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onic acid substituents, which
typically display hydrolysis rates
À6 À1
[9]
of 10
s
at neutral pH. Bind-
ing curves of 2-APBA and AOHA
were generated by monitoring
the changes of UV/Vis absorp-
tion (Figure 1d and Figure S4d
in the Supporting Information).
Data fitting yielded a Kd of ap-
proximately 14 mm, indicating
a more favorable equilibrium for
the formation of oximes than
acylhydrazones.
Similar to the oxime forma-
tion, 2-APBA readily conjugates
with phenylhydrazine (Phzn) to
give a hydrazone as the single
product (Figure S5 in the Sup-
porting Information). The hydra-
zone formation proceeds at
even lower concentrations of the
reactants: 2-APBA was close to
being fully converted to the hy-
drazone even with the reactants
at 10 mm (Figure S5b in the Sup-
porting Information). To analyze
the reaction at lower concentra-
tions, we incorporated the 2-
APBA structure into a fluoro-
phore-labeled peptide by using
Figure 1. Conjugation of 2-APBA to hydrazides and oxyamines. a) UV/Vis characterization of the 2-APBA-acethy-
drazide conjugation, demonstrating its quick reversibility; AHz=acethydrazide. b) Titration curve of the acylhydra-
zone formation generated using H NMR. c) UV/Vis characterization of the oxime formation of 2-APBA demonstrat-
ing its quick reversibility. d) Titration curve of the oxime formation of 2-APBA generated using UV/Vis measure-
ments.
1
an
dubbed AB1) that displays the
-APBA structure as its side
unnatural
amino
acid
(
2
essentially identical UV/Vis spectrum as the one prepared by
directly mixing 20 mm 2-APBA and 0.20 mm acethydrazide (Fig-
ure 1a), demonstrating the rapid reversibility of the reaction.
The binding affinity of 2-APBA to acethydrazide was assessed
by using UV/Vis (Figure S2 in the Supporting Information) and
chain (Figure 2a). The synthesis of AB1 was recently reported
by our group in the application of iminoboronate chemistry
[
3]
for bacterial imaging. A short peptide CGAB1 with the se-
quence of Ac-C*G(AB1)-NH2 was synthesized, where C* de-
notes a cysteine labeled with Alexa Fluor 488 (AF488, Fig-
ure 2a). The conjugation reactions, with 0.2 mm peptide and
Phzn over a range of concentrations, were analyzed by using
LC-MS. With 1 mm Phzn, CGAB1 was over 90% labeled (Fig-
ure S6 in the Supporting Information), demonstrating the high
propensity of 2-APBA to conjugate with Phzn to form hydra-
zones. The peptide labeling was half completed within 7 min
1
H NMR spectroscopy (Figure 1b), which yielded comparable
dissociation constants in the sub-millimolar range (Kd
ꢀ
0.6 mm). In comparison to primary amines, which typically
[
3]
conjugate with 2-APBA at low millimolar concentrations, ace-
thydrazide displays a higher propensity for conjugation with 2-
APBA. Similar behavior was observed for benzhydrazide, for
which a K value of 0.5 mm was obtained (Figure S3 in the Sup-
porting Information).
with 1 mm Phzn (Figure 2b). Fitting the kinetic data yielded
d
3
a rate constant (k ) of 1.7(Æ0.08)10 , which compares well to
1
[
10]
2
-APBA was found to rapidly conjugate with an alkoxyamine
the fastest ligation reactions known in a neutral buffer. Data
À4 À1
(
6-aminoxy hexanoic acid, AOHA) to form oximes as well. NMR
fitting also revealed a rate constant (k ) of 1.210
s
for
À1
and mass-spec characterization reveals that, when AOHA is in
excess, 2-APBA is completely converted into the oxime conju-
gate (Figure S4a–c in the Supporting Information). A dilution
experiment shows that the oxime formation is rapidly reversi-
ble (Figure 1c), suggesting that the boronic acid activates the
oxime for hydrolysis. The quick hydrolysis was also clearly seen
during HPLC analysis of the oxime, in which 2-APBA was re-
generated (Figure S4 f in the Supporting Information). The fast
reversibility differs dramatically from oximes without such bor-
hydrolysis, which is approximatel 100 times faster than struc-
[
2c]
turally similar hydrazones without the boronic acid. In terms
of the thermodynamic equilibrium, the hydrazone formation
gives a K value of 0.07 mm (K =k /k ), which is much more
d
d
À1
1
favorable than the formation of oximes and acylhydrazones.
To further explore the potential of the hydrazone chemistry
for biological applications, we have incorporated AB1 into
villin headpiece subdomain (VHP35), a small model protein
[
11]
that displays a variety of amino acids. The mutant protein
Chem. Eur. J. 2015, 21, 14748 – 14752
14749 ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim