Vinylboronic acid-caged prodrug activation using click-to-release tetrazine ligation

Article abstract of DOI:10.1039/c9ob01881f

Bioorthogonal reactions can be performed selectively in the presence of any biological functional group and are widely used to achieve site-selective chemical modifications of biomolecules. The click-to-release reaction is a bioorthogonal bond-cleavage variant that has gained much interest over the last few years. The bioorthogonal reaction between tetrazines and trans-cyclooctenes or vinyl ethers, for example, initiates the release of a small molecule immediately after the cycloaddition with tetrazines. Recently, our group reported that vinylboronic acids (VBAs) give exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder reaction with tetrazines that are substituted with boron-coordinating ligands. In the present study, we show that VBAs can be used in a click-to-release variant and demonstrate its bioorthogonality with a VBA-protected doxorubicin prodrug. We show that the cytotoxicity of doxorubicin is silenced by the attachment of the VBA, and activity can be largely restored upon the reaction with a tetrazine, inducing cell death.

Full text of DOI:10.1039/c9ob01881f

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Vinylboronic Acid-Caged Prodrug Activation using Click-to-
Release Tetrazine Ligation
Lianne Lelieveldt, Selma Eising, Abel Wijen, Kimberly Bonger
† Department of Biomolecular Chemistry and Synthetic Organic Chemistry, Radboud University
Nijmegen, the Netherlands
email: k.bonger@science.ru.nl
Abstract
Bioorthogonal reactions can be performed selectively in the presence of any biological functional
group and are widely used to achieve site-selective chemical modifications of biomolecules. The
click-to-release reaction is a bioorthogonal bond-cleavage variant that has gained much interest
over the last few years. The bioorthogonal reaction between tetrazines and trans-cyclooctenes
or vinyl ethers, for example, initiate the release of a small molecule immediately after the
cycloaddition with tetrazines. Recently, our group reported that vinylboronic acids (VBAs) give
exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder
reaction with tetrazines that are substituted with boron-coordinating ligands. In the present
study, we show that VBAs can be used in a click-to-release variant and demonstrate its
bioorthogonality with a VBA-protected doxorubicin prodrug. We show that the cytotoxicity of
doxorubicin is silenced by the attachment of the VBA, and activity can be largely restored upon
the reaction with a tetrazine, inducing cell death.

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Introduction
Chemical site-selective protein modifications have become increasingly popular for modification
and control over protein functions in vitro and in living systems. Several reactions have been
developed to decorate biomolecules with a desired functionality such as fluorophores, affinity
probes or reactive tags. All these reactions have some properties in common as they must be
selective over other functional groups in biomolecules, have fast reaction rates and progress in
aqueous media around physiological pH.1 Reactions such as the strain-promoted alkyne–azide
cycloaddition (SPAAC)2 or the inverse electron-demand Diels Alder reaction with tetrazines,3
have been used extensively for modifying purposes. Our group has recently reported a new
bioorthogonal tetrazine ligation utilizing on vinylboronic acids (VBAs).4-7 We showed that by
introducing a boronic acid moiety on an alkene, reaction rates of an inverse electron-demand
Diels-Alder (iEDDA) reaction with pyridyl-containing tetrazines improved several orders of
magnitude compared to the non-modified linear alkene.4 The hydrophilic properties and the
small size of VBAs compared to other bioorthogonal reactants make them attractive for the use
in biomolecular labeling experiments. VBAs were shown to be biocompatible, non-toxic, and
highly stable in aqueous media and cell lysates. In addition, while the number of bioorthogonal
reactions used in situ and in vivo is limited due to the need of toxic reagents (e.g. copper, in
copper (I)-catalyzed alkyne-azide cycloaddition8) or possible side reactions of the reactants (e.g.
strained alkenes or alkynes can react with cysteines9), VBAs appeared useful in cellular
experiments.7 Reactions with vicinal diols, present in carbohydrates for example, were not
observed when performing reactions in living cells.
The reactivity of VBAs depends on the tetrazine and its substituents. Since the boronic acid
coordinates to the pyridyl ring of dipyridyl-s-tetrazine, reaction rates were enhanced in
comparison to tetrazines lacking such potential Lewis base.6 This unique feature allows to
perform orthogonal bioorthogonal reactions with two different tetrazines, one with and one
without substituents allowing for coordination, and using a VBA and a strained alkene.
The interest in bioorthogonal bond-cleavage reactions for activation of protected biologically
relevant molecules in vivo has increased significantly (Figure 1A). A substantial number of so-
called ‘click-to-release’ reactions have been developed in the last years.10–12 These reactions
enable new applications in vitro and in vivo, such as the activation of prodrugs or fluorogenic
compounds. Most reactions are based on the iEDDA reaction between a tetrazine and a (strained)
alkene.11

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Figure 1. A) General scheme of a click-to-release reaction to uncage a prodrug. B) Overview of the inverse electron-
demand Diels-Alder cycloaddition with a tetrazine and a vinyl ether or vinylboronic acid to release an alcohol
functionality.
In 2013, the group of Robillard reported the first bioorthogonal click-to-release reaction based
on the reaction of tetrazines with trans-cyclooctenes (TCOs) that possess a carbamate
substituent at the allylic position (Figure 1A).13 After the iEDDA of the tetrazine and TCO, the 4,5-
dihydropyridazine eliminates the leaving group to liberate both CO2 and the amine.
Unfortunately, part of the dihydropyridazine tautomerizes to the 1,4-dihydropyidazine, which
does not lead to a release reaction and therefore this click-to-release process did not give
complete elimination. Over the last years, the reaction has been optimized14,15 and the scope
was further expanded16 and applied in biological systems for activation of prodrugs17–23, RNA24
or caged proteins.14,25,26 Recently, it was noted that this click-to-release reaction is
environmentally sensitive and improvements on both the TCO and tetrazine resulted in ultrafast
click-to-release reaction rates and the complete elimination of the leaving group.27

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In 2016 and 2017, the groups of Deveraj and Bernardes reported both the bioorthogonal click-
to-release reaction of tetrazines and vinyl ethers (Figure 1A).28,29 These alkenes bear their leaving
group directly on the alkene and give therefore immediately full release of the alcohol after the
iEDDA. The vinyl ethers appeared stable in aqueous solutions for at least 8 hours and were
suitable for the release of a prodrug in living cells.30 The vinyl ethers have the advantage to be
small in size, but have disappointingly low rate constants when reacting with tetrazines.
Since VBAs have proven to be useful in bioorthogonal conjugation, we envisioned that by
introducing a boronic acid group on the vinyl ethers, we could increase the rate of these click-to-
release reactions. In addition, similar to vinyl ethers, we expect that VBA ethers give full release
of the alcohol, or amine when linked via a carbamate, after the iEDDA. The proposed iEDDA click-
to-release reaction mechanism of vinylboronic acids with tetrazines is shown in Figure 1B. Upon
cycloaddition and retro-Diels-Alder reaction, N2, the boric acid and the uncaged drug are
released. We here explore the applicability of the VBA click-to-release strategy and developed a
synthetic route to VBA protected prodrugs, which can be released using tetrazine in the presence
of cells.
Results and Discussion
We first aimed to compare the reactivity of the VBA ether with the unsubstituted vinyl ethers
and synthesized VBA 6 and its primary alkene derivative 9 (Scheme 1). The synthesis of VBA 6
started with the etherification of 4-hydroxybenzyl alcohol 1 with trichloroethylene and
subsequent silyl protection of the primary alcohol. Next, dichlorovinyl ether 3 was treated with
n-BuLi to eliminate hydrogen chloride and to provide alkynyl ether 4. Deprotection of the primary
alcohol and hydroboration of alkyne 5 with pinacolborane yielded VBA pinacol ester 6. In our
kinetic experiments, we used the pinacol ester as precursor for the free boronic acid as it
hydrolyzes quickly in aqueous media (see Figure SI-1). The synthesis of vinyl ether 9 was prepared
following a literature procedure.31 Here, 4-hydroxybenzaldehyde (7) was first reacted with 1,2-
dibromoethane to yield compound 8 which was subsequently eliminated using t-BuOK and the
aldehyde reduced using NaBH4 to yield corresponding benzyl alcohol 9.

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O
O
O
OH
O
B
Cl
c
a
e
HO
O
RO
d
HO
RO
b
Cl
1
2
3
6
: R = H
: R = TBS
4
: R = TBS
5
: R = H
O
OH
O
Br
f
g, h
O
O
HO
H
H
8
7
9
Scheme 1. Synthesis of VBA ether 6 and vinyl ether 9. a) Trichloroethylene, K2CO3, DMF, 70 °C, 16 h, 87%. b) TBSCl,
imidazole, DMF, 2 h, quant. c) n-BuLi, Et2O, -78 to -40 °C, 87%. d) TBAF, THF, 0 °C, 91%. e) Pinacolborane,
RuHClCO(PPh3)3, toluene, 50 °C, 16 h, 76%. f) Dibromoethane, K2CO3, MeCN, reflux, 20 h, 79%. g) t-BuOK, DMSO, r.t.,
10 min. h) NaBH4, MeOH, r.t., 1.5 h, 18%.
Having alkenes 6 and 9 in hand, we examined the second-order rate constants with 3,6-dipyridyl-
s-tetrazine 10 in 75% MeOH in PBS at a controlled temperature of 20 °C (Figure 2A). As we used
the pinacol ester as precursor, we incubated ester 6 for 2 h in the solvent mixture to achieve full
hydrolysis to the free boronic acid before measuring its k2 value. Second-order rate constants
were obtained by following the decrease of the tetrazine UV absorbance in the visible region.
The pseudo first-order constant kobs was first determined by plotting the decay of tetrazine
absorbance against time (minutes) for five different amounts of excess alkene (see also
Supplementary Information). Next, the kobs was plotted against the five different alkene
concentrations and a linear regression fit gave the linear function, of which the slope
corresponded to the k2 (Figure SI-2). The second-order rate constant for VBA 6 was 3.3 × 10-3 M-
1 s-1 and 4-fold higher than the rate constant of vinyl ether 9, clearly indicating a positive effect
of the boronic acid on the reactivity of the alkene.
The observed rate constant of VBA ether 6 was lower than expected as we previously observed
rate enhancements of up to 100-fold compared to the unsubstituted alkene derivatives.45 We
hypothesize that the moderate rate enhancement is due to the ether on the vinylic position.
Since this alcohol is slightly electron-donating, we assume that the alkene becomes more
electron-rich and thereby lowering the acidity of the boronic acid, resulting in less favorable
coordination to the pyridyl substituents of the tetrazine.32

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Figure 2. Click-to-release reactions and reaction rates. A) Scheme of the reaction of 3,6-dipyridyl-s-tetrazine Tz with
VBA ether 6 and vinyl ether 9. k2 values were measured in 3:1 MeOH/PBS; B) 1H-NMR study of the reaction between
tetrazine 10 (∎) and VBA ether 6 (▲) yielding pyridazine 11 () and 4-hydroxybenzyl alcohol 1 (♦) in 3:1
CD3OD/deuterated PBS; C) Similar as B with reaction of Tz with vinyl ether 9 (▼).
Next, we looked in more detail into the click-to-release reaction of 3,6-dipyridyl-s-tetrazine 10
and alkenes 6 and 9 by 1H NMR in 75% CD3OD in deuterated PBS over time (Figures 2B, C). A near
to complete conversion of VBA ether 6 was observed after 7 days, while vinyl ether 9 gave around
50% conversion. Both reactions yielded the clean formation of pyridazine 11 and 4-hydroxybenzyl
alcohol 1. No intermediates were observed showing that the initial cycloaddition step is rate
limiting for both VBA ether 6 as well as vinyl ether 9.

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To further examine the click-to-release reaction with a VBA ether in a more complex system, we
synthesized a VBA-caged cytotoxin doxorubicin and explored the effect on HeLa cells upon
addition of a water soluble tetrazine 12 (Figure 3A). For this, we aimed to couple doxorubicin to
VBA alcohol 6, which acts as a self-cleavable linker by 1,6 elimination upon release (Figure 3B).33
We commenced the synthesis from VBA alcohol 6 and activated the alcohol using
bis(pentafluorophenyl)carbonate and Et3N as a base, yielding successfully VBA-PFP carbonate 13.
Doxorubicin was subsequently added and reacted overnight to yield VBA-caged doxorubicin 14
(VBA-Dox) in reasonable yields.
First, we tested the stability of the VBA-Dox 14 at 100 µM in PBS at 37 °C before performing the
click-to-release reactions with cells. As shown in Figure SI-3, hydrolysis of the pinacol ester was
observed, as expected, while minor decomposition (around 10%) of VBA-Dox 14 was observed
after 48 hours. Decomposition likely results from protodeboronation of the VBA cage.34
Importantly, no free doxorubicin was observed after incubating VBA-Dox 14 in PBS for up to 7
days. Encouraged by these results we next evaluated the cycloaddition with a water soluble
tetrazine 12 and followed subsequent release over time in a buffer system with LCMS.
Deprotection of 14 at 100 µM in PBS at 37 °C, using 1 equivalent of tetrazine 12 resulted in
decaging and release of doxorubicin (Figure SI-4). No intermediate products were observed,
indicating that the cleavage of the linker occured instantaneously after the cycloaddition.
Next, we tested the VBA release and toxicity of doxorubicin on living HeLa cells (Figure 3A). We
first incubated HeLa cells with free doxorubicin and observed a dose dependent toxic effect, as
expected (Figure SI-5). In addition, we incubated VBA-caged doxorubicin in the absence of
tetrazine. No toxicity was observed at 100 nM VBA-doxorubicin 14 while 1 µM VBA-dox did show
some toxicity after 3 days of incubation. As we observed more than 70% cell death with free
doxorubicin at the lower concentration of 100 nM we used these conditions to test the efficiency
of the VBA click-to-release strategy with tetrazine 12.
We incubated VBA-doxorubicin 14 with 10 and 100 equivalents of tetrazine 12 with HeLa cells
for 3 days. To our delight, we observed a dose-dependent toxicity of tetrazine-activated
doxorubicin and an almost similar toxicity level was obtained to that when incubated with free
doxorubin (Figure 3C). Using higher amounts of tetrazine 12 showed also signs of toxicity of the
tetrazine itself, so we omitted this concentration in further cell studies (Figure SI-5).

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Figure 3. Click-to-release of VBA-Dox 14 to yield the free doxorubicin. A) General overview of caging strategy. B)
Synthetic rout of VBA-Dox. a) Bis(pentafluorophenyl)carbonate, Et3N, r.t., 1 h, 84%. b) Doxorubicin HCl, Et3N, DMF
r.t., 24 h, 40%. C) Evaluation of cytotoxicy of VBA-Dox 14 incubated with tetrazine 12 (1 or 10 µM, respectively 10
equiv. or 100 equiv.) for 72h with HeLa cells.
Conclusion
In this work we have shown that VBAs can be used in bioorthogonal click-to-release tetrazine-
ligations. The release was instantaneous after the tetrazine cycloaddition as no reaction
intermediates were observed by NMR experiments. We assume that the slight electron-donating
nature of the ether at the vinylic position of the boronic acid reduces the reaction rate as
compared to VBAs lacking the ether functionality. The boronic acid moiety increases the
reactivity of vinyl ethers in the click-to-release tetrazine ligation several fold when tetrazines
bearing boron-coordinating ligands are used.
The use of VBA-caged prodrugs may be of interest as we have recently shown that this moiety
can orthogonally react with tetrazines bearing a coordinating phenol substituent. These
tetrazines are too electron rich to react with vinyl ethers.5 It would further be interesting to
explore different substituents on the VBA cage, such as carbamates, to study the electronic

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effects in the click-to-release reaction with different coordinating tetrazines. These studies are
currently ongoing in our laboratory.
Acknowledgements
This research was funded by the NWO gravity program ‘Institute for Chemical Immunology’
NWO-024.002.009 and the Institute for Molecules and Materials at the Radboud University. We
thank Selina Thijssen for her pioneering work on this project and Prof. Floris Rutjes for fruitful
discussions.
Conflict of interest
No conflict declared
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