Fluorescent sensing of uronic acids based on a cooperative action of boronic acid and metal chelate

Article abstract of DOI:10.1039/a703471g

A new fluorescent molecular sensor for uronic acids is designed, which features two-point interactions of boronic acid-diol complexation and zinc(II)-carboxylate coordination.

Full text of DOI:10.1039/a703471g

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Fluorescent sensing of uronic acids based on a cooperative action of boronic
acid and metal chelate
Masayuki Takeuchi, Masashi Yamamoto and Seiji Shinkai*
Department of Chemical Science & Technology, Faculty of Engineering, Kyushu University, Fukuoka 812, Japan
A new fluorescent molecular sensor for uronic acids is
designed, which features two-point interactions of boronic
acid–diol complexation and zinc(II)–carboxylate coordina-
tion.
absorption spectra did not change further at higher pH. Using
294 nm as the excitation wavelength we measured the
fluorescence spectra. Fig. 1 shows plots of the fluorescence
intensity (lmax at 375 nm) vs. pH. In the absence of saccharide,
the fluorescence intensity increase at pH 2–6 corresponds to
deprotonation of the 1,10-phenanthroline moiety, which again
gives pKa1 4.4. The fluorescence intensity decrease starting
from pH 5.5 and pH 10 corresponds to deprotonation of the
tertiary amine (pKa2) and OH2 adduct formation with the
boronic acid group (pKa3), respectively. Because of the overlap
of the dissociation groups the pKa2 could be only approximately
estimated to be ca. 7, whereas the pKa3 could not be determined
accurately. In the presence of saccharide ([d-fructose] = 100
mmol dm23, Fig. 1), the fluorescence intensity is increased at
pH 6–11. This is due to the B–N interaction intensified by
complexation of saccharide with the boronic acid group.6 The
association constants (Kass) in the absence of ZnII were
estimated from plots of the fluorescence intensity against the
saccharide concentration. The results are summarized in
Table 1.
Saccharides are nature’s conveyors of energy and play many
crucial roles in metabolic cycles. Uronic acids are known to
appear in the oxidation process of monosaccharides or in the
biosynthetic process of l-ascorbic acid. The quantitative
analysis of uronic acids has so far been accomplished by
classical colorimetic methods such as naphthoresorcine–HCl1
and carbazole–H2SO4.2 Obviously, it seems very important to
develop a new analytical method which is useful under
physiological conditions and in addition, shows high selectivity
and sensitivity towards uronic acids in the presence of
concomitant neutral saccaharides. We have recently demon-
strated the usefulness of the boronic acid function as a
saccharide receptor in a saccharide recognition system.3
Judging from the structure of uronic acids, molecular design
including both a boronic acid moiety for diol binding and a
metal chelate moiety for carboxylate binding within a molecule
should lead to an excellent uronic acid receptor. To achieve high
sensitivity it is desirable to combine this system with the
concept of a photoinduced electron-transfer (PET) sensor.4,5
We previously designed compound LA which selectively binds
1,2- and 4,6-diols of glucose with two boronic acids and reports
the binding event by a fluorescence change in the PET
mechanism.6 Here, we have rationally designed a molecular
sensor L for uronic acids in which one boronic acid group in LA
is replaced by a metal chelation site. We have found that the
zinc(ii) complex of this new molecular sensor selectively
responds to uronic acids in neutral aqueous solution.
Addition of ZnII [added as Zn(NO3)2·6H2O] decreased the
absorbance in the UV region of the absorption spectrum and the
visible region of the fluorescence spectrum and the final spectra
were very similar to those of H2L2+. The results suggest that
ZnII is primarily associated with the phenanthroline moiety.
Since the spectral changes were saturated at [ZnII]/[L] = 4.0,
we fixed the ratio to this value and then added saccharides. The
fluorescence intensity increased with increasing saccharide
concentration and was saturated at high concentration. From the
HO OH
B
OH
HO
B
i
N
N
N
N
N
N
Me
Me
Me
L′
ii
Br
N
OH
HO
B
N
N
N
N
N
N
Me
iii
L
Br
The synthesis of L was achieved from 2-(p-tolyl)-1,10-
phenanthroline7 by the route outlined in Scheme 1. The product
(mp 166–169 °C) was identified by IR and 1H NMR
spectroscopic evidence and elemental analysis.
O
B
HN
OH
B
N
Me
O
Me
HO
Since the water solubility of L was not good, we used a mixed
medium of water–MeOH (1:2 v/v) to avoid self-aggregation.
The absorption maximum of L shifted from 301 to 290 nm upon
a pH change from 2.0 to 7.5 with a tight isosbestic point (at 294
nm). This change corresponds to deprotonation of the chromo-
phoric 1,10-phenanthroline moiety (pKa1 4.4, Scheme 2). The
L
Scheme 1 Reagents and conditions [yields]: i, NBS, AIBN, CCl4, reflux; ii,
MeNH2, CCl4 [30%, calculated from 2-(p-tolyl)-1,10-phenanthroline]; iii,
K2CO3, MeCN, reflux [40%]
Chem. Commun., 1997
1731

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a
Table 1 Association constants (log Kass
)
for saccharides with L and
[ZnIIL]2+
Log Kass (Imax/I0)
N
N
N
N
H
[ZnIIL]2+
+
Saccharide
L
pKa1
b
d-Glucuronic acid
d-Galacturonic acid
Sialic acid
(N-Acetylneuraminic acid)
d-Glucose
—
3.4 (2.11)
3.1 (3.69)
1.9 (1.15)
+
+
HO
B
HO
B
HN Me
HN Me
b
—
—
2.3 (1.83)
HO
HO
b
b
—
d-Galactose
d-Fructose
1.7 (1.12)
2.5 (1.33)
1.3 (1.50)
2.4 (1.90)
H2L2+
HL+
a pH = 8.0 (5.0 mmol dm23 MOPS buffer), water–MeOH (1:2 v/v). b Too
small to determine (log Kass < 1).
pKa2
N
N
N
N
complexes with [ZnIIL]2+: lmax and [q] = 305 nm and 4.0 3
103 ° cm22 dmol21 for d-glucuronic acid and 282 nm and 28.0
3 103 ° cm22 dmol21 for d-galacturonic acid. Job plots using
the CD intensity provided a maximum at [ZnL2+]/ ([ZnL2+] +
[uronic acid]) = 0.50, indicating that the stoichiometry of these
complexes is 1:1.
In conclusion, the present study demonstrates a new
saccharide receptor for uronic acids, which features two-point
interactions of boronic acid–diol complexation and zinc(ii)–
carboxylate coordination. The receptor can discriminate well
between uronic acids and neutral monosaccharides and the
binding event can be sensitively read out by a fluorescence
change. We believe that this is a new molecular design concept
utilizing the cooperative action of boronic acid and the metal
chelate. Further applications are continuing in this laboratory.
This work was supported by a Grant-in-Aid for COE
Research ‘Design and Control of Advanced Molecular Assem-
bly Systems’ from the Ministry of Education, Science and
Culture, Japan (#08CE2005).
pKa3
HO
HO
B
N
Me
N
Me
HO
–
B
HO
HO
L(OH)–
L
Scheme 2
analysis of the plots the Kass values in the presence of ZnII were
determined and are summarized in Table 1.
Examination of Table 1 reveals that as expected,6 d-fructose
gives the largest Kass with L in the absence of ZnII. On the other
hand, uronic acids are scarcely bound to L (except d-
galacturonic acid with Kass = 80 dm3 mol21). In contrast, the
Kass values for three uronic acids are remarkably improved in
the presence of ZnII whereas those for three neutral monosac-
charides are scarcely affected. The difference clearly supports
the view that uronic acids are bound to [ZnIIL]2+ by a
cooperative action of boronic acid–diol complexation and
zinc(ii)–carboxylate coordination.
Footnote and References
* E-mail: seijitcm@mbox.uc.kyushu-u.ac.jp
1 S. S. Cohen, J. Biol. Chem., 1953, 201, 71 and references therein.
2 Z. Dische, J. Biol. Chem., 1953, 204, 983 and references therein.
3 For comprehensive reviews see: T. D. James, P. Linnane and S. Shinkai,
Chem. Commun., 1996, 281; T. D. James, K. R. A. S. Sandanayake and
S. Shinkai, Angew. Chem., Int. Ed. Engl., 1996, 35, 1910.
4 A. P. de Silva, H. Q. N. Gunarathe and C. P. McCoy, Chem. Commun.,
1996, 2399; A. P. de Silva, H. Q. N. Gunaratne, C. McVeigh,
G. E. M. Maguine, P. R. S. Maxwell and E. Q’Hanlon, Chem. Commun.,
1996, 2191; A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson,
C. P. McCoy, P. R. S. Maxwell, J. T. Rademacher and T. E. Rice, Pure
Appl. Chem., 1996, 68, 1443.
It is known that when saccharides interact with the receptor at
two points to form a cyclic structure, the resultant complexes
become CD-active.6,8 In fact, d-glucuronic acid and
d-galacturonic acid with large Kass values gave CD-active
1500
without saccharide
D-fructose
5 A. W. Czarnik, Acc. Chem. Res., 1994, 27, 302; Fluorescent Chem-
osensors for Ion and Molecular Recognition, ed. A. W. Czarnik, ACS
Books, Washington, 1993.
1000
6 T. D. James, K. R. A. S. Sandanayake and S. Shinkai, Angew. Chem., Int.
Ed. Engl., 1994, 33, 2207; T. D. James, K. R. A. S. Sandanayake,
R. Iguchi and S. Shinkai, J. Am. Chem. Soc., 1995, 117, 8982. Although
the distance between the phenanthroline and the amino group is
apparently farther than that in LA, the fluorescence change is as large as
that in LA. Presumably the 2-phenyl group is included in the fluoro-
phore.
I
500
7 M. S. Goodman, A. D. Hamilton and J. Weiss, J. Am. Chem. Soc., 1995,
117, 8447.
8 T. Imada, H. Kijima, M. Takeuchi and S. Shinkai, Tetrahedron Lett.,
1995, 36, 2093; Tetrahedron, 1996, 52, 2817.
0
0
2
4
6
8
10
12
pH
Fig. 1 Fluorescence intensity vs. pH profile of L (1.00 3 1025 mol dm23);
25 °C [d-fructose] = 0 or 100 mmol dm23, lex = 294 nm
Received in Cambridge, UK, 20th May 1997; 7/03471G
1732
Chem. Commun., 1997