One-pot optical sensing of keto acids through the combination of the oxime-click reaction and aggregation-induced emission (AIE)

Article abstract of DOI:10.1246/cl.150153

Keto acids play important roles in vivo. In this paper, a novel one-pot keto acid detection system has been developed by the combination of the oxime-click reaction and aggregationinduced emission (AIE). Particularly high selectivity was observed for pyru

Full text of DOI:10.1246/cl.150153

CL-150153
Received: February 20, 2015 | Accepted: March 19, 2015 | Web Released: June 5, 2015
One-pot Optical Sensing of Keto Acids through the Combination of the Oxime-click Reaction
and Aggregation-induced Emission (AIE)
Daisuke Yoshihara,1 Takao Noguchi,2 Youichi Tsuchiya,1 Bappaditya Roy,2 Tatsuhiro Yamamoto,1 and Seiji Shinkai*1,2,3
1Nanotechnology Laboratory, Institute of Systems Information Technologies and Nanotechnologies (ISIT),
4-1 Kyudaishinmachi, Nishi-ku, Fukuoka 819-0388
2Institute for Advanced Study, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395
3Department of Nanoscience, Faculty of Engineering, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082
(E-mail: shinkai_center@mail.cstm.kyushu-u.ac.jp)
Keto acids play important roles in vivo. In this paper, a
novel one-pot keto acid detection system has been developed by
the combination of the oxime-click reaction and aggregation-
induced emission (AIE). Particularly high selectivity was
observed for pyruvic acid (α-keto acid), but wide applications
for sensing other keto acids are also possible by controlling the
reaction conditions.
emergence of fluorescence emission. These phenomena were
useful for fluorescence sensing of ATP and dicarboxylic acids.
Since keto acids also have the carboxylic group, which can bind
to the guanidinium group, AIE phenomena would be applicable
to keto acid sensing. Taking these lines of precedent reports into
consideration, we designed a keto acid sensing system by the
combination of the oxime-click reaction with AIE. This system
was expected to be useful for the selective detection of keto
acids in situ.
Keto acids are one of the organic acids bearing both ketone
and carboxylic groups. They play various important roles
in vivo. In the TCA cycle, for example, pyruvic acid (pyr,
α-keto acid) is converted to acetyl-CoA through a glycolytic
pathway.1 α-Keto acids are known to be a biomarker for several
diseases. Increase in the phenylpyruvic acid and α-keto-β-
methylvaleric acid concentration in blood is indicative of
phenylketonuria2 and maple syrup urine disease,3 respectively.
The concentration increase in keto acid derivatives in the blood
is induced by diabetic ketoacidosis.4 The regular concentration
of each α-keto acid in human blood is in the range of several tens
of micromolars.5 The concentration of α-keto acids increases up
to hundreds micromolars when some metabolic disorders are
caused.5c Therefore, the detection of keto acids is very important
as a tool for the diagnosis of these diseases. In spite of the
diagnostic usefulness, however, there exist only a few methods
applicable for the detection of keto acids. Some keto acids
were prelabeled and detected by HPLC.6 Acetoacetic acid was
detected through an enzymatic method.7 Thus, exploitation of a
simple, quick, and sensitive in situ method for detecting keto
acids has been strongly demanded.
In this paper, we chose a cyanoOPV12d,13 skeleton as an AIE
fluorophore and synthesized two different molecules: 1 has two
aminooxy groups for complexation with a ketone group and 2
has two guanidinium groups for carboxylic acid recognition
(Figure 1a). In other words, the two functional groups integrated
into keto acid molecules are recognized by two different
fluorophores. The advantages of this two-component system
are (i) synthesis is easy because one can introduce only one
function into each fluorophore and (ii) broad application is
possible by changing the combination of the two fluorophores.
A schematic illustration of the sensing system is shown in
Figure 1b. First, 1 reacts with the keto acid by the oxime-click
reaction, and then the keto acid-appended product can interact
with 2 between the carboxylic group of the product and the
guanidinium group of 2. Aggregation can take place by
(a)
Click chemistry based on the Huisgen cycloaddition is
known as a simple and efficient procedure for synthesizing
various functional molecules.8 Many reactions that can proceed
under physiological conditions have been reported.9 Similarly,
the oxime-click reaction, one of the click reactions that connects
the aminooxy group (-ONH2) with aldehyde or ketone groups,
attracts much attention.10 It was reported that, using the oxime-
click reaction, even the reducing terminal of polysaccharides
could be connected with PEG polymers in a buffered solution.10a
As keto acids have the ketone group, they should work as a
substrate for the oxime-click reaction. Meanwhile, we consid-
ered that aggregation-induced emission (AIE)11 would be
applicable to turn-on-type molecular sensing purposes. We
previously reported the recognition of several bio-related
compounds through AIE.12 Tetraphenylethene (TPE) derivatives
bearing cationic guanidinium groups could bind anionic
phosphoric acids or carboxylic acids and the resultant neutral
complex species aggregated to one another, leading to the
(b)
Figure 1. (a) Structures of 1, 2, and 3a-3d. (b) Schematic
illustration for one-pot keto acid sensing.
812 | Chem. Lett. 2015, 44, 812–814 | doi:10.1246/cl.150153
© 2015 The Chemical Society of Japan

(a)
(b)
(a)
(c)
(d)
(b)
Figure 2. (a) Fluorescence spectra of each compound (100 ¯M)
and mixed samples (100 ¯M) in DMSO-aqueous HEPES buffer
(0.70 M, pH 7.1, 3:7 v/v) at 25 °C with 340 nm excitation. (b)
Fluorescence image of the solution obtained by mixing 2 with 3a
(100 ¯M each). (c) Fluorescence spectra of [1 + 3a] + [2]. Concen-
trations of [1 + 3a] and [2] were fixed to 100 ¯M in DMSO-aqueous
HEPES buffer (0.70 M, pH 7.1, 3:7 v/v) at 25 °C with 340 nm
excitation. (d) Plot of ¦F/F0 versus concentration of 3a. ¦F/F0
means the fluorescence intensity change (¦F = F ¹ F0) and F0 is the
fluorescence intensity of 1 + 2 by plotting the fluorescence intensity
at 515 nm.
Figure 3. (a) Plot of ¦F60min/Fmax in each one-pot system after
60 min reaction at 25 °C. ¦F60min means the fluorescence intensity
change between after 60 min reaction (F60min) and before reaction
(F0, ¦F60min = F60min ¹ F0). Fmax means the fluorescence intensity of
2 + 3a-3d of each compound. These data were collected at 515 nm
with 340 nm excitation. (b) Photo images of each one-pot system
after 60 min under UV light irradiation (365 nm), from left; pyr, val,
ace, lev. Concentration: [1] = [2] = 100 ¯M, [keto acid] = 1.0 mM.
neutralization thorough the cation-anion interaction and by π-π
stacking between the cyanoOPV skeletons, leading to the
appearance of AIE.
To search for the optimum conditions for the one-pot
fluorescence detection of keto acids, we prepared the product of
this system by the reaction of 1 with pyruvic acid (3a, Figure 1).
This reaction finished within a few hours giving good yield (see
Supporting Information for the isolated yield). Figure 2a shows
the fluorescence spectra of each compound (100 ¯M) and the
mixed samples with compound 2 (total concentration, 100 ¯M)
in a DMSO-aqueous HEPES buffer (0.70 M, pH 7.1; 3:7 v/v).
Strong emission was observed at 515 nm only when 3a was
mixed with 2. Furthermore, the fluorescence microscopy image
indicated that aggregates in the range of tens of micrometer in
diameter were generated (Figure 2b). These results clearly show
that the carboxylate group in 3a interacts with the guanidinium
group in 2 and the fluorescent aggregate is formed from a
mixture of 2 and 3a. We evaluated the composition of the
aggregate from a Job plot (Figure S1). The maximum appeared
at 0.5, supporting the 1:1 stoichiometry between 2 and 3a. The
detection limit of the keto acids was estimated from the
concentration dependence of 3a (Figures 2c and 2d): maintain-
ing [1 + 3a] and [2] constant at 100 ¯M, only the percentage of
3a was varied. As shown in Figure 2d, the detection limit of pyr
was estimated to be 30 ¯M.
HEPES buffer (1.0 M, pH 7.1) after 60 min reaction (final
composition of the DMSO-aqueous HEPES buffer was 3:7 v/v).
The resultant photos of 1 + 2 + pyr excited at 365 nm are
shown in Figure S2. Indeed, strong fluorescence was observed
for 1 + 2 + pyr after 60 min reaction. This means that 1 reacts
with pyr and the product interacts with 2, leading to fluorescent
aggregate formation in the one-pot system. To confirm whether
this method really detects only keto acids, we compared pyr
with simple ketones and a carboxylic acid (acetone, acetylace-
tone, and acetic acid). It is clear that significant fluorescence
enhancement was observed only for pyr (Figure S3).
Time dependence of the fluorescence intensity in the one-
pot reaction with keto acids was measured (Figure 3). 1, 2, and a
keto acid (pyr, 2-oxovaleric acid (val, α-keto acid), acetoacetic
acid (ace, β-keto acid), or levulinic acid (lev, γ-keto acid)) were
mixed all at once. The reaction was started in the DMSO-
aqueous HEPES buffer (0.10 M, pH 7.1, 9:1 v/v) at 25 °C
followed by dilution with the HEPES buffer (1.0 M, pH 7.1)
before measurement (final composition of DMSO-aqueous
HEPES buffer was 3:7 v/v). The results are summarized in
Figure 3. When the oxime-click reaction proceeds in the one-pot
system, the products 3a-3d can interact with 2 and fluorescence
should appear. Indeed, strong fluorescence was observed for pyr
and val and the one-pot reaction finished in 10 and 30 min,
respectively (Figure S4). This is short enough for practical use.
These results mean that α-keto acids can be detected more
selectively than β- and γ-keto acids.
In order to confirm the keto acid selectivity, the one-pot
reaction was tested, wherein 1, 2, and the carbonyl derivative
were mixed at once and the reaction was started in a DMSO-
aqueous HEPES buffer (0.10 M, pH 7.1, 9:1 v/v) at 25 °C to
promote the reaction faster, followed by dilution with the
Chem. Lett. 2015, 44, 812–814 | doi:10.1246/cl.150153
© 2015 The Chemical Society of Japan | 813

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30 min at 25 °C. Other keto acids can be detected by changing
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We are currently extending this method to the sensing of various
keto acids that exist in some pathological conditions for the
diagnosis of keto acid related diseases.
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Supporting Information is available electronically on J-STAGE.
814 | Chem. Lett. 2015, 44, 812–814 | doi:10.1246/cl.150153
© 2015 The Chemical Society of Japan