Acylation-Mediated 'Kinetic Turn-On' of 3-Amino-1,2,4,5-tetrazines

Article abstract of DOI:10.1055/s-0036-1591764

The fast and biocompatible ligation of 1,2,4,5-tetrazines with strained alkenes has found numerous applications in biomedical sciences. The reactivity of a 1,2,4,5-tetrazine can generally be tuned by changing its electronic properties by varying the substituents in the 3- and/or 6-position. An increased reactivity of such bioorthogonal probes upon conjugation or attachment to a target molecule has not previously been described. Such an approach would be beneficial, as it would minimize the impact of residual tetrazine reagents and/or impurities. Herein, we describe such a 'kinetic turn-on' of 1,2,4,5-tetrazines upon conjugation. On the basis of the significant increase in reactivity following N-acylation predicted by quantum chemical calculations, we prepared 3-aminotetrazines and their corresponding acetylated derivatives. An investigation of the reaction kinetics indeed revealed a remarkable increase in reactivity upon acylation.

Full text of DOI:10.1055/s-0036-1591764

SYNLETT
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
©
Georg Thieme Verlag Stuttgart · New York
2018, 29, A–F
letter
A
en
Synlett
S. Kronister et al.
Letter
Acylation-Mediated ‘Kinetic Turn-On’ of 3-Amino-1,2,4,5-tetrazines
Stefan Kronister
Dennis Svatunek
Christoph Denk
Hannes Mikula*
R
R
IEDDA
N
N
N
N
'kinetic turn-on'
N
N
N
N
IEDDA
EDG
EWG
NH2
NH
Institute of Applied Synthetic Chemistry, TU Wien,
Getreidemarkt 9/163-OC, 1060 Vienna, Austria
hannes.mikula@tuwien.ac.at
O
Published as part of the Special Section 9th EuCheMS
Organic Division Young Investigator Workshop
Received: 14.11.2017
Accepted after revision: 29.01.2018
Published online: 16.02.2018
terest because of their in vivo compatibility, selectivity, and
exceptionally high reaction rates. In recent years, tetrazine
ligations have been applied in the development of numer-
ous applications in biomedical research, including, but not
DOI: 10.1055/s-0036-1591764; Art ID: st-2017-b0835-l
Abstract The fast and biocompatible ligation of 1,2,4,5-tetrazines
with strained alkenes has found numerous applications in biomedical
sciences. The reactivity of a 1,2,4,5-tetrazine can generally be tuned by
changing its electronic properties by varying the substituents in the 3-
and/or 6-position. An increased reactivity of such bioorthogonal probes
upon conjugation or attachment to a target molecule has not previous-
ly been described. Such an approach would be beneficial, as it would
minimize the impact of residual tetrazine reagents and/or impurities.
Herein, we describe such a ‘kinetic turn-on’ of 1,2,4,5-tetrazines upon
conjugation. On the basis of the significant increase in reactivity follow-
ing N-acylation predicted by quantum chemical calculations, we pre-
pared 3-aminotetrazines and their corresponding acetylated deriva-
tives. An investigation of the reaction kinetics indeed revealed a
remarkable increase in reactivity upon acylation.
7
limited to, (i) bioconjugation; (ii) molecular imaging of pro-
8
–10
11
teins,
surface antigens,
small molecules/modified
drugs,1
2,13
lipids, or glycans; (iii) cell modification with
14
15
16
nanomaterials for clinical diagnostics; (iv) the develop-
ment of smart fluorogenic probes;
17–20
(v) bioorthogonal
approaches to the identification of drug targets in living
cells;21 and (vi) healthcare materials. Additionally, the
outstanding reaction kinetics of tetrazine ligations have led
to an emerging application of bioorthogonal chemistry in
the fields of radiolabeling (in vitro click) and pretargeted
single-proton emission computed tomography or positron
emission tomography (in vivo click), in which high reaction
rates are essential due to the very low concentrations of the
radiolabeled compounds in vivo.2
22
Key words click chemistry, tetrazines, kinetics, bioorthogonal chem-
istry, Diels–Alder reaction, acylation
3–26
Second-order rate constants of up to 3.3 × 10 M s–1
6
–1
The challenge of engineering chemical transformations
that can proceed within the complex environment of living
systems has led to the research field of bioorthogonal
(25 °C, H O) have been reported for the ligation of tetra-
2
zines with highly strained trans-cyclooctenes (TCOs) as
dienophiles;27 this makes the tetrazine/TCO ligation the
fastest bioorthogonal reaction to be discovered so far.
1
chemistry. To enable a bioorthogonal reaction, the chemi-
cal probes that are involved need to exhibit high reactivity,
high selectivity, biocompatibility, and metabolic stability.
N
R2
R2
[
4+2]-
R2
H
N
Cyclo-
reversion
H
H
2
Cycloaddition
The Staudinger ligation and the strain-promoted azide–
N
N
N
N
N
N
N
3
alkyne cycloaddition (SPAAC), both developed by Bertozzi
N
R1
N2
H
and co-workers, were the first bioorthogonal reactions to
be described. The SPAAC ligation is based on Sharpless’s
R1
R1
1
4
click chemistry, but can proceed without toxic copper(I),
Figure 1 Mechanism of the bioorthogonal ligation between trans-cy-
clooctene (1) and 1,2,4,5-tetrazines
1
and is therefore suitable for in vivo applications.
The tetrazine ligation between 1,2,4,5-tetrazines and
strained alkenes such as norbornene or trans-cyclooctene
(
TCO; 1) was first described in 2008 by the groups of Fox
The first 1,2,4,5-tetrazines had already been reported by
the end of the nineteenth century by Adolf Pinner, and
were prepared from imino ester hydrochlorides (Pinner
5,6
and Weissleder. These inverse electron-demand Diels–Al-
der (IEDDA)-initiated ligations (Figure 1) have attracted in-
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–F

B
Synlett
S. Kronister et al.
Letter
(
from left to right) Stefan Kronister graduated from TU Wien in March 2016 with an M.Sc. in Technical Chemistry. He is currently a second–year Ph.D.
student at the Institute of Applied Synthetic Chemistry at TU Wien. His Ph.D. studies focus on the development of bioorthogonal tools and targeted
prodrug activation inside tumor cells through in vivo cleavage reactions.
Dennis Svatunek received his Ph.D. (2016) from TU Wien, working on the kinetics of bioorthogonal ligations and their application in pretargeted
imaging under the supervision of Professor Günter Allmaier. During his Ph.D. studies, Dennis joined the group of Professor Joseph M. Fox at the Univer-
sity of Delaware (2014), working on improved bioorthogonal reactions for rapid radiolabeling. He is currently a postdoctoral researcher at TU Wien,
focusing on investigations on the kinetics of cycloadditions by computational and experimental methods.
Christoph Denk obtained his Ph.D. from TU Wien in 2016, and is currently a postdoctoral researcher at the Institute of Applied Synthetic Chemistry.
His research focuses on radiolabeled bioorthogonal agents for pretargeted nuclear imaging and therapy. Christoph gains ideas and inspiration for his
scientific work while running long distances or while observing life in his saltwater reef tank.
Hannes Mikula obtained his Ph.D. from TU Wien in 2014, working under the supervision of Professor Johannes Fröhlich. He then joined the laboratory
of Professor Ralph Weissleder at the Massachusetts General Hospital/Harvard Medical School as a postdoctoral fellow funded by the Austrian Science
Fund (FWF) within the Erwin-Schrödinger-Program. His postdoctoral studies focused on molecular imaging and the development of bioorthogonal
tools for in vivo chemistry. In 2016, Hannes returned to TU Wien to continue research in the fields of chemical biology, bioorthogonal reactions, in vivo
medical imaging, and the development of diagnostic tools.
salts) and hydrazine, which form an amidrazone intermedi-
ate. This reacts with excess hydrazine to give a dihydro-
tetrazine that is finally oxidized to give a 1,2,4,5-tetrazine
by varying the substituents in the 3- and 6-positions. In
general, electron-withdrawing substituents increase the re-
activity, whereas electron-donating groups decrease it.
Tetrazines bearing an amino group are useful because of
their straightforward conjugation to target molecules or the
ease with which they undergo further modification. An
overview of selected amino-functionalized tetrazines and
their respective second-order rate constants for the reac-
tion with TCO (1) is presented in Figure 3.
36
28
(
Figure 2a).
In recent years, tetrazines have most commonly been
synthesized by condensation of two nitrile molecules with
29,30
hydrazine, followed by oxidation (Figure 2b).
However,
the preparation of the alkyl-substituted tetrazines by this
method often results in low yields. A major improvement
was achieved by Devaraj and co-workers, who used Lewis
acids for the activation of the nitriles, leading to significant-
Aminotetrazine 2 exhibits a low reactivity because of
the electron-donating effect of the NH group directly at-
2
31
ly increased reaction yields. A wide variety of aryl- and
alkyl-substituted tetrazines are accessible by using this ap-
proach. A remaining drawback of the Lewis-acid-mediated
tetrazine synthesis is that a statistical mixture is often ob-
tained when different nitriles are used to prepare asym-
tached to the tetrazine moiety. Dialkyltetrazine 3 shows
only moderate reactivity due to the donating effect of the
alkyl substituents,37 whereas aryl/alkyl-substituted tetra-
zines such as 4 are slightly more reactive.1
0,37
Monosubsti-
tuted tetrazines such as 5 show high reaction rates because
32
38
metrically substituted tetrazines. Alternatively, tetrazines
can be synthesized by nucleophilic aromatic substitution of
precursors mono- or difunctionalized with 3,5-dimethyl-
of a lower steric hindrance; the reaction rates are similar
to those of tetrazines bearing electron-withdrawing hetero-
aryl substituents such as pyridyl or pyrimidyl moieties (e.g.,
33,34
10
pyrazolyl or chloro groups (Figure 2c).
In general, these
6). However, the applicability of highly reactive tetrazines
methods afford tetrazines with decreased or even very low
reactivities. By the 1960s, Takimoto and co-workers had
presented a robust and straightforward method to produce
is often limited because of their low stability in biological
media.39
When a target molecule is modified with a tetrazine
tag, excess reagent needs to be completely removed before
further application of the conjugate, as unbound tetrazines
compete with the tetrazine-labeled molecule in the reac-
tion with TCO (1). We surmised that a tetrazine showing a
significant increase in IEDDA reactivity upon attachment to
a target molecule (‘kinetic turn-on’) might be highly benefi-
35
unsymmetrical 3-aminotetrazines,
in which 3-azido-
1
3
,2,4-triazole-4-amines are thermally decomposed to give
-aminotetrazines in good yields (Figure 2d).
The reaction rate constants of tetrazine ligations can be
tuned within a range of several orders of magnitude by
changing the electronic properties of the tetrazine moiety
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–F

C
Synlett
S. Kronister et al.
Letter
R
R
a)
Cl-
NH2+
[Ox.]
R
HN
HN
N
N
N
N
N
N
OR
R
R
R
R
b)
c)
H2N-NH2
Zn2+, Ni2+
HN
HN
N
N
[Ox.]
N
N
N
N
N
R
R
N
R
N
Cl
NH
N
N
N
N
N
N
N
N
H2N
NH2
N
N
Cl
Figure 3 Overview of various aminotetrazines used in IEDDA ligations
and their respective second-order rate constants for reaction with TCO
(
1) at room temperature under aqueous conditions.3
2,37
Nu
Nu
Nu
Nu
Nu
ple model for conjugation reactions yielding N-acylated 3-
aminotetrazines. Substrates 7 and 8 were chosen because of
their expected higher reactivity compared with the meth-
yltetrazine 2.
N
N
X = Cl or
N
N
N
N
N
N
N
N
or
X
R
‡
Gibbs free energies of activation (ΔG ) for the reaction of
d)
NH
N
N
aminotetrazines 2, 7, and 8 and their respective acetylated
derivatives 9–11 with TCO (1) were calculated by means of
density-functional theory [M06-2X/6-311+G(d,p), gas
DT
N
N
N
N
H2N
NH2
R
N3
N
NH2
‡
NH2
phase, Gaussian 09]. The ΔG values for the reaction of
acetylated compounds were around 4 kcal/mol lower than
those of the corresponding aminotetrazines, resulting in a
predicted increase in reactivity of around 600-fold (Table 1).
Figure 2 Methods for the preparation of 1,2,4,5-tetrazines. (a) First
28
tetrazine synthesis by Pinner; (b) Lewis-acid-mediated tetrazine syn-
thesis;2
9–31
(c) 3,5-dimethylpyrazol-1-yl- or chloro-substituted tetra-
33,34
zines, and subsequent nucleophilic aromatic substitution;
(d)
Synthesis of unsymmetrical aminotetrazines by thermolytic decompo-
35
sition of 3-azido-4H-1,2,4-triazol-4-amines. Ox = oxidation;
Nu = nucleophile.32
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
cial because of the minimalized impact of residual tetrazine
reagent and/or tetrazine impurities. Lengthy purification
procedures could be shortened or even omitted, which
would be of particular importance in cases where short-
lived nuclides (e.g., carbon-11) are involved.
N
N
N
CF3
H
2
7
8
NHAc
O
O
O
N-Derivatization of aminotetrazines 3–6 is likely to have
only a low, or even no, impact on cycloaddition reactivity
because of the limited influence of the electronic properties
of the 1,2,4,5-tetrazine moiety. In contrast, N-acylation of 2
appeared to be likely to have a pronounced influence on the
reaction kinetics. We therefore investigated the kinetic
turn-on of compound 2 and the 3-aminotetrazines 7 and 8
upon N-acylation. Acetylation affording the corresponding
HN
HN
HN
N
N
N
N
N
N
N
N
N
N
N
N
CF3
10
H
9
11
Figure 4 : 3-Aminotetrazines 2, 7, and 8, and corresponding 3-acet-
amidotetrazines 9–11
3
-acetamidotetrazines 9–11 (Figure 4) was chosen as a sim-
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–F

D
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S. Kronister et al.
Letter
Table 1 Predicted Gibbs Free Energies of Activation (ΔG‡) for the
Reactions of 3-Amino- and 3-Acetamidotetrazines with TCO (1)
HCl
NH2
R-COOH,
100 °C
N
N
NH
H NNH
HCl
H2N
N
2
2
NH2
R
N
N
NH2
H2N
NH2
91%
N
N
H
H
H
‡
NH2
ΔG [kcal/mol]
Predicted increase in
reactivity
R1
R2 = H
R2 = Ac
14: R = CH3
5: R = CF3
6: R = H
(
‘kinetic turn-on’)
1
1
1
2
13
R2
Me
23.1
18.9
21.7
18.8
14.9
17.4
660-fold
430-fold
640-fold
R
NH
N
N
CF
3
NaNO2/HCl, 0–5 °C
thermolysis
N
N
N
N
N
N
N
R
N
N3
– N2
H
N
NH2
R1
NH2
1
1
1
7: R = CH3, 30%
8: R = CF3, 42%
9: R = H, 34%
2
7
: R = CH3, 44%
: R = CF3, 53%
This increase can be attributed to the electron-with-
drawing effect of the acetamido group in comparison with
the amino group, as reflected in the calculated molecular-
orbital energies [level of theory: HF/6-311+G(d,p)//M06-
(2 steps from 13)
8: R = H, 49%
Scheme 1 Synthesis of 3-aminotetrazines 2, 7, and 8
2
X/6-311+G(d,p)] for the low-lying unoccupied orbitals in-
yield for compound 9 was due to the formation of a di-
acetylated byproduct (as indicated by LC/MS). The stability
of 11 in phosphate-buffered saline was examined by moni-
toring its absorbance at 525 nm over a period of 24 hours, re-
vealing a recovery of >90% (Supporting Information, Figure S7).
volved in the reaction. The acetylated compounds show or-
bital energies that are 0.6–0.8 eV lower than those of the
corresponding aminotetrazines (Supporting Information,
Figure S8).
In addition, a distortion/interaction analysis40 was per-
formed for the reaction between TCO (1) and the monosub-
stituted tetrazines 8 and 11. As expected, the acetylated de-
rivative 11 shows a lower free energy of activation and an
earlier transition state. Distortion energies are slightly ele-
vated compared with 8; however, interaction energies are
much more favorable over the whole intrinsic reaction co-
ordinate, thus lowering the energy of activation consider-
ably and leading to an earlier transition state (Supporting
Information, Figure S9).
R
R
Ac2O, NEt3,
DMAP, CH2Cl2
N
N
N
N
N
N
N
N
2
: R = CH3
7: R = CF3
: R = H
9: R = CH3, 20%
10: R = CF3, 65%
11: R = H, 55%
NH2
NH
8
O
Scheme 2 Synthesis of 3-acetamidotetrazines 9–11
The reactions kinetics of aminotetrazines 2, 7, and 8 and
those of their corresponding N-acetyl derivatives 9–11 with
TCO (1) in 1,4-dioxane at 25 °C were investigated by
stopped-flow measurements. Pseudo-first-order condi-
tions were used (an excess of 1), and the decrease in the
concentration of the tetrazole was monitored by absor-
bance measurement (Supporting Information). The results
revealed a significant ‘kinetic turn-on’ upon N-acylation
(Figure 5).
3
-Aminotetrazines 2, 7, and 8 were each prepared in
four steps (Scheme 1). Triaminoguanidine hydrochloride
13) was prepared from guanidine hydrochloride (12) and
(
41
hydrazine hydrate. The 3-hydrazino-4H-1,2,4-triazol-4-
amine intermediates 14–16 were synthesized by cyclocon-
densation of 13 with the appropriate carboxylic acid.42 The
crude products were directly converted into the corre-
43
sponding 3-azido-1,2,4-triazol-4-amines 17–19 by diazo-
44
tization of the hydrazino group. Aminotetrazoles 2, 7, and
45
8
were obtained by thermolytic decomposition of the cor-
responding 3-azido-1,2,4-triazoles in overall yields of 12%
3
5
(
(
2), 20% (7), and 15% (8). Notably, anhydrous hydrazine
not commercially available in Europe) was not required for
these syntheses. Although we did not encounter any prob-
lems during this study, all compounds with a high nitrogen
content are potentially energetic materials and should be
handled and stored accordingly.
Acetylation was carried out by applying commonly used
esterification protocols, including (i) acetic anhydride, tri-
ethylamine, and 4-(dimethylamino)pyridine,4 (Scheme 2),
or (ii) acetyl chloride and triethylamine (Supporting Infor-
6
Figure 5 Stopped-flow measurements showing a significantly
increased IEDDA reactivity of N-acetylated aminotetrazoles 9–11 in
comparison with 2, 7, and 8
47
mation), to give the N-acetylated tetrazines 9–11. The 20%
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–F

E
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S. Kronister et al.
Letter
The greatest increase in reactivity of 743-fold was ob-
served for compound 10. The second-order rate constant
for the reaction of the most reactive 3-amidotetrazine 11
(13) Meyer, J.-P.; Adumeau, P.; Lewis, J. S.; Zeglis, B. M. Bioconjugate
Chem. 2016, 27, 2791.
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Ed. 2012, 51, 7476.
–1 –1
and TCO (1) was determined to be 9.5 M s , which is ap-
proximately an order of magnitude greater than that of the
dialkyltetrazines that have been used in many applications
(15) Stairs, S.; Neves, A. A.; Stöckmann, H.; Wainman, Y. A.; Ireland-
Zecchini, H.; Brindle, K. M.; Leeper, F. J. ChemBioChem 2013, 14,
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48–51
in bioorthogonal chemistry.
-Aminotetrazines were prepared by a straightforward
(16) Haun, J. B.; Devaraj, N. K.; Hilderbrand, S. A.; Lee, H.; Weissleder,
3
R. Nat. Nanotechnol. 2010, 5, 660.
(
(
(
17) Carlson, J. C. T.; Meimetis, L. G.; Hilderbrand, S. A.; Weissleder,
method without the need for anhydrous hydrazine, a re-
agent that is not commercially available in Europe. Acetyla-
tion of these compounds by acetic anhydride or acetyl chlo-
ride gave the corresponding 3-acetamidotetrazines. Kinetic
investigations revealed a remarkable ‘kinetic turn-on’ in
agreement with quantum chemical calculations. The most
reactive amidotetrazine 11 was shown to be sufficiently
stable and to react with TCO (1) approximately ten times
faster than dialkyltetrazines. Overall, we are convinced that
the presented concept can be applied in the development of
new bioorthogonal tools, labeling strategies, and improved
protocols.
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(
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S
u
p
p
ortioIgnfrm oaitn
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u
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9) Seitchik, J. L.; Peeler, J. C.; Taylor, M. T.; Blackman, M. L.; Rhoads,
T. W.; Cooley, R. B.; Refakis, C.; Fox, J. M.; Mehl, R. A. J. Am. Chem.
Soc. 2012, 134, 2898.
(
(
(
41) Nhu, D.; Duffy, S.; Avery, M. V.; Baell, J. B. Bioorg. Med. Chem.
Lett. 2010, 20, 4496.
42) Cardillo, P.; Dellavedova, M.; Gigante, L.; Lunghi, A.; Pasturenzi,
C.; Salatelli, E.; Zanirato, P. Eur. J. Org. Chem. 2012, 1195.
43) 3-Azido-4H-1,2,4-triazole-4-amine (19)
(
A suspension of guanidine hydrochloride (13; 2.2 g, 0.014 mol,
(
(
(
10) Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J.
1
equiv) in HCO H (40 mL) was heated to 100 °C for 16 h. The
W. Nat. Chem. 2012, 4, 298.
2
acid was evaporated and the residue was dissolved in 6 N HCl
30 mL) to give a solution that was refluxed for 2 h. Upon
removal of volatiles, the hydrazinotriazolylamine intermediate
6 was obtained as a white crystalline solid and used in the
next step (diazotization) without further purification. A solu-
11) Devaraj, N. K.; Upadhyay, R.; Haun, J. B.; Hilderbrand, S. A.;
Weissleder, R. Angew. Chem. Int. Ed. 2009, 48, 7013.
12) Yang, K. S.; Budin, G.; Reiner, T.; Vinegoni, C.; Weissleder, R.
Angew. Chem. Int. Ed. Engl. 2012, 51, 6598.
(
1
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–F

F
Synlett
S. Kronister et al.
Letter
tion of NaNO2 (0.97 g, 0.014 mol, 1 equiv) in H O (4 mL) was
(46) N-1,2,4,5-Tetrazin-3-ylacetamide (11)
2
added dropwise to a solution of crude 16 in 1 N HCl (20 mL) at
DMAP (7.5 mg, 0.06 mmol, 0.1 equiv), Ac O (292 μL, 315 mg,
2
0
°C. The solution was stirred at 0 °C for 30 min, then allowed to
3.09 mmol, 5 equiv), and Et N (103 μL, 75 mg, 0.74 mmol, 1.2
3
warm to r.t. The mixture was neutralized with to pH 9–10 with
Na CO and extracted with Et O in a continuous extractor for 72
equiv) were added to a solution of amine 8 (60 mg, 0.62 mmol,
1 equiv) in anhyd CH Cl (4 mL), and the mixture was stirred at
2
3
2
2
2
h. The solvent was evaporated, and the crude product was puri-
fied by column chromatography (silica gel, CH Cl –MeOH) to
give a beige solid; yield: 610 mg (34%); mp 53–55 °C. H NMR
r.t. overnight. Purification by column chromatography (silica
gel, hexane–EtOAc) gave a red solid; yield: 47 mg (55%); mp
203–205 °C.
2
2
1
1
(
400 MHz, DMSO-d ): δ = 8.34 (s, 1 H, CH), 5.97 (br s, 2 H, NH ).
H NMR (400 MHz, acetone-d ): δ = 10.18 (s, 1 H, CH), 2.82 (s, 3
6
2
6
13
13
C NMR (100 MHz, DMSO-d ): δ = 148.3 (s, 1 C), 145.1 (d, 1 C).
H, CH3). C NMR (100 MHz, acetone-d ): δ = 168.3 (s, 1 C),
162.1 (s, 1 C), 156.2 (d, 1 C), 24.0 (q, 1 C). MS-ESI: m/z [M + H]
calcd for C H N O : 140.0; found: 139.3.
6
6
+
+
+
MS-ESI: m/z [M + H] calcd for C H N : 126.0; found: 125.4.
2
4
7
+
(
44) Takimoto, H. H.; Denault, G. C.; Hotta, S. J. Org. Chem. 1965, 30,
11.
4
6
5
7
For the syntheses and characterization of the N-(1,2,4,5-tetra-
zin-3-yl)acetamides 9 and 10, see the Supporting Information.
(47) Ośmialowski, B.; Kolehmainen, E.; Dobosz, R.; Gawinecki, R.;
Kauppinen, R.; Valkonen, A.; Koivukorpi, J.; Rissanen, K. J. Phys.
Chem. A 2010, 114, 10421.
(48) Denk, C.; Svatunek, D.; Filip, T.; Wanek, T.; Lumpi, D.; Fröhlich,
J.; Kuntner, C.; Mikula, H. Angew. Chem. Int. Ed. 2014, 53, 9655.
(49) Denk, C.; Svatunek, D.; Mairinger, S.; Stanek, J.; Filip, T.;
Matscheko, D.; Kuntner, C.; Wanek, T.; Mikula, H. Bioconjugate
Chem. 2016, 27, 1707.
(
45) 1,2,4,5-Tetrazine-3-amine (8)
Amine 19 (600 mg, 4.80 mmol, 1 equiv) was suspended in PhCl
(15 mL) and the mixture was refluxed for 18 h. The solvent was
removed under reduced pressure and the residue was purified
by column chromatography (silica gel, hexanes–EtOAc) to give a
red solid; yield: 228 mg (49%); mp 170–172 °C.
1
H NMR (400 MHz, CD Cl ): δ = 9.71 (s, 1 H, CH), 5.74 (br s, 2 H,
2
2
13
NH2). C NMR (100 MHz, CD Cl ): δ = 164.8 (s, 1 C), 154.7 (d,
1
2
2
+
+
C). MS-ESI: m/z [M + H] calcd for C H N : 98.0; found: 97.6.
2 4 5
For the synthesis and characterization of 1,2,4,5-tetrazine-3-
amines 2 and 7, see the Supporting Information.
(50) Versteegen, R. M.; Rossin, R.; ten Hoeve, W.; Janssen, H. M.;
Robillard, M. S. Angew. Chem. Int. Ed. 2013, 52, 14112.
(51) Mejia Oneto, J. M.; Khan, I.; Seebald, L.; Royzen, M. ACS Cent. Sci.
2016, 2, 476.
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–F