Installation of Minimal Tetrazines through Silver-Mediated Liebeskind-Srogl Coupling with Arylboronic Acids

Article abstract of DOI:10.1021/jacs.9b08677

Described is a general method for the installation of a minimal 6-methyltetrazin-3-yl group via the first example of a Ag-mediated Liebeskind-Srogl cross-coupling. The attachment of bioorthogonal tetrazines on complex molecules typically relies on linkers that can negatively impact the physiochemical properties of conjugates. Cross-coupling with arylboronic acids and a new reagent, 3-((p-biphenyl-4-ylmethyl)thio)-6-methyltetrazine (b-Tz), proceeds under mild, PdCl₂(dppf)-catalyzed conditions to introduce minimal, linker-free tetrazine functionality. Safety considerations guided our design of b-Tz which can be prepared on decagram scale without handling hydrazine and without forming volatile, high-nitrogen tetrazine byproducts. Replacing conventional Cu(I) salts used in Liebeskind-Srogl cross-coupling with a Ag₂O mediator resulted in higher yields across a broad library of aryl and heteroaryl boronic acids and provides improved access to a fluorogenic tetrazine-BODIPY conjugate. A covalent probe for MAGL incorporating 6-methyltetrazinyl functionality was synthesized in high yield and labeled endogenous MAGL in live cells. This new Ag-mediated cross-coupling method using b-Tz is anticipated to find additional applications for directly introducing the tetrazine subunit to complex substrates.

Full text of DOI:10.1021/jacs.9b08677

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Communication
Installation of Minimal Tetrazines Through Silver-mediated
Liebeskind-Srogl Coupling with Arylboronic Acids
William Lambert, Yinzhi Fang, Subham Mahapatra, Zhen Huang, Christopher W. am Ende, and Joseph M. Fox
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08677 • Publication Date (Web): 11 Oct 2019
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Installation of Minimal Tetrazines Through Silver-mediated Liebeskind-
Srogl Coupling with Arylboronic Acids
William D. Lambert,† Yinzhi Fang,† Subham Mahapatra,‡ Zhen Huang,§ Christopher W. am Ende,*, ‡
and Joseph M. Fox*,†
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† Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States
‡ Pfizer Worldwide Research and Development, 445 Eastern Point Road, Groton, CT 06340, United States
§ Pfizer Worldwide Research and Development, 1 Portland St, Cambridge, MA 02139, United States
Supporting Information Placeholder
well as in the fabrication and patterning of biomaterials27. Te-
trazines themselves have also found applications in explosives
technology28, in metal-organic frameworks29 and in natural
product synthesis.30
ABSTRACT: Described is a general method for the installation
of a minimal 6-methyltetrazin-3-yl group via the first example
of a Ag-mediated Liebeskind-Srogl cross-coupling. The attach-
ment of bioorthogonal tetrazines on complex molecules typi-
cally relies on linkers that can negatively impact the physio-
chemical properties of conjugates. Cross-coupling with aryl-
boronic acids and a new reagent, 3-((p-biphenyl-4-ylme-
thyl)thio)-6-methyltetrazine (b-Tz), proceeds under mild,
PdCl2(dppf)-catalyzed conditions to introduce minimal, linker-
free tetrazine functionality. Safety considerations guided our
design of b-Tz which can be prepared on decagram scale with-
out handling hydrazine and without forming volatile, high-ni-
trogen tetrazine byproducts.Replacing conventionalCu(I) salts
used in Liebeskind-Srogl cross-coupling with a Ag2O mediator
resulted in higher yields across a broad library of aryl and het-
eroaryl boronic acids and provides improved access to a fluo-
rogenic tetrazine-BODIPY conjugate. A covalent probe for
MAGL incorporating 6-methyltetrazinyl functionality was syn-
thesized in high yield and labeled endogenous MAGL in live
cells. This new Ag-mediated cross-coupling method using b-Tz
is anticipated to find additional applications for directly intro-
ducing the tetrazine subunit to complex substrates.
Conjugates of tetrazines are frequently prepared by amide
bond forming reactions as represented in Figure 1A. A major
limitation of this approach is that large and hydrophobic link-
ers can negatively impact the physiochemical properties of an
attached ligand.6, 17 Complementary new methods for the intro-
duction of minimal tetrazines to small molecules may further
advance their potential as bioorthogonal probes and chemical
reporters. The replacement of bulky derivatives with smaller
tetrazines has resulted in fluorophores with improved fluoro-
genic and cellular wash-out properties31-33, better substrates
for enzyme-catalyzed protein modification,17, 34 and probes for
18F-PET imaging35. However, there are currently few methods
The bioorthogonal reactions of tetrazines have emerged as im-
portant tools for chemical biology over the last decade.1-6 Cy-
cloadditions involving a range of dienophiles including trans-
cyclooctenes1, 7-10, cyclopropenes11-12 and norbornenes13 have
been developed as tools for a variety of applications including
cellular labeling14-17, in vivo imaging18-20, unnatural amino acid
mutagenesis3, 21-22, targeted drug delivery23-25, proteomics26, as
Figure 2: (A) Tetrazine synthesis based on condensation
of nitriles or Pinner reagents with hydrazine (B) Cross-
couplings of tetrazine electrophiles with arylboronic acids
have been limited to N-substituted tetrazines, which are
deactivated for bioorthogonal chemistry applications.
Stille coupling has been used to couple 3-bromo-6-methyl-
tetrazine to fluorophores.
Figure 1: (A) The most common approach to tetrazine
conjugation uses bulky linkers to attach molecules of in-
terest. (B) Cross-coupling approach described here.
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for the direct attachment of ‘minimal’ tetrazine fragments to
shown in Figure 3, the 4-phenylbenzyl derivative b-Tz (1a) was
prepared on large scale via alkylation ofcommercially available
thiocarbohydrazide54 with 4-bromomethylbiphenyl followed
by one-pot condensation with triethylorthoacetate and a novel
Cu(OAc)2-catalyzed air-oxidation of the dihydrotetrazine inter-
mediate. b-Tz was isolated on 27 gram scale with a 47% overall
target molecules.36 Additionally, many approaches to tetrazine
synthesis produce high-nitrogen byproducts and involve harsh
reaction conditions that can limit scalability and scope. Herein,
we describe the decagram synthesis and thermal stability of 3-
((p-biphenyl-4-ylmethyl)thio)-6-methyltetrazine, (b-Tz, 1a)
and a method to directly introduce the 6-methyltetrazin-3-yl
group to arylboronic acids through the first example of a Ag-
mediated Liebeskind-Srogl reaction (Fig 1B).
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Classical tetrazine synthesis involves the condensation of Pin-
ner salts or nitriles with excess hydrazine followed by oxida-
tion.37-38 Catalytic nitrile condensation with neatanhydroushy-
drazine, most notably with Zn(OTf)2 and Ni(OTf)2, has ex-
panded access to unsymmetrical tetrazines.39 Further, thiol ca-
talysis has been shown to promote tetrazine synthesis from ni-
triles using hydrazine-hydrate.40 The most practiced proce-
dures utilize excess acetonitrile or formamidine acetate and
produce volatile tetrazine byproducts with high-nitrogen con-
tent (Fig 2A). Recently, a sulfur-catalyzed reaction of nitriles
with hydrazine hydrate and dichloromethane has been de-
scribed for 3-aryltetrazine synthesis.41 A safety consideration
for all of these procedures is the direct addition of an oxidant
to a reaction mixture containing hydrazine. While these meth-
ods for preparing tetrazines have been transformative to the
field of bioorthogonal chemistry, there is a continuing need for
safer alternatives with complementary functional group com-
patibility.
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Tetrazines have been used in a limited number of metal cata-
lyzed CH activations,42-43 cross-couplings,32-33, 44-51 and in Heck
reactions with in situ generated 3-vinyl-6-methyltetrazine32.
Recently, 3-amino-6-chlorotetrazines have been cross-coupled
under Suzuki conditions (Fig 2B).45 Liebeskind-Srogl cross-
couplings have also been reported with 3-amino-6-thiomethyl-
tetrazines at 200 °C (Fig 2B).46 The 3-aminotetrazine products
of these methods are valuable in medicinal chemistry, but their
utility in bioorthogonal chemistry is attenuated by the deac-
tivating amino substituent.21, 32 The tetrazines most useful to
bioorthogonal chemistry are also sensitive to basic conditions,
making them incompatible with many conditions commonly
associated with cross-coupling chemistry. Currently, there is a
single method of cross-coupling to introduce a 3-methylte-
trazine fragment via Stille coupling with 3-bromo-6-methylte-
trazine, which is prepared from 3-hydrazino-6-methylte-
trazine (Fig 2B).33
We considered that 3-thioalkyl-6-methyltetrazinesmight serve
as useful reagents for the preparation of 3-aryl-6-methylte-
trazines, which are attractive bioorthogonal reagents due to
their balance of rapid kineticstoward dienophilesand high sta-
21, 52
bility in the cellular environment.17,
By modifying a
method for the synthesis of 3-thiomethyl-6-methyltetrazine,53
we prepared compounds 1a-g with the rationale that a sacrifi-
cial S-benzylic substituent could serve to tune cross-coupling
efficiency and improve the safety profile of the tetrazine. As
Figure 4: (A) Rapid decomposition of b-Tz in CuTC. (B)
Proposed Liebeskind-Srogl transmetallation mechanism.
(C) Optimized Pd-catalyzed cross-coupling of tetrazines b-
Tz and 1b with various nucleophiles (yields determined by
GC w/dodecane as a standard). Conditions: (a) Pd2dba3
(12.5 mol%), Cs2CO3 (3.0 eq.), dioxane, 70 °C, 90 min. (b)
[Pd(allyl)Cl]2 (10 mol%), THF, 70 °C, 2 h. (c) Pd(OAc)2 (10
mol%),TBAF(1.0 eq.), dioxane,70 °C, 2.5 h. (d)Pd2dba3 (15
mol%), DMF, 60 °C, 20 h. (e) DMF, 60 °C, 20 h. (D) Screening
of silver(I) and copper(I) additives for condition e. (E)
Screening of tetrazines 1a-g under condition e.
Figure 3: Decagram synthesis and thermal stability of b-
Tz (1a).
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yield after simple silica plug filtration and is a bench-stable
directing groups59-60, and the Cu-catalyzed coupling of aryl-
boronic acids with aromatic thioesters61. To our knowledge, a
Ag-mediated variation of the Liebeskind-Srogl reaction has not
been reported. After extensive optimization (Fig 4C entries 4-
7, S-1 and S-2), PdCl2(dppf) (15 mol%) was found to be espe-
cially effective for cross-coupling of 3-thioalkyl-6-methylte-
trazines with arylboronic acids in polar, aprotic solvents (DMF,
DMSO) at 60 °C. A screening of silver(I) additivesrevealed Ag2O
as the most general promotor, although Ag2CO3 was also effec-
tive (Fig 4D). Substitution of Ag2O by Cu2O gave only trace
product formation. Arylboronic acids are particularly effective
nucleophiles, whereas PhBF3K and PhBPin were both less ef-
fective under identical reaction conditions (Fig 4C entries 8-9).
Further, a series of 3-arylmethyl-6-methyltetrazines 1a-g were
evaluated as coupling partners (Fig 4E). Of these, the 4-phe-
nylbenzyl derivative b-Tz (1a) was identified as the substrate
with both the best cross-coupling yield as well as most favora-
ble thermal stabiliy. We also note that the cost of Ag2O (cur-
rently <$3/g) is similar to the common promotor CuTC, and is
minor in the context of bioorthogonal chemistry reagents
which are typically required only in small amounts.
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crystalline solid (m.p. 141°C). The differential scanning calo-
rimetry (DSC) profile of b-Tz has an onset temperature of 170
°C and a transition enthalpy of 900 J/g and is not flagged as po-
tentially shock sensitive or explosive by a modified Yoshida
correlation (Fig S-11).55
After extensive screening (Fig S-3 thru S-8), we found cop-
per(I)-mediated Liebeskind-Srogl conditions56-57 could pro-
mote cross-coupling of benzylic thioether tetrazines with
PhB(OH)2, PhSnBu3, and PhSi(OMe)3 (Fig 4C entries 1-3). Un-
der Cu-mediated conditions tetrazine 1b was the best sub-
strate; however, the generality under these conditions was
modest. The rapid consumption of tetrazine starting materials
during the reaction led us to test if Cu(I) was causing decompo-
sition of the reagent. Indeed, heating b-Tz with Cu(I)-thiophene
carboxylate (CuTC) at 70 °C resulted in rapid decomposition
and produced 4-phenylbenzaldehyde as the only identifiable
side product (Fig 4A).
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Copper has been proposed to promote the Liebeskind-Srogl re-
action by facilitating transmetallation as shown in Figure 4B.57-
58 We hypothesized that silver(I) salts might be similarly capa-
ble as promotors, whereby transmetallation would be pro-
moted in a dual role by the thiophilic capture of benzylic thio-
late by silver and the borophilic capture by oxygen. Ag(I) addi-
tives have been shown to promote Rh-catalyzed coupling of ar-
ylboronic acids with arylmethylsulfides bearing ortho-
The scope of the Ag-mediated, Pd-catalyzed coupling of b-Tz
with arylboronic acids is summarized in Figure 5A. Successful
reactions were observed for arylboronic acids containing
chloro-, fluoro-, secondary and tertiary amino-, alcohol, ether,
nitro, sulfonyl, thioether, nitrile, aldehyde, ester, ketone, carba-
mate, and styryl groups. Heterocyclic functionality tolerated on
Figure 5: Reaction scope of b-Tz (5A) and 3 (5B). Typical conditions: thioalkyl tetrazine b-Tz or 3 (1.0 equiv.), RB(OH)2 (1.9
equiv.), PdCl2(dppf) (15 mol%), Ag2O (2.5 equiv.), DMF (0.1M), 60°C, 19-21h, average isolated yields of duplicate synthesis (±
5%). a 3.0 equiv. of RB(OH)2. b 3.0 equiv. of RB(OH)2 did not significantly improve yield (< 5%), 1.9 equiv. of RB(OH)2 was used.
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the boronic acid component included quinoline, indole, pyri-
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dine, triazole, N-methylimidazole, furan and thiophene groups.
The protected amino acid 2ae coupled with b-Tz in 96% yield.
Estrone-tetrazine 2ag was also synthesized in 61%. In general,
couplings were carried out using 1.9 equiv. of boronic acid, but
3.0 equiv. were utilized in reactionswhere homocoupling of the
boronic acid was pronounced. ortho-Substituted heteroatoms
had a deleterious impact with a relatively low yield observed
for ortho-methoxy tetrazine 2k and only trace product with N-
Boc-2-aminophenylboronic acid and 2-hydroxyphenylboronic
acid. While protected thiol and amine functionality was well
tolerated (Fig 5), additives with free thiol or primary alkyl
amine groups were not (Fig S-19). Also unsuccessful were 2-
pyridyl- and 4-pyridylboronic acids which are regarded as
problematic across other cross-coupling reactions.62
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This cross-coupling method is not limited to S-benzylic thi-
oethers or methyl-substituted tetrazines. 3-(Methylthio)-6-
phenyl-tetrazine (3) was prepared from triethyl orthobenzoate
and evaluated as a reagent in the synthesis of diaryltetrazines
(Fig 5B). Successful reactions were observed for arylboronic
acids bearing chloro-, alcohol, carbamate, ester, indole and
ether groups with yields comparable to b-Tz. Included is an im-
proved synthesis of 3-(4-hydroxymethylphenyl)-6-phenylte-
trazine (4c), which is used to create cell-contact guiding micro-
fibrous materials for tissue-culture applications.27
We sought to demonstrate the application of b-Tz for the con-
struction of fluorophore-tetrazine conjugates—compounds
that have utility in live cell imaging.15 BODIPY-dye 6 with a di-
rectly attached tetrazine has been developed as ‘superbright’
bioorthogonal probe for fluorogenic labeling in live cells.31 The
condensation of nitriles with hydrazine produces 6 in 8%
yield.31 As shown in Figure 6, compound 6 can be accessed in
78% yield through the Ag-mediated cross-coupling of boronic
acid 5 with b-Tz.
To demonstrate the utility of b-Tz in synthesizing chemical
probes for studying endogenous levels of a protein in a native
biological system, we constructed a tetrazine probe for mono-
acylglycerol lipase (MAGL). MAGL is a serine hydrolase in the
endocannabinoid signaling pathway, and hasattracted increas-
ing interest as a target for neurological and metabolic disor-
ders.63 We designed a MAGL probe (9) by appending a 6-me-
thyltetrazine moiety to a pyrazolylpiperidine scaffold with an
electrophilic hexafluoroisopropyl (HFIP) carbamate warhead
for covalently labeling the active site serine (Fig 7A).64 Synthe-
sis was accomplished by cross-coupling of b-Tz with boronic
acid 7 resulting in a 77% yield of 8. The reactive HFIP carba-
mate was installed by Boc deprotection followed by in situ ad-
dition to a triphosgene and hexafluoroisopropanol mixture,
giving the MAGL reactive probe 9 in 78% yield. The reaction
rate of 9 toward trans-cyclooctene is similar to that of 3-me-
thyl-6-(4-aminomethyl)tetrazine (krel 1.1, Fig S-20).65 Probe 9
inhibited MAGL activity with 31 nM IC50 in an in vitro assay.66
Figure 7: (A) Synthesis of MAGL reactive probe 9. (B) Live
cells were treated with probe 9 for 1 h, followed by 2 μM
TCO-TAMRA for 30 min, cell lysis, andanalysisby in-gel flu-
orescence (C) In-gel fluorescence signals for a dose re-
sponse of probe 9. Probe 9 (32 nM, 1 h) was competed by
pre-treatment with MAGL inhibitor KML29 (300 nM, 1 h).
(D) KML29 also incorporates a HFIP carbamate warhead.
(E) Dose response fitting of the fluorescence signals of
MAGL normalized by the total protein amount indicated by
Coomassie staining. Data are reported as mean ± SEM (n =
2). See Fig S-21 for Coomassie staining.
To test the labeling of endogenous MAGL in live cells, human
brain vascular pericytes were treated with probe 9 for 1 h, fol-
lowed by labeling with 2 µM of TCO-TAMRA for 30 min in live
cells (Fig 7B). After cell lysis, MAGL labeling was assessed with
a gel-based activity-based protein profiling (ABPP) analysis
(Fig 7C-E).67 Strong fluorescence signals were observed for
MAGL with minimal non-specific labeling from TCO-TAMRA.
The labeling by probe 9 was dose responsive with a cellular
IC50 of 8 nM, and was competed by a MAGL inhibitor, KML29.64
The HFIP warhead also labeled an additional protein at ~35
kDa, which is consistent with its reactivity with a/b-hydrolase
domain 6 (ABHD6), and other off-targets at higher concentra-
tions.64, 67
Figure 6: Synthesis of 3-BODIPY-6-methyltetrazine 6.
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(7) Blackman, M. L.; Royzen, M.; Fox, J. M., Tetrazine ligation: fast
bioconjugation based on inverse-electron-demand Diels-Alder
reactivity. J. Am. Chem. Soc. 2008, 130, 13518-13519.
In summary, a method has been described for installing a min-
imal 6-methyltetrazinyl-3-yl group through the first Ag-medi-
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ated Liebeskind-Srogl cross-coupling.
A combination of
(8) Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M., Design and
synthesis of highly reactive dienophiles for the tetrazine-trans-
cyclooctene ligation. J. Am. Chem. Soc. 2011, 133, 9646-9649.
(9) Darko, A.; Wallace, S.; Dmitrenko, O.; Machovina, M. M.; Mehl, R. A.;
Chin, J. W.; Fox, J. M., Conformationally Strained trans-Cyclooctene with
Improved Stability and Excellent Reactivity in Tetrazine Ligation.
Chem. Sci. 2014, 5, 3770-3776.
(10) Lambert, W. D.; Scinto, S. L.; Dmitrenko, O.; Boyd, S. J.; Magboo, R.;
Mehl, R. A.; Chin, J. W.; Fox, J. M.; Wallace, S., Computationally guided
discovery of a reactive, hydrophilic trans-5-oxocene dienophile for
bioorthogonal labeling. Org. Biomol. Chem. 2017, 15, 6640-6644.
(11) Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D. N.; Prescher,
J. A., Functionalized cyclopropenes as bioorthogonal chemical
reporters. J. Am. Chem. Soc. 2012, 134, 18638-18643.
(12) Yang, J.; Seckute, J.; Cole, C. M.; Devaraj, N. K., Live-cell imaging of
cyclopropene tags with fluorogenic tetrazine cycloadditions. Angew.
Chem. Int. Ed. 2012, 51, 7476-7479.
(13) Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A., Tetrazine-based
cycloadditions: application to pretargeted live cell imaging. Bioconjug.
Chem. 2008, 19, 2297-2299.
(14) Devaraj, N. K.; Upadhyay, R.; Haun, J. B.; Hilderbrand, S. A.;
Weissleder, R., Fast and sensitive pretargeted labeling of cancer cells
through a tetrazine/trans-cyclooctene cycloaddition. Angew. Chem. Int.
Ed. 2009, 48, 7013-7016.
(15) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.; Mazitschek, R.;
Weissleder, R., Bioorthogonal turn-on probes for imaging small
molecules inside living cells. Angew. Chem. Int. Ed. 2010, 49, 2869-
2872.
(16) Murrey, H. E.; Judkins, J. C.; am Ende, C. W.; Ballard, T. E.; Fang, Y.;
Riccardi, K.; Di, L.; Guilmette, E. R.; Schwartz, J. W.; Fox, J. M.; Johnson,
D. S., Systematic Evaluation of Bioorthogonal Reactions in Live Cells
with Clickable HaloTag Ligands: Implications for Intracellular Imaging.
J. Am. Chem. Soc. 2015, 137, 11461-11475.
(17) Baalmann, M.; Ziegler, M. J.; Werther, P.; Wilhelm, J.; Wombacher,
R., Enzymatic and Site-Specific Ligation of Minimal-Size Tetrazines and
Triazines to Proteins for Bioconjugation and Live-Cell Imaging.
Bioconjug. Chem. 2019, 30, 1405-1414.
PdCl2(dppf) catalyst and Ag2O mediator was found to be
uniquely effective for coupling 3-thioalkyl-6-methyltetrazines
with arylboronic acids. Safety-testing guided our design of the
reactive substrate b-Tz (1a), which can be synthesized from
commercially available materials on decagram scale in 47%
overall yield. Cross-coupling of b-Tz with boronic acids pro-
ceeds under mild conditions with broad functional group toler-
ance. Alternatively, 3-(Methylthio)-6-phenyl-tetrazine (3) un-
dergoes cross-coupling with arylboronic acids to give 3,6-dia-
ryltetrazines. Application to the synthesis of chemical biology
tools was demonstrated. A BODIPY-tetrazine conjugate was
synthesized in 78% yield—substantially higher than what is
possible using traditional hydrazine-based synthesis. Finally, a
tetrazine-functionalized probe for MAGL was synthesized in
high yield and was shown to covalently label endogenous
MAGL with good selectivity in live cells. We anticipate that this
method for introducing minimal tetrazines to chemical probes
will serve as an important tool for studying protein targets at
endogenous levels in their native environment.
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ASSOCIATED CONTENT
Supporting Information
Optimization tables, DSC data, experimental procedures, kinet-
ics, inhibition assays, and 1H and 13C NMR data are displayed.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: xxxxxxxxxxxxx
AUTHOR INFORMATION
Corresponding Author
*jmfox@udel.edu, *christopher.amEnde@pfizer.com
(18) Rossin, R.; Verkerk, P. R.; van den Bosch, S. M.; Vulders, R. C.;
Verel, I.; Lub, J.; Robillard, M. S., In vivo chemistry for pretargeted tumor
imaging in live mice. Angew. Chem. Int. Ed. 2010, 49, 3375-3378.
(19) Zeglis, B. M.; Sevak, K. K.; Reiner, T.; Mohindra, P.; Carlin, S. D.;
Zanzonico, P.; Weissleder, R.; Lewis, J. S., A pretargeted PET imaging
strategy based on bioorthogonal Diels-Alder click chemistry. J. Nucl.
Med. 2013, 54, 1389-1396.
(20) Wang, M.; Vannam, R.; Lambert, W. D.; Xie, Y.; Wang, H.; Giglio, B.;
Ma, X.; Wu, Z.; Fox, J.; Li, Z., Hydrophilic (18)F-labeled trans-5-oxocene
(oxoTCO) for efficient construction of PET agents with improved
tumor-to-background ratios in neurotensin receptor (NTR) imaging.
Chem. Commun. 2019, 55, 2485-2488.
(21) Blizzard, R. J.; Backus, D. R.; Brown, W.; Bazewicz, C. G.; Li, Y.;
Mehl, R. A., Ideal Bioorthogonal Reactions Using A Site-Specifically
Encoded Tetrazine Amino Acid. J. Am. Chem. Soc. 2015, 137, 10044-
10047.
(22) Hoffmann, J. E.; Plass, T.; Nikic, I.; Aramburu, I. V.; Koehler, C.;
Gillandt, H.; Lemke, E. A.; Schultz, C., Highly Stable trans-Cyclooctene
Amino Acids for Live-Cell Labeling. Chemistry 2015, 21, 12266-12270.
(23) Li, J.; Chen, P. R., Development and application of bond cleavage
reactions in bioorthogonal chemistry. Nat. Chem. Biol. 2016, 12, 129-
137.
(24) Versteegen, R. M.; Rossin, R.; ten Hoeve, W.; Janssen, H. M.;
Robillard, M. S., Click to release: instantaneous doxorubicin elimination
upon tetrazine ligation. Angew. Chem. Int. Ed. 2013, 52, 14112-14116.
(25) Mejia Oneto, J. M.; Khan, I.; Seebald, L.; Royzen, M., In Vivo
Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-
Drug To Treat Soft Tissue Sarcoma. ACS Cent. Sci. 2016, 2, 476-482.
(26) Kang, K.; Park, J.; Kim, E., Tetrazine ligation for chemical
proteomics. Proteome. Sci. 2016, 15, 1-13.
(27) Liu, S.; Moore, A. C.; Zerdoum, A. B.; Zhang, H.; Scinto, S. L.; Zhang,
H.; Gong, L.; Burris, D. L.; Rajasekaran, A. K.; Fox, J. M.; Jia, X., Cellular
interactions with hydrogel microfibers synthesized via interfacial
tetrazine ligation. Biomaterials 2018, 180, 24-35.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We would like to thank Jeffery Sperry for helpful discussions,
Remzi Duzguner for DSC analysis, and Lucy Stevens for the in
vitro potency data on probe 9. This work was supported by NIH
GM132460, NSF 1809612 and Pfizer. Instrumentation was
supported
by
NIH
P20GM104316,
P30GM110758,
S10RR026962, S10OD016267 and NSF CHE-0840401, CHE-
1229234, and CHE-1048367.
REFERENCES
(1) Selvaraj, R.; Fox, J. M., trans-Cyclooctene--a stable, voracious
dienophile for bioorthogonal labeling. Curr. Opin. Chem. Biol. 2013, 17,
753-760.
(2) Patterson, D. M.; Nazarova, L. A.; Prescher, J. A., Finding the right
(bioorthogonal) chemistry. ACS Chem. Biol. 2014, 9, 592-605.
(3) Lang, K.; Chin, J. W., Bioorthogonal reactions for labeling proteins.
ACS Chem. Biol. 2014, 9, 16-20.
(4) Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L., Inverse electron demand
Diels-Alder reactions in chemical biology. Chem. Soc. Rev. 2017, 46,
4895-4950.
(5) Wu, H.; Devaraj, N. K., Advances in Tetrazine Bioorthogonal
Chemistry Driven by the Synthesis of Novel Tetrazines and
Dienophiles. Acc. Chem. Res. 2018, 51, 1249-1259.
(6) Cañeque, T.; Müller, S.; Rodriguez, R., Visualizing biologically active
small molecules in cells using click chemistry. Nat. Rev. Chem. 2018, 2,
202-215.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 7
(28) DeLuca, L. T.; Shimada, T.; Sinditskii, V. P.; Calabro, M., Chemical
electron demand Diels-Alder reaction. Org. Biomol. Chem. 2014, 12,
Rocket Propulsion: A Comprehensive Survey of Energetic Materials.
Springer International Publishing: 2017.
(29) Xue, M.; Ma, S.; Jin, Z.; Schaffino, R. M.; Zhu, G. S.; Lobkovsky, E. B.;
Qiu, S. L.; Chen, B., Robust metal-organic framework enforced by triple-
framework interpenetration exhibiting high H2 storage density. Inorg.
Chem. 2008, 47, 6825-6828.
4177-4185.
1
2
3
4
5
6
7
8
(50) Sołoducho, J.; Doskocz, J.; Cabaj, J.; Roszak, S., Practical synthesis
of bis-substituted tetrazines with two pendant 2-pyrrolyl or 2-thienyl
groups, precursors of new conjugated polymers. Tetrahedron 2003, 59,
4761-4766.
(51) Li, Z.; Ding, J.; Song, N.; Lu, J.; Tao, Y., Development of a New s-
Tetrazine-Based Copolymer for Efficient Solar Cells. J. Am. Chem. Soc.
2010, 132, 13160-13161.
(52) Karver, M. R.; Weissleder, R.; Hilderbrand, S. A., Synthesis and
evaluation of
conjugation. Bioconjug. Chem. 2011, 22, 2263-2270.
(30) Hamasaki, A.; Zimpleman, J. M.; Hwang, I.; Boger, D. L., Total
synthesis of ningalin D. J. Am. Chem. Soc. 2005, 127, 10767-10770.
(31) Carlson, J. C.; Meimetis, L. G.; Hilderbrand, S. A.; Weissleder, R.,
BODIPY-tetrazine derivatives as superbright bioorthogonal turn-on
probes. Angew. Chem. Int. Ed. 2013, 52, 6917-6920.
(32) Wu, H.; Yang, J.; Seckute, J.; Devaraj, N. K., In situ synthesis of
alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell
imaging probes. Angew. Chem. Int. Ed. 2014, 53, 5805-5809.
(33) Wieczorek, A.; Werther, P.; Euchner, J.; Wombacher, R., Green- to
far-red-emitting fluorogenic tetrazine probes - synthetic access and no-
wash protein imaging inside living cells. Chem. Sci. 2017, 8, 1506-1510.
(34) Best, M.; Degen, A.; Baalmann, M.; Schmidt, T. T.; Wombacher, R.,
Two-step protein labeling by using lipoic acid ligase with norbornene
substrates and subsequent inverse-electron demand Diels-Alder
reaction. Chembiochem. 2015, 16, 1158-1162.
(35) Denk, C.; Svatunek, D.; Filip, T.; Wanek, T.; Lumpi, D.; Frohlich, J.;
Kuntner, C.; Mikula, H., Development of a (18) F-labeled tetrazine with
favorable pharmacokinetics for bioorthogonal PET imaging. Angew.
Chem. Int. Ed. 2014, 53, 9655-9659.
(36) For the introduction of bioorthogonal triazines via Suzuki
coupling, see: Kamber, D. N.; Liang, Y.; Blizzard, R. J.; Liu, F.; Mehl, R. A.;
Houk, K. N.; Prescher, J. A., 1,2,4-Triazines Are Versatile Bioorthogonal
Reagents. J. Am. Chem. Soc. 2015, 137, 8388-91.
(37) Pinner, A., Ueber die Einwirkung von Hydrazin auf Imidoäther.
Ber. Dtsch. Chem. Ges. 1893, 26, 2126.
(38) Tolshchina, S. G.; Rusinov, G. L.; Charushin, V. N., 1,2,4,5-
Tetrazines and Azolo[1,2,4,5]tetrazines: Synthesis and Reactions with
Nucleophiles. Chem. Heterocycl. Compd. 2013, 49, 66-91.
(39) Yang, J.; Karver, M. R.; Li, W.; Sahu, S.; Devaraj, N. K., Metal-
catalyzed one-pot synthesis of tetrazines directly from aliphatic nitriles
and hydrazine. Angew. Chem. Int. Ed. 2012, 51, 5222-5225.
(40) Mao, W.; Shi, W.; Li, J.; Su, D.; Wang, X.; Zhang, L.; Pan, L.; Wu, X.;
Wu, H., Organocatalytic and Scalable Syntheses of Unsymmetrical
1,2,4,5-Tetrazines by Thiol-Containing Promotors. Angew. Chem. Int.
Ed. 2019, 58, 1106-1109.
(41) Qu, Y.; Sauvage, F. X.; Clavier, G.; Miomandre, F.; Audebert, P.,
Metal-Free Synthetic Approach to 3-Monosubstituted Unsymmetrical
1,2,4,5-Tetrazines Useful for Bioorthogonal Reactions. Angew. Chem.
Int. Ed. 2018, 57, 12057-12061.
a series of 1,2,4,5-tetrazines for bioorthogonal
9
(53) Fields, S. C.; Parker, M. H.; Erickson, W. R., A Simple Route to
Unsymmetrically Substituted 1,2,4,5-Tetrazines. J. Org. Chem 1994, 59,
8284-8287.
(54) DSC data for commercially available Thiocarbohydrazide: onset
temp. of 172 °C, transition enthalpy of 1634 J/g. As a precaution, all
experiments involving thiocarbohydrazide were carried out below 72
°C (100 °C below the DSC transition onset).
(55) Sperry, J. B.; Minteer, C. J.; Tao, J.; Johnson, R.; Duzguner, R.;
Hawksworth, M.; Oke, S.; Richardson, P. F.; Barnhart, R.; Bill, D. R.;
Giusto, R. A.; Weaver, J. D., Thermal Stability Assessment of Peptide
Coupling Reagents Commonly Used in Pharmaceutical Manufacturing.
Org. Process Res. Dev 2018, 22, 1262-1275.
(56) Cheng, H.-G.; Chen, H.; Liu, Y.; Zhou, Q., The Liebeskind-Srogl
Cross-Coupling Reaction and its Synthetic Applications. Asian J. Org.
Chem. 2018, 7, 490-508.
(57) Liebeskind, L. S.; Srogl, J., Heteroaromatic Thioether-Boronic Acid
Cross-Coupling under Neutral Reaction Conditions. Org. Lett. 2002, 4,
979-981.
(58) Musaev, D. G.; Liebeskind, L. S., On the Mechanism of Pd(0)-
Catalyzed, Cu(I) Carboxylate-Mediated Thioorganic-Boronic Acid
Desulfitative Coupling. A Non-innocent Role for Carboxylate Ligand.
Organometallics 2009, 28, 4639-4642.
(59) Hooper, J. F.; Young, R. D.; Pernik, I.; Weller, A. S.; Willis, M. C.,
Carbon–carbon bond construction using boronic acids and aryl methyl
sulfides: orthogonal reactivity in Suzuki-type couplings. Chem. Sci.
2013, 4, 1568-1572.
(60) Pan, F.; Wang, H.; Shen, P.-X.; Zhao, J.; Shi, Z.-J., Cross coupling of
thioethers with aryl boroxines to construct biaryls via Rh catalyzed C–
S activation. Chem. Sci. 2013, 4, 1573-1577.
(61) Ghosh, P.; Ganguly, B.; Perl, E.; Das, S., A synthesis of biaryl
ketones via the C–S bond cleavage of thiol ester by a Cu/Ag salt.
Tetrahedron Lett. 2017, 58, 2751-2756.
(62) Markovic, T.; Rocke, B. N.; Blakemore, D. C.; Mascitti, V.; Willis, M.
C., Pyridine sulfinates as general nucleophilic coupling partners in
palladium-catalyzed cross-coupling reactions with aryl halides. Chem.
Sci. 2017, 8, 4437-4442.
(63) Blankman, J. L.; Cravatt, B. F., Chemical probes of
endocannabinoid metabolism. Pharmacol. Rev. 2013, 65, 849-871.
(64) Chang, J. W.; Niphakis, M. J.; Lum, K. M.; Cognetta, A. B., 3rd; Wang,
C.; Matthews, M. L.; Niessen, S.; Buczynski, M. W.; Parsons, L. H.; Cravatt,
B. F., Highly selective inhibitors of monoacylglycerol lipase bearing a
reactive group that is bioisosteric with endocannabinoid substrates.
Chem. Biol. 2012, 19, 579-588.
(65) Electron withdrawing aromatic substitutents have an
accelerating effect on Diels-Alder rate constants with tetrazines, see ref
9 and ref 52.
(66) Butler, C. R.; Beck, E. M.; Harris, A.; Huang, Z.; McAllister, L. A.; Am
Ende, C. W.; Fennell, K.; Foley, T. L.; Fonseca, K.; Hawrylik, S. J.; Johnson,
D. S.; Knafels, J. D.; Mente, S.; Noell, G. S.; Pandit, J.; Phillips, T. B.; Piro, J.
R.; Rogers, B. N.; Samad, T. A.; Wang, J.; Wan, S.; Brodney, M. A.,
Azetidine and Piperidine Carbamates as Efficient, Covalent Inhibitors
of Monoacylglycerol Lipase. J. Med. Chem. 2017, 60, 9860-9873.
(67) Chang, J. W.; Cognetta, A. B., 3rd; Niphakis, M. J.; Cravatt, B. F.,
Proteome-wide reactivity profiling identifies diverse carbamate
chemotypes tuned for serine hydrolase inhibition. ACS Chem. Biol.
2013, 8, 1590-1599.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(42) Testa, C.; Gigot, E.; Genc, S.; Decreau, R.; Roger, J.; Hierso, J. C.,
Ortho-Functionalized Aryltetrazines by Direct Palladium-Catalyzed C-
H
Halogenation: Application to Fast Electrophilic Fluorination
Reactions. Angew. Chem. Int. Ed. 2016, 55, 5555-5559.
(43) Mboyi, C. D.; Testa, C.; Reeb, S.; Genc, S.; Cattey, H.; Fleurat-
Lessard, P.; Roger, J.; Hierso, J.-C., Building Diversity in ortho-
Substituted s-Aryltetrazines By Tuning N-Directed Palladium C–H
Halogenation: Unsymmetrical Polyhalogenated and Biphenyl s-
Aryltetrazines. ACS Catalysis 2017, 7, 8493-8501.
(44) Novak, Z.; Kotschy, A., First Cross-Coupling Reactions on
Tetrazines. Org. Lett. 2003, 5, 3495-3497.
(45) Bender, A. M.; Chopko, T. C.; Bridges, T. M.; Lindsley, C. W.,
Preparation of Unsymmetrical 1,2,4,5-Tetrazines via a Mild Suzuki
Cross-Coupling Reaction. Org. Lett. 2017, 19, 5693-5696.
(46) Suzenet, F.; Leconte, N.; Keromnes-Wuillaume, A.; Guillaumet, G.,
Efficient
Palladium-Catalyzed
Synthesis
of
Unsymmetrical
(Het)aryl-tetrazines. Synlett 2007, 2007, 204-210.
(47) Pellegatti, L.; Vedrenne, E.; Leger, J.-M.; Jarry, C.; Routier, S., New
heteroaromatic aminations on 5-aryl-1,2,4-triazines and 1,2,4,5-
tetrazines by palladium catalysis. Tetrahedron 2010, 66, 4383-4389.
(48) Quinton, C.; Alain-Rizzo, V.; Dumas-Verdes, C.; Clavier, G.; Vignau,
L.; Audebert, P., Triphenylamine/tetrazine based π-conjugated
systems as molecular donors for organic solar cells. New J. Chem. 2015,
39, 9700-9713.
(49) Wieczorek, A.; Buckup, T.; Wombacher, R., Rigid tetrazine
fluorophore conjugates with fluorogenic properties in the inverse
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