1,2,4-Triazines Are Versatile Bioorthogonal Reagents

Article abstract of DOI:10.1021/jacs.5b05100

A new class of bioorthogonal reagents, 1,2,4-triazines, is described. These scaffolds are stable in biological media and capable of robust reactivity with trans-cyclooctene (TCO). The enhanced stability of the triazine scaffold enabled its direct use in recombinant protein production. The triazine-TCO reaction can also be used in tandem with other bioorthogonal cycloaddition reactions. These features fill current voids in the bioorthogonal toolkit.

Full text of DOI:10.1021/jacs.5b05100

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Communication
1,2,4-Triazines Are Versatile Bioorthogonal Reagents
David N. Kamber, Yong Liang, Robert J. Blizzard, Fang
Liu, Ryan A. Mehl, K. N. Houk, and Jennifer A. Prescher
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b05100 • Publication Date (Web): 18 Jun 2015
Downloaded from http://pubs.acs.org on June 23, 2015
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1,2,4-Triazines Are Versatile Bioorthogonal Reagents
David N. Kamber,1 Yong Liang,4 Robert J. Blizzard,6 Fang Liu,4 Ryan A. Mehl,6 K. N. Houk,4ꢀ5 and
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Jennifer A. Prescher*,1ꢀ3
Departments of 1Chemistry, 2Molecular Biology & Biochemistry, and 3Pharmaceutical Science, University of California,
Irvine, California 92697, United States
Departments of 4Chemistry and Biochemistry, and 5Chemical and Biomolecular Engineering, University of California, Los
Angeles, California 90095, United States
6Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
Supporting Information Placeholder
While much attention has been paid to strained alꢀ
kenes for bioorthogonal reaction development, less atꢀ
ABSTRACT: A new class of bioorthogonal reagents—
1,2,4ꢀtriazines—is described. These scaffolds are stable
tention has been given to the other half of the IEDꢀDA
in biological media and capable of robust reactivity with
reaction: the electronꢀdeficient dienes. To date, teꢀ
transꢀcyclooctene (TCO). The enhanced stability of the
triazine scaffold enabled its direct use in recombinant
protein production. The triazineꢀTCO reaction can also
trazines have dominated the IEDꢀDA landscape.14 These
moieties react robustly with transꢀcyclooctene (TCO)
and other strained dienophiles in a variety of settings.15
be used in tandem with other bioorthogonal cycloaddiꢀ
Unfortunately, the most rapidꢀreacting tetrazines also
tion reactions. These features fill current voids in the
tend to be the least stable in cells and in vivo.16 Teꢀ
bioorthogonal toolkit.
trazines are prone to hydrolysis and side reactions with
endogenous thiols, limiting their applications in the most
stringent environments (e.g., inside cells).17ꢀ19 More staꢀ
ble tetrazines are being pursued, but these reagents reꢀ
The bioorthogonal chemical reporter strategy has
main large in size.20
emerged as a robust method for visualizing biomolecule
structures and functions.1 This strategy involves the atꢀ
To develop improved bioorthogonal IEDꢀDA reacꢀ
tions, we were drawn to triazine scaffolds. 1,2,4ꢀ
tachment of bioinert functional groups (i.e., chemical
Triazines have been identified in microbial natural prodꢀ
reporters) to biomolecules of interest; the reporters are
ucts and pigments, suggesting that they are stable in
detected in a second step via selective (i.e., bioorthogoꢀ
physiological environments. 21ꢀ23 These motifs also react
efficiently with electronꢀrich alkenes in IEDꢀDA reacꢀ
tions.24ꢀ29 Boger further showed that 1,2,3ꢀtriazines react
with electronꢀrich dienophiles.30 To compare the intrinꢀ
sic DA reactivities of 1,2,3ꢀ and 1,2,4ꢀtriazines with that
of 1,2,4,5ꢀtetrazine, we evaluated the activation free enꢀ
ergies for their reactions with ethylene by density funcꢀ
tional theory (DFT) calculations (Figure 1A, Table
S1).31ꢀ33 The computational analysis suggested that
1,2,4ꢀtriazine is much more reactive than 1,2,3ꢀtriazine
(activation free energy: 29.3 versus 41.0 kcal/mol), but
less reactive than 1,2,4,5ꢀtetrazine (29.3 versus 21.9
kcal/mol). This is consistent with the inverseꢀelectronꢀ
demand nature of the cycloaddition: the LUMO+1 (the
π* orbital that interacts with the dienophile HOMO in
the DA reaction) of 1,2,4ꢀtriazine is increased by 0.49
eV as compared to 1,2,4,5ꢀtetrazine (2.18 versus 1.69
eV, Figures 1A and S1). While highly reactive, tetrazines
nal) reactions with complementary probes.2,3 Numerous
chemical reporters and bioorthogonal reactions have
been reported in recent years, but significant limitations
remain.[4] Many of the reagents are too bulky for general
use or prone to hydrolysis in cellular environments.
Moreover, several popular bioorthogonal reagents crossꢀ
react with one another and cannot be used concurrently
to visualize collections of biomolecules.4
To address these issues and expand the scope of the
chemical reporter strategy, new bioorthogonal reacꢀ
tions—and combinations of reactions—are being purꢀ
sued. In recent years, we and others have developed
compatible chemistries based on cyclopropenes and othꢀ
er strained alkenes.5ꢀ8 These motifs are stable in physioꢀ
logical environments and have been used to target nuꢀ
merous biomolecules in live cells.9ꢀ12 In nearly all cases,
the strained alkenes were detected via inverseꢀelectronꢀ
demand DielsꢀAlder (IEDꢀDA) reactions with 1,2,4,5ꢀ
tetrazines. A handful of tetrazine ligations can also be
are
prone
to
decomposition
by
biological
nucleophiles.18ꢀ20 Seitz and coꢀworkers found that thiols
rapidly decompose tetrazines via 1,4ꢀaddition and subꢀ
sequent release of nitrogen.34 DFT calculations revealed
that formation of the 1,4ꢀadduct of 3ꢀphenylꢀ1,2,4,5ꢀ
tetrazine and methanethiol is endergonic by 23.4
used
simultaneously
with
azideꢀalkyne
cycloadditions,5,11 setting the stage for multiꢀcomponent
bioorthogonal imaging in vivo.8,11,13
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kcal/mol in water, and that the overall barrier for N2 reꢀ
could potentially be exploited for ‘orthogonal’
bioorthogonal cycloaddition development.35ꢀ37
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3
4
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6
7
8
lease is 28.6 kcal/mol (Figure 1B). However, the correꢀ
sponding adduct and transition state of 6ꢀphenylꢀ1,2,4ꢀ
triazine are significantly higher in energy. This implies
that 6ꢀarylꢀ1,2,4ꢀtriazine is quite inert to thiol relative to
monoaryl tetrazine, although both are very similar in
size. Thus, while 1,2,4ꢀtriazine is less reactive in the
IEDꢀDA reaction, considering the extremely fast rates of
the tetrazineꢀTCO cycloaddition (k2 = 102ꢀ104 Mꢀ1 sꢀ1),17
we hypothesized that triazines would be good candidates
for bioorthogonal reaction development based on their
size and stability.
To test these hypotheses, we synthesized a panel of
substituted 1,2,4ꢀtriazines using the reaction sequence
pictured in Scheme 1. In brief, glyoxal was condensed
with aminoguanidine to afford 3ꢀaminoꢀ1,2,4ꢀtriazine
(1). Bromination of this scaffold provided a convenient
handle for diversification, and triazine 2 ultimately unꢀ
derwent Suzuki couplings with a variety of commercialꢀ
ly available boronic acids (Schemes 1 and S1, top). Subꢀ
sequent deamination of the products afforded triazines
4ꢀ9. To access triazines containing nucleophilic substituꢀ
ents (10 and 11), the reverse sequence—
deamination/Suzuki coupling—was employed (Schemes
1 and S1, bottom). This short reaction scheme can be
used to access 6ꢀsubstituted triazines with a broad array
of functionality. By contrast, traditional syntheses of
tetrazines are typically not compatible with free amino
groups owing to the harsh oxidants employed, although
milder conditions have recently been reported.38,39
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Scheme 1. Synthesis of Functionalized 1,2,4-Triazines
Figure 1. (A) DielsꢀAlder reactions of 1,2,4,5ꢀtetrazine,
1,2,4ꢀtriazine, and 1,2,3ꢀtriazine with ethylene.
LUMO+1 energies were computed with HF/6ꢀ
311+G(d,p)//M06ꢀ2X/6ꢀ31G(d), and activation free enꢀ
ergies (in kcal/mol) in water were computed with
CPCM(water)ꢀM06ꢀ2X/6ꢀ311+G(d,p)//M06ꢀ2X/6ꢀ
31G(d). (B) Energetics of 1,4ꢀadduct formation and subꢀ
sequent N2 release transition state for methanethiol and
3ꢀphenylꢀ1,2,4,5ꢀtetrazine or 6ꢀphenylꢀ1,2,4ꢀtriazine. (C)
DFTꢀcomputed activation free energies and predicted
relative rate constants for tetrazine or triazine cycloaddiꢀ
tion with 3ꢀcarbamoyloxymethylꢀ1ꢀmethylcyclopropene,
norbornene, or transꢀcyclooctene, in water at 25 °C.
Table 1. Second-Order Rate Constants for the Triazine-
TCO Ligation
We also predicted relative rate constants for the DA
reactions of 3ꢀphenylꢀ1,2,4,5ꢀtetrazine or 6ꢀphenylꢀ
1,2,4ꢀtriazine
with
3ꢀcarbamoyloxymethylꢀ1ꢀ
methylcyclopropene, norbornene, and transꢀcyclooctene
(Figures 1C and S2). These data suggest that 6ꢀarylꢀ
1,2,4ꢀtriazines react efficiently with TCO, yet remain
inert to other bioorthogonal scaffolds, including cycloꢀ
propene and norbornene. This unique reactivity profile
With the panel of triazines in hand, we analyzed their
reactivities with TCO (Table 1 and Figures S3ꢀS5). The
1
reactions were monitored by HꢀNMR, and oxidized
cycloadducts were observed (Figure S6). As expected,
the most electronꢀpoor triazine 4 exhibited the fastest
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rate (Table 1, entry 8), consistent with the inverseꢀ results are in sharp contrast to monoꢀsubstituted teꢀ
1
2
3
4
5
6
7
8
electronꢀdemand nature of the reaction. No reactivity
was observed when electronꢀrich scaffolds 1 and 2 were
incubated with TCO (Figures S7ꢀS8). The triazineꢀTCO
reaction is significantly slower than many of the teꢀ
trazineꢀTCO ligations,18 but on par with several copperꢀ
free click chemistries,40,41 and some IEDꢀDA reactions
with stabilized tetrazines.20,39 Hammett analysis of the
triazineꢀTCO rate constants gave a slope of ρ = 0.49
(Figure 2). This value is consistent with concerted IEDꢀ
DA reactions and suggests that only partial charge acꢀ
cumulates during the reaction.
trazines that have been observed to hydrolyze and/or
react with cysteine under similar conditions (Figure
S22).17ꢀ20,39
The remarkable stability of the triazine scaffold sugꢀ
gested immediate application in environs that have been
difficult to access with bioorthogonal reagents, including
recombinant protein production in intracellular enviꢀ
ronments. Disubstituted tetrazines and cyclopropenes
have been previously incorporated into recombinant proꢀ
teins and tagged with TCO or tetrazine probes, respecꢀ
tively.9,43 However, monoꢀsubstituted tetrazines and
TCO have been more difficult to incorporate directly
into proteins, due to the length of time required for proꢀ
tein production and the instability of the scaffolds.19,43
Monoꢀsubstituted triazines offer unique advantages in
terms of their size and stability.
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0.4
p-NO2, 4
0.3
pꢀCN, 5
0.2
mꢀCl, 6
0.1
pꢀMe, 8
To demonstrate that monoꢀsubstituted triazines are
compatible with protein labeling and sufficiently stable
for genetic code expansion, we synthesized triazine amiꢀ
no acid 16 (Figure 3A). We screened a panel of seven
Methanocaldococcus jannaschii tyrosyl tRNA syntheꢀ
tase (RS)/tRNACUA pairs for permissivity toward 16,
while maintaining fidelity against canonical AAs (Figure
S23).43,44 The M. jannaschii (RS)/tRNACUA pairs were
previously evolved to incorporate nonꢀcanonical amino
acids (ncAAs) of similar structure (S10, Figure S23) in
response to an amber codon.45 One of the seven
RS/tRNACUA pairs efficiently incorporated 16 in reꢀ
sponse to an amber codonꢀdisrupted GFP gene, resulting
in expression of 18.8 mg/L of GFPꢀ16 in the presence of
16 (Figure 3B, lane 3).
σ
0
ꢀ1
ꢀ0.5
0
H, 7
0.5
1
ꢀ0.1
ꢀ0.2
ꢀ0.3
ꢀ0.4
ꢀ0.5
p-NH2, 10
ρ = 0.495
R² = 0.969
pꢀNMe2,11
log (kX / kH)
Figure 2. Hammet plot for the reactions between TCO
12 and a panel of 1,2,4ꢀtriazines. Sigma (σ) values deꢀ
rive from ref 42
As predicted by our DFT calculations, no ligation was
observed between the more reactive triazines (4 and 9)
and other strained alkenes, including norbornene (14)
and cyclopropene 15 (Scheme 2, Figures S9ꢀS12). Theꢀ
se alkenes do react robustly with the common tetrazine
reagent 13 (Figure S5), suggesting that triazines and
combinations of other bioorthogonal reagents can be
used in tandem.
Scheme 2. Comparison of Tetrazine and Triazine Cy-
cloadditions with Norbornene or 1,3-Disubstituted Cyclo-
propenea
Figure 3. Triazines are suitable for recombinant protein
production. (A) Genetic incorporation of 16 into proteins
and reaction with TCO (B) SDSꢀPAGE analysis of siteꢀ
specific incorporation in response to amber codon 150 in
GFP. (C) MS analysis of GFPꢀ16 shows a single major
peak at 27939.1±1 Da. Reaction of GFPꢀ16 with TCO
shows a single major peak at 28120.1±1 Da, consistent
with the expected mass increase from selective reaction
with TCO.
a The rate constant for 15 + 13 is from ref 8
The triazine scaffold also excels in a key aspect of
bioorthogonality: stability. When monoꢀsubstituted triaꢀ
zines were dissolved in a mixture of d-PBS and CD3CN,
they remained stable for over one week at 37 °C (Figꢀ
ures S13ꢀS16). Triazine scaffolds were also inert to cysꢀ
teine over a similar time period (Figures S17ꢀ21). These
To verify that 16 is stable in complex media and can
be incorporated into recombinant proteins, we compared
the masses of GFPꢀ16 to GFPꢀwt using ESIꢀQ mass
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analysis. Native GFPꢀwt has a mass of 27826.0 ±1 Da
and GFPꢀ16 exhibited the expected increase to
27939.1±1 Da, verifying that 16 is incorporated at a sinꢀ
gle site (Figures 3C and S24). To determine whether the
triazine/TCO ligation is also quantitative on proteins,
pure GFPꢀ16 (10 µM) was incubated with TCO 17 (1
mM) in PBS (pH 7.0). ESIꢀQ mass analysis confirmed
quantitative conversion of GFPꢀ16 to GFPꢀ16+17 (exꢀ
pected 28120.7 Da; observed 28120.1±1 Da, Figure 3C).
These results demonstrate that triazines are stable in
cells and can be incorporated into proteins efficiently
and with high fidelity using genetic code expansion.
Furthermore, the triazine/TCO ligation is suitable for
siteꢀspecific protein labeling applications.
In summary, we identified 1,2,4ꢀtriazines as a new
class of bioorthogonal reagents. These scaffolds are reꢀ
markably stable in aqueous buffers, in the presence of
biological nucleophiles, and in cells. Triazines can be
easily assembled and decorated with diverse functional
groups to tune reactivities. Triazines also react efficientꢀ
ly and selectively with TCO. These features render triaꢀ
zines suitable for a variety of intracellular applications,
and we showed that a triazine amino acid can be effiꢀ
ciently incorporated into recombinant proteins and laꢀ
beled siteꢀspecifically with TCO. Triazines are also
compatible with other strained alkenes, and will enable
different types of IEDꢀDA reactions to be performed in
tandem in cellular environments.
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ASSOCIATED CONTENT
Experimental details, full spectroscopic data for new comꢀ
pounds, and computational details. This material is availaꢀ
ble free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
jpresche@uci.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by UCI, the National Institutes of
Health (R01GM109078 to K.N.H.), the National Science
Foundation (CHEꢀ1361104 to K.N.H. and CHEꢀ1112409 to
R.A.M.), OHSUꢀMRF and Services Core of the Environꢀ
mental Health Sciences Center via grant P30 ES00210 (to
R.A.M.). Calculations were performed on the Extreme Sciꢀ
ence and Engineering Discovery Environment (XSEDE),
which is supported by the NSF (OCIꢀ1053575). We thank
members of the Dong, Jarvo, and Overman labs for providꢀ
ing reagents and equipment, along with members of the
Prescher lab for helpful discussions and manuscript edits.
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