Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups

Article abstract of DOI:10.1039/c9sc04649f

In vivo compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions.

Full text of DOI:10.1039/c9sc04649f

Chemical
Science
View Article Online
View Journal | View Issue
EDGE ARTICLE
Isonitrile-responsive and bioorthogonally
removable tetrazine protecting groups†
Cite this: Chem. Sci., 2020, 11, 169
a
b
c
b
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Julian Tu,
Dennis Svatunek,
Saba Parvez,
Hannah J. Eckvahl,
Minghao Xu, ‡a Randall T. Peterson,c K. N. Houk
and Raphael M. Franzini
b
a
*
In vivo compatible reactions have a broad range of possible applications in chemical biology and the
pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under
very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and
phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is
especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination
under physiological conditions. NMR and computational studies revealed that an imine-tautomerization
step is often rate limiting, and the unexpected cleavage of the Si–C bond accelerates this step in the
case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on
biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity
experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining
tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was
possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry
will open new opportunities towards applications involving multiplexed release schemes and is a valuable
asset to the growing toolbox of bioorthogonal dissociative reactions.
Received 14th September 2019
Accepted 5th November 2019
DOI: 10.1039/c9sc04649f
rsc.li/chemical-science
could be used for the concomitant delivery of synergistic drugs,
in theranostic applications, and in multiplexed detection
Introduction
schemes.
Bioorthogonal chemistry, both ligating and dissociative,
Performing chemistry in living organisms with bioorthogonal
reactions makes it possible to study biological processes in their
natural environments.1,2 Recently, reactions have emerged that
release diverse molecules under physiological conditions.3,4
These reactions have opened unprecedented possibilities in
chemical biology and drug delivery.5,6 Dissociative bio-
orthogonal chemistry has been applied to the on-demand
dissolution of polymers and micelles,7–9 site-specic actuation
of prodrugs,10–12 and control of enzyme activity in vivo.13–15
Although a growing number of “click-to-release” reactions16–23
has provided a solid foundation for applications in the life
sciences, extending the reaction scope will be necessary to
access the full range of capabilities. Moreover, there is a need
for chemistry to allow for the controlled and simultaneous
release of more than one molecule.24 Dual-release reactions
mainly revolves around pericyclic reactions.25,26 In particular,
inverse-electron demand cycloadditions offer rapid reaction
kinetics and high biocompatibility.27,28 1,2,4,5-Tetrazines are the
most prevalent dienes in such reactions.29,30 These heterocycles
react with and subsequently trigger the release of payloads from
allyl-modied trans-cyclooctenes,17,31–33 benzonorbornadiene
derivatives,21,34 and vinyl ethers.7,19,20 Tetrazines also undergo
bioorthogonal cycloaddition reactions with isonitriles,35,36 and we
have recently shown that they can induce the release of payloads
from 3-isocyanopropyl (ICPr) groups (Fig. 1).37,38
Given the prominent role and favorable properties of
tetrazine-based cycloadditions in dissociative bioorthogonal
chemistry, it would be valuable to have tetrazine-based pro-
tecting groups that release a payload upon reaction with some
of these dienophiles. An example of such a molecule was dis-
closed by Wang et al. as demonstrated by a tetrazine-based
prodrug that was activated through a reaction with a cyclo-
octyne modied with a hydroxyl group at the propargylic posi-
tion.23 Such tetrazine derivatives, when combined with
complementary release reagents, could be used for dual-release
applications. Running two bioorthogonal release reactions in
parallel is one possibility to achieve such dual-release as has
been demonstrated by combining the reaction of tetrazines and
aDepartment of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt
Lake City, 84112, USA. E-mail: Raphael.franzini@utah.edu
bDepartment of Chemistry and Biochemistry, University of California, Los Angeles,
California 90095, USA
cDepartment of Pharmacology and Toxicology, College of Pharmacy, University of
Utah, Salt Lake City, 84112, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9sc04649f
‡ Current address: School of Chemistry and Biochemistry, Georgia Institute of
Technology, Atlanta, Georgia 30332, United States
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 169–179 | 169

View Article Online
Chemical Science
Edge Article
and c) consisting of p-nitroaniline (pNA) caged by a Tzmoc
group (Fig. 2a). pNA has a characteristic maximum absorbance
signal at labs ¼ 385 nm, which is hypsochromically shied in
derivatives with acyl-modied amine. 4a was accessed by
a dibutyltin dilaureate-catalyzed reaction of (6-(tert-butyl)-
1,2,4,5-tetrazin-3-yl)methanol (2) with 4-nitrophenyl isocyanate
(Fig. 2b). 2 was prepared in three steps from the nitrile
precursors to obtain 1, followed by deprotection of the methoxy
group by BBr3 (Fig. 2b).
We evaluated the liberation of pNA from 4a upon reaction
with several isonitriles (Fig. 2d). As designed, a primary iso-
nitrile, n-butyl isocyanide (n-BuNC, Fig. 2c), reacted with the
tetrazine and elicited the release of pNA as monitored by the
emergence of the pNA absorbance signal (Fig. 2d). As a control,
we performed the experiment with tert-octyl isocyanide (t-OcNC,
Fig. 2c), which we expected not to release pNA because tertiary
isonitriles form stable 4H-pyrazol-4-imine conjugates.36,38,42
Indeed, pNA-release was undetectable in experiments with t-
OcNC conrming that TzMe-removal follows the designed
release principles. We were interested whether electron-
donating groups adjacent to the isocyano functionality would
accelerate the inverse-electron demand cycloaddition step. We
therefore tested the reaction of 4a with (trimethylsilyl)methyl
isocyanide (TMS-MeNC, Fig. 2c). As predicted, TMS-MeNC
Fig. 1 Proposed reaction to achieve the dual release of biological
effectors from previously reported 3-isocyanopropyl (ICPr) and tet-
razylmethyl (TzMe) derivatives developed herein.
reacted ꢀ3-fold faster with 4a (k2 ¼ 0.344 ꢁ 0.013 Mꢂ1 sꢂ1
)
than did n-BuNC (k2 ¼ 0.117 ꢁ 0.001 Mꢂ1 sꢂ1), and an isonitrile
with an electron-withdrawing substituent (methyl iso-
cyanoacetate) lead to a 2-fold decrease (k2 ¼ 0.05 ꢁ 0.01 Mꢂ1
sꢂ1) in the cycloaddition rate (Table S1†) but still released pNA
(data not shown). Unexpectedly however, the TMS-substituent
also greatly accelerated the release step (Fig. 2d). The rate of
pNA release was ꢀ30-fold faster for TMS-MeNC (k1 ¼ 3.4 ꢃ 10ꢂ4
ꢁ 1.1 ꢃ 10ꢂ6 sꢂ1) than for n-BuNC (k1 ¼ 1.1 ꢃ 10ꢂ5 ꢁ 1.2 ꢃ 10ꢂ7
sꢂ1). Reactions with n-BuNC led to gradual, continuous, elimi-
nation of pNA with a release yield of 35.4 ꢁ 1.0% measured at
the 8 hour time-point in contrast to reactions with TMS-MeNC
leading to near-quantitative release yields in this period as
quantied by the absorbance signal (Fig. 3c). The bimolecular
reaction rates of tetrazines and isonitriles were in the range of
those observed in previous studies (Table S1†).37,38 Under these
conditions, (DMSO : PBS pH 7.4, 4 : 1, v/v at T ¼ 37 ꢄC) the rate
constants of the reactions with 4a ranged from k2 ¼ 0.05–0.38
Mꢂ1 sꢂ1. The water content strongly inuences the kinetics of
the cycloaddition step, and based on previous studies,34,38 we
extrapolate the reaction to be about 10-fold faster in purely
aqueous solutions. These initial results indicate that the TMS-
group promotes pNA release as the faster bimolecular rate of
TMS-MeNC compared to n-BuNC is insufficient to explain the
rapid elimination of pNA for the former.
benzonorbornadienes with that between sulfonyl sydnonimines
and dibenzoazacyclooctyne.39 A second example involved the
reaction between vinyl ethers and tetrazines, which released
alcohols but was limited to generating pyridazine and had slow
reaction kinetics.24 A single pair of reactants that releases two
molecules in a single fast reaction would bring such approaches
to the next level.
Here we describe tetrazylmethyl (TzMe) protecting groups that
can be rapidly removed by a reaction with isonitriles. The ratio-
nale behind our design is based on the precedent that isonitriles
convert tetrazines into 4-aminopyrazoles35,36 and that 5-
membered heterocycles with amine substituents spontaneously
eliminate diverse functional groups.40,41 In a series of experi-
ments, we demonstrated that TzMe-modied molecules reacted
readily with isonitriles to release amines (from tetrazylmethy-
loxycarbonyl (Tzmoc) derivatives) and phenols (Fig. 2a). We
analyzed the reaction mechanism, and in the case of (trime-
thylsilyl)methyl isocyanide (TMS-MeNC), observed an intriguing
C–Si bond cleavage that accelerated release. The reaction was
compatible with living systems, and we demonstrated that when
TzMe-derivatives were combined with ICPr-derivatives (Fig. 1),37
two uorophores could be simultaneously released in zebrash
embryos. This innovative chemistry will open new possibilities
for biomedical research and drug delivery.
Although TMS-MeNC effectively elicits the release of amines
from Tzmoc groups, there are applications for which the rapid
release by simple alkyl isocyanides will be preferred. For
example, TzMe-molecules could be combined with ICPr deriv-
atives37 in dual-release strategies (Fig. 1). Serum albumins
catalyze diverse chemical transformations,43–45 and we hypoth-
Results and discussion
Isonitrile-induced removal of Tzmoc-groups from amines
To prove the concept of isonitrile-induced deprotection of TzMe esized that albumin might also accelerate the release step.
groups, we synthesized a colorimetric reporter probe (4a, Fig. 2b Indeed, both human serum albumin (2 mg mLꢂ1 HSA in 4 : 1,
170 | Chem. Sci., 2020, 11, 169–179
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
Fig. 2 Isonitrile-mediated uncaging of amines and phenols from Tzmoc and TzMe derivatives. (a) Structures of tetrazylmethyloxycarbonyl
(Tzmoc) and tetrazylmethyl (TzMe) groups used to cage amines and phenols, respectively. (b) Synthesis of Tzmoc or TzMe-caged probes
(conditions and yields described in the ESI†). (c) Structures of reporter probes and isonitrile triggers used in this study. (d) Kinetics of pNA release
from 4a triggered by different isonitriles (c(4a) ¼ 0.2 mM, c(R–NC) ¼ 2 mM, DMSO : PBS pH 7.4 (4 : 1, v/v), T ¼ 37 ꢄC, l ¼ 435 nm, n ¼ 3). (e)
Kinetics of pNA release from 4a triggered by n-BuNC catalysed by serum albumin (c(4a) ¼ 8 mM, c(n-BuNC) ¼ 6 mM, c(HSA) ¼ 2 mg mLꢂ1
,
DMSO : PBS pH 7.4 (1 : 4, v/v), T ¼ 37 ꢄC, l ¼ 385 nm, n ¼ 3). (f) Kinetics of O-carboxymethyl fluorescein release from 4b triggered by TMS-MeNC
or n-BuNC (c(4b) ¼ 8 mM, c(R–NC) ¼ 6 mM, DMSO : PBS pH 7.4 (1 : 4, v/v), T ¼ 37 ꢄC, lex ¼ 488 nm, lem ¼ 520 nm, n ¼ 3).
PBS pH 7.4 : DMSO at T ¼ 37 ꢄC) and bovine serum albumin at uorescein (4b, Fig. 2c) and 7-hydroxycoumarin (4b0, Fig. S2†)
2 mg mLꢂ1 (data not shown) greatly accelerated the liberation of that report on TzMe-removal by
a uorescence turn-on
pNA in reactions with n-BuNC (Fig. 2e). n-BuNC was able to signal.46,47 The uorogenic probes were synthesized by ether-
effectively elicit the near-quantitative release of pNA in a little ication of the phenolic dyes with 3-(bromomethyl)-6-(tert-
over an hour while the same reaction without HSA led to less butyl)-1,2,4,5-tetrazine (3), which can be accessed by bromina-
than 20% release during the same timeframe. In contrast, 4a tion (PBr3; 88%) of 2 (Fig. 2b). The reaction of TMS-MeNC with
incubated alone in a solution of HSA (2 mg mLꢂ1 in PBS, T ¼ 37 4b (Fig. 2f) and the 7-hydroxycoumarin derivative 4b0 (Fig. S3†)
ꢄC) did not result in a detectable pNA release signal (data not led to near-quantitative release yields by 2 h as quantied by
shown), indicating that HSA catalyzes the elimination step uorescence emission and HPLC analysis (Fig. S4†). TzMe-
whereas the Tzmoc-probe is stable in the absence of isonitrile. removal was associated with a characteristic uorescence
To differentiate between a catalytic activity of the protein and increase (152-fold for 4b0 and 30-fold for 4b; Fig. S5†). The
simple base-catalysis by its surface amines, we tested the effect kinetics of the release reaction of 4b with TMS-MeNC was
of tris-base (concentration equal to that of surface amines in determined by measuring the uorescence turn-on signal (k1 ¼
HSA experiments; 2 mM) on the release rate of pNA. The base 3.5 ꢃ 10ꢂ3 ꢁ 1.7 ꢃ 10ꢂ4 sꢂ1). The faster release rate compared
had no detectable effect on the isonitrile-induced release of pNA to the release of 4a may in part be explained by the higher water
from 4a (Fig. S1†). It is therefore possible to achieve rapid and content. Surprisingly, the rate of 7-hydroxycoumarin (Fig. S3†)
high-yielding uncaging of amines from stable Tzmoc precursors and O-carboxymethyl uorescein (Fig. 2f) elimination by n-
with simple alkyl isocyanides in serum.
BuNC was signicantly faster than for 4a without the need for
the addition of albumin, an effect that was assessed by NMR
studies. We further evaluated the stability of TzMe-caged
probes. First, we assessed a TzMe-caged 7-hydroxycoumarin
dye (4b0, Fig. S2†) in a human liver microsome stability assay
(Creative Bioarray, USA) and the probe exhibited good stability
(t1/2 ¼ 52.11 min; Clint ¼ 33.36 mL minꢂ1 kgꢂ1; Fig. S6†) even
under these harsh conditions. Second, the TzMe-derivative of
uorescein (4b) used for zebrash studies was stable in serum
Isonitrile-induced removal of TzMe-groups from phenols
Having demonstrated the release of carbamates from Tzmoc-
groups, we aimed to determine whether the chemistry would
be applicable to other functional groups. We were especially
interested in phenols because aromatic hydroxy groups are
present in tyrosine, diverse drugs, and uorophores. For these
experiments, we synthesized a TzMe-caged O-carboxymethyl
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 169–179 | 171

View Article Online
Chemical Science
Edge Article
Next, replacing the C-6 tert-butyl group of 4a by a phenyl
substituent (4e, Fig. 3a) led to a marginally faster release of pNA
triggered by n-BuNC (Fig. S9†). However, the phenyl group
decreased the rate of TMS-MeNC triggered release (Fig. 3b). This
effect may in part be because of a slightly slowed bimolecular
reaction rate (Table S1†), which agrees with the lack of disper-
sion forces between the tert-butyl group and the incoming iso-
cyano group.38 These results show that the substituents on both
the tetrazyl ring and the methylene position are important to
achieve prompt and high-yielding release. These insights
provide guidance for further improvement of probe perfor-
mance in future studies.
TzMe removal by alternative dienophiles
Inspired by the effective release of payloads from TzMe-
derivatives by isonitriles, it was of interest to nd out whether
other dienophiles provide a similar outcome. Tetrazines react
with diverse strained alkenes,29,30 and we tested whether such
dienophiles (methylcyclopropene (Cp), norbornene (Nb), and
trans-cyclooctene (TCO); see Fig. S10† for structures) induce the
release of pNA from 4a. Elimination of pNA occurred; ꢄhowever,
the yields of amine release were modest (t ¼ 8 h, 37 C; Cp ¼
32.4 ꢁ 0.2%; Nb ¼ 31.0 ꢁ 0.3%; TCO ¼ 20.2 ꢁ 4.0%; Fig. 3c).
Addition of HSA, which catalyzed pNA release from 4a (Fig. 2e)
in the reaction with n-BuNC, had an insignicant effect on the
TCO-mediated reaction (data not shown). We further tested the
ability of Cp, Nb, and TCO to elicit the release of O-carbox-
ymethyl uorescein from 4b. Analogous to the results for pNA
release, only a fraction of the product was eliminated (t ¼ 8 h,
37 ꢄC; Cp ¼ 22.1 ꢁ 0.3%; Nb ¼ 12.4 ꢁ 0.5%; TCO ¼ 8.3 ꢁ 1.0%;
Fig. 3c). These experiments demonstrate that isonitriles have
a unique ability to remove TzMe-based protecting groups. In the
case of TCO, the rapid bimolecular reaction with tetrazines may
open opportunities for interesting applications in drug delivery
where the rate of the release step may not be limiting.
Fig. 3 Effect of structural modifications to tetrazines on isonitrile-
induced removal. (a) Structures of modified Tzmoc probes with a pNA
reporter molecule. (b) Half-lives of the TMS-MeNC mediated Tzmoc
deprotection (t1/2 of release of pNA) from probes 4a, 4c, 4d, and 4e
(c(4a–e) ¼ 0.2 mM, c(TMS-MeNC) ¼ 2 mM, DMSO : PBS pH 7.4 (4 : 1,
v/v), T ¼ 37 ꢄC). (c) Release yields of pNA or O-carboxymethyl fluo-
rescein from 4a or 4b, respectively, triggered by several dienophiles
(structures shown in Fig. S10 in the ESI†); pNA release: see Fig. 2d; t ¼
8 h; O-carboxymethyl fluorescein release: c(4b) ¼ 8 mM, c(dienophile)
¼ 2 mM, DMSO : PBS pH 7.4 (1 : 4, v/v), T ¼ 37 ꢄC, lex ¼ 488 nm, lem
520 nm, t ¼ 8 h.
¼
for hours (t1/2 ¼ 19 ꢁ 4 h). The decomposition product was not
the released uorophore and therefore the contribution to
uorescence background is low (Fig. S7†). These experiments
establish that TzMe-groups are removed rapidly and in high
yields from key functional groups.
Studies on the mechanism of TzMe removal
Effect of structural modications on isonitrile-induced TzMe
deprotection
Having established that isonitriles remove TzMe-moieties
from phenols and amines (Fig. 2), we aimed to gain a mecha-
We were interested to determine if the reaction kinetics and nistic understanding of the reaction. Several reaction path-
release yields from isonitrile-induced release from TzMe-groups ways are conceivable. Carbamate release could in principle
could be enhanced by modifying the structure of the tetrazine. occur by heterolytic cleavage of the benzylic C–O bond or by
We designed a series of tetrazyl-derivatives of pNA (Fig. 3a) and a cyclization step involving the attack of a nucleophilic inter-
analyzed such parameters upon reaction with isonitriles n- mediate on the carbonyl (Fig. S11†). To elucidate the reaction
BuNC and TMS-MeNC. Methyl and phenyl groups at the pathway, we performed a time-dependent NMR experiment
methylene position (R0 in Fig. 3a) modestly accelerated the between 4a and n-BuNC (DMSO-d6 : D2O (9 : 1, v/v) at T ¼
release of pNA upon reaction with TMS-MeNC (Fig. 3b) with 25 ꢄC; Fig. 4a–c and S12, S13†). At a lower aqueous content and
minor impact on the bimolecular kinetics (Table S1†). A methyl T ¼ 25 ꢄC, as opposed to the higher water content and T ¼
ꢄ
substituted tetrazine (4c, Fig. 3a) released pNA with a half-life of 37 C we preformed kinetics studies with previously (Fig. 2d),
24 min and a phenyl-substituted derivative (4d, Fig. 3a) with we expected slower reaction kinetics to allow for rigorous
a half-life of 19 min, both near-quantitatively (Fig. 3b). examination of the intermediates formed along the reaction
Intriguingly, modications drastically decreased the ability of n- pathway. As determined by 1H NMR, the formation of one
BuNC to trigger the release of pNA; aer 8 h only 20.2 ꢁ 0.8% equivalent of the 4H-pyrazole intermediate (I1) paralleled the
and 6.9 ꢁ 0.4% of the pNA was deprotected from 4c and 4d, disappearance of 4a in the reaction with n-BuNC (Fig. 4b and
respectively (Fig. S8†). The effect on the bimolecular reaction c). I1 subsequently tautomerized to the 1H-pyrazole interme-
rates did not cause the modest release yields of pNA (Table S1†). diate (I2) that gradually released pNA (Fig. 4b and c). The
172 | Chem. Sci., 2020, 11, 169–179
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
Fig. 4 1H-NMR analysis of reactions between isonitriles n-BuNC or TMS-MeNC and 4a to liberate pNA. (a and d) Proposed intermediates in the
reaction between n-BuNC (a) or TMS-MeNC (d) and 4a leading to the release of pNA. (b and e) Time-dependent 1H-NMR of the reaction
progress between n-BuNC (b) or TMS-MeNC (e) and 4a (c(4a) ¼ 6 mM, c(R–NC) ¼ 15 mM, DMSO-d6 : D2O (9 : 1, v/v), T ¼ 25 ꢄC, expanded
spectra in ESI). (c and f) Normalized amount of starting material (4a), subsequent intermediates, and pNA formed as a function of time.
triplet peak of I2 centered at 7.78 ppm with a normalized n-BuNC portion providing additional support for the struc-
integration value corresponding to one proton is characteristic tural assignment of I2. The observed reaction cascade
for the N]CH–CH2 proton present in the postulated structure mirrored the predicted mechanism (Fig. 1).35,36 Interestingly,
of I2. In reactions between n-BuNC and di-methyl-tetrazine the 1H NMR signals of pNA (d, 2H, 6.60 ppm; d, 2H, 7.94 ppm)
(Fig. S14†) or di-tert-butyl-tetrazine (Fig. S16†), the same emerged before those of the aldehyde (s, 1H, 9.64 ppm). It
characteristic triplet peak at ꢀ7.8 ppm was present (Fig. S15 therefore appears that the elimination step can occur from the
and S17†), which indicated that the signal originated from the imine intermediate I2.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 169–179 | 173

View Article Online
Chemical Science
Edge Article
We proceeded to study the reaction between TMS-MeNC and (Fig. S24†) and according to gHSQC analysis, were bonded to
4a by NMR (DMSO-d6 : D2O (9 : 1, v/v), T ¼ 25 ꢄC; Fig. 4d–f and the same carbon with a chemical shi of 159.7 ppm (Fig. S25
S18, S19†). TMS-MeNC was completely stable for >7 days under and S26†). Furthermore, these protons showed a multi-bond
the experimental conditions (data not shown) ruling out the correlation with one of the aromatic ring carbons in gHMBC
possibility that a decomposition product caused the fast release. (130.9 ppm; Fig. S27†). The spectroscopic data is consistent
Time-dependent 1H NMR spectra revealed a single intermediate with the formation of a methanimine intermediate, which
(I10; Fig. 4e). I10 exhibited a strong coupling peak pattern centered would indicate cleavage of the C–Si bond (Fig. 4d). In agree-
at 7.45 ppm with a normalized integration value corresponding ment, trimethylsilanol was detected in the 1H NMR spectrum (s,
to two protons that was absent from I1 and I2 (Fig. 4b). Repeating 1H, 5.28 ppm; s, 9H, 0.01 ppm; Fig. S23†). The peak corre-
the experiment in DMSO-d6, without the addition of the 10% sponding to the trimethylsilyl protons in the reaction of 4a with
D2O, led to a peak at 5.28 ppm corresponding to one proton, TMS-MeNC remained unaffected as the reaction proceeded to
which could not be assigned to the pyrazole species (Fig. S20 and generate several unidentied side products, further corrobo-
S21†). To further examine the transformation, we performed the rating the formation of trimethylsilanol (Fig. S19†).
reaction between TMS-MeNC and di-tert-butyl-tetrazine in
Cleavage of the C–Si bond under these conditions is
DMSO-d6 at T ¼ 25 ꢄC (Fig. S22–S27†). This reaction provided an surprising as documented cases required harsher conditions.48
adduct with the same strong coupling pattern with a normalized We analyzed this reaction step using density functional theory
integration value corresponding to two protons and this species (DFT) calculations. The analysis was conducted in Gaussian 09
persisted for days in DMSO-d6, making it possible to thoroughly using M06-2X-D3/def2TZV49–51 in water (SMD).52 3,6-Di-methyl-
analyze its structure by various NMR experiments.
1,2,4,5-tetrazine derived intermediates A1 and B1 were used as
The strongly coupled protons that centered at 7.60 ppm in model substances (Fig. 5) with water as the initial nucleophile
1
the H spectrum (Fig. S23†) correlated in the gCOSY spectrum or proton source in all pathways to reect the neutral
Fig. 5 Investigated mechanistic pathways. (a) Cleavage of TMS from A1 by water with subsequent protonation. (b) Tautomerization of B1 induced
by abstraction of a proton by water. (c) Cleavage of TMS from A1 by protonation followed by abstraction by water. (d) Tautomerization of B1
induced by protonation. (e) Predicted SN2 transition state TSA1>A2. (f) Cleavage of TMS from TMS-MeNC by water.
174 | Chem. Sci., 2020, 11, 169–179
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
experimental conditions. The SN2 reaction between A1 and
water was identied as the minimum energy pathway for the
formation of imine A3 going through a highly stabilized anion
A2 making this structure an excellent leaving group, allowing
for low barriers even with weak nucleophiles such as water
(Fig. 5a and e). The barrier was calculated to be 16.4 kcal molꢂ1
and this pathway is therefore in accordance with the fast reac-
tion observed experimentally (Fig. 2d). In contrast, the depro-
tonation of B1 by water to initiate the tautomerization had
a calculated barrier of 29.8 kcal molꢂ1 with the resulting
intermediate B2 being 15 kcal molꢂ1 higher in energy than the
reactants (Fig. 5b). While the omission of tunneling effects may
overestimate barriers for proton transfers calculated with
a classical treatment of the nucleus, it is plausible to assume
that the barrier is above the 16.4 kcal molꢂ1 calculated for the
A1 > A2 transformation, given that the intermediate B2 is
already at +15.2 kcal molꢂ1. This computational prediction
agrees with the experimental observation that the tautomeri-
zation in case of reactions with n-BuNC proceed signicantly
slower than the cleavage of TMS (Fig. 4). Analogous pathways
involving OHꢂ instead of water showed the same trend with
overall lower barriers (Fig. S30†).
Alternative pathways that involve protonation of A1 and B1
followed by transfer of TMS+ to water, or deprotonation,
respectively, were also explored (Fig. 5c and d). Protonation of
A1 or B1 was disfavored by 23.9 and 27.3 kcal molꢂ1, respec-
tively. The barriers for the following abstraction of TMS+ or H+
are lowered considerably compared to the pathways described
above. However, this pathway also favors removal of TMS+ from
protonated A1 over proton abstraction from protonated B1 in
accordance to experimental results.
In addition, stability of TMS-MeNC against nucleophilic
attack of water was investigated computationally. While the
transition state structure could not be located, the trans-
formation is disfavored by over 51.0 kcal molꢂ1 because of the
inability of the adjacent isonitrile group to stabilize a carbanion
at the a carbon, leading to poor leaving group qualities. The
geometry of the anion shows a tetrahedral center at the a methyl
group, consistent with isolation of the negative charge on this
center without any stabilization by the adjacent p-systems
(Fig. 5f). The high barrier and thermodynamically disfavored
nature of this transition corroborate the observed high stability
of TMS-MeNC in aqueous solution.
We further examined the mechanistic steps of phenol release
triggered by n-BuNC. The photospectrometric studies had
revealed a puzzling discrepancy in the rate of carbamate versus
phenol elimination induced by n-BuNC (Fig. 2). To obtain
mechanistic insight into this discrepancy, we analyzed the
reaction of 4b0 and n-BuNC (DMSO-d6 : D2O (9 : 1), T ¼ 25 ꢄC) by
1H NMR (Fig. S28 and S29†). In this experiment, the formation
of the corresponding 4H-pyrazole species, which was noticeable
in the reaction between n-BuNC and 4a (I1; Fig. 4a), was
unobservable. The tautomerization step to the aromatic 1H-
pyrazole following the bimolecular cycloaddition step therefore
seems to proceed substantially more rapidly for the phenol than
for the carbamate.
Fig. 6 TMS-MeNC mediated removal of TzMe-modified molecules on
proteins, in the presence of cells, and in zebrafish embryos. (a) In-gel
analysis of the fluorescent turn-on signal on SNAP protein labelled
with 4b-BG (10 mM) and subsequent deprotection of the TzMe group
with TMS-MeNC (100 mM); lanes 1 and 6 contain the protein ladder (for
an expanded view of the fluorescence and Coomassie-stained gel and
mass spectroscopy verification, see Fig. S34–S37 in the ESI†). (b)
Structure of Tzmoc-caged doxorubicin prodrug (5) and dose–
response curves for cytotoxicity studies with A549 cells after 72 h. (c)
Cartoon representation of experiment to demonstrate the release of
O-carboxymethyl fluorescein and fluorescence turn-on upon incu-
bation with TMS-MeNC. (d) Visualization of the fluorescence signal in
live zebrafish (scale bar ¼ 200 mm) after a 2 h incubation with either 20
mM TMS-MeNC or DMSO.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 169–179 | 175

View Article Online
Chemical Science
Edge Article
The remaining gap in the mechanism is the actual elimi- embryos (Fig. 6c). The non-uorescent TzMe-modied uores-
nation step. We observed a striking dependence of pNA cein derivative 4b was injected into the yolk sac of zebrash
release on the presence of water (Fig. S31†). In anhydrous embryos. The sh were then incubated in either medium con-
DMSO, pNA release was quasi-absent; however, traces of water taining 20 mM TMS-MeNC or only its vehicle (DMSO) for 2
induced the rapid release of pNA. Water therefore participates
in the release step. Several possible release pathways are
conceivable. One possible mechanism could be elimination of
the benzylic leaving group induced by deprotonation of the
pyrazole. Alternatively, water could attack the imine with
concerted electron migrations and elimination of the leaving
group (Fig. S32†).
In summary, through a combination of DFT analysis and
empirical studies, it was possible to establish and validate
a likely reaction mechanism. The reaction cascade largely fol-
lowed the predicted steps of cycloaddition, N2 expulsion, tau-
tomerization, and elimination, with the unexpected cleavage of
the C–Si bond in case of TMS-MeNC.
Demonstration of TzMe-deprotection on biomacromolecules,
in cells, and in living vertebrates
Many potential applications of the presented chemistry would
require it to be compatible with living systems. We aimed to
show that the developed chemistry can be performed under
physiologically relevant conditions. We rst tested whether it
is possible to conjugate a TzMe-modied probe to a protein
and unmask it with TMS-MeNC. For proof of principle, we
used the SNAP-tag system53 for protein labeling. We synthe-
sized an O6-benzylguanine derivative of 4b (4b-BG, Fig. S33†)
and labelled a puried SNAP protein (New England Biolabs)
with it. The SNAP protein-4b-BG conjugate was exposed to
TMS-MeNC (100 mM) and aer 2 h, the uncaging of the uo-
rophore was examined by protein gel analysis (Fig. 6a and
S34†). The protein incubated with TMS-MeNC was visible by
a strong in-gel uorescence signal, whereas the uorescence
signal for controls was low. A 11-fold increase in the uores-
cence signal was measured upon treatment with TMS-MeNC
relative to untreated controls (Fig. S34†). Mass-spectrometry
experiments conrmed the labeling of the SNAP tag to
afford the SNAP protein-4b-BG conjugate and the efficient
removal of the TzMe group by TMS-MeNC (>80%) in the given
time window (Fig. S35–S37†). These results demonstrated that
it is possible to conjugate TzMe-modied groups to bio-
macromolecules and to actuate them by treatment with iso-
nitriles thereaer.
Next, we tested our chemistry with cultured cells. Restoration
of the cytotoxicity of a Tzmoc-caged doxorubicin prodrug
(Tzmoc-Dox (5), Fig. 6b; synthesis described in the ESI†) was
tested with cultured A549 lung adenocarcinoma cells. In the
presence of TMS-MeNC (100 mM) the prodrug was as toxic (EC50
Fig. 7 Dual release of two orthogonal fluorophores from ICPr- and
TzMe-caged dyes in live zebrafish. (a) Cartoon representation of
¼ 0.239 ꢁ 0.014 mM; Fig. 6b) as genuine doxorubicin (EC50
¼
experiment demonstrating the dual release and fluorescence turn-on
0.202 ꢁ 0.025 mM; Fig. 6b). Tzmoc-Dox alone showed almost no of O-carboxymethyl fluorescein and resorufin upon reaction of 4b and
ICPr-rsf, and the corresponding control experiment with non-injected
toxicity below 10 mM, conrming the traceless activation of the
doxorubicin prodrug. Exposure to 100 mM TMS-MeNC for 72 h
caused no cell toxicity (Table S3†).
To demonstrate that TMS-MeNC can activate TzMe-modied
ufin fluorescence signal in non-4b injected control live zebrafish (scale
molecules in vivo, we performed experiments in zebrash bar ¼ 200 mm) after a 2 h incubation with 10 mM ICPr-rsf.
fish. (b) Visualization of fluorescein and resorufin fluorescence signal in
live zebrafish (scale bar ¼ 200 mm) injected with 4b after a 2 h incu-
bation with 10 mM ICPr-rsf. (c) Visualization of fluorescein and resor-
176 | Chem. Sci., 2020, 11, 169–179
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
hours. Subsequently, the sh were washed, and the uores- that the reaction followed the expected mechanism of cycload-
cence turn-on signal analyzed by uorescence microscopy dition and tautomerization to the imine. Unexpectedly, the
(Fig. 6d). Strong green uorescence staining localized to the elimination step could occur from the imine intermediate and
yolk sac was observed for 4b-injected sh incubated with TMS- this step involved the need for water. Furthermore, it was
MeNC, whereas 4b-injected control sh treated with vehicle observed that in reactions with TMS-MeNC, the C–Si bond
(DMSO) exhibited low uorescence (Fig. 6d). A 3.8-fold higher dissociated to generate a methanimine intermediate. TzMe-
uorescence signal was measured in TMS-MeNC treated sh caged uorophore release on a protein, cytotoxicity experi-
relative to untreated controls (Fig. S38†; p-value # 0.001). ments with a doxorubicin-prodrug and cultured cells and uo-
Exposure to 20 mM TMS-MeNC for the duration of the study rophore release in zebrash embryos demonstrated the
caused no developmental issues to the zebrash embryos. potential utility of the reaction in chemical biology and in the
These experiments establish that the reaction of TMS-MeNC context of living systems. It is worth noting that the reaction
and TzMe-groups is suitable for experiments with biomole- with TMS-MeNC generates formaldehyde as side product.
cules and living organisms.
However, endogenous levels of formaldehyde (50–100 mM in
serum and 200–500 mM in cells)54 exceed the levels that would
be released in most foreseeable applications. Furthermore,
metabolic pathways counteract formaldehyde toxicity primarily
mediated by glutathione55 and formaldehyde is diverted to the
one-carbon metabolism.56 The toxicity of aldehyde side prod-
ucts of other isonitriles (e.g. butanal for nBuNC) is typically even
lower.
Combining TzMe-with ICPr-molecules allowed for the rst
time the unmasking of two pro-uorophores by a single bio-
orthogonal reaction. Multiple synergistic drug combinations
would benet from simultaneous and controlled delivery.
Achieving release in a single reaction is important because
controlling the delivery and stability of four individual reactants
required for two reactions occurring in parallel would be chal-
lenging. This versatile protecting group chemistry constitutes
a valuable addition to the dissociative bioorthogonal chemistry
and synthetic methodology toolbox with potential utility for
a broad range of applications. In addition to uses in drug
delivery and controlling biomolecules, it may also be valuable as
a protecting group for the synthesis of sensitive molecules
allowing for late-stage deprotection under extremely mild
conditions.
Dual-release by combining TzMe- with ICPr-modied
molecules
There is considerable interest in developing reaction schemes that
allow for the release of two molecules simultaneously in vivo.24,39
We rationalized that combining the TzMe-release chemistry with
our previously disclosed 3-isocyanopropyl (ICPr) chemistry,37
would liberate two independent payloads. Dual release was rst
tested in vitro. The TzMe-caged uorescein dye 4b was incubated
with ICPr-rsf, an ICPr-caged resorun probe37 (c(4b) ¼ 0.5 mM,
c(ICPr-rsf) ¼ 1 mM, DMSO : PBS pH 7.4 (4 : 1), T ¼ 37 ꢄC, l ¼ 480
nm) and concurrent uorophore release was analyzed by HPLC.
The traceless release of both O-carboxymethyl uorescein and
resorun was observed (Fig. S39†). Dual release from combina-
tions of TzMe/ICPr-reagents was then tested in vertebrates.
Zebrash embryos were either injected with 4b or le untreated
(Fig. 7a). The sh were then incubated in media containing 10 mM
ICPr-rsf for 2 hours, washed, and uorescence turn-on signals
analyzed by uorescence microscopy (Fig. 7b and c). Strong
emission signals were detected in the yolk sac in both green and
red uorescence channels for sh injected with 4b. (Resorun: p-
value # 0.0001; uorescein: p-value # 0.0001; Fig. S38†). Neither
4b (Fig. 6d) nor ICPr-rsf (Fig. 7c) alone produced obvious uo-
rescence signal conrming that it was the reaction between the
isonitrile and the tetrazine that led to the concurrent release of the
two uorophores. While it is acknowledged that precisely
controlling the injected probe volume into the yolk sac is chal-
lenging, the 60-fold higher resorun (p-value # 0.0001) and 3.6-
fold higher uorescein signal (p-value # 0.0001) in zebrash
treated with both reactive species relative to controls indicate
unmasking of a considerable fraction of the uorophores
(Fig. S38†). Conclusively, combining TzMe- and ICPr-reactants can
simultaneously liberate pairs of molecules of interest.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
R. M. F. gratefully acknowledges nancial support from the
University of Utah (VPR seed grant), the Huntsman Cancer
Institute (GU Center Pilot Grant), the American Cancer Society
(129785-IRG-16-190-01-IRG), and the USTAR initiative. K. N. H.
acknowledges nancial support from the National Institute of
General Medical Sciences, National Institutes of Health,
GM-109078. This work was supported by the L. S. Skaggs Pres-
idential Endowed Chair (R.T.P.). J. T. gratefully acknowledges
Conclusions
In summary, the study introduces TzMe-substituents as pro- nancial support from a University of Utah Skaggs Graduate
tecting groups that are removed under physiological conditions Fellowship and from an American Foundation for Pharmaceu-
by isonitriles. In a series of steps, we demonstrated that TzMe- tical Education Pre-Doctoral Fellowship. D. S. gratefully
groups could reversibly cage amines and phenols with near- acknowledges nancial support from the Austrian Science Fund
quantitative release yields. Fast elimination occurred for (FWF, J 4216-N28). We thank Dr Jack J. Skalicky for help with
phenols and for amines could be achieved by the addition of collecting and analysing the NMR data and Sara Augustine for
HSA or the use of TMS-MeNC. NMR and DFT studies revealed assistance in the early stages of this project.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 169–179 | 177

View Article Online
Chemical Science
Edge Article
J. Elyian, M. N. Trinh, O. Koniev, A. Wagner, S. Kolodych
and F. Taran, Angew. Chem., Int. Ed., 2017, 56, 15612–15616.
23 Y. Zheng, X. Ji, B. Yu, K. Ji, D. Gallo, E. Csizmadia, M. Zhu,
M. R. Choudhury, L. K. C. De La Cruz, V. Chittavong,
Z. Pan, Z. Yuan, L. E. Otterbein and B. Wang, Nat. Chem.,
2018, 10, 787–794.
Notes and references
1 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009,
48, 6974–6998.
2 C. S. McKay and M. G. Finn, Chem. Biol., 2014, 21, 1075–1101.
3 J. Li and P. R. Chen, Nat. Chem. Biol., 2016, 12, 129–137.
4 J. Tu, M. Xu and R. M. Franzini, ChemBioChem, 2019, 20,
1615–1627.
24 K. Neumann, A. Gambardella, A. Lilienkampf and
M. Bradley, Chem. Sci., 2018, 9, 7198–7203.
25 T. Carell and M. Vrabel, Top. Curr. Chem., 2016, 374, 9.
26 N. K. Devaraj, ACS Cent. Sci., 2018, 4, 952–959.
27 H. Wu and N. K. Devaraj, Top. Curr. Chem., 2016, 374, 3.
28 B. L. Oliveira, Z. Guo and G. J. L. Bernardes, Chem. Soc. Rev.,
2017, 46, 4895–4950.
29 K. Lang and S. Mayer, Synthesis, 2016, 49, 830–848.
30 H. Wu and N. K. Devaraj, Acc. Chem. Res., 2018, 51, 1249–
1259.
31 J. C. T. Carlson, H. Mikula and R. Weissleder, J. Am. Chem.
Soc., 2018, 140, 3603–3612.
32 R. M. Versteegen, W. Ten Hoeve, R. Rossin, M. A. R. de Geus,
H. M. Janssen and M. S. Robillard, Angew. Chem., Int. Ed.,
2018, 57, 10494–10499.
5 K. Neumann, A. Gambardella and M. Bradley, ChemBioChem,
2019, 20, 872–876.
6 X. Ji, Z. Pan, B. Yu, L. K. De La Cruz, Y. Zheng, B. Ke and
B. Wang, Chem. Soc. Rev., 2019, 48, 1077–1094.
7 K. Neumann, S. Jain, A. Gambardella, S. E. Walker, E. Valero,
A. Lilienkampf and M. Bradley, ChemBioChem, 2017, 18, 91–
95.
8 S. Dadhwal, J. M. Fairhall, S. K. Goswami, S. Hook and
A. B. Gamble, Chem.–Asian J., 2019, 14, 1143–1150.
9 K. Porte, B. Renoux, E. Peraudeau, J. Clarhaut, B. Eddhif,
P. Poinot, E. Gravel, E. Doris, A. Wijkhuisen, D. Audisio,
S. Papot and F. Taran, Angew. Chem., Int. Ed., 2019, 58,
6366–6370.
33 A. J. C. Sarris, T. Hansen, M. A. R. de Geus, E. Maurits,
W. Doelman, H. S. Overklee, J. D. C. Codee, D. V. Filippov
and S. I. van Kasteren, Chem.–Eur. J., 2018, 24, 18075–18081.
34 M. Xu, R. Galindo-Murillo, T. E. Cheatham and
R. M. Franzini, Org. Biomol. Chem., 2017, 15, 9855–9865.
35 B. P. Imming, R. Mohr, E. Miiller, W. Overheu and G. Seitz,
Angew. Chem., Int. Ed., 1982, 21, 20133.
36 H. Stockmann, A. A. Neves, S. Stairs, K. M. Brindle and
F. J. Leeper, Org. Biomol. Chem., 2011, 9, 7303–7305.
37 J. Tu, M. Xu, S. Parvez, R. T. Peterson and R. M. Franzini, J.
Am. Chem. Soc., 2018, 140, 8410–8414.
38 J. Tu, D. Svatunek, S. Parvez, A. C. Liu, B. J. Levandowski,
H. J. Eckvahl, R. T. Peterson, K. N. Houk and
R. M. Franzini, Angew. Chem., Int. Ed., 2019, 58, 9043–9048.
39 Z. Shao, W. Liu, H. Tao, F. Liu, R. Zeng, P. A. Champagne,
Y. Cao, K. N. Houk and Y. Liang, Chem. Commun., 2018,
54, 14089–14092.
10 J. M. Mejia Oneto, I. Khan, L. Seebald and M. Royzen, ACS
Cent. Sci., 2016, 2, 476–482.
11 R. Rossin, R. M. Versteegen, J. Wu, A. Khasanov,
H. J. Wessels, E. J. Steenbergen, W. Ten Hoeve,
H. M. Janssen, A. van Onzen, P. J. Hudson and
M. S. Robillard, Nat. Commun., 2018, 9, 1484.
12 M. Czuban, S. Srinivasan, N. A. Yee, E. Agustin, A. Koliszak,
E. Miller, I. Khan, I. Quinones, H. Noory, C. Motola,
R. Volkmer, M. Di Luca, A. Trampuz, M. Royzen and
J. M. Mejia Oneto, ACS Cent. Sci., 2018, 4, 1624–1632.
13 J. Li, S. Jia and P. R. Chen, Nat. Chem. Biol., 2014, 10, 1003–
1005.
14 G. Zhang, J. Li, R. Xie, X. Fan, Y. Liu, S. Zheng, Y. Ge and
P. R. Chen, ACS Cent. Sci., 2016, 2, 325–331.
15 J. Luo, Q. Liu, K. Morihiro and A. Deiters, Nat. Chem., 2016,
8, 1027–1034.
16 R. M. Franzini and E. T. Kool, J. Am. Chem. Soc., 2009, 131,
16021–16023.
40 C. Jin, Q. Zhang and W. Lu, Eur. J. Med. Chem., 2017, 132,
135–141.
41 X. Ao, S. A. Bright, N. C. Taylor and R. B. P. Elmes, Org.
Biomol. Chem., 2017, 15, 6104–6108.
17 R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen
and M. S. Robillard, Angew. Chem., Int. Ed., 2013, 52,
14112–14116.
42 S. Stairs, A. A. Neves, H. Stockmann, Y. A. Wainman,
H. Ireland-Zecchini, K. M. Brindle and F. J. Leeper,
ChemBioChem, 2013, 14, 1063–1067.
18 S. S. Matikonda, D. L. Orsi, V. Staudacher, I. A. Jenkins,
F. Fiedler, J. Chen and A. B. Gamble, Chem. Sci., 2015, 6,
1212–1218.
43 K. Kikuchi, S. N. Thorn and D. Hilvert, J. Am. Chem. Soc.,
1996, 118, 8184–8185.
19 H. Wu, S. C. Alexander, S. Jin and N. K. Devaraj, J. Am. Chem.
Soc., 2016, 138, 11429–11432.
44 F. Hollfelder, A. J. Kirby and D. S. Tawk, Nature, 1996, 383,
60–62.
45 D. C. M. Albanese and N. Gaggero, RSC Adv., 2015, 5, 10588–
10598.
20 E. Jimenez-Moreno, Z. Guo, B. L. Oliveira, I. S. Albuquerque,
A. Kitowski, A. Guerreiro, O. Boutureira, T. Rodrigues,
G. Jimenez-Oses and G. J. Bernardes, Angew. Chem., Int.
Ed., 2017, 56, 243–247.
´
´
46 R. Perez Carlon, N. Jourdain and J.-L. Reymond, Chem.–Eur.
J., 2000, 6, 4154–4162.
21 M. Xu, J. Tu and R. M. Franzini, Chem. Commun., 2017, 53,
6271–6274.
47 J. Cai, X. Li, X. Yue and J. S. Taylor, J. Am. Chem. Soc., 2004,
126, 16324–16325.
22 S. Bernard, D. Audisio, M. Riomet, S. Bregant, A. Sallustrau,
L. Plougastel, E. Decuypere, S. Gabillet, R. A. Kumar,
48 E. W. Colvin, Chem. Soc. Rev., 1978, 7, 15–64.
178 | Chem. Sci., 2020, 11, 169–179
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
49 F. Weigend and R. Ahlrichs, Phys. Chem. Chem. Phys., 2005, 54 K. J. Bruemmer, R. R. Walvoord, T. F. Brewer, G. Burgos-
7, 3297–3305.
Barragan, N. Wit, L. B. Pontel, K. J. Patel and C. J. Chang,
J. Am. Chem. Soc., 2017, 139, 5338–5350.
55 R. Bateman, D. Rauh and K. M. Shokat, Org. Biomol. Chem.,
2007, 5, 3363–3367.
56 G. Burgos-Barragan, N. Wit, J. Meiser, F. A. Dingler,
M. Pietzke, L. Mulderrig, L. B. Pontel, I. V. Rosado,
T. F. Brewer, R. L. Cordell, P. S. Monks, C. J. Chang,
A. Vazquez and K. J. Patel, Nature, 2017, 548, 549–554.
50 Y. Zhao and D. G. Truhlar, Theor. Chem. Acc., 2007, 120, 215–
241.
51 S. Grimme, J. Antony, S. Ehrlich and H. Krieg, J. Chem. Phys.,
2010, 132, 154104.
52 A. V. Marenich, C. J. Cramer and D. G. Truhlar, J. Phys. Chem.
B, 2009, 113, 6378–6396.
53 A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel
and K. Johnsson, Nat. Biotechnol., 2003, 21, 86–89.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 169–179 | 179