Synergic "click" Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells

Article abstract of DOI:10.1021/jacs.7b08693

Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 μM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low μM concentrations, in neutral buffer, with a rate constant of 9 M^-1 s^-1 as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 μM, thus confirming the potential of this system in biological applications.

Full text of DOI:10.1021/jacs.7b08693

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Article
Synergic ‘Click’ Boronate/Thiosemicarbazone System for
Fast and Irreversible Bioorthogonal Conjugation in Live Cells
Burcin Akgun, Caishun Li, Yubin Hao, Gareth Lambkin, Ratmir Derda, and Dennis G Hall
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 11 Sep 2017
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Journal of the American Chemical Society
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Synergic ‘Click’ Boronate/Thiosemicarbazone System for Fast and Irreversible
Bioorthogonal Conjugation in Live Cells
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Burcin Akgun, Caishun Li, Yubin Hao, Gareth Lambkin, Ratmir Derda, and Dennis G. Hall*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
ABSTRACT: Fast, high-yielding and selective bioorthogonal 'click' reactions employing non-toxic reagents are in high demand for
their great utility in the bioconjugation of biomolecules in live cells. Although a number of click reactions were developed for this
purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or
dual-labeling applications. Recent reports highlighted the potential of boronic ester formation as a bioorthogonal click reaction be-
tween abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process, however it is intrinsically reversible in
aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-
functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and non-toxic to
HEK293T cells at concentrations as high as 50 µM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that
forms rapidly and irreversibly without any catalyst at low µM concentrations, in neutral buffer, with a rate constant of 9 M s as
measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no cross-over side-
products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the con-
jugates is not affected by the presence of biological diols like fructose, glucose and catechol, and the thiosemicarbazide-
functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this
system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent
onto the extracellular domain of Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemi-
carbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was
possible with a reagent concentration as low as 10 µM, thus confirming the potential of this system in biological applications.
-
1 -1
INTRODUCTION
A) ‘Click’ boronate formation14
Me
By exploiting reactions that are inert to native functional
groups, bioorthogonal chemistry has become a powerful tool
to chemically label biomolecules in living cells to understand
the roles of these biomolecules within their native environ-
Me
ON = 7.7 M-1s-1
k
O
O
OH
OH
HO
HO
B
+
B
K
eq = 12 x 104 M-1
O
O
Fast, stable,
but reversible
1
-6
ment. Bioorthogonal chemistry has also been used in other
applications such as installing post-translational modifications
B) o-Boronyl imine/oxime/hydrazone systems17-25
OH
H
N
7
8
X
B
of proteins or constructing antibody drug conjugates. ‘Click’
chemistry
R3
9
,10
HO
OH
HO
R
2
N
is central to the development of bioorthogonal
HO
B
O
B
N
N
reactions because it exhibits fast reactivity in aqueous envi-
2
R1
R1 = H, Me
+
2
NH R
R1
ronment at low concentrations (µM), with high yields and
1
,11,12
R2 = alkyl/NHPh/
OCH Ph/
NHCXNHR
X = O, S)
Fast and high stability,
but still reversible
selectivity without any side products.
Moreover, in view
X = O, S
Stable diazaborine
conjugate
but low conversion
2
of dual-labeling applications, it is desirable for a click reaction
to be orthogonal with other existing bioorthogonal reac-
3
25
(
3
,13
C) Three-component assembly26
tions. Even the most common bioorthogonal reactions dis-
play limitations such as slow rates, use of complex reagents,
side-reactions or lack of mutual orthogonality due to possible
Me
OH
H
2
N
HO
OH
O
B
H
N
B
O
O
HO
MeOH
O
+
+
H
2
,4,13
cross reactivity.
In order to address these challenges, our
H
group recently reported a novel bioorthogonal reaction based
on a ‘click’ boronate formation, which enables fast dynamic
Cooperativity
Higher stability
-1 -1
D) New synergic design
ligation and high stability (kON = 7.7 ± 0.5 M s , Keq = 12 x
4
-1
1
0
M ) between nopoldiol derivatives and 2-methyl-5-
aqueous
media
14
HO
O
B
O
OH
carboxymethyl-phenylboronic acid (Figure 1A). Although
the stability of the resulting boronic ester is high, its reversibil-
ity may be detrimental in live cell imaging since washing steps
to remove excess reagents might lead to undesirable cleavage.
+
B
OH
HO
~pH 7.4
Fast?
n
R2
N
R2
O
N
N
n
NH
2
o/m-R2 = H, Me, Ph
n = ?
Synergy
Higher stability?
Irreversible?
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Page 2 of 8
Figure 1. A) Previously reported ‘click’ boronate formation sys-
Altogether, despite these advances, the current systems
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
tem. B) Enhancement in the rate and stability of the
imine/hydrazone/oxime ligation in the presence of a boronyl unit
in ortho-position. C) Positive cooperativity and higher stability of
a three-component amine/boronic acid/catechol system. D) New
synergic design based on the optimal placement of a hydrazine or
suffer from issues such as stability and toxicity of phenyl hy-
drazine, incomplete conversion to the diazaborine product or
the lack of bioorthogonality of 2-formylbenzeneboronic acid.
Inspired by the iminoboronate concept of Anslyn and James,
synergy was sought in our design based on the judicious
placement of a hydrazine or (thio)semicarbazide unit on a
bifunctional nopol diol reagent, along with a carbonyl handle
(ortho or meta) on the arylboronic acid (Figure 1D). This de-
sign was put to the test as a potentially bioorthogonal, fast and
irreversible click reaction system with stable and benign rea-
gents.
(
thio)semicarbazide on the nopol diol reagent, along with a car-
bonyl handle (ortho or meta) on the arylboronic acid.
Therefore, our ongoing goal is to improve the stability of this
system and ideally achieve a fast and irreversible boronate
click reaction. We hypothesized that such an advance could be
achieved by exploiting a second, synergic interaction.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The most suitable additional covalent interaction must ex-
hibit bioorthogonality and fast reactivity. Moreover, its reac-
tive groups must be small, non-cytotoxic, stable and easy to
install. To this end, imine, hydrazone and oxime ligations at-
tracted our attention. Hydrazone and oxime ligations have
found many applications in bioorthogonal chemistry. Those
RESULTS AND DISCUSSION
Optimization of nopol diol and boronic acid derivatives
Nopol diol derivatives (1a-1e) with two different spacers
(
n = 2, 3) and functional groups (amine/O-hydroxylamine)
were designed and synthesized to test the desired conjugation
with selected boronic acids (2a-2e). Not surprisingly, o-
substituted aryl boronic acids (2a, 2c, 2d) provided the desired
product with full conversion while m-substituted aryl boronic
acids 2b and 2e could only form the boronate product in mod-
erate yield, without any imine/oxime being formed (see Table
S1).
methods, however, tend to suffer from slow rates at neutral pH
1,15,16
and can also exhibit reversibility.
Fortunately, recent re-
ports indicate that the presence of a boronic acid ortho to an
aromatic aldehyde or acetylketone improves both the ligation
rate and stability of the resulting imine, hydrazone and oxime
1
7-20
product due to a dative B–N bond (Figure 1B).
For exam-
ple; Gois and co-workers initially reported an iminoboronate
formation between 2-formylbenzeneboronic acid or 2-
acetylbenzeneboronic acid (0.16 M) and lysine (0.16 M) in
aqueous media with 61 and 71% yield, respectively (Keq = 10
Table 1. Results of initial conjugation attempts of diol de-
rivatives (1a-1f) with aryl boronic acids (2a-2e)
1H NMR & HPLC-MS
analysis
1
7
mM). Gillingham and co-workers designed a rapid alkoxy-
phosphate buffer
pH 7.4/acetonitrile
(65/35 w%)
O
O
iminoboronate condensation between 2-formylbenzeneboronic
OH
OH
B
O
O
B
3
acid and O-benzylhydroxylamine at neutral pH (k = ~11 x 10
1
Y
Y
-
1
-1
-5 -1
8
-1 19
n
n
X
X
n
M s , k-1 = ~4.2 x 10 s , Keq = ~2.6 x 10 M ). Meanwhile,
Gao and co-workers demonstrated a rapid hydrazone ligation
HO
B
X
Desired product
3aa - 3ee
Undesired products
&
controls: 3fa - 3fe
1a - 1f
HO
Y
18
1a; n = 2, X = NH2
b; n = 3, X = NH
2
c; n = 2, X = NHPh
d; n = 2, X = NHCH
1e; n = 2, X = ONH
between phenyl hydrazine and 2-acetylbenzeneboronic acid.
1
1
1
2
a - 2e
Unfortunately, the reversibility of these reactions may limit
their application in living cells. Bane and Gillingham recently
disclosed the formation of 1,2-dihydro-1-hydroxy-2,3,1-
benzodiazaborine as a stable product of click condensation
between 2-formylbenzeneboronic acid and phenyl hydra-
2a; Y = o-CHO
2b; Y = m-CHO
X–Y
2
Ph
2
O
2
2
2
c; Y = o-COMe
d; Y = o-COPh
e; Y = m-COPh
or
1
f; n = 2, X = N
3
N
H
N
N
2
OH –H O
hemiaminal
imine
oxime
2
1,22
zine.
Phenyl hydrazine is, however, susceptible to oxida-
Product
(2 h)
Product
(24 h)
Boronic
acid
tion, and it exhibits cytotoxicity. Furthermore, the bioorthogo-
nality of 2-formylbenzeneboronic acid is problematic due to
Entry
Imine/oxime
2
3,24
(1a-f)
(1 mM)
(1 mM) formation?
its high reactivity towards N-terminal cysteines.
In this
[a]
[a]
3
/1
3/1
regard, Gao and co-workers demonstrated a new bioorthogo-
nal process where a benign and stable (thio)semicarbazide
2
3aa/3aa•H O/1a:
[
b, c]
1
2
(1a) 2a
(1a) 2c
71/21/8 Yes
forms
a
stable
diazaborine
conjugate
with
2-
11/67/22
acetylbenzeneboronic acid (10 minutes, maximum 60% con-
[b]
3ac/1a: 43/57
3ba/1b: 79/21
43/57
79/21
68/32
86/14
100/0
100/0
100/0
73/27
Yes
No
version at 50 µM concentration for semicarbazide, maximum
2
5
2
7% conversion for thiosemicarbazide). According to the
3 (1b) 2a
(1c) 2a
authors, this reaction stalls before completion likely due to the
interaction of the product with the reactants. Remarkably, An-
slyn and James reported a stable three-component assembly
with 2-formylbenzeneboronic acid, catechol and benzyla-
[d]
[d]
[d]
4
3ca/1c: 68/32
3da/1d: 86/14
3ea/1e: 100/0
3ec/1e: 100/0
3ed/1e: 100/0
3fa/1f: 73/27
No
[d]
5 (1d) 2a
6 (1e) 2a
No
[b]
[b]
Yes
Yes
Yes
No
2
6
27
mine or N-hydroxylamines. The authors performed a struc-
tural and thermodynamic analysis of ortho-
7
8
(1e) 2c
(1e) 2d
[b]
iminophenylboronate ester formation in a protic solvent, and
they found that initial binding of either benzylamine or cate-
chol to 2-formylbenzeneboronic acid improves the complexa-
9 (1f) 2a
2
6
[a] The % product (boronate/imine/oxime) conversion was de-
tion of catechol and benzylamine, respectively (Figure 1C).
1
termined by H NMR.
In other words, binding of these two molecules to 2-
formylbenzeneboronic acid becomes cooperative in a protic
solvent.
[b] Imine/oxime formation was also confirmed by HPLC-MS.
[
c] Hemiaminal slowly converted into imine over time.
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d] Because of insufficient solubility at 1.0 mM, the reaction
cause 2c exhibits lower hydrophobicity than 2d, 2c was select-
[
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was performed at 0.5 mM.
ed as the optimal boronic acid.
Boronic acid 2a and diol 1a could initially form the medi-
um-sized ring hemiaminal 3aa•H O, which, according to the
diagnostic –HC=N– resonance in H NMR, slowly converted
into imine 3aa over 24 h in 1.0 mM concentration at rt (Table
Design of fluorescently labeled nopol diol derivative
with hydrazine/(thio)semicarbazide unit.
2
1
Based on the efficiency of oxime derivative 1e, our atten-
tion switched to the synthesis of a diol derivative with a disub-
stituted hydrazine unit. Thus, a nopol diol derivative 1g with a
NBD dye was designed and synthesized (Table 2). A benzyl
linker was employed for the NBD dye since the reactivity of
benzyl bromide towards the in situ synthesized nopol diol
hydrazine is higher than alkyl bromides. Even though the reac-
tion provided high conversion and diol 1g preserved the fluor-
ophore’s intensity, it exhibited low stability and its purifica-
tion led to partial decomposition. Nonetheless, a small amount
of 1g could still be isolated to test its conjugation with 2c via
HPLC-MS analysis. The conjugation reaction again proceeded
1
, entry 1). A comparison of 3aa with 3fa indicated that the
presence of a second interaction (i.e., imine formation) clearly
improved the conversion from 73 to 92% (Table 1, entry 1
and/vs 9). Moreover, boronic acid 2c also formed an imine
with 1a albeit, with a lower conversion (43%) even after 24 h
(Table 1, entry 2). On the other hand, 1b with an additional –
CH spacer unit underwent only boronate formation with 2a,
2
with no imine or hemiaminal formation observed (Table 1,
entry 3, also see Table S1). This result clearly indicates that
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
a, unlike 1b, features the right geometry and size that favor
both boronate and imine formation. Diol 1a contains a simple
primary amine, which could be further functionalized to in-
clude a reporter tag but the resulting secondary amines would
render iminium formation more difficult. On the other hand,
hemiaminal formation may still occur, and hopefully, be suffi-
cient to form a stable conjugate. Thus, 1c and 1d were tested,
however no hemiaminal formation was observed as evidenced
by the lack of the corresponding –RCH(OH)(NHR’) signal at
~6 ppm. Only boronate formation was observed in these cases
(Table 1, entry 4, 5). Next, O-hydroxylamine 1e was designed
and tested with 2a, 2c and 2d. All of these boronic acids
formed the desired boronate-oxime conjugate, in a mixture of
two oxime E/Z isomers, with full conversion in a very short
time (Table 1, entry 6, 7, 8 and Table S1).
through initial boronate formation 3gc•H
conversion into a hydrazone 3gc with two E/Z isomers (Table
, entry 1-4, Figure S11-14,18). After 24 h, a precipitate in
PBS buffer was observed likely due to the low solubility of
gc (Table 2, entry 1, 2). In order to test the stability of the
conjugates 3gc•H O and 3gc, the reaction mixture was sub-
jected to either dilution or introduction of another boronic
acid, 2g, after 4 h. Satisfactorily, both 3gc•H O and 3gc pre-
served their stability under these conditions (Table 2 and Fig-
ure S16). Intermediate 3gc•H O was found to be a stable con-
jugate that transforms slowly into 3gc. The approximate rate
2
O followed by slow
2
3
2
2
2
-
1 -1
constant was estimated as ~8 M s for the formation of
3gc•H O based on the reaction’s half-life as monitored by
2
HPLC-MS analysis (Table 2).
Scheme 1. Stability test of 3aa via a cross-over experiment
HO
OH
Table 2. Results of the aqueous conjugation reaction be-
tween 1g and 2c, and the stability test of conjugate
B
O
H
OH
OH
O
O
B
N
3
2
gc•H O and 3gc via HPLC-MS analysis
2
a (1 mM)
25 mM phosphate buffer/
HO
OH
B
O
acetonitrile (65/35 wt%), 24 h
H
NH
2
3aa
100% conversion
HPLC-MS analysis
1a
1 mM)
~
(
OH
OH
H
N
O
O
slow
B
N
2c
HO
OH
N
B
O
HO
3gc•H2O
acetonitrile/
over
NH2
~24 h
10 mM PBS buffer
(35/65 w%)
or
N
H
N
O
B
O
O
O
B
N
2
f (2 mM)
+
10 mM PBS
1-4 days
N
H
H
Stability test:
*Dilution from
Exchange study
with HPLC-MS
analysis
3af
3aa
NH
NBD
gc
NH
Major product
Cross-over product:
negligible amount
0.25 mM to 0.050 mM
*Exchange study with
N
N
O
HO
OH
B
O
3
NO2
Meanwhile, the stability of 3aa was assessed by a cross-
over experiment where arylboronic acid 2f (2.0 equiv) was
introduced into a buffered solution of 3aa and the reaction
mixture was monitored via HPLC-MS analysis after 1 and 4
days (Scheme 1). Satisfactorily, the major product 3aa was
preserved and a negligible proportion of exchange product 3af
was observed (Scheme 1, see also Figure S8-9). This small
amount of 3af may be due to a small amount of unreacted 1a
in the reaction mixture rather than exchange of ‘parts’ released
from the hydrolysis of 3aa.
1g
Both 3gc•H O and 3gc were stable upon
2
MeO
dilution or in the presence of 2.0 equiv 2g.
2
g
Conc.
of
2
% Proportions of 1g/3gc•H O/3gc
Entry
10
min
40
70
reagents
(mM)
24 h
min
min
[a]
1
2
3
4
0.025
83/12/5
71/23/6
62/31/7
72/18/10 65/22/13 precipitate
54/36/10 49/40/11 precipitate
[a]
0.050
These initial studies led us to choose the most suitable
spacer length for the ideal diol as n = 2 due to its stability as
well as its relatively simple synthesis. Regarding the choice of
boronic acid, 2a was no longer considered due to its reported
[b]
0.050
45/45/10 41/53/12
-
[
c]
0.25
37/37/26 30/22/48 25/15/60 8/4/88
2
3,24
[a] 10 mM PBS buffer was used.
side reactivity with N-terminal cysteines.
Moreover, be-
[b] 10 mM ammonium acetate buffer was used.
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Journal of the American Chemical Society
Page 4 of 8
[
c] Acetonitrile/10 mM PBS buffer (35/65 w%) was used.
In order to improve the chemical stability of the diol rea-
In further experimentation, the stability of conjugate 3hc
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was tested (Figure 2). Firstly, an equimolar concentration of
1h and 2c was incubated for one day, and the reaction mixture
was subjected to dilutions. Conjugate 3hc stayed intact even
after 24 hours (Figure 2, S28B, S31B-C). Subsequently, an-
other boronic acid such as 2g or even the more reactive 2a was
introduced. No breakdown of 3hc occurred since no cross-
over products of these competitors were observed (Figure 2,
S28C, S31D-E). Finally, subjecting 3hc to either acidic (pH 3)
or basic (pH 9) solutions did not affect its integrity significant-
ly (Figure 2, S31F-G).
gent, a (thio)semicarbazide unit, which was recently shown to
be more stable and safer than phenyl hydrazine, was chosen
in the design and synthesis of diol reagent 1h. This reagent
was expected to be more stable, more reactive towards keto
25
25
boronic acids like 2c, and to afford high conversion because
the expected product, 3hc, would not exhibit any inhibitory
interaction with the starting materials that could stall the reac-
tion. Moreover, 1h could be synthesized easily with commer-
cially available fluorescein isothiocyanate and nopol diol hy-
drazine, which was easily prepared in situ from nopol diol
bromide and hydrazine monohydrate, as the precursors. As
expected, the integrity of diol 1h is intact even after one week
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HPLC - 24 h
3
hc
Four separate
stability studies
1
h
R2
* Dilution from 50 µM
to 25 µM or 10 µM
1
O
O
Dilution
O
O
according to H-NMR (Figure S20B).
B
N
to 10 µM
B
N
*
Exchange study with
2g/2a (2.0 equiv)
* Acidify to pH 3
Basify to pH 9
With the more stable diol reagent 1h, in hand, the conjuga-
1
R1
No cross-
tion reaction was examined via both H NMR, HPLC-MS and
N
N
over product
pH 3
*
high resolution ESI-MS analyses (Figure S20-23,30,35). The
reaction appears to proceed through concomitant formation of
the boronate and thiosemicarbazone, with full conversion
achieved within 3.5 hours at 50 µM, while negative controls 1i
and 1j provide diazaborine (27% yield) and no thiosemicarba-
zone ligation, respectively (Table 3, entry 1-3). These results
support the expectation that initial boronate formation is criti-
cal to promote the thiosemicarbazone formation, which occurs
second, by intramolecular fashion.
S
NH
S
NH
3
hc
R1 = Me, R2 = H; 3hc Major Product!
R1 = Me, R2 = OMe; 3hg No cross-over! pH 9
R1 = H, R2 = H; 3ha No cross-over!
=
fluorescein
5
6
7 min
1
Figure 2. Studies on the stability of 3hc via HPLC-MS and H
NMR.
Table 3. Results of the aqueous conjugation reaction of 1h,
and negative controls 1i and 1j, with 2c
Table 4. Examination of the reactivity of 1k, 1l and control
1f with 2c to identify the rate determining step
HO
OH
B
O
OH
OH
O
B
O
OH
OH
O
O
2
c
B
N
NH
2
N
1H NMR
0 mM PBS buffer/D2O
N
N
1
X
NH
2
c
X
NH
NH2
(9/1 v/v) pH 7.4
N
N
0.25 mM
1H NMR
k = 4.2 ± 0.4 M-1s-1
S
NH
S
NH
&
5
0 mM D
2
O phosphate
(65/35 wt%)
1
k; X = O
3kc; X = O
3lc; X = S
HPLC-MS
0 mM PBS buffer pH 7.4
buffer/ACN-d
3
1
h
3hc
1l; X = S
1
OH
OH
What mainly determines
the rate constant?
Negative controls:
O
B
O
NH2
NH2
NH
HN
N
O
N3
3
N
3
S
NH
S
1
f
3fc
=
fluorescein
1
i
1j
% Proportions of 1/3
Conc.
of
% Proportions of 1/3
Entry
1
0
30
60
90
120
min
3.0 h 3.5 h 24 h
Entry
10
30
min
60
120
min
min
min
min
min
reagents
3.5 h 24 h
min
min
1 (1k) 82/18 70/30 55/45 49/51 47/53
-
38/62 0/100
(
mM)
2
3
(1l) 90/10 71/29 53/47 44/56 38/62 30/70
-
-
0/100
47/53
[
a]
a]
1
(1h) 0.050
90/10 86/14 64/36 35/65 0/100 0/100
73/27 (maximal conversion)
100/0 (no product)
(1f) 82/18 67/33 58/42 54/46 52/48 49/51
[
2 (1i) 0.050
[a]
3
4
5
(1j) 0.050
1
[
b]
(1h) 0.25
(1i) 0.25
89/11 81/19 62/38 47/53
72/28 64/36 64/36
O (9/1 v/v) was used.
O phosphate buffer (35/65 w%) was
-
0/100
Using H NMR methodology, the overall rate constant for
the formation of 3hc in 10 mM PBS buffer was measured as
[
b]
-
-
64/36
-1 -1
4
.2 ± 0.4 M s , which is in agreement with our previous study
[
[
a] 10 mM PBS buffer/D
b] ACN-d /50 mM D
2
on rate constant measurements of nopol diol boronate for-
mation (Figure S24-25). It is important to note that the reac-
tion rate was lower in higher buffer concentration (50 mM),
1
4
3
2
used.
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Journal of the American Chemical Society
which is likely due to the competitive effect of salts in boro-
nate formation (Table 3, entry 1 vs 4). Moreover, negative
with 1h/1i/1m (0.10/0.10/0.25 mM) in the presence of a cata-
lyst, p-phenylenediamine (10 mM) (Figure 4). As the reac-
2
8
35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
control 1j without 1,2 cis diol gave no conversion (Table 3,
entry 3). Thus boronate formation is essential in this system,
acting as the overall rate-determining step preceding a fast
intramolecular thiosemicarbazone formation. To understand
the reaction mechanism further, the reactivity of diols 1k, 1l
tions were performed at neutral pH, the catalyst p-
3
5
phenylenediamine was required. These reactions were moni-
tored by HPLC-MS and the analysis showed that 1h is unreac-
tive towards 2i even in the presence of the catalyst while both
1i and 1m afforded the expected thiosemicarbazone and hy-
drazone adducts, 3ii and 3mi, respectively (Figure 4, S44).
1
and 1f with 2c was monitored via H NMR analysis. The re-
sults indicate that both 1k and 1l behave similarly and reach
1
00% conversion with the help of the synergic
2c/2h
(
thio)semicarbazone interaction. In contrast, control diol 1f
(0.1 mM)
OH
OH
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
R
afforded a conversion similar to 1k and 1l in the first 60 min
and then remained in equilibrium (53%) as expected (Table 4,
entry 1-3). Hence, the conversion in this system is determined
mainly by boronate formation. According to our previous
10 mM PBS buffer
HPLC-MS analysis
N
NH2
N
N
1
,2-cis diols (competitors)
0.3 mM fructose
S
NH
S
NH
1
4
8.0 mM glucose
study on nopol diol boronate formation, the reactivity of this
conjugation system could be improved via the placement of e-
withdrawing substituents on 2c. Therefore, a conjugatable o-
acetylphenylpinacol boronate with a p-amide group, 2h, was
designed and synthesized. As predicted, the rate constant in-
1h (0.1 mM)
0.01 µM catechol
50 min, 2 h, 3 h, 4 h
3hc/3hh
similar conversion
=
fluorescein
–
competitors
(50 min)
+ competitors
(50 min)
– competitors
(50 min)
+ competitors
(50 min)
-1 -1
creased to 8.9 ± 0.6 M s (Scheme 2, Figure S26-27). Not
1
h
3hc
3hh
3hh
1
h
surprisingly, the presence of a pinacol protecting group in 2h
3hc
2
6,29
did not impede the formation of conjugate 3hh.
1
h
Scheme 2. Rate constant for the reaction between 1h and
1h
1
2h as measured by H NMR analysis
min
O
OH
O
5
O
HN
O
OH
Figure 3. Competitive effect of biological polyols on the for-
mation of conjugates 3hc/3hh.
B
HN
O
OH
OH
O
O
5
O
B
O
2
h (0.1 mM)
N
NH2
R
O
N
1H NMR
10 mM PBS buffer/D2O
9/1 v/v)
N
OH
OH
OH
HPLC results
after 2 h
H
N
H
OH
S
NH
S
NH
(
O
NHR
O
OH
1h
N
1
h (0.1 mM)
3hh
NH2
k = 8.9 ± 0.6 M-1s-1
N
2i (0.5 mM)
N
2 h
S
NH
S
NH
= fluorescein
10 mM PBS buffer
HPLC-MS analysis
3hi
not observed
1h is unreactive
1
h(0.1 mM)
2
H N
NH2
Studies to assess the bioorthogonality of the boro-
nate/thiosemicarbazone system.
towards reactive aldehydes!
catalyst (10 mM)
More importantly, the effect of biological polyols on the
reaction progress was assessed using competition assays moni-
tored by HPLC-MS analysis. Diol 1h was allowed to form the
irreversible conjugate 3hc/3hh with 2c/2h in the presence of a
mixture of biocompetitors such as glucose (8 mM), fructose
NHR
N
3ii
NH2
HN
HN
O
same conditions
S
NH
S
NH
1
i
1
i (0.1 mM)
3ii
observed
(
300 µM) or catechol (0.1 µM) at concentrations higher than
30,31
those found in the blood stream (Figure 3).
Remarkably,
NHR
O
the formation of the desired conjugates 3hc/3hh was not af-
fected as similar conversion was obtained both in the absence
and the presence of biological polyols (Figure S40,41,43). As
expected, higher conversion was observed for 3hh when com-
pared to 3hc due to the higher reactivity of boronic acid 2h
(Figure 3).
NH2
N
3mi
HN
HN
same conditions
3mi•H2O
3
mi
1
m (0.25 mM)
observed
min
4
5
=
fluorescein
3
2,33
Apart from biological polyols, a recent report
high-
lighted the presence of protein-bound electrophiles caused by
pyruvoyl (ketone) and glyoxylyl (aldehyde) electrophilic post-
translational modifications in human cells (HEK293T cells
and the human breast cancer cell line MDA-MB-231). Thus,
in order to confirm the inertness and bioorthogonality of rea-
gent 1h and its thiosemicarbazide unit towards these electro-
philes, especially the reactive glyoxylyl aldehydes, a readily
Figure 4. Study to confirm the inertness and bioorthogonality of
diol 1h towards model aldehyde 2i, a mimic of protein-bound
glyoxylyl unit.
Cytotoxicity and live cell labeling studies.
With these promising results in hand, the cytotoxicity of
bioorthogonal handles 1l, 2c and 2h was examined. These
reagents were found to be benign towards HEK293T cells
3
4
available model aldehyde 2i (0.50 mM) was allowed to react
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Journal of the American Chemical Society
Page 6 of 8
A)
HO
OH
over 18 hours at 50 and 25 µM concentrations (Figure 5), an
outcome consistent with Gao’s recent report.
B
O
2
5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NH
N
N
O
N
HO
B
OH
O
O
NH
NH2
O
5
HN
2j (10 µM)
Hoechst 33342 (2 µM)
5 min at 37 °C
4
Live HEK293T cell
transiently transfected with
Live HEK293T cells
labeled with boronic acid
on cell surface
pSNAPf-ADRβ2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
B
O
Controls:
1
h (10/30 µM)
i) No labeling without 2j
(only with 30 µM of 1h)
ii/iii) No labeling with 1i/1j
or
N
N
HN
hj
controls: 1i/1j (30 µM)
S
0.5 h or 2.5 h at 37 °C
= fluorescein
3
Live cell labeling on cell surface
Figure 5. Cytotoxicity results of bioorthogonal handles 1l, 2c and
h and on HEK293T cells. Blank is DMSO, and Triton X-100
was used as a positive control.
B)
2
Finally, in order to demonstrate the viability of this irre-
versible boronate/thiosemicarbazone system in live-cell imag-
ing, a SNAP-tag approach was employed to install boronic
acid 2j on the cell membrane of transiently transfected
HEK293T cells with the pSNAP
A). Boronic acid 2j (10 µM) was secured on the cell mem-
brane of HEK293T cells through its reaction with the SNAP
Beta-2 adrenergic receptor. Diol 1h (10/30 µM) or controls
i/1j (30 µM) were then introduced into the cell media and
incubated for 0.5 h or 2.5 h at 37 °C. In parallel, cells without
j were incubated with diol 1h (30 µM) as a negative control.
f
-ADRβ2 plasmid (Figure
6
f
-
1
2
The cells were washed and imaged on a total internal reflec-
tion fluorescence (TIRF) microscope. Whilst controls showed
no labeling in 2.5 h (Figure 6B i,ii,iii), a clear cell surface im-
aging was observed for the HEK293T cells incubated with
both diol 1h and 2j even after only in 0.5 h (Figure 6B iv).
Moreover, a vivid image was possible even when using a low
1
0 µM concentration of 1h (Figure 6B v).
CONCLUSION
In summary, we have successfully developed an irreversi-
ble nopol diol boronate bioconjugation system via a synergic
thiosemicarbazone interaction that provides a stable medium-
sized ring product of double condensation. This binary boro-
Figure 6. Imaging of boronic acid 2j surface labeled
HEK293T live cells with diol 1h. A) Scheme of labeling pro-
cedure. B) Imaging results; i) HEK293T live cells not incubat-
ed with 2j, but only incubated with 1h (30 µM) in 2.5 h. ii)
HEK293T live cells incubated with both 2j (10 µM) and 1i (30
µM) in 2.5 h. iii) HEK293T live cells incubated with both 2j
10 µM) and 1j (30 µM) in 2.5 h. iv) HEK293T live cells in-
cubated with both 2j (10 µM) and 1h (30 µM) in 0.5 h. v)
HEK293T live cells incubated with both 2j (10 µM) and 1h
-1 -1
nate reagent was demonstrated to be efficient (~9 M s ), ro-
bust, compatible with biological polyols and also unreactive
towards possible electrophiles found in human cells. Moreo-
ver, it was successfully utilized in low micromolar concentra-
tion for site selective protein labeling on mammalian
HEK293T cells. Therefore, this irreversible boro-
nate/thiosemicarbazone synergic system is a valuable addition
to the bioorthogonal chemistry toolbox to enable the study of
complex biological processes.
(
(
3
10 µM) in 2.5 h. Scale bar corresponds to 10 µm. Hoechst
3342 was used for live cell nuclear staining.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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Journal of the American Chemical Society
Experimental details, kinetic plots, spectroscopic data for new
compounds, and additional images including Figures S1–S45 and
Table S1.
(13) Shih, H.-W.; Kamber, D. N.; Prescher, J. A. Curr. Opin.
Chem. Biol. 2014, 21, 103–111.
14) Akgun, B.; Hall, D. G. Angew. Chem. Int. Ed. 2016, 55
12), 3909–3913.
15) Lang, K.; Chin, J. W. Chem. Rev. 2014, 114 (9), 4764–
806.
1
2
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1
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2
2
2
2
2
2
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2
2
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6
(
(
(
AUTHOR INFORMATION
Corresponding Author
4
1
(
16) Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science
*
dennis.hall@ualberta.ca
997, 276 (5315), 1125–1128.
Author Contributions
(17) Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A.
V.; Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. J. Am. Chem.
Soc. 2012, 134 (24), 10299–10305.
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
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2
3
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5
6
7
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9
0
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2
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3
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9
0
(
18) Bandyopadhyay, A.; Gao, J. Chem. Eur. J. 2015, 21
(
42), 14748–14752.
Funding Sources
(19) Schmidt, P.; Stress, C.; Gillingham, D. Chem. Sci.
015, 6, 3329–3333.
This work was funded by the Natural Sciences and Engineering
Research Council (NSERC) of Canada (Discovery Grant to DGH)
and the University of Alberta.
2
(20) Gillingham, D. Org. Biomol. Chem. 2016, 14 (32),
7606–7609.
21) Dilek, O.; Lei, Z.; Mukherjee, K.; Bane, S. Chem.
Commun. 2015, 51, 16992–16995.
22) Stress, C. J.; Schmidt, P. J.; Gillingham, D. G. Org. Bi-
ACKNOWLEDGMENT
(
BA thanks Alberta Innovate Technology Future for a Graduate
Scholarship. We thank Mr. Ed Fu (HPLC), Mr. Bela Reiz (Mass
Spectrometry), Mrs. Nupur Dabral and Mr. Mark Miskolzie
(
omol. Chem. 2016, 14 (24), 5529–5533.
(
NMR Facility) for their help and comments. We also thank Prof.
Robert E. Campbell for supplying some cell culture equipment.
(23) Bandyopadhyay, A.; Cambray, S.; Gao, J. Chem. Sci.
016, 00, 1–5.
2
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SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”).
OH
OH
HO
O
NH2
B
OH
O
B
O
N
N
S
NH
N
S
HN
3
0 min
Live HEK293T
labeled with boronic acid
on cell membrane
*Fast (9 M-1s-1), irreversible
*Benign reagents, compatible
with biological polyols
0
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via SNAPtag approach
*Inert towards
biological electrophiles
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