Vinylboronic Acids as Fast Reacting, Synthetically Accessible, and Stable Bioorthogonal Reactants in the Carboni–Lindsey Reaction

Article abstract of DOI:10.1002/anie.201605271

Bioorthogonal reactions are widely used for the chemical modification of biomolecules. The application of vinylboronic acids (VBAs) as non-strained, synthetically accessible and water-soluble reaction partners in a bioorthogonal inverse electron-demand Diels–Alder (iEDDA) reaction with 3,6-dipyridyl-s-tetrazines is described. Depending on the substituents, VBA derivatives give second-order rate constants up to 27 m^?1s^?1in aqueous environments at room temperature, which is suitable for biological labeling applications. The VBAs are shown to be biocompatible, non-toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs can be used orthogonally to the strain-promoted alkyne–azide cycloaddition for protein modification, making them attractive complements to the bioorthogonal molecular toolbox.

Full text of DOI:10.1002/anie.201605271

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International Edition: DOI: 10.1002/anie.201605271
German Edition: DOI: 10.1002/ange.201605271
Bioorthogonal Reactions
Vinylboronic Acids as Fast Reacting, Synthetically Accessible, and
Stable Bioorthogonal Reactants in the Carboni–Lindsey Reaction
Selma Eising, Francis Lelivelt, and Kimberly M. Bonger*
Abstract: Bioorthogonal reactions are widely used for the
chemical modification of biomolecules. The application of
vinylboronic acids (VBAs) as non-strained, synthetically
accessible and water-soluble reaction partners in a bioorthog-
onal inverse electron-demand Diels–Alder (iEDDA) reaction
with 3,6-dipyridyl-s-tetrazines is described. Depending on the
substituents, VBA derivatives give second-order rate constants
up to 27 mÀ1 sÀ1 in aqueous environments at room temperature,
which is suitable for biological labeling applications. The
VBAs are shown to be biocompatible, non-toxic, and highly
stable in aqueous media and cell lysate. Furthermore, VBAs
can be used orthogonally to the strain-promoted alkyne–azide
cycloaddition for protein modification, making them attractive
complements to the bioorthogonal molecular toolbox.
Figure 1. A) Labeling of the protein of interest (POI) using the CL
I
n the last decades, the development of selective reactions
reaction. B) Reagents of the CL reaction. Tetrazine I, trans-cyclooctene
II, norbornene III, cyclopropene IV, N-acylazetine V, terminal alkene VI,
vinylboronic acid VII, and the borate form VIII.
between two reactants that are unaffected by any of the
naturally occurring biological functionalities has been a major
research area in chemical biology.[1] These bioorthogonal
reactions (Figure 1A) make it possible to chemically modify
biomolecules in their native cellular environment and gain
a better understanding of their role in a specific biological
system or process. The bioorthogonal reaction should be high
yielding and rapid, and the reactants and product(s) should be
soluble and stable in aqueous media and non-toxic to the
biological system. The use of small reactants is preferred to
minimize steric interactions with the biomolecule or to
facilitate incorporation by the endogenous cellular machi-
nery.
pene,[6] or N-acylazetine[7] (Figure 1B, II–V). In addition to
strain, the rate of the CL reaction can be significantly
enhanced by the introduction of electron-donating substitu-
ents on the alkene bond.[8] This aspect, however, has been
poorly investigated in relation to bioorthogonal applications.
Vinylboronic acids (Figure 1B, VII) are an interesting
class of compounds with unique electronic properties, which
are due to their vacant p-orbital. As a result of the inductive
effect that is caused by the electronegativity difference of
boron and carbon, the electronic deficiency of boron and the
electron-donating oxygens attached to boron, boronic acid is
considered to be a weak electron-donor.[9] Furthermore,
boronic acids are mild organic Lewis acids, which in basic
aqueous media are in equilibrium with their boronate anion
The Carboni–Lindsey (CL) reaction between an electron-
poor tetrazine (Figure 1B, I) and an alkene has gained
considerable attention for use in bioorthogonal applications.[2]
As linear and unmodified alkenes (Figure 1B, VI) showed
only poor reaction rates with tetrazines,[3] most research has
been directed to the development and use of strained alkenes
such as trans-cyclooctene (TCO),[4] norbornene,[5] cyclopro-
(Figure 1B, VIII), which is
a
strong electron-donor.[9]
Although Diels–Alder reactions are known to proceed
faster in aqueous media, the CL reactions of vinylboronic
acids and alkylboronic acids are reported only in organic
solvents, and high temperatures were often needed for the
reaction to proceed.[10]
In our effort to investigate the electronic effects of the
alkenes in the CL reaction, we found that vinylboronic acids
(VBAs) show impressive reaction rates with 3,6-dipyridiyl-s-
tetrazines in aqueous environments. Herein, we evaluate
VBAs as synthetically accessible, fast reacting bioorthogonal
reaction partners with 3,6-dipyridyl-s-tetrazines. We show
that VBAs are stable, non-toxic, and can be used for protein
labeling in vitro and in cell lysate. The hydrophilic properties
and the small size of VBAs make them attractive for use in
[*] M. Sc. S. Eising, B. Sc. F. Lelivelt, Dr. K. M. Bonger
Department of Biomolecular Chemistry
Institute for Molecules and Materials, Radboud University
Heyendaalseweg 135, 6525 AJ Nijmegen (The Netherlands)
E-mail: k.bonger@science.ru.nl
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201605271.
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial, and no
modifications or adaptations are made.
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protein modifications and other biomolecular labeling appli-
cations.
the (E)-isomer of 4. Addition of a phenyl ring to the VBA
increased the k2 significantly, (E)-phenylvinylboronic acid 5
showed a five-fold higher rate constant than norbornene 2,
comparable to the reported rate constant of the fastest
cyclopropene.[6] For this alkene, the (Z)-isomer of 5 showed
a lower rate constant than (E)-5. Introduction of an electron-
donating substituent on the phenyl ring of 8 increased the rate
constant further to 27 mÀ1 sÀ1.
To evaluate the effect of the boronic acid on the alkeneꢀs
reactivity, we compared the rate constants of the fastest VBAs
(E)-5 and 8 to the alkene derivatives lacking the boronic acid
(Table 1, Entries 4 and 5). Under the selected conditions, we
could estimate a decrease in reaction rate of styrene 6 and p-
methoxystyrene 9 of at least two orders of magnitude,
although because of the reduced reaction rate and low
solubility of the unsubstituted alkene in the aqueous environ-
ment, we could not determine the k2 accurately. Introduction
of an electron-donating alkoxy substituent on the alkene as in
b-methoxystyrene 7 did not result in a significant increase in
reaction rate, clearly indicating that the boronic acid has
a large positive effect on the reactivity of the alkene. A similar
trend in the rate constants of the alkenes 2–9 with 3,6-
dipyridyl-s-tetrazine 10 was observed in 50% MeOH/PBS,
although the constants decreased significantly in these less
aqueous conditions (Supporting Information, Table S1 and
Figures S3 and 4).
The reactions of VBAs 3, (E)-5, and 8 were additionally
studied with 3-phenyl-s-tetrazine and 3-phenyl-6-methyl-s-
tetrazine (Supporting Information, Figure S5). Interestingly,
compared to the reaction rate of these tetrazines with
norbornene 2, the reactions of the VBAs with the tetrazines
are much slower than expected, which shows that the VBAs
are especially suitable in the CL reactions with dipyridyl-s-
tetrazines.
We then studied the reaction of 3,6-dipyridyl-s-tetrazine
10 and the most reactive VBA 8 in more detail (Figure 2A).
We observed quantitative formation of dihydropyradizine 11
as the most abundant isomer, which lacked the boronic acid.
To explore the potential of VBAs as bioorthogonal
reaction partners, we first examined the second-order rate
constants (k2) of 3,6-dipyridyl-s-tetrazine derivative 1 with
several VBAs in 5% MeOH in PBS (Table 1 and the
Supporting Information, Figures S1 and 2) and compared
them to the k2 of norbornene 2, a reagent that is used
extensively in bioconjugation reactions.[2,5] Vinylboronic acid
3 reacted with tetrazine 1 with a rate of 3.0 mÀ1 sÀ1, which is
comparable to the rate of 1 with norbornene 2. A methyl
substituent on the alkene reduced the reaction rate, although
the rate of the (Z)-isomer was almost 3-fold faster than that of
Table 1: Second-order rate constants of tetrazine 1 with several alkenes.
alkene
No.
R
k2 [mÀ1 sÀ1
]
1
2
3
2[a]
–
2.2Æ0.1
3.0Æ0.1
3
B(OH)2
(E)-4
(Z)-4
(E)-5
(Z)-5
6
B(OH)2
B(OH)2
B(OH)2
B(OH)2
H
0.24Æ0.01
0.65Æ0.02
11Æ1
0.59Æ0.03
<0.025[c]
<0.025[c]
27Æ2
4
5
(E/Z)-7[b]
8
OMe
B(OH)2
H
9
0.13Æ0.01
[a] Endo/exo=2:1. [b] E/Z=1:3. [c] k2 could not be determined accu-
rately, owing to the slow reaction rate and low solubility of the alkenes in
water.
Figure 2. The reaction of tetrazine with vinylboronic acids A) Scheme of the reaction of 3,6-dipyridyl-s-tetrazine 10 with VBA 8. B) 1H NMR study of the
reaction between 3,6-dipyridyl-s-tetrazine 10 (&) and VBA 8 (*). C) Proposed reaction mechanism.
2
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No intermediates were observed in the 1H NMR spectra
100 mm for VBA 8 and boric acid, whereas tetrazine 1
during the reaction between 10 and 8, which shows that the
protodeborylation of boric acid is fast (Figure 2B and the
Supporting Information, Figure S6). We therefore propose
a mechanism in which boric acid is released immediately after
the iEDDA cycloaddition, followed by dinitrogen release by
a retro-Diels–Alder reaction (Figure 2C). The dihydropyr-
idazine product 11 is the same as found in the reaction of
tetrazine 10 with p-methoxystyrene 9 (Supporting Informa-
tion, experimental section),[11] and slow isomerization and
oxidization to the pyridazine product[8b] were observed by
1H NMR and UV/Vis absorption (Supporting Information,
Figures S7–9).
and the dihydropyrazine product showed some toxicity at
this concentration after 24 h (Supporting Information, Fig-
ure S12).
The CL reaction between VBAs and 3,6-dipyridiyl-s-
tetrazine was next evaluated for its use for in vitro protein
modification. Vinylboronic acids can be easily synthesized as
the protected ester in one step by, for example, cross-
metathesis between an alkene and a vinylboronic ester[12] or
hydroboration of an alkyne.[13] Hydrolysis of the boronic ester
occurs spontaneously in aqueous media, although many
deprotection strategies to the boronic acid are also avail-
able.[9] For the uniformity of this work, we initially synthesized
the azide-containing VBA-probe 13, containing a free boronic
acid (Figure 3A and the Supporting Information, experimen-
tal section), which was obtained by hydroboration of a phenyl-
acetylene derivative followed by removal of the ester through
the conversion and hydrolysis of the organotrifluoroborate.
We equipped a model protein human serum albumin
(HSA), which contained one free cysteine, with a dibenzocy-
clooctyl (DBCO) function using commercially available
DBCO-maleimide 12 (Figure 3B and the Supporting Infor-
mation, Figure S13). The HSA-DBCO was then reacted with
13, demonstrating that VBAs can be used orthogonally to the
strain-promoted alkyne–azide cycloaddition (SPAAC)
between a DBCO and an azide function. Notably, the VBA
is not orthogonal to a CuI-mediated reaction between an
Next, we examined the biocompatibility of the VBAs
using the most reactive alkene 8. In the presence of most
common functional groups such as amines, alcohols, carbox-
ylic acids, and thiols, 8 was stable for at least seven days as
1
observed by H NMR (Supporting Information, Figure S10).
Additionally, we incubated VBA 8 in cell lysate up to 24 h
before addition of tetrazine 1 and observed only marginal
changes in the reaction rate, which indicates that 8 was stable
in the presence of all biomolecules in the cells (Supporting
Information, Figure S11). For possible future applications of
VBAs in live cells, we assessed the toxicity of VBA 8,
tetrazine 1, boric acid, the side product of the CL reaction,
and the dihydropyridazine product on NIH-3T3 cells. No
toxicity was observed after 24 h for concentrations up to
Figure 3. Evaluation of the CL reaction with HSA functionalized with VBA probe 13. A) The structure of the molecules used for the protein modifications.
B) The route used for individual HSA modification steps using 12, 13, and 16. C) Fluorescent image (top) and Coomassie stain (bottom) of SDS-PAGE gels
showing a) the individual modification steps of HSA (50 mm) as shown in B with 12, 13, and 16 (respectively 2, 10, and 10 equiv), b) the time range of the
CL reaction of HSA-VBA (50 mm) and tetrazine 16 (10 equiv), c) the stability of HSA-VBA (0.5 mm) in cell lysate, and d) a concentration range of HSA-VBA
followed by the CL reaction with tetrazine 16 (10 equiv) in cell lysate. [a] At t=0 min, 1% labeling is expected because a premix of fluorescent tetrazine 16
and tetrazine 10 (1000 equiv) is added.
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azide and an alkyne owing to copper-mediated boronic acid
Acknowledgements
degradation.[14] We used tetrazine-BODIPY probe 16 (Fig-
ure 3A and the Supporting Information, experimental sec-
tion) to visualize the VBA-modified HSA protein. Complete
conversion to the dihydropyridazine (HSA-DHP) product
was observed by mass spectrometry (Supporting Information,
Figure S14), and the appearance of a green fluorescent band
was observed by fluorescence imaging of the SDS-PAGE gel
(Figure 3C, a).
This work was financially supported by the Netherlands
Research Institute for Chemical Biology (NRSCB) and
the Institute of Molecules and Materials (IMM) of the
Radboud University in Nijmegen. We thank Dr. Paul B.
White for expert advice in NMR analysis and Prof. Floris
P. J. T. Rutjes for fruitful discussions and proofreading this
manuscript.
As boronic esters are more synthetically accessible than
the free boronic acids, we evaluated the stability of the
pinacol boronic ester of 13 in aqueous media. Pinacol boronic
ester 14 hydrolyzed to the boronic acid in PBS within 15 min
(Supporting Information, Figure S15). We observed similar
labeling results as with the free boronic acid 13, which shows
the suitability of vinylboronic esters as VBA precursors
(Supporting Information, Figure S16). Additionally, we syn-
thesized the VBA precursor 15, which contained a maleimide
for direct labeling of HSA. With VBA-maleimide 15, similar
labeling intensities were observed after the CL reaction with
16 as in the two-step approach described above using the
SPAAC reaction (Supporting Information, Figure S17).
We evaluated the rate of the CL reaction and the stability
of the HSA-VBA protein in cell lysate. Reaction of HSA-
VBA with 10 equivalents tetrazine-BODIPY probe 16 pro-
vided a clear fluorescent signal on a SDS-PAGE gel within
5 min (Figure 3C, b). For this experiment we incubated HSA-
VBA with 16 for the indicated time points before addition of
a 100-fold excess of non-fluorescent tetrazine 10, which
accounts for the faint band observed at 0 min. In addition,
incubation of 1% HSA-VBA (0.5 mm) in cell lysate
(1 mgmLÀ1) before the addition of the tetrazine-BODIPY
probe 16 did not result in a significant loss of signal after 24 h
(Figure 3C, c), which confirms the high stability of VBAs in
the presence of biomolecules. Finally, a dilution series of
HSA-VBA in cell lysate showed that we could visualize the
modified HSA protein by fluorescence imaging in concen-
trations up to 50 nm. At this concentration, a clear HSA band
could no longer be distinguished on the gel when stained with
Coomassie (Figure 3C, d), indicating the sensitivity of the CL
reaction under these conditions.
Keywords: bioorthogonal reactions · cycloadditions ·
protein modification · tetrazines · vinylboronic acid
´
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Received: May 30, 2016
Revised: August 10, 2016
Published online: && &&, &&&&
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Communications
Bioorthogonal Reactions
Bioorthogonal iEDDA reagents: Vinyl-
boronic acids (VBAs) were studied as
non-strained, synthetically accessible,
and water-soluble bioorthogonal reagents
in the Carboni–Lindsey reaction with
dipyridyl-s-tetrazines. The VBAs were
shown to be biocompatible, non-toxic,
and highly stable in aqueous media and
cell lysate. Furthermore, VBAs were used
orthogonally to the strain-promoted
alkyne–azide cycloaddition for protein
modification.
S. Eising, F. Lelivelt,
K. M. Bonger*
&&&& — &&&&
Vinylboronic Acids as Fast Reacting,
Synthetically Accessible, and Stable
Bioorthogonal Reactants in the Carboni–
Lindsey Reaction
6
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