3,6-Substituted-1,2,4,5-tetrazines: Tuning reaction rates for staged labeling applications

Article abstract of DOI:10.1039/c4ob00280f

Cycloaddition reactions involving tetrazines have proven to be powerful bioorthogonal tools for various applications. Conceivably, sequential and selective labeling using tetrazine-based reactions can be achieved by tuning the reaction rate. By varying th

Full text of DOI:10.1039/c4ob00280f

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3,6-Substituted-1,2,4,5-tetrazines: tuning reaction
rates for staged labeling applications†
Cite this: Org. Biomol. Chem., 2014,
12, 3950
Danzhu Wang, Weixuan Chen,‡ Yueqin Zheng, Chaofeng Dai, Ke Wang, Bowen Ke§
and Binghe Wang*
Cycloaddition reactions involving tetrazines have proven to be powerful bioorthogonal tools for various
applications. Conceivably, sequential and selective labeling using tetrazine-based reactions can be
achieved by tuning the reaction rate. By varying the substituents on tetrazines, cycloaddition rate vari-
ations of over 200 fold have been achieved with the same dienophile. Upon coupling with different di-
enophiles, such as norbornene, the reaction rate difference can be over 14 000 fold. These substituted
tetrazines can be very useful for selective labeling under different conditions.
Received 6th February 2014,
Accepted 10th April 2014
DOI: 10.1039/c4ob00280f
www.rsc.org/obc
Introduction
In the bioorthogonal labeling field, selective labeling via
copper-free click chemistry1–5 is finding increasing appli-
cations. There are many pairs of excellent click reagents used
in this area including strain-promoted azide–alkyne cycloaddi-
tions (SPAACs)6–10 and inverse-electron demand Diels–Alder
reactions (IEDDARs) involving trans-cyclooctene (TCO)11–13 and
tetrazines. The selective labeling at two (or more) positions in
a single biomolecule is more challenging than coupling with
only one reagent. In order to achieve multiplexing, one
approach could be through the use of reagents with distinct re-
action rates. Cycloaddition reactions involving strain-promoted
alkynes and azides typically have second order rate con-
Fig. 1 1,2,4,5-Tetrazine (1) as an electron-poor diene (tetrazine, 1) and
bicycle[6.1.0]nonyne (BCN, 2) as a dienophile.
fine tune the tetrazine-based reaction rates for staged labeling
and multiplexing applications.
In our previous work, we studied the reaction between 3,6-
substituted-1,2,4,5-tetrazines (1) and bicycle[6.1.0]nonyne
(BCN, 2) and found this reaction rate to be tunable (Fig. 1).15
Thus we used the BCN–tetrazine pair as our model reaction
set. Again, it should be noted that tetrazines can react very
quickly with various alkenes and alkynes and thus a “tool set”
for fast labeling can be developed using the tetrazine cyclo-
addition chemistry.16 Herein, we present a comprehensive
follow up study, in which we optimized the procedure for tetra-
zine synthesis and investigated the reactivity between substi-
tuted 1,2,4,5-tetrazines (1) and BCN (2) (Fig. 1).
stants of no more than 1.9 M−1 s−1 14
The IEDDARs involving
.
1,2,4,5-tetrazines with TCO, alkynes, or norbornene, which
have been used for DNA modification and protein labeling,
were developed by the Fox group and the Weissleder group
and have very fast reaction rates (second order rates of up to
22 000 M−1 s−1).11–13 With the extremely fast reaction rates
involving tetrazines, it gives the chance to tune the reaction
rate to allow for staged labeling and yet have a fairly fast reac-
tion rate for each reaction to complete within a reasonable
period of time. Thus we are interested in looking for ways to
Results and discussion
Synthesis of asymmetric 3,6-disubstituted-1,2,4,5-tetrazines
Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for
Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
E-mail: wang@gsu.edu
The study requires the preparation of tetrazines with various
substitutions. Especially important are the asymmetrically sub-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/ stituted tetrazines, which afford a greater degree of tunability
c4ob00280f
as compared to only using symmetric tetrazines. Generally,
3,6-disubstituted-1,2,4,5-tetrazines can be synthesized from
‡Current address: Department of Chemistry and Biochemistry, Georgia Institute
of Technology, 901 Atlantic Drive Atlanta, GA 30332-0400, USA.
commercially available nitriles and hydrazine hydrate
§Current address: Translational Neuroscience Center, West China Hospital,
(Scheme 1, Route A) in a one-pot procedure.17–21 Unfortu-
Sichuan University, No. 37, Guo-Xue Xiang, Chengdu, Sichuan, 610041, P.R.
China.
nately, this method is substituent dependent and only suitable
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Table 2 Reaction yield and time differences between traditional con-
ditions and microwave conditions for conversion of 1,2-dichloromethy-
lene hydrazines to 3,6-substituted-1,2,4,5-tetrazines
Yielda
(reaction time)
Yieldb
(reaction time)
Product
61% (8 h)
81% (30 min)
58% (8 h)
73% (30 min)
80% (30 min)
66% (16 h)
Scheme 1 Synthetic routes to 3,6-substituted-1,2,4,5-tetrazines.
for some symmetric examples. Asymmetric tetrazines with
strong electron withdrawing groups either cannot be prepared
by this method or can only be prepared with extremely low
yields and reproducibility.22–25
52% (5 h)
65% (30 min)
a Yields under traditional reflux conditions. b Yields under microwave
conditions. All yields were isolated yields after column
chromatography.
For the synthesis of asymmetric tetrazines, we optimized
the conditions and improved the yield upon our previous
reported synthetic method (Scheme 1, Route B).26,27 The for-
mation of the 1,2,4,5-tetrazine-ring system involves conden-
sation of 1,2-dichloromethylene hydrazines with hydrazine
monohydrate under microwave conditions. The dihydrotetra-
zine, which is dissolved in acetic acid, can be oxidized with
sodium nitrite at 0 °C. This optimized procedure allows the
preparation of a variety of tetrazines (1, Table 1) with moderate
yields (up to 50%), short reaction time, and easy purification.
We examined whether microwave irradiation could facilitate
the conversion step of 1,2-dichloromethylene hydrazines (3) to
3,6-substituted-1,2,4,5-tetrazines (1). As shown in Table 2, the
reaction time were shortened from up to 24 h to 30 min with
an improvement of the reaction yield in the range of about
20% when microwave was used.
gap.28–30 Our previous work15 analyzed the effect of electron-
withdrawing substituents on decreasing the LUMO energy of
the diene, leading to a decrease in the LUMOdiene–HOMOphil
gap and consequently an increase in the reaction rate. Again
a highly strained alkyne (BCN 2) was used as a model
dienophile. All the reactions proceeded cleanly with N2 as a
byproduct. Side reactions were not observed and yields
were generally high (Table 3). The reaction between a substi-
tuted tetrazine (1) and BCN (2) leads to significant changes in
the UV-vis spectrum of the tetrazine due to its conversion to a
pyridazine product. Hence, the reaction rate constants can be
determined by monitoring the decrease of the UV intensity at
290–300 nm of the reaction mixture (Table 3).
We first examined the reaction between BCN and 3,6-diphe-
nyl-1,2,4,5-tetrazine (1b) in methanol (HPLC-grade) as a refer-
ence. The rate constant was found to be 3.6 M−1 s−1 (Table 3).
Introduction of pyridin-2-yl to the tetrazine structure increased
the reaction rate because of strong electron withdrawing pro-
perty of the pyridyl group. For example, the second order rate
constant of 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (1a) was 118
M−1 s−1, which represents a 37-fold improvement in the reac-
tion rate compared to the reaction of diphenyltetrazine (1b). In
the case of an asymmetric tetrazine, the reaction of 3-(4-fluoro-
phenyl)-6-(pyridin-2-yl)-1,2,4,5-tetrazine (1n) with BCN showed
a rate constant of 23 M−1 s−1, which is lower than that of 1a,
presumably because the 4-fluorophenyl group has less electron
withdrawing ability than pyridine. However, it is still 7-fold
faster than the reference reaction with 1b. To further tune the
reaction rate, 4-(trifluoromethyl)phenyl groups and the pyrimi-
dine ring were attached to the tetrazine ring to give 3,6-bis-
(4-(trifluoromethyl)phenyl)-1,2,4,5-tetrazine (1k) and 3-(pyrimi-
Kinetic studies
[4 + 2] Cycloaddition with electron-poor tetrazines led to the
expected clean pyridazine products. The critical determining
factor of the reaction rate is the LUMOdiene–HOMOphil
Table 1 3,6-Substituted-1,2,4,5-tetrazines (1) prepareda,b
din-2-yl)-6-(4-(trifluoromethyl)phenyl)-1,2,4,5-tetrazine
respectively. The second order rate constants of these two
tetrazines with BCN in MeOH at ambient temperature were
(1p),
a a–e, Symmetric 3,6-substituted-1,2,4,5-tetrazines prepared by the
traditional synthesis route (Scheme 1, Route A). b f–p, 3,6-Substituted-
1,2,4,5-tetrazines prepared by an optimized route (Scheme 1, Route B).
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Table 3 Reactions of 3,6-substituted-1,2,4,5-tetrazines (1) with BCN
(2); rate constants at ambient temperature in MeOH
Table 4 Solvent effect on rate constants of reaction between 3,6-sub-
stituted-1,2,4,5-tetrazines (1) with BCN (2) at ambient temperature
MeOH second
CH3CN second
order rate constant order rate constant
(M−1 s−1
)
(M−1 s−1
)
3.6
1.2
2.7
1.4
Second order
Product
number
Yielda
(%)
rate constant
Tetrazine (R1/R2)
(M−1 s−1
)
0.58
1.4
0.36
0.16
5
97
125
6
7
8
9
98
96
95
97
118
23
Another interesting finding is the reaction rate of 1i being
slightly lower than that of 1b (3.6 M−1 −1) in MeOH, although
10
s
we expected the reaction of 1i to be faster than 1b since fluo-
rine is an electron-withdrawing group (σp = 0.06). The answer
could be in the balance between the resonance effect and the
inductive effect. The same solvent-sensitivity is found in the
reaction of 3,6-bis(4-hydroxylphenyl)-1,2,4,5-tetrazine (1d) and
3,6-bis(4-methoxyphenyl)-1,2,4,5-tetrazine (1e). The reaction
3.6
10
11
12
13
94
92
94
92
2.7
0.58
1.4
2.3
rate for 1d and 1e in MeOH is 0.58 M−1 s−1 and 1.4 M−1 s−1
respectively. However, in acetonitrile, the rates are 0.36 M−1 s−1
and 0.16 M−1 −1. Water could be included as a significant
,
s
part of the solvent with some attenuation of reaction rates.
Norbornene as a dienophile
Norbornene is another click partner for electron-poor
tetrazines,31–35 and has been used in DNA modification and
protein labeling. The reaction with norbornene is much slower
than with TCO or strained alkyne BCN. The Carell group per-
formed a systematic kinetic study of the reactivity of norbor-
nene derivatives and demonstrated that norbornenes with a
non-electron-withdrawing substituent in the exo position are
favored in the reaction.36 Tetrazines with electron-withdrawing
substituents were chosen for our kinetic studies. All reactions
were performed in MeOH at 1 : 10 ratio (tetrazine: 1 mM; nor-
bornene: 10 mM). From the kinetic data (Table 5), we can see
the trend of reactivity being the same as for strained alkyne
BCN. However, the reaction rate constants are far smaller than
that of the reaction between substituted tetrazines and BCN.
For instance, the reaction rate constant of the reference (1b) is
8.5 × 10−3 M−1 s−1, which means this reaction could be com-
pleted in 2 days at 1 mM tetrazine concentration. The fastest
reaction rate observed is that of the asymmetric tetrazine (1p),
which has a pyrimidin-2-yl group and a potential “handle” for
further tethering. The reaction was complete in 2 hours (1 mM
tetrazine concentration). This is also the reason for asym-
metric pyridin-2-yl tetrazine being the most popular tetrazine
used in the norbornene system based on literature reports.33,34
a Isolated yields after column chromatography.
found to be 10 and 125 M−1 s−1, respectively. The rate for
3-(pyrimidin-2-yl)-6-(4-(trifluoromethyl)-phenyl)-1,2,4,5-tetrazine
(1p) is the fastest so far, which represents a 40-fold improve-
ment over that of the reaction involving diphenyltetrazine.
Since increasing the electron density on the tetrazine is
expected to increase the LUMOdiene–HOMOphil gap by increas-
ing the diene LUMO energy, the reaction rate should be lower
than the reference reaction when the tetrazine has an electron-
donating group. Indeed, we tested three tetrazines with elec-
tron-donating substituents (1c, 1d, 1e), and found the reaction
rate constants to be 2.3 M−1 s−1, 0.58 M−1 s−1, and 1.4 M−1 s−1
respectively, in MeOH.
,
We also found the reaction rate between 1,2,4,5-tetrazine
and BCN being moderately sensitive to solvent properties
(Table 4). For example, the reaction of 3,6-bis(4-fluorophenyl)-
1,2,4,5-tetrazine (1i) has a slightly higher rate (2.7 M−1 s−1
)
when performed in MeOH than in acetonitrile (1.4 M−1 s−1).
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Table 5 Reactions of 3,6-substituted-1,2,4,5-tetrazines (1) with norbor-
nene-2-carboxaldehyde (4); rate constants at ambient temperature in
MeOH
Experimental
Materials and methods
All reagents and solvents were of reagent grade or were puri-
fied by standard methods before use. Column chromatography
was carried out on flash silica gel (Sorbent 230–400 mesh). TLC
analysis was conducted on silica gel plates (Sorbent Silica G
UV254). NMR spectra were recorded at 400 MHz for 1H on a
Bruker instrument. Chemical shifts (δ values) and coupling con-
stants (J values) are given in ppm and hertz, respectively, using
solvents (1H NMR) as the internal standard. Symmetric tetra-
zines 1a–1e and BCN (2) were synthesized according to literature
procedures. General synthesis procedure and characterization
data of 3,6-substituted-1,2,4,5-tetrazines 1f–1p, compound 6,
and compound 9 were already reported by our group.15,26
Second order rate
Tetrazine (R1/R2)
constant (M−1 s−1
8.5 × 10−3
)
Kinetic measurements of the reaction of 3,6-substituted-
1,2,4,5-tetrazines 1 with BCN 2 or norbornene-2-carboxalde-
hyde 4. UV/Vis kinetic measurements: Separate solutions of pure
0.039
0.056
0.65
1
tetrazines (1) and pure BCN (2, >95–98% by H-NMR) or nor-
bornene-2-carboxaldehyde (4, 95%, Sigma-Aldrich) were pre-
pared in HPLC-grade MeOH at room temperature. The stability
of tetrazines (1) in MeOH was examined by monitoring their
absorption at 295–330 nm. Solutions containing 1 (25 μM) and
10–18 fold excess of BCN (2) or norbornene-2-carboxaldehyde
(4) were pipetted into quartz cuvettes and thoroughly mixed.
All kinetic runs were the results of triplicates. Curve fitting was
conducted in the Prism5 software.
0.61
General procedure for the strain-promoted inverse electron
demand Diels–Alder reactions with 1,2,4,5-tetrazine. To a
solution of tetrazine (1) in MeOH (1 mL, HPLC-grade), BCN (2)
or norbornene-2-carboxaldehyde (4) in MeOH (1 mL, HPLC-
grade) was added. Reactions were stirred at room temperature
for 5 to 30 min. The progress of the reaction was monitored by
TLC. Upon completion, the reaction mixture was directly
loaded on the flash column chromatograph for purification.
((6aS,7S,7aR)-1-(Pyrimidin-2-yl)-4-(4-(trifluoromethyl)phenyl)-
6,6a,7,7a,8,9-hexahydro-5H-cyclopropa[5,6]cycloocta[1,2-d]pyrida-
zin-7-yl)methanol (5). Compound 5 was prepared according to
the general procedure and purified by column chromatography
(DCM–MeOH, 20 : 1) to give a yellow solid (yield, 97%). 1H
NMR (MeOD): 0.95–1.10 (m, 3H), 1.65–1.75 (m, 2H), 2.10–2.32
(m, 2H), 2.80–2.90 (m, 2H), 2.93–3.10 (m, 2H), 3.68 (d, 2H, J =
6.8 Hz), 7.67 (t, 1H, J = 5.0 Hz), 7.78 (d, 2H, J = 8.0 Hz), 7.90 (d,
2H, J = 8.0 Hz), 9.04 (d, 2H, J = 5.0 Hz). HRMS (ESI): m/z calcd
for C23H21F3N4O [M + H]+ 427.1746, found 427.1744.
From Table 5, we can see that the norbornene system has
relatively low reaction rates. All rate constants are at least 200
fold lower than the BCN system. However, compared to the
Staudinger ligation (2.4 × 10−3 M−1 s−1) and SPAAC (in the
range of 2–70 × 10−3 M−1 s−1),14 the reaction rates for the tetra-
zine–norbornene system are still considered fast and can still
be used for click labeling applications. Thus, among all the
reactions examined, there is an over 14 000-fold difference in
reaction rates (entry 1 in Table 3 and entry 1 in Table 5). Such
a large difference in reaction rates should allow for the selec-
tion of reaction pairs suitable for staged labeling of more than
one site in the same biomacromolecule. If the trans-alkenes
are brought into the picture, there is an even greater reaction
rate difference, which should provide a great deal of flexibility
in selecting reaction pairs for labeling studies.
((6aR,7S,7aS)-1-(4-Fluorophenyl)-4-(pyridin-2-yl)-6,6a,7,7a,8,9-
hexahydro-5H-cyclopropa[5,6]cycloocta[1,2-d]pyridazin-7-yl)-
methanol (7). Compound 7 was prepared according to the
general procedure and purified by column chromatography
Conclusion
Tetrazine cycloaddition as a powerful bioorthogonal tool can (DCM–MeOH, 20 : 1) to give a yellow solid (yield, 96%). 1H
be used for selective labeling studies using reaction pairs with NMR (MeOD): 1.00–1.13 (m, 3H), 1.60–1.70 (m, 2H), 2.10–2.20
large differences in reaction rates. By tuning the substituents (m, 2H), 2.80–2.90 (m, 2H), 2.93–3.10 (m, 2H), 3.67 (d, 2H, J =
of the tetrazine moiety, a large difference of reaction rates can 7.2 Hz), 7.32 (dd, 2H, J1 = 8.8 Hz; J2 = 8.8 Hz), 7.50–7.60 (m,
be achieved. We believe that the availability of reaction pairs 3H), 7.81 (d, 1H, J = 7.6 Hz), 8.06 (dd, 1H, J1 = 1.6 Hz, J2
with tunable reaction rates will be very useful in click-labeling 8.0 Hz), 8.74 (d, 1H, J = 1.6 Hz). HRMS (ESI): m/z calcd for
=
applications.
C23H22FN3O [M + H]+ 376.1825, found 376.1818.
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((6aR,7S,7aS)-1,4-Bis(4-(trifluoromethyl)phenyl)-6,6a,7,7a,8,9-
Notes and references
hexahydro-5H-cyclopropa[5,6]cycloocta[1,2-d]pyridazin-7-yl)metha-
nol (8). Compound 8 was prepared according to the general pro-
cedure and purified by column chromatography (DCM–MeOH,
50 : 1) to give a yellow solid (yield, 95%). 1H NMR (MeOD):
1.09–1.20 (m, 3H), 1.60–1.70 (m, 2H), 2.10–2.20 (m, 2H),
2.80–2.90 (m, 2H), 2.93–3.05 (m, 2H), 3.68 (d, 2H, J = 7.2 Hz),
7.76 (d, 4H, J = 8.0 Hz), 7.90 (d, 4H, J = 8.0 Hz). HRMS (ESI): m/z
calcd for C26H22F6N2O [M + H]+ 493.1715, found 493.1721.
((6aR,7S,7aS)-1,4-Bis(4-fluorophenyl)-6,6a,7,7a,8,9-hexahydro-
5H-cyclopropa[5,6]cycloocta[1,2-d]pyridazin-7-yl)methanol (10).
Compound 10 was prepared according to the general pro-
cedure and purified by column chromatography (DCM–MeOH,
50 : 1) to give a yellow solid (yield, 94%). 1H NMR (CDCl3): 0.80–0.90
(m, 3H), 1.30–1.40 (m, 2H), 1.40–1.70 (m, 2H) 2.40–2.50 (m, 2H),
2.70–2.80 (m, 2H), 2.88–2.95 (m, 2H), 3.44 (d, 2H, J = 6.8 Hz),
7.17–7.22 (m, 4H) 7.49–7.53 (m, 4H). HRMS (ESI): m/z calcd for
C24H22F2N2O [M + H]+ 393.1778, found 393.1762.
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Acknowledgements
Financial support from the NIH (GM086925, GM084933, and 25 C. Larsen, E. Binderup and J. Moller, Acta Chem. Scand.,
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