A Bioorthogonal Reaction of N-Oxide and Boron Reagents

Article abstract of DOI:10.1002/anie.201508861

The development of bioorthogonal reactions has classically focused on bond-forming ligation reactions. In this report, we seek to expand the functional repertoire of such transformations by introducing a new bond-cleaving reaction between N-oxide and boron reagents. The reaction features a large dynamic range of reactivity, showcasing second-order rate constants as high as 2.3×10³ M^-1 s^-1 using diboron reaction partners. Diboron reagents display minimal cell toxicity at millimolar concentrations, penetrate cell membranes, and effectively reduce N-oxides inside mammalian cells. This new bioorthogonal process based on miniscule components is thus well-suited for activating molecules within cells under chemical control. Furthermore, we demonstrate that the metabolic diversity of nature enables the use of naturally occurring functional groups that display inherent biocompatibility alongside abiotic components for organism-specific applications. The bond-cleaving reaction between N-oxide and diboron reagents features second-order rate constants as high as 2.3×10³ M^-1 s^-1. Diboron reagents display minimal cell toxicity at millimolar concentrations, penetrate cell membranes, and reduce N-oxides inside mammalian cells. This new bioorthogonal reaction is thus well-suited for chemically activating molecules within cells.

Full text of DOI:10.1002/anie.201508861

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International Edition: DOI: 10.1002/anie.201508861
German Edition: DOI: 10.1002/ange.201508861
Bioorthogonal Chemistry Very Important Paper
A Bioorthogonal Reaction of N-Oxide and Boron Reagents
Justin Kim and Carolyn R. Bertozzi*
Abstract: The development of bioorthogonal reactions has
classically focused on bond-forming ligation reactions. In this
report, we seek to expand the functional repertoire of such
transformations by introducing a new bond-cleaving reaction
between N-oxide and boron reagents. The reaction features
a large dynamic range of reactivity, showcasing second-order
rate constants as high as 2.3 ” 103 mÀ1 sÀ1 using diboron reaction
partners. Diboron reagents display minimal cell toxicity at
millimolar concentrations, penetrate cell membranes, and
effectively reduce N-oxides inside mammalian cells. This new
bioorthogonal process based on miniscule components is thus
well-suited for activating molecules within cells under chemical
control. Furthermore, we demonstrate that the metabolic
diversity of nature enables the use of naturally occurring
functional groups that display inherent biocompatibility along-
side abiotic components for organism-specific applications.
in vitro;[9d] notably, however, the performance of the reaction
in the context of ligation reactions remains unmatched. In
many ways, current bioorthogonal methods reflect an evolu-
tionary history of selection for bond-forming reactions.
Perhaps simpler, faster, and more tolerant bond-cleaving
reactions could be constructed anew from a bioorthogonal set
outside the ligation-centric compendium. Such reactions
would fulfill the demand for novel triggers, which, in
combination with a generic immolative linker,[9a–c,e] would
comprise a general scheme for the uncaging of biological
effectors. We thus turned to new sources for the development
of bioorthogonal reactions.
Organism-specific variations in biological reactivity allow
the potential discovery of bioorthogonal reaction partners not
just from outside but also within biology. Natural organisms
possess considerable metabolic diversity, such that functional
groups endogenous to one species (e.g., terminal alkynes)[10]
can be orthogonal in another. Trimethylamine N-oxide
(TMAO, 1; Figure 1) provides another case in point. Elasmo-
branchs,[11] deep-sea teleosts,[12] and other osmoconforming
T
he field of bioorthogonal chemistry strives to meet the
demands for methods that can facilitate the molecular
analysis of biological processes. Its foundational applications
were in the chemical targeting of biomolecules with probes
and affinity reagents, both in cultured cells and living
organisms, and in building hybrid biomolecules for therapeu-
tic applications.[1] In the context of these pursuits, chemists
have compiled a reaction compendium that includes the
Staudinger ligation,[2] copper-mediated[3] and metal-free[4]
azide–alkyne cycloadditions, and tetrazine ligations.[5]
An emerging application of bioorthogonal chemistry is
the modulation of the activity of a target molecule under the
control of a small-molecule switch.[6] Small-molecule-based
chemical uncaging strategies are a compelling complement to
photochemical activation methods[7] in applications demand-
ing tissue penetrance, subcellular targeting at the multi-cell
level, or cellular pretargeting. To this end, reactions that
impart new functionality on their targets beyond the attach-
ment of probes are particularly useful but remain relatively
rare in the compendium—recent advances in transition-
metal-[6a,8] and non-metal-mediated[6b, 9] uncaging strategies
notwithstanding.
The tetrazine ligation reaction has recently been adapted
by Chen and co-workers for the bioorthogonal uncaging of
modified lysine residues on target proteins within cells[6b] and
by Robillard and co-workers for the activation of prodrugs
Figure 1. Determination of kinetic parameters. A) The reaction between
TMAO (1) and para-nitrophenylboronic acid (2) features biologically
compatible reagents and byproducts. B) The reaction of N,N-dialkylani-
line-derived N-oxide 6 and phenylboronic acid is three orders of
magnitude faster. C) General scheme for the use of a triggered
reaction for the uncaging of biological effectors.
[*] Dr. J. Kim, Prof. C. R. Bertozzi
Department of Chemistry and Howard Hughes Medical Institute
Stanford University
380 Roth Way, Stanford, CA 94305 (USA)
E-mail: bertozzi@stanford.edu
marine invertebrates acquire high concentrations of urea to
maintain high internal osmolalities. The chaotropic effects of
urea are countered by proportionally high concentrations of
the potent kosmotrope TMAO.[13] In deep-sea fish, where the
kosmotropic effects of TMAO are crucial for countering high
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201508861.
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hydrostatic pressures, its concentration can reach up to
400 mm.[12] Even at extreme levels, N-oxides are inert to
biomolecules. Critically, for most organisms, including
humans, TMAO is not a component of osmoregulation.
Very low concentrations of this analyte (ca. 1 mm) are present
in the serum as a metabolic byproduct of gut-microbiota-
derived trimethylamine.[14] By refining our notion of bioor-
thogonality and looking to nature for inspiration, we have
identified N-oxides as potential reaction partners with
inherent biocompatibility.
Similarly, boron-containing compounds fit the description
that they are sometimes, yet infrequently, found in biological
systems. Indeed, natural-product antibiotics such as boromy-
cin incorporate boron into their structures. Furthermore,
boronic acids are widely appreciated for their relatively
benign toxicology profile[15] and have been incorporated into
chemical biology methods, for example, in fluorogenic
tetraserine-binding[16] and peroxide-responsive probes.[17]
Given the positive qualities of this functional group, we
wished to combine N-oxides with boronic acids to generate
a new bioorthogonal reaction.
The hydroxydeboration reaction between TMAO and
alkyl boranes was discovered by Kçster and Morita in 1967.[18]
Its remarkable functional-group tolerance and quantitative
nature continue to render it a mild, reliable alternative to
hydrogen peroxide mediated deborylative oxidations under
conditions challenging for total synthesis.[19] Kabalka and
Hedgecockꢀs subsequent report that the dihydrate of TMAO
is just as effective as its anhydrous form[20] offered the first
hint that the reaction would work in aqueous media,
a necessary criterion for bioorthogonality.
We first set out to determine the kinetic parameters for
the reaction between TMAO (1) and para-nitrophenylbor-
onic acid (2) in water. The reaction progress was monitored
by measuring the UV/Vis absorption of the para-nitrophenol
product (3) under pseudo-first-order conditions (Figure 1).
We determined a second-order rate constant of 2.83 Æ 0.05 ”
10À6 mÀ1 sÀ1 for the reaction at room temperature, which is
several orders of magnitude below the current standards of
bioorthogonal reactivity[1b–e] but nonetheless a starting point
for kinetic optimization.
We thus turned to the second factor, namely the migratory
aptitude of the boronic acid. We anticipated that significant
gains could be achieved through direct weakening of the
dissociating bond. Accordingly, we considered exchanging the
À
À
migrating C B bond for a B B bond. The reduction of
N-oxides by bis(pinacolato)diboron (B2pin2), first reported by
Carter et al. in 2002,[24] exploits the cleavage of a weak B B
bond (68 kcalmol ) and the formation of strong B O bonds
À
À1
À
(125 kcalmolÀ1) to provide an enthalpic driving force of about
180 kcalmolÀ1. This powerful reaction, which superficially
effects nothing more than deoxygenation, has seen scant
use.[25] We immediately sought to characterize its reaction
kinetics and adapt it for biological systems.
We first evaluated the kinetics of the reaction using
N-oxide 6 (Figure 2A). Impressively, fluorescence measure-
ments with a stopped-flow fluorometer under pseudo-first-
order conditions revealed a second-order rate constant of
8.05 Æ 0.076 ” 102 mÀ1 sÀ1 in PBS (pH 7.4). We also synthesized
the HaloTag linker-bound profluorophore 8, designed for use
in cell-labeling studies (see below), and found the second-
order rate constant for its reaction with B2pin2 to be even
higher at 1.71 Æ 0.043 ” 103 mÀ1 sÀ1, which is likely due to steric
relaxation (Figure 2B).
We then explored the reaction on a biomolecule. The
34 kDa HaloTag protein was ligated to compound 8 to
produce HaloTag–8, which was purified by size-exclusion
chromatography and treated with B2pin2 under pseudo-first-
order conditions. Analysis by stopped-flow fluorometry
revealed a second-order rate constant of 2.30 Æ 0.073 ”
103 mÀ1 sÀ1 (Figure 2C). It should be noted, however, that
pseudo-first-order kinetics obtained under saturating condi-
tions can obscure important information regarding the
deactivation of the diboron reagent through sequestration
or off-target reactivity. To address this issue, a 64 kDa GFP–
HaloTag fusion protein was expressed and ligated to com-
pound 10 to produce GFP–HaloTag–10. Conjugate 11
(500 nm) was then treated with stoichiometric to slightly
superstoichiometric quantities of B2pin2 (1–25 mm) and ana-
lyzed by in-gel fluorescence imaging. Figure 2D shows that 5–
10 equivalents of B2pin2 are necessary to fully reduce the
conjugated fluorophore 10 in < 15 min. Considering that
2 equivalents of reductant are theoretically required, this
experiment validates the robustness of the reaction and
suggests minimal off-target reactivity.
Having confirmed the compatibility of our N-oxide/
diboron reaction with proteins, we explored the viability of
the reaction in mammalian (Jurkat) cell lysate (Figure 2E).
Lysates were made to a final protein concentration of
1 mgmLÀ1, and variable concentrations of B2pin2 were
reacted with a 1 mm solution of probe 6. The fluorescence
intensities were then measured after 30 min. Consistent with
prior kinetic data, adding just 5 equivalents of B2pin2 was
sufficient to drive the reaction to completion in mammalian
cell lysate within the allotted time.
An organism-centric approach to bioorthogonal reaction
development, a central facet of our thesis, enables the
implementation of reactions that would be overlooked
under more stringent searches for abiotic reaction partners.
As a point of emphasis, we also performed the preceding
À
Conjecturing that the concomitant C B bond migration
À
and N O bond cleavage events are rate-limiting (Figure 1A),
we expected that the principal determinants of the reaction
rate would be the leaving-group ability of the tertiary amine
and the migratory aptitude of the boronic acid. Focusing first
on the former, the reaction could indeed be accelerated by
turning to N,N,N-dialkylaryl N-oxides, which produce supe-
rior leaving groups compared to trialkyl amines.[21] The
kinetic parameters for this reaction were measured by
employing the fluorogenic N,N,N-dialkylaryl N-oxide 6,[22]
which was obtained through mCPBA mediated oxidation of
the parent rhodol fluorophore.[23] The reaction of N-oxide 6
with phenylboronic acid in phosphate-buffered saline (PBS,
pH 7.4) proceeded with a second-order rate constant of 1.28 Æ
0.11 ” 10À3 mÀ1 sÀ1 (Figure 1B). While gratified by the rate
acceleration of three orders of magnitude relative to our
baseline reaction using TMAO, we sought even faster rates
for use in biological systems.
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Figure 2. Evaluating the bioorthogonality of diboron reagents. A) B2pin2 rapidly reduces N-oxide 6 in PBS (pH 7.4). The second-order rate constant
was calculated from fluorescence measurements of fluorophore 7 under pseudo-first-order conditions with saturating concentrations of B2pin2.
B) Steric relaxation around the N-oxide enhances the reaction rate, and the second-order rate constant exceeds 103 mÀ1 sÀ1. C) N-Oxide 9
conjugated to a 34 kDa HaloTag protein rapidly reacts with B2pin2 in PBS. The reaction conversions for the lowest and highest B2pin2
concentrations used in the kinetics experiments are displayed. Data represent the mean of triplicate experiments. The vertical dotted lines indicate
the half-lives of the reactions at the respective saturating concentrations of B2pin2. D) Bis(N-oxide) profluorophore 10 (500 nm) was conjugated to
a 64 kDa GFP–HaloTag fusion and treated with stoichiometric to slightly superstoichiometric quantities of B2pin2. The red and green channels
represent the TAMRA and GFP signals, respectively. E) N-Oxide profluorophore 6 (1 mm) was reacted with 0 (black), 5 (light blue), 50 (middle
blue), or 500 mm (dark blue) B2pin2 in Jurkat and E. coli cell lysates containing 1 mgmLÀ1 of protein; the fluorescence was then normalized to the
level of a positive control based on fluorophore 7 (violet). Data represent the mean of triplicate experiments. Error bars represent the standard
deviation. F) Time-dependent fluorescence measurements reveal the stability of N-oxide profluorophore 6 to mammalian cell lysate. Data
represent the mean of triplicate experiments. G) The viability of three mammalian cell lines at a range of B2pin2 concentrations was evaluated
after 24 hours using an MTT assay. Data represent the mean of triplicate experiments. Error bars represent the standard deviation.
experiment in E. coli cell lysate. E. coli, like several other
facultative anaerobes, possess an N-oxide reductase[26] and
were predicted to be incompatible with our method. Indeed,
the results confirmed this hypothesis: Our negative control
containing no B2pin2 displayed high levels of fluorescence
(Figure 2E). Time-dependent fluorescence measurements of
N-oxide probe 6 in E. coli cell lysate confirmed the gradual
reduction of the N-oxides with a half-life of about 15 min
(Figure 2F). Gratifyingly, no degradation was observed in
mammalian cell lysate over 11 hours. Therefore, the N-oxide/
diboron reaction is not suitable for all systems, but should still
be powerful in many.
To ascertain the toxicity of the diboron reagents to cells,
we subjected three mammalian cell lines (HEK293T, HeLa,
and MEF) to the MTT cell viability assay (Figure 2G). Each
cell line was treated with twofold serial dilutions of B2pin2
starting at 1 mm in 0.5% DMSO/DMEM (DMEM = Dulbec-
coꢀs modified eagle medium). The maximum concentration of
B2pin2 was dictated by its solubility limitations in the medium.
The MTT assays indicated that the human cell lines are
relatively insensitive to the diboron reagents, at least at the
concentrations that were tested, and whereas for MEF cells,
some toxicity was observed at higher diboron levels, the
IC50 value was > 1 mm. No aberrations in cell morphology
were observed for HEK293T cells upon treatment with 1 mm
B2pin2 for 24 hours (Figure S5). Furthermore, no gross
changes in the time to confluency were observed between
cells treated with B2pin2 and DMSO vehicles.
Next, we demonstrated the mutual orthogonality of our
N-oxide/diboron reaction with a representative cohort of
bioorthogonal reactions commonly in use today: the amino-
oxy–aldehyde condensation, the azide–cyclooctyne cycload-
dition, and the tetrazine–cyclopropene ligation (Figure 3). To
showcase the robustness of our new reaction, we executed
these experiments amidst the complexity of a cellular system.
Utilizing a combination of chemical, genetic, and metabolic
engineering techniques, four populations of HEK293T cells
were endowed with distinct cell-surface modifications. Pop-
ulation 1 was modified by sodium periodate to display cell-
surface aldehydes derived from sialic acid moieties;[27]
population 2 was modified with azides through the metabolic
incorporation of Ac4ManNAz;[2] population 3 was modified
with a cyclopropene through the metabolic incorporation of
Ac4ManNCp;[28] and population 4 was modified by cell-sur-
face expression of an N-terminal HaloTag–EGFR fusion
protein followed by the ligation of HaloTag-linked bis(N-
oxide) TAMRA profluorophore 10. Each cell population was
labeled with a distinct combination of Hoechst 33342 and
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Figure 3. The N-oxide/diboron reaction is compatible with common
bioorthogonal reactions. A) HEK293T cell surfaces were modified with
aldehydes, N-oxides, azides, or cyclopropenes, mixed, and then treated
with a cocktail of bioorthogonal reagents. Each population was
analyzed by flow cytometry for reaction with aminooxy-Alexa Fluor 488
(light blue), B2pin2 (middle blue), DIBAC-Cy5 (dark blue), and tetra-
zine-Cy7 (violet). The mean normalized fluorescence intensity of each
fluorophore from three biological replicates is shown. Error bars
represent the standard deviation. B) N-Oxide-modified HEK293T cells
were treated with aminooxy, DIBAC, and tetrazine reagents with or
without a diboron reagent. N-Oxides are not reactive towards hydroxyl-
amines, cyclooctynes, or tetrazines.
Figure 4. B2pin2 activates N-oxides on cytosolic proteins in mamma-
lian cells. HEK293T cells transfected with GFP–HaloTag were incubated
with 100 mm of profluorophore 10, washed, treated with 0 or 100 mm
B2pin2, and then imaged by confocal microscopy after 45 min. The
merge is a composite of GFP, TAMRA, and Hoechst 33342 fluores-
cence with a bright-field image.
Fast rates of ligand hydrolysis, even for bulky pinacol
derivatives, enable the utilization of diboron species even
amidst the diols present in biological milieu. B2(OH)4 is as
nontoxic and competent at reducing N-oxides (Figures S2–
S4) as B2pin2. Furthermore, the tolerance of the reaction to
sterically demanding ligand structures on the boron reagents
indicates that even diboron species transiently ligated to
saccharides or other cellular diols may still be competent at
reducing N-oxides.
In conclusion, we have sought new reactions to expand the
functional repertoire of bioorthogonal transformations
beyond bond-forming ligation reactions. We initiated our
search for new bioorthogonal reactivity by relaxing its
definition. Rather than exploring chemical space completely
outside the realm of biology, we focused our search for
molecular components within organisms that are metabol-
ically orthogonal to models of human disease. As a result, we
could readily find novel reaction components with inherent
biocompatibility.
We have thus described a powerful new reaction between
N-oxide and boron reagents. The reaction features fast
reaction kinetics that rival the fastest bioorthogonal reactions
reported to date while introducing functionality that has yet
to gain significant attention. Owing to the benignity of the
reaction conditions towards live cells and its intracellular
compatibility, this reaction holds much promise as a method
for the small-molecule activation of biomolecules in vivo.
We envision that this method will find application in the
spatially and temporally controlled chemical uncaging of
important naturally occurring trialkyl amines, such as the
epigenetically relevant dimethyllysine histone residues.[29]
Furthermore, this reaction acts as a powerful trigger, which,
when interfaced with chemical or biological effectors through
immolative linkers[9] containing an N-oxide, should enable the
small-molecule activation of enzymes and chemotherapeutics.
Syto 41 nuclear stains, combined, then treated with a reagent
cocktail consisting of 10 mm aniline, 100 mm aminooxy-Alexa
Fluor 488, 10 mm DIBAC-Cy5, 20 mm tetrazine-Cy7, and
100 mm B2pin2 in pH 6.7 PBS. B2pin2 was applied at a concen-
tration of 100 mm to rigorously challenge the aminooxy
condensation reaction, the reaction most likely to be per-
turbed by diboron reagents. The mixture of cells was then
analyzed by flow cytometry.
Impressively, each of the cell types was labeled highly
selectively by its corresponding bioorthogonal partner, and
the labeling efficiency was undiminished in the presence of
the complete complement of reactive functional groups
(Figure 3). Notably, aminooxy functional groups are fully
compatible with diboron reagents, and the N-oxide functional
group is neither reduced by nor reactive towards tetrazines,
which are known to be susceptible to degradation through
nucleophilic attack.
Finally, to demonstrate the utility of the N-oxide/diboron
reaction in an intracellular uncaging application, we first had
to determine the membrane permeability of each of the
compounds and evaluate their bioorthogonality in live cells.
We transiently transfected HEK293T cells with a cytosolic
GFP–HaloTag fusion construct and then treated the cells with
100 mm profluorophore 10. After three washes, the cells were
treated with 1 mm, 100 mm, 10 mm, or 0 mm B2pin2 for 45 min
(Figures 4; see also the Supporting Information, Figure S1).
The TAMRA signal increased upon addition of B2pin2 and
also co-localized with the GFP signal. These data indicate that
both profluorophore 10 and the diboron reagent are cell-
permeable, that the reaction can be performed intracellularly,
and, impressively, that fluorophore activation can be
observed at a diboron concentration of 10 mm with full
activation requiring at most 100 mm B2pin2, which is well
below the toxic levels.
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We acknowledge Dr. Paresh Agarwal, Dr. Daniel Liu, Peter
Robinson, and Mason Appel for critical discussions, and Prof.
Susan Marqusee for generous access to stopped-flow instru-
mentation and HaloTag protein. J.K. acknowledges the Miller
Institute for Basic Research in Science at UC Berkeley for
a postdoctoral fellowship and research funding. This research
was supported by the NIH (GM058867).
Keywords: bioorthogonal chemistry · diboron reagents ·
N-oxides · small-molecule activation · uncaging
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Received: September 21, 2015
Published online: November 16, 2015
Angew. Chem. Int. Ed. 2015, 54, 15777 –15781
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 15781