetrazines(6b, 7b, 1).
These results strongly support our hypothesis that the elimination
process is dependent on proton availability and can be catalyzed
at biologically relevant pH (3-7.4) through careful placement of
cationic ammonium functionality acting as an intramolecular
catalyst.
LC-MS analysis
In order to gain additional insight in the course of the “click-to-
release” reaction with the newly developed aminoalkyl tetrazines
and to study the intermediates and possible side products we also
assessed the elimination rates of the key compounds 5 - 7 using
reported LC-MS based approach that uses an ammonium formate
Figure 7. Elimination graphs of tetrazines 5, 3, 6 and 7 showing pH
dependent and pH independent behavior.
pH Dependency of Elimination Rates
+
buffered water-acetonitrile eluent (2.5mM NH4 HCOO-, pH = 8.4)
Following these results we set out to determine whether the fast
releasing aminoethyl functionalized tetrazines retained these
properties over a wide pH-range. To this end, tetrazines 1-3, 21,
4b-7b and 4-7 were exposed to phosphate buffered solutions at
various pH (0.2M [PO42-], 10% DMSO, pH = 3.0 (red lines), 6.0
(green lines), and 7.4(blue lines)). At pH 3.0 (Figure 6A, S16-S18)
all tetrazines showed elimination rates between 30 * 10-5 s-1 and
500 * 10-5 s-1, indicating that the
and taking the first analysis point at t = 5 min.[13b] Known tetrazines
1-3 were analyzed by this method for comparison. The elimination
rates of tetrazines 2 and 3 showed results corresponding to data
reported by Weissleder et al[13b], where tetrazine 3 gives 48%
release at t = 5 min, going up to 72% after 16 hours (Figure 9).
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correspond to the preferential formation of the rapidly releasing
“head-to-head” adduct as predicted by our calculations.
It is noteworthy that although we do observe a multitude of
different intermediates, including putative “head-to-tail”
and ”head-to-head” adducts, in the reaction of 2-TCO-AMC 28
with comparatively slow eliminating tetrazines 1 and 2 (Figure 10,
S19, S20), as well as with tetrazines 3, 5, 6 and 7 (Figure 9, S21-
S24), we do not consistently see formation of dead-end adduct
(End) as reported by others[13b]. Furthermore the amount of
oxidized adduct (Ox) is reduced for tetrazines 1, 6-7 compared to
tetrazines 2, 3, and 5. The results of the LC-MS analysis of the
“click-to-release” reaction of 2-TCO-AMC 28 with tetrazines 1 and
2 are similar to what is published by Robillard and co-workers in
terms of observed intermediates and side products (Figure 9A-
B).[13c] To summarize, the results obtained from the LC-MS
analysis of the key tetrazine derivatives demonstrate that the
method shows reproducible results for the known tetrazines 2 and
3 and that the aminoethyl tetrazines 5-7 do perform as designed
in the “click-to-release” IEDDA reaction.
Conclusions
Figure 9. Time dependent LC-MS analysis of 2-TCO-AMC 28 with
tetrazines 3, 5, 6 and 7 in PBS.
The full kinetic profiling of a focused tetrazine library clearly shows
that the presence of a properly placed cationic ammonium
functionality acting as an intramolecular proton donor gives the
tetrazine mediated “click-to-release” elimination
a
pH
independent character. Asymmetric tetrazines that contain both
this aminoethyl substituent and an electron-withdrawing
substituent on the tetrazine core show unprecedented release
rates combined with nearly complete pH independence over the
whole biologically relevant pH range.
Experimental Section
Full experimental details can be found in the online supporting information.
Acknowledgements
AJCS, DVF and SIVK were funded by the Institute of Chemical
Immunology (NWO Zwaartekracht), MARDG and SIVK were
funded by the European Research Council (ERC-2014-StG-
639005), WD was funded by an NWO BBoL-grant. The authors
thank the SURFsara for the support in using the Dutch national
supercomputer, including the Lisa system.
Figure 10. Time dependent LC-MS analysis of 2-TCO-AMC 28 with
tetrazines in PBS. Aminomethyl coumarin containing molecules: product
“AMC”, adduct “A”, oxidized adduct “Ox” and dead-end adduct “End”. A)
Reaction of 28 with tetrazine 1. B) Reaction of 28 with tetrazine 2. C)
Elimination pathway with possible adducts and products.
The explanation offered by the authors invokes rapidly releasing
“head-to-head” adduct and slowly releasing “head-to-tail” adduct
which are formed in approximately equal amounts.[13b] The data
showed much faster initial release than measured in the
fluorescence assay, and this is likely due to additional “pseudo-
release” or concentration effects during the LC-MS analysis. In
turn, aminoethyl tetrazines 5, 6 and 7, showed 78%, 80% and
70% initial release respectively at t = 5 minutes with only a small
further increase in release over time (Figure 9). This would
Keywords: inverse electron demand Diels-Alder • trans-
cyclooctene elimination • click-to-release • pH independent •
bioorthogonal deprotection
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10.1002/chem.201803839
Chemistry - A European Journal
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Alexi J.C. Sarris, Thomas
Hansen, Mark A.R. de Geus,
Elmer Maurits, Ward Doelman,
Herman S. Overkleeft, Jeroen
D.C. Codée, Dmitri V. Filippov,*
and Sander I. van Kasteren*
Fast and pH independent
elimination of trans-
cyclooctene using aminoethyl
functionalized tetrazines
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