The first fluorescent sensor for D-glucarate based on the cooperative action of boronic acid and guanidinium groups

Article abstract of DOI:10.1039/b300098b

A new fluorescent sensor (1) with a recognition unit consisting of a boronic acid moiety and a guanidinium unit shows selective binding of D-glucarate in aqueous solution.

Full text of DOI:10.1039/b300098b

The first fluorescent sensor for -glucarate based on the cooperative
D
action of boronic acid and guanidinium groups
Wenqian Yang, Jun Yan, Hao Fang and Binghe Wang*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
E-mail: binghe_wang@ncsu.edu; Fax: 1-919-515-3757; Tel: 1-919-515-2948
Received (in Columbia, MO, USA) 7th January 2003, Accepted 4th February 2003
First published as an Advance Article on the web 17th February 2003
A new fluorescent sensor (1) with a recognition unit
length and functional group orientation for binding with
glucarate (Fig. 1).
consisting of a boronic acid moiety and a guanidinium unit
shows selective binding of
D-glucarate in aqueous solution.
The sensor (1)† was synthesized starting with the conversion
of aldehyde 4 to amine 5 through reductive amination (Scheme
1).6k Then the boronic acid moiety and guanidine group were
attached to give 7. Boc-deprotection using HCl gave 1 in about
10% overall yield, which was purified by precipitation from
methylene chloride with the addition of ether. As controls for
the binding studies, compounds 2 and 3 were also synthesized in
similar fashions.
The design of compounds that are capable of selective
recognition of molecules and ions is of great interest in
bioorganic chemistry.1
D-Glucarate is a biologically active
carbohydrate that exists in human serum, vegetables, and
fruits.2 It has been used as a chemopreventive agent in certain
cancers.3 Moreover, the glucarate catabolic pathway is an
attractive field that has been studied extensively.4 The analysis
Fluorescence experiments were conducted to evaluate the
of
D
-glucarate has been achieved by using enzymes.5 Analysis
binding of the sensor (1) and control compounds (2, 3) for -
D
using fluorescent sensors, on the other hand, offers the
advantages of convenience and high sensitivity. Herein, we
wish to report our efforts in developing the first fluorescent
glucarate and other related saccharides in order to examine their
selectivity and sensitivity. Upon addition of glucarate the
fluorescence intensity of the sensor (1) solution indeed
increased by about 4.5-fold at 20 mM (Fig. 2). On the other
hand, control compound 3 did not exhibit any fluorescence
intensity changes upon addition of glucarate (Fig. 3), indicating
that interaction with the guanidinium group alone was not
enough to affect the fluorescence intensity of the sensor. To
examine whether sensor 1 works as designed, it is important to
compare the affinity and selectivity of 1 with the monoboronic
acid compound 2. As expected, 2 also showed fluorescence
intensity changes when glucarate was added. However, the
magnitude of the changes was smaller (Fig. 3).
sensor for -glucarate.
D
Our design uses a two binding site approach with a boronic
acid and a guanidinium recognition moiety separated by a
spacer (1). Such a design takes advantage of the reversible
formation of cyclic esters of boronic acid with diols in aqueous
media6,7 and the known strong interactions between a guanidin-
ium moiety and carboxylate.8 The key in this design is the
appropriate linker length and rigidity, and the relative orienta-
tions of the two recognition moieties. As for the fluorescent
reporter group, an anthracene group was chosen because its
fluorescence intensity can be regulated by a photoelectron
transfer process.6a,b Molecular modelling studies seem to
indicate that attachment of a boronic acid moiety and a
guanidinium group to the 9,10 positions of the anthracene ring
could afford a sensor compound with the appropriate linker
The stability constants of sensors 1 and 2 with different
saccharides were determined in order to quantitatively measure
the effect of the added guanidinium group on the selectivity and
affinity of 1 for glucarate. From Table 1, it can be seen that
Fig. 1 Energy-minimized structure of 1–glucarate complex. The calculation
was carried out using Spartan ’02. Conformational search was conducted
using the MMFF molecular mechanics force field. Conformers within 10
kcal mol21 of the lowest energy conformation were further optimized with
AM1 semi-empirical calculation. The boronic acid was postulated to bind
with 3,4-diol of
D
-glucarate.9 The B–N bond length was set to 1.75 Å based
Scheme 1 Reagents and conditions: i, MeOH, THF, MeNH2 (40%, wt),
then NaBH4, 90%; ii, CH3CN, 8, K2CO3, 60%; iii, 1,3-bis(tert-butox-
ycarbonyl)guanidine, DEAD, Ph3P, 33%; iv; 4 N HCl, dioxane, 55%.
on crystal structural evidence.10 Hydrogen bonds are shown as broken
lines.
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CHEM. COMMUN., 2003, 792–793
This journal is © The Royal Society of Chemistry 2003

nitrogens of the guanidinium group, assuming the boronic acid
bind to glucarate via the 3,4-diol (Fig. 1).9
There are literature precedents that two identical binding
units properly arranged in a synthetic receptor can be used for
the selective recognition of various targets.6d,k,l Compared with
using two identical binding units, the design and synthesis of a
synthetic receptor with two different binding sites is more
challenging. Only a few fluorescent sensors containing two
different binding sites to capture a single species have been
reported so far.11 To the best of our knowledge, compound 1 is
the first fluorescent receptor for -glucarate, which features
D
two-point interactions via boronic acid–diol complexation and
guanidinium–carboxylate recognition. The receptor can dis-
Fig. 2 Fluorescence spectra of 1 (1.0 3 1025 M) upon addition of
D
-
criminate well between
D
-glucarate and other structurally
glucarate (0, 0.01, 0.1, 1, 2, 5, 10, 20 mM) at 25 °C in 50% MeOH/0.1 M
aqueous HEPES buffer at pH 7.4, lex = 370 nm.
similar carbohydrates and the binding event results in sig-
nificant fluorescence changes. Further work utilizing the
concept of multi-point interactions for the design of sensors for
various analytes is under way.
Financial support from the National Institutes of Health
(NO1-CO-27184 and CA88343) and North Carolina Bio-
technology Center (2001ARG0016) is gratefully acknowl-
edged.
Notes and references
† Selected data for 1: Yield 55%. 1H NMR (CD3OD, 300 MHz) d 8.45–8.40
(m, 4H), 7.82–7.56 (m, 8H), 5.45 (s, 2H), 5.41 (s, 2H), 4.73 (s, 2H), 2.77 (s,
3H). MS-ESI: 427.3 (M+ + H). HRMS (ESI) calcd. for C25H27BN4O2 (M+
+
H) 427.2305; found 427.2297. Anal. calcd. for C25H27BN4O-
2·1.4(C4H10O)·2.3HCl: C, 59.78; H, 7.10; N, 9.11. Found: C, 59.82; H, 6.73;
N, 9.09%.
Fig. 3 Relative fluorescence intensity changes of (/) 1, (-) 2 and (:) 3 as
a function of
-glucarate concentration at 25 °C, 1.0 3 1025 M of 1, 2 and
D
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3 in 50% MeOH/0.1 M aqueous HEPES buffer at pH 7.4, lex = 370 nm, lem
= 424 nm.
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compound 2 with only the boronic acid binding site shows the
strongest affinity with
monoboronic acid compounds.7 The order of selectivity for
monoboronic acid 2 is: -sorbitol > -glucarate ≈ -gluconate
-glucuronic acid ≈ -glucose, which reflects the intrinsic
D
-sorbitol, as would be expected with
D
D
D
>
D
D
affinity of a monoboronic acid unit for various sugars.
Compound 1 with two different binding sites, on the other hand,
showed the highest affinity for
all the structurally similar saccharides tested (Table 1). The
binding constant of 1 with -glucarate is increased by about
D-glucarate, as designed, among
D
5-fold compared with that of the monoboronic acid compound
(2), presumably due to the added guanidinium group in 1 for the
recognition of the carboxylate groups of glucarate. Such results
can only be attributed to the cooperative action of boronic acid
and guanidinium units in affording the specific recognition of
glucarate, and, therefore, indicate that 1 binds with glucarate in
a two-point binding mode and the appropriate linker length and
rigidity in 1 afford the selectivity for glucarate. Furthermore, the
fact that glucarate binds more tightly than glucuronic acid
indicates that both carboxylates of glucarate are involved in the
binding, presumably through interaction with the guanidinium
group. This is also consistent with the molecular modelling
studies. As discussed earlier, computer molecular modelling
results indicate that in the lowest energy conformation, the two
carboxylate groups of glucarate can interact with the protonated
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Table 1 Stability constants (M21) for the saccharides with 1 and 2
Saccharide
1
2
D
D
D
D
D
-Glucarate
-Gluconate
-Sorbitol
-Glucuronic acid
-Glucose
5142 ± 267
1452 ± 142
1300 ± 78
46 ± 5
846 ± 82
670 ± 39
1647 ± 141
27 ± 3
62 ± 9
18 ± 3
CHEM. COMMUN., 2003, 792–793
793