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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.
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 15777 –15781