Fast and Tight Boronate Formation for Click Bioorthogonal Conjugation

Article abstract of DOI:10.1002/anie.201510321

A new click bioorthogonal reaction system was devised to enable the fast ligation (k_ON≈340 m^-1 s^-1) of conjugatable derivatives of a rigid cyclic diol (nopoldiol) and a carefully optimized boronic acid partner, 2-methyl-5-carboxymethylphenylboronic acid. Using NMR and fluorescence spectroscopy studies, the corresponding boronates were found to form reversibly within minutes at low micromolar concentration in water, providing submicromolar equilibrium constant (K_eq≈10⁵-10⁶ m^-1). Efficient protein conjugation under physiological conditions was demonstrated with model proteins thioredoxin and albumin, and characterized by mass spectrometry and gel electrophoresis.

Full text of DOI:10.1002/anie.201510321

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International Edition: DOI: 10.1002/anie.201510321
German Edition: DOI: 10.1002/ange.201510321
Bioconjugation
Fast and Tight Boronate Formation for Click Bioorthogonal
Conjugation
Burcin Akgun and Dennis G. Hall*
Abstract: A new click bioorthogonal reaction system was
devised to enable the fast ligation (kON ꢀ 340 mÀ1 sÀ1) of
conjugatable derivatives of a rigid cyclic diol (nopoldiol) and
a carefully optimized boronic acid partner, 2-methyl-5-carbox-
ymethylphenylboronic acid. Using NMR and fluorescence
spectroscopy studies, the corresponding boronates were found
to form reversibly within minutes at low micromolar concen-
tration in water, providing submicromolar equilibrium con-
stant (Keq ꢀ 105–106 mÀ1). Efficient protein conjugation under
physiological conditions was demonstrated with model pro-
teins thioredoxin and albumin, and characterized by mass
spectrometry and gel electrophoresis.
boronate formation in water.[12] This information would
enable a systematic optimization of both diol and boronic
acid partners. As a condensation reaction, boronic ester
formation is intrinsically unfavored in water owing to Le
Châtelierꢀs principle. Thermodynamically, the process is
driven solely by the enthalpy of ring formation because the
À
À
balance of bond enthalpy is neutral (two O H and two B O
bonds are broken to form four similar bonds). It is also well-
established that hindered, pre-organized vicinal diols mitigate
the loss of entropy in the diol substrate, and are thus
preferred.[13] It is therefore unsurprising that pinanediol
affords some of the most hydrolytically robust boronic
esters.[14] There is no knowledge, however, about the rates
of pinanediol boronate formation in aqueous solvents, the
question of its reversibility, and the structural requirements
(sterics and electronics) for an optimal boronic acid partner.
Herein, using the conjugatable pinanediol derivative nopol-
diol, we report the design and proof-of-concept of a fast (kON
ꢀ 340 mÀ1 sÀ1) and tight-binding (Keq ꢀ 105–106 mÀ1) click bor-
onate bioconjugation system (Figure 1).[15]
T
he development of rapid and bioorthogonal chemical
reactions has greatly expanded the utility of bioconjugation
chemistry in the service of chemical biology, even allowing
molecular imaging in live cells or animals.[1] To this end, click
reactions, spontaneous bond-forming processes that can occur
rapidly in aqueous media, are particularly desirable.[2]
Unfortunately, the repertoire of click reactions between
abiotic and biocompatible functional groups is relatively
small, and some of the most popular and efficient reactions
present inconveniences, such as slow rates and side-reactions
that may limit some of their applications in living systems.[3,4]
Most importantly, many common bioorthogonal reactions are
not mutually orthogonal due to possible cross-reactions,[5] and
thus new reactions would provide more options for inter-
rogating cellular processes in tandem. In this context, boronic
ester formation, a process better known in the field of
carbohydrate sensing,[6] has remained an underexplored
option in bioconjugation despite recent applications of
boronic acids in surface immobilization,[7] detection of
reactive oxygen/nitrogen species,[8] and protein modifica-
tion.[9] Simplicity (no catalyst required), bioorthogonality
(boronic acids complex biological diols with low affinities),
fast on-rates, and potential reversibility are many possible
attributes of boronate bioconjugation. Although a recent
phenylboronic acid–salicylhydroxamic acid conjugation
system shows promise, it displays moderate binding affinity
(Keq ꢀ 17800 mÀ1).[10] The design of a tight boronate conjuga-
tion system is hampered by mechanistic ambiguities[11] and
a dearth of comprehensive kinetic data (kON, kOFF, Keq) on
Figure 1. Design of a bioorthogonal click boronate conjugation using
conjugatable nopoldiol derivatives and optimal arylboronic acids in
neutral aqueous media.
To identify fast-forming boronates in aqueous medium,
a model water-soluble nopol-PEG-diol (1a, Table 1) was
synthesized (Supporting Information, Scheme S1).[16] We
1
chose H NMR spectroscopy as the initial method to screen
various arylboronic acids owing to its convenience, even
though its capability to monitor fast reactions is limited.[17]
The low intrinsic sensitivity of NMR makes it difficult to
detect very low concentrations (< 100 mm), therefore it
requires higher reaction concentration at which fast processes
(> 50 mÀ1 sÀ1) are not readily monitored. Moreover, early
rates (< 40 s) cannot be captured owing to the essential
gradient shimming. Thus, because boronate formation is
likely reversible, forward rate data obtained from NMR
kinetics provide an underestimation of rates, which is how-
ever sufficient for a preliminary comparison and identifica-
[*] B. Akgun, Prof. Dr. D. G. Hall
Department of Chemistry, 4-010 CCIS, University of Alberta
Edmonton, Alberta, T6G 2G2 (Canada)
E-mail: dennis.hall@ualberta.ca
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.201510321.
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Table 1: Results of the hydrolytic stability study, forward rate constant
kON, and association constant measurement Keq
tion of promising reagents. In this way, NMR analysis allowed
us to rapidly obtain relative hydrolytic stability data and
forward rate constants (kON, mÀ1 sÀ1) for a large set of
arylboronic acids.[16] It was found that ortho-methyl or
isopropyl groups improved the hydrolytic stability likely
because of their bulkiness, while halogens and other electron-
withdrawing groups decreased boronate stability, which is
probably due to the increased acidity of these arylboronic
acids. Hydrolytic stability showed a near-linear correlation
with the acidity of arylboronics.[11d,16] This is indeed consistent
with a recent report suggesting that more acidic arylboronic
acids tend to form the sp3 trihydroxyborate, a “side-product”
shown to be less reactive compared to a neutral (sp2) boronic
acid, thereby shifting the equilibrium to the left and lowering
boronate stability (Scheme 1).[11g] Moreover, the ortho-methyl
[a]
Entry
(1a/b)
2a–k: R1, R2, R3
Hydrolytic
stability[b]
Keq
kON
”103
[mÀ1sÀ1
]
[d]
(3/1a or 4/1b) [mÀ1
]
[c]
substituent slowed down boronate formation (lower kON
)
1 (1a)
2 (1a)
3 (1a)
4 (1b)
5 (1b)
6 (1b)
7 (1b)
8 (1b)
9 (1b)
2a: Me, H, H
2b: F, H, H
2c: CN, H, H
2a: Me, H, H
2b: F, H, H
2c: CN, H, H
2d: Me, H, CO2Me 4d/1b: 91:9
2e: Me, CO2Me, H 4e/1b: 90:10
3a/1a: 87:13
3b/1a: 78:22
3c/1a: 70:30
4a/1b: 93:7
4b/1b: 84:16
4c/1b: 83:17
–
–
–
180
27
25
120
91
130
330
15
25
40
1.6Æ0.1
18Æ4
while ortho-halogens and especially a cyano group acceler-
ated it to a significant measure. The proposed reaction
mechanism starts with the exchange of one of the two hydroxy
groups of the boronic acid, which is likely the rate-determin-
ing step, followed by a fast cyclization to form the Lewis acidic
boronic ester resting as a tetrahedral hydroxyboronate ion
(Scheme 1).
>50[e]
2.3Æ0.2
33Æ2
>50[f]
6.9Æ0.6
7.8Æ0.7
3.3Æ0.6
1.0Æ0.2
>50[f]
2 f: Me, H, OMe
4 f/1b: 92:8
4g/1b: 94:6
10 (1b) 2g: Me, OMe, H
11 (1b) 2h: F, H, CONMe2 4h/1b: 75:25
12 (1b) 2i: F, H, OMe
13 (1b) 2j: F, OMe, H
14 (1b) 2k: CN, H, CO2Me 4k/1b: 78:22
4i/1b: 82:18
4j/1b: 85:15
>50[f]
18Æ1
12
>50[f]
[a] See the Supporting Information, Table S1 for more detailed screening
conditions. [b] Hydrolytic stability of boronates 3/4 was studied at 1 mm
concentration by 1H NMR in 0.1m D2O phosphate buffer (pD 7.4)/
CD3CN (65:35). The integral ratio of (CH3)3/4/(CH3)1 was monitored after
24 h and recorded as 3/1a or 4/1b. [c] Association constant measure-
ment (Keq, mÀ1) was performed via 1H NMR as described in the
Supporting Information. [d] Second-order rate constant (kON, mÀ1sÀ1
)
was monitored by 1H NMR. Compound 2 (1 equiv, 1 mm) and diol 1a or
1b (1 equiv, 1 mm) were mixed in 0.05m D2O phosphate buffer (pD 7.4)/
CD3CN (70:30) (The disappearance of (CH3)1 was monitored and
converted to a forward rate constant kON). Average of at least three
measurements Æ standard deviation. [e] This reaction showed a circa
50% conversion in 30 seconds at room temperature. It slowed down at
08C and provided kON =49Æ2 mÀ1sÀ1. [f] This reaction could not be
monitored by NMR owing to its very fast conversion.
Scheme 1. Proposed boronate formation mechanism.
Our initial NMR-based screening with diol 1a led to
a selection of three promising boronic acids (Table 1,
entries 1–3). These superior reagents are ortho-tolylboronic
acid (2a), which provided better boronate stability than
phenylboronic acid,[16] ortho-fluorophenylboronic acid (2b),
and ortho-cyanophenylboronic acid (2c), which both showed
excellent kON values. We suspected that the proximal oxygen
of 1a might be detrimental by assisting hydrolysis. Thus,
another nopol-PEG-diol (1b) was synthesized to examine this
reactivity compared to boronic acids 2a–c, and a decrease in
Keq was noted (compare entry 4 vs. 7,8; 5 vs. 11).
On the other hand, whereas an electron-donating group
(OMe) impeded the reactivity (lower kON), it improved Keq
specifically when placed in the para position (compare entry 4
vs. 9,10; 5 vs. 12,13). Even though ortho-fluoro derivatives
2h–2j enabled high reactivity, their lower hydrolytic stability
caused concerns towards their use under the conditions of
bioorthogonal conjugation at micromolar concentrations.
Boronic acid 2k was eliminated because of its lower Keq and
its susceptibility to deboronation. In the end, the ortho-
tolylboronic acid derivative 2d was selected because it best
combines a high reactivity with excellent hydrolytic stability
for the resulting boronates. Moreover, it is easily synthesized
and the carboxyl handle is ideal for conjugation. Yet because
the reaction of ortho-fluorophenylboronic acid derivative 2h
potential issue on the stability and kON.
[16] Boronic acids 2a–c
were then tested with diol 1b. To our satisfaction, their
hydrolytic stability and kON both improved (entries 4–6).
Other arylboronic acids were also evaluated with diol 1b, but
2a–c remained superior. We then proceeded to design,
synthesize, and evaluate conjugatable derivatives of 2a–c
(entries 7–14). Their equilibrium constants (Keq, mÀ1) were
also determined via 1H NMR beside their forward rate
constants (kON). Consistent with our initial screening results,
the presence of an electron-withdrawing group (CO2Me or
CONMe2) at either meta or para positions enhanced the
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was too fast to be monitored by NMR, a more sensitive
fluorescence quenching assay was designed to compare the
forward rate constants of 2d and 2h.
A dansyl-tagged nopoldiol (5) and dabsyl derivatives of
the corresponding boronic acids (6a–b) were synthesized,[16]
and initial forward rate constants (kON) were determined at
10 mm concentration (Figure 2). The results (6a; 340 Æ
Figure 3. Competitive effect of biological polyols on boronate forma-
tion. Boronate conversions were immediately monitored and deter-
mined by HPLC under both UV and fluorescence after diol 5 and 2d/
2h were allowed to react together for 30 minutes.[16]
Figure 2. Kinetic analysis using a fluorescence quenching experiment
to measure the initial rate of boronate formation. Reported kON and Keq
are the average of at least three measurements.[16]
40 mÀ1 sÀ1 and 6b: 1200 Æ 300 mÀ1 sÀ1) are significantly higher
1
than that obtained via H NMR spectroscopy. As indicated
above, it is likely that the NMR method underestimated the
actual rates of these fast reactions. Their rough Keq were also
determined by allowing the corresponding boronate products
(7a,7b) to reach equlibrium in the fluorescence quenching
assay, giving Keq values of about 15 ” 106 and 1.3 ” 106 mÀ1 for
ortho-methyl and ortho-fluoro derivatives, respectively. High
1
Keq values for substrates 5 and 6 relative to H NMR values
(for 2d/2h and 1b) may be due to hydrophobic and p
interactions between fluorescent moieties, dansyl and dabsyl.
Nonetheless, equilibration times obtained from both methods
are consistent, with 6a/7a taking about 20 min to reach
a steady state made of 85% 7a (from both directions, at 10 mm
conc.), whereas 6b/7b needed only 2 min to reach 60% of
7b.[16]
A biological competition assay was designed to determine
whether the optimal nopoldiol boronates compete favorably
with biological polyols such as glucose (4–7 mm), fructose
(8 mm), or catecholamines (ca. 0.0014 mm) found in the blood
stream.[6a,18] Thus, diol 5 was allowed to form boronates with
2d and 2h in the presence of a mixture of these biocompe-
titors used at concentrations higher than that found in the
blood (Figure 3).[16] This experiment was analyzed by HPLC.
Fortunately, 2d preserved its high affinity towards diol 5 in the
presence of the biocompetitors. In contrast, 2h showed none
or little conversion, even in the absence of the biocompetitors.
Although 2h showed promise at higher concentrations, its
conversion to boronate product is too low at micromolecular
concentrations to be useful in bioconjugation.
Figure 4. Labeling of boronic acid modified BSA with nopoldiol-fluo-
rescein-diol (10) and gel results. A) Modification of free cysteine with 8
or 9 and labeling with 10. B) Time-dependent and C) dose-dependent
fluorescent labeling of BSA on gel containing phosphate buffer. Protein
loading was determined by Coomassie blue staining. Compound 9 is
a negative control probe devoid of a boronic acid. D) Fluorescent
labeling of BSA on gel containing tris buffer, a competing triol that
causes breakdown of conjugates.
cysteine, was allowed to react with the boronyl-containing
maleimide 8 to form a boronic acid–BSA adduct (Figure 4A).
Then, click conjugation with fluorescein-derivatized nopol-
diol 10 was conducted in both time and dose dependent
fashion and monitored by gel electrophoresis and gel
fluorescence imaging (Figure 4B,C). As depicted in Fig-
The optimal boronic acid derivative 2d, which fulfills the
criteria of bioorthogonality, reactivity and stability, was used
to demonstrate boronate ligation on proteins. Towards this
end, bovine serum albumin (BSA), which contains one free
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ure 4B, the reaction between 10 and the boronic acid–BSA
conjugate was complete within 5 minutes when 100 mm of 10
was employed. Even at lower concentrations of 8 and 10 (20
and 40 mm), a significant level of conjugation was detected
(Figure 4C). As expected, when a maleimide adduct 9 devoid
of a boronic acid head was used, no labeling was detected.[16]
With this promising result in hand, another selective
protein labeling experiment was performed. This time,
thioredoxin (Trx, 11.7 kDa; 50 mm), a protein with a single
disulfide, was reduced with TCEP (1 mm) overnight followed
by functionalization with boronyl-containing maleimide 8
(100 mm) in ammonium acetate buffer for 30 minutes
(Figure 5). Following the addition of 1b (200 mm) to the
a rigid diol was studied thoroughly in water with an
optimization of both reaction partners, thus providing useful
knowledge of this important condensation process. Measured
kON rates are faster than many existing bioconjugation
systems, and a high Keq enables high conversion with proteins
under physiological conditions. Coupled with the ability to
site-specifically encode boronic acid containing aminoacids in
proteins,[9b,c] reversibility can be an additional asset when
target turnover or time-based profiling are required. This
preliminary account also suggests applications in affinity
purification, surface immobilization, and materials chemistry.
Acknowledgements
This work was funded by the Natural Sciences and Engineer-
ing Research Council (NSERC) of Canada (Discovery Grant
to DGH) and the University of Alberta. BA thanks Alberta
Innovates—Technology Futures for a Graduate Scholarship.
We thank Dr. Sylvain Bernard (preliminary experiments), Ed
Fu (HPLC), Bela Reiz (Mass Spectrometry Laboratory),
Gareth Lambkin (Biological Services), Nupur Dabral and
Mark Miskolzie (NMR Laboratory), and Prof. Ratmir Derda
for their help and suggestions.
Keywords: bioorthogonality · boronates · boronic acids ·
click chemistry · protein labeling
How to cite: Angew. Chem. Int. Ed. 2016, 55, 3909–3913
Angew. Chem. 2016, 128, 3977–3981
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In conclusion, we have developed a click bioorthogonal
reaction system enabling the fast ligation (ca. 340 mÀ1 sÀ1) of
easily synthesized, conjugatable derivatives of nopoldiol and
2-methyl-5-carboxyphenylboronic acid (2d) to form tightly
bound boronates with submicromolar equilibrium dissocia-
tion constant. For the first time, boronate formation with
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Received: November 6, 2015
Published online: February 23, 2016
Angew. Chem. Int. Ed. 2016, 55, 3909 –3913
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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