Stable, Reactive, and Orthogonal Tetrazines: Dispersion Forces Promote the Cycloaddition with Isonitriles

Article abstract of DOI:10.1002/anie.201903877

The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Now it is shown that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure–activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 L mol^?1 s^?1 were accessible by combining bulky and electron-withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles.

Full text of DOI:10.1002/anie.201903877

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Accepted Article
Title: Stable, Reactive and Orthogonal Tetrazines: Dispersion Forces
Promote the Cycloaddition with Isonitriles
Authors: Julian Tu, Dennis Svatunek, Saba Parvez, Albert C Liu, Brian
J Levandowski, Hannah J Eckvahl, Randall T Peterson,
Kendall N Houk, and Raphael Marcel Franzini
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903877
Angew. Chem. 10.1002/ange.201903877
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http://dx.doi.org/10.1002/ange.201903877

10.1002/anie.201903877
Angewandte Chemie International Edition
COMMUNICATION
Stable, Reactive and Orthogonal Tetrazines: Dispersion Forces
Promote the Cycloaddition with Isonitriles
Julian Tu,[a] Dennis Svatunek,[b] Saba Parvez,[c] Albert C. G. Liu,[b] Brian J. Levandowski,[b] Hannah J.
Eckvahl,[b] Randall T. Peterson,[c] Kendall N. Houk,[b] Raphael M. Franzini*, [a]
Abstract: Bioorthogonal reactions are of great value in the life
sciences. The isocyano group is a structurally compact bioorthogonal
functional group that reacts with tetrazines under physiological
conditions. Here we report that bulky tetrazine substituents accelerate
this cycloaddition. Computational studies suggest that dispersion
forces between the isocyano group and the tetrazine substituents in
the transition state contribute to the atypical structure-activity
relationship. Stable asymmetric tetrazines that react with isonitriles at
rate constants as high as 57 M-1s-1 were accessible by combining
bulky and electron-withdrawing substituents. Sterically encumbered
tetrazines react selectively with isonitriles in the presence of strained
alkenes/alkynes, which allows for the orthogonal labeling of three
proteins. The established principles will open new opportunities for
developing tetrazine reactants with improved characteristics for
diverse labeling and release applications with isonitriles.
reactions of isonitriles with tetrazines;[10] accelerating the
cycloaddition without destabilizing the reactants would expand its
utility to study biomolecules with high temporal resolution and in
drug-delivery.[4b, 11] However, making isonitriles more reactive is
challenging because it is impractical to use ring strain, which
allowed increasing the reactivity of cyclic alkenes/alkynes by
orders of magnitude.[2] Enhancing the reactivity of tetrazines is
also problematic because more reactive tetrazines are typically
also less stable.[12] Electron-withdrawing groups make tetrazines
more reactive; however, they also render them susceptible to
nucleophilic attack.[12] While bulky groups protect tetrazines, they
generally obstruct the approach of dienophiles.[5l, 12-13] Here we
report that it is possible to design tetrazines that react rapidly with
isonitriles and are stable in thiol-containing buffer. Furthermore,
sterically encumbered tetrazines react chemoselectively with
isonitriles in the presence of strained alkenes and alkynes.
Bioorthogonal reactions proceed in biological systems without
interference from cellular components.[1] Such reactions are
finding widespread application in the life sciences, for example to
study biomolecules in their native environment[2] and to control the
activity of proteins in vivo.[3] Furthermore, drug-delivery
approaches utilizing bioorthogonal reactions are under
development.[4] Therefore, the pursuance of bioorthogonal
ligation[5] and release[6] reactions with improved attributes is
actively ongoing. The inverse-electron demand Diels-Alder
(iEDDA) cycloaddition between dienophiles and tetrazines has
emerged as an especially impactful reaction because of its fast
rate and high selectivity.[7] As applications are becoming more
complex, there is a need for bioorthogonal groups that can be
orthogonally actuated.[8]
Isonitriles are unique among dienophiles that react with tetrazines
to form adducts[9] or release leaving groups[10] (Fig. 1). The
isocyano group is structurally compact, stable in biological fluids,
synthetically facile to access and has a distinct reactivity profile.
Rate constants of up to k2 = 4 M-1s-1 have been reported for
Figure 1. The cycloaddition of isonitriles and tetrazines is a bioorthogonal
reaction that can form stable conjugates or elicit the release of molecules.
We hypothesized that the linear isocyano group would experience
minimal steric repulsion by bulky tetrazine substituents in the
14]
[4+1] cycloaddition transition-state.[9a,
We synthesized
[a]
[b]
[c]
J. Tu, Prof. R. M. Franzini
Department of Medicinal Chemistry
University of Utah
symmetrical 3,6-dialkyl-1,2,4,5-tetrazines (1a-d) with substituents
of different sizes and measured the rate of the reaction with 2-
phenylethyl isonitrile (PhEtNC, Fig. 2a). Unexpectedly, the
reaction of PhEtNC with tetrazines accelerated with increasing
Taft’s steric parameter (Es)[15] of the alkyl group (Fig. 2a). PhEtNC
reacted 10.8-fold faster with 3,6-bis-tert-butyl tetrazine (1d) than
with 3,6-dimethyltetrazine (1a). This result is remarkable because
1d is highly stable (See Supporting Information) and showed no
reactivity with norbornene under these conditions (DMSO:H2O,
4:1, v/v; T = 37°C; Fig. 2a). Furthermore, as LUMO levels on
tetrazines decreased, reaction rates with both isonitrile and
norbornene increased proportionally (Fig. S1).
30 S 2000 E, Salt Lake City, Utah 84112 (USA)
*E-mail: Raphael.franzini@utah.edu
Dr. D. Svatunek, A. C. G. Liu, Dr. B. Levandowski, H. Eckvahl, Prof.
K. N. Houk
Department of Chemistry and Biochemistry
University of California, Los Angeles
Los Angeles, CA 90095-1569 (USA)
Dr. S. Parvez, Prof. R. T. Peterson
Department of Pharmacology and Toxicology
University of Utah
30 S 2000 E, Salt Lake City, Utah 84112 (USA)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201903877
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To rationalize the unexpected relationship between reactivity and
sterics, we conducted a computational study to calculate the
activation energies required for model compound methylisonitrile
to react with tetrazines 1a-1d. The potential energy surfaces for
these reactions were explored at the M06-2X-D3/def2-TZVP[16]
level of theory in water (SMD)[17] and the first step was identified
as rate determining (Fig. S2-3). In agreement with the
experimental results, a 1.7-2.5 kcal/mol lower Gibbs free energy
of activation was found for the reaction of methylisonitrile with 1d
relative to 1a-1c (Fig. 2b). Furthermore, the ratio of the rate
constants for the reaction between PhEtNC and 1a and 1d is
independent of the solution’s water content (Fig. S4), and it is
therefore unlikely that a hydrophobic effect is responsible for the
observed results. To investigate the effect of the tetrazine
substituents in more detail, calculations using B3LYP/def2-TZVP
with and without Grimme’s D3 dispersion correction were
conducted.[16c] Structures were reoptimized using both methods.
Comparison of the reaction of 1a and 1d with methylisonitrile
demonstrate the importance of dispersion forces (Fig 2c). Without
dispersion correction ΔG‡ is 1.0 kcal/mol higher for 1d compared
to 1a. However, the bulkier t-Bu substituents lead to a 6.0 kcal/mol
stabilization of the transition state through dispersion, in contrast
to 3.1 kcal/mol for 1a, and resulted in an overall 1.9 kcal/mol
lowered free energy of activation. Therefore, not only are steric
repulsions with tetrazine substituents less impactful for isonitriles
than for other dienophiles, but dispersion forces significantly lower
the activation energy. While other interactions may be involved,
these computational studies clearly established that dispersion is
a major causative factor underlying the observed structure-activity
relationship.
These results are significant because they indicate that certain
tetrazine modifications can simultaneously increase the reactivity
towards isonitriles and the stability of the diene. We postulated
that asymmetric tetrazines with both a bulky tert-butyl group and
an electron-withdrawing substituent would react rapidly with
isonitriles and be highly stable in aqueous solutions (Fig. 3a). We
synthesized 3-tert-butyl-6-pyrimidine tetrazines (3a-3c; Fig. 3a)
and measured reaction rates with PhEtNC (DMSO:H2O, 4:1, v/v
at T = 37°C) and stabilities in buffer containing 10 mM glutathione
(GSH) (DMSO:PBS (pH 7.4), 4:1, v/v at T = 37°C). Tetrazines
were synthesized by a zinc-catalyzed reaction between nitriles
and hydrazine followed by oxidation;[18] the preparation of 3c
required an alternative oxidation strategy (BAIB, cat. TEMPO,
See Supporting Information). PhEtNC indeed reacted faster with
these tetrazines than with 3,6-dipyridyltetrazine (DPTz; 0.297 ±
0.012 M-1s-1), which is a commonly-used reactant in iEDDA
reaction studies. 3a and 3b reacted 3.9-fold (1.15 ± 0.20 M-1s-1)
and 5.2-fold (1.53 ± 0.01 M-1s-1) faster than DPTz, respectively,
and also exhibited high stability in the nucleophile-containing
buffer/DMSO solution, whereas DPTz gradually decomposed
under these conditions (Fig. 3b). The CF3-substituent enhanced
the reactivity further and 3c reacted 8.1-fold faster (2.42 ± 0.10 M-
1s-1) than DPTz and showed comparable stability to DPTz in the
GSH-containing buffer/DMSO mixture. The reaction of PhEtNC
with 3c was even faster than the reaction with 3,6-dipyrimidyl
tetrazine (DPmTz, 2.30 ± 0.04 M-1s-1), which is highly unstable
and rapidly degraded in buffer (Fig. 3b, S5).
Figure 2. Dispersion forces increase the reactivity of isonitriles to tetrazines with
bulky substituents. A) Bimolecular reaction rates recorded for PhEtNC and
norbornene as a function of the Taft’s steric parameter of tetrazine alkyl
substituents. B) M06-2X-D3(SMD) calculated transition state geometries and
energies of activation for the rate determining step of the reaction of tetrazines
1a-d with methylisonitrile. Energies in kcal/mol, distances in Å, electronic energy
and Gibbs free energy (in brackets) is shown. C) Gibbs free energies of
activation (kcal/mol) for the reaction of methylisonitrile and tetrazines 1a and 1d
calculated using the B3LYP functional with and without correction for dispersion
forces.
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Figure 3. Asymmetric tetrazines containing t-butyl and pyrimidyl substituents (3a-c) exhibit high stability and robust reactivity with isonitriles. A) Structures of 3a-c
and principles underlying their design. B) Rates of the reaction of tetrazines with PhEtNC (DMSO:H2O = 4:1 , T = 37˚C) and stability of tetrazines in thiol-containing
buffer (t = 3 h, DMSO:PBS (pH 7.4) = 4:1 plus 10 mM GSH, T = 37°C). Experimental values are provided in table. C) Rate of the reaction of 3c and a primary
(PhEtNC) and tertiary (t-OcNC) isonitrile under high-water conditions (DMSO:H2O = 1:4, T = 37˚C).
Water typically accelerates iEDDA reactions,[19] and we measured
the rates of isonitriles reacting with tetrazine 3c in DMSO:H2O,
Table 1. Second-order rate constants of reactions between tetrazines DPTz, 3c,
1d and several dienophiles.
1:4, v/v at T = 37°C. PhEtNC reacted with 3c at a second-order
rate constant of 29.4 ± 0.8 M-1s-1 (Fig. 3c). Tertiary isonitriles are
more reactive than primary ones[9a] (Table S6) and form stable
conjugates with tetrazines for bioorthogonal labeling
applications.[9a] 3c reacted with tert-octyl isocyanide (t-OcNC) at
a rate of 57 ± 5 M-1s-1 (Fig. 3c), and the pyrazole adduct was
stable over 24 h (HPLC analysis; Fig. S6). The reaction of these
optimized tetrazines with isonitriles is therefore faster than
k2,TCO
[M-1s-1]
k2,Nb
[M-1s-1]
k2,Cp
[M-1s-1]
k2,PhEtNC
[M-1s-1]
k2,t-OcNC
[M-1s-1]
Tetrazine[a]
popular bioorthogonal reactions such as the strain-promoted
0.827
0.563
0.297
5e]
azide/alkyne cycloaddition,[5d,
the Staudinger-Bertozzi
DPTz
3c
> 100
1.48 ±0.03
6.27 ±0.05
±0.042
±0.025
±0.012
ligation,[5a] and the iEEDA reactions between tetrazines and
norbornene[5b, 5h] or methylcyclopropenes.[5f, 5g, 5l] The principles
underlying the design of 3a-c should allow for designing tetrazines
with further improved properties.
0.234
0.013
0.588
1.53
±0.002
±0.005
±0.055
±0.01
0.070
2.25e-03
1d
n.d.[b]
n.r.[c]
n.d.[b]
±0.007
±3.54e-05
[a] Conditions: 0.2 mM tetrazine, 2 mM – 20 mM dienophile, DMSO:H2O (4:1),
T = 37°C, triplicate. [b] not determinable; because of slow kinetics, curves could
not be fitted appropriately; k2 < 10-3 M-1s-1; [c] no reaction; k2 < 10-6 M-1s-1
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groups of 1d further deterred the cycloaddition with strained
alkenes, and even with methylcyclopropene reactants, which
retain appreciable reactivity to tetrazines with one t-Bu
substituent[5l] such as 3c. In contrast, the reaction of 1d with
isonitriles proceeds reasonably; however, the kinetic results
indicate a possible steric repulsion between tertiary isonitriles and
the tert-butyl groups of 1d (Fig. S8). Nevertheless, tetrazines
preferentially react with isonitriles or strain-activated alkenes
depending on the bulkiness of their substituents.
Tetrazines that react selectively with isonitriles over other
dienophiles open the possibility for orthogonal labeling and
release applications. It has been previously shown that the
reaction of both TCO and isonitriles with tetrazines is orthogonal
to strain-promoted azide/alkyne cycloadditions.[8, 9b] Considering
the increasing interest in multiplexed labeling[20] and release[6d, 21]
studies employing orthogonal biocompatible reactions, we sought
to determine whether the isonitrile/tetrazine iEEDA reaction could
be used for the simultaneous triple-labeling of biomolecules. As a
demonstration, we attempted to label three model proteins in a
mixture with different fluorophores. Lysine residues on bovine
serum albumin (BSA), ovalbumin (OVA), and lysozyme (LYSO)
were modified with 1d’, azide, and MeTzCOOH (Fig. S9) to
generate BSA-1d’, OVA-N3, and LYSO-Tz (Fig. 4a). The
functionalized proteins were exposed to reactant-modified
fluorophores with different emission wavelengths (Fig. S10)—a
green-fluorescent bodipy-tertiary isonitrile conjugate (BDPY-Fl-
NC), a red-fluorescent bodipy-TCO conjugate (BDPY-TR-TCO),
and a blue-fluorescent AlexaFluor-DBCO conjugate (AF405-
DBCO). Each reactive conjugate labeled one protein selectively
as demonstrated by in-gel fluorescence analysis (Fig. 4b). BDPY-
Fl-NC reacted with BSA-1d’, BDPY-TR-TCO with LYSO-Tz, and
AF405-DBCO with OVA-N3. In a one-pot labeling experiment
combining the three proteins and the three dye-conjugates, each
protein was labeled with the matching fluorophore and with
minimal cross-reaction (Lane 9, Fig. 4b). These results establish
that sterically encumbered tetrazines enable the orthogonal
labeling of biomolecules. Importantly, while there are a handful of
reports using two reactions simultaneously, only few reports of
performing three bioorthogonal reactions concurrently are
available.[22] It should be possible to combine isonitrile/tetrazine
chemistry with additional transformations (e.g. photoinduced click
chemistry[23]) to execute even more reactions in parallel.
Figure 4. Triple-orthogonal labelling of BSA, ovalbumin, and lysozyme proteins.
A) Schematic of the triple-orthogonal protein labelling experiment. B) In-gel
analysis of the fluorescent labelling of BSA-1d’, OVA-N3, and LYSO-Tz through
chemoselective reactions with BDPY-FL-NC (200 µM), AF405-DBCO (200 µM),
and BDPY-TR-TCO (100 µM), respectively. The stained protein gel is shown in
Fig. S10.
Dissociative bioorthogonal reactions that release molecules also
have many potential applications.[4b, 24] We previously developed
tetrazine-responsive 3-isocyanopropyl (ICPr) groups,[10] and we
tested whether the improved tetrazines reported here also
effectively removed ICPr groups. Indeed, 3c near-quantitatively
The observation that the dependence of iEEDA reaction rates on
the size of tetrazine substituents is opposite for isonitriles and
alkene-based dienophiles (e.g. norbornene; Fig. 2a), suggests
that it should be possible to design reactant pairs that are
orthogonal to each other. We investigated the chemoselectivity of
three tetrazines (1d, 3c, DPTz) towards several dienophiles (TCO,
norbornene, methylcyclopropene, primary/tertiary isonitriles;
Table 1, Fig. S7). The mono-tBu tetrazine 3c reacted significantly
faster with the isonitriles than the strained alkenes, TCO and
norbornene. This result is interesting because sterically
unconstrained tetrazines such as DPTz react orders of magnitude
faster with TCO than with isonitriles (Table 1), and this outcome
is in agreement with reports by Devaraj et al.[5l] The two t-Bu
removed ICPr groups from
a
ratiometric 4-hydroxy-1,8-
naphthalimide fluorophore (Fig. S13). Given that 3c reacts
preferentially with isonitriles relative to other dienophiles (Table
1), it could be used to control the release of two different
molecules by separate chemical stimuli.[25] We further tested
whether the optimized tetrazines are compatible with in vivo use
by implanting a bead modified with 3a (Tz-PS) into the yolk sac of
zebrafish embryos and monitoring the release of an ICPr-caged
resorufin fluorophore (Fig. 5a, for in vitro release, see Fig. S15).
Fish implanted with Tz-PS exhibited a significantly higher
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fluorescence signal compared to control fish implanted with
unmodified beads (5.5 fold, t = 4 h; Fig. 5b, 5c). Furthermore,
compared to previously disclosed Tz-beads modified with
MeTzCOOH,[10] a in vivo resorufin release was faster with 3a-
modified beads (Fig. S16).
reactivity and orthogonality. This versatile chemistry may open
doors to diverse applications in chemical biology and therapeutics.
Acknowledgements
R.M.F. gratefully acknowledges financial support from the
University of Utah, the Huntsman Cancer Institute, and the
USTAR initiative. This work was supported by the L. S. Skaggs
Presidential Endowed Chair (R.T.P.). We thank Ethan Lamé
(Juan Diego High School) and Tejita Agarwal (West High School)
for assistance in the early stages of this project. D.S gratefully
acknowledges financial support from the Austrian Science Fund
(FWF, J 4216-N28). K.N.H gratefully acknowledges financial
support from the National Institute of General Medical Sciences,
National Institutes of Health, GM-109078.
Keywords: Bioorthogonal chemistry
•
Cycloadditions
•
Dispersion forces • Chemoselectivity • Bioconjugation
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simulated physiological conditions. Sterically encumbered
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alkenes/alkynes, which enabled the triple-orthogonal labeling of
proteins. Considering the structural compactness and ease of
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10.1002/anie.201903877
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
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COMMUNICATION
Rise of bulky tetrazines: Highly
stable tetrazines that react rapidly with
isonitriles are presented. Bulky
tetrazine substituents sterically attract
the isocyano group in the
J. Tu, D. Svatunek, S. Parvez, A. C. G.
Liu, B. J. Levandowski, H. J. Eckvahl, R.
T. Peterson, K. N. Houk, R. M. Franzini*
Page No. – Page No.
cycloaddition transition state and
block the approach of strained
alkenes. These characteristics enable
rapid bioorthogonal labelling and
release reactions and allow for triple-
orthogonal protein labelling.
Stable, Reactive and
Orthogonal Tetrazines:
Dispersion Forces Promote the
Cycloaddition with Isonitriles
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