Clicking 1,2,4,5-tetrazine and cyclooctynes with tunable reaction rates

Article abstract of DOI:10.1039/c2cc16716f

Substituted tetrazines have been found to undergo facile inverse electron demand Diels-Alder reactions with "tunable" reaction rates.

Full text of DOI:10.1039/c2cc16716f

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Chemical Communications
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Volume 48 | Number 12 | 7 February 2012 | Pages 1709–1824
ISSN 1359-7345
COMMUNICATION
B. Wang et al.
Clicking 1,2,4,5-tetrazine and cyclooctynes with tunable reaction rates

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COMMUNICATION
Clicking 1,2,4,5-tetrazine and cyclooctynes with tunable reaction ratesw
Weixuan Chen,z Danzhu Wang,z Chaofeng Dai, Donald Hamelberg and Binghe Wang*
Received 30th October 2011, Accepted 23rd November 2011
DOI: 10.1039/c2cc16716f
Substituted tetrazines have been found to undergo facile inverse
electron demand Diels–Alder reactions with ‘‘tunable’’ reaction rates.
20 Mꢀ1 sꢀ1 at ambient temperature. This rate is higher than
that of most strained promoted azido-alkyne cycloadditions
(SPAAC) reported thus far.
The reaction between un-substituted tetrazine and cyclooctyne
Click chemistry is playing an increasingly important role in
a variety of areas of research1–8 including bioorthogonal
labeling.9–13 Critical to this field is the availability of reactions
and reagents that allow for facile and specific reactions. Many
reagents have been identified that serve this purpose very well
in different situations. These include difluorinated cyclooctyne
(DIFO),14 dibenzocyclooxtyne (DIBO),5,15 aza-dibenzocyclooctyne
(DIBAC),16 and biarylazacyclooctynone (BARAC).17 All these
belong to one type of reaction: 1,3-dipolar cycloaddition involving
an azido and an alkynyl group. However, this field needs more
such reactions in its arsenal for reasons of multiplexing,
labeling diversity, biocompatibility under different conditions,
and compatibility with other functional groups, as well as
reaction conditions for reagent preparation. In addition, rate
difference among various click reactions can also be exploited
for selective labeling.18 Thus having click reactions with
sufficiently different reaction rates will be very useful. Fox
et al.19,20 and Weissleder6 have developed a reaction using
tetrazine and a strained trans-alkene. The reaction is very fast
(1) has been reported with a stated second order rate constant
of 27.1 Mꢀ1 ꢀ1. However the highly volatile 1,2,4,5-tetrazine
s
is not suitable for any labeling work.24 In our initial studies, we
found the second order rate constant for the reaction between
cyclooctyne (1) and diphenyltetrazine (2) to be 0.07 Mꢀ1 sꢀ1
.
This rate is similar to that of a typical SPAAC reaction8 and is
slower than what we need. Thus we undertook the effort to
explore ways to enhance the reaction rates by performing
modifications guided by computational work.
The reaction between cyclooctyne (1) and a substituted
tetrazine (2) leads to significant changes in the UV-vis spectrum
of tetrazine due to its conversion to a 1,2-diazine product (Fig. 1).
Hence, the reaction can be easily monitored. We first studied
the reaction between cyclooctyne (1) and 3,6-diphenyl-1,2,4,5-
tetrazine (2) in dry methanol as a reference. The rate constant for
the reaction between diphenyltetrazine and cyclooctyne was
found to be 0.07 Mꢀ1 sꢀ1 (Table 1), which is far smaller than
that of the reaction between un-substituted tetrazine and
cyclooctyne. Since the reaction between 1,2,4,5-tetrazines
and a cyclooctyne depends on the LUMOdiene–HOMOphil
gap,25–27 we were interested in exploring ways to lower the
LUMO of the diene or elevate the HOMO of the dienophile in
order to facilitate the reaction. In such a case, it is reasonable to
expect that electron donating substituents, as well as high strain
energy will increase the HOMO energy of the dienophiles, and
electron withdrawing substituents will decrease the LUMO
with a second order rate constant of 2000 to 22 000 Mꢀ1sꢀ1
,
making it very useful for biolabeling applications. In our DNA
labeling work,21–23 we are interested in developing fast click
reactions with tunable reaction rates and reactants that can be
easily prepared. We thus turned to inverse-electron demand
Diels–Alder reactions (IEDDA) involving an alkyne and
tetrazine for several reasons. First, Schuster et al.24 have
demonstrated that un-substituted tetrazine can react very
quickly with alkenes and alkynes. Second, many strained
alkynes have been reported, giving us a set of ‘‘tools’’ for the
intended studies. Third, unlike azido compounds, tetrazine’s
reactivity can be tuned by manipulating its electron deficiency
through the introduction of functional groups at the 3,6-
position. In our case, we need reactions to be fast enough so
that at 10 mM concentrations, the half-life is no more than 2 h.
This requires the second order rate constant to be higher than
energy of the diene, leading to a decrease in the LUMOdiene
–
HOMOphil gap and consequently an increase in the reaction rate.
We then conducted quantum mechanics (QM) calculation
using known strained alkynes and trans-cyclooctene and some
modified tetrazines. From Fig. 2, one can see that relative to
cyclooctyne (1), the HOMO of BCN28 (4) is 1.5 kcal molꢀ1
higher, giving this a chance to have increased reactivity.
Installing electron withdrawing groups on cyclooctynes decreases
the HOMO energy substantially. For example, computational
results indicate that the HOMO energy decreases by 6.1 kcal molꢀ1
for DIFO14 (7) and 11.5 kcal molꢀ1 for fluorocyclooctyne29 (8)
relative to cyclooctyne. Correspondingly, di-pyridine substituted
1,2,4,5-tetrazine 9 lowers the LUMO energy by 2.3 kcal molꢀ1
relative to 3,6-diphenyl-1,2,4,5-tetrazine (2) (Fig. 2). Such
calculations suggest that the BCN–tetrazine pair could have
significantly improved reactivity.
Department of Chemistry, Center for Biotechnology and Drug Design,
and Center for Diagnostics and Therapeutics, Georgia State
University, Atlanta, Georgia 30302-4098, USA.
E-mail: wang@gsu.edu; Tel: +1 404-413-5545
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc16716f
z These two authors made equal contributions.
c
1736 Chem. Commun., 2012, 48, 1736–1738
This journal is The Royal Society of Chemistry 2012

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Fig. 2 Orbital energy of cyclooctynes and tetrazines.
in reaction rate over the tetrazine–cycloctyne pair. On the
other hand, 7 and 8 did not react with tetrazine 9 to an
appreciable degree, as expected. To achieve a deeper under-
standing of the improved reaction rates, Density Functional
Theory (DFT) calculations at the 6-31G** level of theory were
performed to examine the possible transition state(s) and
activation energies. Schematic representation of the energy
profiles for the tetrazine alkyne reactions are shown in Fig. 3.
All of the QM energies are in kcal molꢀ1 and are relative to the
reactants (1 and 2 at the top of Fig. 3, and 4 and 2 at the
bottom of Fig. 3). The activation energy for the first step of the
IEDDA reaction between 2 and 4 via transition state TS10 is
17.6 kcal molꢀ1 as compared to 23.3 kcal molꢀ1 for the reaction
between cyclooctyne 1 and tetrazine 2. The computational results
support the idea that the increased HOMO energy in BCN (4)
compared with cyclooctyne (1) translates into lowered activation
energy and thus increased reaction rate. The intermediate IN1
from the first step of the 1–2 reaction pair quickly loses nitrogen
gas to yield product 3 by passing through a low activation barrier
of 8.6 kcal molꢀ1, with the reaction being strongly exergonic by
ꢀ91.4 kcal molꢀ1. Because of the low barrier for the second step,
the reaction rate is entirely controlled by the first step of the
reaction. For the reaction between BCN (4) and tetrazine 2, the
situation is similar except that the conversion of the intermediate
IN10 to the final product has almost no activation barrier,
with the overall reaction being strongly exergonic by
ꢀ88.1 kcal molꢀ1. All these results indicate that the IEDDA
reaction and the subsequent elimination reaction take place
spontaneously and irreversibly toward the pyridazine products.
With these exciting findings and resulting tunable reaction
rates, we turned our attention to developing the chemistry needed
for bioconjugation. In order to do so, a ‘‘handle’’ needs to be
installed on the tetrazine system. Therefore 4-(6-(pyrimidin-2-yl)-
1,2,4,5-tetrazin-3-yl)benzoic acid30 was prepared. As a model,
isopropyl amine was chosen for conjugation with the benzoyl
chloride tetrazine derivative. The reaction between isopropyl
amide tetrazine 10 and BCN (4) gave a second order rate
Fig. 1 Strain-promoted [4+2] cycloadditions of 1,2,4,5-tetrazine 2
and cyclooctynes 4. Tetrazine: 1 mM, alkyne: 10 mM in dried MeOH.
Table 1 Second order rate constants of cyclooctynes with tetrazines
(7.0 ꢁ 0.7)
3.3 ꢁ 0.4
ꢂ 10ꢀ2 Mꢀ1
Mꢀ1 sꢀ1
sꢀ1
2.0 ꢁ 0.3
44.8 ꢁ 4.9
Mꢀ1 sꢀ1
Mꢀ1 sꢀ1
40.9 ꢁ 13.8
ND
Mꢀ1 sꢀ1
Indeed, when we tested the reactivity of diphenyltetrazine
(2) with 4 at a 1 : 10 ratio (tetrazine: 1 mM; alkyne: 10 mM),
the reaction finished within 10 minutes (Fig. 1), with a second
order rate constant of 3.3 Mꢀ1 sꢀ1 (Table 1). This represents a
47-fold improvement in reaction rate compared to the reaction
between cyclooctyne and diphenyltetrazine. To further accelerate
the reaction, two electron-withdrawing groups were attached
to the tetrazine ring to give 9. The second order rate constant
for the reaction between 3,6-di-2-pyridyl-1,2,4,5-tetrazine (9)
and alkyne 4 in dry MeOH at ambient temperature was found
constant of 40.9 Mꢀ1 ꢀ1, with half-lives of 24 seconds and
s
0.68 hour at mM and 10 mM concentrations, respectively. This is
among the fastest click reactions, and can be used for a variety of
labeling work.
to be 44.8 Mꢀ1 ꢀ1, which represents a 640-fold improvement
s
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1736–1738 1737

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Partial financial support from the National Institutes of
Health (GM086925 and GM084933) and the GSU Molecular
Basis of Disease program is gratefully acknowledged.
Notes and references
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can be tuned to allow for facile conjugation. The reaction rates
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Furthermore, the wide range of reaction rates (over 640-fold
difference) that can be achieved suggests possibilities of using
such chemistry for mutiplexing click labeling. Work is underway
to utilize BCN–tetrazine conjugation reactions for DNA and
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c
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This journal is The Royal Society of Chemistry 2012