A new highly fluorescent probe for monosaccharides based on a donor-acceptor diphenyloxazole

Article abstract of DOI:10.1039/b106763j

A diphenyloxazole substituted with a dimethylamino and a boronic acid group showing intramolecular charge transfer in the excited state undergoes large spectral changes in the presence of monosaccharides.

Full text of DOI:10.1039/b106763j

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A new highly fluorescent probe for monosaccharides based on a
donor–acceptor diphenyloxazole
Nicolas DiCesare and Joseph R. Lakowicz*
Center for Fluorescence Spectroscopy, University of Maryland, School of Medicine, 725 W. Lombard
St., Baltimore, Maryland 21201, USA. E-mail: cfs@umbi.umd.edu; Fax: +1 410 706-8408
Received (in Covallis, OR, USA) 18th July 2001, Accepted 13th August 2001
First published as an Advance Article on the web 12th September 2001
A diphenyloxazole substituted with a dimethylamino and a
boronic acid group showing intramolecular charge transfer
in the excited state undergoes large spectral changes in the
presence of monosaccharides.
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl chlo-
ride, obtained from the commercially available 4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)benzoic acid refluxed in
SOCl2, following by the dehydratation of the product in POCl3.9
Donor–acceptor derivatives of diphenyloxazole are well known
to show high fluorescence quantum yields, long wavelength
emission and to be very sensitive to small variations affecting
the ICT properties of the excited state.9 In this case, the ICT
state is between the boronic acid, the electron-withdrawing
group, and the N,N-dimethylamino group, the electron-donating
group. As the boronic acid group changes to its anionic form, 2
in Scheme 2, the electron-withdrawing properties of the boron
are removed and then the ICT is affected.6 As the pH increases,
we observe a blue shift (results not shown) and a significant
increase in the fluorescence intensity. The emission band of the
neutral form 1 appears at 557 nm with a fF value of 0.03, on the
other hand, the emission of the anionic form 2 appears at 488 nm
with a fF value of 0.95. These important spectral changes are
interpreted by the loss of the ICT property for the anionic form
2. The intensity change following the pH change is shown in
For a decade, the development of synthetic probes for the
recognition and analysis of sugar has attracted much attention.1
Synthetic probes could find useful applications in the food
industry as well as in clinical analysis. Detection and monitor-
ing of glucose is particularly important for diabetics.2 The
development of synthetic sensors could be complementary to
the present technology of glucose testing by using enzymes.3
For example, the use of enzymes shows some limitations in the
development of implantable sensors for continuous glucose
monitoring in blood or in interstitial tissue. Continuous
monitoring of glucose blood level is very important for the long
term health of the diabetics and synthetic probes could lead to
improvements in medical technology such as alarm systems for
hypoglycemia and a control device for an insulin pump.
The boronic acids have been known for a few decades for
their ability to interact with diols.4 They have been used for the
development of receptor and fluorescent probes for sugars.1,5–7
The advantages of using boronic acid as chelator group for
sugars are the fast and reversible interaction with sugars. In
addition, many substituted phenylboronic acids are commer-
cially available allowing the development of a large diversity of
synthetic fluorescent probes for sugars with minimal synthetic
steps. Different mechanisms have been used to induce spectral
change following the interaction of the boronic acid and the
sugars. Among these mechanisms, the use of intramolecular
charge transfer (ICT) involving the boronic acid is very
promising.6,7 ICT is well known to be very sensitive to small
perturbations8 that can result in spectral shifts, intensity changes
and/or lifetime changes. In addition, ICT can be applied to a
very large diversity of fluorophores with no limitation of the
wavelength range and/or lifetime of the fluorophore.
In an attempt to develop highly effective fluorescent probes
for glucose, we synthesized compound 1 (Scheme 1). Com-
pound 1† is readily synthesized from the reaction between the
2-amino-4A-dimethylaminoacetophenone hydrochloride‡ and
Scheme 1 Synthetic route to 1.
Scheme 2 Equilibrium involving pH and sugars.
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Chem. Commun., 2001, 2022–2023
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b106763j

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Fig. 3 Titration curves of 1 against sugars, measured in phosphate buffer–
Fig. 1 Titration curves of 1 against the pH in absence and presence of sugars,
lex = 350 nm.
methanol (2+1 v/v) at pH 7.0, lex = 350 nm. Probe concentration: 5 3 1026
M.
Fig. 1. Nearly a 30-fold increase in the fluorescence intensity
can be observed at 485 nm.
values obtained with donor–acceptor chromophores involving
the boronic acid group.6,7 The higher affinity of the mono-
boronic acid 1 for fructose in comparison with glucose and the
high concentration range of practical usefulness of 1 for glucose
would not be suitable for glucose sensing in biological samples
and/or in the presence of fructose but could find applications in
the food industry and/or in fermentation industry where high
concentrations of glucose are used.
In addition to the observed changes in the steady-state
emission properties of 1, we have also observed changes in the
fluorescence lifetime of the probes. The neutral structure, 1,
shows a monoexponential fluorescence decay with a lifetime of
1.7 ns while the anionic form 2 possesses a lifetime of 3.7 ns,
also monoexponential. At pH 7, the presence of fructose
changes the fluorescence lifetime of the probe from 2.8 to 3.6
ns. Fluorescence lifetime changes are useful for sensing and
monitoring since they are independent of the total intensity and
independent also from the power of the excitation source and
the concentration of the probe.10
Usually, the effects of sugars are observed since the boronic
acid–sugar complex (structure 3 from Scheme 2) has a lower
pKa than the uncomplexed boronic acid. At a selected pH, it is
possible to have a predominance of the neutral form (1) in
absence of sugar and a predominance of the anionic form (4) in
presence of sugar. This conformational change of the boron
group, induced by the presence of sugar, is the origin of the
spectral changes observed. Titration curves of 1 in presence of
the sugar are displayed in Fig. 1. The observed pKa of 1 is 7.8
in absence of sugar, pKas of 5.6 and 6.5 are obtained in presence
of fructose and glucose, respectively. Maximum changes
between the titration curves with and without sugar are obtained
at pH 6.5 and 7.0 for fructose and glucose, respectively. Since
the spectral changes induced by sugar are not so different
between these two pH values, we performed our measurements
at pH 7.0 for all saccharides. It is also interesting to note that
large spectral changes could also be observed at a pH higher
than 9.0 as seen in Fig. 1. This suggests that probe 1 could also
be used for monitoring sugar at high pH.
The effect of fructose on the emission band of 1 is displayed
in Fig. 2. As observed for the pH, the presence of the sugar
induces a blue shift and an increase in the fluorescence intensity.
An isosbestic point is observed at 615 nm showing the
equilibrium of the two conformations, the neutral and anionic
form of the boronic acid. The same isosbestic point was
observed in the pH effects on the emission band. The increase of
the emission intensity is about 5-fold in the presence of fructose
and about 3-fold in the presence of galactose and glucose.
Titration curves of 1 against sugars are displayed in Fig. 3.
Dissociation constants (KD) of 1 were calculated at 1.9 0.1
mM for fructose and 14 1 and 37 3 mM for galactose and
glucose, respectively. KD values are comparable to previous
The authors wish to acknowledge the Juvenile Diabetes
Foundation, 1-2000-546, and the NIH National Center for
Research Resources, RR-08119, for financial support.
Notes and references
† Selected data for
1 5-(4-dimethylaminophenyl)-2-[4-(dihydroxybor-
anyl)phenyl]oxazole: yellow solid, yield 48%, dH(300 MHz; CD3OD) 2.83
(6 H, s), 6.59 (2H, d), 7.05 (1H, s), 7.40 (2H, d), 7.52 (1H, s), 7.67 (1H, s)
and 7.82 (2H, s).
‡ Synthesised from 4A-dimethylaminoacetophenone (TCI America) accord-
ing to the standard procedure described in the litterature.11,12
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Fig. 2 Effect of fructose on the emission of 1, measured in phosphate
buffer–methanol (2+1 v/v) at pH 7.0, lex = 350 nm. Probe concentration:
5 3 1026 M.
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