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Chemistry Letters Vol.35, No.8 (2006)
Glucose-specific Sensing with Boronic Acid Utilizing Enzymatic Oxidation
Ã
Yasumasa Kanekiyo and Hiroaki Tao
National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569
(Received May 15, 2006; CL-060569; E-mail: y.kanekiyo@aist.go.jp)
A novel sensing system for glucose-specific detection has
1
1
1
1
1
1
.6
.5
.4
.3
.2
.1
1
been established based on a combination of a boronic acid and
enzymes. To overcome the inherently low biding affinity of glu-
cose toward boronic acids, glucose is converted into gluconic
acid by the enzymatic reaction using glucose oxidase (GOx),
and then complexed with a fluorescent boronic acid through
the ꢀ-hydroxycarboxylate moiety. According to the present
strategy, glucose concentration is exclusively determined among
other saccharides in aqueous solutions.
Fructose
Gluconic acid
Glucose
Sucrose
no saccharide
0
.9
Growing number of diabetic prompts researchers to develop
glucose-specific sensing systems for the diagnosis and treatment
of patients. Since 1990’s, boronic acid-based sensory system for
saccharides has been extensively studied by the group of Shinkai
3
4
5
6
7
8
9
10
11
pH
ꢁ
Figure 1. Plots of I351=I359 vs pH for 1 at 25 C; [1] = 1 mM,
[saccharide] = 10 mM, ꢁex: 298 nm.
1
and others. These research have proven that boronic acid is a
powerful tool for the molecular recognition of saccharides in
aqueous systems. However, significant drawbacks exist in the
boronic acid-based system, that is, 1; binding affinity toward glu-
cose is the lowest among major saccharides and 2; complexation
with glucose scarcely takes place at physiological pH. Actually,
the stabilities of phenylboronic acid–saccharide complexes
acidic conditions where pH value is between pKa of ꢀ-hydroxy-
carboxylic acids (typically 2.0–4.0) and that of boronic acids. It
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occurred to us that the reaction between boronic acids and ꢀ-
hydroxycarboxylic acids would be useful for glucose-specific
sensing by combining with a selective oxidation process of
glucose converting into gluconic acid, an ꢀ-hydroxycarboxylic
acid.
are in the order: fructose ꢀ galactose > mannose > glucose
2
(
Supporting Information, Table S1). Since glucose is the most
important saccharide in biological systems as an energy source
and hence the primary target for saccharide sensing, the above
mentioned drawbacks greatly limit the usefulness of boronic
acid-based glucose sensing system. To enhance the binding
affinity toward glucose, a series of diboronic acid receptors have
been synthesized. Since glucose has a unique tendency to form
Figure 1 shows the pH profile of the complexation between
9-phenanthreneboronic acid (1) and saccharides in aqueous solu-
tions. Fluorescence spectrum of 1 is significantly changed upon
addition of saccharides, which is attributable to complexation
with saccharides (Supporting Information, Figure S1). Since
the increase in fluorescence intensity at 351 nm is much larger
than that at 359 nm, one can accurately evaluate the complex for-
1:2 complexes in which one glucose molecule is bound by two
3
7
boronic acid moieties, the diboronic acid receptors show fairly
good binding selectivity toward glucose.4
mation by ratiometric measurements (I351=I359). We chose exci-
tation wavelength at 298 nm in order to avoid interferences by
the absorption in shorter wavelength arising from enzymes (Sup-
porting Information, Figure S2). It is seen from Figure 1 that the
complexation behavior of glucose and gluconic acid are totally
different from each other: glucose reacts with 1 only at alkaline
pH, whereas gluconic acid reacts throughout the pH range. This
observation clearly indicates that the binding mode between 1
and saccharides changes depending on pH. In alkaline solutions,
diol unit in both glucose and gluconic acid is responsible for the
binding. In contrast, ꢀ-hydroxycarboxylate unit in gluconic acid
should be the binding site under acidic conditions. Fructose, not
having ꢀ-hydroxycarboxylic acid unit, shows some response
even at neutral pH due to its higher binding affinity toward
boronic acids, but it looses responsiveness at acidic pH. Sucrose,
a disaccharide composed of glucose and fructose, shows no
response even at alkaline pH because it has no cis-diol unit
suitable for complexation. Thus, the result shown in Figure 1
suggests the possibility of glucose-specific detection by selec-
tively converting glucose into gluconic acid. The titration
curve of 1 with gluconic acid at pH 4.7 shows a saturation type
We here report an alternative strategy for a glucose-specific
boronic acid-based sensing system utilizing an enzymatic oxida-
tion reaction of glucose. As illustrated in Scheme 1, boronic
acids reversibly react with diols to form cyclic esters, and the
reaction takes place preferentially at alkaline pH where free
boronic acids take the anionic boronate form. Since pKa values
of boronic acids are usually around 9.0, glucose shows very
weak or no response against boronic acids when pH is equal to
or lower than 7.0. It is noteworthy that boronic acids can bind
ꢀ
-hydroxycarboxylic acids under physiological and weakly
O
O
OH
O
O
O
O
HO
OH
HO
OH
B
B
OH-
H+
B
B
OH HO
O
HO
OH
OH
Scheme 1. Complex formation equilibria of boronic acids with
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diols and ꢀ-hydroxycarboxylic acids.
Copyright ꢀ 2006 The Chemical Society of Japan