Divergent Synthesis of Monosubstituted and Unsymmetrical 3,6-Disubstituted Tetrazines from Carboxylic Ester Precursors

Article abstract of DOI:10.1002/anie.202005569

Since tetrazines are important tools to the field of bioorthogonal chemistry, there is a need for new approaches to synthesize unsymmetrical and 3-monosubstituted tetrazines. Described here is a general, one-pot method for converting (3-methyloxetan-3-yl)methyl carboxylic esters into 3-thiomethyltetrazines. These versatile intermediates were applied to the synthesis of unsymmetrical tetrazines through Pd-catalyzed cross-coupling and in the first catalytic thioether reduction to access monosubstituted tetrazines. This method enables the development of new tetrazine compounds possessing a favorable combination of kinetics, small size, and hydrophilicity. It was applied to a broad range of aliphatic and aromatic ester precursors and to the synthesis of heterocycles including BODIPY fluorophores and biotin. In addition, a series of tetrazine probes for monoacylglycerol lipase (MAGL) were synthesized and the most reactive one was applied to the labeling of endogenous MAGL in live cells.

Full text of DOI:10.1002/anie.202005569

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Divergent Synthesis of Monosubstituted and Unsymmetrical 3,6-
Disubstituted Tetrazines from Carboxylic Ester Precursors
Authors: Joseph M. Fox, Yixin Xie, Yinzhi Fang, Zhen Huang, Amanda
Tallon, and Christopher am Ende
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202005569
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10.1002/anie.202005569
Angewandte Chemie International Edition
RESEARCH ARTICLE
Divergent Synthesis of Monosubstituted and Unsymmetrical 3,6-
Disubstituted Tetrazines from Carboxylic Ester Precursors
Yixin Xie,[a] Yinzhi Fang,[a] Zhen Huang,[c] Amanda M. Tallon,[a] Christopher W. am Ende,*[b] and Joseph
M. Fox*[a]
Abstract: As tetrazines are important tools to the field of
bioorthogonal chemistry, there is a need for new approaches to
Installation of minimal tetrazines from carboxylic esters
synthesize unsymmetrical and 3-monosubstituted tetrazines.
N
N
N
N
Pd(0)
[H]
Described here is a general, one-pot method for converting (3-
methyloxetan-3-yl)methyl carboxylic esters into 3-
H
R
N
N
N
N
CO2R
thiomethyltetrazines. These versatile intermediates were applied as a
platform for the synthesis of unsymmetrical tetrazines via Pd-
catalyzed cross-coupling and in the first example of catalytic thioether
reduction to access monosubstituted tetrazines. The method enables
SMe
N
N
N
N
Pd(0)
R-M
the development of new tetrazines possessing
a favorable
Figure 1. Direct approach for the introduction of tetrazine functionality from
combination of kinetics, small size and hydrophilicity. The chemistry
was applied to a broad range of aliphatic and aromatic ester
precursors and to the synthesis of heterocycles including BODIPY
fluorophores and biotin. In addition, a series of tetrazine probes for
monoacylglycerol lipase (MAGL) were synthesized and the most
reactive one was applied in labeling of endogenous MAGL in live cells.
carboxylic ester precursors.
from carboxylic esters of commercially available 3-methyl-3-
oxetanemethanol. The compounds serve as versatile
intermediates for the divergent synthesis of a broad range of
functionalized tetrazines via Pd-catalyzed cross-coupling and in
the first example of catalytic thioether reduction to form
monosubstituted tetrazines (Figure 1). This ‘carboxylate-to-
tetrazine’ strategy complements known approaches of tetrazine
construction,[12] tolerates a range of heterocycles and functional
groups, and generates classes of tetrazines that are most useful
to bioorthogonal chemistry.
Introduction
Bioorthogonal reactions have become increasingly important to
chemistry and biology since the term was initially coined early in
this century.[1] The bioorthogonal reactions of tetrazines have
become widely used in various fields,[2] including natural product
synthesis,[3] cargo delivery,[4] genetic code expansion,[5]
fluorogenic labeling,[6] radiochemistry,[7] coordination chemistry,[8]
and material science.[9] Coupled to the growing utility of tetrazine-
based bioorthogonal chemistry is the need for mild, safe and
general methods of introducing tetrazine groups to complex
molecules. Carboxylic esters are ubiquitous functional groups that
have been used as handles for the introduction of preformed
tetrazine groups via amide bond forming reactions.[10] For this
approach, linkers are required which can add bulk and
hydrophilicity to the conjugate.[11] The direct preparation of
tetrazines from carboxylic precursors could greatly expand
access to functionalized derivatives. Described here is a new
strategy for the one-pot synthesis of 3-thiomethyltetrazines
The most widely used method of preparing tetrazines involves
condensation of nitriles and hydrazine followed by oxidation
(Figure 2A).[13] Devaraj’s method for Zn(OTf)2 or Ni(OTf)2
catalyzed condensation of nitriles in neat anhydrous hydrazine
has greatly improved access to unsymmetrical tetrazines.[13d]
More recently, thiols have been used as organocatalysts that
function with hydrazine hydrate which is safer and more broadly
available than anhydrous hydrazine.[13e] Mono-functional
tetrazines have been prepared from nitriles, hydrazine and
formamidine acetate under Lewis acid catalyzed conditions[13d, 13f,
13g]
or with dichloromethane under sulfur-catalyzed conditions
(Figure 2B).[14] Safety considerations for these procedures include
the formation of volatile tetrazine byproducts with high nitrogen
content and the direct addition of an oxidant to reaction mixtures
containing hydrazine. Tetrazines have been used in metal
catalyzed Heck-type reactions,[6c] CH activations[15] and cross-
couplings.[6d, 6e, 16] 3,6-Dichlorotetrazine,[2, 17] 3,6-bis(3,5-dimethyl-
pyrazol-l-yl) tetrazine[18], and 3-bromotetrazine[19] have been used
to create thiol, alcohol and amine functionalized tetrazines by
nucleophilic aromatic substitution. While these methods for
tetrazine synthesis have been enabling, there is a continuing need
to broaden the types of precursors that can serve for the direct
installation of tetrazines with improved functional group tolerance.
Carboxylic esters have played only a limited role as precursors
to tetrazines.[12] Unsymmetrical 3-thiomethyltetrazines were
synthesized in low yield through the condensation of S-
methylisothiocarbonohydrazidium iodide (2) with either
trimethylorthoacetate or trimethylorthoformate.[20] Recently, our
group described a procedure for the conversion of trimethyl-
[a]
Y. Xie, Y. Fang, A. M. Tallon and Prof. Dr. J. M. Fox
Department of Chemistry and Biochemistry
University of Delaware
Newark, DE 19716 (USA)
E-mail: jmfox@udel.edu
[b]
[c]
Dr. C. W. am Ende
Pfizer Worldwide Research and Development
Eastern Point Road, Groton, CT 06340 (USA)
E-mail: Christopher.amende@pfizer.com
Dr. Z. Huang
Pfizer Worldwide Research and Development
1 Portland Street, Cambridge, MA 02139 (USA)
This article is protected by copyright. All rights reserved.

10.1002/anie.202005569
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Unsymmetrical Tetrazines from Nitriles with Hydrazine
Table 1. Optimization of condensation between 1a’ and 2.
R'
Me
1) H2N-NH2
excess
catalyst
Me
R'CN
+
MeCN (excess)
N
N
N
N
N
N
N
SMe
N
•HI
NH2
SMe
Me
S
+
N
H2N
HN
N
N
N
N
N
N
N
PIDA
r.t.
2) NaNO2
O
H
Me
Me
NH
2
O
O
volatile high-
nitrogen byproducts
DMF
Mono-substituted Tetrazines from Nitriles
B
OBn
OBn
OBn
3a
1a'
RCN +
AcO–
H
1) H2N-NH2
excess
R
yield
of 3a
equiv
of 1a'
entry additive
temp. time
conc.
+NH2
N
N
N
N
N
N
N
N
catalyst
+
or
CH2Cl2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.25
1.4
r.t.
18%
0
none
6 h
6 h
6 h
7 h
6 h
7 h
1 h
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
1
H
NH2
2) NaNO2
r.t.
NEt3 (1 equiv)
2
(excess)
H
(excess)
H
r.t.
0
DMAP (1 equiv)
Cs2CO3 (1 equiv)
KOtBu (1 equiv)
pyridine (1 equiv)
pyridine (3 equiv)
pyridine (3 equiv)
pyridine (3 equiv)
pyridine (3 equiv)
3
r.t.
0
4
This work: Tetrazines from Ester Precursors
C
r.t.
0
5
Ar
r.t.
24%
23%
54%
75%
87%
6
Pd(dppf)Cl2
Ag2O
N
80 °C
80 °C
80 °C
80 °C
7
N
N
ArB(OH)2
20 min 1.0 M
20 min 1.0 M
20 min 1.0 M
8
N
O
9
R
one pot
10
BF3•OEt2
SMe
H
PdCl2
HSiEt3
Me
N
Me
N
N
N
N
O
•HI
S
N
N
O
Compound 2 is available commercially or can be prepared in
H2N
N
NH2
one step from iodomethane and thiocarbohydrazide.[24] The
differential scanning calorimetry (DSC) profile of 2 has an onset
temperature of 135 °C and a transition enthalpy of 875 J/g (Figure
S3). The OBO orthoester 1a’ was prepared by treating its (3-
methyloxetan-3-yl) methyl ester with BF3·OEt2. Table 1 shows
conditions for optimizing the condensation of 1a’ with 2, with
subsequent oxidation by phenyliodosodiacetate (PIDA)[25] to
provide thiomethyltetrazine 3a. DMF was the only effective
solvent likely due to the limited solubility of salt 2 in other solvents.
Simple combination of 1a’ and 2 at r.t. gave 3a in only 18% yield
(Table 1, entry 1). Given the acid-lability of orthoesters, a number
of basic additives were explored (entries 2-6). Pyridine was found
to be uniquely effective, and further optimization showed that the
temperature could be raised to 80 °C and reaction time shortened
without sacrificing yield (entry 7). Using 3 equiv. of pyridine and
increasing the concentration to 1 M had a significant impact and
raised the yield to 54% (entry 8). Using 1.4 equiv. of 1a’ further
raised the yield to 87% (entry 10).
N
H
N
R
R
3
R
2
1
then PIDA
Pd(PPh3)4
CuTc
OEt
N
OEt
N
R
N
Bu3Sn
N
Figure 2. Selected methods of tetrazine synthesis
orthoacetate to a 3-methyl-6-thioalkyltetrazine that can in turn be
used for the installation of minimal tetrazines through the silver-
mediated Liebeskind-Srogl coupling with arylboronic acids.[21]
This method was limited to trimethylorthoacetate and
triethylorthobenzoate, and we hoped to develop a general
approach for directly converting ester precursors into tetrazines.
However, trimethyl- and triethylorthoesters cannot be prepared
directly from esters but instead require nitrile precursors and
harshly acidic conditions.[22] Thus, our previous method did not
enable the use of carboxylic acids or esters as precursors to
tetrazines. Here, we describe a one-pot procedure for the
conversion of (3-methyloxetan-3-yl)methyl carboxylic esters (1) to
unsymmetrical 3-thiomethyltetrazines (3) via condensation of
oxabicyclo[2.2.2] octyl (OBO) orthoester[23] intermediates with 2
and subsequent oxidation (Figure 2C). The activated esters 3 can
be prepared simply and in high yield from the corresponding
carboxylic acids and inexpensive 3-methyl-3-oxetanemethanol.
We next developed a one-pot method for the synthesis of 3-
thiomethyltetrazines 3 from esters 1, which can be easily
prepared in high yield (>90%) and inexpensive 3-methyl-3-
oxetanemethanol from the corresponding acids by Steglich
esterification. After initial treatment of 1 in CH2Cl2 with BF3·OEt2
to form the OBO orthoester, pyridine and 2 were added. The
solvent was exchanged with DMF, and after heating at 80 °C, the
dihydrotetrazine solution was cooled and directly treated with
PIDA to provide tetrazines 3 in 50–79% yield (Scheme 1).
Aromatic and alkyl substituents with a variety of functional groups
were tolerated including Boc-amino (3b-c, 3n), nitro (3d) and
methoxy (3e) groups. The method also tolerates nitrile (3h) and
ester (3g, 3k-m) groups which are generally not compatible with
tetrazine synthesis. Protected amino acids (3o-p), biotin (3q),
BODIPY-FL (3w) and a number of heterocycles (3i-j, 3s-v) were
also successful and highlighted the ability to directly conjugate
biologically relevant compounds. While the ester of 2-fluoro
These compounds provide
disubstituted tetrazine synthesis and
monosubstituted tetrazines (Figure 2C).
a divergent platform for 3,6-
a new approach to
Results and Discussion
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10.1002/anie.202005569
Angewandte Chemie International Edition
RESEARCH ARTICLE
SMe
benzoic acid provides 3y in 50% yield, esters with bulkier ortho-
substituents (2-nitro or N-Boc-2-amino) were unsuccessful.
one pot
N
N
N
N
N
N
N
N
PdCl2, HSiEt3, THF, 45 °C
then PIDA, r.t.
one pot
N
N
SMe
O
R
4
R
3
N
BF3·OEt2, CH2Cl2
then pyridine, DMF, 80 °C
N
R
O
O
R
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Me
1
N
N
2 (1.0 equiv)
then PIDA, r.t.
3
N
SMe
NH
O
HN
O
R
Me
O
R
N
NHBoc
4a, 82%
COOMe
OMe
NHBoc
NHBoc
4c, 85%
4d, 73%
NO2
OMe
4b, 81%
NHBoc
OBn
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
3a, 72%
BocHN
CO2tBu
3bꢀꢁ56%
3c, 65%
3d, 67%
3e, 69%
NHBoc
COOMe
4h, 66%
4g, 79%
COOMe
Br
CN
4f, 75%
CN
4e, 68%
N
OMe
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
3j, 51%
3i, 61%
3g, 79%
3f, 54%
3h, 67%
N
CF3
4j 71%
N
COOMe
COOtBu
COOMe
NHBoc
4k, 75%
BocHN
CO2tBu
CF3
N
N
N
4i, 78%
N
3m, 60%
3n, 69%
3k, 68%
3l, 60%
BocHN
3x, 72%
N
N
O
Me
Me
F
F
Me
CO2tBu
N
NHBoc
B
N
NH
HN
N
N
H
H
F
H
CO2tBu
Me
H
H
S
4m, 61%
Me
H
3y, 50%
3o, 64%
3p, 70%
3q, 66%
15-fold fluorescence
turn-on with TCO
HO
4l, 70%
H
CF3
N
Me
Me
Scheme 2. Synthesis of 3-monosubstituted tetrazines. Conditions: 3 (1 equiv),
PdCl2 (10 mol %), HSiEt3 (3 equiv.), THF, 45 °C, 24 h; PIDA (1.2 equiv.), r.t., 1h.
H
N
O
H
Me
H
BocHN
H
H
OH
3r, 67%
Table S1), it was found that efficient reduction could be realized
with catalytic PdCl2 (10 mol %) and triethylsilane [27] (3 equiv) in
THF at 45 °C followed by treatment with PIDA to oxidize the
initially formed dihydrotetrazine. The method provides
monosubstituted tetrazines derived from aliphatic and aromatic
esters with a variety of functional groups, including Boc-amino
(4a–b, 4g), methoxy (4c), ester (4d, 4f) nitrile (4e), protected
amino acids (4h-i), and heterocycles (4j-k). Reduction of 3w gave
BODIPY-FL derivative (4m), which shows a 15-fold turn-on of
fluorescence upon reaction with eq-5-hydroxy-trans-cyclooctene
(5-hydroxyTCO).[6a-e] Pyridine derivatives (3i-j, 3t, 3v) gave low
conversions and attempted reduction of 3q was unsuccessful,
possibly due to overreduction of the biotin core.
3t, 55%
3s, 65%
Me
N
F3C
N
N
N
N
N
Boc
B
Me
F
F
3w, 57%
3u, 68%
3v, 64%
Scheme 1. One-pot synthesis of thiomethyltetrazines from oxetane esters.
Monosubstituted tetrazines are valued in bioorthogonal
chemistry for their rapid kinetics and minimal size.[26] We sought
monosubstituted
thiomethyltetrazine reduction (Scheme 2). After optimization (see
Thiomethyltetrazines 3 also served as electrophiles for Ag-
mediated Liebeskind-Srogl coupling reactions
bearing either aliphatic or aromatic groups (Scheme 3). Tolerated
functional groups include ether, chloride, ester, nitrile,
[21]
with thioethers
to
prepare
tetrazines
through
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10.1002/anie.202005569
Angewandte Chemie International Edition
RESEARCH ARTICLE
trifluoromethyl, alcohol, Boc-amino, and protected amino acids as
well as pyridyl and triazole groups. Additionally, the 3-furyl group
was readily introduced to a range of thiomethyltetrazines via Ag-
mediated Liebeskind-Srogl coupling. In line with our previous
observations, electron rich arylboronic acids were the most
efficient nucleophiles.
3,6-Disubstituted aromatic tetrazines have been used broadly
for bioorthogonal chemistry due to their combination of stability
and rapid kinetics but can be limited by their hydrophobicity. We
anticipated that furyl or vinylether groups introduced through
cross-coupling could serve as small and solubilizing alternatives
to phenyl groups. Shown in Scheme 4A are the experimental logP
values for three tetrazine analogs and their rate constants in
reactions with 5-hydroxyTCO (Figure S2A-C). Furyl analog 5l
(logP 1.2) is considerably more hydrophilic albeit 2.9 times less
reactive than phenyl derivative 5b (logP 2.2). Vinylether 6c is the
most reactive and hydrophilic member of the series with logP 0.89
and reactivity that is 4.2 times faster than 5b. Vinylether-
substituted tetrazines represent a new class of dienophile with a
favorable combination of kinetics, small size and hydrophilicity.
Tetrazines 5b, 5l and 6c all display >96% stability after 24 h
incubation in PBS at 25 °C (Figure S1A). To introduce the 2-
ethoxyvinyl group, we employed commercially available stannane
SMe
Ar
Pd(dppf)Cl2 (15 mol %)
Ag2O (2.5 equiv)
N
N
N
N
N
N
N
N
+ ArB(OH)2
DMF
5
R
2
R
NHBoc
OH
OMe
Cl
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
A
OEt
N
O
N
N
N
N
N
N
CO2Me
N
N
NHBoc
N
N
N
5b, 84%c
5a, 78%a
CO2tBu
5e, 78%a
N
CO2Me
CO2Me
CO2Me
CO2Me
5d 81%a
5b
5l
6c
F3C
CO2Me
5c 59%a
CF3
1.0
2.2
0.35
1.2
4.2
krel with TCO
N
O
LogP
0.89
N
N
N
COOMe
CN
B
OEt
OEt
7
SMe
N
N
N
N
Bu3Sn
N
N
N
N
N
N
N
N
N
N
N
Pd(PPh3)4 (15 mol %)
CuTc (2 equiv), dioxane, 100 °C
N
N
N
N
N
N
N
N
N
N
N
N
N
R
R
2
6
15–20 min
BocHN
CO2tBu
CO2tBu
5j 56%c
NHBoc
5h, 69%c
COOMe
5i, 61%c
5g, 65%b
CO2tBu
5f, 60%a
N
N
OEt
N
OEt
N
N
Me
N
O
N
O
Me
H
O
O
N
Me
H
N
N
N
N
N
N
H
H
N
N
N
N
N
N
N
N
N
HO
6b: 60%
N
H
6a, 74%
OEt
BocHN
BocN
OEt
5n, 62%d
CO2Me
BocHN
CO2tBu
OEt
N
N
N
N
5l, 61%d
5m, 56%d
N
N
5k, 60%d
N
N
N
O
N
N
N
O
N
N
N
N
F
F
B
N
N
N
O
N
N
Me
HN
N
N
N
H
N
F3C
NH
H
CO2Me
5p, 48%d
BocHN
N
5o, 47%d
S
6c, 52%
6e, 47%
6d, 57%
Me
Scheme 3. Synthesis of unsymmetrical tetrazines via Ag-mediated Pd-
catalyzed Liebeskind-Srogl coupling. [a] ArB(OH)2 (1.9 equiv.), 60 °C, 20 h. [b]
ArB(OH)2 (3 equiv.), 60 °C, 20 h. [c] ArB(OH)2 (3 equiv.), microwave 100 °C, 3
h. [d] Pd(dppf)Cl2 (30 mol%), Ag2O (5 equiv.), ArB(OH)2 (6 equiv.), microwave
100 °C, 3 h.
Scheme 4. Furyl- and vinylether-subsituted tetrazines with favorable kinetics,
small size and hydrophilicity. (A) Relative reactivity and logP value. (B)
Synthesis via CuTc-mediated Liebeskind-Srogl coupling.
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10.1002/anie.202005569
Angewandte Chemie International Edition
RESEARCH ARTICLE
7. Here, coupling reactions were most efficient under
conventional CuTc-promoted conditions with a brief reaction time
(see Table S2). As shown in Scheme 4B, these cross-coupling
procedures can tolerate a broad range of functional groups and
heterocycles of biological interest.
This divergent approach to synthesizing tetrazine-coupled
probes was applied to the study of MAGL, a serine hydrolase
involved in endocannabinoid signaling.[28] Our previous approach
to studying this drug target was limited by the need for a scaffold
with a relatively uncommon boronic acid group, thus restricting
the types of drug candidates that could be studied.[21] We
hypothesized that a divergent approach could be used to tune
reaction kinetics and/or inhibitory affinity of a new class of MAGL-
tetrazine probes. As shown in Scheme 5, probes 10a-c were
O
O
O
S
S
O
N
H
O
N
N
O
O
CF3
CF3
Me
72%
O
N
O
O
8
O
9
N
H
N
N
CF3
CF3
N
MeS
designed based on
a
4-(arylsulfonamidomethyl)piperidine
67%
54%
74%
scaffold bearing an electrophilic hexafluoroisopropyl (HFIP)
carbamate warhead.[29] Thus, reaction of 2 with the (3-methyl-
O
EtO
H
N
N
N
oxetane-3-yl) methyl ester
8
gave 72% yield of
N
N
N
N
thiomethyltetrazine 9, which served as common intermediate for
the synthesis of furyl-substituted 10a, vinylether-substituted 10b,
and monosubstituted tetrazine 10c. Stopped flow kinetics with 5-
hydroxyTCO showed 10c was 6.5-times more reactive than 10b,
which in turn was 8.6-fold more reactive than 10a. All of these
probes were 3–162 times more reactive than a previously
described MAGL probe based on a different drug scaffold (12[21]
Figure S4).
In an in vitro assay,[30] probes 10a-c inhibited MAGL activity
with IC50’s of 12 nM (10a), 46 nM (10b) and 16 nM (10c). The
most reactive probe 10c was further investigated for labeling of
endogenous MAGL in live cells. Human brain vascular pericytes
were treated with probe 10c for 1 h, followed by labeling with 2
µM TCO-TAMRA for 30 min. After cell lysis, gel-based activity-
based protein profiling[31] (ABPP, Figure 3A) showed strong
labeling of MAGL. The labeling was dose responsive (Figure 3B),
and treating cells with 10c (1 µM, 1 h) followed by incubation with
200 nM TCO-TAMRA gave complete labeling with t1/2 of 6.1 min
N
N
N
N
N
O
O
O
O
O
S
S
S
O
NH
NH
NH
N
O
N
O
N
O
O
CF3
O
CF3
O
CF3
10a
F3C
F3C
10b
F3C
10c
krel with
TCOa
1.0
8.6
56
in vitro
12 nM
)
46 nM
16 nM
potency (IC50
Scheme 5. Divergent synthesis of tetrazine-coupled MAGL-probes 10a-c from
common ester starting material and thiomethyltetrazine intermediate.
N
N
A
MAGL
N
N
N
N
N
10c
N
O
OH
F3C
N
N
O
H
O
F3C
TCO-TAMRA
Live Cells
C
kinetics
Cellular potency
2 µM TCO-TAMRA
100
75
50
25
0
100
75
50
25
0
B
-
-
10c
nM
kDa
-
1 µM 10c
200 nM TCO-TAMRA
37
MAGL
no -10
probe
-9
-8
-7
-6
-5
25
0
10
20
30
40
50
60
log [10c], M
log[probe], M
time (min)
Figure 3. (A) Live cells were treated with probe 10c for 1 h, followed by TCO-TAMRA treatment for 30 min, cell lysis, and analysis by gel-based ABPP.
(B) In-gel fluorescence and dose response fitting of probe 10c. (C) Cellular labeling kinetics of 10c
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(Figure 3C). Similar kinetics were observed when the
concentration of TCO-TAMRA was dropped to 50 nM (Figure S7),
and with a higher concentration of TCO-TAMRA (2 µM), labeling
was complete when the first data point was collected after 2 min.
By contrast, our previous 6-methyltetrazin-3-yl probe for MAGL
displayed slower labeling kinetics (t1/2 of 13 min) with 2 µM TCO-
TAMRA (Figure S8).[21] Together, these experiments illustrate
how modifying the structure of tetrazine-coupled probes can be
used to tune the IC50 and labeling kinetics.
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In conclusion, a one-pot method is described for the conversion
of (3-methyloxetan-3-yl)methyl carboxylic esters into
3
thiomethyltetrazines that can subsequently serve as a platform for
divergent tetrazine synthesis via Pd-catalyzed cross-coupling and
the first example of monosubstituted tetrazine synthesis via
catalytic thioether reduction. The utility of the method was
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Acknowledgements
We would like to thank Jeffery Sperry for helpful discussions,
Remzi Duzguner for DSC analysis, Edelweiss Evrard for
outsourcing support and Lucy Stevens for the in vitro potency data
on probes 10a-c. This work was supported by NIH GM132460
and Pfizer. Instrumentation was supported by NIH awards
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P20GM104316,
P30GM110758,
S10RR026962,
and
S10OD016267 and NSF awards CHE-0840401, CHE-1229234,
and CHE-1048367.
Keywords: tetrazine • bioorthogonal chemistry • minimal probe
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Entry for the Table of Contents
Divergent
platform
Ester-to-
tetrazine
in one pot
N
N
R
R
H
O
SMe
N
N
N
N
N
R
Ar
N
N
N
N
Me
N
O
N
N
N
N
O
R
R
OEt
Divergent Tetrazine Synthesis: Described is a new strategy for the one-pot synthesis of 3-thiomethyltetrazines from carboxylic
ester precursors, providing a platform for the synthesis of unsymmetrical tetrazines via Pd-catalyzed cross-coupling and the first catalytic
thioether reduction to access monosubstituted tetrazines.
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